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PMC548514	Pharmacologic stress echocardiography is an established cost-effective technique for the detection of coronary artery disease \[[@B1]\]. The widespread use in the clinical practice has become possible only after evidence collected through large scale multicenter studies that demonstrated its feasibility, safety, diagnostic and prognostic accuracy \[[@B4]-[@B8]\]. According to the guidelines of ACC/AHA -- pharmacological stress echocardiography with either dobutamine or dipyridamole is a class I indication (of documented effectiveness and usefulness) for the diagnosis of coronary artery disease and for the prognostic stratification of patients with known coronary artery disease \[[@B2],[@B3]\]. Its major limitation is related to a high inter-observer variability and to operator-dependent expertise that might be overcome by an appropriate training and the use of strict reading criteria \[[@B9]-[@B11]\]. Nonetheless the hunt for an objective, operator-independent technique to be applied to the conventional black and white regional wall motion analysis remains a major goal in stress echocardiography. Tissue Doppler Imaging (TDI) provides a quantitative analysis of regional myocardial function through the analysis of myocardial velocities \[[@B12],[@B13]\]. Since velocity imaging is confounded by influence from velocities in other segments, the TDI -- based modalities strain and strain rate imaging have been introduced to measure regional shortening fraction and shortening rate, respectively \[[@B14]\] Is the application of Tissue Doppler Imaging to stress echocardiography the technique that will solve it all? According to major journals the answer is yes: the diagnostic accuracy of stress echocardiography improves with TDI when analyzed in comparison with visual assessment of wall motion analysis for the detection of inducible ischemia. Inducible ischemia quantified in a number without the approximations of visual assessment. However the enthusiasm showed by some investigators is not substantiated by scientific results. In fact, a careful analysis of the data published so far raises more doubts than certainties. What we talk about when we talk about TDI ========================================= The TDI modalities include myocardial velocity imaging, displacement imaging, strain rate imaging and strain imaging. TDI measures velocities by the Doppler shift of reflected ultrasound. Velocities are measured in the conventional imaging planes, from apical views as longitudinal velocities and from parasternal views as radial velocities. When we employ TDI, the velocities within a myocardial segment are the net result of motion caused by contractions in that segment, motion due to tethering to other segments, and overall motion of the heart. This tethering effect is the reason why longitudinal velocities increase progressively from the apex toward the base, when measured in an apical window. Therefore ischemia in the apical region reduces myocardial velocities not only in the apex, but also in the nonischemic basal segments \[[@B15]\]. In practical terms, the reduction of TDI velocities in basal segments is not synonymous of reduction of function in the same segments. The opposite effect, tethering of nonischemic segments might induce increase in velocity of adjacent ischemic segments. These limitations could be overcome by the employment of strain and strain rate. Strain rate reflects how fast regional myocardial shortening or lengthening occurs measured at two locations separated by a distance. This is the reason why some authors define strain rate as a spatial velocity gradient. Strain is calculated as the time integral of strain rate and is a dimensionless quantity. The limitations of TDI have been widely outlined \[[@B16]-[@B18]\] and this is beyond the scope of the present review but they may be synthesized into two main problems: 1 -- the amplitude of the estimated velocity is dependent on the angle at which the region is imaged; 2 -- the overall cardiac motion, rotation, and contraction of adjacent segments will influence regional velocity estimates. Therefore, a more critical approach to this technology would have avoided the inconsistencies of scientific results when it was applied in the clinical arena. TDI and Stress echocardiography for myocardial ischemia detection ================================================================= Feasibility studies have been published demonstrating the applicability of TDI to stress echocardiography \[[@B19]-[@B27]\] but only few studies addressed the issue of its diagnostic accuracy in a clinical context \[[@B28]-[@B31]\]. Cain et al \[[@B28]\] applied myocardial Doppler velocity to dobutamine stress echocardiography in order to assess its diagnostic accuracy when compared to conventional visual assessment. They first identified the normality ranges of myocardial velocities in patients with normal coronary arteries or with a very low pretest probability of having coronary artery disease. Then they selected 114 patient with coronary artery disease assessed at coronary angiography and evaluated the diagnostic accuracy: see Table 1 ([Additional file 1](#S1){ref-type="supplementary-material"}). Neither overall nor vascular territory accuracy was better for myocardial velocity when compared to visual wall motion scoring. The MYDISE Study \[[@B29]\] was the first multicenter study on the absolute value of TDI applied to dobutamine stress echocardiography. The study enrolled 289 patients separated in 3 groups: group 1 (n = 92) healthy volunteers or patients with normal coronary arteries, group 2 (n = 48) patients with known coronary artery disease and group 3 (n = 149) consecutive patients with known or suspected coronary artery disease. Exclusion criteria were: atrial fibrillation, previous myocardial infarction (Q waves on the electrocardiogram, or akinetic segments on the resting echocardiographic images), previous revascularization, unstable angina, complete bundle branch block, significant heart valve disease, contraindication to dobutamine or atropine). The diagnostic criteria were developed by comparing 92 normal subjects with 48 patients with coronary artery disease and applied in a prospective series of 149 patients referred to stress echo laboratory for the suspect of coronary artery disease. Velocity cut-off points were tested and discarded since they did nor perform well when compared to logistic regression models, using systolic velocities at peak stress in 7 myocardial segments and after adjusting for heart rate, age and gender \[[@B29]\]. The main concerns refer to strict stress echocardiographic issues:1.the lack of a comparison between conventional visual assessment of regional wall motion and TDI analysis. In absolute terms the diagnostic accuracy is not striking: see Table 1 ([Additional file 1](#S1){ref-type="supplementary-material"}) and Fig [1](#F1){ref-type="fig"}. Even if we accept the hypothesis of a non-inferiority analysis of TDI versus dobutamine stress echocardiography we have to take into consideration some major limitations outlined by the authors: the optimal diagnostic accuracy was obtained by using peak systolic velocity after adjusting for maximal heart rate, age and gender: \"ignoring these factors reduces both sensitivity and specificity\" \[[@B29]\]. Moreover, authors applied a very complex regression model for diagnostic accuracy assessment. A recent meta-analysis on dobutamine stress echocardiography showed an overall sensitivity of 80% and a specificity of 87% \[[@B32]\] (see fig [2](#F2){ref-type="fig"}). 2 -- The extent and severity of myocardial ischemia as defined by the number of ischemic segments and the pharmacologic load is never provided. The protocol was interrupted only in the presence of secondary criteria, but never for development of myocardial ischemia since the quantitative analysis was performed off-line. It has been demonstrated that diagnostic and prognostic accuracy of stress echocardiography increases when the response is stratified in the time and space domain, i.e. number of ischemic segments, severity of ischemia induced, the time of onset of ischemia and the pharmacologic dose. 3 -- the apical segments have been excluded by the analysis since the systolic velocity is not reliable making the analysis possible only in 11 segments. Nonetheless, the apex and the apical segments are very often the site of stress echocardiographic positivity unless very proximal atherosclerotic lesions are present 4 -- the time for performing analysis is never reported. We are informed that the comparison between systolic velocities at rest and at peak stress is disregarded since it is time consuming and increases the potential for observer variability without increasing diagnostic accuracy. 5 -, apparently, TDI cannot be applied to patients with wall motion abnormalities at rest. The exclusion of this group of patients makes this quantitative approach quite unfeasible for routine clinical application. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Sensitivity and specificity of dobutamine stress echocardiography ::: ![](1476-7120-3-2-2) ::: Voigt et al. \[[@B30]\] used a more realistic approach to the application of TDI to stress echocardiography. They first demonstrated in 44 patients with known or suspected coronary artery disease that strain rate quantitatively differentiates ischemic and nonischemic regional myocardial response to dobutamine stress echocardiography \[[@B30]\] and compared it with conventional visual assessment. The ratio of postsystolic shortening to maximal strain was the best quantitative parameter to identify dobutamine stress induced-ischemia. This quantitative analysis improved sensitivity from 81 (visual assessment) to 86% and specificity from 82% to 89%. The statistical significance is not provided in the manuscript. Then, in the same population of 44 \[[@B31]\], they compared the visual assessment of wall motion abnormalities with different parameters derived from TDI application such as peak systolic velocity, systolic displacement and strain rate imaging. They employed simultaneous perfusion scintigraphy as a gold standard of myocardial ischemia. The stress echocardiographic methodology employed was not a standard one for segmentation of the left ventricle (18 segments instead of 16 or 17), pharmacologic protocol (up to 2 mg atropine instead of 1 mg) and criteria for ischemia (worsening of wall motion only in 1 segment). Also in this case, the overall accuracy is not striking: Table 1 ([Additional file 1](#S1){ref-type="supplementary-material"}). On the basis of these results TDI reduces significantly the diagnostic accuracy of dobutamine stress echocardiography whereas strain rate imaging equals the diagnostic accuracy but it does not improve it. Interestingly enough, the sensitivities and specificities of strain rate imaging are slightly different in the two papers even though the analysis was conducted in the very same set of patients. Moreover, since coronary angiography was performed in all patients, the diagnostic accuracy should have been calculated on this real gold standard instead of perfusion scintigraphy. In a recent \[[@B33]\] article Marwick et al. questioned the hypothesis that false-negative results of dobutamine stress echocardiography reflect the underinterpretation of regional left ventricular function. On the opposite, the quantitative parameters such as strain rate and peak systolic strain rate were no different between false and true negative tests, suggesting that false-negative results are related to lack of ischemia in a functional sense. On the basis of this observation, quantitative markers are unlikely to increase the sensitivity of dobutamine stress echocardiography. Conclusions =========== The quantitative interpretation of stress echocardiography is not superior to expert wall motion assessment. Open issues in the quantitative analysis remain at stake: which technique to be employed among systolic velocities, strain and strain rate, the assessment of normality criteria of myocardial velocities and how to interpret their values, the management of patients with regional and global left ventricular dysfunction, the analysis of the apical segments, the complexity of the analysis in a real clinical environment, the applicability to unselected populations, its unsuitability to exercise, the most widely used stressor in the clinical practice \[[@B34]\]. What is presented as a breakthrough technology should have already answered to these issues and when exported into the clinical arena should have provided an incremental value to the established and more easily accessible methods. It is at this point that we are lost in clinical translation: authoritative journals provide data that cannot be transferred into the daily life of a busy stress echocardiographic laboratory, although the general message is optimistic and tend to ignore flaws and limitations of the technique \[[@B35],[@B36]\]). The advantage/disadvantage balance of a new technology should clearly be stated. The potential advantages should always outweigh the disadvantages related to the higher costs and higher complexity of analysis. Perhaps, the shape of the quantitative technology to come has not been designed yet \[[@B37],[@B38]\]. TDI is one of the tools in our hands but apparently this is not its time. At least not on the basis of this evidence. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### In the left panel sensitivities and specificities in the three main vascular districts and overall sensitivities and specificities without correcting results for age, gender and heart rate. In the right panel, the same data corrected for age, gender and heart rate (modified from ref.29). ::: ![](1476-7120-3-2-1) ::: Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Table 1.TDI vs visual assessment of myocardial ischemia during dobutamine stress echocardiography. ::: ::: {.caption} ###### Click here for file :::	PubMed Central	2024-06-05T03:55:52.729002	2005-1-28	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548514/", "journal": "Cardiovasc Ultrasound. 2005 Jan 28; 3:2", "authors": [ { "first": "Rosa", "last": "Sicari" } ] }
PMC548515	Background ========== Proximity to individuals and society in order to reach out and provide optimal support is basic for patient associations for cancer patients (PACPs) \[[@B1],[@B2]\]. Swedish patient associations for breast cancer patients (PABCPs) offer breast cancer patients\' unlimited meetings with a breast cancer survivor, a contact person (CP). The present study focuses on women with breast cancer and their experiences from having a CP. In Sweden, an association consists of a number of individuals who work together in an organised form towards a common vision \[[@B3]\]. The Scandinavian concept of an \'association\' is different from that in Western Europe and the U.S.\[[@B4],[@B5]\]; in Scandinavia the collective rather than the individual aspect is emphasized with focus on social activities, educational workshops, and support groups \[[@B6],[@B7]\]. However, also individual activities exist within the associations and the CP activities in Swedish PABCPs constitute an example hereof. This activity is inspired by the Reach to Recovery Program(RtoRP), a world-wide rehabilitation program initiated in the U.S. in 1952 and was later established also in Europe. RtoRP is based on the opportunity to meet with individuals with similar experiences and thereby learn different ways of dealing with disease-related feelings and problems \[[@B8],[@B9]\]. Rehabilitation from breast cancer is a requirement for becoming a CP in a PABCP according to the national organisation for Swedish breast cancer associations, (BRO) \[[@B2],[@B10]\]. Listening, supporting, and acting as a partner in conversations and discussions with a breast cancer patient is common to all CPs, although the forms may differ, such as one-to-one meetings or group meetings, between different PABCPs. The 28 of the total 32 PABCPs in Sweden (according to BRO) have appointed specially selected persons to coordinate the CP activities. In addition to self-rehabilitation for breast cancer, the PABCPs also provide education that includes basic psychological knowledge, medical information, organization of and contacts with the health-care system for the CPs \[[@B10]\]. This training program is aimed at preparing the CPs for their task to support individuals in a similar situation. Hence, the CP activity provides an opportunity for cancer patients to meet individuals with similar experiences and to learn different ways of dealing with feelings and problems \[[@B11]-[@B13]\]. Treatment and voluntary action provide different perspectives in studies of self-help groups or mutual support groups in the voluntary sector. We have, in accordance with other researchers\' views, chosen to apply the voluntary action perspective in this study of CPs activities within the voluntary sector \[[@B14]-[@B16]\]. Support in self-help groups, in which the members share and articulate common experiences, is often viewed as a variant of professionally led group therapy \[[@B16]-[@B19]\]. However, the treatment perspective in the latter type of groups is based on the elements of the intervention that lead to cure and on the result obtained. In contrast, the voluntary action perspective of the self-help group is based on a mutual relationship with focus on benefits and aspects related to the individual or to the group \[[@B16],[@B20],[@B21]\]. What experiences do the individuals have in their contact with a CP and what do the individuals (in terms of their breast cancer illness) gain from their contact with the CPs from a PABCP? We applied the voluntary action perspective in this interview study of with the aim to explore how women with breast cancer experienced their contact with a CP from a PABCP. Methods ======= Informants and data collection ------------------------------ Women with breast cancer who had personal experience from CP activities were informants in this study. To qualify for participation, the informants were required not to be involved in treatment (surgery, chemotherapy, or radiotherapy) of the primary tumour. The cancer diagnosis should be within 3 years of the data collection in order to recall the illness and treatments obtained. We approached volunteers responsible for contact and visiting activities in 3 PABCPs who in their turn contacted 8 women who were judged eligible for the study. These women were given information on the purpose of the study and the interview procedure, and confirmation of confidential treatment of the women\'s information was sent by the PACP\'s. The recipients of the letter were informed that it was sent by the PABCP, but they were offered to contact the researcher with questions. The informants were asked to give their decision to the author (CC) by telephone within two weeks and all 8 individuals invited chose to participate in the study. The 8 informants had a mean age of 56 (range 39--69) years and demographic characteristics are presented in Table [1](#T1){ref-type="table"}. Of these individuals, 3 were diagnosed between 3 and 4 years prior to the study, but since these individuals provided extensive narratives they were allowed to stay in the study. Of the 8 individuals, 5 had experience of contact with one single CP, two of several CPs (group sessions), and one had experience of open CP meetings. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Demographic characteristic of informants (n= 8) ::: Characteristics Number ------------------------- ------------ Age: 39 -- 69 (years) 56 (mean) Married and children 8 Time since diagnosis: 18 -- 49 (month) 34 (mean) Treatment: Operation 8 Cytotoxic therapy 4 Radial therapy 7 Hormonal therapy 2 Experience of CP: Open meetings 1 One single CP 5 Several CP 2 ::: All the interviews were held in the home districts of the participants. The data were based on audio-taped narratives and takes into account people\'s natural way of constructing and interpreting experience and provides the opportunity to take relevant contextual factors into account \[[@B22]\]. The personal narrative refers to brief and topically specific discrete stories recapitulating specific events that the narrator had experienced \[[@B22],[@B23]\] (cf Mishler \[[@B24]\] narratives about extra exceptional life events). The informants were asked to tell about their experiences from having a CP. The initial exhortation, \"can you tell me about\...\" was intended to encourage the informants to tell their story in their own way, which would allow analysis from the voluntary action perspective \[[@B16]\]. Each narrative lasted 30 -- 50 minutes, and was transcribed verbatim. Analysis -------- Transcription is a part of the analysis, regardless of whether the emphasis is on the content or the form \[[@B25]\]. This study focused on the contents of the stories. We used Gee\'s analysis structure \[[@B26]\], which is adapted to long oral stories, and gives attention to how a story is told and organizes the narrative into sequences, \"stanzas\" (poetic units) that often consist of four lines and represent a condensation of the narration. The narration also contains small interruptions that embody the basic themes. The basic theme often consists of a single line or sentence, the \"coda\" \[[@B26]\] which gives instant information about the meaning of the narration \[[@B22]\]. To discover the rhythm and to gain an understanding of each narration, the first author (CC) listened to the taped narratives. The two authors CC and KN thereafter read and re-read the transcribed text several times to identify the meanings (the stanzas) and the basic themes (the codas) of the stories (for examples, see Table [2](#T2){ref-type="table"}). This monitoring (Riessman\'s term for reading and interpreting a text) emphasized how the narrative consisted of stanzas and codas \[[@B22],[@B26]\]. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Illustrations of structured transcribed text into stanzas and codas ::: ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Transcribed text Stanza and coda -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------- /\.../ It can have do with prostheses, cytotoxins and radiation therapy, then that I haven\'t had to do but then [there\'s so much we have in common, so the pain over getting the diagnosis and experience of the operation and -- worry about the future]{.underline} Stanza\ There\'s so much we have in common pain over the diagnosis experience of the operation and worry about the future [And it feels incredibly good to be able to talk to someone who has the exact same experience]{.underline}, even though the health care personnel have a lot of experience -- but that is -- that is a different kind of experience, since it\'s more observational or from the outside /\.../ (Informant 8) Coda\ And it feels incredibly good to be able to talk to someone who has the exact same experience ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: The authors examined and discussed the stanzas and codas on the basis of their contents and relevance in relation to the aim. The final monitoring was done to find the informants\' meaning according to their breast cancer when they met another individual with experience of breast cancer, i.e. a CP. The coda was used as the base in the final thematic monitoring \[[@B22]\] and we found 3 themes, including sub-themes, that illustrated the individuals\' experience of CPs. Stanzas were used later in this text to exemplify what emerged in the themes. Ethical permission for the study was obtained from the Ethics committee at Lund University (LU 605-01). Results ======= The thematic contents of the narratives\' stanzas fell into the following themes, which were related to the purpose of the study: 1\. Shared experiences give new perspectives on having cancer 2\. Feelings of isolation are a part of the identity of the illness 3\. Relations with others enable self-help Themes and sub-themes are presented in Table [3](#T3){ref-type="table"}. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Themes and sub-themes ::: Theme Sub-theme --------------------------------------------------------------------- -------------------------------------------------------- 1\. Shared experiences give new perspectives on having cancer 1:1 Feelings of not being alone 1:2 Cancer can be survived 2\. Feelings of isolation are a part of the identity of the illness 2.1 Being different, unique and odd 2:2 One among others like oneself 3\. Relations with others enable self-help 3:1 Talking with others, asking questions and learning 3:2 Right time for making contact 3:3 The contact person provides support 3:4 Indifference in the contact with the CP ::: 1. Shared experiences give new perspectives on having cancer ------------------------------------------------------------ ### 1:1 Feelings of not being alone The meeting with the contact person can have emotional significance in terms of understanding that one is not alone in one\'s illness. Emotions came out in one case by a postcard sent by the CP. The following stanza from the informant exemplifies the CPs\' activity. I got a card from my CP a (certain motif) where she wrote (name) I know what you\'re going through I\'m thinking about you (informant 2) The receiver kept the card and it gave her a warm feeling long after the most difficult time of the cancer illness was over. ### 1:2 Cancer can be survived The contribution of the cancer association and the CPs to giving the women insight about their illness can take place through invitations to rehabilitation activities, such as physical therapy groups. One woman said that, in spite of her having many questions, she did not feel ready to have contact with the association other than coming to the therapy sessions, which gave that woman sufficient and obvious evidence that she was not alone. At the water therapy sessions, she was able to see that it was possible to survive cancer and said \"it\'s nice to be able to, like feel that fellowship\". The following stanza illustrates the activity. when we sit in the sauna -- so everybody is looking - some have no breasts and some they\'ve taken parts of everybody looks a little strange (informant 7) 2. Feelings of isolation are a part of the identity of the illness ------------------------------------------------------------------ ### 2:1 Being different, unique, and odd One woman said that her contact with her CP gave a long-lasting and negative experience of the meeting. After having received advice from a nurse, she had herself contacted the CP. Instead of getting access to other women\'s experiences through the CP she was left with an even stronger feeling of not only being ill but also of being odd. This feeling was caused by the CP not knowing that it was possible to have bilateral breast cancer at the same time (which this woman had). The CP had reacted with alarm -- a reaction that frightened her and made her even more scared. These unpleasant feelings were still present after a year and because of the negative reactions she did not seek further contact with the CP, which led to a sense of embarrassment since she had initiated contact. The narrative illustrates the feelings isolation was strengthened by the CP and how the process of relating to the new disease identity was damaged. Another narrative came from a woman who felt odd because she did not identify herself as being ill. She felt that her cancer was less serious since she did not need further treatment after the operation. This feeling of being isolated gave her a great deal to think about, which she could talk about with her CP. The reaction of being ill came late, according to the informant, and was triggered when she wanted to donate blood. When she filled in the health certificate she realized she was not permitted to do so. The CP was available at that time to talk about the incident and the following two stanzas illustrate the importance that the CP was ascribed: it feels like she \[the CP\] understands what I mean, like she understands that I can float above it and thought like it wasn\'t so serious (informant 8) yes, I think that she took me seriously when I felt like it was hard not to be able to identify myself in the group \[the PACP group\] (informant 8) ### 2:2 One among others like oneself One woman described being one among others like oneself by talking about the special feeling that came over her when she was welcomed into the association. It was not necessary to say that she had had a breast cancer operation since everyone there knew; this was obvious in the association. Another woman described the importance of meeting people in the same age and situation in life, which took place at regular theme meetings for younger women. A younger woman exemplified the importance of being the same age by saying that women of the same age had common questions to discuss -- questions that could not be talked about with health professionals. The following two stanzas illustrate how the women used the CP meetings to talk with women with similar problems: it could for example be how I could make up my eyes now when I don\'t have any eyelashes (informant 6) if there was anyone who had children because the ones I\'d met were only older people who didn\'t have children living at home (informant 6) 3. Relations with others enable self-help ----------------------------------------- ### 3:1 Talking with others, asking questions, and learning At each contact meeting, the women felt that they could gain knowledge through the different experiences that were described about different forms of treatment and their effects on well-being. One woman describes how she could balance her worry in this environment by talking about what she thought about, which is illustrated in the stanzas below: how will I go through this psychologically in the future will I trust this answer or how much will I worry (informant 2) alot of questions like that that I\'ve thought about and that I could talk about with other people (informant 2) Another woman\'s reactions to some of the meetings were feelings of sadness, while other meetings could give her hope for the future. However, while the meetings made her react differently, she felt it was important to come to the CP meetings regularly to see with her own eyes that the other women were alive and led good lives. It came out in another narrative how it is gradually possible to talk with any of the members of the association that have experience even though it is the CP that is the person one initially talks with. Thus in the long run the experiences are felt as being most important, where the CP acts to open the door to them. ### 3:2 Right time for making contact Time, in the sense of a particular phase in the process, is important when the women want to make contact with others with experience of having breast cancer. One woman felt that she was not ready to contact the association and meet others with similar experience despite having many questions. At first, she said, she wanted most just to crawl into herself and manage on her own without involving other people. Nevertheless, this woman was content having received a card from the CP early in the process, telling her about the possibility of establishing a contact. The possibility of getting a contact is thus given higher priority than hearing others\' experiences at an early stage. ### 3:3 The contact person provides support One woman evaluated the meeting with the CP positively in part because the CP was a trained CP and in part because the CP knew what she needed to hear. This is illustrated in the following stanza: it was most of all good to hear \[name of CP\] maybe because she had training knew what you needed to hear (informant 8) Another woman described how she had used her CP as a \"sounding board\" in her choice of surgical method. It comes out in this narrative that the sounding board function and not the counselling function made the woman finally decide in favour of a surgical method that the CP had not recommended on the basis of her own experience. The fact that the CP had given an opinion helped the woman to form an opinion herself. Thus it is not necessary to share the exact same experience; at times it is enough to share the experience of cancer. One of the women said that she would have appreciated there being someone with experience to contact when she was forced to wait for a long period before having her operation. Access to a CP for one\'s own personal problems is illustrated in the following stanza: I know she\'s the contact person which makes me think that I can turn to her and talk about my special problems (informant 8) ### 3:4 Indifference in the contact with the CP Women describe not only the benefit of the CP meetings but also say that there can be a feeling of more or less indifference in the contact with the CP. In one example the CP acted as a guide to the association\'s premises but had otherwise not spoken of anything in particular. Another woman had come into contact with the association\'s CP at an educational class but had never contacted a CP herself. For these women it was sufficient to contact with other women in educational classes organised by professionals. Discussion ========== Since the CP activity is central within the PABCPs, we aimed to investigate how women with breast cancer described their experiences from having contact with a CP. The narratives allowed the women to reflect and formulate themselves through this opportunity to tell their own stories with the aim to analyse their experiences \[[@B24]\]. The strength of the narrative method lies in studying a person\'s identity in times of changing circumstances as in this case, where the individual with a diagnosis of breast cancer meets another individual with the same experience \[[@B22]\]. The experiences described from CP meetings reflect how shared experiences give new perspectives on having cancer and how it is possible to help oneself through the relation to the CP. PABCPs offer meetings with survivors, but it may be difficult to predict the optimal time for each individual to establish such contact since individuals react differently to the diagnosis and to the cancer treatment it requires. The narratives in our study illustrate the importance of meeting women of the same age and in a similar life situation and of being in an environment (CP meeting) that is free of need to explain. Meeting others and experiencing their\' reactions help these women feel normal \[[@B16]\]. \"Catching one\'s breath\" from the isolation that the situation creates seems, according to the respondents, to be needed during certain periods. To become aware of not being alone with the disease, to receive visible evidence there of, and to realize that it is possible to survive appeared most important to the women in our study. The fellowship of existential uncertainty could be discerned in several of the narratives. However, although some women expressed a need to meet with others in order to reduce feelings of isolation, they did not always initiate a contact with the CP. Our results demonstrate that CPs could act as sounding boards in treatment issues and that this could be important for the woman\'s possibility to make her own decision. When these data have been presented during further contacts with PACPB members the findings were perceived as recognizable and the members reported similar experiences (data not shown). However, other aspects and reactions may be identified in other types of patient\'s associations or in other cultures. Indeed, considering the large number of PABCP in Europe multicentre studies with such a focus would be of interest \[[@B27]\]. Even though all patients do not wish to be confronted with the experiences of others via personal contact with a CP, they may be ready to be confronted with the disease in other ways. A confrontation with women who have undergone partial or complete mastectomies can take place in water therapy sessions and in the sauna. The results show that this meeting also gave a feeling of fellowship. This may mean that the person who chooses only this kind of activity is ready only for visual impressions. Breasts mean different things to different women, and only the individual woman can define how great a handicap breast surgery is. Langellier and Sullivan \[[@B28]\] studied illness narratives among breast cancer patients that showed clusters of meanings: the medicalized breast, the functional breast, the gendered breast and the sexualized breast. Compared to other research these results suggest both greater and fewer problems with femininity, sexuality and body image than have earlier been presumed. It is also important that a CP is able to manage timing, in the sense of the phase in the process at which contact is made with others who have experience or when the person is ready to confront others\' experiences. Great sensitivity towards individual needs is required and there are individual variations as to the optimal time for a meeting, which are related to when the individual is ready to encounter other people\'s experiences. In terms of being ready, thoughts arise as to the many visual and auditory impressions that cancer patients receive during a treatment session, e.g. in a waiting room and when given treatment, regardless of whether they are ready for these or not. It is conceivable that some individuals do not have any need to meet with other people and share their experiences since recovery can also occur without involving others. However, it is not only the initial need for contact that varies. Women also have different needs in terms of the duration of the contact. For one person, contact with a CP may simply mean guidance in the association\'s activities while for another a CP can act as a support person for a period of several years and the information-seeking behaviour can also change over time \[[@B29]\]. For the woman who felt great isolation in the illness the meeting with the CP strengthened the feelings of isolation, which demonstrates how the meeting with the CP led to a non-intended consequence. This demonstrates the need for the PABCPs to be meticulous in the choice of CPs; the requirements must not only be that they have been rehabilitated but also that they have substantial knowledge about cancer, treatment possibilities, and psychological reactions to disease. Although self-help may come through a relation with other individuals with a shared experience \[[@B30]\] the associations\' CPs should be aware that patients\' meeting with them does not necessarily provide support, but may strengthen feelings of isolation. The results also demonstrate that some questions could not be answered by health-care professionals, but rather by individuals with personal experience from cancer. Hence, new knowledge developed in the meeting with the CP (cf Borkmans \[[@B14]\] thinking concerning the meaning perspective) and it is therefore important that health-care professionals allow and perhaps also encourage cancer patients to participate in patient\'s associations and thereby share their experiences with others \[[@B31]\]. Worry was balanced by their own experience being reflected in the experience of others, which indicates that CPs have a function in areas that professionals by tradition control, such as in information about treatments and outcome. Our findings confirm results that indicate that support in the form of social relationships with other breast cancer women empowers these women by giving them abilities to cope and adopt supportive roles towards each other during treatment \[[@B32],[@B33]\]. Furthermore the emotional support that is given in connection with other survivors is important for navigating the short and long term impacts of cancer as well as the benefits from rehabilitation \[[@B34],[@B35]\]. Similar observations have been made among individuals with prostate cancer, where shared experiences give reassurance, helps alleviate anxiety, and provides the participants with a more positive outlook \[[@B36]\] and self-help has a potential that could be strengthened in cancer care \[[@B37]\]. Conclusions =========== Our study on patient\'s experience from the CP activity within PABCPs -- based on narratives from 8 patients with breast cancer -- show that shared experiences give new perspectives on having cancer, that feelings of isolation are part of the identity of the illness, and that relations with other individuals may enable self-help. The CP is thus important for the breast cancer patient since it helps these individuals to gain a perspective on their disease, to realize that they are not alone, to provide hope for survival, and support in the feelings of isolation that are part of the identity of cancer. From the patient\'s perspective, it is important that the health care system provides information on the CPs, whose responsibility it is to listen, support and act as conversational partners, and the importance of having access to other persons\' experiences. The CP serves as a counsellor and needs to have an understanding for and knowledge about the patient\'s needs and expectations since self-help may come about in this relation. The CPs should also be aware that their presence and a possible lack of knowledge can sometimes disturb the psychological management of the disease, and contrary to the intention, strengthen feelings of isolation. The CP must also be ready to confront others\' experiences, but also needs to understand that not all individuals have such a need. The CP must be able to offer many different kinds of help related to feelings of isolation, survival, and rehabilitation, and may thereby act as a sounding board for women\'s experiences and a shelter for emotional expressions. Hence, to optimise rehabilitation for individuals with breast cancer, PABCP should carefully select and educate CP\'s, and an exchange of experiences between the PABCP\'s and the health care system may contribute to this process. List of abbreviations ===================== PABCP Patient associations for breast cancer patients CP Contact person Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= CC: study design, data collection, data analysis, and writing the manuscript. MN: supervising the study and participation in writing the manuscript. KN: study design, data analysis, supervising the study, and participation in writing the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6963/5/9/prepub> Acknowledgements ================ We would like to thank the women for generously sharing their experiences and the Scientific Board of the County Council of Halland for their financial support of the study. We acknowledge Professor Ullabeth Sätterlund Larsson (recently deceased) for constructive advice in planning the study.	PubMed Central	2024-06-05T03:55:52.730391	2005-1-25	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548515/", "journal": "BMC Health Serv Res. 2005 Jan 25; 5:9", "authors": [ { "first": "Christina", "last": "Carlsson" }, { "first": "Mef", "last": "Nilbert" }, { "first": "Kerstin", "last": "Nilsson" } ] }
PMC548516	Background ========== Mycobacterium tuberculosis(MTB), the causative organism of tuberculosis, has the distinction of repeatedly being ranked within the top five most commonly laboratory-acquired infections (LAIs) \[[@B1]-[@B3]\]. In 1976, Robert Pike prepared an extensive summary based on both published reports and surveys of 3921 LAIs that included both M. tuberculosisand other pathogens as the infectious agent \[[@B3]\]. He reported that laboratory and mortuary workers that are exposed to tubercle material have a TB incidence rate three times higher than that of the general population and indicated that only 18% of infections could be traced back to a known event. In 1987, a 25 year review at the National Animal Disease Center (NADC) described while only 35% of infections at the strict Biological Laboratory at Fort Detrick, MD, had a reportable, documented cause, the NADC could not account for 73% of LAIs occurring at its own facility \[[@B2]\]. With these reported statistics, it is negligent not to consider aerosol exposure in the absence of a known infecting episode, such as a needle prick \[[@B2]\]. A more recent report from 2003 demonstrated rates from 2 to 6.6 % of TB conversion among heath care workers (HCWs) in New York \[[@B4]\], in spite of current knowledge on precautions and safety measures in place. Furthermore, surveys suggest actual incidence of LAIs with MTB is greater than the amount of reported cases illustrate: these occurrences are likely underestimated due to the nature and length of the disease progression (i.e. workers move or retire before becoming symptomatic), and underreported to the social stigma attached \[[@B1],[@B3],[@B5]\]. Due to the nature of this organism, containment level three (CL3) laboratory operational and physical requirements have been recommended for manipulation of the live organism in North America \[[@B6]\]. Therefore, one would hypothesize that working in a CL3 with personal protective equipment including a respirator would be adequate to protect the worker. However, since conversions are still occurring, it is appropriate to consider the possibility that the procedure one is using to deactivate and extract material from the organism is not 100% efficient. Currently, the application of molecular methodologies for rapid diagnostics of MTB, such as nucleic -acid amplification based identification and subtyping schemes, in addition to extensive genomic and proteomic research in this area, necessitates the removal of material derived from this organism out of a CL3 laboratory to perform the work in a less restrictive containment level 2 (CL2) rated area. Commonly, due to both limited CL3 space, costs and preventative maintenance needs, high-tech equipment such as liquid handling robots and sequencers are shared and can be housed in a central \"DNA core\" CL2 laboratory. To consider the biosafety impact of removing organism material from a CL3 and manipulating it in a CL2, part of a risk assessment undertaken included the review of current literature on decontamination verification and viability testing of M. tuberculosis. The existing literature is limited in regards to viability testing of material derived from MTB with respect to safe manipulation outside of a Biological Safety Cabinet (BSC). To date, no conclusive study has confirmed that this organism is noninfectious after theoretical \'deactivation\' steps. A few reports concerning survival after different heating kill treatments for DNA extractions \[[@B7]-[@B9]\], heat fixing of smears \[[@B10],[@B11]\] and chemical fixation \[[@B12]\] were found. Since, by definition, a risk assessment is based on, but not limited to, the properties of the agent used, personal risk factors, manipulation techniques, and the training and experience of staff, it is necessary to develop a method encompassing these factors to validate the safe removal of material derived from MTB from a CL3 laboratory for use in other laboratory areas. This approach was applied to all our methodologies and took into consideration variables such as interpersonal technique, culture load, temperature fluctuations and statistical significance. Methods ======= The current viability study consisted of the evaluation of a total of 226 material extracts, consisting of 208 initial extracts and 18 extracts tested after revision of faulty protocols (figure [1](#F1){ref-type="fig"}). For each test performed, laboratory staff members sub-cultured a proportion (100 uL) of the resulting material to Bactec 12B radiometric medium (Becton Dickinson, Oakville, ON) that was kept for eight weeks to observe for growth. The vials were incubated at 37°C and the growth index (GI) was read weekly on BACTEC 460 machines. All vials with positive GIs were sub-cultured to tryptic soy agar (TSA) with 5% sheep blood, Middlebrook 7H10 agar and stained with the Kinyoun method for the presence of acid-fast bacilli (AFB). Those positive for AFB were examined for the presence of MTB using a DNA probe specific for M. tuberculosiscomplex, Accuprobe (GenProbe, San Diego, CA). Methods that resulted in viable MTB were revised and retested. All work completed was performed in a CL3 environment, using Class II Type B2 biosafety cabinets (BSCs), and personal protective equipment (PPE) including N100 particulate respirators, double gloves, and protective gowns. The following describe the protocols used in our laboratory at the start of this study. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Overall study flow chart with results after eight weeks incubation of material at 37°C ::: ![](1471-2334-5-4-1) ::: DNA extraction for IS6110 fingerprinting ---------------------------------------- This was performed according to the standard protocol \[[@B13]\]. A total of 125 Bactec 12Bs were inoculated with 0.1 mL of lysate materials. For initial heat deactivation steps, 1.5 mL screw-cap tubes were placed in a water-bath maintained at 80°C. The tubes were not submerged. Three technicians normally performing the procedure inoculated 12B media at different steps of the lysate protocol. Twenty-four samples were processed up to the point of lysozyme and proteinase K addition, prior to CTAB addition, and tested for viability. In 30 other samples the protocol was continued with the addition of choroform:isoamyl alcohol and subsequently centrifuged into organic and aqueous phases. For 10 samples, the organic (bottom) layer that is normally discarded was inoculated into 12B vials and the remaining 20 samples had their aqueous (top) layer planted. Sixty-one samples were processed to completion and inoculated into 12B vials. Finally, 10 extracts from frozen storage (-20°C) were retroactively tested by inoculation to 12B media. Crude lysate preparation ------------------------ A total of 45 lysates used for PCR testing were tested for viability. Thirty-six lysates were prepared from actively growing cultures by three technicians that normally perform the testing procedure. Briefly, a loopful of culture is placed in a pyrex glass bottle containing 1 mL distilled water and glass beads and vortexed in a BSC. Generally this suspension has a turbidity of \> 1 McFarland. This suspension is transferred to a screw-capped vial and is placed in a boiling water bath for 10 minutes followed by transfer into a tube containing 0.5 mm silica beads and mechanically lysed for 2 minutes with a Mini-8-Beadbeater (BioSpec Products, Bartlesville, OK). The resulting lysate is spun down for removal of debris and the supernatant transferred to a new tube to be used for PCR. Lysates were processed in duplicate and the position in water bath was noted (i.e. periphery or centre). A proportion of lysates (100 uL) were planted pre and post bead-beating. An additional nine previously frozen lysates stored at -20°C were also inoculated into 12B media (n =45 lysates). Smear preparation ----------------- Slides were prepared in duplicate according to our standard protocol: a loopful of organism is suspended in water with beads, vortexed and one drop added to a glass slide containing one drop of 0.5% phenol serum (made in-house). The slides were allowed to dry and were placed on a 95°C slide-warmer (Lab-Line Instruments, Melrose Park, IL) for 15, 30, 45 minutes, 1, 1.5 and 2 hours (n =12 slides). The heat fixed slide material was emulsified using a sterile swab and sterile distilled water. The suspension was transferred to a pyrex glass bottle containing 1 mL sterile water with beads, vortexed and 100 μl was inoculated into a 12B media vial. Protein extraction ------------------ Twenty-six extractions of M. tuberculosisorganism were processed as follows. A volume of 25 mL of Middlebrook 7H9 broth was inoculated and incubated at 37°C for 20 days. The culture was centrifuged at 1900 × gfor 15 minutes at room temperature and the pellet was resuspended with 2 ml of ice-cold phosphate buffer solution (PBS) pH 6.8 supplemented with 1% Tween-80. The sample was centrifuged as before at 4°C and the pellet washed twice in ice-cold 1% Tween-80 in PBS. Silicon beads (0.5 mm) and 500μl lysis buffer (2% CHAPS, 2% Triton X-100, 9.5 M urea and 1% DTT/TBP in water) were added to the pellet. The sample was mechanically lysed for 30 seconds and cooled on ice for 30 seconds, repeated eight times. The sample was then filter sterilized with a PES membrane, 0.22 μm Millipore filter unit (Millipore, Etobicoke, ON) into a 2 ml microcentrifuge tube on ice and stored at -80°C. Results ======= In total, 226 samples were tested for viability. As described below, 21 samples grew within the 8-week period, with 16 of those samples from three separate procedures yielding positive growth for M. tuberculosis(figure [1](#F1){ref-type="fig"}). Of the 125 RFLP lysates tested, 2 of 24 sample materials that were tested for viability before addition of CTAB remained viable for M. tuberculosis. Following the complete extraction protocol as described \[[@B13]\], cultured samples yielded 0% viability for M. tuberculosis. Four of ten 12B media vials planted with lysates from frozen storage did show growth, and upon further investigation were determined to be contaminants (Table [1](#T1){ref-type="table"}). All other vials had negative GI readings after 8 weeks of incubation. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Results of positive Bactec 12B vials (n= 21). ::: Description of material Growth on TSA + 5% SBA @ 48 hrs Growth on Middlebrook 7H11 Kinyoun Stain direct from positive 12B Probe Conclusion ----------------------------------------------- ------------------------------------- ------------------------------------------------ ----------------------------------------------------------- ----------- -------------------------------------------------- RFLP lysate -- before CTAB addition (2 vials) - +, MTB morphology AFB+ (4+), serpentine cording ND Viable MTB^1^ RFLP lysate from frozen storage \#1 - +, KS of growth AFB - 4 AFB/slide, morph not consistent with TB MTB- contaminant present -- ubiquitous mycobacteria RFLP lysate from frozen storage \#2 - +, KS of growth AFB - 15 AFB/slide, morph not consistent with TB MTB- contamination present -- ubiquitous mycobacteria RFLP lysate from frozen storage \#3 & \#4 - +, KS of growth AFB - No AFB contamination present Boiled lysate - +, Smooth colonies not consistent with MTB AFB+ 5--10/field, clumping, no obvious serpentine cording MTB- ubiquitous mycobacteria Smear, 1 h at 95°C & Smear, 2 h at 95°C^2^ - +, MTB morphology AFB+ (4+), serpentine cording MTB+ Viable MTB Protein extracts (2) - +, MTB morphology AFB+ (4+), serpentine cording MTB+ Viable MTB ^1^Accuprobe was not performed on these isolates, as MTB was expected due to morphology observed in addition to data from prior studies \[7\]. ^2^All 12Bs from smears (n= 12) were positive, only 2 were chosen as representative for further testing Definitions: KS = Kinyoun stain, AFB = acid-fast bacilli, MTB = M. tuberculosis ::: Forty-five vials of TB PCR lysates were tested for viability. With the exception of one sample that was positive for growth and was later shown to be a contaminant (Table [1](#T1){ref-type="table"}), all were negative for viable M. tuberculosis. All vials containing suspensions from slide material that had been incubated for less than 1 hour on the slide-warmer exhibited identical growth rates, with a positive 12B vial in two weeks. Vials containing suspensions from slides that were incubated greater than 1 hour exhibited identical growth rates and had a positive 12B vials in 3--4 weeks. All 12 12B vials were positive for growth at 4 weeks incubation. Two representative vials were chosen for further analysis (1 hour and 2 hour slide incubations). These 12B culture vials were confirmed positive for M. tuberculosiscomplex using Accuprobe, in addition to displaying colony morphology consistent with TB on subculture to Middlebrook 7H10 agar and being AFB positive with the Kinyoun stain. Two of 25 vials tested for viability from protein extractions became positive. Confirmation of viable MTB in these test vials was confirmed as described above. Based on these results, the methods for slide fixation and protein extraction were modified. Slides were fixed prior to staining using 5% phenol in ethanol according to Chedore et al. \[[@B10]\], and protein extractions samples were centrifuged at 4°C after lysis steps to rid the supernatant of cell debris before filter sterilization. These modified tests were planted again for growth of MTB, resulting in 0% organism viability for every test performed by each laboratory worker (Table [1](#T1){ref-type="table"}). To date, ongoing viability testing of these tests have not shown any growth (data not shown). Discussion ========== There are few publications that review the efficacy of methodologies that render material extracts from a M. tuberculosisculture non-viable \[[@B7]-[@B10]\]. One such study by Bemer-Melchior et al. was prompted by a case of pulmonary tuberculosis acquired by a laboratory technician performing the standard method for IS6110-RFLP in a CL3 mycobacteriology \[[@B7]\]. In this study, placing the organisms in 80°C for 20 minutes gave breakthrough growth, which was also observed in our laboratory. Conflicting information exists which claims 100% loss of viability using this method, however, this report outlines potential reasons for the difference, such as the complete submersion of sample tubes, or volume and density of the suspension \[[@B9]\]. Bemer-Melchior et al. also found that submerging the culture at 100°C for 5 minutes rendered the sample completely non-infectious; data that was supported by work previously done in 1994 by Zwadyk et al.\[[@B8]\]. Although these studies were the first of its kind in verifying the safety of laboratory protocols and addressed issues regarding temperature, timing, cell density and sample volume in deactivating MTB cultures, they clearly demonstrated that certain methods of heating may not be efficient in complete sterilization of a MTB culture. With the unique cell wall characteristics and ability of MTB to clump (or cord), variables to be considered include cell mass density, actual temperature reached and actual time exposed to the heat source. Zwadyk et al.showed that using a 95°C heat block failed to completely inactivate the culture, and in fact, using an internal temperature probe demonstrated that the tube did not reach the intended temperature even after 20 minutes \[[@B8]\]. Another study issued a caution in removing MTB fixed with 0.5 -- 1% glutaraldehyde from a CL3 as the process used to sterilize the culture failed \[[@B12]\]. Again, both the efficacy of rendering MTB material non-viable as well as the effect of the clumping or cording factors not being known was questioned. Therefore, testing all deactivation methods was recommended for all preparations in this laboratory \[[@B12]\]. In preparing a risk assessment for our CL3, it became evident that this was a necessary course of action to reduce the risk of LAIs for both CL3 and CL2 mycobacteriology staff as well as other non-mycobacteriology laboratory staff in the shared CL2 environment. Genomic extractions, the most commonly performed test in our laboratory, are conducted using the standardized method outlined in 1997 by Van Embden et alfor the purpose of RFLP typing \[[@B13]\]. Prior viability testing of this method in our lab showed that lysates contained viable MTB with the initial deactivation steps of this protocol, heating for 20 minutes at 80°C with addition of lysozyme and proteinase K, and thus could not be removed from containment until DNA extraction with CTAB. It is presumable that the small rate of viability observed (2 of 24), which is similar to what was seen by Bemer-Melchior et al., resulted from incompletely submerging sample tubes or cell density within the tube \[[@B9]\]. However, phenol-chloroform extraction such as the CTAB method and heating of specimens should be lethal to mycobacteria since phenolic-based disinfectants have been shown to be tuberculocidal \[[@B14]\], and to date our results reflect this fact. To both preserve the integrity of genomic DNA for the use of fingerprinting and allow the sample to be further processed safely outside a CL3 laboratory, it is recommended to complete DNA extractions with CTAB as per the standard protocol to confirm the complete inactivation of M. tuberculosis. Further sampling of the DNA extractions performed in our laboratory (10% of each lysate batch) is ongoing for quality assurance purposes, i.e. to confirm integrity of reagents as well as to monitor staff performance and adherence to protocol. In addition, continuous sampling attaches statistical significance to the claim that this method ensures that the extracted material is 100% non-viable and guarantees staff safety. For procedures that do not require intact, high quality DNA such as PCR testing, our laboratory depends on the much more expedient lysate method of boiling culture at 100°C for 10 minutes, followed by mechanical lysis for 2 minutes to release DNA. Although crude, this procedure is adequate for our PCR testing needs, has been shown to completely destroy live organism in our laboratory, and is consistent with other studies \[[@B7],[@B8]\]. The study by Zwadyk et al.concluded that inactivating mycobacteria by heat lysing at a temperature of 100°C for 30 minutes did not inhibit its ability to be amplified by PCR or strand displacement amplification \[[@B8]\]. Furthermore, this study has shown that inoculating the boiled lysate alone without mechanical lysis was adequate in rendering the sample non-viable. The viability testing of the two methods outlined above for DNA extractions were performed by various technicians who routinely follow these procedures, lending interpersonal variability to the study. It was demonstrated that the small nuances to procedure, such as the varying density of culture used, did not affect the method employed to deactivate the organism. The last routine test assessed for organism viability was the slide preparation. While phenolics are known to be tuberculocidal, the use of phenol serum alone or in conjunction with heat in slide preparation is not sufficient for killing of the live organism. Flaming of the slide was not an option due to facility requirements that prohibit open flames inside a BSC, despite this, flaming of smear material was found in prior studies to unsuccessfully inactivate smear material \[[@B10]\]. The primary method for slide fixation was a 2-hour incubation at 95°C on the slide-warmer, which was assumed adequate for staining purposes. Slides were then transported to an area where respirator usage was not mandatory. It was discovered that every slide preparation was positive for viable TB. As a consequence of the viability testing and subsequent risk analysis, the protocol was altered to include chemical fixation with a 5% fixative of phenol:ethanol \[[@B10]\]. This allows lab staff to safely remove slides from a CL3 area and examine slides under a microscope without interference by the need to wear a bulky respirator. Examining laboratory protocols for staff and building safety should be an integral part of any CL3 laboratory program. Implementation of a policy to test all procedures used routinely for viability in addition to new methodologies is highly recommended. As an example, applying this policy to a new procedure being utilized in the research arm of our laboratory proved valid: a protein extraction method being developed initially showed that of 2/26 tested samples yielded viable MTB. The researcher concluded that it was due to a clogging of the filter from large particles in the lysed culture used to isolate the proteins and revised the protocol to centrifuge the mixture after final lysis steps to remove cellular debris before filtration. This has proven successful so far, and continuous sampling of extracts is ongoing before removing the material from the CL3. To date, all have been negative. Conclusions =========== It is imperative to evaluate and record the actual rate of viability of DNA lysates from deactivated MTB cultures in individual laboratory settings \[[@B12]\]. This includes all material removed from the CL3 area. This process is vital to establish biosafety validated procedures and practices to protect laboratory workers conducting these procedures \[[@B2]\]. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= KB carried out the studies involving genomic DNA extractions and drafted original and final manuscripts. TB tested all smear preparations, compiled viability data and assisted with the writing of the manuscript. MS contributed by testing protein extractions and writing the method used. CT contributed by testing of crude lysates and editing of manuscript. MS contributed by testing protein extractions and writing the method used. AK and JW conceived of the study, and participated in its design and ongoing coordination. All authors read and approved the final manuscript Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2334/5/4/prepub> Acknowledgments =============== We\'d like to thank support staff Nancy Smart and Alisa Thompson for their contributions to this study.	PubMed Central	2024-06-05T03:55:52.733417	2005-1-24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548516/", "journal": "BMC Infect Dis. 2005 Jan 24; 5:4", "authors": [ { "first": "Kym S", "last": "Blackwood" }, { "first": "Tamara V", "last": "Burdz" }, { "first": "Christine Y", "last": "Turenne" }, { "first": "Meenu K", "last": "Sharma" }, { "first": "Amin M", "last": "Kabani" }, { "first": "Joyce N", "last": "Wolfe" } ] }
PMC548517	Background ========== Gastric reflux of short duration is a normal physiological event for all infants less than six to seven weeks old. When gastric reflux occurs more frequently and past the age of seven weeks, it becomes a clinically significant problem that is diagnosed as pediatric gastroesophageal reflux disease (GERD). Symptoms of pediatric GERD include pain, irritability, frequent spitting-up or vomiting, constant or sudden crying, poor sleep habits and frequent waking. At present, these symptoms represent a clinically significant problem for one of every 500 infants between the ages of six weeks and 18 months \[[@B1]\]. Although these symptoms are not uncommon in childhood, few symptomatic children are treated \[[@B2]\]. The prevalence of symptoms consistent with pediatric GERD varies with age and depends upon the type of symptoms. When referring solely to regurgitation symptoms, over 80% of children experience spontaneous remission by age 18 months \[[@B4]\]. In comparison, findings in the literature indicate that remission of GERD symptoms occurs in 70% of the population at three years of age \[[@B8],[@B17]\]. In fact, research suggests that among children three to nine years of age, only 2.3%, 1.8% and 7.2% will experience symptoms of regurgitation, heartburn and epigastric pain, respectively \[[@B18]\]. Interestingly, despite large variation in prevalence rates by age group, differences in prevalence have not been reported across gender, ethnic groups or socio-economic classes. Many health care professionals (HCPs) specializing in the treatment of GERD feel that published prevalence rates underestimate the extent of the condition \[[@B6],[@B7]\]. Interviews with HCPs suggest that pediatric GERD is under-treated because many pediatricians are not aware of how to effectively diagnose and treat the condition \[[@B8]\]. Furthermore, many specialists and caregivers believe that GERD is often \"missed\" by physicians, since it is \"normal and common for infants to spit up several times a day\" \[[@B4]\]. Failure to properly diagnose, lack of treatment, or sub-optimal treatment of these symptoms can lead to serious complications such as failure to thrive, anemia, esophagitis and respiratory disorders \[[@B4]\]. In addition to having serious consequences for infants and young children, reports from parent advocacy groups suggest that untreated or ineffectively treated pediatric GERD exerts a substantial negative impact on the life of the child\'s primary caregiver \[[@B4]\]. Caregivers of pediatric GERD patients report sleep loss and psychological and physical strain related to the ineffective or inadequate treatment of pediatric GERD \[[@B4]\]. The burden of care appears to affect every facet of the caregiver\'s life, including daily activities, social interactions, professional pursuits and family relationships. This burden results in changes in the caregiver\'s physical and psychological health, quality of life and financial well-being. The current study had two objectives related to the assessment of the impact of pediatric GERD on the caregiver\'s daily life. The first objective was to determine if there was an existing instrument suitable for measuring the impact of caring for an infant or child with GERD. If no instrument could be identified, the second objective was to develop an instrument suitable to quantify the impact of pediatric GERD on caregivers, thereby providing a means to improve public awareness of the issue. Questionnaire development rationale ----------------------------------- A focused literature review was conducted to determine if a caregiver-reported outcome measure that assesses the impact of caring for an infant or child with GERD had been developed. This review included a search of commercial and Mapi Values in-house medical databases of published literature from 1990 to the present in order to identify available instruments and studies relevant to caregiver burden in pediatric GERD. Additionally, the review examined whether generic quality of life measures had been previously applied to the assessment of the impact of caring for a pediatric GERD patient. The literature review uncovered numerous instruments developed for caregivers of adult, elderly or terminally ill patients (i.e., cancer or AIDS patients) \[[@B9]\]. In contrast, few instruments or studies were identified that specifically assess the impact of a child\'s illness on the primary caregiver. Common approaches to assessing the impact of caring for a chronically ill child included asking caregivers open-ended questions about family strain \[[@B10]\], evaluating the effect of the child\'s illness on family resources \[[@B10]\] and measuring the impact of the child\'s illness on the caregiver\'s well-being and quality of life \[[@B11]\]. No instruments that explore the impact on the caregiver\'s quality of life of caring for a child with GERD were identified during this review. Nor were any generic measures identified as having been used to quantify the impact of caring for a pediatric GERD patient on the primary caregiver. Furthermore, no single disease-specific instrument was found that assesses the economic, emotional, psychosocial and physical burden experienced by the caregiver of a chronically ill child. The \"Pediatric GERD Caregiver Impact Questionnaire\" (PGCIQ) was developed in American English and American Spanish to address the need for an instrument to assess the impact of caring for a child with GERD. This instrument was developed for use in observational studies and multi-national clinical trials to systematically assess and document the physical, psychosocial, psychological and financial impact of caring for pediatric GERD patients. In addition, items in the PGCIQ were specifically developed to capture changes in caregiver burden in response to successful treatment. This instrument will allow documentation of the impact of caring for a child with GERD and provide evidence to increase public and physician awareness of the condition. The PGCIQ was developed simultaneously in American English and American Spanish to accommodate the rapidly changing population composition of the United States (U.S.). The U.S. Hispanic population grew by 61% from 1970 to 1980 and by another 53% in the following 10 years \[[@B12]\]. Between 1990 and 2000, Hispanics were the fastest growing ethnic group in the country \[[@B13]\]. Because the PGCIQ is intended for use predominantly in the U.S., it was deemed critical that the instrument be sensitive within Spanish-speaking as well as English-speaking populations. Utilizing the simultaneous development approach reduces risk factors that threaten the validity of cross-cultural comparisons in the two language groups. Although the questionnaire was initially developed in American English and American Spanish, it was designed to be suitable for cross-cultural and linguistic adaptation into multiple languages. Thus, the PGCIQ was developed to provide valuable information in multi-national clinical evaluations regarding the value of different GERD treatments from the caregiver\'s perspective. Information obtained from the PGCIQ can be used to inform and educate health care providers and payers about the needs of caregivers of pediatric GERD patients. Methods ======= Simultaneous questionnaire development -------------------------------------- The development of the PGCIQ followed the methodology of simultaneous questionnaire development. This process was selected to reduce the risk of systematic measurement error at the item level (i.e., item bias) and ensure that construct bias, which occurs when the construct is measured, was identical across all developed language versions. When combining this methodology with a translatability assessment conducted by linguistic experts, the procedure was intended to yield an \"optimal\" measure for adaptation of the American English version into different cultures. Additionally, the process aimed to produce a measure that was less susceptible to cultural differences than a questionnaire developed in one language and followed by translation into other languages \[[@B14]\]. Study participants ------------------ Participants were recruited by pediatric gastroenterologists and pediatricians. The primary caregivers included in the discussions were 18 years or older and were caring for children (newborn to 12 years of age) who were either newly diagnosed with GERD or seeking treatment for a new episode of GERD after a period without treatment. Diagnoses of GERD required that children present with common clinical manifestations of GERD. Neonates and young babies were required to have chronic regurgitation, most commonly demonstrated by vomiting. In young children, diagnosis was confirmed by physical discomfort that manifested as prolonged crying, fussing, arching, or refusal of feedings. As well, all children three months or older were required to have symptoms of GERD requiring acid suppressive therapy. Caregivers were excluded if their child had a history of acute life-threatening events due to manifestations of GERD or a severe unstable illness that could exacerbate caregiver burden. In order to optimize the relevance of the instrument across a wide range of age groups, the study authors attempted to achieve an equal distribution of children within three groups: premature infants to three months, four to 11 months and 12 months to 12 years. Across all three groups, participant eligibility was confirmed by case report forms completed by the treating physician. Concept elicitation ------------------- Four focus group discussions, two in American English and two in American Spanish, were conducted to elicit issues relevant to caring for a child with GERD. For each language group, discussions were conducted on the East and West Coasts to ensure adequate representation of Mexicans, Puerto Ricans and Cubans, the three largest groups of Spanish-speaking Americans currently residing in the US \[[@B15]\]. Each two-hour focus group was conducted using a structured focus group discussion guide that was developed in American English and linguistically adapted into American Spanish. American English and American Spanish focus groups were facilitated by two female researchers both living in the U.S. The native Spanish speaker was from Ecuador. Focus group moderators received identical, in-depth training from Mapi Values, and all focus groups were videotaped to ensure adherence to the discussion guide. Caregivers were asked to discuss how caring for their child has impacted their lives in the following areas: daily activities, social and family life, emotional and physical functioning and financial well-being. At the start of each focus group discussion, caregivers completed a 48-item GERD Caregiver Informational Survey, developed for this study, which contained questions about the caregiver\'s and child\'s demographic, socioeconomic and clinical profile. Qualitative analysis of the focus group discussions was conducted separately for each language group. This analysis included a comprehensive review of verbatim transcripts by native speaking researchers who also conducted the focus group discussions. Participants\' comments were coded to highlight key concepts and psychosocial correlates. Coded comments were subsequently grouped together to elicit the domains and issues important to caregivers. These domains and correlates provided the framework of the conceptual model for questionnaire development (Figure [1](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Conceptual Model ::: ![](1477-7525-3-5-1) ::: Following creation of a conceptual model, global concepts within each language group were compared to evaluate similarities and differences in caregiver perspectives. In addition, responses were evaluated to identify potential differences by the child\'s developmental stage, severity of disease, socio-economic status and demographic profile. Participants\' verbatim quotes from each language group were then consolidated within the common domains. Item generation --------------- Items were simultaneously generated in American English and American Spanish after consolidating caregivers\' verbatim comments. For each item, American English and American Spanish speaking interviewers discussed relevant concepts identified from the focus group discussions and agreed upon conceptually equivalent wording. This process ensured that each item in the questionnaire was pertinent to caregivers in both language groups and formed the basis for Version 1 of the questionnaire. Translatability assessment -------------------------- A panel of linguistic experts conducted a translatability assessment of the PGCIQ to finalize Version 1 of the PGCIQ. The questionnaire was evaluated for its cultural adaptability and translatability in all potential target languages to ensure the cultural equivalence of items. Additionally, this process was used to limit the threat of bias in all current and potential target languages. Items identified as irrelevant for future target languages were considered for deletion or modification in the English and Spanish versions. Content validity testing ------------------------ Content validity interviews were conducted by the native speaking researchers who also conducted the focus group discussions. Researchers interviewed caregivers to assess the ease of comprehension, clarity, cultural equivalence and relevance of the first version of the PGCIQ. Participants were recruited following the same methods and criteria as for the focus group discussions. The interviews aimed to assess the clarity, comprehension and appropriateness of all instructions, questionnaire items and response scales. Interviews were transcribed verbatim in the participants\' native language. Each transcript was comprehensively reviewed, analyzed and coded to highlight caregiver impressions of Version 1 of the PGCIQ. Coded comments were subsequently grouped together to elicit the caregivers\' feedback by item. Items were considered for modification if more than one caregiver in either language had difficulties with or suggestions for the item. The interview results from each language group were analyzed separately and later compared for potential differences. In addition, caregiver responses were evaluated for potential differences by the child\'s developmental stage, severity of disease, socio-economic status and demographic profile. If an item was identified during content validity testing as potentially problematic, the item was reviewed and simultaneously modified by interviewers in both languages. If an item was problematic in at least one of the languages, the interviewers reviewed the caregivers\' verbatim suggestions and attempted to agree upon a conceptually equivalent modification. If no suitable wording could capture the same concept in both languages, the item was deleted from the questionnaire. The final version of the questionnaire aimed to be relevant to caregivers of pediatric GERD patients, conceptually equivalent in American English and American Spanish, free from bias or jargon and easy to understand, interpret and complete within 10--15 minutes. The final version of the questionnaire was also developed to be appropriate for administration for a fifth grade literacy level, as confirmed by a Flesch-Kincaid Grade level test \[[@B17]\] and linguistic adaptability, as confirmed by a translatability assessment. Results ======= Focus group discussion participants ----------------------------------- ### Caregivers characteristics Twenty-seven caregivers, 12 American English-speaking and 15 American Spanish-speaking, participated in the focus group discussions. The final distribution of interview participants by geographic region was East Coast English (n = 7), West Coast English (n = 6), East Coast Spanish (n = 5) and West Coast Spanish (n = 9). The average age of focus group participants was 37 years of age with a range of 18 to 64 years of age. The participants were predominantly female (89%), married (81%), living with their spouse and children (89%) and recipients of a high school diploma (81%). The fact that participants were primarily female reflects the composition of the population of primary caregivers seen by the investigators (referring physicians). Moreover, the study investigators attributed the fewer numbers of single and divorced caregivers in the discussion groups to difficulties in taking time off work. Analysis of the caregiver characteristics also revealed differences by language group. Relative to Spanish-speaking participants, a greater proportion of English-speaking respondents were female (100% vs. 80%) and married (100% vs. 67%). Table [1](#T1){ref-type="table"} contains the characteristics of the focus group participants. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Focus group discussions: caregiver and child characteristics ::: American English n (%) American Spanish n (%) Total n (%) --------------------------- ---------------------------- ---------------------------- ----------------- Number of Caregivers by Language 12 (44%) 15 (56%) 27 (100%) Gender Female 12 (100%) 12 (80%) 24 (89%) Male 0 (0%) 3 (20%) 3 (11%) Age Mean 37 37 37 Range 21--58 18--64 18--64 Marital Status Divorced 0 (0%) 1 (7%) 1 (4%) In long-term relationship 0 (0%) 1 (7%) 1 (4%) Married 12 (100%) 10 (67%) 22 (81%) Single 0 (0%) 2 (13%) 2 (7%) Other 0 (0%) 1 (7%) 1 (4%) Relationship to Child Aunt or Uncle 0 (0%) 1 (7%) 1 (4%) Grandparent 1 (8%) 0 (0%) 1 (4%) Parent 11 (92%) 14 (93%) 25 (93%) Number of Children by Caregiver Language 12 (44%) 15 (56%) 27 (100%) *\Age(years, months) Mean 3.6 2.2 2.8 Median 1.6 3.3 2.5 Range 0.2--11.5 0.5--3.6 0.5--11.5 Gender** Female 6 (50%) 8 (53%) 14 (52%) Male 5 (42%) 7 (47%) 12 (44%) Outlier excluded 1 (8%) 0 (0%) 1 (4%) \outlier age of 18.2 was excluded in determination of mean, median and range values ::: ### Children characteristics The children in this study were primarily female (52%) and their average age was 2.8 years, with a range of six months to eleven years. The average age was determined after excluding an outlier (12 year-olds) from the calculation. In terms of the target age groups, 8% of the sample population was premature up to three month-old infants; 33% were four month to 12 month-old infants; and 55% were 12 month to 12 year-old children. In total, 81% of the children were three years-old or younger, with only five of the children over the age of three. The majority of this sample consisted of children under the age of three years. The age range of this population was consistent with the literature which indicated that the prevalence of GERD symptoms significantly declines with age, with less than 30% of GERD cases persisting past three years of age \[[@B17]\]. According to caregivers\' ratings, their children were primarily in very good (26%), good (26%) or fair health (26%). Analysis of additional clinical measures revealed significant differences in the children\'s health by language group, with Spanish-speakers reporting more hours of fussiness per day, emergency room visits and hospitalizations. Table [1](#T1){ref-type="table"} also contains the characteristics of the children of the focus group participants. Concept elicitation ------------------- Qualitative analysis of the focus group discussion results revealed nine key concepts relevant to caregivers of pediatric GERD patients: experiences related to GERD diagnosis, taking care of the child, daily activities, emotional well-being, household expenses, physical health, social life, relationships, and employment prior to and after GERD diagnosis. Each key concept is discussed in the following sections. ### Experiences related to GERD diagnosis Caregivers recalled feelings of fear, helplessness and guilt associated with the onset of their children\'s GERD symptoms. Many of these feelings were discussed in the context of negative interactions with HCPs. Caregivers shared stories about their struggle to get doctor appointments or referrals and being told that their children\'s symptoms were \"normal\" or that they were \"overreacting\" or \"paranoid.\" These experiences appeared to be particularly stressful for the Spanish-speaking caregivers. In several cases, Spanish-speaking caregivers stayed with their physicians even though they received inadequate medical attention due to their lack of comfort with the U.S. health system. Thus, their children tended to be diagnosed at an older age than children of English-speakers resulting in elevated feelings of fear, helplessness and guilt. Although caregivers stressed the trauma they experienced in order to obtain their child\'s diagnosis, this experience did not appear to be change once the child had received an accurate GERD diagnosis. Therefore, this domain will most likely not be affected by treatment and therefore, has been included as optional. ### Taking care of the child Caregivers discussed four unique parenting issues related to taking care of a child with GERD: using special feeding techniques, putting the child to bed safely, disciplining the child and finding a reliable childcare provider. First, caregivers of children under four years of age discussed the need to use special feeding techniques. These caregivers commented that they needed to feed their children \"small portions,\" feed them \"constantly\" and prepare special meals or formulas. Second, caregivers of infants reported adopting unique bedtime routines. These caregivers used equipment such as wedges, car seats, pillows and monitors to put their children to bed safely. Third, caregivers of children over the age of four cited discipline as a challenging issue. Several caregivers recalled emotional tales of disciplining their children for throwing up \"on purpose,\" while others feared that their children would spit up every time they were \"yelled at\" or \"punished.\" Finally, virtually all of the caregivers cited childcare as a major concern. Most caregivers were unwilling or unable to find childcare providers capable of handling their children\'s special needs. Even those caregivers who found suitable care providers experienced a sense of distress due to concerns that others would not care for their child adequately. For example, one caregiver stated \"No one else is going to give your child the attention \[he/she\] needs\". ### Impact on daily activities The caregivers cited three daily activities that were significantly impaired by their children\'s GERD: mealtimes, housework and bathing. Concerning mealtimes, the caregivers shared stories about spending time \"preparing multiple meals\" for the family (separate meals for their child), lacking time to \"sit down and eat\" and having to eat cereal, frozen meals or take-out for dinner multiple evenings in a row. With regard to housework, the caregivers shared anecdotes about having to do laundry \"every day,\" \"constantly cleaning the carpet\" and putting plastic covers over the furniture to protect it from their children\'s spit-up or vomit. Finally, the caregivers recalled stories about not having time to bathe, struggling to shower while holding the child and feeling too tired to get dressed. Limitations in these three activities appeared to be most pertinent among caregivers of infants and caregivers of children with more severe GERD symptoms. ### Impact on emotional well-being Caring for a child with GERD had a major impact on the caregivers\' emotional well-being. The caregivers mentioned a myriad of feelings including fear, worry, grief and depression. The lingering feelings of fear experienced by caregivers most frequently pertained to the possibility that their children might choke and stop breathing. These feelings of fear were reported primarily by caregivers of infants. Feelings of worry were experienced by caregivers of all age groups and predominantly focused on the child\'s \"failure to thrive.\" These feelings of worry were particularly burdensome for caregivers of children with severe GERD who experienced substantial weight loss and significant developmental delays. Two specific differences between language groups were found in this domain. English-speaking caregivers mentioned feelings of guilt for \"disliking\" or \"not wanting\" their children and feelings of envy upon seeing other \"healthy babies.\" In contrast, Spanish-speakers explained that it was a \"given\" that caregivers would be close to their children and that they did not experience the feeling of \"disliking\" their child. ### Impact on household expenses Caregivers discussed incurring additional expenses for a variety of products and services related to caring for their children including doctor/hospital bills, special infant formula(s), medicine, laundry, childcare, new or replaced furniture, new or replaced carpets, cleaning supplies and special equipment designed for managing GERD (i.e., wedges, car seats and high chairs). During the focus group discussions, the majority of caregivers spontaneously provided information related to their employment and income level when discussing the financial impact of caring for their children. Where possible, this information was noted and recorded by the moderators for analysis. Additionally, the caregivers were asked information about their insurance coverage and typical expenses in the Caregiver Informational Survey. Analysis of these data indicated that the caregivers\' expenses did not differ by their socio-economic status or language group. However, caregivers from lower income households appeared to be more greatly impacted by their additional household expenses and reported greater financial strain. ### Impact on physical health The majority of caregivers indicated that their physical health had declined since their children first exhibited symptoms. Common terms used by the caregivers to describe the state of their physical health were \"tired,\" \"exhausted,\" \"stressed out\" and \"distracted.\" In addition, the caregivers shared numerous stories about carrying their infants all day, sleeping in shifts with their spouses and developing physical ailments such as migraines and skin conditions due to the strain of caring for a child with GERD. These physical health challenges seemed to be particularly burdensome for caregivers of children with more severe cases of GERD. ### Impact on social life Caregivers reported reduced social interactions outside of the home and increased social isolation due to their children\'s GERD. Numerous caregivers commented that they preferred to \"stay at home\" due to the embarrassment of dealing with their children\'s constant \"vomiting\" and \"screaming\" in public and the emotional strain of seeing healthy babies of the same age as their children. For several caregivers, social isolation was exacerbated by difficulties experienced while talking on the telephone due to their children\'s \"vomiting\", \"screaming\" and need for attention. Issues of social isolation appeared to be most relevant for caregivers of younger infants and caregivers of children with more severe symptoms. ### Impact on relationships Caregivers described how caring for their child\'s GERD affected their relationships with their spouse/partner, relatives, other child(ren) in the family, and their child with GERD. Discussions about the caregiver\'s relationship with their spouse/partner focused upon the tension experienced between the couple jointly caring for a child with GERD. Tension was often attributed to disagreements over the child\'s medical care, reduced personal time, reduced desire for physical intimacy and decreased communication. Comments about their relationships with relatives emphasized feelings of frustration with family members who \"just don\'t get it,\" \"complain so much\" and \"always judge.\" Remarks about their relationships with other children in the family reflected reduced time to dedicate to the siblings of the GERD patient. Some caregivers confessed that due to the attention required by their child with GERD, they were \"less patient,\" more \"demanding\" and more \"neglecting\" of their other children. Finally, stories about the impact on the bond between the child with GERD and the primary caregiver reflected a dual-directional relationship. In some cases, the child\'s illness seemed to weaken the bond with the primary caregiver, whereas the illness appeared to strengthen the bond in other instances. No major differences were revealed in the impact on these four relationships by language, age, socio-economic or symptom severity group. ### Impact on employment The impact of caring for a child with GERD on an individual\'s employment was primarily dependent on the caregiver\'s employment situation prior to the child\'s diagnosis. The caregivers who were previously employed mentioned a number of ways that caring for a child with GERD affected their paid employment including: changes in work schedules, ability to take enough time off to care for their child, reduced productivity and changes in long-term career goals. In order to provide sufficient care for their children, many of the caregivers changed their work schedule by leaving their jobs altogether, switching jobs or cutting back their hours. Moreover, several caregivers took more paid or unpaid vacation time in order to take their children to doctors\' appointments, share the burden of care with their spouses or provide more attention to their children. Employed caregivers also described indirect costs in terms of reduced productivity and inability to concentrate at work. Many of those caregivers who were not employed prior to the child\'s diagnosis reported altering or sacrificing their career plans to ensure they had enough time to devote to the child with GERD. The aforementioned employment issues appeared to be relevant to caregivers across language groups, age groups and symptom severity levels. Overall, however, the employment issues appeared to be especially pertinent for individuals from low-income households, many of whom worked in hourly rather than salaried positions. Item generation --------------- Item generation based on nine identified areas and the caregivers\' verbatim responses resulted in 10 domains with a total of 58 items that were simultaneously generated in American English and American Spanish. The first draft assessed the following areas: experiences related to diagnosis, caring for the child, daily activities, emotional well-being, physical health, social life, relationships, household expenses and employment prior to onset of GERD and current employment. The domain assessing the caregivers experiences related to diagnosis was developed for baseline use only. This domain contains six items with \"yes\" and \"no\" response options and has a recall period of present time. Issues about employment were divided into two domains pertaining to the time before the child\'s diagnosis as well as the current time period in order to quantify how the direct and indirect costs associated with caring for a child with GERD change in response to treatment. With the exception of four domains (experiences related to diagnosis, household expenses, employment prior to onset of GERD and current employment), all questions in the remaining domains have ordinal scales evaluating intensity and frequency. These questions are phrased in the first person, for example: \"During the last two weeks, I needed to prepare special meals or formulas for my child.\" The response format, a 5-point Likert scale was chosen, as the upper limit of an individual\'s capacity for discriminate judgments has been shown to be near seven (plus or minus two) \[[@B18]\]. Previous studies have suggested that 5-point Likert scales provide an appropriate level for respondents at a fifth grade level of literacy to discriminate without a loss of information. Furthermore, a Likert scale greater than five-points becomes problematic for translation into other languages. The nine core domains and one optional domain in the first version of the PGCIQ were selected to correspond with the key themes identified from the focus group discussions. To ensure cultural equivalence of items, only those concepts that had been identified in both language groups served as the basis for generating items. Considering that 80% of caregivers had children under the age of three, item generation was predominantly based upon verbatim comments elicited by caregivers of infants and young children. Child\'s age ------------ In the item generation focus group discussions, over 80% of the caregivers had children aged three years or younger. Specific concepts that were noted in the focus group discussions as relevant to caregivers of older children and adolescents, but not to caregivers of infants or toddlers, included discipline issues, concern about the child\'s emotional well-being and concern about the child\'s academic development and self-esteem. In contrast, concepts that were relevant to caregivers of infants and toddlers, but not to caregivers of older children or adolescents, included the need to feed the child frequently, feed the child small portions and spend a significant amount of time feeding the child. In general, infants and toddlers also seemed to require more constant one-on-one attention, thereby exerting a greater impact on the caregivers\' daily activities, social functioning and family lives. Caregiver\'s gender, education and socio-economic status -------------------------------------------------------- The PGCIQ was formulated in caregivers with a range of socio-economic and educational backgrounds. In general, all domains were relevant to caregivers across socio-demographic groups. However, the \"employment\" and \"household expenses\" domains appeared more relevant to caregivers of lower economic and educational status due to a higher prevalence of hourly wage earners. Employees working for hourly wages reported greater difficulties getting time off from work to care for their child, less coworker understanding, more financial strain and more pressure to remain in positions they did not enjoy than employees who were salaried. Those in lower income households also struggled more with additional household expenses due to the child\'s GERD and were more likely to report they could not afford products or services (for example, car seats, wedges, therapy sessions) to improve their children\'s health. Language group -------------- The focus group discussions were conducted in American English and American Spanish. All domains appeared to be relevant to both language groups. Specific issues that were more relevant to the English-speaking groups included experiencing feelings of \"envy,\" feelings of guilt for \"disliking the child\" and feeling \"bonded with the child with GERD.\" In contrast, employment challenges and issues of tension, reduced intimacy and communication in the partner relationship appeared to be more relevant to the Spanish-speaking groups. Relative to the English-speakers, the Spanish-speakers also voiced the following unique challenges: more financial strain, more pressure to be an \"ideal\" parent, more difficulty getting attention from English-speaking doctors, less understanding from co-workers and less access to information about GERD. Translatability assessment -------------------------- The results of the in-depth translatability assessment revealed that 16 of 58 items were potentially problematic for future adaptation of the PGCIQ into other languages. These 16 items were reviewed by native speakers. Four of the items were deleted and 12 of the items were modified to improve the cultural adaptability of the PGCIQ. The modified items were flagged for further testing in content validity interviews. Content validity interviews --------------------------- ### Caregiver and child characteristics Version 1 of PGCIQ was tested in twenty caregivers, 10 English-speaking and 10 Spanish-speaking, who agreed to participate in content validity testing interviews. The final distribution of interview participants by geographic region was East Coast English (n = 4), West Coast English (n = 6), East Coast Spanish (n = 3) and West Coast Spanish (n = 7). The characteristics of the interview population were similar to those of the focus group population. The average age of the participants was 38 years with a range from 24 to 79 years. The majority of the participants were female (70%), married (75%), living with their spouse and children (70%) and recipients of a high school degree (75%). These characteristics resemble those of the focus group population and favor the experiences of married females. Table [2](#T2){ref-type="table"} provides the characteristics of content validity interview participants. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Content validity interviews: caregiver and child characteristics ::: American English N (%)* American Spanish N (%) Total N (%) --------------------------------- ---------------------------- ---------------------------- ----------------- Number of Caregivers by language 10 (50%) 10 (50%) 20 (100%) Gender Female 8 (80%) 6 (60%) 14 (70%) Male 2 (20%) 2 (20%) 4 (20%) Missing Data 0 (0%) 2 (20%) 2 (10%) Age Mean 31 47 38 Range 24--37 30--79 24--79 Relationship to Child Grandparent 0 (0%) 3 (30%) 3 (15%) Parent 10 (100%) 5 (50%) 15 (75%) Missing Data 0 (0%) 2 (20%) 2 (10%) Number of Children by Caregiver Language 10 (50%) 10 (50%) 20 (100%) Child\'s Age(years, months) Mean 0.7 4.2 2.3 Median 0.5 2.0 0.7 Range 0.4--1.4 0.3--10.5 0.3--10.5 Gender Female 4 (40%) 5 (50%) 9 (45%) Male 6 (60%) 5 (50%) 11 (40%) Missing Data 0 (0%) 0 (0%) 0 (0%) ::: The distribution of children was 45% female and 40% male. An additional 15% of the gender data was missing and is not included in these figures. The average age of the population was 2.3 years, with a range from 0.33 to 10.5 years. As noted, the majority of comments used for item generation of Version 1 pertained to infants and toddlers under the age of three years. Thus, an attempt was made to test the relevance of the PGCIQ in caregivers of older children during the content validity interviews. Despite attempts to recruit caregivers of older children, the final age distribution was skewed towards children 12 months-old and younger. Premature through three month-old infants composed 17%, four month-old to 12 month-old infants composed 56% and 12 month-old through 12 year-old children composed 28% of the final distribution. Similar to the age distribution of the focus group discussions, 85% of the interview participants cared for children through the age of three, with only three of the 20 caregivers having children over three years. Table [2](#T2){ref-type="table"} contains characteristics of the children of the interview participants. ### Caregiver Impressions Overall, the respondents had positive impressions of the PGCIQ and described it as \"easy to understand\" and \"easy to answer.\" The respondents were also satisfied with the recall period, length and the format of the questionnaire. The average time of completion was eight minutes, with a range from five to 15 minutes. The caregivers suggested a number of revisions to improve clarity. These suggestions, combined with the results of the in-depth translatability assessment, resulted in wording modification of 25 items and deletion of 10 items. The majority of wording modifications were slight changes to enhance the clarity of the items. For example, one item \"During the last two weeks, I was limited in preparing meals\" was modified to \"During the last two weeks, I was limited in preparing meals for my family.\" Additionally, several caregivers suggested including a new domain to capture their relationships with family members. In response to this feedback, a new domain, \"Your Relationship with Your Family Members,\" was added. Items in this section used the same wording as the items in the \"Your Relationship with Your Partner\" section, with the word \"partner\" replaced by \"family member(s).\" ### Final questionnaire The version of the PGCIQ created after content validity testing contains 49 items assessing 10 core domains: \"Taking Care of Your Child,\" \"Your Daily Activities,\" \"Your Emotional Well-Being,\" \"Your Household Expenses,\" \"Your Physical Health,\" \"Your Social Life,\" \"Your Relationship with Your Partner,\" \"Your Relationship with Your Family Members,\" \"Your Employment Prior to Caring for Your Child with GERD,\" and \"Your Current Employment.\" An additional, optional module with nine items, \"Your Experiences Related to Diagnosis,\" is available for investigative research purposes and for use only at baseline. Discussion ========== The following sections present considerations for the PGCIQ\'s use and potential limitations of the study. The following topics are addressed: \"Your Relationship with Your Family Members\"; symptom severity level; child\'s age and caregiver characteristics. Your Relationship with Your Family Members ------------------------------------------ The new domain, \"Your Relationship with Your Family Members,\" was added following content validity testing interviews based on feedback from participants that additional questions about relationships with family members would provide valuable information. This new domain has not been tested with the wording \"family members.\" As this is a significant change that has not been tested, this domain must be scrutinized during the linguistic validation and psychometric testing process. Symptom severity level ---------------------- The PGCIQ was generated utilizing data from caregivers of children with a range of severity levels. Children with more severe GERD or significant co-morbidities (for example, hiatal hernias or developmental disorders) tended to increase the impact upon the primary caregiver across all nine domains. Conversely, a longer time period since diagnosis tended to reduce the impact on the caregiver. The reduction in impact over time appeared to be the result of two factors. First, many children eventually received successful treatment, which alleviated the GERD symptoms and reduced the strain on the caregiver. Second, the caregivers learned to accept and adapt to their children\'s condition with time. Child\'s age ------------ Given the age distribution of the study population, the PGCIQ is currently recommended for use in caregivers of pediatric GERD patients, newborn through three years old. Despite attempts to recruit across three age groups, ranging from newborn to 12 years of age, 80% of the focus group participants and 85% of the interview participants cared for a child aged newborn through three years of age. Although these age distributions were consistent with findings in the literature that approximately 70% of GERD cases spontaneously remit after age three, we felt that the majority of data favored infants and toddlers. Further testing of this instrument is therefore recommended in order to consider its use in populations of caregivers of children over the age of three. Researchers interested in extending the PGCIQ to children over the age of three are encouraged to consider the issues relevant to a child\'s age presented in the results section. Caregiver\'s gender, education and socio-economic status -------------------------------------------------------- The focus group discussion and content validity participants were primarily married women. From interviews with physicians, it was found that the majority of caregivers who seek treatment for pediatric GERD patients are women. Even though this instrument has been developed in a primarily female population, the identified caregiver issues appear to span gender roles. The men who participated in the focus group discussions were a small, but vocal minority, who elicited the same core domains as the female participants. Particular items of concern when using the questionnaire in men may be those related to domestic daily activities, given that these items tap more stereotypically female responsibilities. However, if the man is the primary caregiver, then it would stand to reason that he would be responsible for domestic daily activities. Future studies should seek to test the validity of the PGCIQ in larger populations of men and women to ensure that the items are equally relevant across gender roles. Conclusions =========== The PGCIQ has been developed in American English and American Spanish following a rigorous methodology of simultaneous item development for use in observational studies and multi-national clinical trials. The instrument is being linguistically validated from the American English version into several other languages following the appropriate methodology. After linguistic validation and psychometric testing is complete, a theoretical scoring model can be developed, and the PGCIQ may be used in international studies. Considering the age composition of the focus group and content validity interview population, the PGCIQ is currently recommended for use only in caregivers of pediatric GERD patients, newborn through three years old. Further testing of this instrument is recommended in order to consider its use in populations of caregivers of children over the age of three. Additionally, the study population was predominantly female, married and of moderate to high educational status. However, analysis of the caregivers\' verbatim comments suggested that the nine domains were equally relevant to men and women, married and unmarried participants and caregivers of different educational levels. Future tests of the PGCIQ should include large samples of men and women of different marital and educational status to confirm the relevance of these domains. The PGCIQ is the first questionnaire to document the multi-dimensional impact of caring for an infant or young child with GERD, making it a valuable tool to gather information and quantify the relevant issues and impacts experienced by caregivers of pediatric GERD patients. Information gathered from this tool can be used to inform and educate physicians, managed health care organizations, pharmacists and other health care providers about the needs of caregivers. In addition, items in the PGCIQ were specifically developed to capture changes in caregiver impact in response to successful treatment. Because caregivers mentioned all nine domains as affected by their child\'s GERD, we conjecture that intervention effects would be associated with changed scores in all nine domains. Thus, the PGCIQ may provide valuable information in multi-national clinical evaluations regarding the value of different GERD treatments from the caregivers\' perspective. This simultaneously generated patient-reported outcome instrument is one of the few examples of simultaneous development identified in the literature. This methodology results in a measure with a reduced risk of factors that might otherwise threaten the successful cross-cultural adaptation of the instrument, making the PGCIQ an appropriate instrument for adaptation into multiple languages. It is important for the PGCIQ to undergo psychometric testing to develop a final scoring model before the instrument can be used to assess the impact of pediatric GERD on caregivers. Academics and researchers interested in obtaining a copy of the PGCIQ should contact the lead author. Authors\' contributions ======================= JK Participated in the study design and coordination DLK participated in the design, coordination and analysis and its interpretation SB participated in the field work and coordination and analysis and interpretation JAC conceived of the study and participated in its design Acknowledgments =============== Crystal Wade, Research Associate and Melba Juez, Research Consultant, Mapi Values, Boston, USA Elizabeth Pulsifer-Anderson, Director and Janice Burns, M. Ed., Associate Director of Pediatric/Adolescent Gastroesophageal Reflux Association, Inc. -- PAGER	PubMed Central	2024-06-05T03:55:52.735474	2005-1-14	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548517/", "journal": "Health Qual Life Outcomes. 2005 Jan 14; 3:5", "authors": [ { "first": "Jennifer", "last": "Kim" }, { "first": "Dorothy L", "last": "Keininger" }, { "first": "Sara", "last": "Becker" }, { "first": "Joseph A", "last": "Crawley" } ] }
PMC548518	Background ========== Inequities in global health continue to be among the major challenges facing the international community \[[@B1]\]. Despite tremendous advances in medicine, the benefits of science and technology have yet to make a major impact on the health and quality of life of majority of the world\'s population. Recognizing its fundamental role as engine for development, the New Partnership for Africa\'s Development (NEPAD) has identified science and technology as a key platform for Africa\'s renewal \[[@B2]\]. A major challenge for Africa, and for the entire international community, is to bring scientific and technological advances to bear on the health priorities of poorer countries \[[@B3],[@B4]\]. Africa in the twenty-first century is faced with a heavy burden of disease, combined with ill-equipped medical systems and underdeveloped technological capacity \[[@B5]\]. The crippling poverty in many countries in the continent contributes to the disease burden, and hampers countries\' ability to address the problem adequately \[[@B6]\]. While Africa\'s response to its health challenges has varied considerably across the continent, with governments traditionally placing less emphasis on developing S&T than other sectors \[[@B7]\], there has been ongoing R&D activity in genomics and related fields of technology over the past several years in various parts of the region. The African Medical Research Foundation (AMREF), Africa\'s largest indigenous health charity, has for nearly half a century made an important contribution to addressing health challenges in Africa through partnerships with local communities, governments and donors \[[@B8]\]. A number of centres of excellence have emerged across the continent in recent decades, including the International Centre of Insect Physiology and Ecology (ICIPE) in Nairobi where important work has been done to uncover the role of insects in the transmission of infection <http://www.icipe.org>, and the Institute for Molecular and Cell Biology-Africa (IMCB-A), founded in 1999 to study the molecular mechanisms of tropical infections. A further example is the new Biosciences Facility for Eastern and Central Africa that was recently launched as part of a NEPAD initiative \[[@B9]\]. NEPAD, which has been adopted by the United Nations General Assembly as Africa\'s development framework, has called \"for the establishment of regional platforms with concrete actions to build and strengthen Africa\'s competence to harness and use new technologies for human development\" \[[@B2]\]. Its strategy acknowledges that Africa will have to overcome considerable challenges, including creating adequate regulatory and biosafety frameworks, building scientific capacity, and developing integrated systems of innovation. In March 2002, the African Centre for Technology Studies (ACTS) and the University of Toronto Joint Centre for Bioethics (JCB) co-organized an intensive five-day Course on Genomics and Public Health Policy in Nairobi, Kenya, bringing together scientists, policy makers, journalists, lawyers and NGOs from ten African countries to discuss, collectively, the question of \"How best to harness genomics to improve health in Africa?\" This course was sponsored by Genome Canada, the International Development Research Centre, and the African Centre for Technology Studies, through the Norwegian Agency for Development Co-operation. The primary goal of the course was to familiarize participants with the potential of genomics and related biotechnologies to address health needs in Africa. This article presents the findings and recommendations that emerged from this process, and suggests how such courses might be more broadly employed as a method for bringing together opinion leaders to share ideas and work collectively to develop practical policy solutions. Methods ======= The programme was planned collaboratively by the African Centre for Technology Studies and the Joint Centre for Bioethics. The basic layout of the sessions and their topics was modelled on a prior course held in Toronto, Canada in May 2002. The programme was organized in line with the objectives outlined in Table [1](#T1){ref-type="table"}. Course participants as well as session leaders were identified on the basis of recommendations from recognized experts in the region and through literature searches. Many session leaders were local experts, well placed to contextualize the \"new science\" of genomics within the frame of concerns and realities particular to Africa. Care was taken to select participants representing a range of interests and backgrounds, including individuals from science, economics, law, government, the press, and non-governmental organizations. Such diversity was sought in recognition of the importance of \"cross-pollination\" on a multifaceted topic like genomics, and consequently the need for multiple actors to be part of the building of policy, as well as mediating the dialogue between policymakers and the public. In total, 30 participants attended; the countries and the institutions they represent are listed in Table [2](#T2){ref-type="table"}. Despite concerted efforts to draw a balanced group, the participant list reveals a markedly high proportion of academics, and indeed no representatives from industry. Moreover, only three of the participants are women. The organizers covered all costs for attending the course (transportation, hotel accommodation, and meals), in order that inability to pay not be an inhibiting factor for those who wished to participate. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Objectives of the course ::: ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- • To familiarize participants with the current status and implications of genomics and biotechnology for health in India, and to provide information relevant to public policy • To provide frameworks for analyzing and debating the policy issues and related ethical questions, and to help understand, anticipate and possibly influence the legal and regulatory frameworks which will operate, both nationally and internationally • To begin developing an opinion leaders network across different sectors (industry, academic, government, and voluntary organizations) by sharing perspectives and building relationships ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Countries and Institutions Represented ::: -------------------------------------------------------------------------------------------------------------- African Centre for Technology Studies, Kenya African Malaria Vaccine Testing Network (AMVTN), Tanzania African Medical and Research Foundation, Kenya Centre for the Development of People (CEDEP), Uganda Chemistry Department, University of Zambia, Zambia Department of Biochemistry, University of Khartoum, Sudan Department of Epidemiology of Parasitic Disease, National School of Medicine and Pharmacy, Mali Department of Obstetrics and Gynecology, Assiut University, Egypt Department of Pathology, Makarere University, Uganda Department of Virology, University of Ibadan, Nigeria Division of Human Genetics, Faculty of Health Sciences, University of Cape Town, South Africa Dysmorphology and Alcohol Pharmacokinetics in Fetal Alcohol Syndrome, South Africa Federal Ministry of Science and Technology, Nigeria Inter-Region Economic Network (IREN), Kenya Journalist Against AIDS (JAAIDS), Nigeria Lawyer, Kenya Maternal, Child and Women\'s Health, Dpt. of Health, Western Cape Province, South Africa Molecular Biology Research Facility, Nelson R Mandela School of Medicine, South Africa National Council for Science and Technology, Kenya National Health Laboratory Service and Division of Human Genetics, University of Witwatersrand, South Africa School of Public Health, University of Ghana, Ghana Science and Development News, and BiotekAfrika, Kenya Science Secretary, Uganda Council for Science and Technology, Uganda The People Newspaper, Kenya -------------------------------------------------------------------------------------------------------------- ::: Because of the diversity of the participants, no background in science was presupposed. The sessions were organized so that participants were first introduced to the subject of the \"new science\" of genomics, and were then instructed in areas including national innovation systems, business models, intellectual property rights, international conventions and regulatory structures, ethics, and the role of networks in facilitating dialogue, advocacy and policy making. A detailed time-table of the programme is shown in Table [3](#T3){ref-type="table"}. Presenters used overhead transparencies or presentation software such as Microsoft Powerpoint. Active participation was encouraged throughout with at least 45 minutes allotted for discussion at the end of each session, on the assumption that each participant brought considerable expertise and valuable practical experience of his or her own. The programme therefore employed a peer-learning environment in which participants could learn from each other, in addition to learning from material presented by instructors. Each participant was provided with a course reader, which included additional background material on session topics; class sessions used a variety of learning methods including lectures, discussions, case analysis, and simulations. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Agenda for the Course on Genomics and Public Health Policy in Africa. ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Time Day 1 Day 2 Day 3 Day 4 Day 5 -------------- --------------------------------------- ------------------------------ -------------------------------------------- ----------------------------------------------------------- ------------------------------- 9.00--10.30 Introduction\ Internet-based Leader\ Intellectual Property Rights I\ Ethics I\ Group Presentations Prof Abdallah Daar, Dr John Mugabe\ Networking: Exercise\ Dr Patricia Kameri-Mbote Dr Peter Singer New Science I : Introduction\ Prof Joseph D\'Cruz Dr Stephen Scherer 11.00--12.30 New Science II\ National Innovation Systems\ Intellectual Property Rights II\ Ethics II\ Group Presentations Continued Dr Stephen Scherer Prof Norman Clark Dr Patricia Kameri-Mbote Prof Abdallah Daar 1.30--3.00 New Science III\ Business Models\ Internet-based Leader Networking: Results\ Science & Innovation Policy in International Conventions\ Prof Onesmo ole-MoiYoi Prof Joseph D\'Cruz Prof Joseph D\'Cruz Dr John Mugabe 3.30--5.00 Genomics and Global Health\ Group Work Group Work Group Work Dr Peter Singer ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ::: Early in the course, participants were divided into small Study Teams consisting of persons with diverse backgrounds, in order to maximize complementary skills. These Study Teams were an integral part of the learning process of the programme. Sessions were intended primarily to provide input for participant Study Teams, which assembled several times during the week. Their primary task was to draw upon the course material and their own experiences to propose recommendations for policy relating to genomics and biotechnology in Africa. Presentations were made on the last day of the course, and the final sessions focused on how to take forward the ideas and proposals generated during the course. This was the first course of its kind in Africa, as well as the first of a series of planned courses on genomics policy to be held in developing countries; evaluation was therefore a key component of the programme. At the end of each day, participants were given a questionnaire to complete, in which they had an opportunity to evaluate the day\'s sessions. At the end of the course, participants were asked to complete a more detailed questionnaire, asking for their feedback on the overall aims and organization of the course. Results ======= The course opened with an Introduction, where Prof. Abdallah Daar and Dr John Mugabe welcomed the participants, explained the course\'s objectives, and then invited each of the participants to introduce him- or herself to the rest of the group. The opening session was led by Dr Stephen Scherer, and was intended to provide a comprehensive overview of the science of genomics and its relevance to health. Several of the participants had a limited scientific background; the presentation therefore include very basic descriptions of the science involved, as well as images and a brief video, and gradually progressed to a discussion of its applications in health research and medicine, both now and in the future. This session was followed by an introduction by Prof. Onesmo ole-MoiYoi, a pioneering Kenyan scientist, to advances in genomics and molecular biology within the African context -- including cutting-edge research at his institute and others on the continent, as well as the broader relevance of genomics and molecular approaches to the health of Africa\'s people, animals and the environment. The first day closed with a session led by Dr Peter Singer who described a five-point strategy to systematically capture the benefits of genomics for the health of citizens in developing countries, through research, capacity-strengthening, consensus-building, public engagement, and an investment fund. Examples of ongoing work by the University of Toronto\'s Canadian Program on Genomics and Global Health in these areas were discussed, including the results of its 2002 study to identify the most promising biotechnologies to improve health in developing countries \[[@B13]\]. Prof. Joseph D\'Cruz opened the second day with a discussion introducing participants to new approaches to forming and expressing opinions about emerging issues using the internet. Leaders in any area are required to develop their own views about new developments in their fields, and the process of forming these views is facilitated by peer discussions. Though traditionally these processes have taken place face-to-face, the internet offers an alternative medium that allows individuals to interact with their peers in other locations at a time and pace suited to each individual\'s commitments, without forcing the group to reach early consensus. Prof. Norman Clark followed with a session aimed at introducing participants to the concept of \'National Systems of Innovation\', a conceptual framework for analysing country-specific factors that influence innovation across sectors. Innovation is understood as processes of generating new ideas, products and production processes, as well as to processes of institutional change and development. Such frameworks can be useful in identifying and analyzing key factors affecting African countries\' ability to engage effectively in biotechnology and genomics for human development. The last session of the day focused on the business life cycle of a genomic product, tracing its development from the laboratory bench to a patented invention that is exploited commercially. The session addressed the strategic issues and choices that firms face at each point in this life cycle, and used a case-study based approach to frame the issues. The last one-and-a-half-hour session of the day was devoted to group work among members of Study Teams, whose members were selected to bring diverse views and experiences to bear on their deliberations. The third day of the course was devoted primarily to the issue of intellectual property rights (IPRs). The two sessions on IPRs were led by Dr Patricia Kameri-Mbote, Kenyan lawyer and scholar. During the first of these, Dr Kameri-Mbote explained the nature and different kinds of IPR protection, and explored how these impact on biotechnology development and technology transfer. She also considered the relationship between IP protection and public health in developing countries, using specific cases that have arisen under the World Trade Organization\'s Agreement on Trade Related Aspects of Intellectual Property Rights (TRIPS). Positions held by different countries and scholars on IP and biotechnology transfer in health were examined, and international, regional and national intellectual property regimes were reviewed. The second session focused on the link between IP, public health and transfer of biotechnology, in addition to the ethical, social and policy implications of the \"Doha Declaration\" on health by WTO ministers intellectual property rights in the area of health under TRIPS. At the end of the third and fourth days, participants again met for 1.5 hours in their Study Teams to prepare their proposals. Day four of the course had a heavy focus on ethical dimensions of emerging technologies like genomics. The first session provided an overview of ethical issues related to genomics and public health policy. Prof. Abdallah Daar led this and the second session on ethics. He described the World Health Organization\'s draft Guiding Principles on Medical Genetics and Biotechnology document, which he co-authored and which provides a broad overview of the ethical principles in this field. During the second session, Prof. Daar and Dr. Singer led the group through a case involving benefit sharing, and introduced the Human Genome Organization\'s principles and statement on benefit sharing. Dr. Singer then described an ethical framework and approach to priority-setting for genomics technologies in health care institutions. The last hour of this session was devoted to providing a forum for participants to share their expertise and experiences in areas related to policy. The final session of the day was led by Dr John Mugabe, then-Director of the African Centre for Technology Studies in Nairobi, Kenya. This session introduced participants to international conventions and protocols that emerged out of the United Nations Conference on Environment and Development (UNCED), and focused on science and innovation issues covered by the Conventions on Biological Diversity and its Cartagena Protocol on Biosafety, and the International Treaty on Plant Genetic Resources for Food and Agriculture. Specific lessons were drawn for international rule-making for health equity, and emphasis was given to biotechnology, risks assessment, technology transfer, sharing benefits of global scientific and technological advances, and technical cooperation. On the last day, each of the four Study Teams presented their proposals, which addressed the overarching question of the course: \"How to harness genomics and related biotechnology to improve health in Africa?\" Study Teams presented one at a time; after each presentation, there was a period for questions and discussion, and afterward an opportunity to consider all proposals together in light of the host of issues raised during the course of the week. The presentations, though prepared independently by each group, demonstrated a number of common themes that tended to be organized in terms of long-term foundational issues of sustainability, and more concrete short-term issues relating to garnering political involvement. Table [5](#T5){ref-type="table"} enumerates the key recommendations that emerged from these sessions. ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Recommended Action-Steps ::: -------------------------------------------------------------------------------------------- Establish a regional network to foster sustained inter-sectoral dialogue Identify champions among politicians Use the New Plan for African Development (NEPAD) as entry point onto political agenda Commission African capacity survey in genomics-related R&D to determine areas of strength Undertake a detailed study of R&D models with demonstrated success in the developing world Establish seven regional research centres of excellence Create sustainable financing mechanisms -------------------------------------------------------------------------------------------- ::: Discussion ========== The following is a synthesis of the participants\' efforts, summarizing and describing key issues that emerged from their presentations and throughout the weeks\' deliberations. It includes several concrete action-steps recommended by the participants, which flow from these considerations. Creating a Platform for Ongoing Dialogue and Advocacy ----------------------------------------------------- The course generated a great deal of enthusiasm and vigorous discussion, and there was consensus among the participants on the need to create a mechanism for capitalizing on this momentum. Course participants and faculty therefore established an e-mail-based network, the African Genome Policy Forum (AGPF), to allow the continued exchange of ideas and the building of consensus on issues related to genomics and public health policy. The group, composed of participants from areas of government, academia, civil society and the media, was created to bring to the table the views of their respective constituencies, and inform their peers of insights gained from the course and through the network. The network may also play an advocacy role in promoting the responsible use of genomics as a tool to improve health and promote development in Africa. ### Concrete Action-Step 1: Establish a regional network to foster sustained inter-sectoral dialogue On the final day of the course, it was decided that a regional network, the \"African Genome Policy Forum\", be established comprising all participants and session leaders; it was further agreed that the Joint Centre for Bioethics would set up a web-site, discussion board, and e-mail based platform to facilitate ongoing discussion and inter-sectoral debate on the issues and proposals raised during the course. Mobilizing Political Support ---------------------------- The success of any major initiative requires sustained dialogue with politicians. It is important to take the time to address their legitimate concerns, by clarifying the specific relevance of genomics and its applicability within the context of their communities. A point of particular relevance is the link between technologies like genomics and Africa\'s development, which has been well described in a number of recent reports \[e.g. \[[@B6],[@B10]\]\]. Participants highlighted the importance of taking back to their colleagues in their respective countries and institutions the lessons drawn from the course; those participants in public office agreed to seize opportunities to raise some of issues and proposals of the course when attending relevant forums. In particular, the nascent New Partnership for Africa\'s Development, adopted in 2001 under the mandate of the Organisation of African Unity, was repeatedly pointed to as an opportunity to bring genomics and its relevance to health in Africa onto the political agenda. Science and technology is among NEPAD\'s seven priority areas; another is human development, which encompasses health \[[@B11]\]. Genomics provides a clear example of how these two areas -- science and technology, and health -- come together, and can serve as a model for considering how science and technology and health concerns can be better integrated to address the continent\'s economic and health needs. ### Concrete Action-Step 2: Identify champions among politicians The most efficient means of garnering political support is often to go directly to the politicians themselves -- those who have been supportive or outspoken of the issues in question -- to put the subject before their colleagues. The course itself represented an important step in this direction, as it brought together a spectrum of stakeholders, including academics, civil society, and government officials. The course, and the subsequently established network, therefore furnished an opportunity for direct communication and dialogue among individuals with a shared vision, including policymakers in a position to \"champion\" the issues and proposals that emerged from the course to their colleagues and others. ### Concrete Action-Step 3: Use the New Plan for African Development (NEPAD) as entry point onto political agenda NEPAD offers a possible forum to bring the subject of genomics-related biotechnology onto the political agenda, and provides a means of informing African leaders of genomics and its relevance to improving health and development in Africa. In particular, the AGPF recommends the establishment by NEPAD of an \'African Genomics Committee\', which would provide a plan for utilizing genomics and other new technologies to enhance health in Africa, advocate for increased investment in S & T, target other relevant stakeholders in individual countries, educate policy makers about the need for a strong R&D base established through partnerships across Africa, and organize steering committees to identify gaps and implement strategies for improvement. Prioritizing Needs ------------------ Participants agreed on the need to consider emerging technologies like genomics in light of Africa\'s specific health challenges, and consequently on the importance of prioritizing these and identifying strategic entry points. Infectious (including sexually transmitted) diseases, genetic and other non-communicable disorders, sanitation, nutrition, environmental pollution and loss of biodiversity were all proposed as areas requiring concerted attention, with a special emphasis on the potential for using genomics-related biotechnology to target the three biggest killers in Africa: malaria, HIV/AIDS and tuberculosis. There are already well-known African-led initiatives to apply scientific innovation to combat important health concerns, such as the Multilateral Initiative on Malaria, and the African Malaria Vaccine Testing Network (AMVTN). It will be important to build on existing success stories, and to identify gaps in terms of priority health areas receiving inadequate attention. This will help to focus efforts and to more efficiently channel limited resource, both financial and human. A regional approach, which has since been adopted by NEPAD, was proposed as a promising mechanism for harnessing existing competence to address local needs. ### Concrete Action-Step 4: Commission African capacity survey in genomics-related R&D to determine areas of strength This survey would identify strategic areas of strength, such as existing centres of excellence, potential areas of improvement, and health priorities receive inadequate attention. It would also serve to identify local and national innovators, and to inform the structuring of Regional Centres of Excellence described below. Capacity Building & Public Engagement ------------------------------------- For several years, genomics has been linked with a number of high-profile, intensely controversial issues like human cloning and genetically modified organisms. While emerging technologies like genomics raise a number of important ethical and social issues that deserve careful consideration \[[@B12]\], a nuanced message takes account of the possibilities as well as the challenges of new approaches. Often, technological applications can complement existing, well-established health approaches \[[@B13]\]. Scientists, policy-makers, and the media have an important part to play in publicizing science, and pointing out its relevance to Africans in a moderate rather than hyperbolic tone \[[@B14]\]. Local leaders can have an important role to play, not only in reflecting the leading-edge opinions of their different constituencies to policymakers, but also by playing a role in raising awareness within their communities. A more informed public is often a more engaged public, which can effectively advocate for the development of policies that reflect legitimate concerns, while leaving space to explore promising avenues of scientific endeavour. Public engagementwas seen to form part of a long-term strategy for capacity building, and raising the overall profile of science and technology in Africa. The discussions reflected a conception of capacity strengthening as intimately linked with quality education -- at all levels, and across disciplines. Core to this debate among course participants was the belief that endogenous capacity must be developed in order that Africa can begin to be self-sufficient, and itself become an innovator. Participants identified the following categories as needing attention: ### Primary, secondary and tertiary education There is a need to introduce innovative techniques to teach science and technology in the classroom, in order to generate interest and aptitude in the subject matter from an early stage in the educational process. Besides contemporary scientific approaches, indigenous knowledge and its applications to health could also be a relevant component to include in the curriculum. ### Policymakers Those in a position to shape policy should be familiarized with codes of ethics pertaining to their field; moreover, they should be educated about how best to capitalize on international frameworks (e.g. WTO\'s Trade-Related Aspects of Intellectual Property Rights; the UN\'s Convention on Biological Diversity) in order to ensure that their countries benefit from such arrangements, and are not exploited. Policy makers should develop strategies for negotiating their interests collectively in international forums, when appropriate, given shared needs and values. ### Media There is a general need to strengthen capacity in the area of communication, in particular on increasing the level of science literacy among the media. This might include integrating journalism and science programs at the college and university levels. There is a corresponding need to improve the ability of scientists to communicate the relevance of their work to the public, and to policy-makers. ### ELSI There is a great need to build capacity in Africa with regard to the ethical, legal and social issues (ELSI) which inevitably accompany the emergence of new technologies. Strategies would in many cases involve sensitizing the public to issues of relevance, such as their rights as patients and participants in research (e.g. informed consent, confidentiality of patient information), encouraging dialogue about the social consequences of introducing new technologies into traditional settings, and putting frameworks in place (e.g. ethics review boards) to ensure that ethical, quality and safety standards before research is undertaken. Partnerships ------------ Along with the need to strengthen the R&D base in science and technology, participants of the course identified a related need to increase the emphasis on commercialization -- not only as a tool for sparking innovation but also to permit the generation of capital necessary to sustain the industry. An important step in the process of moving toward commercialization is the forming of alliances within countries, between universities and industry, sometimes known as \"cross-linking\". The fruitfulness of the Africa course, where people from across sectors and sub-regions came together with a common mission, re-enforced the value and the importance of establishing cross-sectoral networks and collaborations. Networksprovide a means of generating new ideas, pooling the creative energies of individuals, and exchanging advice and expertise around a particular area of focus, in this case genomics and health policy. Such networks could play an advocacy role, combining the voices and the influence of key players from diverse disciplines and sectors, to advance a common aim. Collaborations, at the level of institutions -- both within and between countries and regions -- would facilitate the transfer of both knowledge and technology. During the course, it was pointed out that there is a particular need to encourage linkages between universities and industry to, among other things, facilitate the move from research and development to product generation and commercialization. This could include mechanisms to facilitate relationships between universities undertaking research in biotechnology and local industries. Institutional partnerships and collaborations at all levels, including internationally, can mean the channelling of resources to common areas of focus, and pooling the relative strengths and resources of partner institutions \[[@B15]\]. Such collaborations require very clearly defined roles for partners, and transparency with respect to goals, prioritization of needs, funding, and mechanisms to ensure equitable access to products. Creating sustainable financing mechanisms ----------------------------------------- Ensuring that the benefits of science and technology, including emerging fields like genomics, requires a long-term strategy for sustained investment. ### Concrete Action-Step 5: Design proposals for obtaining sustained investment for both research and development (R&D) in genomics and related biotechnologies to improve health, and the commercialization of the products of R&D Three models were suggested The establishment of an African Science and Technology Fund, dedicated to supporting research and development in the area of health-related biotechnology, would rely upon the contribution of African governments. The establishment of an Investment Fund for genome-related biotechnologies for improving healthwould represent an innovative approach to obtaining capital, providing a further incentive for investors to put money into development by creating a fund that provides a return on investment, as well as furnishing funds for advancement. Such a fund might be dedicated to providing capital for the development of mature, or future, health-related technologies. Capitalizing on existing fundsallocated for research related to diseases afflicting Africa, such as the WHO\'s Global Fund to Fight AIDS, Tuberculosis and Malaria. Genomics and biotechnology represents a powerful set of tools for health improvement, and the World Health Organization through its Genomics and World Health(2002) report has raised it as an important issue deserving international attention. It is important to use this positive emphasis to give weight to the case for the relevance of biotechnology to health in developing countries, particularly for policy makers. Research and Development (R&D) ------------------------------ With respect to R&D, there are already areas of strength on the continent; it is crucial to identify localized expertise, and to establish linkages with centres elsewhere in the region, as well as abroad, to ensure the transfer of knowledge and of technology, and to facilitate human resource development. Infrastructure must be developed to attract qualified African researchers to remain in or to return to Africa -- both to support them, technically, intellectually, and socially and to provide them with similar opportunities for creativity and growth as may be found in other locales. The Biosciences Facility, established in 2003 by NEPAD, takes up this challenge, promoting \"scientific excellence by bringing together a critical mass of scientists drawn from national, regional and international institutions in state-of-the-art facilities where they can undertake cutting-edge research to help solve the most important development constraints faced by the poor in Africa\" \[[@B9]\]. While the new Biosciences Facility is the first of network of centres of excellent focused primarily on using science to help poor farmers, it may be an appropriate model for like initiatives using a regional approach for targeting health challenges. ### Concrete Action-Step 6: Undertake a detailed study of R&D models with demonstrated success in the developing world, i.e. China, India, Cuba, Brazil Developing countries in various parts of the world have proven that they too can have strong technology sectors, and make important contributions in terms of science and innovation. Their successes represent an opportunity to bring to the attention of politicians that there are countries succeeding in genomics. A detailed study of these models can provide important insights into how Africa can capitalize on the promise of genomics and biotechnology, particularly as it relates to health. In 2003, the Joint Centre for Bioethics completed a qualitative study of R&D in biotechnology in South Africa; similar studies are underway in Cuba, Egypt and China. Research of this kind could feed into more systematic efforts in the region to better understand how some developing countries, including those in Africa, have managed to develop S&T research and manufacturing capacity in the health sector. ### Concrete Action-Step 7: Establish Seven Regional Research Centres of Excellence The proposed centres would be distributed across Northern, Southern, Eastern, Western and Central African sub-regions. Each centre would have its own area of focus, in terms of targeted health problems, depending on regional expertise. The Centres would not be the sole preserve of each region, but would in fact use the strengths and specializations of each region to achieve the goal of harnessing genomics to improve health in Africa. These regional centres of excellence need not preclude the existence of national centres of excellence. The Biosciences Facility is modelled on such an approach. Conclusion ========== Analysis -------- The course on Genomics and Public Health Policy in Africa was carefully designed, with inputs from both its Canadian and African co-organizers, to have a programme and participant profile reflecting the inter-disciplinarity of the issue being considered. Genomics cuts across S&T, environmental, development, industrial, education and health policy and generates important ethical, legal and social issues. It therefore requires a genuinely participatory and multi-stakeholder approach, as well as frank discussions about both the potential promise and perils of a relatively new science. The strength of the course, as reflected in the evaluations submitted by participants, was the rare opportunity for discussion and networking among opinion leaders from different sectors. Both during and between sessions, participants exchanged perspectives and experiences with others from different regions of the content, and from different disciplines. Senior political officials, journalists, academics, and civil society representatives worked together in Study Teams to create proposals. Discussions were lively and open, with broad participation from those in attendance. However, a weakness of the course was the absence of industry representatives, who would certainly have contributed an important and valuable point of view. The small number of women participants was also a notable disadvantage. Later courses modelled on the Nairobi offering (i.e. those in Latin America, the Eastern Mediterranean, and India) had greater success in drawing participants from industry and obtaining a better gender balance. Notably, however, the recommendations that emerged from these courses, while reflecting differences due to regional priorities and context, did not vary considerably despite the broader contribution, particularly from the private sector \[[@B20]\]. A major outcome of the Nairobi course, and one which had strong support from participants, was the creation of a virtual network to facilitate ongoing interaction and discussion. Within two weeks of its completion, a website was created for the course <http://www.utoronto.ca/jcb/genomics/html/ACTS_main.htm>, as well as a web-based discussion board. While there was some initial activity on the discussion board, this eventually subsided, and was soon evident that this approach had failed. In an effort to revive the momentum and to solicit ideas from AGPF members about how to best move forward with the network, a short survey was sent to members asking what their needs were, both in terms of the network as well as in terms of the technical facilities at their disposal. The response rate was extremely low; however, those who provided feedback confirmed what the participation level suggested: namely, that information technology facilities in Africa are such that very few individuals, outside of some well-equipped academic or private institutions, have regular access to the internet. The web-based discussion board was, therefore, in practice a highly unsustainable option for the majority of participants. The point was also raised that it was not enough to be connected electronically; there was also a need to share a more tangible goal or project, and to have a more visible leader from within the group, to galvanize efforts and motivate continued interaction. One respondent explained that finding the time to contribute to such networks is extraordinarily difficult for many Africans, who often \"wear many hats\". As a result, a general interest was insufficient to justify diverting time from other tasks; a concrete, realizable goal was essential for engaging individuals who already feel over-stretched. As a consequence of these inputs, an email-based forum was established, since most AGPF members have better access to email than to the internet, and a moderator was temporarily appointed over the group. Activity on the forum improved and continues today, more than two years later, though interventions are irregular and generally extend to the sharing of information or material of interest, rather than discussions about issues. The India course on Genomics and Public Health Policy was held in January 2003, less than one year after the inaugural Nairobi effort. Based on feedback from the previous course, the questionnaire requesting feedback about participants\' technical and substantive needs in relation to the creation of a network was distributed during the course, to permit the creation of a network that was much more responsive to the needs of the participants. Moderators from among the participants were nominated before the course\' end and their roles clarified, to facilitate the sustainability and autonomy of the network. Later in 2003, two further courses were held in Oman and in Venezuela, both of which added a further element demonstrating the learning from the first two courses. On both occasions, the Joint Centre for Bioethics collaborated with the Regional Offices of the World Health Organization; in the first instance, with the Eastern Mediterranean office (EMRO) and in the second, with the Pan-American Health Organization (PAHO). This collaboration ensured that the recommendations of each course had an institutional structure through which they could be channelled, to reach the ear of decision-makers. EMRO and PAHO have extensive links with ministries of health within their regions, as well as with representatives from civil society and industry. This provided an opportunity for the results of the course to have a much wider impact. By contract, the impact of the Nairobi course is very much linked to the efforts of individual participants to engage with their constituencies and with the NEPAD initiative, of which one of their members is now a senior actor. The Forum developed following the Nairobi course has not provided a framework to drive action the way it was initially intended; however, it continues to provide a portal for information-sharing and dialogue. Final Remarks ------------- The executive course on Genomics and Public Health Policy in Africa was the first of its kind to be held on the continent. The response of participants indicated a tremendous enthusiasm for and interest in discussing the emerging technology of genomics and its applications for addressing the health woes of Africans. The sessions covered a spectrum of topics, from basic science, to ethics, business models and international frameworks -- exemplifying the range of intersecting issues relevant to informed discussions about genomics and related policy. The course also was a demonstration of the fruitfulness of a multi-stakeholder approach. An important aim of the course was to encourage network-building and the development of meaningful interactions, as a foundation for sustained dialogue among opinion leaders. Participants were encouraged to develop independent proposals in a collaborative environment, rather than to be passive recipients of \"expertise\" from the session leaders. The result was a series of concrete proposals for action, and the establishment of an e-network to provide a forum for ongoing communication, discussion and elaboration of the issues and proposals raised during the course. Several participants agreed to raise the proposals and themes articulated to their colleagues; the course also generated some publicity, as journalists invited to attend and to participate actively in the meeting reported on the key issues in various media \[\[[@B16],[@B17]\]; see also \[[@B18]\]\]. Since the completion of this course, three more offerings have taken place, one in India in collaboration with the Indian Council for Medical Research (ICMR) in January 2003, another in Oman in August 2003, and a third in Venezuela in 2004. A fourth course is being planned for a venue in South-east Asia. The Nairobi offering demonstrated clearly the receptiveness of African researchers and policy makers to such an initiative, and captured the vision of a cross-section of stakeholders around how to ensure that the new wave of scientific promise does not pass them by, or crush them in its wake, but instead is harnessed for better health and to further economic development in their region \[[@B19]\]. The courses in India and Oman similarly gave rise to regional e-networks \[[@B20]\], which may eventually be connected to form an inter-regional forum for dialogue to form a basis for the sharing of experiences and expertise acrossregions in the developing world. Each of the three executive course held to-date has addressed similar themes in relation to genomics and health; but each has also been adapted to the particular context and interests of the host country or region. This has partly been achieved through active collaboration between the Joint Centre for Bioethics and the host institutions. The electronic networks provide a means of generating a long-term impact, driven by participants who are empowered, in their particular capacities, to take forward the ideas shared and the proposals developed through their interaction. The Nairobi course also highlighted the importance of being proactive in soliciting suggestions from participants about creative means of virtual networking that realistically address the poor information technology infrastructure in most parts of Africa. It also was instructive in demonstrating that a network is not itself self-sustaining; it must be driven by a clear, shared vision among participants, and possibly even a concrete and realizable project. Moreover, ideally a moderator from within the group should take leadership in feeding the forum, and motivating ongoing participation. The New Partnership for Africa\'s Development (NEPAD) has made science and technology (including genomics and biotechnology) a key platform in its plan for economic renewal \[[@B2],[@B9]\]. Indeed, the recommendations outlined above overlap considerably with those described in a recent document detailing the resolutions of the first science and technology workshop of NEPAD, held in February 2003 \[[@B2]\]. The recent establishment of the African Biosciences Facility as a centre of scientific and technological excellence in the region, is further evidence that the recommendations articulated by the AGPF reflect a more widely shared vision. There is a growing recognition in Africa, and internationally, of the role that genomics and biotechnology can play, not only in alleviating health scourges of the poor, but also in addressing some of their economic concerns. With appropriate emphasis on its health needs, incentives for meaningful partnerships, sound regulatory structures, innovation and foresight, Africa could be in a position to benefit from genomics and related fields of biotechnology. The Course and Genomics and Health Policy in Africa had as its overarching goal that of bringing together a vibrant cross-section of individuals to foster dialogue around this timely issue. The African Genome Policy Forum works to build on this foundation, to sustain the momentum of the course, and to fulfill some of the participants\' proposed goals. Perhaps most significantly, this series of courses represents a practical and effective mechanism for drawing together a variety of actors to address an issue of recognized import, which deserves a truly inter-disciplinary approach. Moreover, it is an initiative that generates important debate, but which is ultimately focused around generating concrete proposalsto inform policymaking. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= All authors participated in and contributed to the course. ACS drafted the manuscript. PAS and ASD conceived of the course, refined the manuscript for critical content and approved final version; and with JM, participated the course design and its coordination. AGFP members provided intellectual input, through their lively discussions and proposals during the Course on Genomics & Public Health Policy in Africa, held 4--8 March 2002. Funding ======= The Canadian Program from Genomics and Global Health is funded by several sources listed at <http://www.geneticsethics.net>. This course was funded primarily by Genome Canada and the International Development Research Centre (Canada). PAS holds a Distinguished Investigator Award from the Canadian Institutes for Health Research. ASD is supported by the McLaughlin Centre for Molecular Medicine. The African Centre for Technology Studies, which hosted the course, was supported by the Norwegian Agency for Development Co-operation. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Reading materials. ::: ---- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 1 Scherer, S.W. 2001. The Human Genome Project. Isuma: Canadian Journal of Policy Research Vol. 2, No. 3, 11--19. 2 OWENS, K., KING, M-C. 1999, Genomic views on human history. Science 286, 451--455. 3 ROSES, A.D. 2000, Pharmacogenetics and the practice of medicine. Nature 405, 857--865. 4 Nature, Human Genome Volume, Vol. 409, Feb. 2001. 5 Science, Human Genome Volume, Vol. 291 Feb. 2001. 6 Nature, Human Genome Volume, Vol. 409, Feb. 2001. 7 Science, Human Genome Volume, Vol. 291 Feb. 2001. 8 PA Singer, AS Daar (2001). Harnessing Genomics and Biotechnology to Improve Global Health Equity. Science, 294 pp87--89 9 PA Singer, AS Daar (2000). Avoiding Frankendrugs. Nature Biotechnology, 18(12) 1225. 10 Walter W. Powell (1998). \"Learning from Collaboration: Knowledge and Networks in the Biotechnology and Pharmaceutical Industries\". California Management Review, vol. 40 (3), Spring. 11 Calestous Juma and Norman Clark. \"Technological Catch-up: Opportunities and Challenges for Developing Countries\". SUPRA Occasional Paper, Research Centre for the Social Sciences, University of Edinburgh (February, 2002). 12 Von Hippel, E. 1986. Lead Users: a source of novel product concepts. Management Science, Vol. 32, No. 7, pp. 791--805. 13 OECD, 1998. National Systems of Innovation. OCED, Paris. 14 1\. Stefan Thomke, Ashok Nimgade (2001). \"Millenium Pharmaceuticals, Inc.\" Harvard Business Law Review. 24pp. 15 2\. Ray A. Goldberg. \"Gene Research, the Mapping of Life and the Global Economy\". Harvard Business Review. 58pp. 16 Philippe Cullet. \"Trips and the Human Right to Health in Developing Countries\". International Environmental Law Research Centre. (See <http://www.ielrc.org>) 17 Jean O. Lanjouw (April 2001).\"A Patent Policy Proposal for Global Diseases\". Yale University, Brookings Institution and the NBER 18 Hartley & Hartley. \"Limitations on using existing legal doctrines in addressing changes in technology: the example of the \"Fertility Fraud\" cases at UC Irvine\". See Hartley & Hartley Attorneys at Law (California) at <http://www.hartley.com/technolo.htm> 19 \"Declaration on the TRIPS Agreement and Public Health\" (2001). WTO Ministerial Meeting, Doha, Qatar. 20 A.S. Daar, J.-F. Mattei. Appendix 2: Draft Guiding Principles and Recommendations, with alternative suggestions, after receiving comments. Medical Genetics and Biotechnology: Implications for Public Health. December 1999, World Health Organization. 21 A.S. Daar, J.-F. Mattei. Chapter 6: The Human Genome Diversity Project. Medical Genetics and Biotechnology: Implications for Public Health. December 1999, World Health Organization. 22 A.S. Daar, J.-F. Mattei. Chapter 7: Issues Raised by Conducting Research With Indigenous and Genetically Defined Communities. Medical Genetics and Biotechnology: Implications for Public Health. December 1999, World Health Organization. 23 HUGO Ethics Committee. Statement on Benefit-Sharing. April 9, 2000. 24 B.M. Knoppers, M. Hirtle, S. Lormeau. Statement on the Principled Conduct of Genetic Research. HUGO Ethical, Legal, and Social Issues Committee Report to HUGO Council, March 1996. 25 Statement of the WHO Expert Consultation on New Developments in Human Genetics. World Health Organization, 2000. 26 PA Singer, DK Martin, M Giacomini, L Purdy (2000). Priority setting for new technologies in medicine: qualitative case study. BMJ, 321(7272):1316-8. 27 N Daniels (2000). Accountability for reasonableness. BMJ, 321(7272):1300-1. 28 DK Martin, JL Pater, PA Singer (2001). Priority-setting decisions for new cancer drugs: a qualitative case study. Lancet, 358(9294):1676-81. 29 Mugabe, J. et. al. 1996. Managing Access to Genetic Resources: Strategies for Sharing Benefits. ACTS Press, Nairobi. 30 Mugabe, J. and Clark, N. 1997. Technology Transfer and the Convention on Biological Diversity. ACTS Press, Nairobi. 31 Sanchez, V. and Juma, C. 1993. Biodiplomacy. (Chapter 1). ACTS Press, Nairobi. ---- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: Acknowledgements ================ We wish to specially thank the members of the African Genome Policy Forum: O. Akanni, Y. Amekudzi, E. Amuah, M. Bolo, W. Byarugaba, A.L. Christianson, M. Elbashir, C. Khamala, N. Khaole, W. Kilama, V. Konde, S. Langat, R. Lutalo, H. Maganga, M. Makarem, R. Naidoo, J. Nduba, D. Olaleye, L. Olivier, C.J.G. Orjioke, A. Ouattara, O. Owuor, R. Ramesar, J. Shikwati, D. Viljoen, and N. Wachai.	PubMed Central	2024-06-05T03:55:52.740118	2005-1-24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548518/", "journal": "Health Res Policy Syst. 2005 Jan 24; 3:2", "authors": [ { "first": "Alyna C", "last": "Smith" }, { "first": "John", "last": "Mugabe" }, { "first": "Peter A", "last": "Singer" }, { "first": "Abdallah S", "last": "Daar" } ] }
PMC548519	Background ========== Pulmonary fibrosis is a severe chronic disease with various causes and poor prognosis. Its main histological features include lesions of the alveolar septa, fibroblast and myofibroblast proliferation in lung parenchyma, abnormal reepithelialization, and excessive extracellular matrix macromolecule deposition \[[@B1]-[@B3]\]. Lung fibrosis is associated with chronic inflammation and is characterized by the recruitment of macrophages, neutrophils, and lymphocytes in the airways \[[@B4]\]. During lung inflammation, activated phagocytes release large amounts of reactive oxygen species (ROS), which may be involved in tissue injury and in impeding tissue repair, both of which lead to pulmonary fibrosis \[[@B4]-[@B6]\]. Recent studies show that antioxidant compounds such as N-acetylcysteine and bilirubin protect rats against the tissue damage and pulmonary fibrosis induced by bleomycin, an antineoplastic antibiotic commonly used in such experimental models \[[@B7],[@B8]\]. Because these compounds can attenuate the oxidant burden in tissue, they may prevent the lung damage caused by ROS and subsequent fibrosis. Metalloproteinases (MMPs) and their specific inhibitors, the tissue inhibitors of MMPs (TIMPs), are the hallmark of this fibrogenic microenvironment. MMPs are key enzymes that regulate tissue remodeling through turnover of the extracellular matrix in both normal and pathological conditions (for review see \[[@B9]\]). They play a crucial role in the fibrogenic process, as demonstrated recently through the marked reduction of bleomycin-induced pulmonary fibrosis in mice by batimastat, a selective MMP inhibitor \[[@B10]\]. Gelatinase A (MMP-2) and gelatinase B (MMP-9) are two MMPs that appear to be involved in pulmonary fibrosis, but their specific roles in the process remain unclear \[[@B9]\]. While MMP-9 is released primarily by inflammatory cells, MMP-2 is synthesized by structural cells including fibroblasts and endothelial and epithelial cells. Both may be associated with chronic impairment of tissue remodeling and abnormal collagen deposition \[[@B9]\]. Strong evidence indicates that various MMP/TIMP imbalances are crucial elements in the fibrogenic process. Several authors suggest that a \"nondegrading microenvironment\" induces fibrogenicity, that is, more specifically, that various events cause TIMP-1 levels to rise in lung tissue, which in turn lowers MMP/TIMP ratios \[[@B2],[@B11],[@B12]\]. Bleomycin-induced pulmonary fibrosis, for example, causes the expression of significant levels of TIMP-1 \[[@B13],[@B14]\]. Further study is needed to illuminate the pathway that leads from lung injury, associated with ROS and acute inflammation, to initiation of the fibrogenic process, which involves remodeling mediators such as MMPs and TIMPs. The aim of the present study was to investigate the involvement of the ROS released by inflammatory cells during the development of pulmonary fibrosis and to consider the consequence on MMP/TIMP balances. We therefore examined the fibrogenic response to bleomycin administration in mice deficient for the p47^phox^subunit of NADPH-oxidase \[[@B15]\] and analyzed the variations in the MMP/TIMP balance during this process. Materials and methods ===================== Materials --------- This study used the following materials, from the manufacturers mentioned: bleomycin sulfate from Bellon Laboratories (Montrouge, France); gelatin, Triton X-100, Coomassie Brilliant Blue, EDTA, Tween 20 solution, hydroxyproline, and trypan blue from Sigma (St Louis, MO, USA); May-Grünwald and Giemsa stains from RAL (Paris, France); sodium pentobarbital from Sanofi Santé Animale (Libourne, France); etomidate (Hypnomidate^®^, 2 mg/mL) from Janssen-Cilag (Issy-les-Moulineaux, France); acrylamide, sodium dodecyl sulfate (SDS), Tris, and BSA from Eurobio (Les Ulis, France); ELISA kits for IL-6, TIMP-1, and pro-MMP-9 detection from R&D Systems (Minneapolis, MN, USA); formaldehyde from Merck (Darmstadt, Germany); isopentane from Prolabo (Fontenay-sous-Bois, France); a low-range weight marker for SDS-PAGE from Biorad (Munich, Germany); and an ABEL^®^chemiluminescence kit for measurement of in vitroROS release from Knight Scientific Limited (Plymouth, UK). Bleomycin administration ------------------------ Ten week-old p47^phox^+/+ \"wild-type\" (WT) and p47^phox^-/- \"knockout\" (KO) mice (origin: LHD/NIAID/NIH, Bethesda, MD, USA) with C57BL/6J backgrounds \[[@B15]\] were housed under controlled and ethical conditions that complied with the Interdisciplinary Principles and Guidelines for the Use of Animals in Research, Marketing and Education, New York Academy of Sciences\' Ad Hoc Committee on Animal Research. Pulmonary fibrosis was induced by intranasal (i.n.) instillation (under etomidate anesthesia, 15 mg i.p.) of 0.1 mg bleomycin sulfate in saline solution (50 μL/mouse). Control mice received saline vehicle only. Either 24 h or 14 days after i.n. administration, mice were quickly anesthetized by an i.p. injection of sodium pentobarbital (60 mg/kg) and underwent either bronchoalveolar lavage (BAL) or lung removal. These samples were stored at -80°C until either hydroxyproline measurement or homogenization for zymography. Bronchoalveolar lavage and preparation of tissue homogenates ------------------------------------------------------------ Mice were anesthetized with an i.p. administration (20 mL/kg) of sodium pentobarbital 0.6%. The BAL protocol called for washing the airways 10 times with 0.5 mL of 0.9% NaCl solution at 37°C with a 1 mL syringe. The BAL fluid was centrifuged (600 g for 10 min, 4°C), and the supernatant of the first two fractions (1 mL) divided into aliquots and frozen at -80°C until analysis. The cell pellets were then pooled with the last fractions. Total cells were counted with a Coulter Z2^®^(particle counter and size distribution analyzer, Beckman Coulter). Red blood cells were eliminated by adding 3 mL of distilled water for 30 seconds and then 1 mL of KCl 0.6 M onto the pellets. After centrifugation (600 g for 10 min, 4°C), supernatant was eliminated and the cells were suspended in 1 mL of PBS. They were then cytospun at 700 rpm for 10 minutes (Cytospin 3 ^®^, Thermo Shandon, Ltd, Astmoor, United Kingdom) and stained with the May-Grünwald Giemsa method. Differential cell counts of 200 cells used standard morphological criteria. After in vitro stimulation with phorbol 12-myristate 13-acetate (PMA), 0.8 μM, ROS production was assayed with a chemiluminescence technique that used the ABEL^®^detection kit. At day 14, following BAL processing, lungs were removed and homogenized with an adapted grinder (Fast-Prep FP 120 cell disrupter, QBiogene Inc., Illkirch, France). Lung tissue homogenates were then stored at -80°C until analysis. Zymographic analysis of MMPs ---------------------------- Since MMPs can degrade gelatin, zymographic techniques were used to detect MMPs in both BAL (day 1 and day 14) and lung homogenates (day 14). In nonreducing conditions and in the presence of SDS, as previously described \[[@B10]\], aliquots of BAL fluid or lung homogenate underwent electrophoresis onto a 6% acrylamide stacking gel/10% acrylamide separating gel containing 1 mg/mL gelatin. After electrophoresis, gels were washed twice with 2.5% Triton X-100, rinsed with water, and incubated overnight at 37°C in 50 mM Tris, 5 mM CaCl~2~, 2 μM ZnCl2, pH = 8. The gels were stained with Coomassie Brilliant Blue in a solution of 25% ethanol-10% acetic acid in water and rinsed in an identical solution. Gelatinase activity appeared as clear bands against blue background. We used recombinant protein molecular weight markers (20 kDa-112 kDa) to estimate the molecular weights of the gelatinolytic bands. Relative enzyme amounts were quantified by measuring the intensity of the bands with a densitometric analyzer (Bio-Profile, Vilber-Lourmat, Marne la Vallée, France). Results were expressed as a percentage of the band of migration of one control BAL sample loaded onto each gel. This sample was used as an internal standard to allow further comparisons between gels. Determination of IL-6, TIMP-1 and pro-MMP9 levels in BAL -------------------------------------------------------- The amounts of total IL-6, TIMP-1, and pro-MMP-9 were determined with ELISA methods, performed according to the manufacturer\'s recommendations. Assay sensitivity was 31 pg/mL for TIMP-1, 15 pg/mL for IL-6, and 8 pg/mL for pro-MMP-9. Hydroxyproline measurement -------------------------- Lung tissue was lyophilized, weighed, ground to a fine powder with a mortar and pestle, homogenized in PBS pH = 7.4, and divided into aliquots. After hydrolysis for 45 min in NaOH 2 N, the hydroxyproline content was assayed in duplicate aliquots, as previously described \[[@B16]\]. Expression of the results and statistical analysis -------------------------------------------------- Results were expressed as means ± SEM. The differences between the groups for treatment and strain effects were analyzed with a nonparametric Mann-Whitney U test. Correlations between the BAL analysis data and the MMP levels and activity were assessed with the nonparametric Spearman correlation test. For each analysis, P values less than 0.05 were considered to be statistically significant. Results ======= Reactive oxygen species production and cell recruitment in BAL fluids --------------------------------------------------------------------- At both day 1 and day 14, bleomycin treatment elicited enhanced ROS production by BAL cells from WT mice stimulated in vitrowith PMA (Figure [1](#F1){ref-type="fig"}). In contrast, neither the cells removed from control mice (WT, not treated with bleomycin) nor those from KO mice (regardless of bleomycin treatment) could produce ROS in vitroafter PMA stimulation. No chemiluminescence was detected when BAL cells were not stimulated by PMA in vitro. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Production of reactive oxygen species (ROS) by bronchoalveolar lavage (BAL) cells, 1 day (A) and 14 days (B) after intranasal administration of bleomycin (0.1 mg, BLM) or saline (Control) to mice with the p47^phox^subunit of NADPH-oxidase deleted (p47^phox^-/- knockout mice (KO): solid bars), compared with \"Wild Type\" p47^phox^+/+ mice (WT: blank bars). Cells were stimulated in vitrowith PMA (0.8 μM) and ROS production was evaluated by chemiluminescence. Data were collected at the time of maximum light emission. Results are expressed as relative light units (mean ± SEM). \\\: p \< 0.001 comparison with control mice exposed to saline solution alone. \#\#\#: p \< 0.001 for KO mice compared with WT mice. n = 5--9. ::: ![](1465-9921-6-11-1) ::: At day 1, ROS release by BAL cells from bleomycin-treated WT mice was accompanied by a significant neutrophil influx, but the total BAL cell count did not rise (Table [1](#T1){ref-type="table"}). This bleomycin-induced neutrophil influx was quite noticeable in BAL from KO mice, significantly greater than in WT mice. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Total and differential cell counts of BAL fluid from p47^phox^+/+ WT and p47^phox^-/- KO mice, at day 1 and day 14 after intranasal administration of bleomycin (BLM, 0.1 mg/mouse) or saline vehicle (Control, NaCl 0.9%). Results are presented as the mean (.10^3^cells) ± SEM. n: number of mice. WT: wild-type; KO: knockout; a: P \< 0.05, b: P \< 0.01, c: P \< 0.001 compared with control mice exposed to saline solution only. \: P \< 0.05, \\: P \< 0.01, \\\: P \< 0.001 for KO mice compared with WT mice. ::: Treatment Strain N Total Cells Macrophages Neutrophils Eosinophils Lymphocytes ------------- -------- ---- -------------------- ----------------- --------------------- ------------- ------------- Control \[WT\] 10 540 ± 99 536 ± 97 4 ± 2 0 0 \[KO\] 10 535 ± 105 393 ± 69 129 ± 37 \\\ 1 ± 1 11 ± 5 \\ BLM, day 1 \[WT\] 8 490 ± 77 317 ± 50 164 ± 28 ^c^ 0 0 \[KO\] 9 2208 ± 733 ^b,\ e^ 281 ± 102 1922 ± 634 ^c,^\\ 0 2 ± 2 BLM, day 14 \[WT\] 8 1108 ± 80 ^b^ 991 ± 77 ^a^ 76 ± 15 ^c^ 20 ± 6 ^b^ 24 ± 13 ^b^ \[KO\] 9 793 ± 118 634 ± 97 ^a,^\* 62 ± 12 18 ± 16 80 ± 33 ^a^ ::: Fourteen days after bleomycin administration, the total cell count in the WT mouse BAL increased significantly. Specifically, the alveolar macrophage count rose markedly, as did the neutrophil, eosinophil, and lymphocyte counts, although to a lesser extent. The total cell count in the BAL fluid of the KO mice did not increase significantly, but the number of macrophages and lymphocytes did. The WT mice had significantly more alveolar macrophages than the KO mice (Table [1](#T1){ref-type="table"}). Lung hydroxyproline measurement ------------------------------- Lung hydroxyproline concentration, which reflects collagen deposition in lungs, was measured 14 days after bleomycin administration to quantify pulmonary fibrosis (Table [2](#T2){ref-type="table"}). Hydroxyproline levels did not increase in the KO mice. Moreover, although the hydroxyproline level was similar in both strains of mice at baseline, it was significantly higher in WT than in KO mice at day 14. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Hydroxyproline content (mg/g of dry tissue) in lung homogenate from p47^phox^+/+ WT and p47^phox^-/- KO mice, at day 14 after intranasal administration of bleomycin (BLM, 0.1 mg/mouse) or saline vehicle (Control, NaCl 0.9%). Results are presented as mean ± SEM. WT: wild-type; KO: knockout; \\ : P \< 0.01 for WT mice compared with KO mice. n = 3--6. ::: Strain Control BLM --------------------- ------------- ----------------- p47^phox^+/+ \[WT\] 1.20 ± 0.26 1.74 ± 0.10\\ p47^phox^-/- \[KO\] 1.29 ± 0.24 1.04 ± 0.10 ::: IL-6 levels in BAL fluids ------------------------- IL-6 levels in the BAL fluids of WT and KO mice rose one day after bleomycin administration (figure [2](#F2){ref-type="fig"}) and were significantly higher in WT than KO mice. Fourteen days after bleomycin administration, IL-6 levels were not significantly different from baseline. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Level of IL-6 (pg/mL) in BAL fluids, 1 day after intranasal administration of bleomycin (BLM, 0.1 mg/mouse) or saline vehicle (Control), to p47^phox^+/+ WT mice (blank bars) and p47^phox^-/- KO mice (solid bars). Results are presented as the mean ± SEM. \\ : p \< 0.01 compared with control mice exposed to saline solution alone. \#: p \< 0.05 for p47^phox^-/- KO mice compared with p47^phox^+/+ WT mice. n = 4--9. ::: ![](1465-9921-6-11-2) ::: MMP activity in BAL fluids and lung homogenates ----------------------------------------------- Zymography identified the following gelatinolytic bands as MMP activity: pro-MMP-9 (105 kDa), MMP-9 (86 kDa), pro-MMP-2 (70 kDa), and MMP-2 (64 kDa). At day one, pro-MMP-9 activity was significantly higher in the BAL of bleomycin-treated KO mice than in that of their bleomycin-treated WT counterparts, which in turn was significantly higher than in the control mouse BAL (figure [3](#F3){ref-type="fig"} and figure [4A](#F4){ref-type="fig"}). At day 14, no pro-MMP-9 activity was observed in any of the mice. The active form of MMP-9 (86 kDa) was detected only in KO mouse BAL fluid at day 1 (figure [3](#F3){ref-type="fig"}). Pro-MMP-9 was significantly correlated with the neutrophil count in the BAL fluids of both WT (P = 0.001) and KO (P = 7 × 10^-6^) mice. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Representative gelatin zymogram of BAL supernatant fluids, 1 and 14 days after intranasal administration of bleomycin (BLM, 0.1 mg/mouse) or saline vehicle (Control) to p47^phox^+/+ WT mice (A) and p47^phox^-/- KO mice (B). The following gelatinolytic bands were identified as MMP activity: pro-MMP-9 (105 kDa), MMP-9 (86 kDa), pro-MMP-2 (70 kDa), and MMP-2 (64 kDa). M: molecular weight marker. ::: ![](1465-9921-6-11-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Quantification by densitometry of 105-kDa pro-MMP-9 (A), and 64-kDa MMP-2 (B) gelatinase activity on zymograms of BAL fluid, performed 1 day or 14 days after intranasal administration of bleomycin (BLM, 0.1 mg/mouse) or saline vehicle (Control), to p47^phox^+/+ WT mice (blank bars) and p47^phox^-/- KO mice (solid bars). Results are represented as the mean of relative intensity ± SEM. \* : p \< 0.05, \\ : p \< 0.01, \\\* : p \< 0.001 compared with control mice exposed to saline solution alone. \#\#\#: p \< 0.001 for p47^phox^-/- KO mice compared with p47^phox^+/+ WT mice. n = 8--10. ::: ![](1465-9921-6-11-4) ::: At one day and 14 days after bleomycin administration, MMP-2 activity was observed in both its latent (70 kDa) and active (64 kDa) forms (figure [3](#F3){ref-type="fig"}). Although densitometry analysis could detect only the 64-kD form (figure [4B](#F4){ref-type="fig"}) at day one, bleomycin elicited a significant increase in the 64-kDa MMP-2 activity in both strains, compared with the control; and this activity was substantially stronger in the BAL of KO than WT mice. At day 14, MMP-2 activity remained higher in both strains of bleomycin-treated mice (compared with controls) and did not differ significantly between them (figure [4B](#F4){ref-type="fig"}). MMP activity was also evaluated at day 14 in lung homogenates (figure [5](#F5){ref-type="fig"}). Homogenate from both WT and KO mice showed similar levels of pro-MMP-9 activity levels, unexpected lower than in the homogenate from the control mice. In contrast, MMP-2 activity increased in the lungs of WT mice only (figure [5](#F5){ref-type="fig"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Quantification by densitometry of Pro-MMP-9 (105 kDa, A) and MMP-2 (64 kDa, B) gelatinase activity on zymograms of lung homogenates, performed 14 days after intranasal administration of bleomycin (BLM, 0.1 mg/mouse) or saline vehicle (Control) to p47^phox^-/- KO mice (solid bars) and p47^phox^+/+ WT mice (blank bars). Results are represented as the mean of relative intensity ± SEM. \* : p \< 0.05, \\ : p \< 0.01, compared with control mice exposed to saline solution alone. \#\#: p \< 0.01 for p47^phox^-/- \[KO\] mice compared with p47^phox^+/+ WT mice. n = 3--5. ::: ![](1465-9921-6-11-5) ::: Pro-MMP-9/TIMP-1 ratio in BAL fluids ------------------------------------ We also evaluated pro-MMP-9 as well as TIMP-1 with ELISA (table [3](#T3){ref-type="table"}). The results for pro-MMP-9 confirm those obtained with zymography. In WT mice, TIMP-1 in BAL fluid was markedly higher at day one and day 14 after bleomycin administration than at baseline. In contrast, the TIMP-1 level in the BAL fluid of KO mice was not significantly modified by bleomycin administration at either day 1 or day 14 (table [3](#T3){ref-type="table"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Levels of pro-MMP-9 and TIMP-1, and pro-MMP-9 / TIMP-1 ratio in BAL supernatant fluids, recovered from p47^phox^+/+ \[WT\] and p47^phox^-/- \[KO\] mice, 1 day or 14 days after intranasal administration of bleomycin (BLM, 0.1 mg/mouse) or saline vehicle (Control, NaCl 0.9%). For Pro-MMP-9 and TIMP-1, results are represented as the mean (pg/ml) ± SEM. N : number of mice. a : P \< 0.05, b : P \< 0.01, in comparison to control mice exposed to saline solution only. \* : P \< 0.05, \\ : P \< 0.01, for \[KO\] mice in comparison to \[WT\] mice. ::: Treatment Strain N Pro-MMP-9 TIMP-1 Ratio Pro-MMP-9 / TIMP-1 ------------- -------- --- ---------------------------- -------------------- -------------------------- Control \[WT\] 5 46.3 ± 14.9 38.7 ± 13.5 2.7 ± 1.3 \[KO\] 5 1314.7 ± 798.2 \\ 325.2 ± 173.6 10.4 ± 4.7 BLM, day 1 \[WT\] 7 2645.9 ± 1 011.6 ^b^ 781.8 ± 158.9 ^b^ 3.8 ± 1.3 \[KO\] 8 23646.8 ± 2 359.7 ^b,^\\ 1 213.8 ± 396.4 60.8 ± 23.7 ^a,^\\ BLM, day 14 \[WT\] 7 37.7 ± 9.1 2693.3 ± 378.3 ^b^ 0.02 ± 0.00 b \[KO\] 6 137.9 ± 15.5 ^b^ 567.9 ± 163.5\\ 0.2 ± 0.1 ^b,^\\ ::: Table [3](#T3){ref-type="table"} presents the calculation of the pro-MMP-9/TIMP-1 ratio. At day one after bleomycin administration, this ratio remained stable in the BAL of WT mice, both treated and untreated, but rose significantly in that of the KO mice (table [3](#T3){ref-type="table"}). At day 14, the ratio was significantly lower in both strains of treated mice than in the control mice, and levels in the WT mice were significantly lower than those in the KO mice. Discussion ========== This study shows that mice deficient in the p47^phox^subunit of the NADPH oxidase complex do not develop pulmonary fibrosis after intranasal administration of bleomycin. It also suggests that an imbalance of the molar MMP-9/TIMP-1 ratio may influence the fibrogenic process in this model. Several studies previously reported that antioxidant treatment attenuates the bleomycin-induced oxidative burden and subsequent pulmonary fibrosis \[[@B7],[@B8],[@B17]\]. Moreover, the absence of extracellular superoxide dismutase exacerbates conditions that lead to inflammation and pulmonary fibrosis \[[@B18]\]. Although these studies suggest that ROS contribute to lung damage and fibrosis, they do not clearly indicate the mechanisms of the antioxidant effect. That is, antioxidant compounds may attenuate oxidative damage caused directly by bleomycin \[[@B19]\], or they may limit the impact of ROS produced by phagocytes such as macrophages and neutrophils \[[@B5]\] and thus interfere with the inflammatory process. To clarify the role of ROS produced by phagocytes in the development of pulmonary fibrosis, we induced pulmonary fibrosis by i.n. bleomycin administration to p47^phox^-/- KO mice. Unlike antioxidant compounds, which nonspecifically target all ROS sources in the tissue, knocking out the p47^phox^subunit of the NADPH oxidase complex shuts down only the main pathway of phagocytic ROS production. In this study, in vitroPMA stimulation of BAL cells from KO mice produced no detectable ROS, while BAL cells from WT mice did produce ROS, as previous studies of circulating neutrophils from these mice have shown \[[@B15]\]. Hydroxyproline content did not increase in the lungs of these KO mice. This finding reveals the absence of fibrosis and thus provides strong evidence that phagocytic ROS production is an important component of the fibrogenic environment. Although no ROS production was detected in KO mice, their BAL gave evidence of an acute inflammatory response, as did that from WT mice: bleomycin administration elicited acute inflammation, characterized by an influx of neutrophils and associated with increased pro-MMP-9 activity. Moreover, the response of the KO mice to the bleomycin resembled the abnormal \"exuberant\" inflammation in vivopreviously described in such KO mice \[[@B15],[@B20],[@B21]\]. It is, however, possible that this neutrophil influx and the large amounts of MMP-9 it releases have a protective effect against the development of pulmonary fibrosis. This exaggerated inflammatory response may be caused by defective down-regulation of the inflammatory process in these KO mice, perhaps due to the absence of ROS and the failure to degrade chemotactic signals \[[@B22]\]. This acute inflammation was accompanied by significantly elevated IL-6 levels in the BAL fluids of both strains of mice on the day after bleomycin administration, levels significantly higher in WT than KO mice. Given that IL-6 is secreted primarily by mononuclear cells, specifically macrophages, the difference between strains suggests that these cells are activated more weakly in the KO than in the WT mice. Moreover, in SP-D -/- alveolar macrophages, the NADPH oxidase inhibitor apocynin inactivates NF-kappa B, the transcription factor that regulates numerous proinflammatory responses, including IL-6 release \[[@B23]\]. Similarly, lipopolysaccharide-induced NF-kappa B activation is impaired in nuclear protein extracts of lung tissue from p47^phox^-/- KO mice \[[@B24]\]. This would explain why IL-6 response seems to be redox-sensitive in our experiment. MMP induction, assessed by gelatinase release, has been reported in various cases of pulmonary fibrosis in human and experimental models \[[@B13],[@B25],[@B26]\]. We therefore analyzed the gelatinolytic activities of MMPs, in both BAL fluid and lung homogenate. MMP-9 and MMP-2 activities in the BAL fluid of the KO mice reveal an intense response to bleomycin. MMP-9 levels were highly correlated with neutrophil infiltration, while MMP-2 is known to be produced mainly by epithelial \[[@B27]\] and mesenchymal cells, such as fibroblasts, which are involved in collagen production and deposition \[[@B9],[@B25]\]. This suggests that the exaggerated response observed in the BAL fluid of KO mice involves a wide spectrum of cell types. Surprisingly, at day 14, gelatinase profiles were different in the lung homogenates than in BAL fluids. Although MMP-2 activity was equivalent in BAL of both strains, MMP-2 activity increased substantially in the lung homogenate of the WT mice. One possible explanation is that the increased release of MMP-2 in the inner lung parenchyma may result from downstream events caused by phagocyte activation and ROS production during inflammation. This is consistent with another study, mentioned above, in which apocynin, an inhibitor of NADPH oxidase, also inhibited the release of MMP-9 and MMP-2 in SP-D -/- alveolar macrophages \[[@B23]\]. Moreover, Pardo et al. \[[@B28]\] report increased levels of gelatinases (MMP-2 and MMP-9) in isolated type II alveolar cells from hyperoxic rats; these increases are associated with alterations in the balance between MMPs and TIMPs and finally lead to diffuse alveolitis and its progression to pulmonary fibrosis. It is thus difficult to reach a definitive conclusion at this time about the exact function of MMPs. They play a role in promoting tissue remodeling and counterbalancing excessive matrix deposition, but may also facilitate tissue damage and disruption. Their involvement in bleomycin-induced pulmonary fibrosis has been demonstrated with the inhibition of collagen deposition by bastimastat, a nonselective MMP-inhibitor \[[@B10]\]. Complete understanding of the dynamic process of remodeling nonetheless requires consideration of TIMPs, which are natural MMP inhibitors. Increased TIMP-1 expression has been observed in lung extracts and in BAL fluids after bleomycin administration and after the transfer of the active TGF-beta gene to \"fibrosis-prone\" C57BL/6 mice \[[@B11],[@B14]\]. In humans, increased levels of TIMP protein and RNA are observed in lungs of patients with idiopathic pulmonary fibrosis, and TIMP expression there exceeds that of MMP \[[@B12]\]. A reduced molar MMP/TIMP ratio seems to be a hallmark of pulmonary fibrosis, distinguishing it from other reversible interstitial lung diseases \[[@B26]\] and from chronic obstructive pulmonary disease (COPD) \[[@B29]\]. This ratio might be considered to be a \"snapshot\" of the dynamic matrix remodeling in lung tissue. Interestingly, in our study, the pro-MMP-9/TIMP-1 ratio was significantly higher for KO than WT mice, at both day one and day 14. At day 1 this was due to the lower MMP-9 level and higher TIMP-1 level in the BAL from WT mice, and at day 14, only to the latter. The correlation of these levels with differences in hydroxyproline levels in the lungs of bleomycin-treated mice strongly suggests that a reduced molar pro-MMP-9/TIMP-1 ratio in BAL fluid is associated with collagen deposition, beginning as early as the inflammatory events at day 1 after bleomycin administration. The usefulness of the pro-MMP-9/TIMP-1 ratio as a marker of fibrosis nonetheless requires discussion. Although a molar ratio appears to play a protective role against fibrotic changes, MMP-9 is considered primarily to be an inflammatory mediator released by leukocytes during acute inflammatory events to facilitate their progression across the basement membrane \[[@B30]\]. Moreover, MMP-9 depletion in KO mice does not substantially alter the extent of either pulmonary fibrosis or lung inflammation after bleomycin administration \[[@B31]\]. TIMPs may also counterbalance the activity of MMP-2 or other proteinases, such as collagenases. Ruiz et al. \[[@B32]\] recently observed that MMP-8 and MMP-13 RNA levels decreased and TIMP-1 RNA increased in the paraquat- and hyperoxia-induced pulmonary fibrosis rat model. Matrilysin (MMP-7), which can degrade various substrates, seems to have a crucial role in pulmonary fibrosis \[[@B33]\]. Finally, the exuberant neutrophil influx observed at day one in p47^phox^-/- KO mice could provide great amount of other kind of protease, such as serine proteases. Indeed, neutrophil elastase was shown to have an impact on the severity of bleomycin-induced pulmonary fibrosis \[[@B34],[@B35]\] Conclusion ========== In summary, this study demonstrates that the inability of phagocytes from p47^phox^-/- KO mice to produce large quantities of ROS via the NADPH oxidase pathway inhibits the development of bleomycin-induced pulmonary fibrosis. This inhibition is associated with changes in IL-6 production and in the molar MMP-9/TIMP-1 ratio, both probably key factors in airway remodeling and fibrosis. These rapidity of these differences after bleomycin administration suggests that early inflammatory events and remodeling events may establish a favorable environment for further chronic fibrogenic processes. Abbreviations ============= BAL:Bronchoalveolar lavage IL:interleukin KO:knock out MMP:matrix metalloproteinase TIMP:tissue inhibitor of metalloproteinase ROS:reactive oxygen species WT:wild type Authors\' contributions ======================= BM, SN, OL, IG:and EB have made substantial contributions to acquisition and analysis of data BM, SN, EB and VL have made substantial contributions to conception and design BM, EB and VL have been involved in drafting the article JMP and CPB have been involved in revising it critically for important intellectual content Acknowledgements ================ The authors thank C. Chesné and O. Minella (Biopredic Int., Rennes, France) for supplying the ABEL^®^chemiluminescence kit. This work is supported by a research grant (ref \#2004797) from Conseil Régional de Bretagne and by a collaborative project INSERM/FIOCRUZ.	PubMed Central	2024-06-05T03:55:52.745197	2005-1-21	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548519/", "journal": "Respir Res. 2005 Jan 21; 6(1):11", "authors": [ { "first": "Boris", "last": "Manoury" }, { "first": "Soazig", "last": "Nenan" }, { "first": "Olivier", "last": "Leclerc" }, { "first": "Isabelle", "last": "Guenon" }, { "first": "Elisabeth", "last": "Boichot" }, { "first": "Jean-Michel", "last": "Planquois" }, { "first": "Claude P", "last": "Bertrand" }, { "first": "Vincent", "last": "Lagente" } ] }
PMC548520	Background ========== Photoreceptor cells play a primary role in vision by capturing light energy and converting it into neural stimuli. These sensory neurons are a shared element in all organisms capable of sensing light. In humans, genetic diseases of the eye are common and the primary site of disease is most frequently photoreceptors (for review see \[[@B1]-[@B3]\]). Photoreceptors have been well studied, particularly with respect to the biochemistry and physiology of phototransduction. Insight into the development of vertebrate photoreceptors, however, has lagged behind our understanding of function. Only recently have the first molecular mechanisms regulating photoreceptor development been identified (for review see, \[[@B2],[@B4]\]). Crx (cone-rod homeobox) is an otx-family homeobox gene expressed predominantly in photoreceptors, from early in their development through to the adult ages \[[@B5]-[@B7]\]. Crx gene expression is critically dependent upon Otx2, another member of the same homeobox family which is expressed in early photoreceptor cells \[[@B8]\]. In rod photoreceptors, Crx appears to work in concert with Nrl, a leucine zipper protein that is also restricted in its expression in the retina to rod photoreceptors \[[@B9]\]. Many photoreceptor-specific genes have putative Crx-binding elements in their regulatory regions \[[@B10]\], including rhodopsin \[[@B11]\] and arrestin \[[@B12]\]. Mutations in Crx have been associated with several human diseases that lead to blindness, including cone-rod dystrophy 2 \[[@B6],[@B13],[@B14]\], retinitis pigmentosa \[[@B14]\], and LCA \[[@B14]-[@B16]\]. Based on these data, Crx was hypothesized to play a critical role in the differentiation and maintainance of photoreceptor cells \[[@B5],[@B7]\]. LCA is the most severe genetic disease of photoreceptors (see \[[@B17]\], for recent review). Affected infants exhibit a complete or near complete absence of vision from birth. Mutations in retinal specific genes, such as Crx, have been associated with LCA \[[@B14],[@B15]\], as well as GUCY2D \[[@B18]\], RPE65 \[[@B19]\], AIPL-1 \[[@B20]\], CRB-1 \[[@B21]\], and RPGRIP-1 \[[@B22]\]. There also may be as many as three additional genetically linked loci where genes have not been identified \[[@B23]\]. Crx mutations in LCA are varied, and include a putative dominant mutation that is proposed to encode a dominant-negative form of Crx \[[@B14],[@B15]\]. Recessive mutations also have been reported and at least one allele encodes a protein with decreased DNA-binding activity \[[@B16]\]. Histopathological and ultrastructural studies of LCA should enable a better understanding of the disease process, and the design of suitable therapies. Few such studies exist for human LCA (reviewed in \[[@B17]\]) and the majority of such studies examine the globes of adults with LCA, after the tissue has undergone secondary changes. Only a single study exists where the developing eye of an infant was examined \[[@B24]\]. Animal models for LCA have recently been reported and have already served to broaden our understanding of the pathology of this disease \[[@B25]-[@B28]\]. Since LCA is a clinically and genetically heterogeneous disorder, additional mouse models are in order to allow a full understanding of the many ways in which photoreceptor development can go awry. In addition to their importance as a locus of disease, photoreceptor cells serve as an excellent model for studies in neuronal differentiation. Photoreceptor cells are highly polarized. At their apex, these neurons have a membranous outer segment, which contains proteins involved in the phototransduction cascade. Loss of function mutations in rhodopsin \[[@B29]\], or the structural protein, peripherin \[[@B30]\], result in an inability to form outer segments. At the other extremity, photoreceptors terminate with synaptic endings that make contact with the processes of horizontal and bipolar cells \[[@B31],[@B32]\]. Rod spherules establish an invaginating synapse with rod bipolar dendrites and axonal endings of horizontal cells. This synapse is characterized by the presence of a ribbon in the presynaptic cytoplasm. Cone pedicles make invaginating synapses with the dendrites of on-cone bipolar cells and horizontal cells and basal junctions with the dendrites of off-cone bipolar cells. The factors regulating the formation of the photoreceptor synapses are completely unknown. At least one photoreceptor synaptic protein, HRG4, contains a potential Crx target sequence in its transcriptional regulatory sequence \[[@B33]\]. Few studies of LCA animal models have extended their examination of retinal pathology to the ultrastructural level. Certain features of neuronal differentiation, such as synapse formation, can be detected definitively at this level of analysis. With the hope of understanding the neuropathology of LCA in greater detail, we have analyzed the differentiation of the outer retina in Crx-/- mice at the ultrastructural level. These retinas exhibit several prominent defects. Crx-/- photoreceptors demonstrate a complete block in outer segment formation at the elongation stage. Further, these cells exhibit abnormal synaptic morphology, thereby broadening the function of Crx to photoreceptor synaptogenesis. This represents the first report strongly implicating the process of synapse formation in LCA. Results ======= Multiple pathologies in the outer segment layer in Crx-/- mice -------------------------------------------------------------- A standard knock-out protocol was used to generate mice in which the homeodomain of Crx-/- was targeted and deleted. Generation of these Crx-/- mice has been reported elsewhere \[[@B34]\]. In this study, in order to characterize further the role of Crx in photoreceptor morphogenesis, the outer retinae from Crx-/- mice were examined using transmission electron microscopy. At postnatal day 21 (P21), when Crx+/+ photoreceptors exhibited robust outer segments (Figure [1A](#F1){ref-type="fig"}, os), Crx-/- retinas were without a recognizable outer segment layer (Figure [1B](#F1){ref-type="fig"}). Crx-/- photoreceptors had inner segments, demonstrating at least limited photoreceptor polarization in the Crx mutant, but the inner segments were extremely short (Figure [2](#F2){ref-type="fig"}). Furthermore, the majority of inner segments showed some morphological differentiation, having approximately as many mitochondria as the control (Figure [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}). Occasionally, an inner segment undergoing lysis was noted, appearing swollen or with vacuoles and swollen mitochondria (data not shown). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Transmission electron microscopy of the outer retina at P21 in (A) Crx+/+ and (B) Crx-/- retinas. pe, pigmented epithelium. os, outer segments. is, inner segments. onl, outer nuclear layer with photoreceptor nuclei. Scale bar = 5 μm for A and B. ::: ![](1471-2202-6-5-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Transmission electron micrograph of the outer segment layer of Crx-/- retina at P21. Inner segments of Crx-/- photoreceptors exhibit numerous mitochondria (m indicated by arrow) as in Crx+/+ (Figure 1A). pe, pigmented epithelium. is, inner segments. onl, outer nuclear layer. Scale bar = 2 μm. ::: ![](1471-2202-6-5-2) ::: Photoreceptor inner segments and outer segments are joined by a non-motile connecting cilium that exhibits a characteristic 9 + 0 arrangement of microtubule doublets when viewed in cross-section. At P21, in Crx-/- retinas, numerous cross sections of connecting cilia were noted (Figure [3A](#F3){ref-type="fig"} and [3B](#F3){ref-type="fig"}). Sporadically, connecting cilia contained other than the typical complement of microtubule doublets. For example, in Figure [3A](#F3){ref-type="fig"}, the connecting cilium labelled by arrowhead 1, shows 7 + 0 doublets. The majority exhibited the characteristic 9 + 0 doublets (arrowhead 2 and 3 in Figure [3A](#F3){ref-type="fig"} and Figure [3B](#F3){ref-type="fig"}). These observations indicate that in addition to inner segment formation, ciliogenesis is also largely intact in Crx-/- photoreceptors. Further, in Crx-/- retinas the retinal pigmented epithelium (PE) appeared normal, at least up to P21 (data not shown), the oldest age examined. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Transmission electron micrograph of Crx-/- retina at P21 (A and B), and scanning electron micrograph of Crx-/- at P10 (C) of outer segment layer. (A) Evidence of ciliogenesis in the photoreceptor layer of Crx-/- retina. Nonmotile connecting cilia were observed in cross section (arrowheads 1,2, and 3, for examples). Connecting cilium 1 (arrowhead 1) demonstrated seven microtuble doublets, while cilium 2 and cilium 3 exhibited nine. In A, a displaced cell nucleus (n) appearing pyknotic and abnormal deposition of matrix (mx) material of unknown identity were seen, along with large amounts of membranous vesicles (arrow) which filled the photoreceptor space and appeared to be released from inner segments. Scale bar = 3.7 μm. (B) Nonmotile connecting cilium in cross section, from a Crx-/- photoreceptor, demonstrating characteristic 9+0 radial array of microtubule doublets. Scale bar = 88 nm. (C) Scanning electron micrograph (SEM) of membranous vesicles (arrow shows one example) shed from inner segments of Crx-/- photoreceptors at P10. Figure shows inner segments viewed from the scleral side with the pigmented epithelium removed. Scale bar = 1 μm. ::: ![](1471-2202-6-5-3) ::: In addition to the complete absence of outer segments, Crx-/- retinas exhibited three other notable pathologies in the outer segment layer. First, an abnormal deposition of matrix of unknown identity was noted (Figure [3A](#F3){ref-type="fig"}, mx). Second, sporadically displaced nuclei were found residing in the space abutting the PE. Occasionally, these nuclei appeared pyknotic (Figure [3A](#F3){ref-type="fig"}, n); but, more frequently exhibited the heterochromatin pattern typical of photoreceptors (data not shown), strongly suggesting that they belonged to ectopic photoreceptors. The third pathological entity noted in the outer segment layer were numerous small vesicles (Figure [3A](#F3){ref-type="fig"} arrow) 100 to 200 nm in diameter. They appeared to be emerging from the inner segments, as scanning electron microscopic images showed spherical structures budding from the inner segments (Figure [3C](#F3){ref-type="fig"}, arrow). In order to characterize further the morphogenesis of Crx-/- photoreceptors, the developing outer segment layer was viewed by scanning electron microscopy at P7, P14 and P21 (Figure [4](#F4){ref-type="fig"}). In Crx+/+ retinas, photoreceptor inner segments, connecting cilia, and the first rudimentary outer segment structures were noted at P7. In the Crx-/- retina, only an occassional connecting cilium was noted emerging from inner segments at this stage (Figure [4A](#F4){ref-type="fig"} and [4B](#F4){ref-type="fig"}). This observation was confirmed by comparison with transmission electron micrographs (Figure [5](#F5){ref-type="fig"}). These differences are the earliest noted differences between Crx+/+ and Crx-/- photoreceptors. At P14, elongating outer segments were noted on Crx+/+ photoreceptors, occasionally demonstrating a paddle-like structure at their apex (Figure [4C](#F4){ref-type="fig"}, os). In Crx-/- retinae, the vast majority of photoreceptors at this stage demonstrated connecting cilia without outer segments (Figure [4D](#F4){ref-type="fig"}, cc). Sporadically, Crx-/- photoreceptors would exhibit an irregular structure extending from a connecting cilium (Figure [4D](#F4){ref-type="fig"}, cc\) perhaps representing a malformed outer segment. Such structures were also observed at P21 (Figure [4F](#F4){ref-type="fig"}, cc\). These putative, abnormal outer segments were only rarely noted in Crx+/+ at P14, and never at P21 (Figure [4C](#F4){ref-type="fig"} and [4E](#F4){ref-type="fig"}, cc\). Further, in Crx-/- photoreceptors, unusually long connecting cilia were noted (Figure [4F](#F4){ref-type="fig"}, cc). Serial examination of Crx-/- photoreceptors at P7, P10, P14, and P21 by TEM, demonstrated a distinctive lack of any structure interpretable as orderly stacks of discs or forming discs. These data demonstrate a complete absence of normal outer segment formation in the Crx mutant mouse, and the arrest of development of the photoreceptor appendage at the elongation stage of outer segment formation. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Outer segment morphogenesis in Crx-/- photoreceptors. Scanning electron microscopy of developing photoreceptors viewed from the scleral side with the pigmented epithelium removed at P7, P14, and P21 for Crx+/+ (A, C, and E) and Crx-/- (B, D, F) littermates. In Crx+/+ retina, numerous connecting cilia (A, cc) were evident at P7 with rudimentary outer segments. After P7, in Crx+/+ outer segment elongation occurs. Initially, outer segments have a paddle-like structure (C, os) which is later shed (E, os). In Crx-/- photoreceptors, few connecting cilia were observed at P7 (B, cc). After P7, connecting cilia were more numerous and occasionally a malformed outer segment was noted extending from a connecting cilium (D and F, cc\). These were rarely observed in Crx+/+ and only at P14 (C, cc\). At P21, abnormally long connecting cilia are noted in Crx-/- (F, cc). Scale bars = 10 μm ::: ![](1471-2202-6-5-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Transmission electron micrographs of Crx-/- photoreceptors in the photoreceptor layer at P7. (A) Photoreceptor layer of Crx+/+ retina demonstrating nascent outer segment structures (arrow) emerging from photoreceptor connecting cilia (cc). (B) Crx-/- photoreceptors exhibited connecting cilia (cc) at this early stage, however, nascent outer segment structures were not observed. Scale Bar = 1 μm. ::: ![](1471-2202-6-5-5) ::: Finally, the morphology of the malformed Crx-/- photoreceptors was compared to rhodopsin-/- and peripherin-/- photoreceptors. Rhodopsin and peripherin are two photoreceptor-specific genes whose expression is significantly downregulated in the Crx-/- retinae \[[@B10],[@B34],[@B35]\]. Loss of function mutations in each of these genes separately have been reported to result in a failure to elaborate outer segments \[[@B29],[@B30]\]. Photoreceptors from these two mutant mice examined by SEM from the scleral side appeared highly similar to Crx-/- photoreceptors (compare Figure [4F](#F4){ref-type="fig"} to Figure [6A](#F6){ref-type="fig"} and [6B](#F6){ref-type="fig"}). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Scanning electron microscopy of peripherin-/- (A) and rhodopsin-/- (B) photoreceptors at P21, viewed from the scleral side with the pigmented epithelium removed. cc, connecting cilium. is, inner segment. Scale bar = 10 μm. ::: ![](1471-2202-6-5-6) ::: Crx is necessary for the formation of photoreceptor terminals ------------------------------------------------------------- In a previous study, we demonstrated that forced expression of a dominant-negative allele of Crx in developing rods blocked formation of both rod spherules in the outer plexiform layer (OPL) and outer segments \[[@B7]\]. To expand on these studies, the ultrastructure of photoreceptor synapses was examined in Crx-/- retinas. In Crx+/+ retinas at P21, newly mature rod spherules were abundant (Figure [7A](#F7){ref-type="fig"}). The sperules were blunt or club-shaped structures with a single ribbon associated with a single invaginating synapse (Figure [7A](#F7){ref-type="fig"}, arrow indicates one example; Figure [8A](#F8){ref-type="fig"} and [8B](#F8){ref-type="fig"}). Two processes from horizontal cells were situated on either side of the synaptic ridge (Figure [8B](#F8){ref-type="fig"}, labelled H) and one or more dendrites of rod bipolar cells occupied a central position (Figure [8B](#F8){ref-type="fig"}, bipolar labelled B). Cone terminals are large, flat pedicles that exhibit many invaginating synapses containing separate sets of horizontal and bipolar cell processes. Each pedicle contains numerous ribbons. These terminals were much less common than spherules in Crx+/+ retinas at P21 (Figure [7](#F7){ref-type="fig"}, box). In the OPL of Crx-/- retinas, photoreceptor terminals were highly disorganized at P21 (Figure [7B](#F7){ref-type="fig"}, arrows). Processes containing synaptic vesicles and ribbon-like structures were apparent, suggesting at least limited generation of synapse components. However, well formed spherules and pedicles were not observed. In addition, many terminals appeared to contain multiple ribbons (Figure [8C](#F8){ref-type="fig"} and [8D](#F8){ref-type="fig"}, r) not tethered to the plasma membrane and occasionally in perinuclear positions (Figure [8D](#F8){ref-type="fig"}). ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Transmission electron micrographs of the outer plexiform layer in Crx-/- retinas. (A) In Crx+/+ retina at P21, newly formed rod spherules were abundant (arrow demonstrates one example). The spherules were club-shaped and contained a single invaginating synapse with one ribbon complex. Cone terminals form large, flat pedicles that contain many invaginating synapses with separate ribbon structures. These terminals were more scarce, but apparent in Crx+/+ retinas at P21 (one example in box). (B) In the outer plexiform layer (OPL) of Crx-/- retinas, photoreceptor terminals appeared highly disorganized at P21 (arrows). Well-formed pedicles and spherules were not evident. Putative terminals containing ribbon-like structures were apparent, suggesting at least limited generation of synapse components. Many terminals appeared to contain multiple ribbon-like structures, instead of a singule ribbon. For example, terminal 1 and 2 contained two ribbons each, whereas terminal 3 appeared to contain only one. opl, outer plexiform layer. Scale bar = 2 μm. ::: ![](1471-2202-6-5-7) ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Transmission electron micrographs of the outer plexiform layer in Crx-/- retinas at P21. (A) Crx+/+ rod spherules contained a single invaginating synapse with one ribbon complex. The spherule was a blunt or club-shaped structure. (B) Crx+/+ rod terminals contained a single ribbon structure (r). Two processes, from horizontal cells (h), contacted the rod laterally. An additional process, the postsynaptic bipolar dendrite (b), was situated more centrally. Terminals were filled with synaptic vesicles. One coated vesicle originatinf from the cell membrane was observed (arrow). (C) In the OPL of Crx-/- retinas, photoreceptor terminals appeared highly disorganized. Putative terminals containing synaptic vesicles and ribbon-like structures were apparent (arrows), suggesting at least limited generation of synapse components. However, well formed spherules and pedicles were not observed. Further, many terminals appeared to contain multiple ribbon-like structures (r). The majority of these ribbons were not associated with the synaptic membrane, but instead were found free floating and, in some instances, were perinuclear (D, arrow). H, horizontal cell; B, bipolar cell; N, nucleus; r, ribbon. (A) Scale bar = 500 nm, (B) Scale bar = 250 nm, (C and D) Scale bar = 500 nm. ::: ![](1471-2202-6-5-8) ::: Discussion ========== In this study, an ultrastructural analysis of Crx-/- photoreceptors was carried out. As Crx mutations have been associated with Leber\'s congenital amaurosis, the findings in this study broaden our understanding of the pathology of this disease. Two prominent pathologies were characterized in the Crx-/- retina: (1) An absolute block in outer segment morphogenesis was noted, with the block occuring at the elongation stage of outer segment formation; (2) Crx-/- photoreceptors exhibited a severe perturbation in synapse formation. This represents the first report of a synaptogenesis defect in an animal model of LCA. Crx-/- photoreceptors cannot complete outer segment morphogenesis ----------------------------------------------------------------- Mutations in Crx represent one of a collection of gene mutations that lead to an outer segment formation defect. Homozygous null mutations in the peripherin/RDS gene \[[@B36]\] or in rhodopsin \[[@B29]\] lead to a failure of outer segment formation. The deficits in peripherin-/- and Crx-/- photoreceptor morphogenesis were found to be very similar. Vesicular structures in Crx-/- photoreceptors were observed that were similar to those previously noted in the rds/peripherin-/- mouse. It was initially proposed that these vesicles were due to the breakdown of outer segment membranes that were not properly recruited to the outer segments in the absence of peripherin, or were from the result of the breakdown of the microvilli of Müller cells \[[@B30]\]. Strong support in favor of the former explanation was provided by Nir and colleagues who demonstrated the presence of rhodopsin protein in these vesicles using immunoelectron microscopy against a rhodopsin epitope \[[@B37]\]. Further, as shown here, the vesicles appear to bud from the inner segments themselves. In developing photoreceptors, an extraordinary growth process occurs whereby the outer segment is generated from the nascent connecting cilium (see \[[@B38]\] and references therein). Peripherin/RDS and ROM-1 proteins (localized in disc rims) and the opsin proteins (localized throughout the discs) have important roles in the structural integrity of mature outer segments (see \[[@B39],[@B29]\]). ROM-1-/- mice produce disorganized outer segments with large disks \[[@B40]\]. Crx, by virtue of being a transcription factor, presumably controls genes that are responsible for the building and perhaps maintenance of the outer segment structure, including rhodopsin and peripherin. Using northern blots \[[@B34]\], microarrays \[[@B10]\], and serial analysis of gene expression (SAGE) \[[@B35]\], we have defined a large number of genes that are altered in their expression level in Crx-/- mice. We found that rhodopsin expression was severely diminished in Crx-/- animals, and peripherin mRNA was reduced by approximately 30%. Transgenic mice with variable levels of expression of wild type rhodopsin exhibit rod degeneration \[[@B41]\], indicating the importance of the level of rhodopsin expression. In addition, the timing of rhodopsin expression may be very important, as indicated by studies in Drosophila. In Drosophila, rhodopsin (ninaE) is expressed in photoreceptors R1--R6. In ninaEnull mutants, the rhabdomere, a structure analogous to vertebrate outer segments, fails to develop in R1--R6 photoreceptors \[[@B42]\], reminiscent of the situation in rhodopsin-/- mice \[[@B29]\]. An intriguing experiment by Kumar et al. \[[@B43]\] demonstrated a temporal requirement for rhodopsin expression during rhabdomere development. In ninaEnull flies, a ninaEtransgene under the control of a heat shock promoter was subjected to various temperature shifts during development. Heat shock during the normal time of rhodopsin onset resulted in substantial and long-lasting rescue of photoreceptor structure and transient rescue of photoreceptor physiology. However, expression shortly before or after this critical period failed to rescue, suggesting that rhodopsin expression during a discrete window of time in development is essential for proper rhabdomere morphogenesis. This result is consistent with observations in the rat wherein rhodopsin onset occurs with strict timing in the developmental history of most rods in vivo \[[@B44]\]. Thus, through its regulation of rhodopsin levels, or perhaps through control of the kinetics of the up-regulation of rhodopsin beginning at about P6, Crx may be regulating outer segment morphogenesis. The similarty of the two cases may extend further. At present, the closest Crx relative in Drosophila is Otd, the founding member of the class of homeobox genes to which Crx belongs. Interestingly, in one hypomorphic allele of Drosophila otd, otd^uvi^, photoreceptor morphogenesis is also disrupted \[[@B45]\]. We found that there are many other genes that are dependent upon Crx. Those that are expressed at a lower level in the Crx-/- retina, such as rhodopsin and peripherin, comprise many that are either enriched or specific to photoreceptors in their expression \[[@B35]\]. They include enzymes that are important in lipid metabolism, protein folding and transport, as well as in other processes that one might envision would be important in building a structure such as the outer segment. In situ hybridization using probes from this collection of genes has revealed that some of them have their RNA localized to the inner segment, a finding typical for proteins targeted to the outer segment. Future analyses of the function of these genes might reveal their role in outer segment biogenesis. Finally, polarization of photoreceptors was found to be largely intact, as was ciliogenesis. Another LCA gene, CRB1, and a related gene CRB3, have been implicated in ciliogenesis in in vitromodels \[[@B46]\]. The Drosophila*homologue of CRB1, Crumbs, has been implicated in photoreceptor morphogenesis \[[@B47]\]. However, the spontaneously occurring mouse mutant in CRB1, the Rd8 mouse, develops shortened outer segments that subsequently degenerate \[[@B48]\], suggesting that photoreceptor polarization and synaptogenesis are intact in this mutant. While CRB1 and Crx have been both linked to LCA, further work is necessary to determine if their function is linked in retinal development. Synaptogenesis is perturbed in Crx-/- photoreceptors ---------------------------------------------------- The Crx-/- mouse demonstrates the most severe abnormality of photoreceptor synapses reported to date. The peripherin-/- mouse develops a normal complement of photoreceptor terminals which then degenerate as the photoreceptors are lost \[[@B30]\]. Also, similarly in rhodopsin (Rho) and cyclic nucleotide-gated channel, alpha-3 (CNGA3) double knockout mice (Rho-/-, CNGA3-/-), synapses are reported to form normally \[[@B49]\]. These observations demonstrate that photoreceptor synaptogenesis can occur in the absence of outer segment formation. In keeping with this observation is the fact that some electroretinogram activity is present in peripherin-/- mice, suggesting that minimal phototransduction is present in these mice, enough to drive activity at the photoreceptor synapse. In vitro studies wherein synapse elements are formed in the absence of proper outer segment development and, therefore, possible absence of light-dependent photoreceptor activity, have indicated the independence of phototransduction and synapse formation, at least for the initial stages \[[@B50],[@B51]\]. These data then suggest that the fact that the Crx-/- photoreceptors do not have proper synaptic endings is not due to a lack of outer segment formation. A more likely explanation is that Crx plays a role in photoreceptor synapse formation, perhaps by regulating directly, or indirectly, important genes in this process. Using immunohistochemistry, we examined the expression of common pre-synaptic terminal proteins, including KIF3a, SV2, and synaptophysin, and were unable to observe qualitative differences between Crx-/- and control tissue at P14 (data not shown). Examination of their RNA levels by SAGE showed no significant difference for all 3 genes, though very few tags were recovered from these genes and thus the analysis of RNA levels may not be significant \[[@B35]\]. However, since other genes expressed in photoreceptors were significantly altered in their expression level in the Crx-/- mouse, there are many candidates that could be important for photoreceptor morphogenesis. Tags from three genes from proteins expressed in photoreceptor terminals were found to be decreased in a statistically significant fashion, namely the HGF-regulated tyrosine kinase substrate, the CRIPT protein, and synaptotagmin 1 (Blackshaw and Cepko, unpublished data). An example of a gene that was increased in the Crx-/- retina is HRG4 (a homologue of the C. elegans Unc119 gene) (Blackshaw and Cepko, unpublished data) which encodes a component of the ribbon synapse \[[@B33]\]. The fact that it is upregulated might indicate a response to the lack of proper terminal structures. Several other genes encoding putative cytoskeletal elements also were increased (e.g. microtubule associated protein 4) or decreased (e.g. cofilin 1) in the Crx-/- retina, with P values of \<.005. It is not known whether any of these genes are involved in building or regulating synaptic structures, but they are now genes that might lead to a better understanding of the construction and function of the relatively unique structure of the ribbon synapse. Abnormal photoreceptor terminal formation was noted in a study that examined retinal development in the laminin beta2 chain-deficient mouse \[[@B52]\]. Several pathologies were noted in these mice. First, laminin beta2 chain-deficient mice displayed abnormal outer segment elongation, but a more mild phenotype than that of the Crx-/- mice; the outer segments were reduced by 50% in length. Also photoreceptor terminals were perturbed in laminin beta2 mutants, but again the phenotype was more subtle then that of Crx-/- mice. The outer plexiform layer of the beta2-deficient retinas demonstrated only 7% normal invaginating synapses, while the remainder had various pathologies, including floating synaptic ribbons, as seen here. The mechanistic relationship of these two molecules, if any, in photoreceptor morphogenesis is unknown to date. The mRNA for laminin beta2 was not detected in the SAGE study of the relative RNA levels in Crx-/- and Crx+/+ and thus we cannot comment on whether the levels of RNA for laminin beta2 were altered. Crx-/- mice are a model for LCA ------------------------------- Crx has been implicated in three photoreceptor diseases that result in human blindness, cone-rod dystrophy2, Leber\'s congenital amaurosis, and retinitis pigmentosa (for review, see \[[@B53]\]). The cone-rod dystrophies (CRDs) are characterized by loss of cone-mediated vision in the first decade of life or later, with concomitant or subsequent loss of rod-mediated vision \[[@B54]\]. Conversely, RP is notable for initial loss of rod function, followed by loss of cone-mediated vision \[[@B55]\]. The majority of known genes responsible for human genetic blindness, encode proteins expressed almost exclusively, or exclusively, in photoreceptors, particularly in the outer segment \[[@B35]\]. Many of these proteins are required for phototransduction or outer segment structure. The mechanisms whereby mutations in rod-specific genes, such as rhodopsin, lead eventually to cone degeneration in RP remain obscure. Mutations in Crx were the first, and still one of a very few examples of a transcription factor mutation leading to photoreceptor disease. LCA is a disease in which there is little or no photoreceptor function in infancy; thereby, likely developmental in etiology (\[[@B17],[@B56]\] for review). The Crx-/- mouse may be an excellent model for studying the pathology of this disorder, particularly the subtype of the disorder where Crx mutations are involved. The vast majority of histopathological studies of LCA in human tissue have been derived from adult patients with LCA where secondary changes are likely to be present. Indeed in animal models of LCA, secondary reactive and/or degenerative changes occur early after the abnormal formation of retinal tissue \[[@B57]\]. The only study in human tissue derived from a human 33-week retina with proposed RPE65 mutations was reported to have abnormal retinae at this early stage \[[@B24]\]. These authors report cell loss, including thinning of the photoreceptor layer. In addition, they claim in the text to have seen aberrant synaptic and inner retinal organization, although their examination of photoreceptor synapses unfortunately are not presented in the data section of the paper. Given the scarcity of available human tissue, the characterization of the primary pathology of LCA will require animal models. In the current study, we present data that argue that, in addition to outer segment morphogenesis, synaptogenesis may also be critically impaired in at least a subset of LCA. Methods ======= Mice ---- Crx-/- mice were generated as reported elsewhere \[[@B34]\]. Rhodosin-null mice \[[@B29]\] were obtained from Paul Sieving (University of Michigan). Rds mice were acquired from Jackson Laboratory. Transmission electron microscopy -------------------------------- Littermate Crx-/- and wildtype pups were perfused in 1% formaldehyde and 0.5% glutaraldehyde at various postnatal stages. The eyes were then enucleated, and the cornea and lens were removed. The eye cup was immersed in fixative (1% formaldehyde and 2.5% glutaraldehyde) for 3 to 4 hours at 4°C. The sclera was then partially removed and the retinas were sliced into small pieces and fixed (1% paraformaldehyde and 2.5% glutaraldehyde) overnight at 4°C. These procedures were found optimal for maintaining the structural integrity of the photoreceptor outer segments. After fixing, the tissue was washed 2X in PBS for thirty minutes per wash. The tissue was then postfixed in a 1% osmium tetroxide/1.5% potassium ferrocyanide mixture for 2 hours at 4°C. Staining was carried out for 30 minutes in 1% uranyl acetate in maleate buffer (pH = 6.0) at room temperature followed by 1% tannic acid in 0.1 M cacodylate buffer (pH = 7.4) for thirty minutes. The specimens were then dehydrated and embedded in Epon/Araldite. Thin sections were stained with uranyl acetate and lead citrate, and examined in a Jeol JEM-1200EX electron microscope. Scanning electron microscopy ---------------------------- Specimens used for SEM required removal of the retinal pigment epithelium (PE), enabling visualization of the outer surface of the neural retina. Retinae from Crx-/-, rhodopsin-/-, RDS, or wildtype eyes were dissected free from PE in a dispase solution and fixed in 1.25% glutaraldehyde and 1.0% formaldehyde overnight at 4°C. Tissue was then washed 5X in cacodylate buffer and dehydrated in ascending grades of ethanol. Tissues were subsequently critical point dried in carbon dioxide. All specimens were mounted and coated with sublimated gold-palladium by the sputtering technique. Micrographs were obtained with a Jeol JSM-35CF scanning electron microscope. Authors\' contributions ======================= EM and ER conducted transmission electron microscopy. EM performed scanning electron microscopy. TF and EM generated, characterized and maintained the Crx-/- mouse line. CC participated in the design and coordination of the study and all data analysis. EM and CC drafted the manuscript. All authors read and approved the final manuscript. Acknowledgements ================ We would like to thank Heather Regan and Dr. Susumu Ito for helping with ultrastructural analyses and Dr. David Papermaster for helpful discussions on the formation of outer segments.	PubMed Central	2024-06-05T03:55:52.748349	2005-1-27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548520/", "journal": "BMC Neurosci. 2005 Jan 27; 6:5", "authors": [ { "first": "Eric M", "last": "Morrow" }, { "first": "Takahisa", "last": "Furukawa" }, { "first": "Elio", "last": "Raviola" }, { "first": "Constance L", "last": "Cepko" } ] }
PMC548521	Background ========== Dental caries is one of the most common infectious diseases of humans. The main causative agent is a group of streptococcal species collectively described as the mutans streptococci \[[@B1]\]. Streptococcus mutanshas been identified as the major etiological agent of the disease. Unlike many other diseases, dental caries is as prevalent in the West as it is in developing countries, and therefore attracts significant interest from medical and dental authorities as well as pharmaceutical companies. The first step in the initiation of infection is the attachment of the bacterium to a specific receptor, and this is an ideal point for intervention. Two groups of proteins from mutans streptococci represent primary candidates for a human caries vaccine: i) glucosyltransferase enzymes, which synthesize adhesive glycans and allow microbial accumulation, and ii) cell surface fibrillar proteins that mediate adherence to the salivary pellicle \[[@B2]\]. The bacterial adhesin SAI/II \[[@B3]\], a surface-displayed protein with a molecular mass of 190 kDa, plays an important role in the initial attachment of S. mutansto the tooth surface. Antibodies recognizing this protein prevent colonization of the buccal cavity by the bacterium and could be developed as a vaccine against dental caries. The most suitable vaccination strategy would be passive immunization, in which monoclonal antibodies or fragments thereof are applied to the tooth surface e.g. using toothpaste, mouthwash or chewing gum. This would make active immunization with the S. mutansadhesin unnecessary. The murine monoclonal antibody Guy\'s 13 \[[@B4]\] which specifically recognizes the SAI/II protein of S. mutansand Streptococcus sobrinushas been used successfully to prevent S. mutanscolonization and the development of dental caries in non-human primates \[[@B5]\]. The antibody also prevented bacterial colonization in human clinical trials \[[@B6],[@B7]\]. However, like other murine antibodies, a major limitation in clinical applications may be the human anti-mouse antibody response (HAMA), which can increase the rate of clearance and initiate allergic reactions \[[@B8]\]. The problems associated with murine antibodies can be overcome by replacing murine sequences with their human counterparts, e.g. by chimerization \[[@B9]\], CDR grafting \[[@B10]\] and guided selection using phage display technology \[[@B11]\]. Furthermore, the use of antibody fragments rather than whole antibodies also removes some of the constant regions that may provoke an immune response. There has been a growing interest in the use of single-chain fragment variable (scFv) antibodies, in which the variable regions of the heavy and light chains are combined in the same polypeptide chain (Huston, 1988 \#2785). The advantages of such derivatives are that they can be expressed as single transgenes in various hosts, they fold spontaneously to adopt the correct tertiary structure, and their small size facilitates tissue penetration. The scFv has the heavy and light chain variable regions joined by a flexible peptide linker allowing the two domains to interact, forming a univalent antibody. Alternatively, diabodies have the same structure but the two domains are joined by a shorter, less-flexible linker, forcing dimerization and the formation of divalent antibodies (Holliger, 1993 \#3498). We have generated human derivatives of the murine Guy\'s 13 antibody using a chain-shuffling approach based on human antibody variable gene phage-display libraries. We have taken the variable gene regions of the original Guy\'s 13 monoclonal antibody and created human scFv and diabody derivatives by chain shuffling in human phage-display libraries. Firstly, the heavy chain variable gene of the Guy\'s 13 construct was introduced into a naïve human light chain phage display library to select human light chains that, in combination with the murine heavy chain, showed binding specificity for the SAI/II antigen. Once such chimeric antibodies had been selected, the murine heavy chain gene was replaced with the human counterpart, by introducing the selected human light chain genes into a human heavy chain phage display library. The resulting clones were expressed in bacteria and tested for specificity in ELISAs using both SAI/II antigen and whole S. mutans. The stepwise procedure for generating human antibody chains allows the advantages of scFv and diabody antibody fragments to be exploited without suffering the negative effects of non-human antibodies in a clinical setting. The human antibody fragments were expressed in bacteria as scFv and diabody derivatives and used to aggregate S. mutans in vitro. The diabodies were able to aggregate the bacteria and therefore have the potential to be developed as therapeutic agents to treat and/or prevent dental caries. Results ======= Human recombinant scFv antibodies against SAI/II ------------------------------------------------ Human scFv antibody fragments based on the murine monoclonal antibody Guy\'s 13 were constructed using two consecutive rounds of variable-domain shuffling and phage-library selection (Figure [1](#F1){ref-type="fig"}). First, a chimeric scFv was generated by amplifying the murine Guy\'s 13 heavy chain variable region, and inserting it into a human light chain variable region phage display library. The resulting phage display library had a complexity of 5 × 10^5^. Single chain Fv antibody fragments with appropriate binding activities were selected on purified, immobilized SAI/II antigen. Three rounds of selection were carried out and unique candidate antibodies were identified by ELISA (Figure [2](#F2){ref-type="fig"}). Subsequent sequencing yielded five antibody fragments (chimscFvA1, chimscFvA6, chimscFvA9, chimscFvB4, and chimscFvG4). Sequencing of the human variable genes showed that two of the clones chimscFvA6, chimscFvB4 belonged to family Vκ1, clone chimscFvA6 was homologous to HK137 and chimscFvB4 was homologous to the L12 germline gene family. ChimscFvA9 belonged to family Vκ4 DPκ24. ChimscFvA1 and chimscFvG4 belonged to family Vλ3 DPL16) (data not shown). Inhibition ELISA showed that the binding of all six chimeric scFvs to SAI/II could be inhibited by the murine monoclonal antibody Guy\'s 13. The binding of chimeric scFvs A6, A9, B4 and C6 was inhibited by approximately 80%, suggesting that epitope recognition was maintained (Figure [3](#F3){ref-type="fig"}). The binding of the chimeric scFvs A1 and G4 was only inhibited by approximately 30%, suggesting that these antibodies recognized a different epitope. The selected human V~L~genes were introduced into a human V~H~library (complexity 8 × 10^8^) and a combinatorial library with a complexity of 1 × 10^6^was established. Three rounds of selection were carried out in solution using SAI/II antigen coupled to paramagnetic beads. Eleven human scFvs were identified by ELISA (data not shown). Subsequent sequence analysis identified three human scFvs: clones huscFv B10, huscFv D12 and huscFv H6. Table [1](#T1){ref-type="table"} shows the amino acid sequences of the human scFv antibody fragments. The human V~L~domain in chimeric scFv A6 (Vκ1 HK137) was selected in combination with two different human variable heavy chains, giving human scFvs B10 and H6, respectively. The V~H~domain of human scFv B10 is homologous to V~H~1 family DP10, and the V~H~domain of human scFv H6 is homologous to V~H~3 family DP35. The human V~L~domain in chimeric scFv B4 (Vκ1 L12) was selected in combination with one human variable heavy chain giving the human scFv D12. The V~H~domain of human scFv D12 is homologous to VH5 family DP73. Figure [4](#F4){ref-type="fig"} shows the binding of the three human scFvs to the SAI/II antigen and the pathogenic bacterium S. mutans. Inhibition ELISA showed that the binding of all three human scFvs to SAI/II was inhibited by Guy\'s 13, suggesting that epitope recognition was maintained. Generation of human diabodies and agglutination of S. mutans -------------------------------------------------------------- Recombinant antibody fragments can be engineered to assemble into stable multimeric oligomers of high binding avidity and specificity \[[@B12]\]. A scFv molecule joined by a linker of 3--12 residues cannot fold into a functional Fv domain and instead associates with a second scFv molecule to form a bivalent dimer (diabody, approx. 60 kDa). For the cross-linking of cell surface antigens, at least two binding domains are necessary. The diabody is the smallest bivalent antibody molecule that can fulfill this prerequisite. Through reduced off-rates, which result from multiple binding to two target antigens and to rebinding when one Fv dissociates, the diabody is suitable to facilitate specific agglutination of bacteria. We constructed human diabodies by isolating the variable heavy and light chain genes from human scFvs B10, D12 and H6 and murine scFv Guy\'s 13, amplified by PCR (Table [2](#T2){ref-type="table"}) and inserting them in two consecutive steps into the vector pHenIXdia, containing a 10 amino acid residue linker. The integrity of the clones was confirmed by sequencing and the binding activity was demonstrated by ELISA using both the SAI/II antigen and S. mutanscells (Figure [4](#F4){ref-type="fig"}). Ma et al. \[[@B7]\] reported that bivalent binding of the murine Guy\'s 13 is required for protection against dental caries, since the F(ab\')2 derivative was protective but not the monovalent Fab fragment. S. mutanswas aggregated in a dose dependent manner when grown in the presence of murine diabody Guy\'s 13 and human diabody D12 (Figure [5A](#F5){ref-type="fig"} and [5B](#F5){ref-type="fig"}). Discussion ========== Antibodies recognizing and neutralizing the oral pathogen S. mutansprovide a novel approach for the control and prevention of dental caries. A monoclonal antibody that binds specifically to the SAI/II surface adhesin of S. mutanswas isolated by Smith et al. (20) and has been expressed in plants as a secretory IgA (sIgA) \[[@B13]\]. In ongoing Phase II clinical trials, this recombinant antibody has been shown to prevent recolonization of the mouth by S. mutanswhen coated onto the teeth and gums following eradication of the bacteria. The sIgA is probably the most appropriate format for the topical application of antibodies that inhibit the colonization of the tooth surface by S. mutansbecause this is the predominant form of antibody naturally found in the saliva. However, each sIgA comprises ten polypeptide chains of four different types making it difficult to produce on a large scale in conventional production systems. The more convenient diabody antibody derivatives, which can be expressed in large quantities in microbial culture systems, may be more suitable for the type of production scales that would be required for the routine control of dental caries using this strategy. As a first step in this direction, we have taken the variable gene regions of the original Guy\'s 13 monoclonal antibody and created human scFv and diabody derivatives by chain shuffling in human phage-display libraries. The heavy chain variable gene of the Guy\'s 13 construct was introduced into a naïve human light chain phage display library to select human light chains that, in combination with the murine heavy chain, showed binding specificity for the SAI/II antigen. Once such chimeric antibodies had been selected, the murine heavy chain gene was replaced with the human counterpart, by introducing the selected human light chain genes into a human heavy chain phage display library. The resulting clones were expressed in bacteria and tested for specificity in ELISAs using both SAI/II antigen and whole S. mutans. The stepwise procedure for generating human antibody chains allows the advantages of scFv and diabody antibody fragments to be exploited without suffering the negative effects of non-human antibodies in a clinical setting. Small scFv and diabody antibody fragments are easy to express in large quantities, they penetrate tissues easily and they lack the constant domains that promote often-unwanted and usually superfluous effector functions. However, where such antibodies are murine in origin, they can provoke an immune reaction in the human host, leading to rapid clearance and poor efficacy during long-term treatment. Since dental caries tends to be chronic rather than acute, murine antibodies would be of little benefit to patients in the long term. Two drawbacks of scFvs compared to the ideal sIgA format are monovalency and instability. ScFvs are monovalent because the heavy and light chains are joined by a flexible peptide linker, which allows the two domains to fold and interact with each other. We have addressed this problem by converting the scFv antibodies into diabodies, which is achieved by shortening the linking peptide and forcing the heavy and light chain variable domains to seek interaction partners as part of a dimer. As a consequence of this interaction, the diabody is bivalent like the parent immunoglobulin, and therefore has increased binding avidity. We showed that the bivalent binding of the diabody antibody constructs leads to agglutination of S. mutans. The problem of decreased stability may be more difficult to address, because the efficacy of scFv and diabody molecules used to treat dental caries will depend largely on their persistence and effective concentration. Secretory IgAs include a secretory component, which protects the antibody from proteolytic degradation in the saliva. One possible solution is to include this secretory component in any scFv or diabody format through the use of further gene fusion strategies. However, it is envisaged that antibodies for the prevention of dental caries will be administered in the form of toothpaste or mouthwash, or perhaps chewing gum, which will allow the treatment to be refreshed at regular intervals. Conclusions =========== We have shown that the humanization of a murine monoclonal antibody can be easily achieved using a chain-shuffling approach based on scFv antibody phage display libraries. The human antibody derivatives of the murine Guy\'s 13 antibody can be expressed and isolated from bacteria, recognize SAI/II and S. mutanswith great specificity, and can successfully aggregate S. mutanscells in the dimeric form in a dose dependent manor. These recombinant therapeutic proteins therefore represent the first step towards an inexpensive and convenient general treatment for dental caries. Methods ======= Propagation of S. mutans -------------------------- S. mutans20523 serological group c was purchased from DSMZ (Braunschweig, Germany) and grown in an S2 containment laboratory in trypticase soy yeast extract medium (30 g l^-1^trypticase soy broth, 3 g l^-1^yeast extract, pH 7.0--7.2) at 37°C for 2 d prior to use. Cloning the S. mutansspaP gene encoding surface antigen SAI/II ---------------------------------------------------------------- Nucleotides 214--3048 of the spaP gene \[[@B14]\], which encodes the SAI/II antigen, were removed from pUC18 as a SfiI/NotI fragment and inserted into the bacterial expression vectors pCantab5E (Pharmacia) and pSin1 \[[@B15]\] which had been digested with the same enzymes followed by transformation into E. coliTG1. The pCantab5E vector contained an additional sequence encoding the E-tag, facilitating the detection of expressed proteins using the monoclonal antibody 5E (Pharmacia). The pSin1 vector similarly contained sequences encoding a MYC-tag, facilitating detection with the murine monoclonal antibody 9E10 (ATCC CRL 1729), and a His~6~tag, allowing purification of expressed proteins by immobilized metal-chelate affinity chromatography (IMAC) and detection using a murine Penta-HIS antibody (Qiagen). SAI/II expressed using pSin1 was used for the selection of antibodies from phage-display libraries. SAI/II expressed in pCantab5E was used for enzyme-linked immunosorbent assays (ELISAs). Coating paramagnetic beads with SAI/II -------------------------------------- For the selection of phage-display antibodies, 250 μl of phosphate-buffered saline (PBS)-washed Dynabeads (Dynal Biotech GmbH) was resuspended in 500 μl 0.1 M phosphate buffer (pH 7.4) and mixed gently for 2 min. The beads were collected with a magnet, the supernatant discarded and the beads resuspended in 250 μl of the same buffer, followed by the addition of 500 μl SAI/II antigen (1 mg/ml). After incubation for 16 h at 37°C with slow tilt rotation, the beads were collected with a magnet and the supernatant was discarded. The coated beads were washed four times, twice with 0.13 M NaCl, 1% milk powder in 0.01 M phosphate buffer (pH 7.4) for 5 min at 4°C, once with 0.2 M Tris-HCl (pH 8.5) for 4 h at 37°C and again in the same buffer for 5 min at 4°C. Cloning scFv Guy\'s 13 in pSin1 ------------------------------- The variable region genes of the heavy and light chain of the murine monoclonal antibody Guy\'s 13 were amplified using oligonucleotide primers LMB3 (5\' CAG GAA ACA GCT ATG AC 3\') and fdSeq 1 (5\' GAA TTT TCT GTA TG/AG GG 3\') followed by digestion with SfiI and NotI. The products were inserted into the phagemid vector pSin1, which had been treated with the same enzymes, and the recombinant vector was introduced into E. colistrain TG1. Construction and selection of human SAI/II-specific scFv antibodies ------------------------------------------------------------------- Figure [1](#F1){ref-type="fig"} shows the schematic scheme of the construction and selection of human SAI/II specific scFv antibody approach. The variable heavy chain antibody domain of the murine antibody Guy\'s 13 was cloned as an SfiI/SalI fragment in the bacterial expression vector pHenIX containing a light-chain antibody phage-display library derived from naïve human peripheral blood lymphocytes (8 × 10^8^; R. Finnern, unpublished data). This vector is based on the phagemid vector pHen1 \[[@B16]\] designed to express antibody fragments as N-terminal fusions with the minor coat protein of filamentous bacteriophage M13. An amber stop codon between the two fusion partners allows the expression of both soluble antibody fragments and phage particles displaying recombinant antibodies. The recombinant vectors were introduced into E. colistrain TG1. Three rounds of selection were carried out using immobilized SAI/II antigen as described by Marks et al. \[[@B17]\]. Elution was achieved using the monoclonal antibody Guy\'s 13 to select binders recognizing the same epitope. The expression of soluble scFvs was performed as described by Marks et al. \[[@B17]\] and scFvs specific for the SAI/II antigen were identified by ELISA using SAI/II antigen. The selected variable antibody domain genes of the shuffled human light chains were cloned as ApaLI and NotI fragments in pHenIX containing a human variable heavy chain library (8 × 10^8^; R. Finnern, unpublished data) and introduced into E. coliTG1. This was achieved by PCR amplification of the human light chain genes using primers Vκ4 ApaLI (5\'-TGAGCACACAGTGCACTCGACATCGTGATGACCCAGTCTCC-3\'), Vκ1 ApaLI (5\'-TGAGCACACAGTGCACTCGACATCCAGATGACCCAGTCTCC-3\') and Jκ1 NotI (5\'-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATC/TTCCAC/GCTTGGTCCC-3\'). Three rounds of selection were carried out using SAI/II antigen immobilized on Dynabeads. Briefly, 150 μg of SAI/II-coated beads was blocked for 1 h with 2 ml 2% milk powder. The beads were collected with a magnet, washed in PBS and incubated with the antibody phage display library for 1 h on a turntable. The beads were washed 15 times with PBS containing 0.05% Tween 20 and 15 times with PBS to remove unbound phage. Bound phage were eluted with 100 μl 100 mM triethanolamine for 10 min on a turntable followed by neutralization in 200 μl 1 M Tris-HCl (pH 8.0). Eluted phage were used to infect exponentially growing E. coliTG1 and grown overnight at 30°C on TYE plates containing 100 μg ml^-1^ampicillin, 1% glucose. Selection, phage rescue and induction of soluble scFv expression were carried out as described by Marks et al. \[[@B17]\]. Antigen-specific human scFvs were identified by ELISA using the SAI/II antigen. Propagation of phage display antibody libraries ----------------------------------------------- One litre of 2× TY (supplemented with 100 μg ml^-1^ampicillin, 1% glucose) was inoculated with an aliquot of the phage antibody library glycerol stock. The rescue and induction of the phage was carried out essentially as described in Marks et al. \[[@B17]\]. Phagemid rescue was carried out by the addition of 10^10^units of helper phage VCSM13 (Pharmacia) to the growing phage antibody library. The culture medium was changed to 2× TY containing 100 μg ml^-1^ampicillin and 25 μg ml^-1^kanamycin and incubated on an orbital shaker overnight at 30°C and 250 rpm. Phage were purified twice by polyethylene glycol (PEG) precipitation (20% PEG, 2.5 M NaCl) and resuspended in a final volume of 2 ml PBS. The phage were stored at 4°C until further use. DNA sequencing -------------- The number of unique clones was determined by PCR amplification of the recombinant antibody inserts using primers LMB3 (5\' CAG GAA ACA GCT ATG AC 3\') and fdSeq 1 (5\' GAA TTT TCT GTA TG/AG GG 3\') followed by digestion with the restriction enzyme BstNI (New England Biolabs). The variable antibody genes from two clones of each restriction pattern were analyzed by PCR cycle sequencing using infrared-labeled primers according to the manufacturer\'s instructions (Licor). Sequencing reactions were carried out on a Licor automated DNA sequencer (4000 L) and the sequences were analyzed using Sequencher 3.1 (Gene Codes Corporation). The sequences of the V~H~and V~L~genes were compared with the germline sequences in the V-BASE database (Tomlinson et al., MRC Centre for Protein Engineering, Cambridge, UK). Construction of diabodies ------------------------- The construction of diabodies \[[@B18]\] was carried out by PCR amplification of the variable heavy and light chain antibody regions of the human scFv clones and subcloning these in vector pHenIXdia. The diabody constructs consisted of the variable heavy and light chain antibody domains linked by a ten-amino-acid linker (TGGGGSSSAL), forcing the expressed domains to attach to a complementary chain in solution to create two antigen-binding sites. The primers used for the construction of the diabody antibody format are listed in Table [1](#T1){ref-type="table"}. Upscaled production of recombinant proteins ------------------------------------------- Recombinant proteins were recovered from the bacterial periplasm following induction with 0.5 mM final concentration of isopropyl-β-D-galactopyranoside (IPTG) for 3--4 h at 30°C \[[@B19]\]. After centrifugation (4000 × g, 4°C, 30 min), the pellet was resuspended in 10 ml 30 mM Tris-HCl (pH 8.2) containing 20% sucrose, 1 mM ethylenediaminetretaacetic acid (EDTA), incubated on ice for 15 min and centrifuged as above. The pellet was resuspended in 10 ml 5 mM MgSO~4~, 1 mM EDTA and incubated for 15 minutes on ice before a final centrifugation step as above. Both supernatants were pooled, dialyzed against PBS and stored at 4°C. Recombinant proteins were also expressed in the periplasm under osmotic stress in the presence of compatible solutes as described by Barth et al. \[[@B20]\]. Briefly, bacteria were grown overnight at 26°C in Terrific Broth (TB) (12 g l^-1^bacto-tryptone, 24 g l^-1^bacto-yeast-extract, 4 ml l^-1^glycerol) containing 100 μg ml^-1^ampicillin and 0.5 mM ZnCl~2~. The culture was diluted 30-fold in 200 ml of the same medium. When the OD~600\ nm~of the culture reached 2.0, it was supplemented with 0.5 M sorbitol, 4% NaCl, 40 mM glycine betaine and incubated at 26°C for an additional 30--60 min. Expression was induced with 1 mM final concentration IPTG and growth for 6 h at 26°C. Cells were harvested by centrifugation at 30,000 × g for 10 min. The recombinant antibody fragments were isolated from the periplasmic space as described above. The periplasmic and osmotic shock fractions were pooled and dialyzed against PBS. Phenylmethylsulfonylfluoride (PMSF) was added to a final concentration of 1 mM. Purification of recombinant proteins ------------------------------------ The human scFv and diabody antibody fragments were purified by IMAC using the His~6~tag as described by Griffiths et al. \[[@B21]\]. Briefly, 10 ml columns (BioRad Polyprep chromatography columns) were packed with 500 μl Ni-NTA resin (Qiagen) and washed with five column volumes of PBS prior to loading with the recombinant proteins. The columns were washed with 10 column volumes of PBS containing 10 mM imidazol. Bound proteins were eluted with 250 mM imidazol and collected in 1-ml fractions. Protein concentrations were determined by spectrophotometry assuming that A~280\ nm~= 1 corresponds to a scFv or diabody concentration of 0.7 mg ml^-1^. Gel filtration was used for further purification. A Sephadex 200 column (Pharmacia) was equilibrated with PBS. ScFv or diabody antibody fragments were loaded and run at 1 ml min^-1^. Aprotinin (6500 Da), cytochrome C (12,400 Da), carbonic anhydrase (29,000 Da), bovine serum albumin (BSA) (66,000 Da) and Dextran Blue (2,000,000 Da) were used as molecular weight standards (Fluka). Enzyme linked immunosorbent assay (ELISA) ----------------------------------------- S. mutans, SAI/II antigen or BSA were coated on ELISA plates (Nunc) at a concentration of 1--10 μg per well in PBS overnight at 4°C. The plates were washed three times with PBS and blocked with 2% milk powder in PBS for 2 h at room temperature. The scFvs were tested either at a concentration of 1 μg per well or 100 μl per well of overnight-induced culture. Recombinant antibodies containing the MYC tag were detected with the murine 9E10 monoclonal antibody (ATCC CRL1729). Antibodies containing a His~6~tag were detected using the murine anti-Penta-HIS antibody (Qiagen). The murine antibodies were detected with a goat-anti-mouse (Fc-specific) peroxidase-labeled antibody. The assays were developed with 3,3\',5,5\'-tetramethylbenzidine (TMB) (Sigma). Reactions were stopped by the addition of H~2~SO~4~after 20 min and readings taken at OD~450\ nm~. Between every incubation step, the plates were washed three times with PBS containing 0.05% Tween 20 and three times with PBS. Inhibition ELISAwas carried out by binding of chimeric scFv to SAI/II and replacement with an excess of mAb Guy13. Bound scFv were detected as described above using the murine 9E10 monoclonal antibody detecting the MYC-tag. Agglutination of S. mutans ---------------------------- Cultured S. mutanswas divided into 20-μl aliquots and incubated with serial dilutions of bacterially expressed recombinant antibodies dia mGuy13 and diaD12, respectively for 2 d at 4°C or 1 h at 37°C on Lab-Tek II chamber slides (Nalge Nunc International). As negative control an unrelated human diabody was used. Excess medium was discarded and the cells were air-dried. The bacteria were counterstained with Gram solution (Diagnostica Merck). The slides were mounted with Immunofluor medium (ICN Biomedicals Inc) and photographed with a Zeiss Axioskob microscope. Authors contributions ===================== MBK generated the diabody formats and performed all in vitroassays. MH did the upscaled expression of the antibodies. HS assisted in the analysis of data. JKCM assisted in the analysis and interpretation of the data especially in respect to the data obtained with the murine mAb Guy13. SB assisted in the interpretation of the data. RF assisted in the conception and revision of the project. RF\* generated the human phage display libraries, developed the concept and supervised the project Acknowledgement =============== The authors would like to thank Dr. Neil Emans for the support in generating the images of the S. mutansagglutination. For the helpful discussion and revision of the manuscript we thank Dr. Richard Twyman. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Construction and selection of human SAI/II specific antibody fragments. A: Antibody formats: scFv, in which the variable regions of the antibody heavy and light chains are combined in the same polypeptide chain joined by a (Gly~4~Ser)~3~amino acid linker. The diabody construct consists of the antibody variable heavy and light chain domains linked by a ten-amino-acid linker (TGGGGSSSAL), forcing the expressed domains to attach to a complementary chain in solution to create two antigen-binding sites. scFv presentation as a protein3 fusion of the M13 phage. B: Chain shuffling approach: The variable antibody domain is cloned into a phagemid vector containing a human antibody variable domain library. The selection on SAI/II antigen was carried out for three consecutive rounds. Light chains participating in binding to SAI/II were identified and cloned into a phagemid vector containing a human antibody heavy domain library. Selection on SAI/II antigen for three rounds yielded fully human SAI/II specific scFv antibody fragments. ::: ![](1472-6750-5-4-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Specificity of chimeric scFv (mGuy13VH/huVL) ELISA reactivity of overnight induced bacterial culture supernatant containing chimeric scFv (mGuy13VH/huVL) after 3 rounds of selection on SAI/II antigen. A: 96 individual scFvs on SAI/II coated ELISA plates. B: the same 96 scFvs on BSA coated ELISA plates ::: ![](1472-6750-5-4-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Inhibition of chimeric scFv (mGuy13VH/huVL) to SAI/II by the monoclonal Ab Guy13. Chimeric scFv were allowed to bind to SAI/II followed by replacement with an access of mAb Guy 13. The experiments were carried out in duplicate and the mean values are shown. Bound scFv were detected via their MYC-tag. The murine mAb Guy13 bound to the SAI/II. The scFv Guy13 was not replaced by an unrelated mAb (ctrl.) Binding of A1 and G4 was only inhibited by 36% and 25%, respectively indicating that these scFv might recognize a different epitope than mAb Guy13. The binding of chimeric scFv A6, A9, B4, and C6 were inhibited between 77--84% indicating that they recognize the same epitope as mAb Guy13. ::: ![](1472-6750-5-4-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Binding of human diabodies to SAI/II and S. mutanscoated ELISA plates. One μg SAI/II or 10 ml S. mutansculture were immobilized on the ELISA plate. Binding of 1 μg human diabody B10, D12 and H6 was detected via the His~6~tag. The experiments were carried out in duplicate and the mean values are shown. The three human diabodies recognize the SAI/II antigen in the purified form as well as in the context of the bacterial surface. ::: ![](1472-6750-5-4-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Bacterial agglutination assay. S. mutanswas incubated with serial dilutions of diabody for 1 h at 37°C followed by counterstaining with Gram solution and analysis by microscopy. A: Murine diabody mGuy13. B: Human diabody D12. Neg. ctrl.: Unrelated human diabody ::: ![](1472-6750-5-4-5) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Deduced amino acid sequence of the heavy and light chain variable regions of human scFv and diabody. ::: scFv FR1 CDR1 FR2 CDR2 ------------- ---------------------------------- -------------------- ----------------- ------------------- MuV~H~Guy13 QVKLQESGPDLVKPGASVKISCKASGYTFT DYNIH WVKQSRGKSLEWIG YIYPYNGNTYYNQKFKN MuV~L~Guy13 DIELTQSPAIMSASPGEKVTITC SASSSVSYMH WFQQKPGTSPKLWLY STSNLAS HuV~H~B10 QVQLQESGAEVKKPGLGEGLLQASGGTFS RYALS WVRQAPGQGLEWMG GIIPIFGTTNYAQKFQG HuV~L~B10 DIQMTQSPSSLSASVGDRVTITC RASQGISNYLA WFQQKPGKAPKSLIY AASSLQS HuV~H~D12 QVQLQEXGAEVKKPGESLKIXCKGSGYSFT SYWIG WVRQMPGKGLEWMG IIYPGDXDTRYSPSFQG HuV~L~D12 DIQMTQSPSTLSASIGDRVTITC RASEGIYHWLA WYQQKPGKAPKLLIY EASRLQS HuV~H~H6 QVQLQESGGGVVQPGRSLRLSCAASGFTFS SYAMH WVRQAPGKGLEWVS YISSSGSYIYYADSVKG HuV~L~H6 DIVMTQSPSSLSASVGDRVTITC RASQGISNYLA WFQQKPGKAPKSLIY AASSLQS scFv FR3 CDR3 FR4 MuV~H~Guy13 KATLTVDNSSTSAYMELRSLTSEDSAVYYCAT YFDY WGQGTTVTVS MuV~L~Guy13 GVPARFSGSGSGTSYSLTISRMEAEDAATYYC HQRTSYPYT FGGTKLEIKR HuV~H~B10 RVTIAADESTSTAYLELSSLRSEDTALYYCAK SYDYVWGSYRPNEYGLDI WGQGTMVTVS HuV~L~B10 GVPSKFSGSGSGTEFTLTISSLQPEDFATYYC QELISYPLT FGGGTKLEIKRA HuV~H~D12 QVTISADKSISTAYLQWSSLKASDTAMYYCAR LGLQDDYVWGSXNWFDP WGQGTLVTVSTS HuV~L~D12 GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC LQDFTYPRT FGQGTKVEIKRA HuV~H~H6 RFTISRDNAKNSLYLQMNRLRAEDTAVYYCAR DMAGTSYYYYYMDV WGKGTLVTVSTS HuV~L~H6 GVPSKFSGSXSGTEFTLTISSLQPEDFATYYC QELISYPLT FGGGTKLEIKRA FR = antibody framework region, CDR = complementarity determining region ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Construction of human diabodies. Primer combinations and primer sequences ::: Template V~L~amplification V~H~amplification ----------------------- ------------------- ------------------- --------------- -------------- pHenIX human scFv B10 Vκ1 ApaL1 Jκ1 Not1 VH4 Sfi1/Nco1 JH3 for Sal1 pHenIX human scFv H6 Vκ1 ApaL1 Jκ1 Not1 VH6 Sfi1/Nco1 JH2 for Sal1 pHenIX human scFv D12 Vκ1 ApaL1 Jκ1 Not1 VH4 Sfi1/Nco1 JH2 for Sal1 pHenIX scFv mGuy13 mGuy13 ApaL1 mGuy13 Not1 VH4 Sfi1/Nco1 mGuy13 Sal1 Vκ1 ApaLI: 5\'-TGAGCACACAGTGCACTCGACATCCAGATGACCCAGTCTCC-3\' Jκ1 Not1: 5\'-GAGTCATTCTCGACTTGCGGCCGCACGTTTGATC/TTCCAC/GCTTGGTCCC-3\' V~H~4 Sfi1/Nco1: 5\'-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGCA/ GGAGTCGGG-3\' V~H~6 Sfi1/Nco1: 5\'-GTCCTCGCAACTGCGGCCCAGCCGGCCATGGCCCAGGTACAGCTGCA/ GCAGTCAGG-3\' J~H~3 Sal1: 5\'-GAGTCATTCTCGTGTCGACACGGTGACCATTGTCCC-3\' J~H~2 Sal1: 5\'-GAGTCATTCTCGTGTCGACACAGTGACCAGGGTGCC-3\' mGuy13 ApaL1I:5\'-TGAGCACACAGTGCACTCGACATCGAGCTCACTCAGTCTCC-3\' mGuy13 Not1: 5\'-TTTTCCTTTTGCGGCCGCCCGTTTTATTTCCAACTTTGT-3\' mGuy13 Sal1: 5\'-GAGTCATTCTCGTGTCGACACGGTGACCGTGGTGCCTTGGCCCCAGTAGTCAAAGTAGGT-3\' :::	PubMed Central	2024-06-05T03:55:52.751145	2005-1-25	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548521/", "journal": "BMC Biotechnol. 2005 Jan 25; 5:4", "authors": [ { "first": "Michael B", "last": "Kupper" }, { "first": "Michael", "last": "Huhn" }, { "first": "Holger", "last": "Spiegel" }, { "first": "Julian KC", "last": "Ma" }, { "first": "Stefan", "last": "Barth" }, { "first": "Rainer", "last": "Fischer" }, { "first": "Ricarda", "last": "Finnern" } ] }
PMC548522	Background ========== Fetal growth restriction (FGR) is a major risk factor for illness in the perinatal period and throughout life, with the smallest 7.5 percent of infants accounting for two-thirds of infant deaths \[[@B1]\]. Term, low birth weight infants are at least five times more likely to die in the first year \[[@B2],[@B3]\] and are second only to premature infants in their rates of morbidity and mortality \[[@B4]\]. FGR infants have an increased frequency of hypoglycemia, hypothermia, polycythemia, neurodevelopmental deficits, and cerebral palsy \[[@B5]\]. Later in life, individuals born FGR are at elevated risk of hypertension, cardiovascular disease, and non-insulin dependent diabetes \[[@B6]\]. For example, FGR increases the risk for adult onset of non-insulin dependent diabetes two- to three-fold \[[@B7],[@B8]\]. The ability to both diagnose and treat FGR early in gestation has enormous potential to reduce childhood and adult illness. It is difficult to distinguish the genetic and environmental components of human birth weight variation, but recent studies support a major genetic component to birth weight variation. Clausson et al.\'s \[[@B9]\] study of the offspring of female dizygotic and monozygotic twins (2,009 twin pairs) estimated heritability for human birth weight of 42%, although the confounding influence of shared environmental effects must be considered. In a study of 3,562 captive macaques that minimized environmental heterogeneity, Ha et al. \[[@B10]\] estimated a total heritability for birth weight of 51%, with an additive genetic component of 23%. These findings demonstrate that comparatively simple and readily identifiable genetic factors influence birth weight. In concert with this recent research, FGR tends both to cluster in families and to recur in successive generations \[[@B11]-[@B14]\]. The five genes of the human growth hormone locus reside within about 45 kb on chromosome 17 \[[@B15]\]. Pituitary growth hormone (GH1) is by far the most thoroughly studied of the genes and lies at the 5\' end of the cluster. The remaining four genes, placental growth hormone (GH2) and three chorionic somatomammotropins (CS1, CS2, and pseudogene CS5 or CSHP1), are expressed only from the placenta. The promoter region of GH1 is unusually polymorphic, with 16 SNPs having been identified in a span of 535 bp \[[@B16]-[@B18]\]. Most of these SNPs occur at the comparatively small number of sites that exhibit sequence differences among the five genes of the GH locus, and this has been interpreted as evidence of gene conversion \[[@B16],[@B19]\]. Here we use DNA sequencing to identify and to determine the frequencies of both 12 newly-identified single nucleotide polymorphisms (SNPs) in the promoter and coding region of the GH1 gene and 15 previously reported SNPs \[[@B16],[@B18]\]. Using a case-control design, we identify two SNPs in complete linkage disequilibrium near the start of transcription of the GH1 gene that may predispose to reduced birth weight. Methods ======= Human subjects -------------- DNA was extracted from placental tissue from 125 live births (83 normal birth weight, 42 low birth weight) at Baystate Medical Center (Springfield, MA) in a case-control study of the genetic predisposition to fetal growth restriction. All subjects were Caucasian, both Hispanic and non-Hispanic. Classification of newborns as fetal growth restricted (or IUGR, intrauterine growth retarded) followed the definition of the American College of Obstetricians and Gynecologists as those newborns below the 10^th^percentile of size for gestational age \[[@B20]\]. Our cut-off of 2,500 g at term (\>37 weeks gestation) corresponds to the lowest 7.5 percentile of all US Caucasian deliveries, or the lowest 10^th^percentile of female and lowest 6.5 percentile of male deliveries \[[@B21]\]. Stringent inclusion/exclusion criteria (Table [1](#T1){ref-type="table"}) were employed by a placental pathologist (TKB) to reduce nongenetic contributors to birth weight variation. Ethical approval to conduct this study was obtained from the Human Subjects Institutional Review Board of the University of Massachusetts. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Inclusion and exclusion criteria for the study of fetal growth restriction ::: -------------------- ------------------------------------------------------------------------------------- Inclusion Criteria ≥ 37 weeks gestation (full-term) Mother 17--35 years old Singleton pregnancy Case (fetal growth restricted): \<2,500 g birth weight Control: 3,000 to 4,000 g birth weight Exclusion Criteria Karyotypic abnormalities, including confined placental mosaicism Placental abnormalities Birth defects or syndromic conditions (i.e., Silver-Russell Syndrome) Pregnancy complications Preeclampsia Type 1, Type 2 or gestational diabetes Meconium staining Uterine infection Maternal chronic illnesses (i. e., hypertension, AIDS, hepatitis, endocrinological) Known illicit drug use Rh disease -------------------- ------------------------------------------------------------------------------------- ::: Polymerase chain reaction (PCR) and sequencing ---------------------------------------------- The region from -624 (relative to the start of transcription; GenBank accession J03071) to +1,726 (197 nucleotides after the termination codon) of GH1 was amplified in two overlapping fragments: -624 to +541 (GHN-1F 5\' AGGGACCTGGGGGAGCCCCAGCAA 3\', GHN-1R 5\' TCACCCCTTCCTGCCACCCCTGAT 3\') and +450 to +1,726 (GHN-2F 5\' CCATCGTCTGCACCAGCTGGCCTT 3\'; GHN-2R 5\' GCCCTACAGGTTGTCTTCCCAACT 3\'). Approximately 50 ng of DNA was used as a template in a polymerase chain reaction with 30 cycles of 95°C (1 minute), 62°C (1 minute), and 72°C (2 minutes 30 seconds). PCR products were purified from agarose using a QIAquick PCR Purification kit (Qiagen) and sequenced directly with BigDye v2.0 chemistry (Applied Biosystems) and run on either an ABI Prism 377 or 3100 automated DNA sequencer. PCR products were sequenced with the PCR primers and additional internal primers: 5\' AAGCACAGCCAATAGATTG 3\', -459 to -441; 5\' GCACAAGCCCGTCAGTGGCC 3\', -108 to -89; 5\' GGATTTTAGGGGCGCTTACC 3\', +71 to +90; 5\' CATCTCCCTGCTGCTCATC 3\', +931 to+949; 5\' GCGCTTGGGYACTGTTCCCT 3\', +1280 to +1299. Single nucleotide polymorphism (SNP) genotyping ----------------------------------------------- Sequence traces were aligned and assembled into contigs by the program Polyphred \[[@B22]\]. Contigs were viewed in either the program Consed \[[@B23]\] or Sequencher (Gene Codes Corp.) and polymorphisms confirmed visually. Twenty-six polymorphic sites were identified (Table [2](#T2){ref-type="table"}), including all of the sites identified by Horan et al. \[[@B18]\] in 154 British military recruits with the exception of site -339 which had a minor allele (deletion of G) frequency of 3.6% in their study. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Frequency of alleles at 26 single nucleotide polymorphisms in the promoter and coding region of pituitary growth hormone and the nucleotide present at the homologous site in other members of the human GH locus ::: Frequency GH1 paralogs ------ ---- ----------- -------------- --- --- ---- --- ---------- --------------------- -580 A 0.985 A A A A Constant G 0.015 -476 A 0.012 0.013 A G A G Variant G 0.988 0.987 -360 A 0.972 G G G G Variant G 0.028 -352 G 0.012 T G G G Variant T 0.988 -308 G 0.732 0.753 T C T C Variant T 0.268 0.247 -301 G 0.732 0.753 T T T T Variant T 0.268 0.247 -278 G 0.628 0.601 T A T A Variant NF1 T 0.372 0.399 -168 C 0.024 0.019 T C T C Variant T 0.976 0.981 -75 A 0.900 0.886 G A G A Variant PIT-1 G 0.100 0.114 -57 G 0.687 0.633 G T A T Variant Vitamin D Receptor T 0.313 0.367 -31 G 0.882 0.867 G G \- G Variant Vitamin D Receptor \- 0.118 0.133 -6 A 0.565 0.588 A G A G Variant Transcription Start G 0.435 0.412 -1 A 0.847 0.932 C T A T Variant Transcription Start C 0.044 0.003 T 0.109 0.065 3 C 0.044 0.003 C G G G Variant Transcription Start G 0.956 0.997 16 A 0.976 0.981 G A A A Variant 5\' UTR G 0.024 0.019 25 A 0.980 0.981 C A A A Variant 5\' UTR C 0.020 0.019 59 G 0.072 0.049 G G G G Variant 5\' UTR T 0.928 0.951 69 A 0.968 G C G G Variant Thr/Ala G 0.032 124 A 0.988 G A G A Variant Intron G 0.012 128 A 0.988 C C T C Variant Intron T 0.012 140 A 0.004 G G G G Constant Intron G 0.996 144 A 0.012 G G G G Constant Intron G 0.988 281 C 0.024 T C C C Variant Intron T 0.976 596 C 0.986 T T T T Variant Intron T 0.014 1070 A 0.004 G G G G Constant Synonymous G 0.996 1169 A 0.331 T T T T Constant Intron T 0.669 \* Relative to the start of transcription \\ Polymorphisms in known transcription factor binding sites are shown. Site +69 is part of the signal peptide. ::: Statistical analyses -------------------- The five genes of the human growth hormone locus exhibit high sequence similarity, and the paralogous regions corresponding to the portion of GH1 sequenced in this study (-624 to +1,726) were multiply aligned. Nucleotide positions (Table [2](#T2){ref-type="table"}) were designated as invariant if all five genes had the same nucleotide or the four paralogs of GH1 were identical and matched the major allele at that site in GH1. This categorization explicitly assumes that only the minor alleles in GH1 are the product of gene conversion and that minor alleles not observed in paralogs of GH1 are the result of unique mutations. The proportion of GH1 polymorphisms at invariant versus variant sites was compared by Fisher\'s Exact Test to determine if there was an over-representation of polymorphic sites among variant sites. Logistic regression, using FGR status as the outcome, was performed on gestational age and genotypes at sites with a minor allele frequency above 5% (Table [3](#T3){ref-type="table"}). Sites -301 and -308 (relative to the start of transcription) are in complete linkage disequilibrium and the minor C alleles at sites -1 and +3 are in complete linkage disequilibrium. Therefore, sites -301 and +3 were excluded from logistic regression to avoid multicollinearity. Based on the results from logistic regression, separate ANOVA (Table [4](#T4){ref-type="table"}) was performed on gestational age and the AA versus AC genotypes at site -1 within low birth weight and within normal birth weight subjects. Empirical p values for the F statistic for the genotypic effect, corrected for multiple comparisons, were determined by 2,000 random permutations of the genotypic data \[[@B24]\]. All statistical analyses were performed using the Stata program (Stata Corp., College Station, TX). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Logistic regression on FGR status based on gestational age and SNP genotype for GH1 polymorphisms with minor allele frequency greater than 5% ::: Variable Odds Ratio 95% CI Z Score P-value ----------------- ------------ ------------- --------- --------- Gestational Age 0.42 0.26--0.66 -3.75 \<0.001 -308 2.66 0.54--13.19 1.20 0.23 -278 1.62 0.27--9.80 0.52 0.60 -75 1.70 0.55--5.23 0.93 0.35 -57 3.34 0.62--18.12 1.40 0.16 -31 0.85 0.27--2.65 -0.29 0.78 -6 2.11 0.66--6.74 1.26 0.21 -1 A/T 0.76 0.23--2.44 -0.47 0.64 -1 A/C 0.10 0.01--0.77 -2.21 0.03 +59 1.48 0.32--6.95 0.50 0.62 +1169 1.05 0.38--2.95 0.10 0.92 ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### ANOVA on A/C genotypes at site -1 and gestational age in normal and low birth weight subjects ::: Normal Birth Weight Low Birth Weight --------- --------------------- ------------------ --------- ------- Mean AA 3382.8 2287.9 Mean AC 3230.2 2190.4 ANOVA F 3.75 4.55 3.12 2.25 P-value 0.056\* 0.002 0.073\* 0.099 \* Empirical P value corrected for multiple testing determined by 2,000 random permutations of the genotypic data \[24\] ::: Results ======= Polymorphism in the GH1 gene ---------------------------- Among the 125 subjects sequenced from -624 to +1,726 of the GH1 gene, 26 polymorphic sites were identified (Table [2](#T2){ref-type="table"}). These included all but one of the 15 sites characterized by Horan et al. (2003). In the region of overlap between the two studies, we failed to detect variation at site -339, where there is a minor allele deletion of a single nucleotide with frequency 3.6% in British army recruits, and identified two additional variants at sites -360 and -352 with minor allele frequencies 2.8% and 1.2%, respectively. Therefore, the discrepancies between the two studies in the identification of SNPs can most likely be ascribed to sampling error. Outside the region surveyed by Horan et al. (2003), we detected 10 additional polymorphisms. All of these had minor allele frequencies ≤ 3.2%, except an intron four polymorphism at site +1169 with a minor allele frequency of 33.1%. In general, polymorphisms in the promoter of GH1 tend to be more densely clustered and exhibit higher minor allele frequencies than in the transcribed region. Evidence of gene conversion --------------------------- An alignment of the region of GH1 sequenced in this study with the paralogous sequences of placental growth hormone and chorionic somatomammotropins indicated that there are 1,979 invariant sites and 293 variant sites (excluding 78 sites that could not be unambiguously aligned), as defined in the methods. Of the invariant sites, 5 are polymorphic in GH1, while 21 of the variant sites are polymorphic. A comparison of the proportion of polymorphic sites at invariant and variant sites by Fisher\'s Exact Test is highly significant (P \<\<0.001). This result indicates that there is a strong bias for polymorphisms in GH1 to occur at the minority of sites that exhibit sequence divergence among the paralogous genes. The high correspondence between the sequence of minor alleles in GH1 and nucleotides present in paralogs of GH1 supports previous assertions that the unusually high polymorphism of the GH1 gene is driven by gene conversion \[[@B16],[@B18]\]. A proportion of this bias may be explained by selective constraint, in that sites that are polymorphic within GH1 may be under less selective constraint and thus more free to exhibit sequence divergence among paralogs. However, to entirely explain the bias towards polymorphism at sites of divergence, one must assume that about 1,804 (91%) of the invariant sites are selectively constrained and not free to vary. Given that a substantial proportion (814 bp, \~36%) of the sequence surveyed in this study is composed of introns, this assumption seems unreasonable. Association with fetal growth restriction ----------------------------------------- Logistic regression (Table [3](#T3){ref-type="table"}) was performed on gestational age and SNPs with minor allele frequencies greater than 5%, excluding sites -301 and +3 because they are in complete linkage disequilibrium with other sites, to identify associations with fetal growth restriction. Gestational age was significant because the FGR subjects exhibit a slightly younger average estimate of gestational age (38.1 vs. 39.1 weeks, t = 4.9, p \< 0.0001; gestational ages rounded to nearest week). However, even accounting for the effect of gestational age, the C allele at site -1 was significantly associated with FGR in the combined regression. However, this allele did not retain significance in a regression on only gestational age (p \< 0.001) and the A/C polymorphism at site -1 (p = 0.242). Although the A/C polymorphism at site -1 was not significant in a reduced model, we decided to investigate this site for three reasons. First, the C allele at site -1 is in complete linkage disequilibrium with C at site +3. Second, both of these sites are located at the start of transcription, making them good candidates for affecting the level of transcription of GH1. Third, the only paralog that shares C at these sites is CS-5, a pseudogene, consistent with the possibility that C at sites -1 and +3 is disadvantageous. The C allele at both sites exhibits a much higher frequency in low birth weight (6%) versus normal birth weight (0.4%) subjects. Restricting examination only within the normal or low birth weight subjects, the AC genotype is associated with an average reduction of birth weight of 152 g and 97 g in normal and low birth weight subjects, respectively. However, this difference does not achieve significant in an ANOVA on the A/C polymorphism and gestational age (Table [4](#T4){ref-type="table"}). Discussion ========== Birth weight in humans and other primates exhibits substantial heritability \[[@B9]-[@B13]\]. Although a suite of environmental and genetic/karyological insults are known to cause fetal growth restriction, perhaps as many as 40% of FGR cases have no known etiology \[[@B25]\]. Therefore, identification of the underlying genetic variants that predispose to FGR could have significant medical significance if it allows us to identify early in gestation those pregnancies that are at increased risk of growth retardation. A logical place to begin such a search is among those genes that are known to be major regulators of fetal growth and to exhibit significant differences in circulating protein concentrations between normal and FGR pregnancies, such as the members of the growth hormone-prolactin and insulin-IGF families of hormones, receptors, and binding proteins. Here, we use a stringently selected set of subjects to report suggestive association of SNPs in GH1 with fetal growth restriction. Adjusted for gestational age, the C alleles at sites -1 and +3 of the GH1 gene appear to be associated with reduction in birth weight. The marginal significance of these results may be the result of several factors. First, we examined 125 total subjects, roughly one-third of them FGR, and this number may give inadequate statistical power to identify weak genetic effects. Second, the C allele is low frequency, providing a small number of heterozygotes for the allele and no homozygotes. It is worth noting that among normal birth weight subjects we observed a very similar frequency for the C allele as Horan et al. (2003) did among British army recruits (0.4% vs. 0.3%) but that among the low birth weight subjects the frequency of the C allele is substantially higher (6%). Comparing all the other allele frequencies (Table [2](#T2){ref-type="table"}) between the two studies of Caucasian populations, no other allele shows such a large magnitude of difference, although the proportional difference in frequency of C at -1 and +3 may be somewhat distorted by sampling error at low allele frequencies. Third, FGR undoubtedly has a heterogeneous genetic etiology, and it may be unlikely to find any genetic variant that accounts for more than a small proportion of cases. If the C allele at -1 and +3 exhibits true association with FGR, it unfortunately may be very difficult to determine if the effect is due to one or both sites because they are in complete linkage disequilibrium. Nevertheless, the presence of both alleles at the start of GH1 transcription provides both variants with a biologically plausible possibility to affect the level of transcription of GH1. Future work should be devoted to examining the effect of these alleles on transcription levels both alone and in combination. Site -1 is somewhat unique among single nucleotide polymorphisms in that three alternate alleles exist at that site (the major allele A and the minor alleles C and T). There are two possible explanations for this observation. First, site -1 could be a hotspot for nucleotide mutation, with transversions from A to C/T occurring often. Second, GH1 may be the recipient of gene conversion events from more than one paralog within the GH locus. Among the four other genes of the GH locus, all three alleles observed at site -1 occur (Table [2](#T2){ref-type="table"}). Given the predominant effect that gene conversion \[[@B16],[@B18]\] appears to have on the patterns of nucleotide polymorphism in the GH1 gene, the latter explanation may be more plausible. It must be pointed out that the common wisdom is that GH1 plays no role in regulating fetal growth because the GH receptor is expressed fairly late in gestation and because anencephalic infants or those born without a pituitary achieve nearly normal length \[[@B26]\]. Importantly, we restricted our study to full term deliveries. It is possible that late in gestation, when fetal GH1 is expressed and GHR receptors are present in a wide variety of fetal tissues, pituitary growth hormone begins to have a growth stimulatory role sufficient to account for the 90--150 g difference in birth weight between genotypes for the A/C polymorphism at -1 and +3 that we observed. Conclusions =========== In a stringently selected set of subjects, C alleles at sites -1 and +3 relative to the start of transcription of GH1 have a higher frequency (0.4% vs. 6%) in fetal growth restricted newborns. These two alleles are in complete linkage disequilibrium and their presence is associated with a reduction in birth weight of 152 g in term, normal birth weight subjects and 97 g in term, low birth weight (\<2,500 g) subjects. In combination with environmental, behavioural and other genetic factors, these alleles may contribute to fetal growth restriction. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= RMA conceived the project and directed its design and execution. CC and RV performed the molecular genetic work and participated in preliminary analyses. TKB performed the subject selection. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2393/5/2/prepub> Acknowledgements ================ This work was supported by funding from the Children\'s Foundation Research Center of Memphis at Le Bonheur Children\'s Medical Center, the Center of Genomics and Bioinformatics at the University of Tennessee Health Science Center (both to RMA), and a University of Massachusetts-Baystate Medical Center Collaborative Biomedical Research Grant (to RMA and TKB). We thank Drs. Julia Krushkal and David Hosmer for analytical advice and Jeannette Peeples and Eric Gelke for their technical work.	PubMed Central	2024-06-05T03:55:52.754614	2005-2-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548522/", "journal": "BMC Pregnancy Childbirth. 2005 Feb 3; 5:2", "authors": [ { "first": "Ronald M", "last": "Adkins" }, { "first": "Caroline", "last": "Campese" }, { "first": "Rehana", "last": "Vaidya" }, { "first": "Theonia K", "last": "Boyd" } ] }
PMC548523	Background ========== The last decade has seen increasing interest in the health and well-being of the workforce. This has been driven partly by the increasing burden of direct healthcare costs, but also from a recognition that the economy within the developed world has appreciably changed\[[@B1],[@B2]\]. The relative contribution of industry, compared with the service sector, to gross domestic product (GDP) has steadily declined since 1980. Industry now represents approximately 32% of GDP and services 66%\[[@B3]\]. With the shifting structure of the economy have come new challenges to occupational health physicians and human resource managers alike. A predominantly service-based economy has fewer tangible assets than its industrialised counterparts and the wealth that is generated is almost completely reliant upon the less tangible \"human capital\" of employees. It has therefore become an imperative to ensure that this human factor is optimised in order to meet business demands, especially during times of slow economic growth. In parallel with this greater business emphasis on the human factor has come a greater awareness of \"post-industrialisation\" health issues. These include stress and sleep dysfunction and conditions such as obesity and musculoskeletal pain that have arisen due to greater national wealth and an increasingly sedentary lifestyle \[[@B4]-[@B6]\]. The evidence for the impact of many lifestyle factors upon long-term health is overwhelming. Smoking, excess alcohol intake, poor nutritional status, a sedentary lifestyle and psychological distress have all been associated with numerous diseases \[[@B7]-[@B10]\]. Indeed it has been estimated that about a quarter of all healthcare costs can be attributed to conditions directly resulting from easily modifiable lifestyle factors\[[@B11]\]. As well as the long-term consequences of lifestyle on the genesis of disease, there is increasing evidence of the short-term effects such factors have upon individual performance and productivity. Smoking, high body mass index (BMI) and psychological distress have all been shown to have a major impact upon employee productivity at work \[[@B12]-[@B14]\]. Additionally, it has been shown that those individuals who are physically active in their leisure time are less likely to have short-term illness-related absence or experience musculoskeletal disorders \[[@B15]-[@B17]\]. With these issues gaining greater ascendancy in the corporate world, we saw a need for a short, easy to administer questionnaire that could capture this business critical health status information. By conducting a confidential survey of all employees, aggregated data can be used to provide a first step by which organisations can target and monitor appropriate population-based health interventions within their workforce. A key issue in conducting such surveys is maintaining individual privacy and ensuring confidentiality of information. The majority of US organisations who already conduct annual health surveys of their employee populations do so either via their occupational health departments or external third parties. Although there are a number of general and specific health risk appraisal measures available for corporate use, they are either not well validated, suffer from being too long and cumbersome to administer, or cost an appreciable amount to use. In the case of health related quality of life measures, such as the SF-36, or specific stress indicators such as the general health questionnaire (GHQ), the data that is generated is not specific enough to direct health and well-being interventions within the corporate setting. We report on the development and validation of the health and well-being (HWB) assessment, a free to use twenty item questionnaire. We also describe its use in assessing the impact employee health has upon productivity and performance. Methods ======= Questionnaire development ------------------------- Our principal aim was to develop a questionnaire that focused upon business pertinent health and well-being issues. A secondary aim was that it should be quick and easy to administer with the amalgamated results serving as a baseline from which employers can start to implement appropriate health promotion interventions within their employee populations. We initially surveyed a sample of twelve business managers and executives to ascertain the key issues currently facing their organisations. Interviewees came from four different business sectors, namely (i) Technology (ii) Engineering (iii) Banking and Insurance and (iv) Public sector / Health. Interviews lasted no more than 30 minutes and were semi-structured, asking each interviewee to describe the key issues they faced in their day-to-day operations. We then searched the general and scientific literature for evidence of the effect health parameters have upon the issues identified. The key business issues facing our sample of corporate leaders could be categorised into four separate areas. Table [1](#T1){ref-type="table"} shows these four key areas and summarises how health and well-being can directly impact upon them. Searches of Medline, Embase and PsycINFO were made from 1990 onwards using \"productivity\", \"customer satisfaction\", \"customer service\", \"absence\", \"absenteeism\", \"medical cost\" and \"business risk\" as key words or phrases. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Business Pertinent Health & Well-being Issues ::: ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Business Issue Increasing the productivity of the workforce Improving customer service and satisfaction Reducing the costs of ill-health Reducing potential future business risks and liabilities ---------------------------------------------------------------------- ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------- Modifying effect of employee health and well-being on business issue Many medical conditions and risks (e.g. diabetes, cardiovascular disease, migraine, pain, respiratory disease, high BMI, smoking, excess alcohol consumption) have a direct impact upon the day-to-day productivity of the workforce \[12,14,19,22,33\].\ Physical and mental health are component factors in developing employee commitment, job satisfaction and a \"climate for service\" within an organisation. Via these areas the health and well-being of employees is likely to be an indirect contributor to customer service and satisfaction \[37-40\].\ High risk health status (e.g. poorly controlled medical conditions, sub-optimal nutritional status, lack of physical activity, high levels of psychological distress) are associated with greater medical care expenditure and higher levels of absence \[13,28,41-44\].\ Improving physical fitness within the workforce can reduce voluntary staff turnover \[49\].\ Sleep disturbance and disruption has a significant impact upon an individual\'s performance during the working day \[34\]\ Employee attitude and job satisfaction directly affect sales increases and customer satisfaction. \[37\]. Musculoskeletal issues are the commonest cause of long-term sickness absence in manual workers. \[45\].\ Union backed employee stress-related liability claims have risen four-fold since 1999, posing a significant risk to the business \[50\].\ Psychological distress / stress can have a profound impact upon worker productivity and performance \[21,35,36\]. Corporate health and well-being programmes have been shown to produce a return on investment by decreasing medical care costs, worker compensation costs and absence \[30,31,46-48\]. Early retirement due to illness is placing a significant burden upon pension plans.\ Musculoskeletal and psychological issues are the two most frequent health related reasons for early retirement \[51\]. Domains within HWB assessment that help quantify issue Medical Health\ Overall HWB Score Symptoms of Stress\ Medical Health Nutritional Balance Physical Activity Symptoms of Stress Pain\ Physical Activity Symptoms of Stress\ Pain\ Job Satisfaction Overall HWB Score Pain Body Mass Index\ Smoking Status\ Alcohol Consumption\ Sleep Status\ Symptoms of Stress ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Following interviews with executives and managers the key issues for businesses could generally be classified in one of four main areas; (i) increasing the productivity of the workforce, (ii) improving customer satisfaction, (iii) reducing the costs associated with employee ill-health and (iv) reducing potential future business risks and liabilities. For all four we found evidence for a modifying effect of health and well-being. The table shows the four identified business areas, the impact employee health and well-being has upon these areas and the domains included within the HWB that assess these areas. ::: Initial questionnaire development involved formulating items that corresponded with the health and well-being areas that impact upon the four key business issues. An initial set of 40 questions was tested and discussed in one to one interviews and focus group discussions. Thirty-five employees and managers, from companies in the four industry sectors described previously, participated in these sessions. Question changes and selection were an iterative process, with the final questionnaire formulated after three rounds of small group testing and one round of initial data collection from 100 volunteers. This background research and subsequent refinement led us to construct a 20-item questionnaire covering ten areas of health and well-being (see [additional file 1](#S1){ref-type="supplementary-material"}: Appendix), which we termed sub-indices. The ten areas were: • Medical health status • The presence of pain • Habitual levels of physical activity • Nutritional balance • Sleep status • Symptoms of stress • Job satisfaction • Smoking status • Alcohol consumption • Body mass index We used a combination of 5-point Likert scales and structured multi-choice questions. Six of the ten areas were assessed by single item \"global\" questions, including a modification of the non-exercise estimation of VO~2~max question developed by Jackson and Ross\[[@B18]\]. Body mass index was scored according to desired ranges for the general population, as recommended by the World Health Organisation and the Department of Health. The remaining three areas (nutritional balance, sleep status and symptoms of stress) were assessed by multiple items. The number of possible responses to each of the sleep questions were reduced from an initial five responses to four, as the additional response was not found to be helpful as a discriminator. The checklist for the medical health question was developed according to current best available evidence for medical conditions impacting upon key business issues\[[@B12],[@B19]-[@B23]\]. A single, non-scoring question on self perception of effectiveness at work was also included, not to replicate existing more detailed productivity measures, but to act as a global screening question to examine the relationship between health and work effectiveness in population analysis. The answer to each question was scored on a scale from zero to one-hundred. This was used as the relevant HWB sub-index for single item variables. The question scores for multi-item variables were averaged to give a zero to one-hundred sub-index score (see [additional file 1](#S1){ref-type="supplementary-material"}: Appendix for full scoring algorithm). The overall HWB score was computed by summing and then averaging all ten sub-index scores, giving equal weight to each of the ten areas. Subjects -------- Three thousand full time employees of three UK-based organisations (one insurance company, one telecommunications company and one consumer goods manufacturer) were invited to complete the questionnaire via the internet. All data transmission utilised 128-bit encryption and all data storage was fully compliant with the UK Data Protection Act (1998). All participants were required to electronically sign an agreement for their anonymised data to be used in amalgamated format for purposes of research. A draw with a prize of a weekend break was offered as an incentive to participate for each company group. Thirty employees re-took the questionnaire four weeks after the initial completion date in order to provide test re-test data. As well as completing the newly developed questionnaire, participants were also asked to concurrently complete the Short Form 36 (SF-36) and part B of the World Health Organisation\'s Health and Work Performance (WHO-HPQ) questionnaire in order to assess criterion validity\[[@B24],[@B25]\]. The SF-36 was chosen as it is a \"gold standard\" health-related quality of life measure and because there is some overlap with the HWB assessment in the constructs it assesses. There are a number of well validated productivity measures available for use in the workplace, however the WHO-HPQ was chosen as it is a general productivity measure applicable to both those who have a diagnosed disease and those that do not \[[@B26]\]. Others have shown a clear relationship between health risk and productivity, it was therefore important for the validation of our questionnaire that this was replicated\[[@B12]\]. For each participant in the study details on age, gender, sickness absence in the preceding three months, company position, marital status and weekly working hours were also collected. Data analysis ------------- All data analysis was carried out using Statistica, a statistical software package distributed by Statsoft Inc. (Tulsa, USA. <http://www.statsoft.com>) Results ======= Of the 3000 employees invited to participate in the study, 2224 completed the questionnaires (74% response rate). Online completion ensured that there were no missing data points in completed questionnaires. The mean age was 38.1 years (standard deviation 10.7). Fifty-nine per cent of respondents were female (see table [2](#T2){ref-type="table"}). Age and gender of respondents accurately reflected the demographics of the three company populations as a whole. The average completion time for the HWB assessment was eight minutes. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Participant characteristics ::: Gender Male: 41% ------------------------- ---------------------- ----- Average Age (years) 38.1 (SD: 10.7) Marital Status Single: 34% Married: 59% Separated / Widowed: 7% Weekly Working Hours \<40: 47% 40 -- \<50: 41% 50 -- \<60: 9% 60+: 3% Annual Gross Income (£) \< 10,000: 13% 10,000--19,999: 27% 20,000--29,999: 30% 30,000--49,999: 21% 50,000+: 9% Company Position Junior: 49% Middle: 40% Senior: 11% ::: Questionnaire validation ------------------------ Principal components factor analysis of the three multi-item variables showed that for each the number of factors extracted was 1. Inter-item correlation, as assessed by the Cronbach α value, for each of these three scales was good (see table [3](#T3){ref-type="table"}). General linear model analysis indicated that of age, gender, sickness absence, company position, marital status and weekly working hours the only variables that remained a significant predictor of HWB score were sickness absence and age (p \< 0.0001 for both). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### α values for the multi-item variables of the HWB ::: Scale Number of items Cronbach α --------------------- --------------------- ---------------- Symptoms of stress 6 0.83 Sleep status 3 0.70 Nutritional balance 3 0.73 ::: Comparison with SF-36 scores ---------------------------- Significant correlations were seen between the SF-36 scales that assessed similar areas of health as the HWB sub-indices, namely, bodily pain vs. presence of pain (r = 0.79), mental health vs. symptoms of stress (r = 0.70) and mental component summary measure (MCS) vs. stress (r = 0.71). Additionally, there was a clear association between the overall HWB score and the General Health and Vitality scores of the SF-36 (r = 0.59 and 0.49 respectively). All SF-36 multi-item scales were significantly correlated with the overall HWB score (p ≤ 0.01). WHO-HPQ data ------------ The 2224 individuals who completed the HWB assessment and the SF-36 also completed part B of the WHO-HPQ. The output from the WHO-HPQ is a calculated productivity decrement for each respondent, i.e. the proportion of the week that the individual is not working optimally, either because they are absent or because they are not working effectively (so called \"presenteeism\")\[[@B24]\]. Mean productivity decrement for the population was 26.4% of weekly working time (SD 20.9), median 20% (25^th^percentile was 10% and 75^th^percentile was 33.5%) A negative correlation between the HWB score and calculated productivity decrement was observed (r = -0.4, p \< 0.0001), i.e. better health status, as measured by the HWB assessment, was associated with less weekly productivity decrement. General linear model analysis indicated that age and the overall HWB score were the only two variables that remained as significant predictors of weekly productivity decrement (p \< 0.0001 for both). The 75^th^percentile figure of 33.5% productivity decrement per week was taken as the cut-off for achieving the productivity standard within the current population. Similarly, the lower quartile HWB score of 52.1 was used as the cut-off to define poor health. 2 × 2 table analysis using these cut-offs demonstrates an odds ratio of 3.62 (95% confidence limits, 2.93 to 4.47) for making the productivity standard if HWB score is above the lower quartile value, Chi squares 158.82 (Yates Corrected), p \< 0.0001. There was a significant correlation between the single question on effectiveness contained in the HWB assessment and the productivity decrement, as calculated by the WHO-HPQ, (r = -0.59, p \< 0.0001). Test re-test validity for the HWB assessment -------------------------------------------- Thirty individuals re-took the HWB assessment four weeks after their original completion date. During this time no information or intervention with regard to health and well-being was delivered to them. The correlation between HWB scores at both time points was excellent (r = 0.90), with no significant differences between mean scores or variance of the data sets. HWB scores across the population -------------------------------- Table [4](#T4){ref-type="table"} gives the means, medians, standard deviations and inter-quartile values for the HWB score and sub-indices. The distribution of the HWB score was normal, therefore parametric measures were used to analyse differences between independent groups (t-test). There were no significant differences between the HWB score of males and females or between those who typically worked more than 40 hours and those who did not. There was, however, a significant difference in HWB score between those in senior positions within the company and those within junior positions (mean HWB scores 62.9 and 60.7 respectively, p \< 0.001). Similarly, those who had less than three days sickness absence in the preceding three months had better HWB scores than those who had more sickness absence (means scores 64.0 and 55.2 respectively, p \< 0.0001). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Overall HWB score plus the ten component sub-index scores for the 2224 questionnaire respondents. ::: Mean score Median score Standard deviation 25^th^percentile 75^th^percentile ----------------------- ------------ -------------- -------------------- ------------------ ------------------ HWB score 61.4 62.1 13.7 52.1 71.0 Medical health 62.4 100 41.3 25.0 100 Pain 71.2 75.0 21.9 50.0 75.0 Physical activity 26.3 0 38.1 0 50.0 Nutrition 57.5 58.3 19.0 41.7 75.0 Sleep 62.3 66.7 23.8 50.0 83.3 Stress 55.7 58.3 18.2 41.7 70.8 Job satisfaction 59.0 75.0 30.2 50.0 75.0 Smoking status 77.5 100 41.8 100 100 Alcohol consumption 92.2 100 26.8 100 100 Body Mass Index score 49.7 25.0 42.2 25.0 100 ::: Discussion ========== The association between employee health status and costs incurred by employers is incontrovertible. Numerous studies have clearly shown how health risk factors directly impact upon medical care costs, short- and long-term absence and workers\' compensation\[[@B11],[@B27]-[@B29]\]. Additionally, more recent research is confirming what many of us \"intuitively\" knew; that the health and well-being of the workforce has a direct impact upon work performance\[[@B12]\]. Despite this growing body of evidence, many corporations have been slow to implement appropriate measures to assess, intervene and improve the health of their workforce. The reason for this inertia is unclear, especially as corporate health promotion and management programmes have repeatedly been shown to generate a return on investment (ROI) \[[@B30]-[@B32]\]. A possible explanation may be that whilst medical care costs are inexorably increasing, by focusing solely upon costs and cost savings we miss capturing corporate leaders\' imagination and vision. Combining the message of cost savings with productivity and performance enhancements may just strike the right balance. Measures such as the WHO-HPQ now allow us to objectively measure productivity and, as we have confirmed in this paper, health risk status is an integral component of this construct. As already mentioned, although well-established questionnaires have been extensively validated in many different populations the data that is generated is often of limited value in specifically directing health and well-being interventions. We have presented the first steps of the development and validation of a health risk appraisal measure that has been specifically designed for use in the corporate setting. As well as having good content, criterion and construct validity, the generated data can help health promotion specialists develop appropriate and targeted interventions for the respondent population. The questionnaire provides information on areas such as nutritional choices, levels of habitual physical activity, sleep difficulties and stress symptoms. Amalgamated answers can be used to ensure the correct and most appropriate health interventions are delivered to the population being assessed. In addition, the single question on work effectiveness can be used to confirm the link between the health of the population being studied and their performance, prior to more in-depth evaluations of productivity such as can be made with a specific productivity measure. One would naturally expect those individuals who have taken more time off due to illness to have worse health than those who have been absent for less time. We have demonstrated that sickness absence in the preceding three months is a significant predictor of HWB score and remains so when other variables are controlled for. This is an indication that the HWB assessment is indeed measuring the health and well-being issues that are critical to businesses as a whole. Further confirmation of the discriminant validity of the HWB assessment is needed, however a suggestion that it can detect real differences in health status between groups is also seen in the significantly better scores observed between those with more senior positions as compared with those in junior positions. This difference possibly reflects the better financial rewards, the better access to healthy alternatives and the superior levels of job control associated with more senior corporate positions. The fact that the HWB score and sub-indices were significantly correlated with the broadly similar SF-36 multi-tem scales is an indication that the majority of the constructs assessed by the SF-36 are at least partially reflected in the HWB. Productivity whilst at work can be influenced by a multitude of different factors, however as demonstrated by Burton and colleagues, health is a major contributor\[[@B12]\]. Our study has confirmed this clear relationship between level of health risk and productivity decrement, which remains significant even when other possible confounders are taken into account. Additionally, we have demonstrated that there is an odds ratio of 3.62 of making the productivity standard for those with good health as compared with those with poor health. This information can quite easily be used by corporations to model future productivity gains and to calculate a likely ROI for the institution of a health promotion programme. Although these initial results appear promising, data collection from a larger employee sample, from different sectors and incorporating a wider age range, is necessary in order to confirm that our observations still hold true. Normalising the scoring (as is often performed with SF-36 data) would also make interpretation easier and more user friendly. Additionally, longitudinal data on whether the HWB assessment can be used as a predictive tool for populations, and hence provide businesses with visibility on how their employee health status issues are likely to affect their bottom line, is the logical next step. This process is already underway in four multinational organisations with populations in both the USA and the UK and is being overseen by the Institute for Health and Productivity Management (IHPM). Conclusion ========== In summary we present a new health risk measure, the Health and Well-being Assessment (HWB), which has the following key features: \(i) has been specifically designed for the corporate environment addressing the health and well-being issues that affect key business drivers \(ii) is quick, easy and free to use \(iii) the generated data is useful for guiding future interventions By combining medical health issues with other more \"lifestyle\" and well-being focused areas within a short, easy to use questionnaire we believe that we have created a useful corporate tool. List of Abbreviations ===================== GHQ -- General Health Questionnaire HWB -- Health and well-being ROI -- Return on Investment SF-36 -- Short Form 36 questionnaire WHO-HPQ -- World Health Organisation Health and Work Productivity Questionnaire Competing Interests =================== PM has a part-time salaried role with health and well-being business consultants Vielife. No other financial competing interests No non-financial competing interests. Authors\' contributions ======================= PM performed all of the work that is contained within this paper. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Health & Well-being Questionnaire Questionnaire and scoring algorithms. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ The author would like to thank Dr John Gobble for advice during questionnaire development, Dr Denise Syndercombe-Court for statistical guidance and Ms Jessica Colling for reviewing the manuscript.	PubMed Central	2024-06-05T03:55:52.758572	2005-1-28	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548523/", "journal": "Environ Health. 2005 Jan 28; 4:1", "authors": [ { "first": "Peter R", "last": "Mills" } ] }
PMC548524	Background ========== Protease inhibitors (PIs) used as components of highly active antiretroviral therapy (HAART) have been well documented to reduce both the morbidity and mortality associated with HIV infection \[[@B1],[@B2]\]. However, many PI-based HAART regimens incur treatment-limiting side effects, interactions with concomitant medications, high daily pill burdens, and dietary and/or fluid requirements, making adherence to treatment a challenge \[[@B3]\]. Even more problematic from a long-term treatment perspective are the metabolic adverse effects, such as hyperlipidemia, insulin resistance, and lipodystrophy, which along with HIV infection itself, may constitute major risk factors for the development of coronary artery disease (CAD) \[[@B4]\]. The incidence of hyperlipidemia varies among PIs. The Swiss HIV Cohort Study showed that over 470 days, HIV-infected patients experienced a mean increase in total cholesterol of 77 mg/dL with ritonavir (n= 46), 46 mg/dL with nelfinavir (n= 21), 31 mg/dL with indinavir (n= 26), and 4 mg/dL with non-PI-containing antiretroviral regimens (n= 28) \[[@B5]\]. At the end of the study, total cholesterol exceeded 240 mg/dL in 44% of the ritonavir group, 35% of the indinavir group, 33% of the nelfinavir group, and 14% of the non-PI group. Other research has shown that some of the more recently developed PIs, including fosamprenavir and atazanavir, have a low likelihood of causing significant lipid elevation or adversely affecting the total cholesterol: high density lipoprotein (HDL) cholesterol ratio \[[@B6]-[@B8]\]. Although HIV infection itself is associated with above-normal triglyceride levels due to disease-related reduction in triglyceride clearance and increase in de-novo hepatic lipogenesis \[[@B9]\], some PIs exacerbate this lipid elevation by down-regulating low density lipoprotein (LDL)-receptor expression \[[@B10]\], interfering with the proteasome-mediated degradation of activated nuclear sterol regulatory element-binding protein-1 \[[@B11]\], and reducing triglyceride clearance further \[[@B12]\]. Because PIs have been available for less than a decade, it may be too early to confirm an association between PI usage and a more rapid onset of CAD. Epidemiological studies to date have reported varying findings regarding a PI-CAD link \[[@B13]-[@B15]\]. Until questions about such a possible link are resolved, it appears prudent to treat HIV-infected patients who have CAD risk factors with combinations of antiretroviral agents that produce maximal virologic suppression with the fewest lipid-elevating effects and metabolic adverse events. Unlike most PIs, the nucleoside reverse transcriptase inhibitor (NRTI) abacavir is administered as just one tablet twice daily with no requirements regarding extra hydration or dosing with or without meals \[[@B16]\]. Abacavir is unlikely to be involved in drug interactions because it does not affect CYP3A4 drug metabolism, its use does not affect lipids or metabolic parameters, and it does not cause lipodystrophy \[[@B16]\]. These features of abacavir have prompted its use in substituting for components of HAART regimens in order to simplify treatment and to obviate the risk of drug interactions, fat maldistribution, and metabolic complications \[[@B17]\]. HAART regimens containing abacavir have been shown in direct comparative trials in HIV-infected patients with a baseline viral load \<100,000 copies/mL to be as effective as nelfinavir-and indinavir-containing HAART in suppressing viral load and increasing CD4+ cell counts without inducing hyperlipidemia, insulin resistance, or central adiposity \[[@B18]-[@B21]\]. In patients with a baseline viral load ≥ 100,000 copies/mL, abacavir in combination with lamivudine and zidovudine has been shown to suppress viral load and increase CD4+ cell counts as well as nelfinavir-containing HAART \[[@B20]\], and was as effective as indinavir-containing HAART in one trial \[[@B18]\] but not in another \[[@B21]\]. In view of these findings, we conducted a 28-week, open-label pilot study (ESS40003) to compare the changes in PI-associated hyperlipidemia (fasting serum total cholesterol \>200 mg/dL) in treatment-experienced HIV-infected patients with HIV-1 RNA \<50 copies/mL who either substituted abacavir for the PI component in their regimen or continued the same PI-containing regimen. Methods ======= Patient selection ----------------- Male and non-pregnant, non-lactating female outpatients were eligible for study enrollment if they were at least 18 years of age; had HIV-1 infection (documented by HIV-1 antibody enzyme-linked immunosorbent assay and confirmed by Western blot test of HIV-1 antibody, or positive HIV-1 blood culture, positive HIV serum antigen, or plasma viremia); had fasting serum total cholesterol \>200 mg/dL; were stabilized on a well-tolerated PI-containing antiretroviral regimen for \>3 months (either 2 NRTIs + 1 PI, or 2 NRTIs + 2 PIs if one PI was ritonavir); were naïve to abacavir and all non-nucleoside reverse transcriptase inhibitors (NNRTIs); and if their two most recently reported consecutive plasma HIV-1 RNA values were \<400 copies/mL prior to screening and HIV-1 RNA was \<50 copies/mL at screening. CD4+ cell counts could be of any magnitude. Patients were also eligible if their HAART regimen had changed due to intolerance (one drug substitution, such as a PI for another PI and/or an NRTI for another NRTI) \>12 weeks prior to study start; or if their initial HAART regimen consisted of 2 NRTIs followed within 1 year by the addition of a PI. Patients were excluded from the study if they had genetically-related lipid disorders (familial lipoprotein lipase deficiency, apoprotein CII deficiency, type 3 hyperlipoproteinemia, hypercholesterolemia, hypertriglyceridemia, or combined hyperlipidemia); took a hypolipidemic or antidiabetic drug within 30 days of screening; had an AIDS-defining opportunistic infection or disease within 30 days of study entry; had a history of angina, anginal symptoms, and/or myocardial infarction; were substance abusers; or had a malabsorption syndrome that could interfere with absorption of the study medications. Patients provided written informed consent to participate in the study. Study design and treatment -------------------------- This Phase IV, parallel group, active control, randomized, open-label, multicenter trial was conducted between November 1999 and November 2001 at 44 outpatient sites in the United States. An Institutional Review Board approved the study protocol at each site. To determine study eligibility, study candidates underwent a medical history, physical examination, CDC classification, clinical chemistry, hematology, and β-human chorionic gonadotropin test (women of childbearing age only) at the screening visit within 14 days pre-study. Patients meeting entry criteria at screening were randomized to either continued therapy with their current PI-containing antiretroviral regimen or to the same regimen with abacavir (300 mg twice daily) substituted for the PI(s). Abacavir was supplied as 300-mg tablets of Ziagen^®^(GlaxoSmithKline, Research Triangle Park, North Carolina). In the abacavir-switch arm, patients received both abacavir and the hyperlipidemia-associated PI with their usual two-NRTI background combination for the first 4 weeks, after which the PI component of the regimen was discontinued. Co-administration of abacavir and the PI was done during the first 4 weeks to make sure that there was virologic coverage in case a patient developed a suspected abacavir-related hypersensitivity reaction, which would have necessitated stopping abacavir. Blood was sampled for lipid, viral load, and laboratory value measurements from fasted patients at baseline, and at weeks 4, 8, 12, 20, and 28. Patients who experienced HIV-1 RNA breakthrough (defined as HIV-1 RNA values between 50 and 1000 copies/mL by Roche Amplicor Ultrasensitive Assay) during the study period could receive intensification with efavirenz 600 mg once daily. Assessment of lipids -------------------- A complete fasting lipid panel was obtained from blood samples at baseline and at weeks 4, 8, 12, 20, and 28 to allow measurement of the primary study endpoint (change from baseline in fasting serum total cholesterol) and secondary study endpoints (change from baseline in LDL-cholesterol, very low density lipoprotein (VLDL)-cholesterol, high density lipoprotein (HDL)-cholesterol, and triglycerides). Apolipoproteins B and E, free fatty acids, and LDL subfractions were measured at baseline and week 28. Direct LDL-cholesterol was measured in all patients at baseline, week 4, and week 28, as well as any treatment visit at which time a fasting triglyceride \>400 mg/dL was observed. Fasting serum total cholesterol and triglyceride levels were measured enzymatically by using cholesterol/HP reagent (Roche Diagnostics, Indianapolis, Indiana) and triglyceride reagent (GPO-Trender) (Roche Diagnostics, Ibid). HDL-cholesterol was measured enzymatically in the supernatant formed following centrifugation of serum mixed with a polyanion dextran sulfate/divalent magnesium solution (Roche Diagnostics, Ibid). LDL- and VLDL-cholesterol were estimated by the Friedewald equation \[[@B22]\]. Direct LDL-cholesterol was measured by a nonionic detergent method using alpha-cyclodextrin/4-aminoantipyrin (Roche Diagnostics, Ibid.). Apolipoprotein B was measured using the apolipoprotein B antigen-antibody reaction method employing the Beckman IMMAGE Immunochemistry System (Beckman Instrument, Brea, California), and apolipoprotein E by a phosphate buffer/anti-human apolipoprotein E method employing a Wako Apolipoprotein Calibrator (WAKO Chemicals, Richmond, Virginia). LDL subfractions were measured by electrophoresis (Pacific Biometrics, Seattle, Washington) \[[@B23]\], and non-esterified free fatty acids were measured by the WAKO enzymatic colorimetic method (WAKO Chemicals, Ibid.). Four protocol-defined toxicity grades for hyperlipidemia were assigned during the study for serum cholesterol (Grade 1 for values \>1--1.3 times the upper limit of normal \[ULN\], Grade 2 for \>1.3--1.6 times the ULN, Grade 3 for \>1.6--2 times the ULN; and Grade 4 for \>2 times the ULN) and serum triglycerides (Grade 1 for ULN-399 mg/dL, Grade 2 for 400--750 mg/dL, Grade 3 for 751--1200 mg/dL, and Grade 4 for \>1200 mg/dL). Efficacy assessment ------------------- Changes in HIV-1 RNA levels and CD4+ cell counts were secondary endpoints in this study. Plasma HIV-1 RNA levels were measured in blood samples at screening and at all study visits using both the Roche AMPLICOR PCR Standard 1.0 assay (lower limit of quantitation \[LLOQ\] 400 copies/mL) and the Roche PCR assay Amplicor HIV-1 MONITOR UltraSensitive Version 1.0 (LLOQ 50 copies/mL) (both assays from Roche Diagnostics, Branchburg, New Jersey). Virologic failure was defined as HIV-1 RNA \>1000 copies/mL on two occasions at least 1 week apart. CD4+ cell counts were determined by flow cytometry at baseline, and at weeks 4, 12, 20, and 28. Safety assessment ----------------- Frequency and severity of all clinical and laboratory adverse experiences were assessed at each visit. A cardiovascular disease risk factor assessment was conducted at baseline only. Body mass index (BMI) and waist-to-hip ratio (WHR) were measured at baseline and at weeks 4, 8, 12, 20 and 28. Three types of waist measurements were conducted: mid, minimal, and umbilicus. Insulin resistance measures (C-peptide and fasting insulin to glucose ratio) were assessed at baseline and at weeks 4, 8, 12, 20, and 28. Leptin and lactate were measured at baseline and week 28. Diagnosis of possible abacavir-related hypersensitivity reaction was to be considered if the following multi-organ signs and symptoms appeared in a patient following initiation of abacavir: fever, rash, gastrointestinal symptoms (nausea, vomiting, diarrhea, or abdominal pain), lethargy, or malaise with or without concomitant respiratory symptoms (dyspnea, sore throat, cough), musculoskeletal symptoms (myalgia, myolysis, arthralgia), headache, paresthesia, and edema. No rechallenge of abacavir was permitted in patients developing this syndrome. The definition and usual clinical presentation of abacavir-related hypersensitivity has been defined previously \[[@B24],[@B25]\]. Adherence and health outcomes assessments ----------------------------------------- Adherence was assessed by the Patient Medication Adherence Questionnaire-7 (PMAQ-7) \[[@B26]\]. The PMAQ-7 is a self-reported measure of adherence that patients completed at baseline and at weeks 4, 8, 12, 20, and 28 (or upon permanent discontinuation due to virologic failure or toxicity). In the PMAQ-7, patients were asked to indicate the number of doses of each medication they took during each of the previous 7 days. An overall regimen adherence rate was calculated at each week as the proportion of doses actually taken relative to the number of doses prescribed summed across each medication within the regimen. The PMAQ-7 also measured barriers and motivators to adherence. Health-related quality of life (QOL) was evaluated using the Medical Outcomes Study 36-item Short Form Health Survey (SF-36) \[[@B27]\], which patients completed at baseline and at study weeks 12 and 28. Healthcare resource utilization (total health care visits, emergency room visits, intensive care hospitalizations \[nights\], general ward hospitalizations \[nights\], outpatient clinic visits, home care visits, and long-term care visits \[nights\]) was assessed at weeks 4, 8, 12, 20 and 28. Statistical analysis -------------------- A sample size of 80 patients per treatment arm was deemed necessary for 80% power to detect a difference of 20 mg/dL in fasting total cholesterol between groups using a two-group t-test with a two-sided significance level of 0.05, assuming a dropout rate of 20%. This sample size calculation is based on data from NZTA4002, in which the standard deviation of total cholesterol in the nelfinavir/zidovudine/lamivudine group at baseline was 39.5 mg/dL. The primary efficacy population was the intent-to-treat (ITT) population, which consisted of all patients who were randomized into the study. The safety population consisted of all randomized patients who received at least one dose of study drug. Analysis of covariance (ANCOVA) was used to compare the least squares means (LSM) of the two treatment groups. The LSM represented the mean value adjusted for the average value of the covariate from both treatment groups. LSMs of change from baseline in serum lipids (total, LDL, VLDL, and HDL-cholesterol, LDL subfractions, triglycerides, free fatty acids), apolipoprotein B, apolipoprotein E, leptin, C-peptide, glucose, insulin, and lactate were reported. The ANCOVA model included terms for treatment group, baseline value of the variable of interest, PI strata and gender. Two types of analyses were performed with HIV-1 RNA data: the ITT: observed analysis and the ITT missing equals failure analysis (ITT: M = F). In the ITT: observed analysis, only available assessments were used (no imputation for missing values), regardless of whether the patient was still receiving their original therapy. In the ITT: M = F analysis, all missing values were considered as failure. Proportions of patients with HIV-1 RNA \<400 copies/mL and \<50 copies/mL were compared between treatment groups with a 95% confidence interval (CI) on the difference between proportions. Differences in domain scores to the SF-36 and the PMAQ-7 were compared using the Wilcoxon rank sum test. Differences between treatment arms in the incidence of treatment-related adverse events by body system were evaluated by Fisher\'s Exact test. A Pvalue of 0.05 was considered statistically significant. Results ======= Patient characteristics and disposition --------------------------------------- Of 104 patients enrolled in the study, 52 were randomized to the abacavir-switch arm and 52 to the PI-continuation arm (Figure [1](#F1){ref-type="fig"}). Most of the patients were males (89%), and the median age was 42 years (Table [1](#T1){ref-type="table"}). The study population was ethnically diverse; approximately one-half were Caucasian, one-quarter African American, and one-quarter Hispanic. Mean CD4+ cell counts were 551 and 531 cells/mm^3^in the abacavir-switch and PI-continuation arms, respectively. About two-thirds of the patients were HIV Category A. The abacavir-switch and PI-continuation arms were well matched with respect to mean baseline HIV-1 RNA levels (1.727 vs 1.699 log~10~copies/mL, P= 0.062), total cholesterol (244 vs 241 mg/dL), LDL-cholesterol (149 vs 149 mg/dL), VLDL-cholesterol (68 vs 56 mg/dL), HDL-cholesterol (39 vs 42 mg/dL), triglycerides (340 vs 280 mg/dL), CAD risk factors (Table [1](#T1){ref-type="table"}), and specific PI used immediately pre-study (\>80% receiving either nelfinavir or indinavir). There were no significant differences between treatment arms with respect to baseline BMI, waist-to-hip ratios or insulin resistance. During treatment, the background NRTIs used in the abacavir-switch and PI-continuation arms were similar and included most commonly the lamivudine 150 mg/zidovudine 300 mg combination tablet (Combivir^®^\[GlaxoSmithKline, Ibid.\], 52% vs 47%), stavudine (36% vs 42%), lamivudine (32% vs 40%), and zidovudine (6% vs 2%). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Profile of patient enrollment and discontinuations through 28 weeks of treatment. ::: ![](1471-2334-5-2-1) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics and disposition of the study patients (intent-to-treat population) ::: Characteristic Abacavir-switch arm (N = 52) PI-continuation arm (N = 52) Total study population (N = 104) ------------------------------------------------------------ ------------------------------ ------------------------------ ---------------------------------- Age, y Median (range) 43 (23--64) 42 (25--62) 42 (23--64) Sex, No. (%) Male 46 (88) 47 (90) 93 (89) Female 6 (12) 5 (10) 11 (11) Race, No. (%) Caucasian 26 (50) 28 (54) 54 (52) African American 16 (31) 11 (21) 27 (26) Hispanic 10 (19) 13 (25) 23 (22) Mean HIV-1 RNA, log~10~copies/mL (SD) 1.73 (0.16) 1.69 (0.04) 1.71 (0.06) Mean CD4+ cell count, cells/mm^3^(SD) 551 (226) 531 (233) 541 (229) Mean weight, kg (SD) 79.3 (16.8) 80.4 (16.6) 79.8 (16.7) Mean BMI, kg/cm^2^(SD) 25.9 (4.5) 26.4 (4.9) 26.1 (4.7) Mean waist-to-hip ratio 0.94 (0.06) 0.93 (0.07) 0.94 (0.06) CDC Class, n (%) Category A 33 (63) 34 (65) 67 (64) Category B 10 (19) 11 (21) 21 (20) Category C 9 (17) 7 (13) 16 (15) Mean (SD) total cholesterol, mg/dL 244 (45) 241 (44) Mean (SD) LDL cholesterol, mg/dL 149 (34) 149 (30) Mean (SD) HDL cholesterol, mg/dL 39 (15) 42 (14) Mean (SD) triglycerides, mg/dL 340 (213) 280 (282) Coronary artery disease risk factors, n (%)\* 34 (65) 26 (50) 60 (58) Age 18 (35) 12 (23) 30 (29) Family history 4 (8) 4 (8) 8 (8) Cigarette smoking 19 (37) 13 (25) 32 (31) Hypertension 5 (10) 8 (15) 13 (13) Low HDL 10 (19) 4 (8) 14 (13) Diabetes mellitus 3 (6) 0 3 (3) Antiretroviral medications taken prior to screening, n (%) Any 28 (56) 20 (44) 48 (46) NRTIs Zidovudine 16 (32) 9 (20) 25 (24) Lamivudine 12 (24) 8 (18) 20 (19) Stavudine 5 (10) 2 (4) 7 (7) Didanosine 7 (14) 0 7 (7) Zalcitabine 3 (6) 0 3 (3) NNRTIs Efavirenz 1 (2) 0 PIs Indinavir 11 (22) 8 (18) 19 (18) Nelfinavir 4 (8) 2 (4) 6 (6) Ritonavir 2 (4) 1 (2) 3 (3) Saquinavir 2 (4) 0 2 (2) PI used at screening Nelfinavir 22 (42) 21 (40) 43 (41) Indinavir 21 (40) 22 (42) 43 (41) Saquinavir SGC 6 (12) 6 (12) 12 (12) Amprenavir 2 (4) 2 (4) 4 (4) Ritonavir 1 (2) 1 (2) 2 (2) Premature withdrawal from study, n (%) 14 (27) 11 (21) 25 (24) Adverse event 7 (13) 1 (2) 8 (8) Consent withdrawn 0 9 (17) 9 (9) Lost to follow-up 1 (2) 1 (2) 2 (2) Protocol violation 2 (4) 0 2 (2) Protocol-defined virologic failure 3 (6) 0 3 (3) Other 1 (2) 0 1 (1) \Cumulative coronary artery disease risk factors in the abacavir-switch arm: 0 factors = 18; 1 factor = 16; 2 factors = 12; 3 factors = 5; 4 factors = 1. Cumulative coronary artery risk factors in the PI-continuation arm: 0 factors = 26; 1 factor = 12; 2 factors = 13; and 3 factors = 1. Abbreviations:AIDS = acquired immune deficiency syndrome; ART = antiretroviral therapy; CDC = Centers for Disease Control; HDL = high density lipoprotein; HIV-1 = human immunodeficiency virus type 1; LDL = low density lipoprotein; LSM = least squares mean; SD = standard deviation; SGC = soft gel capsule. ::: A similar number of patients withdrew prematurely from the study for the reasons delineated in Table [1](#T1){ref-type="table"}. Comparatively more patients withdrew due to adverse events in the abacavir-switch arm (7/52 vs 1/52) and due to consent withdrawn in the PI-continuation arm (0 vs 9/52 \[all withdrawals occurred after the patients learned they were not receiving abacavir\]). The difference between the two treatment arms regarding number of premature withdrawals due to the sum of adverse events plus virologic failure was not statistically significant (P= 0.144). No patient required efavirenz intensification of their treatment regimen. Changes in lipids ----------------- In the ITT analysis, the abacavir-switch arm experienced greater reductions in total cholesterol (Figure [2](#F2){ref-type="fig"}) and LDL-cholesterol (Figure [3](#F3){ref-type="fig"}) than the PI-continuation arm over the entire study period, with differences between treatment arms being statistically significant from week 8 onward. At week 28, patients in the abacavir-switch arm had a significantly greater LSM decrease from baseline in total cholesterol (-42 vs -10 mg/dL; P\<0.001), LDL-cholesterol (-14 vs +5 mg/dL; P= 0.016), LDL direct-cholesterol (-15 vs +1 mg/dL; P= 0.028), VLDL-cholesterol (-27 vs -7 mg/dL; P= 0.019), triglycerides (-134 vs -36 mg/dL; P= 0.019), apolipoprotein B (-23 vs -11 mg/dL; P= 0.031), and apolipoprotein E (-2 vs -1 mg/dL; P= 0.021) versus the PI-continuation arm. Over the study period, no significant differences were observed between the abacavir-switch or PI-continuation arms with respect to LDL subfractions (particle size A and major class) or HDL-cholesterol (LSM change from baseline at week 28, +0.2 vs +1.3 mg/dL, P= 0.583; LSM at week 28, 40 vs 41 mg/dL). The abacavir-switch arm showed a trend for greater LSM reduction from baseline in free fatty acids (-0.3 vs -0.2 mEq/L; P= 0.052). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Least squares mean change from baseline in fasting serum total cholesterol. ::: ![](1471-2334-5-2-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Least squares mean change from baseline in fasting serum LDL-cholesterol. ::: ![](1471-2334-5-2-3) ::: The LSM value for total cholesterol was below the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) goal value (\<200 mg/dL) in the abacavir-switch arm (198 mg/dL); however, it remained above this value in the PI-continuation arm (230 mg/dL). The LSM LDL-cholesterol level at week 28 was 133 mg/dL in the abacavir-switch arm and 153 mg/dL in the PI-continuation arm (P= 0.016). A higher proportion of patients in the abacavir-switch arm had shifts to lower toxicity grades as compared to the PI-continuation arm, whereas a lower proportion in the abacavir-switch arm had shifts from baseline in cholesterol to more severe toxicity grades (Figure [4](#F4){ref-type="fig"}) ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Changes in cholesterol toxicity grade. ::: ![](1471-2334-5-2-4) ::: Changes in serum triglycerides in the two treatment groups paralleled the changes in total cholesterol. At week 28, the LSM reduction in triglycerides was significantly greater in the abacavir-switch arm than the PI-continuation arm (-134 vs -36 mg/dL, P= 0.019) (Figure [5](#F5){ref-type="fig"}). A higher proportion of patients in the abacavir-switch arm had decreases in the triglyceride toxicity grade as compared to the PI-continuation arm, whereas a lower proportion in the abacavir-switch arm had increases in triglyceride toxicity grade (Figure [6](#F6){ref-type="fig"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Least squares mean change from baseline in fasting serum triglycerides. ::: ![](1471-2334-5-2-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Changes in triglycerides toxicity grade. ::: ![](1471-2334-5-2-6) ::: Efficacy -------- In the ITT: M = F analysis, no differences were observed between the abacavir-switch and PI-continuation treatment arms at any time during the study regarding proportions of patients achieving undetectable HIV-1 RNA. At week 28, HIV-1 RNA was \<400 copies/mL in 69% \[36/52\] and 77% \[40/52\] of patients in the abacavir-switch and PI-continuation arms, respectively (P= 0.508; 95% CI \[-0.25, 0.09\]), and \<50 copies/mL in 62% \[32/52\] and 75% \[39/52\] of patients in these respective groups (P= 0.206; 95% CI \[-0.31, 0.04\]). The ITT: observed analysis showed that HIV-1 RNA remained \<400 copies/mL in ≥ 90% of patients over the entire study duration. At week 28, the proportion of patients with HIV-1 RNA \<400 copies/ml was 90% \[36/40\] and 100% \[40/40\] in the abacavir-switch and PI-continuation arms, respectively(P= 0.116; 95% CI \[-0.19, -0.11\]) (Table [2](#T2){ref-type="table"}). A significantly higher proportion of patients in the PI-continuation arm achieved HIV-1 RNA levels \<50 copies/ml at week 28 (98% \[39/40\] vs. 80% \[32/40\]; P = 0.029; 95% CI \[-0.31, -0.04\]). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Proportion of patients with HIV-1 RNA \<400 copies/mL and \<50 copies/mL ::: Week HIV-1 RNA \<400 copies/mL, n/N (%) HIV-1 RNA \<50 copies/mL, n/N (%) ---------- ------------------------------------ ------------- ----------------------------------- ------------- ------------- -- ------------ ------------ -- ------------ ------------ Baseline 52/52 (100) 52/52 (100) 52/52 (100) 52/52 (100) 42/52 (81) 47/52 (90) 42/52 (81) 47/52 (90) 4 49/49 (100) 44/45 (98) 49/52 (94) 44/52 (85) 48/49 (98) 43/45 (96) 48/52 (92) 43/52 (83) 8 39/42 (93) 42/42 (100) 39/52 (75) 42/52 (81) 38/42 (90) 40/42 (95) 38/52 (73) 40/52 (77) 12 38/41 (93) 40/40 (100) 38/52 (73) 40/52 (77) 38/41 (93) 39/40 (98) 38/52 (73) 39/52 (75) 20 38/41 (93) 40/40 (100) 38/52 (73) 40/52 (77) 32/41 (78) 38/40 (95) 32/52 (62) 38/52 (73) 28 36/40 (90) 40/40 (100) 36/52 (69) 40/52 (77) 32/40 (80) 39/40 (98) 32/52 (62) 39/52 (75) Abbreviations:ITT = intent to treat; M = F = missing equals failure analysis; PI = protease inhibitor. ::: No differences were observed regarding time to virologic breakthrough (P= 0.961). Three patients (6%) in the abacavir-switch arm were protocol-defined virologic failures. However, one of these patients remained on a 2-NRTI/abacavir regimen after withdrawing from the study and his viral load declined. The second of these patients was noncompliant and discontinued his antiretroviral treatment prior to the confirmation blood draw for virologic failure. The third virologic failure in the abacavir-switch arm had a history of prior dual nucleoside therapy for \>1 year. At baseline, the mean CD4+ cell counts were 551 and 531 cells/mm^3^in the abacavir-switch and PI-continuation arms, respectively. Changes in CD4+ counts over the study period were similar. At week 28, the LSM CD4+ cell counts were 514 and 526 cells/mm^3^, respectively, with the LSM difference from baseline being -13 and -1 cells/mm^3^, respectively (P= 0.597). Adherence and health outcomes assessments ----------------------------------------- PMAQ-7 results showed no apparent differences between the abacavir-switch and PI-continuation arms in overall adherence at week 28 (90% vs 94%) or in the Social Support, Adaptability, Knowledge and Attitudes, and Memory and Recall domains. However, the abacavir-switch arm had significantly higher scores in the Scheduling and Timing domain for all study visits (P≤ 0.027 at each week) and in the Physical Effects domain for all visits (P\< 0.05) except those at weeks 4 and 28. In answer to the PMAQ-7 questions \"it is easy for me to take my medicines at the time I am supposed to\" and \"my medicines are convenient to take\", more patients in the abacavir-switch arm answered \"definitely true\" at week 28 (51% vs 35% and 63% vs 29%, respectively). In the SF-36, the week 28 QOL scores did not differ between treatment arms for most domains, except Vitality (in favor of the PI-continuation arm; P= 0.042) and Health (in favor of the abacavir-switch arm; P= 0.013). There were no differences between treatment arms with respect to any health care utilization parameter. Safety ------ Substitution of PIs with abacavir in HAART regimens was generally well tolerated. No significant differences between treatment arms were observed for cardiovascular, endocrine/metabolic, hepatobiliary tract/pancreatic events, lower respiratory events, musculoskeletal, neurology, psychiatric, or skin adverse events, although more gastrointestinal (P= 0.002) and non-site specific adverse events (P= 0.003) were observed in the abacavir-switch arm. Treatment-related adverse events reported by \>5% of patients in the abacavir-switch arm included nausea (12/50 \[14%\]), fatigue (6/50 \[12%\]), diarrhea (4/50 \[8%\]), and depressive disorders (3/50 \[6%\]). Two patients (4%) in the abacavir-switch arm experienced possible abacavir-related hypersensitivity reactions. Adverse events leading to treatment withdrawal in 7 patients in the abacavir-switch arm included possible abacavir-related hypersensitivity reaction (2), mild nausea (2), mild shortness of breath/tachycardia (1), mild disorientation (1), and combination of mild diarrhea, facial edema/numbness and malaise (1). Hyperlipidemia led to treatment withdrawal in 1 patient in the PI-continuation arm. No differences between the two treatments were observed in body weight, waist-to-hip ratio, BMI, lactate, leptin, glucose, insulin, or C-peptide. Discussion ========== The results of this study indicate that in hyperlipidemic, virologically suppressed, immunocompetent antiretroviral-experienced patients (HIV-1 RNA \<50 copies/mL, CD4+ counts \>500 cells/mm^3^), substituting abacavir for hyperlipidemia-associated PIs in HAART regimens improves lipid profiles and maintains virologic suppression over a 28-week period. The lipid findings are consistent with those of other studies in which abacavir was substituted for PIs in HAART regimens \[[@B28]-[@B33]\]. However, unlike the other studies, ESS40003 evaluated a population consisting entirely of patients who were hyperlipidemic at baseline. Also, unlike two previous studies that reported lipid changes following abacavir substitution \[[@B29],[@B30]\], ESS40003 measured fastinglipids rather than lipids in a non-fasted state (which may confound results). Thus, true differences between abacavir and PI-containing regimens could be determined, and lipid criteria established by the NCEP ATP III could be applied \[[@B34]\]. In addition to monitoring changes in cholesterol and triglycerides, ESS40003 measured changes in other laboratory values known to contribute to atherogenesis (apolipoproteins B and E, LDL subfractions, and indicators of changes in lipid processing \[leptin and free fatty acids\]) to gain a better understanding of the extent of the improvement in lipid profile following the switch to abacavir. Substitution of hyperlipidemia-associated PIs with abacavir was expected to improve lipid profiles because results of direct comparisons of abacavir/NRTI regimens with PI/NRTI regimens (same NRTIs administered) in antiretroviral-naïve patients have shown no significant effects on lipids with abacavir/NRTI-containing regimens \[[@B18]-[@B20]\]. Cross-trial comparisons with other abacavir-switch studies regarding lipid changes are limited by differences between studies in prevalence and severity of hyperlipidemia prior to the switch, specific PIs administered, pre-existing CAD risk factors of the particular patients enrolled, and stipulated dietary and exercise restrictions. Nevertheless, some generalizations can be made between the lipid changes noted in ESS40003 and those reported in other abacavir-switch studies \[[@B28]-[@B33]\]. First, as in these other studies, decreases in total cholesterol, LDL-cholesterol, and triglycerides occurred rapidly following the switch to abacavir, with statistically significant differences noted between treatment arms within 4 weeks. Second, as would be expected, the magnitude of the reduction in total- and LDL-cholesterol and triglycerides at week 28 in our study was markedly greater than that reported in studies that included normolipidemic as well as hyperlipidemic patients. Third, as in the other studies, switching from a PI to abacavir had no significant effect on HDL-cholesterol. The small reduction in lipids observed in the PI-continuation arm may have occurred because patients were aware that their lipids were being monitored, and therefore may have exerted more self-control than usual regarding their dietary fat intake and frequency of smoking, or may have exercised more (not monitored in this study). A similar phenomenon was observed over 48 weeks in another PI-to-abacavir switch study, CNA30017 \[[@B29]\]. As no change in body weight, BMI, or waist-to-hip ratio was observed in our study in either group, anthropomorphic parameters were unlikely to have accounted for the decreases in lipids observed during this study. The 28-week duration of this study may have been too short to see significant changes in BMI or waist-to-hip ratios. However, in the Swiss HIV Cohort Study, substitution of abacavir for a PI was not associated with a change in waist-to-hip ratios even after 48 weeks post-switch \[[@B30]\]. Maintenance of virologic suppression and increases in CD4+ cell count over the 24-week period following the switch to abacavir were expected in view of the lack of change in these surrogate markers previously observed in abacavir-switch studies conducted over 48 weeks to 1 year \[[@B27],[@B29]\]. The abacavir-containing regimen was generally well-tolerated, and the type and incidence of adverse events (notably fatigue and nausea) and rare occurrence of suspected abacavir-related hypersensitivity reactions were consistent with what has been reported in other clinical trials \[[@B16]\]. New adverse events in the PI-continuation arm were not expected as patients had been stabilized on this treatment for \>3 months. This fact may have accounted for the comparatively lower incidence of GI adverse events in the PI-continuation arm than in the abacavir-switch arm, a finding that has not been observed in direct comparisons of abacavir with PIs (indinavir or nelfinavir) administered with the same background antiretroviral drugs \[[@B18],[@B19],[@B21]\]. Results of the PMAQ-7 indicated significantly better Scheduling and Timing scores in the abacavir-switch arm. Significant improvement in this PMAQ-7 dimension was similarly reported at 24 weeks in the PI-to-abacavir switch study, COL30305 \[[@B33]\]. Improved scores in this dimension may have been related to the simpler, twice-daily dosing of abacavir compared to the patients\' previous PI-containing regimens. The proportion of patients reporting 100% adherence previously was shown to be higher at 24 weeks in one PI-to-abacavir switch study (COL30305; 92% vs 68%) \[[@B33]\] and at 48 weeks in another (CNA30017; 91% vs 76%) \[[@B29]\]. In these trials, better adherence with the abacavir-containing regimen was believed to be due at least in part to the relatively lower pill count and absence of special dosing requirements incurred by abacavir-containing HAART. The absence of a significant difference in overall QOL between the abacavir-switch and PI-continuation arms was not unexpected in view of the same finding being demonstrated on the SF-36 at 24 weeks in COL30305 \[[@B33]\]. Switching to abacavir is just one of several switch strategies that have been investigated to date in an attempt to remedy hyperlipidemia in patients receiving PI-containing HAART. It is acknowledged that the ideal candidate for this switch strategy is a patient started on triple therapy, where pre-existing abacavir resistance is unlikely \[[@B30],[@B32]\]. Another switch strategy-replacement of hyperlipidemia-associated PIs with PIs that have a low likelihood of causing significant lipid elevation or adversely affecting the total cholesterol:HDL cholesterol ratio (fosamprenavir or atazanavir)-would be expected to improve lipid profiles in HIV-infected patients \[[@B6]-[@B8],[@B35]\]. Attempts to reduce lipids by switching from a hyperlipidemia-associated PI to NNRTIs have also been investigated \[[@B32],[@B36]-[@B43]\]. More favorable lipid effects appear to occur when a switch is made to nevirapine than to efavirenz \[[@B42],[@B43]\]. This study had several limitations. As patients in this study had mean CD4+ cell counts \>500 cells/mm^3^, they were highly immunocompetent and may not be representative of typical HIV-infected patients presenting to their physician with PI-associated hyperlipidemia. The study did not evaluate the influence of NRTI background drugs on lipid changes because the patients remained on the same baseline NRTIs. This could have affected the results because stavudine is known to elevate cholesterol and triglyceride levels \[[@B44]\], whereas zidovudine and lamivudine do not. Most studies have found that switching therapy tends to be optimally effective in patients whose viral load is fully suppressed for at least 6 months rather than the 3 months in our study \[[@B45]\]. This shorter pre-study time of viral suppression could have biased the virologic results against abacavir, as could allowing prior suboptimal nucleoside therapy. As we did not have information about prior NRTI combinations that many patients received pre-study, we could not assess whether these earlier NRTI combination regimens had been inadequate. Lipid data for study participants prior to their receipt of PI-containing regimens were not available to the investigators; thus, whether PIs were the cause of the patients\' hyperlipidemia could not be verified. Dietary intake and physical activity assessments were not performed in this study to evaluate whether either differed between the treatment arms. Further valuable clinical information could have been acquired from this study had switch agents in addition to abacavir been used as comparators. In the only study of this type that has been conducted to date -- the Nevirapine-Efavirenz-Abacavir (NEFA) Study -- a significantly lower proportion of stabilized HAART recipients (HIV-1 RNA \<50 copies/mL for = 6 months) switching from a PI to abacavir developed fasting plasma triglycerides \>400 mg/dL and plasma cholesterol \>240 mg/dL at 12 months compared to treatment groups in which patients switched from a PI to efavirenz or nevirapine \[[@B32]\]. Kaplan-Meier estimates of the likelihood of reaching the primary treatment end point (increase in HIV-1 RNA levels to ≥ 200 copies/mL, progression to AIDS, or death) in NEFA showed no significant differences between treatments. Median CD4+ cell counts increased above baseline similarly in all three treatment arms (by 39--50 cells/mm^3^at 12 months). However, the abacavir-switch arm had a significantly lower incidence of adverse events than the efavirenz-switch and nevirapine-switch arms (41% vs 57% and 54%, P= 0.03), a lower incidence of neuropsychiatric adverse events than the efavirenz group (9% vs 31%; Grade 3 or 4: 0.7% vs 14%), and significantly fewer cases of discontinuation of study drug due to adverse events (6% vs 17% and 17%, P= 0.01). Overall, NEFA, like our study, indicated that abacavir was an appropriate drug to substitute for PIs in HAART regimens as long as patients were virologically suppressed pre-switch and minimally antiretroviral-experienced. Our study differed from NEFA in that all*participants in ESS40003 had PI-associated hyperlipidemia at baseline (NEFA included only 7--13% with triglycerides \>400 mg/dL and 21--25% with total cholesterol \>240 mg/dL) and because our study used a less stringent definition of virologic failure (HIV-1 RNA \>1000 copies/mL on two occasions at least 1 week apart vs. HIV-1 RNA ≥ 200 copies/mL at ≥ 16 weeks, with subsequent confirmation \[NEFA\]). Conclusions =========== In conclusion, in antiretroviral-experienced patients with HIV-1 RNA \<50 copies/mL and CD4+ counts \>500 cells/mm^3^, substituting abacavir for hyperlipidemia-associated PIs in HAART regimens improves lipid profiles and maintains virologic suppression over a 28-week period, and it simplifies treatment. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= JEH, PHK and VCW conceived the study design, PHK, MGS, EDJ, AR, JFO, VCW, JWS reviewed and approved study design, VCW provided the statistical methods for the study and performed the statistical analysis of the results, EH, VCW, JHW, JWS wrote, reviewed and edited the protocol, GEP drafted manuscript and evaluated lipid data previously published in antiretroviral studies described in Background and Discussion, JWF, JEH, PHK, MGS, EDJ, AR, JFO, JEH, JWF, JHW, JWS, ADS-C, VCW reviewed and edited the manuscript, ADS-C, JWF, PHK, MGS, EDJ, AR, JFO enrolled study subjects, ADS-C, JWF, JEH, JHW monitored the study, ADS-C, JWF, JEH, JWS, VCW evaluated the clinical data from the study, ADS-C set up the study at study sites and JEH contributed to secure funding. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2334/5/2/prepub> Acknowledgements ================ The authors wish to thank the study participants, the clinical investigators and their respective staff members, and the GlaxoSmithKline ESS40003 study team: Heath Hair, Ilisse Minto, J. Phillips, Janice Myers, and Teresa Kauf. The results of this study were presented in part in Poster WePeC6267 at the XIV^th^International AIDS Conference, Barcelona, Spain, July 7--12, 2002.	PubMed Central	2024-06-05T03:55:52.762167	2005-1-12	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548524/", "journal": "BMC Infect Dis. 2005 Jan 12; 5:2", "authors": [ { "first": "Philip H", "last": "Keiser" }, { "first": "Michael G", "last": "Sension" }, { "first": "Edwin", "last": "DeJesus" }, { "first": "Allan", "last": "Rodriguez" }, { "first": "Jeffrey F", "last": "Olliffe" }, { "first": "Vanessa C", "last": "Williams" }, { "first": "John H", "last": "Wakeford" }, { "first": "Jerry W", "last": "Snidow" }, { "first": "Anne D", "last": "Shachoy-Clark" }, { "first": "Julie W", "last": "Fleming" }, { "first": "Gary E", "last": "Pakes" }, { "first": "Jaime E", "last": "Hernandez" } ] }
PMC548665	Background ========== Malaria remains an important health problem in sub-Saharan Africa and in some parts of Asia and South America. Resistance to therapeutic drugs and to insecticides, as well as social and environmental changes, are important factors in this situation. Increasing importance is currently given to antibodies in protection against human malaria, especially directed at erythrocytic stages of Plasmodium falciparum\[[@B1]\]. However, this protection is relatively unstable, and the precise role of specificities remains unclear regarding the antigenic variability of parasite proteins. Although parasite-specific antibodies clearly contribute to protection, it is not evident to which extent protective antibodies in general originate from specific immune responses to parasite antigens. They may also include components of innate immunity and be in part derived from natural antibody repertoires of the host. The remarkable protective properties of natural antibodies have previously been demonstrated in viral and bacterial \[[@B2]-[@B4]\] as well as in Leishmania\[[@B5]\] infection, and it appears conceivable that analogous properties exist toward Plasmodiumparasites, either naturally or selected during the long co-evolution of the human species with these parasites. An example may be autoantibodies to \'band 3\', a host-encoded target on cell membranes, involved in protection against malaria \[[@B6],[@B7]\] as well as physiologic cellular life span regulation \[[@B8]\]. Natural antibodies, immunoglobulins circulating in the absence of particular immunogenic stimuli, emerge from continuous autonomous activity of the immune system which appears largely independent of any external priming, as demonstrated e. g. in germ-free and antigen-free mice \[[@B9],[@B10]\]. They are often multireactive, and a large proportion interacts with endogenous targets and may play roles in internal homeostasis \[[@B11]\]. Reactivity patterns of IgM and IgG natural antibodies to autologous tissue proteins appear established early in life and remain remarkably stable throughout healthy living \[[@B12]\], but are capable of characteristic changes in autoimmune \[[@B13],[@B14]\] and other \[[@B15],[@B16]\] human diseases. Thus, such patterns are likely to reflect stabilized states of physiologic activation, shaped by polymorphic genes relevant for the immune system \[[@B17]\] which can be selected in evolution. The aim of this study was to investigate whether likely natural antibody reactivity patterns measured toward targets not related to parasites, but rather derived from autologous tissue or intestinal flora, could differentiate between asymptomatic and symptomatic forms of malarial infection. Asymptomatic infection is frequent in the Brazilian Amazon \[[@B18]\], even without very long exposure times, although malaria is only hypo- to mesoendemic and transmission is unstable with seasonal fluctuations \[[@B19]\]. Nevertheless, the situation appears analogous to hyperendemic regions, where parasite loads are known to gradually diminish with exposure time, resulting in a state of premunition, in which the still chronically infected subjects nevertheless remain asymptomatic for long periods \[[@B20],[@B21]\]. Asymptomatic infection can also be maintained after clinical cure by antimalarial drugs \[[@B22]\], showing that protection from disease can be very distinct from parasite clearance, which may paradoxically even enhance the risk of clinical relapses \[[@B23]\]. Asymptomatic and symptomatic subjects who are studied here were occasionally detected in a survey study of endemic Brazilian populations. They mainly consisted of migrants who lived in endemic areas for individually different time periods, and may have experienced sequential infections by P. falciparumor Plasmodium vivax, with clinical symptoms of variable degrees of intensity but low reported mortality \[[@B24]\]. Results showed that asymptomatic and symptomatic states could be remarkably well distinguished by multi-specific antibody reactivity, particularly when exposure times were limited, and that the distinction consisted more in a difference in nonspecific polyclonal activation than in a specific immunization effect. Materials and Methods ===================== Study areas and subjects ------------------------ 78 sera analysed here originated from subjects exposed to transmission in the Brazilian Amazon endemic area, who had variable numbers of reported previous episodes of P. vivaxor P. falciparumclinical malaria. The malaria-exposed subjects comprised three distinct groups \[see Additional file 1\]. The principal study group was derived from a screening of 531 miners living in gold-mining areas in the municipality of Apiacás (AP), Mato Grosso, among whom 99 had been found parasitaemic, presenting with positive Giemsa-stained thick-blood smears \[[@B25]\]. Out of these 99 subjects, only 46 had shown classical malaria symptoms within 72 hours after parasite detection, while the other 53 had remained asymptomatic. Symptoms were mainly headache, anorexia and fever. Included in the present study are 48 of these miners, 24 symptomatic and 24 asymptomatic, who had lived for up to 17 years in Apiacás. There was no significant difference between symptomatic and asymptomatic individuals in their time of residence. According to anamnestic reports, numbers of previous malaria episodes were highly variable, but neither significantly different between the groups. The time elapsed since the respective last episode, however, was significantly longer among the asymptomatics (see Table [1](#T1){ref-type="table"}). Within these parasitaemic subgroups, subjects were further distinguished whether or not they were positive for hepatitis B surface antigen (HbSAg) and therefore hepatitis B virus (HBV) carriers. Two further groups included exposed, but aparasitaemic control subjects. The first group (20 subjects) had resided for 10 or more years in Terra Nova Norte (TNN), a small rural community within the endemic region, continuously exposed to malaria. These parasitologically negative, convalescent individuals had been treated for malaria only until two months before the blood collection (Aparasitaemic/Previous malaria). The second group (10 subjects) also lived in TNN, but had no record of previous malarial episodes (Aparasitaemic/No previous malaria). All subjects responded to a questionnaire including information on past malaria and previous treatments. Consent to draw blood was obtained from each individual according to the Fundação Oswaldo Cruz Ethics Committee (MH, November 26^th^, 1994) and to the Universidade Federal de Minas Gerais Ethics Committee (April 15^th^, 1998). Venous blood samples (20 ml per subject) were drawn in Vacutainer™ (Becton Dickinson, Oxnard, CA) heparinized tubes. Giemsa-stained thick-blood smears were examined at this point for blood parasitaemia. Finally, a third control group consisted of 10 healthy adult volunteers from Belo Horizonte (Minas Gerais) who had never been exposed to malaria transmission or visited endemic regions (Aparasitaemic/Not exposed). IgG and IgM to human brain proteins were assessed in the subjects described above. When measuring IgG reactivity to E. coliproteins, two of the Terra Nova and seven of the Apiacas subjects were not included, but replaced by one Terra Nova and eight Apiacas subjects not assayed for reactivity to brain proteins. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Composition of the sample studied ::: Group Origin Nb Age^1^ Residence^1^ Previous malaria^2^ Time elapsed since last episode^1^ ------------------------------------------- -------------- ---- -------------------- ----------------- --------------------- ------------------------------------ Aparasitaemic Not Exposed B. Horizonte 10 27--55 \[31\] 0 Aparasitaemic -- No Previous Malaria Terra Nova 10 16--39 \[27\] 0 Aparasitaemic -- Previous Malaria Terra Nova 20 14--35 \[27.5\]^4^ 1--11 \[3\]^3^ Parasitaemic -- HBV-negative Asymptomatic Apiacas 19 20--47 \[30\] 2--13 \[8\]^4^ 2--70 \[15\] 0--13 \[2 years\]^3^ Parasitaemic -- HBV-negative Symptomatic Apiacas 13 6--66 \[27\] 2--17 \[7\]^3^ 2--50 \[15\] 0--17 \[1 month\] Parasitaemic -- HBV-positive Asymptomatic Apiacas 5 20--50 \[27\] 2--11 \[6\] 7--50 \[15\] 0--4 \[1 year\] Parasitaemic -- HBV-positive Symptomatic Apiacas 11 24--46 \[30\] 5--11 \[10\]^4^ 5--50 \[35\] 0--2 \[2 months\] ^1^In years; range \[median\] ^2^Number of malaria episodes anamnestically reported; range \[median\] ^3^Unknown for one subject ^4^Unknown for two subjects ::: Parasitaemia and anti-P. falciparum reactivities ------------------------------------------------ The parasite density was quantified after examination of 200 microscopic fields at 1.000× magnification under oil-immersion. All slides were examined by three well-trained microscopists. Blood parasitaemia was expressed as the number of parasites per 200 leukocytes. Parasitaemia and reactivity to P. falciparumand MSP1-19 in these populations have been analysed and described previously \[[@B25]\]. Immunoblot Assay ---------------- The assay was done as described \[[@B26]\]. Briefly, protein extracts from human brain and cultured E. coliwere run in a discontinuous SDS-PAGE 10% gel (Mighty Small electrophoresis apparatus, Hoefer Scientific Instruments, San Francisco, CA). After eletrophoresis, proteins were transferred onto a nitrocellulose membrane (Schleicher & Schuell, Germany) in a semi-dry system for one hour at 0.8 mA/cm^2^. After overnight blocking of free binding sites in PBS-Tween 0.2%, membranes were incubated for four hours with sera, diluted 1/20, in incubation units fixing membranes in cassettes with 28 independent channels (Miniblot System C-Shell, Immunetics Inc., Cambridge, MA). Whole membranes were then washed and incubated for 90 minutes at room temperature with secondary anti-human IgM or IgG conjugated with alkaline phosphatase (Southern Biotech, Birmingham, AL). Reactivies were revealed with NBT/BCIP (nitroblue-tetrazolium/bromo-chloro-indolyl-phosphate) substrate (Promega, Madison, WI) for three to five minutes, and membranes scanned in 8-bit grayscale and with 600 dpi resolution in a domestic scanner (Apple Color OneScanner). Thereafter, membranes were stained overnight with colloidal gold (Biorad, Hercules, CA) to reveal total protein and again scanned as described. Data processing and statistical analysis ---------------------------------------- The method of data processing has been described \[[@B26]\]. Briefly, reactivity profiles from the scanned immunoblot image were adjusted for migration irregularities during electrophoresis, using the scan of the same membrane stained for total protein. Special procedures programmed with the software IgorPro (Wavemetrics, Lake Oswego, OR) on a Macintosh computer (Apple computers, Cupertino, CA) were applied in order to represent each serum sample as a profile on a standardized migration scale, with the optical density (OD) as a function of migration distance. After this rescaling, sections were defined by intervals on the standardized migration scale around reactivity peaks, and, after baseline subtraction, reactivity for each section and serum was quantified as the average OD within a respective section. In this way, each serum can be represented by a vector with a dimension equal to the number of sections, containing the respective reactivity quantitation. In order to make these data commensurate across membranes, they were further normalized by the membrane-wise average reactivity of a unique standard (pool of human IgM or IgG), assayed twice on each membrane. These vectors were then analysed by Principal Component Analysis (PCA). PCA is a classic method of multivariate analysis designed to describe multidimensional data with high dimensionality through projection onto characteristic subspaces with lower dimensionality. Principal components are defined in a mathematically strict manner as orthogonal axes fitting maximal information in terms of total variance with decreasing proportion and uncorrelated among each other. This includes no information on experimental groups or other particularities, but provides a completely neutral and unbiased description of the data set as such. The number of bands detected on a respective extract was quantified as the number of sections showing reactivity values two-fold above the average reactivity of the standard used for adjustment. Total reactivity for a given extract was assessed as the average OD over the entire migration scale, from the first to the last section. Only distribution-independent statistics were used: Mann-Whitney rank sum test and Spearman rank correlation. P-values below 0.05 were considered significant. Results ======= IgG reactivity to human brain proteins -------------------------------------- As an example, Fig. [1](#F1){ref-type="fig"} shows one representative out of four total immunoblot membranes on which IgG reactivities to brain proteins were assayed. Most of the reactivity bands appear in samples derived from asymptomatic parasitaemic subjects, with the exception of one symptomatic coinfected with HBV. In Fig. [2](#F2){ref-type="fig"}, three different (however, correlated) ways to evaluate the reactivity quantitatively are shown : (1) by the number of detected bands; (2) by the summation of total reactivity in terms of averaged optical density over the whole migration scale; (3) by the score of the first principal component calculated from standardized optical densities of all reactivity bands. By any of these criteria, asymptomatic parasite carriers from Apiacas showed more reactivity than parasite-free subjects in all groups with high significance (p \< 0.00001). Parasite carriers with symptoms, however, also had less reactivity and, provided that they were not coinfected with HBV, did not significantly differ from parasite-free subjects. Among symptomatics, only HBV-positives showed reactivity levels comparable to the asymptomatics. In the absence of HBV-coinfection, asymptomatic and symptomatic subjects differed with high significance in terms of all three reactivity measures (p = 0.0003, 0.0002 and 0.0001, respectively). Even disregarding the presence of HBV coinfection, this difference was still significant (p = 0.017, 0.008 and 0.005, respectively). Assays of specific anti-parasite reactivity in the same subjects (reported in \[[@B25]\]) are shown in Fig. [2](#F2){ref-type="fig"} for comparison. Although showing the same tendency, these specific assays were less discriminatory, considering either only HBV-negative (IgG anti-P. falciparum: p = 0.034; IgG anti-MSP1-19: nonsignificant) or all parasitaemic individuals (p = 0.017 and p = 0.014, respectively). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### IgG reactivity patterns to brain proteins on one of four membranes. Open circles indicate asymptomatic malaria, closed circles symptomatic malaria in HBV-free parasite carriers, open and closed rhombi analogously in HBV-coinfected subjects. N indicates unexposed (Belo Horizonte), X and + exposed aparasitemic subjects from Terra Nova without and with previous malaria, respectively. Unmarked intermediate lanes contain the standard used for adjustment. ::: ![](1475-2875-4-5-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Comparison of properties of anti-brain IgG immunoblot reactivity andantiplasmodial IgG. A. Immunoblot of IgG to human brain proteins: number of bands, total reactivity and PCA factor-1 scores for IgG reactivities for each patient group. B. Antiplasmodial IgG: equivalent displays of data from the same patients for IgG reactivity to anti-P. falciparumand anti-PfMSP1-19. In both panels, medians for each group are indicated by vertical bars. C. factor loads for PCA factor-1 shown in panel A. ::: ![](1475-2875-4-5-2) ::: The first principal component (PCA factor 1) is, by definition, the linear combination of single reactivity measurements which represents a maximum of information about a multivariate dataset in terms of total variance, here 34%. Since this is the most systematic quantitative representation, the analysis will be continued based on principal components. In PCA, factor loads, i. e., coefficients indicating the relative contribution of measured parameters to the respective factor score, can be interpreted according to whether this factor represents a general level difference or a pattern-related property. Here, the factor-1 score contained only positive loads and, thus, represents a modified measure of total reactivity. Factor-1 scores indeed showed similar groupwise distributions as band number or total reactivity and were most discriminating. Among the first few principal components, factor 1 was the only one with interpretable properties. Further properties of factor-1 scores derived from IgG anti-brain reactivities are shown in Fig. [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}. Parasitaemic subjects had provided information on the time they had spent in Apiacas and in malaria-endemic areas in general. When plotted against these exposure times (Fig. [3](#F3){ref-type="fig"}), factor-1 scores discriminated best between asymptomatic and symptomatic subjects without HBV coinfection who had been exposed for relatively shorter periods. Hence, HBV-free subjects living less than 10 years in Apiacas or less than 20 years in malaria-endemic areas were almost completely separated according to their clinical status by factor-1 scores, but not those with long-term exposure. HBV-coinfected subjects, however, all displayed enhanced scores with no evident relationship to malaria exposure. For instance, both exposure parameters did not differ significamtly between symptomatic and asymptomatic subjects, regardless whether HBV infection is taken into account or not. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Polyclonal reactivity and exposure. Scores of PCA factor-1, derived from IgG reactivity to brain proteins, in relation to the time spent in Apiacas and in malaria-endemic zones, for HBV-negative (left) and HBV-positive subjects (right). Open symbols indicate asymptomatic, closed symbols symptomatic subjects. ::: ![](1475-2875-4-5-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Polyclonal reactivity, parasitemia and anti-PF antibodies. Relations of anti-brain IgG-derived PCA factor-1 scores are shown to parasitemia levels and anti-P. falciparumreactivity (ELISA absorbance, according to ref. 27). X and + indicate exposed aparasitemic subjects without and with previous malaria, respectively. The parameters were not always available from all subjects. Open symbols represent asymptomatic and closed symbols symptomatic subjects, all HBV-free. ::: ![](1475-2875-4-5-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Polyclonal IgG to brain proteins and Plasmodiumspecies in previous and present infections. Association of anti-brain IgG derived PCA factor-1 scores and Plasmodium species detected in the previous clinical Malaria episode (A), or presently (B). Medians calculated for all indicated subjects are shown by vertical bars. Subjects in which both species were detected at the same time were not considered. Crosses indicate exposed aparasitemic subjects with previous malaria, circles HBV-, and rhombi HBV+ parasite carriers. Open symbols represent asymptomatic and closed symbols symptomatic subjects. ::: ![](1475-2875-4-5-5) ::: Fig. [4](#F4){ref-type="fig"} shows the relationship between factor-1 scores and (a) individual parasitaemia and (b) anti-parasite IgG. Within HBV-negative asymptomatic subjects, scores were highest when parasitaemia levels were relatively low, although this association was quantitatively insignificant. A significant correlation was found between factor-1 scores and IgG reactivity to P. falciparum(Spearman rank correlation: +0.57 \[p \< 0.00001\] including parasitaemics and exposed aparasitaemics; +0.37 \[p = 0.011\] within parasitaemics only), or to PfMSP1-19 (Spearman R, considering parasitaemics and exposed aparasitaemics: +0.42 \[p = 0.0002\]; not shown). Nevertheless, as can be seen in Fig. [4](#F4){ref-type="fig"} for IgG anti-P. falciparum, these correlations are mainly due to the fact that all parameters were relatively enhanced in asymptomatic subjects. Considering asymptomatic and symptomatic individuals separately, correlations are no longer evident. Namely, some HBV-free symptomatics had elevated IgG anti-P. falciparum, but without a parallel increase in factor-1 scores. Medical records of previous episodes of clinical malaria were available for both parasitaemic and exposed aparasitaemic subjects. Considering subjects of all groups together, factor-1 scores of individuals with evidence for P. falciparuminfection in their last clinically diagnosed episode were higher than for those who had had P. vivaxmalaria (Mann-Whitney test: p \< 0.01). Qualitatively, this appeared to be the case for parasite carriers as for aparasitaemic subjects with reported previous malaria (Fig. [5](#F5){ref-type="fig"}), although a valid statistical evaluation taking subject groups into account is impossible due to the small sample size. Nevertheless, it is interesting that the Plasmodiumspecies presently detected in the parasitaemic subjects, often not identical with the one ascribed to the previous malaria period, was not associated with a similar difference in respect to factor-1 scores. IgG reactivity to E. coli extract --------------------------------- Patterns of IgG reactivity were also assayed toward an extract of E. coliwhole cultures, which provided an independent and completely different set of target antigens, and, consequently, a different set of reactivity bands. Surprisingly, multivariate analysis of these bands yielded a result very similar to that described above for IgG reactivity to human brain proteins (Fig. [6](#F6){ref-type="fig"}). Although less significantly, the E. coli-derived score shared several properties described above. Analogously, asymptomatic parasitaemics had higher scores for reactivity to E. colithan symptomatics in the absence (p = 0.041), but not in the presence of HBV coinfection. Also similarly to IgG anti-brain, this significance increased when only subjects living less than 10 years in Apiacas were considered (p = 0.002, not shown). As can be seen in Fig. [7](#F7){ref-type="fig"}, factor-1 scores calculated from anti-E. coliIgG reactivities indeed correlated highly with anti-brain-IgG-derived factor-1 scores (Spearman rank correlation: +0.70; p \< 1E-9). Finally, IgG anti-E. coli-derived factor-1 scores were higher in subjects reporting previous infection with P. falciparum, compared to P. vivaxinfection (p \< 0.05; Fig. [8](#F8){ref-type="fig"}). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### IgG reactivity to E. coliproteins. Distributions of PCA factor-1 scores derived from IgG reactivities to E. coliproteins per group. Group medians are indicated by vertical bars. ::: ![](1475-2875-4-5-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### IgG reactivity to different antigenic sources is highly correlated. IgG anti-brain and anti-E. coliderived PCA factor-1 scores, respectively, are displayed in two dimensions. N indicates non-exposed, X and + exposed aparasitemic subjects without and with previous malaria, respectively (B,C). Circles indicate HBV-, rhombi HBV+ parasitemics, open symbols asymptomatic and closed symbols symptomatic subjects. ::: ![](1475-2875-4-5-7) ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Polyclonal IgG to E. coliproteins and Plasmodiumspecies in previous infection. IgG anti-E. coliderived PCA factor-1 scores are displayed against the parasite species detected in the previous malaria episode, in analogy to Fig. 5. Medians indicated by vertical bars include all subjects in the figure. Crosses represent exposed aparasitemic subjects with previous malaria, circles HBV-, and rhombi HBV+ parasite carriers. Open symbols represent asymptomatic and closed symbols symptomatic subjects. ::: ![](1475-2875-4-5-8) ::: When principal components were calculated from IgG reactivities to both brain and E. coliproteins together, the properties described for both respective factor-1 scores fell together in the resulting first principal component (Fig. [9](#F9){ref-type="fig"}), indicating again that they were highly coincident. The difference between HBV-free asymptomatic and symptomatic parasitaemic subjects reached a higher significance level (p = 0.00005) than for factor-1 scores derived from either extract alone. ::: {#F9 .fig} Figure 9 ::: {.caption} ###### IgG reactivity to E. coliproteins. Distributions per group of PCA factor-1 scores derived from IgG reactivities to human brain and E. coliproteins joined together. Group medians are indicated by vertical bars. ::: ![](1475-2875-4-5-9) ::: IgM reactivity to human brain proteins -------------------------------------- IgM reactivities to brain proteins were measured using the same extract as described for IgG (data not shown). Generally, a smaller number of bands was detected. The IgM-derived factor-1 included only positive loads like the IgG-derived one. The scores of both were correlated (Spearman rank correlation: +0.55; p \< 1E-6), and, as for IgG, IgM-derived factor-1 scores were higher in asymptomatic than in symptomatic parasitaemic individuals free of HBV. However, IgM-derived factor-1 did not significantly discriminate between them, except when only individuals living less than 10 years in Apiacas were considered (p = 0.013). No significant difference was found either in respect to previously or presently detected Plasmodiumspecies. In contrast to IgG anti-brain or anti-E. coli, within parasitaemics, a remarkable positive correlation existed between IgM-derived factor-1 scores and individual age (Spearman rank correlation: +0.41; p = 0.005), and also with times spent in Apiacas (+0.34; p = 0.03), but not within aparasitaemic subjects. As for IgG anti-brain, there was no significant correlation with blood parasitaemia levels, but with specific IgG anti-P. falciparum(+0.55; p \< 1E-5). Joint analysis of IgG and IgM reactive to brain proteins reveals distinct components ------------------------------------------------------------------------------------ Finally, principal components were calculated for IgG and IgM reactivities to human brain proteins together (Fig. [10](#F10){ref-type="fig"}). Surprisingly, the properties of the respective first factors calculated for each isotype alone did not coincide in a single principal component, as it had occurred in the co-calculation of IgG anti-brain and anti-E. colireactivities. Instead, the first two principal components (factor-1 and factor-2) of the co-calculation both significantly distinguished between HBV-free asymptomatic and symptomatic parasite carriers (Factor 1: p = 0.013; Factor 2: p = 0.004), and, as above, not between those infected with HBV. Scores of both factors were enhanced in asymptomatic subjects. Since principal components in the same set are by definition uncorrelated, these two factors appear to represent separate effects associated with clinical states. ::: {#F10 .fig} Figure 10 ::: {.caption} ###### Principal components derived from IgG and IgM reactivities to human brain taken together. A. Coefficients of factor 1 and 2, representing weight and direction of section reactivities contributing to the respective factor scores. B. Distributions of factor 1 and 2 scores according to subject groups. Vertical bars represent medians within each group. C. Distribution of factor 1 and 2 scores in respect to Malaria exposure (HBV-free parasite carriers only). Different subject groups are represented by the indicated symbols. ::: ![](1475-2875-4-5-10) ::: Factor-1, characterized by positive loads as the above described, was however positively influenced primarily by IgM reactivity, and properties of factor-1 scores were similar to those of the IgM-derived factor-1. Within parasitaemic subjects, scores increased with age (Spearman R: +0.39; p = 0.007), with exposure times in Apiacas and in endemic areas (+0.29 and +0.26, but not significant), and were correlated with anti-P. falciparumIgG (+0.32; p \< 0.05). However, like IgG-derived factor 1, scores were also elevated in respect to P. falciparumbut not P. vivaxpre-exposure (p = 0.01; not shown). In contrast, factor-2 scores were positively influenced mainly by IgG reactivity, while a number of IgM bands had a negative impact. Thus, factor 2 can be said to represent a certain IgG/IgM relationship. Remarkably, among HBV-free parasite carriers, the highest scores were characteristically found in asymptomatics who had only been exposed to malaria in endemic areas for a limited time. Factor-2 scores showed no significant association with IgG anti-P. falciparumor with pre-exposure to Plasmodiumspecies. Instead, scores were slightly higher in subjects presently infected with P. vivax, but not P. falciparum(p = 0.04; not shown). Neither factor correlated significantly with parasitaemia. Association with reactivity to MSP1-19 -------------------------------------- Global IgG reactivity and subtype-specific reactivity have been previously assayed including some subjects examined here \[[@B25]\]. Among parasitaemic subjects, IgG1, IgG2, IgG3 and IgG4 anti-MSP1-19 all correlated significantly with IgM-anti-brain-derived as well as with IgG/IgM-anti-brain-derived factor-1, in particular IgG3 (Spearman R: +0.68 \[p \< 0.001\] and +0.62 \[p \< 0.01\], respectively). These values, however, did not corrrelate significantly with any IgG-derived factor, nor with IgG/IgM-anti-brain-derived factor-2. Only IgG anti-MSP1-19, measured in a larger number of subjects, correlated positively with IgG-anti-brain and IgG-anti-brain/E. coli-derived factor-1 (not shown). Discussion ========== Autoantibodies detected in the context of malaria infection are an old observation, reviewed in \[[@B27],[@B28]\]. Typically, they were found elevated in long-term exposed subjects and in acute symptomatic infection, while asymptomatic infection has rarely been studied. However, phospholipid-related specificities were found more elevated in asymptomatics than in patients with malaria symptoms \[[@B29],[@B30]\]. All these previous studies focused on antibody specificities otherwise known from autoimmune disease conditions, particularly systemic lupus erythematosus. The findings were often interpreted as results of specific crossreactivity or auto-immunization, and analogies were regularly made to \'autoimmunity\'. This appears confusing, since malaria infection and pathological autoimmunity have little obviously in common and, in fact, the respective pathologies appear to mutually inhibit rather than to facilitate each other \[[@B27]\]. The simultaneous standardized detection of multiple immunoglobulin reactivities on immunoblots, as utilized in the present study, is most likely to represent natural antibody repertoires and, therefore, a way to approach the problem of repertoire description in an unbiased way, avoiding a potentially misleading disease- or otherwise antigen- or specificity-oriented vision. Extracts of human tissue (brain) or whole bacterial cultures were used as ligands for antibodies with the aim of relating eventual changes in binding patterns to clinical status, and comparing whole reactivity patterns by multivariate analysis. The same semiquantitative immunoblot technique has previously demonstrated characteristics of natural IgM and IgG reactivity patterns \[[@B17],[@B31],[@B32]\], among which stability \[[@B12],[@B33],[@B34]\] and independence of physiologic antigenic exposures \[[@B35]\] are most remarkable. IgG patterns, however, change with age \[[@B36]\], during human \[[@B13],[@B14]\] and experimental \[[@B37]\] autoimmune diseases and in murine parasite infection \[[@B38]\]. The present results show that reactivity patterns to human brain and E. coliproteins, unrelated to parasites, could differentiate strikingly between asymptomatic and mild symptomatic malaria infection. Asymptomatic subjects generally showed elevated reactivity, which was concordant but far more characteristic when compared with anti-plasmodial IgG measures in the same sample. Thus, Plasmodiuminfection is able to alter reactivity patterns to target proteins without relation to the parasite particularly in asymptomatic subjects. Only co-infection with HBV, known to be frequent in our study population \[[@B39]\], heightened reactivity regardless of malaria symptoms and obscured this discrimination when disregarded. This effect is most probably unrelated to malaria, since chronic HBV infection itself is known for its association with a variety of autoantibodies \[[@B40]\]. Remarkably similar results were obtained by analyses of IgG immunoblot reactivity against extracts of human brain and of E. coli. In both cases, the same major properties were represented by a single respective principal component, characterized by positive loads. Both were highly correlated and when reactivity to both extracts was analysed together, they coincided in a single PCA factor with maximal discriminatory power, allowing to separate 13/15 (87%) of the HBV-free asymptomatics from symptomatic patients. Thus, IgG reactivity patterns to brain and to E. coliproteins behaved somewhat like fragments of a hologram which, added together, show the same image with increased resolution. Other than previous data on malaria-associated autoantibodies, these observations cannot easily be attributed to specific immune responses, despite a positive correlation with anti-parasite reactivity. Instead, they probably reflect another phenomenon well known for malaria and other parasite infections, that of polyclonal lymphocyte activation. Polyclonal activation, however, is often discussed as an immune evasion mechanism, beneficial primarily for parasites. Our results show in contrast that the production of polyclonal IgG was associated with protection in asymptomatic parasite carriers, and that polyclonal reactions appeared to have at least short-term protective effects. A reason for this discrepancy may be that earlier studies of polyclonal activation \[[@B41]\] assessed peripheral plaque-forming cells, which may not contribute much to circulating antibody repertoires, and not the recruitment of resident plasma cells with a relevant lifespan from which circulating natural antibodies likely originate. When such natural antibodies as they are addressed by us have pre-existent protective properties, their polyclonal recruitment can well be efficient and protective, as it has been demonstrated for other infections \[[@B2],[@B3],[@B5]\]. This could be due to preceding evolutionary selection. Our observation of an effect of the parasite species present in previous clinical episodes further suggests that previous antigenic experience can trigger not only classical recall responses, but also this capacity to react polyclonally. With increasing exposure time, this nonspecific recruitment of natural repertoires could be stepwise complemented by induced parasite-specific antibodies, leading to an increasingly adapted protection as it is observed in long-term exposed subjects. Remarkably, in our study, asymptomatic and symptomatic infections were indeed best discriminated in subjects with relatively short exposure times. In contrast to previous data on autoantibodies, this indicates that the reactivity shift in asymptomatic subjects described here does not result from long-term adaptation to the parasite, but is representative of an intermediate state of adaptation. This interpretation is most clearly supported by the result of joint analysis of IgM and IgG reactivity to brain proteins, where two distinct principal components appeared. Factor-1, representing a general elevation of all reactivities with dominant IgM impact, shows an evident overall time dependency in parasite carriers and may, thus, reflect long-term adaptation. However, factor-2 discriminated asymptomatic from symptomatic infections best and, most characteristically, those asymptomatic subjects who had been least exposed. Factor-2 represented a relative dominance of IgG reactivity against some IgM. Thus, in order to remain asymptomatic, particularly infected subjects without long-term adaptation to the parasite may require a pattern of natural antibody production dominated by IgG but not IgM. This is in principal agreement with known protective effects of antibodies. Classically, passive transfer of hyperimmune IgG to patients infected with P. falciparumhas shown that antibodies play a crucial role in controlling blood stage parasitaemia \[[@B42],[@B43]\]. The gradual acquisition of clinical immunity to malaria after repeated infection is positively correlated with the development of a diverse IgG repertoire, most clearly including IgG reactive to parasitized red blood cells \[[@B1]\]. Such cytophilic antibodies are predominant in clinically immune individuals living in hyperendemic areas \[[@B21]\]. In contrast to IgG, IgM with the same cytophilic properties is associated with rosetting and enhanced pathogenicity \[[@B44]-[@B47]\]. The targets of cytophilic antibodies include diverse and highly variable parasite-encoded proteins, but also host proteins such as \'band 3\' \[[@B6]\], which is involved in parasite entry into red blood cells \[[@B7]\]. Nevertheless, anti-band 3 antibodies may just exemplify the possible relevance of natural repertoires, since they are originally known as physiologic autoantibodies involved in the general clearance of senescent and damaged (including infected) erythrocytes \[[@B8]\]. Intrinsic tuning of the production rate of such pre-existing antibodies may, therefore, contribute as much to the control of parasitaemia that occurs in clinically immune individuals, as do cytophilic antibodies originating from parasite-specific responses. Other natural antibody-mediated effects could act analogously, and an appropriate individual reshaping of the natural repertoire toward such components could well lead to a capability to regulate parasitaemia, triggered by parasite-mediated polyclonal lymphocyte activation. Such reshaping, associated with a shift in internal self-recognition, could also explain the appearance of lupus-like autoantibodies in chronically exposed subjects \[[@B28]\] without, however, indicating autoimmunity. In general, protective natural repertoire components appear to deserve further investigation, since vaccines and other malaria control strategies could possibly be designed to make use of them. Conclusions =========== This study shows that simultaneous detection of IgM and IgG reactivities to a broad range of targets can reveal remarkable properties which are not easily accessible by the usual specificity-focussed approaches. Thus, clinical states in Plasmodiuminfection appear to depend on internal activation states which can be distinguished by different patterns of polyclonal antibody production. Particularly, asymptomatic but not symptomatic infection of HBV-free subjects without extensive pre-exposure to malaria was characterized by elevated polyclonal IgG reactivity in absolute terms and in relation to IgM, furthermore appearing to be triggered by previous P. falciparumbut not P. vivaxinfections. Authors\' contributions ======================= CJFF and EMB did the field work and collected the samples. NMV designed and supervised the study. AC performed preliminary experiments. CF, LFG and ASN did the immunoblots and analysed the results. CF prepared software, performed statistics and wrote the manuscript. Acknowledgments =============== This work was financially supported by FAPEMIG (grants no. CBB 2323/97 and CBB 177/01), CNPq (52.0834/96-8) and Grices/ICCTI (62/00). CF received a fellowship from FCT, Portugal. We thank M. Mota and W. Haas for critical reading.	PubMed Central	2024-06-05T03:55:52.766605	2005-1-20	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548665/", "journal": "Malar J. 2005 Jan 20; 4:5", "authors": [ { "first": "Constantin", "last": "Fesel" }, { "first": "Luis F", "last": "Goulart" }, { "first": "Adolfo", "last": "Silva Neto" }, { "first": "Alysson", "last": "Coelho" }, { "first": "Cor Jesus F", "last": "Fontes" }, { "first": "Erika M", "last": "Braga" }, { "first": "Nelson M", "last": "Vaz" } ] }
PMC548666	Background ========== The P-glycoprotein (P-gp) is an ATP-dependent efflux transporter (ABCB1) encoded by the multidrug resistance gene (MDR1) which extrudes large lipophilic, positvely charged molecules from cells, among them HIV-1 protease inhibitors \[[@B1],[@B2]\]. P-gp is expressed on a variety of cells including human lymphocytes, the target cells of HIV and of antiretroviral substances \[[@B3]\]. 48 single nucleotide polymorphisms (SNP) have been described so far for the MDR1 gene \[[@B4]\]. Though there is still controversy about the biological relevance of these SNPs, there appears to be an association between specific genotypes and mRNA expression, P-gp expression and/or P-gp function (reviewed in \[[@B5],[@B6]\]). Recently, Fellay et al. found lower nelfinavir and efavirenz plasma levels associated with the TT genotype of the SNP C3435T in exon 26 and a greater rise of the CD4 cell count 6 months after initiation of antiretroviral therapy in patients with this genotype \[[@B7]\]. However, the mechanisms underlying this observation remain unclear. In contrast to protease inhibitors (PI), non-nucleoside reverse transcriptase inhibitors (NNRTI) such as efavirenz are not substrates of the P-gp. Therefore, it has been speculated that the P-gp may modulate the clinical course of HIV infection independent from its role in drug transport. Indeed, there have been reports showing inhibition of apoptosis and decreased HIV production in cells overexpressing P-gp \[[@B8]-[@B12]\]. However, these observations from in vitro studies have not been confirmed in vivo when the disease progression before treatment was assessed in HIV infected individuals with different MDR1 genotypes \[[@B13]\]. The genetic variant C3435T in exon 26 is a synonymous polymorphism that does not alter the amino acid sequence. How this variant could affect P-gp expression is still unknown \[[@B6]\]. According to one hypothesis, functional effects of the C3435T SNP may not be genotype- but haplotype-dependent. The exon 26 C3435T polymorphism is in linkage disequlibrium with the polymorphism G2677T in exon 21, which results in the amino acid change Ala893Ser \[[@B6]\]. 2677A leading to Ala893Thr is an infrequent third allel of this SNP. The results of several studies on the functional effects of mutations at position 2677 in exon 21 have shown conflicting results \[[@B6]\]. Because of the unresolved issues surrounding the potential effects of MDR1 polymorphisms and P-gp function in HIV infection, we investigated whether there was an association between the MDR1 polymorphisms 3435 and 2677 and the immunological response in HIV infected individuals after initiation of antiretroviral therapy. Methods ======= Patients -------- Of the HIV 1 infected patients seen at the Department of Infectious Diseases of the University of Würzburg, Germany, 72 patients (18 women, 54 men; mean age 39.5 years, range: 26 -- 59 years; 64 Caucasians, 5 African, 3 Asian), started antiretroviral therapy between 1998 and 2004. The therapy consisted of three nucleoside reverse transcriptase inhibitors (NRTI) (n = 12), two NRTIs plus at least one PI (n = 40; including RTV n = 26) or two NRTIs plus one NNRTI (n = 20). HIV load and CD4 cell count were determined four weeks after initiation of therapy and approximately every three months thereafter. In patients who received NNRTI or PI drugs as part of their therapy, plasma levels were monitored and adjusted to therapeutic drug levels when necessary as previously described \[[@B14],[@B15]\]. The treatment was based on current international treatment guidelines \[[@B16]\], taking into account individual circumstances of each patient (e.g. known intolerabilities, side-effects of previous therapies, concomitant medication). The study was in accordance with the Helsinki Declaration and was approved by the local ethics committee. Patients gave informed consent for the study. Genotype analysis ----------------- DNA was extracted from 200 μl blood and the MDR1 3435 genotype was determined with genotype specific hybridisation probes and melting curve analysis on the LightCycler (Roche, Mannheim, Germany) as previously described \[[@B17]\]. The 2677 genotype was determined in a similar fashion \[[@B18]\]. Briefly, DNA was amplified on the LightCycler with the Quantitect Probe PCR Kit (Qiagen, Hilden, Germany) by using the primers 5\'-gcaggagttgttgaaatgaaaatg-3\' (forward) and 5\'-cgcctgctttagtttgactca-3\' (reverse). Hybridization probes were added to the master mix to a final concentration of 0.05 μM (sensor probe: 5\'-ttcccagTaccttct-fluorescein; locked nucleic acid base in upper case letter) and 0.15 μM (anchor probe: 5\'-LC Red640-ctttcttatctttcagtgcttgtcc-p). Primers and probes were obtained from TIB Molbiol (Berlin, Germany). The melting points were 41°C, 47°C, and 52°C for the T-, G-, and A-alleles, respectively. The results of 30 samples were confirmed by sequencing. Statistical analysis -------------------- Data were analysed by the Kruskal-Wallis test, which as a nonparametric test compairs three or more unpaired groups. Because of the small number of samples a nonparametric test was needed. SPSS version 12.0 (SPSS GmbH, Munich, Germany) was used for statistical analysis. A P-value of 0.05 was considered to be significant. Analogous to \[[@B13]\], the SNPs 3435 and 2677 were analyzed both separately and in combination as composite genotypes: H1: 2677GG and 3435CC (wild type); H2: 2677GT or TT and 3435CT or TT (2677T/3435T haplotype carrier); H3: 2677GG and 3435CT or TT; H4: 2677GT or TT and 3435CC; and H5: 2677AG or AT and any 3435 genotype (2677A carrier). Results ======= At initiation of antiretroviral therapy 8 patients had viral loads of \<10.000 copies/ml, 24 patients between \>10.000 and \<100.000 copies/ml, and 40 patients \>100.000 copies/ml. 39 patients had a CD4 cell count of \<200 cells/μl, 16 patients between \>200 and \<350 cells/μl, and 17 patients \>350 cells/μl. Determination of NNRTI- and PI drug levels indicated compliance of the patients with a NNRTI or PI containing regimen, because the measured drug levels were in the therapeutic range. Genotype analysis of the MDR1 gene at position 3435 in exon 26 revealed 20 patients with the CC genotype, 33 with the CT genotype and 19 with the TT genotype (5 patients of African origin: 4 with CC and 1 with CT genotype; 3 patients o f Asisan origen: 2 with CT and 1 with TT genotye). Analysis of the 2677 polymorphism in exon 21 demonstrated that 24 patients had the GG-, 23 the GT-, 18 the TT-, 4 the AG-, and 3 the AT genotype; the AA genotype was not found in this group (5 patients of African origin: 4 with GG and 1 with GT genotype; 3 patients of Asian origin: 3 with TT genotype). Detailed genotype and haplotype results with respect to the initial viral load and CD4 cell count are presented in table [1](#T1){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Genotype analysis of 72 treatment naive HIV-positive patients with respect to baseline CD4 cell count and viral load. ::: CD4 \[cells/μl\] VL \[copies/μl\] n = 2677 3435 2677/3435^\$^ ------------------ ------------------ ------ ------ ------ --------------- ----- ----- ----- ------ ------ ------ ------ ------ ----- ---- ----- \<200 \>10^5^ 27 7 7 8 3 2 \- 7 10 10 5 15 2 \- 5 10^4^-- 10^5^ 8 4 3 1 \- \- \- 2 6 \- 2 4 2 \- \- \<10^4^ 4 2 1 1 \- \- \- 1 2 1 1 2 1 \- \- 200 \>10^5^ 6 2 3 1 \- \- \- 1 4 1 1 4 1 \- \- -350 10^4^-- 10^5^ 9 3 3 3 \- \- \- 3 3 3 3 6 \- \- \- \<10^4^ 1 1 \- \- \- \- \- 1 \- \- 1 \- \- \- \- \>350 \>10^5^ 7 1 4 2 \- \- \- 1 3 3 1 6 \- \- \- 10^4^-- 10^5^ 7 1 2 2 1 1 \- 2 4 1 1 4 \- \- 2 \<10^4^ 3 3 \- \- \- \- \- 2 1 \- 2 \- 1 \- \- total 72 24 23 18 4 3 0 20 33 19 17 41 7 \- 7 ^\$^composite genotypes: H1: 2677GG and 3435CC (wild type); H2: 2677GT or TT and 3435CT or TT (2677T/3435T haplotype carrier); H3: 2677GG and 3435CT or TT; H4: 2677GT or TT and 3435CC; and H5: 2677AG or AT and any 3435 genotype (2677A carrier) ::: Table [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"} show the median and mean values of the viral load decline and the CD4-cell increase, respectively, determined at week 4, 12, 24 and 48 after initiation of therapy. There were no significant differences of the viral load decline neither between patient groups with different genotypes at positions 2677 or 3435 nor between patient groups with different 2677/3435 haplotypes. As to the CD4-cell response, there were no significant differences between the different genotypes and haplotypes, either. There was a trend of a more pronounced mean CD4-cell increase at week 12 and 24 in patients with the 3435TT genotype. However, this trend did not persist at week 48 and was not confirmed by the corresponding median values. A graphical presentation of the mean viral load decrease and CD4-cell increase with respect to the 2677/3435 haplotypes is given in fig. [1](#F1){ref-type="fig"}. Analysis of patients with different therapy-regimens (only NRTI, NRTI + NNRTI, or NRTI + PI) revealed no differences in the virological and immunological response between the different genotypes either, but was limited by small patient numbers in the subgroups (data not shown). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### VL-decrease \[log copies/ml\] at week 4, 12, 24 and 48 after initiation of antiretroviral therapy with respect to the MDR1 2677 and 3435 genotypes. Statistical analysis was done with the Kruskall-Wallis test. ::: week 4 week 12 week 24 week 48 -------------- ---- -------- --------- --------- --------- --------- ---- -------- -------- ------- ---------- ---- -------- -------- ------- --------- ---- -------- -------- ------- --------- 2677 GG 22 -2,301 -2,210 0,590 \>0.3 22 -3,048 -3,287 0,682 \>0.05 20 -3,943 -3,876 0,748 \>0.9 15 -3,716 -3,835 0,777 \>0.5 GT 21 -2,602 -2,537 0,566 19 -3,786 -3,794 0,635 17 -3,929 -3,933 0,499 16 -3,942 -3,946 0,488 TT 17 -2,495 -2,372 0,651 16 -3,739 -3,602 0,595 17 -4,000 -3,850 0,708 14 -3,977 -3,795 0,615 AG 3 -2,301 -2,689 0,606 § 4 -3,952 -4,167 0,884 § 4 -4,500 -4,537 0,702 § 4 -4,151 -4,362 0,726 § AT 3 -1,875 -2,032 0,493 § 3 -4,176 -3,752 0,690 § 3 -4,176 -4,085 0,223 § 3 -4,176 -4,085 0,223 § 3435 CC 17 -2,301 -2,382 0,501 \>0.6 19 -3,301 -3,416 0,699 \>0.4 17 -4,000 -3,974 0,685 \>0.7 13 -3,716 -3,818 0,709 \>0.6 CT 32 -2,247 -2,363 0,640 29 -3,778 -3,742 0,737 28 -3,812 -3,830 0,704 25 -3,929 -3,984 0,614 TT 17 -2,398 -2,348 0,668 17 -3,602 -3,530 0,595 17 -4,176 -4,054 0,571 15 -4,114 -3,849 0,609 2677/3435^†^ H1 15 -2,398 -2,398 0,531 \>0.8 16 -3,199 -3,361 0,724 \>0.08 14 -3,943 -3,926 0,715 \>0.8 10 -3,659 -3,774 0,787 \>0.4 H2 38 -2,548 -2,463 0,611 35 -3,778 -3,706 0,624 34 -3,954 -3,891 0,614 30 -3,954 -3,875 0,556 H3 7 -1,778 -1,806 0,502 § 6 -2,827 -3,089 0,506 § 6 -3,827 -3,761 0,807 § 5 -4,176 -3,958 0,741 § H5 6 -2,261 -2,361 0,642 § 7 -4,176 -3,989 0,833 § 7 -4,301 -4,343 0,594 § 7 -4,176 -4,243 0,584 § total 66 64 61 52 ^†^composite genotypes: see methods; ^§^excluded from statistical analysis ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### CD4-cell increase \[cells/μl\] at week 4, 12, 24 and 48 after initiation of antiretroviral therapy with respect to the MDR1 2677 and 3435 genotypes. Statistical analysis was done with the Kruskall-Wallis test. ::: week 4 week 12 week 24 week 48 -------------- ---- -------- --------- --------- --------- --------- ---- ------- ------- ------- --------- ---- ------- ------- ------- --------- ---- ------- ------- ------- --------- 2677 GG 24 56,5 69,8 81,5 \>0.3 22 97,0 130,6 132,4 \>0.5 21 107,0 136,1 120,5 \>0.8 15 244,0 235,7 132,1 \>0.1 GT 23 24,0 37,8 82,5 20 84,5 144,3 157,2 17 132,0 180,8 178,0 16 161,5 238,3 218,6 TT 18 9,5 67,4 117,7 16 70,0 143,9 222,3 16 95,0 154,5 162,0 14 107,5 143,9 159,1 AG 4 70,5 85,8 68,5 § 4 82,5 74,8 34,4 § 4 54,5 80,3 81,3 § 4 131,5 126,5 71,0 § AT 3 112,0 105,7 13,4 § 3 176,0 180,7 63,8 § 2 154,5 154,5 54,5 § 3 172,0 248,0 118,2 § 3435 CC 20 76,0 84,8 78,6 \>0.1 19 91,0 123,0 113,0 \>0.9 18 106,5 132,4 112,4 \>0.5 13 191,0 220,1 121,5 \>0.6 CT 34 25,5 47,2 76,1 30 85,0 123,7 135,3 28 131,0 134,2 106,8 25 159,0 187,7 118,4 TT 18 12,0 61,6 119,1 17 74,0 175,9 228,1 16 134,0 212,2 214,7 15 122,0 223,9 259,3 2677/3435^†^ H1 17 72,0 83,1 78,7 \>0.1 16 115,0 132,1 119,8 \>0.8 15 107,0 142,2 113,3 \>0.9 10 194,0 239,6 128,4 \>0.5 H2 41 20,0 50,8 100,6 36 74,5 144,1 188,9 33 130,0 168,1 170,9 30 134,5 194,2 198,8 H3 7 19,0 37,3 79,1 § 6 92,0 126,7 161,2 § 6 122,0 120,8 135,7 § 5 302,0 227,8 138,9 § H5 7 87,0 94,3 53,5 § 7 105,0 120,1 71,9 § 7 100,0 131,0 98,7 § 7 172,0 178,6 111,7 § total 72 65 61 52 ^†^composite genotypes: see methods; ^§^excluded from statistical analysis ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Composite genotypes (see methods) and response to antiretroviral treatment. Suppression of viraemia (lower panel) and CD4-cell count increase (upper panel) are shown. Data are mean values ± standard error. ::: ![](1476-0711-4-3-1) ::: Discussion ========== In an analysis of the virological and immunological response of treatment naïve patients with respect to the MDR1 G2677T/A and C3435T polymorphisms, virus load and CD4 cell count were assessed longitunally after initiation of antiretroviral therapy. We did not find an association between the CD4-cell increase or the HIV load decline and the MDR1 2677 or 3435 genotype during the first year of therapy. Our data are in contrast to a report of Fellay et al. that showed a significantly greater mean CD4-cell rise in patients with the MDR1 3435TT genotype during an observation period of 24 weeks \[[@B7]\]. The number of patients in the previous report (n = 96) was slightly larger than our population (n = 72), but the number of homozygotes of the 3435 polymorphism was very similar (22CC/20TT in \[[@B7]\], 20CC/19TT this study). Though we saw a trend towards a greater mean CD4-cell rise in patients with the 3435TT genotype as well, we doubt that this is indication of a real difference, because this trend was not seen when median values were considered. In our study, neither median values nor mean values were significantly different by Kruskal-Wallis test and ANOVA, respectively. For genotype subgroup sizes as in this study, analysis of the mean values is probably less robust. If there was a real difference between the immunological response of patients with the 3435CC and TT genotype, it did not persist at week 48. At this time point, mean and median values in the two groups were almost identical. A possible explanation for the diverse results is the selection of the study patients. While we included all treatment naïve patients in whom therapy was started between 1998 and 2004, Fellay et al. selected part of the patients on the basis of long-term viral suppression. In both studies, a variety of antiretroviral regimens was used. While all of the patients in the study of Fellay et al. received either nelfinavir or efavirenz, the treatment regimens in our study were more heterogenous and did not allow a meaningful separate analysis. The results of our study are supported by Nasi et al. \[[@B19]\] who analysed data of 149 treatment naïve patients who were treated with a PI-containing regimen (n = 106) or a NNRTI-containing regimen (n = 46) and found no association between the MDR1 genotype at position 3435 and the CD4 cell increases or plasma viral load decreases during the first six months of treatment among individuals with different genotypes. Likewise, in 142 patients enrolled in an open-label, randomized phase IIIb study comparing nelfinavir and efavirenz for treatment of HAART-naïve individuals the CD4 cell counts did not increase to a higher level in individuals with the homozygous variant genotype (TT) at the MDR1 C3435T locus in either the nelfinavir or the efavirenz treatment groups \[[@B20]\]. The MDR1 3435 polymorphism is a synonymous polymorphism. The TT genotype may have a reduced translation efficiency or lead to post-translational differences \[[@B21]-[@B23]\]. It has been shown, that a linkage disequilibirum exists between the exon 26 C3435T and the exon 21 G2677T/A polymorphism \[[@B6]\]. Therefore, we investigated both SNPs in our study. Neither the 2677 polymorphism alone nor the 2677/3435 haplotype was associated with differences in the virological or immunological response of treatment naïve patients. These data are in agreement with those of Haas et al. \[[@B24]\], who showed that the phase 1 and phase 2 viral decay as well as changes in CD4- and CD8-T-cells during triple therapy with ritonavir, zidovudine, and lamivudine in 31 treatment naïve patients were not associated with the MDR1 2677 and 3435 genotypes. In a retrospective study of 455 treatment naïve patients initiating antiretroviral therapy with 40 months of follow-up \[[@B25]\], there was a trend to earlier virological failure in the 3435CC group (p= 0.07) with no effect of the C3435T polymorphism in the MDR-1 gene on immunological failure. However, the difference in the virological response was not observed during the first 10 months. Further follow-up of our patient group is ongoing in order to detect long-term effects that may not have been apparent during the observation period analysed in this report. Conclusions =========== During a follow-up of 48 weeks, we found no evidence for an association between the MDR1 G2677T/A and C3435T polymorphisms and the virological and immunological response to therapy in HIV-positive drug-naïve patients. The individual response to antiretroviral therapy is a complex phenomenon, which is influenced by a large number of biological variables. Further studies on the role of polymorphisms of the MDR1 and other transporters and enzymes involved in drug metabolism are necessary in order to elucidate the role of pharmacogenetic effects in HIV therapy. Competing interests =================== None declared. Authors\' contributions ======================= RW participated in the design of the study and the molecular genetic studies, performed the statistical analysis and drafted the manuscript. PL participated in the design of the study, obtained and reviewed clinical data and helped in the data analysis. MZ participated in the clinical management of the study patients. FT and JS participated in the molecular genetic studies and the virological and immunological analysis. HK participated in the study design and coordinated clinical management of the study patients. BW participated in the study design and coordination and made contributions to the manuscript. All authors read and approved the final manuscript. Acknowledgements ================ This study was supported by a grant from the H. W. & J. Hector foundation, Weinheim, Germany.	PubMed Central	2024-06-05T03:55:52.769785	2005-1-20	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548666/", "journal": "Ann Clin Microbiol Antimicrob. 2005 Jan 20; 4:3", "authors": [ { "first": "Ralf", "last": "Winzer" }, { "first": "Peter", "last": "Langmann" }, { "first": "Michael", "last": "Zilly" }, { "first": "Franz", "last": "Tollmann" }, { "first": "Jörg", "last": "Schubert" }, { "first": "Hartwig", "last": "Klinker" }, { "first": "Benedikt", "last": "Weissbrich" } ] }
PMC548667	Introduction ============ Insulin resistance and hyperinsulinemia are often associated with a group of risk factors such as obesity, dyslipidemia, hypertension and impaired glucose tolerance. This cluster of metabolic abnormalities, first defined as Syndrome X by Reaven in 1988 \[[@B1]\] and supported by additional evidence \[[@B2],[@B3]\], is now more often referred to as the Metabolic Syndrome and has been increasingly recognized as important risk factors for coronary artery disease (CAD). The point of view became institutionalized and although the National Cholesterol Education Program\'s Adult treatment Panel III (ATP III) and the World Health Organization (WHO) have slightly different definitions \[[@B4]-[@B6]\], the Metabolic Syndrome is consistently characterized by a collection of metabolic abnormalities such as insulin resistance, obesity, dyslipidemia, hyperglycemia, and hypertension \[[@B7]\]. Not all of the disorders in the Metabolic Syndrome may be observed in the same individual. Most people with the syndrome have insulin resistance that could lead to glucose intolerance and diabetic hyperglycemia. Although the mechanisms underlying the pathogenesis of the Metabolic Syndrome are not exactly clear, obesity, insulin resistance and other independent factors such as vascular and immunologic origins appear to be involved \[[@B7]\]. The prevalence of the Metabolic Syndrome is more than 20% among the US adults adjusted for age \[[@B8]\], which is far greater than observed in an earlier study with European participants at least partly due to differences in the criteria used to define the condition \[[@B9]\]. Increased cardiovascular and mortality risks are associated with the Metabolic Syndrome \[[@B10]\]. The condition is usually managed with pharmaceutical agents for correcting dyslipidemia, anti-hypertensives, and insulin sensitizing agents or a combination of the above. Most existing agents only treat individual metabolic abnormalities. To date, no single agent can ameliorate all the features of the Metabolic Syndrome. There is an increasing need for novel agents to treat multiple abnormalities of the syndrome. Glucocorticoid (GC) excess has been linked to clinical observations associated with the Metabolic Syndrome. In Cushing\'s syndrome \[[@B11]\], increased secretion of GCs largely due to pituitary adenoma leads to central obesity, hypertension, hyperlipidemia and glucose intolerance, a group of metabolic abnormalities reminiscent of the Metabolic Syndrome. Correction of hypercortisolism by transsphenoidal surgery at least normalizes blood pressure \[[@B12],[@B13]\]. In addition, clinical administration of GCs to treat acute and chronic inflammatory diseases has been associated with metabolic adverse effects such as hypertension, obesity, hyperlipidemia and insulin resistance as seen in the Metabolic Syndrome \[[@B14]-[@B16]\]. These clinical findings suggest that GC action could play a role in the pathophysiology of the Metabolic Syndrome. GC metabolism and action ======================== Cortisol, the principal active GC in humans, is secreted by the adrenal gland and is converted to cortisone, the inert GC, primarily in kidney \[[@B17]-[@B19]\]. Two isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD) are responsible for the tissue-specific interconversion of cortisone and cortisol at the endoplasmic reticulum: type 1 and 2 (11β-HSD1 and 11β-HSD2) \[[@B20]\]. The two isozymes are products of two different genes and have distinct tissue distributions, with 11β-HSD1 expressed primarily in liver, adipose, kidney and brain and 11β-HSD2 mainly in kidney and salivary glands \[[@B20]\]. 11β-HSD1 converts inactive cortisone to cortisol in human or inactive 11-dehydrocorticorsterone (11-DHC) to corticosterone in rodents and 11β-HSD2 catalyzes the opposite reaction. Bidirectional activities (both reductase and dehydrogenase) have been observed with 11β-HSD1 in vitrobut it is mainly a reductase in vivo\[[@B21]\]. Since GC action is largely mediated by the ligand-induced activation of the GC receptor (GR), the local concentration of cortisol (or corticosterone) dictates GR activation. In tissues such as liver and adipose where 11β-HSD1 is expressed, there are two sources for cortisol (or corticosterone) accumulation: the fraction produced by 11β-HSD1 within the tissue and that from the plasma by diffusion. Obviously, 11β-HSD2 activity is responsible for reducing the cortisol level in kidney \[[@B17]-[@B19]\]. In addition, cortisol metabolism in liver is part of the balance maintaining the tissue-specific cortisol concentration. The circulating cortisol level undergoes circadian variations peaking in the early morning at approximately 800 nM and reaching a nadir of about 200 nM at midnight \[[@B22]\]. The plasma cortisone level is much lower and shows no significant circadian rhythm \[[@B22]\]. The salivary cortisol level exhibits a similar trend of diurnal rhythm \[[@B23]\]. Rodents housed under 12-h light, 12-h dark illumination conditions exhibit an opposite pattern of circadian variation with lowest circulating corticosterone levels in the early morning and the peak concentration at the light/dark transition phase before declining to nadir \[[@B24]\]. The plasma GC level is regulated by the activity of the hypothalamic-pituitary-adrenal (HPA) axis, a neuroendocrine feedback circuit that can be activated by physiological stimuli such as stress \[[@B25]\]. Plasma cortisone is largely in the free unbound form but approximately 6% cortisol is bound to albumin and 90% is bound to corticosteroid-binding globulin (CBG), a protein synthesized in liver and secreted in blood \[[@B26],[@B27]\]. Since only free cortisol is active, CBG binding may restrict the access of cortisol to target cells and regulate its bioavailability and metabolic clearance. On the other hand, CBG may act as a carrier protein for cortisol mediating its delivery to sites of inflammation \[[@B28],[@B29]\]. CBG is also present in several tissues and may be involved in the regulation of tissue-specific GC action. For example, the significantly lower CBG level in the adipose tissue of obese Zucker rats may contribute to insulin resistance \[[@B30]\]. CBG levels are down regulated by physiological changes such as stress \[[@B31]-[@B33]\]. Both cortisol and cortisone are metabolized in liver first by the A-ring reductases followed by several steps of further structural transformation catalyzed by other enzymes \[[@B20]\]. The final metabolites, 5α -- and 5β-tetrahydrocortisol (5α -- and 5β-THF) and 5β-tetrahydrocortisone (THE), are eliminated through urinary excretion and are often used as biomarkers for GC metabolism \[[@B20],[@B34]\]. While the total urinary tetrahydro metabolites (THF and THE) may serve as an indicator for GC metabolism or activity, using the ratio of the urinary THF to THE to predict the interconversion of cortisol and cortisone by 11β-HSDs is questionable for the following reasons: First, the ratio is a reflection of the total metabolism of cortisol and cortisone in the whole body instead of one particular tissue because the two isozymes have distinct tissue distribution patterns. Second, other enzymes, including the A-ring reductases and those involved in the subsequent metabolic steps forming THF and THE, also contribute to the balance between cortisol and cortisone. Therefore, the urinary ratio of THF to THE is determined by the combined activities of different enzymes in multiple tissues. Another convenient way to measure GC metabolism is to measure the salivary cortisol levels \[[@B20]\]. GC action is mediated by GR, a nuclear receptor that regulates physiological events through activation or repression of target genes involved in inflammation, gluconeogenesis and adipocyte differentiation \[[@B35],[@B36]\]. Upon activation, a GR dimer binds to GC response elements (GREs), interacts with components of the transcription machinery and activates the transcription of downstream genes \[[@B35],[@B36]\]. The ligand-bound GR could also bind to negative GREs (nGREs) that mediate the repression of gene transcription, or the starting point of transcription and thus interferes with the general transcription machinery \[[@B35],[@B36]\]. Some transrepression effects of GC action are achieved through a DNA binding-independent process, in which GR interacts with transcription factors such as AP-1 and NFκB and represses their activity on gene expression \[[@B37]-[@B39]\]. Repression of NFκB mediated transcription by GC can also be achieved by induction of IκB synthesis \[[@B40],[@B41]\]. Examples of genes regulated by GR and involved in the hepatic gluconeogenesis, adipocyte differentiation, hormonal control, and inflammation are summarized in Table [1](#T1){ref-type="table"}\[[@B39],[@B42]-[@B66]\]. The gene stimulation or suppression effects mediated by activated GR sequentially regulate a myriad of physiological actions in response to GCs. Since the pool of active cortisol or corticosterone is the active ligand for GR, the availability of free cortisol or corticosterone mediated largely by CBG-dependent protein binding and tissue-specific activities of 11β-HSDs are critical for GC action. The role of GC action in obesity and insulin resistance is implicated by the biological or physiological consequences of deficiency or activation of CBG or 11β-HSDs (see below). The GC production and tissue-specific conversions are illustrated in Figure [1](#F1){ref-type="fig"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Examples of genes regulated by GR ::: Gene Names Function Regulation Reference -------------------------------- ------------------------------------------------------------------------- ------------ ----------- Glutamine synthetase Amino acid metabolism Up 42 TAT Amino acid catabolism Up 43, 44 Tryptophan oxygenase Amino acid catabolism Up 45 PEPCK (liver) Gluconeogenesis Up 46 G6Pase Gluconeogenesis Up 47, 48 Angiotensinogen Precursor of angiotensin I; vasoconstriction, electrolyte balance, etc. Up 49 Leptin Energy metabolism Up 50 VLDLR Lipoprotein metabolism Up 51 PEPCK (adipose) Glyceroneogenesis Down 52 aP2 Intracellular lipid shuttling and metabolism Up 53 GLUT4 Glucose transport Up 53 HSL Lipolysis Up 53 LPL Lipid metabolism Up 53 TNF-α Inflammation and apoptosis Down 53 Osteocalcin Marker for mature osteoblasts Down 54, 55 CRH Stress mediated/feedback hormone release Down 56 POMC Precursor of pituitary hormones Down 57, 58 Prolactin Hormone critical for reproduction Down 59 Proliferin Angiogenesis Down 60, 61 Glycoprotein hormone α-subunit Common subunit of gonadotropin hormones Down 62, 63 IL-6 Proinflammatory cytokine Down 64 IL-8 Proinflammatory cytokine Down 65 Collagenase Matrix protease Down 66 ICAM-1 Inflammatory response Down 39 Abbreviations: TAT, tyrosine aminotransferase; PEPCK, phosphoenolpyruvate carboxykinase; G6Pase, glucose-6-phosphatase; VLDLR, very low density lipoprotein receptor; aP2, adipocyte fatty acid binding protein or A-FABP; GLUT-4, glucose transporter 4; HSL, hormone sensitive lipase; LPL, lipoprotein lipase; TNF-α, tumor necrosis factor α; CRH, corticotrophin-releasing hormone; POMC, proopiomelanocortin; IL-6, interleukin 6; IL-8, interleukin 8; ICAM-1, intercellular adhesion molecule 1. ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Glucocorticoid metabolism. The secretion of glucocorticoids by the adrenal gland is regulated by the HPA axis via secretion of ACTH. The main plasma cortisol (F) is protein bound with 4--5% free fraction. The plasma cortisone (E) is in the free unbound form. The equilibrium of cortisol and cortisone between the plasma and tissues are illustrated with the dotted bidirectional arrows. Tissue-specific GC metabolism are also depicted. GCs are metabolized primarily in liver and the metabolites are excreted in the urine. Only tissues relevant to the Metabolic Syndrome are shown. THE, tetrahydrocortisone; THF, tetrahydrocortisol. ::: ![](1743-7075-2-3-1) ::: Clinical association of GC action and the Metabolic Syndrome ============================================================ Accumulating clinical evidence has demonstrated the association of abnormal GC metabolism and the Metabolic Syndrome. The plasma cortisol levels were increased in an elderly cohort with one or more features of the Metabolic Syndrome \[[@B67]\]. Further, a good correlation was observed between total urinary GC metabolites and the number of features of the Metabolic Syndrome in these patients \[[@B67]\]. Both the secretion rate and peripheral clearance of cortisol in these patients were positively correlated with systolic blood pressure, fasting glucose and insulin \[[@B67]\]. In agreement with this finding, stress-related cortisol secretion in a population of 51-yr-old men showed associations with diastolic blood pressure, fasting glucose and insulin \[[@B68]\]. Several additional reports also suggest correlation of increased GC activity with insulin resistance, hyperglycemia and hypertension \[[@B69]-[@B71]\]. Although one study indicated that plasma cortisol levels decreased in obese women due to increased metabolic clearance \[[@B72]\], stress-induced cortisol response is consistently correlated with obesity in independent studies suggesting increased HPA activity in obesity \[[@B73]-[@B77]\]. Higher adrenocortical activity was also observed in children with higher body fat mass \[[@B78],[@B79]\]. Weight loss led to lower plasma cortisol and reduced insulin resistance \[[@B79]\]. A study in the general population indicates that even modestly increased cortisol levels contribute to obesity \[[@B80]\], and insulin resistance is positively associated with cortical activity \[[@B81],[@B82]\]. These clinical findings demonstrate the strong correlation of increased GC activity with the features of the Metabolic Syndrome in humans. The metabolic effects of GCs ============================ The clinical correlation studies raised the possibility that GC action could play a role in the origin of the features of the Metabolic Syndrome. This notion was further established and supported by animal studies to address the metabolic effects of GCs. Adrenalectomy in young ob/obor db/dbmice slowed body weight gain \[[@B83]\]. Upon GC administration, these animals retained body weight gain with concomitant increase in food intake \[[@B83]\]. Likewise, obese Zucker rats lost body fat mass after adrenalectomy and remained so even after exogenous administration of low doses of GCs \[[@B84]\]. The adrenalectomy resulted in significantly reduced plasma insulin, glucose and triglyceride levels \[[@B84]\]. As the doses of administered GCs increased, the plasma insulin and triglyceride levels were elevated \[[@B84]\]. Similar results were observed in another study using adrenalectomized rats with diet-induced obesity demonstrating the effects of GC action on plasma and liver triglyceride levels, plasma insulin, and adipose tissue weight \[[@B85]\]. These effects appear to be minimized when there is restriction on high-energy diet \[[@B86]\], suggesting they may be exerted via mediating the central ingestive behavior. These findings highlight the central role of GCs in the development of obesity and other features of the Metabolic Syndrome. The metabolic effects of GCs are mediated by several mechanisms that are physiologically relevant to hepatic and peripheral insulin resistance, dyslipidemia, obesity and hyperglycemia. Events driven by these mechanisms take place across the tissues contributing to the abnormalities in the Metabolic Syndrome (Fig. [2](#F2){ref-type="fig"}). In liver, GCs increase the activities of enzymes involved in fatty acid synthesis and promote the secretion of lipoproteins \[[@B87],[@B88]\]. The hepatic lipogenic effect of GCs is consistent with clinical findings that GC therapy causes triglyceride accumulation within the liver \[[@B89]-[@B91]\]. Since liver fat appears to be involved in the negative regulation of hepatic insulin sensitivity \[[@B92]\] and is associated with certain features of the Metabolic Syndrome independent of visceral fat mass \[[@B93]-[@B96]\], hepatic fat accumulation promoted by GCs is likely to contribute to the pathophysiology of the Metabolic Syndrome. GCs also induce the hepatic gluconeogenic pathway via the activation of GR, which stimulates the expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase), the rate-limiting enzymes in gluconeogenesis \[[@B97],[@B98]\]. This results in increased hepatic glucose output and hyperglycemia. In adipose tissue, GCs promote the differentiation of pre-adipocytes to adipocytes, which could lead to increased body fat mass \[[@B99],[@B100]\]. However, once differentiated, the adipocytes develop insulin resistance in the presence of GCs with decreased insulin-stimulated glucose uptake without changing their ability to bind insulin \[[@B101]\]. The reduced insulin sensitivity appears to be mediated by GC antagonizing the insulin-stimulated translocation of glucose transporters from intracellular compartments to the plasma membrane \[[@B102]-[@B104]\]. A similar mechanism is likely responsible for the GC-induced insulin resistance in skeletal muscle \[[@B105]\]. GCs also inhibit insulin-stimulated amino acid uptake by adipocytes \[[@B106]\]. Increased lipolysis or lipid oxidation could be also involved in the peripheral insulin resistance induced by GCs \[[@B107],[@B108]\]. GCs inhibit insulin secretion by the pancreatic β cells in animals and perturb high-frequency insulin release in the fasting state in human \[[@B109],[@B110]\]. GC action has been implicated in hypertension as well. GCs are agonists of mineralocorticoid receptor (MR), which upon activation leads to renal salt retention and elevated blood pressure. The expression of both 11β-HSD1 and 11β-HSD2 in kidney suggests the interconversion of inert and active GCs is maintained in a balance so that MR activation can be controlled tissue-specifically \[[@B111]\]. GC excess as a result of either increased 11β-HSD1 activity or reduced 11β-HSD2 activity leads to MR activation and hypertension. GCs also increase aortic vasoconstriction through unknown mechanisms. The expression of 11β-HSD1 in aortic endothelial cells is consistent with such a notion and suggests this could be a second pathway for GC induced hypertension \[[@B112]-[@B114]\]. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### The link between the metabolic effects of glucocorticoids and the features of the Metabolic Syndrome. The major effects in different tissues are summarized and the potential physiological links to the Metabolic Syndrome are shown. ::: ![](1743-7075-2-3-2) ::: These data, both physiologically and mechanistically, suggest that the metabolic effects of GCs are exerted in multiple tissues and increased GC action contributes to the etiology of the Metabolic Syndrome. Through molecular and genetic studies, more information has become available to dissect the role of tissue-specific GC action in the features of the Metabolic Syndrome. Genetic studies with the main players in GC action have been most revealing. Since GR has been well reviewed in other publications, this review will only discuss CBG and 11β-HSD1. Modulation of GC action by CBG is associated with adiposity =========================================================== CBG is not only in the blood but also found in tissues \[[@B115],[@B116]\]. Since CBG is the main GC binding protein, its tissue distribution and local levels play important roles in GC action. Intuitively, CBG level should be negatively correlated with the free cortisol or corticosterone concentration because of its role in restricting free GC fraction. This is especially true in a tissue-specific manner. For example, the reduced adipose CBG level in obese Zucker rat results in elevated free local corticosterone that may have contributed to the obesity and insulin resistance phenotype \[[@B30]\]. In general, in the human population, serum CBG levels are negatively correlated with a variety of parameters important in defining the Metabolic Syndrome: body mass index (BMI), waist to hip ratio (WHR), blood pressure and HOMA \[[@B117]\]. However, over-expression or secretion of CBG in the liver could lead to compensatory activation of the HPA axis and consequently elevated adrenal production of cortisol or corticosterone. This feedback response leads to a global effect of elevated total and free cortisol or corticosterone levels. This was observed in a pig genetic model with high fat deposits and low muscle content, where the hepatic CBG expression was significantly higher than in another population and the total and free cortisol levels were elevated \[[@B118]\]. On the other hand, drastic reduction of CBG concentration or its capacity to bind cortisol or corticosterone can also cause compensatory response by the HPA axis. A familial CBG deficiency led to decreased total and free plasma cortisol levels and hypotension \[[@B119]\]. Likewise, a human CBG polymorphism associated with reduced affinity for cortisol only led to a marginal increase in serum free cortisol, possibly due to the negative regulation of cortisol production by the HPA axis \[[@B120]\]. Together, these data demonstrate the importance of CBG level and its cortisol binding capacity in modulating GC action and origination of the Metabolic Syndrome. Further, these studies also suggest that the variation in CBG level or capacity may trigger compensatory response of the HPA axis to balance plasma free cortisol concentrations. Despite the compensatory response by the HPA axis to balance the plasma free cortisol or corticosterone concentrations under conditions of CBG reduction or deficiency, tissue-specific GC excess can still occur. This is especially important with respect to GC-stimulated differentiation of pre-adipocytes and insulin resistance of mature adipocytes, with the former effect increasing fat content and the latter reducing the tissue sensitivity to insulin. For instance, preadipocytes from an individual with CBG deficiency had increased proliferation and enhanced differentiation compared to normal cells \[[@B121]\], which may be responsible for the increased adiposity in CBG deficiency. This notion was observed in genetic models of obesity and insulin resistance. The CBG capacity the white adipose tissue of Zucker rat is lower than that in its lean counterpart \[[@B30],[@B122]\], suggesting increased GC action in the obese adipose tissue that could contribute to the obese and insulin resistance phenotype. 11β-HSD1 and obesity and insulin resistance =========================================== Both 11β-HSD1 and 11β-HSD2 are located at the endoplasmic reticulum (ER) but with distinct topologies. 11β-HSD1 has one short N-terminal transmembrane region with the catalytic domain protruding into the ER lumen; in contrast, the N-terminus of 11β-HSD2 is lumenal with the catalytic domain facing the cytoplasm \[[@B123]-[@B125]\]. The primary role of 11β-HSD2 is to prevent renal GC excess and consequent MR activation by inactivating cortisol or corticosterone, as mice deficient in 11β-HSD2 had hypokalemia and hypertension \[[@B126]\]. Given the growing interest in 11β-HSD1 and its role in the Metabolic Syndrome, this section will primarily focus on this isozyme. Dysregulation of tissue-specific 11β-HSD1 expression and activity has been observed in obese diabetic animal models and humans. Compared with their lean littermates, ob/obmice have reduced hepatic 11β-HSD1 activity but higher corticosterone level in liver due to their elevated plasma corticosterone \[[@B127]\]. As a result, the liver PEPCK expression is elevated at least partly contributing to hyperglycemia. However, the hepatic 11β-HSD1 activity is marginally increased in db/dbmice \[[@B128]\]. As in ob/obmice, the 11β-HSD1 activity is decreased in liver but increased in omental fat in obese Zucker rats \[[@B129],[@B130]\]. Although both impaired hepatic regeneration of cortisol by 11β-HSD1 and elevated adipose 11β-HSD1 activity were observed in obese humans \[[@B131],[@B132]\], the association of adipose 11β-HSD1 activity with obesity, insulin resistance and other features of the Metabolic Syndrome has been consistently observed in different groups of obese subjects, including obese men and women \[[@B131],[@B133],[@B134]\]. However, no difference in 11β-HSD1 activity was detected between obese type 2 diabetics and their obese controls, suggesting the dysregulation of 11β-HSD1 is better associated with obesity than the diabetic phenotype \[[@B135]\]. In-situ hybridization revealed that 11β-HSD1 mRNA is increased in both subcutaneous and visceral fat in obese subjects \[[@B136]\]. The association of adipose 11β-HSD1 with BMI and other features of the Metabolic Syndrome was also found in populations of different ethnic backgrounds \[[@B137]\]. In a group of young adult monozygotic twins, the intrapair differences in BMI are positively correlated with those in adipose 11β-HSD1 expression \[[@B138]\]. This association is clearly established on the same genetic background, confirming the direct link of adipose 11β-HSD1 activity and adiposity. Most of these association studies were done with subcutaneous fat. It is important to note that 11β-HSD1 activity is higher in omental fat and subject to stimulation \[[@B139]\]. The activity of 11β-HSD1 in adipocytes is relevant for the correlation since the activity in cultured preadipocytes does not appear to be correlated with obesity \[[@B140]\]. These association studies suggest that the adipose 11β-HSD1 may be a contributing factor to obesity and insulin resistance. In agreement with this conclusion, treatment of obese Zucker rats with carbenoxolone slightly improved lipid profile but had no effect on obesity and insulin resistance, because only the hepatic 11β-HSD1 but not that in adipose tissue was inhibited \[[@B141]\]. It is important to note that carbenoxolone also inhibits 11β-HSD2 and further studies with selective 11β-HSD1 inhibitors are needed to confirm this observation. In contrast to increased adiposity in the Metabolic Syndrome, some human immunodeficiency virus (HIV)-infected patients treated with combined highly active antiretroviral therapy (HAART) develop a lipodystrophic syndrome. The condition is characterized with loss of subcutaneous fat, accumulation of abdominal fat, hypertriglyceridemia and insulin resistance \[[@B142]\]. The condition is also referred to as pseudo-Cushing\'s syndrome because the distribution of fat accumulation in these patients is similar to that in Cushing\'s syndrome but their circulating cortisol levels are not elevated \[[@B143]\]. Interestingly, patients with lipodystrophy were shown to have higher levels of subcutaneous adipose 11β-HSD1 expression and higher ratios of urinary cortisol:cortisone metabolites than non-lipodystrophic patients \[[@B144]\]. These findings suggest that 11β-HSD1 could play a role in mediating the metabolic abnormalities of the HAART-associated lipodystrophy with the almost complete loss of subcutaneous fat. This further suggests that the expression of 11β-HSD1 seems to be more important to the metabolic state than the amount of subcutaneous fat though further investigation is required. Genetic studies using animal models support the findings in the clinical studies. In mice deficient in 11β-HSD1 generated through targeted gene disruption, there was no conversion of the inert 11-dehydrocorticosterone to corticosterone and attenuation of the hepatic activities of PEPCK and G6Pase, two key gluconeogenic enzymes \[[@B145]\]. These mice consumed more calories but were resistant to high fat diet-induced obesity, insulin resistance and hyperglycemia with improved lipoprotein profile \[[@B145]-[@B147]\]. Concomitant with these phenotypes, the adipose expression of TNF-α decreased, and adiponectin, PPARγ, and UCP-2 increased indicating insulin sensitization \[[@B146]\]. There were no bone marrow adipocytes in these knockout animals but bone formation appeared to be normal, suggesting that intracellular GC action may not play a role in bone formation \[[@B148]\]. The HPA axis appears to be activated in the 11β-HSD1 knockout mice. There was compensatory adrenal hyperplasia, increased secretion of corticosterone and exaggerated ACTH and corticosterone response to stress \[[@B145],[@B149]\]. The plasma CBG levels were slightly reduced \[[@B149]\]. These findings with 11β-HSD1 deficiency suggest inhibition of this enzyme could help ameliorate some of the features of the Metabolic Syndrome. However, compensatory response from the HPA axis and the induced adrenal activity can occur. Interestingly, 11β-HSD1 knockout ameliorated age-related learning impairments but the underlying mechanism is not clear \[[@B150]\]. The importance of 11β-HSD1 in the Metabolic Syndrome was also demonstrated with 11β-HSD1 transgenic animals. Mice with adipose-specific overexpression of the rat 11β-HSD1 had increased adipose levels of corticosterone and acquired features of the Metabolic Syndrome: diet-induced visceral obesity, insulin resistance, hyperlipidemia and hyperphagia \[[@B151]\]. The transgenic mice also developed hypertension, at least in part due to the increased adipose expression of angiotensinogen and the consequent activation of the rennin-angiotensin system (RAS) \[[@B152]\]. In contrast, selective overexpression of 11β-HSD1 in liver only caused mild insulin resistance with no effect on fat depot mass \[[@B153]\], although impaired hepatic lipid clearance and hypertension were observed in these animals. These transgenic studies demonstrate that both the hepatic and adipose 11β-HSD1 activities contribute in some way to insulin resistance and other features of the Metabolic Syndrome. However, the adipose activity appears to be correlated with a stronger phenotype of obesity and insulin resistance and therefore is likely the primary target for the treatment of insulin resistance. The hepatic 11β-HSD1 activity, although secondary, appears to be more important in improving lipid metabolism and controlling blood pressure. Several cases of human 11β-HSD1 deficiency have been reported. The ability of these subjects to convert cortisone to cortisol upon dexamethasone suppression was apparently compromised \[[@B154]-[@B158]\]. These patients appeared to be normal except for mild adrenal hyperplasia in some cases, and hirsutism, and elevated plasma cortisol levels \[[@B154]-[@B158]\]. Unfortunately, insufficient insulin sensitivity data have been reported with these patients. Although both obese and lean patients with 11β-HSD1 deficiency have been identified, it is not clear if the body weight is associated with 11β-HSD1 deficiency. However, polymorphisms in the 11β-HSD1 gene have been linked to adiposity in association studies with human subjects \[[@B159],[@B160]\]. Inhibitors of GC action ======================= Given its important role in the Metabolic Syndrome, antagonizing GC action has been taken as an approach to treat some features of the Metabolic Syndrome. Targeting GR is a direct approach to antagonize the GC action. The global effect on GC action by this approach could lead to the activation of the HPA axis as well as blocking the anti-inflammatory function of GCs. Inhibition of 11β-HSD1 activity offers more tissue specificity due to the limited expression pattern of this enzyme. Inhibitors for both 11β-HSD1 and GR include naturally occurring and pharmaceutically developed compounds. The expected effects of 11β-HSD1 inhibition include reduced hepatic PEPCK and G6Pase expression to reduce hepatic glucose output; reduced adiposity and improved peripheral insulin sensitivity. Since 11β-HSD1 mediated GC action inhibits glucose-dependent insulin secretion \[[@B161]\] and the expression of 11β-HSD1 is significantly increased in diabetic islets \[[@B162]\], 11β-HSD1 inhibitors can potentially help reduce postprandial glucose excursion. Several inhibitors of 11β-HSD1 were described in the literature prior to the pharmaceutical targeting of this enzyme in recent years but none of them is selective and highly potent. Metyrapone, often used in the diagnosis of adrenal corticoid-related disease such as Cushing\' syndrome, is a weak competitive inhibitor of 11β-HSD1 \[[@B163]\]. Other inhibitors include liquorice derivatives carbenoxolone (CBX) and glycyrrhetinic acid (GE) \[[@B164]\]. GE is more potent against the dehydrogenase activity and CBX is almost equally potent against activities of both directions (dehydrogenase and reductase). Although far more potent than other inhibitors, CBX and GE are not selective because they also inhibit 11β-HSD2. Chenodeoxycholic acid (CDCA) inhibits 11β-HSD1 with a potency of micromolar range but studies of its activity against 11β-HSD2 have generated conflicting results \[[@B165]-[@B167]\]. Although not selective, CBX has been used in human studies where it reduced glucose production during hyperglucagonemia largely due to its suppressive effect on glycogenolysis in lean male patients with type 2 diabetes \[[@B168]\]. Interestingly, CBX also improved verbal frequency and memory in healthy elderly men and patients with type 2 diabetes \[[@B169]\]. This is consistent with findings in 11β-HSD1 knockout mice \[[@B150]\]. Selective 11β-HSD1 inhibitors have been developed for pharmaceutical use in recent years. These inhibitors have been shown to be efficacious in diabetic animal models \[[@B170]-[@B173]\]. GR antagonists were developed on the rationale that activated GR stimulates PEPCK and G6Pase, the two key enzymes in hepatic gluconeogenesis that increases the hepatic glucose output \[[@B97],[@B98],[@B174]\]. Since hepatic gluconeogenesis in diabetics is increased \[[@B175]\], inhibition of hepatic GR action is expected for glucose lowering in diabetics. A well-known GR antagonist is RU-486, which was also found to have agonist activities \[[@B176]\]. Although efficacious \[[@B177]\], long-term systemic treatment with a GR antagonist may activate the HPA axis and increases cortisol secretion \[[@B178]\]. Other GR antagonists were also reported but without resolving the issue of HPA activation \[[@B179]\]. Selective inhibition of the hepatic GR activation in a non-systemic manner could provide advantages with no undesirable side effects. Liver selective targeting of the drug appears to be a good strategy \[[@B180]\]. Conclusions =========== GCs are stress hormones with a wide spectrum of physiological effects and have been implicated in the pathophysiology of the Metabolic Syndrome. This notion has been supported by animal studies and clinical findings. The GC action appears to mediate certain aspects of the Metabolic Syndrome. In that regard, targeting key players in the GC action is expected to be a viable approach to treat some or all the features of the Metabolic Syndrome. However, cautions should be taken because the GC metabolism is regulated by the HPA axis and inhibition of GC pathways could lead to the activation of HPA axis and elevated adrenal cortisol secretion. To avoid the compensatory feedback response, efforts to separate the effect of GC modulators from HPA activity is needed. Although challenging, this could be achieved by tissue-specific modulation of GC action by targeting drugs to tissues of interest while sparing others, especially the CNS where HPA activation occurs. The availability of small molecule compounds will facilitate this type of studies in animal models to further dissect the regulatory function of the HPA axis and help assess whether tissue selective modulation of GC action without triggering the HPA axis is achievable. Declaration of competing interests ================================== The author is an employed researcher in a biopharmaceutical company.	PubMed Central	2024-06-05T03:55:52.773324	2005-2-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548667/", "journal": "Nutr Metab (Lond). 2005 Feb 2; 2:3", "authors": [ { "first": "Minghan", "last": "Wang" } ] }
PMC548668	Background ========== After the tsunami hit Sri Lanka on 26 December 2004, news reports and public health agencies warned against the possibilities of an increase of vector borne diseases, in particular malaria and dengue. Immediately after the disaster, an estimated 860,000 people were displaced and more than 820 emergency camps established throughout the affected areas \[[@B1]\]. By 14 January, approximately 440,000 people were still sheltered in approximately 460 emergency camps \[[@B2]\]. Maps of the tsunami affected area, are presented elsewhere \[[@B3]\]. Malaria in Sri Lanka is of a highly unstable nature and has historically fluctuated greatly over the years and with significant seasonal differences. Sixty-five to eighty percent of the malaria cases are caused by Plasmodium vivaxand the remainder by Plasmodium falciparum\[[@B4]\]. Recently, an overview of the spatial and temporal distribution of malaria in Sri Lanka over the period 1995 -- 2002 was published in this journal \[[@B5]\]. The present publication aims at providing an update on the recent malaria situation, to October 2004 inclusive, and to discuss factors of relevance which may help in assessing the potential of the tsunami and ensuing events for exacerbating the malaria situation. Methods ======= Malaria maps were based on monthly records over the period January 2004 -- October 2004 (the most recent month for which data recording was complete at the time of writing) of microscopically confirmed malaria parasite positive blood smear readings, at district spatial resolution. These were collected by the Anti Malaria Campaign (AMC) Directorate of the Ministry of Health from aggregated disease records reported by governmental hospitals and mobile clinics. Additionally, in the temporal analysis, monthly data by district for the period 2001 -- 2002, and data by sub district for 1995 -- 2000 as described by Briët et al. \[[@B5]\] were used. The quality of routinely collected information on malaria is described elsewhere \[[@B5]\]. As denominator for the incidence calculations, population estimates for 2001 and beyond were made by exponential interpolation (and extrapolation to December 2004) (Figure [1](#F1){ref-type="fig"}) as follows. For the districts Mannar, Vavuniya, Trincomalee and Batticaloa, that were not or incompletely enumerated in the 2001 census because of limited access of the government to these conflict affected areas, the 2001 mid-year population was taken from data posted by the North East Provincial Council \[[@B6]\]. For all other districts, the 2001 mid-year population was taken from data posted by the Department of Census and Statistics \[[@B7]\]. The natural annual (mid-2001 to mid-2002 and mid-2002 to mid-2003) population growth rates for Jaffna, Kilinochchi, Mullaitivu, Mannar, Vavuniya, Trincomalee and Batticaloa were taken as the average annual growth rates of all the other districts, calculated from mid year population statistics estimated by the Department of Census and Statistics. For all other districts, these growth rates were calculated for each district separately. For mid 2003 to mid 2004 and beyond, the growth rates for mid-2002 to mid-2003 were used. Further, the number of internally displaced persons (IDPs) was taken into account \[[@B8]\]. For each month and for each district, the net number of immigrants was calculated as the total number of IDPs moved to or within a district since 2001, minus the number of IDPs moved from or within that district. This net number of immigrants was then distributed over the months proportionately to the monthly statistics of IDPs moved to or within a district. Additionally, the number of monthly immigrants from India was taken into account. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Population.Map of population by divisional secretariat division in Sri Lanka estimated for mid December 2004. One dot represents 1,000 people. Sources: Department of Census and Statistics <http://www.statistics.gov.lk/>, North East Provincial Council <http://www.nepc.lk/> and UNCHR <http://www.unhcr.lk>. ::: ![](1475-2875-4-8-1) ::: A focused review of literature has been performed, identifying crucial information for the outbreak preparedness and control during the emergency phase. The intent was not to present a complete review of malaria in Sri Lanka but to provide information useful for an assessment of the current situation. A general review of malaria in Sri Lanka can be found in Konradsen, Amerasinghe et al. \[[@B4]\]. Results and discussion ====================== Present malaria situation and parasite reservoir ------------------------------------------------ The country-wide malaria incidence increased from January 1996 to January 2000, with the typical seasonality of high peaks around January and lower peaks around June -- July, but it has decreased dramatically since January 2000 (Figure [2](#F2){ref-type="fig"}). Figure [3](#F3){ref-type="fig"} shows that the recent decrease in the overall malaria incidence in the country is predominantly due to a decrease in incidence in the districts of Vavuniya and Kilinochchi in the north. The decrease was least in the district of Ampara, making it the most malarious district during January to October 2004 (Figures [4](#F4){ref-type="fig"} and [5](#F5){ref-type="fig"}). Although districts on the east coast which were badly affected by the tsunami had been relatively malarious in 2004 as compared to the rest of the country, the maximum of around 1 case per 1000 people over a 10 month period in these districts is remarkably low. The total number of malaria cases in 2003 was 10,510, the lowest since the resurgence of malaria in 1968 when the eradication campaign failed \[[@B9]\]. The year 2004 promises to be three times lower with only 3,037 cases recorded up to October, as opposed to 9,682 cases recorded during January -- October 2003. The low incidence is not related to a decline in collection effort, which has decreased only marginally (Figure [2](#F2){ref-type="fig"}). At the time of writing, malaria incidence information for the months of November and December was still incomplete. In November 2004, without the figures for the non endemic districts Gampaha and Kalutara, and data from a few medical institutions in Mannar and Mullaitivu missing, thus far only 230 cases were recorded. In the malaria endemic districts, December, January and February are normally the months with the highest malaria incidence \[[@B5]\], so a rise in case numbers may normally be expected. However, neither the district authorities nor the Epidemiology Unit of the Ministry of Health have reported any malaria cases from the affected areas for 30 December 2004 -- 13 January 2005, based on the spot checks performed and the review of available health information \[[@B10]\]. Asymptomatic infections of P. falciparumand P. vivaxand dormant stages of P. vivaxnormally provide the parasite reservoir for bridging periods of low seasonal transmission (with unsuitable conditions for mosquito vectors). Under the present policy of administering primaquine in addition to chloroquine (see section on diagnosis and treatment), the reservoir of dormant stages of P. vivaxwill be low and this will delay a possible outbreak. It must be emphasized that the low level of malaria transmission in the recent past does not guarantee that localized or even island wide epidemics will not occur. In the past, even after periods of very low levels of malaria transmission, outbreaks have occurred, often due to constraints placed on the public health system, by unusual rainfall patterns or by yet unexplained factors. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Monthly parasite and blood smear examination incidence patterns.Monthly parasite incidence patterns of P. falciparumand P. vivaxmalaria combined per 1000 population (red line on logarithmic scale), blood smears examined per 1000 population (black line on logarithmic scale), and percentage of blood smears positive for malaria (blue line) from January 1995 to October 2004 in Sri Lanka. ::: ![](1475-2875-4-8-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Trends of parasite incidence.Trends of parasite incidence of P. falciparum(red bars) and P. vivax(blue bars) malaria over the years November 1995 -- October 1996 (bar on far left) to November 2003 -- October 2004 (bar on far right), at district resolution. The height of the bars in the legend represents an annual parasite incidence of 10 cases per 1000 persons. ::: ![](1475-2875-4-8-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### *Parasite incidence of Plasmodium vivax.**Map of the districts of Sri Lanka with P. vivaxmalaria cases per 1000 population over the period January -- October 2004. ::: ![](1475-2875-4-8-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Parasite incidence of Plasmodium falciparum.**Map of the districts of Sri Lanka with P. falciparummalaria cases and mixed infections of both P. vivaxand P. falciparumper 1000 population over the period January -- October 2004. ::: ![](1475-2875-4-8-5) ::: Capacity of health care services and disease surveillance --------------------------------------------------------- An important factor to consider in the current situation is the capacity of the existing health care service. Following the tsunami the Sri Lanka Ministry of Health reported 22 hospitals and nine administrative buildings damaged or completely destroyed, mostly in Ampara and Trincomalee districts \[[@B11]\]. It has been reported that at least 40 doctors and hundreds of other medical staff have died as a consequence of the tsunami and a much higher number injured or in other ways affected by the disaster \[[@B12]\]. However, both the central government departments and organizations in the field report sufficient medical staff. Even in the conflict affected areas in the north and east, the AMC has been able to monitor malaria and react timely with control measures to outbreaks since the peace process started in 2002. Also, the AMC has long standing experience with mobile clinics for malaria detection and treatment in remote areas. Lack of co-ordination among the many government departments, international aid agencies, non-governmental organizations and private individuals involved in the first phase of the emergency continues to be an important issue weeks into the disaster. According to the Ministry of Health media reports, more than 600 foreign doctors are now working in the affected areas, but few, if any, are registered with the Sri Lanka Medical Council or other relevant authorities \[[@B13]\]. With doctors from many countries, language barriers are also a perceived problem. In some places, central stocks of medical supplies were destroyed, including the Regional Medical Supply Division in the Ampara District. However, sufficient drugs have been imported during the days and weeks following the disaster. The World Health Organization has drawn up plans for antimalarials, insecticides and spray equipment to be made available on request. Although the increased capacity at the district and provincial levels has improved the co-ordination, a risk remains that local needs for health care are not adequately covered in spite of the availability of significant resources. In some parts of the island, especially areas in the east, affected both by the destruction caused by the tsunami and by exceptionally heavy rainfall in the weeks following, distribution of drugs has been problematic and this has left certain communities vulnerable. Whereas the overall capacity to provide treatment and routine malaria control activities, in general, has not been severely hampered, the routine health information system will have been constrained by the large number of autonomous health camps set up, and their lack of integration with the established surveillance system. It is essential to establish a system for monitoring malaria in the affected areas. Many people are moving back to their old place of residence trying to rebuild livelihoods and it will be essential for the public health authorities to keep contact with these communities to prevent an increase in malaria going unnoticed. Diagnosis, treatment and drug resistance ---------------------------------------- In Sri Lanka, microscopy on blood smears or use of rapid diagnostic test kits have been the standard diagnostic procedure, and precedes the prescription of drugs to the patient. In the current situation, with the many small health clinics established within emergency camps, it is likely that the use of rapid diagnostic kits would be the more feasible means of confirmation. The first line drugs recommended for malaria treatment in Sri Lanka is still a chloroquine and primaquine (PQ) combination for cases of P. vivaxas well as P. falciparuminfection. Primaquine is not administered to children below one year, and those with known G-6PD enzyme deficiency, and for pregnant mothers. So far, there have been no reports of chloroquine-resistant P. vivaxinfections in Sri Lanka. The first chloroquine-resistant P. falciparumcase was reported in 1984 \[[@B14]\]. Up to 62% in vivochloroquine resistance has been recorded in malarious areas \[[@B5],[@B15]-[@B17]\]. For chloroquine resistant cases of P. falciparumthe government recommended drug is sulphadoxine-pyrimethamine (SP). However, SP is not recommended for the last trimester of pregnancy, first six weeks of lactation and for children below two months of age. The first SP-resistant case of P. falciparumwas reported in 1992 in Polonnaruwa district. Up to 1999, five to six cases have been reported by the AMC. More recently (January -- June 2002), SP resistant P. falciparumhas been documented in the Northern Province \[[@B17]\]. For SP resistant cases quinine is recommended, but only as an in-patient treatment. In the current emergency situation, with many (foreign) doctors working autonomously, the diagnosis and treatment practices may depart from the established government guidelines and new antimalarials are also likely to be brought in. Moreover, the current practice of restricting SP to government hospitals will be difficult to enforce. Similarly, introduction of low quality and obsolete drugs will be difficult to counter at community level at the current stage of supervisory capacity and co-ordination level. Drugs have been reported stolen from warehouses, allegedly finding their way to private trade establishments \[[@B18]\]. Overall, it is crucial that the development of drug resistance is monitored closely and inappropriate drugs are actively phased out of the market to avoid later complications in case management. Environmental changes and vector breeding ----------------------------------------- The seawater brought inland by the tsunami has mixed with monsoon rainwater to form puddles of varying salinity. Also, thousands of muddy surface water puddles have been created as a result of destruction and rehabilitation activities that are already underway. The brackish puddles are expected to favour the breeding of Anopheles subpictussibling species B, which is a well-known coastal breeding species in Sri Lanka. However, it has not been directly incriminated as a field vector in Sri Lanka, despite its susceptibility to P. falciparum\[[@B19]\]. Nevertheless, Abhayawardana et al. \[[@B20]\] found peak malaria transmission in coastal areas of Puttalam in the presence of An. subpictussibling species B and the complete absence of Anopheles culicifacies(the main malaria vector in Sri Lanka), and suggested that this An. subpictussibling may have a role in transmission. It is noteworthy, that freshwater An. subpictus(which is now known to consist of a mixture of species A, C and D), which breeds in muddy rain fed puddles, has been consistently incriminated in malaria transmission in many inland areas of Sri Lanka \[[@B4]\]. Another species that is likely to breed prolifically in muddy rain-fed pools is Anopheles vagus. This species has been linked as a vector responsible for a malaria outbreak in southern Sri Lanka \[[@B4],[@B21]\]. On present evidence, neither An. subpictusnor An. vagus, are likely to cause major malaria epidemics but could, at high density, be responsible for focal outbreaks that need quick action. Thus, it is important that an entomological monitoring programme be set up in the period leading up to and during the south west monsoon that is expected during May to June 2005 in the tsunami affected western and southern Sri Lanka. It should be noted that the infamous Asian brackish water breeding malaria vector Anopheles sundaicus, which is a threat in the tsunami-affected areas in Indonesia, Myanmar, and the Andaman and Nicobar islands \[[@B22]\], does not occur in Sri Lanka. The main vector in Sri Lanka is An. culicifaciestype E \[[@B23],[@B24]\], which breeds mainly in pools formed in river and stream beds, and therefore, its density is mostly dependent on temporal and spatial variations in rainfall and river flow. Anopheles culicifaciesalso breeds in abandoned gem mining pits, agricultural wells and to a lesser extent in pools in agricultural water reservoirs \[[@B4]\]. It is unlikely that the rubble constituting a major part of the landscape in the affected areas creates breeding opportunities for An. culicifacies, unless it blocks waterways and creates pooling. Post-tsunami development activities may revive banned sand mining practices in rivers. If this happens, clear water pools created by these sand mining activities may serve as breeding sites for An. culicifacies\[[@B4]\]. Overall, it is very unlikely that the principal vector of malaria in Sri Lanka will breed prolifically in brackish water habitats or other habitats that may be created during the post tsunami reconstruction phase. Similarly, the principal dengue vector in Sri Lanka, Aedes aegypti, does not breed in saline water \[[@B25]\]. However, it may find plenty of rainwater-filled containers amidst the rubble created by the disaster for it to breed. Vector control strategies and insecticide resistance ---------------------------------------------------- The Colombo based Head Office of the AMC gives the overall guidelines for island wide vector control, while each province works out a plan for control activities based on the distribution and level of malaria transmission. Several malaria vector control interventions are currently employed within the country. In all districts, residual insecticide spray activities are focused on areas where malaria transmission has been established by confirmed malaria cases. The control of anopheline larvae using mostly chemicals focuses on sites close to human habitation. Small-scale application of larvivorous fish and environmental modifications are also carried out. Since 1997, mosquito nets, which are biannually treated with insecticide, are distributed free of charge in malarious areas. During the last two years, the main control effort has been through these nets. Since January 2004, 80,000 nets with long lasting insecticide have been distributed. Also, nets are available for purchase from outlets in most parts of the country. Studies in Sri Lanka over the 1990s on An. culicifaciesand a range of potential secondary vectors such as An. subpictusand An. vagushave shown high level of resistance to either organochlorines, organophosphates or to both groups of insecticides \[[@B4],[@B26]-[@B28]\]. DDT and Malathion are no longer recommended since An. culicifaciesand An. subpictushas been found resistant. Currently, synthetic pyrethroids such as Cyfluthrin, Deltamethrin, Etofenprox, and Lambda-cyhalothrin are being used in the country. At present, Fenitrothion is the only organophosphate used for vector control. A study conducted by Abhayawardana from 1990 to 1992 on An. subpictusfound 68% and 54% susceptibility to Malathion and Fenitrothion, respectively, for inland species (sibling species A), whereas for coastal species (primarily sibling species B) it was 100%. However, the latter was found resistant to permethrin \[[@B20]\]. From several districts it was reported that, as a result of the tsunami, organisations have brought in insecticides not normally used or no longer recommended for vector control in Sri Lanka (P. Amerasinghe, personal communication). Vector resistance, in the light of the introduction of new insecticides, needs to be monitored and if necessary action should be taken. Exposure of the affected community ---------------------------------- The majority of the people initially affected by the disaster are still living in emergency camps or in other places close to the coast. At the time of writing, to the best of our knowledge, relatively few people have moved from areas of low or no malaria transmission to areas of high transmission. However, during the next phases, when people may be resettled in semi-permanent and later in permanent housing, communities may be relocated from areas where they have had no malaria experience to malarious areas. In these situations, the communities\' capacity to cope with malaria infection will be low. Despite distribution of nets to many camps, and intensified vector control in some areas, people in the emergency camps (schools, temples, mosques, etc.) and those returning to damaged houses are more exposed to mosquito bites than in pre-disaster housing, due to the open nature of the shelter. Additionally, most families will have lost mosquito nets or other means to protect against mosquito bites. It is more difficult to assess the protective effect of tents that have been set up in most of the semi-permanent camps established. The location of semi permanent and permanent settlements may have a significant effect on the risk of infection. Epidemiological studies from other parts of Sri Lanka have shown that people living within 750 m of a stream with An. culicifaciesbreeding, were at significantly higher risk for malaria than people living further away \[[@B29]\]. Conclusions =========== This paper provides maps of both P. vivaxand P. falciparummalaria incidence distribution on the island of Sri Lanka at district resolution in the 10 months preceding the tsunami, and an analysis of monthly malaria incidence in the country since January 1995. The malaria incidence was historically low, which implies a limited parasite reservoir in the human population. In spite of the fact that the months of December - February are normally the peak period for transmission, given the transmission level in the months leading up to the disaster, the risk of a large-scale outbreak seems to be limited. However, the low transmission levels over the past years may also have made people less alert to possible outbreaks, and the population would have less protective immunity towards the disease. The environmental changes resulting from the tsunami do not create particular opportunities for breeding of the principal malaria vector An. culicifaciesbut potential does exist for less important species such as An. subpictusand An. vagus*. People living in emergency camps or returning to pre-disaster areas of residence are at higher risk of mosquito bites than normal. In spite of the emergency, the capacity of the public health authorities to perform malaria preventive and curative interventions remains high and essential supplies and staff capacity is not a problem. However, co-ordination of assistance and maintaining a strong surveillance system remain significant areas of concern. Increased attention to the establishment of a monitoring system including both parasitological and entomological parameters is recommended. Likewise, the large inflow of donated drugs and insecticides outside government control will potentially have long term implications on malaria control and case management, and especially the quality of administered drugs and the development of drug resistance requires careful monitoring. Authors\' contributions ======================= GNLG collected the malaria data. OJTB checked the data, calculated incidence, made the maps. FK, FPA and PHA performed the focused literature review. All authors helped write, read and approved the final manuscript. Acknowledgements ================ OJTB is funded through the NOAA, NSF, EPA and EPRI Joint Program on Climate Variability and Human Health. We acknowledge the Directorate of the AMC and Regional Malaria Officers for making malaria data available.	PubMed Central	2024-06-05T03:55:52.776836	2005-1-27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548668/", "journal": "Malar J. 2005 Jan 27; 4:8", "authors": [ { "first": "Olivier JT", "last": "Briët" }, { "first": "Gawrie NL", "last": "Galappaththy" }, { "first": "Flemming", "last": "Konradsen" }, { "first": "Priyanie H", "last": "Amerasinghe" }, { "first": "Felix P", "last": "Amerasinghe" } ] }
PMC548669	Background ========== CLC (cardiotrophin-like cytokine) shares homology with CNTF (ciliary neurotrophic factor) and CT-1 (cardiotrophin-1) and requires co-expression with either CLF (cytokine-like factor-1) or the soluble form of the CNTFR to be secreted \[[@B1],[@B2]\]. The CLC-CLF heterodimer displays activities only on cells expressing a functional CNTF receptor \[[@B1]\] and therefore CLC is likely to be part of the developmentally important second ligand for CNTFR. The existence of such a second ligand has been suggested by the phenotype of mice lacking any of the three receptor subunits comprising the functional CNTF receptor complex (LIFRβ, gp130 and CNTFR) which exhibit significant reductions in motoneuron number \[[@B3]-[@B5]\] whereas CNTF-deficient mice have no motoneuron loss during development \[[@B6]\]. There are however two prerequisites for CLC to play a major role in motoneuron development: 1) CLC must be expressed in the environment of motoneurons during development. 2) As it cannot be secreted alone, it must be co-expressed with either CLF or sCNTFR, in the same cell. Results and Discussion ====================== Developmental expression of clc ------------------------------- Since the expression of clchas only been studied in adult mouse tissues \[[@B7]\], we first examined the expression of genes encoding CLC or its co-secreted proteins, CLF and CNTFR in various embryonic tissues using reverse transcription and quantitative real-time polymerase chain reaction (RT-PCR). In all tissues tested from E16.5 and E18.5 (Table [1](#T1){ref-type="table"}), the level of expression of clcis very low when compared with that of clfor cntfr. The highest level of clcexpression was observed in the muzzle, a very heterogeneous region containing different positive tissues, as described below. Clcexpression is also observed in lung, kidney, brain and skeletal muscles such as the tongue or limb muscles. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### RT-PCR analysis of clc, clfand cntfrexpression^a^ ::: *clc* *clf* *cntfr* ----------------- -------------- -------------- --------------- -------------- --------------- -------------- Skeletal muscle 0.113 ± 0.02 0.936 ± 0.12 0.232 ± 0.003 24.84 ± 2.13 90.25 ± 2.71 6.72 ± 0.76 Heart NS^c^ NS NS NS NS NS Tongue 3.12 ± 0.12 1.2 ± 0.09 495 ± 17.1 77.1 ± 4.64 28.3 ± 10.9 2.6 ± 0.83 Muzzle 11.4 ± 0.75 9.25 ± 0.79 1050 ± 65.3 524 ± 85.8 425 ± 47.5 57.9 ± 4.02 Lung 4.08 ± 0.65 16.4 ± 0.48 2290 ± 490 2240 ± 184 43.8 ± 15.6 ND^b^ Kidney 5.55 ± 0.12 6.75 ± 1.15 61.4 ± 4.59 ND 51.8 ± 9.49 19.8 ± 3.12 Liver 0.191 ± 0.02 0.129 ± 0.03 0.071 ± 0.001 0.034 ± 0.09 0.047 ± 0.002 0.038 ± 0.03 Brain 1.04 ± 0.12 0.317 ± 0.09 150 ± 3.39 74.3 ± 16.4 216 ± 2.01 236 ± 16.1 Spinal cord ND 0.183 ± 0.01 0.123 ± 0.01 61.2 ± 6.25 6.13 ± 0.03 109 ± 27.4 ^a^Expression of clc, clfand cntfrwas determined using reverse transcription and quantitative real-time PCR as detailed in Experimental Procedures and expressed as fM of cDNA/μg total RNA. ^b^not determined ^c^not significant ::: To further assess the potential involvement of CLC in the development of motoneurons, we performed in situhybridization experiments to determine the pattern of expression of clcin the environment of developing motoneurons and compare it with the expression of both clfand cntfr. Motoneuron death occurs between E14.5 and E18.5 in mice lacking in the ability to produce a functional CNTF receptor complex \[[@B5]\], suggesting that expression of CNTFR and its relevant ligands is critical between these timepoints. We therefore studied clcmRNA expression levels at E16.5. Clcis expressed in muscles along the whole rostro-caudal axis, at the brachial level (Fig. [1A](#F1){ref-type="fig"}) as well as at the lumbar level (Fig. [1E](#F1){ref-type="fig"} and \[[@B8]\]. It is also expressed in the tongue (Fig. [1C](#F1){ref-type="fig"}) like clf (Fig. [1D](#F1){ref-type="fig"}). The identity of muscle cells (Fig. [1G](#F1){ref-type="fig"}) was confirmed by double staining performed on transgenic mice with the nlacZreporter gene under the control of the muscle-specific MLC promoter \[[@B9]\]. All clc-positive muscle fibers also stained positive for clf(Fig. [1E](#F1){ref-type="fig"}, [1F](#F1){ref-type="fig"}, [1G](#F1){ref-type="fig"}, [1H](#F1){ref-type="fig"} and \[[@B8]\]). clcexpression was not detected in certain clf-positive muscles however, such those around the vibrissae (Fig. [1I](#F1){ref-type="fig"} and [1J](#F1){ref-type="fig"}). Since the level of clcexpression is generally low, this could reflect the limited sensitivity of the in situhybridization technique used. To determine the onset of clcand clfexpression in the muscles, the motoneuron targets, we performed in situ hybridizations at different stages. Clcand clfare expressed, although at low levels, as soon as the muscles develop and are clearly observed at E14.5 (Fig. [1K](#F1){ref-type="fig"} and [1L](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### *In E16.5 mouse embryos clcis expressed in muscles.Cryostat sections (A, E-L) or vibratome sections (B-D) from E16.5 (A-J) or E14.5 (K, L) were hybridized to clc(A, C, E, G, I and K) or clf(D, F, H, J and L). The control clcsense probe gave rise to very faint staining (B). Transverse section through the forelimb (A, K and L), the hindlimb (E and F) and saggital sections through the tongue (C and D) showing expression of clcand clfin muscles. The identity of the clc-positive cells such as muscle fibers was confirmed by double staining and compared to clf-positive cells. In situhybridization using Dig-labeled probes for clcand clf(cytoplasmic blue staining) was performed on sections through shoulder muscles (G, H) or vibrissae (I, J) from E16.5 MLCnlacZmice, which express the nlacZreporter gene under the control of a muscle-specific MLC promoter. Subsequently, the sections were processed for immunohistochemical detection of β-galactosidase (nuclear brown staining). Arrows indicate double-labeled cells. Transverse section through the muzzle (I and J) shows that vibrissae (arrowheads) are positive for clcand clfwhereas only clfis detected in muscles (asterisks) surrounding vibrissae. c, cartilage; m, muscle. Scale bars are 200 μm in A, E and F, 25 μm in G and H and 100 μm in I-L. ::: ![](1478-811X-3-1-1) ::: Clcis also expressed in several organs in which reciprocal epithelial-mesenchymal interactions are essential, such as the developing vibrissae (Fig. [1I](#F1){ref-type="fig"} and [2I](#F2){ref-type="fig"}), tooth, kidney, and lung. In the kidney, clcis expressed in the comma-shaped body (Fig. [2A](#F2){ref-type="fig"}). Strikingly, CLF and CNTFR are expressed in different structures, clfbeing synthesized in the tips of the ureteric (Fig. [2B](#F2){ref-type="fig"}) and cntfrbeing synthesized by mesenchyma cells surrounding these structures (Fig. [2C](#F2){ref-type="fig"}). In the lung, both clcand cnftrare expressed faintly in distal airway epithelium whereas clfis strongly expressed in distal and proximal epithelia (Fig. [2D](#F2){ref-type="fig"}, [2E](#F2){ref-type="fig"} and [2F](#F2){ref-type="fig"}). Sections through molar tooth germs (Fig. [2G](#F2){ref-type="fig"} and [2H](#F2){ref-type="fig"}) show that clfis expressed in both the mesenchyma surrounding the dental follicle which gives rise to alveolar bone and the inner enamel epithelium whereas clcis expressed only in the former. Clcand clfare also co-expressed in the epithelium bordering the mandibles and the lips although clfis also expressed in mesenchyma (Fig. [2I](#F2){ref-type="fig"} and [2J](#F2){ref-type="fig"}). Together these results are in agreement with the expression pattern described for both clf\[[@B10]\] and cntfr\[[@B11]\]. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### *Clcis expressed in epithelia*Transverse sections from E16.5 mouse embryos were hybridized to clc(A, D, G and I), clf(B, E, H and J) or cntfr(C and F). Sections through the kidney (A-C) show that clcis expressed in developing nephrons (arrows), clfin ureteric tips (arrowheads) and cntfrin nephrogenic mesenchyme. Sections through the lung (D-F) show that whereas clfis strongly expressed in both distal (arrowheads) and proximal (arrows) epithelia, clcand cntfrare weakly expressed in distal epithelium. Boxed areas are shown in higher magnification in the corner of each panel. Sections through molar tooth germs (G, H) show that mesenchyma (arrows) surrounding the dental follicle is positive for both clcand clfand that the inner enamel epithelium (arrowheads) expresses only clf. Coronal sections through muzzle (I, J) show that both clcand clfare expressed in the epithelium bordering the mandibles and in between the lips and mandibles (arrow) as well as in follicles of vibrissae (arrowheads); in addition, clfis expressed in mesenchyma (asterisks). a, pulmonary artery; dp, dental papilla; de, dental epithelium; oc, oral cavity; uli, upper lip; lli, lower lip. Bars: 100 μm in A-H, 200 μm in I and J. ::: ![](1478-811X-3-1-2) ::: Co-expression of clc, clfand cntfrin the developing muscle ---------------------------------------------------------------- In transfected cells CLC requires either CLF or sCNTFR to be secreted \[[@B1],[@B2]\]. This cooperative effect requires the expression of genes for both factors in the same cell. To ascertain whether a single muscle cell can express at least CLC and CLF or CLC and sCNTFR, we studied co-expression on hind-limb muscle sections. We performed double in situhybridization of clcand clfand of clcand cntfr. Most muscle cells expressed both clc, (revealed using NBT/BCIP; Fig. [3A, C](#F3){ref-type="fig"}) and clfor cntfr(Fig. [3B, D](#F3){ref-type="fig"}; revealed using Fast Red). Co-expression was observed at the single cell level demonstrating that in vivoCLC could be co-secreted either with CLF or sCNTFR. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Double-labeling detects co-expression of clcand clfor clcand cntfrin individual muscle cells.Single sections of E16.5 muscles were hybridized with two probes. Dig-labeled clc(A-D) and Fluo-labeled clf(A, B) or cntfr(C, D). Anti-Dig antibodies were applied first and stained using NBT/BCIP to reveal cells expressing clc(A, C). Anti-Fluo antibodies were then applied and detected using Fast red to reveal cells expressing clf(B) and cntfr(D), after removal of the first red reaction product. Most muscle cells express clcand clfor clcand cntfr(examples indicated by arrows). Bars: 100 μm. ::: ![](1478-811X-3-1-3) ::: Conclusions =========== Clcis expressed in developing muscles during the period of motoneuron loss in mice lacking a functional CNTF receptor and it is co-expressed with both CLF and CNTFR. This expression pattern is in favor of the hypothesis that CLC is an important player in the signal triggered by the CNTF receptor and that is required for motoneuron development. In addition, our results show that in the kidney, clcis expressed in cells neighboring those expressing clfor cntfrbut it is not co-expressed with these genes suggesting either the possible existence of an additional protein capable of inducing secretion of CLC or that CLC is not secreted in these cells and therefore not functional. Because genetic deletion of cntffails to perturb neuronal development before birth, we can hypothesize some functional redundancies in vivothat will require the analysis of double or triple knockout mice for CNTFR ligands to clarify their respective involvement in mouse neural development. Methods ======= RT and real time PCR -------------------- Total RNA was extracted using Trizol reagent (Invitrogen) from E16.5 or E18.5 mouse tissues according to the manufacturer\'s instructions. Complementary cDNA was synthesised from 2 μg of RNA by random hexamer priming using MMLV reverse transcriptase (Promega). Quantitative PCR was performed using a capillary real-time LightCycler (Roche Diagnostics), and the data analysed using \"Fit Point Method\" (Roche Diagnostics). For comparison of gene expression levels, all quantifications were normalized to endogenous gapdhto account for variability in the initial concentration of RNA and for differences in the efficiency of the reverse transcription reactions. The following primers were designed to amplify mouse clc: 5\'-GCTACCTGGAGCATCAACT-3\', 5\'-GGTGACTGTACGCCTCATAG-3\'; clf: 5\'-CAGTCAGGAGACAATCTGGT-3\', 5\'-ACGTGAGATCCTTCATGTTC-3\'; cntfr: 5\'-CTACATCCCCAATACCTACA-3\', 5\'-GTGAATTCGTCAAAGGTGAT-3\'; gapdh: 5\'-TGCGACTTCAACAGCAACTC-3\', 5\'-CTTGCTCAGTGTCCTTGCTG-3\'. Results are expressed in fmole of cDNA/μgRNA. Probes ------ Plasmid cDNA clones were linearized and transcribed with T7 or T3 polymerase using digoxigenin (Dig) or fluorescein (Fluo)labeling reagents (Roche Diagnostics). Probes were used at a concentration of 500 ng/ml. The cntfrclone was as previously described \[[@B12]\] and the mouse clf\[[@B13]\] and clcprobes corresponded to the isolated cDNAs. In situ hybridization --------------------- In situhybridization was performed as described previously \[[@B14]\] on 20 μm-thick frozen transverse cryostat sections prepared from mouse embryos fixed with 4% paraformaldehyde in PBS, and cryopreserved in 15% sucrose in PBS before embedding in OCT compound (Miles). Alternatively, 100 μm-thick vibratome sections were prepared from fixed embryos embedded in glutaraldehyde/gelatin. After hybridization overnight at 70°C with Dig-labeled riboprobes, the slides were washed twice in 1X SSC, 50% formamide at 70°C for 30 min and blocked in the presence of 4% blocking reagent (Roche Diagnostics) and 20% inactivated sheep serum. The slides were then incubated with anti-Dig-alkaline-phosphatase (AP)-conjugated antibody (1/5000, Roche Diagnostics), washed and revealed by NBT/BCIP staining. In order to confirm that muscle fibers, per se, express clcand clf, double in situhybridization / immunohistochemistry was carried out as described \[[@B15]\] on sections from E16.5 MLCnlacZmice, which express the nlacZreporter gene under the control of a muscle-specific myosin light chain promoter. After in situhybridization, slides were rinsed in PBT (PBS, 0.1% Triton), and sections were successively incubated for 1 h with blocking solution containing 2% BSA, 2% heat-inactivated donkey serum in PBT and then overnight at 4°C with rabbit anti-β-galactosidasel (1/1000, Cappel). After three washes in PBT, slides were incubated 1 h at RT with a biotin donkey anti-mouse secondary antibody. Slides were then washed in PBS, and TBS (50 mM Tris-HCl, 0.15 M NaCl, pH 7.6), and incubated for 30 min at RT in ABC streptavidin/HRP in TBS. Staining was revealed with DAB (D4293, Sigma) in the presence of H~2~O~2~. Double in situhybridization was performed as described previously \[[@B14]\]. Briefly, Dig- and Fluo-labeled probes were mixed in hybridization buffer and applied to sections. After hybridization at 70°C overnight and washing at 65°C, the first probe was revealed using a 1:2000 dilution of anti-Fluo-alkaline phosphatase (AP)- conjugate (Roche Diagnostics) and Fast Red (Sigma) as a substrate. Sections were photographed at this stage. After AP inactivation with 0.1 M glycine, pH 2.2, the second probe was revealed using a 1:5000 dilution of anti-Dig-AP and NBT/BCIP staining. Fast Red precipitates were then removed by incubating the slides in increasing concentrations of ethanol culminating in two final incubations in 100% ethanol for 10 min before cleaning with Histoclear and mounting with Eukitt (VWR, Strasbourg, France). Photomicrographs of the NBT/BCIP results were then taken for comparison with those showing the Fast Red results on the same sections. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= BB performed in situ hybridizations whereas DD and HG performed RT-PCR analyses. GE and JFG provided the clcand clf*probes before publication. OL participated in the experimental design and coordination of the research. All authors read and approved the final manuscript. Acknowledgements ================ We thank members of INSERM U.623 and U.564 for many helpful discussions and encouraging support. This work was funded by INSERM, CNRS, the Association Française contre les Myopathies (AFM), the post-genome program from Région Pays-de-la-Loire, the Canadian Institutes of Health Research (IRSC) and the Multiple Sclerosis Scientific Research Foundation (SP). Damien Derouet was supported by INSERM and the Région Pays-de-la-Loire.	PubMed Central	2024-06-05T03:55:52.779038	2005-1-31	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548669/", "journal": "Cell Commun Signal. 2005 Jan 31; 3:1", "authors": [ { "first": "Béatrice", "last": "de Bovis" }, { "first": "Damien", "last": "Derouet" }, { "first": "Jean-François", "last": "Gauchat" }, { "first": "Greg", "last": "Elson" }, { "first": "Hugues", "last": "Gascan" }, { "first": "Odile", "last": "deLapeyrière" } ] }
PMC548670	Background ========== The inflammatory response is in the first instance a mechanism of self-defense, set by the innate immune system against endogenous and exogenous insults, and essential for the survival of the organism. Inflammation must be tightly regulated as deficiency as well as excess in its response will result in pathological conditions, such as immunodeficiency or chronic inflammatory diseases \[[@B1]\]. In the last decade increasing evidence has highlighted the role of inflammation in most brain pathologies, including immune-mediated diseases such as multiple sclerosis, acute neurodegeneration following ischemia or trauma, and, more recently, chronic neurodegenerative diseases \[[@B2]\]. Among the endogenous mechanisms that regulate the inflammatory response, cross-talk between the immune and nervous systems play an important role. In particular, it has been shown that electric stimulation of the vagus nerve attenuates the inflammation during endotoxemia in rats \[[@B3]\], and that acetylcholine (ACh), the main parasympathetic neurotransmitter, effectively deactivates peripheral macrophages and inhibits the release of pro-inflammatory mediators, including the cytokine tumor necrosis factor-α (TNF-α). The ACh-dependent macrophage deactivation is mediated by the α7 subunit of the nicotinic ACh receptor (herein referred as α7 subunit), which is expressed in peripheral macrophages and has been described as essential for the so called \"cholinergic anti-inflammatory pathway\" \[[@B4],[@B5]\]. Neuronal acetylcholine receptors (nAChRs) are ligand-gated ion channels, which belong to a large family of neurotransmitter receptors that includes the GABA~A~, glycine and 5-HT~3~receptors \[[@B6]\]. Each nAChR consists of five homologous or identical subunits arranged around a central ion channel whose opening is controlled by ACh, nicotine and other receptor agonists \[[@B6]\]. At least 8 α subunits (α2--9) and three β subunits (β2--4) have been identified and the combinatorial association of different α and β subunits results in a variety of nAChRs \[[@B7]\]. In addition to neurons and peripheral macrophages, several studies have demonstrated the expression of nAChRs in cell types both within and outside the nervous system \[[@B8]\]. In the CNS, the presence of nAChRs has been demonstrated in O~2~A-oligodendrocyte precursor cells but not in adult differentiated oligodendrocytes, suggesting that receptor expression is developmentally regulated \[[@B9]\]. Cultured hippocampal astrocytes express functional α7 receptors \[[@B10]\] and cortical astrocytes express both nicotinic and muscarinic receptors \[[@B11]\]. A functional α7 nicotinic receptor has been recently described in murine microglial cells \[[@B12]\]. In peripheral organs, human and rat epithelial and endothelial cells express functional α7 receptors, as well as other nicotinic subunits such as α3, α5, β2 and β4 \[[@B13],[@B14]\]. Acute or chronic exposure to nicotine has been shown to influence cell viability and motility of bronchial epithelial and endothelial cells \[[@B13]\]. Furthermore, nicotine has been shown to suppress the antimicrobial activities of murine alveolar macrophages \[[@B15]\]. Lymphocytes present both muscarinic and nicotinic receptors and it has been demonstrated that the interaction with antigen presenting cells enhances the synthesis and release of ACh \[[@B16]\]. These observations suggest that ACh might function as an important modulator of cellular interactions and immune functions. Epidemiological studies indicate that nicotine, besides its immunosuppressive effects, may be protective against the development of neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson\'s disease (PD) \[[@B17]\], in which a local inflammatory response is sustained by microglial cells, the largest population of phagocytes associated with the CNS. In normal healthy brain, microglial cells show a typical down-regulated or \"resting\" phenotype when compared to other tissue macrophages, but they rapidly react in response to a number of acute and chronic insults. Activated microglial cells could cause neuronal damage via liberation of free radicals as well as cytokines and toxic factors. Alternatively, microglia can exert neuroprotective functions by secreting growth factors or diffusible anti-inflammatory mediators, which contribute to resolve inflammation and restore tissue homeostasis \[[@B18],[@B19]\]. Thus, understanding the molecular mechanisms governing microglial activation is essential to prevent tissue damage related to excessive activation. Since nicotine and ACh have been recently reported to inhibit TNF-α production in mouse microglial cultures, the aim of our study was to extend our knowledge on the effect of α7 subunit stimulation on the functional state of activated microglia. We first confirmed that rat microglia express the α7 subunit and we demonstrated that, in addition to inhibit TNF-α, the α7 agonist nicotine significantly up-regulated COX-2 expression and PGE~2~synthesis. Other important microglial products, such as interleukin-1β (IL-1β), nitric oxide (NO) and interleukin-10 (IL-10) were not affected or moderately decreased. Materials and methods ===================== Reagents -------- All cell culture reagents were from Gibco (Grand Island, NY, U.S.A) and virtually endotoxin free (less then 10 E.U./ml as determined by the manufacturer). BCA protein assay was from Pierce (Rockford, Illinois). ELISA-kits for rat TNF-α and IL-10 were from Endogen Inc. (Woburn, MA). ED-1 monoclonal antibody was from Serotec (Oxford, UK). (±) Nicotine, α-bungarotoxin, FITC-α-bungarotoxin and lipopolysaccharide LPS (from Escherichia coli, serotype 026:B6) were from Sigma Chemical (St.Louis, MO). Rabbit polyclonal antibody against alpha 7 subunit was from Santa Cruz Biotechnology. Cell cultures ------------- Microglial cultures were prepared from 10--14 day mixed primary glial cultures obtained from the cerebral cortex of 1-day-old rats, as previously described \[[@B20]\] and in accordance with the European Communities Council Directive N. 86/609/EEC. Microglial cells, harvested from the mixed primary glial cultures by mild shaking, were resuspended in Basal Eagle\'s Medium (BME) supplemented with 10 % fetal calf serum, 2 mM glutamine and 100 μg/ml gentamicin, and plated on uncoated plastic wells at a density of 1.25 × 10^5^cells/cm^2^. Cells were allowed to adhere for 20 min and then washed to remove non-adhering cells. After a 24 h of incubation, the medium was replaced with fresh medium containing the substance(s) under study. Cell viability was greater than 95%, as tested by Trypan Blue exclusion. Immunostaining, performed as previously described \[[@B20]\], revealed that cultures consisted of ≥ 99% positive cells for the microglia/macrophage marker ED1. Microglial cells were pre-stimulated for 30 min with nicotine and then stimulated for 24 h in the presence of 10 ng/ml LPS. A rat pheochromocytoma cell line, PC12, was propagated and maintained in RPMI-1640 medium supplemented with 5% heat-inactivated fetal bovine serum (FBS) and 10% heat-inactivated horse serum (HS) 100 U/ml penicillin, 100 μg/ml of streptomycin, and 2 mM L-glutamine. The cells were plated in 12-well plates for 24 h before performing RNA extraction. Cytokines nitric oxide and PGE~2~determination ---------------------------------------------- At the end of the incubation time, cell supernatants were collected, centrifuged, and stored at -70°C until tested. The levels of TNF-α and IL-10 were assayed by specific ELISAs, following the manufacturer\'s instructions. The ranges of determination were: 31--2500 pg/ml for TNF-α, 10--1000 pg/ml and 8--500 pg/ml for IL-10. The production of NO by measuring the content of nitrite, one of the end products of NO oxidation, as previously described \[[@B21]\]. PGE~2~content was quantified using a specific radioimmunoassay \[[@B21]\]. The assay detection limit was 25 pg/ml and cross-reactivity of the antibody for PGE~2~with other prostaglandins less than 0.25%. Immunostaining of microglial cells with α-bungarotoxin and western blot analysis -------------------------------------------------------------------------------- Microglial cells were plated on uncoated glass coverslips (2.5 × 10^5^cells/cm^2^), allowed to adhere for 20 min and then washed to remove non-adhering cells. After a 24 h of incubation, the complete BME medium was replaced with fresh BME medium without serum. Cells were incubated at 4°C for 15 min with FITC-labeled α-bungarotoxin at 1.5 μg/ml. Where indicated, nicotine was added at the concentration of 500 μM for 10 min, in order to saturate all the binding sites before the addition of FITC-labeled α-bungarotoxin. Cells were washed 3 times with BME medium and then fixed with 4% paraformaldehyde at room temperature for 15 min. After fixation, coverslips were washed twice with PBS solution, mounted in PBS:glycerol and examined using a fluorescent microscope. Cell culture lysates from microglial cells and PC12 cells (used as positive control) were analyzed for α7 subunit expression. Total protein content was estimated using the Bio-Rad protein assay. An aliquot corresponding to 50 μg (microglia cells) and 20 μg (PC12 cells) of total protein for each sample was separated by sodium dodecyl sulphate polyacrylamide gel elecrophoresis (SDS-PAGE) and transferred electrophoretically to nylon membranes. Membranes were blocked with 10% non-fat milk and incubated with a rabbit policlonal antibodies against α7 subunit (1:2000) overnight at 4°C. Horseradish peroxidase conjugated anti-rabbit IgG (1:5000, 1 h at 25°C) and ECL reagents were used as detection system. RNA extraction and semiquantitative RT-PCR analysis --------------------------------------------------- Total RNA was prepared from rat microglia, PC12 cells and rat hippocampus using Trizol reagent according to manufacturer\'s protocol. Two μg of denatured total RNA were converted into first-strand cDNA using the SuperScript™synthesis system (Life Technologies™) in a total reaction volume of 20 μl following the conditions provided by the manufacturer\'s protocol. Oligonucleotide primers with similar Tm were designed to generate a PCR fragment of 754 bp for the α7 subunit. PCR conditions (number of cycles and cDNA and primer concentration) that ensure the data to be obtained within the exponential phase of amplification of each template were carefully assessed. The amplification of the β-actin, COX-2 and α7 subunit within the exponential phase of amplification was achieved with 25, 30 and 40 cycles respectively. Five μl, 15 μl and 40 μl of diluted cDNAs were amplified for β-actin, COX-2 and α7 respectively. PCR-amplification was done in a final volume of 50 μl containing 1x PCR buffer, the four dNTPs (0.2 mM), MgSO~4~(2 mM), 1 Unit of Platinium Taq DNA polymerase High Fidelity (Invitrogen). The primers were: α7 subunit (Gene bank accession number S53987), sense 5\'-TCT GTG CCC TTG ATA GCAC, antisense 5\'-CTT CAT GCA ACC AGG ATC AG, product length 754; COX-2 \[[@B22]\], sense 5\'-TGA TGA CTG CCC AAC TCC CATG; antisense 5\'-AAT GTT GAA GGT GTC CGG CAGC, product length 702 bp; β-actin (accession number NM031144) sense 5\'-GTC GAC AAC GGC TCC GGC ATG; antisense 5\'-CTC TTG CTC TGG GCC TCG TCGC, product length 158 bp. A sample containing all reaction reagents except cDNA was used as PCR negative control in each experiment. The absence of genomic DNA was verified using 2 μg of RNA from microglia that was reverse-transcribed without the enzyme (-RT). The PCR conditions for COX-2 were as follows: initial denaturation at 94°C for 2 min followed by 30 cycles of 94°C for 30 sec, 58°C for 45 sec, 68°C for 1 min, and an additional cycle with extension at 72°C for 7 min. The PCR conditions for β-actin were as follows: initial denaturation at 94°C for 5 min followed by 25 cycles of 94°C for 30 sec, 68°C for 30 sec, 68°C for 45 sec and an additional cycle with extension at 72°C for 1 min. The PCR conditions for α7 subunit were as follows: initial denaturation at 94°C for 5 min followed by 40 cycles of 94°C for 30 sec, 57°C for 1 min, 68°C for 45 sec and an additional cycle with extension at 72°C for 7 min. PCR products were analyzed by electrophoresis, stained with ethidium bromide and photographed. Transcript levels were analyzed by Fluor-STM Multimager analyser (Biorad). For each experiment, the ratio between optical density (arbitrary units) of bands corresponding to COX-2 and β-actin (used as internal standard) was calculated to quantify the level of the transcripts for COX-2 mRNAs. Statistical analysis -------------------- Data are expressed as mean ± SEM with the number of independent experiments, run in duplicate, indicated in figure legends. Comparison between treatment groups was made by Student\'s t-test. A two-tailed probability of less than 5 % (i.e. p \< 0.05) was taken as statistically significant. Results ======= Expression of α7 subunit mRNA in microglial cultures ---------------------------------------------------- The expression of the mRNA for α7 subunit in rat microglial cells was investigated by RT-PCR. As shown in Figure [1A](#F1){ref-type="fig"}, we detected a band of the expected size of 754-bp, which was then confirmed to correspond to α7 subunit by sequencing (M-Medical, Pomezia, I). The absence of genomic DNA contamination was demonstrated amplifying 2 μg of total RNA from microglia that was reverse-transcribed without the enzyme (Fig. [1B](#F1){ref-type="fig"}). As positive controls, we analyzed the expression of α7 subunit mRNA in rat hippocampus and PC12 cells (Fig. [1C](#F1){ref-type="fig"}), known to express the α7 subunit at high levels \[[@B23],[@B24]\]. The expression of α7 subunit at protein level was established by western blot analysis using a specific antibody for the α7 subunit, which recognized a clear band with a molecular mass of approximately 55 kD from both microglial cells and PC12 cells, used as a positive control (Fig. [2A](#F2){ref-type="fig"}). The expression of the receptor was confirmed by labeling microglial cells with FITC-labeleled-α-bungarotoxin (α-Bgtx), a selective nicotinic antagonist. Microglial cells were pre-treated for 10 min in the absence (Fig. [2B](#F2){ref-type="fig"}, left panel) or in the presence (Fig [2B](#F2){ref-type="fig"}, right panel) of nicotine (500 μM) before adding 1.5 μg/ml FITC-α-Bgtx. As shown in Figure [2](#F2){ref-type="fig"}, a strong binding of α-Bgtx was observed on the cell surface of microglial cells (left panel), while nicotine pre-treatment resulted in a marked reduction of the intensity of the fluorescent signal (right panel). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### α7 nAChR subunit is expressed in rat microglial cultures. Semiquantitative RT-PCR analysis of α7 nAChR mRNA expression in rat microglial cells (A) and in PC12 cells and rat hippocampus (C). A 754-bp band corresponding to α7 nAChR was specifically amplified (acc. number S53987; amplified region: 906--1660). Expression of β-actin is shown as internal control. No contamination of genomic DNA was present as shown in panel B (-RT: RNA from microglia that was reverse transcribed without the enzyme and amplified for α7 subunit). ::: ![](1742-2094-2-4-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Western blot and fluorescent immunostaining of α7 nAChR in rat microglial cultures. A: Proteins from microglial cultures and PC12 cells were analysed by western blot (50 ug/lane) using specific polyclonal anti AChRα7 antibodies. B: microglial cells were pre-incubated in the absence (B, left panel) or presence of 500 μM nicotine (B, right panel) for 10 min and then incubated with FITC-labeleled-α-Bgtx (1.5 μg/ml) for 15 min at 4°C. A strong binding of α-Bgtx was observed on the cell surface of microglial cells. Nicotine pre-treatment resulted in a marked reduction of the intensity of binding. ::: ![](1742-2094-2-4-2) ::: Effects of nicotine and α7 subunit activation on TNF-α release by rat microglial cells -------------------------------------------------------------------------------------- Once we had demonstrated the presence of α7 subunit mRNA and protein in microglial cells, we studied the functional consequences of receptor activation using the specific agonist nicotine. Microglial cells were pre-treated for 30 min with increasing concentrations of nicotine and then incubated for 4 or 24 h in the absence or the presence of 10 ng/ml LPS. In resting microglial cultures nicotine did not affect the basal level TNF-α(data not shown). As previously demonstrated using mouse microglial cultures, nicotine pre-treatments dose-dependently inhibited the release of TNF-α(Fig. [3](#F3){ref-type="fig"}). At 1 μM concentration, nicotine reduced the release of TNF-α after 4 h of LPS stimulation by approximately 35%, an effect similar to that recently reported for murine microglial cultures \[[@B12]\]. The inhibitory effect of nicotine on TNF-α release was still significant in microglial cultures exposed to LPS for 24 h (data not shown). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Effects of specific α7 nAChR agonist and antagonist on TNF-α production by activated rat microglial cultures. Microglial cells were subcultured for 24 h in 10% FCS-containing medium, which was replaced with fresh medium before stimulation. Nicotine (0.1--1 μM) and/or α-Bgtx were added 30 min before LPS stimulation (10 ng/ml). Supernatants were collected after 4 h and analyzed for TNF-α content. Data are shown as mean ± SEM for 3 independent experiments, run in duplicate. \p \< 0.03 vs LPS. ::: ![](1742-2094-2-4-3) ::: To verify that the effect of nicotine was mediated by α7 subunit, we measured the level of TNF-α in activated microglial cells exposed to nicotine in the presence or in the absence of α-Bgtx. The addition of 0.01 μM α-Bgtx almost totally prevented the inhibitory effect of nicotine (Fig. [3](#F3){ref-type="fig"}). In addition to TNF-α, we also analyzed the release of two important microglial mediators such as NO and IL-1β and we found that nicotine pre-treatment only slightly reduced the release of NO (9 ± 4 and 14 ± 6 % of inhibition vs LPS activated microglia; n = 9; p \< 0.04, for 1 and 10 μM nicotine, respectively) and did not modify the release of IL-1β (data not shown). Effects of nicotine and α7 subunit activation on interleukin-10 and prostaglandin E~2~synthesis by rat microglial cells ----------------------------------------------------------------------------------------------------------------------- We then analyzed the effects of nicotine on the production of interleukin-10 (IL-10) and prostaglandin E~2~(PGE~2~), two important local mediators with anti-inflammatory and immunoregulatory functions. Nicotine pre-treatment only moderately reduced (18.6 ± 7% of inhibition vs LPS activated microglia; n = 4; p \< 0.03, for 1 μM) the level of IL-10 in the culture media of microglia cells stimulated for 24 h with LPS (data not shown). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Effect of specific α7 nAChR agonist and antagonist on PGE~2~synthesis by activated rat microglial cultures. Microglial cells were subcultured as in Fig. 3, and nicotine (0.1--1 μM) added 30 min before LPS stimulation (10 ng/ml). Supernatants were collected after 24 h and analyzed for PGE~2~content. Data, with induction expressed as a percentage of LPS-induced PGE~2~production, are shown as mean ± SEM for 5 independent experiments, run in duplicate. The levels of PGE~2~were undetectable in basal conditions, and were 24 ± 6 ng/mg protein after LPS-stimulation for 24 h. \p \< 0.05 vs LPS; \\p \< 0.02 vs LPS. ::: ![](1742-2094-2-4-4) ::: By contrast, nicotine pre-treatments dose-dependently enhanced the synthesis of PGE~2~in LPS-activated microglial cells. The presence of 0.01 μM α-Bgtx, blocked the nicotine-dependent increase of PGE~2~released by LPS-activated microglia (Fig. [4](#F4){ref-type="fig"}). At this concentration, α-Bgtx did not by itself affect basal (not shown) or LPS-induced PGE~2~. We investigated the molecular mechanism underlying the increased synthesis of PGE~2~induced by α7 subunit stimulation by measuring by RT-PCR the levels of COX-2 mRNA. COX-2 is the enzyme responsible for the first committed step in prostaglandin synthesis, and is known to be readily induced by LPS in both peripheral macrophages and microglia \[[@B25]\]. As expected, COX-2 mRNA was expressed at low levels in resting microglial cultures and was remarkably increased after 7 h and 24 h of LPS treatment (Fig. [5](#F5){ref-type="fig"}). The basal COX-2 mRNA level was not significantly altered by nicotine pre-treatment at any tested concentration (0.1 μM and 1 μM) or incubation time (7 and 24 h). However, nicotine pre-treatment strongly increased the levels of COX-2 mRNA induced by 7 h treatment with 10 ng/ml LPS; the maximal effect was reached at 0.1 μM concentration (Fig. [5A](#F5){ref-type="fig"}). The enhancing effect of nicotine pre-treatment persisted after 24 h of LPS-treatment, although the increase was significant only at the lower concentration of nicotine (Fig. [5B](#F5){ref-type="fig"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Semiquantitative RT-PCR analysis of COX-2 mRNA. Representative semi-quantitative RT-PCR analysis of COX-2 mRNA in microglial cultures, subcultured as in Fig. 3, pre-treated with nicotine (Nic, 0.1--1 μM) for 30 min and stimulated for 7 h (A, upper panels) or 24 h (B upper panels) with LPS (10 ng/ml). The amount of COX-2 mRNA, expressed as the ratio of densitometric measurement of the sample to the corresponding internal standard (β-actin), is shown in the lower panels. Data are shown as mean ± SEM for 3 to 4 independent experiments, with the exception of 1 μM nicotine, panel A (n = 2); all run in duplicate. \* p \< 0.05 vs fcs; \\p \< 0.05 vs fcs. ::: ![](1742-2094-2-4-5) ::: Discussion ========== The present study provides evidence that supports the existence of a cholinergic control of microglial activation. First, we have confirmed using rat microglial cells previous data showing that murine microglia express the α7 subunit and that their exposure to the specific agonist nicotine reduces LPS-induced release of the pro-inflammatory molecule TNF-α, thus suggesting that these events are not species specific. Furthermore, we extended the analysis of α7 subunit activation to other important microglial functions, including the synthesis of mediators possessing anti-inflammatory and immunomodulatory activities. We found that in LPS-activated microglial cells, the interaction of α7 subunit with its agonist nicotine had moderate or no effect on the release of NO, IL-1β and IL-10. By contrast, nicotine treatment significantly increased the expression of COX-2 and the synthesis of PGE~2~. The effect of nicotine on the LPS-induced PGE~2~release was significantly reversed by the specific antagonist of α7 subunit, α-bungarotoxin, demonstrating the involvement of α7 nicotinic receptors in the induction of PGE~2~production by activated microglial cells. COX-2 is the inducible isoform of the enzyme responsible for the first committed step in PGE~2~synthesis, one of the major prostaglandins produced during inflammatory response and potent modulator of several macrophage and lymphocyte functions \[[@B26]\]. Within the brain, COX-2 activity and PGE~2~production, depending on their levels of induction, have been associated with both protective and harmful effects on neurons and glial cells \[[@B27]\]. In microglial cells, COX-2 is the major isoform, rapidly induced by LPS stimulation or interaction with apoptotic neurons \[[@B28]\]. The constitutive isoform COX-1 is only moderately expressed by these cells and is not up-regulated during their activation \[[@B25],[@B27]\]. PGE~2~has been found to be neuroprotective in several experimental settings. At nanomolar concentrations, PGE~2~protects hippocampal and cortical neuronal cultures against excitotoxic injury or LPS-induced cytotoxicity \[[@B29]-[@B32]\]. In hippocampal neuronal and organotypic cultures, the protective effect of PGE~2~against glutamate and oxygen deprivation is mediated by the activation of the EP2 receptor, one of the four PGE~2~receptor subtypes whose activation leads to cAMP formation \[[@B31]\]. The protective effect of EP2 receptor activity has been confirmed in vivo, in a model of transient forebrain ischemia, in which the genetic deletion of this PGE~2~receptor exacerbates the extent of neuronal damage \[[@B31]\]. On the other hand, at concentrations in the μM -- mM range, PGE~2~contributes to neuronal death and stimulates release of glutamate by astrocytes \[[@B33]-[@B35]\]. PGE~2~has also been shown to down-regulate microglial activation and expression of pro-inflammatory genes, including TNF-α, both in vitro and in vivo \[[@B36],[@B37]\]. We have recently found that the interaction of microglial cells with apoptotic neurons promotes the synthesis of PGE~2~along with neuroprotective and immunoregulatory molecules such as TGF-β and NGF \[[@B38],[@B28]\]. In this system, the release of PGE~2~is triggered by the specific interaction between phosphatidylserine, a phospholipid exposed on the cell surface during the initial phase of apoptosis, with its cognate receptor expressed by microglia \[[@B39]\], consistent with previous studies on peripheral macrophages \[[@B40]\]. It has been suggested that the PGE~2~, released by macrophages engulfing apoptotic cells, contributes to one of the main features of apoptotic cell death, namely the efficient removal of dying cells without eliciting inflammation in the surrounding tissue \[[@B41]\]. It is therefore tempting to speculate that the α7 subunit-dependent increase of PGE~2~in activated microglia cells is part of an anti-inflammatory pathway regulated by the cholinergic system. The detection of microglial cells, astrocyte processes and choline acetyltransferase- (ChAT-) positive fibers around β-amyloid plaques in transgenic APP~SW~mice suggests a close connection between cholinergic terminals and microglial cells \[[@B42]\]. A deficit in ACh level due to loss of cholinergic neurons associated with AD as well as aging could contribute to the establishment of chronic inflammation rendering microglial cells more susceptible towards environmental changes and orientating them towards a pro-inflammatory phenotype. However, to date there is no definitive evidence of a causal link between loss of cholinergic neurons and increased levels of pro-inflammatory cytokines such as TNF. In the last few years, several lines of evidence have suggested that activation of α7 subunits plays an important role in the maintenance of cognitive functions in several neurodegenerative disorders \[[@B43]\]. Epidemiological studies have shown that cigarette smoking can be protective against the development of AD, PD and other types of dementia, suggesting that chronic inhalation of nicotine may slow the progression of these neurodegenerative diseases or improve some cognitive responses in AD patients \[[@B44],[@B17]\]. Loss of nAChRs has been reported in patients with diverse forms of dementia \[[@B45]\]. In particular, a reduction in α7 subunit number was detected in AD and PD brain tissue specimens \[[@B46]\]. The administration of ligands targeting nicotinic receptors in animal models of neurodegeneration, as well as in humans, induced cognitive improvement \[[@B47]\] and conferred neuroprotection against several neurotoxic agents \[[@B48],[@B49]\]. Furthermore, cholinesterase inhibitors used in the symptomatic treatment of AD have been reported to exert additional benefits through the increased density of specific nicotinic receptor subunits (including the α7) \[[@B50]\]. This effect could be relevant in view of the anti-inflammatory role suggested for the α7 subunit. As mentioned in the introduction, the presence of α7 subunit on immune cells as well as on other non-excitable cells has provided a molecular basis for a non-neuronal cholinergic pathway that might function as an essential regulator of inflammation as well as immune responses \[[@B4],[@B5]\]. Primary cultures of astrocytes and microglia show ChAT activity and synthesize acetylcholine \[[@B51]\]. Accordingly, we have found the expression of ChAT mRNA in both resting and activated microglia cells (unpublished results). This suggests that this neurotransmitter may act as a local hormone and contribute to the regulation of microglial functions. It should be noted that although our study focused on the effects of nicotine on the process of microglial activation induced by LPS, our findings may have broader implications since other microglial activators, such as pro-inflammatory cytokines and fibrillogenic peptides, share some common signaling pathways with LPS \[[@B52],[@B53]\]. In addition, it has been recently reported that the LPS receptor CD14 interacts with fibrils of Alzheimer amyloid peptide and a deficiency of this receptor significantly reduces fibril-induced microglial activation \[[@B54]\]. At present, the signaling pathways downstream to α7 subunit activation and leading, in particular, to COX-2 and PGE~2~up-regulation is under investigation. Shytle et al.\[[@B12]\] have reported that either ACh or nicotine inhibit LPS-induced phosphorylation of the mitogen-activated protein kinases p44/42 and p38 in murine microglia. We have recently found a reduction of p38 phosphorylation in two experimental settings in which exposure of microglial cells to phosphatidylserine vesicles -- mimicking apopototic neurons -- or to chronic activation stimuli, resulted in downregulation of pro-inflammatory cytokines and in enhancement of PGE~2~synthetic pathway \[[@B55],[@B56]\], thus suggesting that p38 may also have a role in α7 dependent up-regulation of COX-2. Conclusions =========== Activation of α7 nicotinic receptors in microglial cells by nicotine controls some important microglial functions, thus preventing chronic inflammation. Since microglial activation and chronic inflammation have been associated with most neurodegenerative pathologies \[[@B57]\] the understanding of the molecular pathway(s) triggered by α7 subunit activation in microglial cells will offer new venues for potential pharmacological regulation of microglial activation in neurodegenerative diseases. At the same time, the development of molecules able to stimulate the α7 subunit may represent a potential promising approach for the treatment of these disorders. List of abbreviations ===================== Lipopolysaccharide (LPS) Acetylcholine (ACh) Neuronal acetylcholine receptors (nAChRs) Tumor necrosis factor-α (TNF-α) Prostaglandin E~2~(PGE~2~) Interleukin-1β (IL-1β) Nitric oxide (NO) Interleukin-10 (IL-10) Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= RDS conceived of the study, participated in its design and coordination, produced the primary microglial cultures, performed the ELISA and the immunofluorescence staining, was primarily responsible of the RT-PCR review the data and drafted the manuscript. MAAC participated in the design and coordination of the study, produced the primary microglial cultures, performed the ELISA and the immunofluorescence staining, was primarily responsible for western blot analysis and review the data. DC participated in the production of the primary microglial cultures and in RT-PCR. LM contributed to the design of the study, guided data interpretation and presentation and assisted in the preparation of the manuscript. Acknowledgements ================ This work has been supported by the Istituto Superiore di Sanità (Ricerca Intramurale), grant no. C3A4 to RDS.	PubMed Central	2024-06-05T03:55:52.780472	2005-1-25	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548670/", "journal": "J Neuroinflammation. 2005 Jan 25; 2:4", "authors": [ { "first": "Roberta", "last": "De Simone" }, { "first": "Maria Antonietta", "last": "Ajmone-Cat" }, { "first": "Daniela", "last": "Carnevale" }, { "first": "Luisa", "last": "Minghetti" } ] }
PMC548671	Background ========== The protein expression of the homeobox gene NK3 transcription factor related locus 1 (NKX3.1)is highly specific for the prostate and the testis \[[@B1]-[@B3]\], and is frequently lost in cancers of these two tissue types \[[@B1],[@B4],[@B5]\]. NKX3.1is located in chromosome band 8p21 \[[@B2],[@B6],[@B7]\], a region that undergoes frequent allelic imbalance in prostatic intraepithelial neoplasia (PIN) and prostate carcinomas \[[@B8],[@B9]\]. In mice, targeted disruption of Nkx3.1leads to prostatic epithelial hyperplasia and dysplasia \[[@B10],[@B11]\], and over-expression of exogenous NKX3.1suppresses growth and tumorigenicity in human prostate carcinoma cell lines \[[@B12]\]. However, the expression levels and possible role for NKX3.1 during prostate cancer progression in humans is still being debated \[[@B13]-[@B15]\]. No gene mutations of NKX3.1have been found \[[@B6]\], and NXK3.1is therefore believed to be epigenetically inactivated in the cases with loss of protein expression \[[@B1],[@B5],[@B16]\]. Only one study has reported NKX3.1 protein expression in testicular germ cell tumors (TGCTs), however the series analyzed was large, including a total of more than 500 samples, and NKX3.1 was found absent in all embryonal carcinomas and present in only 15--20% of the seminomas as well as among the differentiated histological subtypes of germ cell tumors \[[@B5]\]. During the last decade, epigenetic changes in cancer have been frequently reported and are now recognized to be at least as common as genetic changes \[[@B17]\]. The best characterized epigenetic mechanism is DNA hypermethylation, in which cytosines located within selected CpG sites in the gene promoters become methylated, thereby inactivating gene expression. Several tumor suppressor genes are inactivated by such promoter hypermethylation in various cancer types \[[@B18],[@B19]\]. In the present study we have performed methylation-specific PCR (MSP) and bisulphite sequencing of the NKX3.1promoter in TGCTs and prostate adenocarcinomas to examine whether this mechanism may explain the commonly observed loss of NKX3.1 protein. Results ======= Only one out of 54 TGCTs and none of the prostate adenocarcinomas (n = 20), intratubular germ cell neoplasias (n = 7), normal testis tissues (n = 4), or the cell lines (n = 6) displayed methylation when analyzed with MSP (Figure [1a](#F1){ref-type="fig"}). Bisulphite genomic sequencing of the tumors and cell lines showed that all cytosines at non-CpG sites were converted to thymine (Figure [1b](#F1){ref-type="fig"}). Only one sample demonstrated overall methylation in the NKX3.1sequence, and this was the same sample that was positive for methylation from the MSP analysis. Interestingly, all the samples that were sequenced, including the normal blood, unmethylated cell lines, and primary tumors, displayed some extent of methylation (the majority below 25%) at the cytosine in CpG number 21 (base 1914762, +1 bp from transcription start). We detected a possible polymorphism in base 1914730 (+33 bp from transcription start and 15 bp upstream of the coding sequence). In previous sequences this site has been described as a guanine (Gene bank accession number NT\_023666, and AF24770). In the cell lines, 5/6 contained adenosine in this position, but all except the germ cell tumor cell lines NCCIT and TERA2 were heterozygotes. In contrast, all 5 primary tumors sequenced were homozygous for the adenosine allele. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### *Representative results of the methylation analyses of the NKX3.1promoter.(A) Methylation-specific PCR. A visible PCR product in Lanes Uindicates the presence of unmethylated alleles whereas a PCR product in Lanes Mindicates the presence of methylated alleles. The left panel illustrates the methylation status of selected TGCTs and the testicular cancer cell lines. Note the methylation of sample \# 2110. The right panel shows the unmethylated status of primary prostate cancers and prostate cancer cell lines. Abbreviations: NB, normal blood (positive control for unmethylated samples); IVD, in vitromethylated DNA (positive control for methylated samples); neg, negative control (containing water as template); U, lane for unmethylated MSP product; M, lane for methylated MSP product. (B) Bisulphite sequencing. The bisulphite sequence allows a positive display of 5-methyl cytosines in the gene promoter as unmethylated cytosines appear as thymines, while 5-methylcytosines appear as cytosines in the final sequence. The left chromatogram represents a part of the unmethylated NKX3.1promoter in the germ cell tumor cell line TERA2, including 11 CpG sites marked by underlined letters. The right chromatogram represents the unmethylated prostate cancer cell line DU145. Both sequences have been generated by reversing the respective anti-sense sequences by use of the software \"Chromas\". ::: ![](1476-4598-4-8-1) ::: Discussion ========== We have previously reported that the protein expression of NKX3.1 is virtually ubiquitously lost in TGCTs \[[@B5]\]. This was done using a tissue microarray containing 510 testicular tissue samples. NKX3.1 expression is known for 25 of the TGCTs now analyzed for promoter hypermethylation and 22 showed complete absence of protein. The down regulation of NKX3.1 in TGCT has also been detected at the mRNA level, both by quantitative RT-PCR \[[@B5]\] and from an oligonucleotide microarray study including 20 of the present TGCTs (Skotheim et al., submitted). This simultaneous down regulation of both protein and mRNA levels of NKX3.1is consistent with epigenetic regulation, which is further supported by the fact that mutations have not been detected in the NKX3.1gene \[[@B6]\]. DNA promoter hypermethylation is the best-characterized epigenetic change in cancer, and can be associated with gene silencing. It was therefore of interest to analyze the methylation status of the NKX3.1promoter in TGCT and prostate cancer samples. However, with the exception of a single TGCT, the NKX3.1promoter was unmethylated in the samples analyzed. The methylated TGCT was classified as a yolk sac tumor, and has also been demonstrated to have promoter hypermethylation of several other genes that are generally unmethylated in TGCTs (Lind et al., unpublished). We therefore consider this sample not to be representative for the general TGCT epigenotype, nor for the general epigenetic profile of yolk sac tumors. Thus, we do not regard promoter hypermethylation as the general mechanism of NKX3.1 down-regulation neither in TGCT nor in prostate carcinomas. We also studied cell lines since it can be argued that presence of normal cells as well as tumor heterogeneity may mask cancer specific methylation in primary tumors. LNCaP cells have previously been demonstrated to express NKX3.1, in contrast to PC-3 and DU-145, which do not express NKX3.1 since they lack a functional androgen receptor \[[@B2]\]. The lack of NKX3.1expression in PC-3 and DU-145 cells is not due to methylation. This was also the case with the germ cell tumor cell lines. From Western analysis, the cell line NCCIT had strong expression of NKX3.1 whereas both TERA1 and TERA2 had no expression (data not shown). The polymorphism in NKX3.1that we detected 15 bases upstream of the coding sequence has to our knowledge not been described previously. It was identified by bisulphite sequencing of the cell lines and a subgroup of primary tumors, thus caution should be taken when concluding from these results, since regular sequencing analysis is the recommended approach for describing sequence changes. As the polymorphism is located in the promoter region of NKX3.1, it has no influence on the protein structure. However, it can still have a potential role in the transcriptional regulation of NKX3.1. A polymorphism in the coding sequence is also reported for NKX3.1\[[@B6]\]. All samples analyzed with bisulphite sequencing, including cell lines expressing NKX3.1, as well as non-expressing cell lines, demonstrated some degree of methylation in the cytosine in CpG number 21. As this site-specific methylation included only one CpG site, it is unlikely that it will have any regulating effect on gene expression. However, considering its intriguing location immediate upstream of the transcription start point, this possibility should not be excluded. There is also the possibility that the apparent methylation could be due to a less efficient bisulphite conversion for this site. In general, the bisulphite sequencing results showed that all cytosines at non-CpG sites were converted to thymine (Figure [1b](#F1){ref-type="fig"}), but sequence-specific partial resistance to this conversion may lead to methylation artifacts, but only in rare cases, as has been reported previously \[[@B20]\]. Conclusions =========== In summary, these data show that the previously reported down-regulation of NKX3.1 in TGCTs and prostate carcinomas is not caused by promoter hypermethylation. Even though the NKX3.1promoter is unmethylated, the simultaneous down-regulation of mRNA and protein levels in TGCTs and the absence of mutations still make other epigenetic mechanisms, such as modulation of chromatin structure or modifications of histones, possible explanations for loss of NKX3.1 expression in testicular- and prostate cancers. Materials and Methods ===================== Primary tumors and cell lines ----------------------------- Included in the present study are primary TGCTs (n = 55), intratubular germ cell neoplasias (also called carcinoma in situ; n = 7), normal testis tissue (n = 4), germ cell tumor cell lines (TERA1, TERA2, and NCCIT), prostate adenocarcinomas (n = 20), and prostate cancer cell lines (LNCaP, PC-3, and DU-145). The primary TGCTs include all histological subtypes: seminomas, embryonal carcinomas, teratomas, yolk sac tumors, and one choriocarcinoma, classified according to the WHO\'s recommendations \[[@B21]\] by a germ cell tumor reference pathologist using light microscopic examination of hematoxylin and eosin stained tissue sections. From our previous comparative genome hybridization analysis, about half of the TGCTs had a low-level copy number gain at chromosome 8, but only rarely 8p deletions \[[@B22]\]. Primary prostate adenocarcinomas obtained from radical prostatectomy specimens were graded according to the Gleason grading system \[[@B23]\] using routinely stained tissue sections. The median Gleason score of prostate adenocarcinomas was 7 (range: 4 -- 8). The prostate carcinomas were all of pTNM stage 2 and 3, and included 10 samples with 8p deletions (among other cytogenetic aberrations), 3 samples with copy number changes not involving the 8p region, and 7 samples with no copy number changes (Ribeiro et al., submitted). Methylation-specific PCR ------------------------ The DNA samples were initially bisulphite modified \[[@B24],[@B25]\], which converts unmethylated but not methylated cytosines to uracil. All samples were subsequently submitted to MSP analysis \[[@B26]\] using PCR primers specific to methylated and unmethylated sequences: NKX3.1unmethylated sequence, sense: 5\'GGAAAGTGAAAGTGGTGTGGGTT3\', antisense: 5\'CTACACACCATCCCACAAAATATC3\', methylated sequence, sense: 5\'AAAGTGAAAGCGGTGCGGGTC3\', antisense: 5\'ACGCGCCGTCCCGCAAAATAT3\' (MedProbe AS, Oslo, Norway). The two fragments were amplified by the Fast Star DNA polymerase (Roche Ltd, Basel, Switzerland) in a reaction containing 1.5 mM Mg^2+^. We used a 58°C annealing temperature for both primer sets. Bisulphite treated DNA from normal blood (NB) and Sss1methyltransferase (New England Biolabs Inc., Beverly, MA, USA) in vitrotreated placenta DNA (IVD; Sigma Chemical Co., St. Louis, MO, USA) represented the unmethylated positive control and the methylated positive control, respectively. Water, replacing bisulphite treated template, was the negative control in both reactions. Bisulphite sequencing --------------------- Bisulphite sequencing allows a positive display of 5-methyl cytosines in the gene promoter after bisulphite modification as unmethylated cytosines appear as thymines, while 5-methylcytosines appear as cytosines in the final sequence \[[@B27]\]. A subset of the samples (n = 11) were bisulphite sequenced, including all 6 cell lines, 3 TGCTs, and 2 prostate adenocarcinomas. Additionally, NB and IVD were bisulphite sequenced as positive controls for unmethylated and methylated sequence, respectively. The NKX3.1bisulphite sequence fragment (Gene bank accession number NT\_023666 (minus strand), bases 1914526 to 1914961) was 436 bp long and covered 52 CpG sites in the promoter and first exon of the gene. We designed bisulphite sequencing primers (MedProbe) with the following sequences; sense: 5\'ATTGGGGAAGGAGAGGGAATTG3\', antisense: 5\'CCTCTAACTCTAACTCTAACTCC3\'. The Mg^2+^content of the reaction was 1.3 mM, the enzyme used was HotStarTaq™ DNA polymerase (QIAGEN Inc., Valencia, CA, USA), and the annealing temperature 52°C. The PCR fragments were eluted from a 2% agarose gel (BioRad Laboratories Inc, CA, USA) containing ethidium bromide, by the MinElute™Gel Extraction kit (QIAGEN), and sequenced with the dGTP BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA) in an ABI Prism 377 Sequencer (Applied Biosystems). The bisulphite sequencing results were scored according to Melki et al*. where the amount of methylcytosine of each CpG dinucleotide is quantified by comparing the peak height of the cytosine signal with the sum of the cytosine and thymine peak height signals \[[@B28]\]. Authors\' contributions ======================= GEL performed the experimental analyses and statistics, interpreted the results, and drafted the manuscript. RIS did an independent scoring of the results, and contributed to manuscript preparation. MFF designed the primers used for the MSP and bisulphite treatment and contributed to manuscript preparation. VMA and RH were reference pathologists for the testicular cancer tissues and prostate tissues, respectively. FS was responsible for the Western Blot studies of the cell lines and participated in the writing of the manuscript. Parts of this work were done in the lab of ME who also contributed with scientific discussions. MRT provided the relevant selected series of primary prostate carcinomas with known genetic profiles, and contributed to manuscript preparation. RAL conceived the study, was responsible for its design and coordination, and contributed in the evaluation of the results and in preparation of the manuscript. Acknowledgements ================ The study was funded by grants from the Norwegian Cancer Society supporting GEL as a Research Fellow and RIS as a Post-Doctoral Fellow (A95068 RAL). The authors are grateful to Gunnar Brunborg at the Norwegian Institute of Public Health, Oslo, Norway for providing the normal testis DNA from organ donors.	PubMed Central	2024-06-05T03:55:52.782930	2005-2-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548671/", "journal": "Mol Cancer. 2005 Feb 3; 4:8", "authors": [ { "first": "Guro E", "last": "Lind" }, { "first": "Rolf I", "last": "Skotheim" }, { "first": "Mario F", "last": "Fraga" }, { "first": "Vera M", "last": "Abeler" }, { "first": "Rui", "last": "Henrique" }, { "first": "Fahri", "last": "Saatcioglu" }, { "first": "Manel", "last": "Esteller" }, { "first": "Manuel R", "last": "Teixeira" }, { "first": "Ragnhild A", "last": "Lothe" } ] }
PMC548672	Background ========== Intrauterine malnutrition, as reflected by birth weight and abnormal thinness at birth, has been associated with an increased incidence of risk factors for arterial disease, i.e. hypertension, impaired glucose tolerance, diabetes and to a lesser extent hyperlipidemia and body fat distribution in adulthood \[[@B1]-[@B10]\]. This observation has become known as the \'fetal origins of adult disease\' or \'Barker hypothesis\', which suggests that several of the major diseases of later life, including coronary heart disease, stroke and cardiovascular death, originate in impaired intrauterine growth and development \[[@B11],[@B12]\]. In cohort studies, Barker \[[@B13]-[@B15]\] in England and Finland, Rich-Edwards et al. \[[@B16]\] as part of the Nurses\' Health Study in the USA and Leon et al.\[[@B17]\] from Uppsala in Sweden showed an inverse relationship between birth weight and the clinical endpoint ischemic heart disease. Leon et al.\[[@B17]\] found a significant relationship only among male singletons and adjusted their results for gestational age and socioeconomic confounding. The association was not found in a cohort study from Gothenburg \[[@B18]\]. Our aim was to investigate the association in a case control study among Dutch women. Methods ======= The RATIO (Risk of Arterial Thrombosis In relation to Oral contraceptives) study is a population-based case-control study on myocardial infarction in relation to oral contraceptive use among women aged 18 to 49 years in the Netherlands \[[@B19]\]. An additional standardized questionnaire was sent to all 218 patients and 769 controls from whom also blood samples had been taken for determination of metabolic risk factors (diabetes and hypercholesterolemia). Questions elicited information on birth weight, waist and hip circumference and data on the menstrual cycle. For 13 women no current address could be found (12 patients, 1 control). Four women had died since the index date (2 patients, 2 controls), which was the date of the first myocardial infarction for the patients and the midyear for the controls. One hundred and fifty two patients (71%) and 568 controls (75%) responded to the questionnaire and women were asked to measure their waist and hip circumference. Body mass index (BMI) was calculated as body weight (kg) divided by height squared (m^2^). Waist-hip-ratio was calculated as waist circumference divided by hip circumference. Multiple linear and unconditional logistic regression were used to analyze the data. Odds ratios for the relationship between birth weight and myocardial infarction were calculated and 95% confidence intervals (95%CI) were derived from the models. Birth weights were categorized according to quintiles in control women in order to investigate an association between birth weight and the risk of myocardial infarction later in life. These were \<3000 g, 3000 to 3199 g, 3200 to 3499 g, 3500 to 3883 g, and \>3884 g, respectively. To determine whether women with a lower birth weight had a higher risk for a myocardial infarction, patients were divided in a group with a birth weight equally or higher than 2000 g and a group with a birth weight lower 2000 g \[[@B20]\]. Odds ratios were adjusted for age, education level, body mass index, waist-hip ratio, hypertension, diabetes, hypercholesterolemia, smoking, and family history of cardiovascular disease, when appropriate. Interaction between low birth weight and low education level was investigated by computing a dummy variable. Results ======= The characteristics of 152 women with myocardial infarction and 568 control women at the index date are shown in Table [1](#T1){ref-type="table"}. At the moment of completing the questionnaire, patients were aged 32--59 years (mean 50), and control women 25--60 years (mean 47). The mean body mass index was 25.1 kg/m^2^for the patients and 23.4 kg/m^2^for control women, mean difference 1.76 kg/m^2^(95%CI 1.05--2.47), p \< 0.001. Ninety-seven patients (64%) and 415 (73%) controls could give their birth weight. Compared with control women, patients had a significantly lower mean birth weight (3214 vs. 3370 g, mean difference -156.3 g (95%CI -9.5 to -303.1). The odds ratio for myocardial infarction for children with a low birth weight (\< 2000 g) compared to a birth weight ≥ 2000 g was 2.4 (95%CI 1.0 to 5.8). After adjustment for putative confounders (age, education level, body mass index, waist-hip ratio, hypertension, diabetes, hypercholesterolemia, smoking, and family history of cardiovascular disease) the odds ratio did not change. Odds ratios for myocardial infarction in different categories of birth weight as compared to the reference category (birth weight higher than 3884 g) were 1.3 (95%CI 0.5--3.3) for a birth weight 3500 to 3883 g, 1.4 (95%CI 0.6--3.4) for a birth weight 3200 to 3499 g, 1.7 (95%CI 0.6--5.1) for a birth weight 3000 to 3199 g, and 2.3 (95%CI 1.0--5.4) for a birth weight lower than 3000 g (Table [2](#T2){ref-type="table"}). The risk of myocardial infarction was 6.2 fold increased (95%CI 2.7--13.9) among women with low birth weight and a low educational level compared to women with a high birth weight and a high educational level (reference category). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of patients with a first myocardial infarction and control women ::: Characteristic Patients (N = 152) Control women (N = 568) ---------------------------------------------- ------------------------ ----------------------------- Age -- yr (SD) 42.1 (0.5) 38.6 (0.3) Caucasian ethnicity (%) 142 (93) 538 (95) Educational level \- Primary school or less (%) 83 (55) 160 (28) \- Secondary school (%) 52 (34) 257 (45) \- Higher education or university (%) 17 (11) 149 (26) Current smokers (%) 128 (84) 218 (39) History of hypertension (%) 35 (23) 35 (6) History of hypercholesterolemia (%) 16 (11) 14 (3) History of diabetes (%) 8 (5) 7 (1) Family history of cardiovascular disease (%) 98 (66) 194 (36) Birth weight-gram \- Mean (SD) 3214 (676) 3370 (659) \- Median (range) 3150 (1500--5010) 3500 (1500--5800) Body Mass Index -- kg/m^2^-- Mean (SD) 25.1 (0.4) 23.4 (0.2) Waist circumference -- cm (SD) 89.5 (13.1) 83.0 (10.2) Waist/hip ratio (SD) 0.85 (0.006) 0.81 (0.008) Premenopausal (%) 132 (87) 480 (85) ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Odds ratios (95%CI) for myocardial infarction in quintiles of birth weight as compared to the reference category ::: Birth weight (g) Odds Ratio (95% CI) ---------------------- ------------------------- \> 3884 1\* 3500--3883 1.3 (0.5--3.3) 3200--3499 1.4 (0.6--3.4) 3000--3199 1.7 (0.6--3.4) \< 3000 2.3 (1.0--5.4) \* Reference category ::: Discussion ========== In this case control study we have found that women with a low birth weight had a higher risk of myocardial infarction than women with a higher birth weight. The data confirm the association found in cohort studies \[[@B13]-[@B17]\] and as such support the \'fetal origins of adult disease\' or \'Barker hypothesis\'. A limitation of the study is that we have no information on gestational age. However, recently it has been demonstrated that both children who had been born prematurely and children who are small for gestational age had a reduction in insulin resistance \[[@B21],[@B22]\]. The self-report of birth weights as well as the rather high percentage of missing values for birth weight may also limit the study, but the random events among cases and controls cannot explain our results. Socio-economic factors associated with low birth weight are also associated to risk factors for arterial disease later in life. As pointed out by several others, it will be nearly impossible to disentangle these effects \[[@B16],[@B17]\]. In the present study we confirmed an interactive effect between low birth weight and a low educational level on the risk of myocardial infarction in women. However, when we adjusted for age, education level, body mass index, waist-hip ratio, hypertension, diabetes, hypercholesterolemia, smoking, and family history of cardiovascular disease, factors of which some may partly been seen as (the result of) socio-economic/environmental and genetic factors, we still observed an association between low birth weight and a higher risk of myocardial infarction. This is in agreement with others who also found that genetic and socio-economic circumstances at birth and in adult life can not completely explain the association between low birth weight and disease late in life \[[@B10],[@B16],[@B17],[@B23]-[@B25]\]. Among the proposed underlying biological mechanisms to explain the association is impaired endothelial development. Already at very young age, individuals with low birth weight exhibit endothelial dysfunction that persists into childhood and adult life, suggesting that endothelial dysfunction precedes the development of vascular related diseases later in life and represents the link between low birth weight and these diseases \[[@B26]-[@B32]\]. Furthermore, Smith et al. \[[@B33]\] found that mothers, who once gave birth to thin babies, have a higher risk of developing ischemic heart disease later in life. Therefore, these mothers seem to have, just as their children, an impaired endothelial function. If the impaired endothelial function is already manifest during the process of implantation, it might lead to inadequate development of the vasculature in the maternal part of the placenta, which enfeebles the function of the placenta resulting in low birth weight. Conclusions =========== In conclusion, our study shows that a low birth weight (\<2000 g) is associated with a 2.4 fold higher risk of myocardial infarction before the age of 50 as compared with a birth weight ≥ 2000 g. Because the risk of cardiovascular disease is known to increase with an increasing number of risk factors, women with a low birth weight should try to avoid acquired risk factors, like smoking and obesity. In addition, extra attention should be given in detecting diabetes or hypertension at a later stage in life and in detecting exaggerated growth during childhood, as these individuals seem most prone to develop disease later on in life \[\[[@B34]-[@B36]\]\]. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= BT participated in the design, execution and analysis of the RATIO- study and drafted the manuscript. KK analyzed the data and drafted the manuscript. RH collected the data and performed statistical analyses. FR initiated the study and helped to draft the manuscript. FH participated in the design of the study and the writing of the paper. Acknowledgements ================ This work was supported by the Netherlands Heart Foundation (97-063).	PubMed Central	2024-06-05T03:55:52.784802	2005-1-10	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548672/", "journal": "Reprod Health. 2005 Jan 10; 2:1", "authors": [ { "first": "Bea C", "last": "Tanis" }, { "first": "Kitty", "last": "Kapiteijn" }, { "first": "Ronella M", "last": "Hage" }, { "first": "Frits R", "last": "Rosendaal" }, { "first": "Frans M", "last": "Helmerhorst" } ] }
PMC548673	Background ========== Magnetic materials have been used with grain sizes down to the nanoscale for longer than any other type of material \[[@B1]\]. This is attributable to a number of factors including a large surface area to volume ratio and the possibility of immobilizing a biological entity of interest \[[@B2]\]. In the last decade increased investigations and development were observed in the field of nanosized magnetic particles \[[@B2]\]. Here the term nanoparticles is used to designate particulate systems that are less than 1μm, and effectively below 500 nm \[[@B2]\]. Due to their magnetic character, magnetite (Fe~3~O~4~) nanoparticles can be attracted by a magnetic field and are easily separable in solution. Similarly, substances to which they have been attached can be separated from a reaction medium, or directed by an external magnetic field to site specific drug delivery targets \[[@B2]\]. Magnetic nanoparticles have been widely used in the immobilization of many bioactive substances such as proteins, peptides, enzymes \[[@B3]-[@B6]\], and antibodies \[[@B7]\]. Magnetite is one of the most commonly used magnetic materials because it has a strong magnetic property and low toxicity \[[@B4]\]. The binding of magnetic particles to bioactive substances involves a number of interactions including the interactions between organic ligand, and the interactions between the amino acid side chains of proteins and the metals centers. Such bindings pave the way for the coupling of biomolecular entities of enhanced stability. Recently reported work in the area of enzyme immobilization has described the catalytic activity of yeast alcohol dehydrogenase \[[@B3]\] and lipase \[[@B4]\] directly bound to magnetite nanoparticles, via carbodimiide activation without the use of a ligand. This binding method offers tremendous scope because of its simplicity and high efficiency. Cholesterol oxidase is a flavin-enzyme (with a FAD prosphetic group) that produces hydrogen peroxide according to the reaction 1. Cholesterol+ O~2~→ 4 - Cholesten- 3 - one+ H~2~O~2~ (1) The structure of cholesterol oxidase reveals deeply buried active sites occupied by water molecules in the absence of its substrate steroids \[[@B8]\]. Cholesterol oxidase is industrially and commercially important for application in bioconversions for clinical determination of total or free serum cholesterol \[[@B9]-[@B12]\] and in agriculture \[[@B13]\]. Its activity can be determined by following the appearance of the conjugated ketones, the formation of hydrogen peroxide in a coupled test with peroxidase, or by measuring the oxygen consumption polarographically \[[@B13]\]. Several studies on its kinetic properties have appeared \[[@B13]-[@B15]\]. More recently, Cholesterol biosensor based on entrapment of cholesterol oxidase in a silicic sol-gel matrix at a Prussian Blue modified electrode has been developed \[[@B15]\]. However, this method of enzyme immobilization raises concerns on reduced surface area for enzyme binding and pore-diffusion resistance \[[@B2]\]. Immobilization of enzymes onto inorganic material surfaces is of vital importance in enzymatic reactions, especially in biosensor applications. Information on the activity and availability of cholesterol oxidase bound to Fe~3~O~4~magnetic nanoparticles will contribute to the basic understanding of its activity and function. The present study proposes to investigate the direct binding of cholesterol oxidase to Fe~3~O~4~magnetic nanoparticles. The sizes and structure of the nanoparticles were characterized using TEM and FTIR spectroscopy. The stability, activity, and kinetic behavior of bound cholesterol were also examined. Results and discussions ======================= Particle size and structure --------------------------- TEM micrographs of \"bare\" magnetic nanoparticles and CHO-functionalized magnetic nanoparticles are shown in Figure [1a](#F1){ref-type="fig"} and [1b](#F1){ref-type="fig"}. The \"bare\" particles were very fine with a diameter ranging from 9.7 to 56.4 nm. The size of the particles after binding to CHO was globally the same as the \"bare\" particles. Figure [2](#F2){ref-type="fig"} shows the size distribution of the particles. However, some spots of agglomerated particles were visible as seen in figure [1b](#F1){ref-type="fig"}. These agglomerates cause an increase in maximum particle size. The overall sizes of the particles after binding to magnetic nanoparticles were between 9.7 and 166 nm suggesting a perceptible agglomeration in association with the binding process. A possible explanation is that the binding of magnetic nanoparticles was not only a monomolecular process but may involve the binding of several CHO molecules on a single Fe~3~O~4~particle. It could also be envisaged that CHO molecules formed aggregates to bind several magnetic nanoparticles. Another possible factor in the agglomeration process is the centrifugation process involved in the separation of the supernatant from the Fe~3~O~4~-CHO. It is obvious that the centrifugation tend to bring particles together as a compact material. The effect of agglomeration at this stage can be reduced by separating the Fe~3~O~4~-CHO by an external magnetic field. Since the particles are released after removal of the magnetic field, they may fall separately apart from each other, and are less likely to agglomerate. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Transmssion Electron micrographs of Fe~3~O~4~magnetic nanoparticles (a) and Fe~3~O~4~-CHO (b). ::: ![](1477-3155-3-1-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Distribution of the particle sizes on the electron micrographs. The values denote the averages of duplicate measurements. ::: ![](1477-3155-3-1-2) ::: Binding efficiency ------------------ The unbound enzyme was determined by assaying the protein content in the supernatant. It was found that the percentage of cholesterol oxidase bound was between 98 and 100%, irrespective of the amount of particles. The amounts of Fe~3~O~4~nanoparticles used were 14.4, 17.2 and 20 mg/mL, corresponding to CHO/Fe~3~O~4~weight ratios of 0.01, 0.08 and 0.007, respectively. These results show that in all the binding operations, there were sufficiently available amount of particles to bind the enzymes till complete saturation. In a previous study \[[@B4]\], it was found that increasing the amount of Fe~3~O~4~nanoparticles, that is reducing the weight ratio of CHO to Fe~3~O~4~below 0.033 caused an increase in lipase binding up to 100%. This was not observed in this study, possibly because of the difference in the binding mechanism, due to differences in the structure of the enzyme. However, the percentage of bound CHO (98--100%) shows that the binding process was successful. Binding confirmation -------------------- The binding of CHO to magnetic nanoparticles was confirmed by FTIR analysis. Figure [3](#F3){ref-type="fig"} (a, b, and c) shows the FTIR spectra for \"bare\" Fe~3~O~4~, Fe~3~O~4~-CHO, and CHO in water, respectively. A characteristic band of NH~2~was observed at 1618 cm^-1^in the \"bare\" Fe~3~O~4~nanoparticles. The NH~2~group can be associated with NH stretch at 3400 cm^-1^which is not visible here, because of a possible hindrance by OH stretch from water. However, this band was not apparent in the spectra of Fe~3~O~4~-CHO suggesting that the binding of CHO to the nanoparticles involved this amino group and the carboxylic groups of CHO after being activated by Carbodiimide, as suggested by \[[@B4]\]. Peaks at 3032 cm^-1^and 1445 cm^-1^are more visible in Figure [3a](#F3){ref-type="fig"} (bare particles) and perceptible in Figure [3b](#F3){ref-type="fig"} (Fe~3~O~4~-CHO) and could be assigned to traces of residual ammonium hydroxide. The characteristic bands of proteins at 1647 and 1541 cm^-1,^and 1645 and 1541 cm^-1^, in the spectra of Fe~3~O~4~-CHO, and CHO, respectively shows that cholesterol oxidase was effectively present in the samples, confirming the binding of cholesterol oxidase to Fe~3~O~4~nanoparticles. The negative peak at 3400-2799 cm^-1^is possibly due to a reduced amount of water in the sample compared to the water used for background subtraction. The characteristic bands of proteins in the Fe~3~O~4~-CHO spectra were very weak compared to those in the spectra of cholesterol oxidase in water. The weakness of the peaks is due to the limited amount of CHO bound to the nanoparticles, in comparison to the amount dispersed in water. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### FTIR spectra of Fe~3~O~4~magnetic nanoparticles (a) in nanopure water and Fe~3~O~4~-CHO (b), and pure CHO (c) prepared in phosphate buffer and then dissolved in nanopure water for FTIR analysis. ::: ![](1477-3155-3-1-3) ::: Cholesterol oxidase activity and binding kinetics ------------------------------------------------- The kinetic parameters of the enzymatic reactions estimated by the Lineweaver-Burk plots of the initial rates of cholesterol oxidase from experimental data are presented in Figure [4](#F4){ref-type="fig"}. The Michaelis-Menten constants V~max~and K~m~for CHO were determined to be 0.67 μmol/min mg and 2.08 mM for the free enzyme and 1.64 μmol/min mg and 0.45 mM for the immobilized enzyme, respectively. The V~max~value of the bound CHO was 2.4 fold higher than that of the free, and the K~m~value of the bound CHO was 4.6 fold lower than that of the free CHO. The low K~m~reflects the high affinity to substrate \[[@B4]\]. The high affinity of the enzyme to the substrate may be explained by the fact that when binding onto the surface of the nanoparticles, the enzyme rearranged itself to present a better conformation. Since the secondary and tertiary structure of cholesterol oxidase play important roles in its activity \[[@B9]\], the rearrangement in structure and conformation may result in better availability of its active sites. The increase in affinity of the enzyme to the substrate upon binding to Fe~3~O~4~nanoparticles contributed to an enhancement of the activity of the enzyme. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Lineweaver Burk plots of the initial rates of CHO (■) and (◆) Fe~3~O~4~-CHO at pH 7.4, from experimental data. ::: ![](1477-3155-3-1-4) ::: Effect of pH ------------ The effect of pH on the activities of the free and bound CHO was investigated in the pH range of 6--8.5 at 25°C and presented in Figure [5](#F5){ref-type="fig"}. In the pH range between 6 and 7.4 the activities of the free and bound CHO were quite similar and reached a maximum at pH 7.4. The activity then decreased from pH 8 to 8.5. In this range, the activity of the bound CHO was much higher than its free counterpart. This shows that the bound enzyme showed better tolerance to the variation of solution pH. The similarities in these activities in the pH range of 6 to 7.4 indicate that in these conditions, CHO did not suffer from any major activity constraint. Rather, this pH range appears to be suitable for CHO activity. It is well known that the ability of the amino acids at the active sites of the enzyme to interact with the substrate depends on their electrostatic state \[[@B16]\]. The decrease in activity observed at pH 8 and 8.5 shows that CHO faces some limitations as the pH increased toward more alkaline conditions. If the pH is not appropriate, the charge on one or all of the required amino acids is such that cholesterol can neither bind nor react properly to produce 4-cholesten-3-one. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Effect of pH on the activities of free (■) and bound CHO (◆). ::: ![](1477-3155-3-1-5) ::: Thermal stability ----------------- The thermal stability of free and bound CHO was investigated after 40 min of storage in the temperature range of 25--70°C (Figure [6](#F6){ref-type="fig"}). There was no apparent change in activity in the free CHO as well as in the bound CHO, in the temperature range of 25--37°C. Above this temperature range, the residual activity decreased in both systems. However, the bound CHO showed higher retained activity than the free CHO. The remaining activity at 60°C was about 2 fold that of the free CHO. This proved that the thermal stability was significantly improved upon binding of CHO to magnetic nanoparticles. Table [1](#T1){ref-type="table"} shows the inactivation rates constants (k) at temperatures where the inactivation experiments were observed. The rate constants increased with increasing temperature and were higher for the free CHO than for bound CHO. As stated above, the binding to nanoparticles suggests a better resistance of the enzyme to temperature. We hypothesize that the bound enzyme could possibly undergo a conformational change and a spatial rearrangement that could slow down the folding process and denaturation of the enzyme. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Thermal stability of free CHO (■) and Fe~3~O~4~-CHO (◆) at pH 7.4. The samples were stored at 50, 60, or 70°C for 40 min and the activities were then measured at 25°C. ::: ![](1477-3155-3-1-6) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Inactivation rate constants (k) of the \"bare\" and bound CHO at various temperatures ::: Temperature (°C) Free CHO Fe~3~O~4~-CHO ------------------ -------------- --------------- k(min^-1^) k(min^-1^) 50 3.4 × 10^-2^ 4.6 × 10^-3^ 60 9.3 × 10^-2^ 5.6 × 10^-2^ 70 2.8 × 10^-1^ 1.9 × 10^-2^ ::: Effect of temperature on enzyme activity and stability ------------------------------------------------------ The effect of temperature on the activity of the free CHO was examined by measuring its relative activity when stored at various temperatures (Figure [7](#F7){ref-type="fig"}). It can be observed that at 37°C, the enzyme retained its activity for about 80 minutes before showing a slight decrease. At 50°C the activity decreased continuously to 35% after 110 min. A more severe decrease in activity occurred at 60 and 70°C, resulting in a complete loss of activity after 60 and 70 min, respectively. The decrease in activity may be attributed to a dramatic change in the structure of the enzyme that hindered the availability of the active sites, with a possible denaturation of the enzyme itself. The effect of temperature on the activities of free and bound CHO at pH 7.4 are displayed in the Arrhennius plots (Figure [8](#F8){ref-type="fig"}). Only temperatures (50, 60 and 70°C) at which perceptible changes in activity were observed were studied. The activation energies were calculated to be 13.6 and 9.3 KJ/mol for free and bound CHO, respectively. The low activation energy related to the bound CHO suggests that when bound to the magnetic nanoparticles, CHO seems to acquire a better orientation that reduces the energy barrier for activity. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Effect of various temperatures on the activity Fe~3~O~4~-CHO at pH 7.4. ::: ![](1477-3155-3-1-7) ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Arrhennius plots of the initial plots of the oxidation rates of cholesterol by free CHO (■) and Fe~3~O~4~-CHO (◆) for samples at 50, 60, or 70°C. ::: ![](1477-3155-3-1-8) ::: Storage stabilities ------------------- The stability and activity of the enzyme are naturally reduced during storage. Figure [9](#F9){ref-type="fig"} shows the storage stabilities of free and bound CHO at 25°C at pH 7.4. After 15 days, no residual activity was observed in free CHO. However, the residual activity of bound CHO was 59% during the same time period, and 27% after 30 days indicating a considerable enhancement on its stability. It has been argued that this higher stability of the bound enzyme was due to its fixation on the surface of magnetic nanoparticles, preventing the auto-digestion and thermal inactivity \[[@B3]\]. Another plausible explanation is that the binding of CHO on Fe~3~O~4~nanoparticles might allow a better spatial orientation of the FAD prosphetic groups and the side chains of CHO providing a better stability to the enzyme. ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Storage stability of free CHO (■) and Fe~3~O~4~-CHO (◆). The activities measurements were performed at pH 7.4, at 25°C ::: ![](1477-3155-3-1-9) ::: Materials and methods ===================== Materials --------- Cholesterol oxidase (EC 1.1.3.6), Nocardiasp. was purchased from VWR international (Pittsburgh, USA). Carbodiimide-HCl (1-ethyl-3-(3-dimethyl-aminopropyl), ammonium hydroxide reagent, Triton X-100, TRIS (Hydroxymethyl) aminomethane HCL, 4-cholesten-3-one, bovine serum albumin (BSA), iron (II) chloride tetrahydrate 97 %, and iron (III) chloride hexahydrate 99% were obtained from Sigma-Aldrich, St Louis (USA). The Biorad Protein Assay Dye Reagent Concentrate was purchased from Biorad Laboratories (Hercules, CA). Acetonitrile was obtained from EMD Chemicals, (New Jersey, USA). Preparation of magnetic nanoparticles ------------------------------------- Magnetic nanoparticles (Fe~3~O~4~) were prepared by chemical co-precipitation of Fe^2+^and Fe^3+^ions in a solution of ammonium hydroxide followed by a treatment under hydrothermal conditions \[[@B4],[@B5]\]. Iron (II) chloride and iron (III) chloride (1:2) were dissolved in nanopure water at the concentration of 0.25 M iron ions and chemically precipitated at room temperature (25°C) by adding NH~4~OH solution (30%), at a control pH (10--10.4). The suspensions were heated at 80°C for 35 min under continuous mixing and separated by centrifuging several times in water and then in ethanol at 2800 rpm. The purification step was used to remove impurities from Fe~3~O~4~nanoparticles. The particles were finally dried in a vacuum oven at 70°C. The dried particles exhibited a strong magnetic attraction to a magnetic rod. Attachment of cholesterol oxidase onto magnetic nanoparticles ------------------------------------------------------------- 50--70 mg of magnetic nanoparticles was added to 1 mL of phosphate buffer (0.05 M. pH 7.4). The mixture was sonicated for 15 min after adding 0.5 mL of carbodiimide solution (0.02 g/mL in phosphate buffer (0.05 M. pH 7.4). Following the carbodiimide activation, 2 mL of cholesterol oxidase (0.25 mg/mL) was added and the reaction mixture was sonicated for 30 min at 4°C in a sonication bath and the mixture was centrifuged at 3000 rpm \[[@B17]\]. The precipitates containing Fe~3~O~4~nanoparticles and Fe~3~O~4~bound cholesterol oxidase (Fe~3~O~4~-CHO) were washed with phosphate buffer pH 7.4 and 0.1 M Tris, pH 8.0, 0.1 M NaCl and then used for activity and stability measurements. NaCl was added to enhance the separation of the magnetic nanoparticles \[[@B3]\]. Determination of immobilization efficiency ------------------------------------------ The amount of protein in the supernatant was determined by a colorimetric method at 595 nm using the Biorad Protein Assay Reagent Concentrate with bovine serum albumin (BSA) as the protein standard. The amount of bound enzyme was calculated from: A= (C~i~- C~s~)\*V (2) Where Ais the amount of bound enzyme, Ciand Csis the concentration of the enzyme initially added for attachment, and in the supernatant, respectively (mg-mL^-1^), Vis the volume of the reaction medium (mL). Characterization ---------------- The size of Fe~3~O~4~nanoparticles and Fe~3~O~4~-CHO was characterized by transmission electron microscopy (TEM, JEM 1200 EXII, JEOL USA) and structure by Fourier Transform Infrared (FTIR) spectroscopy (Biorad FTS 6000, Cambridge, MA). The samples for TEM analysis were prepared by placing a drop of the magnetic nanoparticles dispersed in nanopure water onto a copper grid and evaporated in air at room temperature. Before preparing a sample onto the copper grid, the dispersed solution was sonicated for 4 min to obtain better particle dispersion. The binding of CHO onto the magnetic nanoparticles was investigated using FTIR. CHO and Fe~3~O~4~-CHO samples in phosphate buffer and Fe~3~O~4~particles were dissolved in nanopure water for FTIR analysis. Activity measurement -------------------- The activity of bound CHO was determined by measuring the initial oxidation rates of cholesterol by cholesterol oxidase at given temperature following the increase of 4-cholesten-3-one concentration at 240 nm, using a Beckman Du Spectrometer. A solution of cholesterol was prepared by dissolving 4.8 g of cholesterol in 10 mL of 2-propanol. A phosphate buffer solution (0.05 M. pH 7.4) containing 4% of Triton-100 was added to the mixture to result in a 0.26 M cholesterol solution. The mixture was gently heated until the solution was clear. To start the enzymatic reaction, 5 ml of cholesterol solution was added to 15 mL centrifuge test tubes containing Fe~3~O~4~-CHO, and mixed by vortex. A solution of free CHO of the same concentration was used to evaluate the activity of the free enzyme. The solution was incubated at various temperatures (25--70°C) at specific intervals of time (1 h) and centrifuged at 3000 rpm for 5 min to separate the supernatant from Fe~3~O~4~-CHO. 10 μL aliquots of the supernatant were then taken and the concentration of 4-cholesten-3-one was assessed. Before measuring the amount of 4-cholesten-3-one in a sample, the activity of the free enzyme was stopped by adding an equal volume of acetonitrile to the reacting solution \[[@B18]\]. Each kinetic measurement was the average of duplicate replications. Thermal stability of free and immobilized enzyme ------------------------------------------------ The thermal stability of free and Fe~3~O~4~-CHO were determined by measuring the residual activity of the enzyme at 25°C, after being exposed to different temperatures (25--70°C) in phosphate buffer (0.05 M, pH 7.4) for 40 min. Aliquots of the reacting solution were taken at time intervals (every 30 min for 7 hours) and assayed for enzymatic activity as described above. The first order inactivation rate constant, kwas calculated from the equation: In A= In A~0~- kt (3) where A~0~is the initial activity, Ais the activity after a time t (min), kis the reaction constant. Effect of temperature on enzyme activity ---------------------------------------- The effect of temperature on the free CHO and Fe~3~O~4~-CHO was estimated by determining the concentration of 4-cholesten-3-one in samples at various temperatures. A solution of cholesterol was added to the various centrifuge test tubes containing bound or free enzymes. The test tubes were stored in a water bath at specific temperatures (25, 37, 50, 60, and 70°C). At time intervals, the concentration of 4-cholesten-3-one was determined by spectrophotometric analysis. Storage activity ---------------- The storage stability was evaluated by determining the concentration of 4-cholest-en-3-one at room temperature at time intervals (5 days). Test tubes containing Fe~3~O~4~-CHO or free enzyme solution were stored at 25°C in phosphate buffer (0.05 M. pH 7.4) for 30 days. Thereafter, 5 mL of cholesterol was added. The storage stability of the free and bound cholesterol oxidase was determined by assaying for their residual activity. Determination of kinetics parameters ------------------------------------ The kinetic parameters of free CHO and Fe~3~O~4~-CHO, K~m~ and V~max~ were determined by measuring initial rates of oxidation of cholesterol (1.3--5.2 mM) by CHO (0.25 mg/mL) in phosphate buffer pH 7.4 at 25°C. Conclusions =========== Magnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chlorides. The binding of CHO to the particles was confirmed by FTIR spectroscopy and the size characterized by TEM. The binding efficiency was between 98 and 100% irrespective of the amount of particles used. Kinetic studies of the free and bound CHO revealed that the stability and activity of CHO were significantly improved upon binding to nanoparticles. Furthermore, the bound enzyme exhibited a better tolerance to pH, temperature and substrate concentration. The activation energy indicated that the binding of CHO onto Fe~3~O~4~magnetic nanoparticles reduced the energy barrier for CHO activity. As a result of the binding to the magnetic nanoparticles, the storage stability of CHO was considerably enhanced. This higher stability of the Fe~3~O~4~-CHO is attributable to its possible fixation on the surface of the particles preventing auto-digestion and thermal inactivity. In addition, the binding on Fe~3~O~4~nanoparticles might allow a better spatial orientation of the FAD prosphetic groups and the side chains of CHO to provide better stability to the enzyme. The overall improvements observed in activity, stability, and functionality of CHO resulted from structural and conformational changes of the bound cholesterol oxidase. The study may be useful in improving the stability and activity of cholesterol oxidase, and will contribute to more efficient use of this enzyme. List of Abbreviations used ========================== CHO: Cholesterol oxidase TEM: Transmission electron microscopy FTIR: Fourier Transform Infrared BSA: Bovine serum albumin Authors\' contributions ======================= Drs Gilles K Kouassi and Joseph Irudayaraj were the primary authors. They were responsible for the concept, experimental plan, and analysis. Dr Gregory McCarty was the secondary author and contributed to the overall effort. Acknowledgements ================ The authors acknowledge the 2003 USDA challenge grant program for partial funding of this research. Dr Chen Xu is also acknowledged for the TEM images.	PubMed Central	2024-06-05T03:55:52.785818	2005-1-20	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548673/", "journal": "J Nanobiotechnology. 2005 Jan 20; 3:1", "authors": [ { "first": "Gilles K", "last": "Kouassi" }, { "first": "Joseph", "last": "Irudayaraj" }, { "first": "Gregory", "last": "McCarty" } ] }
PMC548674	Background ========== Breast cancer is the most frequent cancer in the female population of industrialized countries. Metastasis of breast cancer cells to the skeleton occur in \>70% of patients with progressive disease, resulting in debilitating symptoms such as severe bone pain, fractures, hypercalcaemia and spinal cord or nerve compressions due to extensive bone loss and tumour cell growth and expansion. Such bone loss occurs as a result of increased bone matrix resorption but the mechanisms by which cancer cells mediate this increased degradation have not been fully elucidated. Obviously, tumour expansion in bone requires the removal of the extracellular matrix (ECM) that is particularly abundant in bone. Cancer cells express matrix metalloproteinases (MMPs) and the plasminogen activator system (PAS) \[[@B1]-[@B3]\] and their levels of expression increase with progression of the tumour. The matrix metalloproteinases (MMPs) constitute a large family of structurally related matrix degrading proteases that have pivotal roles in development, tissue remodelling, and cancer \[[@B4]-[@B6]\]. The gene family of MMPs includes the interstitial collagenases (MMPs-1 and -13), gelatinase A (MMP-2), gelatinase B (MMP-9), the stromelysins (MMPs-3, 10 and 11) and the membrane type-matrix metalloproteinases (MT-MMPs 14,15,16,17, 24 and 25) \[[@B6]\]. The MMPs have the combined ability to degrade the major components of the ECM including type I collagen, the principal organic constituent of bone \[[@B4]\]. The PAS comprises: tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA), their inhibitors, and receptors. T-PA is thought to be more important in fibrinolysis, due to its fibrin binding capacity, whilst u-PA, especially when it is bound to its specific cell surface receptor (u-PAR), is thought to be involved in tissue remodelling and cell migration processes \[[@B7]\]. Whereas uPA alone recognizes a narrow range of substrates, the enzyme can catalyze the conversion of the circulating zymogen, plasminogen to plasmin. Plasmin, in turn, is a broad- spectrum proteinase that can directly degrade multiple ECM targets and can also cooperate with other ECM-degrading enzymes including members of the MMP gene family. Regulation of the PA/plasmin system is achieved mainly via plasminogen activator inhibitor (PAI) type-1 and type-2 and by agents that stimulate bone resorption, e.g. parathyroid hormone (PTH) and interleukin (IL)-1 \[[@B8]\]. To date, emphasis has focused on the ability of breast cancer cells to stimulate the formation and activity of osteoclasts, the cell primarily responsible for bone resorption under physiological conditions. The ability of osteoclasts to degrade bone lies in their ability to secrete protons and specialized collagenolytic proteinases, the cysteine proteinases in the acidic microenvironment that underlies osteoclasts during bone resorption \[[@B9]\]. Experimental studies showing that increased expression of MMPs and the PAS is associated with increased cellular invasion in vivosupport the idea that they play an important role in metastasis of tumour cells \[[@B10],[@B11]\]. Obviously tumour expansion in bone necessitates the removal of the ECM that is particularly abundant and resistant to degradation. Synthetic inhibitors of MMPs have been developed and two recent reports on their use on in vivobreast cancer metastasis to bone show promise when given as a preventive treatment to mice \[[@B12],[@B13]\]. However, the role of MMPs and the PAS in mediating breast tumour bone collagen dissolution has not been addressed. We have therefore assessed the ability of three human breast cancer cell lines, MDA-MB-231 (MDA-231), ZR-75-1 and MCF-7 to degrade bone collagen in vitrousing matrix degradation assays and compared their effects with those of a normal breast epithelial cell line, HME. We correlated the degradation activity of the breast cancer cells with their expression of MMPs and the PAS and we assessed the ability of group-selective proteinase inhibitors to prevent degradation of the organic aspect of bone by breast cancer cells. Results ======= Type I collagen degradation --------------------------- Bone degradation involves an initial phase of removal of the unmineralized type I collagenous layer followed by degradation of the mineralized matrix which also comprises type I collagen. The fibrillar integrity of the collagen layer was confirmed by incubation with collagenase that degraded the film whilst the collagen fibres were resistant to degradation by both trypsin and plasmin (data not shown). When the breast cancer cells (MDA-231, ZR-75-1 or MCF-7) were stimulated with TGFβ(10^-10^M) and cultured in the presence of 10% FCS the cancer cells induced significant degradation of type I collagen (range 70--80%), whereas minimal degradation was observed in the absence of serum (Fig. [1](#F1){ref-type="fig"}). To investigate the possible role of the PAS in collagen breakdown by breast cancer cells, plasminogen was depleted from FCS by lysine-Sepharose chromatography \[[@B14]\]. Interestingly, the depletion of plasminogen from serum also completely blocked breast cancer cell mediated collagen dissolution, implicating the PAS in breast cancer-mediated collagen degradation (Fig. [1](#F1){ref-type="fig"}). In accordance with this finding, the breast cancer cells degraded collagen under serum-free conditions only when supplemented by exogenous plasminogen (Fig. [1](#F1){ref-type="fig"}). The TGFβ-stimulated normal breast cell line, HME cultured in the presence of 10 % FCS demonstrated low type I collagenase activity (Fig. [1](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Degradation of ^14^C-labelled type I collagen films by breast cancer cells.Breast cancer cells (10^5^cells/well) stimulated with TGFβ (10^-10^M) were cultured for 24 h on ^14^C-labelled type I collagen under the following conditions: serum-free conditions; presence of 10% serum; 10% plasminogen (Plg)-depleted serum; serum-free medium supplemented with 2 μg/ml of human Plg. After 24-h incubation, collagen degradation was measured as described in Materials and methods. 8--800 mU (Ploug units) of pure human uPA in 1 ml of serum-free medium with or without 2 ug/ml of plasminogen, incubated as a control in parallel wells in the absence of cells, released 2--4% of the total radioactivity. This experiment was repeated twice. The results are expressed as percentage release of ^14^C. Each bar is the mean ± S.E.M of six wells. The stimulatory effects of plasminogen and TGFβ on breast cancer cell mediated ^14^C release were statistically significant \\\P \< 0.001 compared with the unstimulated controls and the HME cells. ::: ![](1475-2867-5-1-1) ::: The ability of the cancer cells to degrade the type I collagen is consistent with the expression of proteolytic activity. However, the midpoint melting temperature of reconstituted firbrillar type I collagen (47°C) is lower than that of authentic type I collagen in tissues (55°C to 60°C) \[[@B4]\]. Because the proteinase resistance of type I collagen can be compromised at temperatures within 10°C of that at which the helix reversibly unfolds \[[@B4]\], reconstituted fibrillar collagen may provide a less resistant substrate to proteolytic activity. Hence to determine whether breast cancer cell mediated degradation of type I collagen could be extended to a more physiological system, cancer cells were cultured on bone matrix as described below. Bone matrix degradation by breast cancer cells ---------------------------------------------- The described sequential gene expression of differentiating osteoblasts \[[@B15]\] was verified in MC3T3-E1 cell cultures so that non-mineralized matrix production was prepared after collagen production had commenced but before mineralization started. Mineralization of the matrices was confirmed by von Kossa staining (not shown). When TGFβ-stimulated breast cancer cell lines were cultured as a monolayer on either non-mineralized (Fig. [2A](#F2){ref-type="fig"}) or mineralized (Fig. [2B](#F2){ref-type="fig"}) bone matrix over a 24 h culture period there was significant degradation of the non-mineralized matrix (range 65--75%; Fig. [2A](#F2){ref-type="fig"}) and to a lesser extent the mineralized matrix (range 40--45%; Fig. [2B](#F2){ref-type="fig"}) only in the presence of plasminogen. In contrast, the TGFβ-stimulated breast cancer cell lines achieved a minimal amount of degradation of either matrix in the absence of plasminogen (Fig. [2A](#F2){ref-type="fig"} and [2B](#F2){ref-type="fig"}). The normal breast cell line, HME achieved a minimal amount of degradation of both matrices (5--15%) in the presence of plasminogen. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Degradation of ^3^H-non-mineralized (A) and ^3^H-mineralized (B) bone matrix by breast cancer cells. Breast cancer cells (10^5^cells/well) stimulated with TGFβ (10^-10^M) were cultured for 24 h on ^3^H-labelled extracellular matrices in the presence (+) and absence (-) of 2 μg/ml of human plasminogen. After 24-h incubation, bone matrix degradation was measured as described in Materials and methods. 8--800 mU (Ploug units) of pure human uPA in 1 ml of serum-free medium with or without 2 μg/ml of plasminogen, incubated as a control in parallel wells in the absence of cells, released 3--4% of the total radioactivity. This experiment was repeated twice. The results are expressed as percentage release of ^3^H labelled bone matrix. Each bar is the mean ± S.E.M. of six wells. The stimulatory effects of TGFβ on breast cancer cell mediated ^3^H release were statistically significant \\\P \< 0.001 compared with the unstimulated controls and the HME cells. ::: ![](1475-2867-5-1-2) ::: Expression of mRNA for MMPs and the PAS by breast tumour cells -------------------------------------------------------------- To characterize the profile of MMPs expressed constitutively and upon stimulation with TGFβ, breast cancer cells were cultured on type I collagen. Total RNA was isolated from 24-h cultures and screened by reverse transcription-PCR for MMP-1 through MMP-17. Under these conditions, four secreted MMPs were identified: MMP-1, MMP-3, MMP-9, and MMP-13 and the membrane-anchored MMP, MT1-MMP in all 3 breast cancer cell lines (Fig [3A](#F3){ref-type="fig"}; MDA-231 cells shown, results similar in all 3 breast cancer cell lines). TGFβ upregulated MMP expression in all 3 cancer cells lines (Fig. [3B](#F3){ref-type="fig"}; MDA-231 cells shown, results similar in all 3 breast cancer cell lines). The normal epithelial cell line, HME was found to express low levels of MMP-1 and MMP-3 upon stimulation with TGFβ (Fig. [3C](#F3){ref-type="fig"}) ::: {#F3 .fig} Figure 3 ::: {.caption} ###### RT-PCR of MMPs in breast cancer cells.Breast cancer cells were cultured as described in the Material and methods in the absence (A) and presence (B) of TGFβ (10^-10^M). Total RNA was isolated and RT-PCR performed with specific primers for MMPs-1,2,3,7,8,9,10,11,12,13,14,15,16,17. The housekeeping gene GAPDH was used as a positive control. Representative results for MDA-231 cells are shown (A) and (B): Lane 1 MMP-1; lane 2 MMP-13; lane 3 MMP-3; lane 4 MMP-9; lane 5 MMP-14; lane 6 GAPDH. The normal breast cell line HME stimulated with TGFβ is shown in (C). Band intensities were quantified by scanning densitometry and data expressed as a ratio (MMP/G3PDH) of the average optical density (OD) × area. The ratio of the intensity of the MMP mRNA band over the intensity of the G3PDH mRNA was arbitrarily designated as 1.0. ::: ![](1475-2867-5-1-3) ::: All 3 breast cancer cell lines expressed u-PA and u-PAR (Fig. [4A](#F4){ref-type="fig"}; ZR-75-1 cells shown, results similar with all 3 breast cancer cell lines) and upon stimulation with TGFβ there was increased expression of both u-PA and u-PAR by the breast cancer cells (Fig. [4B](#F4){ref-type="fig"}; ZR-75-1 cells shown, results similar with all 3 breast cancer cell lines.). The intensity of the signal was greater for the breast cancer cells than the normal breast cell line, HME (Fig. [4C](#F4){ref-type="fig"}). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### RT-PCR of uPA and uPAR in breast cancer cells.ZR-75-1 breast cancer cells were cultured as described in the material and methods in the absence (A) and presence (B) of TGFβ (10^-10^M). Total RNA was isolated and RT-PCR performed with specific primers for uPA, tPA and uPAR. The housekeeping gene GAPDH was used as a positive control. Lane 1 uPA; lane 2 uPAR; lane 3 GAPDH. The normal breast cell line HME stimulated with TGFβ is shown in (C). Band intensities were quantified by scanning densitometry and data expressed as a ratio (uPA or uPAR/G3PDH) of the average optical density (OD) × area. The ratio of the intensity of the uPA or uPAR mRNA band over the intensity of the G3PDH mRNA was arbitrarily designated as 1.0. ::: ![](1475-2867-5-1-4) ::: MMP and uPA production by breast cancer cells --------------------------------------------- To analyze the functional activities of MMPs and PAs expressed in breast cancer cells, collagenase activity in BCCM was measured by the degradation of FITC labelled type I collagen in the presence and absence of plasminogen. TGFβ markedly stimulated collagenase activity only in the presence of plasminogen (Fig. [5](#F5){ref-type="fig"}). uPA production was high in all cancer cells which degraded bone matrix but not in HME cells (Table [1](#T1){ref-type="table"}) that did not degrade bone at all, suggesting that uPA may be necessary to accomplish this task. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Collagenase activity in breast cancer conditioned mediaBreast cancer cells were cultured for 24 h in serum-free medium in the presence and absence of 2 μg/ml of human plasminogen and TGFβ (10^-10^M). Conditioned media were collected and incubated for 4 h with FITC-labelled type I collagen to detect collagenase activity, as described in Material and methods. The data are expressed as means SEM of 4--6 independent experiments, significantly different from control (\\, P \< 0.01; \\\* P \< 0.001). ::: ![](1475-2867-5-1-5) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Production of uPA by Breast Cancer Cells ::: Cells uPA (U/1 × 10^5^cells/24 h) --------- ----------------------------- MDA-231 2.3 ZR-75-1 1.9 MCF-7 1.6 HME Undetected uPA was measured by a chromogenic assay (see Materials and methods) in serum-free conditioned medium collected over a 24 h period. Breast cells were cultured in the presence of plasminogen (2 ug/ml) and TGFβ (10^-10^M). uPA was not detected in unstimulated breast cancer cells. ::: Roles of MMPs and PAS in breast cancer cell mediated bone degradation --------------------------------------------------------------------- We examined the effects of inhibitors of MMPs and the PAS on TGFβ-stimulated MDA-231 cell-mediated degradation of non-mineralized bone matrix under serum free conditions supplemented with plasminogen. The MMP inhibitors, CT1166 and TIMP-1 completely prevented TGFβ-stimulated breast cancer cell mediated bone collagen degradation (Fig. [6](#F6){ref-type="fig"}). When aprotinin, which inhibits both plasmin bound to the cell surface and plasmin in solution, was added, collagen degradation was also completely blocked (Fig. [6](#F6){ref-type="fig"}). Similar inhibitory effects were seen with function blocking antibodies to uPA or PAI-1 (Fig. [6](#F6){ref-type="fig"}). In contrast, the serpin α~2~-antiplasmin, which is a poor inhibitor of cell surface bound plasmin but an excellent inhibitor of plasmin in solution, did not prevent collagen degradation (Fig. [6](#F6){ref-type="fig"}). Since in the absence of cells, plasmin had no collagenolytic activity (see legend to Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}) and none of these inhibitors was cytotoxic, (data not shown), these results showed that bone collagen degradation by human breast cancer cells is dependent upon plasminogen activation and MMP activity. Western blot analysis demonstrated that neither CT1166 nor aprotinin influenced the production of MMPs (Fig. [7A](#F7){ref-type="fig"}) or uPA (Fig. [7B](#F7){ref-type="fig"}). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Effects of MMP and PAS inhibitors on MDA-231 cell mediated degradation of non-mineralized matrix.MDA-231 breast cancer cells (10^5^cells/well) were cultured for 24 h on ^3^H-labelled extracellular matrices in the presence of 2 ug/ml of human plasminogen, TGFβ (10^-10^M) with and without CT1166 (10^-5^M), TIMP-1 (50 ug/ml), aprotinin (10^-5^M), antibodies to human uPA (50 μg/ml) or human uPAR (50 μg/ml). After 24-h incubation, bone matrix degradation was measured as described in Materials and methods. This experiment was repeated twice. The results are expressed as percentage release of degradation of ^3^H labelled bone matrix. Each bar is the mean ± S.E.M. of six wells. The effects of the inhibitors were statistically significant \P \< 0.05;\\\P \< 0.001. ::: ![](1475-2867-5-1-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Immunological characterization of MMPs and uPA in Breast Cancer Cells.Breast cancer cells (10^5^cells/well) stimulated with TGFβ (10^-10^M) were cultured for 24 h in serum-free medium in the presence of 2 μg/ml of human plasminogen and CT1166 and aprotinin. Western blot analysis was undertaken as described in the Materials and methods section. Lane 1, MDA-231 cells; lane 2, ZR-75-1cells; lane 3 MCF-7 cells; lane 4, HME cells. Pro- and active forms of collagenase-1 gelatinase-B, and stromelysin-1 and proform of collagenase-3 were detected (A). Pro and active forms of uPA are shown (B). ::: ![](1475-2867-5-1-7) ::: Discussion ========== The data presented in this paper clearly demonstrate that human breast carcinoma cell lines have the capacity to degrade the organic aspect of bone matrix in vitro, and there is a dependency on the PA system for the cell-mediated collagen degradation. Furthermore, we have shown that plasmin associated with the cell surface is responsible for activating the fibrillar collagenase, MMP-13. The ECM in our experiments was produced by MC3T3-E1 mouse calvarial-derived cells. These cells display osteoblast-like characteristics, providing a suitable model of osteogenesis analogous to in vivobone formation \[[@B17]\]. The bone nature is evident from the appearance of mineralization, resulting in the production of a solid sheet of mineralized matrix. Our evidence showing that breast cancer cells degrade bone matrix is in agreement with that of Eilon and Mundy \[[@B18]\] who reported that MCF-7 cells were capable of degrading the organic aspect of devitalized murine bone explants in vitro. More recently it has been demonstrated that prostate cancer cells and melanoma cells directly degrade mineralized bone matrix and that the degradation was reduced by generalized inhibition of MMP activity \[[@B19],[@B20]\]. Whereas the induction of MMPs in TGFβ stimulated breast cancer cells that are actively engaged in tumour osteolysis has not been examined previously, this growth factor has been reported to increase the expression of MMPs-1, -3 and -9 \[[@B21]\]. In our study TGFβ induced a complex MMP expression profile that included MMPs-1, -3, -9 -13, and -14 as the principle products. Furthermore, MMPs are able to release and activate TGFβ, a very abundant bone matrix-bound factor \[[@B22]\]. The fact that breast cancer cells degraded the non-mineralized bone matrix to a greater extent than the mineralized substrate, suggests that access of the matrix degrading proteinases to the organic phase of the matrix may have been restricted by the mineralized phase. This investigation has demonstrated a significant difference in the ability of breast cancer cells and normal breast epithelial cells to degrade the organic aspect of bone matrix in vitro. Variations in the profile and amounts of proteinases expressed by the different cell lines may be responsible. Since the MMP inhibitors used in this study (CT1166 and TIMP-1) inhibit the activities of all MMPs, except the membrane-type MMPs in the case of TIMP-1, we cannot identify the contribution of individual MMP members to bone matrix degradation. However, our demonstration of a direct requirement of plasmin in the activation of proMMP-13 suggests that this fibrillar collagenase may play a prominent role in the degradative activity of breast cancer cells. However, the possibility of other known MMPs also being involved in plasminogen-dependent collagenolysis by breast cancer cells cannot be excluded. The participation of the membrane-type MMPs such as MMP-14, -15 and -16 is improbable due to the ability of TIMP-1 to effectively block collagen dissolution by breast cancer cells. MMP-2 and -- 8 were not expressed in detectable amounts by breast cancer cells, as assessed by RT-PCR. The existence of very small, but functionally important, amounts of MMP-2 or -- 8 however cannot be excluded unequivocally by the expression studies. MMP-1 is expressed by the human breast cancer cells in this study and may contribute to plasmin-dependent fibrillar collagen dissolution. Currently, little is known with regard to the physiological mechanisms by which MMPs undergo activation in intact cell systems \[[@B23]\]. However, a primary function of the uPA-uPAR couple is to focus the processing of the plasminogen zymogen to active plasmin on the cell surface. It has recently been demonstrated that binding of u-PA to its receptor increases pro-MMP-2,-9 \[[@B23]\] and -13 \[[@B16]\] activation, and that in the absence of cells, plasmin not only fails to activate pro-MMPs, but rapidly degrades them \[[@B23]\]. This would explain why interfering with only one element, plasmin or MMP activity, has such powerful biological effects. It should be stressed, however, that few of the cascades or activities have been unequivocally documented in intact cell systems. Nonetheless, because MMP-specific inhibitors are not yet available, the individual role of each of the cell-derived MMPs awaits the use of interventions based on RNA inhibition. The increased bone resorption that accompanies breast cancer cell infiltration of bone may arise as a result of (1) breast cancer cells stimulating osteoblast and osteoclast activity and (2) the production by breast cancer cells of proteinases that participate in degrading the organic aspect of bone matrix which would also facilitate the access of osteoclasts to the underlying mineralized matrix. Even if tumour-derived MMPs do not directly digest mineralized matrix, they may participate in osteolysis via a mechanism that is analogous to a known osteoblast function. It is believed that all endosteal surfaces are covered with a layer of nonmineralized matrix. In areas of bone formation, this layer is called osteoid and it is easily observed in stained sections \[[@B24]\]. Evidence is accumulating that degradation of this layer must occur before the attachment of osteoclasts to the underlying mineralized matrix \[[@B25]\]. In normal remodelling, digestion of this layer is apparently accomplished by MMPs produced by osteoblasts \[[@B25]-[@B27]\]. It is conceivable that breast cancer cells in bone adopt this osteoblast function; thus, the possibility that MMPs produced by cancer cells enhance osteoclastic degradation by prior removal of the overlying unmineralized layer. Furthermore, removal of this layer may be partially responsible for the recruitment and activity of osteoclasts due to the release of osteoclast attractants/stimulants. Bone resorption by osteoclasts is augmented experimentally by coating mineralized matrix with collagenase-cleaved collagen fragments \[[@B28]\]. As nonmineralized matrix is degraded, osteoclasts may be exposed to extracellular proteins, such as fibronectin, vitronectin, osteopontin or other cryptic epitopes. Osteoclasts bind to these proteins via integrins a process that may enhance bone resorption \[[@B29]\]. Conclusion ========== In conclusion, we have shown that breast cancer cells can degrade the organic aspect of bone matrix in contrast to non-tumourogenic breast epithelial cells. There is an absolute requirement for both the PA system and MMPs in the degradation process. Materials and methods ===================== Reagents -------- The MMP inhibitor CT-1166 was a generous gift from Dr A Docherty, Celltech (Slough, UK). The PAS inhibitor aprotinin, plasminogen and TGFβ were purchased from Sigma. The human mammary epithelial cell (HMEC) line, Mammary Epithelial Basal Medium (MEBM), insulin, hydrocortisone, gentamicin/amphotericin-B and Bovine Pituitary Extract (BPE) were purchased from Clonetics (Walkersville, MD, USA). Human breast tumour cell lines MDA-231, ZR-75-1 and MCF-7 were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). Gelatinase-A, TIMP-1 and polyclonal sheep antibodies to human MMP-1, -2,- 3, -9 and -13 were gifts from Dr J Reynolds, Strangeways Research Laboratory, Cambridge, UK. Neutralizing mAbs against human uPA or human uPAR were from American Diagnostica, Greenwich, CT, USA. Cell cultures ------------- Cell lines were cultured in 75-cm^2^plastic tissue culture flasks (Costar Corning, Cambridge, MA, USA) as follows: ZR-75-1, MDA-231, MCF-7 and MC-3T3-E1 in α-MEM and HME cells in MEBM medium. All media were supplemented with 10% heat inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 ug/ml streptomycin (Sigma Chemical Co.). Cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% carbon dioxide and subcultured every third day. Type I collagen degradation assay --------------------------------- ^14^C-labelled collagen films were prepared as described previously \[[@B30]\]. Breast cells were seeded into collagen coated 16 mm tissue culture wells at a density of 1 × 10^5^/well. After 4 h incubation at 37°C in MEM supplemented with 10% FCS, the cells were washed twice with phosphate buffered saline (PBS) to remove residual FCS, and incubated for 24 h with 1 ml/well of serum-free MEM with or without the indicated concentrations of plasminogen, TGFβ and the proteinase inhibitors to be tested. At the end of the culture period the media were centrifuged (15 min, 1200 × g) to remove any collagen fibrils, and radioactivity released during collagen degradation quantified by liquid scintillation counting. Residual collagen was digested with bacterial collagenase (50 μg/ml) and assayed for radioactivity. Collagenolysis was expressed as radioactivity released from the films as a percentage of the total ± SEM. Formation of ^3^H-labelled, non-mineralized and mineralized bone matrix ----------------------------------------------------------------------- The murine calvarial-derived MC3T3-E1 is a well characterized osteoblast culture system providing a suitable model of osteogenesis analogous to in vivobone formation \[[@B15]\]. The cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% carbon dioxide. Culture medium was changed on the first day after seeding and then every 72 h. The cells were split after 7--9 days in culture, before they reached confluence, and plated at a density of 1 × 10^4^cells/well on collagen-coated 24-well plates (Becton Dickinson, MA, USA). After 4--5 days, when the cultures had reached confluence and the formation of an ECM had started, fresh medium was added containing 1 μCi/ml ^3^H amino acid mixture for 7 days (Amersham International, Aylesbury, UK) to create a non-mineralized radiolabelled ECM. In order to create a mineralized bone matrix, the medium was supplemented with 10 mM β-glycerol phosphate and the cells cultured for 14 days and the radiolabelled medium changed every 3 days. Unincorporated ^3^H-radiolabelled amino acids were washed from the remaining ECM using 2 water washes and 75% (v/v) ethanol. The matrices were dried and stored at -20°C until use. Von Kossa staining for mineralization ------------------------------------- Mineralization of matrices was determined by von Kossa staining. The matrices were rinsed with cold PBS and fixed in 10% neutral buffered formalin for 15 min, rinsed with distilled water and left in distilled water for 15 min. Matrices were stained with 2.5 % silver nitrate solution for 30 min at room temperature. The silver nitrate was removed and matrices were rinsed with distilled water before the addition of sodium carbonate formaldehyde for 5 min. After rinsing, the matrices were counterstained with toluidine blue, rinsed with tap water and air dried. Bone matrix degradation assay ----------------------------- Breast cancer cells (10^5^/16 mm culture well) were seeded onto the bone matrix as for the type I collagen degradation assay. After 4 h incubation at 37°C in MEM supplemented with 10% FCS, the cells were washed twice with phosphate buffered saline (PBS) to remove residual FCS, and incubated for 24 h with 1 ml/well of serum-free MEM with or without the indicated concentrations of plasminogen, TGFβ and the proteinase inhibitors that were tested. After 24 h incubation, the media were removed and the extent of degraded ^3^H-radiolabelled matrix released into the medium was determined by liquid scintillation counting. The remaining matrix was degraded as for the type I collagen assay and matrix degradation expressed as above. PCR and RT-PCR -------------- Confluent breast tumour cells were stimulated with TGFβ (10^-10^M) in serum-free medium for 24 h. For PCR analysis, oligonucleotide primers were synthesized based on the published sequences for 14 MMPs (i.e., MMP-1, -2, -3, -7,-8,-9,-10, -11, -12, -13,-14,-15, -16, and -17) \[[@B4]\]. t-PA, u-PA and, u-PAR oligonucleotides were designed using Designer PCR (Research Genetics, AL, USA) and primers purchased from Life Technologies Ltd. The housekeeping gene GAPDH was used as a positive control for the RT-PCR methodology. Enzymes and buffers for the reverse transcriptase and PCR reactions were obtained from Perkin Elmer (CA, USA). Reverse transcriptase reactions were done according to the manufacturer\'s protocol using 1 μg of total RNA collected from untreated or TGF-β treated breast cells. PCR reactions were performed in an automated DNA thermal cycler (Perkin Elmer) for 25 cycles of denaturation (95°C, 1 min), annealing (variable time and temperature) and polymerisation (60°C, variable time). Amplification of the house-keeping gene, G3PDH mRNA (35 cycles), provided an internal control for the efficiency of the RT-PCR process. RT-PCR products were analyzed against molecular weight standards (pBR322 HaeIII digest) on a 2.5% agarose gel stained with ethidium bromide, electrophoresed in 0.5× TBE buffer at 100 V for 90 minutes. Gels were examined under ultraviolet light and photographed. The authenticity of the PCR products was verified by sequencing \[[@B31]\]. Western Blot analysis --------------------- To identify MMP proteins and uPA present in breast cell conditioned medium, Western blot analysis was performed using their specific antibodies as previously described \[[@B32]\]. Briefly, samples were separated by 8.5% SDS-PAGE, transblotted on to PVDF membranes (Millipore Corp., MA, USA) and immunoblotted with polyclonal sheep antiserum to MMPs or polyclonal goat antiserum to uPA and secondary peroxidase-conjugated anti-sheep / anti-goat antibodies. Labelled proteins were detected with ECL detection solution and exposed to autoradiographic film (Hyperfilm ECL, Amersham International, UK). Assay of collagenase activity ----------------------------- To measure collagenase activity, TGFβ (10^-10^M)-stimulated breast cells were cultured in the presence/absence of 2 ug/ml of human plasminogen for 24 h. Collagenase activity was measured by the degradation of fluorescein isothiocyanate (FITC)-labelled type I collagen using type I activity assay kits. One unit of these activities degrades 1 μg of collagen per min at 37°C. uPA Assay --------- Cells were plated in 6-well trays and grown to near confluence, the medium was removed, and the cells were washed and incubated with 2 ml of serum-free medium for 24 hr. The medium was collected, centrifuged, and frozen until assayed. The cells were lysed in 300 ul of 0.1 M Tris (pH 8.1) and 0.1% Triton X-100. The u-PA (10 ul of conditioned medium and 10 μg of cell protein) was assayed as previously described \[41\] using plasminogen and S2251, the chromogenic substrate for plasmin. Statistical Analysis -------------------- Differences between control and treatment groups were determined by the Mann Whitney U-test.	PubMed Central	2024-06-05T03:55:52.787931	2005-2-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548674/", "journal": "Cancer Cell Int. 2005 Feb 8; 5:1", "authors": [ { "first": "Hayley", "last": "Morgan" }, { "first": "Peter A", "last": "Hill" } ] }
PMC548675	Background ========== One of the potential use of sugar and sugar intermediate binding proteins and binders of intermediate sugar metabolites derived from microbes is in new and expanding area of environmental biotechnology particularly in carbon dioxide fixation bioprocess reactors \[[@B1],[@B2]\]. Accelerated consumption of fossil fuels and other anthropogenic activities have resulted in increased atmospheric levels of the greenhouse gas carbon dioxide (CO~2~). Sustained increase of atmospheric CO~2~has already initiated a chain of events with unintended ecological consequences \[[@B3]-[@B7]\]. The reduction in atmospheric carbon dioxide level is highly desirable lest it will have a catastrophic impact upon both the environment and the economy on a global scale \[[@B5]-[@B7]\]. Biotechnological process with recombinant catalytic proteins offer contained handling of carbon dioxide and could be one method of abatement of carbon dioxide pollution \[[@B8],[@B9]\]. Recent advances in biotechnological methods makes possible efficient capture \[[@B10]\] and fixation of CO~2~at emission source/site into concatenated carbon compounds \[[@B9],[@B11]\]. Such a process starts with initial capture of the carbon dioxide solublized as carbonic acid or bicarbonate \[[@B10]\]. After adjustment of pH using controllers and pH-stat the solution is fed to immobilized Rubisco reactors \[[@B12]\] where acceptor D-Ribulose-1,5-bisphosphate (RuBP) after CO~2~fixation is converted into 3-phosphoglycerate \[[@B8],[@B9]\]. We have invented a novel scheme which proceeds with no loss of CO~2~(unlike cellular biochemical systems) in 11 steps in a series of bioreactors \[[@B13]\]. For starting up the process, however, a different scheme was used to generate RuBP from D-glucose rather than from 3-PGA \[[@B14]\]. The linear combination of reactors in the 11 step RuBP regeneration process requires large volume and weight and are unsuitable for use in mobile CO~2~emitters leaving only the stationary source of emission to be controlled using this technology \[[@B8],[@B9]\]. These problems are circumvented in a new scheme where enzyme-complex reactors instead of linear combination of purified single enzyme reactors were proposed \[[@B1],[@B2]\]. In this scheme, the catalytic enzymes have been used as functionally interacting complexes/interactomes. The four reactors harboring enzymatic complexes/mixtures \[[@B2]\] replace the current 11 reactors for conversion of 3PGA into RuBP \[[@B8],[@B9]\] and are termed as enzyme-complex reactors (Figure [1](#F1){ref-type="fig"}). As an alternate to immobilized enzyme-complexes, in this scheme, successive conversion in radial flow with layers of single and purified but uniformly oriented enzymes in concentric circle with axial collection flow system with required combination of enzymes in individual reactors could also be used \[[@B1]\]. This arrangement in preliminary experiments shows promise and leads to a faster conversion rate and requires less volume and material weight. Sugar and sugar intermediate metabolite removal in enzyme-complex reactors at key steps is necessary for proper and specific driving of forward reactions. This requires in-situ separation of sugar or intermediate metabolites by specific binding entities. The compounds such as 3-phosphoglycerate (3PGA) and 3-phosphoglyceraldehyde (3PGAL) must be separated at key steps for proper functioning of these enzyme-complex reactors \[[@B1],[@B2]\]. Sugar and their intermediate metabolites binding proteins derived from microbial and other sources despite being used for various applications \[[@B15],[@B16]\] have not yet been used in environmental biotechnological applications. However, they are potentially applicable in RuBP recycling. In this report we demonstrate the utility of two inactive mutants of enzymatic proteins: phosphoglycerate mutase (PGDM) and enolase. The inactive mutants of yeast enzymes PGDM \[[@B17]-[@B19]\] and enolase \[[@B20],[@B21]\] were characterized for properties that may render them potentially useful in reactors. We report determination of the enzymatic activity, sugar binding capacity, specificity in binding for different sugar and their metabolites and reversibility in binding with respect to changes in physicochemical factors and stability on repeatedly subjecting to these changes using purified proteins. Results ======= Purity of proteins ------------------ The inactive PGDM mutant \[[@B17],[@B18]\] was received as purified protein and analyzed on a 10% SDS-PAGE (Figure [2](#F2){ref-type="fig"}). The S39A mutant of yeast enolase 1 \[[@B20],[@B21]\] was purified from recombinant yeast using ammonium sulphate precipitation and chromatographic purification on CM cellulose (Figure [2](#F2){ref-type="fig"}). The yeast enolase mutant of H159A was also purified using a similar protocol (data not shown). The purified proteins were tested for modifying activity on 3-phosphoglycerate, 3-phosphoglyceraldehdye, glucose and sucrose (Figure [3A](#F3){ref-type="fig"}). Chromatographic detection of sugars and intermediate metabolites ---------------------------------------------------------------- Paper chromatography was used for initial qualitative detection of sugars or sugar metabolites (Figure [3A](#F3){ref-type="fig"}) subsequently thin layer chromatography (Figure [3B](#F3){ref-type="fig"}) was used for detection and determination of modification as well as for measurements. Relative measurements of spot area with respect to the controls allowed determination of binding as described in methods. Binding constants ----------------- Binding constants for mutant proteins were determined and presented in Table [1](#T1){ref-type="table"}. Dissociation constant (Kd) is a useful measure to describe the strength of binding or affinity between the proteins (P) and the ligands (L) and serves as an indicator as to how easy it is to separate the complex PL. With both PGDM and enolase mutants high micromolar (μM) concentration L is required to form PL indicating that the strength of bindings is rather moderate or even low. The smaller Kd, the stronger is the binding. The dissociation constant for 3-phosphoglycerate was found to be lower by both PGDM (655 ± 33 μm) and yeast enolase mutants S39A (676 ± 28 μm), H159A (797 ± 47 μm) compared to that for 3-phosphoglyceraldehde (822 ± 42, 835 ± 38 and 966 ± 31 μm respectively). The H159A had the weakest binding among other proteins for 3-phosphoglycerate. In qualitative measurements both proteins showed release or lack of binding when subjected to low pH (pH 4.0). In addition EDTA at concentrations that leads to chelation of Mg^2+^ions was very effective for release of ligands from enolase as well as from PGDM (Table [1](#T1){ref-type="table"}). Reversibility in binding and repeated use ----------------------------------------- The PGDM showed binding with 3PGA and 3PGAL at pH 7.5 in presence of 50 mM NaCl. The binding was completely lost when the protein was exposed to pH 4.0 (Table [1](#T1){ref-type="table"}). Binding was restored if the pH value was brought back to 7.5 within 30 min exposure to lower pH. The S39A mutant of yeast showed binding to 3PGAL as well as to 3PGA in presence of 10 mM NaCl and 10 mM MgCl~2~. Both 3PGA and 3PGAL failed to bind the protein and is released when subjected to 15 mM EDTA. The binding is restored upon removal of EDTA and addition of NaCl and MgCl~2~. Both proteins retained more than 50% initial binding upon repeated use, in this study we did not use more than 30 min incubation at lower pH during any single use. As shown in Table [2](#T2){ref-type="table"}, the proteins retained qualitatively more than 50% binding for at least 8 cycles of repeated changes between pH 4.0 and pH 7.5. PGDM was less stable (8 cycles) than S39A or H159A mutants (10 cycles) each. This is important for reactor applications. While it is possible to control reactor operations where in-situseparation reactors can be bought back to pH immediately upon binding and for small reactors less than 30 min of residence time can be useful, more optimization studies are needed to establish stability at low pH for these proteins. For MgCl~2~cycling, the proteins retained more than 85% activity even after 20 cycles and were not studied any further. Discussion ========== Microbes (as well as higher organisms) produce a number of binding and metabolizing proteins for sugar and other intermediate metabolic products of sugar metabolism pathways. Complex conversion necessitated using binding entities to enhance reactor performance as well as obtaining converted compounds in purified state. In the biocatalytic CO~2~fixation bioprocess, three modular reactors enables continuous fixation. The first module is Rubisco reactor where CO~2~is fixed onto RuBP and converted into 3PGA, the second involves regeneration of ATP which acts as a cofactor for subsequent process and the third being RuBP regeneration \[[@B8],[@B9]\]. Recently an added module has been devised for efficient capture of CO~2~, which is constructed before first module \[[@B10]\]. However, the generation of RuBP from converted 3PGAL requires a series of conversion in 11 reactors \[[@B8],[@B9]\]. In many reactors in this series, the sugar or intermediate metabolites are generated that are not substrates for immediate subsequent reactors. Thus a specific capture and delivery will help eliminate dilution as well as interference in reactors where sugar intermediates are not direct substrate. Towards this goal, three different entities divided into biological and chemical moieties have potential for use. The biological entities include lectins or sugar binding proteins or non-immunogenic origin and inactive mutants of sugar binding enzymes. The chemical entities include the entities that recognize and binds aldehyde, ketones and alcohols. However, most of the biological entities provides high specificity, strong binding, reversible with respect to select physico-chemical conditions. They are also compatible with buffer systems with respect to conversion reactors in the loop where they are likely to be used. For 3PGAL and 3PGA as test sugars we have attempted using PGDM and inactive yeast enolase mutants (S39A, H159A) here. The determination of binding strength by these entities will enable their pilot tests in novel reactors in close loop with 3PGA to RuBP conversion reactors. It appears that the PGDM and enolase mutants are reversible binders and are stable with respect to repeated use-cycles. However, the binding is moderate at best (Kd values are in the range of 655--966 μM). Similar measurements for enolase provides 500 ± 28 and 673 ± 32 μM for binding with 3PGA and 3PGAL indicating that wild type enolase has slightly higher binding affinity for these metabolites. Further enhancement in binding by screening other different inactive enzyme mutants or that from different sources may help find more suitable entities. Conclusions =========== In the present report we demonstrate binding without chemical modification of 3PGA and 3PGAL by inactive yeast mutant enzymatic proteins PGDM and enolase. The binding seems to be specific as the proteins were not found to bind other sugars (glucose, fructose or sucrose in mixtures that were subjected to incubation). The binding is reversible with respect to pH and EDTA and proteins retain activity even after repeated use. The MgCl~2~cycling seems to have less effect on protein stability with respect to binding and release and could be more suitable for use in larger reactors. While these criteria are suitable for use of the proteins for in-situseparation of sugar metabolites in reactors, but high micromolar dissociation constants (655--966 μM) suggest the moderate or even low binding strength. Finding inactive enzymes with high binding affinity or engineering them for this purpose will improve their utility. Methods ======= Protein purification -------------------- Purified yeast PGDM and recombinant cultures for inactive yeast enolase mutants (S39A and H159A) were obtained from Dr. J. Nairn and Prof. J. M. Brewer as research gift respectively. The enolase mutants were purified following suitable modifications of published protocols \[[@B20],[@B21]\]. Briefly, the overnight grown yeast cultures were harvested when absorbance values reached 12. The cells were harvested by centrifugation at 4000 × g and disrupted using sonication for 10 min with 20 sec gap and burst cycles and centrifuged at 12000 × g for 20 min. This crude protein solution was subjected to ammonium sulphate precipitation at 75% saturation. After ammonium sulphate precipiration the protein was dialyzed for 16 hours in a dialysis bag (molecular weight cutoff 3000) in 0.1 M Tris-Cl (pH8.5) containing 0.1 M NaCl and 0.1 mM MgCl~2~, with three changes in buffer solution. Enolase mutants were subjected to ion exchange chromatography for further purification on Acta prime protein purification system (Amersham Pharmacia Biotech, CA) using Q Sepharose column at a flow rate of 0.5 ml /minute at 4°C. Protein was eluted using a NaCl gradient (0.1 M to 0.3 M), about 95 fractions containing 8.6 ml each were collected. The proteins eluted in about conductivity equivalent of 0.1--0.15 M NaCl (fractions 5 to 9). This purified protein was temporarily stored at 4°C and used for subsequent experiments. Proteins were estimated using Bradford\'s method \[[@B22]\] using BSA (1 mg/ml) as standard. 10%SDS-PAGE was prepared and subjected to Coomassie blue staining. Paper and thin layer chromatography ----------------------------------- Paper and thin layer chromatography was performed to demonstrate that the purified mutant proteins lacked any sugar modification capacity. Sugar mixtures in appropriate concentration were incubated with Proteins (PGDM or enolase mutants S39A and H159A) for up to 16 hours. At the end of incubation (every 2 hours), the mixtures were centrifuges and the aliquots from experimental mixtures were spotted onto the filter paper chromatograms (Whatman; 3 mm) or on TLC plates. The TLC analyses were performed on Plastic backed 20 cm × 20 cm Silica Gel 60 F254 plates with 0.2 mm layer thickness (Merck). After spotting with an applicator the samples were air-dried and placed in a TLC tank (27 cm × 24 cm × 7 cm) containing the solvent system. For both chromatography, the spots were air-dried and the chromatograms were dipped in the solvent system \[60% v/v n-propanol/30% v/v conc.ammonia/10% v/v distilled water\] and allowed to run for 5 hours. The chromatograms were removed from the solvent system and subjected to staining. Three different staining techniques were used to detect sugars, ammonium molybdate, silver nitrate and alpha-naphthol staining \[[@B23],[@B24]\]. For ammonium molybdate staining, the paper chromatogram was dipped in the solution contianing: 5 ml 60 per cent w/w perchloric acid,10 ml and 0.1 N hydrochloric acid,25 ml, 0.4 per cent w/v ammonium molybdate, and made the volume to 100 ml with distilled water. The paper, after drying in a current of warm air for a few minutes to remove excess water, was subjected to heating at 85°C for 7 min in a water-jacketed oven. The spots in the chromatogram were also visualized using alkaline silver oxide reagent. This reagent was composed of two parts: first part containing 0.1 ml saturated aqueous silver nitrate plus 19 ml acetone and second part containing 0.5 g NaOH dissolved in 5 ml water and finally these two parts are added to 100 ml with ethanol. First part was mixed immediately before use and a few drops of water were added, with stirring, until all the AgNO~3~is dissolved. The dried chromatogram was then dipped through the silver reagent and allowed to air dry for 10 min and subsequently dipped into ethanolic sodium hydroxide and again allowed to air dry. After the spots are visible, the paper was soaked in dilute (5 mg/l) sodium thiosulfate for 1 minute and rinsed in tap water. The last step dissolves the dark background and allows obtaining a permanent record. For staining with alphanapthol, paper chromatogram was dipped in the following solution: 1 per cent w/v alpha-napthol, 10 per cent v/v orthophosphoric acid and distilled water to make up the volume. The air-dried paper or TLC plate was heated for a few minutes at 85°C in a water-jacketed oven for color development. Determination of binding parameters ----------------------------------- The dissociation constants for protein-sugar binding was estimated by measurements of area in chromatograms. For this purpose covalently immobilized protein A sepharose beads (Pharmacia Biotech, CA) was used. The proteins were immobilized on protein A using Amino link kit (Pierce Chemicals, CA). The known concentration of protein was incubated at room temperature (25°C) with varying concentration of sugar in the range of 1 μM to 1 mM in a 100 μl fixed volume. At the end of incubation (10 min), the mixture was centrifuged at 10000 × g and an aliquot of supernatant was spotted and chromatogram (TLC) was developed. A similar mixture but with BSA coupled beads served as control. Area calibration using varying concentration of sugar with a fixed aliquot spot volume was recorded under identical conditions. From the measurement of area in control and experimental set the free sugar was calculated such bound sugar is control minus sugar left in experimental set. The experimental data was used to draw a Scatchard type plot from where dissociation constant was calculated, represented by P as free protein, L as ligand and PL as the ligand-bound-protein, the dissociation constant is defined as Kd = \[Pfree\] \[Lfree\]/ \[PL\]. The dissociation constant Kd values for PGDM and S39A enolase for 3PGA and 3PGAL were calculated using experimental data using MS excel program. Authors\' contributions ======================= DB and DB carried out purification of enolase mutants, MK and SM carried out the binding assays. MTS, SC, AG and VG participated in design of the study and performing analyses, MTS also helped to draft the manuscript. SKB conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Acknowledgements ================ We thank Dr. Nanasaheb P. Chougule, Mrs. Vaijyanti Thambane and Gauri Bhatt for their help with the experiments. We thank Mr. R. N. Ghosh for his timely administrative assistance. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Arrangement of enzymes in four reactors with indicated enzyme complexes enabling internal channeling, greatly reduces volume and weight for regenerating reactors with faster overall conversion rate to RuBP starting with 3PGA making the system compatible for application in mobile devices in addition to stationary emitters \[2\]. The reactors may use the sugar binding entities at indicated positions, the solid symbols represent metabolite-bound matrix (binding protein entities), the plus, circle, cylinder and box are symbols for 3PGA, DHAP, X5P and RuBP binders respectively. ::: ![](1475-2859-4-5-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### The crude lysate and purified proteins. About 5--10 μg of proteins were loaded on a 10% SDS-PAGE as indicated. The inactive PGDM and mutant yeast enolase 1 S39A and H159A has been indicated. The gel was stained with Coomassie blue. ::: ![](1475-2859-4-5-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### A.Representative paper chromatographic detection of sugars metabolites. The sugars or intermediate metabolites (0.1--0.5 mM) were incubated with BSA (control), PGDM or enolase mutants overnight at room temperature, proteins removed by centrifugation at 10000 × g and an aliquot of supernatant was spotted. a. d. The chromatogram for enolase mutant S39A stained with silver nitrate reagent, b. c. chromatogram for enolase mutant S39A stained with ammonium molybdate reagent. B.Representative thin layer chromatography of sugar metabolites (3-phosphoglycerate, 3-phosphoglyceraldehdye). About 0.1 mM substrate (0.1 mM of each mixture component) as indicated was incubated overnight with purified S39A enolase or BSA (control) has been shown. The chromatogram was developed using silver nitrate reagent following protocol as described in methods and image has been converted to greyscale. ::: ![](1475-2859-4-5-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### The binding constants of proteins for sugar metabolites. ::: Protein Condition 3-Phosphoglycerate 3-Phosphoglyceraldehyde ---------------- ------------------------------------------------------------ ------------------------ ----------------------------- ---- ------------- Kd (μm) Kd (μm) PGDM 10 mM Tris-Cl pH 7.5, 50 mM NaCl +++ 655 ± 33 ++ 822 ± 42 10 mM Tris-Cl pH 4.0, 50 mM NaCl R R Enolase, S39A 10 mM Tris-Cl pH 7.5, 10 mM NaCl, 10 mM MgCl~2~ +++ 676 ± 28 ++ 835 ± 38 10 mM Tris-Cl pH 7.5, 10 mM NaCl, 1 mM MgCl~2~, 15 mM EDTA R R Enolase, H159A 10 mM Tris-Cl pH 7.5, 10 mM NaCl, 10 mM MgCl~2~ ++ 796 ± 23 \+ 966 ± 31 10 mM Tris-Cl pH 7.5, 10 mM NaCl, 1 mM MgCl~2~, 15 mM EDTA R R The binding constant was estimated from scatchard type plots using experimental data. The results provided here are from 3 replicates ± standard deviations. The substrates used in different concentration were analyzed with respect to BSA control for binding using TLC as described in methods. A silver nitrate protocol was used for detection and spot area determination. Plus signs denote the strength of ligandbinding; R indicates ligand release. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### The stability of proteins with use-cycles. ::: Protein Number of Cycle when still active\* ---------------- ----------------------------------------- PGDM 8 Enolase, S39A 10 Enolase, H159A 10 \* The retaintion of at least of 50% initial activity was considered as active. The cycle refers changes between pH 4.0 and 7.5 in 10 mM Tris buffer containing 50 mM NaCl. The values shown here are concurrent values from five independent experiments. :::	PubMed Central	2024-06-05T03:55:52.790491	2005-2-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548675/", "journal": "Microb Cell Fact. 2005 Feb 2; 4:5", "authors": [ { "first": "Debojyoti", "last": "De" }, { "first": "Debajyoti", "last": "Dutta" }, { "first": "Moloy", "last": "Kundu" }, { "first": "Sourav", "last": "Mahato" }, { "first": "Marc T", "last": "Schiavone" }, { "first": "Surabhi", "last": "Chaudhuri" }, { "first": "Ashok", "last": "Giri" }, { "first": "Vidya", "last": "Gupta" }, { "first": "Sanjoy K", "last": "Bhattacharya" } ] }
PMC548676	Background ========== Mosquito sampling is a prerequisite to most vector population studies \[[@B1]\]. The entomological parameter being studied and the behaviour of the mosquito species being sampled determine the choice of a sampling method \[[@B2]\]. However, most of the available mosquito sampling methods may not allow for such rational choices to be made, as there are major limitations associated with their use \[[@B3]\]. Therefore, new tools for sampling mosquito vector populations must be continuously developed. Nonetheless, even these new sampling tools must be calibrated against the existing ones in different vectorial systems \[[@B4]\] if they are to be adopted for conventional use. A new trap, the Mbita trap has been developed \[[@B5]\] and separately evaluated in quite different vectorial systems in Western Kenya and Madagascar \[[@B6],[@B7]\] with varying degrees of success. The Mbita trap was originally developed in semi-field systems with an Anopheles gambiaecolony originating from southern Tanzania \[[@B5]\] and proved a sensitive and representative way to sample An. gambiae, Anopheles arabiensisand Anopheles funestusin Western Kenya \[[@B6],[@B7]\]. In sharp contrast, the Mbita trap proved highly insensitive for catching An. funestus and An. arabiensisin rice-growing communities in the highlands of Madagascar \[[@B6],[@B7]\]. In this study, the performance of the Mbita trap compared to the CDC light trap hung adjacent to a human-occupied bednet and the human landing catches in the sampling of An. arabiensis An. funestusand culicines species of mosquitoes in a rice-growing community in western Kenya with relatively high mosquito densities is reported. Methods ======= Description of the study area ----------------------------- These studies were carried out in a village adjacent to the Ahero rice irrigation scheme in western Kenya. Populations of An. arabiensis and An. funestus\[[@B8]\] as well as culicine species \[[@B1]\] are predominant in this area. The characteristics of the mosquito population and malaria vectorial system in this area have been described in detail elsewhere \[[@B1],[@B9]\]. ### Sampling In Ahero, three houses were selected upon receiving consent from the household heads. Occupants were given a non-impregnated bed net per sleeping space and trained in their correct use. With informed consent, three young men who had earlier been trained in mosquito sampling \[[@B6]\] were recruited to act as bait in the three alternative mosquito collection methods. On each experimental night, one of the three subjects slept in the Mbita trap (BNT), another slept in a bed net with a CDC light trap suspended beside it (CDC) and the third conducted a human landing catch (HLC) \[[@B3]\]. Both the Mbita trap and the CDC light trap-bed net system were set on mattresses placed on mats laid on the floor and not on beds. In all the experiments, a standard miniature CDC light trap (Model 512; John W. Hock Company, Gainesville, Florida, USA) with an incandescent light bulb was used. The trap was hung beside the bed net on the foot side of the sleeping person with its shield touching the side of the net and its inlet about 25 cm above the sleeping person \[[@B10]\]. Each of the three sampling methods was allocated to one of the three houses on a given night in a 3 × 3 randomised Latin square experimental design replicated 3 times. The human baits did not move around the sites so that the effects of a particular site and the attractiveness of the human bait associated with it were combined for simplified statistical analysis. Sampling was carried out from 20.00 hrs to 06.00 hrs between October and November 2002. Ethical considerations ---------------------- Informed consent was obtained from all the participants. Thick and thin blood smears were regularly taken from the participants to examine for the presence of malaria parasites and, when found positive, they were treated with pyrimethamine-sulfadoxine (Fansidar^®^). A follow-up was made to ensure that any parasitaemia was fully cleared. If parasitaemia did not clear, the participants were referred to hospital for further treatment with second line drugs. The Kenya Medical Research Institute (KEMRI) through the KEMRI/National Ethical Review Committee granted ethical approval (KEMRI/7/3/1) for this study. Mosquito processing ------------------- Mosquitoes were taken to the laboratory and killed by suffocation with chloroform vapour. They were counted and identified morphologically using taxonomic keys \[[@B11],[@B12]\] and then desiccated over anhydrous copper sulphite and kept at room temperature until further processed. Abdomens of An. gambiaesensu lato were analysed by PCR for sibling species identification \[[@B13]\]. Statistical methods ------------------- The simple expedient of adding one to each mosquito count in order to cater for zero counts can be misleading \[[@B14]\]. Therefore, Winbugs version^®^1.4 was used to fit regression-based models to the data. The conceptual basis of this Bayesian regression has been described in detail elsewhere \[[@B15]\]. The following models were fitted to the data: ### Scenario A: A linear model for sampling proportionality E(y~i~) = α~t~β~c~E(x~i~) Where: E(y~i~) is the expected number of mosquitoes caught using the method being tested; E(x~i~is the expected number of mosquitoes caught using the human landing method (assuming the same mosquito collector as bait); α~t~is a multiplication factor corresponding to trapping method tin relation to the reference trapping method which was human landing catch; and β~c~is a multiplication factor corresponding to human bait ccompared to the reference catcher, assigned number 1, whose value is set to 1. ### Scenario B: A non-linear model for sampling proportionality E(y~i~) = α~t~β~c~(E(X~i~))^yt^ All the terms for model B are identical to model A except that it includes y~t~which is the exponent corresponding to trapping method t. A value of y~t~different from 1 indicates a lack of proportionality between the methods. Both models assumed Poisson errors in the numbers of mosquitoes caught by any of the three methods. Results ======= Overall, the Mbita trap, the human landing collection and the CDC light trap-bednet method caught 135, 576, and 474 An. arabiensisand 309, 427, and 470 An. funestusrespectively. The corresponding figures for culicines mosquitoes (mainly Culexspecies) were 32, 121 and 578. Also, 30 male mosquitoes were caught, 29 of them by the CDC light trap and one in the landing collections. The parameter estimates from our models (Table [1](#T1){ref-type="table"}, Figure [1](#F1){ref-type="fig"}) indicate the Mbita trap caught about 17%, 60%, and 20% of the number of An. arabiensis, An. funestus, and culicine species caught in the human landing collections respectively. There was consistency in the sampling proportionality between the Mbita trap and the human landing catch for both An. arabiensisand the culicine species whereas for An. funestus, the Mbita trap portrayed some density-dependent sampling efficiency. More specifically, the Mbita trap appears more sensitive than human landing catch at low mosquito densities. The CDC light trap, on the other hand, caught about 60%, 120%, and 552% of the number of An. arabiensis, An. funestus, and culicine species caught in the human landing collections respectively. There was consistency in the sampling proportionality between the CDC light trap and the human landing catch for both An. arabiensisand An. funestus, whereas for the culicines, there was no simple relationship between the CDC light trap catches and the landing catches (Table [1](#T1){ref-type="table"}, Fig. [1](#F1){ref-type="fig"}). From PCR identification, all the successfully amplified specimens of An. gambiaes.l. were found to be An. arabiensis. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Point estimates and 95% confidence intervals for model parameters. ::: An. arabiensis An. funestus Culicines --------- -------------------------------- ------------------- ------------------- ------------------- Model A α~t~: BNT versus HLC 0.17 (0.14, 0.21) 0.73 (0.62, 0.85) 0.20 (0.13, 0.29) α~t~: CDC versus HLC 0.56 (0.49, 0.66) 1.19 (1.03, 1.37) 4.84 (3.81, 6.21) β~c~: Person 2 vs person 1 0.38 (0.32, 0.45) 0.89 (0.77, 1.02) 0.54 (0.41, 0.72) β~c~: Person 3 vs person 1 0.54 (0.47, 0.62) 0.77 (0.66, 0.90) 0.59 (0.42, 0.80) Model B α~t~: BNT versus HLC 0.80 (0.52, 1.12) 0.39 (0.30, 0.50) 0.71 (0.00, 2.02) α~t~: CDC versus HLC 1.04 (0.79, 1.42) 0.84 (0.66, 1.06) 5.52 (3.08, 7.46) ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Numbers of female mosquitoes caught by the three sampling methods in 9 nights in western Kenya. Regression lines (unbroken) depict the fitted simple proportionality model (Model A) and the non-proportional (broken lines), density-dependent sampling efficiency model (Model B). ::: ![](1475-2875-4-7-1) ::: Discussion ========== The results obtained from this study indicate a three-fold decrease in efficiency for both the Mbita trap and the CDC light trap when used to sample An. arabiensiscompared to their reported performance for nearby An. gambiaes.l. population that comprised of roughly equal numbers of An gambiae sensu stricto and An. arabiensis\[[@B6]\]. However, the consistency in the proportionality of their catches relative to the human landing collections was maintained. Several factors might explain this observation. First, this area is dominated by An. arabiensis, a mosquito species that is usually largely zoophagic but endophilic \[[@B16]\]. Therefore, protecting all the people in a bednet, as was the case in the houses where the Mbita trap and the CDC light trap were used, might have prompted the indoor resting An. arabiensisto seek alternative hosts outdoors. For the case of the human landing collection, the human bait was more readily available for such indoor resting mosquitoes. Second, unlike in Lwanda \[[@B6]\], where no cattle were present in any of the homesteads that were sampled, large numbers of cattle were present in all the homesteads sampled at Ahero. Therefore, there was an alternative source of blood meal to this more flexible species, which can utilize both domestic \[[@B16]\] and wild bovids \[[@B17]\]. The availability of cattle could possibly account for the reported poor performance of the Mbita trap in sampling An. arabiensisin the highlands of Madagascar \[[@B7]\]. Other studies in Ahero have reported similar CDC light trap to human landing catch ratios \[[@B1]\] for An. arabiensisas this study found but with no correlation between the two methods. The performance of the Mbita trap relative to the human catch for An. funestusin Ahero was similar to that reported for Lwanda \[[@B6],[@B7]\] but showing density dependent sampling efficiency as density increased. Specifically, it appears that the Mbita trap may be more sensitive at low densities (Figure [1](#F1){ref-type="fig"}). It was considered whether this could be caused by the lowered attentiveness of individuals conducting tedious human landing catches when few mosquitoes are present. However, this was not found to be the case as the sampling efficiency of the CDC light trap, relative to the human landing catches showed no density dependence. The efficiency of the CDC light trap for An. funestuswas about 2.5-fold that in Lwanda. The relatively high densities of this species in Ahero compared to Lwanda, might, at least partly, account for these observations. At Lwanda, where the densities of An. funestuswere low, no density-dependent sampling efficiency was noted for the Mbita trap while some density-dependent sampling efficiency was noted for the CDC light trap \[[@B6]\] suggesting that the Mbita trap is more sensitive in low densities while the CDC light trap is better at higher densities of this species. Many studies have evaluated the performance of the CDC light trap relative to the human landing catch but it is very difficult to compare the results due to the different methodologies and sampling procedures applied. In this study, the three methods were used concurrently in different houses on the same night while in other studies \[[@B2],[@B18],[@B19]\] the methods were used in the same houses but on different nights. Small differences in sleeping arrangements, availability of alternative hosts, temperatures, humidity, and wind speed and direction between the different days might introduce some sampling bias in this case. Furthermore, the procedures used for conducting human landing catch also vary appreciably: some studies have used one human per house to perform landing catches \[[@B20]\] while others \[[@B2],[@B18]\] have used two catchers in the same house. There is, therefore, a need to standardize the operational conditions and sampling procedures used if valid comparisons between various studies in are to be made Conclusions =========== Although the Mbita trap is less sensitive than either the human landing collections or the CDC light trap, for a given investment of time and money \[[@B5]\], it is likely to catch more mosquitoes over a longer period, larger number of sampling sites or both. Adult mosquito densities are highly aggregated in space and time, resulting over 80% of transmission occurring in 20% of places and time \[[@B21]\], and the importance of catching them across large numbers of sampling points and frequent intervals to obtain representative samples of the vector population has recently been emphasized \[[@B22]\]. The Mbita trap may therefore be very useful for enabling community members in collecting large numbers of samples that are representative of the overall vector population at a less cost \[[@B5]\] than a smaller number of light traps/human catchers. Used in this way, rather than as a direct replacement for the CDC light trap-bednet method, this trap will surely find a place in community-based malaria vector surveillance. It might be important to note that some community members in Rusinga, an island adjacent to ICIPE-Mbita point where the trap was developed, have adopted this trap for passive mosquito surveillance with some encouraging results. However, this trap might not work in all epidemiological settings \[[@B7]\] and therefore more mosquito behavioural studies should be carried out in order to gain more insight to guide further development of mosquito sampling and control tools. The human landing catch should be maintained as the standard reference method for use in calibrating new methods for sampling the human biting population of mosquitoes. Authors\' contributions ======================= EMM designed the trap, designed the study and drafted the manuscript. GOM & DOO supervised the fieldwork and recorded the data. LWI & PNN were involved in the study design and drafting the manuscript, TAS carried out the data analysis and helped in the interpretation of results. GFK guided the experimental design, data analysis and drafting of manuscript. BGJK conceived the initial idea of developing the trap and solicited for funds used in the trap development and trials. All authors read and approved the final manuscript. Acknowledgements ================ The authors would like to thank the people of Ahero for allowing us to use their premises for the study. Christian Abuya, Steve Abong\'o and James Nderitu are thanked for accepting to participate in this study. Lawrence Omukuba, Basilio Njiru, Joseph Mwangi, Lucy Njeri, Cannon Maina, Sammy Maina and Jackton Arija offered valuable technical assistance. Dr. Ulrike Fillinger offered financial and logistic assistance. This work was supported by WHO/WORLD BANK/UNDP/Special Programme for Research and Training in Tropical Diseases through project ID No. 980794, the National Institutes of Health, NIH-ICIDR project No. 5U19AI45511 and the Fogarty International Centre, through ABC grant number ID43TWØ1142.	PubMed Central	2024-06-05T03:55:52.792982	2005-1-25	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548676/", "journal": "Malar J. 2005 Jan 25; 4:7", "authors": [ { "first": "Evan M", "last": "Mathenge" }, { "first": "Gedion O", "last": "Misiani" }, { "first": "David O", "last": "Oulo" }, { "first": "Lucy W", "last": "Irungu" }, { "first": "Paul N", "last": "Ndegwa" }, { "first": "Tom A", "last": "Smith" }, { "first": "Gerry F", "last": "Killeen" }, { "first": "Bart GJ", "last": "Knols" } ] }
PMC548677	Background ========== It has become increasingly evident that the central nervous system is an immunocompetent organ \[[@B1]\]. Microglia are the primary immune effector cells of the brain parenchyma and functionally resemble tissue macrophages elsewhere in the body \[[@B1],[@B2]\]. The brain ventricles are also under immune surveillance by intraventricular macrophages which patrol the cerebrospinal fluid (CSF), choroid plexus, and supraependymal surface \[[@B3]\]. At the interface of the CSF and brain proper is the ciliated neuroepithelial ependymal cell which lines the ventricular system of the brain and spinal canal. The ependyma not only functions as a physical barrier preventing foreign proteins and organisms from entering the brain from the CSF, but also displays immunological effector ability such as phagocytosis of fluorescent beads injected into the CSF \[[@B4]\] and upregulation of MHC-II in response to interferon gamma challenge in vivo\[[@B5]\]. These diverse cell types may work in concert establishing the basis for the innate immune system of the CNS. Importantly, a population of resident subventricular microglia (SVMs) are found in the subependymal zone \[[@B6],[@B7]\] suggesting the ependyma and microglia may cooperate to prevent invasion of the CNS from the ventricular system. Juxtaventricular microglia have been shown to react to both direct periventricular/ependymal damage as well as the mere presence of cytokines in the CSF. For instance, the intracerebroventricular (ICV) injection of lentiviral tat protein in low nanomolar quantities is sufficient to kill ependymal cells and cause a periventricular inflammatory reaction including the characteristic microglial nodules of human HIV-1 encephalopathy (HIVE) \[[@B8]\]. Alternatively, Kong et al \[[@B9]\], noting the high CSF levels of inflammatory cytokines in multiple sclerosis (MS), demonstrated a vigorous periventricular activation of microglia with ICV injection of IFN-γ alone or in combination with endotoxin or TNF-α. In these cases, microglia were activated in the absence of primary tissue damage, but were thought to contribute secondarily to immune-mediated periventricular damage via potentiation of cytokine release and bystander effect. Of note, both HIVE and MS often present with enigmatic periventricular inflammatory lesions in humans \[[@B10]-[@B13]\]. The reaction of SVMs to periventricular damage has not been described in detail. Specifically, the functional repertoire displayed by activated SVMs including phagocytosis and long-distance migration have not been investigated. Here, we directly examine SVM function by exploiting a novel methodology which selectively activates and labels SVMs in vivocombined with confocal timelapse techniques for dynamic analyses in adult mouse brain tissue. We hypothesized that SVM activation is a general consequence of periventricular insults that can be caused by diverse circulating substances in the CSF known to damage the ependyma. Our work indicates that activated, phagocytic SVMs are capable of infiltrating deep within the parenchyma. Methods ======= Animals ------- Adult male C57bl/6 mice (6--8 wks) were obtained commercially from Harlan (Indianapolis, IN) and cared for in accordance with Public Health Service and University of Virginia guidelines. Surgical procedures ------------------- To damage the ependyma 2--3 μl of 0.2% Sp-DiI (D-7777, Molecular Probes) in DMSO; 1:20 rhodamine-conjugated latex microspheres (Lumafluor, Naples, FL) in sterile PBS; 0.25 U Neuraminidase (Sigma); or 2.0 nM recombinant HIV-1 tat protein in 100 mg/ml BSA, 0.1 mM DTT in PBS were stereotaxically injected into the left lateral ventricle at the following coordinates: L, 1.5 mm; P, -0.5 mm; D, 2.0 mm. GFP-expressing adenovirus (10^9^plaque forming units); 100 mg/ml BSA, 0.1 mM DTT in PBS, or deactivated tat \[[@B14]\] were used as volume-matched controls. Forebrain stab lesion for deafferenting lesion of the contralateral hippocampus was performed as previously described \[[@B15]\]. Briefly, mice were anesthetized with a Xylazine/Ketamine mixture and placed in a stereotaxic head holder (Benchmark, myneurolab.com). Temperature was maintained with a ventral heat pad. A right parietal craniectomy extending 3--4 mm from midline and spanning Lambda to Bregma was created with a microdrill. Beginning at the level of Bregma, a 3 mm lesion was created in the sagittal plane 1.5 mm from midline at a depth of 3.5 mm with a sterile no. 11 scalpel blade held in the stereotaxic device. After achieving hemostasis, the bone was replaced and sealed with carboxylate cement (Durelon, Fisher Sci). This right sided lesion results in deafferentation injury to primarily stratum oriensof the left hippocampus. Static analyses --------------- Brains were collected and processed for histopathology as previously described \[[@B15]\]. All antibodies and staining procedures have been described previously \[[@B15]\] except for rat anti-cd11b/MAC-1 (1:50), mouse anti-foxj1 (1:1000), mouse anti-nestin/rat-401 (1:100), and rat anti-F4/80 (1:10). Histochemistry for biotinylated Griffonia simplicifolialectin IB~4~was performed 1:100 in PBS overnight at 4°C and visualized with either 1:200 FITC- or Alexa Fluor 350-conjugated streptavidin (Sigma and Molecular Probes, respectively). Dynamic analyses ---------------- 200--400 μm live slices were prepared from adult C57BL/6 mice as described previously \[[@B16],[@B17]\]. Briefly, mice were acutely anesthetized in a chamber with halothane and decapitated. The brain was rapidly removed, blocked, and covered with 10% agar at 37°C in a specimen mold (Tissue-Tek 4566, Fisher). Live slices were obtained with a vibratome and placed individually on Millicell-CM inserts (Millipore PICM03050, Fisher). Culture medium consisted of CCM1 (Hyclone, Logan, UT) with 20% heat-inactivated normal horse serum. Vital labelling of microglia in tat experiments was performed with Alexa 488 or 568 IB~4~(Molecular Probes) \[[@B17]\]. Laser-scanning confocal images were acquired on a Nikon IX-70 inverted microscope with Fluoview 300 software (Olympus). A z-series stack covering 40 μm of slice thickness was taken every 1.5--4 minutes, creating a three-dimensional timelapse data set. To create timelapse movies from the data set, 4 to 6 z-plane images were collapsed as 2D projections using ImageJ 1.31 u and compiled into quicktime movies with Quicktime Pro 6.3. Movies were analyzed for migration speed and distance as described \[[@B18]\]. Values represent the mean ± SEM. Statistical analysis was performed with ANOVA or student\'s t-test. Pairwise post-hoc analysis was performed with a t-test and the Bonferroni correction factor. A p\< 0.05 was considered statistically significant. Rose plots were created in Kaleidagraph 3.0. Results ======= Selective ependymal death is induced by high-dose rhodamine dyes ---------------------------------------------------------------- Rhodamine dyes such as SP-DiI and rhodamine latex microbeads (RhoB) have been classically used for tract tracing studies in vivoand in fixed tissues \[[@B19],[@B20]\]. Recently, intracerebroventricular (ICV) injection of these dyes into the CSF has also been shown to selectively label ependymal cells \[[@B4],[@B21]\]. In pilot studies for other projects, we discovered doses of these dyes that, in addition to labelling, result in selective death and denudation of the ependyma (not shown). To investigate the timecourse of ependymal damage in response to rhodamine dyes we injected a toxic bolus of SP-DiI (0.2% in DMSO) or rhodamine latex microbeads (1:20 in PBS) into the left lateral ventricle of mice. Overt ependymal damage was appreciated beginning by 12 h (Fig. [1a](#F1){ref-type="fig"}, left panel) post-injection and progressed rapidly by 24 h (Fig. [1a](#F1){ref-type="fig"}, middle panel) where the ependyma appeared swollen and ragged with frequent pyknotic profiles (Fig. [1b](#F1){ref-type="fig"}). At these doses, damage was largely restricted to the lateral ventricle ipsilateral to the injection and the third ventricle (not shown) while sparing the contralateral lateral ventricle (Fig. [1b, c](#F1){ref-type="fig"}) suggesting a dose- and diffusion-dependent toxicity. Near complete ependymal cell loss occured in regions of the lateral ventricle proximal to the injection site within 3 days with both dyes (Fig. [1A](#F1){ref-type="fig"}, right panel &[1D](#F1){ref-type="fig"}). Mild subependymal astrogliosis as revealed by nestin immunohistochemistry was also evident by 3d (Fig. [1E](#F1){ref-type="fig"}). The loss of ependyma was further confirmed by chronic loss of immunoreactivity for the ciliated cell-specific transcription factor foxj1 at 1 month after injection (Fig. [1f](#F1){ref-type="fig"}). Animals injected with equal volumes of GFP-reporter adenovirus (Fig. [1g](#F1){ref-type="fig"}) or low-dose rhodamine microbeads in PBS (1:50, not shown) demonstrated no ependymal loss or activation of subventricular microglia (SVMs, not shown). Thus, rhodamine dyes rapidly and selectively damage ependymal cells at high doses. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Ependymal damage with rhodamine dyes. (A) Timecourse of ependymal death in the lateral ventricle after rhodamine dye injection demonstrated with digital subtraction. Damage to the ependyma was evident at 12 h and rapidly progressed by 24 h. (B) Histology at 24 h demonstrates swollen ependyma with numerous pyknotic profiles in injected, but not the contralateral, hemisphere. e, ependyma; lv, lateral ventricle; p, parenchyma. RHO fluorescence overlaid on brightfield hematoxylin images. (C) Low-power view of lateral ventricles 3 d after injection demonstrates halo of rhodamine-positive cells around injected ventricle (white arrow). The contralateral ventricle demonstrates labelled ependyma in the absence of damage. (D) By 3 d, near-complete loss of the ependyma was evident. This coincided with the appearance of dye-laden SVMs, black arrowheads. The ependyma remained intact in the contralateral hemisphere (right panels). e, ependyma; lv, lateral ventricle; p, parenchyma; RhoB, rhodamine beads. RHO fluorescence overlaid on brightfield hematoxylin image (RhoB) and photoconverted DiI counterstained with hematoxylin. (E) Periventricular reactive astrocytes (black arrows) visualized with nestin immunohistochemistry (IHC) at 3d post-injection at wall of injected ventricle (left), but not in the contralateral hemisphere (right). lv, lateral ventricle; sp, septum. (F) IHC for ciliated cell-specific foxj1 28d after dye injection demonstrates persistent loss of ependyma in injected hemispere (left). cc, corpus callosum, cp, caudate/putamen; sp, septum. (G) Equivalent volume control injection of GFP-reporter adenovirus demonstrates no ependymal damage 3 weeks after injection. e, ependyma; lv, lateral ventricle; p, parenchyma. GFP fluorescence overlaid on brightfield hematoxylin image. ::: ![](1742-2094-2-5-1) ::: SVMs phagocytose ependymal debris --------------------------------- Loss of the ependyma in the above areas coincided with the appearance of dye-laden periventricular cells resembling macrophages (Fig. [1d](#F1){ref-type="fig"}, arrowheads). At low power, affected ventricles were surrounded by a halo of these rhodamine-positive (RHO+) cells (Fig. [1a](#F1){ref-type="fig"}, last panel; [1c](#F1){ref-type="fig"}). To determine the identity of the RHO+ cells we performed transmission electron microscopy (Fig. [2a](#F2){ref-type="fig"}), immunohistochemistry for microglial/macrophage markers F4/80 (Fig. [2b](#F2){ref-type="fig"}) and MAC-1/cd11b (not shown), and histochemistry for IB~4~lectin from Griffonia simplicifolia(Fig. [2b](#F2){ref-type="fig"}). These techniques demonstrated periventricular RHO+ cells to be microglia. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Selective labelling of SVMs with rhodamine dyes. (A) RHO+ cells are microglia. Transmission electron microscopy demonstrates dye-laden inclusions (white arrows) in a SVM. n, nucleus. (B) Immunohistochemistry for F4/80 (top) and histochemistry for lectin IB~4~(bottom) demonstrate double-labelled periventricular cells, white arrows. (C) Time-lapse confocal microscopy in live brain slices demonstrates SVM (white arrow) extending (time 0\' and 9\') and retracting (time 4.5\' and 13.5\') a process toward ependymal debris (yellow star) highly suggestive of phagocytosis. See also Video 1. lv, lateral ventricle; p, parenchyma. (D) Neuraminidase injection following sublethal ependymal labelling similarly results in RHO+ SVMs (black arrows). e, ependyma; lv, lateral ventricle; p, parenchyma. Left panels, hematoxylin; Right panels, RHO fluorescence overlaid on hematoxylin. ::: ![](1742-2094-2-5-2) ::: SVMs may become RHO+ as a result of phagocytosis of the dye-labeled ependymal debris. To provide direct evidence of this hypothesis we performed confocal time-lapse microscopy in living slices from adult mice given dye injection 24 h prior to sacrifice. Grossly, we observed a dramatic increase in RHO+ periventricular cells over several hours suggesting active clearance of labelled debris (not shown). Further, SVMs displayed dynamic behavior consistent with phagocytosis of ependymal debris (Fig. [2c](#F2){ref-type="fig"}, Video 1(Additional file [1](#S1){ref-type="supplementary-material"})). Therefore, SVMs became rhodamine positive after high-dose dye injection due to phagocytosis of labelled ependymal debris. To determine if SVM activation is a general response to periventricular damage we injected animals with a sublethal dose of RhoB to label ependymal cells without causing damage. 24 h later we injected 0.25 U neuraminidase to damage the ependyma \[[@B22]\] via an alternate mechanism. Histological examination of sections at 7 d revealed loss of the ependyma and the presence of RHO+ SVMs (Fig. [2d](#F2){ref-type="fig"}, left panels). Control injection of PBS vehicle alone resulted in no ependymal damage or SVM labelling (Fig. [2d](#F2){ref-type="fig"}, right panels). Thus, ependymal cell damage of diverse etiologies incites a reactive response by SVMs including phagocytosis of debris. Long-distance infiltration by SVMs after parenchymal injury ----------------------------------------------------------- Unpurturbed, SVMs remained in the immediate periventricular vicinity with no deeper parenchymal migration (Fig. [1c](#F1){ref-type="fig"}). Indeed, his population was stable in animals sacrificed up to 30d following dye injection (not shown). Periventricular lesions in HIVE and MS often extend deep into the parenchyma suggesting long-distance infiltration of reactive cells. Microglia have been shown to migrate in vitroin response to many chemokines and growth factors present in brain lesions and plaques \[[@B23]-[@B26]\]. In order to investigate whether activated SVMs can migrate towards parenchymal brain damage in vivowe gave mice a deafferenting lesion (FSL) of the hippocampus 24 hours following rhodamine dye injection and allowed survival for up to 28 days. Invasion of the parenchyma by RHO+ cells occurred in the stratum oriensof the denervated hippocampus (cSO) in 21/21 animals but not in sham animals (0/4) (Fig. [3a](#F3){ref-type="fig"}). Infiltrating cells were found an average of 849 ± 34 μm from the lateral ventricle after FSL compared to 210 ± 16 μm in uninjured mice (p \< 0.01, Fig. [3b](#F3){ref-type="fig"}). Based on population distribution histograms, greater than 75% of RHO+ cells in sham animals were found within 300 μm of the ventricles (maximum: 860 μm) whereas greater than 75% were found beyond 400 μm (maximum: 2377 μm) in injured mice. Temporal quantification of RHO+ cell infiltration in the cSO demonstrates that this event commences between 1 and 3 days post-injury (PI) and peaks at 5 days PI (Fig. [3c](#F3){ref-type="fig"}). This timecourse mirrors that of the appearance of degeneration debris (Fig. [3d](#F3){ref-type="fig"}, GSD), activated resident hippocampal microglia (Fig. [3d](#F3){ref-type="fig"}, IB4), and reactive gliosis in the cSO (Fig. [3d](#F3){ref-type="fig"}, pERK \[[@B15]\]) supporting migration of RHO+ cells towards injury cues \[[@B24],[@B25]\]. 94.6% of all RHO+ cells in the cSO were immunoreactive for F4/80 confirming the infiltrating cells are microglia. Finally, BrdU-positive/RHO+ cells were observed in the cSO maximally at the 3 day timepoint suggesting mitosis occurred primarily after the SVMs had migrated to the hippocampus (not shown). Therefore, activated SVMs are capable of infiltrating deep into the parenchyma in response to brain injury. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Infiltration of parenchyma by SVMs after injury. (A) SVMs infiltrated the stratum oriensof the hippocampus in injured mice (right panel) but not in sham animals (left panel). cSO, contralateral stratum oriensof hippocampus; ffx, fimbria/fornix; lv, lateral ventricle; th, thalamus. (B) SVMs migrate significantly farther into parenchyma of injured animals compared to sham injury (\p \< 0.01). (C) Infiltration of hippocampus begins days after injury and cells remain for weeks (\p \< 0.05 compared to sham). (D) Temporal pattern of infiltration corresponds to neuropil degeneration (black punctate staining, bottom left) activation of resident microglia (shown by increased IB4 staining, bottom middle) and glial activation (indicated by phospho-ERK immunoreactivity, bottom right). GSD, Gallyas silver degeneration stain; pERK, phospho-extracellular signal-related kinase. ::: ![](1742-2094-2-5-3) ::: To provide direct evidence for the migration of SVMs into the parenchyma and characterize their general migratory behavior we prepared live brain slices from dye-injected/lesioned mice and rendered confocal time-lapse movies in the cSO (Fig. [4a](#F4){ref-type="fig"}; Video 2 (Additional file [2](#S2){ref-type="supplementary-material"})). RHO+ cells migrated in a directed fashion from the periventricular region into the cSO (Fig. [4b](#F4){ref-type="fig"}) and demonstrated an average speed of 80 ± 6 μm/h. Migrating cells displayed polarized morphologies with a prominent leading protrusion demonstrating numerous side branches (Fig. [4c](#F4){ref-type="fig"}). We conclude that activated SVMs are able to migrate long distances into the brain parenchyma towards damaged regions in vivoand in situ. We have named this event \"infiltrative microgliosis\" (IMG). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Dynamics of infiltrative microgliosis. (A) 2D projections of confocal images demonstrate three migratory cells (large and small white arrows) migrating into the cSO. white arrowhead, non-migratory cell for reference. e, ependyma; cSO, stratum oriens. See also Video 2. (B) Migration was highly directed from ventricle to hippocampus, five representative cells from a single experiment. lv, lateral ventricle; black stars, cell origin (C) Highly polarized, migratory morphologies of RHO+ cells as demonstrated by confocal 3D reconstruction. cb, cell body; lpr, leading process; tpr, trailing process. ::: ![](1742-2094-2-5-4) ::: ICV injection of HIV-1 tat protein causes IMG --------------------------------------------- Lentiviral tat protein has been shown to be neurotoxic \[[@B8]\], stimulate microglial migration in vitropossibly by mimicking, and inducing expression of, chemokines \[[@B27],[@B28]\], and soluble tat protein is released from HIV-infected cells \[[@B29]\]. Further, ependymal lesions were found in 16% of AIDS patients at autopsy \[[@B30]\] and HIV-1 tat has been shown to damage the ependymal layer of mice in low nanomolar concentrations \[[@B8]\]. To establish IMG as an event relevant to neurologic disease we tested the idea that ependymal damage caused by an ICV injection of 2.0 nM recombinant HIV-1 tat protein in mice would cause activation, and possibly intraparenchymal migration, of SVMs. 24 hours post-injection mice demonstrated ependymal cell damage (Fig. [5a](#F5){ref-type="fig"}, top left) and extensive activation of SVMs (Fig. [5a](#F5){ref-type="fig"}, bottom left). No damage or SVM activation was seen after injection of deactivated tat (Fig. [5a](#F5){ref-type="fig"}, right panels). Interestingly, activated microglia could often be found several hundred microns from ventricular surfaces as demonstrated by IB~4~histochemistry (not shown). To determine whether this was due to migration of SVMs into the parenchyma or spreading activation of stationary cells we performed timelapse confocal analysis of live brain slices taken from animals 24 h after ICV tat injection. We found that nearly all activated periventricular microglia were motile and many were locomotory after tat injection (Video 3 (Additional file [3](#S3){ref-type="supplementary-material"}). Injection of deactivated tat did not result in migration (Video 4 (Additional file [4](#S4){ref-type="supplementary-material"})). Velocities of HIV-1 tat activated microglia averaged approximately 500 μm/h. Further, we observed many microglia which migrated deep into the parenchyma from the periventricular zone (Fig. [5b](#F5){ref-type="fig"}, Video 5 (Additional file [5](#S5){ref-type="supplementary-material"})). Intense microglial activity at the ependyma suggestive of phagocytosis was also observed (Video 5). We conclude that nanomolar concentrations of ICV-injected HIV-1 tat protein alone is sufficient to cause ependymal damage, SVM activation, and diffuse IMG. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### HIV-1 tat injection activates SVMs and incites IMG. (A) Ependymal loss (top left) and subventricular microgliosis (bottom left) 24 h following injection of 2.0 nmol tat protein but not in animals injected with deactivated tat (right panels). (B) To determine if tat-activated SVMs migrated in situwe rendered timelapse confocal movies 24 h post-injection. Colored arrowheads demonstrate three SVMs which migrate from the region near the ventricle (green line) deep into the parenchyma (colored dashed lines). Field measures \~200 μm horizontally. See also Video 5. ::: ![](1742-2094-2-5-5) ::: Discussion ========== We have shown that SVMs represent a pool of microglial cells which are highly reactive to periventricular damage and are responsible for clearance of resulting cellular debris. Further, activated SVMs are capable of migrating away from the ventricle towards injury cues from damaged regions in the parenchyma several hundred microns away. We confirmed these findings dynamically in acute slice preparations from adult mice. Both the juxtaventricular origin and the extensive migratory capacity of activated SVMs have important implications for neurobiology and disease. Periventricular/subependymal microglia have been noted by histologists since microglia were first identified as a distinct cell type (reviewed in \[[@B31]\]). Little attention has been paid to these cells in the literature until recently due to their intimate arrangement among the subventricular neural progenitors \[[@B6],[@B7]\]. Possible phenotypic differences between SVMs and parenchymal microglia have not been investigated, however, the location of SVMs among stem cells and within close proximity to ependymal cells and ventricular CSF is unique. A few neurological diseases demonstrate altered CSF constituents and often pathological involvement of the periventricular tissues \[[@B8]-[@B13]\]. Therefore, the specific function of microglia in these specialized regions of the brain under normal and pathological conditions deserve further investigation. While evaluating rhodamine dyes for selective labeling of ependymal cells \[[@B21]\] for other studies we discovered that the ependyma of the injected hemisphere became rapidly damaged after dye uptake. Upon death of the ependyma, SVMs phagocytosed the ependymal debris, and thereby became rhodamine-positive. This serendipitous finding results in rapid and selective labelling of SVMs allowing study of this specific cell population in vivo. For instance, in this study we were able to demonstrate phagocytosis, mitosis, and migration of activated SVMs using histopathological techniques alone. We have shown these cellular activities can be confirmed in situwith timelapse confocal microscopy further validating this versatile protocol. Mechanistic investigations are possible by combining our in vivoand in situprotocols with genetic or pharmacological techniques. We found SVMs only infiltrated the brain after selective ependymal damage with rhodamine dyes if a distant lesion was also present, likely providing a gradient of chemoattractive cues. CC chemokines are known to be upregulated rapidly after deafferenting injury of the hippocampus \[[@B26]\]. The extensive migration of SVMs in response to HIV-tat injection, on the other hand, may be due to a direct effect of tat on microglia or possibly an indirect effect due to upregulation of chemokines by neurons and glia \[[@B27],[@B28]\]. Further, that ICV injection of recombinant HIV-1 tat protein aloneis sufficient to damage the ependyma, activate SVMs, and incite infiltrative microgliosis supports the \"cytokine dysregulation hypothesis\" \[[@B8],[@B22]\] of damage in HIV-1 encephalitis whereby overactivation of microglia/monocytes may be more critical than actual CNS viral load \[[@B33],[@B34]\]. Conclusions =========== In summary, we have shown that SVMs are a highly reactive pool of cells which, when activated, can infiltrate the parenchyma in response to injury cues from damaged brain regions or exposure to HIV-1 tat. These findings provide new in vivoand in situmodels for the study of SVM function, further insight into microglial dynamics after brain injury, and novel hypotheses for the role of microglia in periventricular reactions in neurological diseases. List of abbreviations ===================== BrdU, 5-bromo 2-deoxyuridine; BSA, bovine serum albumin; CSF, cerebrospinal fluid; cSO, stratum oriensof the hippocampus contralateral to stab lesion; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; GFP, green fluorescent protein; GSD, modified Gallyas silver degeneration stain; HIVE, human immune deficiency virus 1 encephalitis; ICV, intracerebroventricular; IFN-γ, interferon gamma; IMG, infiltrative microgliosis; MS, multiple sclerosis; PBS, phosphate-buffered saline; pERK, phosphorylated/activated extracellular signal-regulated kinase; PI, post-injury; RHO+, rhodamine-positive; RhoB, rhodamine latex microbeads; SEM, standard error of the mean; Sp-DiI, 1,1\'-dioctadecyl-6,6\'-di(4-sulfophenyl)-3,3,3\',3\'-tetramethylindocarbocyanine; SVM, subventricular microglia; TNF-α, tumor necrosis factor alpha. Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= WSC conceived of and designed the study, carried out all experiments, performed data analysis, and drafted the manuscript. S-IM participated in study design especially with regards to the timelapse experiments. AFH participated in study design and coordination and provided the confocal facilities, equipment, and expertise for timelapse experiments. JWM participated in study design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 SVM phagocytosis of ependymal debris. Activity of SVMs suggestive of phagocytosis of dye-labeled ependymal cell debris 24 h following injection. SVMs can be seen extending processes towards debris. Examples of ependymal debris are pseudocolored yellow. Each frame is a 2D projection representing a stack of 6 images 8 μm apart. Original magnification, 40×. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 Dynamics of infiltrative microgliosis. Infiltrative microgliosis of SVMs into the hippocampal stratum oriensfrom the subependymal region of the posterior lateral ventricle. Note highly directed migration into the hippocampus. Each frame is a 2D projection representing a stack of 4 images 10 μm apart taken every 3 minutes. Original magnification, 20×. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 3 SVM dynamics in response to HIV-1 tat protein. Extensive migratory activation of periventricular microglia in response to 2.0 nM ICV HIV-1 tat protein. This migratory reaction extends several hundred microns into the parenchyma. v3v, ventral third ventricle. Each frame is a 2D projection representing a stack of 6 images 8 μm apart taken every 90 seconds. Original magnification, 20×. Field measures 700 × 700 μm. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 4 Control video for HIV-1 tat protein. Lack of activation of SVMs and migration with ICV injection of deactivated HIV-1 tat protein (compare to Video 3, similar field). The paucity of IB4 labeling indicates the limited microglial activation. Note blood vessel endothelial cell labeling and gradual photobleaching. Each frame is a 2D projection representing a stack of 6 images 8 μm apart taken every 90 seconds. Original magnification, 20×. Field measures 700 × 700 μm. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 5 HIV-1 tat incites infiltrative microgliosis. HIV-1 tat activated subventricular microglia infiltrate the parenchyma. Three highlighted cells correspond to those in Figure [5b](#F5){ref-type="fig"}. Note also intense activity of SVMs at ventricle (red line) suggestive of phagocytosis of ependymal cell debris. Each frame is a 2D projection representing a stack of 6 images 8 μm apart taken every 90 seconds. Original magnification, 40×. ::: ::: {.caption} ###### Click here for file ::: Acknowledgments =============== We thank Stephen Brody (Washington University) for the gift of foxj1 antibodies; Rooshin Dalal and Claire Brown for expert assistance with confocal microscopy; John Stock for expert assistance with electron microscopy, Sean Aeder and Isa Hussaini for the gift of the Ad-GFP; Isa Hussaini, Akira Sakakibara, and Scott Vandenberg for helpful discussion; and Margo Roberts for comments on this manuscript. The following reagents were obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: TAK-779 and HIV-1 Tat protein from Dr. John Brady and DAIDS, NIAID. This research was supported by National Institutes of Health Grants NS-047378 (to J.W.M.) and GM-232442 and GM-064346 (to A.F.H.). W.S.C. was supported by the UVA Medical Scientist Training Program, a Raven Society Fellowship, and an award from the National Neurotrauma Society.	PubMed Central	2024-06-05T03:55:52.794701	2005-1-28	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548677/", "journal": "J Neuroinflammation. 2005 Jan 28; 2:5", "authors": [ { "first": "W Shawn", "last": "Carbonell" }, { "first": "Shin-Ichi", "last": "Murase" }, { "first": "Alan F", "last": "Horwitz" }, { "first": "James W", "last": "Mandell" } ] }
PMC548678	Background ========== During skeletal development and growth, bone formation occurs either by intramembraneous or endochondral bone formation. In endochondral bone formation, which occurs at the growth plates of long bones, cartilage is formed first, then the chondrocytes undergo a proliferative phase followed by hypertrophy, changes in gene expression, and matrix calcification, after which the cartilage is replaced by bone. Although generally referred to as chondrocyte hypertrophy, cell enlargement is just one manifestation of the more complex process of chondrocyte maturation, which can be considered an end-stage of chondrocyte differentiation. It is important to define the mechanisms that induce chondrocyte maturation, not only to understand bone development, but also to help prevent hypertrophy and ossification during cartilage tissue engineering. Hypertrophic chondrocytes are characterized by their increased levels of alkaline phosphatase (ALP), reduced levels of type II and IX collagens, and the emergence of type X collagen (Col X), which is a specific marker of hypertrophy \[[@B1],[@B2]\]. Ascorbate and bone morphogenetic proteins (BMPs) are among the factors previously shown to be inducers of ALP gene expression in chondrocytes. Either of these inducers alone will elevate ALP activity in chondrocytes derived from pre-hypertrophic regions of avian cartilage, but the combined effect of BMP and ascorbate is more than additive \[[@B3]\]. In early studies with avian chondrocytes, ascorbate-induced increases in type X collagen expression appeared to parallel increasing alkaline phosphatase activity, suggesting that both Col X and ALP might be controlled by common pathways \[[@B4]\]. However, analyses of BMP-stimulated hypertrophy suggested that ALP activity gradually increased over a 3 day period, while Col X mRNA reached maximal levels within 24 h. Experiments in which pre-hypertrophic chick chondrocytes were transfected with luciferase constructs regulated by sequences from the avian type X collagen gene demonstrated that a \"b2\" region 2.6-2.0 kilobases upstream of the ColX transcription start site, when joined to 640 base pair (bp) region of the proximal promoter, was transcriptionally activated by BMP-2, -4, and -7 \[[@B5]\]. Northern blot analyses after cyclohexamide treatment showed that new protein synthesis is not required for BMP-induced Col X expression \[[@B3]\]. Additional studies indicated that the mechanism for type X collagen promoter regulation probably involves BMP-activated Smads interacting with a Runx2/Cbfa1 transcription factor \[[@B6]\], and that retinoic acid stimulation of Col X expression is via the same 316 bp region \[[@B7],[@B8]\]. Although long-term (4--7 day) treatment of chondrocytes with ascorbate results in increased levels of type X collagen mRNA \[[@B9]\], there is no data concerning the ability of ascorbate to regulate the type X collagen promoter. In osteoblastic cells, BMPs and ascorbate have been shown to operate via mechanisms that at least partly involve mitogen activated protein kinases (MAP kinases). For example, ascorbate promotes extracellular matrix production which, in turn, activates the extracellular-signal regulated kinases, (ERK1/2 or p42/ p44) in an osteoblastic cell line \[[@B10]\]. MAP kinases including ERK1/2 \[[@B11],[@B12]\], p38 \[[@B13]\] and PI3 (phosphatidylinositol 3-) kinase \[[@B14]\] have also been reported to be required for BMP-dependent induction of osteoblast differentiation. However, these pathways can act oppositely in certain BMP induced processes such as osteocalcin synthesis by osteoblasts \[[@B15]\]. In general, MAP kinase pathways involving ERK1/2 stimulate proliferation, growth and differentiation, whereas those that stimulate p38 kinase lead to differentiation and apoptosis \[[@B16]\]. In early stages of chondrocyte differentiation, an increase in p38 and decrease in ERK1/2 activity is required for the progression to cartilage nodule formation in chick limb buds \[[@B17]\]. In hypertrophying chondrocytes p38 has been shown to be required for Col X mRNA synthesis \[[@B18]\]. In apoptosis of articular chondrocytes \[[@B19]\] and other cell types \[[@B20]\], ERK1/2 inhibits and p38 stimulates the apoptotic pathway. Chick sternal chondrocytes are a popular model for the study of chondrocyte maturation because under normal development chondrocytes from the cephalic portion of the sternum undergo hypertrophy followed by mineralization and bone formation, whereas the caudal portion remains as cartilage \[[@B3],[@B8]\]. In this study we investigate the roles of ERK1/2, p38 and two upstream pathways, protein kinase C (PKC) and PI3 kinase, in the maturation of chick prehypertrophic sternal chondrocytes induced by BMP-2 and ascorbate. Results ======= ERK signaling inhibits transcription of the BMP-2 responsive type X collagen promoter but is not involved in the regulation of alkaline phosphatase activity ------------------------------------------------------------------------------------------------------------------------------------------------------------ Studies with the ERK1/2 inhibitor U0126 indicated that blocking ERK1/2 signaling increased the activity of the type X collagen promoter but had no effect on alkaline phosphatase (ALP) activity in chick cephalic sternal chondrocytes (Fig. [1](#F1){ref-type="fig"}). Cells transfected with luciferase reporter plasmid containing the BMP-responsive b2/640 region of the Col X promoter showed a 3-fold (p \< 0.05) increase in luciferase expression, as a ratio to the pRL null control vector, after the addition of 4 μM U0126, both with and without exogenous BMP-2 (Fig [1A](#F1){ref-type="fig"}). In contrast neither basal nor BMP-stimulated ALP activity were significantly changed in the presence of U0126 (Fig. [1B](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Effects of BMP-2 and the ERK inhibitor, UO126, on the Col X promoter and alkaline phosphatase activity in chick cephalic sternal chondrocytes.A:Activity of the b2/640 type X collagen promoter; 24 hrs after seeding cells were transfected with PGLb2/640 and pRLnull luciferase vectors, 5 hrs after transfection 4 μM U0126 or vehicle (DMSO) was added. BMP-2 was added to selected wells after a further hour. Values are mean ± S.D of the mean ratio of promoter to empty vector fluorescence units, for 6 experiments assayed in triplicate. B:Alkaline phosphatase activity; 24 hrs after seeding, medium was changed and 4 μM U0126 or vehicle was added. BMP-2 was added to selected wells after a further hour. Cell extracts were prepared 72 hrs later. Data was obtained using 5 different isolates of chondrocytes assayed in triplicate. Values are mean ± SEM of 12--15 samples normalized to within experiment controls treated with BMP-2 but no inducers, \:p \< 0.01 group differs from non BMP-2 treated group within inhibitor treatment, +: p \< 0.05 that group differs from group with no UO126. ::: ![](1478-811X-3-3-1) ::: ALP activity was highly variable between cell isolates and is expressed here normalized to BMP-2 treated controls for the purpose of combining experiments, typical ALP values ranged from approximately 0.5--2 nmol/min/μg DNA in controls to between 4 and 12 nmol/min/μg DNA in BMP-2 treated cultures. The effects of altered ERK1/2 signaling on Col X promoter activity in chick sternal chondrocytes was further studied both by transfection with mutant kinases and by treatment with additional kinase inhibitors. Col X promoter activity was increased, both in the presence and absence of BMP-2, when the mitogen-stimulated ERK pathway was suppressed by transfecting chondrocytes with dominant negative ERK-2 (Figs. [2A, 2B](#F2){ref-type="fig"}). Conversely, stimulating the ERK1/2 pathway by over-expressing constitutively active MEK1, an upstream kinase of ERK1/2, decreased promoter activity by 50% (p \< 0.05) and in BMP-2 treated cells it eliminated any BMP response. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Effects of MAP kinase manipulation on b2/640 type X collagen promoter activity, in chick cephalic sternum chondrocytes.Aand B: Mutant kinases; 24 hrs after seeding, cells were transfected with luciferase vectors and 0.5--1 μg mutant kinase DNA. After 5 hrs medium was changed (A) or medium changed and BMP-2 added (B). Cand D: Inhibitor treatments; 5 hours after transfection with luciferase vectors, medium was changed and kinase inhibitor or vehicle (DMSO) was added as indicated. In D30 ng/ml BMP-2 was added one hr after medium change. Data obtained using at least 2 independent isolates of chondrocytes, assayed in triplicate. Values are mean ± SEM of luciferase ratios of type X collagen promoter activity to control vector, normalized to BMP-2 treated controls. \:p \< 0.05 that luciferase ratio differs from non BMP-2 treated group, +: p \< 0.05 that luciferase ratio differs from group with no MAP kinase manipulation. ::: ![](1478-811X-3-3-2) ::: As seen with the ERK1/2 inhibitor U0126, treatment with the more specific ERK1/2 inhibitor PD098059 increased b2/640 Col X promoter activity, in the presence of BMP-2 (Fig. [2D](#F2){ref-type="fig"}). Dose response experiments indicated that concentrations of PD98059 as low as 10 μM significantly increased luciferase expression 2-fold (p \< 0.001) in BMP-treated cells, but not in the absence of BMP-2 (Fig. [2C](#F2){ref-type="fig"}). At a higher does, 50 μM, of PD90859 luciferase levels in BMP-2 treated cells were 10--20 fold higher (p \< 0.005) than BMP-containing cultures without inhibitor (Fig. [2D](#F2){ref-type="fig"}), at this dose PD90859 also stimulated the promoter in the absence of BMP-2 (Fig. [2C](#F2){ref-type="fig"}). p38 MAP kinase signaling contributes to the response of the type X collagen promoter to BMP-2 --------------------------------------------------------------------------------------------- Transfection with dominant negative p38 caused a decrease in Col X promoter activity in BMP-2 treated cephalic chondrocytes, reducing activity to half of that seen in BMP-2 treated controls (p \< 0.005) and eliminating the BMP-2 response (Figs. [2A](#F2){ref-type="fig"} and [2B](#F2){ref-type="fig"}). Similarly, 1 μM SB 203580, an inhibitor of p38, significantly decreased BMP-stimulated promoter activity (Fig. [2D](#F2){ref-type="fig"}), but had little effect on promoter activity in the absence of BMP-2 (Fig. [2C](#F2){ref-type="fig"}). Inhibiting PKC and PI3 kinases increases type X collagen promoter activity -------------------------------------------------------------------------- Addition of either PI3 kinase inhibitor or PKC inhibitor resulted in similar stimulation of the collagen type X promoter. Calphostin C, a PKC inhibitor, increased activity in BMP-2 treated cells more than 2-fold, an effect similar to that seen with the ERK1/2 inhibitor PD98059 at 10 μM (Fig. [2D](#F2){ref-type="fig"}). Similarly, 50 μM LY294002, a PI3 kinase inhibitor, stimulated the b2/640 promoter approximately 2-fold (Fig. [2D](#F2){ref-type="fig"}). However, both of these inhibitors also increased transcription of the collagen type X promoter in non-BMP-2 treated cells to levels as high as seen with the combination of BMP-2 and the respective inhibitor treatment (Fig. [2C](#F2){ref-type="fig"}). Kinase inhibitor effects on viable cell number ---------------------------------------------- To assess the possible effects of protein kinase inhibitors on cell proliferation and survival, we measured relative numbers of live cells using a tetrazolium (MTS) assay. The results indicated that all cultures treated with inhibitors, with and without BMP-2 and/or ascorbate, had cell numbers within 10% of untreated controls (data not shown). Ascorbate has no effect on the type X collagen promoter and stimulates alkaline phosphatase activity regardless of kinase inhibitor treatment --------------------------------------------------------------------------------------------------------------------------------------------- We examined the effect of 75 μM ascorbate-2-phosphate on the activity the Col X promoter in cultures treated with kinase inhibitors. Col X promoter activity was unaffected by addition of ascorbate, and 4 μM of the ERK1/2 inhibitor U0126 increased promoter activity to comparable levels both with and without ascorbate (Fig. [3A](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Effects of ascorbate and MAP kinase inhibitors on the Col X promoter and alkaline phosphatase activity in chick cephalic sternal chondrocytes.A:Activity of the b2/640 type X collagen promoter; 24 hrs after seeding cells were transfected with luciferase vectors, 5 hrs after transfection 4 μM U0126 or vehicle were added. Ascorbate was added to selected wells after a further hour. Values are mean ± S.D of the mean ratio of promoter to empty vector fluorescence units, for 3 experiments assayed in triplicate. B:Alkaline phosphatase activity; 24 hrs after seeding, medium was changed and 4 μM U0126 or vehicle was added. Ascorbate or ascorbate with BMP-2 was added to selected wells after a further hour. Cell extracts were prepared 72 hrs later. Data was obtained using 5 different isolates of chondrocytes assayed in triplicate. Values are mean ± SEM of 12--15 samples normalized to within experiment controls treated with BMP-2 but no inducers. \: p \< 0.05 that group differs from non-supplemented group within inhibitor treatment. The increase in ALP caused by BMP-2 addition is shown, this increase is significantly smaller in UO126 treated cells, +:p \< 0.05. ::: ![](1478-811X-3-3-3) ::: The increase in alkaline phosphatase activity caused by adding BMP-2 to ascorbate treated cultures is reduced by ERK inhibitors ------------------------------------------------------------------------------------------------------------------------------- ALP activity in the absence of exogenous BMP was stimulated at least 2-fold in ascorbate-treated cultures without inhibitors, as previously reported, and this stimulation was not significantly affected by addition of either ERK1/2 or p38 inhibitors (Fig. [3B](#F3){ref-type="fig"}). In cultures treated with ascorbate and BMP-2 addition of ERK1/2 inhibitors resulted in ALP levels that were \<60% of the level seen in cells without inhibitor (Fig. [3B](#F3){ref-type="fig"}). The increase caused by BMP-2 addition, relative to ascorbate only-treated cultures was significantly reduced by treatment with U0126 (p \< 0.05). The p38 inhibitor SB203580 did not cause a statistically significant inhibition of alkaline phosphatase activity. PI3 kinase and PKC inhibitors had no significant effects on ALP activity (data not shown). Discussion ========== The present studies demonstrate that ERK1/2 inhibition increases activity of the BMP responsive region of the type X collagen promoter. This indicates that ERK1/2 signaling interferes with the ability of BMP-induced signals to stimulate type X collagen transcription. Interestingly ERK1/2 has also been shown to inhibit type I collagen expression in an osteoblastic cell line \[[@B21]\] suggesting there may be a common pattern of ERK1/2 inhibition of collagen transcription pathways. In contrast to the stimulatory effects of inhibiting the ERK pathway, p38 inhibition blocked BMP-stimulated Col X promoter activity. Zhen et al. \[[@B18]\] and Beier and Luvalle \[[@B22]\] also showed that p38 signaling is important for regulation of Col X expression, Beier and Luvalle suggested that the proximal promoter contained a site for p38 action. Here, we have confirmed these results and narrowed the region of p38 responsiveness to within the region of the Col X promoter that is also BMP responsive. While the classical pathway for BMP signaling is via activation of R-Smads, there is also evidence for BMP signaling via a TGF-activated kinase (TAK1) leading to p38 signaling \[[@B23]-[@B26]\]. However, in preliminary experiments Smad1 over-expression increased BMP-stimulated Col X promoter activity even in the presence of DN-TAK1 (data not shown). This suggests that BMP-activated Smad signaling and not TAK1 signaling is the major factor in Col X promoter regulation. Taken together these data suggest that the role of p38 is as a co-activator of Smads or Runx-2 rather than a downstream effector of BMP signaling. Inhibiting either protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI3 kinase) increased type X collagen promoter activity both in BMP-2 treated cultures and controls. Both PKC and PI3 kinase have been reported to negatively regulate p38 \[[@B18]\] and positively regulate ERK1/2 \[[@B20],[@B27],[@B28]\]. The complex effects in which these kinases stimulate basal Col X promoter activity but inhibit BMP-2 from stimulating additional activity may be due to their simultaneously affecting both of these pathways. Although alkaline phosphatase expression, like type X collagen expression, increases during chondrocyte hypertrophy and is stimulated by BMPs, its regulation clearly differs from Col X in several respects. ERK1/2 inhibition has little effect on the ALP activity induced by BMP-2 or ascorbate acting alone and reduces the ability of BMP-2 to further stimulate ALP activity in ascorbate treated cultures. Inhibiting p38 does not have a clear effect on BMP-stimulated alkaline phosphatase activity in this model although it has been shown to reduce ALP activity in long-term (12--15 day) micromass cultures \[[@B29]\]. Preliminary studies with cyclohexamide indicate that new protein synthesis is required for the up-regulation of alkaline phosphatase mRNA in response to BMP-2. We propose that these differences reflect a direct Smad-mediated effect of BMPs on type X collagen expression and an indirect effect on ALP expression. A mechanism which could account for the observed effects of ERK and p38 signaling on expression of type X collagen and ALP is presented in Fig. [4](#F4){ref-type="fig"}. The simplest explanation for our observation that a decrease in ERK1/2 signaling causes increased type X collagen promoter activity is that ERK1/2 can phosphorylate the linker region of BMP-activated Smads, preventing nuclear translocation of activated Smads, as suggested by Kretzschmar et al. \[[@B30]\]. Alternatively, products of ERK1/2 signaling may act directly on a silencer within the type X collagen promoter such as the region identified by Beier et al. \[[@B31]\] at 2864-2410 base pairs which would overlap with our b2-containing construct (2649 -2007). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Possible mechanism for kinase involvement in BMP stimulated type X collagen expression.ERK1/2 is proposed to suppress activated Smad translocation to the nucleus as described by Kretschmer et al. \[30\] and thereby inhibit Col X transcription. ERK-specific Runx2 phosphorylation is required for regulation of typical osteoblastic genes but may not be required for Col X expression. However p38 facilitates Col X transcription, possibly via Runx2 phosphorylation and although was not shown to be involved in short term increases in ALP in our model, has been shown to be involved in long term increases in ALP over 15 days of chondrocyte differentiation in culture (question mark) \[29\]. ::: ![](1478-811X-3-3-4) ::: Evidence that BMP stimulation of type X collagen requires both activated R-Smads and Runx2 has been previously reported \[[@B5],[@B7]\]. Little is known concerning kinase phosphorylation of Runx2, except for the report that ERK phosphorylates Runx2 and increases its binding to the osteocalcin promoter \[[@B32]\] in osteoblasts. If ERK phosphorylation of Runx2 were required for BMP-stimulated type X collagen transcription, we might expect ERK1/2 inhibition to decrease the activity of the Col X promoter. However, as ERK1/2 inhibition increases Col X promoter activity while partially inhibiting ALP we propose that the Runx2-Smad complex binding to the Col X promoter may not be phosphorylated by ERK1/2, but that ALP expression does require Runx2 phosphorylated by ERK1/2. As well as its demonstrated role in Col X expression as found here and by \[[@B18],[@B31]\], there are also reports that p38 inhibitors block osteoblast differentiation \[[@B33],[@B34]\]. Because Runx2 plays an important role in both osteogenesis and chondrocyte maturation, we have suggested that p38 may function in Runx2 expression, activation or nuclear translocation. However, there are many other possible roles, including the suggestion that p38 is downstream of BMP-activated Smad signaling \[[@B35]\]. Retinoic acid \[[@B7],[@B8]\], another stimulator of chondrocyte hypertrophy has also been shown to act at the BMP-2 responsive b2 region of the type X collagen promoter, however ascorbate, which does produce an increase in type X collagen mRNA expression \[[@B4]\], does not seem to have any effect on this promoter region. These results, in combination with previous findings that Col X mRNA expression only occurs after 4--9 days stimulation with ascorbate \[[@B9]\], suggest that the effects of ascorbate on regulation of type X collagen expression are via a separate mechanism than BMP stimulation and are probably indirect. Conclusions =========== Elucidating the signaling pathways by which chondrocytes are driven to hypertrophy is necessary in order to better understand skeletal development, cartilage disease and improve the design of tissue engineered cartilage. We showed here that the ERK1/2 pathway inhibits type X collagen production by either directly or indirectly acting at the BMP responsive region of the promoter. p38 kinase signaling stimulates type X collagen transcription at the same promoter region, probably in conjunction with BMP-2 activated Smads. The factor upstream of p38 in this stimulatory pathway is unknown. Alkaline phosphatase activity is likely to be regulated in a different way from type X collagen since MAP kinases do not contribute in the same way to this pathway. Although ascorbate and BMPs both induce hypertrophy in chondrocytes ascorbate does not act at the same region of the Col X promoter as BMPs. Methods ======= Inhibitors and plasmids ----------------------- The ERK1/2 inhibitor PD98059, which blocks the upstream kinase (MEK1) of ERK1/2, the p38 inhibitor SB203580, and the PKC inhibitor Calphostin C were obtained from Sigma (St. Louis, MO). UO126, also a MEK1 inhibitor, was obtained from Biomol (Plymouth Meeting, PA) and LY294002, a phosphatidylinositol 3- (PI3) kinase inhibitor from Cell Signaling Technology (Beverly, MA). Plasmids were kindly donated as follows: constitutively active MEK1 from Michael Webber (University of Virginia); dominant negative p38 \[[@B36]\] from Roger Davis at the Howard Hughes Medical Institute, University of Massachusetts Medical School, dominant negative ERK2 \[[@B37]\] from Melanie Cobb at University of Texas Southwestern Medical Center. Cell Culture ------------ Chondrocytes were cultured as previously described \[[@B3]\]. Cephalic and caudal sternal chondrocytes were isolated from 15 day chick embryos and cultured for 5 days. Dissection of chick cartilage was performed under a University of Pennsylvania IACUC exemption. On day 5 non-adherent cells were removed and plated in 12 well plates at 300,000 cells /well in DMEM supplemented with 10% NuSerum, 2 mM L-glutamine, 100 U/ml penn/strep and 4 U/ml hyaluronidase, to promote cell attachment. Transfection of cephalic (pre-hypertrophic) sternal chondrocytes ---------------------------------------------------------------- On day 1 of secondary culture (24 hrs after plating) the cell layer was washed in HBSS and the media changed to DMEM supplemented with 10% FBS in place of NuSerum. Cells were co-transfected with pGL2 plasmid containing the b2/640 type X collagen promoter region attached to a firefly luciferase reporter (0.2--1 μg/well) (Promega, Madison, WI) and pRL null plasmid attached to a renilla*luciferase reporter (0.4 μg/well) (Promega) which served as a transfection control \[[@B5]\]. When appropriate, mutant plasmids were added at 0.5 or 1 μg/well along with the luciferase vectors. Luciferase and mutant kinase plasmids were transfected either using CaPO~4~precipitation or Fugene transfection reagent at 6 μl/ml (Promega). Since preliminary experiments using green fluorescent protein showed that Fugene was more effective in terms of numbers of cells transfected, this method was used for the majority of the experiments; however, relative outcomes between controls and treated cells were not affected by the transfection method. Transfection proceeded for 5 hrs after which the cell layer was rinsed twice in HBSS and cultured with serum free medium (DMEM with 2 mM L-glutamine, 100 U/ml penn/strep, 4 U/ml hyaluronidase, 60 ng/ml insulin, 1 mM cysteine, and 10 pM triiodothyronine). Some wells were supplemented with 75 μM ascorbate-2-phosphate (Wako, Takara, Japan) or 30 ng/ml human recombinant BMP-2 (Wyeth, Cambridge, MA). Where inhibitors were used they were added at this point and cells incubated for 1 hr before the addition of BMP-2. Cells were cultured for a further 48 hours, then lysed and assayed using a dual luciferase assay kit (Promega). Alkaline Phosphatase Assay -------------------------- For alkaline phosphatase assays, cells were switched to serum free medium on day 1 of secondary culture, inhibitors were added and cells incubated for 1 hr before the addition of ascorbate or BMP-2, as described for the luciferase assay. Cells were cultured for a further 72 hrs and then rinsed twice in HBSS. Cells numbers were assayed either by DNA quantification (CyQUANT cell proliferation assay kit, Molecular Probes, Eugene, OR) or by MTS tetrazolium salt assay of mitochondrial activity (Cell Titer 96 AQueous One Solution Cell Proliferation Assay, Promega). When MTS was used, a 1:10 dilution of MTS was applied in phenol red-free media for 30--60 minutes, 200 μl of media plus MTS was transferred to a 96 well plate and assayed in a \'Multiskan ascent\' plate reader (Thermolabsystems, Franklin, MA). The cell layer was then washed twice in HBSS and extracted with 0.15 M Tris, pH 9 with 0.1 mM ZnCl~2~, 0.1 mM MgCl~2~and 1% Triton X-100 for 30 mins at 37°C, followed by overnight storage at 4°C. A sample of the cell lysate was reacted with p-nitrophenyl phosphate substrate in 1.5 M Tris buffer pH 9 with 1 mM ZnCl~2~and 1 mM MgCl~2~. Phosphatase activity was measured specrophotometrically at 410 nm with 1 absorbance unit equivalent to 64 nmol of product. For DNA analysis, cells were trypsinized and a subsample of cell suspension centrifuged, the cell pellet lysed with the CyQUANT lysis buffer and the fluorescent DNA dye added. The resulting solution was transferred to a 96 well plate and DNA assayed fluorometrically. The remaining cells were extracted for the alkaline phosphatase assay as above. Alkaline phosphatase enzyme levels were calculated as nmol p-nitrophenol product per minute normalized to MTS units or μg DNA. Statistical analysis -------------------- Statistics were performed using Minitab™ software. After expressing results as a ratio of experimental/control within each experiment, the data from at least 3 experiments were combined. The combined data was tested for normality using the Anderson-Darling test. Normally distributed or non-parametric data were tested for differences between treatments using two-sample Students t-test or the Mann-Whitney test respectively. Where groups of experiment means were compared a paired t-test was used. List of Abbreviations ===================== ALP -- Alkaline Phosphatase BMP -- Bone Morphogenetic Protein bp -- base pairs CA -- constitutively active cbfa -- core binding factor alpha 1 Col X -- Collagen type X DN -- dominant negative ERK -- extracellular signal regulated protein kinase MAP -- Mitogen activated protein MEK -- ERK kinase MTS -- 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt PI3 -- phosphhatidylinositol 3 PKC -- Protein Kinase C Runx -- Runt associated protein TAK -- Transforming growth factor beta activated kinase Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= GCR was involved in experimental design, performed 90% of the experiments, analyzed the data and wrote the first draft of the manuscript. EBG isolated chondrocytes, performed preliminary experiments and contributed to trouble-shooting of the methods. GG-K, performed preliminary experiments and developed methodology. PSL was involved in experimental design and data analysis, wrote portions of and edited the whole manuscript and was the project supervisor. Acknowledgements ================ This work was supported by NIH grant DE13800, and BMP-2 was kindly donated by Wyeth/Genetics Institute. We thanks Drs. Hyun-Duck Nah-Cederquist, Hydar Ali and Richard Assoin of the University of Pennsylvania for sharing information and techniques regarding mutant MAP kinase constructs.	PubMed Central	2024-06-05T03:55:52.796910	2005-2-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548678/", "journal": "Cell Commun Signal. 2005 Feb 3; 3:3", "authors": [ { "first": "Gwendolen C", "last": "Reilly" }, { "first": "Eleanor B", "last": "Golden" }, { "first": "Giovi", "last": "Grasso-Knight" }, { "first": "Phoebe S", "last": "Leboy" } ] }
PMC548679	Background ========== Helicobacter pylori, occurring throughout the world and causing gastroduodenal diseases, is one of the most common chronic bacterial agents in humans \[[@B1]\]. Although the number of peptic ulcers unrelated to H. pyloriis increasing, most ulcers are related to H. pyloriinfection \[[@B2],[@B3]\]. For most of the patients with gastrointestinal symptoms apply to general practitioners (GPs), non-invasive \"test and treat\" policies for H. pyloriinfection have been promoted in order to improve early detection and treatment of ulcers in dyspeptic patients \[[@B4],[@B5]\]. The successful isolation of H. pyloriinfection in patients with chronic gastritis and peptic ulcer disease in 1983 has fundamentally changed concept of the etiologic, pathogenesis and management of upper gastrointestinal (UGI) diseases \[[@B6]\]. This has led to an explosion of H. pylorirelated information and the development and publication of international, regional and national guidelines \[[@B7]\]. Subsequently, numerous educational initiatives have been undertaken to educate health care professionals regarding the appropriate diagnosis and management of this infection. However, results from several recent surveys conducted in different countries have revealed that significant confusion exists and discrepancies are present in the thinking among GPs with respect to the understanding of the relationship between H. pyloriinfection and the pathogenesis, diagnosis and treatment of UGI diseases \[[@B6]\]. The major uncertainties surround the management of patients with dyspepsia where the GPs needs to make a decision whether to test for H. pyloriinfection and treat if positive, and when to refer patients to a specialist \[[@B8],[@B9]\]. It is thought that too many patients with dyspeptic symptoms apply to GPs in Turkey. This study was performed a survey of GPs\' to assess their knowledge and practices pertaining to H. pyloriinfection. Methods ======= A cross-sectional study was conducted in all of 19 primary health care centres (PHCC) in Samsun, Turkey, between November 1 and December 31, 2003. The questionnaire was sent to all GPs (n = 124). 109 of 124 (87.9 %) GPs from different PHCCs completed the survey. The material used was adapted from the questionnaire devised by Sharma et al. \[[@B10]\] and translated into Turkish. The questionnaire was designed in a self-administered format with closed answers being provided to questions. Demographic variables such as gender, age and working year were assessed. The survey questionnaire includes a multiple-choice question relevant to sources of medical information. The H. pyloriknowledge section contained 6 questions. The items assessed respondents\' knowledge of diagnosis of infection, case selection for treatment and treatment options in H. pylori. Participating GPs were asked to offer the type(s) of diagnostic tests such as ELISA, histology, biopsy urease test (BUT), urea breath test (UBT) or culture of biopsy specimen. A list of 7 different clinical presentations was given. It was asked whether the respondents would offer testing for H. pyloriand also treat the infection when the test results were positive for H. pylori. The respondents were asked to select a regimen for the management of H. pyloriinfection among the list of four drug combination regimens included proton pump inhibitor (PPI)-based triple therapies. \[[@B7],[@B11],[@B12]\]. GPs were also asked about their choice of treatment duration. The authors did not make any educational program focusing on H. pyloriinfection before or after the survey in this study period. Data were given as mean ± standard deviation (SD) and percentage. Results ======= Sociodemographic characteristics -------------------------------- The mean age and working year of the GPs was 31.7 ± 5.4 and 7.2 ± 5.0 years, respectively; 59 (54.1%) of the GPs were women. Sources of information ---------------------- Medical journals were the most frequently used source of information on H. pylori, being cited by 86 (78.9%) of GPs. Pharmaceutical company-sponsored symposia (70.6%), textbooks (64.2%), conferences (20.2%) and on-line sites (6.4%) were the other major source of information used by the GPs. These numbers add up to more than 100% because the GPs had been checked more than one item. Diagnostic tests for H. pylori -------------------------------- Ninety-two (84.4%) of the GPs reported having used one or more tests and 17 (15.6%) never used any test for the diagnosis of H. pyloriinfection. Of those, 44.1% had used UBT, 34.5% had used BUT, 23.8 % had used ELISA and 9.8% had used the stool antigen test. The practitioners included in the survey had not equally access to all diagnostic tests mentioned in the study. Testing and treatment choices for H. pyloriinfection ------------------------------------------------------ The proportions of GPs who would test patients for H. pyloriinfection in the 9 different clinical situations and the proportions of those who would offer treatment based on a positive test result, were summarized in Table-[1](#T1){ref-type="table"}. 92 (84.4%) of the GPs answered this section. GPs reported that they would prescribe symptomatic treatment without ordering diagnostic tests for 29 (26.6%). 54.1% of the GPs explain that they send patients with H. pyloriinfection to a specialist. Treatment of H. pyloriinfection in patients with confirmed H. pylori-positive --------------------------------------------------------------------------------- Treatment regimens of choice are listed in Table-[2](#T2){ref-type="table"}. Most used a triple drug regimen containing a PPI. Of the GPs 3.7% would treat patients for 7 days, 4.6% for 10 days, 80.7% for 14 days, 3.7% for 21 days, 5.5% for 28 days and 1.8% for more than 28 days. Discussion ========== UGI symptoms are common reasons for patients to visit GPs. In recent years, the development of non-invasive H. pyloridetection methods, including ELISA and the UBT, has enabled GPs to diagnose and treat H. pyloriinfection. The inadequate treatment of peptic ulcer disease results in therapy failures, high recurrence rates, the emergence of resistant bacterial strains, and increased health care costs, therefore clinical application of current knowledge is crucial \[[@B13],[@B14]\]. There are several tests used for diagnosis of H. pyloriinfection \[[@B12],[@B13],[@B15],[@B16]\]. 84.4% the GPs surveyed used one or more tests for H. pyloriinfection same as Huang J et al.\'s study \[[@B6]\]. However this was higher than reported in a recently preliminary survey of GPs, of whom 48% used one or more tests \[[@B17]\]. UBT is the most (44.1%) used tests in this study. On the other hand, stool antigen test, a useful test for diagnosis, was the least ordered test. It is thought that GPs may not have enough knowledge about the importance of stool antigen test or possibility of usage of this test. Diagnosis of infection should be done by using UBT or stool antigen test. It is always recommended to test by UBT, or endoscopy-based test if endoscopy is clinically indicated, for successful eradication. On the other hand stool antigen test is the alternative if UBT is not available \[[@B14]\]. It is worrisome that considerably fewer perceived a need for testing and subsequent treatment in patients with a new diagnosis and past history of duodenal ulcer (Table [1](#T1){ref-type="table"}). Both the recent America College of Gastroenterology (ACG) publications \[[@B8]\] and Centers for Disease Control and Prevention (CDC) \[[@B7]\] clearly state that a new diagnosis and past history of duodenal ulcer disease is a definite indication for testing and, if positive, for treatment. Testing and treatment of H. pyloriinfection are recommended following resection of early gastric cancer and for low-grade gastric MALT lymphoma. Retesting after treatment may be prudent for patients with bleeding or otherwise complicated peptic ulcer disease \[[@B7]\]. Although it is not recommended to test the asymptomatic individuals for H. pyloriinfection, 64.1% of GPs reported that they would offer testing for H. pyloriinfection and 31.5% of them reported that they would treat H. pyloriinfection based on a positive test result in asymptomatic individuals. These findings suggest that GPs have not sufficient knowledge about the difference between symptomatic and asymptomatic individuals. On the other hand, in any person testing positive for the infection, treatment may be offered after a full discussion about its potential risks and benefits \[[@B4],[@B18]\]. Anti-H. pyloritherapy was almost never recommended for suspected ulcer disease without the prior use of diagnostic tests \[[@B13],[@B18]\]. In this study it was found that 26.6% of GPs treat the initial onset of a suspected ulcer empirically without ordering diagnostic tests for H. pyloriinfection. 54.1% of the GPs, whether ordering diagnostic tests or not, explain that they send patients with suspected or diagnosed H. pyloriinfection to a specialist. In the light of these findings, it is thought that GPs preferred to treat the patients with suspected ulcer, empirically or to send them to a specialist because of the limited diagnostic conditions, the lack of rapidly diagnostic tests at PHCCs, or they thought that they should be treated by a specialist. H. pyloripeptic ulcers are treated with drugs that kill the bacteria, reduce stomach acid, and protect the stomach lining. Antibiotics are used to kill the bacteria. Two types of acid-suppressing drugs might be used: H~2~blockers and PPI. H~2~blockers and PPI have been prescribed alone for years as a treatment for ulcers. When used alone, these drugs do not eradicate H. pyloriand, therefore, do not cure H. pylori-related ulcers. Bismuth subsalicylate, a component of Pepto-Bismol, is used to protect the stomach lining from acid. It also kills H. pylori\[[@B4],[@B7],[@B12],[@B19]\]. The highest eradication rates are achieved with the following regimens: a PPI, clarithromycin, and either amoxicillin or metronidazole for 2 week; ranitidine bismuth citrate, clarithromycin, and either amoxicillin, metronidazole, or tetracycline for 2 week; a PPI, bismuth, metronidazole, and tetracycline for 1 to 2 week \[[@B7],[@B11],[@B12],[@B20]\]. There is good evidence for the efficacy of 14-day triple regimens including a PPI or RBC. Possibility of shortening the duration of PPI-based triple therapies between 7 and 10 days will depend on further results from US-based studies. Seven-day duration may be too short. Some trials have failed to show a statistically significant difference in eradication rates between 7-day and 14-day duration \[[@B20]\]. 84.4% GPs test for H. pyloriinfection but 15.6% never test. Urea breath test is a commonly used investigative tool for H. pyloriinfection. Triple therapy consisting of a proton pump inhibitor, clarithromycin and amoxicillin is the most commonly used treatment combination for H. pyloriinfection. It was found that most of the information was being obtained in traditional teaching formats such as medical journals, pharmaceutical company-sponsored symposia, textbooks and conferences. Other studies (\[[@B10],[@B13],[@B17]\]) confirmed our finding that medical journals were the most important source of information on H. pyloriinfection among GPs. Our data suggested that pharmaceutical company-sponsored symposia were used very frequently among GPs. Patients with dyspeptic complaints are mostly managed in primary care. The most prescriptions for dyspepsia are empirical without testing due to limitations of diagnostic facilities around the world \[[@B21]\]. Similar results were reported that there were significant gaps related to testing and treating H. pyloriinfection \[[@B13],[@B17]\]. Conclusions =========== The diagnosis of and treatment of peptic ulcer disease related to H. pyloriis not adequate. The choice of optimal therapeutic decision depends on the appropriate definition of the disease. In order to provide accurate diagnosis and treatment of H. pyloriinfection, it\'s suggested that efforts to educate the GPs about the algorithms regarding the management of H. pyloriinfection during post-graduation period should be improved in PHCCs. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= SC participated in the design and coordination of the study; ATS provided drafted the manuscript and performed the statistical analysis. YP drafted the questionnaire and participated in study design and coordination. HL conceived the study, participated in its design and drafted the manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-230X/5/4/prepub> Figures and Tables ================== ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### The Proportions of GPs who would test patients for H. pyloriinfection in various clinical situations and those who would offer treatment based on a positive test (n = 92) ::: *Would you offer testing for H. pyloriinfection* *Would you treat H. pyloriinfection based on a positive test result* Current recommendation ----------------------------------------------- ------------------------------------------------------ -------------------------------------------------------------------------- ---------------------------- -------- ----- ----- Peptic ulcer 73 79.3 74 80.4 yes yes New diagnosis of duodenal ulcer 67 72.8 73 79.3 yes yes Past history of duodenal ulcer 47 51.1 59 64.1 yes yes Asymptomatic; spouse has H. pyloriinfection 59 64.1 29 31.5 no no GERD\* 38 41.3 50 54.3 no no Gastric cancer 30 32.6 19 20.7 no no Patient on long term PPI\\ treatment 43 46.7 29 31.5 no no \GERD = Gastroesophageal reflux disease \\* PPI = proton pump inhibitor ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Treatment regimens ::: Regimen Number\* % --------------------------------------------------------------------------------------------------------------- -------------- -------- PPI\* + clarithromycin + amoxicillin 100 91.7 PPI + clarithromycin + metranidazole 7 6.4 Ranitidine bismuth citrate + clarithromycin + amoxicillin or metronidazole, or tetracycline 1 0.9 Ranitidine bismuth citrate + clarithromycin + amoxicillin or metronidazole, or tetracycline+PPI OR omeprazole 13 11.9 \The total number is higher than the number of the participants for the participants checked more than one item. \\* PPI = proton pump inhibitor :::	PubMed Central	2024-06-05T03:55:52.798980	2005-1-26	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548679/", "journal": "BMC Gastroenterol. 2005 Jan 26; 5:4", "authors": [ { "first": "Sevgi", "last": "Canbaz" }, { "first": "Ahmet Tevfik", "last": "Sunter" }, { "first": "Yildiz", "last": "Peksen" }, { "first": "Hakan", "last": "Leblebicioglu" } ] }
PMC548680	Background ========== Multiple lines of evidence suggest that a dysfunction in glutamatergic neurotransmission might be involved in the pathophysiology of schizophrenia \[[@B1]-[@B6]\]. The amino acid glutamate plays a central role in nitrogen metabolism and participates in multiple biochemical pathways. Released glutamate is taken up by glia, where it is converted to glutamine, transported back to the presynaptic neuron, and reconverted to glutamate \[[@B6],[@B7]\]. Thus, it seems that glutamate-glutamine cycle plays a role in the neuron-glia communication in the synapse, and that impairment of glutamate-glutamine cycle may be implicated in the pathophysiology of schizophrenia \[[@B1]-[@B6]\]. By means of in vivoproton magnetic resonance spectroscopy (MRS), a significant increase in glutamine level was detected in the medial prefrontal cortex of never-treated schizophrenic patients compared with controls \[[@B8]\]. In addition, a recent 4.0 T MRS study demonstrated that levels of glutamine in the left anterior cingulate cortex and thalamus of the never-treated patients with schizophrenia were significantly higher than those of healthy controls \[[@B9]\]. In contrast, significant lower levels of glutamine were found in the left anterior cingulate cortex of medicated patients with chronic schizophrenia than in the healthy controls, suggesting the role of chronic medication \[[@B10]\]. Thus, it is possible that the glutamate-glutamine cycle between neuron and glia may play a role in the glutamate hypothesis of schizophrenia. Although Kim et al. \[[@B11]\] first reported reduction of cerebrospinal fluid (CSF) levels of glutamate in patients with schizophrenia, the findings of subsequent studies are inconsistent, with several report of no alteration in CSF levels of glutamate \[[@B12]-[@B14]\]. Furthermore, it was reported that a gradient for glutamate and glutamine in CSF was lack, and that there were significant correlations between the CSF and serum levels of glutamate (r = 0.67, p \< 0.05) and glutamine (r = 0.84, p \< 0.01)\[[@B15]\]. Moreover, sodium-dependent neutral amino acids transporters, located in the abluminal membranes of the blood brain barrier, are capable of actively removing neutral amino acids from the brain \[[@B16]\]. These findings suggest that concentration of neutral amino acids in the extracellular fluid of brain are maintained at \~10% of those of the blood \[[@B15],[@B16]\]. In this study, we investigated whether levels of glutamate and glutamine or ratio of glutamine to glutamate in CSF of first episode and drug naive schizophrenic patients are different from those of age-matched healthy normal controls. Methods ======= Twenty-five male patients with schizophrenia (mean age 26.1 years, range 18--39) and 17 age-matched male healthy subjects (mean age 27.3 years, range 22--44) with no psychiatric disease were enrolled in Uppsala University and Linkoping University Hospital, Sweden. All patients diagnosed according to the DSM-III-R were first episode and drug naive, i.e. they had never been treated with antipsychotic drugs. In the morning (8:00--9:00) from May 1997 to January 1998, CSF of subjects was obtained by lumbar puncture (L4-L5), and twelve to eighteen μL of CSF was collected with a 0.9 mm needle and the samples were immediately frozen at -80°C, as reported previously \[[@B17]\]. The ethics committee of each institute approved the present study, and we received the informed consent from the participants of the study. Measurement of glutamate and glutamine levels were carried out according to established methods \[[@B18]\] with a slight modification using a high performance liquid chromatography (HPLC) system with fluorescence detection (Shimadzu Corporation, Kyoto, Japan). Ten μL of the human CSF was added with 10 μL of 0.1 M borate buffer (pH 8.0) and 30 μL of 50 mM 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F; Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan) in CH~3~CN. The reaction mixture was then heated at 60°C for 2 min, and immediately supplemented with 100 μL of H~2~O/CH~3~CN (90/10) containing 0.1 % trifluoroacetic acid (TFA) to stop the reaction. Ten μL of the resultant solution was injected into the HPLC system. A reversed-phase ODS column (TSKgel ODS-80Ts, Tosoh Corporation, Tokyo, Japan) was used for the separation and quantification of glutamate and glutamine, and the gradient elution of the mobile phase was kept at a constant flow rate of 0.8 mL/min. Mobile phase 1a consisted of H~2~O/CH~3~CN (90/10) containing 0.1 % TFA, and phases 1b and 1c, of H~2~O/CH~3~CN (10/90) containing 0.1 % TFA and CH~3~CN, respectively. The time program for gradient elution was programmed as follows: 0--50.5 min 1a: 1b : 1c = 95 : 5 : 0, 50.5--55.5 min 1a : 1b : 1c = 0 : 100 : 0, and 55.5--57 min, 1a : 1b : 1c = 0 : 0 : 100. The column temperature of all columns was maintained at 35°C. Fluorescence detection was made at 530 nm with an excitation wavelength at 470 nm. Differences between two groups were analyzed using the Mann-Whitney U-test. The relationship between two variables was examined using Spearman correlation coefficients. A p \< 0.05 was considered significant. Results ======= The CSF levels (421.7 μM (median), 468.1 ± 146.1 μM (mean ± S.D.), 254.0--775.1 (range)) of glutamine in the patients were not different (z = -1.038, p = 0.299) from those (410.5 μM (median), 405.6 ± 108.6 μM (mean ± S.D.), 219.8--689.0 (range)) of normal controls. The CSF levels (4.17 μM (median), 4.25 ± 1.77 μM (mean ± S.D.), 2.22--8.88 (range)) of glutamate in the patients were not different (z = -1.307, p = 0.191) from those (5.26 μM (median), 4.73 ± 1.29 μM (mean ± S.D.), 2.54--6.51 (range)) of normal controls. However, the ratio (126.1 (median), 117.7 ± 27.4 (mean ± S.D.), 42.0--161.6 (range)) of glutamine to glutamate in the CSF of patients was significantly (z = -3.29, p = 0.001) higher than that (81.01 (median), 89.1 ± 22.5 (mean ± S.D.), 59.7--134.0 (range)) of controls (Table 1). Furthermore, we found significant correlations between glutamate and glutamine in normal controls (r = 0.549, p = 0.022) or patients (r = 0.780, p \< 0.001). Discussion ========== In this study, we found that the ratio of glutamine to glutamate in the CSF of first episode and drug naive schizophrenic patients was significantly higher than that of normal controls although each level of glutamine and glutamate in the CSF of patients was not significantly different from that of normal controls. To our knowledge, this is a first report demonstrating that the ratios of glutamine to glutamate in the first episode and drug naive patients are significantly higher than those of normal controls. In contrast, it was supposed earlier that alterations in CSF levels of glutamate are not so prominent compared with those in the brain \[[@B14]\]. Therefore, it is likely that a difference in glutamate (or glutamine) levels between our CSF study and MRS studies may be due to the difference between CSF samples and specific corticolimbic regions. However, it should be noted that alterations in the ratio of glutamine to glutamate are detected in the CSF samples of first episode and drug naive schizophrenic patients, suggesting an abnormality of the glia-neuronal glutamate-glutamine cycle in the brain of patients with schizophrenia. In general, glutamine is synthesized in astrocytes from glutamate by the enzyme glutamine synthetase, found exclusively in brain glia cells. Glutamine then crosses the astrocytes to be transported into nerve cell terminals, where it is converted again into the neurotransmitter glutamate by glutaminase. It is reported that activities of glutaminase and glutamic acid decarboxylase (GAD; the rate-limiting enzyme in the synthesis of GABA by decarboxylation of glutamate) are significantly greater in the dorsolateral prefrontal cortex (DLPFC) of schizophrenia than in the control group, whereas activities of glutamate dehydrogenase, glutamine synthetase, and GABA transaminase in the DLPFC of schizophrenia are not different from the control group \[[@B19]\]. These findings suggest that metabolism of glutamate and GABA might be altered in the DLPFC of schizophrenic patients. Furthermore, it has been reported that activity of glutamine synthetase and glutamate dehydrogenase, the key enzymes involved in glutamate-glutamine cycle between neuron and glia, were markedly altered in the prefrontal cortex of schizophrenic patients, suggesting abnormalities in the function of glutamate-glutamine cycle in schizophrenic brain \[[@B20]\]. It is also well known that the glutamate-glutamine cycle between neuron and glia is tightly related to glutamate neurotransmission, glutamatergic function, and their regulation in human brain \[[@B7]\]. Taken together, it is likely that a dysfunction in glutamate-glutamine cycle in the brain may play a role in the pathophysiology of schizophrenia, supporting the glutamate hypothesis of schizophrenia. As described in introduction, sodium-dependent amino acids transporters, located in the abluminal membranes of the blood brain barrier, are capable of actively removing amino acids from the brain \[[@B16],[@B20],[@B21]\]. Sodium-dependent amino acids transporter are capable of pumping both glutamine (system N) and glutamate (glutamate transporters EAAT-1, 2, and -3) from the extracellular fluid into endothelial cells \[[@B20],[@B21]\]. The luminal facilitative carriers for both glutamate and glutamine can then transport them to the blood \[[@B16],[@B20],[@B21]\]. Therefore, the concentrations of naturally occurring amino acids in the CSF \[presumably similar to the extracellular fluid of brain\] are \~10% of those of the blood \[[@B15],[@B16]\]. Taken together, it seems that alteration in the transport mechanisms regulating levels of glutamate and glutamine in CSF may be implicated in elevated glutamine/glutamate ratio in CSF of schizophrenic patients although further study is necessary. Conclusion ========== Our findings suggest that a dysfunction in glutamate-glutamine cycle between neuron and glia may play a role in the pathophysiology of schizophrenia, supporting the glutamate hypothesis of schizophrenia. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contribution ====================== KH conceived of the study, its design and coordination, and edited the manuscript. GE participated in the design of the study. CN and LHL recruited subjects and collected CSF samples. ES and MI assisted HPLC analysis and data analyses. All authors read and approved the final manuscript. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Levels of glutamine and glutamate, and ratio of glutamine to glutamate in CSF of normal controls, and first episode and drug naive schizophrenic patients. (A) CSF levels of glutamine in patients were not different from those of normal controls. (B) CSF levels of glutamate in patients were not different from those of normal controls. (C) Ratios of glutamine to glutamate in patients were significantly higher than those of normal controls. ::: ![](1471-244X-5-6-1) ::: Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-244X/5/6/prepub> Acknowledgement =============== This study was supported in part by Grant-in Aid (A03) for Scientific Research on Priority Areas on \"Elucidation of glia-neuron network mediated information processing systems\" from Ministry of Education, Culture, Sports, Science and Technology (KH) and the Research Grant (15B-1) for Nervous and Mental Disorders from the Ministry of Health, Labor and Welfare (KH).	PubMed Central	2024-06-05T03:55:52.800981	2005-1-31	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548680/", "journal": "BMC Psychiatry. 2005 Jan 31; 5:6", "authors": [ { "first": "Kenji", "last": "Hashimoto" }, { "first": "Göran", "last": "Engberg" }, { "first": "Eiji", "last": "Shimizu" }, { "first": "Conny", "last": "Nordin" }, { "first": "Leif H", "last": "Lindström" }, { "first": "Masaomi", "last": "Iyo" } ] }
PMC548681	Background ========== Citizens increasingly use email in personal communication \[[@B1]\]. It is not however clear to what extent physicians utilize it for patient communication. In reports from the 1990s 1--14% of doctors in the USA and Norway used email in patient work \[[@B2],[@B3]\]. In recent studies up to 73% of physicians had used email for patient communication \[[@B4]-[@B6]\]. International and national recommendations and guidelines have been published on email use between doctor and patient; the contents of the Finnish guidelines are well in line with European guidelines \[[@B7],[@B8]\]. These guidelines emphasize the suitability of email for only certain limited purposes and stress the risks to information security. Use of email between doctor and patient has been studied in one controlled and randomized and three cross-sectional studies \[[@B4],[@B5],[@B9],[@B10]\]. In a study by Katz and colleagues the number of email contacts of physicians in their study group (46 email messages/100 scheduled visits) was greater than that in the control group (9/100) \[[@B5]\]. Houston and associates found that the majority of doctors received daily 1--5 email messages from their patients \[[@B9]\]. According to Sittig, physicians received daily in average 2.6 messages, and monthly an average of 40 per 140 visits \[[@B10]\]. Gaster and colleagues noted that physicians on average received 7.7 email messages in a month from their patients. Physicians in university clinics were most active in email use, while those in municipal primary health care were least active. Of physicians 58% reported in the questionnaire that the email contacts with patients were for the most part not registered in patient records \[[@B4]\]. Among Finnish citizens of working age young adults are the most active users of email and Internet \[[@B1],[@B11]\]. University students use these electronic net services even more actively than the young adult population as a whole. In a study from 2002 99% of students reported using email and Internet at least weekly \[[@B12]\]. All students have an email address at the university and their health providers at the FSHS can be reached by email. The student health care system can be seen as an appropriate setting to use email for patient communication \[[@B13]\]. The students represent a young, well educated, relatively healthy part of population which has been identified to be the most active to use email in patient-doctor communication \[[@B2],[@B5],[@B14]\]. The Finnish Student Health Service ---------------------------------- The Finnish Student Health Service (FSHS) provides primary health care services to approximately 140.000 university students in Finland. The FSHS has health stations in 16 university cities. Services include health promotion, consultations with general practitioners and with other clinical specialists, mental health care, and dental care. Since 1993 FSHS has provided health counseling in Internet. Since 1999 all physicians have had an email account at their disposal in health stations and an email address of type: <firstname.surname@yths.fi>. Principles of communication by email with FSHS\' employees and of other forms of electronic services (email service for cancellation of appointments, health counseling service on the Internet, and email service for feedback) are available at the FSHS\' website. The Social Insurance Institution, the university cities, the State of Finland, and the students themselves finance FSHS services. Students pay an annual obligatory health care fee as a part of the Student Union\'s membership fee. There is no other fee for preventive services, visits to general practitioner or public health nurse, and laboratory or X-ray examinations prescribed during these visits. Use of Internet services is also free of charge. The FSHS employs 560 persons and 63% of the physicians are general practitioners. In this paper general practitioners also include specialists of general practice/family medicine, whereas \"specialists\" refers to clinical specialists other than psychiatrists or oral surgeons. Aims of the study ----------------- The aim of the study was to seek answers to the following questions: 1\. How actively did physicians at student health care use email in communication with their patients? 2\. How much did they use email compared to phone calls and patient visits? 3\. Who were the active doctors using email with patients? 4\. What proportion of visits and phone calls could be candidates for substitution by email communication? 5\. Did the volume of visits, phone calls and email messages documented in the EPR of the FSHS during the study period differ from that of visits, phone calls and email messages registered in the study? Methods ======= All physicians (n = 82) in the FSHS\' functionary register in April 2003 received a questionnaire by email. We excluded six physicians, who were not any more working for the FSHS and took exception to the two authors. The actual number of survey population was 74. The questionnaire (see [Additional file 1](#S1){ref-type="supplementary-material"}) included background factors and a registration (in form of daily tally) of numbers of patient contacts, phone calls and email messages over one working week. Respondents were also asked to assess the number of visits and calls replaceable by email, and the number of email messages including a request, which could not be fulfilled without face-to-face contact. Also doctors\' attitudes toward email use for patient communication were asked. The first mailing of questionnaire took place 28.4.2003 and a reminder was sent 5.5.2003. Recipients were asked to print the survey form, fill it in by hand, and return it by internal mail. Overall 52 out of 74 (70%) physicians returned a completed survey. Respondents were grouped according to age, location, speciality licence, and type of employment (Table [1](#T1){ref-type="table"}). Facts on years of birth were collected from the register book \"Finland\'s Doctors 2002\" and other background factors from the FSHS\' functionary register. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Background variables of all physicians at the FSHS and of the respondents. ::: All physicians Respondents ----------------------- ---------------- ------------- (n = 76) (n = 52) \% \% Gender Male 34 29 Female 66 71 Age (years) 45 or under 33 31 46--55 39 42 56 or over 28 27 Location Capital city region 38 42 Turku and Tampere 28 27 Other^1)^ 34 31 Speciality licence General practitioner 63 69 Specialist 37 31 Type of employment Permanent 68 85 Non-permanent^2)^ 32 15 Distribution (%) of background variables of all physicians at the Finnish Student Health Service and of the respondents of the survey. ^1)^Small university towns ^2)^Vicars and fee-based working doctors ::: All physicians at the FSHS utilize EPR (Medicus^®^). We collected the numbers of patient contacts documented at Medicus^®^for the study period using the statistical software Cognos^®^. We entered data using Microsoft Excel^®^software and performed statistical analysis using StatsDirect Statistical^®^(3,2,7 -version) software. Statistical analyses were conducted using the proportion test for two independent groups, the χ^2^test, Fisher\'s exact test, the unpaired t-test, the Mann-Whitney test and the Kruskal-Wallis test. All tests were made two-sided and p-values below .05 were regarded as statistically significant. Results ======= Respondents ----------- Of all respondents 29% were men and 71% women (Table [1](#T1){ref-type="table"}). The respondents and all doctors at the FSHS were compared according to the background variables. The doctors who answered represented well the overall body of physicians working in the FSHS. The mean age of male respondents was 52.8 (range 34--65) and of female 48.5 years (range 29--65). This was in the same range as the mean age of all doctors at the FSHS (men 51.3 and women 48.3 years). Activity of using email ----------------------- In one working week 79% of doctors had used email and 98% the phone for patient communication. Respondents reported 2296 patient visits, 948 phone calls and 449 email contacts. They had on average 8.6 email contacts and 18.2 phone calls per week with their patients (Table [2](#T2){ref-type="table"}). They reported a mean percentage for \"email per visit\" of 20%, and phone calls in proportion to visits (\"phone call per visit\") averaging 40%. Eleven doctors (21%) reported more email contacts than phone calls. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Characteristics of the use of email. ::: Characteristic Mean Median Minimum Maximum -------------------------------------------- ------- -------- --------- --------- n^1)^ n^1)^ n^1)^ n^1)^ Type of patient contact Visit 44.2 46.5 7 78 Phone call 18.2 18.0 0 59 Email 8.6 3.5 0 96 \% \% \% \% Proportion of the respective contact types Phone call / visit 40 36 0 100 Email / visit 20 12 0 185 Email / phone call 55 22 0 600 Characteristics of the use of email in one week (5.5. -- 9.5.2003) among physicians (n = 52) at the Finnish Student Health Service. ^1)^ Number of contacts ::: We tested the variables showing email usage and phone calls with respect to background factors. There were no statistically significant differences by gender, age group, speciality licence or type of employment. Of physicians in the capital city area 41% reported more email contacts than phone calls. Among doctors working in Turku and Tampere the proportion was 7.1%, and among those working in small towns 6.3%. The difference between capital city region and other locations was statistically significant (p = .015). Respondents\' general attitude toward email for patient communication was evaluated by the statement \"Email contacts with patients facilitate my work.\" The group of \"positives\" was formed of the 56% of respondents who on a five step Likert scale replied: \"same opinion\" or \"nearly same opinion.\" The \"positives\" reported more email contacts than others (email per visit: median18% versus median 3%, p \< .001). All eleven who had reported more email contacts than phone calls belonged to the \"positive\" group. Patient visits and phone calls replaceable by email --------------------------------------------------- Doctors estimated that 2% (57/2296) of patient visits could have been replaced by email. Of phone calls 21% (196/948) could have been substituted with email. Respondents estimated that 10% (45/449) of patients\' email messages required a personal consultation. Documentation in the EPR ------------------------ The number of visits registered in the survey did not differ from the presumed number documented in the EPR (Table [3](#T3){ref-type="table"}.). The difference noted between registered and presumed email contacts shows that 73% of email contacts were not entered in the EPR. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Patient contacts documented in the EPR and registered in the survey. ::: Type of patient contact Number of patient contacts Statistical significance of the difference between expected and registered contacts^2^) ------------------------- ---------------------------- ----------------------------------------------------------------------------------------- ------ ---------- n n n p Visit 3098 2107 2296 0.237 Phone call 1115 758 948 \< 0.001 Email 178 121 449 \< 0.001 Patient contacts documented by all physicians at the Finnish Student Health Service (n = 76) in the electronic patient record (Medicus^R^) and registered by respondents (n = 52) in a survey over one working week (5.5. -- 9.5.2003), as well as the statistical significance of the difference in contact numbers. ^1)^Based on the proportion of respondents among all physicians ^2)^Proportion test for two independent groups ::: Discussion ========== Even if university students do not represent the whole population, they can act as \"pilot population\" representing adults of working age of a future information society. Our study population -- doctors taking care of students -- was small with only 52 respondents. Thus the results of the study cannot be indiscriminately generalized. Because of the small study population comparison of the subgroups may not be reliable. Although the study group was small, it well represented all the doctors at the FSHS, and the response rate was high. A further strength was that we compared the number of patient contacts documented in the questionnaires during the study week to the statistical data of contact numbers from the EPR at the same time. In other studies no corresponding comparison has been made. The doctors were asked to keep a daily tally of visits, phone calls and email messages, and to evaluate how many visits or phone calls could have been replaced by email. Many doctors undoubtedly did this simultaneously with patient work. Some doctors might have been in a hurry, they probably supplemented the questionnaire at the end of the day. To achieve a more accurate evaluation of visits and phone calls replaceable by email, a continuous assessment (visit by visit, phone call by phone call) could have been stressed even more in the instructions. The doctors at the FSHS do not have a specific electronic communication system focused on patient communication. They use their general, unprotected email system also to communicate with patients. A specific communication system used only for patient communication would enable an automatic collection of the patient communication data and create a more accurate database than our data collection method. Katz and colleagues have made the only controlled and randomized study concerning physicians\' use of email \[[@B5]\]. Our own results on the average number of doctors\' email contacts and email usage are of the same magnitude as those referred to above and in other recent studies in the USA \[[@B4],[@B9],[@B10]\]. In the present study 79% of respondents used email for patient communication. The proportion of those who had used email is clearly larger than in older studies, and at the same level as reported in recent international studies \[[@B2]-[@B6]\]. Our study revealed individual differences in the use of email in patient work. Differences in physicians\' activity in using email have previously been reported in only one study \[[@B4]\]. Deriving of our small study group only especially glaring association between subgroups of respondents could be verified. Our findings still support the results published by Gaster and associates. Physicians working in the capital area were more active email users than their colleagues elsewhere in Finland. Physicians reckoned that email could replace only 2% of visits. This confirms Sittig\'s evaluation in 2003 that email could possibly cover a small percentage of visits \[[@B10]\]. Increasing the use of email can thus not considerably reduce the number of patient visits. On the other hand it could make physicians\' crowded telephone hours easier \[[@B15]\]. When we compared contacts in the EPR with contacts registered daily on the questionnaires we found that the majority of email contacts were not registered in the EPR. This finding is supported by Gaster and colleagues who asked physicians themselves to describe how often they usually registered email contacts in patient records \[[@B4]\]. FSHS provides specific electronic services for focused issues (email service for cancellation of appointments, health counseling service on the Internet, and email service for feedback). Principles of recommended issues to use email between health providers and patients are available for students at FSHS\' websites. We have had a presumption that email messages between FSHS\' physicians and their patients mainly handle patient care. Nyström\'s congress report from 2004 supports our presumption. He explored 139 email messages from 103 individual patients at his GP practice at the FSHS and noticed that 77 % of email messages handled medical tests, and 16 % handled follow-ups of symptoms or illnesses \[[@B16]\]. Thus the information in email communication should be entered in patient records. All university students in Finland have access to Internet and email at their universities. Use of email as communication method in health care does not in their case cause inequalities in health. A general tendency in the societies to provide also health services widely in electronic form (in Internet or by email) can contribute to inequalities for those who are not able to use modern technologies \[[@B13],[@B17]\]. Conclusions =========== Doctors at the FSHS had an average of 8.6 email contacts with their patients during one week. The proportion of email contacts per visit was 20%. Physicians estimated that email contacts could substitute 2% of patients\' visits and 21% of phone calls. Of email contacts 73% were not documented in the EPR. Our study indicates that email communication really constitutes a part of patient work. This should be taken into account in planning working time and daily timetables. Use of software not integrated with the EPR increases the physician\'s registering load and currently involves extra work. It is not possible to confirm the patient\'s identity reliably using two separate systems. A system allowing retrieval of patient\'s identity safely and with no need to register separately the email communication in the EPR would promote the patient\'s adequate treatment and reduce the physician\'s medico-legal risks. There is a need for a larger study on email utilization between patient and physician which better covers the medical profession. The consumer point of view should also be better taken into account. A content analysis of email messages for patient communication combined with assessments of email documentation in the EPR could have strengthened present study regarding the importance of email registration in patient records. In Finland the Ministry of Social Affairs and Health has instigated a major project to safeguard health care \[[@B18]\]. One part of this programme demands that the whole public health care field should be using EPR by 2007 \[[@B19]\]. More research data are needed on electronic communication with patients and on users\' experiences. The future EPRs should include a purposeful, safe and secure means of patient communication. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= JC participated in the design of the study, coordinated it, keyed data into computer, performed the statistical analysis and drafted the manuscript with co-authors. MN conceived of the study, participated in the design of the study and wrote parts of manuscript in English. IV participated in the design of study and drafted the manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6947/5/2/prepub> Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Questionnaire ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ We gratefully acknowledge the financial support of Finnish Student Health Foundation.	PubMed Central	2024-06-05T03:55:52.802083	2005-1-27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548681/", "journal": "BMC Med Inform Decis Mak. 2005 Jan 27; 5:2", "authors": [ { "first": "Johanna", "last": "Castrén" }, { "first": "Marja", "last": "Niemi" }, { "first": "Irma", "last": "Virjo" } ] }
PMC548682	Background ========== Nosocomial infections are frequent complications of hospitalization and also an important public health problem in developing countries, as well as in developed ones. The socioeconomic impact, ie, prolongation of hospitalization, mortality, and cost of these infections adversely effects patients and nations\' economic well-being \[[@B1]\]. The cost of nosocomial infections includes increased length of hospital stay, staff time, laboratory cultures of pathogens and antimicrobial treatment \[[@B2]-[@B4]\]. Although, cost of antimicrobial treatment is an important part of health expenditure, data on this subject are extremely limited in Turkey. The aim of our study was to determine daily antibiotic cost of nosocomial infection per infected patient in a university hospital. Methods ======= The hospital setting -------------------- Akdeniz University Hospital is a 600-bed tertiary referral centre in Antalya, Turkey, treating 27 000 patients per year. The study was conducted in six adult medical and surgical intensive care units (ICUs) with a total of 51 ICU beds. Neonatal ICU was not included in the study. Since 1993, the institutional policies of hospital infection control have been implemented by infection control team. Definitions and study population -------------------------------- In our hospital, routine prospective, active surveillance of nosocomial infections in all ICUs is performed by one infection control nurse, supervised by an infection control physician. Nosocomial infections are defined using the Centers for Disease Control and Prevention criteria \[[@B5],[@B6]\]. We do not follow patients for signs of infection after discharge unless they are readmitted to the hospital. Between January 1, 2000 and June 30, 2003, all inpatients hospitalized in one of the adult ICUs were included in this study. Data on antimicrobial treatment were recorded for patients aged 15 or above presenting with only one nosocomial infection. For all patients included in the study, the following were recorded: age, sex, infection site, microbiologic data, antimicrobial therapy and antibiotic cost. Measurement of costs -------------------- In our ICUs, all antimicrobial prescriptions are recommended by an infectious disease consultant. Antimicrobial agents prescribed only for therapeutic indications were recorded. The daily antibiotic cost was calculated in US dollars based on June 2003 prices of antimicrobial agents provided by the hospital pharmacy. The daily antibiotic cost per infected patient was calculated by the multiplication of box price and number of daily doses that was used for that infection. Two costs were calculated for each antimicrobial; the minimal cost (min) was based on the lowest recommended parenteral daily dose and the maximal cost (max) was based on the highest recommended parenteral daily dose. Results ======= Between January 1, 2000, and June 30, 2003, a total of 8460 patients were admitted to the adult ICUs. Overall, 817 patients developed 1407 episodes of nosocomial infections, accounting for an infection rate of 16,6%. Among them, 233 patients (mean age:50,1; sex ratio female:male 0,49) had only one nosocomial infection. Mean daily antibiotic cost was found \$89,64 per infected patient (\$8,56 to \$359,28). Among the sites of nosocomial infections, urinary tract infections had the lowest daily antibiotic cost per infected patient (Table [1](#T1){ref-type="table"}). The mean daily antibiotic cost for pneumonia was the highest of all sites, but patients with bloodstream infection reached the highest range of daily cost (\$31,31 to \$359,28). In addition, mean daily antibiotic cost was found \$162,35 for seventeen other infections including postoperative meningitis, mediastinitis, and empyema. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Daily antibiotic costs according to the sites of nosocomial infections. ::: Site of nosocomial infections Number of patients Range of daily cost per infected (US\$) Mean (US\$) ------------------------------- -------------------- ----------------------------------------- ------------- Pneumonia 111 10,02--250,74 99,02 Bloodstream infections 28 31,31--359,28 94,32 Surgical site infections 11 17,12--204,74 94,31 Urinary tract infections 66 8,56--228,68 52,37 ::: Out of 233 patients, 206 patients had microbiologically documented infections, 177 patients were infected by a single pathogen while 29 patients were diagnosed as having polymicrobial infections (Table [2](#T2){ref-type="table"}). Pseudomonas aeruginosawas the most prevalent bacteria followed by Klebsiellaspp. and Acinetobacterspp.. P. aeruginosainfections had the highest overall daily antibiotic cost per infected patient than other pathogens. Among 21 Staphylococcus aureusstrains 15 (72%) were resistant to methicillin. The overall daily antibiotic cost of methicillin resistant S. aureus(MRSA) infections was two to three times higher than infections with susceptible strains. However, median daily cost per infected patient for MRSA infections were lower than susceptible strain infections. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Daily antibiotic cost according to the pathogens. ::: Pathogens No. Overall daily cost (US\$) Range of daily cost per pathogen (US\$) Median (US\$) -------------------------------- ----- --------------------------- ----------------------------------------- --------------- Pseudomonas aeruginosa 60 5567,06 17,98--204,74 100,04 Acinetobacterspp 36 4951,06 17,98--359,28 92,47 Stenotrophomonas maltophilia 4 310,99 31,31--100,04 89,82 Klebsiellaspp 37 3104,22 10,02--179,64 79,6 Enterobacterspp 10 961,79 60,42--139,08 77,28 Escherichia coli 23 1652,57 10,02--179,64 60,41 Proteusspp 3 49,04 13,24--49,04 49,04 Staphylococcus aureus Methicillin-susceptible (MS) 6 476,22 10,02--142,2 74,52 Methicillin-resistance (MR) 15 1188,1 35,88--142,2 71,1 CoNS\* MS-CoNS 1 49,64 16,34--49,64 49,64 MR-CoNS 3 148,92 17,52--49,64 49,64 Enterococcusspp 16 1141,25 32,29--142,2 49,64 Candidaspp 21 235,52 8,56--38,64 8,56 No pathogen identified 27 3166,75 10,02--359,28 114,6 \* Coagulase negative Staphylococcus ::: Among the 350 antibiotic prescriptions for nosocomial infections, piperacillin-tazobactam and amikacin were the most prescribed antibiotics (Table [3](#T3){ref-type="table"}). Carbapenems especially meropenem were the most expensive drugs. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Daily cost of antimicrobial agents for nosocomial infections. ::: Antimicrobial agents Number of prescriptions Daily cost per infected (US\$) --------------------------------- ------------------------- -------------------------------- -------- Betalactams Ampicillin-sulbactam 20 23,92 71,76 Piperacillin-tazobactam 48 85,95 114,6 Ticarcillin-clavulanate 4 14,9 22,35 Carbapenems Imipenem 31 100,04 150,06 Meropenem 23 179,64 359,28 Cephalosporins Cefepime 26 38,64 77,28 Ceftazidime 18 35,82 71,64 Cefoperazone-sulbactam 16 40,28 80,56 Cefazoline 10 10,02 20,04 Ceftriaxone 3 19,7 39,4 Aminoglycosides\* Amikacin 52 \- 7,96 Netilmicin 21 \- 24,48 Tobramycin 3 \- 18,56 Fluoroquinolones Ciprofloxacin 18 49,04 98,08 Ofloxacin 2 25,74 51,48 Levofloxacin 1 41,07 82,14 Glycopeptides Vancomycin 13 49,64 87,27 Teicoplanin 12 71,1 142,2 Other antibiotics Clarithromycin 4 11,57 23,14 Trimethoprim-sulphamethoxazole 2 4,48 8,96 Metronidazole 2 5,51 10,04 Antifungal agents Fluconazole 21 8,56 34,24 Total 350 8,56 359,28 \* dosage given as once-daily. ::: Disscussion =========== Cost is an important factor which determines the physician\'s choice of medication to treat patients in spesific stiuations. In this study, we tried to demonstrate the daily cost of antimicrobial treatment of nosocomial infections according to site of infection, pathogen and antimicrobial agent. In different studies, economical analysis regarding costs attributable to nosocomial infections has been evaluated and reported between \$1018 to 2280 per infected patient \[[@B7]-[@B9]\]. Jarvis et al reported that the estimated average costs of nosocomial infections were \$558 to 593 for each urinary tract infection, \$2734 for each surgical site infection, \$3061 to 40000 for each bloodstream infection, and \$4947 for each pneumonia \[[@B1]\]. Daily cost of antimicrobial treatment has been reported to be a significant extra cost attributable to nosocomial infections. In this study, we found an average daily antibiotic cost of \$89,64 per nosocomial infection. It is clear that cost of overall antibiotic treatment for a period of approximately 10--15 days is \$900 to \$1350. Prolongation of hospital stay has been the major extra cost attributable to nosocomial infections in many reports \[[@B2]-[@B4]\], but in comparative case-control study from our country, Yalcin et al. \[[@B8]\] found that cost of antibiotic therapy of \$1190 per infected patient, accounted for about 75% of the total extra cost. This finding may be due to the high prices of antibiotics in Turkey. To calculate the true costs of antibiotic therapy, hidden costs arising from intravenous administration, labor, serum antibiotic assay, monitoring hematological and biochemical indices and adverse effects of antibiotics must be considered \[[@B10]\]. The present study does not include these relevant \"hidden costs\" that could substantially modify the total cost of an antibiotic treatment. Although, hidden costs were not calculated, an average daily antibiotic cost of a single nosocomial infection is found to be markedly high in our hospital. This result is within the limits reported by other large economic studies, suggesting that our data is comparable to those found in other countries and with other assessment methods. In a French prevalence survey, Astagneau et al.\[[@B11]\] reported an average daily antibiotic cost between FF 520 to 1085 (about \$86 to \$160) per nosocomial infection. French et al.\[[@B12]\] and Haley et al.\[[@B13]\] reported an average cost of antibiotic treatment of \$190 and between \$72 to \$128 per nosocomial infection, respectively. In Turkey, Yalcin et al.\[[@B14]\] found that daily antibiotic cost of nosocomial infections was \$70 per patient. The daily antibiotic cost varies markedly according to site of infection. Our study has demonstrated that pneumonia and bloodstream infections were associated with the highest daily antibiotic costs as reported in other studies \[[@B11],[@B13],[@B14]\]. Surgical site infections had also high daily antibiotic cost in our study. In their case-control study, Coello et al. reported that antibiotic therapy for surgical patients was the second most significant contributor to cost \[[@B15]\]. In the present study, nosocomially infected patients that had only one nosocomial infection were considered for analysis. Clearly, antimicrobial treatment of patients with multiple nosocomial infections might be much more expensive. P. aeruginosainfections had the highest daily antibiotic cost followed by other non-fermentative bacilli. Infections caused by P. aeruginosaare difficult to treat because of its virulence and relatively limited choice of effective antimicrobial agents, so, these infections often require combination therapy. Emergence of resistance in P. aeruginosahas been associated with increased morbidity, mortality, and costs \[[@B16]\]. On the other hand, although the overall antibiotic cost of MRSA infections was higher than infections with susceptible strains, the daily antibiotic cost per infected patient with MRSA was lower with susceptible strain infections. MRSA infections are treated by glycopeptides which cost less than beta-lactams in our country. Astagneau et al reported that the daily antibiotic cost of multi-resistant bacterial infections such as multi-resistant P. aeruginosainfections, was 20% higher than susceptible infections, but the daily antibiotic cost per infected patient for MRSA infections was not higher than for susceptible strain infections \[[@B11]\]. Expensive antibiotics, such as piperacillin-tazobactam, carbapenems, cefepime, ciprofloxacin, teicoplanin were prescribed more commonly than the cheaper agents such as ampicillin-sulbactam, ceftriaxone or ofloxacin in our ICUs. These expensive antibiotics were mainly prescribed for resistant and severe gram-negative nosocomial infections, such as ventilator-associated pneumonia and postneurosurgical meningitis in ICU. Physicians may be forced to choose empirical antibiotic therapy with broad spectrum antimicrobials by increasing bacterial multi-resistance. In conclusion, mean daily antibiotic cost was found \$89,64 per nosocomial infection in our ICUs and nosocomial pneumonia had the highest daily antibiotic cost per infected patient. It is clear that cost of antibiotic therapy of nosocomial infections is an important part of extra cost attributable to nosocomial infection. Approximately one third of nosocomial infections are preventable by full implementation of the current infection control guideline recommendations \[[@B17]\]. Each institution should develope empirical antibiotic guidelines according to its own local nosocomial infections data. Infection control measures, such as education of health care workers regarding antimicrobial agents and resistance; isolation of patients infected with multi-resistant organisms, should be implemented to reduce infections and expensive antibiotic prescriptions. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' conributions ====================== DI collated and analyzed the data, participated in the study design and was principal writer of manuscript. ANY conceived the study. GO carried out the laboratory studies. RS, FG, OT and LM participated in the patient management. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2334/5/5/prepub> Acknowledgments =============== The authors thank Ms. Sevim Keskin, an infection control nurse, for her valuable collaboration. This study was supported by Akdeniz University Scientific Research Project Unit.	PubMed Central	2024-06-05T03:55:52.804532	2005-1-31	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548682/", "journal": "BMC Infect Dis. 2005 Jan 31; 5:5", "authors": [ { "first": "Dilara", "last": "Inan" }, { "first": "Rabin", "last": "Saba" }, { "first": "Filiz", "last": "Gunseren" }, { "first": "Gozde", "last": "Ongut" }, { "first": "Ozge", "last": "Turhan" }, { "first": "Ata Nevzat", "last": "Yalcin" }, { "first": "Latife", "last": "Mamikoglu" } ] }
PMC548683	Background ========== Hematopoietic differentiation is a complex process whereby multiple functionally and morphologically distinct cell types arise from a population of pluripotent hematopoietic stem cells (PHSCs) \[[@B1]\]. The accurate and efficient regulation of hematopoietic development is controlled by a large number of regulatory proteins that have been identified over the past few decades. These regulatory molecules include the hematopoietic growth factors (HGFs), soluble proteins that recognize specific receptors on the surface of sub-populations of hematopoietic cells, thereby initiating signal transduction pathways that modulate the differentiation, proliferation, and/or survival of target cells \[[@B2]\]. The identification of regulators of hematopoiesis has been an ongoing effort for many years and has benefited from the existence of accessible cell line models as well as the characterization of genes affected by somatic mutations associated with specific human leukemias \[[@B3]\]. We have used a pair of factor-dependent murine cell lines to identify novel genes expressed within distinct hematopoietic lineages as an approach to the identification of novel candidate genes for development of diagnostic and therapeutic approaches to leukemia. The EML and MPRO cell lines were both established by infecting murine bone marrow cells with a retrovirus expressing a dominant negative retinoic acid receptor α (RARα) protein \[[@B4],[@B5]\]. The infected cells were selected in the presence of either stem cell factor (SCF) or granulocyte/macrophage colony stimulating factor (GM-CSF). EML are SCF-dependent and resemble uncommitted hematopoietic progenitor cells. They can be induced to differentiate to the promyelocyte stage of granulopoiesis in the presence of interleukin-3 (IL-3) and high doses of all trans retinoic acid (atRA) \[[@B4],[@B6]\]. Terminal neutrophil differentiation of EML cells can be induced by replacement of IL-3 and SCF with GM-CSF. MPRO cells are GM-CSF-dependent and can be induced to differentiate to neutrophils by adding high doses of atRA to the culture medium. The expression patterns of a number of genes expressed during hematopoiesis have been examined in EML and MPRO cells and generally agree with the patterns observed in other cell systems and in primary hematopoietic cells. Thus, EML and MPRO provide a powerful system for the identification and characterization of novel genes expressed within the hematopoietic lineage. We have employed the representation difference analysis technique \[[@B7]\] to identify cDNAs representing genes expressed at higher levels in EML cells 72 hours after induction of differentiation than in uninduced cells. We describe the identification of a clone derived from an uncharacterized putative secreted protein. We have performed a comparative genomics analysis and determined that this protein is the founding member of an extended family of highly related proteins. This family contains three members in mammalian species, one or two members in invertebrate or simple vertebrate species and five or six members in fish. We have determined that one family member is a secreted glycoprotein and describe the expression pattern of the human and mouse genes in tissues and during hematopoietic differentiation. Results ======= Identification of differentially expressed genes by representational difference analysis (RDA) ---------------------------------------------------------------------------------------------- Total RNA was prepared from EML cells grown in the presence of SCF alone (0 hour) or in medium supplemented with IL-3 and atRA for 72 hours. The RNA was converted to cDNA and subjected to three rounds of RDA as previously described \[[@B6]\]. Six differentially-expressed clones were identified \[[@B6],[@B8]\]. Clone number 1623 was chosen for further analysis and the differential expression of this gene was confirmed by Northern blot analysis. 1623 mRNA was essentially undetectable in the 0 hour sample but readily detectable in the 72 hour sample (data not shown and see figure [10](#F10){ref-type="fig"}). Sequence analysis of clone 1623 ------------------------------- The initial cDNA isolated by RDA was a 273 bp fragment that appeared to contain the coding sequence of the C-terminus of a protein that was not present at that time in public databases. To identify the full open reading frame of this cDNA, we first performed rapid amplification of cDNA ends (RACE) in both the 5\' and 3\' directions. Extension in the 3\' direction revealed the presence of a consensus polyadenylation signal located 154 nucleotides downstream of the putative translation stop codon. Extension in the 5\' direction yielded an additional 443 bp of sequence containing a contiguous ORF. Comparison of this extended sequence to public databases identified a cDNA (NM\_017565) that was identical to clone 1623 in the region of overlap. This cDNA was isolated from a mouse mammary tumor but no functional analysis had been performed. We designed PCR primers based on the published sequence and confirmed that the cDNA isolated from EML cells was identical to the published sequence. The full length ORF was 1623 bp in length and encoded a protein of 541 amino acids (Figure [1A](#F1){ref-type="fig"}). The protein did not contain any recognizable motifs when examined using domain mapping software such as SMART or Profilescan (see Methods). However, a putative amino terminal signal sequence was identified using the SignalP analysis program. The cDNA mapped to an 11 exon gene located on mouse chromosome 11E1 (Figure [1B](#F1){ref-type="fig"}). The gene spanned approximately 60,000 bp of genomic sequence and the relatively large size of the gene is primarily due to the fact that the first intron is greater than 44,000 bp in length (Figure [1C](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Characterization of the full length mouse 1623 (Fam20a) cDNA and genomic sequence.A. The full length cDNA derived from the original RDA clone was isolated using a combination of 5\' and 3\' rapid amplification of cDNA ends (RACE) procedures, comparisons to public databases, and amplification of putative full length clones by PCR. The full open reading frame was 1623 bp in length and encoded a 541 amino acid protein. The locations of regions conserved within the subsequently identified FAM20 family are indicated using underlines. Eight cysteine residues that are also conserved within the family are indicated in bold and four putative N-glycosylation sites are indicated in red type. B. The distribution of the 11 exons of the mouse Fam20a gene is shown with the exons indicated using numbers. A consensus polyadenylation signal is located downstream of the terminal exon. C. The sizes of the 11 exons and 10 introns of the Fam20a gene are shown. ::: ![](1471-2164-6-11-1) ::: Identification of a family of related genes ------------------------------------------- The full length cDNA and the encoded protein were compared to sequences in public databases. We reported previously a weak similarity to a protein named Fjx1, which is the mouse orthologue of a Drosophila protein named four-jointed\[[@B8]\]. However, the degree of sequence identity between these proteins was low (16%) and thus the search was extended to include uncharacterized proteins. We first identified two other mouse proteins that displayed significant similarity to, but were distinct from, the query sequence. One protein (Accession number NP\_663388 aka Riken C530043G21) was 409 amino acids in length and displayed 27% identity to the query sequence while the second (NP\_085042) was a truncated version of a 579 amino acid protein that displayed 40% identity to the query sequence. We have subsequently discovered that these proteins are members of a highly related family and this family has received the official name \"family with sequence similarity 20\" (FAM20) from the Human Genome Organization Gene Nomenclature Committee. The protein derived from our original 1623 cDNA is named Fam20a in mouse and the other two family members are named Fam20b and Fam20c, respectively. Continued searches of public databases revealed the existence of related proteins in several other species. Each mammalian genome contains genes encoding three members that are orthologous to the three mouse proteins mentioned above. The accession numbers for the relevant cDNAs in human and rat are listed in Table [1](#T1){ref-type="table"} and we have also identified the same number of related sequences in other mammalian genomes, including the pig, cow and dog (data not shown). However, most of these sequences are incomplete and will not be described in detail here. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Accession numbers for vertebrate FAM20 family members^1^ ::: ----------------------------------------------------------------------------------------------------------------------------------- Family Member Human Mouse Rat Fugu Zebrafish Ciona --------------- ------------ ------------ ----------------------- -------------------------------- --------------------- ---------- FAM20A NM\_017565 NM\_153782 XM\_221067 (BK001521) SINFRUP00000146987 (BK001515) ND ND FAM20B NM\_014864 NM\_145413 XM\_222770 SINFRUP00000138548 (BK001520) CAI11712 AK115425 FAM20C NM\_020223 NM\_030565 XM\_221975 (BK001522) SINFRUP00000140879 (BK001516)\ ENSDARP00000009272\ ND SINFRUP00000141732 (BK001518)\ ENSDARP00000005688\ SINFRUP00000163431 (BK001517)\ ENSDARP00000028589 BK001519 ----------------------------------------------------------------------------------------------------------------------------------- 1\. Accession numbers refer to nucleotide sequences in GenBank except in the Fugu and Zebrafish listing where the accession numbers refer either to predicted peptides in the Ensembl database (entries beginning SINFRUP or ENSDARP) or Genbank (entry beginning CAI). Entries beginning with BK refer to predicted sequences in the Third Party Annotation database arising from this study. ND: None detected. ::: In order to gain further information concerning the origin of the FAM20 family, we searched for related sequences in several other invertebrate and vertebrate organisms. The ascidian Ciona intestinalisis a model for a basal chordate organism and has emerged as a powerful model for evolutionary and developmental studies \[[@B9],[@B10]\]. In particular, many gene families or subfamilies are represented by single members in C. intestinalisand thus the identification of an orthologue in this organism can provide useful information for evaluating the evolutionary origin of the members of a gene family. Consistent with this concept, we identified a single cDNA and the corresponding genomic locus in C. intestinalisthat displayed significant sequence similarity to the mammalian FAM20 genes and proteins (Table [1](#T1){ref-type="table"}). Complete genome sequences are also available for several invertebrate species and two related sequences were identified in Drosophila melanogasterand Anopheles gambiaewith one family member in Caenorhabditis elegans(Table [2](#T2){ref-type="table"}). Finally, analysis of genomic cDNA and protein databases for the pufferfish (Fugu rubripes) and zebrafish (Danio rerio) revealed the presence of six and five family representatives, respectively (Table [1](#T1){ref-type="table"}). The gene numbers in these various species are listed on an idealized evolutionary tree in Figure [2](#F2){ref-type="fig"} and suggest that the FAM20 gene family has undergone a complex set of gene duplications in both the invertebrate and chordate lineages. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Accession numbers for invertebrate FAM20 family members^1^ ::: ------------------------------------------------------ Family Member Drosophila Mosquito C. elegans --------------- ------------- ----------- ------------ FAM20A ND ND ND FAM20B NM\_170079\ EAA08010\ NM\_078126 NM\_206490 EAA13434 Fam20C ND ND ND ------------------------------------------------------ 1\. Accession numbers refer to nucleotide sequences in GenBank. ND: None detected. ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Evolutionary distribution of FAM20 gene number.An idealized evolutionary tree (modified from \[10\]) is shown with the number of FAM20 genes identified in several genomes as described in the text. The gene numbers are supportive of a single gene duplication event occurring in invertebrates (at least in insects) and multiple gene duplication events occurring in higher vertebrates. ::: ![](1471-2164-6-11-2) ::: Assignment of subfamily relationships ------------------------------------- To elucidate the nature of these putative gene duplications, we sought to assign the individual sequences from the various species into subfamilies based on protein sequence and gene structure, specifically using the number and size of exons in the latter case. Initially, the exon distribution of each of the Fam20 members in three mammalian species (human, mouse and rat) was compared and revealed obvious inter and intra orthologue similarities (figure [3A](#F3){ref-type="fig"}). Each FAM20A gene contained 11 exons and exon sizes were identical in these three species. Likewise, each FAM20B gene contained 7 exons that were identical in size in human, mouse and rat. The FAM20C genes each contained 10 exons and only exon 1 displayed any variation in size amongst these three species. In intra-orthologue comparisons, the exons in FAM20B and FAM20C genes clearly aligned with exons in the FAM20A genes, with small variations (in multiples of three bases) in the size of the internal exons in FAM20B. FAM20B lacks exons corresponding to exons 2--4 of FAM20A while FAM20C lacks exon 3. In addition, exons 8 and 9 in FAM20A and FAM20C are represented by a single exon in FAM20B that is identical in size to the combined exons in the other two genes. Thus, the three mammalian genes are highly evolutionarily related and presumably are derived from a common ancestral gene. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Assignment of FAM20 family members to subfamilies.A. Exon size and distribution of mammalian FAM20 members. The exons within each FAM20 gene in human, mouse and rat are indicated with the number of base pairs indicated within each exon. The sizes of exons that differ in size from the FAM20A genes are indicated. B. A dendrogram showing the relationships between FAM20 proteins from human (Hs), mouse (Mm), rat (Rn), Fugu rubripes(Fr), Danio rerio(Dr), D. melanogaster(Dm), A. gambiae(Ag), C. intestinalis(Ci) and C. elegans. The accession numbers of the cDNA sequences from which each protein sequence was derived are shown in parentheses except in the case of the mosquito family members where the accession number is used as the gene/protein name. Accession numbers for zebrafish peptide sequences are listed in Table 1. The FAM20 nomenclature has not been extended to the invertebrate sequences and the previous gene names have been used for Drosophila and C. elegans family members. The subfamily assignment of each family member is shown on the right. C. Exon number and size distribution within Fugu Fam20 members. The accession number of each sequence within the Third Party Annotation database is shown at left and family assignment based on dendrogram position and exon distribution is shown on the right. ::: ![](1471-2164-6-11-3) ::: To assign the genes identified in other species to these three subfamilies, we performed a global comparison of the peptide sequences derived from 25 of the identified family members listed in Tables [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}. One zebrafish protein (from FAM20A) was omitted as its sequence is incomplete. A dendrogram showing the results of this comparison is presented in Figure [3B](#F3){ref-type="fig"}. As expected, the mammalian orthologues clustered together and thus defined the subfamilies. All of the invertebrate proteins and the single protein identified in C. intestinalisclustered with FAM20B proteins, suggesting that this represents the ancestral branch of the FAM20 family. A single protein from Fugu and zebrafish clustered with the FAM20A and FAM20B family members while two Fugu and two zebrafish proteins clustered with FAM20C members. However, two Fugu proteins and one zebrafish protein clustered on a separate branch between FAM20A and FAM20B. In order to determine the subfamily to which these proteins belonged, we made use of the high degree of conservation of exon size and number noted in the mammalian genes (Figure [3C](#F3){ref-type="fig"}). The exon number and size of the Fugu and zebrafish genes encoding the two proteins assigned to FAM20A and FAM20B were consistent with their membership in these families. The only variations noted were a slightly larger exon 2 in the Fugu Fam20a gene and the division of exon 1 into two exons in the Fugu Fam20b gene. As in the mammalian family members, the sizes of the terminal exons varied more than the internal exons. The other four Fugu genes displayed exon distributions consistent with membership in FAM20C, despite the clustering of two of the encoded proteins between FAM20A and FAM20B. We have assigned each of these proteins to FAM20C with number suffixes (c1, c2, etc.) to designate individual genes and proteins. Each of these genes maps to distinct genomic loci and thus represents independent genes and not splicing variants of a smaller number of genes (data not shown). The gene structures of the three zebrafish family members were also consistent with this family assignment (data not shown). Comparisons of the derived protein sequences within each subfamily are shown in figures [4](#F4){ref-type="fig"},[5](#F5){ref-type="fig"},[6](#F6){ref-type="fig"}. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Sequence alignment of FAM20A protein sequences.The complete protein sequences of FAM20A members were compared using the AlignX component of the VectorNTI sequence analysis suite of programs. Identical amino acids are outlined in yellow, and similar residues are indicates in light blue. Conserved regions 1, 2 and 3 are underlined (see below). Gaps are indicated with dashes and the sequences are from human (H), mouse (M), rat (R) and puff erfish (F). ::: ![](1471-2164-6-11-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Sequence alignment of FAM20B protein sequences.The complete protein sequences of FAM20B members are presented as described in figure 4. The sequences are from human (H), mouse (M), rat (R), pufferfish (F), zebrafish (D) and C. intestinalis(Ci). ::: ![](1471-2164-6-11-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Sequence alignment of FAM20C protein sequences.The complete protein sequences of FAM20C members are presented as described in figure 4. The sequences are from human (H), mouse (M), rat (R), pufferfish (Fcl-4) and zebrafish (Dcl-3). ::: ![](1471-2164-6-11-6) ::: Features of FAM20 proteins -------------------------- All of the identified FAM20 protein sequences contain putative signal sequences at their amino termini but no other functional domains were unambiguously detected using several different annotation search software programs. In order to search for potential functional domains, we compared the sequences of all family members. These comparisons revealed that the greatest similarity was located within the carboxy-terminal two thirds of each protein (Figure [7A](#F7){ref-type="fig"}). We have named this region the conserved C-terminal domain (CCD) and it overlaps with a domain listed in the CDD database at NCBI as DUF1193. The CCD contains three distinct regions that are more highly conserved within all members of the family than the surrounding sequences (named conserved regions 1, 2 and 3 in figure [7A](#F7){ref-type="fig"}) and the consensus sequences for each conserved region were derived (figure [7B](#F7){ref-type="fig"}). Amino acids that are essentially invariant in all family members have been indicated in bold type and the heptapeptide DRHHYE in CR2 is the longest contiguous sequence that is conserved in all members of the family. A set of eight cysteine residues is also perfectly conserved within the CCD of each family member that may participate in inter-or intramolecular disulphide bond formation. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Schematic representation of the structural features of FAM20 family members.A. Structural features of FAM20A showing domains and residues conserved within the entire family. Key: SS: signal sequence; CCD: conserved C-terminal domain; CR: conserved region; Cys: cysteine residues conserved within CCD (indicated with asterisk). B. Consensus sequences were derived for CR1, CR2 and CR3 using a global comparison of all the family members listed in Tables 1 and 2. Residues that are invariant or only differ in one sequence are indicated in bold. Non-conserved residues are indicated with an x and positions with more than one common residue are shown below the main sequence. ::: ![](1471-2164-6-11-7) ::: Fam20a is a secreted protein ---------------------------- As the putative signal sequence was the only known domain identified in all family members, we next tested whether this sequence is functional. Signal sequences are commonly found on proteins that are directed to the endoplasmic reticulum (ER) and either retained there or processed and transported into the Golgi apparatus and secreted from the cell. Many proteins are glycosylated during their transit through the ER and Golgi apparatus and the mouse Fam20a protein contains four potential sites for N-glycosylation (indicated in red type in figure [1](#F1){ref-type="fig"}). As Fam20a does not contain an ER retention signal, we predicted that it should be detected in the medium of expressing cells. A mammalian expression vector was constructed that contained the full length mouse Fam20a coding sequence fused to a C-terminal Myc epitope tag and a hexahistidine sequence to permit purification. The plasmid was transfected into monkey kidney COS-1 cells and total protein was isolated from both the cells and the cell medium. Proteins in the cell medium were first processed on a Nickel column to isolate and concentrate the recombinant Fam20a protein and both protein samples were analyzed by immunoblotting using an antiserum specific for the Myc epitope. The predicted molecular weights of the full length and processed forms of Fam20a are 61,500 and 57,500, respectively, and a recombinant form of the protein synthesized in rabbit reticulocyte lysates was run alongside as a molecular size marker. The recombinant protein migrated just below the 62,000 mol.wt. size marker (Figure [8A](#F8){ref-type="fig"} and [8B](#F8){ref-type="fig"}, lane 5); however, the proteins detected in both the cell medium and cell extract migrated slower (lane 3). To test whether this slower migrating band represented a glycosylated form of Fam20a, the protein samples were treated with the enzyme N-glycosidase F (PNGaseF). The protein detected after enzyme treatment migrated more rapidly than the untreated protein and comigrated with the recombinant form of the protein (compare lanes 3, 4 and 5). We noted a second band that migrated slightly more slowly than the recombinant protein in the PNGase F treated cell extracts that may represent an alternatively modified form of Fam20a (Figure [8B](#F8){ref-type="fig"}, lane 4). To confirm that Fam20a is a secreted protein, we also exposed Fam20a-expressing cells to Brefeldin A, a fungal metabolite that specifically blocks transport from the ER to the Golgi apparatus, and examined the effects on Fam20a secretion. Brefeldin A treatment resulted in a consistent decrease in the amount of Fam20a detected in the cell medium (Figure [8C](#F8){ref-type="fig"}, compare lanes 5 and 6). Thus, Fam20a is a secreted glycoprotein. ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Fam20a is a secreted protein.COS-1 cells were transfected with either an empty expression vector (-) or one encoding mouse Fam20a with a C-terminal myc epitope tag and proteins were isolated from either the medium (panel A) or the cells (panel B). The proteins were analyzed by immunoblotting using a Myc tag-specific antiserum. Samples in lanes 2 and 4 of each blot were pre-treated with protein N-glycosidase prior to analysis to remove glycosyl groups. A recombinant form of Fam20a synthesized in rabbit reticulocyte lysates (TnT) was included on each gel as a size marker. The position of glycosylated and deglycosylated Fam20a is indicated using arrowheads and cross reacting material detected in the medium is indicated using asterisks. The location of molecular size markers is shown on the left of each gel. C. Protein samples from the medium of transfected cells that were untreated or treated with Brefeldin A were analyzed by immunoblotting using the Myc tag-specific antiserum. As Brefeldin A was resuspended in DMSO, the untreated cells were exposed to DMSO alone as a vehicle control. The amount of Fam20a detected in the medium of Brefeldin A treated cells was consistently lower than that observed in untreated cells (indicated using an arrowhead). ::: ![](1471-2164-6-11-8) ::: Fam20a secretion requires a functional signal sequence ------------------------------------------------------ We next tested whether the integrity of the signal sequence was required for Fam20a secretion. Signal sequences typically contain a high proportion of hydrophobic amino acids and 19 of the first 34 amino acids of Fam20a are hydrophobic (Figure [9A](#F9){ref-type="fig"}). Therefore, we expressed a Fam20a protein lacking the first 23 amino acids (FAM20a(Δ23)) in COS-1 cells and examined secreted and intracellular proteins by immunoblotting (Figure [9B](#F9){ref-type="fig"}). Glycosylated FAM20a(Δ23) protein was not detected in the medium (compare lanes 2 and 3) and immunoreactivity that comigrated with the unglycosylated recombinant protein was detected in the cell extract (lane 6). We also compared the subcellular location of the FAM20a(Δ23) protein to the wild type protein using GFP fusion proteins (figure [9C](#F9){ref-type="fig"}). The wild type Fam20a-GFP proteins displayed perinuclear and cytoplasmic staining consistent with ER localization. In contrast, the Fam20a (Δ23)-GFP protein was absent from the cytoplasm and appeared to be exclusively localized within the nucleus. To ensure that this effect was not a consequence of a gross change in protein structure due to the deletion of 23 amino acids, we also constructed an expression vector encoding a Fam20a protein with a two amino acid substitution within the putative signal sequence (Figure [9A](#F9){ref-type="fig"}). These changes (Leu^14^--Leu^15^to Asp-Glu) were predicted to disrupt the signal sequence without grossly altering the protein structure. Again the mutant protein displayed nuclear staining and was absent from the ER (Figure [9D](#F9){ref-type="fig"}). These results confirm that an intact signal sequence was necessary for secretion of Fam20a and that secretion was accompanied by prominent localization of the protein to the ER. ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Secretion of Fam20a requires an intact signal sequence.A. Schematic representation of the putative signal sequence of Fam20a. The predicted cleavage site is indicated with a red arrowhead. The two amino acid substitutions introduced in the SSmut construct and the sequence remaining in the Δ23 mutant construct are shown. B. Immunoblot analysis of Fam20a and Fam20a(Δ23) protein levels in transfected COS-1 cells. The position of the glycosylated form of Fam20a (which is absent in Fam20a(Δ23) transfected cells is indicated with an arrowhead. C. Fluorescence images of COS-1 cells expressing either Fam20a-GFP or Fam20a(Δ23)-GFP. The wild type protein was observed within the cytoplasm, predominantly in a structure that is likely to be the ER. The mutant protein was primarily localized to the nucleus. D. Immunofluorescence images of Fam20a and Fam20a (SSmut) proteins as detected by antiserum directed against the C-terminal Myc epitope. The wild type protein was again detected in the ER and the mutant protein primarily in the nucleus. The cells have been counterstained with DAPI to delineate the nucleus. ::: ![](1471-2164-6-11-9) ::: ::: {#F10 .fig} Figure 10 ::: {.caption} ###### RT-PCR analysis of mouse Fam20 mRNA levels during differentiation of EML and MPRO cells.Total RNAs were prepared from EML (panel A) or MPRO (panel B) cells at the indicated timepoints during myeloid and granulocytic differentiation. cDNAs prepared from each sample were amplified using primer pairs specific to each mouse family member. The PCR products were analyzed by agarose gel electrophoresis and stained using Gelstar SYBR Green DNA stain. GAPDH was used as a loading control. ::: ![](1471-2164-6-11-10) ::: Expression patterns of FAM20 genes during myeloid differentiation ----------------------------------------------------------------- We originally identified Fam20a as a differentially expressed mRNA in EML cells induced to differentiate along the myeloid lineage. To determine whether Fam20b and Fam20c are also expressed during hematopoiesis, we performed RT-PCR analysis of cDNAs prepared at various times during experimentally-induced differentiation of EML and MPRO cells using primers specific to each family member. Fam20a mRNA levels were low in uninduced EML cells maintained in the presence of SCF and increased during the subsequent 72 hours of incubation in atRA and IL-3 (Figure [10A](#F10){ref-type="fig"}). EML cells mature to the promyelocyte stage of neutrophil differentiation under these conditions and can subsequently be differentiated into neutrophils by adding GM-CSF in place of SCF and IL-3. Fam20a mRNA levels decreased during terminal neutrophil differentiation in EML cells and also in MPRO cells induced to undergo the same differentiation process in the presence of atRA (Figure [10A](#F10){ref-type="fig"} and [10B](#F10){ref-type="fig"}). Fam20b and Fam20c mRNAs were readily detected in both cell lines and their levels did not vary dramatically during the differentiation process in either cell line (Figure [10A](#F10){ref-type="fig"} and [10B](#F10){ref-type="fig"}). Expression patterns Of FAM20 genes in human tissues --------------------------------------------------- Although we originally isolated Fam20a from a hematopoietic cell line, cDNAs and ESTs derived from each of the FAM20 family members have been isolated from non-hematopoietic tissues (data not shown). Therefore, we examined the expression patterns of the three genes in a panel of cDNAs derived from various human tissues. FAM20A displayed the most restricted expression pattern with high levels in lung and liver and intermediate levels in thymus and ovary (Figure [11](#F11){ref-type="fig"}). Low levels of FAM20A mRNA were detected in several other tissues. FAM20B and FAM20C were expressed in a wider variety of tissues and their expression patterns were very similar. ::: {#F11 .fig} Figure 11 ::: {.caption} ###### RT-PCR analysis of human FAM20 mRNA levels in human tissues.A panel of commercially available human cDNAs prepared from the indicated tissues was analyzed by PCR using primer pairs specific for each of the human FAM20 family members. GAPDH was again used as a loading control although large variations were observed in the GAPDH signal in the different tissues. ::: ![](1471-2164-6-11-11) ::: Discussion ========== Several classes of secreted proteins, including the colony stimulating factors or hematopoietins, are important regulators of hematopoietic differentiation and function \[[@B11]\]. These molecules are of clinical significance due to their use in stimulating hematopoiesis in patients with neutropenias and other hematological disorders \[[@B11]\]. Consequently, the identification of novel secreted proteins that display specific spatiotemporal expression patterns in hematopoietic cells is of great interest. In this report, we describe the identification and initial characterization of a new family of secreted glycoproteins expressed within the hematopoietic lineage as well as several other cell types and tissues. The family has been named FAM20 to indicate the fact that the members are related by sequence similarity rather than a specific shared function and contains three members in mammals. We anticipate that the members will acquire new names as their specific functions are determined. The family contains three separate subfamilies which are referred to as FAM20A, FAM20B and FAM20C in humans. FAM20 proteins: features and potential functions ------------------------------------------------ At the present time, the specific function(s) of the FAM20 proteins is unknown. Analysis of the sequences of the proteins from various species failed to reveal obvious similarities to known functional domains except for an N-terminal signal sequence. Expression studies clearly demonstrated that the mouse Fam20a protein is a secreted protein and that disruption of the signal sequence prevented the detection of the glycosylated form of the protein in cell media. Surprisingly, disruption of the signal sequence resulted in redistribution of intracellular Fam20a from a cytoplasmic compartment that is likely to be the ER to the nucleus. It is unclear whether this redistribution is functionally significant and is presumably due to the presence of a cryptic nuclear localization signal (NLS) in the protein. NLSs are generally comprised of stretches of basic residues \[[@B12]\] and several candidate regions rich in basic amino acids are present in Fam20a that could direct the mislocalized protein to the nucleus. Overall, the FAM20 proteins vary in length between 400--670 amino acids with the FAM20B family members being the shortest and the FAM20C members generally being the longest. The variation in length is due to differences in the length of the less conserved N-terminal region. The FAM20 proteins are further characterized by a highly conserved C-terminal region of approximately 350 amino acids that we named the conserved C-terminal domain (CCD). We noted the presence of three regions that are more highly conserved amongst all family members, with the most invariant extended sequence being the DRHHYE heptapeptide within conserved region 2. The function of the peptide is as yet unknown but three possible functions can be proposed. First, the histidines within this sequence could be involved in the coordination of metal ions that may be required for FAM20 protein function. Second, FAM20 proteins may be enzymes and this sequence may be a component of the active site of the enzyme. Interestingly, a weak match was detected between this region of certain FAM20 family members and a conserved domain within the phosphatidylinositol 3- and 4-kinases \[[@B13]\]; however additional experiments must be performed to address the relevance of this similarity. Third, the highly charged nature of this peptide suggests that it may be located on the surface of FAM20 proteins, where it may participate in protein:protein interactions. Although we have been unable to identify any other proteins in public databases that contain the exact sequence, the sequence RHHYE is found between amino acids 41--45 in the N-terminal region of the viral infectivity protein (Vif) from human immunodeficiency virus 1 (Accession number AAQ09611) \[[@B14]\]. Vif enhances HIV-1 infectivity by blocking the antiviral activity of the nucleotide editing enzyme APOBEC3G \[[@B15]\]. Vif exerts its inhibitory effect by binding to and inducing the degradation of APOGEC3G and also by blocking its translation \[[@B16],[@B17]\]. The APOBEC3G interacting domain is located within the N-terminal region that contains the FAM20-related pentapeptide although the specific involvement of this sequence has not yet been investigated \[[@B16]\]. Thus, this sequence within conserved region 2 may be a site for protein:protein interaction. However, APOBEC3G or related proteins are unlikely candidate binding partners as they are intracellular proteins. We also noted that one of the Drosophila FAM20 proteins (NM\_170079; CG31145) was identified as a protein that interacted with the Dynein light chain protein Dlc90f \[[@B18]\]. Dyneins are motor proteins involved in intracellular transport \[[@B19]\]. It appears unusual that a secreted protein would directly interact with a motor protein; therefore, this may represent a specific interaction of this particular family member in fruitfly. Evolution of the FAM20 gene family ---------------------------------- Tremendous progress has been made over the past decade in the sequencing of genomes from species at different positions on the evolutionary tree \[[@B20]-[@B25]\]. This vast amount of information can be used for comparative studies to elucidate the evolutionary origin of members of gene families. We have reported here the identification of orthologues of the FAM20 members in two insect species (D. melanogaster and A. gambiae), a simple chordate (C. intestinalis), three mammals (H. sapiens, M. musculus and R. norvegicus) and two fish (F. rubripes and D. rerio). In each case, the identification of these genes as bona fidetranscription units is supported either by direct experimental evidence for the human and mouse genes, or by the existence of multiple EST sequences in public databases. A single FAM20 gene was identified in C. intestinalis, which is considered to be a representative of a basal chordate related to the common ancestor of humans and other higher chordates \[[@B10]\]. The C. intestinalisgenome encodes approximately 16,000 genes and generally contains single representatives of superfamilies in higher vertebrates, thereby permitting the elucidation of likely evolutionary origins of genes within these families \[[@B9]\]. The C. intestinalis FAM20 protein clustered with the FAM20B subfamily members, as did the family members identified in invertebrate species. Therefore, we propose that the FAM20B subfamily contains the direct descendents of the ancestral FAM20 gene and that the FAM20A and FAM20C subfamilies result from duplication and subsequent evolution of this ancestral gene \[[@B26]\]. This pattern is consistent with the 2R hypothesis of genome evolution proposed by Ohno in 1970 \[[@B27]\]. In the FAM20 case, the loss of one gene at some stage of higher vertebrate evolution would need to be hypothesized to explain the final paralogue number of three in mammals. A single round of gene duplication appears to have occurred subsequent to the divergence of the nematode and insect lineages, giving rise to the two paralogues in fruit fly and mosquito that both cluster within the FAM20B subfamily. A further round of gene duplication has been proposed to have occurred in fish \[[@B28]\] and the existence of five to six FAM20 genes in pufferfish and zebrafish is consistent with this hypothesis. However, it is interesting that the expansion appears to have occurred exclusively in the FAM20C subfamily. This pattern could be explained either by two successive rounds of gene duplication of a small genomic region that included the original FAM20C representative, or by two rounds of duplication of larger genomic segments followed by gene conversion of FAM20A and FAM20B descendents (or parents) to yield four FAM20C members. Analysis of the genomic regions surrounding each of the FAM20C genes in fish will be necessary to distinguish between these two possibilities. Nevertheless, the expansion of the FAM20C subfamily in fish suggests that these proteins have acquired specific functions required within these species. FAM20 expression patterns in mammalian cells and tissues -------------------------------------------------------- Expression analysis of the three FAM20 family members in mammalian tissues and hematopoietic cells showed that FAM20A is expressed in a much more restricted pattern that the other two members. Importantly, Fam20a was also the only member to display obvious differential expression in hematopoietic cells undergoing myeloid differentiation. Fam20a mRNA levels were highest during intermediate stages of differentiation of EML cells, at a time when many cells are becoming committed to the myeloid lineages and undergoing extensive proliferation \[[@B4],[@B8],[@B29]\]. EML cell cultures also give rise to a small number of cells from B cell and erythroid lineages under these conditions but these cells do not survive after SCF and IL-3 is removed from the medium and replaced with GM-CSF. Thus, the decrease in Fam20a mRNA levels after the 72 hour timepoint could be interpreted in two ways. First, Fam20a may be expressed in one of the lineages that cannot survive in GM-CSF. Second, Fam20a may be expressed specifically in cells committed to the myeloid lineage and its expression may decrease during terminal granulocytic differentiation. The similarities in Fam20a expression patterns in EML cells after the 72 hour timepoint and in MPRO cells suggests that the second explanation is most likely. Therefore, we propose that Fam20a is primarily expressed in cells committed to the granulocytic lineage and presumably plays a role in either lineage commitment of cell proliferation. The hypothesis is currently under investigation using gene disruption techniques and through further characterization of the Fam20a protein. Methods ======= Cell culture and Representational Difference Analysis (RDA) ----------------------------------------------------------- EML and MPRO cells were cultured as described previously \[[@B6],[@B30]\]. Briefly, EML cells were cultured in Iscove\'s modified Dulbecco\'s medium containing 20% horse serum (Atlanta Biochemical, Norcross, GA) supplemented with 10% BHK-MKL conditioned medium (CM) as a source of stem cell factor (SCF). The cells were differentiated by adding 10% of WEHI-3 CM as a source of IL-3 and all trans retinoic acid (atRA, 1 × 10^-5^M, Sigma) for 72 hours. Terminal granulocytic differentiation was induced by removing IL-3 and SCF and adding 10% BHK-HM5 CM as a source of granulocyte/macrophage-colony stimulating factor (GM-CSF). MPRO cells were cultured in Dulbecco\'s modified Eagle\'s medium supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT) and 10% BHK-HM5 CM. Terminal differentiation was induced by adding 1 -- 10^-5^M atRA to the culture medium. For collection of RNA samples at different timepoints, 10 cm dishes were seeded with 150,000 cells and cultured under identical conditions. Cells were harvested from individual wells at 24 hour intervals for RNA preparation. Total RNA was prepared from isolated cells using a modified guanidium isothiocyanite/phenol extraction procedure as described previously \[[@B31]\]. Poly A^+^RNA prepared from EML cells at zero hours (stem cell stage) and 72 hours (promyelocyte stage) was converted to double stranded cDNA and three rounds of RDA was performed as described previously \[[@B6]\]. cDNA clones representing six putative differentially expressed genes were identified and named according to clone number. Initial characterization of these clones was described earlier \[[@B6],[@B8]\] and clone number 1623 was examined further in this study. Cos-1 cells were maintained in Dulbecco\'s modified Eagle\'s medium (DMEM, BioWhittaker, Walkersville, MD) containing 10% FBS and Penicillin/Streptomycin (BioWhittaker) under standard cell culture conditions. For transfections, cells were plated at a density of 2.5 × 10^4^per well in six well plates and transfected using the Effectene Transfection Reagent (Qiagen, Valencia, CA) under conditions recommended by the manufacturer. Isolation of a full length 1623 cDNA ------------------------------------ Analysis of the sequence of the original 1623 cDNA isolated from the RDA experiments revealed that it contained the coding sequence from the C-terminus of an uncharacterized protein. 5\' and 3\' rapid amplification of cDNA ends (RACE) was performed using Marathon-Ready cDNA from mouse spleen using the Advantage cDNA PCR kit (both from Clontech) as described previously \[[@B30]\]. The RACE reactions were performed using gene specific primers designed based on the original 1623 sequence or on sequences identified in early RACE reactions and adaptor primers provided with the cDNA. The 5\' RACE products were compared to sequences in public databases and identified a mouse cDNA (NM\_017565; aka DKFZp434F2322) that was identical to the extended 1623 sequence. PCR primers were designed that overlapped the putative initiation and stop codons of the ORF encoded by NM\_017565 and used to PCR amplify products from cDNAs prepared from undifferentiated MPRO cells. The product of this PCR reaction was subcloned and sequenced in its entirety. The sequence was identical to the original clone (which was isolated from mammary tissue) indicating that the same mRNA is expressed in both tissues. The full length clone (now named Fam20a) encoded a 541 amino acid protein. Sequence analysis ----------------- All basic sequence manipulations were performed using the VectorNTI suite of sequence analysis programs (Informax/Invitrogen Corp., Carlsbad, CA). Identification of FAM20 members from different species was initially achieved using standard BLAST searches at NCBI <http://www.ncbi.nlm.nih.gov/BLAST/>. Genome specific searches were performed either through NCBI or Ensembl <http://www.ensembl.org/> or through genome specific websites (Ciona intestinalis: <http://genome.jgi-psf.org/ciona4/ciona4.home.html>; Fugu rubripes: <http://aluminum.jgi-psf.org/prod/bin/blast.fugu3.cgi>). Searches were performed either as nucleotide-nucleotide (blastn) searches or as protein-translated nucleotide (tblastn) searches. In general, these searches were sufficient to identify gene regions encoding the most highly conserved regions of the proteins, particularly in the fish and Ciona genomes. To assemble complete genes, genomic regions containing the identified regions of similarity were downloaded into VectorNTI and searches were performed for individual exons, initially derived from mammalian genes but subsequently, from potential orthologues in fish or lower vertebrates, depending on the sequence being examined. Putative exons were examined for the presence of consensus splice donor and acceptor sites and complete sets of exons were assembled into cDNAs for translation and further comparisons. These results were compared against genes assembled by two gene prediction programs, FGENESH: <http://www.softberry.com/berry.phtml> and GENSCAN: <http://genes.mit.edu/GENSCAN.html>, however, these programs often made incorrect assignments that required manual assessment of the results. The protein sequences derived from each of the examined species were primarily used in the comparisons to define subfamily assignments and alignments were performed in the AlignX program in VectorNTI using the blosum62 scoring matrix. Protein domain searches were performed using the Simple Modular Architecture Research Tool (SMART) <http://smart.embl-heidelberg.de/> and/or Profilescan <http://hits.isb-sib.ch/cgi-bin/PFSCAN>. Signal sequence searches were performed using the SignalP analysis program <http://www.cbs.dtu.dk/services/SignalP/>\[[@B32]\]. Sequences derived by annotation of genome sequences in this study have been submitted to the Third Party Annotation database at NCBI under accession numbers BK001515 to BK001522. The FAM20 family name has been approved by the Human Genome Organization nomenclature committee. Plasmid construction -------------------- The Fam20a and Fam20aΔ23 were constructed in the pEGFP-N1 vector by PCR cloning. XhoI and HindIII restriction sites were engineered into the PCR primers (Fam20a: 5\'GGCCTCGAGGCCATGCCCGGGCTGCGCAGG3\', 5\'GGCAAGCTTGCTCGTCAGATTAGCCTG3\'; Fam20aΔ23: 5\'GGCCTCGAGGCCATGTACTTCCACCTCTGGCCG3\' and reverse primer was as above). Similar strategy was used to clone Fam20a and Fam20aΔ23 in the pcDNA3.1 vector (Forward primers were same as above; Fam20a and Fam20aΔ23 reverse primers: 5\'GGCAAGCTTGGGCTCGTCAGATTAGCCTG3\'). The Fam20a(SSmut) was generated using a PCR-based site-directed mutagenesis kit (Quickchange, Stratagene). All of the sequences were verified by sequencing using an ABI automated sequencer. Western blotting ---------------- Whole cell extracts were prepared from transfected COS-1 cells by lysing directly in 2X Laemmli Sample Buffer as described previously \[[@B31]\]. Purified His-tagged proteins were prepared as described below and mixed with 2X Laemmli Sample Buffer (120 mM Tris-HCl (pH 6.8), 10% Glycerol, 3.3% SDS, 0.2 M dithiothreitol, 0.004% Bromophenol Blue). Equal amounts of total cellular and purified secreted proteins were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes (Micron Separations, Westborough, MA). Membranes were blocked in 5% non-fat milk in TBST (100 mM Tris-HCl (pH 7.5), 0.9% NaCl, 0.1% Tween-20) for 1 hour. Myc-tagged Fam20a proteins were detected using an anti-myc mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Horseradish peroxidase-conjugated donkey anti-mouse antibody (Promega, Madison, WI) was used for the secondary antibody. The immune complexes were detected using SuperSignal chemiluminescence detection kit (Pierce, Rockford, IL). In vitro transcribed and translated Fam20a was generated using the TnT T7 coupled reticulocyte lysate system (Promega) as described previously \[[@B33]\]. Purification of His-tagged proteins from cell media --------------------------------------------------- COS-1 cells were transfected as described above and media were collected after 48 hours and mixed with freshly made 10X binding buffer (500 mM NaH~2~PO~4~-H~2~O (pH 8.0), 150 mM NaCl, 10 mM Imidazole). His-tagged proteins were purified using Ni-NTA Magnetic Agarose Beads (Qiagen, Valencia, CA) according to manufacturer\'s recommendations. Following the purification step they were either used directly in western blot analysis or for deglycosylation experiments. Purified proteins were subjected to enzymatic deglycosylation using the GlycoPro deglycosylation kit (ProZyme, San Leandro, CA) following the manufacturer\'s protocol. Immunofluorescence ------------------ COS-1 cells were plated on glass coverslips and transfected with the indicated constructs the following day. After 48 hours of incubation, the cells were fixed in 1% formaldehyde for 30 minutes at room temperature. Coverslips were incubated in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na~2~HPO~4~(pH 7.3)) containing 1% Triton X-100 for 10 minutes to permeabilize the cell membranes. They were then transferred to PBST (PBS containing 0.1% Tween-20) and incubated for 30 minutes. Mouse monoclonal anti-Myc antiserum (Santa Cruz) was used at 1:2,000 dilution in PBST containing 1% bovine serum albumin (1 hour at room temperature). Coverslips were then washed with PBST three times and incubated with the secondary antibody (Alexa Fluor 488 \[Molecular Probes, Eugene, OR\]) for 1 hour. Coverslips were washed three times in PBST and once in water for 15 minutes each, and were mounted on glass slides. The cells were observed by epifluorescence microscopy using an Axiovert 135 TV microscope (Zeiss, Gottingen, Germany). Images were captured with a Kodak DC290 zoom camera and analyzed with MetaMorph 6.0 (Universal Imaging Corporation, Downingtown, PA). Reverse transcription-polymerase chain reaction ----------------------------------------------- Total RNA was purified as described above from EML and MPRO cells at 24 hour timepoints during the differentiation process of each cell line The primers were designed using the Vector NTI sequence analysis suite of programs (InforMax, Frederick, MD) and were follows: MmFam20a forward 5\'-catagaggcccacggcgagcg-3\' and reverse 5\'-atggaatggggcaacag gggc-3\'; Mmfam20b forward 5\'-tggacaggattctgggtttc-3\' and reverse 5\'-ccagggatgtcgatgtttct-3\'; MmFam20c forward 5\'-agcagacgagagagcaggag-3\' and reverse 5\'-cggatctccttggtcatgtt-3\'; HsFAM20a forward 5\'-ctggcaggaaaagagtg-3\' and 5\'-cccgaacttggtgaacatct-3\', HsFAM20b forward 5\'-ccctgaagagacaccagaagagc-3\' and reverse 5\'-gaaacccagaatcctgtcca-3\', HsFAM20c forward 5\'-ggctcacgttccacattggt-3\' and reverse 5\'-aaagtcagggggtgtctcct-3\'.; mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward 5\'-aatggtgaaggtcggtg tgaac-3\' and reverse 5\'-gaagatggtgatgggcttcc-3\'; human (GAPDH) forward 5\'-tgaaggtcggagtcaacggatttggt-3\' and reverse 5\'-catgtgggccatgaggtccaccac-3\'. GAPDH was used as control for cDNA integrity. Single stranded cDNA was reverse transcribed from 2 μg of total RNA using 400 Units of Superscript RNase H^-^Reverse Transcriptase (Invitrogen, Carlsbad, CA), 0.125 mM dNTPs, 10 mM DTT, and 1 μM oligo (dT)~15~in a total volume of 50 μl for 1 hour at 37°C. 2 ul of the RT reaction was then mixed with 1 ul of 10X PCR Buffer (500 mM Tris-HCl (pH8.3), 2.5 mg/ml crystalline BSA and MgCl~2~at 10, 20 or 30 mM (Idaho Technology Inc., Idaho Falls, ID), 0.2 mM each dNTP, 0.05 μM of each primer and 1.25 Units Taq DNA polymerase (Fisher Scientific, Pittsburgh, PA) in a total volume of 10 μl. Reactions were loaded into a capillary tube and PCR cycles were carried out using the Rapidcycler Thermal cycler (Idaho Technology). Annealing temperatures and Mg^2+^concentrations were initially optimized for each primer. A commercial set of tissue cDNAs, (Human Multiple Tissue cDNA (MTC) Panels, Clontech, Palo Alto, CA) was used for the RT-PCR analysis of human FAM20 family members. List of Abbreviations ===================== FAM20: family with sequence similarity 20; EML: Erythroid, myeloid, lymphoid cell line; MPRO: mouse promyelocyte cell line; GM-CSF: granulocyte/macrophage-colony stimulating factor; PHSC: pluripotent hematopoietic stem cell; HGF: hematopoietic growth factor; RAR: retinoic acid receptor; atRA: all trans retinoic acid; SCF: stem cell factor; IL-3: interleukin-3; RDA: representational difference analysis; RACE: rapid amplification of cDNA ends; CCD: conserved C-terminal domain; GFP: green fluorescent protein; CM: conditioned medium. Author Contributions ==================== DN isolated full length Fam20a cDNA, performed transfection experiments and drafted the manuscript. HY performed RT-PCR assays. IN assisted in construction of expression vectors. SS performed genomics analyses. EC and EGB provided reagents and assistance in performing RT-PCR assays. YD performed the original representational difference analysis and SCW directed the project and performed genomics analyses. SCW also wrote the final draft of the manuscript and all authors read and approved this version. Acknowledgements ================ This work was supported by grants from The [CH]{.underline} Foundation and Texas Tech University Health Sciences Center. SCW is an Established Investigator of the American Heart Association. We appreciate the assistance of Susan San Francisco and Ruwantha Wettisinghe at the Center for Biotechnology and Genomics at Texas Tech University in sequencing and sequence analysis. We also thank Dan Hardy for critical reading of the manuscript and Patricia Frisbie for technical and editorial assistance.	PubMed Central	2024-06-05T03:55:52.807587	2005-1-27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548683/", "journal": "BMC Genomics. 2005 Jan 27; 6:11", "authors": [ { "first": "Demet", "last": "Nalbant" }, { "first": "Hyewon", "last": "Youn" }, { "first": "S Isil", "last": "Nalbant" }, { "first": "Savitha", "last": "Sharma" }, { "first": "Everardo", "last": "Cobos" }, { "first": "Elmus G", "last": "Beale" }, { "first": "Yang", "last": "Du" }, { "first": "Simon C", "last": "Williams" } ] }
PMC548684	Background ========== In germinal centers (GC), B cells undergo clonal expansion, somatic hyper-mutation in the variable region of antibody genes \[[@B1]-[@B3]\] and class switch recombination (CSR) from IgM to IgG, IgA, and IgE \[[@B4]-[@B8]\], processes that are dependent on helper T cells \[[@B9]-[@B11]\]. Antibodies to the CD57 epitope (HNK-1) have been used to identify a T cell type in germinal centers in human tonsils, spleen and lymph nodes. These cells are CD4^+^T cells \[[@B12]-[@B14]\], exhibit a memory phenotype (CD45RO^+^CD45RA^-^) \[[@B15]\] and are not cytolytic \[[@B16]\]. CD57^+^GC-Th cells proliferate only when they are TCR-activated in the presence of IL-2 \[[@B17],[@B18]\]. CD57^+^GC-Th cells express the B-cell zone homing chemokine receptor CXCR5 but not the T cell zone homing chemokine receptor CCR7, a pattern consistent with their specific localization in GC \[[@B19]\]. Based upon their non-polarized cytokine profile, localization in GC and potential helper activity, it has been proposed that CD57^+^GC-Th cells may constitute a novel effector T cell subset distinct from other well known effector T cell subsets such as Th1 and Th2 cells \[[@B20]\]. Using a gene expression profiling study, we determined that CD57^+^GC-Th cells are remotely related to other memory/effector T cells in global gene expression \[[@B21]\]. The microarray study also revealed that CD57^+^GC-Th cells have the unique capacity to produce CXCL13, a follicle chemokine implicated in recruitment of CXCR5^+^cells \[[@B22],[@B23]\] and development of follicles/GCs \[[@B24]\]. Because of their specific localization in germinal centers, the activities of CD57^+^GC-Th cells on B cell proliferation and antibody production have been studied by several groups of scientists \[[@B19],[@B25]-[@B27]\]. The results of these previous studies reveled unique features of CD57^+^GC-Th cells, but, when combined, they are inconclusive and widely vary from negative to neutral or positive in assessing the helper activities of CD57^+^GC-Th cells. To clarify and gain more insight into their function in helping B cells, we systematically investigated the capacity of human tonsil CD57^+^GC-Th cells in inducing B cell Ig synthesis in naïve vs. germinal center B (GC-B) cells in comparison with other T cell subsets in human tonsils. We show that CD57^+^GC-Th cells are more efficient than other germinal center or interfollicular T cells in supporting B cell production of Ig. CD57^+^GC-Th cells, when compared to other T cells, have better helper activity for GC-B cells than for naïve B cells. CD57^+^GC-Th cells induced the expression of activation-induced cytosine deaminase (AID) and CSR in developing B cells. CD40L, but not other major cytokines, is critical for the helper activity of CD57^+^GC-Th cells. IL-10 positively and TGF-β1 negatively regulate the helper activity of CD57^+^GC-Th cells. Results ======= Distribution and identification of T helper cell subsets in tonsils ------------------------------------------------------------------- We examined the distribution of T helper cell subsets in human tonsils based upon the expression of CD4, CD57 and CD69. As reported previously \[[@B12],[@B19],[@B28],[@B29]\], most CD57^+^CD4^+^T cells are located in germinal centers surrounded by IgD^+^naïve B cells (Fig. [1](#F1){ref-type="fig"}). Small numbers of CD57^+^T cells were also present in the interfollicular areas (IFA or T cell-rich zone) surrounding GC. Although some are found in IFA, CD69^+^CD4^+^T cells were also preferentially found in GC (Figure [1C](#F1){ref-type="fig"}). In contrast, the T cells in IFA were mostly negative for CD69 expression. Therefore, CD57, CD69 and CD4 are useful markers to identify CD57^+^GC-Th cells and other T cell subsets differentially localized in tonsils: CD4^+^CD57^+^cells (mainly in GC), CD4^+^CD57^-^CD69^+^cells (mainly in GC and a minor proportion in IFA), and CD4^+^CD57^-^CD69^-^cells (mainly in IFA). CD57^+^GC-Th cells are highly efficient in supporting Ig production by B cells ------------------------------------------------------------------------------ Based upon the information obtained in Figure [1](#F1){ref-type="fig"}, the total tonsil CD4^+^T cell population was fractionated into CD57^+^GC-Th cells (all of these cells are CD69^+^), total CD57^-^T cells, CD57^-^CD69^+^T cells and CD57^-^CD69^-^T cells (Figure [2](#F2){ref-type="fig"}). We compared the B cell helping activity of CD57^+^GC-Th cells with that of other CD4^+^T cell subsets. We co-cultured each of the isolated T cell subsets with syngeneic tonsil CD19^+^B cells in the presence of SEB, a superantigen that conjugates MHC class II molecules and TCR (Figure [3](#F3){ref-type="fig"}). B cell receptors were cross-linked by Ab to human Ig μ chain and human Ig (H + L) chain prior to culture to provide BCR activation signals mimicking the antigen signals in vivo. CD57^+^GC-Th cells were most efficient in inducing B cell production of IgM, IgG, IgA and IgE among the T cell subsets examined. CD57^-^CD69^+^T cells, many of which are located in GC in a manner similar to CD57^+^GC-Th cells, were able to induce the production of antibodies but at significantly lower levels compared to CD57^+^GC-Th cells (Figure [3A](#F3){ref-type="fig"}). T cell stimulation, in this study by SEB, was required for efficient induction of the B cell helper activity as it enhanced Ig production up to \~1000 (not shown). GC-B cells are the preferred target cells for the helper activity of CD57^+^GC-Th cells --------------------------------------------------------------------------------------- Because of their specific localization in germinal centers, the physiological target cells for CD57^+^GC-Th cells would be GC-B cells rather than naïve B cells. We compared the helper activities of CD57^+^GC-Th cells and CD57^-^CD69^+/-^CD4^+^T cell subsets for B cells. In this study, we fractionated CD19^+^B cells into two groups: IgD^+^CD38^-^naïve B and CD38^+^IgD^+/-^GC B cells as shown in Figure [2B](#F2){ref-type="fig"}. CD57^+^GC-Th cells, when co-cultured with GC-B cells, were significantly more efficient than CD57^-^CD69^+^T cells in inducing the production of all four isotypes of Ig (Figure [3C](#F3){ref-type="fig"}). However, when co-cultured with naive B cells, CD57^+^GC-Th cells were not significantly different from CD57^-^CD69^+^T cells in their induction capacity of Ig (Figure [3B](#F3){ref-type="fig"}). Again, the helper activities of total CD57^-^T cells and CD57^-^CD69^-^T cells for naïve and GC-B cells were very low. The relative composition of IgM, IgG, IgA and IgE produced in response to CD57^+^GC-Th cells in the cultures with GC-B vs. naïve B cells was determined. CD57^+^GC-Th cells drove the production of IgM, IgG, IgA and IgE in descending order (Figure [3D](#F3){ref-type="fig"}). Class-switched Ig isotypes such as IgG and IgA were more produced in GC-B cell cultures than in naïve B cell cultures. There was no statistically significant difference between the two T cell subsets (CD57^+^GC-Th cells and CD4^+^CD57^-^CD69^+^T cells) in the composition of Ig that they induced. CD57^+^GC-Th cells induce AID expression and class switch recombination in B cells ---------------------------------------------------------------------------------- AID expression in the maturating B cells in GC is necessary for CSR and somatic hypermutation. We examined whether CD57^+^GC-Th cells have the capacity to induce AID in B cells. Naïve B cells were co-cultured with CD57^+^GC-Th cells, and AID expression was examined (Figure [4A](#F4){ref-type="fig"}). CD57^+^GC-Th cells induced AID in activated B cells with peaks around days 3--4. CD57^+^GC-Th cells were able to induce the expression of productive V~H~DJ~H~-C~H~Ig transcripts. The major subtypes of Ig transcripts in response to CD57^+^GC-Th cells were IgG3, IgG2, IgG1 and IgA1 (Figure [4B](#F4){ref-type="fig"}). When the peak expression levels of AID and the productive V~H~DJ~H~-C~H~IgG3 transcript (the most readily detected Ig transcript) were compared, AID expression preceded the expression of IgG3 transcript by 1--2 days in culture (Figure [4C](#F4){ref-type="fig"}). Ig class switch recombination between tandemly repeated S regions located 5\' to each C~H~gene generates switch circles. We used a nested PCR technique designed to specifically detect switch circles but not genomic Ig sequences. Freshly isolated GC-B, but not naïve B cells, contained switch circles, which were detected as smeared multiple bands on agarose gels as expected. Naïve B cells cultured with CD57^+^GC-Th cells generated detectable switch circles in a time-dependent manner (Figure [4D](#F4){ref-type="fig"}). We also used a DC-PCR technique \[[@B30]\] to detect γ3 and α1/2 switch circles (Figure [4E](#F4){ref-type="fig"}). Again, GC-Th cells induced switch circles in the naïve B cells cultured with GC-Th cells. CD40L signal is necessary for, while cytokines modulate, the helper activity of CD57^+^GC-Th cells -------------------------------------------------------------------------------------------------- Cytokines and CD40L regulate B cell maturation and Ig production. We examined whether CD40L, IL-4, IL-10 and IFN-γ play any roles in the CD57^+^GC-Th cell-driven B cell production of Ig. In cultures with naïve B cells, the blockage of CD40L by neutralizing antibody completely suppressed the helper activity of CD57^+^GC-Th cells in inducing the B cell production of IgM, IgG1, IgA and IgE (Figure [5A](#F5){ref-type="fig"}). In this case, IgG1 was measured instead of total IgG to avoid cross-reaction of the polyclonal capturing antibody for IgG with the neutralizing antibodies to cytokines. Blockage of IL-4 partially but specifically suppressed the production of IgE, but it did not significantly suppressed other isotypes. In contrast, blockage of IFN-γ enhanced the production of IgM, IgG1 and IgA but not IgE. Since CD40L is essential for the helper activity of CD57^+^GC-Th cells, we examined CD57^+^GC-Th cells and other T cells for the expression of surface CD40L. Freshly isolated CD57^+^GC-Th cells expressed CD40L, which became up-regulated upon T cell activation within hours (data not shown), whereas CD4^+^CD57^-^CD69^-^interfollicular T cells did not express CD40L at significant levels. There was no significant difference in the expression of surface CD40L between CD57^+^and CD57^-^CD69^+^cells. In the cultures with GC-B cells, blocking of CD40L again completely suppressed the B cell helping activity of CD57^+^GC-Th cells (Figure [5B](#F5){ref-type="fig"}). However, IL-4 neutralization did not significantly affect the IgE production induced by CD57^+^GC-Th cells, an activity different from that for naïve B cells. For GC-B cells, IFN-γ neutralization significantly increased the production of IgA as it did for naive B cells. The effects of IFN-γ neutralization on other Ig isotypes were smaller. While a slight decrease of IgE production in the cultures of GC-B cells and CD57^+^GC-Th cells was observed, neutralization of endogenous IL-10 did not have any statistically significant effect on CD57^+^GC-Th cell-driven Ig production in the cultures of either naïve or GC-B cells. Exogenously-added IL-10 enhances while TGF-β1 completely suppresses the B cell helping activity of CD57^+^GC-Th cells --------------------------------------------------------------------------------------------------------------------- To further examine the regulatory effect of cytokines, IL-4, IL-10, IFN-γ and TGF-β1 were exogenously added to the cultures of CD57^+^GC-Th cells with B cells (Figure [5C](#F5){ref-type="fig"} and [5D](#F5){ref-type="fig"}). In cultures of CD57^+^GC-Th cells with naïve B cells, exogenously added IL-4 enhanced the production of some subsets of Ig, but this effect was small and not statistically significant (Figure [5C](#F5){ref-type="fig"}). However exogenously added IFN-γ significantly suppressed the production of IgG, IgA and IgE. IL-10, when added exogenously, was highly efficient in enhancing the production of the four subsets of Ig. TGF-β1 completely suppressed the B cell-helping capacity of CD57^+^GC-Th cells for naive B cells. In cultures of CD57^+^GC-Th cells with GC-B cells, IL-10 was again highly effective in enhancing the helper activity of CD57^+^GC-Th cells, while TGF-β1 completely suppressed it (Figure [5D](#F5){ref-type="fig"}). IFN-γ partially but significantly suppressed the production of IgM, IgG, IgA and IgE. Exogenous IL-4 added to the cultures had no effect on the CD57^+^GC-Th cell-driven Ig production in this condition (Figure [5D](#F5){ref-type="fig"}), which is in line with the negligible effect of anti-IL-4 on GC-B cells in Figure [5B](#F5){ref-type="fig"}. Discussion ========== CD57^+^GC-Th cells are unique CD4^+^T cells. They express the follicle homing receptor CXCR5 but lack the T cell area localization receptor CCR7 \[[@B19]\], and reside specifically in germinal centers \[[@B12]-[@B14]\]. CD57^+^GC-Th cells proliferate only when appropriate signals such as TCR, CD28 and IL-2 are provided \[[@B17],[@B18]\]. GC-Th cells are widely disseminated and diverse in their TCR sequence \[[@B31]\]. CD57^+^GC-Th cells can express CD40L, ICOS and CXCL13 but are non-polarized T cells in their cytokine profile \[[@B21]\]. It has been controversial and unclear whether CD57^+^GC-Th cells are intrinsically more efficient in helping B cells than other T cells or they are simply localized in germinal centers without any significant differences from other T cells in their capacity as helpers. In this report, we systematically investigated the effector function of CD57^+^GC-Th cells in regulation of B cell immunoglobulin production and its regulation. When compared for their helper activities in inducing Ig synthesis by total B cells, CD57^+^GC-Th cells were most efficient among the T cell subsets in tonsils. CD57^+^GC-Th cells were particularly more efficient in their helper activity for GC-B cells vs. naïve B cells. CD57^-^CD69^+^T cells were equally efficient to CD57^+^GC-Th cells in inducing naïve B cell differentiation for Ig production, while they were less effective than CD57^+^GC-Th cells in helping GC-B cells. This preference of CD57^+^GC-Th cells for GC-B cells is physiologically relevant, since both the helper T cell subset and target B cells are specifically present in germinal centers. Therefore, CD57^+^GC-Th cells would constitute an ideal T helper subset that can drive GC-B cell differentiation in germinal centers. The effects of cytokines such as IL-4, IL-10, IFN-γ and CD40L on B cells in humans and mice have been well documented. It is considered that CD40L is a critical factor \[[@B4],[@B11],[@B32]-[@B37]\], and IL-4 and IL-10 are positive factors in regulation of B cell Ig production \[[@B38]-[@B44]\]. IFN-γ induces class switch to certain isotypes while it inhibits to others \[[@B45],[@B46]\]. In this study of the helper activity of CD57^+^GC-Th cells, the positive role of IL-4 in promoting Ig production was valid only for IgE, but not IgG and IgA in the cultures of naïve B cells with CD57^+^GC-Th cells (Figure [5](#F5){ref-type="fig"}). GC-B cells were even more resistant to the neutralization of IL-4 than naïve B cells in CD57^+^GC-Th-cell driven Ig production. This smaller than expected effect of IL-4 may be due to the fact that there is not much IL-4 to neutralize in the cultures of GC-Th cells. This also suggests that GC-Th cells may provide helper signals to GC-B cells that are not significantly affected by IL-4. AID \[[@B47]\] is a molecule essential for somatic hypermutation, CSR and Ig gene conversion \[[@B48]-[@B54]\]. We showed in this study that CD57^+^GC-Th cells can induce AID expression (Figure [4A](#F4){ref-type="fig"}). This capacity is consistent with their ability to induce class switch recombination, which can be detected within a few days in the cultures of naïve B cells with CD57^+^GC-Th cells. CD57^+^GC-Th cells can induce the expression of productive IgG1-3 and IgA1 transcripts. However, CD57^+^GC-Th cells were inefficient in induction of IgE (Figure [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"} and [5](#F5){ref-type="fig"}), which is consistent with their poor production capacity of IL-4 \[[@B19]\]. CD40L appears to be essential for the helper activity of CD57^+^GC-Th cells. CD40L was required for the synthesis of all Ig isotypes in all the conditions tested regardless of whether the target B cells for CD57^+^GC-Th cells were naïve or GC-B cells. While neutralization of IL-10 did not have any significant effect on the CD57^+^GC-Th cell-driven Ig synthesis, exogenous IL-10 was highly effective in enhancing the Ig synthesis in our study. This could be due to insufficient neutralization of the IL-10 produced by CD57^+^GC-Th cells, which are known to produce IL-10 upon TCR activation \[[@B19]\]. Another possibility is that higher concentration of IL-10 than the level produced by CD57^+^GC-Th cells may be necessary to significantly enhance the Ig response. Exogenous IFN-γ negatively regulates the CD57^+^GC-Th cell-driven Ig synthesis, suggesting the potential roles of Th1 cells or other IFN-γ producing cells in regulation of the CD57^+^GC-Th cells\' helper activity. TGF-β1 plays dual roles: it is a switch factor for IgA and a potent immunosuppressive cytokine that inhibits Ig synthesis \[[@B55]\]. We did not detect any switching effect but were able to detect its suppressive activity for the CD57^+^GC-Th cell response. This could be due to the fact that the culture conditions (e.g. the saturating concentration of TGF-β) employed in our study appear to favor the detection of the suppressive function of TGF-β. Taken together, these results imply that Th1, Th2 and regulatory T cells, if present in germinal centers, could positively or negatively control the function of CD57^+^GC-Th cells in regulation of humoral immune responses. Indeed, there are regulatory T cells in GCs that express surface TGF-β and can effectively suppress the function of CD57^+^GC-Th cells \[[@B56]\]. Conclusions =========== Our results demonstrated the capacity of CD57^+^GC-Th cells in supporting CSR and Ig synthesis in B cells, and revealed the factors that regulate their activity, thereby substantiating the so-far inconclusive function of CD57^+^GC-Th cells. The fact that these T cells have preferential and efficient helper activity for GC-B cells and are specifically localized in GCs in large numbers suggests that CD57^+^GC-Th cells are probably the major T helper subset responsible for supporting B cell differentiation for Ig production in germinal centers. Methods ======= Cell isolation -------------- Mononuclear cells were prepared by density gradient centrifuge on histopaque 1077 (Sigma-Aldrich, St. Louis) from human tonsil pathological specimens obtained from young patients (3--10 yr) undergoing tonsillectomy to relieve obstruction of respiratory passages and improve drainage of the middle ear at Sagamore Surgical Center (Lafayette, IN). The use of human pathological specimens in this study was approved by the institutional review board at Purdue University. CD4^+^T cells (purity \>97%) were isolated by depleting non-CD4^+^T cells using a magnetic bead depletion method (Miltenyi Biotec, Auburn, CA). After staining of the isolated CD4^+^T cells with appropriate antibodies, CD57^+^GC-Th cells (purity \>95%) were isolated by a positive magnetic selection method (Miltenyi Biotec). CD4^+^CD57^-^CD69^+^and CD4^+^CD57^-^CD69^-^T cell subsets (purity \>95%) were further isolated from the CD57^-^T cell fraction by magnetically selecting CD69^+^T cells (Miltenyi Biotec). Total B cells were isolated by rosetting with 2-amino-ethylisothiouronium bromide (AET)-treated sheep red blood cells followed by CD4^+^T cell depletion (CD19^+^cells \> 99.5%). Naïve B cells (CD19^+^IgD^+^cells \>99%) were isolated from the total B cell fraction by depleting CD38^+^T cells followed by positive magnetic selection of IgD^+^B cells. CD19^+^CD38^+^IgD^+/-^GC-B cells (purity \>95%) were isolated from the tonsil CD19^+^B cells as described before \[[@B57]\] using anti-CD44, anti-IgD antibodies and pan-mouse IgG beads (Dynal, Brown Deer, WI). Cell culture ------------ All cell cultures were performed in RPMI1640 medium supplemented with 10% FBS, gentamycin, streptomycin, and penicillin. To cross-link the B cell receptors, isolated B cells were incubated for 2 h at 4°C with Sepharose-conjugated rabbit Ab to human Ig μ chain and human Ig (H + L) chain (Irvine Scientific, Santa Ana, CA; mixed 1:1 at 2 μg/ml), and then washed with cold PBS. 10^5^T and 10^5^B cells were co-cultured, unless indicated otherwise, in each well of 48-well plates in the presence of Staphylococcal enterotoxin B (SEB; 1 μg/ml, Sigma-Aldrich, St. Louis, MO). Cells were incubated in 5% CO^2^incubators at 37°C for 3--8 days. Recombinant IL-4, IL-10, and TGF-β1 were purchased from R&D systems (Minneapolis, MN). Recombinant IFN-γ was obtained from BD Pharmingen (San Diego, CA). Purified CD154-blocking antibody (24--31) was obtained from Ancell Corporation (Bayport, MN). IL-4-blocking antibody (MP4-25D2) was purchased from BD Pharmingen. Blocking antibodies for IFN-γ (25718.111) and IL-10 (23738.111), and IgG1 isotype control antibody (11711.11) were purchased from R&D systems. All antibodies and reagents added to culture were azide-free. Cytokines were added at saturating concentrations: IL-4 (40 ng/ml), IL-10 (40 ng/ml), IFN-γ (200 ng/ml) and TGF-β1 (10 ng/ml). Neutralizing antibodies were added at following concentrations: anti-CD40L (20 μg/ml), anti-IL-4 (5 μg/ml), anti-IL-10 (5 μg/ml), anti-IFN-γ (2.5 μg/ml) and isotype antibody (5 μg/ml). Flow cytometry analysis ----------------------- T cells were stained with anti-human CD57 (NK-1; FITC, BD Pharmingen), anti-human CD69 (FN-50; FITC, BD Pharmingen), anti-human CD4 (S3.5; R-PE, Caltag Laboratories, Burlingame, CA), and anti-human CD3 (UCHT1; APC, BioLegend, San Diego, CA). B cells were stained with anti-CD19 (4G7; PerCP, BD Pharmingen), anti-human IgD (IAb-2, FITC, BD Pharmingen), anti-human CD38 (HTT2; R-PE, BD Pharmingen), and anti-human CD3 (UCHT1; APC, BioLegend). Stained cells were analyzed using a 4-color FACSCalibur™ (BD Biosciences). In situ fluorescent immunohistochemistry ---------------------------------------- Frozen sections of tonsils were acetone-fixed and stained using antibodies to CD57 (BD Biosciences -- Pharmingen; clone NK-1, labeled with FITC), CD69 (BD Biosciences -- Pharmingen; clone FN50, labeled with FITC), IgD (BD Biosciences -- Pharmingen; clone IA6-2, labeled with PE) and/or CD4 (Caltag Laboratories; clone S3.5, labeled with APC). Stained sections were analyzed using a confocal microscopy system (Bio-Rad MRC 1024UV and Nikon Diaphot 300 microscope) at Purdue Cytometry Lab. ELISA ----- Culture supernatants were assayed by ELISA as previously described \[[@B19]\]. The sensitivity of this ELISA system is greater than 5 ng/ml, 300 pg/ml, 30 pg/ml, 600 pg/ml, and 15 pg/ml for IgM, IgG, IgG1, IgA and IgE, respectively. Detection of productive V~H~DJ~H~-C~H~Ig transcripts and reciprocal DNA recombination products ---------------------------------------------------------------------------------------------- Total RNA was extracted from cultured cells with Trizol reagent (Invitrogen, Carlsbad, CA), and was reverse-transcribed into cDNAs with SuperScript™ First-Strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer\'s protocol. The primer pairs used in this study were designed by Cerutti et al. \[[@B37]\]: IgM, FR3 forward (5\'-GAC ACG GCT GTG TAT TAC TGT GCG-3\') and Cμ reverse (5\'-CCG AAT TCA GAC GAG GGG GAA AAG GGT T-3\'); IgG1, FR3 forward and Cγ1 reverse (5\'-GTT TTG TCA CAA GAT TTG GGC TC-3\'); IgG2, FR3 forward and Cγ2 reverse (5\'-GTG GGC ACT CGA CAC AAC ATT TGC G-3\'); IgG3, FR3 forward and Cγ3 reverse (5\'-TTG TGT CAC CAA GTG GGG TTT TGA GC-3\'); IgG4, FR3 forward and Cγ4 reverse (5\'-ATG GGC ATG GGG GAC CAT TTG GA-3\'); IgA1, FR3 forward and Cα1 reverse (5\'-GGG TGG CGG TTA GCG GGG TCT TGG-3\'); IgA2, FR3 forward and Cα2 reverse (5\'-TGT TGG CGG TTA GTG GGG TCT TGC A-3\'); IgE, FR3 forward and Cε reverse (5\'-CGG AGG TGG CAT TGG AGG-3\'); human β-actin, actin forward (5\'-ATG TTT GAG ACC TTC AAC AC-3\') and actin reverse (5\'-CAC GTC ACA CTT CAT GAT GG-3\'). PCR reactions were performed on serially diluted cDNA samples using an Eppendorf master cycler (denaturation at 95°C for 15 s, annealing at 55°C for 45 s and extension at 72°C for 30°C; 30--35 cycles). Extrachromosomal switch circles were detected by a nested PCR strategy as previously described by others \[[@B37]\]. Briefly, genomic/extrachromosomal DNA was isolated from fresh or cultured B cells using a QIAamp DNA Mini Kit (Qiagen, Valencia, CA) and was used as templates for amplification of Sγ1-Sμ, Sγ2-Sμ, Sγ3-Sμ, Sγ4-Sμ and Sα-Sγ. The PCR products were subject to second PCR using internal forward 5\' Sγ or 5\'Iα1/2i and reverse 3\'Sμi or 3\'γi primer pairs. This method has been verified for specificity using positive controls \[[@B37]\]. Additionally, we amplified genomic β-actin gene as a control using 5\'-GTA CCA CTG GCA TCG TGA TGG ACT-3\' (G-actin-forward-1 primer) and 5\'-ATC CAC ACG GAG TAC TTG CGC TCA-3\' (G-actin-reverse-1) for the first PCR; and 5\'-AGA AGA GCT ACG AGC TGC CTG AC-3\' (G-actin-forward-2) and 5\'-TGA GGA CCC TGG ATG TGA CAG CT-3\' (G-actin-reverse-2) for the second PCR. Additionally, we used a DC-PCR technique \[[@B30],[@B58]\] to demonstrate the presence of switch circles (γ3 and α1/2) in human B cells. Please see the reference \[[@B30]\] for primer sequences. RT-PCR analysis for AID expression ---------------------------------- Total RNA was extracted from freshly isolated or cultured cells using Trizol reagent (Invitrogen, Carlsbad, CA), and was reverse-transcribed into cDNAs with SuperScript™ II Reverse Transcriptase. RT-PCR amplification of AID was performed using the two primers: AID-forward (5\'-GAT GAA CCG GAG GAA GTT TC-3\') and AID-reverse (5\'-TCA GCC TTG CGG TCC TCA CAG-3\'), which generated a specific 351 bp PCR product after 30 cycles of PCR reaction (30 s at 94°C, 30 s at 60°C, and 60 s at 72°C). β-actin was also amplified as a control. Statistical analysis -------------------- Student\'s paired t-test was used. P values smaller than 0.05 were considered significant. List of abbreviations used ========================== GC, germinal center; GC-Th cells, germinal center T helper cells; GC-B cells, germinal center B cells; AID, activation-induced cytosine deaminase; CSR, class switch recombination; SEB, staphylococcal enterotoxin B. Authors\' contributions ======================= CHK conceived, coordinated the study, analyzed the results and wrote the text. JRK, HWL and SGK participated in experiments, data analysis, making figures and proofreading the manuscript. PH provided specimens and helped perform the study. All authors read and approved the manuscript. Acknowledgments =============== We thank Dr. Meenakshi Roy (UCLA) and Dr. Harm HogenEsch (Purdue University) for their help/advice, and thank Nancy Petretic for her excellent assistance in flow analysis. This study has been supported from grants from the Leukemia and Lymphoma Society, the Leukemia Research Foundation, and the American Cancer Society (\#IRG-58-006-44) to CHK. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Distribution of CD4^+^T helper cells in tonsils. Frozen tonsil sections were stained with anti-IgD (PE, red) or anti-CD57 (FITC, green) and isotype control antibodies in panel group A to show the background staining of the system. In panel group B, sections were stained with anti-CD57 (FITC), anti-IgD (PE) and anti-CD4 (APC). In panel group C, sections were stained with anti-CD57 (FITC), anti-CD69 (PE) and anti-CD4 (APC). Two different sections were shown in each group of panels. Stained sections were analyzed with a confocal microscope. GC-Th cells can be divided into CD57^+^and CD57^-^T cells, both of which are CD69^+^. A few CD69^+^or CD57^+^T cells are found outside of GC. Most CD4^+^T cells in the interfollicular areas (IFA or T cell-rich zone) are CD57^-^and CD69^-^. GCs are surrounded by the ring of mantle zones (MZ) filled with IgD^+^cells. A representative set of images from three different specimens are shown. ::: ![](1471-2172-6-3-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Isolation of human tonsil T cell and B cell subsets examined in this study. T cell subsets and B cell subsets were isolated from tonsils as described in the materials and methods section and were used in this study. The frequencies of the populations in total tonsil CD4^+^T or CD19^+^B population are 15--25% for CD4^+^CD57^+^GC-Th cells, 50--60% for CD57^-^CD69^+^T cells, 20--30% for CD57^-^CD69^-^T cells, 40--60% for CD19^+^CD38^-^IgD^+^naïve B cells and 30--40% for CD19^+^CD38^+^IgD^-^GC-B cells. ::: ![](1471-2172-6-3-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### CD57^+^GC-Th cells are more efficient than other tonsil CD4^+^T cell subsets in helping B cells. (A) CD4^+^T cell subsets were cultured with total tonsil CD19^+^B cells for 7 days in the presence of SEB. Naïve B cells (C) or GC-B cells (D) were cultured with equal numbers of CD57^+^GC-Th cells or other T cell subsets (CD57^-^, CD57^-^CD69^+^and CD57^-^CD69^-^T cells) for 7 days followed by ELISA for IgM, IgG, IgA and IgE. Data from 5 independent experiments were combined and the averages are shown with standard errors. Relative production levels to CD57^+^GC-Th cells are shown. \Significant differences from CD57^+^GC-Th cells. The absolute Ig production levels (ng/ml) in panel A (GC-Th + Total B cells) were 5737 ± 1764 (IgM), 2111 ± 1185 (IgG), 577 ± 186 (IgA), and 4.8 ± 2.1 (IgE). The absolute Ig production levels (ng/ml) in panel B (GC-Th cells + naïve B cells) were 2045 ± 697 (IgM), 63 ± 21 (IgG), 40 ± 23 (IgA), and 2.9 ± 1.2 (IgE). The average levels (ng/ml) of Ig produced in the cultures of GC-Th cells and GC B cells were 750 ± 279 (IgM), 175 ± 52 (IgG), 51 ± 13 (IgA), and 1.0 ± 0.5 (IgE). (D) Isotype composition of the Ig induced by CD57^+^GC-Th cells. Naïve B cells or GC-B cells were cultured with equal numbers of CD57^+^GC-Th cells or CD57^-^CD69^+^T cells for 7 days followed by ELISA for IgM, IgG, IgA and IgE. Data from 4 independent experiments were combined and the averages are shown with standard errors. \Significant differences between naïve and GC-B cells, but not between the two T cell subsets, were observed. ::: ![](1471-2172-6-3-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### CD57^+^GC-Th cells have the capacities to induce AID expression and to support CSR in B cells. IgD^+^CD38^-^naïve B cells were cultured with CD57^+^GC-Th cells for indicated time periods followed by RT-PCR analysis for (A) AID expression and (B) CSR. The sizes of specific PCR products are 152 bp (IgM); 416 bp (IgG1, G2, G3), 904 bp (IgG4); 904 bp (IgA1); 891 bp (IgA2); and 179 bp (IgE). Shown are productive recombination products. (C) The expression kinetics of AID and productive IgG3 transcripts over an 8 day period are shown together in a graph. In this panel, normalized expression levels calculated after dividing the levels of AID amplification by β-actin levels are shown. The time gap to reach the peak levels of the expression between AID and productive IgG3 transcripts is shown by an arrow. Representative data from at least three independent experiments are shown (panels A and B). (D) Identification of extrachromosomal reciprocal DNA recombination products. Naïve B cells were cultured with CD57^+^GC-Th cells for indicated time periods and were processed to isolate genomic DNA. Fresh GC-B cells were examined for positive controls. The switch circles were detected by a nested PCR method. Representative data out of three independent experiments are shown. (E) Detection of switch circles by a DC-PCR technique. Naive B cells, CD38^+^GC-B cells and naïve B cells cultured with GC-Th cells for 5 days were examined for the presence of γ3 and α1/2 switch circles. ::: ![](1471-2172-6-3-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### CD40L and cytokines in regulation of the helper activity of CD57^+^GC-Th cells. (A and B) Effects of endogenous CD40L and cytokines on the helper activity of CD57^+^GC-Th cells were determined. In cultures of CD57^+^GC-Th cells with naïve or GC-B cells, neutralizing antibodies to IL-4, IL-10, IFN-γ or CD40L or control antibodies (mouse IgG1) were added. \Significant differences from the control group (control antibody). (C and D) Effects of exogenously added cytokines on the helper activity of CD57^+^GC-Th cells were determined. To cultures of CD57^+^GC-Th cells with naïve or GC-B cells, IL-4, IL-10, IFN-γ and TGF-β1 were added separately. Cells were cultured for 7 days followed by ELISA for IgM, IgG, IgA and IgE. Relative Ig secretion levels (the medium control = 1) obtained from 9 independent experiments were combined, and averages and standard errors are shown. \Significant differences from the control group (medium). ::: ![](1471-2172-6-3-5) :::	PubMed Central	2024-06-05T03:55:52.812426	2005-2-4	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548684/", "journal": "BMC Immunol. 2005 Feb 4; 6:3", "authors": [ { "first": "Jong R", "last": "Kim" }, { "first": "Hyung W", "last": "Lim" }, { "first": "Seung G", "last": "Kang" }, { "first": "Peter", "last": "Hillsamer" }, { "first": "Chang H", "last": "Kim" } ] }
PMC548685	Proteolytic cleavage of precursor proteins is an essential process in the life cycle of many viruses, including vaccinia virus (VV). The cysteine proteinase encoded by the VV I7L gene, was originally identified based on a sequence comparison with the African Swine Fever virus proteinase and an ubiquitin-like proteinase in yeast \[[@B1],[@B2]\]. We have previously shown through transprocessing assays that the I7L gene product is capable of cleaving the core protein precursors p4a, p4b, and p25K at conserved AG/X sites and have used reverse genetics to identify active site residues \[[@B3],[@B4]\]. To determine the role that the I7L proteinase plays in the VV replication cycle, we report here the construction and in vivoanalysis of a VV mutant in which the expression of the I7L gene can be conditionally regulated. While this work was in progress, Ansarah-Sobrinho and Moss \[[@B5]\] published a report demonstrating that the I7L proteinase, in an inducible mutant virus regulated by the lacoperator and driven off of the T7 promoter, was responsible for cleaving the A17L membrane protein as well as the L4R core protein precursor. In this work, we show that I7L proteinase, in a different inducible mutant virus, this one regulated by the tetracycline (TET) operator/repressor system and driven off of the I7L native promoter, is responsible for cleaving the other core protein precursors (p4a and p4b). We also demonstrate that expression of the I7L gene from its native promoter appears to be important for optimal viral assembly and replication. To investigate the role of the I7L proteinase in the viral life cycle, an inducible mutant virus was constructed in which the expression of the I7L gene could be regulated by the presence or absence of TET using the components of the bacterial tetracycline operon \[[@B6],[@B7]\]. This system has been shown to be successful in the regulation of the vaccinia virus G1L \[[@B8],[@B9]\] and A14L \[[@B10]\] genes. A plasmid was constructed containing the tetO just upstream of the I7L open reading frame (ORF) in order to regulate expression of I7L proteinase with TET in the presence of a tetracycline repressor (TetR). Also included was the genomic DNA sequence from 250 bp upstream of the I7L ORF, to include the native promoter, and to aid in homologous recombination. This plasmid was used to create the recombinant virus vtetOI7L using the transient dominant selection method \[[@B11]\]. A commercially available cell line, T-Rex-293 (Invitrogen), expressing the TetR was used to regulate the expression of the I7L gene from the infecting recombinant virus. This conditional-lethal expression system has recently been used to show that the enzymatic activity of the VV G1L metalloproteinase is essential for viral replication \[[@B9]\]. The conditional-lethal phenotype of the recombinant virus was shown by plaque assay (Fig. [1](#F1){ref-type="fig"}), in which the formation of plaques from vtetOI7L is dependent on the presence of TET, while the wild-type virus is unaffected by either the presence or absence of TET. To determine the optimum TET concentration required for replication of vtetOI7L, TREx-293 cells were infected with vtetOI7L in the presence of varying concentrations of TET, harvested 24 h later, and the titer determined on BSC~40~cells \[[@B12]\]. A 2-log increase in viral yield was observed with 1 μg/ml TET (data not shown). To confirm that expression of the I7L gene was essential for viral replication, TREx-293 cells were infected with vtetOI7L at a multiplicity of infection (MOI) of 0.1, 0.5, 5, or 10 in the presence or absence of TET, harvested 24 h later, and the titer of the virus infected cell lysates determined on BSC~40~cells. At an MOI of 0.1 or 0.5 there was an average reduction of 99.1% of infectious virus particles (Fig. [2](#F2){ref-type="fig"}). At an MOI of 5 there was an average reduction of 95.7%, and at an MOI of 10 there was an average reduction of 90.3% (Fig. [2](#F2){ref-type="fig"}). This multiplicity-dependent breakthrough of viral replication is likely due to gene copy overwhelming the amount of TetR being expressed by the TREx-293 cell line. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Effect of TET on plaque formation.TREx-293 cells were infected with vtetOI7L or wild-type virus in the presence or absence of 1 μg/ml TET and harvested 24 hpi. BSC~40~cells were then infected and stained with crystal violet 48 hpi. ::: ![](1743-422X-2-4-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Effect of TET on viral replication and rescue of the vtetOI7L mutant.TREx-293 cells were infected with vtetOI7L in the absence (-) or presence of 1 μg/ml TET at an MOI of 0.1, 0.5, 5, or 10. Infected cells were harvested 24 hpi and titrated on BSC~40~cells. ::: ![](1743-422X-2-4-2) ::: To test whether the insertion of the TET operator just upstream of the I7L ORF had an effect on the viral growth kinetics, a one-step growth curve was conducted. TREx-293 cells were infected with wild type virus or vtetOI7L in the presence or absence of TET and infected cell lysates were harvested at the indicated times and the titer determined on BSC~40~cells (Fig. [3A](#F3){ref-type="fig"}). In the presence of TET, the recombinant virus grew to the same yield and with the same kinetics as wild type virus while in the absence of TET the production of infectious virus was much lower indicating that the presence of the TET operator did not have an effect on the growth kinetics of the inducible mutant virus. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Panel A: One step growth curve.TREx-293 cells were infected with wild-type virus (circle) or vtetOI7L in the presence (square) or absence (triangle) of 1 μg/ml TET. Infected cells were harvested at the indicated times and the titer determined on BSC~40~cells. Panel B: Rescue of replication.TREx-293 cells were infected with vtetOI7L and transfected with either vector alone (pRB21), plasmid with wild-type I7L driven off of a synthetic early/late promoter (pI7L), plasmid with mutant I7L, mutated in the putative active site, driven off of a synthetic early/late promoter (pI7LH241A), or wild-type I7L driven off of its native promoter (pCB26) in the absence of TET. Infected cells were harvested 24 hpi and the titer determined on BSC~40~cells. Transfection of plasmid borne wild-type I7L but not of mutant I7L or vector alone partially rescued the replication of vtetOI7L. ::: ![](1743-422X-2-4-3) ::: To demonstrate that the replication defect of the vtetOI7L mutant virus in the absence of TET was due to the I7L gene we tested whether viral replication could be rescued by the introduction of a plasmid-borne I7L gene. TREx-293 cells in 6-well plates were transfected with 1.8 μg of plasmid DNA (containing either no insert, a wild type I7L gene under the control of the synthetic early-late promoter, a I7L gene with the catalytic His241 mutated to Ala, or the I7L gene under the control of its native promoter) and infected with vtetOI7L at an MOI of 0.2 plaque-forming units per cell in the absence of TET. Cells were harvested 24 hours post infection (hpi) and the titer determined on BSC~40~cells. As an additional control, TREx-293 cells were mock transfected and infected with vtetOI7L in the presence of 1 μg/ml TET to compare growth conditions. A partial rescue of viral replication was observed when cells were transfected with the I7L gene under the control of the synthetic early/late promoter, but not when cells were transfected with plasmid alone or with a mutant I7L gene (Fig. [3B](#F3){ref-type="fig"}). This was an approximate 5-fold increase in virus replication compared to the pRB21 or pI7LH241A transfected controls. When the I7L gene was driven off of its own promoter in pCB26 and transfected in, there was a much higher level of rescue (Fig. [3B](#F3){ref-type="fig"}), suggesting that the timing and amount of I7L gene expression has important implications for the viral life cycle. We have previously shown through transient expression assays that the I7L proteinase is capable of cleaving the p4b, p4a, and p25k core protein precursors \[[@B3],[@B4]\] which are products of the A3L, A10L, and L4R open reading frames respectively. Here we were interested to see whether the I7L proteinase in the conditional lethal mutant system was also capable of cleaving these proteins in the presence but not the absence of TET. First, to see whether I7L protein was expressed at the same time from the mutant virus as from the wild type virus, TREx-293 cells were infected in the presence of TET and cells harvested at various time points. Proteins in the crude cell extracts were separated by SDS-PAGE and detected by Western blot with anti-I7L antisera. I7L enzyme from both viruses appeared at late times after infection, around 8 hpi and increased as time progressed (data not shown). To determine the effect of TET on I7L protein expression, cells were infected and treated with 0 to 5 μg/ml TET. After 6 h, the infected cells were labeled with 60 μCi/ml ^35^S-met and harvested after 24 h. Extracts were immunoprecipitated with I7L antisera and protein detected by autoradiography. With wild type virus, I7L protein was expressed at each TET concentration (data not shown). However, in the mutant virus, expression of I7L enzyme was repressed in the absence of TET and increased with the addition of TET. To determine the effect of TET concentration on p4b core protein precursor processing, cells were infected in the presence of 0 to 5 μg/ml TET, harvested 24 hpi, and the extracts immunoblotted with anti-4b antisera. With wild type virus p4b was processed at each TET concentration as expected, however with the mutant virus, p4b processing was repressed in the absence of TET (data not shown). The slight processing in the absence of TET is likely due to slight leak-through of I7L gene expression in this system. The same results were seen for the processing of p4a, with processing in each of the wild type virus lanes, repressed processing with the mutant in the absence of TET and increased processing in the presence of TET (data not shown). Kane and Shuman \[[@B13]\] have previously shown that I7L protein is located in the virus core. To verify that the I7L protein from the inducible mutant was localized correctly, purified virions were treated with DTT and NP-40 to separate the envelope fraction from the core fraction and protein from each sample was separated by SDS-PAGE and detected by Western blot with anti-I7L antisera. As expected, the I7L enzyme from the inducible mutant was detected in the core sample, as was the wild type virus (data not shown). The morphogenesis of vtetOI7L under nonpermissive conditions was analyzed via electron microscopy. TREx-293 cells were infected with vtetOI7L at an MOI of 1 in the presence or absence of TET and harvested 24 h later. In the presence of TET, cells contained a variety of both immature and mature forms of the virus (Fig. [4](#F4){ref-type="fig"}, panels A-C), which were indistinguishable from cells infected with wild type virus (not shown). However, in the absence of TET, no mature virions were observed in any of the infected cells observed. There appeared to be an accumulation of immature viral particles, some with nucleoids, as well as the appearance of crescent shaped particles (Fig. [4](#F4){ref-type="fig"}, panels D-F), similar to those observed by Ansarah-Sobrinho et al\[[@B5]\]. Also observed were numerous dense virus particles. Virion morphogenesis appears to arrest at a stage prior to core condensation. The observation that there is still some processing of p4b in the absence of TET and yet the morphology of the mutant virus in the absence of TET shows only immature virus particles suggests the hypothesis that there is a requirement for the processing threshold of the core protein precursors to be achieved before morphogenesis can proceed. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Electron microscopy of cells infected with vtetOI7L.TREx-293 cells were infected with vtetOI7L at an MOI of 1 in the presence (panels A, B, and C) of 10 μg/ml TET or in the absence (panels D, E, and F) of TET. Cells were harvested at 24 hpi, immediately fixed and prepared for transmission electron microscopy. The bar in panels A, B, D, E, and F represents 400 nm. The bar in panel C represents 200 nm. ::: ![](1743-422X-2-4-4) ::: Taken together, the data we have presented here, as well as analysis of the VV G1L conditional lethal mutant \[[@B9]\], suggests a morphogenesis model in which these two putative proteases operate sequentially to regulate assembly. According to this model, if we assume that both I7L and G1L are associated with the immature virus along with the accompanying DNA and other viral proteins, then activation of I7L leads to the process of core protein precursor cleavage and the initiation of core condensation. Following this activity, the activation of G1L completes core condensation and allows progression to the formation of intracellular mature virus. If the activity of the I7L proteinase is blocked, viral morphogenesis arrests prior to core condensation. If the activity of G1L proteinase is blocked, viral morphogenesis arrests at a stage subsequent to this but still prior to complete core condensation. To test this model, it will be of interest to isolate biochemically active I7L and G1L enzymes and determine the series of events that lead to their activation. Competing Interests =================== The author(s) declare that there are no competing interests. Authors\' contributions ======================= CMB conducted all the experiments and wrote the manuscript. DEH conceived the study, coordinated the research efforts and edited the paper. Both authors read and approved the final manuscript. Acknowledgements ================ We kindly thank Dr. Paula Traktman for the vTetR virus strain and for helpful discussions, Tove\' Bolken and Dr. Marika Hedengren-Olcott for helpful discussions, and Dr. Michael Nesson for the electron microscopy analysis. This work was supported by National Institute of Health grant UO1 A1 48486.	PubMed Central	2024-06-05T03:55:52.814821	2005-2-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548685/", "journal": "Virol J. 2005 Feb 8; 2:4", "authors": [ { "first": "Chelsea M", "last": "Byrd" }, { "first": "Dennis E", "last": "Hruby" } ] }
PMC548686	Background ========== Teak (Tectona grandisL.) is a very valuable timber species, and is a member of the moist deciduous and dry deciduous forest types. Teak plantations are threatened by two major pests: the Teak Defoliator (Hyblaea pueraCramer) Lepidoptera: Hyblaeidae and the Teak Skeletonizer (Eutectona machaeralis(Walker) syn.). H. puerais less widely distributed in the tropics: in Oriental and Australian regions (India, Sri Lanka, Burma, Java, Papua New-Guinea, Northern Queensland in Australia, Solomon Islands); in the West Indies; and in South and parts of East Africa \[[@B1]\]. The Teak defoliator is of major concern since it is involved in complete defoliation of trees during the early part of the growing season. Defoliation does not kill the trees, but does lead to huge timber loss. Recent studies have shown that the defoliation leads to an average loss of 44% of the potential volume increment in four to nine year-old teak plantations. It has been estimated that in the Nilambur teak plantation during the study period, protected trees increased by an annual increment of 6.7 m^3^/ha compared with 3.7 m^3^/ha for unprotected trees, a gain of 3 m^3^/ha per annum \[[@B2]\]. Teak defoliator outbreaks are a regular annual feature in teak plantations in Kerala, India. It is difficult to predict the exact time and place of these outbreaks. Evidence gathered from the past decade on the population dynamics of H. pueraindicates habitual, short range movements of emerging moth populations, suggesting that these spread to larger areas, generation after generation, affecting entire teak plantations \[[@B3]\]. Earlier studies also indicated that the outbreaks begin as small epicenters during the pre-monsoon season \[[@B4]\]. Populations were classified as \'endemic\', \'epicenter\' and \'epidemic\', based on their time of occurrence and the density of the population as represented by the area it infests. Endemics are insects belonging to the low-density population level; epicenters are patchy, medium density outbreaks that occur during the pre-monsoon season, whilst epidemic represents large area, high-density outbreak populations. An understanding of the origin and spread of the epidemic of this pest, which erupt suddenly following the pre-monsoon rain each year, is an important prerequisite for developing appropriate control strategies. If progenies of the epicenter populations cause the larger epidemics, control of these could prevent major outbreaks. On the other hand, if immigrant moths were involved, it would be difficult to control major outbreaks. Thus, understanding the cause and effect relationship between initial small outbreaks and large outbreaks that occur later in the year is crucial for the control of the pest. Recently, molecular markers have been used to enhance understanding of insect displacements, especially including estimates of movement of particular genotypes and/ or biotypes, reproductive strategy and success. Such approaches have also been used to study founder events \[[@B5]\], geographical invasions \[[@B6]\], small and large scale displacements \[[@B7],[@B8]\], including movement of entire population demes \[[@B9]\], and even altitudinal movements related to habitat patchiness and persistence \[[@B10]\]. Molecular data can yield valuable information when integrated with information from ethology, field ecology, comparative morphology, systematics and palaeontology \[[@B11]\]. Use of direct and indirect methods of tracking insects along with description of the role and utility of various molecular markers -- protein and DNA -- in monitoring insect dispersal, has been extensively reviewed \[[@B12]\]. Arbitrarily-primed DNA markers, and involving the polymerase chain reaction (PCR), have proved very useful for genetic fingerprinting and for facilitating positional cloning of genes. This class of markers are particularly important for less studied species, for which genome sequence information is generally not known. These technologies include randomly amplified polymorphic DNA (RAPDs) \[[@B13],[@B14]\], DNA amplification fingerprinting (DAF)\[[@B15]\], and amplified fragment length polymorphisms (AFLPs) \[[@B16]\]. In this study, we used a variant of the RAPD approach involving various nuclear and mitochondrial gene specific primers to trace the origin of teak defoliator outbreaks. It is expected that the molecular data would provide the necessary information to elucidate the origin of the epidemic population. Such information should prove valuable in planning and implementing measures to control these pests. Therefore, the aim of the present study was to identify the relationship among the three apparent populations -- endemic, epicenter and epidemic. Results ======= The nuclear and mitochondrial gene specific primers chosen did not produce any amplification product when used in combination with the corresponding primers as described in the UBC primer set kit \[[@B17]\]. This resulted in our devising a novel PCR, which we have named RAGEP-PCR. In RAGEP-PCR, we used single nuclear and mitochondrial gene encoding primers at low stringency annealing temperatures. Unlike RAPDs, in RAGEP longer nuclear (21--26 nucleotide) and mitochondrial (19--26 nucleotide) gene encoding primers were utilised, and which we have here extensively employed to evaluate the species taxonomic specificity/reproducibility and to discriminate the endemic, epicenter and epidemic populations of teak defoliator from one another. RAGEP markers were first tested for polymorphisms, species-specificity and repeatability. Similar fingerprinting pattern were observed in subsequent PCRs for the same individual using the same primers (Fig [1](#F1){ref-type="fig"}), which displayed overall robustness and repeatability with RAGEP-PCR. It was also possible to discriminate various moth species based on their species-specific DNA fingerprint pattern (Fig [2](#F2){ref-type="fig"}). The bands scored for each nuclear RAGEP used in the present study were of a size range 200 bp to 1500 bp. With nuclear RAGEP markers, an average of 2--3 monomorphic bands were observed, except for primer CK6-5\'. In each marker, the average number of bands scored varied from 7--16. The maximum number of bands was detected using primer cytC-B-3\', while the maximum number of monomorphic bands were detected using primer EFS599. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Reproducibility of RAGEP fingerprints. mt1-3, nu1-3 depicts reproducibility of RAGEPs using mitochondrial and nuclear primers respectively ::: ![](1472-6785-5-1-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Species specificity of RAGEP fingerprints. M1-4 depicts variability in Lepidopteran species. M1-Eutectona machaeralis, M2-Sylepta derogata, M3-Cnaphalocrocis medinalis, M4-Bombyx mori ::: ![](1472-6785-5-1-2) ::: Each individual RAGEP marker gel was screened and a similarity matrix was generated. Subsequently similarity matrixes of all experimental patterns were combined to generate a UPGMA (Unweighted pair-group mathematical average) tree. While evaluating the similarity matrix based on the Dice coefficient for all nuclear specific RAGEP markers and whilst constructing a UPGMA tree, it was observed that the various population groups of H. puerafall in two clusters, which are further divided into two major sub clusters. Average similarity between the two major clusters was 20%, while that between the two sub clusters was 34%. In one of the major clusters, we observed all the endemic insects clustering together with some of the populations from the epicenter insects; however, both populations fall in two distinct sub-clusters (Fig [3](#F3){ref-type="fig"}). Similarly in the second major cluster, the remaining populations from the epicenter and entire epidemic insect populations were likewise seen to fall into two distinct sub-clusters. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### RAGEP fingerprint patterns generated by nuclear gene specific DNA markers in individuals of three populations (Panel B) and UPGMA dendrogram showing clustering of different insect populations of Hyblaea puera (Panel A) ::: ![](1472-6785-5-1-3) ::: Using the mitochondrial RAGEP markers, the average numbers of bands scored for each primer ranged from 6--15. All bands scored were of size range 300 bp to 1600 kb. The maximum numbers of bands detected was found using primer SR-J-14233, the minimum numbers using marker N4-N-8924. Among mitochondrial markers, an average of 1--2 monomorphic bands were observed. The maximum number of monomorphic bands was observed using marker CB-N-10920. Two distinct clusters were observed in the UPGMA dendrogram for mitochondrial markers. Similarity between the two clusters was only 20%. One of these clusters comprised the majority of the endemic samples with a few samples from epicenter insects, whilst the other cluster was comparatively larger and had the two major sub clusters. Both these sub-clusters have insects from epicenter and epidemic populations (Fig [4](#F4){ref-type="fig"}). From this dendrogram, it may be deduced that all the seven epidemic population samples tested in the study shared the same gene pool with sets of epicenter populations. In contrast, the endemic populations are genetically distant from the epicenter populations. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### RAGEP fingerprint patterns generated by mitochondrial gene specific DNA markers in individuals of three populations (Panel B) and UPGMA dendrogram showing clustering of different insect populations of Hyblaea puera (Panel A) ::: ![](1472-6785-5-1-4) ::: Discussion ========== The first teak plantation in India was started as early as 1842 in Nilambur, Kerala State, India. Preliminary information on the life history of H. pueraand the nature of its damage was published in 1898 \[[@B18]\]. H. pueraoutbreaks have been reported to begin in small epicenters and later spread to larger areas. It was then suspected that population build-up in the early outbreak epicenters might account for the subsequent widespread epidemic. However, a study using the time lapse (developmental time) between two epidemics to determine whether an earlier epidemic was responsible for causing the subsequent outbreak showed that all subsequent outbreaks could not be attributed to previous outbreaks, thereby indicating the possibility of migrant populations being involved \[[@B19]\]. Several technical advancements on the DNA fingerprinting methodologies have been established to resolve the taxonomic uncertainties and address the issue on species variability and migration \[[@B13]-[@B16],[@B20],[@B21]\]. The RAGEP-PCR method described here uses gene-specific primers and randomly amplifies the nuclear and mitochondrial-like gene products. Longer mitochondrial (19--26 nucleotide) gene encoding primers are likely to increase the reproducibility and specificity when compared to RAPD technique. This method was found to be efficient, simple and highly reproducible. Here it has been effectively used to discriminate the various population groups of H. puerainfesting teak plantations in South India. It can also be used to discriminate taxonomically various closely -- related moths to the species level. Mitochondrial RAGEP fingerprints are derived from the randomness of RAGEP-PCR. It is difficult to predict with certainty that the bands are diagnostic feature of the mitochondrial genome, but since RAGEP PCR uses gene specific primers, the PCR products could therefore be a result of amplification of homologous genes or pseudogenes which could represent nuclear mitochondrial DNA (NUMTs). Mitochondrial DNA sequences are frequently transferred to the nucleus-giving rise to NUMTs, which are considered to be common in eukaryotes \[[@B22]\]. Very high rate of horizontal transfer between organellar and nuclear genomes has been reported in the brown mountain grasshopper, Podisma pedestris(L.)\[[@B23]\]. Age groups, sexes, life history variants, etc. and the processes including birth, death, immigration and emigration as different phenotypic classes have been very well defined \[[@B24]\]. While studying the differentiation process of grain aphid, Sitobion avenae(F.) populations across agricultural ecosystems using DNA fingerprinting \[(GATA)~4~\] and RAPDs, it was possible to discriminate the micro- and macro geographical heterogeneity \[[@B25]\]. Highly diagnostic banding patterns in individuals of S. avenaeon wheat and cocksfoot grass, Dactylis glomerata(L.) were observed during the early months of infestation, which declined as the season progressed, largely as a result of genetic drift and local movement between adjacent host species \[[@B26]\]. Monophyly and a strong biogeographic pattern of each biotype have been reported in whitefly, Bemisia tabaci(Gennadius) populations studied throughout the world \[[@B27]\]. While evaluating the genetic structure in introduced population of the fire ant, Solenopsis invicta(Buren) using different classes of markers, it was confirmed that both mitochondrial and nuclear markers display the same hierarchical structure \[[@B28]\]. Distinct mitochondrial and nuclear DNA sequence divergence patterns for phylogenetic inference has been established among nymphalid butterflies \[[@B29]\]. The present study using RAGEP-PCR provides a tool for a logical continuation of the earlier work to trace the relationship of endemic, epicenter and epidemic populations of the teak defoliator. The dendrogram produced from nuclear RAGEP clearly indicates that the endemic insects are not involved in causing the epidemic; however, they are apparently involved in the localized spread by building up small epicenter populations. Similarly, while evaluating the observation based on mitochondrial RAGEP\'s, it is further apparent that endemic populations were not involved in causing the epidemic. This suggests that all the epidemic insects, which are spatially distinct, but temporally co-occurring, share the same gene pool. Randomness of genome amplification methods have been efficiently used in constructing the phylogenetic history in the weevil, Aubeonymus mariafranciscae(Roudier), which had diverged recently \[[@B5]\], whilst the origin of the Argentine stem weevil, Listronotus bonariensis(Kuschel) in New Zealand, was traced to the eastern coast of South America \[[@B30]\]. Use of RAPDs to examine, for example, population subdivision of the saw toothed grain beetle, Oryzaephilus surinamensis(L.) \[[@B31]\], characterization and identification of Asian and North American gypsy moth, Lymantria dispar(L.) \[[@B32]\], host based genotype variation in S. avenae\[[@B33]\], and genotypic variation among different phenotypes of asexual adult winged and wingless of some clones of cereal aphid species \[[@B34]\], has been well documented. Earlier reports involving molecular DNA markers mention the use of these markers in the detection of sibling species of black flies, Simuliumspp. \[[@B35]\], whilst the dynamics of colonization of Drosophila subobscura(Collin) \[[@B36]\] in the west coast of North America and its impact in the sibling species Drosophila athabascaSturtevant and Dobzhansky, and Drosophila aztecaSturtevant and Dobzhansky has been extensively studied by allozymes, mitochondrial DNA (mtDNA) and RAPD markers. With the Teak defoliator, earlier studies based on temporal and spatial distribution of the larvae indicated that the epicenters were not constant over the years and did not represent highly favourable local environments \[[@B3]\]. The present study found little evidence to show that the aggregation of moths belonging to the endemic populations cause the epicenter populations. On the other hand, the findings do suggest the alternate hypothesis, i.e., that immigration of moths from distant teak plantations cause the epidemic, and that there is a continuous inflow of moths during the infestation period. This suggests that under a single demographic structure, two phenotypic classes of H. pueracoexist during the outbreak season. The degree of variability observed for RAGEPs also argues that this technique could be useful for a variety of questions, including individual identification, strain identification and phylogenetic analysis. Conclusions =========== The present results appear to validate the hypothesis, that control of H. pueraepicenter populations would help prevent large-scale outbreaks of the teak defoliator in teak plantations. Therefore, appropriate strategies should be adopted to control the epicenter populations, which occurs in a smaller area. This appears to be a more practical and economical approach for teak defoliator management when compared with management of the pest in the total plantation area covering thousands of hectares. Thus the molecular markers detected using RAGEP-PCR can enhance the understanding of insect population dynamics and aid in tracing the spread and cause of epidemics. Methods ======= Sample collection ----------------- Based on the spatial pattern of infestation in the past, the area was divided into convenient observation units of approximately 50 ha, based on natural boundaries of streams, roads and footpaths. The canopy of teak is continuous within in the observation area. Each area was monitored every 15 days, which was precisely based on the life cycle of H. puera. Larval samples were collected from the infestation sites. Whenever fifth instar larvae were available, ten larvae were preserved in 70 % alcohol and stored deep frozen at -20°C. If only lower stages were available, i.e., third or fourth instar, they were reared up to 5th instar in the laboratory. Ten 5th instar larvae were preserved for DNA isolation from each sample site, whilst the remaining larvae were reared into the next generation. Each sample was assigned a code number containing the details of Year / Month / Date / Block / Grid / Generation for further reference. Using the duration of each instar (egg -- one day; 1^st^and 2^nd^instars -- two days each, 3^rd^to 5^th^instars -- three days each; pre-pupa -- one day and pupa -- four days), the temporal data on outbreaks were examined to see whether each subsequent epidemic could be explained on the basis of a previous outbreak. The details of location of pest incidence and extent of infestation were later transferred to the field map in order to understand the spatial pattern of infestation (Fig [5](#F5){ref-type="fig"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Landscape of Nilambur teak plantation showing distribution of the endemic, epicenter and epidemic populations of Hyblaea puera ::: ![](1472-6785-5-1-5) ::: Populations were classified as \'endemic\', \'epicenter\' and \'epidemic\', based on their time of occurrence and the density of the population as represented by the area it infests. Five endemic populations, twenty six epicenter populations and seven epidemic populations for the year 2002 were included in the study. Earlier studies had indicated that outbreak begin as small epicenters in February during the pre-monsoon season and end by June. Endemic samples were collected throughout that year based on their stray occurrences in various life stages, whilst epicenter samples from each aggregated patch were collected only from the insects that attained the same stage of its life cycle at the time of collection in that patch. Similarly the epidemic samples were also collected from insects representing the same life stages at the time of collection from each aggressive patch. The temporal relationship between the endemic population and the epicenter populations and that of the epicenter populations with the large-scale epidemics were first worked out. The larval samples that were geographically close and had a difference of one complete life cycle stage between the population groups were subjected to molecular studies to evaluate their relatedness. DNA isolation ------------- DNA extraction was performed with a minor modification of isolation and purification protocol as described earlier \[[@B37]\] being extracted from whole larvae and quantified spectrophotometrically using a spectrophotometer at 260 nm (Shimadzu). The quality of the DNA was checked spectrophotometrically by taking the absorbance ratios of 260/280 nm. Polymerase chain reaction ------------------------- Both nuclear and mitochondrial DNA RAGEP amplifications were performed in a total volume of 30 μl. Each reaction consisted of 1x Taq buffer with 1.5mM MgCl~2~, 1.2 U of Taqpolymerase (BG), 0.25 mM of dNTPs (Amersham) and 12 pM of primer per reaction. Primers were initially screened for polymorphism and repeatability. Amplifications were performed in similar cycling conditions in a Thermocycler (Biorad) programmed as follows: initial denaturation at 95°C for 5 min., followed by 45 cycles of cycle denaturation at 94°C for 1 min., annealing at 36°C for 1 min., extension at 72°C for 2 min. and final extension at 72°C for 5 mins. The amplification products were separated using 1.2% agarose gel in 0.5 × TBE buffer with ethidium bromide staining to visualize the product separation using a Bio-Rad\'s Fluor S imager. The molecular weight of each band was estimated by comparing with a co-migrating 100-bp ladder (Amersham). RAGEP fingerprints of each sample from different regions were then interpreted using Fingerprint type module of Bionumerics software (Applied Maths Kortrijk Belgium, ver.2.0). A preliminary screening with 50 nuclear RAGEPs and 37 mitochondrial RAGEP primers were evaluated for polymorphism and repeatability. Only 11 nuclear (Table-[1](#T1){ref-type="table"}) and mitochondrial RAGEPs (Table-[2](#T2){ref-type="table"}) from each group was selected for the study, as they showed constant repeatability of highly polymorphic patterns. Species specificity was evaluated by comparing the banding patterns in H. puerawith those from the Teak skeletonizer, E. machaeralis(Walk.), Leaf roller, Sylepta derogata(F.), Leaf folder, Cnaphalocrocis medinalis(Guenée), and the Silkworm, Bombyx mori(L.). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Insect nuclear gene specific primer sequences used in nuclear RAGEP-PCR ::: S.No Primer name Sequence ------ ------------- ------------------------------------------------ 1 EFS599 ATC TCC GGA TGG CAC GG(CT) GAC AA 2 22.5drc GAA CCA (AG)TT (AG)AC (AG)TG (AG)AA GAT C 3 LEPWG1 GA(AG)TG(CT)AA(AG)TG(CT)CA(CT)GG(CT)ATGTCT GG 4 CK6-5\' GAC CAC CTC CGA GTC ATC TC(CG) ATG 5 CK7-3\' CAG GTG CTC GTT CCA CAT GAA 6 CytC-B-3\' CAT CTT GGT GCC GGG GAT GTA TTT CTT 7 EF1-5\' GAC AAC GTT GGC TTC AAC GTG AAG AAC G 8 Tub3-5\' GAT TTG GAG CC(AGCT) GG(AGCT) ACC ATG GA 9 18S-A1984 TCC CTG GTT GAT CCT GCC AGT A 10 S1124 AGC GTA TGG C(AC)T C(AG)A AGAACT G 11 rcM4 ACA GC(CGA) AC(GT) GT(TC) TG(CT) CTC AT(AG) TC ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Insect mitochondrial gene specific primer sequences used in mitochondrial RAGEP-PCR ::: S.No Primer name Sequence ------ ------------- ------------------------------------ 1 C1-J-2183 CAA CAT TTA TTT TGA TTT TTT GG 2 TL2-J-3034 AAT ATG GCA GAT TAG TGC A 3 C2-N-3661 CCA CAA ATT TCT GAA CAT TGA CCA 4 TK-N-3785 GTT TAA GAG ACC AGT ACT TG 5 N4-N-8924 AAA GCT CAT GTT GAA GCT CC 6 CB-J-10612 CCA TCC AAC ATC TCA GCA TGA TGA AA 7 LR-J-12887 CCG GTC TGA ACT CAG ATC ACG T 8 LR-J-13417 ATG TTT TTG TTA AAC AGG CG 9 LR-N-13398 CGC CTG TTT AAC AAA AAC AT 10 SR-J-14233 AAG AGC GAC GGG CGA TGT GT 11 CB-N-10920 CCC TCA GAA TGA TAT TTG TCC TCA ::: Analysis -------- The polymorphic content for nuclear and mitochondrial primers were analyzed using Bionumerics software \[[@B38]\]. Band search parameters were kept constant as 5% minimum profiling for all the gels. The position tolerance for selection of bands in constructing a dendrogram was kept constant at 1% through out the interpretations. Only bands showing clear and reproducible patterns were included in the final analysis and these were scored. Real-time normalization of gel electrophoresis patterns and band position for all the gels was based on the reference system for the species-specific bands. Normalization helped us to control the brightness and streakiness of bands without altering the lighter bands and also control the inter-gel mobility shifts. Subsequently a data matrix of similarity values was produced for each individual for each marker. The Dice coefficient was used to analyze the similarities of the banding patterns. Consensus similarity matrix and dendrogram based upon individual matrices from different markers were used for pair wise clustering based on unweighted pair group method (UPGMA) with average linkages (11). The UPGMA dendrogram prevails on the assumption that nucleotide substitution rates are same across all branches. It employs a sequential clustering algorithm, in which local topological relationships are identified in order of similarity, and the phylogenetic tree is built in a stepwise manner. All analysis was done using Bionumerics software V-2. Authors\' contributions ======================= CN and MB performed the molecular studies and are responsible for the interpretation of molecular data whilst TV and VVS performed the field data collection and are responsible for spatial and temporal data interpretation. All authors read and approved the final manuscript. MB and VVS conceived and designed the study. Acknowledgements ================ We thank the Department of Biotechnology, Government of India for financial support. The authors are thankful to Dr. Hugh D Loxdale for his valuable suggestions on the manuscript of this paper.	PubMed Central	2024-06-05T03:55:52.816113	2005-2-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548686/", "journal": "BMC Ecol. 2005 Feb 2; 5:1", "authors": [ { "first": "N", "last": "Chandrasekhar" }, { "first": "TV", "last": "Sajeev" }, { "first": "VV", "last": "Sudheendrakumar" }, { "first": "Moinak", "last": "Banerjee" } ] }
PMC548687	Background ========== Apoptosis is a genetically programmed form of cell death employed to regulate cell number, spatial organisation and remove infected and damaged cell populations that may compromise the integrity of the organism \[[@B1]\]. Aberrant or unscheduled apoptotic responses are thought to contribute to various diseases including Alzheimer\'s disease, rheumatic diseases and neoplastic growth \[[@B2]\]. Induction of apoptosis in virally infected cells is a key weapon in the immune system\'s arsenal against viral attack. Many viruses have evolved multiple strategies to counteract host-mediated apoptosis in order to facilitate infection, replication or persistence \[[@B3],[@B4]\]. Epstein-Barr virus (EBV) a human γ-herpesvirus that latently infects the majority of adults, was the first virus shown to influence the survival of infected cells \[[@B5]\]. In vivo, EBV is associated with a number of benign and malignant proliferative diseases, such as infectious mononucleosis, Burkitt\'s lymphoma and nasopharyngeal carcinoma \[[@B6],[@B7]\]. Latent infection of B cells in vitroleads to outgrowth of apoptosis resistant lymphoblastoid cell lines (LCLs), which phenotypically resemble activated B-cells. Although B cells can produce progeny virus, epithelial cells are also known to be permissive for viral replication. Studies have shown that EBV has at least two mechanisms of suppressing apoptosis. During latent infection latent membrane protein 1 (LMP1) is the key mediator of protection. LMP1, a CD40 mimic, up-regulates cellular Bcl-2 \[[@B8]\] and A20 \[[@B9]\] via the NFκB pathway. The virus also encodes two structural homologues of Bcl-2: BHRF1 \[[@B10],[@B11]\] and the less well characterised and more controversial BALF1 \[[@B12],[@B13]\] which are thought to play their major roles during lytic infection. Whilst many other viruses also have Bcl-2 homologues (v-Bcl-2s) \[[@B14]\], EBV and its mammalian analogues appear unique in having two v-Bcl-2s. Despite being considered dispensable for EBV replication or cellular transformation in vitro\[[@B15],[@B16]\], BHRF1 is extremely highly conserved in distinct geographical isolates of EBV \[[@B17]\], and its role in vivoremains unclear. BHRF1 is thought to act in lytic cycle to delay apoptosis during replication in terminally differentiating epithelium \[[@B18]\]. However, some EBV-positive B-cell lymphomas have been found to be positive for BHRF1 transcripts \[[@B19]\] and latent BHRF1 transcripts have been detected in T-cell lymphomas \[[@B20]\] and in EBV-transformed tightly latent B-cell lines in vitro\[[@B21]\]. Additionally, EBV BHRF1 has been shown to suppress apoptosis in lymphoid cells in response to a range of triggers including serum deprivation \[[@B11]\], DNA damaging agents and chemotherapeutic drugs \[[@B22]\] and cytokines \[[@B23]\]. It is possible then, that BHRF1 may play a role at some stage during latent infection, which warrants the study of this protein in B cells, the target for latent infection. Analogues of EBV are found in the higher primate species. Herpesvirus papio(H. papio) and Herpesvirus pan(H. pan) infect baboons and chimpanzees respectively. H. papioencodes a BHRF1 homologue that shares 65% amino acid identity (81% similarity) with EBV BHRF1, whilst the H. panBHRF1 is even more similar at 82% identity and 90% similarity \[[@B24]\]. H. papioBHRF1 has been shown to confer resistance to cisplatin induced apoptosis in human keratinocytes akin to EBV BHRF1 \[[@B25]\], but this study represents the first functional analysis of a primate virus BHRF1 in a lymphoid cell background. Here we show that H. panBHRF1 exhibits similar anti-apoptotic activity to that of EBV BHRF1 in the human Burkitt\'s lymphoma cell line Ramos-BL in response to apoptosis induced by either serum withdrawal, etoposide treatment and the previously untested trigger, ultraviolet radiation. These results show that H. panBHRF1 is biologically active in human B-lymphocytes in vitrosuggesting a conserved role for BHRF1 during in vivoinfection of B-lymphocytes. Results ======= Construction of BHRF1 expression vectors and selection of positive clones ------------------------------------------------------------------------- As the senior author has previously shown that EBV BHRF1 suppresses apoptosis in response to serum reduction in various BL cell lines \[[@B11]\], we initially elected to compare the abilities of H. panBHRF1 and EBV BHRF1 to regulate serum reduction induced apoptosis in the EBV negative Ramos-BL cell line to ascertain whether the genes are functional, as well as structural, homologues. In order to produce expression vectors, the H. panand EBV BHRF1 reading frames were amplified by PCR from template vectors pHVPanTW4 and pDH222 respectively using primers incorporating XhoI restriction sites. The PCR fragments were introduced into the pCR2.1Topo^®^vector and the XhoI fragment was released by restriction digestion of the TopoBHRF1 constructs and cloned into the XhoI restriction site of the pcDNA4/HisMax mammalian expression vector (InVitrogen), which produces an N-terminal fusion protein with 6His and Xpress epitopes. Ramos-BL cells were electroporated with either control vector, EBV BHRF1 or H. panBHRF1 and cloned by limiting dilution in medium containing 150 μg/ml zeocin. There was no obvious difference in the numbers of drug resistant clones that were produced by transfection with the vector or either of the BHRF1 constructs. Drug resistant clones were screened by RT-PCR for BHRF1 expression and approximately 50% of the clones were shown to be expressing detectable BHRF1. The RT-PCR profiles of two clones each of the three transfected populations which were selected for further analysis, are shown in Figure [1](#F1){ref-type="fig"}. RT negative samples were included (lower panel) to rule out DNA contamination of RNA samples. Expression of the fusion proteins was confirmed by Western blot analysis (not shown) and by indirect immunofluorescence using an antibody directed against the 6His epitope (Figure [2](#F2){ref-type="fig"}). Some diffuse fluorescence is seen in the His C controls (A and B), but this is to be expected as the fusion part of the protein is at the amino terminus and the vector therefore can produce a small protein containing the 6His epitope. There is a distinctive crescent-like pattern of fluorescence in clones transfected with the EBV BHRF1 (C and D) and H. panBHRF1 (E and F), indicative of BHRF1 expression. Determination of sensitivity to serum withdrawal induced apoptosis ------------------------------------------------------------------ To determine the sensitivity of the transfected clones to the induction of apoptosis by serum reduction, EBV clones EBV 1B and EBV 3B, H. panclones Pan 4 and Pan 5 and BHRF1 null vector control clones His C12 and His C13 were seeded in medium containing either 10% or 0.1% serum and the percentage of apoptotic cells was determined at 24-hourly intervals, using acridine orange to distinguish between apoptotic and viable cell nuclei. Necrotic nuclei that appeared after several days due to secondary necrosis of cells already lost by apoptosis were excluded from the counts. Apoptotic death was also confirmed by DNA laddering (not shown). We found that when maintained in medium containing 10% serum (Figure [3A](#F3){ref-type="fig"}), both the EBV BHRF1 clones exhibited a slight drop in viability after 48 hours and thereafter viabilities increased to above 90%. A similar drop in viability was seen in the two vector clones, but after an increase at 72 hours, both clones began to show a decline in viability by 96 hours. Both the H. panBHRF1 expressing clones, maintained viability above 90% throughout the 96 hours period. In contrast, the BHRF1 null vector controls showed higher rates of cell death compared to either the EBV or H. panBHRF1 expressing clones from 24 hours onwards in medium containing 0.1 % serum (Figure [3B](#F3){ref-type="fig"}). After 96 hours in culture, approximately 15% viability was observable in the BHRF1 null vector controls whilst the viability of the BHRF1 clones ranges from 35 to 49%. Clone EBV IB was the most resistant to apoptosis induced by serum deprivation with 49% viability after 96 hours. Two-way ANOVA and Tukey\'s pairwise analysis of arcsine transformed data showed that at all time points from 48 hours onwards there were significant differences (p \< 0.001) between the BHRF1 expressing clones and the vector control clones, but not between the Pan and EBV BHRF1 clones, except at 96 hours when clone EBV 1B showed significantly higher viability than all other clones (eg p = 0.0001 when compared to EBV 3B). Therefore our data indicates that 0.1% serum is sub-optimal for survival of Ramos-BL since a high degree of apoptotic cells are observable at this concentration. Clearly however, both EBV and H. panBHRF1 expression were able to delay the apoptotic response, and thus promote the survival of these cells which would otherwise undergo apoptosis. Determination of sensitivity to etoposide induced apoptosis ----------------------------------------------------------- We then investigated whether or not the proteins could regulate apoptosis induced by the DNA damaging agent etoposide. The transfected clones were seeded in medium containing either no etoposide or 200 ng/ml etoposide. The percentage of viable versus apoptotic cells was determined every 24 hours by acridine orange fluorescence. We found that, in the absence of etoposide, viability was maintained at above 90% in all clones (Figure [4A](#F4){ref-type="fig"}). In the presence of etoposide (Figure [4B](#F4){ref-type="fig"}), both the control clones showed a steady decline in viability from 24 hours onwards, with clone His C13 maintaining higher viability than His C12, whilst the BHRF1 clones remained viable for 48 hours before starting to show a decline in viability. Two way ANOVA and Tukey\'s pairwise comparisons of arcsine transformed data showed that the differences between the control and BHRF1 clones were significantly different from 24 hours onwards. Whilst clone His C13 was significantly different to the BHRF1 clones at all time points, it was also significantly different to His C12 at the 72 (p = 0.359) and 96 (p = 0.002) hour time points. There were no significant differences between the EBV and Pan BHRF1 clones. Determination of sensitivity to ultraviolet induced apoptosis ------------------------------------------------------------- Ultraviolet radiation has been shown to induce apoptosis in other cell lines within a 24 hour period. Having first determined the UV conditions necessary to induced apoptosis in the parent cell line, we then exposed the transfected clones to UV radiation at 30 Jm^-2^. Acridine orange counts were taken after 24 hours and the results are shown in figure [5](#F5){ref-type="fig"}. Statistical analysis was performed on both the transformed and untransformed data. The untransformed data is shown in Figure [4](#F4){ref-type="fig"}, as the error bars were so small in the transformed data that they were not visible. The results are highly significant with p \< 0.000001. Mapping the amino acid changes on to the 3D structure ----------------------------------------------------- During the course of this study, the structure of BHRF1 was solved \[[@B26]\]. Using RasMol, v2.6, the freely available molecular visualisation software created by Roger Sayle, we have produced a 3D image of BHRF1, highlighting the regions where the EBV and H. panproteins differ, as shown in Figure [6A](#F6){ref-type="fig"}. Non-conservative changes, shown in yellow, tend to be found at the end of helices, whereas conservative replacements tend to be within the helices. There are changes within the known functional BH domains (shown in red). The aligned primary amino acid sequences, with the BH domains and changes highlighted using the same colour scheme are shown in Figure [6B](#F6){ref-type="fig"}. Discussion ========== In this study we present evidence that the BHRF1 protein encoded by H. panis functionally homologous to the analogous protein in the EBV in human B cells. BHRF1 is one of two Bcl-2 homologues encoded by EBV. Previously the EBV BHRF1 protein has been shown to protect transfected B-cells from apoptosis \[[@B11]\] and delay the differentiation of epithelial cells in vitro\[[@B18]\]. The primate virus analogues of BHRF1 have now been cloned by us and others \[[@B24],[@B25]\] and have been shown to be highly homologous in their primary structure both at the DNA and protein level. BHRF1 is normally expressed at high levels in during lytic replication in epithelial cells and the first functional studies of a primate BHRF1, namely the baboon virus BHRF1, were carried out in a keratinocyte system. Whilst a role for BHRF1 during latent infection has not been proven, circumstantial evidence that BHRF1 transcripts are found in certain lymphoid malignancies and that BHRF1 can suppress apoptosis in lymphoid cells suggests that BHRF1 has the potential to contribute to malignant transformation. Whilst infection of B cells is largely latent, lytic replication in B cells also plays an important role in EBV\'s lifecycle by providing virus to re-infect epithelial cells, ensuring the continued shedding of virus in the oropharynx. We therefore decided to investigate whether the functional homology extended to a lymphoid cell background by comparing EBV BHRF1 with the previously uncharacterised BHRF1 from H. pan. We used 3 triggers known to induce apoptosis at differing rates. Serum withdrawal is a slow trigger, with low levels of apoptosis for 48 hours followed by a precipitous drop in viability. The kinetics of etoposide activity are very different with a steady decline in viabilty over the 4 day period, whilst UV irradiation (never previously reported for BHRF1) induces apoptosis within hours. The EBV and H. panBHRF1s afforded significant protection against the induction of apoptosis by all three triggers, compared to the vector controls. There was no statistical difference between the EBV and H. panBHRF1s, (except for clone EBV1B at 96 hours in 0.1% serum), suggesting that H. panBHRF1 is indeed a true functional homologue of EBV BHRF1. The difference between clone EBV1B and the other BHRF1 clones at 96 hours in low serum could possibly be attributed to slightly higher levels of BHRF-1 expression in this clone, although it is difficult to quantify absolute expression levels from the fluorescence images shown in Figure [2](#F2){ref-type="fig"}. Clone His C13 maintained higher viability than clone His C12 against all three triggers and when exposed to etoposide this is statistically significant from 72 hours onwards. This possibly reflects genetic changes accumulated by the clones during selection and passage and highlights the importance of including more than one clone in assays such as these. When one looks more closely at the regions of the protein that differ between the human and chimpanzee viruses (Figure [6](#F6){ref-type="fig"}), it is perhaps not surprising that the two proteins are functional homologues, despite the changes in the amino acid sequence. The non-conservative changes are almost always at the end of a helix where they are least likely to disrupt the overall structure of the protein, whilst the conservative changes are mainly within the helices or loops. Notably there are some changes within the BH domains, known to be critical for the function of Bcl-2 family proteins, which are clearly tolerated in terms of conservation of function. None of the observed changes are likely to significantly alter the conformation of the protein, highlighting the fact that functional conservation of BHRF1 is subject to 3D structural constraints. These observations emphasise the importance of combining structural information with homology studies such as this one to identify regions of the protein that need to be conserved to retain structural integrity. That the two proteins appear to be so similar at the 3D level implies they probably interact with cellular proteins that are themselves highly conserved in the two species. There are only a few reports of proteins known to interact with BHRF1, including PRA1 \[[@B27]\] and although some studies have shown interactions with several Bcl-2 family proteins \[[@B28],[@B29]\], the reported differences in the 3D structures of BHRF1 and Bcl-2 (namely the lack of a binding groove for pro-apoptotic Bcl-2 family members) \[[@B26]\] suggests that the anti-apoptotic activity of BHRF1 does not exactly parallel that of Bcl-2. This warrants further study into the viral and cellular proteins which interact with BHRF1, not only to yield more insight into the interplay between virus and host, but also to further our knowledge of the fundamental mechanisms of apoptosis. Conclusions =========== Our results are the first demonstration of functional homology between a primate BHRF1 (H. pan) and EBV BHRF1 in a human lymphoid cell background. Both proteins protect against apoptosis induced by two previously described triggers and also a new trigger, UV radiation, against which BHRF1 has never been reported to provide protection. Comparison of the EBV and H. panproteins at the 3D structural level reveals that none of the changes is likely to significantly alter the structure of the protein. Methods ======= Construction of expression vectors ---------------------------------- The 573 bp coding sequences of EBV BHRF1 and H. panBHRF1 were amplified by PCR from pDH222 \[[@B11]\] and pHVPanTW4 \[[@B24]\] respectively using primers which incorporated XhoI sites flanking the translational start and stop codons. We used Promega\'s PCR mastermix with 50 pmol each primer. The sequence of the forward primer was TTGCAGCTCGAGATGGCCTATTC and the reverse primer sequence was GAAAATCTCGAGATTAGTGTCTTCC. The PCR parameters were 35 cycles of 95°C (30 sec), 55°C (30 sec) and 72°C (60 sec). The PCR products were introduced by Topo TA cloning into pCR2.1-Topo^®^(Invitrogen) and the XhoIfragments were released by digestion with XhoIand cloned into the Xho1 site of pcDNA4/HisMaxC (Invitrogen). The plasmids were sequenced to verify that the sequences had joined in frame and contained no PCR induced errors. Transfection of Ramos-BL cells ------------------------------ 6 × 10^6^Ramos-BL cells were independently transfected with 20 μg endotoxin-free EBV BHRF1, H. panBHRF-1 or vector control constructs (pEBV9, pPan4 and pcDNA4/HisMax respectively) by electroporation in a BioRaD Gene Pulser II at 0.45 kV, 125 μF. The cells were seeded in 96-well plates at a density of 5 × 10^4^cells/ well in RPMI 1640 supplemented with 10% FetalClone III calf serum (HyClone) 2 mM L-glutamine (Sigma) 0.4% gentamycin (Sigma) and 200 μg/ml Zeocin (Invitrogen). Wells showing outgrowth of drug resistant cells were cloned by limiting dilution and clones were expanded into 2 ml cultures and maintained in medium containing 150 μg/ml Zeocin. All cultures were fed twice weekly by replacing 90% of the culture with fresh medium. RT-PCR ------ Total RNA was purified from 5 × 10^6^cells using the RNA Safekit (Q Biogene) according to manufacturers instructions. Reverse transcription of 100 ng RNA with oligo dT primer using the Improm II kit (Promega) in a total volume of 20 μl according to manufacturer\'s instructions was followed by PCR (parameters as above) using 5 μl of the template cDNA and 50 pmols each of the forward and reverse primers CTGTACGACGATGACGATAAG and GTGTCTTCCTCTGGAGATA respectively, to amplify a 655 bp product. Indirect immunofluorescence --------------------------- Cells were suspended in PBS and spotted onto multiwell glass slides and air dried for 60 minutes. Slides were fixed for 20 minutes in methanol at -20°C followed by 5 minutes in acetone at -20°C. The cell spots were rehydrated in 20% normal rabbit serum (in PBS) for 20 minutes then incubated with a 1 in 500 dilution of the monoclonal anti-poly histidine antibody (Clone HIS-1, Sigma-Aldrich) for 1.5 hours at room temp. After three 5 minute washes with PBS, the cells were incubated with a 1 in 50 dilution of FITC goat anti-mouse IgG (Biomeda) for 1 hour at room temp. Thereafter the slides were washed thrice in PBS, a drop of DABCO was added and coverslips were placed over the cell spots. The cells were viewed with by fluorescence microscopy and photographed with a Leica digital camera using a 1.27 sec exposure time and 15× magnification. Apoptosis assays ---------------- For the serum reduction assay cells were washed 3 times in PBS and then 0.5 × 10^6^cells (BHRF1 or vector clones) were seeded in either medium containing 10% serum or 0.1% serum and the percentage of viable versus apoptotic cells was determined by acridine orange flourescence at 24 hour intervals. For the etoposide assays, 0.5 × 10^6^cells (washed 3 times) were seeded in either routine medium containing DMSO (equivalent to the volume added to the etoposide samples) or medium containing 200 ng/ml etoposide (diluted from a 1 mg/ml solution in DMSO) and the % of apoptotic cells versus viable cells was determined every 24 hours by acridine orange fluorescence. For the UV induction assays, 1 × 10^6^cells in 10 ml of medium were placed in a Petri dish and exposed to 30 Jm^-2^UV. The cells were then pelleted by centrifugation and resuspended at 2 × 10^5^cells/ml in medium and returned to culture flasks. Each clone was tested in triplicate and each experiment was repeated 3 times. Acridine orange fluorescence ---------------------------- 10 μl acridine orange (100 μg/ml) was added to 100 μl of cell culture containing the individual clones. At least 200 cells were counted using a haemocytometer at 15 × magnification and the number of viable versus apoptotic cells in the population noted. List of abbreviations ===================== EBV -- Epstein Barr virus, H. pan-- Herpesvirus pan, PBS phosphate buffered saline, PCR -- polymerase chain reaction, UV -- ultraviolet, v-Bcl-2 -- viral Bcl-2 homologue Authors\' contributions ======================= MH did the RT-PCR and UV assay, TW constructed the expression vectors and carried out some of the statistical analyses and SAH produced the transfected clones, carried out the serum withdrawal and etoposide assays, some of the statistical analyses and did the structural comparisons. Acknowledgements ================ Thanks to Dr Tony Polwart for advice on the statistical analyses. This work was supported by BBSRC grant no: 323/C09500 and Wellcome grant no 06581/Z/01/Z (SAH), a School of Life Sciences research studentship (TW) and AICR grant no 01-106 (MH). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### RT-PCR analysis of transfected clones.The upper panel shows RT-PCR analysis of transfected clones using primers designed to amplify a 655 bp fragment of the BHRF1 transcripts. From left to right samples are 1) Promega kilobase ladder, 2) vector control clone His C12, 3) vector control clone His C13, 4) Blank 5)EBV BHRF1 clone EBV 1B, 6) EBV BHRF1 clone EBV 3B, 7) Blank 8) H. panBHRF1 clone Pan 4 9) H. panBHRF1 clone Pan 5 10) Blank 11) Ramos-BL 12) negative RT control 13) pEBV9 14) Promega 100 bp ladder. The lower panel shows the same samples (except 13 -- negative PCR control) in which the RT has been omitted in the RT step, to exclude DNA contamination in the RNA samples. ::: ![](1471-2180-5-6-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Detection of BHRF-1 fusion protein by indirect immunofluorescence.Cell smears were incubated with Clone His-1 monoclonal anti-polyhistidine antibody followed by FITC-conjugated goat anti-mouse antibody, viewed by fluorescence microscopy and photographed at 15× magnification (1.27 s exposure) using a digital camera. Key A -- His C12; B -- His C13; C -- EBV 1B; D -- EBV 3B; E -- Pan 4 and F -- Pan 5. ::: ![](1471-2180-5-6-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Determination of apoptosis levels in reduced serum conditions.The percentage viability of EBV and H. panBHRF1 Ramos-BL transfectants compared to vector controls in medium containing A) 10% serum or B) 0.1% serum over a 4 day period. The % viability was determined daily by acridine orange fluorescence. The data plotted are the means three experiments carried out in triplicate ± standard error. Statistical analysis was performed using ANOVA and Tukey\'s pairwise comparison on data normalised by arcsine transformation. ::: ![](1471-2180-5-6-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Determination of apoptosis levels in etoposide treated cells.The percentage viability of EBV and H. panBHRF1 Ramos-BL transfectants compared to vector controls in medium containing A) DMSO control or B) 200 ng/ml etoposide over a 4 day period. The % viability was determined daily by acridine orange fluorescence. The data plotted are the means three experiments carried out in triplicate ± standard error. Statistical analysis was performed using ANOVA and Tukey\'s pairwise comparison on data normalised by arcsine transformation ::: ![](1471-2180-5-6-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Determination of apoptosis levels in UV irradiated cells.The effect of 30 Jm^-2^UV irradiation (solid bars) on EBV BHRF1 and H. panBHRF1 transfected Ramos-BL clones compared to vector controls. Cell viability was assayed after 24 hours by acridine orange fluorescence. The data plotted are the means three experiments carried out in triplicate ± standard error. Statistical analysis was performed using ANOVA and Tukey\'s pairwise comparison on data normalised by arcsine transformation ::: ![](1471-2180-5-6-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### *Mapping of amino acid changes in H. panBHRF1 on the EBV BHRF1 structure.The structure of the first 160 amino acids of EBV BHRF1 was visualised by importing the structural data file (1q59.pdb) into RasMol v2.6, which converts the structural data into a 3 D image (Figure 6A) Using the RasMol edit commands we have highlighted the BH domains in red, whilst the remainder of the protein is shown in dark blue. Conservative replacements in the H. pan*protein are highlighted in light blue and non-conservative replacements are shown in yellow. For comparison the aligned amino acid sequenced are shown in Figure 6B, using the same colour coding as in Figure 6A. ::: ![](1471-2180-5-6-6) :::	PubMed Central	2024-06-05T03:55:52.818858	2005-2-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548687/", "journal": "BMC Microbiol. 2005 Feb 3; 5:6", "authors": [ { "first": "Melanie", "last": "Howell" }, { "first": "Tracey", "last": "Williams" }, { "first": "Sheila A", "last": "Hazlewood" } ] }
PMC548688	Background ========== Antihypertensive drug therapy can reduce cardiovascular morbidity and mortality by 25--30% \[[@B1]-[@B3]\] in those with hypertension. The 2004 Canadian hypertension guidelines \[[@B4]\] recommend pharmacotherapy in those patients where there is proven cardiovascular benefit from randomized controlled trial evidence. However, many of these \'at risk\' patients with hypertension remain untreated even though they have a higher risk for cardiovascular events \[[@B5]\]. This lack of treatment is a major public health problem as hypertension is a modifiable risk factor. It remains unclear as to how much of this lack of treatment is because of failure to diagnose hypertension or failure to initiate drug treatment for those with a diagnosis of hypertension. Further understanding would help to strategize public health care initiatives in controlling high blood pressure. The primary aim of this study was to determine the proportion of those untreated individuals who would be recommended to start drug therapy for control of blood pressure among those aware or unaware of their diagnosis of hypertension. Methods ======= From the 2004 Canadian Hypertension Guidelines, pharmacotherapy is recommended if the diastolic blood pressure is \> 100 mmHg; the systolic pressure is \> 160 mmHg; or the diastolic blood pressure is \>90 mmHg in the presence of cardiovascular heart disease or risk factors. To determine the proportions of individuals who satisfied the guidelines for initiation of drug treatment for hypertension, we used the Canadian Heart Health Surveys (1986 -- 1992). This national, cross-sectional descriptive survey (n = 23 129) contains data on individual blood pressure measurements using standardized technique as well as information on cardiovascular risk profile, antihypertensive medication use and self reported awareness of hypertension. Further details of this survey have been published elsewhere \[[@B6]\]. We then calculated for both aware and unaware subgroups, the number of untreated hypertensive individuals that would be recommended drug therapy/ those untreated, hypertensive individuals. Patients with diabetes mellitus were excluded from the analysis because they require antihypertensive drug therapy at a much lower threshold. Descriptive statistics were used to calculate proportions and 95% confidence intervals in this study. Results ======= Of those with untreated hypertension (= 140/90 mmHg), only 37% (n = 897 786) were aware of their diagnosis. Among those aware and unaware of their diagnosis of hypertension, 35% vs. 36% were female; 14% vs. 12% had any cardiovascular disease and 31% vs. 75% were under the age of 65 years respectively. The proportions of those who would be recommended drug therapy among those who are aware and unaware are presented in Table [1](#T1){ref-type="table"}. Table [1](#T1){ref-type="table"} demonstrates that compared to the unaware subgroup, a greater proportion of those aware of their hypertensive status needed drug therapy but were untreated. Among both aware and unaware groups, the largest proportion needing drug therapy was less than age 65 years. Of note, among those who were unaware, there was a considerably lower proportion that needed drug therapy over the age of 65 years. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Proportions of those aware and unaware who require drug therapy (95% CI) ::: ---------------------------------------------------------------------------------------------------- Awareness of hypertensive status \>65 years 18--64 years Total ---------------------------------- --------------------- --------------------- --------------------- Aware 71 477/ 138 342\ 592 186/ 759 444\ 663 663/ 897 786\ 51.7% (51.5--51.9%) 78% (77.9--78.1%) 73.9% (73.8--74%) Unaware 129 398/ 376 861\ 738 542/ 1 143 872\ 867 940/ 1 520 733\ 34% (33.8--34.2%) 64.6% (64.5--64.7%) 57.1% (57--57.2%) ---------------------------------------------------------------------------------------------------- ::: Discussion ========== In both unaware and aware subgroups, the majority of patients with untreated hypertension would benefit from antihypertensive drug therapy according to the 2004 Canadian Hypertension recommendations. This analysis extends the findings from other studies \[[@B5],[@B7],[@B8]\] by determining those \'at risk\' individuals that would be recommended pharmacotherapy based on awareness of their hypertensive status. The proportion of untreated patients that still needed drug therapy was higher among those who were aware compared to those who were unaware. Specifically, among the elderly, most of the patients who are unaware that they are hypertensive would not actually be recommended to take drug therapy. These study findings suggest that a significant gap in hypertension control may be initiating drug therapy among those known to have hypertension. Possible barriers to initiating drug therapy may arise from patients, physicians \[[@B9]\] or the health care system. There are several limitations with this study. First, the Canadian Heart Health Survey data are 12 years old and there is some evidence to suggest that prescription rates of antihypertensive drugs have improved since then \[[@B10]\]. However, this data set represents the best population data available on patients with hypertension in Canada since it is of high quality and captures a large cohort of Canadians. Another limitation is that the awareness or unawareness of hypertensive status is based on self report and not from physician records. Conclusion ========== This study found a considerable number of both unaware and aware patients with untreated hypertension require drug therapy. However, the majority of those who needed drug therapy were actually aware that they had a diagnosis of hypertension compared to those who were not aware. This finding suggests that the major gap in hypertension control may be in initiating drug therapy rather than in diagnosing hypertension. Further studies are needed to confirm these results to ultimately help strategize public health efforts in controlling hypertension. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' Contribution ====================== NK, DW and NC contributed to the design of the project. NK contributed to data analysis. NK, DW and NC contributed to writing the manuscript and for substantive, intellectual editing contributions. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2261/5/4/prepub> Acknowledgements ================ Dr. Khan is supported by Canadian Institute Health Research and Michael Smith Foundation for Health Research postdoctoral fellowship awards. The authors would also like to thank Vicki Stagg for her biostatistical support.	PubMed Central	2024-06-05T03:55:52.821140	2005-2-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548688/", "journal": "BMC Cardiovasc Disord. 2005 Feb 3; 5:4", "authors": [ { "first": "Nadia A", "last": "Khan" }, { "first": "Dennis", "last": "Wardman" }, { "first": "Norman RC", "last": "Campbell" } ] }
PMC548689	Background ========== The adult behavioral lifestyle theory of cardiovascular disease (CVD) describes an adult\'s lifestyle choices and levels of physiologic risk factors as the primary predictors of CVD risk \[[@B1]-[@B3]\]. This approach is supported by a relatively consistent literature demonstrating an inverse, graded relationship between SES and CVD \[[@B4]-[@B12]\]. There is also a growing literature that focuses upon the influence of SES at different points in life on adult CVD risk. A force behind the interest in the impact of early-life conditions on adult health is the fetal origins (Barker) hypothesis. This hypothesis, which has been met with considerable support and criticism, posits that poor nutrition during fetal and early infant development (\"critical periods\") can increase risks for adult disease. While the dominance of the adult lifestyle model of chronic disease development may not be threatened by evidence for the fetal origins hypothesis, a reconsideration of the primary importance of adult behaviors and risk factors is underway. A life course approach to chronic disease proposes that the combination, accumulation, and/or interaction of the social environments and biological insults experienced throughout the life course impact current and future events, environments, and health conditions and thus ultimately impact adult health \[[@B13],[@B14]\]. Various interrelated theories have been put forward, \[[@B14]-[@B23]\] and many study designs have been utilized to examine the impact of life course SES \[[@B13],[@B17],[@B19],[@B23]-[@B30]\]. This review describes the major groups of conceptual life course SES models, and then categorizes and summarizes studies that examine the associations between life course SES and CVD risk. Studies are grouped by the basic life course design utilized; summaries include methodologic critiques and descriptions of certain key studies. Evidence supporting each conceptual life course model is considered and future research directions are discussed. Life course SES conceptual models --------------------------------- The different extant hypotheses on the influence of life course SES on CVD can be grouped into four broad conceptual models: the latent effects, pathway, social mobility, and cumulative life course models. Most studies reviewed tested the influence of life course SES on CVD outcomes via the operation of one or more of these models. ### The latent effects model The \"latent effects\" life course conceptual model hypothesizes that adverse early life experiences increase the risk of CVD in later life, independent of intervening SES, lifestyle, or traditional CVD risk factors. Power & Hertzman (1997) describe a latent effects model wherein certain early life events may have strong independent effects on adult health \[[@B14]\]. Kuh and Ben-Schlomo (1997) propose the related concept of biological chains of risk, wherein prenatal and early life socioeconomic factors affect biologic resources and directly influence adult health \[[@B13]\]. A recent formulation suggests the operation of biological or developmental influences during early \"sensitive periods\" which permanently impact the organism \[[@B28]\]. ### The pathway model In this life course model, early life events and environments influence later life experiences, opportunities, and health risk factors. Hertzman et al. (2001) propose a developmental process linking early-life psychosocial environments with adult health risk via pathway effects, wherein early experiences place an individual onto a certain \"life trajectory,\" eventually impacting adult health \[[@B28]\]. Similarly, Kuh et al. (1997) use the concept of \"social chains of risk,\" whereby early events influence later life experiences, thus impacting adult disease risk \[[@B20]\]. Blane (1999) describes an \"ongoing social process\" wherein a continuity of social circumstances are linked and may create a \"chain of disadvantage \[[@B31]\].\" While a pathway life course model is intuitively appealing, its operation is difficult to test empirically. Life course studies typically collect information on participants at two or three time points, which does not permit the continuous, lifelong operation of pathway effects to be observed. ### The social mobility model Social mobility theories all hypothesize that SES mobility across the life course impacts adult health, although the different proposed theories posit different health effects. Forsdahl (1978) hypothesized that deprivation in early life followed by later affluence combine to produce elevated CHD mortality risk, partly via elevation of adult cholesterol levels \[[@B32]\]. Others proposed that natural \"health selection\" occurs, wherein less healthy individuals tend to have downward social mobility and healthier individuals tend to be upwardly mobile \[[@B30],[@B33]\]. In contrast, the \"health constraint\" hypothesis contends that socially mobile individuals possess health characteristics of both the SES group they leave and the one they join, so that social mobility minimizes the health differences between SES groups \[[@B15],[@B34]\]. ### The cumulative model A cumulative SES life course model hypothesizes that psychosocial and physiological experiences and environments during early and later life accumulate to influence adult disease risk. Davey-Smith et al. (2002) suggest that if factors operating at different life stages are combined, large differences in CVD risk will be observed \[[@B35]\]. Kuh et al. (1997) describe an individual\'s biological resources accumulated over the life course as their \'health capital\' \[[@B20]\], which describes and influences current and future health. The \"accumulation of risk\" model described by Ben-Schlomo and Kuh (2002) proposes that the impacts of different life course events accumulate but do not interact \[[@B26]\]. Methods ======= We conducted a MEDLINE search using keywords and MeSH terms related to SES, life course, early life, longitudinal studies, CVD and CVD risk factors. References identified and books on life course research were also examined for study citations. Inclusion criteria were: (1) publication date between January 1966 and July 2003; (2) SES or related measures as independent variables; (3) outcomes of subclinical CHD, CVD morbidity and/or mortality, or traditional CVD risk factors. Behavioral risk factors were limited to: adult diet, physical activity, alcohol consumption, and smoking. Physiological outcomes were: measures of obesity, blood pressure, intima-media thickness, fibrinogen, insulin resistance, dyslipidemia, and lung function. Ecologic studies, studies primarily evaluating the fetal origins hypothesis, and studies only examining the impact of birth weight, height, upper leg length, patterns of growth, household crowding, geographic mobility or numbers of siblings were excluded. Forty-nine studies met the inclusion criteria. Quantitative summarization of the study findings is problematic due to varied study populations, SES measures and CVD outcomes evaluated, time points evaluated, and designs utilized. Moreover, a tally approach to summarizing across studies based on statistical significance can be misleading due to the different strengths, weaknesses, power and biases of each study (p. 671) \[[@B36]\]. Summaries therefore consist primarily of qualitative information on the types and directions of associations observed and the findings reported by authors. Although we avoid focusing on statistical significance, in most studies the authors\' decision to declare a positive finding was tied to a null-hypothesis significance test using a p-value of \< 0.05. Categorization of life course studies by life course study design ----------------------------------------------------------------- Studies were categorized by their life course study design(s) and study question(s) into four groups: early SES → outcome, early SES → risk factor, social trajectory, and cumulative SES studies. Several studies appear in multiple categories, as they employed more than one of these designs. Table [1](#T1){ref-type="table"} describes the research questions and hypotheses typically considered by each design. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Life course study designs: hypotheses tested and typical study questions posed ::: Life course study design Typical chronological analysis set-up used in study design Typical study question ------------------------------ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------- Early SES → Outcome Early-life SES variable(s) used to predict CVD (e.g., CHD, stroke). Adjusted for later-life events, behaviors, risk factor levels to determine \"direct\" effect of early life SES. Is there a significant independent effect of early-life (childhood) SES on the adult risk of CVD after adjusting for later-life SES and risk factors? Early SES → Risk Factor Early-life SES variable(s) used to predict adult CVD risk factors levels. How does early-life SES affect later-life levels of behavioral and physiological CVD risk factors? Social Trajectory Inter- or intragenerational movement from one SES level to another (i.e., Low SES to High SES) used to predict adult CVD risk factors levels or CVD outcomes. How does social mobility from one point to another during the life course affect the risk of CVD? Cumulative SES A summary variable indicating number of negative SES events/environments over the life course used to predict adult CVD risk factors levels or CVD outcomes. How does the accumulated number of negative SES-related exposures across the life course influence the risk of CVD? CHD = Coronary heart disease. ::: Results ======= Early SES → outcome life course studies: overview ------------------------------------------------- These studies examined the effect of childhood and/or adolescent SES on risk of adult CVD (e.g., incident myocardial infarction (MI), CVD death, incident/fatal stroke) \[[@B37]-[@B56]\] and typically tested the latent effects hypothesis (i.e., examining the \"direct\" effect of early-life SES on adult CVD risk). They commonly measured the independent effect of childhood SES by statistically adjusting for later-life SES and CVD risk factors in regression models. Early SES → outcome studies: summary of results ----------------------------------------------- Table [2](#T2){ref-type="table"} lists these 20 studies according to CVD outcome utilized (see [Additional file 1](#S1){ref-type="supplementary-material"} for greater detail on these studies). All 19 studies conducting unadjusted or age-adjusted analyses reported a point estimate consistent with an inverse association between early-life SES and risk of one or more of the adult cardiovascular outcomes. Authors of 14 studies concluded that their findings supported some or all of their study hypotheses \[[@B39],[@B41]-[@B45]\], [@B47]\[[@B49]-[@B55]\]. Fourteen of 16 studies adjusting for adult SES and/or CVD risk factors reported some indication of an inverse association; however, in only five did the authors suggest that their results support the hypothesis of an inverse association between early life SES and CVD. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Summary of SES -- CVD life course studies using an early SES → outcome design ::: ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 1^st^Author, year & reference number\ Early life SES measures Variables adjusted for other than age Study name ------------------------------------------- ------------------------------------------------------------------------------------------- ------------------------------------------------ Acute/ Survived MI, CVD Burr 1980 \[49\]\ Father\'s occup (RG, 3 groups), father unemployed (\> 1 year), family size Current SES South Wales hospital cohort Notkola 1985 \[40\]\ 5-level index: father\'s occup & farm size Current SES, CVD RF\'s East-West Study Coggon 1990 \[37\]\ Father\'s occup (RG, 5 groups), height, sibling death Current SES, smoking Stoke-on-Trent & Newcastle study Hasle 1990 \[48\]\ 8 variables (yes/no) on parent\'s occup, health, household, residence, edu, illness None Danish worker\'s union study Kaplan 1990 \[43\]\ Factor analysis of edu, occup, farm, farm size, perceived wealth CVD RF\'s Kuopio Study Lundberg 1993 \[51\]\ 4 yes/no variables: economic hardship, large family, broken family, dissension Current SES, gender Swedish population cohort study Gliksman 1995 \[39\]\ Father\'s occup at 16 years Current SES, CVD RF\'s Nurses\' Health Study Lamont 2000 \[41\]\ Birth: father\'s occup; 5 & 10 years: parent\'s occup, housing, \# of adverse life events Current SES, CVD RF\'s Newcastle 1,000 Families Cohort Marmot 2001 \[46\]\ Childhood: father\'s occup (RG, 4 groups), age at leaving edu\ Current & child SES Whitehall II Study Labor force entry: Occup Wamala 2001 \[45\]\ Early-life SES disadvantage index (0--3) of 3 variables: large family, born last, low edu Current SES, CVD RF\'s Stockholm Study Stroke Gliksman 1995 \[39\]\ Father\'s occup (4 groups) at 16 years Current SES, CVD RF\'s Nurses\' Health Study Coggon 1990 \[37\]\ Father\'s occup (RG, 5 groups), height, sibling death Current SES, smoking Stoke-on-Trent & Newcastle study Davey Smith 1998 \[44\]\ Father\'s occup (RG, 4 groups), also mnl vs. non-mnl Current SES, CVD RF\'s Collaborative Study Frankel 1999 \[54\]\ Father\'s occup (RG, 5 groups) Townsend area deprivation score Boyd Orr Cohort Dedman 2001 \[56\]\ Persons/room, tap water (yes/no), toilet type, ventilation, cleanliness (3 levels each) Childhood SES, area deprivation score Boyd Orr Cohort CHD Mortality Notkola 1985 \[40\]\ 5-level index using father\'s occup & farm size Current SES, CVD RF\'s East-West Study Lynch 1994 \[38\]\ SES index (3 groups), by parents\' edu, occup, farm, perceived wealth Current SES Kuopio Study Vagero 1994 \[50\]\ Occup of head of household (mnl, non-mnl, unemployed) Current SES Uppsala Birth Cohort Study Gliksman 1995 \[39\]\ Father\'s occup (4 groups) at 16 years Current SES, CVD RF\'s Nurses\' Health Study Davey Smith 1998 \[44\]\ Father\'s occup (RG, 4 groups), also divided into mnl vs. non-mnl Current SES, CVD RF\'s, area deprivation Collaborative Study Hart 1998 \[42\]\ Early SES: father\'s occup; Labor force entry: Occup; At screening: occup None Collaborative Study Frankel 1999 \[54\]\ Father\'s occup (RG, 5 groups) Townsend area deprivation score Boyd Orr Cohort Davey Smith 2001 \[47\]\ Father\'s social class, (RG, 5 groups) CVD RF\'s Glasgow Alumni Cohort Dedman 2001 \[56\]\ Persons/room, tap water (yes/no), toilet type, ventilation, cleanliness (3 levels each) Childhood SES, Townsend area deprivation score Boyd Orr Cohort Davey Smith 2002 \[52\]\ Father\'s occup (mnl/non-mnl) Current SES, CVD RF\'s Collaborative Study Claussen 2003 \[53\]\ Index of housing conditions items Current SES Oslo Mortality Study Osler 2003 \[55\]\ Father\'s social class (3 groups) by occup Birth weight, IQ at age 12 Project Metropolit ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- CHD = Coronary heart disease; Edu = Education; IHD = Ischemic heart disease; MI = Myocardial infarction; Mnl = Manual occupational class; Non-mnl = Non-manual occupational class; Occup = Occupation; RF = Risk factor; RG = Registrar General\'s social class categories. ::: For studies with CHD and stroke outcomes limited to early-life SES (i.e., not controlling for adult SES or other risk factors) almost all included a point estimate consistent with an inverse association. However, in adjusted analyses, authors of only one study with a CHD-related outcome and fewer than half the studies of CVD mortality reported inverse adjusted associations. Thus, the existence of a \"direct effect\" of early-life SES after adjusting for adult risk factors was not strongly supported. Early SES → outcome studies: methodologic issues ------------------------------------------------ To prevent statistical confounding by adult conditions, most studies employed statistical models that adjusted the association between childhood SES and adult CVD risk for adult SES and behavioral/ physiological CVD risk factors. This may be an over-adjustment, as certain risk factors (e.g., BMI, smoking) are part of the pathways through which low childhood SES may influence CVD risk. Little information is usually available on how participants\' early-life SES may have influenced levels of adult CVD risk factors. Additionally, if unknown variables influence both the distribution of a CVD risk factor adjusted for and the distribution of the outcome measure, then adjustment will result in an incorrect partitioning of the total effect of early-life SES into \"direct\" and \"indirect\" components \[[@B57]-[@B59]\]. Thus, early SES → outcome studies which seek to determine the \"direct,\" adjusted effect of early-life SES on CVD risk may incorrectly estimate this effect \[[@B60]\], leading to questions about the accuracy of such estimates. Some studies examined and qualitatively compared the effects of adjustment for different classes of CVD risk factors. Gliksman et al. (1995), for example, analyzed the association between father\'s occupational class and adult CVD in a series of models adjusting for different classes of potential mediators or confounders. This allowed for a more complete description of the impact of adult environment, behavior, and physiologic risk factors versus the latent impact of early life socioeconomic factors \[[@B39]\]. Studies utilizing more than one life course study design (\"mixed-design studies\") take a related approach and are considered in the Discussion section. Early SES → risk factor studies: overview ----------------------------------------- Life course studies in the early SES → risk factor group evaluated the influence of early- and/or mid-life SES or living conditions on later-life behavioral or physiologic CVD risk factors \[[@B14],[@B40],[@B44],[@B52],[@B61]-[@B73]\]. These designs are often similar to those used in the early SES → outcome studies group except that CVD risk factors are the outcomes. The life course hypotheses typically examined, however, include both the latent effects and pathway conceptual models. The typical study design involves a statistical model where early-life SES measures predict the levels of CVD risk factors in adulthood. Early SES → risk factor studies: summary of results --------------------------------------------------- Table [3](#T3){ref-type="table"} outlines the 17 studies reviewed (see [Additional file 2](#S2){ref-type="supplementary-material"} for detailed summaries). Five of six studies reported associations between low early-life SES and little or no adult leisure-time physical activity \[[@B14],[@B61],[@B63],[@B64],[@B66]\], five of five found associations with high adult alcohol intake \[[@B52],[@B61],[@B63],[@B68],[@B73]\], and eight of twelve studies reported associations with higher smoking rates as study findings \[[@B14],[@B44],[@B52],[@B63],[@B65]-[@B68]\]. Nine of 11 studies reported (unadjusted) associations between lower early-life SES and elevated BMI or WHR \[[@B14],[@B44],[@B63],[@B64],[@B67],[@B68],[@B70],[@B71],[@B73]\]. The impact of a statistical adjustment for other CVD risk factors varied, including no impact \[[@B59]\], attenuation with a persistence of the significant effect \[[@B46],[@B55]\], and strong attenuation with associations no longer apparent \[[@B54],[@B78]\]. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Summary of SES -- CVD life course studies using an early SES → risk factor design ::: ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 1^st^Author, year & reference number\ Early life and/or adult SES measures Variables adjusted for other than age CVD risk factor(s) measured Study name ------------------------------------------- ------------------------------------------------------------------------------------------------------------- ------------------------------------------- ---------------------------------------------------------------------------------- Arnesen 1985 \[65\]\ Early-life: 4-level index of household economic conditions CVD RF\'s Cholesterol, SBP, glucose, BMI, smoking, more Tromso Heart Study Notkola 1985 \[40\]\ Early-life: 5-level index using father\'s occup & farm size; Adult: occup (6 groups) None Smoking, cholesterol, SBP East-West Study Wadsworth 1985 \[98\]\ Early-life: index of father\'s occup & parents\' edu; Adult: occup (RG) & employment CVD RF\'s SBP, DBP Medical Research Council\ National Survey of Health Study Braddon 1986 \[71\]\ Early-life: 2 social class indices of father\'s occup; Adult: occup (RG, 8 groups), edu (high/low) Adult SES, CVD RF\'s Obesity (BMI \> 30.0 for men, \> 29.1 for women) British 1946 Birth Cohort Peck 1994 \[66\]\ Early-life: father\'s occup (7 groups); Adult: occup (7 groups) None Smoking, physical activity Swedish census cohort study Blane 1996 \[64\]\ Early-life: father\'s occup (RG, 4 groups); Adult: occup (RG, 4 groups) Adult SES DBP, physical activity, smoking, BMI, FEV1, more Collaborative Study Lynch 1997 \[99\]\ Child: SES index (3 groups); Adolescent: edu (3 groups); Adult: occup, income, possessions, more Energy intake Smoking, drinking, obesity, physical activity, diet Kuopio Study Power 1997a \[14\]\ Child: father\'s occup (RG, 4 groups); At 23 years: occup, edu; At 33 years: occup None BMI (obesity) 1958 British Birth Cohort Power 1997b \[90\]\ Child: father\'s occup (RG, 4 groups); At 23 years: occup, edu; At 33 years: occup None Smoking, BMI (obesity) 1958 British Birth Cohort Davey Smith 1998 \[44\]\ Early-life: father\'s occup (4 groups); Adult: occup (6 groups) None Smoking, DBP, cholesterol, BMI, FEV1 Collaborative Study van de Mheen 1998 \[63\]\ Early-life: father\'s occup (6 groups); Adult: occup (6 groups) Adult SES BMI, smoking, alcohol, leisure physical activity Longitudinal Study, Netherlands Brunner 1999 \[67\]\ Early-life: father\'s occup (RG, 4 groups); Adult: occup (Civil Service grade, 4 groups) Adult SES Smoking, activity, HDL, BMI, fibrinogen, more Whitehall II Study Davey Smith 2002 \[52\]\ Early-life: father\'s occup (mnl/non-mnl); Adult: occup (mnl/non-mnl) None Smoking, alcohol, area deprivation Collaborative Study Lawlor 2002 \[68\]\ Early-life: father\'s occup (RG, 6 groups); Adult: current occup (RG, 6 groups) Adult SES Insulin resistance, SBP, cholesterol, BMI, smoking, triglycerides, alcohol, more British Women\'s Heart Study Poulton 2002 \[73\]\ Early-life: parental occup (6 groups) at 0, 3, 5, 7, 9, 11, 13 & 15 years; Adult: current occup (3 groups) Adult SES BMI, WHR, SBP, smoking, alcohol, more Dunedin Multidisciplinary Study Lawlor 2003 \[69\]\ Early-life: father\'s longest occup (mnl / non-mnl); Adult: longest occup (RG, 6 groups) CVD RF\'s HOMA, SBP, HDL, triglycerides British Women\'s Heart Study Parker 2003 \[70\]\ Birth: father\'s occup & housing; 5 & 10 years: same, plus adverse life events; Adult: wage earner\'s occup None CMS, BMI, WHR, fasting insulin, triglycerides, HDL Newcastle 1000 Families Study ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- BMI = Body mass index; CMS = Central metabolic syndrome; DBP = Diastolic blood pressure; Edu = Education; FEV1 = Forced expiratory volume in 1 second; HOMA = Homeostasis model assessment score; Mnl = Manual occupational class; Non-mnl = Non-manual occupational class; Occup = Occupation; RF = Risk factor; RG = Registrar General\'s social class categories; SBP = Systolic blood pressure; WHR = Waist-to-hip ratio. ::: Early SES → risk factor studies: methodologic issues ---------------------------------------------------- As with the early SES → outcome studies, concerns that lack of adequate adjustment or over-adjustment may lead to biased estimates are germane to this group of studies. The issue of covariate adjustment is complex in these studies, given that many CVD risk factors are considered. Certain physiologic risk factors (e.g., insulin resistance, dyslipidemia) may be linked to early-life SES through latent physiologic effects of negative childhood exposures, continuous exposure to negative physiologic stimuli, or other physiologic or life course pathway mechanisms \[[@B14],[@B63],[@B70],[@B74]\]. In contrast, the links between early-life SES and levels of behavioral risk factors (e.g., smoking, diet), and certain physiologic risk factors (e.g., BMI, hypertension), may operate principally through learned behaviors, behavioral responses to negative psychosocial stimuli, or other psychosocial life course pathway effects. The associations may develop through the operation of multiple physiologic and psychosocial pathways, making \"correct\" covariate adjustment challenging within a single model. Thus, statistical adjustment should take into account the specific life course pathways hypothesized to be operating \[[@B39]\]. Previous findings should be used to generate a priori hypotheses; nonetheless, there is considerable opportunity for inappropriate adjustments to be made. Social trajectory studies: overview ----------------------------------- Studies evaluating the social mobility life course model typically considered the impact of inter-generational or intra-generational social mobility on CVD and CVD risk factors \[[@B38],[@B44],[@B49],[@B64],[@B72],[@B73],[@B75]-[@B79]\]. Inter-generational mobility was usually determined by contrasting the participant\'s father\'s occupational SES to the participants\'. Intra-generational SES was typically defined as a change in occupational SES from early adulthood to later adulthood. Six of 11 studies evaluated adult CVD risk factors as outcomes \[[@B49],[@B64],[@B72],[@B73],[@B75],[@B77]\]; seven evaluated CVD mortality or CHD \[[@B38],[@B44],[@B75]-[@B79]\]. Four adjusted only for age \[[@B38],[@B49],[@B76],[@B80]\]. Social trajectory studies: summary of results --------------------------------------------- Table [4](#T4){ref-type="table"} outlines these studies (see [Additional file 3](#S3){ref-type="supplementary-material"} for greater detail). Of 10 studies carrying out statistical analyses \[[@B38],[@B44],[@B49],[@B72],[@B73],[@B75]-[@B79]\], six did not report associations between upward or downward mobility and either elevated levels of CVD risk factors or increased CVD morbidity or mortality when compared to stable low-SES or high-SES trajectories \[[@B38],[@B44],[@B49],[@B75],[@B78],[@B79]\]. Nine studies, however, reported the suggestion of inverse, although not always statistically significant, relationships between social mobility and a CVD-related outcome \[[@B38],[@B44],[@B72],[@B73],[@B75]-[@B79]\]. Two studies found marked differences in CVD mortality risk between upwardly mobile individuals and individuals maintaining the same SES across time. However, one study reported increased CVD risk among the upwardly mobile \[[@B77]\] and one reported decreased mortality risk \[[@B79]\]. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Summary of SES -- CVD life course studies using a social trajectory design ::: ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 1^st^Author, year & reference number\ SES measures Variables adjusted for other than age CVD risk factor(s) / outcomes measured Study name ------------------------------------------- --------------------------------------------------------------------------------------------------------------------------------------- --------------------------------------------------- ------------------------------------------------ Kaplan 1971 \[76\]\ Early-life: father\'s occup (7 groups); Early Adult & Adult: occup & social class (5 classes) None MI, chronic IHD, AP, sudden death Evans County Heart Study Gillum 1978 \[77\]\ Early-life: by parental occup (blue/white collar); Adult: assumed to be at least middle class A confounder summarizing score AP, HTN, fatal or non-fatal CHD or MI Harvard Alumni Cohort Burr 1980 \[49\]\ Father\'s occup (RG, 3 groups), father unemployed (\> than 1 year), family size, current occup (RG, 3 groups) None Survived MI South Wales hospital cohort Wadsworth 1985 \[72\]\ Early-life: index of father\'s occup and parents\' edu; Adult: occup (RG), employment & edu for women Smoking, edu, father\'s CVD, BMI, more SBP, DBP British 1946 Birth Cohort Faresjo 1994 \[78\]\ Early-life: father\'s SES (3 groups) by occup, social class; Adult: same; all mobility relative to child SES SBP, cholesterol, smoking MI Swedish Study of Men Born in 1913 Lynch 1994 \[38\]\ Early-life: SES index (3 groups); Adult: current income (2 groups) None CVD mortality Kuopio Study Blane 1996 \[64\]\ Early-life: father\'s occup (RG, 4 groups); Adult: occup (RG, 4 groups) BMI, cholesterol, DBP, smoking, activity, FEV1 BMI, cholesterol, DBP, smoking, activity, FEV1 Collaborative Study Hart 1998 \[75\]\ Early-life: father\'s occup (mnl/non-mnl); Labor force entry: occup (mnl/non-mnl); Adult: occup (mnl/non-mnl), area deprivation index Smoking, DBP, cholesterol, FEV1, angina, ischemia CVD mortality & risk factors listed Collaborative Study Davey Smith 1998 \[44\]\ Early-life: father\'s occup: (RG, mnl/non-mnl); Adult: occup: (RG, mnl/non-mnl) Smoking, DBP, BMI, area deprivation, more Mortality from CHD & stroke Collaborative Study Poulton 2002 \[73\]\ Early-life: parental occup (6 groups) at 0, 3, 5, 7, 9, 11, 13 & 15 years; Adult: current occup (3 groups) Infant health index, gender, adult SES BMI, WHR, SBP, smoking, alcohol dependence Dunedin Multidisciplinary Study Pensola 2003 \[79\]\ Early-life: father\'s occup (mnl vs. non-mnl); Adult: current occup (mnl vs. non-mnl) None CVD mortality Finnish census cohort ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- AP = Angina pectoris; BMI = Body mass index; CHD = Coronary heart disease; DBP = Diastolic blood pressure; Edu = Education; FEV1 = Forced expiratory volume in 1 second; HTN = Hypertension; IHD = Ischemic heart disease; MI = Myocardial infarction; Mnl = Manual occupational class; Non-mnl = Non-manual occupational class; Occup = Occupation; RG = Registrar General\'s social class categories; SBP = Systolic blood pressure; WHR = Waist-to-hip ratio. ::: Four studies examined differences in CVD risk between stable low-SES trajectory and stable high-SES trajectory individuals \[[@B38],[@B44],[@B75],[@B79]\]. Three reported that individuals with stable low-SES trajectories had a greater CVD risk than stable high-SES trajectory individuals \[[@B44],[@B75],[@B79]\]; the fourth reported a marginally significantly greater risk \[[@B38]\]. These results are similar to those of most cumulative SES studies (described below) in that greater exposure to low SES was associated with increased CVD risk. Social trajectory studies: methodologic issues ---------------------------------------------- In these studies the unit of analysis is a trajectory, permitting the impact of change over time to be examined. However, the socioeconomic trajectories in most reviewed studies were limited to two time points, and groups compared tended to share the same SES at one of these time points. These similarities may partly explain why seven of ten studies did not report an association between social mobility and risk of CVD risk factors or events. Social trajectory studies incorporating SES at three or more time points allow for the analysis of more informative trajectories than studies evaluating SES at only two points \[[@B75]\]. Yet, even with large studies, analysis becomes cumbersome if more than two or three SES levels at three time points are measured. Additionally, the impact of uncommon (e.g. downward) trajectories are difficult to study due to the small numbers of individuals who typically comprise them. Cumulative SES studies: overview -------------------------------- These studies tested the operation of the cumulative life course conceptual model, typically by summing the number of times participants experienced unfavorable SES situations during early, middle or later life, and creating SES indices representing the accumulation of these experiences \[[@B45],[@B52],[@B53],[@B68],[@B79],[@B81],[@B82]\]. For example, three of these studies summed the number of times a participant (or their parents during the participant\'s childhood) had been in a manual occupation to create an index of accumulated low-SES exposure \[[@B68],[@B81],[@B82]\]. Cumulative SES was also measured using indices of occupational class, socioeconomic categories, exposures to negative socioeconomic experiences/conditions, income, and housing conditions. \[[@B45],[@B52],[@B53],[@B68],[@B81],[@B82]\]. Cumulative SES studies: summary of results ------------------------------------------ Table [5](#T5){ref-type="table"} summarizes the seven cumulative SES studies (see [Additional file 4](#S4){ref-type="supplementary-material"} for greater detail). All authors reported that participants\' cumulative life course exposure to low SES conditions was associated with increases in CVD outcome, supporting the cumulative life course SES hypothesis. Several studies indicated that cumulative SES was a more powerful predictor of CVD morbidity and/or mortality than adult or early-life SES alone \[[@B45],[@B53],[@B79],[@B81]\]. In studies that adjusted for CVD risk factors, graded associations were attenuated but remained strong in two studies \[[@B81],[@B82]\] and were greatly attenuated in another \[[@B45]\]. ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Summary of SES -- CVD life course studies using a cumulative SES design ::: ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 1^st^Author, year & reference number\ Cumulative SES measure(s) Variables adjusted for other than age CVD risk factor(s) / outcomes measured Study name ------------------------------------------- ----------------------------------------------------------------------------------------------------------------------------------- ---------------------------------------------------------------- ---------------------------------------------------------------------- Davey-Smith 1997 \[82\]\ Sum of \# of times at mnl vs. non-mnl SES using father\'s, own first, & own current occup class BMI, DBP, FEV1, cholesterol, smoking, AP, ischemia, more CVD, mortality, AP, smoking, BMI, DBP, cholesterol, more Collaborative Study Heslop 2001 \[81\]\ Sum of \# of times at mnl vs. non-mnl SES using father\'s, own first, & own current class DBP, BMI, FEV1, cholesterol, activity, smoking, alcohol CVD mortality, DBP, BMI, FEV1, exercise, smoking, alcohol, more Collaborative Study Wamala 2001 \[45\]\ Sum of \# of instances (0--6) of SES disadvantage (large family, born last, low edu, blue-collar/ housewife, economic hardship) Height, HDL, HTN, marriage, fibrinogen, obesity, smoking, more Cases: CHD event (acute MI, unstable/ recurrent AP) Stockholm Study Davey-Smith 2002 \[52\]\ Sum of \# of risks (0--6): Father mnl SES, left edu at \< 15 years, current mnl SES, smoking, high alcohol, high deprivation area None CVD mortality Collaborative Study Lawlor 2002 \[68\]\ Cross-classification of father\'s longest occup and current occup (RG, mnl/non-mnl) None Insulin resistance, HTN, smoking, triglycerides, LDL, HDL, BMI, more British Women\'s Heart Study Claussen 2003 \[53\]\ Early life: Index of housing conditions (scored 0--7) Adulthood: standardized income (7 groups) None CVD mortality Oslo Mortality Study Pensola 2003 \[79\]\ Sum of \# of times in mnl vs. non-mnl class, by father\'s occup & own occup at 30--34 None CVD mortality Finnish census cohort ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- AP = Angina pectoris; BMI = Body mass index; CHD = Coronary heart disease; DBP = Diastolic blood pressure; Edu = Education; FEV1 = Forced expiratory volume in 1 second; HTN = Hypertension; MI = Myocardial infarction; Mnl = Manual occupational class; Non-mnl = Non-manual occupational class; Occup = Occupation; RG = Registrar General\'s social class categories. ::: Davey-Smith et al. (2002) employed a unique cumulative SES measure, combining early and later-life occupational class experience with the CVD risk factors of smoking and heavy alcohol consumption \[[@B52]\]. They reported a marked, graded relationship between the number of negative SES exposures and risk of CVD mortality. Only a large cohort study of Norwegians reported a statistically significant supra-multiplicative interaction between early- and later-life SES on risk of CVD \[[@B53]\]. Another study reported marginally statistically significant supra-multiplicative interaction effects between early and later life \[[@B45]\]. Cumulative SES studies: methodologic issues ------------------------------------------- The reported associations between cumulative SES and CVD risk are more consistent than those reported in the other life course study designs. However, three of the seven studies evaluated were based on the same cohort \[[@B52],[@B81],[@B82]\]. Cumulative life course SES variables include current SES, and therefore may be conflating the effect of current SES with that of SES over the life course. For example, in Pensola et al. (2003), social mobility analyses suggested that the observed gradient in CVD mortality associated with cumulative manual occupational class was driven primarily by the impact of current occupational status. Additionally, cumulative indices implicitly assume that a specific negative life experience or situation has the same impact regardless of when it occurs in an individual\'s lifetime, with no distinction made between the impact of childhood versus adult events on risk of disease \[[@B45]\]. Furthermore, some cumulative SES studies combined disparate measures into a single, lifetime index variable, or summed the number of times different negative events or exposures occur \[[@B45],[@B52]\]. This approach assumes that different types of exposures have an equivalent impact on the risk of CVD. Evidence supporting these assumptions is not provided in these studies. Discussion ========== General life course SES study issues ------------------------------------ Certain general limitations and assumptions of life course SES studies should be considered. First of all, SES is a theoretical construct operationalized using various measures (e.g., income, occupation, education) that tap into different components of this construct. Despite several proposed SES evaluation schemes \[[@B83]-[@B85]\], there is no overarching theory in the biomedical literature providing a rationale for the use of specific SES measures. Occupational status, the SES measure most commonly used in the studies reviewed, is assumed to be an adequate proxy of SES, but in fact represents only one component of SES. As associations of a given SES measure with CVD risk factors are not always consistent with those of other SES measures \[[@B47],[@B60]\], it is important to consider that results may depend upon the proxy SES measure employed \[[@B64],[@B81]\]. Most life course studies used retrospective cohorts or a case-control design, relying on participants\' recall of early life SES. There has been little systematic evaluation of the validity of recalled early life circumstances or of the potential for such recall errors to bias associations. In studies directly comparing the impact of childhood and adult SES on adult CVD risk, greater error in childhood (vs. adult) SES measures may underestimate the true impact of child SES \[[@B86],[@B87]\]. Additionally, selection bias due to either loss to follow-up or selective survival may distort findings. Studies using cohorts followed from birth or early life probably do not have these limitations \[[@B14],[@B28],[@B47],[@B62],[@B71],[@B72],[@B77],[@B88]-[@B93]\] and may avoid the problem of changing occupational status and workplace functions across time if they establish SES scales at the time of measurement and update them as appropriate. As more studies analyze data from prospective cohorts starting in early life, concerns about unequal measurement error and the changing status of specific occupations should decrease \[[@B14],[@B54],[@B73],[@B89],[@B93],[@B94]\]. Most studies were limited to white males. While the number of studies including women has increased in recent years, minority groups remain underrepresented in most life course studies. These groups may interact differently with the economic and/or social systems of the majority group; there are also established associations between minority status, low SES, and increased CVD risk \[[@B5],[@B84],[@B85]\]. The lack of information on the manner in which life course SES relates to adult CVD risk in minorities should be addressed. Support for life course effects on CVD risk ------------------------------------------- The majority of the early SES → outcome group studies\' results describe an inverse association between early life SES and risk of adult CHD, non-fatal MI, and CVD mortality. Similarly most early SES → risk factor studies suggest that low early-life SES negatively impacts levels of adult CVD risk factors. Most studies reported relationships between lower early-life SES and elevated alcohol intake, BMI/WHR, and insulin resistance, as well as decreased leisure physical activity. Together, the early SES → outcome and early SES → risk factor studies support the hypothesis that low early-life SES is inversely associated with adult risk of CVD. However, support for the hypothesis of a \"direct effect\" of early-life SES on risk of CVD mortality is equivocal as findings were less consistent after risk factor adjustment. Additionally, reported associations between early-life SES and adult health behaviors detract from the evidence supporting a latent effects life course model, as they suggest that CVD risk may be driven by learned behaviors. The operation of social chains of risk or life trajectories described by a pathway life course model are difficult to observe or test, since almost all early SES → risk factor studies consider only two points during the life course. However, significant associations between early-life SES and the adult risk factors suggest behavioral, psychosocial or environmental links that may be best explained as a pathway effect. As described below, the pathway model will need further examination through the use of series of linked studies. While nine of 10 social trajectory studies reported some relationship between inter- or intra-generational social mobility and CVD-related outcomes, only four were identified as providing evidence in favor of a social mobility hypothesis. Conversely, most studies comparing stable low- and high-SES trajectory groups reported markedly increased CVD mortality among the low-SES group, with many socially mobile groups having a CVD risk between the stable high and low groups. Determining whether these associations are a function of social mobility, or whether they are due to exposure to low SES at some point in the life course and not to mobility, is problematic. Future studies should focus on differentiating between the influences of cumulative SES and social mobility. Of the conceptual models discussed, the cumulative life course model was the most consistently supported. However, the weight of these findings is limited as much of the data came from one cohort \[[@B57],[@B60],[@B61]\]. Also, specific methodological concerns (e.g., equal weighting of life course periods, conflation of current and life-course SES) need to be considered when interpreting these findings. Future studies conducted in other cohorts that address these concerns will help clarify the viability of cumulative life course SES hypotheses. Directions for future research ------------------------------ Researchers have noted the limitations of life course SES study designs examining only one potential pathway linking SES and CVD. Accordingly, recent studies tend to use a combination of SES measures, CVD risk factor and/or outcomes and life course study designs \[[@B40],[@B44],[@B45],[@B52],[@B53],[@B68]\]. These mixed-design studies allow for a comparison of how well different life course SES conceptual models fit the patterns observed in the same data. Such studies are also less likely to overlook patterns of association in the data than studies which only evaluate one possible relationship between SES and CVD. As modern life course cohorts are followed from childhood, longer prospective studies or series of cross-sectional studies that evaluate associations between early-life experiences and risk factor levels at several time points may provide the opportunity to observe the operation of pathway life course effects. For example, two studies in the early SES → risk factor group \[[@B14],[@B90]\] compared levels of CVD risk factors at two points in young adulthood (22 and 33 years of age). The authors observed an association between childhood SES and adult obesity, which was smaller for participants at 33 than at 23 years of age \[[@B14]\], suggesting a decreasing impact of early life SES on adult health with increasing age. Such information is only obtained through longer ongoing studies or series of interrelated studies. Individuals\' socioeconomic environments may impact their CVD risk factor levels and CVD risk. Community socioeconomic conditions may influence individuals\' ability to engage in leisure physical activity or eat a healthy diet \[[@B95]-[@B97]\]. Some life course studies have evaluated the community socioeconomic environment, primarily through the use of indicators of neighborhood deprivation \[[@B44],[@B48],[@B54],[@B81],[@B82]\]. Future studies should likewise consider the effect of physical and psychosocial environmental factors. Life course SES studies have generally failed to consider the length of exposure to the various socioeconomic conditions measured. As this may influence the impact of negative SES experiences on adult health, future cumulative life course studies may benefit from evaluating the effect of length of exposure into their indices. Conclusions =========== The wide range of populations, analysis designs, exposures, and outcomes used in the life course studies reviewed precludes a simple, quantitative analysis of the impact of life course SES on CVD risk. Nevertheless, the results thus far modestly support the existence of life course SES effects on risk of adult CVD. The cumulative life course model is more consistently supported by extant studies than other models. However, the different methodologic issues of each study design make direct comparisons of the relative support for each conceptual model difficult. Analyses utilizing multiple life course designs within the same study offer the best approach to testing which theories best describe the links between life course SES and CVD risk. The inclusion of minority participants, different SES measures, and data from early and middle adulthood in large, prospective, mixed-design studies will allow more informed and generalizable statements to be made about the impact of life course SES on adult disease risk. While this area of research needs methodologic refinement, it offers a promising and informative perspective from which to understand the development of chronic disease. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= R. A. Pollitt conducted the literature search, reviewed and categorized the articles and had primary responsibility for writing the manuscript. K. M. Rose assisted in the categorization and summarization of the papers reviewed, and helped to revise the manuscript in response to the reviewer\'s comments. All authors participated in interpreting the studies\' results and preparing the methodologic criticism, provided input on the various drafts, and read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2458/5/7/prepub> Supplementary Material ====================== ::: {.caption} ###### Additional File 1 SES -- CVD life course studies using an early SES → outcome design ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 SES -- CVD life course studies using an early SES → risk factor design ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 3 SES -- CVD life course studies using a social trajectory design ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 4 SES -- CVD life course studies using a cumulative SES design ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ R. A. Pollitt was funded by National Heart, Lung, and Blood Institute (NHLBI) Training Grant 5T32-HL007055-27, and by the Life Course SES, Social Context and Cardiovascular Disease (LC-SES) study (NHLBI grant 5-RO1-HL064142-01-04).	PubMed Central	2024-06-05T03:55:52.822101	2005-1-20	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548689/", "journal": "BMC Public Health. 2005 Jan 20; 5:7", "authors": [ { "first": "Ricardo A", "last": "Pollitt" }, { "first": "Kathryn M", "last": "Rose" }, { "first": "Jay S", "last": "Kaufman" } ] }
PMC548690	Background ========== Humans have two enzymes with α-galactosidase activity and an acidic pH optimum, α-N-acetylgalactosaminidase (α-NAGA) (previously called α-galactosidase B) and α-galactosidase A (α-GAL). Hereditary deficiency of each of the hydrolases causes a distinct lysosomal storage disorder in humans, Schindler and Fabry diseases, respectively \[[@B1],[@B2]\]. Early studies suggested that both human enzymes were glycoforms with similar substrate specifities. Purified enzymes had similar physical properties, including subunit molecular mass (\~46 kDa), homodimeric structure, and amino acid sequences. However, additional studies showed kinetic, structural, and immunologic differences proving that α-GAL and α-NAGA were products of two different genes \[[@B3],[@B4]\]. The two genes differed in the number of exons (7 and 9, respectively) and also in the number, placement, and orientation of Alu repeats. Exons 2 -- 7 of the α-NAGA gene showed high similarity to the first six exons of α-GAL gene. Because of the remarkable amino acid identity (49%) and similarity (63%) between the two genes and the similar intron placement, Wang \[[@B5]\] and co-workers suggested that a duplication event occurred during the evolution of both enzymes. Both enzymes belong to the glycoside hydrolase family 27 clan D \[[@B6]\]. Glycoside hydrolase family 27 clan D orthologs have been identified in a broad spectrum of prokaryotes and eukaryotes, including plants. Members of the family have a highly similar active site and share the same reaction mechanism. The structures of chicken α-NAGA, human α-GAL and rice α-GAL have been determined by X-ray crystallography \[[@B7]-[@B9]\]. Chicken and human enzymes have a homodimeric quarternary structure whereas rice α-GAL acts as a monomer. The monomer units are composed of two distinct domains. Domain I contains the active site and adopts a (β/α)~8~barrel structure, a domain fold observed commonly in glycosidases. Domain II has eight antiparallel β strands, packed into two β sheets in a β sandwich fold containing a Greek key motif \[[@B8]\]. The physiological importance of both enzymes is evidenced by the severe presentation of α-NAGA and α-GAL deficiencies in humans \[[@B1],[@B2]\]. Our recent study on degradation of blood group A and B glycolipids in Fabry cells indicated a high residual activity in Fabry cells toward natural substrate glycolipid B-6-2 \[[@B10]\] although α-galactosidase activity was completely absent when measured in vitro by routine procedures using artificial substrates. We proposed that another enzyme, different from α-GAL, contributes in vivo to hydrolysis of α-galactosides. We suggested α-NAGA as the most likely candidate. Human α-NAGA is known to accept α-galactosides albeit with a high K~m~\[[@B11],[@B12]\]. Its activity must be inhibited when measuring α-GAL in tissues with high α-NAGA activity \[[@B13]\]. We investigated the phylogenesis of a single C. elegansα-[GA]{.underline}L and α-[NA]{.underline}GA ortholog (gana-1) to both human genes. We present evidence suggesting that this gene has indeed evolved from the α-GAL/α-NAGA ancestral gene before the duplication event which resulted in separate α-NAGA and α-GAL genes in higher metazoans. We further performed structural analysis of the GANA-1 3D model acquired by homology modeling. We determined the spatial and temporal expression of the gene in transgenic worms using a translational reporter and examined the effect of RNA interference (RNAi) as a first step in the possible use of C. elegansas a model organism for Schindler and Fabry diseases. Results and discussion ====================== cDNA amplification and sequencing --------------------------------- The complete C. elegansgenome \[[@B14],[@B15]\] contains only one open reading frame, designated gana-1(R07B7.11) that has sequence similar to human genes encoding α-GAL and α-NAGA. Similar results were obtained from searching the available C. briggsaegenome sequence \[[@B15]\]. The gana-1gene consists of 5 exons and 4 introns and is annotated as an ortholog of human α-NAGA. Several EST clones for this open reading frame (ORF) have been reported and open-reading-frame sequence tag (OST) is present in the Worfdb database \[[@B16]\]. Available public database data are in agreement with our findings. We verified the gene structure by sequencing the PCR products from genomic DNA and cDNA (Figure [1](#F1){ref-type="fig"}). The analyzed region spanned the entire coding region and the 3\' and 5\' untranslated regions (UTR). The 5\' UTR SL1 element suggests that the gene is either the only gene transcribed from the promoter or is the first gene in an operon including gana-1and the two predicted downstream genes R07B7.12 and R07B7.13. Although this region has not been reported as an operon, the physical distances between this cluster of three genes are suggestive of an operon \[[@B17]-[@B19]\]. No alternative splicing was found using RT/PCR, a feature similar to both human and mouse orthologs. RNA editing was reported in the 3\' UTR of human α-GAL, a finding that another group was unable to confirm \[[@B20]\]. We noted no signs of RNA editing in clones derived from the gana-1cDNA. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **gana-1gene structure.*Schematic representation of gana-1gene structure. The length of genomic DNA from start to stop codons is 1681 bp. The spliced cDNA consists of 1356 bp + 91 bp of 3\' UTR. ::: ![](1471-2121-6-5-1) ::: Phylogenetic studies -------------------- We aligned GANA-1 protein sequence with other melibiase family members (Figure [2](#F2){ref-type="fig"}) and constructed a phylogenetic tree (Figure [3](#F3){ref-type="fig"}). The alignment showed a striking similarity of GANA-1 to all other included sequences. GANA-1 had the highest sequence similarity with Anopheles gambiaeGAL (46%), the lowest similarity was observed with Streptomyces avertimilisGAL (22%). The results of our phylogenetic analysis are in accordance with generally accepted evolutionary concepts. The analysis identified four main clades: animal NAGAs, animal GALs, plant/lower organisms GALs and the clade containing sequences of Drosophila melanogaster, Anopheles gambiaeand Caenorhabditis elegans. The branch including C. elegansis positioned between higher animal GALs and NAGAs and plant/lower organisms GALs. This position in the tree infers the evolutionary ancestrality of gana-1gene to both animal GALs and animal NAGAs. However, this conclusion is not in complete agreement with the presence of pairs of genes in Drosophilaand Anophelesgenomes annotated as α-GALs and α-NAGAs. The presence of these genes in the Caenorhabditis/Drosophila/Anophelesbranch (and not in the GAL and NAGA clades) could be due to low divergence of these sequences from a common ancestral gene or to independent gene duplication in the Drosophila/Anophelesancestral organism. It is also important to note that the phylogenetic analysis by maximum parsimony algorithm placed the Caenorhabditis/Drosophila/Anophelesbranch into the neighborhood of the animal NAGAs branch \[[@B8]\]. In this case the computational algorithm probably favored the lower number of necessary sequence changes (parsimony) between GANA-1 and NAGA clade sequences. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Multiple alignment.Multiple alignment of 29 sequences homologous with GANA-1. These sequences represent animal, plant and protozoan kingdoms. The SwissProt/TrEMBL codes are part of the sequence names. Predicted signal peptides are shown in brown letters. In cases where two signal sequence prediction algorithms gave different results the difference is marked by amber color. The residues forming active site pocket of GANA-1 are indicated by arrowheads above the alignment. The catalytic domain I is indicated by green band above the alignment. ::: ![](1471-2121-6-5-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Cladogram of GANA-1 orthologs.Cladogram of GANA-1 orthologs. The numbers at the branch nodes represent bootstrap values. ::: ![](1471-2121-6-5-3) ::: In our opinion, the phylogenetic analysis provides evidence that the GANA-1 evolved from a common ancestor of α-GAL and α-NAGA enzymes. However, the topology of the tree could also be explained by a loss of the second gene during the evolution of C. elegans. In this case the enzyme found in C. eleganswould probably be the ortholog of human α-NAGA and the lost gene would likely be the ortholog of human α-GAL. The likelihood of these two hypotheses depends on functional divergence of duplicated gene products and their dispensability for organism\'s metabolic pathways \[[@B21]\]. Homology modeling ----------------- The best Squared Root of Mean Square Deviations value (RMSD), found between GANA-1 backbone atoms and the chicken α-NAGA template \[[@B7]\], was 0.52 Å. The structural model of the enzyme molecule has a two-domain structure (Figure [4A](#F4){ref-type="fig"}). Domain I, which contains the predicted active site, adopts a (β/α)~8~barrel structure which represents a common motif in many glycoside hydrolases. Less conserved is domain II that has a β domain with β sandwich structure containing a Greek key motif. The active site pocket of GANA-1 is formed by the same twelve amino acids (W31, D76, D77, Y118, C126, K152, D154, C156, S186, A189, Y190 and R211) (Figure [4B](#F4){ref-type="fig"}) as in chicken α-NAGA. This finding infers their identical function in catalytic reactions as described for chicken α-NAGA \[[@B7]\]. D134 carboxyl starts the initial nucleophilic attack and D215 carboxylic oxygen serves as a subsequent donor and acceptor of the proton during the reaction cycle. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### GANA-1 protein model.A) Ribbon representation of GANA-1 monomer model. A two-domain structure is apparent in the left picture. The N-acetyl-D-galactosamine (inhibitor) is placed into the active site. Dots represent VdW radii of surface atoms. B) Stereo picture of the active site pocket with N-acetyl-D-galactosamine (inhibitor) and amino acid labels. The viewing angles for stereo representation of the protein structure are ±2 degrees from the central axis. ::: ![](1471-2121-6-5-4) ::: Residues forming the \"N-acetyl recognition loop\" in the chicken α-NAGA \[[@B8]\] (S172, A175, Y176) have the closest contact with the N-acetyl moiety of the ligand. These residues are completely conserved between human and chicken NAGAs, but in human α-GAL serin 172 is replaced by glutamine and alanine 175 is replaced by leucine. The replacements with bulkier residues apparently discriminate between α-GAL and α-NAGA substrates. While NAGAs can accommodate α-galactose and can have some α-GAL activity, GALs do not have α-NAGA activity and are not inhibited by N-acetylgalactosamine. The corresponding residues of GANA-1 in the NAGA recognition loop are S186 and A189 and are characteristic for NAGAs. According to Garman \[[@B8]\] the key residue in the dimer interface in human α-GAL is F273. Residues corresponding to this position in other orthologs can serve as predictive markers of the protein quartenary structure. Phenylalanine or tyrosine is present in enzymes that act as homodimers while glycin indicates a monomeric structure \[[@B8]\]. The equivalent residue to human α-GAL F273 in GANA-1 is lysine at position 257 which is suggestive of homodimeric structure due to its sterical properties. The homology modeling showed that a groove opposing K257 is formed by residues T260, L261, D262, M263, I389, V390 and V391 of the other monomer unit of GANA-1. In the case of chicken α-NAGA these residues are equivalent to S246, Y247, E248, Q249, N375, P376 and S377 (for details see [Additional file 1](#S1){ref-type="supplementary-material"}). Biochemical studies ------------------- Standard bacteria/nematode separation protocol previously used by other authors \[[@B22],[@B23]\] while evaluating lysosomal enzyme activites is based on sucrose flotation approach. We avoided standard sucrose flotation of worms because we could not exclude unpredictable artifacts caused by this compound, which is known to induce artificial lysosomal storage in eukaryotic cells and to alter lysosomal gene expression at concentrations significantly lower \[[@B24]\] than those used in flotation protocols. We found both α-GAL and α-NAGA enzymatic activities in the homogenates from C. elegansN2 strain using 4-methylumbelliferyl (MU) substrates. The α-NAGA activity was dominant over the α-GAL activity. The activity of α-NAGA measured at 37°C was 430 nmol.mg^-1^.h^-1^with MU-α-N-acetylgalactosaminide compared to the activity of α-GAL with MU-α-galactopyranoside of 43 nmol.mg^-1^.h^-1^(about 10% of that of α-NAGA). In the assay of α-GAL, the degradation of the MU-α-galactopyranoside was inhibited up to 95% in the presence of N-acetyl-D-galactosamine (D-GalNAc), whereas in the presence of D-galactose (D-Gal) the degradation of the same substrate was inhibited up to 75%. In the assay of α-NAGA, the degradation of the MU-α-N-acetylgalactosaminide was inhibited up to 97% by D-GalNAc and up to 90% by D-Gal. No inhibition of α-NAGA and α-GAL activity by D-glucose was observed. According to published observations in human enzymes, D-GalNAc has no inhibitory effect on α-GAL activity. On the other hand, human α-NAGA activity is inhibited by both, D-GalNAc and D-Gal \[[@B25]\]. The model of GANA-1 predicts only one active site per monomer of the enzyme. If the enzyme had activity toward both substrates, MU-α-D-galactopyranoside and MU-α-N-acetylgalactosaminide, it is to be expected that D-GalNAc and D-Gal would inhibit both activities. The strong inhibitory effect of D-GalNAc on the α-GAL activity, which is not present in human α-GAL, supports the hypothesis that C. eleganshas only one enzyme with both α-GAL and α-NAGA activities. Nevertheless, these experiments were not conducted with the pure enzyme and thus do not provide absolute proof of this hypothesis. RNA interference ---------------- RNA interference assays directed against the whole coding region of gana-1, employing combination of microinjection and feeding approaches, did not reveal any abnormal morphological phenotypes. Nevertheless, measurement of α-GAL and α-NAGA activities in four different experiments showed a simultaneous decrease of both enzymatic activities in RNAi-treated worms (Table [1](#T1){ref-type="table"}) as compared with control animals. In all RNAi experiments, both α-GAL and α-NAGA activities decreased proportionally, usually by tens of percent of activity of appropriate controls. The activity of the control enzyme (β-hexosaminidase) did not differ between the RNAi-treated nematodes and controls (data not shown). This finding supports the specificity of gana-1RNAi. The differences between individual experiments are not surprising due to the well-known variability in the efficiency of RNAi \[[@B26]\]. The results of RNAi experiments further support the hypothesis that GANA-1 has both enzymatic activities. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### α-GAL and α-NAGA activities after gana-1RNAi. The table shows a proportional parallel decrease of both enzymatic activities (α-GAL and α-NAGA) after gana-1RNAi compared to controls. ::: experiment sample α-GAL α-GAL (% of control) α-NAGA α-NAGA (% of control) α-NAGA/α-GAL (% of control) ------------ -------------- ------- ---------------------- -------- ----------------------- ----------------------------- 1 control 1.78 100 53.13 100 gana-1RNAi 1.19 67 26.63 50 0.75 2 control 13.26 100 221.68 100 gana-1RNAi 11.1 84 195.13 88 1.05 3 control 3.43 100 61.68 100 gana-1RNAi 1.02 30 11.75 19 0.63 4 control 9.6 100 212.1 100 gana-1RNAi 2.9 30 50.69 24 0.80 ::: Both enzymatic activities were lower in RNAi-treated and control worms cultured on the bacterial strain HT115 \[[@B26]\] compared to a N2 strain cultured on the OP50 strain. RNAi previously provided sufficient level of inhibition of structural lysosomal proteins for development of abnormal phenotypes in the worm \[[@B27],[@B28]\]; however, it is apparently not efficient enough for lysosomal catalytic proteins. Expression of gana-1* ---------------------- To study the expression of gana-1in C. elegans, we created transgenic worms with a reporter gene containing the entire coding region of gana-1C-terminally tagged with green fluorescent protein (GFP) under the control of a 3 kb region of the gana-1hypothetical promoter. The presence of the gana-1::GFPtransgene in the worms was confirmed on the level of genomic DNA, cDNA and protein. However, no GFP signal was observed by fluorescence microscopy under the standard laboratory conditions. As Western blotting showed the presence of fusion protein of the expected size (data not shown), we assumed that the absence of the GFP signal was caused by a pH-dependent quenching of GFP fluorescence, which has neutral to alkaline optimum (pH 5.5--12) \[[@B29]\]. To study the tissue and intracellular distribution of the fusion protein, we resorted to immunofluorescence detection of the transgene product. Immunofluorescence detection of GFP fusion protein showed a specific and coarsely granular cytoplasmic pattern of fusion protein expression. This transgene product was limited to body wall muscle cells (30% of population) (Figure [5A](#F5){ref-type="fig"}, [Additional file 2](#S2){ref-type="supplementary-material"}) or found in a broader tissue distribution that included body wall muscle cells, intestinal cells and coelomocytes (3--5% of population) (Figure [5B](#F5){ref-type="fig"}, [Additional file 3](#S3){ref-type="supplementary-material"}). This latter staining pattern is consistent with the GFP detection in NH~4~Cl and concanamycin A (CON A) experiments (Figure [6](#F6){ref-type="fig"}) discussed below. The expression of the transgene was observed in about 30% of the population which corresponded to usual expression efficiency of extrachromosomal array transgenes \[[@B30],[@B31]\]. The immunofluorescence staining protocol resulted in a significant decrease of inherent intestinal granular autofluorescence previously assigned to secondary lysosomes \[[@B32]\]. The decrease of autofluorescence intensity together with its poorly defined emission spectra hampered co-localization study. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Immunofluorescence detection of GANA-1::GFP.A) A coarsely granular cytoplasmic distribution of immunopositivity (green) in body-wall muscle cells (arrowheads). Two non-transgenic worms are shown in the background (asterisks) for comparison. Nuclei are counterstained in red. B) Detailed view of two body wall muscle cells with coarsely granular cytoplasmic distribution of immunopositivity (arrowheads) and a coelomocyte (asterisk), both pictures were acquired by 3D rendering of initial confocal Z-stacks. Note: compare with figure [6](#F6){ref-type="fig"}. ::: ![](1471-2121-6-5-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Alkalization of transgenic worms using CON A.Two coelomocytes showing a GFP signal in a membrane bound vesicular compartment (arrowheads) after 24 hour incubation in 50 nM CON A. DIC/fluorescence merged image. ::: ![](1471-2121-6-5-6) ::: To confirm that the absence of the GFP signal was due to the quenching of fluorescence by low pH in the acidic cellular compartment, we used two agents specifically alkalizing acidic cellular compartment \[[@B33],[@B34]\] to enhance the GFP emission. Soaking of gana-1::GFPtransgenic animals in NH~4~Cl or CON A resulted in a distinct GFP signal in a vesicular compartment of endocytically active coelomocytes in a small proportion of worms (3--5% of population). The GFP signal intensity was dependent on the time of incubation and the concentration of the alkalizing agent used. The first visible GFP signal was observed after 8 hour incubation in 100 mM NH~4~Cl and within 2 hours of incubation in 50 nM CON A. Lower concentrations of both NH~4~Cl and CON A did not result in visible GFP signal even after prolonged incubation of up to 24 hours. The reappearance of the GFP signal after treatment of the worms with compounds increasing the acidic compartment pH indirectly confirms lysosomal localization of the fusion protein. The GFP signal in coelomocytes had the same coarsely granular pattern as that observed after immunostaining. Limited access of alkalizing agents to the tissues can explain the differences between the results of immunofluorescence and alkalization studies. Conclusions =========== Our findings showed that gana-1is the only ortholog of human α-NAGA and α-GAL in C. elegans. Based on phylogenetic and homology modeling analyses we speculate that GANA-1 most probably developed from a hypothetical ancestral gene before the duplication event which gave rise to separate α-NAGA and α-GAL genes. We also speculate that gana-1gene product has both α-NAGA and α-GAL activities as detected in C. eleganshomogenates. Importantly, both activities in the worm were inhibited by D-galactose and N-acetyl-D-galactosamine, which is a specific inhibitor of human α-NAGA and does not inhibit α-GAL. The GANA-1::GFP fusion protein had a pattern of distribution that is compatible with lysosomal subcellular localization. The lysosomal localization of the fusion protein was also supported by pH sensitive fluorescence of GFP that was detectable only after alkalization of the acidic cellular compartment. Not suprisingly, RNAi of gana-1yielded no abnormal morphological phenotypes, most likely because it did not provide sufficient knockdown of enzymatic activities, necessary for development of lysosomal storage as observed in human pathology states. Nevertheless, gana-1RNAi resulted in a partial decrease of both enzymatic activities supporting the notion that this gene encodes both of them. It is possible that a deletion allele of gana-1may provide more insight into the function of gana-1and efforts are underway to isolate such alleles. Deletion alleles of lysosomal hydrolases may serve as valuable models of human lysosomal storage disorders. Methods ======= C. elegansmethods, strains and nomeclature -------------------------------------------- The wild type Bristol N2 strain was used for all experiments and was handled under standard laboratory conditions as described previously \[[@B35]\]. Standard methods were used for DNA microinjection \[[@B36]\] and dsRNA synthesis and microinjection \[[@B37]\]. Nomenclature is in agreement with available Genetic Nomenclature for Caenorhabditis elegans\[[@B15]\] and has been approved prior to manuscript submission. BLAST search ------------ Wormbase (2002--2004 versions and freeze versions \[[@B15],[@B38],[@B39]\]) databases were repeatedly searched for human α-GAL and α-NAGA orthologs using the BLASTP \[[@B40]\] program set at default values. Amino acid sequences of human lysosomal α-GAL and α-NAGA (acc. no. NP\_000160 and acc. no. NP\_000253 \[[@B41]\]) were used as query sequences. cDNA amplification and sequencing --------------------------------- Total RNA was isolated from mixed stages of N2 cultures \[[@B42]\] and reverse transcribed with an oligodT-T7 (5\'-AATACGACTCACTATAG) primer and Superscript reverse transcriptase (Invitrogen). The entire coding region of R07B7.11 was PCR amplified in two overlapping PCR products, with intragenic primers designed according to available Wormbase \[[@B15]\] and Worfdb \[[@B16]\] data. SL1 primer (5\'GGTTTAATTACCCAAGTTTGAG) and SL2 primer (5\'GGTTTTAACCCAGTTACTCAAG) \[[@B17]\] together with gene specific primer (5\'ATCCTGATTAATTTTAATTGC) were used to amplify 942 bp of the 5\' part of the cDNA and to evaluate the mode of transsplicing; the 1142 bp fragment of the 3\' end of cDNA was amplified with T7 primer and a gene specific primer (5\'CTTAAGTTTGGAATTTATGAA). The dominant PCR products were cloned with TOPO TA cloning kit (Invitrogen) into the pCR 2.1 vector. Positive clones were sequenced using the Li-Cor automated fluorescent sequencer and sequences were aligned with R07B7 reference cosmid sequence in the AlignIR software (Li-Cor) to evaluate splicing boundaries and overall gene organization. Multiple alignment and phylogenetic analyses -------------------------------------------- Confirmed or predicted amino acid sequences of melibiase family members \[[@B43]\] representing plant, unicellular, and animal kingdoms were aligned using ClustalW algorithm \[[@B44]\] and Blosum62 matrix. The SwissProt/TrEMBL \[[@B45]\] accesion code and source organism of the sequences are depicted in Figure [2](#F2){ref-type="fig"}. The sequence alignment was used for phylogenetic analysis with the software package PHYLIP \[[@B46]\]. The phylogenetic tree is based on 100 bootstraped input alignments and was constructed by maximum likelihood method with Jones-Taylor-Thornton matrix model \[[@B47]\]. Sequence identities between species were calculated without signal sequence in EMBOSS by Needleman-Wunsch global alignment algorithm with Blosum62 matrix, gap penalty -- 10 and gap extension penalty -- 0.5 \[[@B8],[@B48],[@B49]\]. Signal peptides were predicted at the SignalP server \[[@B50]\] both by algorithms using neural networks and Hidden Markov Models. The results were compared to known signal sequences. The differences between signal peptides predicted by the algorithms are depicted in Figure [2](#F2){ref-type="fig"}. The 3D model of GANA-1 is based on the X-ray structure of chicken α-NAGA, rice α-GAL and human α-GAL (PDB codes 1ktcA, 1uas and 1r47, respectively) \[[@B7]-[@B9],[@B51]\]. The model was created using the automated homology modeling server SwissModel with structure refinement and model evaluation in the DeepView program \[[@B52]\]. The print quality figures (Figure [4](#F4){ref-type="fig"}) and animations ([Additional file 1](#S1){ref-type="supplementary-material"}) were ray traced using PovRay software package \[[@B53]\]. Transgenic GFP expression ------------------------- The entire coding region of the gana-1gene, including 3 kb of its 5\'upstream sequence, was amplified from N2 genomic DNA through a nested PCR reaction using DyNAzyme EXT™ PCR system (Finnzymes) and two pairs of primers: the external pair (5\'GTGAGAGTGGGGAGATAGAA and 5\'TCAATTTGCTTGAGGTACATA) and the internal primers, with overhangs containing SphI and SalI restriction sites respectively (5\'ACATGCATGCAACTTTCACAGGAACACAAC and 5\'CGACGTCGACAATTGAACTCTATTGGTTCTCAA). The amplified DNA fragment (4709 bp) was cloned using TOPO-XL cloning kit (Invitrogen) into the pCR-XL-TOPO vector. The SphI and SalI gana-1restriction fragment was recloned into the GFP reporter vector pPD95.67 (supplied by A. Fire, Stanford University). The in-frame nature of the insert was confirmed by sequencing. The green fluorescent protein (GFP) fusion construct pJH3 (50 ng/μl) and pRF4 plasmid (50 ng/μl) used as the dominant marker were co-injected into the gonads of young adult N2 worms. Transgenic animals were screened for GFP signal. Nikon Eclipse E800 with C1 confocal module and 488 nm and 543 nm lasers and differential interference contrast (DIC) optics was used for specimen examination. EZ-C1 software (Nikon) was used for picture analysis and 3D rendering (Additional Files [2](#S1){ref-type="supplementary-material"}, [3](#S3){ref-type="supplementary-material"}). Alkalization of acidic cell compartment --------------------------------------- Mixed stage pJH3 and N2 (control) cultures were harvested from NGM OP50 plates and washed with water. Worms were pelleted by centrifugation (max. 1000 RPM, 2 min.) between the washes. Worms were treated with either one of two agents (NH~4~Cl, concanamycin A -- CON A) \[[@B33],[@B34]\], that are known to specifically increase pH in the cellular acidic compartment. For the NH~4~Cl method, animals were suspended in 0, 10, 25, 50, 75 and 100 mM aqueous solutions of NH~4~Cl. Small aliquots of worms were examined after 30 min, 2, 4, 6, 8 and 24 hours. For CON A, animals were suspended in 0, 10, 20, 50, 100, 150, 200 nM solutions of CON A in aqueous media. Small aliquots of worms were examined after 1, 3, 6 and 24 hours. Microscopical examination was performed as described above. Immunofluorescence ------------------ The fixation and immunofluorescence staining procedures were based on the approaches of Nonet et al. \[[@B54]\]. Mixed stage N2 cultures were harvested from NGM OP50 plates and washed thoroughly in M9 buffer to remove intestinal bacteria. Worms were pelleted by centrifugation (1000 RPM, 2 min.) between the washes. Worms were fixed overnight in 4% paraformaldehyde in 100 mM sodium/potassium phosphate buffer. Afterwards the pellets were washed three times in 1 × PBS, and incubated in 1% Triton X-100, 100 mM Tris (pH 7.0), 1% β-Mercaptoethanol overnight at 37°C to reduce the cuticle. After 5 washes in 1 × PBS, the worms were incubated for 5 hours in 900 U/ml collagenase type IV (Sigma) diluted in Krebs-Ringer solution (119 mM NaCl, 25 mM NaHCO~3~, 11.1 mM glucose, 1.6 mM CaCl~2~·H~2~O, 4.7 mM KCl, 1.2 mM KH~2~PO~4~, 1.2 mM MgSO~4~·7H~2~O, pH 7.4). The reduction/digestion step was performed twice. Pellets were washed 3 times with 1 × PBS and stored for further processing in AbA buffer (1 × PBS, 0.1% Triton X-100, 1% BSA, 0.05% NaN~3~). AbA buffer was used for antibody dilution. Primary antibody (polyclonal rabbit anti-GFP IgG (Abcam)) was diluted 1:500. Secondary antibody (goat anti-rabbit IgG Alexa Fluor 488 labeled (Molecular Probes)) was diluted 1:1000. Both incubations were performed overnight at room temperature, with AbB buffer (1 × PBS, 0.1% Triton X-100, 0.1% BSA, 0.05% NaN~3~) washes in between. Nuclei were counterstained with SYTOX orange (Molecular Probes) and the microscopic evaluation was performed as described above. Western Blotting ---------------- Mixed stage pJH3 and N2 cultures were harvested from NGM OP50 plates. Worms were homogenized by sonication and the concentration of protein was measured by the Hartree method \[[@B55]\]. The proteins (equivalent of 25--50 μg of protein per lane) were separated by SDS-PAGE gradient gel (4% to 20% polyacrylamide) and transferred onto nitrocellulose membrane by semi-dry blotting. The membrane was treated according to a common Western blotting protocol with chemiluminiscence detection (SuperSignal, West Pico) \[[@B56]\]. Rabbit polyclonal anti-GFP IgG (Abcam, dilution 1:5000) was used as the primary antibody, the secondary antibody was goat anti-rabbit IgG/Px (Pierce, diluted 1:20 000). RNA mediated interference ------------------------- The PCR product containing the entire gana-1cDNA was cloned into pCRII-TOPO vector (Invitrogen) and L4440 double promoter vector for microinjection and feeding RNAi respectively. In-vitro transcription employing T7 and SP6 RNA polymerases (Promega) was used to generate antisense single stranded RNA molecules, which were annealed to generate double stranded RNA (dsRNA). dsRNA was microinjected into N2 worms which were fed on HT115 \[[@B26]\]E. colistrain carrying L4440 plasmid with gana-1insert. The F~1~and early F~2~progeny was screened for morphological phenotypes. N2 worms microinjected with water and fed on HT115 E. colitransformed with L4440 vector without insert were used as a control. 5--7 worms were microinjected both with dsRNA and water in each of 4 separate experiments, single worm progeny reaching 110--150 individuals. Determination of α-GAL and α-NAGA and β-hexosaminidase activities ----------------------------------------------------------------- Prior to all activity measurements worms were washed from culture plates and repeatedly (6 times) washed and centrifuged in M9 buffer and finally pelleted by centrifugation. 4-methylumbelliferyl (MU)-α-D-N-acetylgalactosaminide (1 mM), 4-MU-α-D-galactopyranoside and 4-MU-β-D-glucopyranoside in theMcIlvaine buffer (0.1 M citrate/0.2 M phosphate buffer at acidic pH) were used as enzyme substrates. Reaction mixtures (sample and enzyme substrate) were incubated at 37°C and reactions were stopped by 600 μl of 0.2 M glycine/ NaOH buffer (pH 10.6) \[[@B13],[@B57]\]. Fluorescence signal of the 4-methylumbelliferone was measured on the luminiscence spectrofotometer LS 50B (Perkin Elmer) (emission 365 nm and excitation 448 nm). Inhibitors (N-acetyl-D-galactosamine, D-galactose and D-glucose) were used in 0.1 M final concentration. All measurements were performed in doublets. Authors\' contributions ======================= JH carried out molecular, RNAi, expression and biochemical analyses and wrote the first draft of the manuscript. JS participated in the design of all experiments and participated on the bioinformatic, molecular, RNAi and expression analyses and wrote the final version of the manuscript. RD performed phylogenetic analyses including homology modeling. HP participated on the biochemical analyses. MK and JL participated on the coordination of the project. MH conceived the project and provided fundraising. All authors read and approved the final version of the manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Structure of GANA-1 dimer. The color of the backbone represents differences of amino acids between GANA-1 and chicken NAGA. Blue color represents identical residues and orange stands for non-conservative changes. The colors from cyan to green represent different degrees of conservation. The surface of one monomer unit at the interface area is rendered with colors representing electrostatic potential. N-acetyl-D-galactosamine (inhibitor) is placed in the active site pocket of both monomer units (D-GalNAc arrowhead). K257 arrowhead depicts predicted dimerisation residue. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 Immunofluorescence detection of GANA-1::GFP in muscle cells. 3D volume rendered and animated image corresponding to Figure [5A](#F5){ref-type="fig"} ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 3 Immunofluorescence detection of GANA-1::GFP in muscle cells and coelomocytes. 3D volume rendered and animated image corresponding to Figure [5B](#F5){ref-type="fig"} ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ This work was supported by a grant 303/02/1324 from the Czech Science Foundation and partially also from grant VZ111100003 from Ministry of Education of the Czech Republic (institutional support). We would like to thank Michael Krause, Ph.D. (NIDDK) and Zdeněk Kostrouch, Ph.D. (Institute of Inherited Metabolic Disorders) for critical reading of the manuscript and Eva Brožová, Kateřina Šimečková, Hana Prouzová and Lenka Brtvová (Institute of Inherited Metabolic Disorders) for technical advice and assistance.	PubMed Central	2024-06-05T03:55:52.826510	2005-1-27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548690/", "journal": "BMC Cell Biol. 2005 Jan 27; 6:5", "authors": [ { "first": "Jana", "last": "Hujová" }, { "first": "Jakub", "last": "Sikora" }, { "first": "Robert", "last": "Dobrovolný" }, { "first": "Helena", "last": "Poupětová" }, { "first": "Jana", "last": "Ledvinová" }, { "first": "Marta", "last": "Kostrouchová" }, { "first": "Martin", "last": "Hřebíček" } ] }
PMC548691	Background ========== Chicken is a model organism in various fields of biology, such as embryology or immunology. It is also the only bird species for which the genome has been studied in detail and a lot is expected from its use in comparative genome analyses. This will help to detect sequences conserved between species, which should correspond to unknown exons and to regulatory or other functional regions. Such analyses will therefore be essential for the annotation of other genomes, including that of human \[[@B1],[@B2]\]. Chicken is also actually the only major agricultural species for which a draft assembly of the genome sequence is available <http://www.ensembl.org/Gallus_gallus/>\[[@B3],[@B4]\]. Thanks to a significantly lower rate of interspersed repetitive elements and to the use of a highly inbred bird for sequencing, this draft is probably more accurate than the first one published for human three years ago \[[@B5],[@B6]\]. Nevertheless, previous comparisons of RH mapping data with the sequence data showed that some sequence segments are in wrong positions of the genome assembly \[[@B7]\]. Therefore the integration of all available chicken mapping resources will be essential for improving the quality of the assembly, building a more reliable and informative resource. In addition to the genetic and BAC contig maps that have already been used, the RH map will thus provide an independent source of data to assist the chicken genome sequence assembly process towards a finished quality sequence. RH panels are available for several domestic animals: cow \[[@B8]\], pig \[[@B9]\], horse \[[@B10]\], dog \[[@B11]\] and cat \[[@B12]\]. The successful production of a RH panel in chicken is quite recent \[[@B13]\] and therefore only a limited number of chicken chromosomes have been studied to date, namely: GGA4 \[[@B14]\], GGA5 \[[@B15]\], GGA7 \[[@B16]\], GGA14 \[[@B7]\], and GGA15 \[[@B17]\]. We present here the radiation hybrid map of chicken chromosome 2, built by using markers chosen from the GGA2 genetic map and a substantial number of additional markers developed from chicken EST data. Markers from the genetic map are essential to anchor the RH map onto GGA2. Markers developed from EST data were chosen on the basis of existing comparative mapping information data, indicating conservation of synteny with HSA1, HSA3, HSA6, HSA7, HSA8, HSA9, HSA10, HSA18 and HSA22. They were used primarily to saturate the map in markers and also to increase the precision of comparative maps. The GGA2 RH map obtained was aligned with the genomic sequence assembly to detect eventual discordances. Results and discussion ====================== Development of EST markers -------------------------- Eighty eight microsatellite markers and 10 genes were selected from available data in the literature or the databases. In addition to this, 219 markers were developed from EST data to saturate the map. At the time this study was initiated, comparative mapping data suggested conservation of synteny of GGA2 with 9 human chromosomes and therefore genes were selected from regions of HSA1 (9 markers), HSA3 (28 markers), HSA6 (22 markers), HSA7 (10 markers), HSA8 (84 markers), HSA9 (19 markers), HSA10 (7 markers), HSA18 (35 markers), HSA22 (5 markers). Primers were developed using the Iccare web server \[[@B18]\] with the constraints specific to RH mapping. One hundred and seventy eight out of 219 markers (81 %) amplified successfully. This high success in primer design using EST is comparable to what was achieved in other studies using the Iccare web server \[[@B16],[@B19],[@B20]\]. GGA2 RH map (figure [1](#F1){ref-type="fig"}) --------------------------------------------- ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Radiation hybrid map of chicken chromosome 2 and comparison the draft sequence assembly. The GGA2 RH map (left) is 2794 cR long and is aligned to the genome sequence assembly (right). The limits between the 5 framework groups of the RH map are indicated by double slashes. Markers present only in the comprehensive map are indicated with their most likely position and the confidence interval, to the left of the map. The coloured framed boxes indicate the results from the BLAST analysis, for the markers that were not found on the chromosome 2 sequence assembly <http://www.ensembl.org/Gallus_gallus/>. ::: ![](1471-2164-6-12-1) ::: Altogether, genotyping data were obtained for a total of 270 markers. Two-point analysis with CarthaGene using a LOD threshold of 4 enabled the construction of 5 GGA2 linkage groups containing a total of 198 markers, including all the microsatellite and gene markers from the genetic map. Out of the remaining markers, two (LOC51059and ALDH5A1) should have been integrated into the GGA2 RH map, given their location within the sequence assembly. One explanation, concerning the lack of these 2 markers, could be that the retention frequency, that is unusually low for these two markers and indicative of PCR problems, did not allow attaining a LOD score value significant for linkage. The other 70 markers map either to other chromosomes or to unknown regions. They correspond to the external boundaries of the regions of conserved synteny with human, from which EST were chosen for marker development. The final RH map of chicken chromosome 2 is 2794 cR~6000~long and comprises 86 markers. A one-to-one comparison with the genetic map shows a good overall agreement, with a few improvements over the genetic map; for example markers ADL114and MCW310are mapped with a higher precision than previously on the GGA2 linkage group. At the end of the chromosome (position 2794 cR), the 3 markers (MCW157, MCW189, and MCW0073/ HSF1) were in the same order in the linkage group, but its orientation is different from the genetic map. These markers are mapped on the comprehensive RH map (less significant than the framework one), so the orientation given in the genetic map may be the right one. The corresponding comprehensive map comprises a total of 198 markers (figure [1](#F1){ref-type="fig"}). Comparative maps (figure [2](#F2){ref-type="fig"}) -------------------------------------------------- ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Comparison of gene orders between the GGA2 RH map and the homologous human regions.The GGA2 RH map (this study) is compared to the order of homologous genes in human (left) Each colour (plain boxes) corresponds to a human chromosome. Arrows on the right indicate the gaps between the 5 framework maps. ::: ![](1471-2164-6-12-2) ::: As a result of our map construction strategy, based in great part on the development of EST markers, we found a few observations worthy of comment. Based on the information that the microsatellite MCW0189, identified as being in the LIMK2gene, mapped to the GGA2 genetic linkage group\[[@B21]\], 5 chicken EST markers were developed, corresponding to 8 Mb of HSA22q12.2 containing the homologous gene. Recently, Jennen et al indicated that the mapping of LIMK2was erroneous and that it was located on the GGA15 RH map \[[@B17]\]. We confirm here this result, as all of our five markers mapped to the GGA15 RH linkage group (data not shown). The MCW0189marker remains on the GGA2 RH map, at the position corresponding to where the former LIMK2would have been expected. The result of the BLAST search of MCW0189in the genome assembly indicated a homology with a sequence fragment of unknown location. No similarity with a LIMKsequence could be found in this fragment. A recently published paper describing the mapping of IL1Bon GGA2 suggested a conservation of synteny with HSA2q \[[@B22]\]. The IL1Bprimers we tested failed to amplify correctly, but other chicken EST markers located near IL1B, whose position is 113.3 Mb on HSA2q, had been used in another mapping project in our laboratory \[[@B16]\]. Of these, ACTR, positioned at 111.9 Mb and BIN1at 125.1 Mb on HSA2, were mapped to GGA7. If IL1B, located between these two markers in human, is indeed on GGA2, the fragment of conserved synteny must be very small. The sequence of IL1Bin the chicken genome assembly is in a fraction of unknown location. Previous data suggested that the RYR2gene, mapped on HSA1, was located on GGA2 \[[@B23]\]. We have therefore tested 9 chicken EST markers orthologous to HSA1 genes, but only one (FH) could be localized on the GGA2 RH map. As this localisation could be questioned, the identity of the fragment was confirmed after sequencing of the PCR product. As already observed for other chicken chromosomes \[[@B7],[@B15]-[@B17],[@B24]-[@B26]\], the GGA2 regions investigated here, corresponding to regions of human chromosomes 3, 6, 7, 8, 9, 10 and 18 showed a high number of intra-chromosomal rearrangements within the regions of conserved synteny. Alignment of the RH map to the genomic sequence (figure [1](#F1){ref-type="fig"}) --------------------------------------------------------------------------------- A first draft chicken genome assembly was recently deposited into public databases by a team led by R. Wilson and W. Warren, from the Washington University School of Medicine in St. Louis (1st March, 2004, <http://www.ensembl.org/Gallus_gallus/>). The sequence coverage is 6.6X. For GGA2, 147.590.765 bp were sequenced, 166 known genes were identified, and a total of 1432 genes were defined altogether when including results from prediction programs. For each gene or microsatellite marker from our GGA2 RH map, we compared the fragment sequence with the chicken genome sequence assembly by using the BLAST algorithm \[[@B27]\]. The position for each marker in the sequence is indicated in figure [1](#F1){ref-type="fig"}. The agreement between the RH map and the sequence order is almost perfect, with only a few local inversions that can be put on the account of genotyping errors or other artefacts that may remain despite all precautions taken, such as the double genotyping process. In the upper part of the chromosome, several markers absent in the sequence assembly could be localized on the RH map: sequences corresponding to CENTG3and CSPG5genes are aligned with \"Unknown\" sequence (not assigned to known chromosomes) and ABR0336is found on the GGA27 sequence. The RH two point LOD scores between CENTG3, CSPG5, ABR0336and their neighbours on GGA2 (MCW082, ACVR2Bor PAXIP1L) are higher than 6. In addition, the genetic map confirms the localization of ABR0336on chicken chromosome 2. In the same region of the RH map, MCW071(a microsatellite in the EN2gene) belongs to a genomic region with no sequence available (no blast hit); the genetic map confirms the RH position of this marker. These results suggest possible improvements to be made in the sequence assembly for this region of chromosome 2. On the sequence assembly, the TSGgene, at position 116.4 Mb, is close to EYA1and STAU2, whereas on the RH map TSGmaps at 1660 cR, close to PTPN2and LEI0147, located at positions 96.4 and 97.3 Mb respectively. At the same position as TSGon the RH map, the VAPAgene corresponds to a sequence fragment of unknown location. Moreover, the RH map in this region is in agreement with comparative mapping, so we suspect there may be problems in the sequence assembly in the TSGregion. Around position 2100 cR, two local inversions between the RH map and the sequence orders are observed. The first concerns PRKDCand MAPRE2, and the second SDCBPand ADL114. In both cases, EST markers mapped close to these genes are missing in the GGA2 sequence assembly: CEBPDnear PRKDCand ASPHnear SDCBPare in the fraction of unassigned sequence. However, in the case of the PRKDC-MAPRE2region, the sequence assembly is in agreement with the gene order on human chromosome 8. The order of the markers in the RH map is supported in both cases by a difference of LOD greater than 8, when compared to the alternate order. In the lower part of the chromosome, several markers localized on the RH map are absent from the GGA2 sequence assembly: the HEYgene at position 2330 cR is localized on GGA26 in the assembly, whereas the two-point LOD scores with its neighbours on the GGA2 RH map are trust-worthy (11.5 with IMPA1, 8.8 with PKIA). The MATN2and NCALDgenes are in the unknown fraction of genomic sequence, whereas the 2-point LOD scores with their neighbours, around 2550 cR, are higher than 6. At the end of the chromosome (position 2690 cR), we observed an inversion between the RH map and the sequence concerning the NOVgene and the microsatellite LEI228. The order in the RH map is supported by a difference of LOD greater than 4 when compared to the order in the sequence assembly. Altogether, out of 198 markers localized on GGA2 through RH mapping, only one, labelled \"no hit\" in figure [1](#F1){ref-type="fig"}, could not be found by BLAST in the chicken genome sequence; 10 were assigned to existing genomic sequence of unknown location; 2 were assigned to a wrong chromosome and 4 were not localized at the same position when both maps were compared. With less than 10% of markers either missing in the sequence or for which the two maps disagree, these results confirm the high quality of the genome assembly, with only few finishing improvements to be made in the near future. The GGA2 RH map is available on the ChickRH web server <http://chickrh.toulouse.inra.fr/> also used for RH genotyping data collection. Conclusions =========== We have built a high resolution radiation hybrid map of chicken chromosome 2 using the chickRH6 panel. Our goal was to provide jointly a source of potential polymorphic markers and of candidate genes for QTL mapping on this chromosome. In the course of our work, the first draft chicken genome assembly was released and we aligned it to our GGA2 RH map. Although the sequence assembly is globally in good agreement with our data, a limited number of discrepancies and the mapping of sequence fragments of unknown location or of markers not present in the genomic sequence, show that RH mapping it still useful. Future developments of the chicken RH map will now be based on the genomic sequence, using it for choosing STS markers in selected regions to develop RH framework maps and thereby detect eventual problems in the genomic sequence assembly. This is clearly needed in the regions for which the genetic map is still not complete, such as some microchromosomes, but also for parts of macrochromosomes, as shown in this study. Methods ======= Radiation hybrids ----------------- The generation of the 6000 rads chicken RH panel has already been described \[[@B13]\]. This panel, named ChickRH6, consists of a total of 90 hybrids. The mean retention frequency of markers is 21.9%. Gene selection and primers design --------------------------------- Seventy-seven microsatellite markers well distributed along chicken chromosome 2 were selected from the published chicken genetic map. In addition, 11 other microsatellite markers and 10 genes were selected from other published data \[[@B28]\]. Primer information for these markers can be found in the ARKdb farm animal database <http://www.thearkdb.org/browser?species=chicken> and the ChickAce database <https://acedb.asg.wur.nl/>. Primers for 219 EST were designed using the Iccare web server \[[@B18]\], <http://genopole.toulouse.inra.fr/bioinfo/Iccare/>. All publicly available chicken EST (\> 420,000) were collected in a local database and compared to the human genome sequence. EST selection targeted towards GGA2 was based on available comparative mapping data with the human genome. Long introns, whose positions could be predicted in chicken on the basis of their position in the human genes, were avoided and primers were chosen in the most divergent regions of the chicken/human sequence alignments, to avoid cross-amplification of the hamster DNA present in the hybrids. Information on primers for EST markers is given in the additional table. PCR conditions -------------- PCR were carried out in 15 μl reactions containing 25 ng hybrid DNA, 2 mM MgCl~2~(Life technologies: Carlsblad, CA, USA), 0.3 U Taq DNA polymerase (Life technologies), 1X buffer (Life technologies), 200 μM of each dNTPs (Life technologies), 0.25 μM of each primer, and 1X loading buffer (350 mM sucrose and 0.2 mM cresol). After a 5 min denaturation step, 30 to 35 cycles (depending on the primers) of 30 sec at 94°C, 30 sec at annealing temperature, 30 sec at 72°C, were performed, followed by a final elongation step of 5 min at 72°C. Chicken DNA was used as positive control; hamster DNA and TE were used as negative controls. PCR amplification results were scored on 2% agarose gels. Each marker was genotyped twice. Map construction ---------------- The CarthaGene program \[[@B29]\] was used to build the RH map for chromosome 2 <http://www.inra.fr/bia/T/CarthaGene/>. First, linkage groups were constituted by using a two-point LOD threshold of 4, after which a 1000:1 framework map (a map whose likelihood is at least 1000 fold higher than the next possible highest likelihood using the same markers in alternate orders) for each group was built under a haploid model. Then, the framework maps obtained were aligned on the genetic map and the orientation and distance between each group were estimated using the \"printbestmap\" option in the Carthagene program. Finally, after this final validation, all distances between markers were re-evaluated under a diploid model. Markers not included in the framework map, but displaying two point Lod scores higher than 4 with framework markers, were mapped relative to the framework map by using the CarthaGene \"buildfw\" option which calculates the most likely position of a marker and gives a confidence interval. The map was drawn using the MapChart software \[[@B30]\]. Maps comparison --------------- Data on the location of the chicken genes in the sequence assembly was obtained by BLASTN searches using the Ensembl <http://www.ensembl.org/> browser. Data on human gene order were obtained from the Iccare web server. Authors\' contributions ======================= SL, MD, SB, FV and KF carried out the molecular study. SL built the RH map, and drafted the manuscript with FP. MM made the ChickRH6 panel. AV coordinated the project and finalized the manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Description of the markers. The GCTXXXX markers were developed using the Iccare web server. The EXTXXXX markers were chosen from published information. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ We wish to thank Denis Milan for helpful discussions over the use of Carthagene. This work was financially supported by the Génopole de Toulouse Midi-Pyrénées and the GIS AGENAE.	PubMed Central	2024-06-05T03:55:52.829763	2005-2-4	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548691/", "journal": "BMC Genomics. 2005 Feb 4; 6:12", "authors": [ { "first": "Sophie", "last": "Leroux" }, { "first": "Mélanie", "last": "Dottax" }, { "first": "Suzanne", "last": "Bardes" }, { "first": "Florence", "last": "Vignoles" }, { "first": "Katia", "last": "Fève" }, { "first": "Frédérique", "last": "Pitel" }, { "first": "Mireille", "last": "Morisson" }, { "first": "Alain", "last": "Vignal" } ] }
PMC548692	Background ========== The importance of comprehensive search strategies to identify \'all relevant articles\' when conducting systematic reviews has been long documented \[[@B1]\]. Comprehensive strategies include systematic searching of multiple databases as well as hand searching and contacting relevant experts. These strategies, however, may yield thousands of citations from which only a small number is eventually included in the review. Knowing which sources yield a reasonable number of relevant studies on a health topic may help those planning and conducting systematic reviews in that particular area. At the World Health Organization (WHO), we have conducted a systematic review of prevalence/incidence of maternal mortality and morbidities from 1997 to 2002. The primary objective of this review is to assist in mapping the burden of reproductive ill-health by providing a comprehensive, standardized and reliable tabulation of data on the prevalence/incidence of maternal morbidity and mortality \[[@B2]\]. This article evaluates the usefulness of different sources in identifying data for the systematic review. We also discuss limitations of the databases and implications for future systematic reviews of observational studies. Methods ======= The methodology of the systematic review and the search strategy have been described elsewhere \[[@B3]\]. In brief, we searched for reports of maternal mortality and morbidity across various study designs (cross-sectional, cohort, census, RCTs, etc.). The search included multiple electronic databases: MEDLINE, Popline, EMBASE, CINAHL, CAB Abstracts, Econlit, Sociofile, LILACS, BIOSIS and PAIS International. Specialist librarians together with the reviewers developed database-specific search strategies according to the particular subject headings and searching structure of the databases (See Additional file [1](#S1){ref-type="supplementary-material"}). Given the importance of retrieving data from developing countries, we also identified and searched databases in developing countries such as Index Medicus of the Eastern Mediterranean Region (IMEMR) \[[@B4]\]; African Index Medicus (AIM) \[[@B5]\]; IndMED \[[@B6]\], a bibliographic database of Indian biomedical journals; and HELLIS.ORG \[[@B7]\], a network of health science libraries across Asia. However, the search for some of these databases did not yield complete results due to limited functionality of these systems (e.g. lack of essential information from the citation, inconsistencies) and their results, except for IMEMR, are not presented independently but under \'other\' in Table [1](#T1){ref-type="table"}. The search also included hand searching of journals not indexed in major databases and available at WHO\'s library, government reports, screening of reference lists of retrieved articles, congress abstract books, and contacting experts active in the field for unpublished datasets. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Results of the various types of searches ::: Source Number identified Number included Number unique included\* Sensitivity (%) Precision (%) ---------------------------------- ------------------- ----------------- -------------------------- ----------------- --------------- MEDLINE 38986 1590 400 61.6 4.1 Popline 3255 297 91 11.5 9.1 EMBASE 23133 1135 154 43.9 4.9 CINAHL 9090 263 9 10.2 2.9 CAB Abstracts 4317 190 19 7.4 4.4 Econlit 188 4 1 0.2 2.1 SocioFile 1618 32 8 1.2 2.0 LILACS 1456 137 117 5.3 9.4 BIOSIS 3313 436 21 16.9 13.2 PAIS International 705 21 3 0.8 3.0 IMEMR 109 11 5 0.4 10.1 *All Electronic databases\\* 64098 2093 NA 81.1 3.3 Other^§^ 487 487 487 18.9 NA TOTAL^±^ 64585 2580 2580 NA 4.0** \* Refers to the number of citations that were exclusively identified by each database. \\ Refers to all citations identified through the electronic databases above after duplicate entries are removed. ^§^Includes hand searching, contacting experts, conference proceedings, reference lists, library collections of journals, and databases in developing countries. ^±^Refers to all citations identified through all methods after duplicate entries are removed. NA: Non-applicable ::: We downloaded all citations identified by electronic databases into Reference Manager^®^software. Selection of eligible studies involved two stages; the first stage consisted of screening of title and/or abstracts from the citations downloaded. Citations were excluded if any of the following applied: (i) data collection dates were not reported, (ii) data were collected only before 1990, (iii) part of the data were collected before 1980 and disaggregation by year was not possible (in order to exclude data before 1990), (iv) number of study participants was less than 200, (v) the study design was case-control and incidence/prevalence estimates from the defined population cannot be calculated, (vi) the methodology was not described. The second stage consisted of full-text evaluation of those citations that were not excluded in the first step applying the same criteria. For practical reasons, studies identified by other sources were only entered in Reference Manager^®^if they were eventually included in the review. We recorded the source or sources of each citation. For each source, we calculated number of citations identified, included, and number included that were unique to the source. The sensitivity of each source was defined as the number of included citations identified by the source over the total number included. The precision was defined as the number of included citations identified by a source over the number of both included and excluded citations identified by that source. This review had no language restrictions and we recorded the language in which the report was written. Detailed results regarding languages will be reported separately. Two reviewers performed the screening for all citations. In order to estimate the level of disagreement between the two reviewers within 2.5% of the true value, they independently screened title/abstracts for a sample of citations (560). This sample size assumes a 95% confidence interval and that the level of disagreement between the two reviewers will not exceed 10% \[[@B8]\]. The percentage of agreement was 88.9% (95% CI 86.0% to 91.4%). The inter-observer agreement beyond chance was calculated using the Kappa statistics and found to be 0.60 (95% CI 0.52 to 0.69). This value corresponds to moderate to substantial agreement between the reviewers. Results ======= For the time period from 1997 to 2002, 64098 different citations from electronic databases were identified of which 2093 were included. Additionally, 487 citations were included from other sources. There were 92 citations for which we could not obtain full text and, therefore, they could not be assessed (2% of those that required full-text evaluation). Table [1](#T1){ref-type="table"} shows a breakdown of the results by source including, for each source of data, number of citations identified, number included, number included unique to each source, sensitivity and precision. Overall, electronic databases identified four fifths (81.1%) of the included citations. MEDLINE and EMBASE which identified about 62% and 44% of the included citations respectively were most sensitive. Considering electronic citations alone (2093), almost 20% were identified uniquely by MEDLINE (400), 7.4% uniquely by EMBASE (154), and 5.6% uniquely by LILACS (117). About 60% of the electronic citations included were identified by 2 or more databases. Overall, in terms of precision, we included 1 in 25 screened citations (4%). The most precise database was BIOSIS where 13.2% of the screened citations were included; IMEMR, LILACS and Popline followed this with 10.1%, 9.4% and 9.1%, respectively (see Table [1](#T1){ref-type="table"}). MEDLINE and EMBASE were similarly precise, 4.1% and 4.9%, respectively. Preliminary analysis regarding languages revealed that about 20% of the included studies were published in other languages than English. Spanish and French were, after English, the most used languages for reporting. Discussion ========== Failure to identify relevant information in systematic reviews can result in bias \[[@B9]\]. The importance of including other sources of data in addition to electronic databases in general and MEDLINE in particular has been documented, especially for clinical or randomised controlled trials \[[@B1],[@B9]-[@B11]\]. On the other hand, search strategies for systematic reviews of observational data on morbidities are less precise, more difficult to narrow the focus and have been studied to a lesser extent \[[@B12]\]. This led us to perform for our systematic review an extensive search strategy that is highly sensitive but barely precise. From 64098 citations identified only 2580 were included which represents 4% of the scrutinized articles. Although MEDLINE identified about 62% of all citations and 76% of electronic citations relevant for this review, sources of data other than the major electronic databases are confirmed to be crucial. Some 487 (one fifth) citations were identified by reference lists of articles, expert contacts, congress proceedings, abstract books, hand searching of journals available in libraries that are not indexed in electronic databases, and other emerging databases in developing countries. As expected, there has been a large overlap between databases: 60% were identified by two or more databases and about 44% were identified by MEDLINE and EMBASE together. These two databases also provided the largest number of unique citations and both are considered necessary. PAIS International and Econlit only identified 3 and 1 citations, respectively, that were not identified by any other database and they could probably be disregarded in future reviews. The nature of this systematic review with its focus on settings where burden of disease is highest necessitates extensive searching of developing country sources. However, literature from developing countries is difficult to access and it is not well represented in MEDLINE or other well-known electronic databases \[[@B13],[@B14]\]. An editorial by Zielinksi in 1995 stated that only 2% of the journals indexed in MEDLINE or the Science Citation Index were from developing countries \[[@B15]\]. In 2004, the situation was similar. We calculated the number of journals published in developing countries and also indexed in MEDLINE to be about 6%. In 1996, the whole Latin American continent accounted for 0.39% of the total number of articles included in MEDLINE, down from a high of 2.03% in 1966 \[[@B16]\]. One of the reasons for this is the indexing of journals on a priority system where the impact factor of a journal influences its chances of being indexed. This results in country bias since western journals have in general higher impact factors, and they are therefore more likely to be indexed than those from developing countries. The value of LILACS database to improve the quality of systematic reviews has been previously reported \[[@B17]\]. Our analysis confirms LILACS as a unique source of information for the Latin America and the Caribbean region that is not covered in other databases (117 unique citations included). Unfortunately, specific databases for other less developed regions like Asia and Africa, are just emerging or their access and functioning limited (e.g. AIM, IMEMR, IndMED, HELLIS.ORG). Although these regional databases are included in the review, the results are not presented individually but under \'other\' in Table [1](#T1){ref-type="table"}. With these databases, we experienced language barriers, difficulty in obtaining abstracts and full-text reports, inconsistencies, lack of essential information from the citation (e.g. year or title missing) and other technical problems. We believe that the low number of citations identified by IMEMR (see Table [1](#T1){ref-type="table"}) is due more to the limitations mentioned above than to lack of data. These regional databases provide unique relevant citations and incomplete access limits their usefulness. Strengthening the functionality and improving the search facility of these databases could provide substantial relevant information. A limiting factor for identifying citations is related to late indexing of journals in electronic databases. Search strategies for this review were conducted in early 2003 to identify articles published in 2002 or earlier. While only few articles published in 2002 could be expected not to be in the databases by 2003, some articles published in 1997 were only appearing in the databases as late as 2003. Traditionally, EMBASE has been found to index faster than MEDLINE, thus supporting the argument to search multiple databases \[[@B18]\]. Furthermore, each database producer has a particular schedule that the searcher needs to be aware of. For example, MEDLINE available through OVID, due to the updating of the MeSH terms by the National Library of Medicine, will cease entry of new citations in November and only update the database in January of the following year. These factors need to be considered in assessing the yield from different databases. It is necessary to determine how to capture these \'late indexed\' citations whether by delaying the running of the search or building into follow-up studies the need to capture these citations. The electronic search for this review would have probably captured more citations had it been run in 2004. This systematic review involved significant financial and human resources over a 3-year period \[[@B3]\]. Screening of a large number of citations and retrieving the full text of about 5000 articles have resource implications that need to be balanced with the benefits of the results. For this type of reviews, decisions on the extent of the comprehensiveness of the search strategy should take the resource implications into account. A careful selection of databases to be used and tailored search strategies for each database would help to maximise the benefits compared to costs. Conclusions =========== 1\. In contrast with RCTs, guidelines for search strategy of systematic reviews of observational studies in general, and incidence/prevalence studies in particular, need to be developed. 2\. Searching beyond the major electronic databases such as MEDLINE or EMBASE is necessary to identify studies in journals from less developed countries. 3\. Regional databases indexing citations from local journals not reaching MEDLINE are especially relevant for this type of systematic review. They need to be fully functioning and made worldwide accessible. A network for accessing the full text of these articles would be helpful. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= APB, LS, AMG, TA and LH developed the search strategy for the systematic review. APB and LS analysed the data and wrote the manuscript. AMG, TA and LH participated in the interpretation of results and provided key comments on the manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2288/5/6/prepub> Supplementary Material ====================== ::: {.caption} ###### Additional file 1 Detailed search strategy for electronic databases used for the systematic review. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ We thank Dr Paul Van Look for his comments and support.	PubMed Central	2024-06-05T03:55:52.831666	2005-1-28	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548692/", "journal": "BMC Med Res Methodol. 2005 Jan 28; 5:6", "authors": [ { "first": "Ana P", "last": "Betrán" }, { "first": "Lale", "last": "Say" }, { "first": "A Metin", "last": "Gülmezoglu" }, { "first": "Tomas", "last": "Allen" }, { "first": "Lynn", "last": "Hampson" } ] }
PMC548693	Background ========== This study was undertaken in a 28 -- bedded gynaecological ward catering for female patients aged 16 and above. There were 21 nurses based in this ward of whom 14 were qualified and the remaining were health care assistants, with experience ranging from 1 ^1/2^-- 33 years. The shift handover in this ward was conducted as \"a ritual inheritance,\" \[[@B3]\] distant from patients hearing and vision, such as the ward manager\'s office or the nurses\' station, thus excluding patients participation in their care. The traditional handover used to consist of one-way communication, where the nurse in charge gave the relevant information and instructions to the nurses resuming their shift. A very salient feature of the handover was the absence of individual care planning and where all information about patients was either written in the ward diaries or in the patient files or nursing notes. The sample size of patients involved in the evaluation part of the study was 58. The verbal handover was derived from written information on the office white board which included the patient\'s name, bed number, medical diagnosis and the treating doctor. This was in line with the findings of Sherlock \[[@B12]\] who argued that the shift handover was characterized by a focus on the biomedical model and marginalized the psychosocial aspects of care. The same style of reporting was repeated from one shift to another. As a result, the contents would sometimes degenerate into irrelevant and outdated statements, unrelated to the patient progress and often judgmental in nature with the likelihood of leading to omissions in care. It was therefore not uncommon that nurses were questioned on their practice by the ward manager or the treating doctor which gave rise to a blaming culture among nurses. There was also a level of dissatisfaction among patients who felt that they were not being involved enough in their care. Diagnose need for change ------------------------ The root cause of the problem identified was the model of handover used to communicate clinical information. As a benchmark, the findings from evidence on the bedside handover were used to give meaning and strength to the proposed change. Bedside handovers offer an immediate solution to the many problems that are associated with the traditional handover \[[@B5],[@B15],[@B16]\]. It has further advocated that bedside handover lay more emphasis on individualized patients care whereas bedside reporting is the most frequently used model of handover \[[@B5]\]. It puts the patients central to all care activities and does not rely purely on verbal information but which combines the key principle of patients/clients involvement and participation. In the same context, patients involved in handovers gain access to information that is thought to provide them with comfort and speed recovery \[[@B10]\]. Bed-side reporting makes it possible for nurses starting their shift to obtain a better insight into the care each patient requires \[[@B6]\]. Patients can discuss their health by asking questions and it was found to improve the consistency and continuity of patients care. The information style of bedside handover was informative, personal, shorter and comprehensive. In the light of the above findings, bedside handover had become a valid option for change in this ward. Methods ======= Theories underpinning the change process ---------------------------------------- An adaptation of Spradley\'s 8-step model and Lewin\'s 3-step model of Unfreezing, Movingand Refreezingprovided us with useful frameworks for our change management \[[@B9],[@B14]\]. Unfreezing ---------- Unfreezing is about encouraging people think about the current situation and helping them recognise the need for change \[[@B5]\]. Change to be initiated requires a sense of direction and considerable power of leadership \[[@B8]\]. The authors were also guided by the work of Swansburg and Swansburg, \[[@B15]\] who argued that \"transformational leaders are seen in health care organizations as a commitment to excellence.\" The first move therefore was to create awareness by communicating the proposed change to all those who were going to be affected by the new practice: the nurses, patients and the ward manager so that they all had a shared vision of an improved handover system. A goal-seeking behavior with a clear logical sequence of action, were demonstrated throughout the process as advocated by Lancaster and Lancaster \[[@B8],[@B17]\]. Research based articles were also used to demonstrate how this system was successfully implemented in different areas of the health care system. The proposed change was announced in advance by using different communication channels, e.g. personal contact with individual nurses, staff information/notice board by the authors. This initiated informal discussion among nurses of the ward by creating a cognitive dissonance which led to a quest for more information about the new handover. This consultation phase allowed the nurses to discuss various clinical scenarios and analyse the constraints and benefits of the new proposal in the local context. They were also involved in group work to identify and make proposals on how to deal with some of the problems that we may encounter in our local context e.g. handover coinciding with ward rounds or emergency situations and patients too distressed to talk. Case studies and research articles on this topic were used for discussion and to further reinforce the beliefs of staff of the ward that the current practice had shortcomings and could be improved. The status quo was therefore unsettled and this enabled us to rule out the first resistance through a normative re-educative strategy. A group of senior nurses who had experience in this particular area agreed take turn to act as mentors in order to facilitate this process and offer support to their junior colleagues in the first week until they become confident to carry out the process without supervision. Analysing the alternative options --------------------------------- The extensive literature search also provided us with options for alternatives to bedside handover. These were thoroughly debated before reaching a decision. The options considered were the following: 1\) Tape recorded handover 2\) Computer generated handover using information technology 3\) Bedside handover, based on individualized care plan The \'SMART\' criteria were used to evaluate the feasibility of the alternatives to bedside of handover. The tape recorded handover would require a tape recorder being taken around to each of the patients and the interaction recorded. An informal discussion with the patients revealed that this method was distractive and the majority of them did not feel comfortable about their conversation being recorded. With regards to the computer generated handover using information technology, the patients felt this system will not enable them to engage fully in the process. It was also felt that since the first two options required extra financial, technical resources for implementation, these would not be feasible in the first instance whereas the bedside handover gained unanimous support from both patients and staff. This was also more realistic in term of its applicability in our practice area. It was specific, measurable in terms of its performance and achievable within existing resources and a defined time frame. Its foundation rested on evidence base practice, which showed theoretical soundness. Selecting the change -------------------- There was a shared vision about the worth of the proposed change by the team and consequently bedside handover was logically considered as the best option for change. The vision formulated was that in three months\' time, bedside handover would become the normal shift handover process of the ward. The mission statement agreed was \"all handovers would be carried out at the patients\' bedside between the incoming and outgoing nursing staff with the patients\' involvement.\" Force-field analysis -------------------- A force field analysis, as shown in table [1](#T1){ref-type="table"} was carried out to evaluate the driving and the restraining forces for the change as per Lewin\'s model \[[@B9]\]. The driving forces resided in the support of the ward manager, peers, evidence based arguments and our determination to see the change happen. The restraining forces were mostly related to a lack of information and uncertainty surrounding the change process. Other significant issues that were identified to cause resistance to the change were lateness at work, non-overlapping of shifts and maintaining confidentiality of patient\'s information. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### A force field analysis using Lewin\'s (1951) driving and restraining forces ::: ------------------------------------------------------------------------------------------------------------------------------------- DRIVING FORCES RESTRAINING FORCES ----------------------------------------------------------------- ------------------------------------------------------------------- • Critical incidents on the increase\ • Ritualism and tradition\ • Care given predominantly biomedical in orientation\ • Fear that this may lead to more work\ • Complaints from patients, doctors and relatives on the rise.\ • Lack of confidence on the part of some nurses\ • Increase in discharge against medical advice\ • Fear of increased accountability\ • Staff knowledgeable in change management\ • Problems associated with arriving late at work\ • Ward manager\'s and peer lending support\ • Problems associated with disclosure of confidential information • Familiarity with ward culture ------------------------------------------------------------------------------------------------------------------------------------- ::: Planning the change ------------------- Careful planning is essential if trauma is to be minimized \[[@B2]\]. It was quite important for us to provide information so as to unlock the status quo. This was done by drafting a protocol, (table [2](#T2){ref-type="table"}) on a six points systematic step on how to proceed in practice with the change. This protocol was piloted over 2 morning and 2 evening handover sessions to ensure validity and reliability. There were no changes required to the protocol following the pilot study. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Results of protocol with 6 criteria based on observational data on 10 handovers ::: ---- --------------------------------------------------------------------------------------------------------------------- ------ 1. Outgoing and incoming nurses meet in the office to get a report on confidential matters. 100% 2 Outgoing and incoming nurses then move on to the patient\'s bedside. 100% 3 Nurses introduce themselves to the patient and initiate handover from patient\'s him/herself in the first instance. 100% 4 Patient\'s progress is reviewed as per care plan with a discussion of the future care of the patient. 100% 5 Any other queries from patient is dealt with 100% 6 Session with patient is concluded satisfactorily 90% ---- --------------------------------------------------------------------------------------------------------------------- ------ ::: The time frame earmarked to implement the change was three months starting from the 8^th^of February 2003 up to 8^th^May 2003. One-month time was judged sufficient to unfreeze the situation and the remainder to implement and evaluate the change. Selecting strategies for change ------------------------------- Choosing a strategy for the change process depended upon various factors and good interpersonal relationship was a critical factor. It has been proposed that strong leadership and excellent communication skills were essential if an atmosphere of trust was to be engendered \[[@B7],[@B8]\]. With this in mind, the change was announced in advance to encourage the nurses. It also offered the opportunity to share the reaction of colleagues where some valuable proposals were proposed, for example, how to cater for lateness at work, non-overlapping of shift as well as dealing with confidentiality of information. Confidential issues related to matters that the patients brought up during the admission procedure and during their stay, certain issues that were brought up during ward rounds and from the patients own requests. In cases of occasional lateness in resuming work, the handover would proceed with the other patients in first the instance and if the staff was still late, then some other colleague would step in her place. Reassurance was given with respect to \'no substantial overlapping\' of shift in that it would not have major bearing on the handover process by explaining that shorter handover can reduce the likelihood of information overload and result in concise and pertinent information being exchanged as per care plans. There was a general agreement that fifteen minutes as officially allocated for handover would be sufficient for this purpose. Assurance was also given that confidentiality of patients\' clinical information would be taken into consideration in drafting a protocol for bedside handover, as shown in table [2](#T2){ref-type="table"}. Empowering the staff -------------------- Several meetings were organized with different groups of nurses to explain and clarify any shortcomings and to reach a consensus. This approach was recommended by Driscol \[[@B4]\], as it empowers the team to make the change for itself, without instruction or oversight and is believed to be a strategy for an effective and lasting transformation in a team spirit. The empirical rational strategy was used to convince others of the veracity of the change by making reference to evidence base documentation on the positive outcomes that bedside handover might bring, for example, increase patient satisfaction. Nurses within the ring of informal leaders were gradually encouraged to take some of the ownership of the change by entrusting role model responsibilities to them. This proved to be quite successful as a strategy to encourage participation to create attitudinal and behavioral change. Eventually, there was more acceptance and collaboration on the part of the team to implement the change. In keeping with Skinner\'s theory \[[@B13]\], positive reinforcement, was used to praise and encourage staff. The ward manager helped in the reinforcement process by complimenting the whole team for their excellent effort to bring the change during the weekly meeting of staff. The strategy of facilitation also involved providing training in the new skill demanded by the change. Mocked handover exercises were demonstrated with the different steps of bedside handover to different groups of nurses. This was done by adopting a democratic leadership style engendering a participative approach, which in turn generated a degree of enthusiasm for the change. Moving stage ------------ Following a pilot handover session involving senior staff in a participant and an observer capacity over 2 morning and 2 evening handover sessions, which did not require any major changes, implementation of the bedside handover was started on 8^th^of March 2003. For the first week, six senior staff who had experience in this area volunteered and took turn to continue to be present in as many handovers as observers and participants, to monitor and reinforce the established protocol step by step. They also provided clarification and support to staff in cases of difficulty, and helped evaluate the extent of change that had taken place in an effective manner. The nurses present during the handover had no difficulty in adapting to this new situation, using a care plan incorporating a more psychosocial and patient-centered approach to bedside handover with the patients\' participation. Results ======= Evaluation of the change ------------------------ The evaluation of the implementation of bedside handover was carried out in two distinct phases. A protocol, as shown in table [2](#T2){ref-type="table"}, was developed which included 6 criteria was duly filled after every shift handover. As a benchmark, a good handover was one where at least five of the criteria were strictly followed. The data collection consisted of ten non-participant observation handovers. Semi-structured interviews, using a questionnaire derived from a focus group of staff and patients as shown in table [3](#T3){ref-type="table"}, with 40 patients were carried out to get their perceptions of the new handover. This was done randomly, consisting of both morning and evening handovers over a period of a week by a staff specifically chosen for this job from another ward to prevent bias from the hawthorn effects and ensure validity. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Evaluation of bedside handover from patients\' perspectives -- Results of semi-structured interviews with 40 patients ::: ---------------------------------------------------------------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------- 1\. Do the outgoing and incoming nurse come to your bedside to handover in the morning and in the evening during the change of shifts? 95% -- yes 2\. How do you feel about their presence at your bedside to discuss your care? 100% -- ok and most of them said it was a good thing 3\. Do the nurses involve you in your care planning? 80% -- yes, 10% -- to a certain extent, 10% not sure 4\. Are you satisfied with the way information about your care is passed on and followed by the incoming nurses? 100% -- no problem 5\. How do you feel issues of confidentiality are handled? 100% -- sensitively 6\. Any other comments you would like to make to improve on shift handovers? 1\) Satisfied -- 100%\ Other comments:\ 2) Doctors and other professionals to adopt this approach\ 3) Nurses should spend more time talking to them, not during the handover period only\ 4) Would like this to happen in all wards ---------------------------------------------------------------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------- ::: Analysis of results of the observational data on 10 handovers, Table [2](#T2){ref-type="table"}, showed that the first 5 criteria were met at 100% and the 6^th^criteria at 90%. In one of the sessions, the nurse had left the patient whilst the bedside handover was in progress to attend to another patient without explaining the reason for this short absence to him which accounts for the 90%. Analysis of the results of semi-structured interviews with 40 patients, Table [3](#T3){ref-type="table"}, show that a 96% overall satisfaction level was achieved. This was beyond our expectations, as we had targeted a success rate to be 80% initially. We had to be cautious about the result for it could be either most of the staff had accepted the change or just doing it in this euphoric phase. Refreezing phase ---------------- The result was evaluated at a full staff meeting and the ward manager and colleagues recognized the change. Despite unlearning of the old practice had taken place two nurses still displayed some difficulties with the new handover as they were always eager to report everything themselves rather than allowing the patients to have a say. After a reassessment of the situation, accurate feedback was given to them. With the group support, they became used to the new system by observing their colleagues in action during the handover and doing it in turn. After a couple of sessions they became fully conversant with the new system. By this time, this project was ready for the refreezing stage. Discussion ========== One of the major difficulties encountered was to rally everybody behind this project. The normative re-education, in line Bernhard and Walsh \[[@B1]\], was used in order to help nurses value the new knowledge and create a readiness for learning. Various tasks identified for the future, for example on how to deal with issues of confidentiality, patients who would keep talking endlessly making the process drag on for a long time were allocated to members of the team according to their expertise to prepare so that they could be discussed in depth during the next meeting. A flexible and humanistic in approach was adopted in dealing with conflict, and resistance was not underestimated. Suggestions were treated with respect and dignity. Considerable effort was made to maintain good interpersonal relationship and to highlight motivating factors and safety needs. Constructive feedback was provided on their level of performance. Positive behaviors were rewarded equally in terms of recognition and praise and often with a simple and genuine \"thank you\". Application of this knowledge was reinforced from day one when this new handover became operational into the practice area through continuous coaching, supervision and mentorship. Conclusion ========== Managing change in a hospital set up is a daunting task as it involves a change in the attitude and behavior of staff in a complex environment in order to gain their collaboration. The concept of no pain no gain was very evident throughout the process. Lewin\'s 3 -- stage model was useful in implementing the change in a planned and structured way. Resistance was overcome by creating a climate which encouraged open communication. The support of the ward manager and key stakeholders were significant. Evaluation has shown that the new system of handover is working well but monitoring will be ongoing with evaluation of a larger sample of patients. This change has been an enriching experience for the staff, and has generated enthusiasm and given them confidence to question some of the practices on the ward. This new approach to handover can therefore be implemented in other areas of practice and evaluated to ensure that they are meeting patients\' satisfaction. Further studies can be undertaken to explore how the multidisciplinary team could further consolidate this process. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= HKK taught this module and supervised this change management project. ZBJ implemented this change in the gynaecological ward as part of her assessment in the \"Management Development\" module. Both authors wrote this manuscript, read and approved the final version. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6955/4/1/prepub> Acknowledgements ================ The authors are grateful to all the staff and patients who participated in this project	PubMed Central	2024-06-05T03:55:52.833225	2005-1-28	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548693/", "journal": "BMC Nurs. 2005 Jan 28; 4:1", "authors": [ { "first": "Hemant K", "last": "Kassean" }, { "first": "Zaheda B", "last": "Jagoo" } ] }
PMC548694	Background ========== Tetanus is still a major health problem in developing world. Care of this potentially fatal, though preventable, disease has been revolutionized with advent of mechanical ventilation. However, still a significant minority, at least 10%, would die despite every effort \[[@B1]\]. Respiratory arrest used to be a common problem during preventilation era, however, ANS dysfunction has been emerged as a major problem in these patients in post ventilation era \[[@B1]\]. ANS dysfunction can cause a variety of arrhythmias which can potentially be lethal. There are reports that certain arrhythmias e.g. tachycardia carry poor prognosis \[[@B2]\], however, ANS dysfunction has been attributed to severe tetanus, which carry poor prognosis anyways. Violent autonomic disturbances, severe hypertension and tachycardia alternating with hypotension and bradycardia is reported in relation to severe tetanus. The association of so called milder forms i.e. stages I and II on Ablett\'s criteria \[[@B3]\] to ANS dysfunction and its affect on outcome is not well established. Our study highlights the importance of this previously not well-known aspect of tetanus. Methods ======= All patients admitted to two tertiary care centers in Karachi, Pakistan over a period of 10 years, were identified through ICD-9 coding system of the hospital medical records. The charts were reviewed retrospectively. The demographic, clinical, laboratory data was recorded and analyzed. The autonomic dysfunction was defined as presence of labile or persistent hypertension (\>140/90 mmHg) or hypotension (\<90/60 mmHg) and persistent sinus tachycardia (heart rate \>100 bpm), tachyarrhythmia or bradycardia (heart rate \<50 bpm) alternating with tachycardia on EKG. These values were recorded at resting state, not during tetanic spasm. Brief episodes of tachycardia or hypertension are common in ICU and could be related to pain or anxiety. The values in ICU were mostly recorded in sedated and relaxed position. Continuous automated blood pressure and heart rate monitoring was done in all ICU patients. Blood pressure and heart rate were checked every four hour for the ward patients. The data regarding arterial lines is not available. The values recorded during tetanic spasms were not included in the study. The decision to admit these patients was based mostly on availability of ICU beds. Patients with only locked jaw without any autonomic abnormalities on admission were electively admitted to ward. Severity of tetanus was classified based on Ablett criteria. The poor outcome was defined as either death or partial recovery at the time of discharge from the hospital. Partial recovery was defined as neck pain, back pain, walking difficulty and abnormal gait at discharge, in various combinations. Complete recovery was defined as absence of all of the symptoms and normal gait on examination at discharge. The data was analyzed on SPSS version 10.0. Statistical analysis employed descriptive and univariate (chi-square and ttest) methods. Results ======= Ninety six patients were identified. Sixty four (67%) were men and 32 (33%) were women. Their mean age was 44 (11--75) years. Identifiable risk factors were present in only 49 (51%) patients, recent trauma being most common which was noted in 27(28%) of the patients. Other risk factors were skin wounds, either infected or uninfected; 10 (11%), needle injury; 8 (9%) and recent surgery; 8 (9%). Most common clinical features were trismus; 89 (93%), neck stiffness; 73 (76%), muscle spasms; (67) 70%, followed by dysphagia; 57 (59%), back stiffness; 42 (44%), dysarthria; 19 (20%), abdominal pain; 10 (11%) and subjective breathing difficulty; 7 (8%). All patients received TIG (500--8000 international units) and almost all (99%) received benzodiazepine infusion. About one third (34%) of the patients received paralytics and 4 (5%) were also given magnesium sulphate. Majority of patients (71; 72%) were given antibiotics. The EKG abnormalities include sinus tachycardia in 44 (46%) patients, cardiac ischemia in 5 (5%), sinus bradycardia in 2 (2%) and heart block in 1 (1%) patient. Blood pressure abnormalities include persistently elevated blood pressure in 8 (8%) patients, persistent hypotension in 2 (2%) and labile hypertension in 14 (15%). Forty five (47%) patients recovered fully, 27 (28%) had partial recovery and 24 (25%) patients died. Major cause of death was cardiac arrhythmias (14/24; 58%) followed by respiratory arrest (7/24; 29%). All of the patients who had respiratory arrest were in general wards and never intubated. Fifty eight (60%) patients admitted to ICU. 43 (44%) required mechanical ventilation. Mean hospital stay was 17 (1--63) days. The hospital stay was prolonged in patients who required ventilation (25 vs 9 days) ANS group had 31 (32%) patients while non ANS group comprised of 65 (68%) patients. Severity of tetanus in ANS group was mild to moderate (Ablett I and II); 12 patients and severe/very severe (Ablett III and IV); 19 patients. 15 patients out of these 19 with severe tetanus required mechanical ventilation. Fourteen of 31 (47%) patients died in ANS group as compared to 10/62 (15%) in non ANS group (p= 0.002) (Table [1](#T1){ref-type="table"}). Out of 14 patients who died, six had mild to moderate tetanus and eight patients had severe tetanus. Cause of death was cardiac arrhythmias (13 patients) and sepsis (1 patient) in ANS group while in non ANS group 7 patients died of respiratory arrest, one died of sepsis and one died of myocardial infarction. Cause of death was not known in one patient. None of these patients were witnessed to die during tetanic spasm. All patients who died were receiving benzodiazepines but not at a dose that could cause respiratory failure. All patients in ICU and majority of patients in the ward were receiving DVT prophylaxis in form of subcutaneous heparin and compression stockings. Ambulatory, non-bedridden patients (considered low risk for DVT) were not given DVT prophylaxis. Pulmonary embolism was not considered as a cause of death in any of the patients because these patients had no clinical evidence of DVT. The possibility that these patients died of acute autonomic dysfunction or arrhythmia could not be ruled out. Autopsy was not performed in any of the patients. Ten of 31 (33%) patients had complete recovery in ANS group as compared to 35/62 (48%) in non ANS group (p= 0.05). Both groups were similar in other aspects (Table [2](#T2){ref-type="table"}) ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Tetanus severity, autonomic dysfunction and mortality. ::: Tetanus severity ANS dysfunction (n = 31) Non-ANS dysfunction (n = 65) ------------------ -------------------------- ------------------------------ \>Mild/Moderate 12 (6; died) 40 (1; died) severe 19 (8; died) 25 (9; died) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Comparison of Autonomic neuropathy versus no autonomic dysfunction in patients with tetanus. ::: Variable ANS group (n = 31) Non ANS group (n = 65) P value ------------------- -------------------- ------------------------ --------- Mean age 39 years 46 years 0.2 Male: Female 23:8 41:24 0.1 Locked jaw 89 (93%) 89 (93%) 0.5 Dysphagia 18 (58%) 39 (60%) 0.12 Ventilation 15 (48%) 28 (43%) 0.09 Death 15 (48%) 10 (15%) 0.002 Complete recovery 10 (32%) 35 (54%) 0.05 Partial recovery 7 (23%) 20 (31%) 0.25 ::: Discussion ========== Tetanus is disease of antiquity. It was first described in Egypt over 3000 years ago \[[@B1]\]. It is caused by tetani clostridium which is an obligate non aerobe and ubiquitous organism. Tetanus kills approximately one million people every year world wide and is a major health problem in developing world \[[@B1]\]. The clinical presentation of our patients is similar to that described in literature \[[@B2],[@B4],[@B5]\], most common presenting features being, trismus, neck stiffness and spasms (vide supra). Prognosis of patients with tetanus has been variable. Overall mortality is approximately 10--50% \[[@B1],[@B6]\], however, it is as high as 90--95% in certain select groups i.e. neonates \[[@B7]\] and tetanus associated with intramuscular quinine \[[@B8]\]. Various factors have been known to affect the prognosis. The poor prognostic factors include shorter incubation and onset periods \[[@B2],[@B9]\], fever \[[@B9]\], tachycardia \[[@B2]\], fluctuating blood pressure \[[@B2]\], tetanus associated with intramuscular injections specially quinine \[[@B8]\], extreme of ages \[[@B1],[@B7]\], hypoxia and acidosis at admission \[[@B10]\]. The major complication used to be respiratory arrest before advent of artificial ventilation, however, nosocomial infections and autonomic disturbances are major complications in post ventilator era \[[@B1]\]. Though the autonomic instability has been recognized as a major complication in the post ventilator era, this has been known since 1960s \[[@B11]\] and reported to occur in about a third of the tetanus patients \[[@B4]\]. An electrocardiographic study from India showed the incidence of sinus tachycardia as high as 85% \[[@B12]\]. The autonomic disturbance often develops a few days later in course. Pathogenesis of autonomic disturbances is unclear, however, several theories have been put forward, including damage to brain stem and hypothalamic nuclei \[[@B5]\] and direct disturbances in autonomic nerves \[[@B5],[@B13]\]. The role of tetanus induced damage to brain stem nuclei has been discarded by many authorities. \[[@B14]\] Initially these were considered to be sympathetic overactivity \[[@B11]\], however, later studies with hemodynamic monitoring showed that both sympathetic as well as parasympathetic systems are involved \[[@B13]\]. The autonomic disturbances can be fatal. Though sudden cardiac arrest is most devastating complication, various tachy and bradyarrhthmias can be fatal. We also noted that the autonomic system dysfunction was associated with higher mortality (table [I](#T1){ref-type="table"}). In our cohort the major cause of death in patients with ANS dysfunction was cardiac arrhythmias. The plausible mechanisms for higher mortality in patients with ANS dysfunction include fatal tachyarrhthmias e.g. VF, cardiac arrest, myocardial infarction. Complications of intervention for ANS dysfunction is another potential mechanism of catastrophic end result as these patients are exquisitely sensitive to pharmacotherapy. To avoid these complication careful hemodynamic monitoring and cautious and judicious use of fluids and drugs like beta blockers, atropine may improve outcome in these patients. Morphine has been reported to control autonomic dysfunction. \[[@B15]\] It was used in only a few patients in our series in ICU patients, however, not as first line therapy. Data prior to use of morphine was used for presence or absence of autonomic dysfunction. The ANS disturbances are considered to be part of severe tetanus \[[@B1]\], however, we did note the disturbances even in milder cases. As mentioned earlier the ANS dysfunction carries poor prognosis but this has been considered to be part of severe tetanus \[[@B1]\], which carries poor prognosis anyways. The association of so called milder forms i.e. stages I and II on Ablett\'s criteria \[[@B3]\] to ANS dysfunction and its affect on outcome is not well established. In our cohort 40% of the patients with ANS dysfunction were in grade I or II. Our study is limited by its retrospective nature and paucity of hemodynamic and autonomic testing. However, based on the results we conclude that the presence of autonomic nervous dysfunction irrespective of the respiratory/ventilatory status, predicts poor outcome in tetanus. Studies have shown that reduced tetanus mortality in the recent era is most probably due to advances in ICU management. \[[@B16]\] We also strongly think that the ANS dysfunction is inherent to tetanus even in milder cases so all patients with tetanus should be carefully monitored in ICU settings to avoid mortality from this deadly but potentially reversible disease. Further prospective studies are required to confirm our findings Conclusions =========== Autonomic dysfunction is not uncommon in patients with Tetanus. It was present in one third of our tetanus population. 40% patients with ANS dysfunction had only mild to moderate tetanus. ANS dysfunction, irrespective of the need of mechanical ventilation or severity of tetanus, predicted poor outcome. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= MW: conceptualization, data analysis, manuscript preparation. BAK: data entry and analysis, statistical analysis, manuscript preparation. NT: data acquisition, manuscript preparation. RS: data acquisition, data analysis, manuscript preparation. NAS: conceptualization, manuscript preparation. NS: conceptualization, manuscript preparation. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2377/5/2/prepub> Acknowledgements ================ This study was presented in preliminary form at American Neurological Association meeting, New York in October 2002.	PubMed Central	2024-06-05T03:55:52.835500	2005-1-31	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548694/", "journal": "BMC Neurol. 2005 Jan 31; 5:2", "authors": [ { "first": "Mohammad", "last": "Wasay" }, { "first": "Bhojo A", "last": "Khealani" }, { "first": "Naasha", "last": "Talati" }, { "first": "Rohmah", "last": "Shamsi" }, { "first": "Nadir A", "last": "Syed" }, { "first": "Naseem", "last": "Salahuddin" } ] }
PMC548695	Background ========== Genomes sizes (as measured by the DNA mass per diploid nucleus) are smaller on average in birds than in other tetrapod classes, and genome sizes within the class Aves show less variation than those of other tetrapod classes \[[@B1],[@B2]\]. It has been proposed that reduced genome size in birds represents an adaptation to the high rate of oxidative metabolism in birds, which results primarily from the demands of flight \[[@B1]-[@B4]\]. Cell size and nuclear genome mass are correlated in vertebrates, and cell sizes of birds are smaller than those of mammals \[[@B1]\]. Smaller cells are advantageous in an animal with a high rate of oxidative metabolism because a smaller cell has a greater surface area per volume of cytoplasm, thus facilitating gas exchange. An alternative to the hypothesis that the reduced genome size is adaptive is the hypothesis that it resulted from an event of genomic DNA loss that was fixed in the ancestor of all birds due to genetic drift. The fixation of even a deleterious mutation is possible if the population undergoes an extreme bottleneck \[[@B5]\]. Some authors have argued that such a bottleneck may have occurred in the ancestor of birds at the end of the Cretaceous period \[[@B6]\], although this conclusion is not consistent with recent molecular evidence placing the radiation of the avian orders well prior to that time \[[@B7]\]. In order to decide whether genome reduction in birds was adaptive or due to a random event, Hughes and Hughes \[[@B8]\] compared the lengths of corresponding introns of orthologous chicken (Gallus gallus) and human (Homo sapiens) genes. They found that corresponding introns were significantly shorter in chickens, indicating that numerous independent deletions have occurred in the introns of birds. These results support the hypothesis that genome size reduction in birds is adaptive, since it is unlikely that such a large number of independent deletion events were due to chance alone. Additional evidence in support of the adaptive hypothesis is provided by the observation that a secondary increase in genome size has occurred in avian lineages which have become flightless or have reduced flying ability \[[@B9]\]. It has been suggested that an important factor in genome size reduction in birds has been that birds have lower levels of repetitive DNA than other vertebrates \[[@B10]\]. Genomes of mammals and reptiles are estimated to consist of about 30--50% repeats, while those of birds have been estimated to consist of only 15--20% repeats \[[@B10]-[@B12]\]. In birds chromosomes are of two types: a minority of macrochomosomes (3--6 μm in length) and a larger number of microchromosomes (0.5--2.5 μm in length). In the chicken, there are six pairs of macrochromosomes, and thirty-three pairs of microchromosomes \[[@B13]\]. There is a high rate of chiasma formation on avian microchromosomes, and this may be an adaptation that ensures correct pairing of these chromosomes during meiosis and mitosis \[[@B14]\]. Burt \[[@B10]\] proposed that the avoidance of repeats in the avian genome may in turn be an adaptation that enhances the probability of chiasma formation between homologous microchromosomes. This hypothesis and the hypothesis that genome size reduction represents an adaptation to flight are not mutually exclusive, since both factors may be at work simultaneously. Consistent with Burt\'s hypothesis, Wicker et al. \[[@B15]\] reported that in the chicken genome the ratio of repeats to protein-coding genes is higher on macrochromosomes than on minochromosomes. The sequencing of a substantial portion of the chicken genome has made it possible to examine quantitatively the distribution of repeating sequences on different chromosomes in the genome. Here we compare the distribution of repeats on 28 sequenced autosomes of chicken with that on the 22 human autosomes in order to test the hypothesis that reduction in repeat density in the avian genome has occurred as a result of adaptive evolution. Results ======= The characteristics of repeat arrays on the 28 sequenced chicken chromosomes are summarized in Table [1](#T1){ref-type="table"}. The number of repeat arrays varied from 319 on chromosome 16 to 283,761 on chromosome 1; and the percent of the chromosome occupied by repeats varied from 4.1% on chromosome 32 to 14.9% on chromosome 1. In spite of the considerable variation among chicken chromosomes with respect to the percent of the chromosome occupied by repeats, the overall percentage of the chicken genome occupied by repeats (10.3%) was less than one quarter the percentage of the human genome occupied by repeats (44.9%) (Table [2](#T2){ref-type="table"}). Even the most repeat-rich chicken chromosome, chromosome 1, had a repeat density less than one third that of the human genome (Tables [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}). The range of repeat array lengths was much narrower ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### DNA sequence repeats on the assembled portion of the chicken genome. ::: Chromosome Chromosome length (bp) No. repeat arrays Total repeat length (bp) (% of sequence) ------------ ------------------------ ------------------- ------------------------------------------ 1 188,239,860 283,761 27,978,835 (14.9%) 2 147,590,765 214,512 19,430,497 (13.2%) 3 108,638,738 151,571 12,198,434 (11.2%) 4 90,634,903 121,663 8,905,732 (9.8%) 5 56,310,377 69,048 4,638,645 (8.2%) 6 33,893,787 38,873 2,468,824 (7.3%) 7 37,338,262 41,189 2,397,200 (6.4%) 8 30,024,636 33,974 2,086,343 (6.6%) 9 23,409,228 24,255 1,384,475 (5.9%) 10 20,909,726 19,914 1,075,555 (5.1%) 11 19,020054 20,514 1,095,858 (5.8%) 12 19,821,895 19,419 1,116,593 (5.6%) 13 17,279,963 16,894 1,015,160 (5.9%) 14 20,603,938 21,588 1,417,684 (6.9%) 15 12,438,626 11,830 640,595 (5.2%) 16 239,457 319 18,614 (7.8%) 17 10,632,206 9,508 554,602 (5.2%) 18 8,919,268 8,312 574,276 (6.4%) 19 9,463,882 8,635 491,763 (5.2%) 20 13,506,680 12,826 766,482 (5.7%) 21 6,202,554 6,001 359,040 (5.8%) 22 2,228,820 2,636 183,334 (8.2%) 23 5,666,127 4,932 234,823 (5.7%) 24 5,910,111 5,435 356,373 (6.0%) 26 4,255,270 3,385 188,003 (4.4%) 27 2,668,888 2,833 252,335 (9.5%) 28 4,731,479 5,183 446,256 (9.4%) 32 1,018,878 806 42,242 (4.1%) ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Features of DNA sequence repeats on human and chicken autosomes. ::: Human Chicken ---------------------------------------------------------- --------------------------------------- ---------------------------------- No. chromosomes analyzed 22 28 Total sequence length (bp) 2,864,255,932 901,598,378 No. repeat arrays 4,698,717 1,160,319 Total repeat length (bp) (% of sequence) 1,287,381,310 (44.9%) 92,440,122 (10.3%) Mean repeat array length (bp) \[median\] (range) 274.0 \[188.0\] (7--160,603) 79.7 \[25.0\] (6--7,096) Mean no. repeat arrays per chromosome \[median\] (range) 213,578 \[219,247\] (57,109--409,783) 41,440 \[14,860\] (319--283,761) ::: In the chicken genome, there was a significant positive correlation (r = 0.847; P \< 0.001) between chromosome length and the percentage of the chromosome occupied by repeats (% repeats)(Figure [1](#F1){ref-type="fig"}). The four largest chicken chromosomes (chromosomes 1--4) contributed strongly to this positive correlation. In the case of the four largest chromosomes, there was a clear linear relationship between chromosome length and % repeats (Figure [1](#F1){ref-type="fig"}). In the human genome, there was also a positive, but non-significant correlation (r = 0.412; n.s.) between chromosome length and % repeats (Figure [1](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The percentage of the chromosome occupied by repeats (% repeats) as a function of chromosome length in human (blue dots) and chicken (red dots). ::: ![](1471-2148-5-12-1) ::: As illustrated in Figure [1](#F1){ref-type="fig"}, % repeats values for human chromosomes were considerably higher than those for chicken chromosomes, even when the chromosome length were similar. An analysis of covariance was used to compare % repeats between the two species, with chromosome length as a covariate. There was a significant difference between species (P \< 0.001) and a significant effect of chromosome length (P \< 0.001), but there was not a significant interaction between species and chromosome length. These results imply that there is a similar slope to the linear relationship between chromosome length and % repeats in the two species, but that the % repeats values for human are significantly higher than those for chicken. Comparison of summary statistics human and chicken genomes showed that both mean and median repeat array lengths were considerably greater in the former species than in the latter (Table [2](#T2){ref-type="table"}). In order to provide a statistical test of this difference that was not biased by the presence of different array types in the two genomes, we conducted paired tests on the 294 simple sequence repeat types shared by the two genomes (Table [3](#T3){ref-type="table"}). Both the mean array length and the maximum array length were significantly greater in human than in chicken (Table [3](#T3){ref-type="table"}). By contrast, the minimum array length did not differ significantly between chicken and human (Table [3](#T3){ref-type="table"}). In order to test whether these differences between the two species were due mainly to the influence of the smaller chicken chromosomes, we repeated the analysis using only the four largest chicken chromosomes (chromosomes 1--4). In this case also, both the mean array length and the maximum array length were again significantly greater in human than in chicken, while the minimum array length was not significantly different between species (Table [3](#T3){ref-type="table"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Mean (± S.E.) of variables describing simple sequence repeat types shared between human and chicken. ::: Human Chicken P (paired-sample t-test) ---------------------------------------------- -------------- ------------- -------------------------- All chicken chromosomes (294 repeat types): Mean array length (bp) 83.6 ± 2.8 58.1 ± 1.5 \< 0.001 Minimum array length (bp) 22.1 ± 1.6 22.1 ± 0.7 n.s. Maximum array length (bp) 457.9 ± 25.0 193.5 ± 8.1 \< 0.001 Chicken chromosomes 1--4 (286 repeat types): Mean array length (bp) 83.8 ± 2.9 56.4 ± 1.7 \< 0.001 Minimum array length (bp) 21.7 ± 1.6 24.8 ± 1.1 n.s. Maximum array length (bp) 466.7 ± 25.5 153.3 ± 6.9 \< 0.001 ::: Discussion ========== Tabulation of DNA repeat arrays in the assembled portion of the chicken autosomes showed the overall percentage of repeats to be 10.3%. This value is similar to, but slightly lower than, previously published estimates (about 15%) based on reassociation kinetics \[[@B11],[@B12]\]. By contrast, a similar tabulation in the human autosomes showed the overall percentage of repeats to be 44.9%. Because the value for chicken is substantially lower than the mammalian value, the results support the hypothesis that a relative scarcity of repeating DNA is a major factor in causing the relatively compact size of the avian genome \[[@B15]\]. Moreover, when simple sequence repeat array types shared between chicken and human genomes were compared, mean repeat array length and maximum repeat array length were significantly lower in the chicken than in human. The fact that these differences occurred consistently in nearly 300 distinct array types is evidence that the reduction in repeat arrays in the chicken has involved numerous independent evolutionary events. Mutational changes to simple sequence repeat arrays typically involve slippage events that either decrease or increase the number of repeat units \[[@B16]\]. The fact that simple sequence repeat arrays are shorter in the chicken than in the human implies that mutational events increasing array length have been eliminated by selection in the chicken to a greater extent than in human. Such long arrays might have included some that were inherited from the ancestors of birds and others that arose due to mutational events within Aves. In either case, the evidence for numerous, independent events of elimination of long arrays implies that reduction of DNA repeat length and thus of overall genome size in birds has occurred as a result of adaptive evolution. There were substantial differences among chicken chromosomes with respect to the percentage of the chromosome occupied by repeats, and % repeats increased significantly as a function of chromosome length. This trend implies that the avian genome is characterized by an especially pronounced avoidance of longer repeats on the smaller chromosomes. This finding is consistent with the hypothesis of Burt \[[@B10]\] that the reduction of repeating DNA in avian genomes is adaptive in permitting chiasma formation and alignment of microchromosomes. However, even the largest chicken chromosomes had repeat densities much lower than human chromosomes of similar length (Figure [1](#F1){ref-type="fig"}). This implies that avoidance of repeats on microchromosomes cannot be the sole factor at work in repeat avoidance in avian genomes. This interpretation is further supported by the fact that mean repeat array length and maximum repeat array length of repeat types shared between chicken and human genomes were significantly lower on the largest four chicken chromosomes than in human. Thus, the largest chicken chromosomes, like the rest of the chicken genome, showed a pattern indicating adaptive reduction of repeat array length. Our results imply that some other selective factor besides the need for alignment of minichromosomes contributes to genome size reduction in birds. Together with previous evidence \[[@B9]\], the results are thus consistent with the hypothesis that genome size reduction in birds is adaptive in that it leads to reduction of cell size and thus is advantageous in view of the energetic demands of flight. Methods ======= The chicken (Gallus gallus) genome assembly (May 2004 freeze, release 25.1b.1) was downloaded from Ensembl web site at <http://www.ensembl.org/Gallus_gallus/>. Only autosomes were used in the analyses; data were available for chromosomes 1 through 24, 26, 27, 28 and 32. We extracted Ensembl annotations of the features of repeat arrays (including repeat name, start and end positions on the chromosome, and orientation). The human genome assembly (May 2004 freeze, build 35 (hg17)) was downloaded via the UCSC Genome Browser <http://genome.ucsc.edu/>. Repeat information based on the RepeatMasker annotations (repeat name, start and end positions on the chromosome and orientation) was extracted from the UCSC genome annotation database. Only autosomes (chromosomes 1 through 22) were used. For both chicken and human, repeats tallied included simple sequence repeats, class I elements, class II elements, low-complexity regions, and satellite regions. In addition, we compared between genomes a set of 294 simple sequence repeats which were present in both genomes; i.e., repeats of the same short nucleotide motif were present in both genomes. For these 294 repeat types, mean, minimum and maximum length of repeat arrays were compared in pairwise fashion between human and chicken. Authors\' contributions ======================= HP gathered and summarized the data. ALH conducted statistical analyses and wrote the manuscript. Both authors read and approved the final manuscript. Acknowledgments =============== This research was supported by grant GM066710 from the National Institutes of Health to A.L.H.	PubMed Central	2024-06-05T03:55:52.837302	2005-2-4	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548695/", "journal": "BMC Evol Biol. 2005 Feb 4; 5:12", "authors": [ { "first": "Austin L", "last": "Hughes" }, { "first": "Helen", "last": "Piontkivska" } ] }
PMC548696	Background ========== Literature review \[[@B1]\] indicated that local control, laryngeal preservation, and survival rates of larynegeal cancer patients were similar after transoral laser resection, open partial laryngectomy, and radiotherapy. Open partial laryngectomy was reserved for patients with locally recurrent tumors. There are still some unanswered questions. Will radiation combined with surgery give a better result than single modality treatment alone? Will treatment results from the community centers follow published data from prestigious centers? After radiotherapy, radio-resistant cells theoretically may take some time to grow before recurrence. Short-term data may not reflect long-term local control and survival rates. We attempt to address these questions in the present study. The lognormal distribution is defined as the distribution of a random variable whose logarithm is normally distributed. The purpose of this study is to validate the use of the lognormal model \[[@B2]-[@B4]\] by estimating the long-term survival from short-term follow-up data of laryngeal cancer treated by three different treatment methods: radiation and surgery, radiation alone, and surgery alone. We have previously validated the application of the lognormal model for small cell lung cancer \[[@B5]\], glottic laryngeal cancer \[[@B6]\], prostate cancer \[[@B7]\] and breast cancer \[[@B8]\]. This model may be useful for randomized clinical trials because it allows the prediction of long-term survival rates several years earlier than is possible by using the standard actuarial life table/Kaplan-Meier method of calculation \[[@B9]\]. The idea that long-term survival rates can be estimated from short-term follow-up data is attractive because this method shortens the delay in further research to improve cancer treatment. The validation of the lognormal model has two phases. Phase 1 tests the goodness of fit to a lognormal distribution of the survival times of those cancer patients who died with disease. Phase 2 attempts to verify the lognormal model, which uses short-term follow-up data to predict long-term survival rates. These survival rates are then compared with values calculated by the Kaplan-Meier life table method from available long-term data. The second phase has been difficult to implement because of the general lack of large number of patients with sufficiently long follow-up information. With the SEER database \[[@B10]\], the validation of the lognormal model is now possible. Methods ======= We analyzed a total of 907 cases of laryngeal cancer treated by three treatment methods: radiation and surgery (248 patients), radiation alone (345 patients), and surgery alone (314 patients), registered in two registries, Connecticut and Metropolitan Detroit, from 1973--1977 with known survival status up to 1999 extracted from the SEER database. Fourty-seven patients with unknown survival time and 165 patients with missing treatment methods were excluded. A table of the patient characteristics was listed in Table [1](#T1){ref-type="table"}. The cause-specific survival time was defined as the interval from the date of diagnosis to the date of death from laryngeal cancer or the date of last follow-up for censoring purposes, if the patient was alive and still being followed at the time of analysis. Patients who died of other causes were also censored at date of death. Phase 1 -- Test of goodness of fit for lognormality --------------------------------------------------- Testing for lognormality was done separately for laryngeal cancer patients who died with disease (as distinct from those who died of an intercurrent disease) treated by: radiation and surgery, radiation alone, and surgery alone. For Phase 1, a minimum chi-square method was used to estimate the standard deviation S and mean M of the log~10~(survival time) of the distribution of patients who died of the cancer. The minimum chi-square test was run by a Microsoft Excel program. A range of S-values and M-values were tested to reduce the chi-square values to a minimum. In order to determine if the observed survival times for a given cohort are lognormally distributed, the results are given in terms of probability levels of significance P for the chi-square estimates which correspond to a minimization of chi-square and hence a maximization of P. The test statistic of the minimum chi-square test was minimized by varying the parameters and the P-value gave the significance of the test. The class intervals were in the powers of 2 in months of the survival time, such as 0--2, \>2--4, \>4--8, \>8--16, and so on. The number of cases in each interval should not be less than 5. The null hypothesis being tested is H~0~: that there is no difference between the observed survival times and the expected survival times calculated from a lognormal distribution with a specified S and M. If P \< 0.05 the null hypothesis is rejected. Phase 2 -- Validation of the lognormal model -------------------------------------------- A second computer program for the Phase 2 of the study was also run using Microsoft Excel to estimate the cured fraction C by using a maximum likelihood method described by Boag \[[@B4]\]. Using the lognormal model, the standard deviation (S) was fixed, and only the two remaining parameters, the mean (M) and the cured fraction among all patients (C), were kept floating. Multiple iterations converged to a stable solution for C. The cause-specific survival rate (CSSR) at time τ = \[C + (1-C) × Q\] × 100%, where Q = the integral of the lognormal distribution between the limits τ and infinity, τ = long-term survival time, C was calculated by the maximum likelihood method. The five-year cohort extended from 1973 to the end of 1977. Prediction of the long-term CSSR were made after one year follow-up, i.e. at the end of 1978, for the laryngeal cancer patients with three different treatments: radiation and surgery, radiation alone, and surgery alone. The predicted CSSR were then validated by comparing with the results calculated by the Kaplan-Meier method using the actual follow-up data available up to 1999. Log-rank tests were performed for the three different treatment groups over the whole time period in order to see if there was a difference in CSSR between treatments. Results ======= Using data from 1973--1977, the minimum chi-square tests verified that the survival times of the patients who died of laryngeal cancer and who were treated by three treatments followed three different lognormal distributions. At minimum chi-square, the S and M values, the numbers of patients N and the P values of the minimum chi-square tests for the three different treatments are listed in Table [2](#T2){ref-type="table"}. As in a prospective trial at interim analysis, Phase 2 was performed for each of the five-year cohort periods after one year of follow-up. Phase 2 predicted, using the maximum likelihood method, the long-term 10-, 15-, 20-, and 25-year CSSR. An S value was selected for the maximum likelihood method, so that the prediction curve obtained by the lognormal model would best fit the Kaplan-Meier graph at one yearafter the five-year cohort period. Hence at the time of prediction by the lognormal model, the long-term data were not known yet. Table [3](#T3){ref-type="table"} lists the results for each treatment arm and their comparison with calculation obtained by the Kaplan-Meier method. The predictions were validated by comparing them with the Kaplan-Meier calculation using available data up to 1999. The 25-year CSSR were predicted by the lognormal model after only short term follow-up to be 72%, 68% and 65% for treatments by radiation and surgery, radiation alone, and surgery alone respectively. The 25-year CSSR were found to be 72+/-3% (one standard error), 68+/-3% and 66+/-4% respectively by Kaplan-Meier method. Long-term survival rates at other years, e.g. 10-, 15-, and 20-year CSSR, were all within one standard error compared with the Kaplan-Meier calculations. There were no statistically significant differences between the CSSR of the three different treatment groups for all stages combined (p = 0.35 by log-rank test for the three treatments). Figures [1](#F1){ref-type="fig"}, [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"} show the comparisons of the three different treatments: radiation and surgery, radiation alone, and surgery alone respectively for laryngeal cancer in the Kaplan-Meier graph at the year 1999 compared with the lognormal model prediction curve which could be obtained at 1978. The SEER database provides localized and regional disease stages, instead of T1N0 or T2N0 stages, etc. The lognormal model prediction of the 25-year CSSR for 454 patients with localized stage disease were 84%, 75%, and 80% for treatments by radiation and surgery, radiation alone, and surgery alone respectively. The 25-year CSSR calculated by the Kaplan-Meier method were 83+/-4%, 75+/-4%, and 77+/-6% respectively (p = 0.08, log-rank test for the three treatments). For 286 patients with regional stage disease, the 25-year CSSR were 62%, 58%, and 52% respectively, and compared with Kaplan-Meier method were 63+/-6%, 58+/-7%, and 46+/-6% respectively (p = 0.76, log-rank test for the three treatments). Discussion ========== The current study demonstrates the application of the lognormal model to a population based study. The lognormal model is being applied in a manner that can be applied to prospective trials in practice. Our previous publication about the application of the lognormal model was for different stages of laryngeal cancer patients treated by one radiation oncologist, Dr. M. Lederman in the Royal Marsden Hospital, United Kingdom \[[@B6]\]. The current study shows that the lognormal model is applicable in different scenarios. Detroit and Connecticut registries were chosen because they have the earliest data starting from 1973 and they include a large population of both white and black patients. Generally cause-specific death rates underestimate the mortality associated with a diagnosis of the specific cancer, because some patients died of other causes\[[@B11]\]. Gamel et al.\[[@B12]\] found that the follow-up time should be one standard deviation beyond the mean of the survival time so as to obtain stable results. In the current study, five-year cohorts extended from 1973 to the end of 1977 were used. Stable results for prediction of the long-term CSSR were obtained after one year of follow-up. In this study, for localized stage disease 5-year CSSR were 91%, 83%, 95% for treatments by radiation and surgery, radiation alone, and surgery alone respectively with Kaplan-Meier method. Jones et al.\[[@B13]\] found that the 5-year tumor-specific survival for those treated by radiation was 87% and for those treated by surgery was 77% (p = 0.1022). Both radiation and surgery are equally effective for treating early stage laryngeal carcinoma. In the current study, there were marginal difference in the results of the three treatments for localized stage patients. Jorgensen et al.\[[@B14]\] found that among patients with T1 glottic carcinomas the 5-year locoregional control rate was 88%, i.e. 88% of patients were cured by radiotherapy alone. The 5-year disease-specific survival (DSS) was 99%, i.e. salvage surgery added approximately 11% to the survival of T1 glottic patients. Only 4% (12/312) of T1 glottic patients underwent laryngectomies. Locoregional control among T2 glottic patients was 67% and the DSS 88%, and 18% (41/233) of patients underwent laryngectomies. The corresponding results among T3 glottic patients were 30% and 59%, about 50% of patients underwent laryngectomies. For T3 glottic carcinomas, initial surgery did not produce better survival rates. Franchin et al.\[[@B15]\] studied T1 and T2 glottic carcinoma. The 5-year and 10-year overall survival rates were 83% and 63.5%, respectively. The overall 10-year local control rate for patients with T1 and T2 glottic carcinoma was 89%. In this study, the treatment results were marginally different for localized stage (p = 0.08, log-rank test for the three treatments) and the treatment results were similar for regional stage (p = 0.76, log-rank test for the three treatments). For localized stage combination radiation and surgery may have more certainty of disease control and hence long-term survival benefit. The treatment results were similar for regional stage because the patients were diagnosed late and disease control may be more difficult. The predicted survivals were within one standard error of the Kaplan-Meier estimations for both localized and regional stages. It shows that the prediction method can work for both good and poor prognosis cases. The practical value of this study is that this lognormal model may be used for prediction of the results of prospective trials earlier than the Kaplan Meier method. This lognormal model may become a useful tool in research about outcomes. Use of this lognormal model could result in more rapid advances in cancer treatment and have the potential benefit of a reduction of cost in cancer research. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' constributions ======================== PT: Data analysis and writing of the manuscript. EY, RS: Critical appraisal of the manuscript. JT: Data analysis and critical appraisal of the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/5/13/prepub> Acknowledgments =============== PT: Saskatchewan Cancer Agency Research Grant Award 2792. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Cause-specific survival for all patients with laryngeal cancer treated by radiation and surgery estimated by the lognormal model at 1978 versus actuarial survival calculated by the Kaplan-Meier method at 1999. ::: ![](1471-2407-5-13-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Cause-specific survival for all patients with laryngeal cancer treated by radiation alone estimated by the lognormal model at 1978 versus actuarial survival calculated by the Kaplan-Meier method at 1999. ::: ![](1471-2407-5-13-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Cause-specific survival for all patients with laryngeal cancer treated by surgery alone estimated by the lognormal model at 1978 versus actuarial survival calculated by the Kaplan-Meier method at 1999. ::: ![](1471-2407-5-13-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Patient Characteristics ::: Total number of patients 907 -------------------------- ------------------- --------- Median age 62 (range 21--96) Patient Percent Race: White 828 91% Black 75 8% Others 4 1% Sex: Male 780 96% Female 127 14% Stage: Localized 454 50% Regional 286 31% Distant 71 8% Unstaged 50 6% In Situ 46 5% ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Phase 1 results for the three different treatments ::: Treatment method S M N P ----------------------- -------- -------- ---- ------ Radiation and surgery 0.3623 1.3413 57 0.26 Radiation alone 0.5279 1.2740 86 0.66 Surgery alone 0.6938 1.5057 73 0.32 Abbreviations: S = standard deviation of the lognormal distribution; M = mean of the lognormal distribution; N = number of patients; P = P value for the minimum chi-square test. ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Cause-specific survival rates after one year of follow-up as predicted by the lognormal model compared with the actuarial results as calculated by the Kaplan-Meier method ::: ----------- ------------------------ ---------------------------------- -------- -------- -------- ---------- (A) Radiation and Surgery: Cause-specific survival rate (%) Method Year 10 y 15 y 20 y 25 y C × 100% Lognormal 1978 74 73 72 72 72 KM 1999 74 ± 3 72 ± 3 72 ± 3 72 ± 3 \-- (B) Radiation alone: Cause-specific survival rate (%) Method Year 10 y 15 y 20 y 25 y C × 100% Lognormal 1978 71 69 68 68 67 KM 1999 71 ± 3 68 ± 3 68 ± 3 68 ± 3 \-- (C) Surgery alone: Cause-specific survival rate (%) Method Year 10 y 15 y 20 y 25 y C × 100% Lognormal 1978 74 70 67 65 61 KM 1999 77 ± 3 72 ± 3 68 ± 3 66 ± 4 \-- ----------- ------------------------ ---------------------------------- -------- -------- -------- ---------- y, year; C × 100%, cured rate estimated by the lognormal model; KM, Kaplan-Meier method ± one standard error. :::	PubMed Central	2024-06-05T03:55:52.839254	2005-1-31	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548696/", "journal": "BMC Cancer. 2005 Jan 31; 5:13", "authors": [ { "first": "Patricia", "last": "Tai" }, { "first": "Edward", "last": "Yu" }, { "first": "Ross", "last": "Shiels" }, { "first": "Jon", "last": "Tonita" } ] }
PMC548701	Background ========== Recent scientific and technical advances in the field of experimental biology, particularly in genomics, have produced large amounts of biological data, which has posed new conceptual challenges. The visualization and visualization-driven analysis of these experimentally derived data has become a key component of the scientific process. Until recently, these needs were typically approached by designing applications specialized in a set of well-defined specific tasks. So, for example, popular applications for molecular visualization include, but are not limited to, Molscript \[[@B1]\], PyMol \[[@B2]\], Rasmol \[[@B3]\] and Swiss-PdbViewer \[[@B4]\]. However, the analysis of these molecular data frequently requires novel approaches to visualization and integration with a variety of data types. Therefore, the ability to quickly prototype and develop software suitable for diverse tasks becomes paramount. Hence, libraries, like the MBT described here, are particularly useful, especially given that many applications can be accessed through the Web. This paper, aimed at bioinformatics software developers, provides a concise presentation of the design and capabilities of the toolkit, presents a number of models for its usage, and illustrates its performance with several applications. The paper is organized as follows. The following section provides a general overview of the application programming interface (API). The Results and Discussion section presents details about core components of the toolkit and illustrates its capabilities with two applications. The Conclusions section summarizes the main functionality of the toolkit, discusses availability and documentation, presents performance data and outlines future development plans. Implementation ============== The application-programming interface (API) that serves as the foundation of the toolkit has emerged from the exploration of the typical needs encountered by a researcher in the analysis of a documented biological molecule. The list of requirements includes reading the data file and converting the information to data objects that are easily accessible by applications, extracting subsets of chemical components according to different chemical or biological properties of interest (e.g. chains, residues, CA atoms, ligands, etc.), deriving information that is not explicitly present in the original input data (e.g., covalent and hydrogen bonds, torsion angles, secondary structures) and visualizing chemical or physical properties of different subsets of the molecule, or the entire molecule. Based on the requirements identified above, we have arrived at the multi-layered design illustrated in Fig. [1](#F1){ref-type="fig"}. The bottom (input/output) layer contains facilities for importing molecular data from a variety of sources. The StructureFactoryclass offers a uniform approach to loading structural data, independent of the format of the source. The class makes use of a set of loaders that can import data from a variety of sources, either on the same machine or located on a remote server. Moreover, this allows the developer to write applications that do not have to uniquely specify the source of data. Instead, a number of methods in the StructureFactoryclass enable loading of structures based on a series of source descriptors: file name, PDB id code, URL location, etc. The first loader capable of handling the source descriptor is used thus providing a data access and retrieval mechanism that is transparent to the user. The output of the load methods in the StructureFactoryis a Structureobject, which is effectively an interface to a summary of raw primary and secondary structure data. The Structureobject is not restricted to holding structural information and can contain any other data relevant to the organization of the molecular entity. For instance, it can store the description of single or multiple protein sequences preserving the ordering and alignment of the amino acid residues and their properties. The StructureMapclass builds the internal data model of the simple or complex molecule and provides hierarchical access to both raw data and derived information generated from the input. This includes access to chains, residues, atoms, bonds, nucleic acid components, ligand atoms, fragments associated with secondary structure components, or features defined by the user. The StructureStylesclass provides information about rendering, coloring and selection attributes associated with any structure component produced by the StructureMap. A wide variety of methods in this class allow any module of an application, in particular any viewer, to set and retrieve the information describing the visually represented parameters of any structure component. The StructureDocumentis designed as a container class that maintains a log of all loaded structures and viewers that are instantiated by an application in a given session. It also has the role of generating events associated with the addition or removal of structures and viewers. The next level of the API contains graphical user interface (GUI) elements. This portion of the package contains high level constructs, such as windows and panels designed to display 3D graphics, protein or DNA sequences or tree representations of the chemical components of the molecule, as well as lower level components that can be used to build a molecular scene according to a developer\'s preferences. Finally, at the application level, a number of applications are provided that can be used to illustrate the features of the toolkit, or as starting templates for new application development. For example, the applications illustrate how to use methods in the StructureMapclass to retrieve all secondary structures within a given molecule, or how to obtain a list of atoms or residues. This is the level that most developers would modify in order to create custom applications tailored to their specific needs. Note that care has been taken to further enable application developers to use different components of the toolkit to build purely analytical applications that have no visualization component. For example, a developer could write a command-line tool that simply loads a molecule and gathers statistics about the structure data without presenting any graphical interface. Such an application could be part of a back-end process that runs in a web-server environment. Results and discussion ====================== Core components --------------- The MBT was developed as an object-oriented Java-based environment and hence is flexible, modular and lightweight, which facilitates maintenance, web deliverability and limits the required computer resources. Moreover, the toolkit can be easily extended and having been written completely in Java is effectively platform-independent. The internal data model describes the hierarchical organization of a protein molecule -- Structure, Chain, Residue, and Atom. We have implemented this data model by designing a class hierarchy to efficiently encode its elements. The components of this class hierarchy (Fig. [2](#F2){ref-type="fig"}) are built around a Structureobject (Fig. [1](#F1){ref-type="fig"}). Structure is a container designed to hold all raw information pertaining to the given unit of biological information: protein sequence, genomic sequence, taxonomy information, experimental data and so on. The remainder of the class hierarchy represents the components of the macromolecule. The toolkit is not inherently limited to operating on biological molecules, and can easily be used to manipulate small organic and inorganic molecules. Selection, rendering and coloring attributes for all biological molecules are handled by StructureStyles. The Fragmentclass contains, for example, one of the four known conformations: α-helix, β-strand, turn or random coil, but is sufficiently general to define ranges of residues grouped according to any property of interest. In addition, the data model contains classes that describe other objects associated with derived information, such as covalent or hydrogen bonds. In order to provide uniform style features across the toolkit a StructureStylesclass has been implemented. This class maintains a representation of the rendering characteristics of all structure component objects so that any application module has access to the style data for any given object that needs to be visually represented. The set of style parameters maintained by the StructureStylesclass is comprehensive -- it is the union of the sets of style parameters required by any known viewer. Communication between different components of the toolkit is enabled by a flexible event handling mechanism. Changes in the data, rendering styles, addition or removal of viewers and many other actions with toolkit-wide impact generate descriptive events, which are recursively propagated across the toolkit components, allowing an automatic synchronization of the state of different active parts of an application. Version 1.0 of the MBT provides three standard viewers: a 3D structure viewer, a primary sequence viewer, and a tree viewer. The 3D viewer is implemented using the Java3D™ extension. The use of Java3D for visualization was motivated by the convenience of the availability of high-level constructs for building complex 3D scenes. Analysis of the performance aspects \[[@B5]\] of Java3D has shown that some performance issues can be overcome through a careful organization of the molecular scene. Existing applications indicate that the visualization of most molecules using typical desktops and graphics cards is fast and fully interactive. For example, typical protein data sets with four to five thousand atoms (e.g., PDB identifiers 4 HHB, 10 MH, 6 GEP) load and display in four or five seconds on a Pentium III 1.2 GHz laptop computer. A schematic representation of the data structure used by the 3D viewer is shown in Figure [3](#F3){ref-type="fig"}. For each primary or secondary structure element, a geometry object (GeometryEntity) is built, which is then attached as a BranchGroupnode to a SceneGraphObject(the common superclass for all graph objects in Java3D) representing the three-dimensional image of the molecule. The PsGeometry(primary structure) and SsGeometry(secondary structure) classes provide a number of methods that build complete 3D scenes from a given set of primary or secondary structure data object (See Fig. [2](#F2){ref-type="fig"}). The geometry engine of the 3D viewer uses a flexible approach to generate ribbon-like surfaces. It allows the construction of ribbons using an extrusion with any shape of the cross section. A few most commonly used shapes are immediately available as core components of the toolkit. However, the developers could easily implement and register with the toolkit any additional shapes that may be of interest in their specific applications. The quality of geometry can be controlled either directly by setting individual geometry rendering parameters, or indirectly by a general quality parameter that optimizes the number of facets/vertices used in the construction of different geometric shapes. This allows for an easy adjustment of the application parameters in a wide performance-quality range, from very fast line-only drawing, to a somewhat slower, publication-quality rendering. The sequence viewer is a module designed to display primary sequences of proteins and nucleic acids that are either derived from the loaded structures or acquired from individual sequence files or sequence alignments. The sequence viewer uses AWT drawing methods, which does not impose any specific requirements on the client system, as they are part of the standard Java distribution. The viewer is designed as a full-featured module capable of performing most of the sequence analysis tasks, including basic statistics, pattern and motif searching and display of secondary structure mapping onto the sequence. The viewer is capable of displaying an unlimited number of sequences and provides multiple representation options. The latter include residue coloring by several criteria with a possibility of an easy extension, setting sequence display to any of the available system fonts, a flexible selection system, and more. As stated, the integrated event handling of the toolkit allows for simultaneous updates of the presentation layer for any participating viewers. Hence, the toolkit has the built-in support for common selection and common coloring across all registered viewers. This offers an important visual cue to many applications, linking for example sequence and structural components. The tree viewer offers a hierarchical view of all components of a given molecule. It reflects the logical organization of the derived StructureMapdata including Molecule, Chain, Residue, and Atomobjects. The tree view provides a convenient mechanism to select portions of a molecule based upon the biological relationship between atoms, residues, and chains. Finally, the MBT provides a repository of data and methods that can be used for the retrieval and/or derivation of physical, chemical and structural information associated with the molecules loaded by an application. For example, the package contains a periodic table with physico-chemical properties of the elements, as well as methods for the derivation of the secondary structure information, using the Kabsch-Sander algorithm \[[@B6]\]. Full details of all these features are provided with the documentation. Applications built using the MBT -------------------------------- Applications can be explored and downloaded from the MBT Website \[[@B7]\]. They have been tested on a variety of UNIX, Windows and MAC OS X platforms. The Ligand Explorer \[[@B8]\] (a.k.a. LigPro; Figure [4](#F4){ref-type="fig"}) is an integral part of the reengineered RCSB Protein Data Bank (PDB) \[[@B9],[@B10]\], which is currently in beta testing. In the present PDB, a user interested in protein-ligand interactions must download the structure, decide on a graphics program and likely learn a scripting language to provide details of hydrophilic and hydrophobic interactions between protein and ligand at different cutoff distances. Ligand Explorer achieves this at the push of a button. This produces a view with all ligands highlighted. The user then selects a ligand for a review of detailed interactions. Ligand Explorer can be downloaded as a separate application and used to access local files or files on the PDB\'s servers. The protein kinase exploration tool (Fig [5](#F5){ref-type="fig"}.) is part of the new protein kinase resource \[[@B11]\]. It uses the 3D viewer supplied by the toolkit with a few modifications that allow more extensive coloring and rendering options. The multiple sequence viewer presents the multiple sequence alignments resulting from the multiple structure alignments which are stored in the database. Another viewer displays the superfamily relationship of the sequences present in the database. Conclusions =========== The molecular biology toolkit (MBT) provides a set of pluggable and extensible classes for use by application developers interested in the visualization and analysis of macromolecular data. The MBT provides a set of pre-written data loaders, viewers, a common data model and the means to add to and customize the toolkit for specific applications delivered as applets through the Web or as standalone applications. Base functionality and comparable tools (where applicable) are as follows: • Classes to load raw data from a number of common protein structure and sequence data sources (PDB, mmCIF, FASTA, etc.) and a means to easily add new \"loader\" modules independent of the applications they might serve. • A common data model to which raw information is imported, mapped, and indexed. A number of data record types (StructureComponentobjects) are provided (e.g., Atom, Bond, Residue, Chain) and new data types are easily registered. Further the data model provides an extensible means to describe viewable or visible attributes (e.g., color, radius, drawing style) of these objects. • Written entirely in Java, programs may be embedded (using Java WebStart or the Java Plug-In) directly inside web pages. This enables the deployment of tightly coupled interactive web content much like the popular MDL Chime plug-in. • Applications are not restricted to the features provided by the MBT APIs. The Programmers Guide details how to extend the system. • With source code provided, core features of the toolkit may be directly modified or extended for independent use (though, this may cause your code to diverge from and become incompatible with subsequent releases of MBT). However, adding code within the existing framework and contributing it back for others to use is encouraged. The source code has been extensively commented to produce a rich and complete set of hyper-linked javadoc API documents. • A set of pre-written viewers (Sequence, Structure, Tree) that can be extended, replaced, omitted, or augmented with completely new viewers that implement entirely new visualization techniques. • The 3D StructureViewermodule provides visual representations similar to RasMol and Molscript such as balls-and-sticks, CPK spheres, split-bonds, extrusion/ribbon-style backbone traces, and secondary structure cartoons (Helix, Turn, Coil, Strand). It also provides 3D labeling. • A scripting interface is being developed for MBT to enable toolkit functionality to be command-line or even script-driven (similar to MDL Chime or PyMol). Version 1.0 of the toolkit and sample applications, including those described here, are available for download from the project web page \[[@B7]\]. The same site contains links to various documentation pages (Project Introduction, Talk/Presentation Slides, Related Links, Installation Guide, Build Guide, Programmers Guide, Examples Source, and Toolkit API). The MBT has been tested on common hardware and UNIX, Windows and MAC OS X operating systems. A good consumer-level graphics card is recommended. The loading and generation of a 3D scene when representing a typical protein structure takes a few seconds. Large structures from the PDB \[[@B9]\] that contain over 10^5^atoms require physical memory in excess of 500 MBytes and on a notebook computer with a 1.2 GHz processor can take nearly one minute. Efforts at optimization are on-going. New applications are on-going including the generation of high quality images for all structures in the Protein Data Bank and new ways of visualizing protein-protein interactions. We invite contributions to the MBT by sending mail to <mbt@sdsc.edu>. Bugs may be reported to a bug tracker available on the project web site \[[@B7]\]. Availability and requirements ============================= • Project Name:Molecular Biology Toolkit (MBT) • Project Home Page:<http://mbt.sdsc.edu> • Operating System:Platform independent • Programming Language:Java • Other requirements:Java 1.3.1 or higher, Java3D • License:Free for educational, research and non-profit purposes • Any restrictions to use by non-academics:Contact the University of California at San Diego\'s Technology Transfer Office (<invent@ucsd.edu>, 1-858-534-5815) Authors\' contributions ======================= JLM is one of the designers of the API and co-developer of the toolkit. AG designed and implemented the geometry generation modules, implemented the algorithms for secondary structure generation and bond detection, and drafted the paper. OVB developed the PKR Explorer. QZ developed the Ligand Explorer. PEB coordinated the whole project, suggesting the general functionality and scientific objectives of the toolkit. Acknowledgements ================ We are very grateful to Drs. Paul Craig, John Westbrook and members of the developer and user community at the San Diego Supercomputer Center for their suggestions and valuable feedback. This work was supported by NIH grant 1-P01-GM63208. SB is supported by the Protein Kinase Resource project NSF grant DBI-0217951. QZ is supported by the RCSB Protein Data Bank, a multi-agency project led by the NSF. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The architecture of the toolkit. ::: ![](1471-2105-6-21-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### The MBT data model. ::: ![](1471-2105-6-21-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### The class hierarchy of the geometry engine. ::: ![](1471-2105-6-21-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Ligand Explorer. The structure of cAMP dependant protein kinase (PDB id 1ATP) showing the hydrophilic interactions of the bound ATP to the protein and bound inhibitor which mimics substrate in the phosphotransfer reaction from the gamma phosphate of ATP. ::: ![](1471-2105-6-21-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Protein kinase explorer. An alternative view of a cAMP dependant protein kinase with residues from the multiple structure alignment mapped to the corresponding aligned sequences and to a single template structure from the set. ::: ![](1471-2105-6-21-5) :::	PubMed Central	2024-06-05T03:55:52.841226	2005-2-6	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548701/", "journal": "BMC Bioinformatics. 2005 Feb 6; 6:21", "authors": [ { "first": "John L", "last": "Moreland" }, { "first": "Apostol", "last": "Gramada" }, { "first": "Oleksandr V", "last": "Buzko" }, { "first": "Qing", "last": "Zhang" }, { "first": "Philip E", "last": "Bourne" } ] }
PMC548793	Introduction {#s1} ============ The World Trade Center and anthrax terrorist attacks in 2001, as well as the recent West Nile virus and SARS outbreaks, have motivated many public health departments to develop early disease outbreak detection systems using non-diagnostic information, often derived from electronic data collected for other purposes ("syndromic surveillance") \[[@pmed-0020059-b1],[@pmed-0020059-b2],[@pmed-0020059-b3],[@pmed-0020059-b4],[@pmed-0020059-b5],[@pmed-0020059-b6],[@pmed-0020059-b7],[@pmed-0020059-b8],[@pmed-0020059-b9],[@pmed-0020059-b10],[@pmed-0020059-b11],[@pmed-0020059-b12],[@pmed-0020059-b13],[@pmed-0020059-b14],[@pmed-0020059-b15],[@pmed-0020059-b16],[@pmed-0020059-b17]\]. These include systems that monitor the number of emergency department visits, primary care visits, ambulance dispatches, nurse hot line calls, pharmaceutical sales, and West Nile--related dead bird reports. The establishment of such systems involves many challenges in data collection, analytical methods, signal interpretation, and response. Important analytical challenges include dealing with the unknown time, place, and size of an outbreak, detecting an outbreak as early as possible, adjusting for natural temporal and geographical variation, and dealing with the lack of suitable population-at-risk data. Most analytical methods in use for the early detection of disease outbreaks are purely temporal in nature \[[@pmed-0020059-b18],[@pmed-0020059-b19],[@pmed-0020059-b20],[@pmed-0020059-b21],[@pmed-0020059-b22]\]. These methods are useful for detecting outbreaks that simultaneously affect all parts of the geographical region under surveillance, but may be late at detecting outbreaks that start locally. While purely temporal methods can be used in parallel for overlapping areas of different sizes in order to cover all possible outbreaks, that approach leads to a severe problem of multiple testing, generating many more false signals than the nominal statistical significance level would indicate. First studied by Naus \[[@pmed-0020059-b23]\], the scan statistic is an elegant way to solve problems of multiple testing when there are closely overlapping spatial areas and/or time intervals being evaluated. Temporal, spatial, and space--time scan statistics \[[@pmed-0020059-b24],[@pmed-0020059-b25],[@pmed-0020059-b26],[@pmed-0020059-b27]\] are now commonly used for disease cluster detection and evaluation, for a wide variety of diseases including cancer \[[@pmed-0020059-b28],[@pmed-0020059-b29]\], Creutzfeldt-Jakob disease \[[@pmed-0020059-b30]\], granulocytic ehrlichiosis \[[@pmed-0020059-b31]\], sclerosis \[[@pmed-0020059-b32]\], and diabetes \[[@pmed-0020059-b33]\]. The basic idea is that there is a scanning window that moves across space and/or time. For each location and size of the window, the number of observed and expected cases is counted. Among these, the most "unusual" excess of observed cases is noted. The statistical significance of this cluster is then evaluated taking into account the multiple testing stemming from the many potential cluster locations and sizes evaluated. To date, all scan statistics require either a uniform population at risk, a control group, or other data that provide information about the geographical and temporal distribution of the underlying population at risk. Census population numbers are useful as a denominator for cancer, birth defects, and other registry data, where the expected number of cases can be accurately estimated based on the underlying population. They are less relevant for surveillance data such as emergency department visits and pharmacy sales, since the catchment area for each hospital/pharmacy is undefined. Even if it were available, the catchment area population would not be a good denominator since there can be significant natural geographical variation in health-care utilization data, due to disparities in disease prevalence, access to health care, and consumer behavior \[[@pmed-0020059-b34]\]. One option when evaluating data that are affected by utilization behavior is to use total volume as the denominator. For example, one may use total emergency department visits as a denominator when evaluating diarrhea visits \[[@pmed-0020059-b7]\], or similarly, all pharmacy sales as the denominator when evaluating diarrhea medication sales \[[@pmed-0020059-b4]\]. This may or may not work depending on the nature of the data. For example, changes in total drug sales due to sales promotions or the allergy season could hide a true signal or create a false signal for the drug category of interest. In this paper we present a prospective space--time permutation scan statistic that does not require population-at-risk data, and which can be used for the early detection of disease outbreaks when only the number of cases is available. The method can be used prospectively to regularly scan a geographical region for outbreaks of any location and any size. For each location and size, it looks at potential one-day as well as multi-day outbreaks, in order to quickly detect a rapidly rising outbreak and still have power to detect a slowly emerging outbreak by combining information from multiple days. The space--time permutation scan statistic was gradually developed as part of the New York City Department of Health and Mental Hygiene (DOHMH) surveillance initiatives, in parallel with the adaptation of population-at-risk-based scan statistics for dead bird reports (for West Nile virus) \[[@pmed-0020059-b13]\], emergency department visits \[[@pmed-0020059-b7]\], ambulance dispatch calls \[[@pmed-0020059-b6]\], pharmacy sales \[[@pmed-0020059-b4]\], and student absentee records \[[@pmed-0020059-b3]\]. In this methodological paper, the space--time permutation scan statistic is presented and illustrated using emergency department visits for diarrhea, respiratory, and fever/flu-like illnesses. Methods {#s2} ======= New York City Emergency Department Syndromic Surveillance System {#s2a} ---------------------------------------------------------------- The New York City Emergency Department syndromic surveillance system is described in detail elsewhere \[[@pmed-0020059-b7]\]. In brief, participating hospitals transmit electronic files to the DOHMH seven days per week. Files contain data for all emergency department patient visits on the previous day, including the time of visit, patient age, gender, home zip code, and chief complaint---a free-text field that captures the patient\'s own description of their illness. As of November 2002, 38 of New York City\'s 66 emergency departments were participating in the system, covering an estimated 75% of emergency department visits in the city. Data are verified for completeness and accuracy, concatenated into a single dataset, and appended to a master archive using SAS \[[@pmed-0020059-b35]\]. To categorize visits into "syndromes" (e.g., "diarrhea syndrome"), a computer algorithm searches the free-text chief complaint field for character strings indicating symptoms consistent with that syndrome. The goal of data analysis, which is carried out seven days per week, is to detect unusual increases in key syndrome categories. To run the space--time permutation scan statistic we have written a SAS program that generates the necessary case and parameter files, invokes the SaTScan software \[[@pmed-0020059-b36]\], and reads the results back into SAS for reporting and display. Two sets of analyses are performed, one based on assigning each individual to the coordinates of their residential zip code and the other based on their hospital address. With 183 zip codes versus 38 hospitals, the former utilizes more detailed geographical information, while the latter may be able to pick up outbreaks not only related to place of residence but also to place of work or other outside activities (if people go to the nearest hospital when they feel sick). Residential zip code is not recorded by the hospital for about 3% of patients, and for the analyses described here, these individuals are only included in the hospital-based analyses. The performance of the prospective space--time permutation scan statistic was evaluated using both hospital and residential analyses. We used historical diarrhea data to mimic a prospective surveillance system with daily analyses from 15 November 2001 to 14 November 2002. For each of these days, the analysis only used data prior to and including the day in question, ignoring all data from subsequent days. This corresponds to the actual data available at the DOHMH 8--12 h after the end of that day, when that analysis would have been conducted if the system has been in place at that time. We also present one week of daily prospective analyses conducted in November 2003, where the daily analysis was run about 12 h after the conclusion of each day, as part of the regular syndromic surveillance activities at the DOHMH. The Space--Time Permutation Scan Statistic {#s2b} ------------------------------------------ As with the Poisson- and Bernoulli-based prospective space--time scan statistics \[[@pmed-0020059-b27]\], the space--time permutation scan statistic utilizes thousands or millions of overlapping cylinders to define the scanning window, each being a possible candidate for an outbreak. The circular base represents the geographical area of the potential outbreak. A typical approach is to first iterate over a finite number geographical grid points and then gradually increase the circle radius from zero to some maximum value defined by the user, iterating over the zip codes in the order in which they enter the circle. In this way, both small and large circles are considered, all of which overlap with many other circles. The height of the cylinder represents the number of days, with the requirement that the last day is always included together with a variable number of preceding days, up to some maximum defined by the user. For example, we may consider all cylinders with a height of 1, 2, 3, 4, 5, 6, or 7 d. For each center and radius of the circular cylinder base, the method iterates over all possible temporal cylinder lengths. This means that we will evaluate cylinders that are geographically large and temporally short, forming a flat disk, those that are geographically small and temporally long, forming a pole, and every other combination in between. What is new with the space--time permutation scan statistic is the probability model. Since we do not have population-at-risk data, the expected must be calculated using only the cases. Suppose we have daily case counts for zip-code areas, where c~zd~ is the observed number of cases in zip-code area z during day d. The total number of observed cases (C) is For each zip code and day, we calculate the expected number of cases μ~zd~ conditioning on the observed marginals: In words, this is the proportion of all cases that occurred in zip-code area z times the total number of cases during day d. The expected number of cases μ~A~ in a particular cylinder A is the summation of these expectations over all the zip-code-days within that cylinder: The underlying assumption when calculating these expected numbers is that the probability of a case being in zip-code area z, given that it was observed on day d, is the same for all days d. Let c~A~ be the observed number of cases in the cylinder. Conditioned on the marginals, and when there is no space--time interaction, c~A~ is distributed according to the hypergeometric distribution with mean μ~A~ and probability function When both Σ~z~ ~εA~ c~zd~ and Σ~d~ ~εA~ c~zd~ are small compared to C, c~A~ is approximately Poisson distributed with mean μ~A~ \[[@pmed-0020059-b37]\]. Based on this approximation, we use the Poisson generalized likelihood ratio (GLR) as a measure of the evidence that cylinder A contains an outbreak: In words, this is the observed divided by the expected to the power of the observed inside the cylinder, multiplied by the observed divided by the expected to the power of the observed outside the cylinder. Among the many cylinders evaluated, the one with the maximum GLR constitutes the space--time cluster of cases that is least likely to be a chance occurrence and, hence, is the primary candidate for a true outbreak. One reason for using the Poisson approximation is that it is much easier to work with this distribution than the hypergeometric when adjusting for space by day-of-week interaction (see below), as the sum of Poisson distributions is still a Poisson distribution. Since we are evaluating a huge number of outbreak locations, sizes, and time lengths, there is serious multiple testing that we need to adjust for. Since we do not have population-at-risk data, this cannot be done in any of the usual ways for scan statistics. Instead, it is done by creating a large number of random permutations of the spatial and temporal attributes of each case in the dataset. That is, we shuffle the dates/times and assign them to the original set of case locations, ensuring that both the spatial and temporal marginals are unchanged. After that, the most likely cluster is calculated for each simulated dataset in exactly the same way as for the real data. Statistical significance is evaluated using Monte Carlo hypothesis testing \[[@pmed-0020059-b38]\]. If, for example, the maximum GLR is calculated from 999 simulated datasets, and the maximum GLR for the real data is higher than the 50th highest, then that cluster is statistically significant at the 0.05 level. In general terms, the p-value is p = R/(S + 1) where R is the rank of the maximum GLR from the real dataset and S is the number of simulated datasets \[[@pmed-0020059-b38]\]. In addition to p-values, we also report null occurrence rates \[[@pmed-0020059-b8]\], such as once every 45 d or once every 23 mo. The null occurrence rate is the expected time between seeing an outbreak signal with an equal or higher GLR assuming that the null hypothesis is true. For daily analyses, it is defined as once every 1/p d. For example, under the null hypothesis we would at the 0.05 level on average expect one false alarm every 20 d for each syndrome under surveillance. Because of the Monte Carlo hypothesis testing, the method is computer intensive. To facilitate the use of the methods by local, state, and federal health departments, the space--time permutation scan statistic has been implemented as a feature in the free and public domain SaTScan software \[[@pmed-0020059-b36]\]. Implementation for New York City Syndromic Surveillance {#s2c} ------------------------------------------------------- Depending on the application, the method may be used with different parameter settings. For the syndromic surveillance analyses we set the upper limit on the geographical size of the outbreak to be a circle with a 5-km radius, and the maximum temporal length to be 7 d. This means that we are evaluating outbreaks with a circle radius size anywhere between 0 km (one zip code only) and 5 km, and a time length (cylinder height) of 1 to 7 d. The latter restriction is a reflection of the belief that the main purpose of this syndromic surveillance system is early disease outbreak detection, and if the outbreak has existed for over 1 wk, it is more likely to be picked up by reporting of specific disease diagnoses by clinicians or laboratories. Another practical choice is the total number of days to include in the analysis. One option is to include all previous days for which data are available. We chose instead to analyze the last 30 d of data, adding one day and removing another for each daily analysis. We believe this time frame provides enough baseline beyond the 1- to 7-d scanning window to establish the usual pattern of visits while avoiding inclusion of data that may no longer be relevant to the current period. To reduce the computational load, we limited the centers of the circular cylinder bases to be a collection of 446 zip-code area centroids and hospital locations in New York City and adjacent areas. This ensures, among other things, that each zip-code area may constitute an outbreak on its own. The last parameter that we need to set is the number of Monte Carlo replications used for each analysis. For the daily prospective analyses we chose 999, which meant that the smallest p-value we could get was 0.001, so that the smallest null occurrence rate possible for a signal was once every 2.7 y. In our system, signals of that strength clearly merit investigation. For the historical evaluation, in order to obtain more precise null occurrence rates, we set the number of replications to 9,999. Adjusting for Space by Day-of-Week Interaction {#s2d} ---------------------------------------------- The space--time permutation scan statistic automatically adjusts for any purely spatial and purely temporal variation. For many syndromic surveillance data sources, there is also natural space by day-of-week interaction in the data that is not due to a disease outbreak but to consumer behavior, store hours, etc. For example, if a particular pharmacy has an exceptionally high number of sales on Sundays because neighboring pharmacies are closed, we might get a signal for this pharmacy every Sunday unless we adjust for this space by day-of-week interaction. This can be done through a stratified random permutation procedure. The first step is to stratify the data by day of week: Monday, Tuesday,..., Sunday. The space--time permutation randomization step is then done separately for each day of the week. For each zip code and day combination, the expected is then calculated using only data from that day of the week. For each cylinder, both the observed and expected number of cases is summed over all day-of-week strata, zip code, and day combinations within that cylinder. The same technique can be used to adjust for other types of space--time interaction. The underlying assumption when calculating these expected numbers is now that the probability of a case being in zip-code area z, given that it was observed on a Monday, is the same for all Mondays, etc. All our analyses were adjusted for space by day-of-week interaction. Missing Data {#s2e} ------------ Daily disease surveillance systems require rapid transmission of data, and it may not be possible to get complete data from each provider every single day. When we first tried the new method in New York City, a number of highly significant outbreak signals were generated that were artifacts of previously unrecognized missing or incomplete data from one or more hospitals. This is a good reflection on the method, since it should be able to detect abnormalities in the data no matter what their cause, but it also illustrates the importance of accounting for missing data in order to create an early detection system that is useful on a practical level. Depending on the exact nature of the missing data, there are different ways to handle it. We used a combination of three different approaches. (1) If a hospital had missing data for all of the past 7 d (all possible days within the cylinder), we completely removed that hospital from the analysis, including all previous 23 d. (2) If a hospital had no missing data during the last 7 d, but one or more missing days during the previous 23 baseline days, then we completely removed the baseline days with some missing data, for all of the hospitals. (3) If a hospital had missing data for at least one but not all of the last 7 d, then we removed those missing days together with all previous days for the same hospital and the same day of week. That is, if Monday was missing during the last week, then we removed all Mondays for that hospital. This removal introduces artificial space by day-of-week interaction, so this approach only works if it is implemented in conjunction with the stratified adjustment for space by day-of-week interaction. For some analyses, more than one of these approaches were used simultaneously. Note that, since the missing data depend on the hospital, the solution is to remove specific hospitals and days rather than zip codes and days, even when we are doing the zip-code-based residential analyses. If there are many hospitals with missing data, then the second approach could potentially remove all or almost all of the baseline days. To avoid this, one could sometimes go further back in time and add the same number of earlier days to compensate. Another option is to impute into the cells with missing data a Poisson random number of cases generated under the null hypothesis. Given the completeness of our data, neither of these methods were employed (94% of analyses were conducted with four or fewer baseline days removed). Results {#s3} ======= Evaluation Using Historical Data: Diarrhea Surveillance {#s3a} ------------------------------------------------------- We first tested the new method by mimicking daily prospective analyses of hospital emergency department data from 15 Nov 2001 to 14 Nov 2002, looking at diarrhea visits. Signals with p ≤ 0.0027 are listed in [Table 1](#pmed-0020059-t001){ref-type="table"} and depicted on the map in [Figure 1](#pmed-0020059-g001){ref-type="fig"}. That is, we only list those signals with a null occurrence rate of once every year or less often. ::: {#pmed-0020059-g001 .fig} Figure 1 ::: {.caption} ###### Locations and Dates of Detected Diarrhea Outbreak Signals, Using Historical Data from 15 November to 14 November 2002 The three stronger hospital-based signals are depicted with thicker lines/circles. The stronger residential-based signal was signal C. Note that all the zip-code areas in the residential signal E are also part of signal C. ::: ![](pmed.0020059.g001) ::: ::: {#pmed-0020059-t001 .table-wrap} Table 1 ::: {.caption} ###### Analyses of Emergency Department Visits from 15 November 2001 to 14 November 2002 Due to Diarrhea ::: ![](pmed.0020059.t001) This historical analysis mimics a real-time surveillance system with daily analyses. Geographical coordinates of the patient\'s residence and the visited hospital, respectively, were used in separate analyses. Only signals with p ≤ 0.0027 are listed, corresponding to a null occurrence rate of one expected false signal per year ::: For the residential zip-code analyses, there were two such signals. For the hospital analyses, there were six, two of which occurred in the same place on consecutive days. It is worth noting that at the false alarm rate chosen, none of the residential signals correspond to any of the hospital signals. For the residential analysis, the strongest signal was on 9 February 2002, covering 17 zip-code areas in southern Bronx and northern Manhattan. This signal had 63 cases observed over 2 d when 34.7 were expected (relative risk = 1.82). With a null occurrence rate of once every 5.5 y, a spike in cases of this magnitude is unlikely to be due to random variation. The signal immediately preceded a sharp increase in citywide diarrheal visits from 10 February to 20 March ([Figure 2](#pmed-0020059-g002){ref-type="fig"}). In both the localized 9 February cluster and the citywide outbreak, the increase was most notable among children less than 5 y of age. The weaker 26 February hospital signal and the 7 March residential signal that were centered in northern Manhattan occurred at the peak of this citywide outbreak. Laboratory investigation of the citywide increase in diarrheal activity indicated the rotavirus as the most likely causative agent. ::: {#pmed-0020059-g002 .fig} Figure 2 ::: {.caption} ###### The Daily Temporal Pattern of Emergency Department Diarrhea Syndrome Visits in New York City, 1 November to 14 November 2002 For the citywide line (blue), daily counts are provided for the whole year. For each local area with a signal, daily counts are provided for the 1-mo period leading up to and including the day of the signal. The four stronger signals are depicted with thicker lines. ::: ![](pmed.0020059.g002) ::: The two hospital signals on 1 November and 2 November 2002, were at the same three hospitals in southern Bronx and northern Manhattan, with null occurrence rates of 1.6 and 3.4 y, respectively. These signals immediately preceded another sharp increase in citywide diarrheal activity, this time among individuals of all ages ([Figure 2](#pmed-0020059-g002){ref-type="fig"}). This citywide outbreak lasted approximately 6 wk and coincided with a number of institutional outbreaks in nursing homes and on cruise ships. Laboratory investigation of several of these outbreaks revealed the norovirus as the most likely causative agent. A similar citywide outbreak of norovirus in 2001 began shortly before the 21 November 2001 hospital signal in northern Bronx, which had a null occurrence rate of once every 3.4 y. For the hospital analyses, the strongest signal was a 1-d cluster at a single hospital in Queens on 11 January 2002, with ten diarrhea cases when only 2.3 were expected, which one would only expect to happen once every 3.9 y. Being very local in both time and space, it is different from the previously described signals preceding citywide outbreaks. While examination of individual-level data revealed a predominance of infants under the age of two, this cluster could not be associated with any known outbreak, and retrospective investigation was not feasible. As shown in [Table 1](#pmed-0020059-t001){ref-type="table"}, at the p = 0.0027 threshold there were six and two signals for the hospital and residential analyses, respectively, compared to one expected in each. [Figure 3](#pmed-0020059-g003){ref-type="fig"} shows the number of days on which the p-value of the most likely cluster was within a given range. Had the null hypothesis been true on all 365 d analyzed, the p-values would have been uniformly distributed between zero and one. The fact that in our data there were more days with low rather than high p-values is an indication that there may be additional true "outbreaks" that are indistinguishable from random noise. These could be very small disease outbreaks, for example, due to spoiled food eaten by only a few people, or they could be artifacts caused by, for example, changes in the hours of operation at an emergency department or coding differences between the emergency department triage nurses. ::: {#pmed-0020059-g003 .fig} Figure 3 ::: {.caption} ###### The Number of Days from 15 November 2001 to 14 November 2002 when the p-Value of the Most Likely Emergency Department Diarrhea Cluster Fell within the Interval Indicated for Both the Hospital (Top) and Residential (Bottom) Analyses ::: ![](pmed.0020059.g003) ::: Daily Prospective Surveillance {#s3b} ------------------------------ Since 1 November 2003, the space--time permutation scan statistic has been used daily in parallel with the population-at-risk-based space--time scan statistics \[[@pmed-0020059-b7]\] as part of the DOHMH Emergency Department surveillance system. For respiratory symptoms, fever/flu, and diarrhea, the results for the last week of November are listed in [Tables 2](#pmed-0020059-t002){ref-type="table"} and [3](#pmed-0020059-t003){ref-type="table"}. For diarrhea or respiratory symptoms there were no strong signals warranting an epidemiological investigation, and all had null occurrence rates of more often than once every month. This reflects a very typical week. ::: {#pmed-0020059-t002 .table-wrap} Table 2 ::: {.caption} ###### Real-Time Analyses of Emergency Department Visits Due to Diarrhea, Fever/Flu, and Respiratory Syndromes on Selected Days in November 2003, Using the Geographical Coordinates of the Hospital ::: ![](pmed.0020059.t002) ::: ::: {#pmed-0020059-t003 .table-wrap} Table 3 ::: {.caption} ###### Real-Time Analyses of Emergency Department Visits Due to Diarrhea, Fever/Flu, and Respiratory Syndromes on Selected Days in November 2003, Using the Geographical Coordinates of the Patient\'s Residence ::: ![](pmed.0020059.t003) ::: For fever/flu there was a strong 7-d hospital signal in southern Bronx and northern Manhattan on 28 November with a null occurrence rate of once every 2.7 y. On each of the following 2 d, there were again strong hospital signals in the same general area as well as residential zip-code signals of lesser magnitude. These signals started 12 d into a gradual citywide increase in fever/flu that continued to grow through the end of December, driven by an unusually early influenza season in New York City. Discussion {#s4} ========== In this paper we have presented a new method for prospective infectious disease outbreak surveillance that uses only case data, handles missing data, and makes minimal assumptions about the spatiotemporal characteristics of an outbreak. When using historical emergency department chief complaint data to mimic a prospective surveillance system with daily analyses, we detected four highly unusual clusters of diarrhea cases, three of which heralded citywide gastrointestinal outbreaks due to rotavirus and norovirus. Three of four weaker signals also occurred immediately preceding or concurrent with these citywide outbreaks. If we assume that all of these clusters were associated with the citywide disease outbreaks, then the method generated at most two false alarms at a signal threshold where we would have expected one by chance alone. For disease outbreak detection, the public-health community has historically relied on the watchful eyes of physicians and other health-care workers. However, the increasing availability of timely electronic surveillance data, both reportable diagnoses and pre-diagnostic syndromic indicators, raises the possibility of earlier outbreak detection and intervention if suitable analytic methods are found. While it is still unclear whether systematic health surveillance using syndromic or reportable disease data will be able to quickly detect a bioterrorism attack \[[@pmed-0020059-b39],[@pmed-0020059-b40]\], the methods described here can also be applied to early detection of outbreaks of other, more common infectious diseases. There are other alternative ways to calculate expected counts from a series of case data. One naive approach is to use the observed count 7 d ago in a zip-code area as the expected count for that same area today, and then apply the regular Poisson-based space--time scan statistic. When applied to the New York City diarrhea data described above, such an approach generated at least one "statistically significant" outbreak signal on each of the 365 d evaluated. The basic problem with this is that there is random variation in the observed counts that are used to calculate the expected, which is not accounted for in the Poisson-based scan statistic. If we based the expected on the average of multiple prior weeks of data, we would get less variability in the expected counts and fewer false signals, but the problem would still persist, and as the number of weeks increase beyond a few months other problems may gradually arise due to, for example, seasonal trends or population size changes. Computing time depends on the size of the dataset and the analysis parameters chosen. With 999 replications, the hospital analyses with 38 data locations take 7 s to run on a 2.5-MHz Pentium 4 computer, while the residential analyses using 183 zip-code area locations take 11 s. The same numbers for 9,999 replications are 27 and 57 s, respectively. There are a number of limitations with the proposed method. The method is highly sensitive to missing or incomplete data. Our first implementation of the method resulted in a number of false alarms, and highlights the need for systematic data quality checks and the analytic adjustments described above. When excellent population-at-risk data are available, we expect the Poisson-based space--time scan statistic that utilizes this extra information to perform better than the space--time permutation scan statistic. If, however, the population-at-risk data are of poor quality or nonexistent, which is often the case, then the space--time permutation scan statistic should be used. Since the space--time permutation scan statistic adjusts for purely temporal clusters, it can only detect citywide outbreaks if they start locally, but not if they occur more or less simultaneously in the whole city. Hence, it does not replace purely temporal surveillance methods, but rather complements them. Finally, it is important to note that the geographical boundary of the detected outbreak is not necessarily the same as the boundary of the true outbreak. Since we used circles as the base for the scanning cylinder, all detected outbreaks are approximately circular. Other shapes of the scanning window are also available \[[@pmed-0020059-b36]\], but it has been shown that circular scan statistics are also able to detect noncircular outbreak areas \[[@pmed-0020059-b41]\]. The less geographically compact the outbreak is, though, the less power (sensitivity) there is to detect it. For example, using circles we cannot expect to pick up an outbreak that is very long and narrow such as a one-block area on each side of Broadway, stretching from southern to northern Manhattan. The emergency department data used in this study also have some limitations. In addition to the citywide outbreaks, there were several institutional gastrointestinal outbreaks reported to DOHMH during the historical 1-y period but not detected in emergency department data using the space--time permutation scan statistic. One reported outbreak involved school children that went to the emergency department of a nonparticipating hospital. Other outbreaks went undetected because medical care was not sought in emergency departments. Most people with diarrhea do not go to the hospital emergency department. Rather, they call or go to their primary care physician, they visit the pharmacy to buy over-the-counter medication, or they may have symptoms that are so mild that they do not seek medical care. Further studies are needed to evaluate the strengths and weaknesses of different data sources. The geographic units of analysis used were residential zip code and hospital location. It may be hard to detect outbreaks that affect only a small part of a single zip code, especially if the background rate of the syndrome is fairly high. Where available, the exact coordinates of a patient\'s residence can be used to avoid problems introduced when aggregating data. Furthermore, some outbreaks may not be clustered by place of residence, as in the case of an exposure occurring at the place of work or in a subway. Using the location of the hospital rather than residence may provide higher power to detect workplace-related outbreaks, but the only way to fully address this issue may be to conduct workplace surveillance. In spite of these limitations, we have presented a new method for the early detection of disease outbreaks and illustrated its practical use. The primary advantages of the method are that it is easy to use, it only requires case data, it automatically adjusts for naturally occurring purely spatial and purely temporal variation, it allows adjustment for space by day-of-week interaction, and it is capable of handling missing data. While the method was developed and applied in the context of syndromic surveillance, it may also be used for the early detection of diagnosed disease outbreaks, or for detecting changes in the pattern of chronic diseases, when population census information is unavailable, unreliable, or not available at the fine geographical resolution needed. The ability to perform disease surveillance without population-at-risk data is especially important in developing countries, where these data may be hard to obtain. The space--time permutation scan statistic could also be used for similar early detection problems in other fields, such as criminology, ecology, engineering, social sciences, and veterinary sciences. Patient Summary {#sb1} --------------- ### {#sb1a} #### Background {#sb1a1} Detecting disease outbreaks early means that health officials are better able to fight and contain them. Electronic patient records that can be analyzed with statistical methods in computer programs should help with disease surveillance and make it possible to detect outbreaks early without raising too many false alarms. #### Why Was This Study Done? {#sb1a2} The researchers who did this study have developed and operated real-time disease surveillance systems. In any such system, there will always be more disease cases in some places and time periods than in others, for example, because there are more people living there, or because there are more people of a certain type living there, like older people or children, who are more prone to get sick. The researchers were trying to develop a method that can discover outbreaks without the need to know about the structure of the population under surveillance. #### What Did the Researchers Do? {#sb1a3} They modified an existing method to make it work without data on the structure of the population under surveillance. They also found a way to deal with incomplete data, when, for example, one hospital did not report any data for a particular day. #### What Did They Find? {#sb1a4} When they applied the method to emergency room data from New York City, they found that it performs well: it seems to be able to detect real outbreaks early and not result in many false alarms. #### What Are the Limitations of the Method? {#sb1a5} The method can detect only outbreaks that start locally, not those that occur more or less simultaneously in the whole surveillance area. For some outbreaks---for example, those caused by exposure to an infectious agent in the subway---patients will not necessarily live in the same neighborhood or go to the same emergency room. The method will not detect outbreaks with very few cases, such as one case of small pox or three cases of anthrax, such as the anthrax bioterrorism attacks in the fall of 2001. And the method only works for diseases with early symptoms severe enough that people go to the emergency room. Efficient disease surveillance will need the parallel use of different methods, each with their own strengths and weaknesses. #### What Next? {#sb1a6} The method was developed as part of the New York City Department of Health and Mental Hygiene surveillance initiatives and is now being used every day to analyze emergency department records from 38 hospitals in the city. To facilitate wider use, the method has been integrated into a more diverse software called SaTScan that is freely available. #### Where Can I Find Out More? {#sb1a7} The following websites provide additional information on this and other methods. Details on SaTScan and software for downloading: <http://www.satscan.org/> United States Centers of Disease Control and Prevention Web page on electronic disease surveillance: <http://www.cdc.gov/od/hissb/act_int.htm> National Syndromic Surveillance Conference: <http://www.syndromic.org/index.html> National Bioterrorism Syndromic Surveillance Demonstration Program: <http://btsurveillance.org/> The Real-Time Outbreak and Disease Surveillance Open Source Project: <http://openrods.sourceforge.net/> This work was supported by a grant from the Alfred P. Sloan Foundation. The funders had no role in the study design, data analysis, decision to publish, or manuscript preparation and content. Valuable and insightful comments by the reviewers are gratefully acknowledged. Citation: Kulldorff M, Heffernan R, Hartman J, Assunção R, Mostashari F (2005) A space--time permutation scan statistic for disease outbreak detection. PLoS Med 2(3): e59. DOHMH : New York City Department of Health and Mental Hygiene GLR : generalized likelihood ratio [^1]: Competing Interests: The authors have declared that no competing interests exist. [^2]: Author Contributions: MK and FM designed the study. MK, RA, and FM developed the statistical methodology. MK and RH analyzed the data. MK, RH, JH, RA, and FM contributed to writing the paper.	PubMed Central	2024-06-05T03:55:52.843218	2005-2-15	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548793/", "journal": "PLoS Med. 2005 Mar 15; 2(3):e59", "authors": [ { "first": "Martin", "last": "Kulldorff" }, { "first": "Richard", "last": "Heffernan" }, { "first": "Jessica", "last": "Hartman" }, { "first": "Renato", "last": "Assunção" }, { "first": "Farzad", "last": "Mostashari" } ] }
PMC548794	For disease outbreak detection, the public-health community has historically relied on the watchful eyes of doctors and other health-care workers, who have reported individual cases or clusters of cases of particular diseases to health-care and other authorities. The increased availability of electronic health-care data, however, raises the possibility of more automated and earlier outbreak detection and subsequent intervention. Besides diagnoses of known diseases, pre-diagnostic syndromic indicators---such as the primary complaints of patients coming to the emergency room or calling a nurse hotline---are being collected in electronic formats and could be analyzed if suitable methods existed. Martin Kulldorff and colleagues have been developing such methods, and now report a new and very flexible approach for prospective infectious disease outbreak surveillance. Their method, which they call the "space--time permutation scan statistic," is an extension of a method called scan statistic. All previously developed scan statistics require either (i) a uniform population at risk (with the same number of expected disease cases in every square kilometer), (ii) a control group (such as emergency visits not due to the disease of interest), or (iii) other data that provide information about the geographical and temporal distribution of the underlying population at risk, such as census numbers. The new method, because of a different probability model, can be used for the early detection of disease outbreaks when only the number of cases is available. It also corrects for missing data and makes minimal assumptions about the spatiotemporal characteristics of an outbreak. To make it widely accessible, the method has been implemented as a feature of the freely available SaTScan software. ::: {#pmed-0020065-g001 .fig} ::: {.caption} ###### Disease surveillance in New York City ::: ![](pmed.0020065.g001) ::: In their article, Kulldorff and colleagues illustrate the utility of the new method by applying it to data collected from hospital emergency departments in New York City. The researchers analyzed diarrhea records from 2002, and did both a "residential analysis" (based on the home address of the patients) and a "hospital analysis" (based on hospital locations). The former has more detailed geographical information, the latter maybe be better able to detect outbreaks not primarily related to place of residence but, for example, school or workplace. They found four highly unusual clusters of diarrhea cases, three of which heralded citywide gastrointestinal outbreaks due to rotavirus and norovirus. Since November 2003, the space--time permutation scan statistic has been used daily to analyze emergency department data in New York City in parallel with other methods, and it seems to perform well. As the authors discuss, as any other surveillance method, theirs has limitations. Because it adjusts for purely temporal clusters, the method can only detect outbreaks if they start locally (not simultaneously across the entire surveillance area). The less geographically compact an outbreak is, the less power there is to detect it. And some outbreaks, for example, those caused by exposure to an infectious agent in the subway, will be hard to cluster by place of residence or choice of emergency department. In the present study, Kulldorff and colleagues have applied their method to infectious disease surveillance in a metropolitan area in the United States. As they state, however, "the ability to perform disease surveillance without population-at-risk data is especially important in developing countries, where these data may be hard to obtain."	PubMed Central	2024-06-05T03:55:52.846990	2005-2-15	{ "license": "Creative Commons Zero - Public Domain - https://creativecommons.org/publicdomain/zero/1.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548794/", "journal": "PLoS Med. 2005 Mar 15; 2(3):e65", "authors": [] }
PMC548940	Background ========== The aim of this paper is to provide data on the equity of general medical practitioner (GP) prescribing rates for coronary heart disease (CHD) drugs. Since CHD represents a major cause of premature mortality in the Western world, it is vital that those populations with the highest need for CHD drugs actually receive them. Whilst there is a large literature on inequities in the provision of a number of other health care services and treatments, the equity of GP practice prescribing has received little attention. Therefore, this study was an attempt initiate the development of an evidence-base and to provide data on the equity of GP practice prescribing rates. Conceptualisation and definition of the equity of prescribing ------------------------------------------------------------- There are large literatures around how to define, operationalise and measure equity in relation to health care services \[[@B1]-[@B3]\], although equity is generally taken to mean \'fair\' or \'just\'. Equity has been divided into three domains: equal accessto health care for people in equal need; equal treatmentfor people in equal need; and equal outcomesfor people in equal need \[[@B1]\]. Whilst this is a simplification of the nature of equity, it is useful in delineating the various domains in which inequities may arise. The current paper is focussed around the equal prescribing (i.e. equal treatment) for people in equal need. Using the example of the current study, an analysis of equity would assess the differences in prescribing rates provided to the population of one GP practice compared to another GP practice, weighted to take account of the levels of need for CHD drugs in their patient populations. Therefore, it would be equitable to have higher prescribing rates for populations with higher levels of health care need and lower prescribing rates for populations with lower levels of health care need. However, it would be inequitable to have higher prescribing rates for populations with lower levels of health care need and lower prescribing rates for populations with higher levels of health care need. The identification of populations where prescribing was deemed inequitable could then be targeted for further resources aimed at redressing the balance between prescribing rates and health care need. Previous research on the equity of GP practice prescribing ---------------------------------------------------------- A recent paper by the authors questioned the equity of GP practice prescribing rates for a range of CHD drugs\[[@B4]\] and highlighted the contemporary relevance of the \'inverse care law\'\[[@B5]\] in the context of GP prescribing. That paper presented the findings of bivariate correlations between prescribing rates and healthcare needs indicators (HCNIs). One of the inherent problems with bivariate analysis is that prescribing rates are likely to be associated with a number of HCNIs. Therefore, the purpose of this paper is to present the findings of multivariate regression analyses between prescribing rates and the HCNIs and ultimately to examine the independent associations between prescribing rates and HCNIs. In doing so, this paper sets a benchmark for future studies aimed at assessing the effectiveness of the National Service Framework for CHD in developing CHD services commensurate with healthcare need \[[@B6]\]. There is a growing body of research which has highlighted large variations in overall prescribing rates between GP practices, which are only partially explained by factors other than health care need \[[@B4],[@B7]-[@B11]\]. Statin prescribing has been shown to vary between health authorities and GPs \[[@B12]-[@B15]\] and between patients on the basis of gender \[[@B13],[@B16]-[@B18]\], demographics \[[@B13],[@B19]\], ethnicity \[[@B20]\] and deprivation \[[@B21]\]. Prescribing rates of beta-blockers have also been found to be lower in patients aged over 75 years and in minority ethnic groups \[[@B22],[@B23]\]. Studies attempting to \'explain\' the variation in statin prescribing rates have been modest, with most studies explaining around 20 per cent of the variation \[[@B13],[@B15],[@B21],[@B24]\]. In addition, one study explained around 40 per cent of the variation in prescribing rates for all cardiovascular drugs \[[@B21]\]. The prevalence of CHD explained 12 per cent of the variation in statin prescribing in men, and 7 per cent in women \[[@B13]\], deprivation explained 14 per cent \[[@B15]\], and a combination of nitrate prescribing rates and population aged between 35 and 74 years explained 18 per cent \[[@B24]\]. In addition, the indicative prevalence of CHD was moderately correlated with prescribing rates for statins, and was the most important variable in the multiple regression model \[[@B21]\]. However, characteristics of GP practices such as their training status, the number of GPs, or their single-handed status (i.e. whether or not they are \'lone\' GPs or work in a multi-partner practice) have been found to have no relationship with prescribing rates for statins \[[@B15],[@B24]\]. Therefore, the majority of variations in statin prescribing rates in addition to prescribing rates for other CHD drugs remain unexplained. Context and setting for the study --------------------------------- The planning and provision of health care to local populations in England is now the role of primary care trusts (PCTs). Essentially, PCTs are organisations whose main responsibilities are around developing, commissioning and providing services, which are targeted to the needs of local people, and ultimately to improve the health (and reduce health inequalities) of local people \[[@B25],[@B26]\]. PCTs have taken over these responsibilities from health authorities, which no longer exist \[[@B27]\] and are responsible for spending 75 per cent of the overall NHS budget in England \[[@B28]\]. This study was undertaken in 4 PCTs in England (referred to as PCT1, PCT2, PCT3, and PCT4 throughout this paper), which included 132 GP practices (PCT1 had 50 GP practices, PCT2 had 24, PCT3 had 31 and PCT4 had 27). In terms of patient populations, we excluded patients aged under 35 years, since prevalence of CHD is particularly low in this age group. In total, there were 353,897 registered patients aged over 35 year across all 4 PCTs. Methods ======= In order to analyse the equity of prescribing for CHD drugs, we firstly needed to gather data on, and then develop rates for both GP practice prescribing and health care need (called health care needs indicators (HCNIs) in this paper). The data sources and methods used to develop prescribing rates and HCNIs have been outlined in previous papers by the authors \[[@B4],[@B29],[@B30]\], although a brief précis will be presented here. Local Research Ethics Committee approval was sought and granted for this study. Developing prescribing rates ---------------------------- When an NHS prescription is dispensed in primary care, the prescription form (FP10) is sent to the Prescription Pricing Authority (PPA) for processing. The PPA collates these data and provides them to GP practices and PCTs in the form of Prescribing Analysis and Cost (PACT) data. PACT data are available for all GP practices in England, and allow detailed interrogation in terms of drugs prescribed along with their dosages, pack sizes and formulations. For example, for a specific time period, we can collect data on which statins were prescribed by a GP practice in addition to the dosages and pack sizes. This allows for a complex and timely analysis of PACT data. Useful critiques of PACT data can be found elsewhere \[[@B31],[@B32]\]. PACT data were obtained for all GP practices in the 4 PCTs for the 12-month period October 1999 to September 2000. These data were collected for statins, ACE-inhibitors, beta-blockers, aspirin, and bendrofluazide (a full list of drugs obtained are listed in Appendix A). These drug groups were chosen because they represent major drug groups recommended for the prevention (primary and secondary) of coronary heart disease (CHD) in the United Kingdom (UK) \[[@B6]\]. Using prescribing rates for aspirin was potentially difficult, since it can also be purchased over the counter (OTC) in community pharmacies and therefore may not represent the totality of aspirin use within populations \[[@B33],[@B34]\]. It has also been found that non-prescription aspirin use was higher in men aged under 65 years, and also in more affluent areas \[[@B35]\]. Therefore, PACT data may underestimate actual aspirin use within the community. Nevertheless, given the importance of aspirin within the management and prevention of CHD \[[@B17],[@B36],[@B37]\], it was postulated that prescribing rates for aspirin may reflect CHD prevalence within GP practice populations, at least as well or even better than prescribing rates for the other CHD drugs. The numerator in all prescribing rates was based on a measure of prescription volume, as opposed to prescription cost. The validity of using the number of prescription items or total cost as a measure of prescribing volume has been called into question \[[@B38],[@B39]\] since it does not specify the quantity of prescription medication (e.g. number and/or dosage of tablets). Therefore, a measure of prescription volume which calculates the total number of grams prescribed is much more useful. The main options available are defined daily doses (DDDs) \[[@B40],[@B41]\] and Average Daily Quantities (ADQs) \[[@B40],[@B42],[@B43]\]. The Prescribing Support Unit website provides up to date lists of DDDs <http://www.psu.co.uk/ddds.html> and ADQs <http://www.psu.ac.uk/drugs/adqind.html> for all drugs for which they have been developed. Within this study, total ADQs were used as the unit of analysis since they represent prescribing practices in the UK, as opposed to DDDs which represent prescribing practices internationally. The denominator was the total registered (and resident) patient population aged over 35 years. This age group was chosen since the prevalence of CHD is particularly low in people aged less than 35 years \[[@B44]\]. Developing health care needs indicators (HCNIs) ----------------------------------------------- In total, 24 HCNIs were developed for each GP practice in this study, and all of these were entered into multiple regression models (all HCNIs are outlined in Appendix B). These HCNIs reflect patient demographics, ethnicity, socio-economic status, long term limiting illness, CHD mortality and CHD morbidity. However, the only HCNIs discussed here are those included in the final regression models (see Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Descriptions of statistically significant variables in final regression models ::: Descriptor used in text and Table 4 Description ----------------------------------------- --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- CHD HES rate 6-year crude rate of CHD hospital procedures per 1000 patients (i.e. proxy for CHD morbidity). Data source was the General Practice Research Database. \% patients aged 55--74 Proportion of patients in GP practice population aged 55--74 years. Data source was the individual GP practices. \% patients aged \>75 Proportion of patients in GP practice population aged over 75 years. Data source was the individual GP practices. LISI score Low Income Scheme Index (LISI) score which is the proportion of prescriptions in GP practices which were exempt from payment due to low income (i.e. proxy for deprivation). Data source was the individual GP practices. Ethnicity Proportion of patients in GP practice population defined as South Asian. Data source was the 1991 Census. ::: In order to assess the equity of prescribing for CHD drugs, we needed to develop a set of variables which measured (or acted as proxies for) the health care need in GP practice populations. In terms of coronary heart disease (CHD), there are a number of identifiable risk-factors, all of which increase the risk of CHD morbidity and/or mortality. However, readily available data are not available for a number of these risk-factors, and therefore could not be explored within this study. Nevertheless, data were available on the following risk-factors: age, gender ethnicity, socio-economic status. Evidence on the importance of these risk-factors for assessing health care need is presented below. Rates of CHD related morbidity and mortality increase dramatically with age \[[@B45],[@B46]\]. For example, the prevalence of angina in England was 1 per cent in people aged 16--44 years, 3 per cent in those aged 45--54 years, although this increased to 10 per cent in those aged 55--64 years, 16 per cent in those aged 65--74 years and 18 per cent in those aged over 75 years \[[@B47]\]. Rates of CHD mortality and morbidity in the UK are generally seen to be higher for people from South-Asian groups, than from white-English people, and much lower for people born in the Caribbean. It has been suggested that the mortality rate for South Asians is 50 per cent higher than for the general population \[[@B48]\]. In the UK, the standardised mortality rates for CHD in South-Asian men was 146, compared to 100 for all men and just 46 for men born in the Caribbean \[[@B49]\]. It is also recognised that there are differences within South Asian groups, especially between Indians, who generally experience better health, and Pakistanis or Bangladeshis, who have generally worse health \[[@B50]-[@B53]\]. However, reliable data on CHD morbidity or mortality down to these more complex ethnic groupings are not readily available, and any estimates are, at best, imprecise \[[@B50]\]. Nevertheless, data on the ethnic minority profiles of GP practice populations may prove very useful in determining the need for CHD drugs. There is an extensive literature on socio-economic inequalities in CHD mortality and morbidity \[[@B54]-[@B57]\]. Whilst rates of CHD have been declining in the UK for almost 20 years, they have not been falling as fast as countries such as Australia and the United States \[[@B46]\]. Declining CHD mortality rates are only partially explained by reductions in established cardiovascular risk factors \[[@B58]-[@B60]\] and it is possible that general social and economic improvement over time has contributed to this trend \[[@B61]\]. However, it is noteworthy that these benefits have not been observed by all of the socio-economic groups within the UK \[[@B62]\]. For example, deaths from CHD in men in the highest social class have halved in the past 20 years, but remain almost unchanged among men in the lowest social class \[[@B60]\]. Between 1986 and 1992, people from the highest social classes had a rate of 160 CHD deaths per 100,000 population, whereas the rate for the lowest social classes was 266 per 100,000 \[[@B49]\]. In addition, a 22-year follow-up study on the relationship between socioeconomic status and CHD in middle-aged men \[[@B63]\] found that irrespective of length of follow-up, lower social classes had a clearly increased risk of fatal CHD -- after 8 years they had a 69 per cent increased risk, which dropped to 67 per cent after 15 years and 59 per cent after 22 years. Therefore, socio-economic status is significantly related to CHD mortality and morbidity, and as such, represents an important indicator of health care need for CHD drugs. Overall, the age, ethnicity and socio-economic status are all important factors in shaping the epidemiology of CHD in the UK, and as such, are important variables for use in developing the HCNIs in this study. The actual development of HCNIs relating to these risk factors are outlined below, in addition to additional HCNIs related more directly to CHD morbidity and mortality. Some HCNIs were collected directly from GP practice lists (proportion of patients aged over 75 years and proportion of patients aged 55--74 years), some have been calculated for GP practices (Low Income Scheme Index (LISI) score) \[[@B64]\], and others were calculated from data directly attributable to GP practices (data based on hospital episode statistics). However, due to the lack of information available from GP practices on variables such as socio-economic status and ethnicity (in this study, this refers to South Asian groups) of patients, a number of HCNIs were developed which estimated this from data such as the 1991 Census and Local Authority statistics. The method of patient weighted attribution was used to develop these estimates using data at enumeration district level (small geographical areas), whereby the postcodes of patients were linked to enumeration districts. Data on the enumeration districts of all registered patients were then aggregated for each GP practice and divided by the total registered population in order to provide a patient-weighted score. In this way, data from the Census were directly applied to GP practice populations. Further information and critiques of this method can be found elsewhere \[[@B65]-[@B67]\]. Whilst 1991 Census data may be regarded as rather old now (and has since been superseded by 2001 Census data), these were the only data available at the time of the study, since GP practices do not routinely collect data on the ethnicity of patients. Nevertheless, the HCNI relating to ethnicity may be seen with caution in this paper. Demographic HCNIs were developed directly from GP practice list data, and these relate to the proportion of patients aged 55--74 years, and the proportion aged over 75 years. Both of these demographic groups are indicators of health care need for CHD drugs \[[@B44]\]. The Low Income Scheme Index (LISI) was used as a proxy for low income since it represents the proportion of prescriptions which are exempt from prescription charges due to low income \[[@B64]\]. The proportion of patients from South Asian groups was estimated for each GP practice using data from the 1991 census. Data were also obtained from hospital episode statistics (HES) on specific hospital procedures (coronary artery bypass graft (CABG), percutaneous transluminal angioplasty (PTCA), and coronary angiogram) and diagnoses (primary diagnosis of CHD at discharge). Although HES relate to the supply, as opposed to need for health care services \[[@B68]\], it was hypothesised that in the absence of other CHD morbidity data, they may represent a useful proxy of CHD morbidity in GP practice populations. Due to the low numbers of procedures and diagnoses within a GP practice population, data were aggregated for 6 years (1995 to 2000 inclusive). Crude rates (per 1000 patients aged over 35 years) were calculated for CHD HES, which represents the CHD procedures + CHD diagnoses. As outlined in Appendix B, a number of other variables relating to CHD mortality and morbidity were entered into the regression models, although these were not statistically significant. Indeed, as outlined in a previous paper by the authors \[[@B4]\], bivariate analyses revealed that associations between prescribing rates and standardised mortality rates for CHD were often not statistically significant and in some cases had a negative association. Therefore, prescribing rates and CHD mortality rates do not exhibit a strong relationship, adding to the suggestion of an inequity in prescribing rates. Data analysis ------------- Multiple linear regression modelling was undertaken for each drug group in each PCT in addition to the combined dataset. The dependent variable in each model was the respective prescribing rate and the independent variables were all 24 HCNIs developed in the study. The forward-stepwise approach was taken but not slavishly adhered to since considerations of model coherence and plausibility were taken into account when seeking the most parsimonious models. The final regression models only contained the HCNIs which had statistically significant (p \< 0.05) independent associations with the dependent variable, and each model was checked for collinearity and normality of residuals. Overall, for each multiple regression model, all 24 HCNIs were entered as independent variables, and the final model included only those variables that were statistically significant and added to the fit of the model. Results ======= Descriptive findings will be presented first, in order to contextualise the findings from the multiple regression analyses. Variations in prescribing rates and health care needs indicators (HCNIs) ------------------------------------------------------------------------ Table [2](#T2){ref-type="table"} provides details of variations between primary care trusts (PCTs) in prescribing rates for each of the study drug groups. PCT1 generally had the highest median prescribing rates, followed by PCT2, with the lowest prescribing rates occurring in PCT3 and PCT4. The median ADQs per patient over 35 years for all study CHD drugs combined were 105 in PCT1, 93 in PCT2, 90 in PCT3, and 84 in PCT4. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Variation in prescribing rates by primary care trust (PCT) ::: Drug group PCT1 PCT2 PCT3 PCT4 ------------------------- ------------ ---------- ---------- ---------- ---------- Statins Min 11.05 9.56 6.86 6.44 Max 47.23 32.12 46.60 45.76 Median 22.88 19.65 18.08 17.36 IQR 8.20 11.20 8.40 7.24 Aspirin Min 14.62 15.34 4.94 9.6 Max 44.97 46.40 46.63 45.65 Median 29.27 27.35 25.33 30.87 IQR 7.40 16.5 7.60 18.85 Beta-blockers Min 5.04 6.38 3.94 3.43 Max 23.92 29.04 23.91 26.19 Median 12.84 12.38 12.32 12.64 IQR 4.61 7.70 7.41 10.11 ACE inhibitors Min 15.47 9.85 4.20 4.69 Max 48.40 39.25 32.97 35.69 Median 26.43 19.76 16.59 19.19 IQR 8.82 11.17 9.37 11.29 Bendrofluazide Min 4.19 1.63 1.63 0.60 Max 35.61 29.55 32.90 22.91 Median 14.42 10.85 11.04 9.01 IQR 6.98 8.15 12.70 7.28 All study CHD drugs Min 71.70 57.28 25.89 24.76 Max 156.82 154.21 136.64 150.68 Median 105.37 92.78 90.33 83.65 IQR 28.25 57.07 32.08 51.18 ::: PCT1 had the highest median prescribing rates for all drugs except aspirin, where PCT4 had the highest median rate. PCT3 had the lowest median prescribing rates for aspirin, beta-blockers and ACE inhibitors, and PCT4 had the lowest median prescribing rates for statins, bendrofluazide, and all study CHD drugs combined. The difference between the median prescribing rates for PCT1 and PCT4 for bendrofluazide, statins and all CHD drugs was around 4, 5, and 21 ADQs per patient over 35 years. Therefore, an average GP practice in PCT1 with 3000 registered patients over 35 years, prescribed 12000 more ADQs for bendrofluazide, 15000 more ADQs for statins, and 63000 more ADQs for all study CHD drugs. In addition, a similar GP practice in PCT1 also prescribed almost 30000 more ADQs for ACE inhibitors than a comparably sized GP practice in PCT3. An initial overview of the health care needs of the PCTs suggests that PCT4 had the highest levels of CHD related health care needs within the study, whereas PCT1 had the lowest health care needs. PCT4 was the most deprived of all PCTs, had the highest proportions of patients aged over 75 years, and the highest median score for standardised mortality rates (SMR) for CHD. In contrast, PCT1 may be seen as the \'least needy\' of all PCTs on the basis of the HCNIs developed in this study. PCT1 was the least deprived, had the lowest proportions of South Asian groups, had the lowest median SMR and the lowest median rate of CHD hospital procedures. These overall descriptive findings are indicative of inequitable prescribing rates, whereby PCT1 had high prescribing rates and low comparative health care needs, whereas PCT4 had low prescribing rates and high comparative health care needs. However, these are purely descriptive and do not take into account the multiple risk factors for CHD or the interactions between HCNIs. Therefore, findings from the multiple regression analyses are presented below. Multiple regression analysis ---------------------------- Table [3](#T3){ref-type="table"} presents the percentage of variation explained in each of the models (i.e. R^2^\* 100), Table [1](#T1){ref-type="table"} presents a description of the HCNIs included in the final regression models and Table [4](#T4){ref-type="table"} presents details for the combined dataset in terms of the HCNIs included in each model, their standardised beta coefficients and their contribution (in terms of percentage variation explained) to the total R^2^of the model. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Percentage of variation in prescribing rates explained by HCNIs ::: PCT1 PCT2 PCT3 PCT4 Combined -------------------- ---------- ---------- ---------- ---------- -------------- Statins 24.5 58.3 31.3 50.5 24.9 Aspirin 44.2 88.2 62.3 51.7 51.2 ACE inhibitors No Model 55.3 26.6 45.8 31.6 Beta-blockers 28.3 42.4 46.1 41.1 27.1 Bendrofluazide 15.6 53.9 40 54.5 24.1 ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Regression models for combined dataset ::: Drug group R^2^ Needs indicator Beta coefficient % explained -------------------- ---------- ------------------------- ---------------------- ----------------- Statins .249 CHD HES rate .350 14.7 \% patients aged \>75 -.240 4.2 Ethnicity -.233 3.1 \% patients aged 55--74 .199 2.9 Aspirin .512 CHD HES rate .579 32.9 LISI score -.261 10.4 \% patients aged \>75 .347 8.2 ACE inhibitors .316 Ethnicity -.395 25.3 \% patients aged \>75 -.170 3.4 \% patients aged 55--74 .248 2.9 Beta-blockers .271 CHD HES rate .411 19.7 \% patients aged 55--74 .274 7.4 Bendrofluazide .241 LISI score -.261 13.1 CHD HES rate .171 6.6 \% patients aged 55--74 .274 4.4 All CHD drugs .454 LISI score -.303 22.7 CHD HES rate .461 20.0 \% patients aged 55--74 .242 2.7 ::: Regression models generally explained much more variation in prescribing rates in PCT2 than any other PCT. For example, in PCT2, modelling explained around 55 per cent of the variation in prescribing rates for ACE inhibitors and bendrofluazide, almost 60 per cent for statins and almost 90 per cent of variation in aspirin prescribing rates. Multiple regression models explained around 25 to 55 per cent of the variation in prescribing rates in the combined dataset, around 25 to 60 per cent in PCT3, and around 40 to 55 per cent in PCT4. PCT1 had the lowest R^2^values in general, and no model was derived for ACE inhibitors. Given the reduced explanatory power of the regression models in PCT1, when regression modelling was undertaken for the combined dataset, the R^2^values were much lower than those for PCT2, and generally lower than those for both PCT3 and PCT4. In general, regression modelling explained more variation in prescribing rates for aspirin than for the other drug groups. Not including PCT1, regression modelling explained between 50 and 90 per cent of the variation in aspirin prescribing rates. Comparative percentages were between 30 and 60 per cent for statins, 30 and 45 per cent for beta-blockers, and 25 and 55 per cent for ACE inhibitors and bendrofluazide. Table [4](#T4){ref-type="table"} presents details of the regression models derived for each drug group in the combined dataset. Many of the models in Table [4](#T4){ref-type="table"} are quite similar in terms of the HCNIs included within them, with all models (except for ACE inhibitors) including the rate of CHD HES (positive association) and all models (except for aspirin) including the proportion of patients aged between 55 and 74 years (positive association). In addition, the LISI score was included in the models for aspirin, bendrofluazide and all CHD drugs (negative association). The models for statins, aspirin and ACE inhibitors also included the proportion of patients aged over 75 years although this indicator had a negative association with prescribing rates for statins and ACE inhibitors, and a positive association with prescribing rates for aspirin. This suggests that GP practices with higher proportions of patients aged over 75 years have lower prescribing rates for statins and ACE inhibitors, and higher prescribing rates for aspirin. The CHD HES rate varied considerably between models, in terms of the amount of variation explained. For example, it explained 33 per cent of the variation in aspirin prescribing rates, 20 per cent of the variation in prescribing rates for all CHD drugs and beta-blockers, 15 per cent of the variation in statin prescribing rates, and 7 per cent of the variation in prescribing rates for bendrofluazide. The LISI score explained 23 per cent of the variation in prescribing rates for all CHD drugs, 13 for bendrofluazide and 10 per cent for aspirin. In addition, the proportion of patients aged between 55 and 74 years explained between 2.5 and 7 per cent of the variation for statins, ACE inhibitors, beta-blockers, bendrofluazide, and all CHD drugs. Discussion ========== The rate of CHD hospital procedures (CHD HES) explained some of the variation in prescribing rates and therefore, prescribing rates in these instances were positively related to health care need (i.e. equitable). These findings concur with other studies whereby positive relationships were found between rates of PTCAs and both aspirin prescribing \[[@B36]\] and statin prescribing \[[@B36],[@B69],[@B70]\]. These may suggest that prescribing rates are positively related to healthcare need, although as already outlined, HES may actually reflect supply or demand, rather than need per se. Nevertheless, within this study, HES were the best data available as proxies for CHD in the GP practices, and therefore, we suggest that these prescribing rates reflect CHD prevalence, and hence healthcare need. GP practices in the UK are now developing CHD registers, which may be used in the future as proxies of CHD prevalence, although again, these will only reflect treated CHD, as opposed to the true prevalence of CHD in the community. The proportion of patients aged 55--74 years was generally positively related to prescribing rates, suggesting that prescribing rates are related to health care need, since people in this age group have a higher prevalence of CHD \[[@B49]\]. A number of studies have found that statin prescribing is higher in this age group, than in older age groups \[[@B13],[@B16],[@B17],[@B70]\], with one study finding that the proportion of patients aged 35--74 years explained just 5 per cent of the variation in statin prescribing rates between GP practices \[[@B24]\]. Whilst the previous finding is not unexpected given the higher CHD prevalence in this age group, the negative relationship between the proportion of patients aged over 75 years and some prescribing rates should be of great interest. The proportion of patients aged over 75 years had a negative relationship with statins and ACE inhibitors and a positive association with aspirin. A number of studies found that older patients were much less likely than younger patients to undergo a CABG or a PTCA\[[@B71],[@B72]\] or to receive a prescription for a statin \[[@B13],[@B16],[@B17],[@B70]\], which may result from the lack of research evidence on the efficacy of statins in elderly populations or judgements about likelihood of benefit based on age. Therefore, the negative associations between the proportion of patients aged over 75 years and rates of ACE inhibitor and statin prescribing are in line with other seemingly inequitable health care. Although the proportion of patients aged over 75 years is a proxy for CHD prevalence, it may not represent a useful proxy of the potential to benefit from statins. However, it may be suggested that prescribing rates for aspirin are higher in this age group since they are more cost-effective than statins \[[@B73]\]. Therefore, these drugs may be more readily prescribed to patients for whom the benefits (both clinical and cost-effectiveness) of statins have not been studied. Conclusion ========== Overall, this study has found that prescribing rates may be explained (to differing degrees between primary care trusts (PCTs) and between drug groups) by a mixture of health care need indicators (HCNIs). Whilst prescribing rates were generally positively related to the rates of hospital procedures and diagnoses, they were also negatively associated with proxies of deprivation and ethnicity and with the proportion of patients aged over 75 years. However, the relatively low R^2^values reveal a large amount of unexplained variation in prescribing rates. Indeed, even with the models for aspirin and all CHD drugs, there is still around 50 per cent of unexplained variation in prescribing rates. This study cannot be used to infer inequitable prescribing by GPs, since the lower prescribing rates in areas with high proportions of South Asian and deprived groups may be due to lower utilisation of primary health care services due to social, economic or cultural barriers \[[@B74],[@B75]\]. Therefore, further work needs to be undertaken in areas of deprivation and with high proportions of minority ethnic groups in order to understand the reasons for the low prescribing rates and ultimately to make CHD prescribing commensurate with healthcare need. In addition, the HCNIs used in this study are proxies for healthcare need, and may not reflect actual healthcare need. Therefore, future studies may attempt to verify our findings by using either morbidity or mortality data gathered from GP practices. Whilst the clinical and epidemiological data on these CHD drugs has allowed for the development of evidence-based guidelines and evidence-based prescribing, this paper suggests that in practice, actual prescribing rates may not be related to health care need. Further research needs to concentrate on verifying or falsifying these claims on a more micro-level analysis (eg clinical audit in specific GP practices identified in the study) and on exploring the reasons why such a relationship exists (e.g. qualitative studies with GPs, practice nurses and patients in the identified GP practices). In addition, more work is required to understand the differences between PCTs in terms of the explanatory power of the regression models (i.e. much more of the variation in prescribing rates were explained in PCT2 than any other PCT). Such a strategy may enable educational tools to be developed which would facilitate more evidence-based prescribing, but may also identify particular patient groups who do not present symptoms of CHD (ie unmet need) and therefore may require educational outreach or targeted screening in order to increase their consultations and ultimately prescribing to these groups. Although we have focussed on drugs used in the prevention of CHD, a similar approach may be taken across different therapeutic groups of drugs, in different health care settings and in different countries in order to generate more rounded, grounded and extensive evidence on the equity of prescribing in general. Appendix A -- List of drugs used in this study ============================================== • Aspirin (75 mg) • Bendrofluazide^1^(2.5 mg) • Statins (Atorvastatin, Cerivastatin, Fluvastatin, Pravastatin, Simvastatin) • ACE inhibitors^2^(Captopril, Enalapril, Lisinopril, Ramipril, Trandolapril) • Beta-blockers^3^(Atenolol, Co-tenidone^4^) ^1^In some countries, this may also be known as (among other names) Neo-NaClex, Bendroflumethiazide, Aprinox, Berkozide, Naturetin, Pluryl, Polidiuril, Salural, Urizde. ^2^The 5 ACE inhibitors represent the majority of prescribing for all ACE inhibitors ^3^Atenolol represents the majority of prescribing of all beta-blockers ^4^Co-tenidone is a combination product containing both a beta-blocker (atenolol) and a diuretic (chlorthalidone). Appendix B -- List of health care needs indicators (HCNIs) developed during the study ===================================================================================== • Proportion of patients aged between 55 and 74 years • Proportion of patents aged over 75 years • Proportion households with no car • Proportion males who are economically inactive • Townsend Score • Proportion of households receiving council tax benefits • Proportion unemployment • Index of Multiple Deprivation • Income Deprivation Index • Low Income Scheme Index (LISI) score • Standardise mortality rate (SMR) for CHD under 75 years • 6-year crude rate of coronary artery bypass grafts (CABGs) per 1000 patients • 6-year crude rate of percutanious transluminal angioplasty (PTCAs) per 1000 patients • 6-year crude rate of coronary angiograms per 1000 patients • 6-year crude rate of CHD hospital procedures (CABGs + PTCAs + angiograms) per 1000 patients • 6-year crude rate of CHD hospital diagnoses per 1000 patients • 6-year crude rate of CHD prevalence (diagnoses + procedures) per 1000 patients • Regionally specific prevalence, age and sex standardised prescribing units per patient over 35 years (PASS-PUs) • Proportion of population defining themselves as \'non-white\' • Proportion of population defining themselves as \'South Asian\' • Proportion of population over 30 with a limiting long-term illness (LLI) • Health Deprivation Index • Proportion of households with more than 2 cars • Access Deprivation Index Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= PW was awarded funding for this study, was involved in the conception, design and managed the day to day running of the study, undertook all data collection and analysis, and wrote the paper. PN and ASL were involved in the conception and design of the study, made active contributions in project meetings, were involved in an advisory capacity in all aspects of the project and advised on drafts of the paper. PW is the guarantor. Acknowledgements ================ Paul Ward received a Health Services Research Training Fellowship from the North West NHS Executive to carry out the study on which this paper is based. We thank all health authority, PCT and Local Authority staff who provided access to PACT data, GP practice list data, hospital episode statistics and a variety of other data sources. We also thank Professor Barbara Starfield for her helpful comments on an earlier draft of this paper.	PubMed Central	2024-06-05T03:55:52.847458	2005-2-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548940/", "journal": "Int J Equity Health. 2005 Feb 8; 4:3", "authors": [ { "first": "Paul R", "last": "Ward" }, { "first": "Peter R", "last": "Noyce" }, { "first": "Antony S", "last": "St Leger" } ] }
PMC548951	How do we relate human thought processes to measurable events in the brain? Mental chronometry, which has origins that date back more than a century, seeks to measure the time course of mental operations in the human nervous system \[[@pbio-0030051-b1]\]. From the late 1800s until 1950, the field was built almost entirely around a single method: measuring and comparing people\'s reaction times during simple cognitive tasks. As far back as 1868, Franciscus Donders \[[@pbio-0030051-b2]\] subtracted the time taken to make a single response to an unvarying stimulus---what he called an instructed reflex---from the time it took to make the same response to one of two events, obtaining the time required to discriminate between the two stimuli. Further, he subtracted the time to discriminate two stimuli from a situation in which there were also two possible responses in order to obtain the time required for choice. In the 1950s, studies of reaction time were combined with the then-developing mathematical theory of information \[[@pbio-0030051-b3]\] to address issues such as the maximum transmission rate of the human nervous system \[[@pbio-0030051-b4]\] and how coding in the brain of stimuli and their responses could influence these limits \[[@pbio-0030051-b5]\]. These studies revealed that reaction time alone was not sufficient to elucidate the exact processes by which the brain achieved the human ability to process information. However, when combined with other methods, the latency of responding can help connect brain studies to the behavior of humans in real situations. Recording the average event-related electrical potentials from scalp electrodes became a research tool in the 1960s, with the advent of analog and then digital computers to accomplish the recording and averaging. It became clear that components of the event-related potential could be systematically related to sensory and motor stages of information processing. For instance, a visual stimulus was found to evoke a short-latency scalp response from the primary visual cortex at about 60 milliseconds, followed by positive and negative voltage changes in neighboring visual areas. Similarly, scalp recorded potentials from the frontal cortex could be recorded in relation to motor activity. It was now possible to observe some of the sensory and motor stages that were inferred from Donders\'s subtractive method (see \[[@pbio-0030051-b6]\] for a review). Saul Sternberg \[[@pbio-0030051-b7]\] developed a much-improved method for dividing reaction time into successive or serial stages, called the additive factors method. Subjects were asked to determine whether or not a probe digit had been present in a just previously presented series of digits. Sternberg argued that the time to complete the task could be divided into a sensory stage, dependent on stimulus parameters such as the intensity or clarity of the probe; a comparison stage, dependent only on the number of items in memory; and a response stage that reflected the difficulty of the specified response. Factors that influenced one stage (e.g., stimulus intensity) would be additive with those that influenced another stage (e.g., motor output). With this simple framework, it was now possible to determine at which stage(s) a new factor (e.g., nicotine, sleep deprivation, or Parkinson\'s disease) had its influence. In the 1950s, the advent of microelectrode recordings of single neurons from anesthetized monkeys allowed for an even finer resolution of neurophysiological processes and seemed to provide support for the view that the brain does indeed process information in serial stages. Hubel and Wiesel \[[@pbio-0030051-b8]\] argued that successive levels of the visual system could be seen as accomplishing successive analyses of input. The microelectrode strategy was quickly adopted to alert animals, making it apparent that higher level brain areas involved in operations upon input might feedback their influences on earlier processing stages \[[@pbio-0030051-b9],[@pbio-0030051-b10]\]. These control systems, often called attention, posed something of a problem for completely serial views of information processing. However, they also provided evidence of localized brain areas within the parietal lobe of the monkey that could be systematically related to processing operations involved in attention---which were then being investigated by mental chronometry in patients with parietal and other lesions \[[@pbio-0030051-b11]\]. In the late 1980s, neuroimaging experiments made possible the examination of activity in localized brain areas, first through the use of injected radionuclides detected by positron emission tomography (PET) \[[@pbio-0030051-b12]\] and later through the use of an externally imposed magnetic field in functional magnetic resonance imaging (fMRI) \[[@pbio-0030051-b13]\]. Over the last ten years, fMRI has improved in spatial and temporal resolution and can now provide evidence of quite specific brain areas, in the millimeter range, that are involved in cognitive tasks. Most studies have shown a small number of widely distributed brain areas that must be orchestrated to carry out a cognitive task. Although, as in all sciences, there are disagreements, the convergence of results in areas of attention and language seem to me particularly impressive. When the fMRI method for localization is brought together with methods that can accurately measure timing of the same activity (i.e., electrical or magnetic event-related potentials) they can provide considerable insight into the nature of thought. Consider the simple task of deciding whether a presented digit is above or below five \[[@pbio-0030051-b14]\]. Dehaene argued that the task could be divided into four stages. The first involves obtaining the identity of the probe input (encoding), the second making a comparison against the stored representation of the digit five, the third selecting a response, and lastly, checking the output for error. According to additive factor theory, a variable that effects overall reaction time by varying the time to complete one stage will be additive with the effects of variables that affect other stages. The input or encoding stage was varied by using either Arabic or spelled digits (e.g., "3" or "three"). The comparison stage was varied by comparing digits close to five (e.g., six) with those far from five (e.g., nine). The response stage was varied by specifying a response from either the dominant or non-dominant hand and error monitoring was examined by comparing error with correct trials. Each of these variables influenced only the appropriate stage and was additive in its effect with each of the other variables (see [Figure 1](#pbio-0030051-g001){ref-type="fig"}). ::: {#pbio-0030051-g001 .fig} Figure 1 ::: {.caption} ###### Reaction Time for Various Conditions People were asked to judge whether a presented digit was greater or less than five. The time to respond (reaction time) varied systematically as a function of notation (Arabic digits vs. spelledout numbers), distance (closer or farther in sequence from five), and responding hand. The three effects are additive, indicating the likelihood of serial stages of cognitive processing. (Adapted from \[[@pbio-0030051-b14]\]) ::: ![](pbio.0030051.g001) ::: Moreover, scalp-recorded, event-related potentials showed a separate scalp distribution and latency for each variable \[[@pbio-0030051-b14]\]. Subsequent fMRI data has confirmed and increased the precision of the anatomy proposed for each of these stages. Of course, not all human activities involve a set of exhaustive and independent serial stages that can be shown to add up to the overall reaction time. While tasks like the number comparison discussed above can be usefully divided into stages, some components may deal with simultaneous operations and may be limited only by a total capacity of central mechanisms. We know that many situations involve parallel processing and feedback loops at many levels. Sternberg has attempted to apply a modified version of additive factor theory to brain systems using neuroimaging that allows for some of these possibilities \[[@pbio-0030051-b15],[@pbio-0030051-b16]\].[](#pbio-0030051-g002){ref-type="fig"} ::: {#pbio-0030051-g002 .fig} Figure 2 ::: {.caption} ###### Regions of the Brain Involved in a Number Comparison Task Derived from EEG and fMRI Studies The regions represented correspond to those showing effects of notation used for the numbers (pink and hatched), distance from the test number (orange), choice of hand (red), and errors (purple). (Illustration: Giovanni Maki; adapted from \[[@pbio-0030051-b18]\]) ::: ![](pbio.0030051.g002) ::: Laboratory studies often use the simultaneous execution of two different tasks (dual tasks) to simulate the more realistic situations where humans time-share activities. In this issue of PLoS Biology, Sigman and Dehaene \[[@pbio-0030051-b17]\] provide a model that further extends the additive factor logic to the dual task situation. They propose that for tasks that can be broken down into three consecutive stages---perception, decision based on noisy integration of evidence, and response---the perceptual and motor stages can operate both simultaneously with and independently of stages of another task and are thus easily amenable to additive factor analysis. The decision stage, however, appears to represent a kind of "cognitive bottleneck" for which the reaction times of the two tasks become interdependent. The model adds considerably to the range of situations to which an additive factor approach can be applied, allowing investigators to seek more information about how new variables influence hidden processing stages. Many cognitive and emotional tasks studied with neuroimaging have implicated a small number of brain areas that are consistently active. Mental chronometry plays a role in suggesting the cognitive operations that each of these areas performs and how they are organized in real time. The toolkit of new techniques provides the basis for further tests to evaluate whether a chronometric model reveals a crucial set of connected computations (circuit) for carrying out the task. For example, using a magnetic pulse delivered outside the skull, it is now possible to induce a reversible lesion at the time of a particular computation to determine whether the specific computation assigned to a given area is truly needed to carry out the task. Studies using diffusion tensor imaging can examine whether there are large-scale connections between neural areas posited by a particular model. In describing the links between brain and behavior, mental chronometry is still a cornerstone that binds psychology to the techniques of neuroscience. Citation: Posner MI (2005) Timing the brain: Mental chronometry as a tool in neuroscience. PLoS Biol 3(2): e51. Michael I. Posner is with the Department of Psychology at the University of Oregon, Eugene, Oregon, United States of America. E-mail: <mposner@darkwing.uoregon.edu> fMRI : functional magnetic resonance imaging PET : positron emission tomography	PubMed Central	2024-06-05T03:55:52.850754	2005-2-15	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548951/", "journal": "PLoS Biol. 2005 Feb 15; 3(2):e51", "authors": [ { "first": "Michael I", "last": "Posner" } ] }
PMC548952	The study of genetic material from ancient specimens was, in its early years, dominated by a race to sequence DNA from extinct species like the dodo and the woolly mammoth. Now that the supply of these crowd-pleasing curiosities has run dry, scientists are starting to ask new questions of ancient DNA (aDNA) that are revealing how the genetic make-up of prehistoric populations changed through time. These findings look set to trounce assumptions about how evolution really unfolded. However, there is still concern that many studies are not paying enough attention to the exacting protocols needed to overcome the technical challenges of the discipline and to defend it from the ridicule that has plagued it in the past. In 1994, while Jurassic Park was still taking in millions of dollars at the box office, scientists claimed to have extracted and sequenced DNA from an 80-million-year-old dinosaur \[[@pbio-0030056-b1]\]. When sceptical researchers took a look at the sequence, it turned out to be of human rather than dinosaur origin. "To make that mistake, you\'d have to try really, really hard," says Alan Cooper, head of the Henry Wellcome Ancient Biomolecules Centre at Oxford University in the United Kingdom. If you think you\'ve sequenced some dinosaur DNA, the first thing you\'d do is run a phylogenetic analysis on it, he says. "Had they done that properly, with any mammal at all involved in the tree,...they would have found that their sequence was grouping with the mammals and not with the reptiles or the birds," says Cooper. Perhaps they\'d watched Michael Crichton\'s inventive fiction one too many times, he suggests. Setting the Standards {#s2} ===================== It was this kind of bungling study that highlighted the need for an exacting protocol that would steer researchers around the significant pitfalls posed by DNA decay and contamination. A list of "authenticity criteria" emerged during the 1990s, aimed at preventing similarly bogus claims from entering the literature \[[@pbio-0030056-b2]\]. This list includes stringent laboratory controls; cloning of products amplified by polymerase chain reaction (PCR); replication of results from a second, independent extract; and, for really new or unexpected results, replication of results by an independent research group. Such requirements have allowed work on aDNA to move on and mature. Now, it\'s possible to focus on the really interesting questions that aDNA can answer. "What we\'re able to do with ancient DNA is really look at evolution," says Cooper. The fossil record can only hint at how evolution unfolded. "It just shows you there\'s a bear and then there\'s not a bear," he says. "It doesn\'t show you where it came from or what the relationship between the groups is." By contrast, aDNA can do just that, giving researchers a window onto the population genetics of the past and revealing how evolution really played out. And the signs are that descriptions of the evolutionary process based on the fossil record and modernday gene pools are far too simple. "The modern data is clearly misleading us," says Cooper. "Evolution is much, much more complex and dynamic than we would hope." Thinking Big {#s3} ============ Because of the decay that occurs with time, there is a limit to how far back aDNA can gaze [(Box 1)](#box1){ref-type="boxed-text"}. "Your ideal preservation conditions are something that falls under ice, freezes instantly, and stays frozen until you get it," says Cooper. "As soon as we get up to 2 million \[years ago\] we can\'t get anything to work, and that\'s even under deep-frozen conditions." But within the past 60,000 years, there are several major evolutionary events that are worth studying---including a glacial maximum around 18,000 years ago, the invasion of the New World by humans about 12,000 years ago, and a global mass extinction about 11,000 years ago. These relatively recent events should be a good model for working out how similar events affected genetic diversity throughout evolutionary history. Box 1. Decay ------------ As soon as an organism dies, nucleases get the better of repair enzymes and rapidly digest strands of DNA. Under certain conditions, such as rapid desiccation, freezing, or high salt concentrations, the nucleases are inactivated before the damage is done. Even if some DNA is left intact, however, radiation, oxidation, and hydrolysis can still cause damage. These processes mean that ancient DNA specimens are like a four-letter alphabet soup, says Cooper. Very little of the original DNA remains, which is why most aDNA studies focus on mitochondrial rather than nuclear DNA: there are simply more copies of mitochondria, so the chances of getting an aDNA sequence are that much higher. Furthermore, chemical changes to the DNA fragments that remain cause additional problems: the PCR is often fooled into inserting inappropriate bases when it is copying an ancient template strand. "Amplification of DNA molecules older than one million years of age is overly optimistic," note Pääbo and his colleagues \[[@pbio-0030056-b11]\]. In spite of these difficulties, several groups are hoping to work out ways to spot aDNA damage and set about repairing it. Since the 1980s, Svante Pääbo and Tomas Lindahl have made several stabs at removing glitches in aDNA using purified repair enzymes, filling in the gaps between sequences and then joining them together to resurrect something like an original sequence. "Sometimes it has seemed to help, but nothing really reproducible has come out of it," admits Pääbo. One approach has, however, been successful at repairing DNA damage that occurs with time. Cross-links can form between reducing sugars and amino groups, he says ([Figure 4](#pbio-0030056-g004){ref-type="fig"}). Such cross-links can sometimes be broken using the chemical N-phenacylthiazolium bromide, releasing PCR fragments that would otherwise be tied up. The amount of repair that\'s possible will never be able to restore the DNA sequence of an extinct species Jurassic Park--style, but it should allow researchers to ask even more profound questions of aDNA. "In five years, I think we\'ll see some repair methods really get going," says Cooper. Cooper\'s latest work has analysed DNA from over 400 bison fossils from Beringia---the frozen wastes between eastern Siberia and the Canadian Northwest Territories \[[@pbio-0030056-b3]\]. "What we\'ve done is carbon-date a shitload of bison and get DNA out of them." It\'s the largest aDNA study to date, he says ([Figure 1](#pbio-0030056-g001){ref-type="fig"}). The icy conditions mean that good quality mitochondrial DNA could be extracted from most of the specimens. The bison could also be dated accurately. This allowed Cooper and his colleagues to trace the changes in the bison genetic diversity from 150,000 years ago to the present. It was even possible to predict the effective population size throughout this period of bison evolution. "Our analyses depict a large diverse population living throughout Beringia until around 37,000 years before the present, when the population\'s genetic diversity began to decline dramatically," they note. ::: {#pbio-0030056-g001 .fig} Figure 1 ::: {.caption} ###### How Did Bison Really Evolve? \(A) A modern bison (Bison bison), (B) the skull of an extinct bison ancestor, and (C) extraction of aDNA from a bison bone. (Images: \[A\] Steve Malowski, United States Fish and Wildlife Service, \[B and C\] Henry Wellcome Ancient Biomolecules Centre, Oxford University) ::: ![](pbio.0030056.g001) ::: This finding challenges some common assumptions. It has been argued that modern bison are descended from Beringian bison, but Cooper\'s data suggest otherwise. "All modern bison belong to a clade distinct from Beringian bison," he and his colleagues report. Furthermore, the dramatic decline in the numbers of bison occurs long before humans arrive on the scene, scuppering the idea that hunting pressure was primarily responsible for the demise of the bison. As the glacial maximum approached 18,000 years ago, the cooler, dryer conditions were probably responsible for the downturn in the bison population, argues Cooper. "Climate change is giving the animals an absolute whacking," he concludes. A similar analysis of brown bear DNA excavated from permafrost and cave deposits in the Arctic is also challenging conventional evolutionary wisdom \[[@pbio-0030056-b4]\]. Being able to get both a radiocarbon date and some DNA from a specimen pins a particular genetic sequence to a particular moment in time. These data suggest that genetically and geographically distinct groups of bear have replaced each other relatively often during the last 60,000 years. Regional extinctions and replacements seem to be tied to climate change and competition with the much larger short-faced bears, the authors argue ([Figure 2](#pbio-0030056-g002){ref-type="fig"}). ::: {#pbio-0030056-g002 .fig} Figure 2 ::: {.caption} ###### What Are the Real Evolutionary Origins of the Brown Bear (Ursus arctos)? (Image: John Nickles, United States Fish and Wildlife Service) ::: ![](pbio.0030056.g002) ::: Recent analysis of aDNA from Haast\'s eagle has also thrown up a surprising result. This New Zealand giant had a wingspan of up to three metres and a weight of around 14 kilograms, says Michael Bunce, an anthropologist at MacMaster University in Ontario, Canada. Analysis of aDNA from 2,000-year-old specimens indicates that this extinct creature is closely related to the little eagle from Australia and New Guinea, which typically weighs less than one kilogram. The common ancestor of these two eagles lived as recently as 1 million years ago, he and his colleagues estimate \[[@pbio-0030056-b5]\]. "It means an eagle arrived in New Zealand and increased in weight by 10--15 times over this period," says Bunce. "Such rapid size change is unprecedented in terrestrial vertebrates." In addition to illuminating these natural events, the study of aDNA can also show changes in the frequency of key genes that occurred during the domestication of crops and animals. For example, aDNA from samples of early maize reveals when certain desirable traits appeared \[[@pbio-0030056-b6]\]. "It\'s the first study of ancient DNA that looks at phenotype," says Svante Pääbo, an evolutionary anthropologist at the Max Planck Institute in Leipzig, Germany. "One can actually look at specific genes that early humans selected during domestication of an important crop." Pääbo\'s analysis suggests that the alleles typical of contemporary maize were already present in Mexican maize 4,400 years ago, so just a couple of thousand years after its initial domestication from the wild grass teosinte ([Figure 3](#pbio-0030056-g003){ref-type="fig"}). "Quite early on, properties were selected that were not only the structure of the plant but also the biochemistry," he says. ::: {#pbio-0030056-g003 .fig} Figure 3 ::: {.caption} ###### Domesticating Maize \(A) Maize cob from the Ocampo Caves in Mexico dated to 3,890 years before the present. aDNA can reveal the selection of traits during early maize domestication that cannot be observed in the fossil record. \(B) Examples of modern maize. (Images: \[A\] Svante Pääbo, Max Planck Institute, \[B\] Keith Weller, USDA Agriculture Research Service) ::: ![](pbio.0030056.g003) ::: aDNA is also being used to decipher human origins. Mitochondrial DNA from Neanderthals looks quite different from the mitochondrial DNA of early modern humans \[[@pbio-0030056-b7]\]. This lends support to the hypothesis that modern humans have a "single African origin" rather than the alternative hypothesis of "multiregional evolution", where the ancestors of modern humans bred with Neanderthals. aDNA could also, in principle, be used to shed light on the evolutionary position of the 18,000-year-old "hobbit" recently unearthed on the Indonesian island of Flores \[[@pbio-0030056-b8]\]. Both Cooper and Pääbo have offered to have a go at isolating DNA from the "hominid" skeleton, but the early signs are that DNA has not survived. "The somewhat moist and tropical preservation conditions make the recovery of DNA improbable," says Peter Brown, the paleoanthropologist at the University of New England in Armidale, Australia, who led the hobbit study. Efforts to extract DNA from other bones collected at the same site as this tiny hominid have not produced results. "We have made attempts with Stegodon molars," he says, "but so far without success." Ongoing Controversy {#s4} =================== However, in spite of the authenticity criteria and this transition towards testing the big questions in evolutionary biology, aDNA research continues to invite controversy. In 2000, a team of United States researchers claimed to have cultured a bacterium sealed inside a 250-million-year-old salt crystal \[[@pbio-0030056-b9]\]. For Cooper, this is the sort of study that should require replication by an independent laboratory before publication. "When we repeated that work with the same primers, we were pulling up halobacteria from everywhere," he says. "We took some dust from the top of the natural history museum in Oxford, extracted \[DNA\], used their supposedly halospecific primers and extracted a whole bunch of sequences, including some that fell within their diversity." This strongly suggests, says Cooper, that the bacterium that was cultured was a modern bacterium, rather than an ancient specimen. "I can\'t see any logic for having 250 million years without any evolution." But Russell Vreeland, a microbiologist at West Chester University in Pennsylvania and first author of the salt-crystal study, is adamant that his methods were exacting. "The probability of having a contaminant in our sample was one chance in a billion," he calculates. "If you use a Band-Aid today on your skin or your children, you are 1,000 times more likely to have an infection from that Band-Aid than I am to have a contaminant." It\'s completely unscientific to argue that the cultured bacterium was a result of contamination simply because it resembles modern bacteria, says Vreeland. "That\'s throwing out the baby with the bathwater. If you can show that nothing has penetrated your sample and the DNA is inside, then the age of the DNA has to be equal to the age of that rock," he says. "I think you can make your criteria so stringent that you miss reality." Others are alert to this danger. Sticking rigidly to the authenticity criteria can be a problem, argues Tom Gilbert of the Department of Ecology and Evolutionary Biology at the University of Arizona. "\[The criteria\] can both hinder the publication of good studies that do not adhere to all the criteria, and also enable the publication of erroneous results that adhere strictly to them," he says. Part of the problem is that many referees of aDNA papers do not have a background working with aDNA, so are inclined to use the authenticity criteria as a checklist rather than critically evaluating each bit of research on a case-by-case basis. For example, he says, a recent high-profile study that followed all the criteria found that aDNA from two Cro-Magnon-type humans was very similar to DNA from modern humans \[[@pbio-0030056-b10]\]. But this could just mean that the specimens were contaminated by modern humans. "As no information was provided on the sample\'s handling history," says Gilbert, "it becomes impossible for a reader to decide whether the sequences are authentic or contaminant" [(Box 2)](#box2){ref-type="boxed-text"}. Such papers will continue to appear as long as the authenticity criteria are used by authors and referees as a checklist, he says. This does not mean the criteria should be relaxed, he adds, but they should be used in a more intelligent way. Box 2. Contamination -------------------- Most tissues under the scrutiny of the aDNA researcher will contain not only DNA from the organism of interest, but also DNA from bacteria, fungi, and all sorts of other organisms. Of course, most of this confusion can be cleared up using a speciesspecific primer in the PCR to amplify DNA from just one genome. "You\'re after a needle in a haystack," says Alan Cooper, "but fortunately that\'s what the PCR technique is so good at doing--- sorting through that haystack for you." A lot of focus has been placed on contamination in the laboratory. This "external contamination" is a particular problem because low levels of aDNA can easily be outnumbered by just a tiny amount of high-quality modern DNA floating around the lab. One of the criteria of authenticity---separating the site of DNA extraction from the site of PCR amplification---is designed to minimise this. In addition, there is "innate contamination" that has occurred to the sample before it even reaches the laboratory. This is much harder to control and is a real problem for those working on human origins because it\'s difficult to tell what is ancient human DNA and what is a modern contaminant. Bone is like sponge. "In life, bone is about 8% air. In death,...40% or 50% of it is air," says Gilbert. So if an archaeologist digs up and washes a bone from some human ancestor, it\'s almost certain that modern DNA will find its way into the centre of the bone \[[@pbio-0030056-b12]\]. Some archaeologists have even been known to lick bones to determine their porosity, he says. "If they\'re licking bones, God knows what else they\'re doing to things." This variable and unrecorded handling history of many museum specimens, means that contamination is difficult to rule out. This explains why many aDNA researchers focus on unusual species like the bison or the cave bear: these animals are extinct, so the chances of contamination are nil. It\'s easier to come up with real results that the scientific community will accept, says Gilbert. There will always be a temptation to work on humans, however, because it\'s these studies that grab the headlines, he says. But allowing authors the freedom to use the criteria as they see fit could come at a cost, says Cooper. "The trouble with a case-by-case basis is that it basically equates to no standards, because then people will do what they feel like doing and we\'re back to the 1990s again," he says. Agreement {#s5} ========= This ongoing disagreement over how aDNA studies should be judged does, however, stem from a common concern. As more and more biologists come to appreciate the unique ability of aDNA to probe the evolutionary process, it is more important than ever to stress the immense challenges of working with just a few fragments of degraded DNA that might have come from several different sources. It is obvious why an archaeology lab might want to set up its own aDNA facility. But this is like creating molecular biologists without a license, says Pääbo. "You wouldn\'t buy an accelerator and say \'I will now start doing my own carbon dating,\'" he says. "You have to really have experience working with low copy number." However, in spite of the continual problem of eager but inexperienced biologists trying to extract DNA from specimens in the university museum, there is a sense that aDNA is starting to fill in the gaps in our understanding of key moments in evolutionary history. So at the start of 2005, as aDNA research enters its 21st year, the discipline is, perhaps, coming of age. ::: {#pbio-0030056-g004 .fig} Figure 4 ::: {.caption} ###### Cross-Linked DNA Extracted from 4,000-Year-Old Liver of an Ancient Egyptian Priest Called Nekht-Ankh (Image: Svante Pääbo, Max Planck Institute) ::: ![](pbio.0030056.g004) ::: Citation: Nicholls H (2005) Ancient DNA comes of age. PLoS Biol 3(2): e56. Henry Nicholls is a freelance science writer based in London, United Kingdom. E-mail: <henry.nicholls@absw.org.uk> aDNA : ancient DNA PCR : polymerase chain reaction	PubMed Central	2024-06-05T03:55:52.852036	2005-2-15	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548952/", "journal": "PLoS Biol. 2005 Feb 15; 3(2):e56", "authors": [ { "first": "Henry", "last": "Nicholls" } ] }
PMC548953	The excitement of scientific research and discovery cannot be fully conveyed by didactic lectures alone. Several recent initiatives and proposals, therefore, have supported a more participatory, discovery-based instruction for undergraduate science education \[[@pbio-0030059-b1],[@pbio-0030059-b2]\]. In functional genomics, we have found an ideal platform to simultaneously benefit students and contribute to scientific discovery. The sequencing of eukaryotic genomes has facilitated the identification of complete sets of genes in humans and model genetic organisms. This has allowed many forms of high-throughput analyses of transcriptional profiles, protein interactions, structural motifs, and even genome-wide knock-downs in cell lines or in selected organisms. However, one of the best tools to provide functional information about gene action--- obtaining in vivo evidence about the phenotype resulting from heritable loss of function---is difficult and less amenable to high-throughput research. We were able to achieve a large-scale in vivo analysis with a significant number of undergraduate students at UCLA, called the UCLA Undergraduate Consortium for Functional Genomics. This work, a practical manifestation of policy positions proposing discoverybased education, is described in summary form here (and in [Box 1](#box1){ref-type="boxed-text"}) and in detail online at <http://www.bruinfly.ucla.edu>. This effort combines professional-quality research with a strategy for research-based undergraduate education. Box 1. Scientific Results ========================= The Drosophila eye is an intricate neurocrystalline lattice of approximately 800 individual ommatidia arrayed in a very precise order ([Figure 3A](#pbio-0030059-g003){ref-type="fig"}) \[[@pbio-0030059-b3]\]. Minor perturbations in ommatidial development can be easily detected, making it a very sensitive system for functional genomic screens \[[@pbio-0030059-b3]\]. Our study utilized 1,375 unique recessive lethal transposable element (P-element) insertion stocks from the 2nd and 3rd chromosomes of Drosophila to characterize their later role in eye development. To avoid early lethality, the FLP/FRT system was used to generate homozygous mutant tissue specifically in the eye \[[@pbio-0030059-b4]\]. Of the mutations analyzed, 501 (36%) displayed a mutant eye phenotype, providing the first genome-wide estimate of the fraction of essential genes that are also involved in eye development. Adult eye phenotypes were classified into three broad classes: rough, cell lethal, and glossy ([Figure 3](#pbio-0030059-g003){ref-type="fig"}). The genes responsible for these phenotypes were assigned into 19 different functional categories, which are summarized in [Table 1](#pbio-0030059-t001){ref-type="table"}. Signal transduction components previously established to be important for eye development (e.g,. EGFR, pointed, Star, tramtrak, Delta) were identified, validating the effectiveness of our screen. In addition, our genomics approach has shown that a number of novel classes of genes are involved in eye development that have not been previously described ([Table 1](#pbio-0030059-t001){ref-type="table"}). ::: {#pbio-0030059-g003 .fig} Figure 3 ::: {.caption} ###### Summary of Phenotypes Determined Light (left panels) and scanning electron (right panels) micrographs of mosaic Drosophila eyes. Large homozygous mutant clones are orange (arrowheads); heterozygous tissues are dark red. Examples are shown of lethal mutations that give a (A) wild-type (63.4% of lethal mutations), (B) rough (disordered ommatidia, 18.2%), (C) cell lethal (absence of homozygous mutant tissue, 14.5%), and (D) glossy (loss of lens structure, 3.9%) phenotype. For details on how clones are generated, see <http://www.bruinfly.ucla.edu>. ::: ![](pbio.0030059.g003) ::: ::: {#pbio-0030059-t001 .table-wrap} Table 1 ::: {.caption} ###### Summary of Genes Involved in Adult Eye Development ::: ![](pbio.0030059.t001) Molecular information is available for 315 of the 501 phenotypic mutants. Of these, 291 were identified as unique genes and categorized into 19 classes, based solely on the insertion site data from FlyBase ::: We have created a novel curriculum with three main components: didactic, computer, and laboratory. The only prerequisite for this course is high school advanced-placement-level biology; all other knowledge necessary for the course is taught within it. Since there are no other prerequisites for the course, a majority of the students enrolled are freshmen and sophomores, enabling us to educate them in this novel way early in their undergraduate career. Approximately 30 students take this course each quarter, and it is offered every quarter through the school year, allowing for a broad impact. In the lecture series, students are exposed to interactive lectures on background material, basic concepts of genetics, research ethics, and career options. For their "midterm," each student proposes an experiment in a grant proposal formatted according the National Institutes of Health requirements. The "final" is written as a scientific paper summarizing the student\'s own results. In the computer section, students perform research with a "virtual fly lab" to help them understand more about their crosses in the laboratory section. In addition, they learn about modern genomic resources available on the Internet, and utilize some of the genomics tools available (e.g., BLAST) to help them determine the identity and function of their disrupted genes. The main component of the class, however, is the laboratory portion. In the laboratory, the students perform all the necessary work to manipulate the genotypes of their stocks to determine what effect homozygous mutation of their target genes has in the adult Drosophila eye ([Figure 1](#pbio-0030059-g001){ref-type="fig"}). To accomplish this, the students perform five-generation Drosophila crosses that nicely fit into a ten-week quarter. Each student is assigned about ten mutants to work with. During the quarter, students are able to recombine each mutation onto a flippase (FLP) recombination target (FRT) chromosome, generate mutant somatic clones, and record details of the adult eye phenotype with both light and scanning electron microscopic techniques. The students then upload their data into an online database (<http://www.bruinfly.ucla.edu>). ::: {#pbio-0030059-g001 .fig} Figure 1 ::: {.caption} ###### Representative Pictures from the Laboratory Section of the Course ::: ![](pbio.0030059.g001) ::: Our database contains pictures of the mutant eyes for all of the stocks examined, as well as other information pertinent to that stock, including the gene disrupted, the exact genomic location of the P-element insertion, and whether an excision of the P-element has been performed and its results. A sample Web page of the database is shown in [Figure 2](#pbio-0030059-g002){ref-type="fig"}. Following the introductory course, a small number of students continue to analyze the developmental basis for select mutations in future quarters in more advanced laboratory classes. In these advanced classes, the students perform P-element excision experiments to determine whether the mutant phenotype observed is indeed derived from the P-element. These students have performed 294 excision experiments, the results of which indicate that 72% of the stocks successfully revert to wildtype phenotype when the P-element is removed. Over the last two years, we have educated 138 students in the introductory course. Advanced classes have totaled 96 student-quarters (46 students, each working two or more additional quarters). ::: {#pbio-0030059-g002 .fig} Figure 2 ::: {.caption} ###### Example of the Type of Data Available from the Online Database (<http://www.bruinfly.ucla.edu>) ::: ![](pbio.0030059.g002) ::: In summary, discovery-based experiments in functional genomics are well suited for undergraduate education: they actively engage a large number of students in research without compromising their didactic training. The sense of ownership developed from this research amplifies the students\' learning experience. For the research community, the online database and the large collection of newly generated FRT-lethal lines represent a valuable resource for future experiments in eye development. Furthermore, the stocks developed can be used to create mutant clones in an investigator\'s tissue of choice. This novel approach for performing research, for which functional genomics is very amenable, not only encourages many students in new ways of thinking but also generates professional-quality results and resources for the scientific community. Supporting Information {#s2} ====================== Figure S1 ::: {.caption} ###### A Compilation of the Undergraduates and Some of Their Fly Mutants (1.2 MB JPG). ::: ::: {.caption} ###### Click here for additional data file. ::: UB is a Howard Hughes Medical Institute (HHMI) Professor, and this undergraduate research course was funded by the HHMI Professorship Program. We thank the Bloomington and Szeged stock centers and Barry Dickson for fly stocks. We thank the University of California at Los Angeles for providing support and infrastructure for this project. Citation: Chen J, Call GB, Beyer E, Bui C, Cespedes A, et al. (2005) Discovery-based science education: Functional genomic dissection in Drosophila by undergraduate researchers. PLoS Biol 3(2): e59. J. Chen and G. B. Call are instructors and U. Banerjee is a Howard Hughes Medical Institute professor in the Departments of Molecular, Cell, and Developmental Biology and Biological Chemistry in the Molecular Biology Institute at the University of California, Los Angeles, California, United States of America; J. Chen and G. B. Call contributed equally to this program. G. Alkin, from the Life Sciences Division, was the Web applications and database developer for the UCLA Undergraduate Consortium for Functional Genomics. S. Go, D. M. Huff, H. Minye, E. Paul, N. Villarasa, and A. Milchanowski were/are teaching assistants for the UCLA Undergraduate Consortium for Functional Genomics. The remaining authors are UCLA Undergraduate Consortium for Functional Genomics students, listed alphabetically in two groups representing the length of their participation (first group, three or more quarters; second group, two or fewer quarters).	PubMed Central	2024-06-05T03:55:52.854257	2005-2-15	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548953/", "journal": "PLoS Biol. 2005 Feb 15; 3(2):e59", "authors": [ { "first": "Jiong", "last": "Chen" }, { "first": "Gerald B", "last": "Call" }, { "first": "Elsa", "last": "Beyer" }, { "first": "Chris", "last": "Bui" }, { "first": "Albert", "last": "Cespedes" }, { "first": "Amy", "last": "Chan" }, { "first": "Jenny", "last": "Chan" }, { "first": "Stacy", "last": "Chan" }, { "first": "Akanksha", "last": "Chhabra" }, { "first": "Peter", "last": "Dang" }, { "first": "Artemis", "last": "Deravanesian" }, { "first": "Brenda", "last": "Hermogeno" }, { "first": "James", "last": "Jen" }, { "first": "Eunha", "last": "Kim" }, { "first": "Eric", "last": "Lee" }, { "first": "Gemma", "last": "Lewis" }, { "first": "Jamie", "last": "Marshall" }, { "first": "Kirsten", "last": "Regalia" }, { "first": "Farnaz", "last": "Shadpour" }, { "first": "Aram", "last": "Shemmassian" }, { "first": "Kristin", "last": "Spivey" }, { "first": "Maggie", "last": "Wells" }, { "first": "Joy", "last": "Wu" }, { "first": "Yuki", "last": "Yamauchi" }, { "first": "Amir", "last": "Yavari" }, { "first": "Anna", "last": "Abrams" }, { "first": "Amanda", "last": "Abramson" }, { "first": "Latiffe", "last": "Amado" }, { "first": "Jenny", "last": "Anderson" }, { "first": "Keenan", "last": "Bashour" }, { "first": "Elena", "last": "Bibikova" }, { "first": "Allen", "last": "Bookatz" }, { "first": "Sarah", "last": "Brewer" }, { "first": "Natalie", "last": "Buu" }, { "first": "Stephanie", "last": "Calvillo" }, { "first": "Joseph", "last": "Cao" }, { "first": "Aileen", "last": "Chang" }, { "first": "Daniel", "last": "Chang" }, { "first": "Yuli", "last": "Chang" }, { "first": "Yibing", "last": "Chen" }, { "first": "Joo", "last": "Choi" }, { "first": "Jeyling", "last": "Chou" }, { "first": "Sumit", "last": "Datta" }, { "first": "Ardy", "last": "Davarifar" }, { "first": "Poonam", "last": "Desai" }, { "first": "Jordan", "last": "Fabrikant" }, { "first": "Shahbaz", "last": "Farnad" }, { "first": "Katherine", "last": "Fu" }, { "first": "Eddie", "last": "Garcia" }, { "first": "Nick", "last": "Garrone" }, { "first": "Srpouhi", "last": "Gasparyan" }, { "first": "Phyllis", "last": "Gayda" }, { "first": "Chad", "last": "Goffstein" }, { "first": "Courtney", "last": "Gonzalez" }, { "first": "Mariam", "last": "Guirguis" }, { "first": "Ryan", "last": "Hassid" }, { "first": "Aria", "last": "Hong" }, { "first": "Julie", "last": "Hong" }, { "first": "Lindsay", "last": "Hovestreydt" }, { "first": "Charles", "last": "Hu" }, { "first": "Farid", "last": "Jamshidian" }, { "first": "Katrin", "last": "Kahen" }, { "first": "Linda", "last": "Kao" }, { "first": "Melissa", "last": "Kelley" }, { "first": "Thomas", "last": "Kho" }, { "first": "Sarah", "last": "Kim" }, { "first": "Yein", "last": "Kim" }, { "first": "Brian", "last": "Kirkpatrick" }, { "first": "Emil", "last": "Kohan" }, { "first": "Robert", "last": "Kwak" }, { "first": "Adam", "last": "Langenbacher" }, { "first": "Santino", "last": "Laxamana" }, { "first": "Chris", "last": "Lee" }, { "first": "Janet", "last": "Lee" }, { "first": "So-Youn", "last": "Lee" }, { "first": "To Hang S", "last": "Lee" }, { "first": "Toni", "last": "Lee" }, { "first": "Sheila", "last": "Lezcano" }, { "first": "Henry", "last": "Lin" }, { "first": "Peter", "last": "Lin" }, { "first": "Julie", "last": "Luu" }, { "first": "Thanh", "last": "Luu" }, { "first": "Will", "last": "Marrs" }, { "first": "Erin", "last": "Marsh" }, { "first": "Sarah", "last": "Min" }, { "first": "Tanya", "last": "Minasian" }, { "first": "Amit", "last": "Misra" }, { "first": "Miles", "last": "Morimoto" }, { "first": "Yasaman", "last": "Moshfegh" }, { "first": "Jessica", "last": "Murray" }, { "first": "Cynthia", "last": "Nguyen" }, { "first": "Kha", "last": "Nguyen" }, { "first": "Ernesto", "last": "Nodado" }, { "first": "Amanda", "last": "O'Donahue" }, { "first": "Ndidi", "last": "Onugha" }, { "first": "Nneka", "last": "Orjiakor" }, { "first": "Bhavin", "last": "Padhiar" }, { "first": "Mara", "last": "Pavel-Dinu" }, { "first": "Alex", "last": "Pavlenko" }, { "first": "Edwin", "last": "Paz" }, { "first": "Sarah", "last": "Phaklides" }, { "first": "Lephong", "last": "Pham" }, { "first": "Preethi", "last": "Poulose" }, { "first": "Russell", "last": "Powell" }, { "first": "Aya", "last": "Pusic" }, { "first": "Divi", "last": "Ramola" }, { "first": "Meghann", "last": "Ribbens" }, { "first": "Bassel", "last": "Rifai" }, { "first": "Desiree", "last": "Rosselli" }, { "first": "Manyak", "last": "Saakyan" }, { "first": "Pamela", "last": "Saarikoski" }, { "first": "Miriam", "last": "Segura" }, { "first": "Ramnik", "last": "Singh" }, { "first": "Vivek", "last": "Singh" }, { "first": "Emily", "last": "Skinner" }, { "first": "Daniel", "last": "Solomin" }, { "first": "Kosha", "last": "Soneji" }, { "first": "Erika", "last": "Stageberg" }, { "first": "Marina", "last": "Stavchanskiy" }, { "first": "Leena", "last": "Tekchandani" }, { "first": "Leo", "last": "Thai" }, { "first": "Jayantha", "last": "Thiyanaratnam" }, { "first": "Maurine", "last": "Tong" }, { "first": "Aneet", "last": "Toor" }, { "first": "Steve", "last": "Tovar" }, { "first": "Kelly", "last": "Trangsrud" }, { "first": "Wah-Yung", "last": "Tsang" }, { "first": "Marc", "last": "Uemura" }, { "first": "Mary", "last": "Unkovic" }, { "first": "Emily", "last": "Vollmer" }, { "first": "Emily", "last": "Weiss" }, { "first": "Damien", "last": "Wood" }, { "first": "Sophia", "last": "Wu" }, { "first": "Winston", "last": "Wu" }, { "first": "Qing", "last": "Xu" }, { "first": "Kevin", "last": "Yackle" }, { "first": "Will", "last": "Yarosh" }, { "first": "Laura", "last": "Yee" }, { "first": "George", "last": "Yen" }, { "first": "Grant", "last": "Alkin" }, { "first": "Sheryllene", "last": "Go" }, { "first": "Devon M", "last": "Huff" }, { "first": "Helena", "last": "Minye" }, { "first": "Eric", "last": "Paul" }, { "first": "Nikki", "last": "Villarasa" }, { "first": "Allison", "last": "Milchanowski" }, { "first": "Utpal", "last": "Banerjee" } ] }
PMC548954	Despite the biases that a single author inevitably brings to a subject, only one or a few closely interacting authors can bring coherence, synthesis, and vision to a broad and complex topic. A symposium volume just doesn\'t do the job. Few topics in biology are as simultaneously encompassing, complex, and controversial as the origin of species, i.e., speciation. Speciation is, after all, the process responsible for biological diversity, at least of sexual organisms, so it is hardly a minor topic. But even though recent years have seen the publication of symposia on speciation and books on the ever-contentious issue of species concepts, it has been 23 years since Verne Grant\'s authoritative Plant Speciation \[[@pbio-0030062-b1]\] and 41 years since Ernst Mayr\'s magisterial and highly influential Animal Species and Evolution \[[@pbio-0030062-b2]\]---the last syntheses of research on speciation. Now, two outstanding new books not only treat speciation as a conceptually unified topic in both plants and animals for the first time, but also provide rich review and analysis of a vast subject that has progressed at least as much since Mayr and Grant wrote as in the century that preceded their work. These books are very different, but wonderfully complementary. Gavrilets reviews and adds to mathematical theories and simulation studies of speciation and related issues, such as fitness landscapes and selection in heterogeneous environments. A deep reading of his book will require considerably more mathematical competence than most evolutionary biologists (including this reviewer) have, but Gavrilets provides excellent verbal explanations of the models\' assumptions and conclusions, as well as comparisons and critiques of related models. Gavrilets cites empirical studies (with which he has very broad familiarity) plentifully, but as a theoretician, he does not evaluate them or describe them in depth. That task is undertaken by Coyne and Orr, who introduce most topics with a verbal overview of theory, review empirical evidence and its bearing on hypotheses, and conclude with incisive assessments of what they think we know and what remains uncertain or unexplored. Like Gavrilets, they offer a number of novel ideas or suggestions about how to proceed. Coyne and Orr have both worked mostly on speciation genetics in Drosophila, so it is hardly surprising that their treatment of speciation bears a strong genetic emphasis and draws heavily on Drosophila work (perforce, since this is almost the only source of evidence on some topics, such as the genetics of hybrid sterility and inviability). Even the most drosophilophobic readers, however, will be pleased by the extent to which Coyne and Orr have conscientiously scoured the literature on nonmodel animals and plants. To appreciate the landmark status of these books, consider what has happened in speciation studies since Mayr and Grant published theirs. Mayr and Grant articulated positions on species and speciation that had developed during and soon after the Modern Synthesis of the 1930s and 1940s, when modern evolutionary theory developed from a reconciliation of genetics, systematics, and paleontology. Mayr and Grant drew on abundant systematic data on patterns of divergence and experimental data on genetic differences between related species. They rightly identified reproductive isolation (RI) as a critical, even defining, property of species, and allopatric divergence (i.e., in disjunct geographic areas) as the major geographic mode of speciation. They recognized that trait differences between species, including RI, usually have a polygenic basis, and that different coadapted (epistatically interacting) sets of genes underlie incompatibility (e.g., hybrid sterility). They emphasized the role of ecological selection as a driving force in speciation, largely by extrapolation from the primacy of selectionist thinking that developed during the Synthesis. They accepted that natural selection can reinforce prezygotic isolation (i.e., lack of mating or zygote formation) between species and thereby reduce production of unfit hybrids, even if Mayr did not share Dobzhansky\'s belief that this was the norm. Mayr combined selection with genetic drift in his theory of founder-effect speciation (divergence in populations founded by just a few individuals), which became widely accepted. (It became Eldredge and Gould\'s \[[@pbio-0030062-b3]\] theoretical foundation for punctuated equilibrium nine years after Mayr\'s book appeared.) Mayr and Grant wrote against a background that almost entirely lacked any mathematical theory of speciation (which I suspect neither of them would have drawn on even if it had been developed), any relevant molecular data (other than early allozyme studies by the time Grant published), and any rigorous phylogenetic methodology. Kimura\'s neutral theory of molecular evolution \[[@pbio-0030062-b4]\] had not yet been published when Mayr wrote, and had not been vindicated when Grant wrote, so genetic drift and a neutral (nonselectionist) interpretation of molecular data were still suspect. Detailed analysis of genetic architecture was decades away, and of course insights into selection and historical demography from DNA sequence data were a dim dream at best. Coyne and Orr and Gavrilets analyze a new world of speciation studies. Theoretical studies of speciation, for example, now include more than 100 papers on one topic alone, the evolution of prezygotic isolation. (Gavrilets laments that the theoretical work suffers from domination by simulation rather than analysis, so that it is often hard to draw general conclusions from models that use different assumptions, but he nevertheless draws some fairly strong conclusions, as I note below.) Molecular studies have provided important data on such issues as the absolute dates of speciation events, the duration of speciation, and the time course of the evolution of RI. The field of molecular phylogeography, which documents the history of spatial isolation and geographic expansion of populations, has developed. The relation between range overlap of related species and their molecularly dated time of divergence provides some evidence on the role of geographic versus sympatric speciation (i.e., speciation without geographic segregation). In all these areas and others, our knowledge has increased steadily. For instance, molecular markers enable more detailed dissection of the genetic architecture of species differences, and support the conclusion that they are usually rather highly polygenic, but that much of the variance can be explained by a few major gene substitutions. We now have good evidence, as Coyne and Orr emphasize, that at least in animals, hybrid infertility is caused by differences in gene action, not by structural chromosome differences or failure of meiosis. Regarding the mechanisms of speciation, evidence for the role of divergent ecological selection in allopatric speciation is sparse, because this crucial topic has been unaccountably neglected until recently. Very different kinds of data, ranging from DNA sequences to correspondence between RI and ecological divergence, support natural selection, but there is hardly enough evidence, in my opinion, to support Coyne and Orr\'s strong conclusion that "at least one important debate has been settled: selection plays a much larger role in speciation than does drift" (p. 410). Even more astonishing than the paucity of studies of the role of ecological selection in speciation is the fact that the likely role of sexual selection was not even recognized until almost 20 years after Mayr\'s book. I agree with Coyne and Orr that the theory and evidence for speciation by sexual selection is one of the most important advances in speciation studies, but it is important to recognize that the evidence consists mostly of correlations between diversification rates and indices of the likely strength of sexual selection; as Coyne and Orr note, there are no cases in which we understand just how sexual selection has caused speciation. This is a rich, largely unexplored area. Gavrilets feels that divergent evolution by sexual conflict (in which females evolve resistance to males\' advances) is a potentially important process, whereas Coyne and Orr are skeptical that this will prove widespread. Coyne and Orr remark that populations may diverge in male signals because of intrasexual selection (competition among males), and that female mate preference may follow. Quite so, but even though Berglund et al. \[[@pbio-0030062-b5]\] summarized many examples in which male signals appear to serve both inter- and intrasexual functions, this topic has been almost ignored in the literature of both speciation and sexual selection. On a related theme, an important speciation process appears to be the extraordinarily rapid evolution of male reproductive proteins (e.g., sperm surface proteins), which may contribute to the "faster male evolution" that is a cause of "Haldane\'s rule" (that hybrid sterility and inviability first appear in the sex that has two unlike sex chromosomes). Despite their very different approaches, Coyne and Orr and Gavrilets arrive at rather similar conclusions on some of the most controversial issues in speciation. One such is the role of genetic drift in speciation. Gavrilets analyzes founder-effect speciation (which combines drift and selection), agrees with most other theoreticians (e.g., Barton and Charlesworth) \[[@pbio-0030062-b6]\] that it is very improbable, and argues instead for his model of evolution on "holey landscapes," whereby allopatric populations can evolve by genetic drift along ridges of roughly equal fitness to different, incompatible gene constitutions. He admits that the time to speciation under this process will ordinarily be very long unless selection is involved. Coyne and Orr fully accept both Barton and Charlesworth\'s critique and Gavrilets\'s alternative model. But while admitting the plausibility of Gavrilets\'s models of speciation by genetic drift, they nevertheless maintain that "the models seem unnecessary when compared to adaptive ones" (p. 398). Coyne and Orr appear to adopt selection as the null hypothesis for speciation, whereas drift is generally taken as the null hypothesis in much of evolutionary genetics, for the simple reason that drift operates at all loci in all finite (i.e., real) populations, whereas selection need not. The burden of demonstrating that selection is not responsible for an evolutionary event (i.e., demonstrating a negative) is, of course, far heavier than the burden of demonstrating selection; indeed, Coyne and Orr do not address the difficult question of what would constitute evidence for drift. Having, perhaps, stacked the deck, Coyne and Orr find almost no evidence that drift has contributed to speciation in nature, but conclude that there is "considerable evidence" that selection has done so. However, the amount of evidence is hardly on a par with, say, the evidence for allopatric speciation. It consists of only about eight studies of ecological selection, indications that diversification rates are associated with greater scope for sexual selection, selection signatures in a few genes that underlie genetic incompatibility, and a paucity of molecular evidence for bottlenecks (i.e., opportunities for founder events) in the history of recently formed species. But the evidence on the role of sexual selection is very indirect, and the high levels of genetic variation revealed in molecular studies argue against past bottlenecks only if this is ancestral variation, rather than variation generated anew since a possible bottleneck---a question that has been addressed in only a few cases. Assuming that experiments with laboratory populations can be validly extrapolated to natural speciation processes, founder-effect speciation may indeed be a moribund hypothesis, but I do not believe long-term genetic drift can yet be ruled out, and cannot agree that this "important debate has been settled" (p. 410). The geography of speciation continues to be one of the most difficult and contentious topics, and undoubtedly will remain so despite the careful analyses by these authors. They agree that parapatric speciation (evolution of RI between neighboring populations that exchange genes) is theoretically plausible, but Gavrilets notes that although it has become clear that its likelihood is sensitive to several model parameters, parapatric speciation is difficult to model and has been shamefully neglected. Coyne and Orr do not doubt that it is a fairly common mode of speciation, yet "it is almost impossible to demonstrate parapatric speciation in nature" (p. 118), and no cases have been well documented. Gavrilets provides an exhaustive analysis of the many models of sympatric speciation, and identifies some key issues that have been underemphasized. For example, the sympatric evolution of behavioral isolation by "matching traits" (e.g., genetically independent male signal and female preference) is generally much more difficult than "similaritybased" mating (in which females choose males that have the same phenotypic trait as themselves). Just how common the latter is in animals is an open question that Coyne and Orr unfortunately do not address. Gavrilets also identifies the cost of female choosiness as a critical issue: many models of sympatric speciation depend on the assumption that females always succeed in mating even if the male type they prefer is rare, so their choosiness has no cost. Gavrilets criticizes some popular models of sympatric speciation on these and other grounds, and while granting that sympatric speciation by divergent habitat or host preference is plausible, he concludes that it need not be faster than allopatric speciation and that "contrary to common claims in recent theoretical papers, conditions for sympatric speciation are not wide and sympatric speciation does not occur easily" (p. 404). For their part, Coyne and Orr feel that the prevalence of sympatric speciation is an empirical issue (but a very difficult one), and undertake a broad, detailed review. They identify three examples of completed speciation in which a sympatric scenario "seems plausible." I see no reason to accept one of these cases, a pair of sister species of "parasites" (fig wasps) on the same host species, since allopatric speciation of a widespread parasite need not be accompanied by speciation of its host. Moreover, Coyne and Orr note weaknesses in all three cases, as well as in examples of "host races" that have been advanced as species in statu nascendi. Coyne and Orr\'s conclusion echoes Gavrilets\'s: "It is hard to see how the data at hand can justify the current wave of enthusiasm for sympatric speciation" (p. 178). Bravi! I have indicated some disagreements with Coyne and Orr, and could certainly cite others. But whatever weaknesses their book may have (more ecology and phylogeny, anyone?) are much less important than its strengths. The strengths of Speciation are not only Coyne and Orr\'s comprehensive, scholarly coverage of an exceedingly broad subject, but also, and especially, their rigorous, incisive analysis, coupled with strongly stated conclusions and suggestions for how to resolve controversies. Many readers will have a visceral reaction against their position on sympatric speciation, reinforcement, founder-effect speciation, or other issues---but can these readers counter Coyne and Orr\'s arguments with equally cogent analysis? Or are these subjects that simply require more, and perhaps more imaginative, research? Together, these books provide a comprehensive, thoughtful synthesis of our current understanding of one of the most important processes in evolution. They are required reading for anyone who studies species and speciation. I recommend Speciation and the nonmathematical final chapter ("General Conclusions") of Fitness Landscapes and the Origin of Species to all evolutionary biologists, students, and professionals alike. It may not take another two decades for the next foundational books on speciation to appear, but these books will fill that role for a long time to come. Citation: Futuyma DJ (2005) Progress on the origin of species. PLoS Biol 3(2): e62. Douglas J. Futuyma is Distinguished Professor in the Department of Ecology and Evolution at the State University of New York, Stony Brook, New York, United States of America. E-mail: <futuyma@life.bio.sunysb.edu> RI : reproductive isolation	PubMed Central	2024-06-05T03:55:52.855924	2005-2-15	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548954/", "journal": "PLoS Biol. 2005 Feb 15; 3(2):e62", "authors": [ { "first": "Douglas J", "last": "Futuyma" } ] }
PMC548955	Biological databases offer access to formalized facts about many aspects of biology---genes and gene products, protein structure, metabolic pathways, diseases, organisms, and so on. These databases are becoming increasingly important to researchers. The information that populates databases is generated by research teams and is usually published in peer-reviewed journals. As part of the publication process, some authors deposit data into a database but, more often, it is extracted from the published literature and deposited into the databases by human curators, a painstaking process. Research literature and scientific databases fulfil different needs. Literature provides ideas and new hypotheses, but is not constrained to provide facts in formats suitable for use in databases. By contrast, databases efficiently provide large quantities of data and information in a standardised schema representing a predefined interpretation of the data. While the acceptance of a paper can enforce the submission of data to a central data repository, such as EMBL ([www.ebi.ac.uk/embl/](www.ebi.ac.uk/embl/)) or ArrayExpress ([www.ebi.ac.uk/arrayexpress/](www.ebi.ac.uk/arrayexpress/)), nobody receives credit for the submission of a fact to a database without an associated publication. As long as this practice continues, curation will be necessary to add the (re)formalised facts to biological databases. Given that publications are not about to be replaced with routine deposition of data into databases, is it possible to develop software tools to support the work of the curator? Could we automatically analyse new scientific publications routinely to extract facts, which could then be inserted into scientific databases? Could we tag gene and protein names, as well as other terms in the document, so that they are easier to recognise? How can we use controlled vocabularies and ontologies to identify biological concepts and phenomena? Fortunately, there are many groups that are now seeking to answer these questions, precisely with a view to extracting facts from text. Part of the motivation for this effort in text mining technology is the inexorable rise in the amount of published literature ([Figure 1](#pbio-0030065-g001){ref-type="fig"}). This massive growth, coupled with the current inefficiencies in transferring facts into other data resources, leads to the unfortunate state that biological databases tend to be incomplete (for example, DNA sequences without known function in genetic databases), and there are inconsistencies between databases and literature. ::: {#pbio-0030065-g001 .fig} Figure 1 ::: {.caption} ###### Medline Article Deluge This figure shows the exploding number of articles available from Medline over the past 65 years (data retrieved from the SRS server at the European Bioinformatics Institute; [www.ebi.ac.uk/](www.ebi.ac.uk/)). In 2003, about 560,000 articles were added to Medline, and from 2000 to 2003, 2 million articles. (Articles already registered for 2005 are given as well.) ::: ![](pbio.0030065.g001) ::: In theory, text mining is the perfect solution to transforming factual knowledge from publications into database entries. But computational linguists have not yet developed tools that can analyse more than 30% of English sentences correctly and transform them into a structured formal representation \[[@pbio-0030065-b1],[@pbio-0030065-b2]\]. We can analyse part of a sentence, such as a subphrase describing a protein--protein interaction or part of a sentence containing a gene and a protein name, but we always run into Zipf\'s law whenever we write down the rules for how the extraction is done ([Figure 2](#pbio-0030065-g002){ref-type="fig"}) \[[@pbio-0030065-b3]\]. A small number of patterns describe a reasonable portion of protein--protein interactions, gene names, or mutations, but many of those entities are described by a pattern of words that\'s only ever used once. Even if we could collect them all---which is impossible---we can\'t stop new phrases from being used. ::: {#pbio-0030065-g002 .fig} Figure 2 ::: {.caption} ###### Zipf\'s Law Zipf\'s eponymous law is illustrated by the analysis of 30,000 Medline abstracts (4,952,878 occurrences of words; 144,841 different words). Frequent terms account for a large portion of the text, but a large fraction of terms appear at a low frequency and often only once (69,782 words appear only once). Zipf was a linguistic professor at Harvard University \[[@pbio-0030065-b3]\]. ::: ![](pbio.0030065.g002) ::: Curators---The Gold Standard {#s2} ============================ Hand-curated data is precise, because the curator is trained to inspect literature and databases, select only high-quality data, and reformat the facts according to the schema of the database. In addition, curators select citations from the text as evidence for the identified fact, and those citations are also added to the database. Curators read and interpret the text at the same time, and if they don\'t understand the meaning of a sentence, they can go back and pick a new strategy to analyse it---they can even call the authors to iron out any ambiguities. Curators can also cope with the high variability of language described by Zipf\'s law. At present, no computer-based system comes close to matching these capabilities. In particular, it is difficult to convert all the curators\' domain knowledge into a structured training set for the purposes of machine learning approaches. Curators fulfil a second important task: they know how to define standards for data consistency, in particular, the most relevant terminology, which has led to the design of standardised ontologies and controlled vocabularies (see [Box 1](#box1){ref-type="boxed-text"} for an explanation of these and related terms). Examples of these include Gene Ontology (GO; [www.geneontology.org/](www.geneontology.org/)), Unified Medical Language System ([www.nlm.nih.gov/research/umls/](www.nlm.nih.gov/research/umls/)), and MedDRA ([www.meddramsso.com/NewWeb2003/index.htm](www.meddramsso.com/NewWeb2003/index.htm)) \[[@pbio-0030065-b4]\]. These terminological resources help to relate entries in bioinformatics databases to concepts mentioned in scientific publications and to link related information in databases using different schemas. Text miners would love such standards to be used in text, but there is an understandable reluctance to impose and use standards that might limit the expressiveness of natural language. Box 1. Glossary --------------- Controlled vocabulary: A set of terms, to standardise input to a database. F-measure: A statistic that is used to score the success of NE recognition by text mining tools. The F-measure is an average parameter based on precision (how many of the entities found by the tool are correct identifications of an entity) and recall (how many of the entities existing in the text did the tool find). Machine learning: The technology and study of algorithms through which machines (computers) can "learn", or automatically improve their systems through data gathered in the past (experience). Ontology: A set of terms with clear semantics (language), clear motivations for distinction between the terms, and strict rules for how the terms relate to each other. Curation and Text Mining---In Partnership {#s3} ========================================= The problem with curation of data is that it is time consuming and costly, and therefore has to focus on the most relevant facts. This compromises the completeness of the curated data, and curation teams are doomed to stay behind the latest publications. So, is it possible for curation and text mining to work together for rapid retrieval and analysis of facts with precise postprocessing and standardisation of the extracted information? There are several software tools that perform well in the identification of standardised terms from the literature. Examples include Textpresso and Whatizit \[[@pbio-0030065-b5],[@pbio-0030065-b6],[@pbio-0030065-b7],[@pbio-0030065-b8]\]. Extensive term lists come from the Human Genome Organization ([www.gene.ucl.ac.uk/hugo](www.gene.ucl.ac.uk/hugo); 20,000 gene and protein names), GO (almost 20,000 terms), Uniprot/Swiss-Prot ([www.ebi.uniprot.org/index.shtml](www.ebi.uniprot.org/index.shtml); about 200,000 terms), and other databases. In addition, terms describing diseases, syndromes, and drugs are available from the Unified Medical Language System. Altogether, about 500,000 terms constitute the basis of domain knowledge in life sciences. To gain some perspective of this figure: an average individual handles 2,000 to 20,000 terms in his or her daily language, and Merriam-Webster\'s Collegiate Dictionary provides definitions for 225,000 terms ([www.merriam-webstercollegiate.com/](www.merriam-webstercollegiate.com/)). The identification of all terms by a text mining system still sets challenging demands. All variants of a term have to be taken into account, including syntactical variants and synonyms. In the case of ambiguities, relevant findings have to be distinguished from other findings---a process referred to as disambiguation. Depending on the curation task, it might therefore be advantageous to select only part of the terminological resources and thus restrict the domain of the terminology to the curators\' needs ([Figure 3](#pbio-0030065-g003){ref-type="fig"}). ::: {#pbio-0030065-g003 .fig} Figure 3 ::: {.caption} ###### GOAnnotator The illustrated software tool brings together data from text mining and from databases to support curators in the GO annotation of proteins (Couto FM, Lee V, Dimmer E, Camon E, Apweiler R, et al., unpublished data). Here a protein is shown in conjunction with the GO terms that have been gathered from various databases and attributed to the protein through electronic annotation. Both are evaluated against similar GO terms extracted from text documents. The curator looks into the evidence and decides whether any of the GO terms extracted from the documents should be assigned to the protein. ::: ![](pbio.0030065.g003) ::: Available text mining solutions are concerned with named entity (NE) recognition (entities are, for example, proteins, species, and cell lines), with identification of relationships between NEs (such as protein interactions), and with the classification of text subphrases according to annotation schemata in general (thyroid receptor is a thyroid hormone receptor) \[[@pbio-0030065-b9],[@pbio-0030065-b10],[@pbio-0030065-b11],[@pbio-0030065-b12],[@pbio-0030065-b13],[@pbio-0030065-b14],[@pbio-0030065-b15]\]. Whilst the identification of a curation team\'s terminology in the scientific text under scrutiny is immensely valuable, there is still a long way to go before this becomes routine. Some Immediate Challenges {#s4} ========================= Not all terms used in the literature (NEs) can actually be found in some kind of database (perhaps because of an author error, or an alternative name for an entity adopted by the community). Text mining methods therefore have to detect new terms and map the term to known terminology \[[@pbio-0030065-b16]\]. If several mappings are possible, the correct version has to be selected (disambiguation). Over the past several years text mining research teams have presented various approaches that train a software tool to locate representations of gene or protein names (for example, BioCreative, [www.pdg.cnb.uam.es/BioLINK/BioCreative.eval.html](www.pdg.cnb.uam.es/BioLINK/BioCreative.eval.html), and JNLPBA, [www.genisis.ch/\~natlang/JNLPBA04/](www.genisis.ch/~natlang/JNLPBA04/)) \[[@pbio-0030065-b17],[@pbio-0030065-b18]\]. These tools are scored with a statistic known as the F-measure, with the best methods scoring about 0.85. At the level of 0.85, curators still tend to be unhappy. However, analyses have shown that this score is in the range of curator--curator variation (unpublished data, measured as part of the project work for \[[@pbio-0030065-b19]\]), which suggests that such methods produce useful results. Additional information-extraction methods have been proposed, for example, for the documentation of mutations in specific genes and for the extraction of the subcellular location of proteins \[[@pbio-0030065-b11],[@pbio-0030065-b13]\]. An even larger number of tools focus on the identification of appropriate terminology for the annotation of genes (GO terms) \[[@pbio-0030065-b7]\]. The evaluation of their usefulness depends on the demands of the user groups. Finally, another way to support curation teams would be to provide information-retrieval methods to guide the team members towards documents containing relevant information. For example, in 2002, the participants in the Knowledge Discovery and Data-Mining Challenge Cup ([www.cs.cornell.edu/projects/kddcup/](www.cs.cornell.edu/projects/kddcup/)) had to select documents from a given corpus that contained relevant experimental results about Drosophila \[[@pbio-0030065-b20]\]. How Can Publishers Contribute? {#s5} ============================== For all automated information-extraction methods, it is obvious that access to literature is crucial. Electronic access has, of course, already had a huge impact, but the structure and organisation of manuscripts could also be improved. For example, semantic tags could be integrated into the text. The markup would not appear on web pages or when the document is printed, but it would help software to deal with semantic aspects of the document. Inserting tags, for example, to mark protein names would allow retrieval software to find documents about proteins even if they look like common English words, such as "you" or "and". Retrieval engines currently often ignore such terms. In addition, explicit tags would enable text mining methods, for example, when looking for protein--protein interactions, to use the correct semantic interpretation. Text mining systems already available today, such as Whatizit, can integrate semantic tags during submission, which have to be verified by the author. Text mining is ready to deliver tools whereby information is passed back to the authors about the proper use of terminology within their documents. If the use of a term raises conflicts or ambiguities or if the use of a term is wrong, the author is asked to provide feedback. The curation effort is resolved at the earliest possible time-point. Author, publisher, reviewer, and reader profit from consistent information representation, which leads to better dissemination of documents and journals and easily offsets the additional cost in the generation of an article. Publishers and authors have to agree on standards though. Is Text Mining Ready to Deliver? {#s6} ================================ Text mining solutions have found their way into daily work, wherever fast and precise extraction of details from a large volume of text is needed. We have to keep in mind, however, that any text mining tool, just like other bioinformatics resources, will only be suitable for a limited number of tasks. For example, the same text may serve curators from different communities who extract different types of facts, depending on their domain knowledge. Furthermore, different communities have different expectations for accuracy. For example, curators dealing with a small set of proteins prefer tools with high recall, whereas curators dealing with a large number of proteins prefer tools with high precision. Although text mining cannot dissect English sentences completely, and cannot extract the meaning and put the facts into a database, text mining tools are becoming increasingly used and valued. Text mining is ready to deliver handling of complex terminology and nomenclature as a mature service. It is only a matter of time and effort before we are able to extract facts automatically. The consequences are likely to be profound. Not only will we have a more effective approach for the mining of knowledge from the literature, our approach to the publication process itself might change. If a fact is clear enough for automatic extraction, it could be reported in a fact database instead of a publication. As methods improve, authors will see more and more of their text being analysed and formalised in a database. If appropriate quality control is provided, and if authors receive due credit for their deposition of facts into databases, we might well see a shift towards original papers describing new creative ideas and visions rather than just listing facts. Citation: Rebholz-Schuhmann D, Kirsch H, Couto F (2005) Facts from text---Is text mining ready to deliver? PLoS Biol 3(2): e65. Dietrich Rebholz-Schuhmann and Harald Kirsch are at the European Bioinformatics Institute, Cambridge, United Kingdom. Francisco Couto is in the Departamento de Informática, Faculidade de Ciências, Universidade de Lisboa, Portugal. GO : Gene Ontology NE : named entity	PubMed Central	2024-06-05T03:55:52.857576	2005-2-15	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548955/", "journal": "PLoS Biol. 2005 Feb 15; 3(2):e65", "authors": [ { "first": "Dietrich", "last": "Rebholz-Schuhmann" }, { "first": "Harald", "last": "Kirsch" }, { "first": "Francisco", "last": "Couto" } ] }
PMC548956	Viruses are intracellular pathogens that are subject to intense selective pressures during their ongoing battles within the host. To propagate successfully, they must exploit numerous machineries of the infected cell. Thus, studies of their replicative cycles have yielded fundamental insights into eukaryotic biology. A prime example is the human immunodeficiency virus (HIV), which is a lentivirus that causes the acquired immunodeficiency syndrome (AIDS). Unlike simpler oncoviruses that rely exclusively on host cell machinery, lentiviruses code for additional accessory and regulatory proteins that act as molecular switches at different stages of viral entry and exit from the infected cell. Studying the actions of these viral proteins has yielded understanding of diverse cellular functions such as the innate immunity against retroviruses, control of transcriptional elongation, export of macromolecules from the nucleus to the cytoplasm, and intracellular trafficking of proteins (reviewed in \[[@pbio-0030076-b1]\]). The transcriptional transactivator (Tat) is a key regulatory protein of HIV. It is expressed early after the virus integrates into the cell, and stimulates the elongation of RNA polymerase II (RNAPII). This type of transcriptional control had not been previously appreciated; thus, work on Tat established a new paradigm in the field of eukaryotic biology. Moreover, these findings impacted greatly studies of cotranscriptional processing of nascent mRNA. To understand these processes better, we need to start with the basics of transcriptional control. RNAPII is the enzyme that transcribes protein-coding genes in eukaryotic cells. Elegant studies in vitro first suggested that the simple recruitment of RNAPII to transcription units was not sufficient for the copying of genes and cotranscriptional processing of their transcripts. Rather, distinct steps could be defined, which began with the assembly of the preinitiation complex (PIC), promoter clearance, pausing, and arrest, and ended with efficient elongation of transcription (reviewed in \[[@pbio-0030076-b2]\]). The central component of PIC is the general transcription factor (GTF) TFIID, which contains the TATAbox- binding protein (TBP) and 12 to 15 TBP-associated factors (TAFs). TFIID acts as a "landing pad" for other GTFs and RNAPII to nucleate PIC assembly. Moreover, TAFs serve as coactivators to a diverse set of activators. Both an ordered stepwise assembly and the recruitment of the 100-plus-subunit "holoenzyme" have been proposed to be critical for the positioning of RNAPII at start sites of transcription. Next, the GTF TFIIH unwinds the DNA, opens the transcription bubble, and phosphorylates serines at position 5 in the C-terminal domain (CTD) of the RPB1 subunit of RNAPII (reviewed in \[[@pbio-0030076-b2]\]). This phosphorylation is critical for the recruitment of complexes that put a 7-methylguanylate cap on the 5′ end of nascent transcripts. After the transcription complex clears the promoter, the negative transcription elongation factor (N-TEF) is recruited to the RNAPIIa (reviewed in \[[@pbio-0030076-b3]\]). It consists minimally of 5,6- dichloro-1-β-D-ribofuranosylbenzimidazole riboside (DRB)- sensitivity-inducing factor (DSIF) \[[@pbio-0030076-b4]\] and negative elongation factor (NELF) \[[@pbio-0030076-b5]\]. They bind and arrest RNAPII distal to the promoter cooperatively. Such arrested transcription complexes have now been found on many inducible genes in Drosophila melanogaster (reviewed in \[[@pbio-0030076-b6]\]) and humans \[[@pbio-0030076-b7]\]. The transition to robust elongation depends on the positive transcription elongation factor b (P-TEFb) (reviewed in \[[@pbio-0030076-b3]\]). P-TEFb contains the cyclin-dependent kinase 9 (CDK9) and one of four possible C-type cyclins. When recruited to stalled transcription complexes, P-TEFb phosphorylates serines at position 2 in the CTD \[[@pbio-0030076-b8]\], the Spt5 subunit of DSIF \[[@pbio-0030076-b9]\], and the RD subunit of NELF \[[@pbio-0030076-b10]\]. These modifications result in heavily phosphorylated RNAPII (RNAPIIo), the recruitment of the Elongator, which contains splicing and polyadenylation machineries, and the conversions of DSIF and NELF into elongation factors. RNAPIIo now copies the gene and directs the cotranscriptional processing, i.e., splicing and polyadenylation, of primary transcripts. Upon successful polyA addition, the CTD phosphatase FCP1 dephosphorylates RNAPIIo. RNAPIIa dissociates from DNA, and the transcription cycle starts all over again (reviewed in \[[@pbio-0030076-b2]\]). Tat is unique among transcriptional activators in eukaryotic cells in that it functions via RNA rather than DNA promoter elements ([Figure 1](#pbio-0030076-g001){ref-type="fig"}). It binds the transactivation response element (TAR) that forms a stable RNA stem loop at the 5′ end of all viral transcripts. Thus, Tat requires minimally the transcription of TAR before it can stimulate HIV transcription from the long terminal repeat (LTR). Indeed, in the absence of Tat, RNAPIIa clears the HIV LTR successfully but soon arrests, yielding predominantly short viral transcripts \[[@pbio-0030076-b11]\]. Tat binds the 5′ bulge in TAR via its arginine-rich motif from positions 49 to 57, where a central arginine (R52) is key for this interaction. However, this binding is not sufficient for Tat\'s function in vivo. Adjacent to the arginine-rich motif lie N-terminal core and cysteine-rich regions, which form the activation domain of the protein. This activation domain binds cyclin T1 (CycT1) from P-TEFb, whose partner is CDK9 \[[@pbio-0030076-b12]\]. As a consequence, P-TEFb and Tat bind TAR cooperatively. The final proof that P-TEFb is the cellular cofactor for Tat came from studies of HIV transcription in murine cells, where the introduction of the human CycT1 protein restores Tat function \[[@pbio-0030076-b12]\]. The same effect can be achieved by substituting just the tyrosine with the cysteine at position 261, such as are found in murine and human CycT1 proteins, respectively \[[@pbio-0030076-b13]\]. A paper in this issue of PLoS Biology suggests that Tat and P-TEFb can also recruit TAF-independent transcription complexes to the HIV LTR \[[@pbio-0030076-b14]\] ([Figure 1](#pbio-0030076-g001){ref-type="fig"}). Possibly, this assembly reflects interactions between CycT1 and the unphosphorylated CTD of RNAPIIa \[[@pbio-0030076-b15]\]. ::: {#pbio-0030076-g001 .fig} Figure 1 ::: {.caption} ###### Activation of HIV Transcription by Tat Activators (red circles) that bind the HIV LTR promoter (light-blue rectangle) assemble the PIC and recruit RNAPIIa to the start site of transcription. For simplicity, only RNAPIIa in the PIC is presented. The yellow sphere with two open circles, depicting serines at position 5 and 2 within the CTD (S5 and S2, respectively), represents the unphosphorylated CTD of RNAPIIa (white sphere). TFIIH, which performs DNA-helicase and CTD-kinase activities, melts the DNA and phosphorylates S5 (red circle in the CTD; P-S5), resulting in promoter clearance. RNAPIIa transcribes TAR (red hairpin) and is paused by the binding of N-TEF, DSIF, and NELF, which are presented as blue spheres. The RD subunit of NELF binds the bottom stem in TAR. P-TEFb (comprising the red \[CDK9\] and pink \[CycT1\] spheres), which binds TAR together with Tat (small red sphere), phosphorylates S2 (red circle in the CTD; P-S2) to form elongating RNAPIIo (large red sphere). It also phosphorylates Spt5 in DSIF and RD in NELF, which become elongation factors, with the latter dissociating from TAR. In addition, P-TEFb, possibly independent of its kinase activity, assembles PIC via recruitment of TBP and RNAPIIa (dotted arrow). The phosphorylated CTD in RNAPIIo now binds the Elongator, which contains splicing machinery and polyadenylation factors. The red sphere at the 5′ end of the HIV transcript (red line) represents its cap. Finally, p300 acetylates Tat (magenta circle) and dissociates it from TAR. Acetylated Tat binds P-CAF and transfers it to RNAPIIo, possibly facilitating chromatin remodeling. Collectively, efficient RNAPII elongation of viral transcription ensues. ::: ![](pbio.0030076.g001) ::: The assembly and disassembly of the complex between PTEFb, Tat, and TAR is a regulated process in vivo. Whereas the phosphorylation of CDK9 strengthens this complex \[[@pbio-0030076-b16]\], the acetylation of the lysine at position 50 in Tat weakens it \[[@pbio-0030076-b17]\]. Upon this disruption, acetylated Tat is liberated from P-TEFb and recruits the p300/CREB-binding protein-- associated factor (P-CAF) to the elongating RNAPIIo, most likely facilitating chromatin remodeling. In this issue of PLoS Biology, Pagans et al. now demonstrate that acetylated Tat is deacetylated by SIRT1 \[[@pbio-0030076-b18]\] ([Figure 1](#pbio-0030076-g001){ref-type="fig"}). In this way, Tat can reassemble with P-TEFb on TAR. Clearly, P-TEFb plays a key role in the control of transcriptional elongation. Although Tat was the first activator known that could recruit P-TEFb to initiating RNAPII, additional members of this group were soon identified. They include the androgen receptor, c-Myc, the class II transactivator (CIITA), myoblast determination protein (MyoD), and nuclear factor κ-B (NF-κB). The last one is of great interest as it explains how the HIV genome can be transcribed before the synthesis of Tat \[[@pbio-0030076-b19]\]. Cellular activation triggers the nuclear translocation of NF-κB, where it binds the HIV enhancer, leading to the stimulation of viral transcription. It is not surprising that proviral latency, in which low levels of transcription or only short HIV transcripts containing TAR are observed, would in large part reflect the absence of these activators. Indeed, in many of these latently infected cells, the induction of NF-κB or the addition of Tat leads to the reactivation of viral replication and spreading of the infection \[[@pbio-0030076-b20],[@pbio-0030076-b21]\]. Recently, important aspects of the regulation of P-TEFb have been revealed ([Figure 2](#pbio-0030076-g002){ref-type="fig"}). Of interest, P-TEFb exists in two complexes in cells \[[@pbio-0030076-b22],[@pbio-0030076-b23]\]. The larger measures approximately 500 kDa and contains the hexamethylene bisacetamide (HMBA)--induced protein 1 (HEXIM1) and 7SK small nuclear RNA (snRNA) in addition to P-TEFb \[[@pbio-0030076-b24],[@pbio-0030076-b25]\]. In this large complex, Cdk9 is enzymatically inactive. HEXIM1 was identified as the inducible gene following the exposure of vascular smooth muscle cells to a potent differentiating agent, HMBA \[[@pbio-0030076-b26]\]. 7SK snRNA is one of the most abundant snRNA species, whose function remained a mystery for over a decade. Of interest, targeting of P-TEFb by HEXIM1 and 7SK snRNA contributes significantly to the control of cell growth and differentiation. For example, growth signals liberate P-TEFb from the large complex in the course of cardiac hypertrophy in mice, a disease characterized by the enlargement of myocytes due to a global increase in mRNA synthesis \[[@pbio-0030076-b27]\]. Also, following stress, ultraviolet light, or the administration of actinomycin D and DRB to cells, the large complex is converted to the small complex to stimulate transcription \[[@pbio-0030076-b22],[@pbio-0030076-b23]\]. ::: {#pbio-0030076-g002 .fig} Figure 2 ::: {.caption} ###### Inhibition of P-TEFb by the Coordinate Actions of HEXIM1 and 7SK snRNA HEXIM1 (blue sphere) binds the 5′ half of 7SK snRNA (red structure with multiple hairpins). Upon this binding, P-TEFb joins this RNA--protein complex and becomes enzymatically inactive, depicted by CDK9 as a black sphere. For simplicity, only the CDK9/CycT1 heterodimer is presented. Multiple stimuli, including stress, ultraviolet light, actinomycin D, DRB, and hypertrophic signals, dissociate HEXIM1 and 7SK snRNA from P-TEFb, possibly by preventing the RNA--protein interaction. In this way, P-TEFb is rendered active, depicted by CDK9 as a red sphere. ::: ![](pbio.0030076.g002) ::: How central is P-TEFb to eukaryotic transcription? In Saccharomyces cerevisiae, there are two candidates for PTEFb, CTDK-1 and Bur1/2. CTDK1-negative but not Bur1/Bur2-negative yeasts still grow, albeit poorly and only on rich media (reviewed in \[[@pbio-0030076-b2]\]). In Caenorhabditis elegans, genetic inactivation of CDK9 or CycT1 and CycT2 resulted in the inhibition of all RNAPII transcription \[[@pbio-0030076-b8]\]. Moreover, in D. melanogaster, following heat shock, PTEFb is recruited upstream of activated promoters \[[@pbio-0030076-b28]\]. Although no murine knockouts of subunits of P-TEFb have been reported, DRB and flavopiridol, two ATP analogs that inhibit the kinase activity of CDK9, can inhibit nearly all transcription by RNAPII in human cells \[[@pbio-0030076-b29]\]. Indeed, as P-TEFb is a coactivator of potent activators that mediate effects of enhancers and can itself activate transcription when placed on sites distal to promoter elements \[[@pbio-0030076-b15]\], it might mediate many more signaling events than those of heat shock, ultraviolet light, stress, and hypertrophy. Conversely, the inhibition of P-TEFb could explain the mode of action of some transcriptional repressors. Indeed, the global transcriptional repressor PIE-1, the regulator of embryogenesis in C. elegans, binds the histidine-rich stretch in CycT1, thus decoying P-TEFb away from RNAPII and blocking the elongation of transcription \[[@pbio-0030076-b30]\]. These are exciting findings and suggest a plethora of future experiments, including the genetic inactivation of subunits of P-TEFb and isoforms of HEXIM1 in the mouse. Of special interest are questions as to where to place this mechanism of transcriptional regulation in the hierarchy of competing or complementary processes. What roles do different P-TEFb complexes play in the transcription of specific genes? How central will the regulation of P-TEFb be to cellular growth, proliferation, and differentiation, and what roles will it play in normal development and disease states? As to HIV, how can we use our knowledge of P-TEFb to slow down viral replication and/or to eliminate the state of proviral latency in the host? Obviously, we are only at the beginning of this journey, which promises to change radically our view of eukaryotic transcription. MB is supported by a fellowship from the American Foundation for AIDS Research. This work was supported by a grant from the National Institutes of Health (RO1 AI49104). Citation: Barboric M, Peterlin BM (2005) A new paradigm in eukaryotic biology: HIV Tat and the control of transcriptional elongation. PLoS Biol 3(2): e76. Matjaz Barboric and B. Matija Peterlin are in the Departments of Medicine, Microbiology, and Immunology, Rosalind Russell Medical Research Center, University of California, San Francisco, California, United States of America. AIDS : acquired immunodeficiency syndrome CDK9 : cyclindependent kinase 9 CIITA : class II transactivator CTD : C-terminal domain CycT1 : cyclin T1 DRB : 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole riboside DSIF : 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole riboside-sensitivity-inducing factor GTF : general transcription factor HEXIM1 : hexamethylene-bisacetamideinduced protein 1 HIV : human immunodeficiency virus HMBA : hexamethylene bisacetamide LTR : long terminal repeat NF-κB : nuclear factor κ-B N-TEF : negative transcription elongation factor P-CAF : p300/CREB-binding protein--associated factor PIC : preinitiation complex P-TEFb : positive transcription elongation factor b RNAPII : RNA polymerase II snRNA : small nuclear RNA TAF : TATA-boxbinding protein--associated factors TAR : transactivation response element Tat : transcriptional transactivator TBP : TATA-box-binding protein	PubMed Central	2024-06-05T03:55:52.859166	2005-2-15	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548956/", "journal": "PLoS Biol. 2005 Feb 15; 3(2):e76", "authors": [ { "first": "Matjaz", "last": "Barboric" }, { "first": "B. Matija", "last": "Peterlin" } ] }
PMC548957	In PLoS Biology, volume 2, issue 12. [10.1371/journal.pbio.0020380](10.1371/journal.pbio.0020380) In Materials and Methods, the sentence "The magnetic beads were added to the extract to a ratio of about 8 ×10^9^ beads per g of cells" contains an error. The correct amount of beads is 8 × 10^8^, i.e., ten times less. Published February 15, 2005 Citation: (2005) Correction: Components of coated vesicles and nuclear pore complexes share a common molecular architecture. PLoS Biol 3(2): e80.	PubMed Central	2024-06-05T03:55:52.860744	2005-2-15	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548957/", "journal": "PLoS Biol. 2005 Feb 15; 3(2):e80", "authors": [ { "first": "Damien", "last": "Devos" }, { "first": "Svetlana", "last": "Dokudovskaya" }, { "first": "Frank", "last": "Alber" }, { "first": "Rosemary", "last": "Williams" }, { "first": "Brian T", "last": "Chait" }, { "first": "Andrej", "last": "Sali" }, { "first": "Michael P", "last": "Rout" } ] }
PMC548958	Published February 15, 2005 In PLoS Biology, volume 2, issue 12. [10.1371/journal.pbio.0020449](10.1371/journal.pbio.0020449) The art credits were missing from the image accompanying this synopsis. The image caption should read as follows:[](#pbio-0020449-g001){ref-type="fig"} ::: {#pbio-0020449-g001 .fig} ::: {.caption} ###### Reconstruction of Neanderthal woman (Photo: Bacon Cph; makeup: Morton Jacobsen) ::: ![](pbio.0030090.g001) ::: Citation: (2005) Correction: Cro-Magnons conquered Europe, but left Neanderthals alone. PLoS Biol 3(2): e90.	PubMed Central	2024-06-05T03:55:52.861015	2005-2-15	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548958/", "journal": "PLoS Biol. 2005 Feb 15; 3(2):e90", "authors": [] }
PMC549028	Background ========== α1-antitrypsin (AAT) is a glycoprotein, which is the major inhibitor of neutrophil elastase and proteinase 3 \[[@B1],[@B2]\]. AAT is mainly produced in liver cells, but also in extrahepatic cells, such as monocytes, macrophages and pulmonary alveolar cells \[[@B3],[@B4]\]. The average concentration of AAT in plasma in healthy individuals is 1.3 mg/ml, with a half-life of 3 to 5 days. AAT is an acute phase protein, and its circulating levels increase rapidly to concentrations exceeding 2 mg/ml in response to inflammation or infection \[[@B5]\]. Individuals with plasma AAT values below 0.7 mg/ml are considered to be AAT deficient \[[@B6],[@B7]\]. Over 75 alleles of AAT have been identified to date, of which at least 20 affect either the amount or the function of the AAT molecule in vivo \[[@B6]-[@B8]\]. A very common deficiency allele is termed Z, which differs from the normal M in the substitution of Glu 342 to Lys \[[@B7],[@B9],[@B10]\]. This single amino acid exchange causes spontaneous polymerization of the AAT, markedly impeding its release into the circulation \[[@B11]\]. The retained material is associated with hepatic diseases \[[@B12]\], while diminished circulating levels lead to antiproteinase deficiency and higher susceptibility to elastase mediated tissue injury \[[@B13],[@B14]\]. The alleles of AAT are inherited in an autosomal codominant manner \[[@B2]\]. Therefore, individuals heterozygous for the Z allele (MZ) have 30--40% whereas individuals homozygous for the Z allele (ZZ) have only 10--15% of normal plasma AAT levels \[[@B15]-[@B17]\]. Tobacco smoke and air pollution have long been recognised as risk factors for the development of chronic obstructive pulmonary disease (COPD); the only proven genetic risk factor, however, is the severe Z deficiency of AAT \[[@B18],[@B19]\]. Cigarette smokers with AAT-deficiency develop COPD much earlier in life than smokers with the normal AAT genotype \[[@B8],[@B10],[@B11]\]. The pulmonary emphysema that is associated with inherited AAT deficiency is intimately linked with the lack of proteinase inhibitor within the lungs that is available to bind to, and inactivate, neutrophil elastase. On the basis of clinical observations involving patients with inherited AAT deficiency and various experimental studies, the elastase-AAT imbalance hypothesis became widely accepted as the explanation for lung tissue destruction in emphysema \[[@B20],[@B21]\]. There is now increasing evidence that an excessive activity of various proteolytic enzymes in the lung milieu, including members of the serine, cysteine and metalloprotease families, may damage the elastin network of lungs \[[@B14]\]. Since the severe ZZ and intermediate MZ AAT deficiency accounts for less than 1--2% and 8--18% of emphysema cases, it is believed that the protease-antiprotease hypothesis provides a rational basis for the explanation of the development and progression of emphysema in general \[[@B22],[@B23]\]. Based on the protease-antiprotease hypothesis, augmentation therapy of emphysema with severe AAT deficiency was introduced during the 1980s \[[@B24]\]. Intravenous administration of a pasteurized pooled human plasma AAT product (Prolastin; Bayer Corporation; Clayton, North Carolina) is used to increase AAT levels in deficient individuals \[[@B25]\]. The major concept behind augmentation therapy is that a rise in the levels of blood and tissue AAT will protect lungs from continuous destruction by proteases, particularly neutrophil elastase \[[@B26]\]. For example, anti-elastase capacity in the lung epithelial lining fluid has been found to increase to 60--70% of normal in homozygous Z AAT-deficient individuals subjected to augmentation therapy \[[@B26],[@B27]\]. Whether this biochemical normalization of AAT levels influences the pathogenic processes of lung disease is still under debate. The most recent results, however, suggest that Prolastin therapy may have beneficial effects in reducing the frequency of lung infections and reducing the rate of decline of lung function \[[@B28],[@B29]\]. There is growing evidence that AAT, in addition to its anti-proteinase activity, may have other functional activities. For example, AAT has been demonstrated to stimulate fibroblast proliferation and procollagen synthesis \[[@B30]\], to up-regulate human B cell differentiation into IgE-and IgG4-secreting cells \[[@B31]\], to interact with the proteolytic cascade of enzymes involved in apoptosis \[[@B32],[@B33]\] and to express contrasting effects on the post-transcriptional regulation of iron between erythroid and monocytic cells \[[@B34]\]. AAT is also known to inhibit neutrophil superoxide production \[[@B35]\], induce macrophage-derived interleukin-1 receptor antagonist release \[[@B36]\] and reduce bacterial endotoxin and TNFα-induced lethality in vivo\[[@B37],[@B38]\]. We recently demonstrated, in vitro, that both native (inhibitory) and non-inhibitory (polymerised and oxidised) forms of AAT strongly inhibit lipopolysaccharide-induced human monocyte activation \[[@B39]\]. AAT appears to act not just as an anti-proteinase, but as a molecule with broader anti-inflammatory properties. Data presented in this study provide clear evidence that Prolastin, a preparation used for AAT deficiency augmentation therapy, significantly inhibits bacterial endotoxin-induced pro-inflammatory cell responses in vitro, and suppresses nasal IL-8 release in lipopolysaccharide-challenged individuals, in vivo. Materials and Methods ===================== α1-antitrypsin (AAT) preparations --------------------------------- α1-antitrypsin (Human) Prolastin^®^(Lot 26N3PT2) was a gift from Bayer (Bayer Corporation, Clayton, North Carolina, USA). This vial of Prolastin contained 1059 mg of functionally active AAT, as determined by capacity to inhibit porcine pancreatic elastase. Prolastin was dissolved in sterile water for injections provided by manufacture and stored at +4°C. Purified human AAT was obtained from the Department of Clinical Chemistry, Malmö University Hospital, Sweden. Native AAT was diluted in phosphate buffered saline (PBS), pH 7.4. To ensure the removal of endotoxins, AAT was subjected to Detoxi-Gel AffinityPak columns according to instructions from the manufacturer (Pierce, IL, USA). Purified batches of AAT were then tested for endotoxin contamination with the Limulus amebocyte lysate endochrome kit (Charles River Endosafe, SC, USA). Endotoxin levels were less than 0.2 enzyme units/mg protein in all preparations used. The concentrations of AAT in the endotoxin-purified batches were determined according to the Lowry method \[[@B40]\]. Polymeric AAT was produced by incubation at 60°C for 10 h. Polymers were confirmed on non-denaturing 7.5% PAGE gels. Monocyte isolation and culture ------------------------------ Monocytes were isolated from buffy coats using Ficoll-Paque PLUS (Pharmacia, Sweden). Briefly, buffy coats were diluted 1:2 in PBS with addition of 10 mM EDTA and layered on Ficoll. After centrifugation at 400 gfor 35 min, at room temperature, the cells in the interface were collected and washed 3 times in PBS-EDTA. The cell purity and amount were determined in a cell counter Autocounter AC900EO (Swelabs Instruments AB, Sweden). The granulocyte fractions were less than 10%. Cells were seeded into 12-well cell culture plates (Nunc, Denmark) at a concentration of 4 × 10^6^cells/ml in RPMI 1640 medium supplemented with penicillin 100 U/ml; streptomycin 100 μg/ml; non-essential amino acids 1×; sodium pyruvate 2 mM and HEPES 20 mM (Gibco, UK). After 1 h 15 min, non-adherent cells were removed by washing 3 times with PBS supplemented with calcium and magnesium. Fresh medium was added and cells were stimulated with lipopolysaccharide (LPS, 10 ng/ml, J5 Rc mutant; Sigma, Sweden) in the presence or absence of various concentrations of Prolastin (0--16 mg/ml), constant concentration of native or polymerised AAT (0.5 mg/ml) for 18 h at 37°C, 5% CO~2~. Neutrophil isolation and culture -------------------------------- Human neutrophils were isolated from the peripheral blood of healthy volunteers using Polymorphprep TM (Axis-Shield PoC AS, Oslo, Norway) as recommended by the manufacture. In brief, 25 ml of anti-coagulated blood was gently layered over the 12.5 ml of Polymorphprep TM and centrifuged at 1600 rpm for 35 min. Neutrophils were harvested as a low band of the sample/medium interface, washed with PBS, and residual erythrocytes were subjected to hypotonic lysis. Purified neutrophils were washed in RPMI-1640- Glutamax-1 medium (Gibco-BRL Life Technologies, Grand Island, NY) supplemented with 0.1% bovine serum albumin (BSA) and resuspended in the same medium. The neutrophil purity was more than 75% as determined on an AutoCounter AC900EO. Cell viability was \> 95% according to trypan blue staining. Neutrophils (5 × 10^6^cells/ml) were plated into sterile ependorf tubes. Zymosan was boiled, washed and sonicated. Opsonized zymosan was prepared by incubating zymosan with serum (1:3) in 37°C water bath for 20 min. After, zymosan was centrifuged, washed with PBS and re-suspended at 30 mg/ml. Cells alone or activated with zymosan (0.3 mg/ml) were exposed to various concentrations of Prolastin (0--8 mg/ml), and native or polymerised AAT preparations (0.5 mg/ml) for 18 h at 37°C 5% CO~2~. Cell free supernatants were obtained by centrifugation at 300 gfor 10 min, and stored at -80°C until analysis Cytokine/chemokine analysis --------------------------- Cell culture supernatants from monocytes and neutrophils stimulated with LPS or zymosan alone or in combination with Prolastin, native or polymerised AAT were analysed to determine TNFα, IL-1β and IL-8 levels by using DuoSet ELISA sets (R&D Systems, MN, USA; detection levels 15.6, 3.9, and 31.2 pg/ml, respectively). Subjects -------- Seven subjects (four females and three males) of 26--50 (median 38) years of age, non-smokers, non-allergic volunteers participated in the study. All subjects gave written informed consent before participation in the study. None of the subjects has a history of respiratory disease and none took any medication at the study time. Study Design ------------ At 2-week intervals each subject was submitted to a nasal challenge with sterile saline, LPS and LPS-Prolastin combination. All experimental sessions were done in the same room. On each provocation day, the nose was inspected and cleaned with 8 ml of isotonic NaCl. Between nasal lavages the subjects stayed in the same building and asked to keep away from known sources of nasal irritants. The night was spent in their own homes. All participants completed a symptom questionnaire. In the first session, the baseline lavage was taken after instillation to each nostril of 8 ml of sterile isotonic NaCl. In the next session, the subjects were challenged with LPS from Escherichia coli serotype 026:B6, Lot 17H4042 (Sigma-Aldrich, USA). The provocation solution was prepared prior to use. LPS was added to 8 ml of sterile 0.9% NaCl to obtain a final concentration of 250 μg/ml, and 100 μl of the provocation solution was sprayed into each nostril, using a needle-less syringe. In the third session, the subjects were first challenged with LPS, as described above, and after 30 min with 2.5 mg of Prolastin into each nostril. Lavage samples were taken with instillation to each nostril of 8 ml of sterile isotonic NaCl after 2, 6 and 24 h followed by assessment of symptoms by a questionnaire. All subject completed a symptom questionnaire with questions about nasal and eye irritation, and throat and airway symptoms. None of the participants reported symptoms of nasal, eye or throat irritations, and no general symptoms such as muscle pain, shivering, were mentioned. Nasal Lavage ------------ The procedure for nasal lavage was performed according to a method described by Wihl and co-workers \[[@B41]\]. Each nasal cavity was lavaged separately with a syringe (60 ml) to which a plastic nasal olive was connected for close nostril fitting. To prevent lavage spilling into the throat, the subject was bent forward at an angle of 60° during the procedure. Equilibrium was maintained between the mucosal lining and the lavage fluid by injecting the saline gently into the nasal cavity and drawing it back five times into the syringe. The lavage was performed in both nostrils and samples were collected into a test tube. The samples were then centrifuged at 1750 rpm, 6°C for 10 min and immediately frozen at -80°C. The protein concentration in the lavage fluids was measured by Lowry method and IL-8 levels were determined by DuoSet ELISA sets (R&D Systems, MN, USA; detection levels 31.2 pg/ml). Statistical Analysis -------------------- Statistical Package (SPSS for Windows, release 11.5, SPSS Inc., Chicago) was used for the statistical calculations. The differences in the means of cell culture experimental results were analysed for their statistical significance with the one-way ANOVA combined with a multiple-comparisons procedure (Scheffe multiple range test). The equality of means of experimental results in healthy volunteers were analysed for statistical significance with independent two sample t-test and repeated measures of ANOVA using the SPSS MANOVA procedure <http://www.utexas.edu/cc/docs/stat38.html>. Tests showing p \< 0.05 were considered to be significant. Results ======= Concentration-dependent effects of Prolastin on LPS-induced cytokine release from human monocytes ------------------------------------------------------------------------------------------------- Various concentrations of Prolastin (0--16 mg/ml) were added to adherent-isolated human monocytes with or without LPS (10 ng/ml). Cells stimulated with LPS alone served as a positive control, while PBS stimulated monocytes served as negative controls. As illustrated in figures [1A](#F1){ref-type="fig"} and [1B](#F1){ref-type="fig"}, simultaneous incubation of monocytes with LPS and Prolastin resulted in a reduction in TNFα and IL-1β release compared to the cells stimulated with LPS alone. Inhibition of LPS-induced cytokine release by Prolastin was concentration-dependent and was typically observed over a concentration range of 0.5--16 mg/ml. At 16 mg/ml the inhibitory effects of Prolastin appeared to be maximal for both TNFα (10.7-fold, p \< 0.001) and IL-1β (7.3-fold, p \< 0.001), compared to LPS alone. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### A concentration-response inhibition of lipopolysaccharide-stimulated TNFα (A) and IL-1β (B) release by Prolastin in human blood monocytes. Isolated blood monocytes were treated with LPS (10 ng/ml) alone or together with various concentrations of Prolastin (0--16 mg/ml) for 18 h. TNFα and IL-1β levels were measured by ELISA. Data are the means of quadruplicate culture supernatants ± S.E. and are representative of three separate experiments. ::: ![](1465-9921-6-12-1) ::: Inhibitory effects at 0.5 mg/ml of AATs on LPS-mediated IL-1β and TNFα release ------------------------------------------------------------------------------ We recently found that simultaneous incubation of monocytes with LPS and either the inhibitory (native) or non inhibitory (polymeric) form of AAT resulted in a reduction in TNFα and IL-1β release compared to the cells stimulated with LPS alone. At 0.5 mg/ml the effects of native and polymerised AAT appeared to be maximal (41). Therefore, we selected a 0.5 mg/ml concentration of Prolastin, native and polymerised AAT, and compared their effects on LPS-stimulated cytokine release at 18 h. As shown in figures [2A](#F2){ref-type="fig"} and [2B](#F2){ref-type="fig"}, LPS triggered a significant release of TNFα and IL-1β (p \< 0.001 v medium alone) by monocytes. At 0.5 mg/ml, native and polymerised AAT remarkably inhibited LPS-induced TNFα and IL-1β release (p \< 0.001) (Fig. [2](#F2){ref-type="fig"}). The inhibitory effect of Prolastin (0.5 mg/ml) on LPS-stimulated TNFα release was comparable in magnitude to that of native or polymeric AAT, whereas its inhibitory effect on LPS-induced IL-1β release did not reach significance. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Comparisons of the effects of native (nAAT), polymeric (pAAT) and Prolastin on lipopolysaccharide -- stimulated TNFα (A) and IL-β (B) production by human blood monocytes isolated from four healthy donors. Isolated blood monocytes were treated with LPS (10 ng/ml) alone or together with 0.5 mg/ml nAAT, pAAT or Prolastin for 18 h. TNFα and IL-1β levels were measured by ELISA. Each bar represent the mean ± S.E. \\\* p \< 0.001. ::: ![](1465-9921-6-12-2) ::: Concentration-dependent effects of Prolastin on neutrophil IL-8 release ----------------------------------------------------------------------- The effects of Prolastin (0--8 mg/ml) on human neutrophil IL-8 production are shown in Figure [3A](#F3){ref-type="fig"}. Neutrophils stimulated with opsonized zymosan (0.3 mg/ml) released a large amount of IL-8 (p \< 0.001), compared to controls. Prolastin inhibited IL-8 release by neutrophils stimulated with opsonized zymosan (Fig [3A](#F3){ref-type="fig"}). This inhibition was concentration-dependant, with maximal suppression of IL-8 release (5.3-fold, p \< 0.001 compared to zymosan treated cells) at 8 mg/ml. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Effects of AATs on neutrophils activated with zymosan. (A) Concentration-dependent effects of Prolastin on IL-8 release from neutrophils activated with opsonised zymosan. Freshly isolated blood neutrophils were treated with zymosan (0.3 mg/ml) alone or together with various concentrations of Prolastin (0--8 mg/ml) for 18 h. IL-8 levels were measured by ELISA. Data are the means of quadruplicate culture supernatants ± S.E. and are representative of three separate experiments. (B) Effects of opsonised zymosan alone or together with native (nAAT), polymeric (pAAT) AAT or Prolastin on IL-8 release from neutrophils. The release of neutrophil IL-8 was measured in cell free supernatants as described in Materials and methods. Neutrophils were treated for 18 h with a constant amount of zymosan (0.3 mg/ml) alone or together with nAAT, pAAT or Prolastin (0.5 mg/ml) for 18 h. IL-8 levels were measured by ELISA. Each bar represents the means ± S.E. of three separate experiments carried out in duplicate repeats. \\\* p \< 0.001 ::: ![](1465-9921-6-12-3) ::: Inhibitory effects at 0.5 mg/ml of native, polymeric AAT and Prolastin on zymosan-mediated IL-8 release ------------------------------------------------------------------------------------------------------- Neutrophils were stimulated with zymosan (0.3 mg/ml) or AATs (0.5 mg/ml) either alone or in combination for 18 h and IL-8 protein determined. As illustrated in figure [3B](#F3){ref-type="fig"}, polymeric and native AAT and Prolastin significantly inhibited the release of IL-8 protein by activated neutrophils. In terms of maximal effect, native AAT \>polymerised AAT\>Prolastin. It must be noted that native, polymeric AAT and Prolastin alone showed no effect on neutrophils, relative to non-treated buffer controls (data not shown). Inhibition of the LPS-induced increase in nasal IL-8 release by Prolastin ------------------------------------------------------------------------- To assess the effect of Prolastin on LPS-induced nasal provocation, IL-8 levels in nasal lavages were measured. Nasal instillation 25 μg per nostril of LPS alone or in combination with 2.5 mg/ml of Prolastin was performed in non-smoking and non-allergic volunteers (n = 7, 4 females and 3 males). The IL-8 release in response to LPS challenge increased over time compared to baseline levels (Fig. [4](#F4){ref-type="fig"}). The levels of IL-8 increased already after 2 h of LPS challenge (245.7% ± 87) and remained higher after 24 h (310 ± 77.5) compared to baseline (100% ± 19.2). By contrast, when IL-8 levels were examined in LPS-Prolastin-treated lavage samples, no significant changes in IL-8 release were observed compared to baseline. In the presence of Prolastin, the LPS effect on IL-8 release was inhibited (p \< 0.05) (Fig. [4](#F4){ref-type="fig"}). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### IL-8 analysis in nasal lavage of subjects challenged with LPS alone or LPS+Prolastin combination. Seven healthy volunteers were treated with LPS (25 μg/nostril) or with LPS followed 30 min later with Prolastin (2.5 mg/nostril), nasal lavage was collected at different time points (0, 2, 6 and 24 h) as described in Material and Methods. The concentration of IL-8 (pg/ml) was measured by ELISA. IL-8 values are expressed as a ratio of IL-8 concentration at selected time point and the basal level. Independent two sample t-test shows after 6 and 24 h significantly higher levels of IL-8 in subjects treated with LPS compared to LPS+Prolastin. \* p \< 0.05 ::: ![](1465-9921-6-12-4) ::: Disscussion =========== There is now, however, ample evidence that serine proteinase inhibitors (serpins), in addition to their well established anti-inflammatory capacity to regulate serine proteinases activity, may possess broader anti-inflammatory properties. Several studies have shown that the biological responses of bacterial lipopolysaccharide (endotoxin) in vivomay be sensitive to serpins. For example, the serpin antithrombin, has been shown to protect animals from LPS-induced septic shock and also to inhibit IL-6 induction by LPS \[[@B42],[@B43]\]. Our recent study provided first in vitroevidence that native (inhibitor) and at least two modified (non-inhibitory i.e. polymeric and oxidised) forms of AAT can block the release of an array of chemokine and cytokines from LPS-stimulated monocytes \[[@B39]\]. These studies therefore further support a central role of serpins in inflammation, not only as the regulators of proteinase activity, but also as the suppressers of endotoxin induced pro-inflammatory responses. In line with these findings, we demonstrate here that Prolastin, a preparation of human AAT which is used for augmentation therapy, significantly inhibits endotoxin-induced pro-inflammatory effects in vitroand in vivo. Stimulation of human monocytes and neutrophils with bacterial endotoxin results in the release of a range of inflammatory mediators including the pro-inflammatory cytokines (e.g.IL-6, IL-1β and TNFα) and the chemokines (e.g.MCP-1 and IL-8) \[[@B44]-[@B46]\]. Together, these play a crucial role in the recruitment and activation of leukocytes and the subsequent release of harmful proteases that may further perpetuate the inflammatory process. We found that Prolastin significantly inhibits endotoxin-induced IL-1β and TNFα release by monocytes and IL-8 release by neutrophils in vitro. The Prolastin exhibited these anti-inflammatory properties in a concentration-dependent manner. Its maximal effects were observed with 16 mg/ml in the monocyte model and with 8 mg/ml in the neutrophil model, since doubling these concentrations did not significantly modify the intensity of the effects. Indeed, Prolastin markedly prevented endotoxin-induced cell activation at 0.5--4 mg/ml concentrations, implying that these lower concentrations of Prolastin might also be sufficient to inhibit endotoxin effects. It is worth noting that in order to reduce a potential risk of transmission of infectious agents the Prolastin preparation is heat-treated in solution at 60° ± 0.5 for not less than 10 h. Data from in vitrostudies show that heat-treatment results in AAT polymerization and loss of its inhibitory activity \[[@B47],[@B48]\]. Therefore, in our experimental model we compared anti-inflammatory effects of Prolastin with those of native and heat treated (60°C 10 h) AATs. At concentrations used (0.5 mg/ml), no significant difference was found between the effects of Prolastin and native or heat-treated (polymeric) AAT on endotoxin-induced monocyte TNFα and neutrophil IL-8 elevation. The median concentrations of endotoxin-stimulated IL-1β levels also decreased in the presence of Prolastin but failed to reach statistical significance. In general, inhibitory effects on endotoxin-stimulated monocyte IL-1β and neutrophil IL-8 release were better pronounced by native AAT compared to polymeric AAT or Prolastin. Similarly, in our previous study we found that in terms of maximal effect, native AAT \>polymerised AAT\>oxidized AAT were efficient in inhibiting LPS-stimulated TNFα and IL-1β, and IL-8 release from monocytes \[[@B39]\]. Further studies will be necessary to better evaluate how temperature, pH or other physicochemical challenges may influence anti-inflammatory effectiveness of AAT preparations. To explore our hypothesis that AAT functions as a potent inhibitor of endotoxin-induced effects, we examined whether Prolastin also inhibits responses to LPS in the nasal airway, in vivo. In particular, we were interested in concentrations of the neutrophil chemoattractant, IL-8. Endotoxin (or LPS) from gram-negative bacteria is a common air contaminant in a number of occupational conditions, especially those in which exposure to animal waste or plant matter occurs \[[@B44],[@B49]-[@B51]\]. Levels of LPS in such environments may exceed 20 μg/m^3^air and may be associated with respiratory symptoms and nasal inflammation in exposed persons \[[@B52]\]. For example, nasal inflammation as evaluated by an increased influx of inflammatory cells into the nasal airway and increased IL-8 levels, has been described in persons occupationally exposed to LPS \[[@B51]\]. Moreover, it has been suggested that constitutive levels of IL-8 might further enhance responses to an inflammatory stimulus, such as LPS \[[@B53]\]. A number of experimental studies have shown that a nasal instillation of LPS causes the cytokine and chemokine reaction \[[@B54],[@B55]\]. In our pilot study we also showed that instilled defined amounts of endotoxin (25 μg/per nostril) induce time-dependent nasal IL-8 release in normal subjects. Two hours after LPS instillation the IL-8 levels in nasal lavage reached more than twice the basal level and remained higher during all the times studied. However, during the next session, when 30 min after challenge with LPS, Prolastin (2.5 mg/ per nostril) was instilled, no induction of nasal IL-8 release was found compared to the basal levels. Furthermore, the protective ability of Prolastin did not disappeared over study time. We cannot determine from these experiments whether Prolastin is directly suppressing IL-8 release or suppressing another inflammatory response that leads to IL-8 release; nonetheless, our finding suggests that effects of Prolastin directed against endotoxin-stimulated inflammatory responses may be beneficial. Thus, data from both in vitroand in vivoexperiments provide novel evidence that the Prolastin preparation is a potent inhibitor of endotoxin effects. The major concept behind augmentation therapy with pooled plasma-derived AAT has been that a rise in the level of AAT in subjects with severe inherited AAT deficiency would protect the lung tissue from continued destruction by proteinases (i.e. primarily leukocyte elastase) \[[@B7],[@B56],[@B57]\]. Recent findings provide evidence that augmentation therapy with AAT reduces the incidence of lung infections in patients with AAT-related emphysema \[[@B28],[@B58]\]. Furthermore, Cantin and Woods have reported that aerosolized AAT suppresses bacterial proliferation in a rat model of chronic Pseudomonas aeruginosalung infection \[[@B59]\]. Stockley and co-workers demonstrated that a short-term therapy of AAT augmentation not only restores airway concentrations of AAT to normal, but also reduces levels of leukotriene B4, a major mediator of neutrophil recruitment and activation. Interestingly, authors have suggested that the efficacy of AAT augmentation may be most beneficial in individuals with the most inflammation \[[@B29],[@B60]\]. Data presented in this study clearly show that Prolastin inhibits endotoxin-stimulated pro-inflammatory responses, and thus provides new biochemical evidence supporting the efficacy of augmentation therapy. The current findings also suggest that Prolastin may, in fact, be used for broader clinical applications than merely augmentation therapy. Abbreviations ============= AAT, α1-antitrypsin; COPD, chronic obstructive pulmonary disease; LPS, lipopolysaccharide; ZZ, homozygous AAT-deficiency variant; MM, wild type AAT variant; PBS, phosphate buffered saline; EDTA, ethylenediaminetetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Authors\' contribution ====================== Izabela Nita, performed cell culture experiments, made contribution to acquisition of data; Camilla Hollander, made substantial contribution to patient study design, material collection and analysis; Ulla Westin, contributed to study design and data interpretation; Sabina Janciauskiene, contributed to conception and study design, data interpretation and wrote the article Acknowledgments =============== This work was supported by grants from the Swedish Research Council, and Department of Medicine, Lund University, Sweden.	PubMed Central	2024-06-05T03:55:52.861421	2005-1-31	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549028/", "journal": "Respir Res. 2005 Jan 31; 6(1):12", "authors": [ { "first": "Izabela", "last": "Nita" }, { "first": "Camilla", "last": "Hollander" }, { "first": "Ulla", "last": "Westin" }, { "first": "Sabina-Marija", "last": "Janciauskiene" } ] }
PMC549029	Background ========== Transcription factors function uniquely at the interface of the genome and the proteome. A significant portion of transcription factors serve not only as executors of gene transcription programs, but also as biochemical sensors of extracellular stimuli. For instance, members of the nuclear receptor family are directly activated by lipophilic extracellular ligands, and transcription factors of the Smad and Stat families are activated by phosphorylation in response to cytokine stimulation of cell surface receptors. Chromatin-bound transcription factors that act both as sensors of extracellular cues and as transcriptional effectors carry exceptional instructive value about the biological state of individual cells. Their high biological information value makes such factors particularly attractive for use as markers to predict disease activity and outcome, as well as predictive markers of disease-responsiveness to drugs. Based on related and broader rationale, the second phase of the human genome project, ENCODE (ENCyclopedia Of DNA Elements), has been initiated with the ambitious goal of identifying all regulatory elements of the human genome, including chromatin binding sites for individual transcription factors \[[@B1]\]. In fact, several transcription factors are prognostic biomarkers in cancer, including estrogen and progesterone receptors \[[@B2]\] and signal transducers and activators of transcription (Stats) \[[@B3],[@B4]\]. However, as a result of tumor-specific alterations in chromatin accessibility and structure, individual transcription factors may regulate distinct gene sets in different tumor specimens. Specifically, genes that are actively regulated vary as a result of chromatin structure, DNA methylation, histone modifications, and the presence of additional cofactors. Tumor-specific patterns of transcription factor binding to target chromatin are expected to enhance diagnostic information beyond what is achieved through protein and mRNA analyses. Such added diagnostic information may directly improve disease prognostication and prediction of tumor responsiveness to therapy. Our laboratory is particularly interested in the role of transcription factor Stat5 in human breast cancer, which is associated with favorable prognosis, especially in early stage malignancy \[[@B3]\]. Stat5 belongs to the Stat transcription factor family, which represents latent cytoplasmic transcription factors that are activated by phosphorylation of a conserved tyrosine residue in response to extracellular cytokines and hormones, such as prolactin, growth hormone, erythropoietin, and several interleukins. Basal activation of Stat5 has been shown in healthy breast epithelial cells \[[@B5]\] and in many early stage breast cancers, but is gradually lost during metastatic progression \[[@B3]\]. Furthermore, active Stat5 correlated with higher histological differentiation and reduced mitotic rate \[[@B6]\]. Additional evidence suggests that Stat5 may actively inhibit metastatic progression by promoting homotypic adhesion and inhibiting tumor cell scattering \[[@B7]\]. Based on technical progress with immunocapture of transcription factor-bound chromatin fragments, genome-wide mapping of interaction sites may be achieved either by hybridization of captured DNA to linear microarrays of genomic DNA, or by cloning and sequencing of captured chromatin fragments. Microarray-based hybridization has been successfully used in the small yeast genome \[[@B8]\], but high cost and technical hurdles remain for human genome-wide DNA arrays at sufficient nucleotide resolution. Early efforts have focused on medium resolution arrays of restricted portions of the genome, such as the small chromosome 22 \[[@B9]\], arrays of classical promoter regions immediately upstream of transcriptional start sites \[[@B10]\], and arrays that contain CpG island clones \[[@B11]\]. In contrast, generation of a genome-wide library of transcription factor-bound chromatin fragments, a chromatin library, represents an inclusive and unbiased approach to the entire human genome \[[@B12]\]. Chromatin libraries also hold the potential to identify transcription factor binding to abnormal, tumor-specific neochromatin arising from genomic instability. However, progress has been hampered by a high degree of non-specific capture of irrelevant chromatin fragments and lack of methods for effective validation of captured sequences. The purpose of the work described here is to optimize parameters to generate and validate a chromatin library for genome-wide identification of Stat5 target chromatin in human breast cancer. We identify novel Stat5 binding sites from a genome-wide chromatin library and validate the sites by prolactin-inducible Stat5 binding by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Results and Discussion ====================== In contrast to transcription factors that bind to chromatin in a constitutive manner, Stat5 is a latent cytoplasmic transcription factor that is activated by tyrosine phosphorylation and binds tightly to DNA in response to extracellular cytokines, such as prolactin \[[@B13]\]. We used the well-differentiated, estrogen receptor positive T-47D human breast cancer cell line, which maintains robust prolactin-induced Stat5 activation \[[@B5],[@B14]\], to generate a library of Stat5-bound chromatin fragments. Sonication is necessary to shear genomic DNA into fragments that can be easily manipulated for PCR amplification, cloning, and sequencing. Fragments of approximately 400 base pairs (bp) allow for a complete sequencing read-through and are of sufficient size to localize the fragment within the human genome with a high degree of statistical certainty. Optimal shearing of chromatin from formaldehyde-fixed T-47D cells into approximately 400 bp fragments was established empirically (Figure [1A](#F1){ref-type="fig"}, lane 5). This target size of chromatin fragments was confirmed by agarose gel electrophoresis of two parallel sets of sonicates of cells treated with or without prolactin for 30 min (Figure [1B](#F1){ref-type="fig"}), prior to immunocapture of Stat5-bound fragments. An antibody that recognizes the highly homologous Stat5a and Stat5b isoforms \[[@B13]\] was used to capture Stat5-bound chromatin as detailed in Methods. Before subcloning of the captured chromatin fragments, the specificity of immunocapture was verified by analysis of binding to known human Stat5 target chromatin. In particular, we took advantage of earlier work that has identified a group of Stat5 regulated genes that have been shown to contain the Stat5 consensus sequence, TTCNNNGAA, in the traditional promoter element \[[@B13],[@B15]\]. Aliquots of the captured chromatin pool were amplified by PCR using oligonucleotide primers flanking Stat5 binding sites within the gene promoters of Cytokine-Inducible SH2 Protein(CISH), β-Casein, and α2-Macroglobulin. Due to the average chromatin fragment size of 400 bp, primers were designed to yield shorter PCR products of 200 -- 300 bp. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Specificity of immunocapture of Stat5-bound chromatin in T-47D human breast cancer cells. A. Optimization of sonication parameters for shearing genomic DNA in T-47D cells. Sonication of formaldehyde-fixed cells for two 30 s pulses yielded \~400 bp chromatin fragments. The number and time of sonication pulses are listed above each lane. B. Prolactin (PRL) pretreatment did not affect chromatin fragmentation. T-47D cells were treated with (+) or without (-) 10 nM PRL for 30 min, fixed, sonicated (2 × 30 s), and a portion of the pre-IP samples were separated by agarose gel electrophoresis. C-E. Validation of quality of immunocaptured, Stat5-bound chromatin pool by ChIP analysis of known Stat5 target genes. Cells were incubated with (+) or without (-) 10 nM PRL for 30 min, and processed as described in Methods. Non-specific IgG was used as a negative IP control and PCR was performed using primers designed to specifically amplify the known Stat5 response element. PCR products shown are representative of at least two separate amplifications of at least two independently generated pools of Stat5-chromatin complexes. (-) PCR ctrl indicates a negative (no template) PCR control, (+) PCR ctrl Pre-IP indicates a positive PCR control performed on the pre-immunoprecipitated (input) DNA, and (+) PCR ctrl Gen indicates a positive PCR control performed on purified T-47D genomic DNA. PRL activated Stat5 specifically associated with the promoter of CISH(C) and β-Casein(D -- upper panel), but not α2-Macroglobulin(D -- lower panel), unless cells had been pretreated for four days with glucocorticoid (E -- 1 μM dexamethasone; Dex). ::: ![](1476-4598-4-6-1) ::: Stat5 was inducibly associated with the promoter of the CISHgene response element in T-47D cells (Figure [1C](#F1){ref-type="fig"}). The capture was specific, since binding was only detected in prolactin-stimulated cells, and only when Stat5 antibody and not control IgG was used. PCR amplification of intact genomic DNA is shown as a control to verify the specificity of the PCR reaction, in addition to amplification of the pre-immunoprecipitation chromatin fragment pool (input DNA). Likewise, Stat5 was inducibly and specifically bound to the β-Caseingene promoter in T-47D cells (Figure [1D](#F1){ref-type="fig"}, upper panel). In contrast, Stat5 did not associate with the Stat5-response element of the α2-Macroglobulingene (Figure [1D](#F1){ref-type="fig"}, lower panel), a gene reportedly responsive to Stat5 in liver \[[@B16]\], uterine stromal cells \[[@B17]\], and ovarian cells \[[@B18],[@B19]\]. However, pretreatment of T-47D cells with the glucocorticoid hormone analog, dexamethasone, for four days prior to Stat5 activation made the α2-Macroglobulinpromoter accessible to Stat5 binding (Figure [1E](#F1){ref-type="fig"}). It has been well established that glucocorticoids play a vital role in many cell types and cell processes, including mammary differentiation. In fact, several genes have been shown to be regulated by cooperative Stat5-glucocorticoid receptor interactions \[[@B20]-[@B23]\]. In summary, based on Stat5 inducibility and antibody specificity in testing of known Stat5 chromatin interaction sites, we concluded that the conditions for effective immunocapture of Stat5-bound chromatin from T-47D cells were established. The enriched, Stat5-bound chromatin pool was then cloned into a bacterial vector to generate a chromatin library. Because sonication generates random overhangs in double stranded DNA \[[@B24]\], T4 DNA polymerase was first used to blunt-end DNA fragments. Subsequently, a single 3\' adenosine residue was added using Taqpolymerase, and the resulting fragments were ligated into the pCR2.1 TA cloning vector. Transformed bacteria were plated on ampicillin- and S-gal-containing selection plates for blue/white screening. PCR amplification of inserts was performed directly on white bacterial colonies with common primers flanking the vector cloning site. A PCR reaction under standard conditions was used to lyse the bacteria and inactivate endogenous nucleases, cycled 36 times, and the products were separated by agarose gel electrophoresis. Initial analysis of 389 white colonies yielded 185 (48%) insert-containing PCR products. Figure [2](#F2){ref-type="fig"} shows a display of PCR products from a run of 17 clones, in which three clones did not contain an insert and 14 clones had inserts of a median size of approximately 300 bp. PCR products containing an insert were purified and sequenced directly without plasmid minipreps in a cost-effective and time-saving manner. BLAST analysis was used to localize sequences to the human genome. Of 185 inserts, 31 (17%) sequences could be unambiguously matched to a location within the human genome. Sequences that could not be localized were either repetitive or did not produce a statistically significant homology to the published human genome. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Cloned Stat5-bound chromatin fragments from PRL-stimulated T-47D breast cancer cells. Immunoprecipitated chromatin fragments were modified for cloning into a TA cloning vector and were transformed into electrocompetent bacteria. PCR was performed directly on white bacterial colonies using primers flanking the cloning site and was cycled 36 times. Samples were run on an agarose gel and analyzed for the presence of insert. (+) denotes vector control (no insert yields a vector-derived 172 bp product); (-) denotes negative PCR control. ::: ![](1476-4598-4-6-2) ::: To validate the quality of the chromatin library, ten clones were randomly selected and first tested for ability to bind activated Stat5 from nuclear extracts of T-47D cells in vitroby EMSA. Stat5 binds to the consensus sequence TT(N5)AA and to relatively conservative variations, with TTC(N3)GAA considered to be optimal \[[@B13],[@B15]\]. Typically, Stat5-DNA interaction by EMSA is performed on synthetic oligonucleotides of 20--40 bp size \[[@B25]\]. To effectively determine whether Stat5 interacts directly with large immunocaptured chromatin fragments, we established conditions for rapid validation by EMSA on chromatin fragments up to 400 bp in length by reducing gel polyacrylamide concentration to 3% and using amplification and isotope labeling by PCR directly from the bacterial clones. Stat5 binding in vitrowas detected in seven of the ten chromatin fragments, as evidenced by prolactin-inducible DNA-binding complexes that could be supershifted by a specific anti-Stat5 antibody, but not by non-specific IgG (Figure [3A](#F3){ref-type="fig"}). Negative data are only presented for one cloned chromatin fragment (CCF \#30) of the three non-binding fragments (CCF \# 21, \# 25, and \#30). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Validation of cloned Stat5-bound chromatin fragments from PRL-stimulated T-47D breast cancer cells. A. PRL-activated Stat5 bindsin vitro to 7 of 10 (8 shown) Cloned Chromatin Fragments (CCF) obtained from a Stat5-chromatin library as shown by EMSA. Nuclear extracts from confluent T-47D cells overexpressing Stat5 and incubated with (+) or without (-) 10 nM human PRL for 30 min were used. Prolactin-induced protein-DNA complexes are indicated by the retarded migration of probe in the (+) PRL lanes for each of the CCFs. Supershift reactions, or band depletions, are indicated by the antibody (Ab) added and signify a specific Stat5-DNA interaction when compared to the supershift control, non-specific, purified IgG. B. Validation ofin vivo Stat5 binding to immunocaptured Cloned Chromatin Fragments (CCFs) by ChIP assay. T-47D cells were incubated with (+) or without (-) PRL as described and Stat5-DNA complexes were enriched by chromatin immunoprecipitation. PCR primers specific for each CCF were designed and used for amplification of two independently generated pools of Stat5-chromatin interaction sites and PCR was performed twice on each pool; a representative experiment is shown. CCFs \#5, \#18, \#23, \#28, \#29, and \#30 were consistently positive for in vivoStat5-DNA binding (upper panel). Positive PCR controls were performed on pre-IP template (lower panel). ::: ![](1476-4598-4-6-3) ::: As a second and independent means to validate the quality of the Stat5 chromatin library, the same randomly selected fragments were analyzed for inducible Stat5 binding in vivousing the ChIP assay. Independent pools of immunocaptured chromatin fragments from T-47D cells were analyzed in which Stat5 was either inactivated by serum deprivation or activated by prolactin. Densitometry of the PCR products was used to verify at least a 2-fold increase in intensity between the (-) prolactin and (+) prolactin samples. In all cases there was no detectable product from the replicate samples that had been immunoprecipitated with a non-specific IgG antibody (data not shown), indicating a specific Stat5-mediated capture of genomic elements. All PCR amplifications were performed at least twice on at least two separate pools of immunoprecipitated genomic elements. Of the seven chromatin fragments that were positive for in vitroStat5 binding by EMSA, five (CCFs \#5, \#18, \#23, \#28, and \#29) were also consistently positive for in vivoStat5 binding by ChIP assay (Figure [3B](#F3){ref-type="fig"}, upper panel). In addition, CCF \#30 was positive by ChIP, but not by EMSA, possibly reflecting indirect binding via other proteins. Conversely, CCFs \#11 and \#14, which bound Stat5 in vitroby EMSA, were both negative by ChIP. Corresponding PCR products from the pre-IP DNA is shown (Figure [3B](#F3){ref-type="fig"}, lower panel) and no product was detected in samples immunoprecipitated with non-specific IgG (data not shown). Due to sequence complexity, fragment-specific flanking primers could not be designed for fragments CCF \#21 and \#25, neither of which bound Stat5 in EMSA. The localization data and binding validation of the ten clones are summarized in Figure [4](#F4){ref-type="fig"}. Each of the CCFs that bound Stat5 by EMSA contained at least one broad consensus TT(N5)AA site as expected. Correspondingly, of the three EMSA-negative CCFs, \#21 and \#25 lacked consensus binding sites, while a single TT(N5)AA site was present in CCF \#30. Furthermore, the five CCFs verified to bind both by EMSA and ChIP may be involved in transcriptional control of nearby genes (Figure [4](#F4){ref-type="fig"}), or alternatively, control transcription of small regulatory RNAs \[[@B26]\]. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Genomic localization of cloned Stat5-chromatin interaction sites from T-47D human breast cancer cells. A karyogram of the human genome illustrates the loci of the cloned Stat5 binding sites. Each site is designated by the CCF number and is localized to its specific area of the genome, based on BLAST alignment. CCFs positive by EMSA are labeled \"E\", and CCFs positive by ChIP are labeled \"C\". The genomic localization and two nearest genes of each verified CCF is given by the genomic position of the centrally located nucleotide: CCF\#5: 8q12.1:58980112 (LOC389663-similar to hypothetical protein; MGC39325-hypothetical protein); CCF\#18: 3q29:193835263 (FGF12-fibroblast growth factor 12; LOC151963-similar to BcDNA:GH11415 gene product); CCF\#23: 5q33.1:147357743 (LOC391839-similar to elongation factor 1 gamma; SPINK5-serine protease inhibitor, Kazal type, 5); CCF\#28: 6q24.1:142177080 (LOC340149-hypothetical protein; NMBR-neuromedin B receptor); CCF\#29: 6q13:74774694 (CD109 antigen); SLC17A5-solute carrier family 17 (anion/sugar transporter) member 5). Other CCFs include: CCF\#11: 7q31.1:111547940; CCF\#14: 15q11.2:21265800; CCF\#21: 18q21.2:49639649; CCF\#25: Xq28:153691722; CCF\#30: 1p34.1:43842775. ::: ![](1476-4598-4-6-4) ::: Conclusions =========== While the present work was being completed, an independent report also identified Stat5 binding sites using a genome-wide approach in mouse lymphoma cells \[[@B27]\], although direct binding by EMSA was not verified. We conclude that unbiased, genome-wide strategies can now be used to identify novel Stat5 binding sites by cloning immunocaptured chromatin fragments. At the current efficacy, approximately 20% of cloned Stat5-immunocaptured fragments from T-47D breast cancer cells could be localized within the normal human genome, and Stat5 binding in vitroand in vivowas confirmed in approximately half of those. Further reductions in capture of non-specific chromatin, combined with refinements in cloning procedures \[[@B28]\], reduced sequencing cost, direct and high-throughput sequencing from bacterial colonies, and direct labeling of PCR products for EMSA testing, will allow streamlining of the procedure for genome-wide identification of Stat5-chromatin interaction sites. Ongoing efforts are also exploring whether some of the clones with only weak homology to the normal human genome represent Stat5-bound neochromatin unique to the cancer cells as a result of genomic instability. Methods ======= Cell Culture and Hormones ------------------------- The human breast cancer cell line T-47D was grown to confluence in RPMI-1640 medium (Biofluids, Rockville, MD) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin-streptomycin (50 IU/ml and 50 μg/ml, respectively) at 37°C with 5% CO~2~. Human prolactin was kindly provided by Dr. A.F. Parlow under the sponsorship of the NIDDKs National Hormone & Peptide Program. Dexamethasone (Dex) was purchased from Sigma Chemical Co. (St. Louis, MO). Chromatin Immunoprecipitation ----------------------------- After grown to confluence (\~2 × 10^7^cells/T175 cm^2^flask), T-47D cells were serum starved for 24 h and then treated with or without 10 nM hPRL for 30 min. For prodifferentiation experiments with glucocorticoid pretreatment, confluent cultures were maintained in serum-free RPMI-1640 with 1 μM Dex dissolved in DMSO or in DMSO alone for 96 hours, then stimulated with or without PRL for 30 min. Proteins were then crosslinked to the chromatin by the addition of formaldehyde (Fisher Scientific, Fairlawn, NJ) to a final concentration of 1% and incubated for 30 min at 37°C. The cells were rinsed, scraped, and pelleted in ice-cold PBS with 1 mM PMSF, 2 μg/ml aprotinin, and 2 μg/ml pepstatin A. Cell pellets were resuspended in 400 μl lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0, 1 mM PMSF, 2 μg/ml aprotinin, and 2 μg/ml pepstatin A) and the lysates were sonicated using a Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA), fitted with a tapered microtip set to an amplitude of 50% and were pulsed twice for 30 s. Debris was pelleted by centrifugation for 10 min at 13,200 RPM at 4°C. The supernatants were then diluted 10-fold in IP buffer (0.1% SDS, 1.1% Triton-X, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 16.7 mM NaCl, 1 mM PMSF, 2 μg/ml aprotinin, and 2 μg/ml pepstatin A) and 1% was set aside for Pre-IP or input sample. The solution was then pre-cleared with beads (50% protein A-sepharose, 1 mg/ml poly dI-dC, 0.1% BSA, in TE, pH 7.4) for 30 min at 4°C. Next, the samples were incubated overnight with Stat5 antibody (N-20, Santa Cruz Biotechnology) or IgG IP-control followed by immunoprecipitation with pre-incubated beads. The samples (beads) were then washed once with each of these buffers in the following order: Wash Buffer 1 (0.1% SDS, 1% Triton-X, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl), Wash Buffer 2 (0.1% SDS, 1% Triton-X, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 500 mM NaCl), Wash Buffer 3 (0.25 M LiCl, 1% NP-40, 1% Na-deoxycholate, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA), then twice with Wash Buffer 4 (TE, pH 8.0). The samples were eluted from the beads in 1% SDS and 0.1 M NaHCO~3~. Next, the cross-links were reversed with 0.2 M NaCl overnight at 65°C followed by protein digestion with proteinase K for 1 hour at 45°C. The DNA was recovered by phenol:chloroform extraction and ethanol precipitation. This pool was then used as PCR template or could be blunted with T4 DNA polymerase (New England Biolabs, Beverly, MA) at 37°C for 5 minutes under standard conditions for uniform cloning. TaqDNA polymerase (New England Biolabs) was then used to add a 3\' A overhang for cloning into a TOPO TA cloning kit (Invitrogen, Carlsbad, CA). The ligated vectors were transformed into electrocompetent bacteria, DH-5αE (Invitrogen), using the manufacturer\'s recommendations for transformation in a Bio-Rad Gene-Pulser (Hercules, CA), and plated on S-Gal/Ampicillin plates (Sigma Chemical Co., St. Louis, MO). Positive clones by blue-white screening were then analyzed directly by colony PCR in a standard reaction with vector-specific M13 and T7 primers that flank the cloning site and were incubated at 94°C for 4 min and cycled at 94°C for 30 s, 55°C for 45 s, and 72°C for 30 s. Colonies with quantifiable inserts were then purified (QiaQuick PCR purification kit, Qiagen, Valencia, CA), sequenced, and localized using BLAST (NCBI, NIH, Bethesda, MD). Validation using known Stat5 responsive genes --------------------------------------------- The following primers were designed to flank the Stat5 binding site in the promoter of the following genes: α2-Macroglobulinforward 5\' TTT AGC CCT CCA GGG ATT CT 3\', reverse 5\' CAA TCC ATC TGG TCC CAA AC 3\'; β-Caseinforward 5\' GGA GAA ACA GTT TGC CTC ACA 3\', reverse 5\' CCT AGT GGG GCC TTG AGA TT 3\'; CISHforward 5\' CTA TTG GCC CTC CCC GAC 3\', reverse 5\' AGC TGC TGC CTA ATC CTT TG 3\'. PCR amplifications shown are representative of at least 2 separate amplifications of at least 2 independently generated immunocaptured pools of Stat5-chromatin complexes. Preparation of cellular extracts for EMSA ----------------------------------------- After reaching confluence, T-47D cells were infected with adenovirus containing wild type Stat5 at a Multiciplity Of Infection (MOI) = 6.67, as described previously \[[@B29]\]. Parallel samples of T-47D cells were not exposed to adenovirus as a mock infection (standard control). After infection, the cells were cultured in serum-free medium for 24 hours, then stimulated for 30 min with 10 nM hPRL, the culture medium was removed, and the cells were collected as described above. Nuclear lysates were collected as described previously \[[@B5],[@B25]\]. Generation of radiolabeled DNA probes ------------------------------------- Radiolabeled products were generated by PCR in a 10 μl reaction under standard conditions with the addition of 0.25 μl α^32^P dATP (10 mCi/ml; Amersham-Pharmacia, Piscataway, NJ). Initially the samples were incubated at 94°C for 1 min, then cycled 36 times at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The reaction was then held at 72°C for 5 min following the cycling to allow for product fill-in and addition of a 3\' terminal \"A\". After completion of the cycling, the PCR products were purified using the Qiagen PCR purification kit, according to manufacturer\'s instructions. The final products were eluted in 50 μl and stored at -20°C until use. DNA-protein binding reaction ---------------------------- The DNA-protein binding reactions were performed in a 10 μl mixture containing 3 μl of nuclear extract from the respective sample, and 1 μg of double-stranded poly dI:dC (Boehringer Mannheim, Indianapolis, IN), as previously described \[[@B25]\]. After 1 h on ice, samples (with 1 ng specific anti-Stat5 antibody (Santa Cruz Biotechnology), or 1 ng non-specific, purified IgG (Sigma), or no antibody) were incubated with 2.0 μl ^32^P-labeled PCR probe and incubated for 20 min at room temperature. The samples were then resolved in a 3% non-denaturing polyacrylamide gel, as previously described \[[@B25]\]. ChIP in vivo validation ----------------------- As an independent validation technique, we designed primers specific to each cloned chromatin fragment (CCF) then performed PCR on a separately-generated enriched pool of immunoprecipitated Stat5-chromatin interaction sites. The following primers were used for PCR: CCF \#5forward 5\' TGA CAT CAG TGA GAG TGG AGG 3\', reverse 5\' TGA GGC TGT AAT GTC ACT CAG AA 3\'; CCF \#11forward 5\' GGA CAC ATC CAC TAC TGC CA, reverse 5\' AAA AGA AGT GCA GTT CAG GAT AA 3\'; CCF \#14forward 5\' GCC TGT GTG ATA TCA TAT GGA AAG 3\', reverse 5\' TTT CCA AGA ACT TAT CAG AAT GAC TT 3\'; CCF \#18forward 5\' TCA GTC TTC CTC TTT CCC CA 3\', reverse 5\' CGG CTT ACA TTC TGT GTC GC 3\'; CCF \#23forward 5\' CAT ATC TGT CTA CAA ATA ACA GTT CCC 3\', reverse 5\' AAT AGA GGG CAG AGT TAA GAA TCA AA 3\'; CCF \#28forward 5\' AAG CCT GAA AAG AAA AAT CTC A 3\', reverse 5\' CCT GAC ACA TCC AGA CTT GG 3\'; CCF \#29forward 5\' CAG ACG TTT GCT GGA AGA TAT G 3\', reverse 5\' TTG TTC CAT GAA ACC AGG G 3\'; CCF \#30forward 5\' AAG GCC ATT GAA GTG AGG TG 3\', reverse 5\' CCA TCA GCC AGT CAT TGA AG 3\'. The PCR was performed with standard conditions using template immunoprecipitated from cells treated with or without prolactin to indicate Stat5 inducibility and was executed at least twice on each of 2 separate pools. Authors\' contributions ======================= MJL carried out the experimental studies, participated in the design of the study, and drafted the manuscript. JX participated in the design of the study and scientific discussion of the results. HR conceived and participated in the design of the study, contributed in the evaluation of the results, and in the preparation of the manuscript. All authors read and approved the final manuscript. Acknowledgements ================ This work was supported in part by NIH grants DK52013 and CA10184.	PubMed Central	2024-06-05T03:55:52.863901	2005-2-1	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549029/", "journal": "Mol Cancer. 2005 Feb 1; 4:6", "authors": [ { "first": "Matthew J", "last": "LeBaron" }, { "first": "Jianwu", "last": "Xie" }, { "first": "Hallgeir", "last": "Rui" } ] }
PMC549030	Background ========== Allergic rhinitis is a common illness, which affects approximately 15 % of the population \[[@B1]\] and has a large impact on the quality of life of the patients. In some studies the symptoms of allergic rhinitis have shown a circadian rhythm with morning symptoms being most prominent in a majority of patients \[[@B1]-[@B6]\]. Antihistamines are important medications in the treatment of allergic rhinitis. One should expect that the effect of an antihistamine is best near or shortly after peak serum level is attained. If this also coincides with the peak in allergy symptoms, an optimal treatment effect should be expected. In one study evening dosing of the antihistamine mequitazine (half-life of 38--45 hours and time to peak serum level about 6 hours) gave better symptom relief than morning dosing on morning symptoms \[[@B4],[@B7]\]. Desloratadine has as mequitazine a rather long half life of 27 hours, and the time to peak serum level at about 3 hours \[[@B8]\]. Evening dosing of this antihistamine may be expected to give better symptom relief than morning dosing on peak morning symptoms. Some studies have also confirmed a circadian variation in efficacy of some antihistamines on histamine induced skin reactions \[[@B9],[@B10]\]. The aim of this study was to examine the efficacy of the antihistamine desloratadine at different time points during the day and to evaluate whether the time of dosing of desloratadine has any impact on the treatment efficacy in seasonal allergic rhinitis (SAR). Methods ======= This was a randomized, open label, parallel group, multicenter study of two weeks duration in patients with SAR during the birch or grass pollen season. Eighty one medical centers in the Nordic countries participated. The inclusion criteria were: patients 18 years or above with a minimum of two years history of SAR confirmed by either a positive skin prick test or a positive serologic allergen test to the relevant seasonal allergen; clinically symptomatic with SAR at baseline/inclusion with a minimum total nasal symptom score (rhinorrhea, congestion, itching and sneezing) of at least 6 and rhinorrhea being minimum 2 (moderate); willingness to adhere to dosing and visit schedule. Females of childbearing potential had to use medically accepted methods of birth control and written informed consent had to be obtained from all patients. The exclusion criteria were: pulmonary disease, perennial rhinitis, sinusitis, rhinitis medicamentosa, pollen desensitization during the last 6 months, respiratory tract infection within the last two weeks, structural nasal abnormalities (including polyps), use of oral, nasal, ocular decongestants, corticosteroids in any form (except mild dermatological group I corticosteroids allowed in only small areas), other antihistamines (oral or topical), any investigational drug during the last 30 days, pregnant or nursing females. The patients were randomized into one of two treatment groups with dosing of 5 mg desloratadine tablets either in the morning between 07 -- 09 (AM-group) or evening between 19 -- 21 (PM-group) in a 1:1 ratio. Randomizing was computer generated for the whole study population using SAS version 6.12 and performed in blocks of eight. Each subject unit (bottle with medication) was labelled with randomization number. Physicians in the different Nordic countries recruited the patients. They assigned the medication in consecutive order. The study was monitored by Schering-Plough. The following symptoms were assessed using a scale from 0 to 3 (0=none, 1=mild, 2=moderate, 3=severe): rhinorrhea, nasal congestion, sneezing, itching nose and eye symptoms (itching, burning, tearing, redness). These symptoms were recorded in a patient diary every morning (AM 12 hours reflective and AM last hour) and evening (PM 12 hours reflective and PM last hour) both at baseline and during the 2 weeks treatment period. Interference with sleep and interference with daily activity were also assessed by the patients every day using the same scale from 0 to 3. In addition, the number of hours spent outdoors was recorded. Visit 1 was at day 0 at the start of baseline, visit 2 after one week and visit 3 after two weeks. A wash-out period prior to Visit 1 was necessary if the patient had been on any drugs which could interfere with the study results (e.g. no other commonly used antihistamines allowed during the prior 10 days). Baseline symptoms were recorded in the evening at day 0 and the following morning (day 1) after which the patients started taking the study medication as randomized. A physical examination was performed at visit 1 and 3. All adverse events were recorded. The study period was from April 11^th^2001 to September 2^nd^2002. Pollen counts were not recorded. The primary objective was to evaluate the efficacy of 5 mg desloratadine taken orally once daily in the morning versus evening. The primary efficacy variable was the mean change from baseline for the AM last hour Total Symptom Score (TSS) over the 2 weeks treatment period. TSS is the sum of the individual symptom scores for the following symptoms that in prior studies \[[@B2],[@B3]\] have shown a circadian rhythm: rhinorrhea, nasal stuffiness/congestion, sneezing and eye symptoms (maximum score 12). Since nasal itching had shown little circadian rhythm in these studies, this symptom was omitted from the TSS. AM last hour was chosen as primary time point since the symptoms had in the same studies shown to be worst in the morning. The study was designed to enrol 700 patients in order to have 600 evaluable patients. This sample size was chosen to detect with 90 % power and 5 % significance level, a difference between treatment groups of 0.6 units or more in mean change from baseline diary TSS, assuming a pooled SD of 2.25 units. In a study on morning vs. evening dosing of the antihistamine mequitazine the differences in dosing-time-related efficacy increased in the sub-group of patients having predominantly morning symptoms \[[@B4]\]. A sub-group analysis was therefore performed on patients with a higher TSS (≥ 1 point difference at baseline) in the morning (AM last hour) than in the evening (PM last hour) and patients with higher TSS in the evening than in the morning in this study. A comparison was then done on the treatment efficacy seen 12 hours and 24 hours after dosing (AM vs. PM dosing) in these patients. All patients receiving at least one dose of study drug and having at least one post dose registration were included in the efficacy analysis (intention-to-treat, ITT), and confirmatory analysis were based on evaluable patients with no protocol violations. Statistical analyses were made with 2-way ANOVA. For evaluation of response of therapy Wilcoxon rank sum test was used. Adverse events were tabulated. The study protocol and the patient informed consent form were approved by Ethics Committees and Health Authorities in each of the participating countries. Results ======= Patients -------- Six hundred and sixty-three patients were randomized at baseline; 336 to the AM-group and 327 to the PM-group. The two groups were comparable with respect to demographics and baseline characteristics (Tab 1). To assess the primary parameter 310 in the AM and 294 in the PM group fulfilled the criteria for ITT. Of the AM group 259 and of the PM group 254 patients completed the study without any violation. Mean baseline TSS varied between 4.64 and 6.10, a difference of 31%; highest during daytime (PM 12 hours reflective) and lowest at night (AM 12 hours reflective). The circadian variation at baseline was more evident for sneezing (around 60% difference between night and day), rhinorrhea and eye symptoms, less so for nasal itching and hardly noticeable for nasal congestion. Fig. [1](#F1){ref-type="fig"} shows total and individual symptom scores at baseline and during two weeks treatment. The circadian variation was much less apparent during treatment with desloratadine (Fig. [1](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Symptom scores.Total and individual symptom scores at baseline and over two weeks treatment period Baseline:○ AM-group; Δ PM-group, Treatment:● AM-group; ▲ PM-group ^1^Max score = 12, ^2^Max score = 3 ::: ![](1476-7961-3-3-1) ::: Efficacy -------- During the two weeks period the mean reduction in TSS ± SE for AM last hour (primary efficacy variable) was 1.63 ± 0.17 (30 %) for the AM-group and 1.80 ± 0.17 (35 %) for the PM-group. There was no statistically significant difference (ITT-analysis) between the groups at this time point (p = 0.456) or at any other time points. The reduction in TSS was highest (2.5 -- 41%) for day time symptoms (PM previous 12 hours) and lowest at night. This was evident for all individual symptoms except for nasal congestion. In the subgroup analysis comparing TSS AM last hour and PM last hour at baseline, 32 % of the patients had more severe symptoms in the morning (≥ 1 point difference in TSS) than in the evening, and 37 % had more severe symptoms in the evening. Looking at these two sub-groups, no difference in treatment efficacy on TSS was seen 12 or 24 hours post dosing (Fig. [2](#F2){ref-type="fig"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Sub-group Total Symptom Score.These sub-groups consists of patients with higher (one or more score points) morning TSS (AM last hour) than evening TSS (PM last hour) at baseline and of patients with higher evening TSS (PM last hour) than morning TSS (AM last hour) at baseline. There was no statistical significant difference in the treatment efficacy between the AM-group and the PM-group. ::: ![](1476-7961-3-3-2) ::: According to their diaries the patients spent in average more than 3.5 hours outdoors daily. The score for the interference of SAR on the patients\' sleep and daily activity at baseline and throughout the study is shown in Fig. [3](#F3){ref-type="fig"}. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Sleep and daily activity.The score for interference with sleep and daily activity at baseline and during treatment shows that there is a higher interference with daily activity at baseline and during treatment than with sleep. ::: ![](1476-7961-3-3-3) ::: Safety ------ The incidence of treatment related adverse events were comparable between the groups, 20 % in the AM-group and 18 % in the PM-group, headache being most frequent, 7 % and 4 % respectively. Discussion ========== This study was randomized but without a placebo control. Since this study was a comparison between two different dosing times of the same medication, a placebo control was superfluous. The study was not blinded as there is no reason to believe that neither the patients nor the physicians should have a biased opinion as to the time of dosing. To blind such a study, the patients need to take study medication from different boxes in the morning and evening. However, this method was not used since this may complicate the study and impair patient compliance. A circadian rhythm has been found in many diseases, also in allergic rhinitis \[[@B1]-[@B6]\]. The effect of an antihistamine may be modulated \[[@B9]-[@B13]\] by variations in allergen exposure, hormonal activity, organ sensitivity and plasma concentration of the drug. In this study we have shown that desloratadine maintains its effect at different time points throughout the day and thus the effect appears unaffected by a modulating factor. The baseline period in this study lasted 24 hours which is the same as in the study on mequitazine \[[@B4]\] and other studies \[[@B16],[@B17]\]. In some studies of the effect of antihistamines the baseline period has been longer \[[@B14],[@B15]\]. It would have been difficult to keep patients in the Nordic countries off medication for more than one day in addition to any washout period during the pollen season. We do not believe that the duration of baseline influenced the results of this comparative study. The circadian rhythm at baseline found in this study with maximum symptoms during the day differs from some other studies \[[@B1]-[@B6]\] where more patients had the most severe symptoms in the morning. This difference may partly be due to patient selection. Patients with perennial rhinitis were excluded from our study. Thus indoor allergens do not influence symptom variation. The patients spent several hours outdoors during the day in the pollen season. It seems likely that this exposure would influence the symptoms. The circadian variation was not apparent during treatment, possibly because the suppression of symptoms by desloratadine is more observable when symptoms are most prominent. The best effect of mequitazine was obtained after evening dosing (12 hours before peak of symptoms) compared to morning dosing (24 hours before peak of symptoms). In our study no difference in treatment efficacy was seen 12 or 24 hours after dosing in the sub-group analysis of patients with higher baseline morning or evening TSS. Whatever the cause for this discrepancy between these two antihistamines, other antihistamines may show a variation in effect during the day not only on dermal symptoms \[[@B9],[@B12]\] but also on nasal ones. Thus studies on the effect of other antihistamines in allergic rhinitis should be encouraged. The adverse events recorded were of a magnitude and nature as seen in other studies of desloratadine and other antihistamines \[[@B14]-[@B17]\]. Many patients have circadian variations in symptoms. The peak of symptoms can be at different time points from patient to patient. Individual dosing time of medication may improve symptom relief. Desloratadine, however, apparently shows no circadian variation in effect. Conclusions =========== A circadian rhythm was seen for most SAR symptoms at baseline, being most distressing during daytime, possibly due to long outdoor exposure. This circadian variation is less apparent after treatment with desloratadine. No statistically significant difference in efficacy was seen whether desloratadine was given in the morning or in the evening. This gives the patients more flexibility in choosing dosing time. Competing interests =================== The study was funded by Schering-Plough in the Nordic countries. None of the authors will gain financially from the publication. There are no patents pending. There are no other competing interests. Authors\' contributions ======================= RH participated in the design of the protocol and is the main author of the article. KH participated in the design of the protocol and as investigator. OB participated as principal investigator in Sweden and enrolled most patients in the study. SF participated in the statistical analysis, drafted the tables and figures and participated in drafting the manuscript. TØ was project leader and participated in the design of the protocol, the statistical analysis and drafting of the manuscript. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Demographics ::: Treatment groups --------------------------------------------------- ---------------------- -------------- ---------- [Age (years)]{.underline} Mean (SD) 35.4 (11.0) 36.5 (12.0) 0.232^a^ Min-Max 18--69 18--75 [Age Group (years)]{.underline} 18--29 115 (34.2 %) 113 (34.6 %) 30--39 113 (33.6 %) 95 (29.1 %) 40--49 70 (20.8 %) 66 (20.2 %) ≥ 50 38 (11.3 %) 53 (16.2 %) [Sex]{.underline} Male (%) 152 (45) 163 (50) 0.235^b^ Female (%) 184 (55) 164 (50) [Duration of SAR History (years)]{.underline} Mean (SD) 14.2 (9.2) 14.4 (10.3) 0.662^a^ Min-Max 2--50 1--55^c^ ^a^t-test for differences between the means of the two treatment groups ^b^χ^2^-test for the distribution between the two groups ^c^One patient in the PM-group had only one year duration of SAR history although the inclusion criterion was 2 years ::: Acknowledgements ================ Participating Investigators Denmark:J. Arnved, J. Boserup, J. Blokkebak, B. Smidt Hausted, J. Holm-Pedersen, H. Isaksen, F. Ourø Jensen, N. Kobborg, N.O. Nielsen, E. Olafsson, I. Haugaard Rasmussen, K. Reuther, J-M. Sannig, L. Malte Sehestad, P. Skyttebo, L. Stievano, J. Lyngfeldt Thomsen, L.G. Aagaard. Finland:J. Antila, T. Pirilä, M. Rautiainen, J. Seppä. Iceland:D. Gislason, P. Stefansson. Norway:U. Andruchow, T. Eikeland, H. Fonneløp, B. Fossbakk, S. Gangstø, A.C. Geheb, H. Helvig, K. Hjelle, A.S. Hølland, K. Høye, I. Jørum, A. Kaisen, M. Killi, R. Kleiven, S.A. Lønning, A. Mangersnes, T. Mohn, M. Mundal, I.O. Myrbakken, B. Nicolaisen, D. Niklasson, P.S. Norheim, S. Rognstad, S. Rokstad, P. Schrøder, H.K. Sveaas, A. Visted, I.M. Warlo, E. Øvstedal. Sweden:B. Barr, O. Berg, M. Bergstedt, E-L. Birkebo, T. Bremer, A. Bylander-Groth, J.O. Eklöf, J-E. Friis-Liby, P. Gemryd, S. Boes Hansen, S. Henriksson, M. Hochermann, U. Kingstam, B. Lie, M. Lundgren, D. Nelker, H. Nordell, A. Nordkvist, P. Rahnster, J. Sobin, S. Stemer, C. Stenström, L. Sundén, C. v. Sydow, M. Ungerstedt, S. Wollin, M. Åberg.	PubMed Central	2024-06-05T03:55:52.866747	2005-2-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549030/", "journal": "Clin Mol Allergy. 2005 Feb 2; 3:3", "authors": [ { "first": "Rolf", "last": "Haye" }, { "first": "Kjetil", "last": "Høye" }, { "first": "Olof", "last": "Berg" }, { "first": "Sissel", "last": "Frønes" }, { "first": "Tone", "last": "Ødegård" } ] }
PMC549031	Background ========== Responsiveness, a concept introduced in the mid-eighties by bio-medical researchers, is considered to be an essential measurement property of health status questionnaires, distinct from reliability and validity \[[@B1]\]. However, it can be questioned whether an instrument\'s sensitivity to differences between health status changes over time, which refers to responsiveness, is different from an instrument\'s sensitivity to differences in health among subjects at one point in time, which refers to the psychometric concept of parallel forms reliability from the framework of classical test theory \[[@B2]\]. A number of theorists have argued that responsiveness is not a separate psychometric attribute of health status instruments, but merely some form of construct validity \[[@B3]\]. The aim of the study is to provide empirical evidence of this notion by investigating the relationship between instrument responsiveness and the traditional psychometric concept of parallel forms reliability as embodied by the internal consistency coefficient \[[@B2],[@B3]\]. Methods ======= We used three datasets comprising the scores of patients on three widely used health status instruments on two moments in time in order to assess health changes. The first dataset was from a randomized clinical trial investigating the effects of arm and leg rehabilitation training on the functional recovery of stroke survivors using the 10-item Barthel (basic) activities of daily living scale \[[@B4]\]. Patients (n = 89) were rated one week and 12 weeks after stroke. A Barthel scale score ranges from zero to 20 points with higher scores indicating more independent functioning. The second dataset comprised the scores on the 45-item physical component of the Sickness Impact Profile (SIP) of 227 patients with myocardial infarction \[[@B5]\](on average 2 year interval between assessments), 120 patients with stroke \[[@B6]\] (3 year interval between assessments) and 141 patients scheduled for a carotid endartectomy surgical procedure \[[@B7]\] (3 months time interval). The SIP physical items are scored on a dichotomous scale with 1 point for each endorsed item statement. The scale ranges from 0 to 45 points with higher scores indicating higher levels of sickness related dysfunction. The third dataset contained the scores of 164 patients with Graves\' ophthalmopathy scored on the 8-item psychosocial dimension of the Graves\' ophthalmopathy quality of life (GO-QOL) instrument \[[@B8]\]. The GO-QOL scale items are scored on a 1 to 3 point rating scale. Overall scores are transformed to a 0--100 scale with higher scores indicating better psychosocial functioning. Patients completed the instrument before and three or six months after radiotherapy or eye surgery. Only subjects with no missing values at baseline or follow-up were included in the analysis. The Barthel dataset had no missing values, the SIP datasets had 13 % (16/120), 28% (64/227) and 0.7% (1/141) missing values respectively and the GO-QOL had 0.6% (1/164) missing values. For the SIP datasets, there was a mean deterioration in health (1 point), for the Barthel and GO-QOL scales patients were improved at follow-up (Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Score statistics and reliabilities (α) at baseline and follow-up. ::: Barthel N = 89 SIP N = 407 GO-QOL N = 163 ----------------------------- ---------------------- ------------------- ----------------------- Baseline score (SD, IQR^1^) 7.98 (4.52, 5--12) 5.12 (6.28, 1--7) 59.32 (24.64, 44--81) Follow-up score (SD, IQR) 14.25 (5.06, 10--19) 6.13 (7.57, 0--9) 65.22 (24.17, 50--81) Mean change score (SD)^2^ 6.27 (3.21) 1.01 (4.36) 5.90 (17.13) SRM^3^ 1.95 0.23 0.34 Chronbach\'s α time 1 0.86 0.92 0.83 Chronbach\'s α time 2 0.89 0.94 0.84 1\) Distribution of change scores was approximately normal for all three datasets 2\) IQR = interquartile range 3\) SRM = Standardized Response Mean (see statistical analysis section) ::: Statistical analysis -------------------- The analysis aimed to assess the strength of the relationship between internal consistency reliability (Cronbach α or Kuder-Richarson-20) reflecting sensitivity to differences in health status among patients \[[@B3]\], and the Standardized Response Mean effect size (SRM) indicating an instrument\'s sensitivity to change. The SRM is calculated as the mean of the change scores divided by the standard deviation of the change scores \[[@B3],[@B9]\]. In a stepwise procedure, we reduced the baseline internal consistency by removing the item contributing most to the internal consistency coefficient until 0.60 was reached, which was considered as the minimum standard for reliability \[[@B10]\]. For the 45-item SIP physical scale, two items were removed at every step. For the other instruments, one item was removed at each step. Using the remaining items at each item reduction step, we calculated the SRM. The thus decreasing internal consistency coefficients and associated SRMs were plotted and the strength of the relationship was calculated using Spearman rank correlation coefficients. All analyses were performed with SPSS 11.0, a commercially available software package. Results ======= Figures [1](#F1){ref-type="fig"}, [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"} show the spearman rank correlations between the internal consistency coefficients and the SRM using the scores on the Barthel, the SIP and the GO-QOL respectively. The spearman rank correlations ranged between 0.90 for the Barthel index to 1.00 for the GO-QoL indicating strong to perfect relations between internal consistency and responsiveness. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Barthel -- Relation between the internal consistency reliability (alpha) and the SRM(Spearman\'s r = 0.90) ::: ![](1477-7525-3-8-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### SIP -- Relation between the internal consistency reliability (alpha) and the SRM(Spearman\'s r = 0.99) ::: ![](1477-7525-3-8-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### GO-QOL -- Relation between the internal consistency reliability (alpha) and the SRM(Spearman\'s r = 1.00) ::: ![](1477-7525-3-8-3) ::: Discussion ========== Our results contradict the conviction that responsiveness is a separate psychometric property of health scales. Internal consistency reliability, reflecting a scale\'s sensitivity to cross-sectional differences in health, closely coincided with the instruments\' sensitivity to change as measured with the standardized response mean. Our results also reflect what is already known within the framework of classical test theory. A test score cannot correlate more highly with any other variable than its own true score \[[@B2]\]. This implies that the maximum correlation between an observed test score and any other variable, i.e. its validity, is the square root of its reliability \[[@B2]\]. Thus, the more reliable a test, the more potential for validity, in this case responsiveness, there exists. We used nested versions of the same test, which are highly correlated with each other, to illustrate this phenomenon. It is likely, however, that the results will also apply with different instruments measuring similar health constructs that are highly inter-correlated. It should also be noted that the results apply to one-dimensional psychometric scales and not to instruments containing so-called \"causal\" variables, for example disease symptoms \[[@B3]\] since these instruments are not strictly one-dimensional. We used the SRM effect size that uses the standard deviation the change scores and therefore includes all information about the changes on the selected instruments. The results can not generalized to alternative effect sizes such as Cohen\'s effect size or Guyatt\'s responsiveness statistic \[[@B1]\] because these largely depend on the variability of scores at baseline or the variability in scores obtained from a separate, not improved, sample. In a frequently cited paper, Guyatt et al. \[[@B1]\] made the distinction between discriminative instruments, whose purpose it is to measure differences between subjects and evaluative instruments, designed to examine change over time. This in contrast to most of the scales used in clinical medicine (blood pressure, cardiac output), which are assumed to work well in both discriminative and evaluative roles. To corroborate his arguments, he used the hypothetical example of two health status instruments designed to evaluate therapeutic interventions in patients with chronic lung disease that were presented to the same patient sample (Table [2](#T2){ref-type="table"}). \"Evaluative\" instrument A showing poor test-retest reliability because of small between subject score variability but excellent responsiveness, and \"discriminative\" instrument B with excellent reliability because of large between-subject score variability and poor responsiveness. From Table [2](#T2){ref-type="table"}, however, it can be seen that this representation of instrument behaviour in clinical research is logically inconsistent, since it does not explain how two instruments, both measuring the same health construct show such divergent score distributions at baseline. According to instrument A the sample is highly homogeneous, while it is highly heterogeneous according to instrument B. In Appendix 1 (see [additional file 1](#S1){ref-type="supplementary-material"}), we show that the above representation is not impossible, but highly unlikely since it occurs only in extreme situations. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Representation of the scores on \"evaluative\" instrument A and \"discriminative\" instrument B in a randomized clinical trial \[1\] ::: Instrument A Time 1 Time 2 Intervention Time 3 Difference score Exercise test Result -------------- -------- -------- -------------- -------- ------------------ ---------------------- Subject 1 8 9 Verum 15 +6 Much improved Subject 2 9 8 "" 15 +7 Much improved Subject 3 8 9 "" 15 +6 Much improved Subject 4 9 8 "" 15 +7 Much improved Subject 5 8 9 Placebo 8 -1 Unchanged Subject 6 9 8 "" 9 +1 Unchanged Subject 7 8 9 "" 8 -1 Unchanged Subject 8 9 8 "" 9 +1 Unchanged Instrument B Time 1 Time 2 Time 3 Difference score Exercise test Result Subject 1 5 5 Verum 5 0 Much improved Subject 2 9 9 "" 9 0 Much improved Subject 3 13 13 "" 13 0 Much improved Subject 4 17 17 "" 17 0 Much improved Subject 5 5 5 Placebo 5 0 Unchanged Subject 6 9 9 "" 9 0 Unchanged Subject 7 13 13 "" 13 0 Unchanged Subject 8 17 17 "" 17 0 ::: During the past 20 years, clinimetric research has resulted in about 25 definitions and 30 measures of instrument responsiveness, sometimes referred to as sensitivity to change or longitudinal validity \[[@B11]\]. Moreover, it is evaluated in literally hundreds of published papers on the validation of health status instruments. Our results show that responsiveness, as measured with the SRM, mirrors the traditional concept of parallel test reliability as embodied by the internal consistency coefficient. When comparing instruments measuring similar health constructs, an instrument sensitive to health differences among subjects is also likely to be sensitive to therapy-induced change as well. However, further empirical data will be needed to confirm the relationship between internal consistency and responsiveness, e.g., by reviewing studies in which health status instruments were compared on their responsiveness. Authors\' contributions ======================= Robert Lindeboom conceived the idea for the manuscript, Mirjam Sprangers and Robert Lindeboom wrote the manuscript, Koos Zwinderman provided the mathematics and rewrote parts of the manuscript in earlier drafts Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Appendix 1: Is it possible to have one high reliable scale with low \'responsiveness\', and a low reliable scale with high \'responsiveness\' measuring the same construct? ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ The authors would like to thank Dr. Caroline Terwee for her critical comments on earlier drafts of the manuscript.	PubMed Central	2024-06-05T03:55:52.868445	2005-2-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549031/", "journal": "Health Qual Life Outcomes. 2005 Feb 3; 3:8", "authors": [ { "first": "Robert", "last": "Lindeboom" }, { "first": "Mirjam A", "last": "Sprangers" }, { "first": "Aeilko H", "last": "Zwinderman" } ] }
PMC549032	Introduction ============ We have developed an electric field hypothesis for the mechanism of transmission of excitation from one cell to the next that does not require gap-junction channels \[[@B1]-[@B4]\]. In the electric field hypothesis, the electrical voltage that develops in the narrow junctional cleft (V~jc~) when the prejunctional membrane generates an action potential, serves to depolarize the postjunctional membrane its threshold, by a patch-clamp-like effect. The parameters that affect the magnitude of Vjc include the size of R~jc~, the transverse resistance of the junctional cleft. This results in excitation of the postjunctional cell, after a brief junctional delay. The total propagation time consists primarily of the summed junctional delays. This results in a staircase-shaped propagation, the surface sarcolemma of each cell firing almost simultaneously \[[@B2]\]. There are no low-resistance connections between the cells in several different cardiac muscle and smooth muscle preparations (reviewed in \[[@B3],[@B4]\]. Propagation by mechanisms not requiring low-resistance connections have also been proposed by others \[[@B5]-[@B8]\]. Propagation has been demonstrated to be discontinuous (or saltatory) in cardiac muscle \[[@B9]-[@B12]\]. Fast Na^+^channels are localized in the junctional membranes of the intercalated discs of cardiac muscle \[[@B13]-[@B15]\] Sperelakis, 1995, a requirement for the EF mechanism to work \[[@B3],[@B4],[@B1],[@B2],[@B13]\]. In connexin-43 and Cx40 knockout mice, propagation in the heart still occurs, but it is slowed \[[@B15]-[@B19]\], as predicted by our PSpice simulation study \[[@B20]\]. It was reported that the anisotropic conduction velocity observed in the heart is not a result of cell geometry \[[@B21]\]. Subsequently, we published a series of papers on the longitudinal and transverse propagation of action potentials in cardiac muscle and smooth muscle using PSpice analysis \[[@B22],[@B20],[@B24]\]. In the review process for our recent paper \[[@B24]\], one of the referees asked us to determine the effect of introducing strong cell coupling via gap-junction (g-j) channels between cells within each chain on the transverse propagation in our 5 × 5 model (5 parallel chains of 5 cells each). Unexpectedly, we found that strong cell coupling (10,000 or 1,000 g-j channels per junction) actually inhibitedtransverse propagation. This fact was briefly mentioned as an unpublished observation in that paper. The purpose of the present study was to do a thorough investigation of that strange phenomenon. The results showed that, in cardiac muscle, transverse propagation was inhibited when many g-j channels were added between cells of each chain. This was true when either a single cell in the first chain was stimulated (cell A1) or the entire chain (A-chain) was stimulated simultaneously. Methods ======= Details of the methods used and modeling of the PSpice analysis were given in our previous papers, including the limitations \[[@B21]-[@B24]\]. The full version of the PSpice software for circuit analysis/design was obtained from the Cadence Co. (Portland, OR). The assumptions made were given previously, including the entire circuit that was used \[[@B22]\]. An abbreviated version of the circuitry is given in the first two figures. The surface membrane of each myocardial cell was represented by 2 units and each junctional membrane by 1 unit (Figs. [1](#F1){ref-type="fig"}, [2](#F2){ref-type="fig"}). The values for the circuit parameters used under standard conditions were given previously for both the surface units and junctional units in cardiac muscle \[[@B22]\]. The myocardial cell was assumed to be a cylinder 150 μm long and 16 μm in diameter. The myocardial cell capacitance was assumed to be 100 pF, and the input resistance to be 20 MΩ. A junctional tortuosity (interdigitation) factor of 4 was assumed for the cell junction. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The 5 × 5 model for cardiac muscle, consisting of 5 parallel strands (A-E) of 5 cells each (1--5) (total of 25 cells). Each muscle cell was represented by a block of 4 basic units: 2 units representing the surface membrane (one upward-facing and one downward-facing) and one unit for each of the two junctional membranes. For simplicity, the lumped resistance of the gap-junctions is not indicated here, but is shown in Fig. 2. Transverse propagation is sequential activation of chains A to B to C to D to E. ::: ![](1475-925X-4-7-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Blow-up of a small portion of the 5 × 5 model to show the electrical circuit for each basic unit, including the \"black-box\" required for excitability. R~ol2~represents the longitudinal resistance of the interstitial fluid between the parallel chains; the higher the resistance, the tighter the packing of the chains. Depolarizing current (0.25 nA) is applied to the interior of either the first cell or the entire chain (A-chain) simultaneously. When gap-junction channels were added, a resistor (R~gj~) was inserted across each cell junction, from the interior of one cell to the interior of the next one. ::: ![](1475-925X-4-7-2) ::: The circuit used for each unit was kept as simple as possible, using only those ion channels that set the resting potential (RP) and predominate during the rising phase of the AP. We wanted to only inscribe the rising phase of the APs to study propagation in the 2-dimensional sheet of myocardial cells. It was not possible to insert a second or third \"black-box\" into the basic excitable units, because the system became erratic, therefore the dynamic behavior of the cardiac cell membrane was an approximation. The RP was -80 mV, and the overshoot potential was +30 mV (AP amplitude of 110 mV). Propagation velocity was calculated from the measured total propagation time (TPT) (measured as the difference between when the APs of the first cell and last cell crossed -20 mV) and cell length. Because the PSpice program does not have a V-dependent resistance to represent the increase in conductance for Na^+^ions in myocardial cells during depolarization and excitation, this function had to be simulated by a V-controlled current source (our \"black-box\") in each of the basic circuit units (Fig. [2](#F2){ref-type="fig"}). The current outputs of the black-box, at various membrane voltages, were calculated assuming a sigmoidal relationship between membrane voltage and resistance between -60 mV and -30 mV. The V values used in the GTABLE were those recorded directly acrossthe membrane. The excitabilities of the basic units were the same as in our previous papers \[[@B21]-[@B24]\]. The upper chain of cells was assumed to be bathed in a large volume of Ringer solution connected to ground. The external resistance (R~o~) of this fluid was divided into two components: a radial (or transverse) resistance (R~or~) and a longitudinal resistance (R~ol~). The longitudinal resistance values (R~ol2~) between the upper chain and interior chains were increased over a wide range to reflect packing of parallel chains into a bundle of fibers, with different degrees of tightness of the packing (Fig. [1](#F1){ref-type="fig"}). The higher the R~ol2~value, the tighter the packing of the chains, i.e., the lower the cross-sectional area of the ISF space. The applicable equation is R~ol2~= ρ (Ω-cm) × L (cm) /Ax (cm^2^), where ρ is the resistivity of the ISF, L is the length, and A~x~is the cross-sectional area of the ISF space. The transverse resistance of the interstitial fluid (ISF) space (R~or2~) also reflects the closeness between the chains; the lower the R~or2~value, the closer the chains are packed. In the present 5 × 5 model, there were five parallel chains (chains A, B, C, D, and E) of five cells each (cells 1--5). Electrical stimulation (rectangular current pulses of 0.25 nA and 0.50 ms duration) was applied to the inside of either the first cell of chain A (cell A1) or all 5 cells of the A-chain. Under initial conditions, the cells in each chain were not interconnected by low-resistance pathways (gap junction channels), so that transmission of excitation from one cell to the next had to be by the EF developed in the narrow junctional cleft. Then gj-channels were added (100, 1,000 and 10,000 channels per junction) to determine what effect they would have on transverse propagation. Since the number of functional gj-channels per junction have been estimated to be about a 1000 \[[@B12]\], we varied the number over a very wide range. The resistances of the gap-junction channels were lumped into one equivalent resistances because they are all in parallel. As shown in Figure [1](#F1){ref-type="fig"}, there were two surface membrane units in each cell (one facing upwards and one inverted) and one unit for each of the junctional membranes (intercalated disks of cardiac muscle). To improve clarity, in some runs the V-recording markers were placed on only one chain at a time. When all cells in a model were being recorded simultaneously (25 cells), the V markers were removed from most of the basic units to minimize confusion. That is, the voltage was recorded from only one surface unit (upward-facing) in each cell. The junctional cleft potential (V~jc~) was recorded across R~jc~, the radial (or transverse) resistance of the narrow and tortuous junctional cleft. Under standard conditions, R~ol2~was 200 KΩ, R~or2~was 100 Ω, and R~jc~and was 25 MΩ (50 MΩ ÷ 2). Results ======= The 5 × 5 model (5 parallel chains of 5 cells each) of cardiac muscle was used to examine whether addition of gap-junction (gj) channels between the cells in each chain would affect transverse propagation of simulated (PSpice) action potentials. The number of gj-channels was increased from zero (standard conditions; resistance of the gap junction (R~gj~) of infinite) to 100 (R~gj~= 100 MΩ), 1000 (R~gj~= 10 MΩ), and 10,000 (R~gj~= 1.0 MΩ). Experiments were done with electrical stimulation (0.5 nA, 0.5 ms) of only the first cell of the first chain (A; cell A1) and with simultaneous stimulation of all 5 cells of the A-Chain (each cell receiving 0.5 nA current). This second method of stimulation was done to obtain a more accurate assessment of strictly transverse propagation. Figure [3](#F3){ref-type="fig"} illustrates some of the results with stimulation of only cell A1. Panel Ashows the standard conditions: R~gj~= ∞ (0 gj-channels) and R~ol2~of 200 KΩ (the longitudinal resistance of the interstitial space between the chains). As shown, all 5 chains (25 cells) responded. The total propagation time (TPT) was 4.2 ms (measured as the elapsed time between when the first and last APs crossed -20 mV). When 10,000 g-j channels were inserted into the cells of each chain (R~gj~= 1.0 MΩ), then the last 2 chains (D and E) failed to respond (panel B). The 5 cells of each chain (A, B, and C) that responded now fired simultaneously because of the high degree of cell coupling, thereby giving only three AP traces, as shown. However, raising R~ol2~to 10 MΩ allowed all 5 chains to respond (Panel C), and propagation velocity was increased. These data are summarized in Table [1](#T1){ref-type="table"}, part A. Hence greatly elevating R~ol2~could overcome the impaired transverse propagation caused by the gj-channels. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Transverse propagation of simulated action potentials (APs; rising phase) for cardiac muscle (5 × 5 models) with stimulation of one cell only (cell A1; first cell of the A-chain). A: R~gj~= ∞ (0 channels). R~ol2~= 200 KΩ. Standard conditions. All 25 cells responded. B:R~gj~= 1.0 MΩ (10,000 channels). R~ol2~kept unchanged. The last 2 chains (D, E) failed to respond. All 5 cells of each chain that responded (A, B, C) fired simultaneously because of the strong cell coupling. C:With R~gj~held at 1.0 MΩ, raising R~ol2~to 10 MΩ (representing tighter packing of the parallel chains) now allowed all 5 chains to respond. Thus, adding gj-channels inhibited transverse propagation, but this inhibition could be overcome by raising R~ol2~. ::: ![](1475-925X-4-7-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Summary of simulation data on cardiac muscle with stimulation either of single cell or entire chain. ::: R~gj~(MΩ) R~ol2~(MΩ) TPT~5c~(ms) TPT~4c~(ms) TPT~3c~(ms) TPT~2G~(ms) \# of chains responding ----------- ------------ ------------- ------------- ------------- ------------- ------------------------- ∞ 0.2 4.2 3.5 2.7 5 100 0.2 2.4 2.3 2.2 5 10 0.2 \-\-\-- \-\-\-- 2.1 3 1.0 0.2 \-\-\-- \-\-\-- 2.1 3 ∞ 1.0 3.8 3.2 2.6 5 100 1.0 3.6 3.1 1.9 5 10 1.0 \-\-\-- \-\-\-- 1.6 3 1.0 1.0 \-\-\-- \-\-\-- 1.5 3 ∞ 5.0 2.7 2.6 2.3 5 100 5.0 2.0 1.6 1.2 5 10 5.0 \-\-\-- \-\-\-- 1.0 3 1.0 5.0 \-\-\-- \-\-\-- 0.9 3 ∞ 10 2.4 2.2 2.0 5 100 10 1.5 1.3 1.0 5 10 10 2.6 1.2 0.8 5 1.0 10 2.9 1.3 0.8 5 ∞ 0.2 3.6 2.7 2.0 1.2 5 100 0.2 2.5 2.4 2.4 1.2 5 10 0.2 \-\-\-- \-\-\-- \-\-\-- 1.1 2 1.0 0.2 \-\-\-- \-\-\-- \-\-\-- 1.1 2 ∞ 1.0 2.6 2.1 1.5 0.9 5 100 1.0 2.7 2.6 1.6 0.8 5 10 1.0 \-\-\-- \-\-\-- 1.6 0.8 3 1.0 1.0 \-\-\-- \-\-\-- 1.6 0.7 3 ∞ 5.0 1.7 1.4 0.9 0.6 5 100 5.0 1.6 1.4 0.9 0.6 5 10 5.0 \-\-\-- 1.3 0.8 0.5 4 1.0 5.0 \-\-\-- 1.3 0.8 0.5 4 ∞ 10 1.4 1.1 0.8 0.6 5 100 10 1.3 1.0 0.8 0.5 5 10 10 1.2 0.9 0.7 0.4 5 1.0 10 1.2 0.9 0.7 0.4 5 ::: Figure [4](#F4){ref-type="fig"} illustrates some of the results with simultaneous stimulation of all 5 cells of the A-chain. Panel Ashows the standard conditions: R~gj~= ∞ and R~ol2~= 200 KΩ. As shown, all 5 chains responded (TPT was 3.6 ms). Note that all 5 cells of the B -- E chains did not respond simultaneously. When 10,000 gj-channels were added to the contiguous cells of each chain (R~gj~= 1.0 MΩ), then the last 3 chains (C, D, and E) failed to respond (Panel B). The 5 cells of each chain that responded (A, B) now fired simultaneously because of the strong coupling. However, elevating R~ol2~to 10 MΩ allowed all 5 chains to respond (Panel C). These data are summarized in Table [1](#T1){ref-type="table"}, part B. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Transverse propagation of simulated APs for cardiac muscle with stimulation simultaneously of the entire A-chain. This was done as a better assessment of transverse propagation for comparison with stimulation of only one cell of the A-chain. A:R~gj~= ∞ (0 channels). R~ol2~= 200 KΩ. Standard conditions. All 5 chains responded. B:R~gj~= 1.0 MΩ (10,000 channels). With R~ol2~kept at 200 KΩ, chains C, D, and E failed to respond. C: With R~gj~held at 1.0 MΩ, raising R~ol2~to 10 MΩ (representing tighter packing of the chains) now allowed all 5 chains to respond. All 5 cells of each chain responded simultaneously because of the strong coupling. Thus, adding gj-channels inhibited transverse propagation, but this inhibition was overcome by raising R~ol2~. ::: ![](1475-925X-4-7-4) ::: Figure [5](#F5){ref-type="fig"} is a graphic summary of the results for both stimulation of only one cell (A1) (Panel A) and stimulation of the entire A-chain (Panel B). The data include R~gj~values and R~ol2~values not illustrated in Figures [3](#F3){ref-type="fig"} and [4](#F4){ref-type="fig"}. As such, this figure is complementary to Table [1](#T1){ref-type="table"}, but Table [1](#T1){ref-type="table"} lists the TPT values as well. As shown, progressive addition of gj- channels reduces the number of chains that respond, with both ways of stimulation, and progressive elevation of R~ol2~reverses this inhibition. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Summary of the transverse propagation experiments. Graphic plot of the number of chains responding as a function of R~gj~for cardiac muscle, with stimulation of only one cell (cell A1) (panel A) or with stimulation of the entire A-chain (panel B). Data for various R~ol2~values (0.2, 1.0, 5.0, and 10 MΩ) are indicated. Four different R~gj~values were tested: ∞ (0 channels), 100 MΩ (100 channels), 10 MΩ (1,000 channels), and 1.0 MΩ (10,000 channels). Thus, transverse propagation was depressed when there were many gj-channels (1000 or 10,000), but elevation of R~ol2~could overcome this depression. ::: ![](1475-925X-4-7-5) ::: Discussion ========== The results demonstrated that, in a cardiac muscle model, the insertion of many gj- channels, between abutting cells in each longitudinal chain of cells, actually inhibited transverse propagation of excitation between the parallel chains. This was true both for stimulation of only a single cell of the first chain (A-chain; cell A1) and for stimulation simultaneously of the entire 5 cells of the A-chain. The inhibition produced by the gj-channels could be overcome by greatly increasing the value of R~ol2~(the longitudinal resistance of the narrow interstitial space between the parallel chains), reflecting tighter packing of the chains. This finding surprised us at first. We were expecting either that there would be no effect on transverse transmission of excitation or that transverse transfer of excitation would be enhanced. But on further refection, the inhibition produced might be predicted. This is based on the fact that, with strong longitudinal coupling between cells, the entire chain of 5 cells must be simultaneously stimulated to threshold. Therefore, if the transverse transfer energy available is limited and is near threshold for a give chain, it is likely that some chains will fail to fire. Thus, the problem of strong coupling is not in the chains that are already excited, but rather in the quiescent adjacent chain which is in process of trying to become excited (D-chain in case of Fig. [3](#F3){ref-type="fig"} and C-chain in Fig. [4](#F4){ref-type="fig"}). Strong coupling in the \"in-process\" chain requires more energy transfer from the \"already-activated\" chain. If the \"in-process\" chain were not coupled by gj-channels, then if only one cell of the chain received enough stimulating energy to become activated, excitation would spread from it to the remaining cells of that chain. This idea was tested, and the results shown in Figure [6](#F6){ref-type="fig"} are consistent with the mechanism proposed above. In panel A, in which R~gj~was 1.0 MΩ (10,000 gj-channels) uniformly in all 5 chains, chains D and E failed to fire. When R~gj~of just chains D and E were changed to infinity (0 channels), then all 5 chains fired (panel B). Therefore, the inhibition of transverse propagation can be reversed by uncoupling the cells of the chains that failed. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Special experiment to test why presence of many gap-junction channels inhibits transverse propagation. Stimulated Cell A1 only. R~ol2~of 1.0 MΩ. A:Uniform value for R~gj~of 1.0 MΩ (10,000 channels) in all 5 chains. Chains D and E failed to respond. Compare with Fig. 3B for the standard R~ol2~of 0.2 MΩ. B:The R~gj~values in chains D and E only were changed to infinity (0 channels). Now chains D and E responded. See text for details. This demonstrates that removing the gj-channels in the two chains awaiting excitation (D, E) increased the safety factor for transverse propagation. ::: ![](1475-925X-4-7-6) ::: Consistent with the argument presented in the paragraph above, the transverse propagation velocity was not inhibited by insertion of gj-channels, up through the last chain activated (C-chain in case of Fig. [3](#F3){ref-type="fig"} and B-chain in Fig. [4](#F4){ref-type="fig"}). If anything, the transverse velocity was increased slightly (TPT lowered). The TPT values (for differing amount of transverse spread of excitation, e.g. 2-chains, 3-chains, 4-chains, or all 5-chains) are listed in Table [1](#T1){ref-type="table"} for different degrees of cell coupling. Note that, in part A, the largest change in TPT value is between R~gj~of ∞ (0 channels) and R~gj~of 100 MΩ (100 channels); adding more channels (Rgj of 10 MΩ (1000 channels) or 1.0 MΩ (10,000 channels)) did not further decrease TPT. Similar results were found when the entire A-chain was simulated (part B of Table [1](#T1){ref-type="table"}). The reader should be alerted to the limitations of the PSpice program. It was not possible to insert a second or third \"black-box\" in the basic excitable units (because the system went berserk). Therefore, the dynamic behavior of the cardiac cell membrane was only a close approximation. We previously found (unpublished observation) that the transverse velocity was greater when size of the model was increased (eg, 3 × 4, 5 × 5, 7 × 7) up to a presumed maximum, indicating that the boundary conditions affected behavior of the model. Therefore, the present experiments should be repeated on larger-sized models. But we believe that the qualitative findings would be much the same. In addition, 2-dimensional activation maps should be made in the future to better elucidate how the wavefront spreads. These findings might have important clinical implications, especially for the genesis of arrhythmias in pathophysiological situations. Any pathology that altered the number of functioning gj-channels, not only would affect longitudinal velocity of propagation, but also the transverse propagation ability and velocity. Therefore, the genesis of some arrhythmias, e.g., the reentrant type, could be promoted under such conditions. Acknowledgements ================ The authors thank Cara Stevens for typing the manuscript. The author also thanks Bijoy Kalloor, a graduate student in the Department of Electrical Engineering and Computer Science, assisted with the PSpice Program.	PubMed Central	2024-06-05T03:55:52.869747	2005-1-28	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549032/", "journal": "Biomed Eng Online. 2005 Jan 28; 4:7", "authors": [ { "first": "Nicholas", "last": "Sperelakis" }, { "first": "Lakshminarayanan", "last": "Ramasamy" } ] }
PMC549033	Background ========== Despite many attempts in the last few years to target cancer-specific antigens, a breakthrough in terms of clinical response has yet to be achieved mainly because of a scarcity of effective genuine cancer antigens, immunological evasion, or an immunosuppressive state. Melanoma-associated antigens are categorized as class I human leukocyte antigen (HLA)-restricted cancer/testis antigens \[[@B1]\] which are considered to be tolerable to the immune system because they are also expressed in normal tissues. However, malignant melanoma is the most well known cancer in which multiple tumor-specific antigens have been defined and utilized in vaccination strategies as peptide vaccines or peptide-pulsed DC vaccines \[[@B2]-[@B9]\]. From a clinical point of view, several vaccination strategies for stage IV melanoma using a combination of several (more than 3) peptides with a restriction to HLA-A2 have been reported to date \[[@B10],[@B11]\]. However, little immunotherapeutic study regarding HLA-A24-restricted multiple peptides has been conducted because HLA-A24 is not a common allele in Caucasians. Several studies have demonstrated the identification of many HLA-A24-restricted CTL epitopes from various cancer-related antigens including p53, CEA, telomerase, tyrosinase, MAGE proteins etc. \[[@B12]-[@B18]\]. When it comes to melanoma, our group demonstrated the feasibility of using a combination of 5 melanoma-associated peptides with restriction of HLA-A24 (peptide cocktail) as a specific cancer vaccine in an immunotherapeutical trial (Akiyama et al, Anticancer Res., 2004). Based on basic research results, a phase I clinical trial of HLA-A2 or A24-restricted melanoma peptide cocktail-pulsed dendritic cell-based immunotherapy has been performed. Here we describe the safety and efficacy of DC-based immunotherapy against metastatic melanoma. Materials and methods ===================== Patient characteristics and eligibility criteria ------------------------------------------------ Nine patients with metastatic melanoma were enrolled in a phase I clinical trial of a peptide cocktail-pulsed DC-based vaccine approved by the Institutional Review Board (No. 12--93 and 12--94) of the National Cancer Center, Tokyo. All patients gave written informed consent. All patients had received prior surgery, chemotherapy and radiation (Table [1](#T1){ref-type="table"}). Three subjects had metastatic lesions in the brain and been given radiation to control them. Inclusion criteria were: i) biopsy-proven stage IV metastatic melanoma, ii) age ≥ 18 years, iii) performance status ≤ 2, iv) HLA-A2 or A24 phenotype and v) measurable target lesions. Exclusion criteria were : i) prior therapy \< 4 weeks before trial entry, ii) untreated CNS lesion, iii) pregnancy, iv) autoimmune disease, and v) concurrent corticosteroid/immunosuppressive therapy. All the patients, who gave written informed consent, received subcutaneously (s.c) 3 DC vaccines at the inguinal region weekly and toxicity was checked. DCs were injected in dose-escalation design at a dose level per cohort of 1.0, 2,0 and 5.0 × 10^7^/body/shot (Table [1](#T1){ref-type="table"}). The injected DC number was calculated from the percentage of Lin^-^HLA-DR^+^gated populations in a FACS analysis. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Phase I study of DC-based therapy against melanoma ::: Patient No. Age Sex Previous therapy Measurable lesions DC injection (times) Side effect [DTH]{.underline} Response ----------------- --------- --------- ---------------------- ------------------------ -------------------------- ----------------- ----------------------- -------------- -------- 1 41 F ST, CT, RT, IFNβ lung, LN 1 × 10^7^(10) Hepatic (II) \- ++ PR 2 75 M ST, CT, IFNβ LN 1 × 10^7^(10) \- \+ \+ SD 3 49 F ST, CT, IFNβ, RT lung, liver 1 × 10^7^(3) \- \- \- (PD)\* 4 49 M ST, CT lung, liver 2 × 10^7^(6) \- \- \- PD 5 50 M ST, CT, IFNβ lung, liver, LN 2 × 10^7^(6) Hepatic (I) \- \- PD 6 69 M ST, CT, IFNβ LN 2 × 10^7^(10) \- \+ \+ CR 7 61 M ST, CT, RT liver, LN 5 × 10^7^(8) Hepatic (I) \+ ++ PD 8 64 F ST, CT, RT lung 5 × 10^7^(3) Fever (I) \- \- (PD) 9 66 F ST, CT, lung, LN 5 × 10^7^(3) \- \- \- (PD) \* The (PD) patients represent those who received fewer than 4 DC injections because of an early progression of the disease. ::: Preparation of DCs and peptides ------------------------------- Leukapheresis products from 7 L of processed blood were washed and centrifuged using density-adjusted OptiPrep™ (Axis-Shield PoC, Oslo, Norway), then the monocyte layer at the top was retrieved. Cells were transferred to an X-fold culture bag (Nexell, Irvine, CA) and cultured in the presence of GM-CSF at 50 ng/ml (CellGenix, Freiburg, Germany) and IL-4 at 50 ng/ml (CellGenix) in X-VIVO15 serum-free medium (Biowhittaker, Walkersville, MD). After 7 days, harvested cells were pulsed with a cocktail of 5 melanoma-specific synthetic peptides (25 μg/ml each) restricted to HLA A2 or A24 and KLH (25 μg/ml, Intracell, Frederick, MD). DC-enriched cells were washed and cryopreserved in Cryocyte bags (Baxter Healthcare Co., Deerfield, IL) until used. The purity of CD14^+^cells was evaluated with a flow cytometer (FACSCalibur, Becton-Dickinson Co., CA) before and after OptiPrep™ separation. The percentage of DCs was rated as the lin^-^HLA-DR^+^population (lineage antibodies including CD3, CD14, CD16, CD19, CD20, CD56 ; Becton-Dickinson Co.). The additional DC-related markers were determined on gated lin^-^HLA-DR^+^cells. The following peptides restricted to HLA-A2 or A24 were synthesized according to GMP standards by Multiple Peptide Systems, CA. HLA-A2: MART-1~27--35~(AAGIGILTV), gp100~209--217~(IMDQVPFSV), tyrosinase~368--376~(YMDGTMSQV), MAGE-2~157--166~(YLQLVFGIEV), MAGE-3~271--279~(FLWGPRALV) ; HLA-A24: gp100~152--160~(VWKTWGQYW), tyrosinase~206--214~(AFLPWHRLF), MAGE-1~135--143~(NYKHCFPEI), MAGE-2~156--164~(EYLQLVFGI), MAGE-3~195--203~(IMPKAGLLI). Characterization of tumor specimens before DC vaccines ------------------------------------------------------ Skin metastatic lesions were obtained from patients who gave written informed consent. The expression of melanoma tumor antigens was investigated using RT-PCR as described previously \[[@B19]\]. HLA protein expression was also evaluated using an immunohistochemical (IHC) analysis with anti-HLA-A2 or A24 monoclonal antibody (One Lambda Inc., Canoga Park, CA). A phenotypical analysis of lymphocytes infiltrating the tumor site was also performed using IHC. Clinical and immunological monitoring ------------------------------------- Adverse effects were evaluated according to the NCI Common toxicity criteria after 3 DC injections. Standard conventional definitions of major (complete or partial) objective responses were used. Stable disease (SD) was defined as less than a 25% change in size with no new lesions lasting at least 4 weeks. Clinical response was rated as maximal through the DC vaccinations. The patients received up to 10 injections on the condition that at least one measurable lesion showed more than stable disease (SD) response and/or an ELISPOT assay performed after 4 injections indicated a positive response for more than 1 melanoma-associated peptides. PBMC samples were harvested before and 29, 78, 134 and 190 days after the 1 st DC injection, and frozen prior to use for immunological monitoring tests. All patients were followed up for 2 years after the enrollment into the study. ELISPOT assay ------------- The ELISPOT assay was performed using in vitro re-stimulations. Briefly, PBMCs were incubated in a 24-well culture plate at 4 × 10^6^per ml and divided into non-adherent and adherent cells. Adherent cells were treated with a peptide cocktail and β2-microglobulin for 2 hrs, and co-cultured with non-adherent cells in the presence of IL-2 at 15 U/ml and IL-7 at 10 ng/ml. On day 7, non-adherent cells were re-stimulated with peptide-pulsed adherent cells. On day14, responder cells (1 × 10^4^/well) were incubated with peptide-pulsed target cells (1 × 10^5^/well; .221A201 cells for HLA-A2 peptide or TISI cells for HLA-A24 peptide) in a 96-well culture plate coated with anti-IFN-γ antibody (MABTECH AB, Nacka, Sweden) overnight. Finally positive spots stained with anti-IFN-γ antibody were measured using the KS ELISPOT system (Carl Zeiss AG, Overkochen, Germany). HLA-A2-restricted Influenza M1 peptide (GILGFVFTL) or HLA-A24-restricted EBNA3A peptide (RYSIFFDY) was used as a negative control. Tetramer staining ----------------- PBMCs were re-stimulated twice in vitro and utilized for tetramers staining. CD8^+^-enriched T cells were obtained by the depletion of CD4^+^T cells using Dynabeads M-450 CD4 (Dynal, Oslo, Norway) and used for tetramers staining. The staining was performed according to the method reported by Kuzushima et al \[[@B20]\]. The PE-labeled tetramers used in the present study were as follows: HLA-A\0201 MART1 (Beckman Coulter Inc., San Diego, CA), HLA-A\0201 gp100, HLA-A\2402 tyrosinase, HLA-A\2402 MAGE-1, HLA-A\2402 HIV (RYLRDQQLL) and HLA-A\0201 Influenza M1 tetramers (MBL, Nagoya, Japan). Intracellular cytokine staining ------------------------------- PBMCs were stimulated with 25 ng/ml of PMA (Sigma) and 1 μg/ml of ionomycin (Sigma) for 5 hrs in a 96-well culture plate. Breferdin A (10 μg/ml) was also added to cultures in the last hour. After the stimulation, cells were stained with FITC-anti-CD4 MoAb, and subsequently intracellular staining was performed with fix/permealization buffer and PE-labeled anti-IFN-γ or anti-IL-4 MoAb (Pharmingen, San Diego, CA). Finally, the ratio of Th1 (IFN-γ^+^) and Th2 (IL-4^+^) was calculated in PBMC samples obtained before and after DC vaccination. DTH reactions ------------- The HLA-A2 or A24 peptide cocktail solution diluted to a dose of 5 μg/ml (each peptide) and KLH (50 μg/ml) were injected intradermally on the patient\'s forearm and the redness and induration at the injection site was measured. PPD was used as a positive control. Statistical analysis -------------------- Statistical differences were analyzed using Student\'s paired two-tailed t-test. Values of p\< 0.05 were considered significant. Results ======= DC characterization ------------------- The mean percentage of DCs rated as lin^-^HLA-DR^+^in melanoma patients was 46.4 ± 15.6 %, not different from that in healthy volunteers (data not shown). The frequencies of the DC-related markers were determined on gated lin^-^HLA-DR^+^cells : HLA-class I 97.5 ± 0.9 %, CD80 87.6 ± 6.9 %, CD86 85.5 ± 7.4 %, CD1a 55.2 ± 24.2 %, CD83 29.9 ± 13.3 %, CCR7 32.4 ± 13.7 %, DC SIGN 78.2 ± 19.3 %, CD11c^+^HLA-DR^+^90.6 ± 6.0 %, CD123^+^HLR-DR^+^0.99 ± 1.3 %. Most of DCs expressed high level of co-stimulatory molecules and type1 phenotype (CD11c^+^HLA-DR^+^), while a moderate number of mature DCs with CD83 and CCR7 positive were contained in DC products. On the other hand, the T cell-stimulating activity of DCs investigated in the MLR assay using allogeneic T cells was as strong as that of DCs obtained from healthy volunteers (data not shown). Characterization of tumor specimen ---------------------------------- An analysis of melanoma antigen expression by RT-PCR was performed in 3 cases. The expression of more than 2 antigens in the tumor was verified in all cases. HLA protein expression was positive in 5 out of 9 cases (Table [2](#T2){ref-type="table"}). Patient 1 who showed a remarkable clinical response (PR), was representative of HLA protein-positive cases (Fig. [1](#F1){ref-type="fig"}). In contrast, in patient 7, HLA-A2 protein expression in the tumor was lost in the course of treatment. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Immunological monitoring in melanoma patients ::: Patient No. HLA Tumor antigen, HLA expression ELISPOT Tetramer Th1/Th2 balance ----------------- --------- ----------------------------------- ------------------ -------------------- --------------------- 1 A\2402 3/5(Tyr,M1,M2), A24(+) 3/5(Tyr,M1,M2) Tyrosinase (0.34%) 5.19 (1.45)^a^ 2 A\0201 A2(+) 2/5(MART1,gp100) MART1 (0.64%) 3.68 (1.49) 3 A\2402 A24(-) N. D^b^ N. D. \- 4 A\0206 A2(-) 2/5(MART1, M2) \- 3.05 (2.57) 5 A\0201 A2(-) 2/5(MART1, M2) MART1 (1.48%) 2.83 (3.68) 6 A\2402 2/5(M2,M3), A24(+) 2/5(M2, M3) \- 3.76 (2.00) 7 A\0201 4/5(MART1,Tyr,gp100,M2), A2(+) 2/5(gp100, M2) \- 2.64 (1.79) 8 A\2402 A24(-) N. D. N. D. \- 9 A\0201 A2(+) 1/5(gp100)^c^ N. D. N. D. ^a^The value in the parenthesis shows Th1/Th2 ratio prior to DC vaccination. ^b^N. D. ; not done. ^c^The value shows the one obtained prior to DC vaccines. ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Immunohistochemical analysis of metastatic tumor tissue from responder patient 1 and non-responder patient 7. A; H-E stain and B; anti-HLA-A24 MoAb from patient 1. C; anti-HLA-A2 MoAb before DC vaccination and D; anti-HLA-A2 MoAb after 4 DC injections from patient 7. Magnification × 200. ::: ![](1479-5876-3-4-1) ::: ELISPOT assay ------------- CTL precursors of more than 2 melanoma peptides were recognized after DC vaccines in 6 of 9 cases. Two HLA-A2^+^cases (patients 5 and 9) showed HLA-A2 peptide-specific CTL responses before the vaccination. Patients 1 and 6, which showed remarkable clinical responses, exhibited many CTL precursors against a HLA-24 restricted peptide-cocktail (Table [3](#T3){ref-type="table"}, Figure [2](#F2){ref-type="fig"}). Notably, in patient 1, a remarkable increase in the CTL response to the HLA-A24 peptide cocktail was seen in accordance with the regression of metastatic tumor of the lung (Fig. [3](#F3){ref-type="fig"}). On the other hand, patient 7 also demonstrated a high CTL precursor frequency, but showed no significant clinical response. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Peptide cocktail-specific CTL precursor frequency during DC vaccination ::: Spot No./CD8^+^T cell (%)^a^* --- --------------- -------------------------------------- ----------- ----------- ----------- ----------- 1 1 × 10^7^(10) 1.19/0.45 6.96/0.06 8.82/0.63 8.81/0.08 5.4/0.08 2 1 × 10^7^(10) 0.07/0.05 0.07/0.2 0.02/0 0.02/0 0.29/0.03 3 1 × 10^7^(3) N.D.^b^ N.A.^c^ N.A. N.A. N.A. 4 2 × 10^7^(6) 0.39/0.53 1.29/0.03 1.12/0 N.A. N.A. 5 2 × 10^7^(6) 1.74/0.05 0.51/0.2 1.25/0.04 N.A N.A. 6 2 × 10^7^(10) 0.21/0.27 0.31/0.28 1.18/0.24 7.80/0.19 9.82/0.30 7 5 × 10^7^(8) 0.62/0.20 6.52/0.1 7.33/0.11 N.A. N.A. 8 5 × 10^7^(3) N.D. N.A. N.A. N.A. N.A. 9 5 × 10^7^(3) 3.09/1.24 N.D. N.A. N.A. N.A. The percentages represent IFN-γ-positive spot No. divided by total CD8^+^cell No. from 1 × 10^4^PBMCs. ^a^Each value represents the percentage with peptide cocktail/without peptide cocktail. ^b^N.D. ; not done, ^c^N.A. ; sample not available. ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### CTL responses in the course of DC injections in 6 evaluable cases. Patients 1, 2 and 6 were responders and patients 4, 5 and 7 were non-responders. Responders (cases 1,6) showed remarkable CTL expansion in PBLs compared with before DC vaccination. In contrast, non-responders (patients 4,5) showed no significant CTL responses except in patient 7. ::: ![](1479-5876-3-4-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Impact of DC vaccines on metastatic lesions of the lung in responder patient 1. Upper and lower panels show a lung and hilar lymph node metastatic lesion (arrow), respectively. The CT scan was made before therapy and after 4, 7 and 10 DC vaccinations. ::: ![](1479-5876-3-4-3) ::: Tetramer staining ----------------- After CD4^+^T cell depletion, the frequency of CD8^+^cells was more than 85%. The proportion of PE-labeled tyrosinase-HLA-A24 tetramer-positive cells among gated CD8^+^cells was 0.34% in patient 1 (Table [2](#T2){ref-type="table"}). HIV-A24 tetramer (negative control)-positive cells were not detected. The percentage of PE-labeled MART1-HLA-A2 tetramer-positive cells was 0.64% and 1.48% in patients 2 and 5, respectively. On the other hand, that of Influenza M1-HLA-A2 tetramer (negative control)-positive cells was 0.04%. Th1 and Th2 balance after DC vaccination ---------------------------------------- In 5 of 6 evaluable cases, the balance of Th1 and Th2 shifted more to Th1 after 4 DC injections compared with prior to vaccination. (Table [2](#T2){ref-type="table"}). The amplitude of the shift seemed to be larger in clinical responders (patients 1, 2, 6) than non-responders (patients 4, 5, 7) (% of ratio increase; 264 ± 86 vs. 114 ± 35). DTH --- Three of 6 evaluable cases showed positive DTH to a peptide-cocktail after DC injections (Table [1](#T1){ref-type="table"}). On the other hand, 4 of 6 cases developed a DTH response to KLH protein. There were stronger reactions to KLH in patients 1 and 7. Adverse effects of DC vaccine ----------------------------- Safety was assessed after 3 DC injections in all 9 cases. Three of 9 patients developed mild hepatic dysfunction (grade I-II), however it was only transient and disappeared in spite of the continuance of DC injections. Rheumatoid factor and anti-nuclear antibody were negative before the injection, but increased to 1:160 and 1:40, respectively after the injections finished in patient 1. No clinical symptoms of autoimmune disease were found in patient 1 (Table [1](#T1){ref-type="table"}). Clinical response ----------------- Clinical response was rated as maximal through the DC vaccinations. In 6 evaluable cases except for 3 cases of early PD cases due to a rapid progression of the disease, 1CR (patient 6), 1PR (patient 1), 1SD and 3 PD were obtained (Table [1](#T1){ref-type="table"}). Large metastatic lesions in the lung and hilar nodes in patient 1 dramatically decreased in size after 4 DC injections, and almost disappeared after treatment finished (Fig. [3](#F3){ref-type="fig"}). Moderate sized cervical metastatic lesions in patient 6 finally started to decrease after 8 DC injections and disappeared surprisingly rapidly after the finish of DC therapy. In contrast, patient 7 who exhibited good immunological responses in the ELISPOT assay and DTH, showed no shrinkage of the tumor, resulting in cessation after 6 DC injections. Characterization of infiltrated lymphocytes in the tumor -------------------------------------------------------- IHC analysis of infiltrated lymphocytes in the tumor after DC vaccines was performed only in patient 1 and 7. The obvious infiltration of a larger number of CD4^+^or CD8^+^T cells and a small number of CD20^+^B cells were shown in patient 1 (Fig. [4](#F4){ref-type="fig"}). In contrast, no significant cell infiltration was seen in patient 7 who did not develop any therapeutical effect on the tumor (data not shown). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Phenotype analysis of lymphocytes infiltrating the tumor site in responder patient 1. Obvious infiltration of a larger number of CD4^+^or CD8^+^T cells and a small number of CD20^+^B cells is shown. Indirect staining using anti-CD4, CD8, CD20 or CD56 MoAb as primary Ab and goat anti-mouse Ab as secondary Ab was performed. Magnification × 200. ::: ![](1479-5876-3-4-4) ::: Discussion ========== Clinical trials of specific immunotherapy against metastatic melanoma using peptide-pulsed Mo-derived DCs have been performed in mainly Western countries, and some fruitful results were obtained \[[@B7],[@B10],[@B11]\]. In those cases, most of the patients belonged to the HLA-A\0201 type. In the present study, we investigated the effect of peptide-pulsed DCs on 4 cases of HLA-A\2402^+^metastatic melanoma patients besides 4 cases of HLA-A\0201^+^patients in a clinical phase I trial. This is the first report to demonstrate that peptide-pulsed DCs were effective in some HLA-A24^+^melanoma patents in Japan. It is well known that HLA-A\2402 is a common genotype and around 60% positive in Asians. There was one case of HLA-A\0206 patient among 5 HLA-A2^+^patients (Table [2](#T2){ref-type="table"}). Sidney et al. \[[@B21]\] demonstrated that over 70% of the peptides that bound A0201 with high affinity were found to bind at least two other supertype molecules like A\0202, A\0203 or A\0206. Taking it into considerations, the HLA-A\0206 patient was finally enrolled into the study. With regard to other HLA-A24^+^solid cancers, stomach, colon and bladder cancers have been treated with peptide (MAGE-3)-pulsed DC vaccines, and showed a limited response \[[@B22]-[@B24]\]. Considering that melanoma is highly immunogenic and probably a good model for tumor-specific immunotherapy despite being an unusual tumor in Asian countries, it deserves a phase I study using peptide-pulsed DCs. In our study, peptide cocktails combining 5 peptides for each HLA type (HLA-A2 or A24) were prepared and used for DC pulsing. Our clinical study revealed positive ELISPOT responses against more than 2 peptides in all 6 evaluable cases. In previous reports, clinical DC therapy using more than 3 melanoma peptides demonstrated the induction of a specific CTL response against multiple melanoma peptides \[[@B10],[@B11]\]. However,, there is still some controversy over the efficacy of multiple epitope-based vaccinations and Smith et al.*\[[@B25]\] demonstrated that, although polyepitope vaccines are an effective way of priming polyvalent CTLs, continual stimulation with polyepitope vaccines might restrict CTL induction as a result of immunodominance. The results of our study are thought to answer that question, but testing of the peptide cocktail vaccine in more patients will be needed. To refine the quality and protocol of the tumor-specific immunotherapy for clinical trials, the prediction of clinical response in an individual is important \[[@B26]\] and should be discussed. In our study, the correlation between immunological parameters and clinical response was investigated in a limited number of cases. First of all, as to HLA expression in the tumor, patients 1, 2, 6 and 7 were positive, and patients 4 and 5 were negative. HLA-negative cases showed a progression of the tumor. Even in positive cases, patient 7 turned negative in the course of DC therapy, showing tumor progression. Loss of HLA expression in melanoma is reported to be a complex phenomenon associated with melanoma antigen loss \[[@B27]\], β2-microglobulin gene mutation \[[@B28]\] or loss of heterozygosity (LOH) in chromosome 6 and may lead to tumor progression and metastasis. As to patient 7, considering that the melanoma antigen expression was maintained, the functional expression of β2-microglobulin should be investigated. All the other HLA-positive cases showed CR, PR and SD, respectively. There was a tendency for HLA expression to be associated with tumor response, and some researchers reported a positive correlation of HLA-expression to tumor response in immunotherapy against melanoma. However, despite the positive correlation of HLA-expression in the tumor with anti-tumor response, Nestle et al. demonstrated that HLA-expression in the tumor did not correlate to survival in melanoma patients \[[@B29]\]. Second, the amplitude of the CTL response in the ELISPOT assay seems to be another key factor predicting anti-tumor response. Patients 1, 6 and 7 showed large responses to peptide cocktail in ELISPOT, and patients 2, 4 and 5 showed small responses. The former exhibited a remarkable regression of tumor except patient 7. On the other hand, the latter showed a poor response. There was a likely tendency that the amplitude of the CTL response was associated with tumor regression. Also, it was difficult to predict when immunological responses like CTL induction start to be activated in vivo during DC vaccination, and this question needs to be answered. In the present study, because of a limited number of patients given DC vaccines, the tendency that HLA-class I protein expression in the tumor and the amplitude of ELISPOT responses are seemingly associated with tumor regression is not convincing. Finally, in order to improve tumor response in the present study, there are still some issues regarding clinical DC preparation. First of all, the purity of CD14^+^cells after Opti-prep separation is still low and may not be reproducible. Therefore, other clinical grade-monocyte separation methods using an elutriator or negative selection with CD2 and CD19 MoAbs \[[@B30]\] should be tried. Second, considering that the amplitude of the CTL response was associated with tumor regression, and that even a remarkable increase of CTL frequency inevitably diminished in spite of the repetition of DC vaccinations, it seems to be crucial to maintain increased CTL frequency in blood leading to TIL in the tumor and expand more than enough to develop a substantial number of memory CD8^+^CTL in lymph nodes. Such a novel method will be needed to develop an effective cancer vaccine. Conclusions =========== In the present study, we investigated the effect of dendritic cell (DC)-based immunotherapy on metastatic melanoma patients with HLA-A2 or A24 genotype. Nine cases of metastatic melanoma were enrolled into a phase I study using HLA-A2 or A24-restricted peptide cocktail-pulsed DCs. All 6 evaluable cases showed positive immunological responses to more than 2 melanoma peptides in an ELISPOT assay. Clinical response through DC injections was as follows : 1CR, 1 PR, 1SD and 6 PD. All 59 DC injections in the phase I study were safely administered to patients. These results suggested that peptide cocktail-treated DC-based immunotherapy had the potential for utilizing as one of therapeutic tools against HLA-A2 or A24^+^metastatic melanoma. Abbreviations ============= DC, dendritic cell ; HLA, human leukocyte antigen ; GM-CSF. granulocyte macrophage-colony-stimulating factor ; IL, interleukin ; KLH, Keyhole limpet hemocyanin ; CTL, cytotoxic T cell ; DTH, delayed-type hypersensitivity ; CR, complete remission : PR, partial remission ; SD, stable disease ; PD, progressive disease ; RT-PCR, reverse transcription-polymerase chain reaction ; IFN, interferon ; PBMC, peripheral blood mononuclear cell. Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= YA participated in the design of the study and drafting the manuscript and were responsible for completing the study. RT, NI, MS, YH carried out apheresis and cell processing and were responsible for DC production. AY and NY were responsible for the clinical side of the study. IK, IN, KT and KM participated in the design of the study and performed biological assays. YT and KY reviewed the manuscript. All authors read and approved the final manuscript. Acknowledgements ================ We would like to thank Ms. Takikawa, Mr. Kuromi and Ms. Kajimura for their excellent technical assistance. This work was partly supported by Grants in Aid from the Ministry of Health, Labour and Welfare for Cancer Research (9--32 and 10--28) and the Second-Term Comprehensive 10-year Strategy of Cancer Control.	PubMed Central	2024-06-05T03:55:52.872458	2005-1-28	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549033/", "journal": "J Transl Med. 2005 Jan 28; 3:4", "authors": [ { "first": "Yasuto", "last": "Akiyama" }, { "first": "Ryuji", "last": "Tanosaki" }, { "first": "Naoki", "last": "Inoue" }, { "first": "Makiko", "last": "Shimada" }, { "first": "Yukie", "last": "Hotate" }, { "first": "Akifumi", "last": "Yamamoto" }, { "first": "Naoya", "last": "Yamazaki" }, { "first": "Ichiro", "last": "Kawashima" }, { "first": "Ikuei", "last": "Nukaya" }, { "first": "Kazutoh", "last": "Takesako" }, { "first": "Kouji", "last": "Maruyama" }, { "first": "Yoichi", "last": "Takaue" }, { "first": "Ken", "last": "Yamaguchi" } ] }
PMC549034	Background ========== Approximately one to three per 1000 children are born with at least moderate, bilateral hearing disorders \[[@B1]-[@B4]\]. Children with congenital hearing impairment benefit from early detection and treatment of their hearing loss \[[@B5]\]. The neurological development of hearing abilities requires acoustic stimulation in the first 18 months of life. Deficits due to lack of acoustic stimulation within the first two years are not or not easily recovered by later rehabilitation. The consequences include delayed development of speech and other cognitive and social functions. This delay is already measurable in the first 3 years of life \[[@B6]\]. If disorders are detected and treated in time, either by the use of a hearing device or cochlea implant, most of the children develop normally and do not need additional speech therapy \[[@B7],[@B8]\]. Early diagnosis and treatment within the sensitive time frames are therefore essential. The German consensus conference on neonatal hearing screening proposed diagnosis in the first 3 months and the start of treatment in the first 6 months of life. Various tests and test combinations with acceptable sensitivity and specificity are available. Transient evoked oto-acoustic emissions (TEOAE) or brainstem evoked responses (BERA) can be measured. One common strategy consists of a two-step TEOAE with the first measurement within the first days of life and a second test a few days later \[[@B9]\] (chapter 2.2.7). While modern screening techniques for early detection are available there is still a gap between the consensus on detection as early as possible and the current situation. Only 50% to 60% of children with permanent hearing impairment are diagnosed before their second birthday with traditional health care services \[[@B2]\]. While congenital hearing loss is a serious health problem, there is little evidence to support the use of routine universal screening because of the following factors: • as the prevalence is very low, the positive predictive value of the tests is low • screening technologies are still in development • possible costs and consequences are not sufficiently known • benefits of early intervention are frequently expressed in qualitative terms without presenting unbiased measures of outcome. In 1995 the US Preventive Services Task Force found insufficient evidence in favour of universal neonatal hearing screening (UNHS) \[[@B10]\]. The Task Force proposed selective screening of new-borns with risk factors to improve the predictive value of the test. In the UK a national neonatal hearing screening programme aimed at detecting bilateral moderate to severe hearing impairments has been recommended \[[@B11]\] and partially implemented within a pilot project \[[@B12]\]. Several studies have modelled the outcomes of UNHS versus risk factor screening \[[@B13]-[@B15]\]. Keren et al \[[@B16]\] presented a cost-effectiveness analysis based on a decision tree model which reported short-term effectiveness of UNHS compared to risk factor screening. However, there is presently no model explicitly quantifying the effectiveness of UNHS versus other programme alternatives in terms of early diagnosis, nor has it been taken into account that diagnosis of hearing impairment within the first few months of life is more \"valuable\" for the child\'s further development than diagnosis later in life. Children identified within the developmentally sensitive time frame should therefore be given more weight compared to children with delayed diagnosis. The objective of this clinical decision analysis was to systematically compare two screening strategies for the early detection of new-born hearing disorders: UNHS and risk factor screening; with the option of no systematic screening regarding their influence on early diagnosis. Our specific objectives were to show differences between strategies expressed as the number of quality weighed detected child months (QCM), the number of true positive cases at the age of 6 and 12 months, and the number of false positive cases. We also wanted to investigate which parameters had the most influence on the reported differences and how likely these differences were. Methods ======= We developed a clinical decision model to compare two different screening strategies with the option of no systematic screening. Parameters were extracted from the literature, empirically derived from a representative patient survey, and estimated by experts. Univariate and multivariate sensitivity analyses were performed on all relevant parameters. The decision model was used to predict absolute and incremental effectiveness of two new-born hearing strategies compared with the option of no screening in new-born infants. For the modelling of effectiveness the recommendations of the Panel on Cost-Effectiveness in Health and Medicine were followed \[[@B17]\]. Three possible strategies for neonatal hearing screening (NHS) were evaluated: \- Universal neonatal hearing screening (UNHS): Every hospital-born baby is screened during the first days of life. \- Risk factor screening (RS): The prevalence of hearing disorders is estimated to be higher at risk groups such as children with a positive family history, with congenital infections, cranofacial abnormalities, low APGAR-Score or low birth weight. This strategy screens all children with one or more risk factors for hearing disorders. \- No systematic screening (NS): Children undergo the usual distraction test when presented to the paediatric service during the routine visit at the age of 12 weeks. Some neonates are screened in the hospital, but not in a systematic way. This reflects the present situation in Germany. UNHS is performed in some maternity hospitals and outpatient paediatric services with pilot screening projects in several regions. The target population of this analysis was all newborn infants. Health effects are expressed as quality weighed number of detected child months (QCM), and as true positive and false positive cases at certain developmentally important ages (6 and 12 months); for example, if a hearing impairment was diagnosed briefly after birth, the infant contributed six QCM at the age of six months. If the infant\'s hearing loss was diagnosed at the age of five months, the infant added only one detected child month at the age of six months. The term \'quality\' is to reflect the idea that the early detection of impairment is a better and desired outcome, although there is no data on quality of life gained by this early detection. QCM, true positives and false positives are reported at the age of 6 and 12 months and with a time horizon of 120 months. Child months which were added up until the age of 6 months were multiplied with a weight of 1, child months added after the age of 6 months were multiplied with decreasing weighting. The derivation of this weight index is described in the section \'data and assumptions\' below. We assumed that all children with hearing impairment would be detected before the age of 72 months, the age of school entry, regardless of the kind of screening strategy. In order to give outcomes in the present more weighting, compared to outcomes in the future, future effects have been discounted at an annual rate of 3%. Discounting reflects the higher value of money spent now as opposed to in the future. Similarly, discounting also weights outcomes experienced now (e.g., being diagnosed as true positive) more heavily than those experienced in the future. Table [1](#T1){ref-type="table"} gives the model parameters and their references. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Model parameters ::: Parameter Base case (in %) Range for sensitivity analysis Source ------------------------------------------------------------------- -------------------------------------------------------- -------------------------------- ------------------------------------------------------ Prevalence of new-born hearing impairment 0.15 0.09--0.3 \[3\], \[28\], \[29\], \[4\], \[30\], \[31\], \[25\] Prevalence of one or more risk factors for hearing impairment 20 \- \[2, 25, 32\] Prevalence of hearing impairment \- In children with risk factors 0.38 \- Author\'s calculation, \[33\] In children without risk factors 0.09 \- Author\'s calculation Prevalence of risk factors in children with hearing impairment 50 48--56 \[1, 28, 34\] Sensitivity of screening 96 96--100 \[11, 32, 35\] Specificity of screening 89 77--96 \[11, 32, 35\] Sensitivity of diagnostic testing 98 \- Specificity of diagnostic testing 98 \- Coverage of screening 90 85--95 Author\'s estimate Follow-up after screening 80 75--85 Author\'s estimate Healthy children under suspicion of hearing impairment 0.1 \- Author\'s estimate Discounting factor 3 per year 0--5 Weighs for quality adjustment Time to diagnosis ≤ 6 months of age 1 \- Experts\'estimate Time to diagnosis \> 12 months of age 0.875 Time to diagnosis \> 6 months and ≤ 12 months linear extrapolation Probability of \"natural\" discovery without systematic screening Weibull Distribution Median age at diagnosis 18 months \- Empirical data \) \) author\'s calculations, derived from a representative survey, covering all diagnosed cases and the age of diagnosis in Upper Bavaria in 1998 and 1999 \[20\] ::: The decision model ------------------ We developed a state-transition (Markov) model \[[@B18]\] with monthly cycles to reflect the course of disease and diagnosis under the three screening strategies (figure [1](#F1){ref-type="fig"}). Probabilistic modelling has been performed by Monte Carlo simulations. The following health states were possible: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Health states framework of the Markov model. Arrows indicate the possible transitions.\"Unknown status\" is the initial state, \"True Positive\" and \"True Negative\" are final (absorbing) states. ::: ![](1471-2458-5-12-1) ::: \- Unknown status \- Healthy (hearing) confirmed by diagnostic test and/or screening -- true negative \- Healthy (hearing) not confirmed by diagnostic test \- Hearing impaired confirmed by diagnostic test and/or screening -- true positive \- Thought to be healthy (hearing) but hearing impaired -- false negative \- Thought to be hearing impaired but healthy (hearing) -- false positive \- Not compliant/not followed up The baseline cohort consisted of infants with a certain prevalence of hearing impairment but unknown status regarding this disorder. Screened and impaired children are detected with the sensitivity of the screening test, whereas screened and healthy children are classified as healthy with the specificity of the screening test. All children with a positive screening test (i.e., true positives and false positives) undergo a second confirmatory diagnostic test, unless they did not adhere to the screening or did not present for the following tests (lost to follow-up). Impaired children who have not been screened in the first cycle can be diagnosed in the subsequent cycles according to the \"natural history\" of diagnosis, that is, because they don\'t develop speech adequately or become apparent during the routine visits to the paediatrician. In each cycle children can move to other health states according to the transition probabilities. QCM are only attributed to impaired children in whom hearing impairment is detected. Ultimately, all impaired children of the model cohort are diagnosed as impaired and all healthy children are either classified as healthy or remain unclassified. Data Professional (TreeAge Inc., Williamstown, MA) was used to construct and run the Markov model and Excel for Windows (Microsoft Corp.) was used to validate the model and to perform the Monte Carlo simulations. Data and assumptions -------------------- A pre-defined and externally reviewed literature search was performed on new-born hearing screening using all relevant electronic databases. Search strategy and methods have previously been reported in detail \[[@B9]\]. In brief, we searched 13 medical databases including MEDLINE, EMBASE, Current Contents for published papers and HTA databases for published HTA reviews. Relevant articles were identified by a combined text word and thesaurus search. The references of the retrieved articles were then checked for further relevant articles. We restricted our search to publication dates from 1990 to September 2001. Reviewed publications were scored for study quality according to a standardised questionnaire developed by the German Scientific Working Group Technology Assessment for Health Care \[[@B19]\] and then either included or excluded. All model parameters are shown in Table [1](#T1){ref-type="table"}. A two-step screening strategy of Transient Evoked Oto-Acoustic Emissions (TEOAE) was chosen as the model for screening strategies, as this is one of the most widespread and commonly used technologies \[[@B9]\]chapter 2.2.7. The prevalence of congenital hearing disorders in children with risk factors was calculated using the prevalence of children born with one or more risk factors (20%) and the prevalence of risk factors in children with congenital hearing disorders (50%) using Bayes theorem. The probability of presentation with a falsely suspected hearing disorder in hearing children was estimated by a panel of four clinical experts. The probability of detection at a certain age without screening was calculated from a representative survey, covering all diagnosed cases and the age of diagnosis in Upper Bavaria in 1998 and 1999 \[[@B20]\]. A Weibull function for the probability to diagnosis, was fitted to the empirical data. The slope of the weight function has been previously estimated by experts making the following assumptions: each month detected before the age of 6 months is weighted with 1, assuming that children detected (and treated) within the first 6 months of life can develop normal speech and language abilities. If not detected within the first 12 months, profoundly and severely impaired children will conclude with a weight of 0.85, and moderately impaired children with a weight of 0.90. Presuming that 50% of the children with permanent congenital hearing disorders are moderately impaired, gives a weight of 0.875 for every month which is detected after the first birthday. The weights between 6 and 12 months were extrapolated in a linear fashion. Model assumptions ----------------- Screening procedures and diagnostic procedures are based on different biological and clinical testing principles. We therefore assumed conditional independence of screening procedures and subsequent diagnostic procedures. Sensitivity analysis -------------------- In order to investigate the influence of the parameter estimates on the outcome measures, one-way sensitivity analyses were performed on all relevant parameters. Ranges used for sensitivity analyses were derived from literature searches and are shown in table [1](#T1){ref-type="table"}. Multi-way probabilistic sensitivity analysis was performed on prevalence, sensitivity, specificity, coverage and follow-up using the Monte Carlo technique with 1,000 trials. Point estimates and 95% confidence limits were obtained by counting the number of trials in which a certain strategy has previously been found to be superior to the other strategies \[[@B21]\]. As we assumed that UNHS will always yield more QCM than RS or NS, we also calculated these confidence limits as a function of the difference between strategy effectiveness. This function results in a curve showing the cumulative relative frequency of trials (vertical axis) yielding a certain difference in QCM (horizontal axis) between two alternative strategies. The relative frequency gives an estimate of the probability of a certain difference in clinical effectiveness. The ranges for probability estimates, derived from the literature, assumed beta distribution. Model validation ---------------- The decision model was validated on three levels: \(i) Technical validation: The model was tested independently with two different software packages (TreeAge Data Professional and MS Excel). Routine tests (e.g., replacing the Weibull function by a constant, setting screening probability equal for all strategies) yielded the expected results. \(ii) Internal validation: All data used to derive model parameter values, were reproduced exactly by the model (e.g., number of detected children at model end point). \(iii) External validation: The derived values are consistent with external projections and estimates of recently published studies which were not used in our model \[[@B16],[@B22]\]. for this section. Results ======= Base-case analysis ------------------ Table [2](#T2){ref-type="table"} presents the results of the base-case analysis. With the base-case prevalence of 150 per 100,000 in a hypothetical cohort of 100,000 children a maximum of 900 QCM would have been accumulated at the age of 6 months, 1800 at the age of 12 months, 18,000 at the age of 120 months (discounted: 892, 1771, 15503), if all children born with hearing impairment had been discovered at birth. UNHS discovered 644 weighted child months before the age of 6 months (72,2% of the expected value). RS yielded 393 child months (44,1%), no systematic screening 152 child months (17,0%). UNHS yielded 74,3% and 86,7% weighted child months at 12 and 120 months respectively, RS 48,4% and 73,3%, NS 23,7% and 60,6%. At the age of 6 months UNHS identified approximately 75% of all children born with hearing impairment, RS 50% and NS 25%. At the time of screening UNHS marked 10% of screened healthy children for further testing (false positives), RS 2%. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Results of modelling, base case assumption, for a hypothetical cohort of 100,000 children (QCM discounted at an annual 3%) ::: Alternative strategies Expected value ---------------------------- ------------------------ ------ ------- ------ ------ ------ ---------------- \% \% \% QCM at 6 months 644 72.2 393 44.1 152 17.0 892 QCM at 12 months 1315 74.3 858 48.4 420 23.7 1771 QCM at 120 months 13436 86.7 11367 73.3 9394 60.6 15503 TP at 6 months 112 74.7 74 49,3 38 25.3 150 Incremental TP at 6 months 38 36 \- TP at 120 months 150 150 150 150 FP after screening 9885 1973 \- \- UNHS = universal neonatal hearing screening RS = risk factor screening NS = no systematic screening QCM = quality weighed detected child months TP = true positives FP = false positives. ::: Sensitivity analyses -------------------- Results of sensitivity analyses are presented in table [3](#T3){ref-type="table"}. All sensitivity analyses are reported for 120 months follow up. Resulting QCM strongly depended on the prevalence of hearing disorders. Very low prevalence decreased the incremental benefit of UNHS versus RS. Comparing UNHS vs. RS, a prevalence of 9 per 1000 children yielded a gain of 1241 QCM, a prevalence of 15 per 1000 yielded a gain of 2027 QCM. The results were insensitive to varying assumptions about test parameters and the proportion of children lost to follow up. A decrease in slope of the linear weighting function resulted in decreasing incremental QCM. If detected child months were not weighted according to time of diagnosis, UNHS would still be superior to RS and RS to NS in terms of detected child months (data not shown). Figure [2](#F2){ref-type="fig"} shows the frequency distributions of QCM per strategy as a result of multi-way probabilistic sensitivity analysis. QCM was higher in UNHS compared to RS or NS in 100% of the 1000 performed trials. Figure [3](#F3){ref-type="fig"} shows the cumulative probability of obtaining a given fixed incremental value of QCM. With a probability of 95%, UNHS resulted in a gain of at least 1200 QCM compared with RS and a gain of at least 2500 QCM compared with NS. A gain of 1200 QCM means, for example, that 200 impaired children would be positively diagnosed at birth instead of at the age of 6 months, or that 1200 impaired children would be positively diagnosed before the age of 5 months instead of before the age of 6 months etc. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### One-way sensitivity analyses for QCM at 120 months. The model was evaluated with a range of different values for one parameter while the other parameters were held constant. The ranges of the parameter values are given in table 1 ::: Parameter Strategy Lower estimate Upper estimate ------------------------------------------------- ---------- ---------------- ---------------- Prevalence UNHS 8061 26872 RS 6820 22733 NS 5636 18787 Sensitivity of screening UNHS 13436 13608 RS 11367 11453 NS 9394 9394 Specificity of screening UNHS 13436 13436 RS 11367 11367 NS 9394 9394 Prevalence of risk factors in impaired children UNHS 13436 13436 RS 11284 11615 NS 9394 9394 Coverage of screening UNHS 13158 13714 RS 11204 11530 NS 9394 9394 Follow up after screening UNHS 13177 13694 RS 11237 11496 NS 9394 9394 Discounting factor UNHS 15725 12124 RS 13447 10180 NS 11276 8327 UNHS = universal neonatal hearing screening RS = risk factor screening NS = no systematic screening QCM = quality weighed detected child months ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Distributions of quality weighted detected child months (QCM, 100,000 screened children, 120 months) for newborn hearing screening strategies evaluated by Monte Carlo simulation (UNHS = Universal Newborn Hearing Screening).For example, if a hearing impairment was diagnosed briefly after birth, the infant contributed six QCM at the age of six months. If the infant\'s hearing loss was diagnosed at the age of five months, the infant added only one detected child month at the age of six months. ::: ![](1471-2458-5-12-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Probability of a fixed level of incremental quality weighed detected child months (QCM) between strategies. These graphs are the results of a multi-way sensitivity analysis where all model parameters were varied simultaneously within the ranges described in table 1. They give the probability that the incremental gain of QCM between two screening strategies exceeds a certain value (given on the horizontal axis).This reads as follows: With a probability of 95% the difference between UNHS and RS will be 1200 QCM or more. Results of 1000 trials of a Monte Carlo simulation with a time horizon of 120 months. (UNHS = Universal Newborn Hearing Screening, RS = Risk Screening, NS = No Screening). ::: ![](1471-2458-5-12-3) ::: Discussion ========== We developed a decision-analytic Markov model for the evaluation of the effectiveness of different new-born hearing screening strategies. In our model, UNHS identified 72% of all detectable QCM at the age of 6 months, RS identified 44% QCM, the control group with no systematic screening identified 17% QCM. UNHS shows higher effectiveness even under a wide range of additional relevant parameters. QCM was introduced as a dynamic time-to-event measure which takes into account that the age of confirmation of congenital hearing impairments is important for further language development. The model results were not sensitive to test the accuracy of parameters within the assumed range but varied with the prevalence of hearing impairment. We have shown that UNHS leads to an earlier age of confirmed diagnosis compared to selective RS. The cost effectiveness of UNHS has already been modelled along secondary data \[[@B16],[@B23],[@B24]\]. This model is the first to establish a time-dependent and quality-weighted outcome, to introduce empirical data of the natural history of discovery and to present the results within a probabilistic framework. The strength of the presented model is that detected child months are multiplied by a weighted function which adds more benefit per month to children that were diagnosed before 6 months compared to those with late detection. In the existing literature the most interesting outcome, the proportion of children detected early enough, has not been modelled \[[@B16]\]. Our findings are consistent with study results and other projections of effectiveness. The Wessex Universal Neonatal Hearing Screening Trial Group found that 71 more babies with moderate or severe hearing loss per 100 000 target population were diagnosed before the age of 6 months during periods with neonatal screening than during periods without \[[@B25]\]. We found that UNHS would yield 112 true positive cases per 100 000 as compared to 38 without systematic screening, which is a difference of 74 babies. In our model UNHS identified 28% more children in time compared to RS. Thompson et al. estimated this difference to be between 19% and 42% \[[@B22]\] and stated that 77% of hearing impaired children would be identified before 10 months. Keren et al. assumed that UNHS detects 77% and RS 52% of hearing impaired children at the age of 6 months \[[@B16]\]. Any differences may be due to the fact that our function of discovery without systematic screening is more pessimistic. Our study has several limitations. Firstly, it does not differentiate between bilateral and unilateral hearing loss or moderate and profound impairment. We believe, however, that even with a more refined model the differences in effectiveness would not be substantial. Variations in the degree of impairment would result in variations in sensitivity of the test, and the model was rather insensitive to changes of test parameters. Secondly, even though QCM has been weighted, it is a surrogate parameter for the actual burden of disease for the child. Preference-based utilities have not been measured and the weighting for the impact of early or late identification of hearing loss used in our analysis were estimated by experts. Sensitivity analyses, however, revealed that without weighing, the difference in effectiveness would still be substantial. Thirdly, effectiveness was measured as a function of time to diagnosis. In routine health care, adequate treatment does not necessarily start immediately after the diagnosis is made. However, as knowledge on the consequences of delayed intervention is limited, including time to intervention as another variable in the model would have resulted in decreased precision \[[@B22]\]. Fourthly, our empirical data do not differentiate between congenital and acquired hearing impairment. The rather pessimistic estimation of the detection rate without screening, which might be due to the lack of differentiation between congenital and acquired hearing impairment, may bias the results in favour of UNHS. Similar data, however, have been published (\[[@B2],[@B26]\], presenting a median age at diagnosis 18 months). Our estimate of discovery rate in a setting without screening might be biased by regional differences but can be easily replaced by a constant or a different rate function adapted to other local settings. Fifthly, the impact of late diagnosis on delayed language development, is not yet sufficiently known \[[@B22]\]. The economic effects on society in terms of lost productivity \[[@B24]\] and the quality-of-life effects on individuals have been discussed \[[@B27]\]. There is a lack of evidence concerning health care utilization due to hearing loss and the proportion of children following a regular school and professional career after timely fitting of a hearing aid. Further research should be undertaken to investigate the effect of the age of diagnosis and intervention, on the development of hearing impaired children and on their quality of life. Conclusions =========== The value of this modelling exercise on effectiveness, lies in the facilitation and provision of information to decision makers- by quantitatively projecting available data, making explicit and transparent statements about assumptions and the degree of uncertainty involved in this area. The probability of timely intervention increases with UNHS. UNHS can reduce age of confirmation to a much greater extent than RS. In further studies, our model can be used to predict costs of real life situations to evaluate whether programme implementation costs would surpass cost-effectiveness thresholds. Policy makers can also base their decisions on the incremental effectiveness of UHNS, by introducing a screening programme. The model shows how likely an outcome is under the assumption of parameter uncertainty. In our model UNHS showed higher clinical effectiveness compared to RS and NS. The strength of our model lies in the naturalistic and generic structure, which makes it useful as a model for further evaluation. The model gives explicit, transparent and quantitative information about the effectiveness of different screening strategies for policy makers who have to decide on the potential impact of neonatal screening. The model presented here is easily adaptable to different settings. It will and should be verified and tested with longitudinal data of ongoing trials and model projects. This can be one of the first steps towards the needed transparency concerning universal new-born hearing screening. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= EG developed the decision model, carried out the statistical analyses and drafted the manuscript. FH designed and coordinated the study and participated in the development of the decision model and in the writing process. US participated in the statistical analyses, in the development of the decision model and in the writing process. PS-I, SK and AN performed the systematic review and data extraction. AN participated in the design of the study. JW conceived of the study and participated in its design and coordination. All authors revised the manuscript and read and approved the final version. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2458/5/12/prepub> Acknowledgements ================ This study was supported by a grant from the German Federal Ministry of Health and Social Security (BMGS).	PubMed Central	2024-06-05T03:55:52.876025	2005-1-31	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549034/", "journal": "BMC Public Health. 2005 Jan 31; 5:12", "authors": [ { "first": "Eva", "last": "Grill" }, { "first": "Franz", "last": "Hessel" }, { "first": "Uwe", "last": "Siebert" }, { "first": "Petra", "last": "Schnell-Inderst" }, { "first": "Silke", "last": "Kunze" }, { "first": "Andreas", "last": "Nickisch" }, { "first": "Jürgen", "last": "Wasem" } ] }
PMC549035	Background ========== The inference of phylogenies from molecular sequence data, like most other quantitative problems in science, is most powerful within a model-based statistical framework. Sophisticated models are available to describe how sequences change along branches of a tree, and how the rate of sequence change varies among sites. Statistical measures describe both the quality of inferred trees, and the confidence that can be assigned to the existence and position of subtrees. Likelihood-based approaches have proven especially powerful for inferring phylogenetic trees \[[@B1],[@B2]\] but are computationally expensive owing both to the form of the likelihood function itself, and to the need to search the multidimensional space of possible outcomes (tree space) for optimal trees. This computation then must be repeated, typically 100--1000 times, if the nonparametric bootstrap \[[@B3]\] is used to estimate the support for specific subtrees. As a result, maximum-likelihood inference can be prohibitively slow for problems that involve large numbers of aligned sequences, comprehensive search of tree space, and/or many bootstrap replicates. The much faster RELL approximation \[[@B4],[@B5]\] can in principle replace the bootstrap, although so far it has not been extensively investigated with large datasets \[[@B6]\]. At the same time, the ongoing success of genomic sequencing -- new microbial genome sequences are now appearing at the rate of at least one per week -- is yielding a wealth of ever-larger gene and protein datasets suitable for large-scale analysis of deep issues in comparative and evolutionary genomics, e.g.the relative contributions of vertical and lateral gene transfer to genomic diversity \[[@B7],[@B8]\]. However, these datasets are too numerous, and many of them too large, for ready analysis by likelihood inference. For example, using an automated phylogenetics pipeline \[[@B9]\] we have generated more than 22400 protein datasets having up to 144 sequences each, for which we must infer trees. There is consequently much interest in approaches that offer improved search efficiencies while remaining within a model-based statistical framework. Among the most interesting of these is Bayesian inference, in which the posterior probability of a hypothesis (i.e.a tree) is associated with its probability of being correct, given the prior probability, model and data \[[@B2],[@B10]\]. Although posterior probabilities cannot be computed analytically for interestingly large datasets, Markov chain Monte Carlo (MCMC) methods can be used to find and examine equilibrium distributions of trees, on the basis of which we can make probability statements about the true tree \[[@B10]-[@B14]\]. Bayesian inference of phylogeny supports sophisticated evolutionary models, while MCMC, particularly with heated chains (Metropolis-coupled MCMC), recovers from the posterior probability distribution a sample of topologies within which the empirical relative frequency of a given topology converges to its corresponding marginal posterior probability \[[@B15]\]. The topology with highest relative frequency in this sample is typically reported, and posterior probabilities of subtrees can be estimated by consensus from the topologies visited \[[@B10],[@B13]\]. Bayesian phylogenetic inference has been applied to simulated \[[@B16]-[@B18]\] as well as empirical nucleotide datasets (see below). The results establish the applicability and computational efficiency of the Bayesian MCMC approach to molecular phylogenetic inference. However, concerns have arisen about (1) finding optimal trees, (2) overly liberal confidence estimates on subtrees \[[@B19]-[@B24]\], and (3) the possibility that Bayesian inference can resolve topological features (e.g.internal edges, hence subtrees) that do not actually exist \[[@B16]\]. Certain other issues have not been systematically addressed with nucleotide data, notably the robustness of Bayesian inference to relative branch-length differences and to model violation. Much less is known about the behaviour of Bayesian inference with protein-sequence data. While there is no a priorireason that protein-sequence data should be more or less problematic than nucleotide data for Bayesian phylogenetics, gene and protein sequences have distinct statistical properties, and are subject to different selective constraints; so it is not inconceivable that, in practice, the corresponding models of sequence change might tend to fail in different ways, or to different extents. Bayesian inference has been applied to inference of phylogenetic trees for cytochrome b\[[@B25]\], elongation factor 1α \[[@B26]\], hydroperoxidases \[[@B27]\], 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) \[[@B18]\], membrane-intrinsic protein \[[@B28]\], and concatenated mitochondrial protein \[[@B29]\] and larger \[[@B30]\] datasets. Douady et al.\[[@B18]\] report a linear, if noisy, correlation between bootstrap proportion and Bayesian posterior probability for a 15-taxon HMGR protein dataset. As in the case of nucleotide data, the robustness of Bayesian inference to branch-length differences and model violation with protein-sequence datasets remains unexplored. To better characterize the behavior of Bayesian phylogenetic inference with protein-sequence data, we have applied MrBayes \[[@B31],[@B32]\] to both empirical and simulated data. Based on the analysis of 21 empirical protein datasets, we compare maximum likelihood bootstrap proportion and Bayesian posterior probability as estimates of subtree confidence. From analyses of simulated data known to contain phylogenetic signal, we address the fidelity with which the correct topology is recovered under progressively extreme ratios of branch-length differences, both under the correct model of sequence change (the model under which the data were evolved) and under a model that incorporates different amino acid substitution probabilities. Given our ongoing research on lateral gene transfer (above), we were particularly interested in the number of discrete events (edits: \[[@B33]\]) separating inferred from known trees. In this work we compare and contrast results obtained using two popular software programs, PROML \[[@B34]\] and MrBayes \[[@B31]\], as well-developed implementations of the ML and Bayesian approaches to phylogenetic inference respectively. Although the comparison is illustrative, it would be an oversimplification to view these two approaches as diametric opposites, or even as fundamentally mutually exclusive. Both likelihood and Bayesian are general statistical frameworks, with high-level decision criteria (the Akaike Information Criterion, or AIC \[[@B35]\] and Bayesian Information Criterion, or BIC \[[@B36]\], respectively: see also \[[@B37]\]) and associated apparatus for e.g.examining solution space, estimating support, and assessing stability to stochastic error. Only a subset of these broad bodies of theory and practice has so far been applied to phylogenetic inference, and even less implemented in platform-independent software. If we apply BIC to alternative trees and assume equal prior probabilities, it becomes possible to estimate Bayesian posteriors from their likelihood differences, linking the two approaches at this level \[[@B6],[@B38]\]. Stochastic approaches related to MCMC, including simulated annealing \[[@B39]\] and the generalised Gibbs sampler \[[@B40]\], can be used to search tree space in ML. The nonparametric bootstrap, more typically applied in conjunction with parsimony and ML, has proven useful in assessing subtree support in Bayesian inference \[[@B6],[@B18]\]. The application of likelihood in hybrid methods \[[@B41]-[@B43]\], the likelihood ratchet \[[@B44]\], and a metapopulation genetic algorithm \[[@B45]\] lie farther beyond the scope of this discussion, but illustrates the potential for further development of both of these phylogenetic approaches beyond the specific implementations used in this study. Results ======= Empirical data -------------- ### Topology We inferred maximum likelihood (ML) and Bayesian (B) trees for the 21 empirical protein-sequence datasets. For 7 of these datasets, every combination of approach and model that we investigated (ML-JTT-HMM, ML-JTT-gamma, B-JTT, B-EQ: see Empirical dataunder Methods) yielded the same topology. Interestingly, for these, the bootstrap consensus ML trees were topologically identical to the ML and Bayesian trees, indicating that the sequences in these datasets show a high degree of internal consistency across positions (i.e.bear few homoplasies). For another 10 datasets, one or more of these four approaches yielded a tree that differs slightly (edit distance ≤ 2) from the others. No pattern was obvious among these disagreements: the differences do not, for example, systematically separate ML from Bayesian trees. For these 10 datasets, the differences are simple edits, e.g.-(A(BC)) to -(B(AC)), or -((AB)(CD)) to -(A(B(CD))). For the remaining 4 datasets, one or more of the four approaches yielded a tree that differed more substantially (edit distance ≥ 3). Over these examples, the datasets that yield more-conflicted trees are slightly larger (mean, 12.25 sequences each) than those yielding slightly conflicted (mean, 11.50 sequences each) or identical trees (mean, 10.86 sequences each), although the numbers of datasets involved are too few for this observation to be generalized. ### Support for subtrees We compared PROML bootstrap proportions (BPs) with Bayesian posterior probabilities (PPs) separately for all subtrees among the three groups of trees inferred from these 21 empirical datasets: the 7 trees for which all four sets of approaches and models (ML-JTT-HMM, ML-JTT-gamma, B-JTT, B-EQ) yielded the same topology, the 10 for which one or more approach yielded a slightly different tree, and the 4 for which one or more tree differed more substantially. In Figure [1](#F1){ref-type="fig"}, we show the relationship between BP (from PROML using the 8-category gamma distribution: see Methods) and PP for subtrees in these three groups of trees; results for PROML using the hidden Markov model (HMM) are very similar (results not shown). Where ML and Bayesian approaches yield the same topology, the relationship between BP and PP can most simply be fit by a straight line (P-values for linearity are between e-10 and e-13 depending on the subset of data examined). With very few exceptions, however, the PP values are greater, and almost all of the BP values above 80% correspond to 100% PP (Figure [1](#F1){ref-type="fig"}, panel A). For the 14 datasets for which at least one of the four approaches yields a conflicting tree, the relationship between BP and PP appears much more complex (Figure [1](#F1){ref-type="fig"}, panels B,C), although for the subset of non-conflicting subtrees among these 14 datasets (Figure [1](#F1){ref-type="fig"}, panel D) the relationship between BP and PP is similar to that for topologically identical trees (Figure [1](#F1){ref-type="fig"}, panel A). Panel E combines data for all non-conflicting subtrees (panels A and D). In all of these views on the data (Figure [1](#F1){ref-type="fig"}, panels A-E), however, most points lie above and to the left of the diagonal (Table [1](#T1){ref-type="table"}), indicating that for empirical protein-sequence datasets, as for DNA-sequence datasets (see Introduction), Bayesian PPs tend to be more generous than nonparametric BPs as estimates of confidence in subtrees. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Empirical data: relationship between ML consensus bootstrap proportion and Bayesian posterior probability.Comparison of PROML bootstrap proportions (horizontal axes) with Bayesian posterior probabilities (vertical axes) for all internal nodes in trees inferred from 21 empirical protein-sequence datasets. Data are for trees inferred by gamma-corrected ML under JTT, versusthose inferred by gamma-corrected Bayesian inference under JTT (open diamonds) or under EQ (closed squares), (A) for the 7 datasets for which the two ML and two Bayesian trees (see text) are topologically identical, (B) for the 10 datasets for which at least one ML or Bayesian tree (see text) differs slightly (edit distance ≤ 2) from the other three, (C) for the 4 datasets for which at least one tree differs more substantially (edit distance ≥ 3), (D) for the subset of internal nodes, within the latter 14 non-identical trees, that subtend identical subtrees, and (E) for data in panels (A) and (D) plotted together. ::: ![](1471-2148-5-8-1) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Linear fit equations for data in Figure 1. Slope, y-intercept, significance, and R^2^values for linear equations relating bootstrap proportion and Bayesian posterior values shown in panels A, D and E of Figure 1, i.e.for all nodes subtending identical subtrees among the 21 empirical protein-sequence datasets, regardless of whether the corresponding full ML and Bayesian trees are topologically identical or not. ::: [Data]{.underline}^1^ [Panel]{.underline} [Slope]{.underline} [SE^2^]{.underline} [Signif]{.underline} [**y-Intcpt]{.underline} [SE]{.underline} [Signif]{.underline} [Mult R^2^]{.underline} [Adj R^2^*]{.underline} --------------------------- ------------------------- ------------------------- ------------------------- -------------------------- ------------------------------ ---------------------- -------------------------- ----------------------------- ---------------------------- JTT model, all data A 0.4993 0.0536 0.001 51.823 4.568 0.001 0.6207 0.6136 D 0.5150 0.0398 0.001 50.125 3.441 0.001 0.5279 0.5247 E 0.5101 0.0322 0.001 50.625 2.773 0.001 0.5508 0.5486 EQ model, all data A 0.4557 0.0572 0.001 55.661 4.871 0.001 0.5452 0.5367 D 0.4517 0.0352 0.001 56.269 3.050 0.001 0.5227 0.5195 E 0.4531 0.0298 0.001 56.077 2.569 0.001 0.5297 0.5274 JTT model, BP \<85% A 0.7536 0.1771 0.001 38.180 10.819 0.01 0.4880 0.4610 D 0.7816 0.1258 0.001 34.931 8.115 0.001 0.4081 0.3976 E 0.7694 0.1020 0.001 36.112 6.487 0.001 0.4251 0.4176 EQ model, BP \<85% A 0.6909 0.1809 0.01 43.124 11.054 0.001 0.4342 0.4044 D 0.6837 0.1099 0.001 43.086 7.089 0.001 0.4088 0.3982 E 0.6843 0.0922 0.001 43.174 5.866 0.001 0.4170 0.4095 ^1^All data: values based on all data shown in the respective panel in Figure 1; BP \<85%: values based on only those data for which the value of the PROML bootstrap proportion is less than 85%. ^2^SE, standard error; Signif, significance (probability level that estimate \>\\|t\\|); y-Intcpt, y-intercept; Mult R^2^, multiple R^2^; Adj R^2^, adjusted R^2^. ::: From our data, it is not possible to reject the hypothesis that the relationship between BP and PP has the same slope whether the Bayesian inference is conducted using JTT, or EQ, as the model of sequence change. Analysis of covariation (ANCOVA) yields probabilities 0.579 (Panel A), 0.235 (Panel D) and 0.195 (Panel E) that the lines described in Table [1](#T1){ref-type="table"} differ in slope between the JTT and EQ models. When data having \>85% BP are removed from analysis, the probabilities become 0.806, 0.559 and 0.537 respectively, but equivalence still cannot be rejected. Given the limitations of these data, we did not attempt a more-complete analysis, e.g.involving minority subtrees (those not in the extended 50% majority-rule consensus) or higher-order (sigmoidal) fit curves. Because for these trees the true molecular phylogeny is unknown, these results do not speak to the accuracy of the inferred topologies. For this, it is necessary to examine inferences based data simulated on trees of known topology. Simulated data -------------- ### Topology We first examine cases where tree inference was carried out under the same model (JTT) as that used to generate the data, and where a single branch was progressively extended in length (see Methods). When trees were inferred using gamma-corrected ML, the correct tree was recovered in 100% (50/50) of the cases in which the relative branch-length difference was 5-, 10- or 20-fold (Robinson-Foulds symmetric distance in Figure [2](#F2){ref-type="fig"}, panel A, and edit distance in Figure [3](#F3){ref-type="fig"}, panel A). The frequency of inaccurately reconstructed trees increased with further increase in relative branch-length difference, and the inaccurately reconstructed trees were increasingly inaccurate as judged by Robinson-Foulds symmetric distance (which measures the number of bipartitions involved in topological incongruence) although not by edit distance (which measures the number of break-and-reanneal differences without reference to bipartitions). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Comparative performance with simulated data: correct model, single long branch, symmetric distancePerformance at different branch-length ratios of ML and Bayesian inference with simulated protein-sequence data evolved on a tree having a single long branch, measured as Robinson-Foulds symmetric distance. The JTT model was used for both sequence evolution and tree inference. Number (out of 50) of accurately reconstructed topologies (vertical axes) versusbranch-length ratio (horizontal axes), where inference was by (A) gamma-corrected PROML, (B) Bayesian uncorrected for ASRV, with uniform prior, (C) gamma-corrected Bayesian with uniform prior, and (D) gamma-corrected Bayesian with exponential prior. Shading codes for each different distance are shown in the small box at the right of each panel (A-D). Thus the right-hand bar in panel B shows that using Bayesian inference uncorrected for ASRV and assuming a uniform prior, with a dataset generated on a tree in which one branch was lengthened 70-fold, 33 of 50 independent trees recovered the correct topology (Robinson-Foulds symmetric distance zero); 6 differed topologically in ways that involved a single node (distance two); 2 differed in ways that involved two adjacent nodes (distance four); 4 were at distance six; and the remaining 5 were at the maximum symmetric distance, eight. See text for explanation of dual bars in Panel A. ::: ![](1471-2148-5-8-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Comparative performance with simulated data: correct model, single long branch, edit distance.Performance at different branch-length ratios of ML and Bayesian inference with simulated protein-sequence data evolved on a tree having a single long branch, measured as edit distance. The JTT model was used for both sequence evolution and tree inference. Models, panels and axes are as in Figure 2. ::: ![](1471-2148-5-8-3) ::: We investigated two ways of assessing the performance of ML inference. In panel A of Figures [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}, a pair of bars is shown at each value of branch-length difference. The left-hand bar shows performance assessed over 50 single ML reconstructions (one from each of the 50 datasets evolved at that relative length increment), while the right-hand bar shows performance assessed over 50 consensus trees (each of which summarizes 10 bootstrap replicates for each of the same 50 datasets). For datasets having a single long branch, the two representations yield very similar results, with the individual ML results usually showing slightly better performance. By contrast, the situation was reversed for datasets with two long branches. Although consensus is an appropriate way to summarize bootstrap results, nonparametric bootstrap proportions do not measure support for subtrees in a simple, direct and unbiased manner \[[@B46]-[@B48]\]. For this reason, one might question whether an approach based on bootstrap and consensus appropriately summarizes the performance of ML for comparison with Bayesian inference, as Bayesian posteriors dodirectly measure subtree probabilities (given the priors, model and data). The similarities we observe in both magnitude and trend for the two approaches demonstrate that the comparison we are making between ML and Bayesian inference does not, in these cases at least, depend on whether or not the performance of ML is assessed using an approach that involves the nonparametric bootstrap. With Bayesian inference, inaccurately reconstructed trees were also first seen at the 30-fold branch-length ratio (Figure [2](#F2){ref-type="fig"}, panels B-D, and Figure [3](#F3){ref-type="fig"}, panels B-D). Compared with the ML consensus result (panel A, right-hand bar), Bayesian inference almost always yielded a higher frequency of accurate reconstructions. However, unless correction was made for ASRV, the inaccurate trees, although fewer in number, could be more inaccurate as judged by symmetric distance. Gamma correction for ASRV greatly reduced the frequency of the most inaccurate reconstructions, yielding results (Figure [2](#F2){ref-type="fig"}, panels C,D) noticeably better than with gamma-corrected ML. In our simulations, use of an exponential prior (Figure [2](#F2){ref-type="fig"}, panel D) gave slightly fewer inaccurate trees at the most-extreme branch-length ratios, although the difference is not statistically significant (Wilcoxon matched-pairs signed-rank test). In Figures [4](#F4){ref-type="fig"} and [5](#F5){ref-type="fig"} we present the results of tree inference carried out under the same model (JTT) as that used to generate the data, but where two branches were progressively extended relative to the others. For each of the four sets of approaches and models considered, the first inaccurate tree reconstruction was observed at 20-fold relative difference. At higher branch-length ratios, relative performance among the four suites of approaches and models is much more striking than was seen when only a single long branch was present. With gamma-corrected ML, for example, by 50-fold ratio only 9/50 tree topologies are accurately inferred, and at 70-fold ratio only 1/50 (Figure [4](#F4){ref-type="fig"}, panel A). ASRV-uncorrected Bayesian inference (Figure [4](#F4){ref-type="fig"}, panel B) performs even worse, with no accurate inferences at branch-length ratios 50 or greater. However, gamma correction (Figure [4](#F4){ref-type="fig"}, panels C,D) yielded a much higher frequency of accurate reconstructions, with the uniform prior performing better than the exponential prior at the more extreme ratios (Wilcoxon P ≤ 0.003906 at 70-fold) as judged by symmetric distance. About three-quarters (74.9%) of the inaccurate topologies inferred in the case of two differentially lengthened branches showed long-branch attraction, i.e.the long branches were topologically adjacent in the reconstructed tree. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Comparative performance with simulated data: correct model, two long branches, symmetric distance.Performance at different branch-length ratios of ML and Bayesian inference with simulated protein-sequence data evolved on a tree having two long branches, measured as Robinson-Foulds symmetric distance. The JTT model was used for both sequence evolution and tree inference. Models, panels and axes are as in Figure 2. ::: ![](1471-2148-5-8-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Comparative performance with simulated data: correct model, two long branches, edit distance.Performance at different branch-length ratios of ML and Bayesian inference with simulated protein-sequence data evolved on a tree having two long branches, measured as edit distance. The JTT model was used for both sequence evolution and tree inference. Models, panels and axes are as in Figure 2. ::: ![](1471-2148-5-8-5) ::: ### Support for subtrees In Figure [6](#F6){ref-type="fig"} we compare the quantitative support for subtrees, in trees inferred from these simulated datasets by ML and Bayesian approaches, as assessed by bootstrap proportion and posterior probability respectively. Panels A-C show the comparisons based on 1600 extended majority-rule consensus trees for datasets with one long branch (50 ML trees at each of eight branch-length ratios, compared with 50 Bayesian trees at each of the same ratios, over three combinations of ASRV correction and prior probability distribution), and panels D-F are based on 1600 consensus trees for datasets with two long branches. The values shown were derived by summation of BP, and of PP, values over all internal nodes only for the trees that were accurately inferred (i.e.identical with the known topology). By structuring the comparison in this way, we avoid cases where the ML consensus might be topologically different from the best component tree, and avoid dealing with the plethora of cases and sub-cases that arise in comparing topologically non-congruent trees. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Simulated data: relationship between ML consensus bootstrap proportion and Bayesian posterior probability.Relationship between bootstrap proportion for ML consensus trees, and posterior probability for Bayesian trees, for datasets with one (A-C) or two (D-F) branches of relatively greater length. Bayesian trees were inferred (A and D) without ASRV correction and with a uniform prior, (B and E) with gamma correction for ASRV and with a uniform prior, and (C and F) with gamma correction and with an exponential prior. Panel D does not show data at relative branch-length ratios ≥ 50 because none of the trees inferred at these branch-length ratios recovered the known topology. ::: ![](1471-2148-5-8-6) ::: For all three combinations of ASRV correction and prior (corresponding to panels B-D of Figures [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}), the relationship between BP and PP, structured in this way, is best fit by a smooth curve that reaches 100% PP only at BP 99.75% (Figure [6](#F6){ref-type="fig"}, panels A-D) or BP 100% (panels E-F), i.e.shows little or no \"saturation\". For both the single- and two-long-branches cases, the PP is greatest, compared to BP, for Bayesian trees inferred without correction for ASRV, and least generous for gamma-corrected trees where the prior distribution was assumed to be uniform. Unsurprisingly, the lower values of subtree support, as measured both by BP and by PP, arise from the trees with the most extreme relative branch length differences. ### Performance under model violation We next compared the performance of ML and Bayesian inference under violation of the model of sequence change, by evolving datasets under a mammalian mitochondrial model (mtmam), but inferring trees under the JTT model (see Methods). Performance of each of the four sets of approaches and methods was assessed by comparing four measures: the branch-length ratio at which inaccurate trees were first observed; the total number of steps (summed over the eight ratios) by which the 400 trees differ from the true topology; the weighted sum (\"burden\") of these steps; and the mean number of steps by which each inaccurate tree differs from the known tree. The latter two measures were each calculated using both Robinson-Foulds symmetric distance, and edit distance, yielding six comparisons in all. A more-complete description is provided at footnote 2 of Table [2](#T2){ref-type="table"}. Performance in the case of one long branch is summarized in Figures [7](#F7){ref-type="fig"} and [8](#F8){ref-type="fig"}, and in the case of two long branches in Figures [9](#F9){ref-type="fig"} and [10](#F10){ref-type="fig"}. In Table [2](#T2){ref-type="table"} we summarize and compare the performance of ML and Bayesian inference with these datasets under the correct, and an incorrect, model. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Simulated data: comparative performance under correct and incorrect models. Performance of maximum-likelihood and Bayesian phylogenetic inference without, and with, violation of the model of protein sequence change, for trees with one, or two, relatively long branches. ::: One long branch* ----------------------- ----------------------------- -------------------------- --------- --------- --------- --------- ------- ------- --------- --------- --------- --------- *No model violation^1^* *Model violation^1^* First^2^ Wrong Burd SD Mean SD Burd ED Mean ED First Wrong Burd SD Mean SD Burd ED Mean ED ML^3^ 30 56 144 2.57 56 1.00 20 65 164 2.52 65 1.00 BUU 30 46 232 5.04 46 1.00 20 50 222 4.44 50 1.00 BGU 30 36 96 2.67 36 1.00 20 39 118 3.03 39 1.00 BGE 30 28 88 3.14 28 1.00 20 39 116 2.97 39 1.00 Two long branches *No model violation* *Model violation* First Wrong Burd SD Mean SD Burd ED Mean ED First Wrong Burd SD Mean SD Burd ED Mean ED ML 20 186 1124 6.04 273 1.47 20 174 1166 6.70 207 1.19 BUU 20 237 1854 7.82 326 1.38 10 244 1900 7.79 299 1.23 BGU 20 87 270 3.10 104 1.20 20 105 468 4.46 119 1.13 BGE 20 86 314 3.65 101 1.17 20 115 650 5.65 131 1.14 ^1^Protein-sequence data were evolved under the Jones et al.(JTT) or, alternatively, mammalian mitochondrial (mtmam) model of sequence change, and trees were inferred assuming the JTT model. ^2^Performance was measured by six indices: First, the lowest investigated branch-length ratio at which at least one inaccurately reconstructed tree was found; Wrong, the number of inaccurately inferred trees out 400 (8 branch-length ratios × 50 replicates at each ratio); BurdSD, the bipartition burden, calculated as the Robinson-Foulds symmetric distance by which each tree differs from the known tree, summed over the 400 trees; MeanSD, the mean Robinson-Foulds symmetric distance per inaccurate tree; BurdED, the edit burden, calculated as the edit distance by which each tree differs from the known tree, summed over the 400 trees; and MeanED, the mean edit distance per inaccurate tree. ^3^Inference methods: ML, protein maximum likelihood with gamma ASRV correction; BUU, Bayesian inference, uncorrected for ASRV, uniform prior; BGU, Bayesian inference, gamma ASRV correction, uniform prior; and BGE, Bayesian inference, gamma ASRV correction, exponential prior. See text for further details. ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Comparative performance with simulated data: incorrect model, one long branch, symmetric distance.Performance at different branch-length ratios of ML and Bayesian inference with simulated protein-sequence data evolved on a tree having a single long branch, measured as Robinson-Foulds symmetric distance. Data were evolved under the mtmam model, but trees were inferred under the JTT model. Panels and axes are as in Figure 2. ::: ![](1471-2148-5-8-7) ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Comparative performance with simulated data: incorrect model, one long branch, edit distance.Performance at different branch-length ratios of ML and Bayesian inference with simulated protein-sequence data evolved on a tree having a single long branch, measured as edit distance. Data were evolved under the mtmam model, but trees were inferred under the JTT model. Models, panels and axes are as in Figure 2. ::: ![](1471-2148-5-8-8) ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Comparative performance with simulated data: incorrect model, two long branches, symmetric distance.Performance at different branch-length ratios of ML and Bayesian inference with simulated protein-sequence data evolved on a tree having two long branches, measured as Robinson-Foulds symmetric distance. Data were evolved under the mtmam model, but trees were inferred under the JTT model. Models, panels and axes are as in Figure 2. ::: ![](1471-2148-5-8-9) ::: ::: {#F10 .fig} Figure 10 ::: {.caption} ###### Comparative performance with simulated data: incorrect model, two long branches, edit distance.Performance at different branch-length ratios of ML and Bayesian inference with simulated protein-sequence data evolved on a tree having two long branches, measured as edit distance. Data were evolved under the mtmam model, but trees were inferred under the JTT model. Models, panels and axes are as in Figure 2. ::: ![](1471-2148-5-8-10) ::: For datasets in which a single branch was of relatively greater length, violating the model of sequence change degraded performance of the four approaches (Table [2](#T2){ref-type="table"}). In each case, inaccurate trees were first observed at 20-fold branch-length ratio, earlier than the 30-fold ratio seen in the absence of model violation. Inaccurate trees were more numerous, in comparison with inference under the correct model. With ML, each inaccurate tree was about as inaccurate under the incorrect model as under the correct one, as measured by symmetric distance (Table [2](#T2){ref-type="table"}). With Bayesian inference, inaccurate trees produced under the wrong model were, unexpectedly, sometimes less inaccurate than those inferred under the correct model (Table [2](#T2){ref-type="table"}), and in one case (no correction for ASRV, uniform prior distribution) the total burden of changes was less, as measured by symmetric distance. Exclusion of results from the 70-fold data (results not shown) demonstrated that this effect is not due to a loss of dynamic range at extreme values. For datasets containing two long branches, model violation affected performance of ML and Bayesian inference differently. With ML, inference under the wrong model produced a somewhat lower frequency of topologically inaccurate trees, although each inaccurate tree was more inaccurate as judged by symmetric distance (Table [2](#T2){ref-type="table"}). With Bayesian inference, use of the wrong model increased the frequency of inaccurate trees, and each inaccurate tree tended to be more inaccurate as measured by symmetric distance. With Bayesian inference uncorrected for ASRV and using a uniform prior, the first inaccurate tree appeared at a ratio of only 10, and no accurate trees were recovered at ratios 50 or higher; although by most indices the performance was not further degraded by violation of the model of sequence change, performance was already quite poor, and not much dynamic range remained available. Use of an exponential prior again made a significant difference only with two long branches and assessment using Robinson-Foulds symmetric distance (Wilcoxon P ≤ 0.00097 and P ≤ 0.00003 for degraded performance at 60- and 70-fold branch-length ratios respectively). Discussion ========== Unlike the situation with established approaches based on pairwise distances, parsimony or maximum likelihood, relatively little experience has accumulated so far on the application of Bayesian approaches to phylogenetic inference, especially for protein-sequence datasets. In this work we (a) extend the comparison of Bayesian posterior probabilities with nonparametric bootstrap proportions as measures of confidence in subtrees, (b) systematically investigate the robustness of ML and Bayesian inference to branch-length differences, and (c) compare the behavior of these two approaches to one specific violation of the model of sequence change. We used two measures to compare topologies (Robinson-Foulds symmetric distance, and edit distance), and it is clear that they captured different facets of topological incongruence. Support for subtrees -------------------- Using 21 empirical protein-sequence datasets, we compared Bayesian posterior probabilities with bootstrap proportions based on ML as measures of support for subtrees. To make this comparison as fair as possible, we restricted our analysis to a model of sequence change (JTT) and a correction for ASRV (discrete approximation to the gamma distribution) available in both PROML and MrBayes. We did not optimize models separately for each approach or for each dataset, as JTT+gamma represents the most-parameterized combination that these two programs support in common. It is therefore possible that some of the difference observed between the two measures results from differential sensitivity of ML and Bayesian inference, as implemented in these programs, to deviation of JTT and the discrete gamma distribution from an optimal description of the processes of sequence change that actually gave rise to these sequences (but see the final paragraph under Model violation, below). As it is unlikely that any existing model -- certainly any that fails to account for lineage-specific processes and temporal variations along these lineages -- fully represents the historical complexity of molecular evolution, the same criticism could be levelled, albeit perhaps in lesser degree, against all current applications of statistically based phylogenetic inference to empirical datasets. The data presented in Figure [1](#F1){ref-type="fig"} demonstrate that, at least for these protein-sequence datasets, Bayesian PPs tend to offer a more-generous estimate of subtree reliability than does the nonparametric bootstrap combined with ML. This result supports and extends previous studies with DNA- \[[@B16]-[@B18],[@B20]-[@B22],[@B49]\] and protein-sequence data \[[@B18],[@B19]\]. Bayesian PPs and nonparametric bootstrap BPs are not commensurate \[[@B17],[@B48]\] and may be seen as \"potential upper and lower bounds of node reliability\" respectively (page 248 of \[[@B18]\]). (Being more-generous than a too-conservative measure does not, of course, imply that Bayesian PPs must be too-generous.) Our results strongly suggest that the interpretation of BPs and PPs being developed for nucleotide sequences will be applicable, as well, to protein sequences. For sets of consensus trees inferred from simulated protein-sequence data (Figure [6](#F6){ref-type="fig"}), Bayesian PPs tend to be more generous than nonparametric BPs in estimates of subtree support. However, whereas for empirical protein-sequence data (and nucleotide-sequence data: see references cited immediately above) PPs tend to \"saturate\", i.e.reach 100% at BP values less than 100% (here around 80%), with our simulated data the relationship between BP and PP resembles a smooth curve reaching 100% PP only at BP greater than 99%. Further studies will be required to disentangle why little or no saturation was observed; possibilities include the structure of our simulated trees (e.g.their symmetry, or an usually regular spacing of internal nodes), the way that data were evolved on these trees (e.g.assuming strict independence among sites, or rigorous adherence to the JTT model), and/or the way we summarize the support data for ML (viaextended majority-rule consensus trees). Relative branch-length differences ---------------------------------- Dissimilar sequences (represented in phylogenetic trees as long branches) create difficulties in phylogenetic analysis. The issue has been most extensively explored in parsimony analysis, where branch length can be an important consideration, e.g.in selection of outgroups and resolution of topologically problematic regions. Parsimony analysis is particularly susceptible to \"long branch attraction\" (LBA) artefacts, in which two or more branches are resolved adjacent in a tree solely because they are highly divergent from the others \[[@B2]\]. ML inference can be more robust against LBA, although to our knowledge this has been not been specifically examined for protein-sequence data. We are unaware of any systematic examination of the degree to which Bayesian phylogenetic inference is robust against branch length-based artefact. Our results (Figures [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}) indicate that for protein-sequence datasets of this size, both gamma-corrected ML and Bayesian inference can be robust to artefact arising from the levels of dissimilarity likely to be encountered in empirical biological data. Both ML and Bayesian inference can be fully robust (within our limits of detection) to at least a 20-fold relative length ratio for a single branch, and both perform nearly as well when two branches are relatively lengthened. When a single branch is lengthened, performance (accurate retrieval of the known topology) degrades slowly as relative branch length increases thereafter; Bayesian inference with gamma correction for ASRV performs best among these alternatives. When two branches are relatively lengthened, the performance of ML, and of ASRV-uncorrected Bayesian inference, falls off much more rapidly, whereas in our simulations ASRV-corrected Bayesian inference was more robust than ML. These performance characteristics have been demonstrated only for protein-sequence datasets of the size, length, composition, divergence and tree shape we examined, and for these implementations of ML (PROML) and Bayesian inference (MrBayes). Applicability to larger, longer, and more divergent protein-sequence datasets, to more-diverse tree shapes, and to different implementations seems highly probable, although further nuance will doubtlessly emerge, and scope may remain for further optimization. Model violation --------------- Both the mammalian mitochondrial (mtmam) and JTT models embody empirical probabilistic models of amino acid substitution. Codon usage is highly skewed in mitochondrial genomes compared with the cognate nucleocytoplasmic components \[[@B50]\], and the amino acid transition probabilities in mtmam differ correspondingly from those in JTT. Nonetheless, for the datasets we examined, both ML and Bayesian inference perform well, at biologically reasonable ratios of branch-length difference, even when the JTT model is used to infer trees from protein datasets evolved under mtmam (Figures [7](#F7){ref-type="fig"}, [8](#F8){ref-type="fig"}, [9](#F9){ref-type="fig"}, [10](#F10){ref-type="fig"}). With one exception, the first inaccurately reconstructed trees were observed at the 20-fold ratio (Table [2](#T2){ref-type="table"}). Model violation increased the inaccuracy of reconstruction (as measured by the total number of inaccurate trees over the eight branch-length ratios) by 8 to 39% (mean 18%) in the case of one differentially extended branch, and by -6 to 34% (mean 13%) where two branches are lengthened (Table [2](#T2){ref-type="table"}). In the former case, the total burden of these inaccuracies was 16% and 18% as assessed by symmetric and edit distances respectively. The effect of model violation on accuracy for trees with two differentially lengthened branches was more variable; little change (or even a reduction in burden) was observed for ML and Bayesian inference without ASRV correction, whereas violating the model greatly decreased the accuracy of reconstruction by gamma-corrected Bayesian inference. Nonetheless, even at this reduced accuracy, gamma-corrected Bayesian inference performed much more-accurately than either ML or uncorrected Bayesian inference at branch-length ratios of 20-fold and greater. Particularly in simulations where a single branch was differentially lengthened (Table [2](#T2){ref-type="table"}), using the wrong model of sequence change sometimes improved some aspects of performance. Thus with ML inference, inference under the wrong model increased both the total number of inaccurate trees and the bipartition burden over the 8 branch-length ratios (400 trees), but each inaccurate tree was, on average, slightly less inaccurate (as assessed by symmetric distance) than those inferred under the correct model of sequence change. The same phenomenon was observed with Bayesian inference using gamma ASRV correction and an exponential distribution of prior probability over branch lengths. With Bayesian inference uncorrected for ASRV, both the total bipartition burden, and the mean inaccuracy of inaccurate trees as assessed by symmetric distance, were lessened under the wrong model. In simulations with two long branches as well (Table [2](#T2){ref-type="table"}), we observed that with ML inference, model violation reduces the number of inaccurate trees and the burden of edits required to generate them, although the latter was not seen when using symmetric distance as the metric. Others have reported situations in which using the wrong model improves the performance of ML (\[[@B51]-[@B53]\] and pp. 272--274 of \[[@B2]\]). Some of these cases appear to result from the specific placement of long branches in the \"anti-Felsenstein zone\", where biased estimation can increase the efficiency of finding the correct topology \[[@B52],[@B54]\]. However, this does not explain our results, as we separated the long branches from each other. The degree of insensitivity to model violation we observe for gamma-corrected ML and Bayesian inference goes some way toward mitigating possible concern (see above under Support for subtrees) that the relative performance of these approaches as reported herein might, in part, reflect their differential sensitivity to sub-optimality in the models used. Measures of tree comparison --------------------------- Our results (Figures [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}, Figures [7](#F7){ref-type="fig"}, [8](#F8){ref-type="fig"}, [9](#F9){ref-type="fig"}, [10](#F10){ref-type="fig"} and Table [2](#T2){ref-type="table"}) illustrate how Robinson-Foulds symmetric distance and edit distance provide non-identical, complementary views of topological incongruence. The former metric enumerates the number of internal nodes that must be collapsed to make two topologies identical, whereas the latter counts the number of break-and-reanneal operations needed to convert one topology into another. The scores are identical if all incongruent subtrees can be reconciled by collapse through, or transfer across, a single internal node, but diverge from each other to the extent that incongruent subtrees are positioned more distantly (i.e.across more internal nodes) from each other. Our results also illustrate the difference in dynamic range offered by these metrics, while simulation studies \[[@B17],[@B55],[@B56]\] indicate their differential sensitivity to overall tree shape and/or local topology. Other tree-comparison metrics are available and may offer advantages, e.g.in distinguishing transformations that affect large numbers of termini from those that affect small numbers of termini, in robustness against displacement of particular termini, or in application to very large trees \[[@B57]-[@B59]\]. Conclusions =========== Bayesian inference can be as robust as ML against relative branch-length differences of 20-fold or greater in inference of correct topologies from protein-sequence data, although details depend on the number of relatively long branches, the presence or absence of an effective correction for ASRV, and (presumably) other factors. One might doubt that sequences so dissimilar as to produce a 20-fold (or more) difference in branch lengths could be believably recognised as homologous, or reliably aligned. Bayesian inference can also be as robust as ML to violation of the model of amino acid transition probability. For empirical protein-sequence data that might reasonably be encountered in biological research, then, both gamma-corrected ML and gamma-corrected Bayesian inference perform well in recovering the correct topology. As Bayesian inference is typically very much faster than even a single ML run, not to mention than e.g.100 or 1000 replicate runs required to estimate bootstrap proportions, ASRV-corrected Bayesian inference must be seen as an important alternative for statistically based phylogenetic analysis of protein-sequence data when computational resources are limiting. It appears that the interpretation of bootstrap proportions and posterior probabilities being developed for nucleotide sequences will apply as well to protein sequences. Our interest in lateral genetic transfer (LGT) \[[@B7]-[@B9]\] led us to investigate different measures with which to characterise topological difference among trees. Whether LGT tends to occur primarily among closely related lineages, or alternatively whether the frequency of transfer depends more critically on some other factor (oligonucleotide frequency, common environment) -- or indeed is purely random -- remains an open question. Attention has recently been focused on hypotheses that accord to close-range LGT the central role in metabolic and physiological innovation \[[@B60]\] and in shaping organismal phylogeny \[[@B61]\]. A statistic that captures both the number of transfer events (as does edit distance), and the topological breadth of transfer (as does symmetric distance), would thus be valuable in elucidating the pattern and significance of LGT. For such a statistic to be meaningful in a biological context, it must be sensitive to the annotation (specific phyletic value) of the subtrees involved. Implementation of this, and of a broader range of tree-comparison metrics, in platform-independent software should be a matter of some urgency. Methods ======= Simulated data -------------- Simulated data were evolved using the \"evolver\" program within PAML version 3.13a \[[@B62],[@B63]\]. First, we generated random trees, each with 7 species (sequences), using the settings birth rate 0.2, death rate 0.2, sampling fraction 0.5, and mutation rate 0.5. In one set of runs, 8 additional trees were then produced, in which 1 of these 7 branches (selected at random) was progressively lengthened to be longer than the others by the factors 5, 10, 20, 30, 40, 50, 60 and 70. In a second set of runs, 8 other trees were produced, in which 2 branches were progressively lengthened by these same factors (each long branch in a given tree was extended by the same factor). The branches to be lengthened were selected to be as distant from each other as possible in the tree; for a strictly bifurcating tree with seven termini (leaves), this means that it would have a Robinson-Foulds symmetric distance (\[[@B64]\]; see below) of 8 if its long branches were forced to become adjacent. For the trees with 1 or 2 branches differentially lengthened 70-fold, branch lengths were reduced proportionally (very slightly) to maintain all absolute values less than 10, so as not to exceed bounds set on Bayesian prior distributions (below). Protein data sets were then generated on each of the 16 trees with differentially lengthened branches, using the \"evolver\" program in PAML. On each tree we evolved 100 replicate protein datasets under the JTT model of sequence change \[[@B65]\], with among-sites rate variation (ASRV) modelled as an 8-category discrete approximation to a gamma distribution with alpha (shape) parameter 0.5. In a second set of runs, we similarly evolved 100 datasets under the mtmam \[[@B62],[@B63]\] model, originally named REV \[[@B66]\], estimated from a set of mammalian mitochondrial proteins. Each protein dataset was of initial length 1000 amino acids. From each of the 32 sets of 100 replicate protein datasets, we then selected 50 replicate protein datasets at random for further analysis. ### Maximum likelihood inference All maximum-likelihood (ML) trees were inferred using PROML version 3.6a3 in Felsenstein\'s PHYLIP package \[[@B34]\] implemented on an 8-processor SGI Origin 2100 under IRIX, a 128-processor SUN Netra-1 cluster under Linux, and a 16-processor IBM p690 Regatta under AIX. In all ML inference we assumed the JTT model of sequence change, randomized (jumbled) the order of sequence addition, used global rearrangements, and selected the \"not rough\" analysis option in PROML. More information on these settings is available online \[[@B34]\]. We assumed an 8-category discrete approximation to a gamma distribution, with values for the gamma shape parameter estimated separately for each dataset using Tree-Puzzle \[[@B67]\], but frequencies for each category estimated by PROML; rates were assumed to be uncorrelated at adjacent sites. For both empirical and simulated data, models and parameter values were selected to facilitate, as much as possible, a fair comparison of ML and Bayesian approaches. Some details of ML inference differed for empirical vssimuated data. Here we present methods for the simulated data; methods specific to the empirical data are given below. For simulated data, we report results from both (1) single ML inference runs based on each of the 50 replicate protein datasets at each branch-length ratio increment, and (2) bootstrapping (N= 10) each of the 50 replicate datasets, as described in the preceding paragraph. ### Bayesian inference Bayesian inference (B) was carried out using MRBAYES version 2.01 \[[@B31]\] implemented on a 16-processor IBM p690 Regatta under AIX, and on a 508-processor Compaq ES45 cluster under Linux. (Version 3.0 of MRBAYES was not used because, at the time these analyses were carried out, no documentation was available on how to force the shape parameter to remain fixed after initialization). Priors were defined over the branch-length interval (0.0,10.0), and the JTT model of sequence change was assumed (or known to be correct) for all analyses. For empirical data, trees were inferred using two models of sequence change: JTT, and a variant (\"equalin\", EQ) of the F81 model of Felsenstein \[[@B1]\]. ASRV was modeled as an 8-category gamma distribution, and the shape parameter was optimized by MRBAYES. The prior distribution on branch lengths was assumed to be uniform, and following initial trials (data not shown) the Markov chain temperature was set to 0.2000. For each dataset, 8 Markov chains were propagated for 30,000 generations each and sampled every 100 generations. As preliminary analyses showed convergence within a few thousand generations, burn-in was conservatively set at 10,000 generations. Posterior probabilities were obtained using allcompat(i.e., extended 50% majority-rule consensus) among these sampled trees. For simulated data, we examined three models of different complexities: (1) a uniform prior distribution over branch lengths, and a single rate category; (2) a uniform prior, and an 8-category gamma model of ASRV; and (3) an exponential prior, and an 8-category gamma. The gamma shape parameter was, as above, estimated using Tree-Puzzle, and was fixed (i.e.did not merely serve to initialize estimation by MRBAYES). In runs where an exponential prior was used, the value of the exponent was estimated from the simulated data, and differed according to sequence-change model: under JTT, 0.10 for datasets with both one and two long branches, and under mtmam, 1.04 for one long branch, and 0.60 for two long branches. ### Comparing topologies and subtree reliabilities For trees inferred from the 16 sets of simulated data (1 or 2 long branches, 8 ratios of branch-length difference), topologies were compared against that of the (known) tree on which the data had been evolved. For this we employed two metrics: (1) the minimum number of break-and-reanneal edits required to convert one tree into the other. This metric goes under various names, including subtree prune and regraft distance\[[@B33]\]; we refer to it simply as edit distance; and (2) the Robinson-Foulds symmetric distance \[[@B64]\] as implemented in TREEDIST in the PHYLIP package \[[@B34]\]. The values of these metrics were not normalized (cf. \[[@B68]\]) because all simulated trees have the same number of internal edges. Subtree support was assessed as bootstrap proportion (BP) for ML, and as posterior probability (PP) for Bayesian inference. Empirical data -------------- Methods and procedures followed those for simulated data (above), except as described subsequently here. Aligned protein sequence datasets (see [Additional file 1](#S1){ref-type="supplementary-material"}) were obtained from Dr Nick Goldman (EBI). We selected 21 datasets (240 sequences in total, mean 11.4 sequences per dataset), requiring each to be of interestingly large size (minimum 8 sequences) but not too large for analysis by bootstrapped protein likelihood, given the computational resources available to us (maximum 16 sequences). These were reformatted for further analysis, assigning new designators to anonymize individual sequences and to avoid the use of characters that are not supported within the rule sets of the software programs we used (\"illegal characters\"). For empirical data, we inferred ML trees in two ways: (1) using a user-defined hidden Markov model (HMM) with 8 categories, each set to 12.5% of sites, and with rates in each category estimated using Tree-Puzzle version 5.0 \[[@B67]\]; and (2) assuming an 8-category discrete approximation to a gamma distribution, with values for the gamma shape parameter estimated using Tree-Puzzle (but frequencies for each category estimated by PROML), rates at adjacent sites assumed to be uncorrelated, and the among-sites rate variation (ASRV) gamma-shape parameter estimated for each dataset using Tree-Puzzle. In all our use of Tree-Puzzle, we assumed 8 rate categories (each covering one-eighth of the aligned positions) and JTT. For simulated data, ML trees were inferred using only the 8-category discrete approximation to a gamma distribution. ML analyses were bootstrapped (N= 100 for the 21 empirical data sets, N= 10 for the 1600 simulated datasets) with preservation of rate-class information as described in the SEQBOOT documentation. Nonparametric bootstrap proportions (BPs) were computed under extended majority rule consensus using CONSENSE. Both SEQBOOT and CONSENSE are in PHYLIP \[[@B34]\]. For the 21 sets of empirical data, no \"true\" tree is available. Topologies resulting from the different inference approaches and models (ML-JTT-HMM, ML-JTT-gamma, B-JTT, B-EQ) were therefore compared amongst themselves, using edit distance (determined manually) as the comparison metric. Topologies of the bootstrap ML consensus trees were compared as well, although as consensus trees they do not necessarily reflect most-likely topologies. Availability of data -------------------- The 21 empirical protein-sequence datasets from Dr Nick Goldman, and our simulated datasets with one or two long branches, are available for download at \[[@B69]\]. Authors\' contributions ======================= JCM was responsible for data simulation and analysis. TJH was responsible for high-performance computing, and generated the figures. MAR initiated and supervised the project, and wrote the manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Description of 21 empirical datasetsThis PDF file contains information on each of the 21 empirical datasets provided by Dr Nick Goldman, including: number of sequences, GenBank ID (gi number) of first sequence in dataset, key words from description line of first sequence, PAUP\* parsimony score of dataset, number of internal nodes, and number of zero-length internal edges observed with PROML to have support in three non-overlapping intervals: P \< 0.01, P \< 0.05 but not P \< 0.01, and worse than P \< 0.05. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ We thank Nick Goldman for the empirical protein-sequence datasets, John Huelsenbeck and Fredrik Ronquist for assistance with earlier versions of MrBayes, Rob Beiko for statistical advice, J. Peter Gogarten for helpful discussions, Ben Evans for parallelising MrBayes on the APAC National Facility, and reviewers for helpful comments. Supported by Australian Research Council grants DP0342987 and CE0348221, and by the Australian Partnership in Advanced Computing (APAC) Merit Allocation Scheme.	PubMed Central	2024-06-05T03:55:52.879114	2005-1-28	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549035/", "journal": "BMC Evol Biol. 2005 Jan 28; 5:8", "authors": [ { "first": "Jessica C", "last": "Mar" }, { "first": "Timothy J", "last": "Harlow" }, { "first": "Mark A", "last": "Ragan" } ] }
PMC549036	Background ========== The American Thoracic Society (ATS) and European Respiratory Society (ERS) have jointly proposed standards \[[@B1]\] for the diagnosis, treatment and spirometric classification of patients with chronic obstructive pulmonary disease (COPD). According to the GOLD (Global Obstructive Lung Disease) guideline \[[@B2]\], the goals of clinical control in patients with COPD include health-related quality of life goals (improved exercise tolerance and emotional function) and clinical goals (prevention of disease progression and minimization of symptoms). The Clinical COPD questionnaire (CCQ) \[[@B3]\] is the first practical clinical instrument to be used for routine evaluation of clinical control (symptom, functional state and mental state) concerning patients with COPD, in general practice. The development and validation study has been published in this journal and data were collected from 119 subjects. The aim of the present study is the validation of the CCQ in Italian in specific pulmonary disease clinical practice. In this practice, the ATS/ERS \[[@B1]\] recommended routine measurements, in all patients with COPD, are the following: forced expiratory volume in one second (FEV~1~) and forced vital capacity (FVC), body mass index (BMI) and functional dyspnoea (Medical Research Council -- MRC). Methods ======= Subjects -------- Healthy subjects were selected in social meeting places. Subjects were asked, individually, to answer a simple questionnaire after the study had been explained to them. Only subjects over 40 years of age were interviewed. We excluded subjects with any disease symptoms, or any limitation in daily activities for any reason, or who mentioned suffering from disabling chronic diseases (COPD, asthma, arthritis, angina or heart insufficiency). All subjects gave their informed written consent for baseline spirometry and questionnaires administration, as approved by the local Medical Ethics Committee. We enrolled 55 subjects, 52 non-smokers and 3 ex-smokers. Subject data are shown in Table [1](#T1){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics and results of the study population in subgroups ::: Healthy subjects Mild-moderate COPD-class I-II Severe COPD-class III Very severe COPD-class IV ------------------------- ------------------------ ----------------------------------- --------------------------- ------------------------------- N 55 40 55 25 Males (%) 62.0 85.0 63.6 72.0 Age (yr) 70^abcd^(41--82) 72^abcd^(58--84) 71^abcd^(41--86) 71^abcd^(42--86) LTOT (%) 0.0 0.0 32.7 72.0 HMV (%) 0.0 0.0 7.2 8.0 BMI (kg/m^2^) 25.7^abcd^(18.0--30.0) 26.7^abcd^(18.6--37.8) 25.1^abcd^(16.4--36.4) 26.6^abcd^(16.2--34.6) FEV~1~/FVC (%) 79.2 (70.4--94.5) 59.7 (40.4--68.2) 44.2 (27.9--66.2) 35.1 (21.1--57.3) FEV~1~(% predicted) 108.0 (69--132) 69.5 (51.4--117.1) 40.7 (30.2--49.8) 26.4 (16.4--29.7) MRC functional dyspnoea 0.6 ± 3.4 (0--1) 1.1 ± 0.8 (0--2) 1.6 ± 0.7 (0--4) 2.3 ± 0.9 (0--4) CCQ symptom 0.5 (0.0--4.0) 1.3^b^(0.0--4.0) 1.5^b^(0.3--5.8) 2.5 (0.3--3.8) CCQ functional state 0.5^a^(0.0--5.3) 1.0^a^(0.0--3.5) 1.5 (0.0--5.3) 3.0 (0.3--5.0) CCQ mental state 0.0 (0.0--4.5) 0.0 (0.0--5.0) 1.0^c^(0.0--6.0) 1.5^c^(0.0--6.0) CCQ total 0.4 (0.0--3.8) 0.9 (0.0--3.5) 1.4 (0.3--5.2) 2.6 (0.4--4.3) CCQ = Clinical COPD Questionnaire; range 0--6; 0 indicating best possible control and 6 indicating worst clinical control. LTOT = long term oxygen therapy. HMV = home assisted mechanical ventilation during night. BMI = body mass index. FVC = forced vital capacity. FEV~1~= forced expired volume in one second. MRC = Medical Research Council. Healthy: normal spirometry, no chronic symptoms (cough, sputum production and/or dyspnoea). COPD classification by post-bronchodilator spirometry according to GOLD guidelines: mild-moderate FEV~1~/FVC \<= 0.70 and FEV~1~\>= 50% predicted, severe FEV~1~/FVC \<= 0.70 and FEV~1~\>= 30% predicted, very-severe FEV~1~/FVC \<= 0.70 and FEV~1~\< 30% predicted. Medians not sharing a common superscript (a,b,c,d) are significantly different at p \< 0.05 after Mann-Wittney U test. MRC data are reported as mean value with standard deviation and range. ::: Patients with COPD were consecutively enrolled in the outpatient section of our Division during medical consultation. According to the guidelines \[[@B1],[@B2]\], COPD was defined by the presence of chronic cough, sputum production and/or dyspnoea. Patients with airways obstruction (FEV~1~/FVC \<= 0.70) were classified as mild (FEV~1~post-bronchodilator (pb) \>= 80% predicted), moderate (FEV~1~pb \>= 50% predicted), severe (FEV~1~pb \>= 30% predicted) and very severe (FEV~1~pb \< 30% predicted). We excluded COPD patients with: a) significant improvement of FEV~1~pb (\>= 15 % and/or 200 ml) compared with baseline, b) disease exacerbation in the previous four weeks, c) asthma, chronic heart failure, obstructive sleep apnoea syndrome, cancer or other disabling diseases except COPD. We enrolled 120 patients (77 ex-smokers, 19 smokers). In 1999, the local health service authority approved the standard evaluation procedures used in our outpatient clinic for patients with COPD. The patients\' data are shown in Table [1](#T1){ref-type="table"}. Forty-six patients with COPD (exclusion criteria as mentioned above) were enrolled in a continuous pulmonary rehabilitation program, 31 males, 30 ex-smokers, 6 smokers, 13 in long-term oxygen therapy (LTOT), 2 in home-assisted mechanical ventilation during the night (HMV), median age 72 (range 41--83), median FEV~1~pb 46 % predicted (range 18--68). In 1999, the local health service authority approved our continuous pulmonary rehab program for patients with COPD. Cross sectional validity ------------------------ The CCQ was administered to all subjects. They were instructed to recall their experiences during the previous week. The CCQ is self-administered and contains only 10 items, subdivided into three domains: symptom (item 1--2--5--6), functional state (item 7--8--9--10) and mental state (item 3--4). Subjects responded to each question using a 7 point scale from 0 = asymptomatic or no-limitation, to 6 = extremely symptomatic or totally limited. The overall clinical COPD control score and the score of the three domains was calculated by adding all the scores together and dividing the sum by the number of questions. The Italian translation of the copyrighted questionnaire and permission for use was obtained from T. van der Molen \[[@B3]\] in February 2004 by one of the team (SD). Lung function (FEV1 and FVC) was measured according to ERS guidelines \[[@B4]\] using a portable turbine spirometer (Pony, Cosmed, Italy) in base condition (all subjects) and 20 minutes after metered inhalation of 200 mcg of salbutamol (COPD patients only). The copyrighted Italian validated version \[[@B5]\] of the 36-item Short Form Health Survey (SF-36) \[[@B6]\], a generic health-related quality of life questionnaire, was administered to 120 patients with COPD and 55 healthy subjects. The validated Italian version of SF-36 and permission for use was obtained from GlaxoSmithKline in June 2002 by one of the team (SP). Functional dyspnoea was assessed in all subjects using the Medical Research Council (MRC) scale as proposed by ATS/ERS guidelines \[[@B1]\]: 0 = not subject to breathlessness except with strenuous exercise, 1 = subject to shortness of breath when hurrying or walking up a gradually sloping hill, 2 = walks slower than people of the same age due to breathlessness or has to stop for breath when walking at a normal pace on a level, 3 = stops for breath after walking about 100 m or after a few minutes on a level, 4 = too breathless to leave the house or breathless when dressing or undressing. BMI was calculated by dividing weight (in kg) over height (in m^2^), for all subjects. Longitudinal validity --------------------- The CCQ was re-administered after 2 weeks (where there was no variation of the previous therapy or introduction of new therapy) in 112 subjects (53 healthy and 59 patients with COPD), 75 males, median age 71 years (range 41--84), median FEV~1~60 % predicted (range 19--117). We tested the CCQ responsiveness in patients with COPD undergoing continuous pulmonary rehabilitation. Patients were treated in four successive groups in our hospital following a standard three-week protocol. According to guidelines \[[@B7]\], the program was individually tailored and designed to optimize physical and social performance and autonomy, and to be integrated into overall patient treatment. It was a mix of physical retraining, thoracic and general physiotherapy, education, self-monitoring. At the end of the three-week hospitalization period, patients received: a) their individual continuous pulmonary rehabilitation home program together with optimized pharmacological therapy, b) the next three-month appointment for the outpatient evaluation visit, c) the next six-month appointment for successive inpatient three-week pulmonary rehabilitation. The CCQ was administered, on admission and on discharge from hospital, to 46 COPD patients. Patients were submitted to a 6-minute walking test (6 MWT) on hospital admission and discharge, according to guidelines \[[@B8]\]. In each occasion, we measured distance-walked and breathlessness at the end of 6 MWT, using the standard Borg rating scale \[[@B9]\]. This is a category scale in which simple verbal expressions, that describe increasing degrees of breathlessness in exercise, are linked to numbers (range from 0 = nothing at all to 10 = maximal). The CCQ was also administered after two more weeks during home-based comprehensive treatment. All patients gave their informed written consent for re-administration of CCQ, at home or in the outpatient section. Statistical Analysis -------------------- We applied the same analysis undertaken in the original English validation study \[[@B3]\], taking for granted the same a priori assumptions. Data analysis was performed using SPSS version 12.0 (SPSS Inc, USA). Data are expressed as median (range) unless otherwise stated. CCQ internal consistency was evaluated by calculating Cronbach\'s alpha coefficient (for the three domains and the total). Non-parametrical testing (Mann-Whitney U test) was used to determine the discriminant validity of the CCQ to differentiate between healthy subjects and COPD patients with different degrees of airways obstruction (mild, moderate, severe, very severe). Spearman\'s rank correlations were used to examine convergent and divergent validity. Test-retest reliability analysis was done by calculating the Intra-class Correlation Coefficient (ICC). Responsiveness was tested using Wilcoxon U test. A value of p \< 0.05 was considered as statistically significant. Results ======= Score distributions ------------------- The distributions for all domains and the overall scores were skewed. In the population study, 12 subjects (7%) scored optimally (= 0) in the total score, whereas 87 subjects (50%) scored optimally in the mental state domain. In the COPD group (120 subjects), 3% of the patients scored optimally in the total score, whereas 35% scored optimally in the mental state. Internal consistency -------------------- Cronbach\'s alpha was 0.89 for the total score. Internal consistencies of symptom, functional state and mental state were 0.71, 0.88 and 0.80, respectively. Discriminant validity --------------------- Healthy subjects had significantly lower (better) CCQ score only in the symptom domain (p = 0,05) compared with the small number (6 subjects) of patients with mild COPD (FEV~1~pb \>= 80% predicted). At the same time, this small group did not differ significantly (all CCQ scores) from the group (34 patients) with moderate COPD (FEV~1~pb \< 80% and \>= 50% predicted). For this reason, Table [1](#T1){ref-type="table"} shows CCQ scores in subgroups of healthy, mild-moderate COPD, severe COPD, and very severe COPD subjects. Healthy subjects had significantly lower CCQ scores than patients with mild-moderate COPD with respect to total score (p = 0.001), symptom domain (p = 0.000), mental state domain (p = 0.005), except functional state domain. Patients with mild-moderate COPD had better CCQ values compared with patients with severe COPD, with respect to total score (p = 0.041), functional state (p = 0.017), mental state (p = 0.037), except symptom domain. Patients with severe COPD had lower CCQ scores than patients with very severe COPD, with respect to total score (p = 0.007), functional state domain (p = 0.003), symptom domain (p = 0.032) except mental state domain. The healthy subjects group had a significantly (p = 0.003) lower (better) MRC score than patients with mild-moderate COPD. Patients with severe COPD had a significantly (p = 0.003) higher (worse) MRC score than patients with mild-moderate COPD. Patients with very severe COPD had a significantly (p = 0.000) worse MRC score compared with those with severe COPD (Table [1](#T1){ref-type="table"}). We did not find (Table [1](#T1){ref-type="table"}) any significant difference in BMI between healthy and diseased subjects and among patients with increasing airways obstruction. In agreement with Celli BR et al. \[[@B10]\], we considered 21 as a cut-off BMI value for COPD patients\' clinical control. Table [2](#T2){ref-type="table"} shows the data of 120 patients with COPD subdivided into three different classes: subjects having BMI \<= 21 (low-range), BMI \<= 28 (acceptable-range) and BMI \> 28 (high-range). In these three groups, no significant difference was found for FEV~1~pb % predicted, age and MRC score. CCQ scores were higher in both low and high BMI groups, with respect to the acceptable BMI range group. CCQ scores did not indicate any significant difference between acceptable-range and high-range groups, except in the CCQ mental state domain (p = 0.02). On the other hand, there was a statistically significant difference between low-range BMI and acceptable-range BMI groups for CCQ total (p = 0.01), CCQ symptom (p = 0.01), CCQ mental state (p = 0.04) except CCQ functional state. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Characteristics and results of 120 patients in subgroups by BMI ::: BMI \<= 21 BMI \<= 28 BMI \>28 ------------------------- ----------------------- ------------------------ ----------------------- N 15 66 39 Males (%) 60.0 78.8 66.7 Age (yr) 71^abc^(42--86) 72^abc^(41--86) 71^abc^(50--82) BMI (kg/m^2^) 19.7 (16.2--20.8) 25.0 (21.3--27.8) 29.9 (28.0--37.8) FEV~1~/FVC (%) 39.0^abc^(21.1--64.8) 49.3^abc^(23.6--68.0) 50.3^abc^(27.5--68.2) FEV~1~(% predicted) 43.5^abc^(18.9--68.1) 44.5^abc^(19.5--117.1) 40.8^abc^(16.4--85.9) MRC functional dyspnoea 1.8 ± 1.4^abc^(1--4) 1.4 ± 0.4^abc^(0--4) 1.7 ± 0.9^abc^(0--4) CCQ symptom 2.5 (0.3--5.8) 1.3^a^(0.0--4.0) 1.5^a^(0.0--5.0) CCQ functional state 2.3^a^(0.0--5.0) 1.3^ab^(0.0--5.3) 1.9^ab^(0.0--4.5) CCQ mental state 2.0 (0.0--5.5) 0.5 (0.0--6.0) 1.5 (0.0--6.0) CCQ total 2.2 (0.4--5.2) 1.2^a^(0.0--4.3) 1.7^a^(0.0--4.6) BMI = body mass index. FVC = forced vital capacity. FEV~1~= forced expired volume in one second. CCQ = Clinical COPD Questionnaire. MRC = Medical Research Council. Medians not sharing a common superscript (a,b,c) are significant different at p \< 0.05 after Mann-Wittney U test. ::: Convergent and divergent validity --------------------------------- The CCQ score showed significant correlations with all SF-36 components except the pain component(Table [3](#T3){ref-type="table"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Correlations between CCQ, SF-36, FEV~1~and functional dyspnoea ::: CCQ Symptom CCQ Functional state CCQ Mental state CCQ Total ---------------------------- ----------------- -------------------------- ---------------------- --------------- SF-36 Physical functioning -0.51\\ -0.78\\ -0.45\\ -0.72\\ SF-36 Social functioning -0.36\* -0.40\\ -0.40\\ -0.43\\ SF-36 Role physical -0.34\* -0.38\\ -0.43\\ -0.43\\ SF-36 Role emotional -0.31\* -0.30\* -0.39\* -0.36\* SF-36 Mental health -0.35\* -0.47\\ -0.54\\ -0.48\\ SF-36 Vitality -0.47\\ -0.58\\ -0.44\\ -0.57\\ SF-36 Pain -0.15 -0.23 -0.05 -0.20 SF-36 Health perceptions -0.56\\ -0.58\\ -0.49\\ -0.64\\ MRC functional dyspnoea +0.52\\ +0.64\\ +0.44\\ +0.63\\ FEV~1~(% predicted) -0.51\\ -0.50\\ -0.51\\ -0.57\\ SF-36 = Medical Outcome Survey Form-36 (higher score indicates better health status); FEV~1~= forced expired volume in one second; MRC = Medical Research Council functional dyspnoea; \p \< 0.05; \\p \< 0.01, Spearman\'s rank correlation. ::: The CCQ scores and the FEV~1~% predicted values correlated significantly with respect to the whole population, the highest correlation (Figure [1](#F1){ref-type="fig"}) being that of CCQ total score (rho = -0.57; p \< 0.01). The correlation (rho = -0.41) was highly significant (p \< 0.01) even if only the group of 120 COPD patients is considered. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Correlation between CCQ and FEV1 %predicted in 175 subjects.CCQ = Clinical COPD Questionnaire. FEV~1~= forced expired volume in one second. ::: ![](1477-7525-3-9-1) ::: The functional dyspnoea MRC score correlated strongly with CCQ total (rho = 0.63), functional state (rho = 0,64), symptom (rho = 0.52) and mental state (rho = 0.44). No significant correlation was found between BMI and all the CCQ scores. Test-Retest Reliability and Responsiveness ------------------------------------------ The intra-class correlation coefficient was 0.99 for the overall CCQ score. In table [4](#T4){ref-type="table"} we see the results concerning responsiveness to change of the CCQ, as tested in 46 COPD patients undergoing pulmonary rehabilitation. The group\'s CCQ scores significantly (p = 0.000) improved after three weeks of pulmonary rehabilitation in hospital. A statistically significant (p = 0.000) improvement was found also for walked distance and Borg breathlessness rating at the end of 6 MWT. At the same time, no significant change was found for the FEV~1~pb %. After two successive weeks of individualized home rehabilitation there was a worsening of CCQ scores compared with the scores when hospital discharge took place. Nevertheless, CCQ scores were still significantly better than in baseline condition (hospital admission) for total (p = 0.01), functional state (p = 0.01), symptom (p = 0.02) and mental state (p = 0.03). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Changes of CCQ scores in 46 patients submitted to pulmonary rehabilitation ::: ---------------------- ---------------- ------------------ ------------------ Baseline* HospitalR HomeR CCQ functional state 2.2 (0.5--5.0) 1.5 (0.0--4.7)\* 1.7 (0.0--4.7)\* CCQ symptom 1.8 (0.2--4.7) 1.3 (0.0--3.5)\* 1.7 (0.0--4.2)\* CCQ mental state 2.0 (0.5--4.0) 1.0 (0.0--3.5)\* 1.5 (0.0--4.5)\* CCQ total 2.0 (0.0--3.9) 1.3 (0.0--3.8)\* 1.7 (0.0--4.0)\* Distance\_walked (m) 264 (104--380) 306 (156--459) \-\-\-\-- Borg end-walking 2 (1--9) 1 (0--8) \-\-\-\-- ---------------------- ---------------- ------------------ ------------------ CCQ = Clinical COPD Questionnaire, HospitalR = end of hospital pulmonary rehabilitation, HomeR = during home rehabilitation. Borg = breathlessness rating scale. \*p \< 0.05 after Wilcoxon U test (HospitalR or HomeR versus Baseline) ::: Discussion ========== The validated Italian version of SF-36 was used as an instrument to measure the convergent validity of the clinical COPD questionnaire. Moderate to high correlations were found in the present study supporting the convergent validity in the Italian version, reflecting the original English development and validation study \[[@B3]\]. FEV~1~was used to measure divergent validity with the same a priori assumption behind the original English version (range from -0.20 to -0.4). The correlation was stronger than expected also in the Italian version, concerning the whole population study and the COPD population alone. In addition to the FEV~1~, also MRC functional dyspnoea has proved to be useful in predicting outcomes in patients with COPD, thus MRC functional dyspnoea measurement is recommended in the routine handling and evaluation of these patients \[[@B1]\]. In the present study, both MRC score and CCQ scores values were able to discriminate healthy subjects and COPD patients with different degree of airways obstruction (from mild-moderate to very severe). We had the opportunity of testing the correlation between CCQ scores and MRC score and it was statistically highly significant. We found no significant difference, as far as the CCQ functional state is concerned, between healthy subjects and mild-moderate patients with COPD. This reflects COPD guidelines \[[@B1]\], which state that restrictions in daily living activities only become significantly apparent once the FEV~1~falls below 50% predicted, i.e., as a result of the transition from mild-moderate to severe airways obstruction in patients with COPD. BMI calculation has also proved to be useful in the routine handling of patients with COPD \[[@B1]\]. In the present study, BMI does not differ significantly between healthy and diseased subjects and among groups of COPD patients with different degrees of airways obstruction. According to Celli BR et al. \[[@B10]\], BMI \<= 21 is associated with poor prognosis in patients with COPD. Therefore, this condition can be considered an indication of less than optimal clinical control in patients with COPD. In our study, CCQ scores were able to discriminate the patients with COPD and BMI \<= 21 in the COPD population. The relation between BMI and CCQ scores in our COPD population is non-linear, since scores tend to be worse with both decreasing BMI values below 22 and increasing values above 28. This trend, which is statistically significant only in the low BMI range, explains the non-significant overall correlation that was found between BMI values and CCQ scores in patients with COPD. Only the CCQ mental state score is significantly worse in the overweight group, compared with the acceptable BMI range group. This would suggest the presence in these subjects of emotional problems, possibly related also to overfeeding. We have been able, by means of the CCQ scores, to detect significant changes in response to the inpatient portion of a comprehensive and continuous standard pulmonary rehabilitation program for patients with COPD. Disease control improvement is also documented with independent outcome measurements of variables at the end of 6 MWT. It is a well-known fact that improvements in clinical disease control and health status occur with pulmonary rehabilitation, despite a minimal effect on pulmonary function measurement, i.e., FEV1 % predicted \[[@B1],[@B7],[@B11]\]. The present study wishes to validate the Italian language version of the CCQ questionnaire; it does not intend to validate the optimal duration of a time-limited pulmonary rehab program. The GOLD guidelines \[[@B2]\] state that there is type B scientific evidence for two-month duration of a time-limited pulmonary rehab program in patients with COPD. In our clinical practice, we have never succeeded in obtaining the compliance of patients with stable COPD over such a long pulmonary rehab hospitalization period. The comparison of our data with the results presented in the original CCQ article \[[@B3]\] show similar CCQ scores as far as the healthy subjects group is concerned (total score \< 1). A separate comparison for severe and very severe groups of patients with COPD was impossible since the original CCQ article \[[@B3]\] presents data in these patients, grouped according to the classification criteria available in January 2003. Indeed, the classification of patients into different groups has been changed from one based upon the relation between airways obstruction and clinical features (respiratory failure or clinical signs of heart failure) \[[@B12]\] into another based upon airways obstruction alone \[[@B1],[@B2]\]. However, we excluded from the study the patients with signs of heart failure or acute respiratory failure. In our study, a separate comparison between mild and moderate COPD was impossible, since patients with mild COPD seldom refer to our specialized outpatient clinic. Furthermore, in our clinical setting, we have been unable to find any subject presenting symptoms of COPD in the absence of airways obstruction (subjects who risk developing COPD). We believe such patients are more typical in a general practice setting, as is the case in the original CCQ development and validation study \[[@B3]\]. Conclusions =========== The clinical COPD questionnaire is the first to have been specifically developed and validated to measure clinical control in patients with COPD in general practice \[[@B3]\]. The validation of the questionnaire, in Italian and in specific pulmonary disease clinical practice, confirms strong discriminative properties, test-retest reliability and responsiveness. Furthermore, the CCQ scores are highly correlated with the usual functional dyspnoea MRC scale and are able to discriminate COPD patients with already known poor prognosis according to the critical BMI index. Authors\' contributions ======================= SD designed the study, analyzed the results, performed the statistical analysis and drafted the manuscript. SP participated to the study design and organization, collected and elaborated the SF-36 data and helped to draft the manuscript. CB-VF-CR-RR-FR participated in the organization of the study, in the individual subjects clinical selection and in the results discussion. All Authors read and approved the final manuscript. Acknowledgements ================ This study was funded by University Milano-Bicocca, Italy	PubMed Central	2024-06-05T03:55:52.884728	2005-2-7	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549036/", "journal": "Health Qual Life Outcomes. 2005 Feb 7; 3:9", "authors": [ { "first": "Salvatore", "last": "Damato" }, { "first": "Chiara", "last": "Bonatti" }, { "first": "Vinicio", "last": "Frigo" }, { "first": "Silvana", "last": "Pappagallo" }, { "first": "Rita", "last": "Raccanelli" }, { "first": "Claudio", "last": "Rampoldi" }, { "first": "Francesco", "last": "Rodi" } ] }
PMC549037	Background ========== ADAMTS (A Disintegrin-like and Metalloprotease with Thrombospondin motifs) proteins have homology with the metalloprotease region of the ADAM proteases, but also have at least one of the Thrombospondin type 1 Sequence Repeat (TSR) motifs that are common in extracellular matrix proteins. Since the discovery of a gene encoding ADAMTS1 in 1997 \[[@B1]\], a total of 19 similar genes have been found in the human genome \[[@B2]\], numbered ADAMTS1-20; there is no ADAMTS11because early reports of an ADAMTS11\[[@B3]\] were later found to describe ADAMTS5. Many of these genes have been implicated in a variety of diseases, including connective tissue disorders \[[@B4]\], cancer \[[@B5]-[@B7]\], osteoarthritis \[[@B3],[@B8]\], and possibly Alzheimer\'s disease \[[@B6],[@B9]\]. Recently, an autosomal recessive form of Weill-Marchesani syndrome (WMS) has been attributed to null mutations of the ADAMTS10gene \[[@B10]\]. The symptoms characteristic of this syndrome (short stature, brachydactyly, joint stiffness, and anomalies of the eye lenses), together with its expression patterns, suggest a role for the gene encoded by this protein in normal growth and in skin, eye, and heart development. ADAMTS proteins are characterized by a pro-domain, a metalloprotease domain, the so-called disintegrin-like and spacer domains, and a tail of TSR repeats. The pro-domain of ADAMTS1 and -4 is cleaved at the RX(K/R)R furin cleavage site \[[@B11]\] in the Golgi \[[@B12],[@B13]\], releasing an active protein \[[@B14]\]. There are clearly conserved furin cleavage sites for most human ADAMTS proteins (positions 578--581 of the alignment) \[[Additional File 2](#S2){ref-type="supplementary-material"}\]. While this site was less well conserved in ADAMTS10 and ADAMTS12, the pro-domain of ADAMTS12 was also shown to be removed by a furin-mediated process \[[@B7]\]. On the basis of this combined evidence, it is commonly believed that furin cleavage of the pro-domain might occur for all ADAMTS proteins. The metalloprotease domain of ADAMTS proteins is shared with the related ADAM proteins, and the catalytic Zn2+-binding motif HEXGHXXXXXHD \[[@B15]\] is well conserved, shown at amino acid positions 761--772 \[[Additional File 2](#S2){ref-type="supplementary-material"}\]. While the metalloprotease domain of ADAM proteins is followed by a disintegrin domain which binds integrins at a conserved X(D/E)ECD site \[[@B16],[@B17]\], the corresponding amino acids in the disintegrin-like domain of ADAMTS proteins are not well conserved. We also note that the so-called spacer domain following this disintegrin-like domain (amino acids 1060--1400) \[[Additional File 2](#S2){ref-type="supplementary-material"}\] in fact has many highly conserved residues, despite its comparatively reduced overall conservation of amino acid sequence. There are four matrix metalloprotease (MMP) cleavage sites in the spacer domain of ADAMTS1 \[[@B14],[@B18]\], including the highly conserved IPAGA site at amino acid positions 1229--1233 \[[Additional File 2](#S2){ref-type="supplementary-material"}\] (L. Iruela-Arispe, personal communication). Further proteolytic processing within this domain has been demonstrated for ADAMTS1, -2, -5, and -12 \[[@B3],[@B6],[@B7],[@B19]\]. For ADAMTS1, this second proteolytic step is mediated by several MMPs, and results in removal of the C-terminal TSRs that interact with the extracellular matrix (ECM). This leads to release of the protein from the endothelial cell membrane, reducing its ability to inhibit endothelial cell proliferation and probably reducing its anti-angiogenic potential \[[@B14]\]. Release of ECM-bound proteins viaproteolytic removal of their TSR domains may be a common theme, as we see similar proteolytic removal of the C-terminal TSRs of the unrelated neuronal guidance protein F-spondin by plasmin, releasing it from ECM binding \[[@B20]\]. While the exact mechanism of the proteolytic processing of ADAMTS proteins remains somewhat controversial, there is an intriguing possibility that regulation of the ratio of ECM-bound vs. free ADAMTS protein could be mediated by MMPs. The region containing these sites is conserved to varying degrees in the newly discovered ADAMTS proteins, suggesting variable (perhaps tissue-specific) MMP processing of these proteins. ADAMTS4, which lacks a TSR tail, may not have an ECM-bound form. The region between the metalloprotease domain and the TSR repeat tail was demonstrated to be necessary for gon-1, a Caenorhabditis ADAMTShomolog, to mediate cell migration during gonadogenesis \[[@B21]\]. A variant of this region that lacks the conserved amino acids upstream of the classic TSR but maintains the highly conserved spacer residues is found in papilin, where it has been implicated in influencing cell rearrangements during organogenesis \[[@B22]\] and in the Manduca sextalacunin protein which plays a role in basal lamina remodeling during morphogenesis \[[@B23]\]. It will be interesting to investigate whether there is to be a common theme of organogenesis function among proteins that contain this configuration of domains. There is evidence that several mammalian ADAMTS proteins are expressed organogenesis. For example, mutations in the mouse ADAMTS20gene have been found to cause the belted white-spotting mutation, resulting from a defect in melanocyte development or migration during embryogenesis \[[@B29]\], the ADAMTS1 protein is necessary for mouse gonadogenesis \[[@B30]\], ADAMTS12 is specifically expressed in fetal lung \[[@B7]\], and several of the newly described ADAMTS proteins \[[@B28]\] are expressed solely or primarily in fetal tissue. ADAMTS proteins contain a single \"classic\" TSR motif (WXXWXXW) in the disintegrin-like domain, and a variable number of variant TSRs within the C-terminal tail of the protein, which contain 4 amino acids between tryptophan residues (W4XW) rather than two. TSRs can be divided in several structural groups, based on the presence and spacing of cysteines \[[@B24]\]. The precise function of each type of TSR has not yet been determined, although it is known that the sequence CSVTCG in one of the thrombospondin-1 TSR\'s mediates endothelial cell apoptosis through binding to CD36 \[[@B25],[@B26]\]. About 70 proteins in the human genome contain TSRs \[[@B27]\] and many of them are matrix binding proteins. ADAMTS1 and -8 inhibit angiogenesis \[[@B31]\], and gene expression profiling suggests that ADAMTS4 also has a role in angiogenesis \[[@B32]\]. Several of these proteins (ADAMTS1, -4, and -5) have also been shown to cleave aggrecan, the proteoglycan that makes cartilage elastic and compressible \[[@B19],[@B33],[@B34]\], and ADAMTS4 was recently shown to cleave cartilage oligomeric matrix protein (TSP5) \[[@B35]\]. The ADAMTS2, -3, and -14 proteins appear functionally related. ADAMTS3 is a procollagen II N-propeptidase, and ADAMTS14 appears also to be an aminoprocollagen peptidase \[[@B36]\]. ADAMTS2 is an aminoprocollagen peptidase of procollagen I and II, and deficiency of this protein causes Ehlers-Danlos syndrome type VIIC \[[@B4]\]. ADAMTS13 is a von Willebrand factor-cleaving protease. Mutations in the ADAMTS13gene result in inappropriate platelet activation, leading to the blood disorder thrombotic thrombocytopenic purpura (TTP) \[[@B37]-[@B40]\]. Recently, an intriguing link has been discovered between ADAMTS metalloproteases. The proinflammatory cytokines IL17 \[[@B41]\], IL1β \[[@B42]\] and TGFβ \[[@B43]\] induce expression of ADAMTS4. TGFβ also induces ADAMTS2\[[@B44]\] and TNFα was found to up-regulate ADAMTS1, ADAMTS6, and ADAMTS9in ocular cells \[[@B45]\], suggesting a role for these proteases in inflammatory eye disease. Similarly, TNFα produced a marked induction of ADAMTS1in endothelial cells \[[@B46]\]. As several ADAMTS proteins, ADAMTS4 in particular, are implicated in rheumatoid arthritis, and TNFα inhibitors have been recently been used with great success in its treatment \[[@B47]\], we speculate that part of the effect of the TNFα inhibitors is an indirect downregulation of the ADAMTS proteins that break down connective tissues. As TNFα inhibitors are not without inherent risks \[[@B48],[@B49]\], transcriptional inhibition of specific ADAMTSgenes may ultimately provide similar benefits with fewer risks. To better understand the multiple functions of the ADAMTS proteins, we carried out the most detailed and comprehensive analysis to date of the phylogenetic relatedness and intron/exon organization of all human ADAMTSgenes, including their comparison with invertebrate and chordate ADAMTShomologs. Prior analyses included fewer species and did not address the sequence of genomic events that resulted in the current ADAMTSgenomic structure \[[@B2],[@B28],[@B50]\]. Our analysis reveals distinct sub-families with unique functions and reveals a history of gene duplications, retrotransposition, and the loss and gain of introns during animal evolution. For example, ADAMTS1, -4, -5, -8, and -15genes all derive from a retrotransposition event that occurred prior to the divergence of vertebrates and the urochordate Ciona intestinalis, and from subsequent gene duplications that occurred prior to the divergence of mammals and the pufferfish, Fugu rubripes. This ADAMTS protein subfamily encompasses proteins that share aggrecanase and angiogenesis-related activities. Results & Discussion ==================== Gene discovery -------------- To perform a phylogenetic analysis of the ADAMTS gene family we first examined several genome databases to have a comprehensive survey of all ADAMTS genes in humans and other species. When we started this analysis in 2002 only 12 ADAMTS proteins were known. We predicted coding sequences of the human ADAMTSgenes based on several sequence databases (see Material and Methods) and found them to be identical to those subsequently published by Cal, et al\[[@B28]\] based on cloned cDNAs. This work confirmed that a total of 19 ADAMTS genes exist in the human genome, with various isoforms. Since there were several cases in which alternative splicing or different exon predictions resulted in ADAMTS proteins of varying lengths, the most complete (longest) translations were considered in our analyses, and their Genbank accession numbers are indicated \[[Additional File 2](#S2){ref-type="supplementary-material"}\]. ADAMTS9has three known splice variants, of which the long variant that we used for analysis was NM\_182920. ADAMTS20has two known splice variants, of which we used NM\_025003. ADAMTS18has two known variants, of which we used NM\_199355, minus the final exon. ADAMTS13has four known variants, of which we used NM\_139025. ADAMTS10and ADAMTS6each have a single known coding sequence, and we found evidence of others. The variant of ADAMTS6that is published (NM\_014273) is a short form, and contains a non-consensus exon immediately following the metalloprotease catalytic site, while the variant of ADAMTS10that is published (NM\_030957) has a consensus exon at this location, and is in the long form. We predict a long form of ADAMTS6, as well as a consensus exon for ADAMTS6and a non-consensus exon for ADAMTS10. We used the consensus exons of both genes in our analyses. Phylogenetic analyses --------------------- We compared the 19 known human ADAMTS protein sequences with ADAMTS homologs from invertebrates (Drosophilaand Caenorhabditis) from which entire genome sequences were recently determined, to elucidate their evolutionary relationships (Figure [1A](#F1){ref-type="fig"} and [1B](#F1){ref-type="fig"}) \[[Additional File 1](#S1){ref-type="supplementary-material"}\]. This revealed a series of gene duplications among human ADAMTSgenes, of uncertain affinity to these invertebrate relatives. With the goal to further elucidate this gene duplication history, the human and invertebrate ADAMTSorthologs were compared with ADAMTSorthologs from Mus, Fuguand Ciona, which diverged between humans and invertebrates, and with an additional invertebrate, the honeybee, Apis mellifera(Figure [2](#F2){ref-type="fig"}) \[[Additional File 2](#S2){ref-type="supplementary-material"}\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Phylogenetic analysis of human ADAMTS proteins and invertebrate homologs. Unambiguously aligned amino acids were analyzed by distance (Protdist+NJ), maximum parsimony (MP) and maximum likelihood (ML) methods. The trees shown are the ML distance topologies. Numbers at the nodes represent the percent of bootstrap replicates of distance (NJ) and parsimony (MP), and the percent of quartet puzzling steps (QP) in support of each group. (A) Phylogenetic tree of human and distantly related invertebrate ADAMTS homologsinferred from a 359-amino acid alignment, with α = 1.42 and proportion of invariable sites (pI) = 0.09. (B) Phylogeny of human and invertebrate ADAMTS homologs with long branches removed,inferred from 543 aligned amino acids, with α = 1.48 and proportion of invariable sites (pI) = 0.10. For reference, Genbank GI numbers for the sequences are provided \[[Additional File 2](#S2){ref-type="supplementary-material"}\]. ::: ![](1471-2148-5-11-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Phylogenetic analysis of animal ADAMTS homologs.This is the consensus maximum likelihood tree topology determined from 900 trees with the highest posterior probabilities inferred by Bayesian analysis of protein sequences. 571 aligned amino-acid sites were analyzed, with mean α = 1.59 (1.38 \< α \< 1.83), pI = 0.10 (0.07 \< pI \< 0.13) and lnL = -37875.26. Numbers at nodes represent Bayesian posterior probabilities for that relationship, with the best-supported posterior probabilities (1.00) represented by bullets (•). The percent of 1000 bootstrap replicates in support of the nodes, as found by distance and parsimony analyses, are also reported. Accession numbers and scaffold numbers for sequences are provided \[[Additional File 2](#S2){ref-type="supplementary-material"}\]. ::: ![](1471-2148-5-11-2) ::: To perform this analysis we inferred the coding sequence of sixteen ADAMTS proteins in the Fugu rubripesgenome \[[@B52]\], and drew from Genbank nine representative mouse orthologs, three orthologs from the Drosophilagenome and four from Caenorhabditis(see Figures [1A](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}). Other Mus(and Rattus) ADAMTSorthologs that were unannotated at the time of our initial work have since been identified by others \[[@B51],[@B53]\], and some are annotated in the MEROPS database\[[@B82]\]. However, they were not included for our final analyses presented here since they are so similar to the human sequences (69--99% identical, \[[@B53]\]) that they offered no help in elucidating the gene duplication history. The invertebrate genomes were surveyed extensively for additional ADAMTSgenes, and those most closely related to the vertebrate ADAMTSorthologs were retained in the analyses presented herein. The most divergent Drosophilaand CaenorhabditisADAMTS homologs represented in Figure [1A](#F1){ref-type="fig"} were removed from further analyses in attempt to avoid systematic bias known as \"long branch attraction\" where divergent but putatively unrelated sequences group together because of their divergence rather than due to shared characters \[[@B54]\]. All ADAMTS proteins introduced here contain the same basic domain structure as previously described ADAMTS proteins. A complete alignment of all human and invertebrate ADAMTS protein sequences, and representative Ciona, Fuguand Musorthologs, annotated with intron positions and phases, is available \[[Additional File 2](#S2){ref-type="supplementary-material"}\]. Intron position and phase ------------------------- We compared the positions of introns and their phases between Homo, Fugu, Ciona, Drosophilaand Caenorhabditishomologs of ADAMTS genes, in attempt to corroborate and further elucidate their evolutionary relationships, as shown in Figure [3](#F3){ref-type="fig"}. The term intron phase refers to the position of the splice site with respect to the codon, where phase 0 describes a splice site 5\' of the codon, phase 1 describes a splice site between the first and second base of a codon, and phase 2 describes a splice site between the second and third base of a codon. Introns at the same position that have the same phase in homologous genes are considered to be shared characters that were conserved during evolution. The lack of an intron at a conserved position may either suggest that the gene lost an intron at that position during its evolution, or that it never had that intron, and the intron conserved in the other homologs was gained after those homologs diverged from the intron-lacking homolog. In combination with the phylogenetic analysis of the ADAMTS protein sequences, a parsimonious interpretation of the data summarized in Figure [3](#F3){ref-type="fig"} that invokes the fewest changes should help to distinguish between older and more recent gene duplication events in this gene family. Three of our most striking observations of the intron distribution are that (i) some intron positions are shared between worm, fly, chordate and vertebrates, (ii) recently duplicated genes share similar patterns of introns, and that (iii) the complete absence of ancient introns and the presence of introns at new positions in ADAMTS1, 4, 5, 8and 15of vertebrates and Cionareveals that this subgroup of genes evolved by retrotransposition early during chordate evolution, was repopulated by new introns (in some cases, separately in vertebrates and Ciona), and subsequently underwent gene duplication during the evolution of vertebrates. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### The phase and position of introns in ADAMTS genes support the phylogeny of ADAMTS proteins.Intron positions 16 -- 87, found in the conserved region of the multiple alignment, are numbered consecutively from the 5\'\>3\' locations in the ADAMTS-coding regions of genes. The presence of an intron is indicated according to its phase (0, 1, 2), absence of an intron indicated by \"-\" and missing data is blank. Unambiguously aligned intron positions are highlighted in bold, and conserved intron positions shared between chordates and DrosophilaCG4096 or CG6107 or chordates and Caenorhabditisare indicated by \$, \# or \* symbols respectively. The phases and positions of introns summarized here are also individually highlighted in the multiple sequence alignment \[[Additional File 2](#S2){ref-type="supplementary-material"}\]. ::: ![](1471-2148-5-11-3) ::: The phylogenetic analysis of animal ADAMTS homologs reveals that proteins that are known to have similar biological activities are closely related, and that they have evolved by a series of gene duplication events (Figure [2](#F2){ref-type="fig"}). Since the functions of only some ADAMTS proteins have been empirically tested, estimates of evolutionary relatedness amongst the entire family may imply closer functional relatedness, and thus guide the future design of more specific empirical tests of protein functions. An interesting property of the vertebrate ADAMTS proteins are the extensive sequence similarity between many pairs of sequences, as indicated in Figures [1](#F1){ref-type="fig"}, [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"} \[and [Additional file 2](#S2){ref-type="supplementary-material"}\]. Although in many cases little is known about the functions of these proteins, we can speculate that the two proteins in each pair may share similar biological activities due to their shared primary sequence. It is also possible that these ADAMTS proteins act as heterodimers, in a manner similar to the ADAM proteins fertilin α and β \[[@B55]\]. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Proposed scenario for the evolutionary history of ADAMTS proteins.During chordate evolution a series of gene duplications resulted in six ADAMTS proteins present in the Cionagenome, while an early retrotransposition event gave rise to the \"angiogenesis clade\" of ADAMTS proteins. This proliferation of ADAMTS proteins did not occur in invertebrates, and there is evidence of the loss of one ADAMTS ortholog from Caenorhabditis. More recent duplications that occurred early during vertebrate evolution resulted in the paired sets of ADAMTS proteins present in the human genome. The chromosomal locations of the human ADAMTS genes are indicated in parentheses and the exon structure of each human gene is diagrammed to the right of its position in the schematic phylogenetic tree, and shown in more detail in the Additional Files. ::: ![](1471-2148-5-11-4) ::: As shown in Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"} and summarized in Figure [4](#F4){ref-type="fig"}, animal ADAMTShomologs have undergone several gene duplications. Assuming that the Ecdysozoa hypothesis is true and arthropods and nematodes are united as a group \[[@B56]-[@B62]\], our results indicate that a single ADAMTSgene duplication preceded the divergences of Ecdysozoa and chordates. At least 3--4 ADAMTSgene duplications occurred in chordates prior to the divergence of Cionaand vertebrates, followed by additional gene duplications in vertebrates prior to the divergence of Fuguand mammals (Figures [2](#F2){ref-type="fig"} and [4](#F4){ref-type="fig"}). Ciona intestinalis, the urochordate sea squirt, was found to have at least six ADAMTSgenes (Figure [2](#F2){ref-type="fig"}), which correspond to six of the seven major groups of vertebrate ADAMTS homologs. CionaADAMTS6 is the sister of the group comprised of vertebrate ADAMTS6 and -10, indicating that ADAMTS6 and -10 evolved by gene duplication early during vertebrate evolution, preceding the divergence of pufferfish and mammals, but after their divergence from urochordates. Similarly, CionaADAMTS16 is a sister to the group comprised of vertebrate ADAMTS16 and -18, CionaADAMTS7 is a sister to the group comprised of vertebrate ADAMTS7 and -12, CionaADAMTS3 is a sister to the group comprised of vertebrate ADAMTS2, -3 and -14, CionaADAMTS9 is a sister to the group comprised of vertebrate ADAMTS9 and -20 and CionaADAMTS15 is the sister to the group comprised of vertebrate ADAMTS1, -4, -5, -8 and -15. This reveals that both gene duplications early in chordate evolution as well as subsequent gene duplications early during vertebrate evolution have contributed to the proliferation of ADAMTS genes studied in growing depth in mammalian model systems. ADAMTS2, -3, and -14have been recognized as evolutionarily closely related genes, encoding proteins with a common functionality as procollagen aminopeptidases. They are as a group most closely related to a single gene in Ciona, and appear to have evolved by gene duplications that occurred prior to the divergence of pufferfish and mammals but after the divergence of urochordates and vertebrates. They are most closely related to ADAMTS13, suggesting a gene duplication from a common ancestor (Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}). ADAMTS13appears to have originated early in vertebrate evolution as it has a closely related homolog in the pufferfish Fugu rubripesbut is apparently absent in Ciona, fly and worm genomes. The pufferfish ADAMTS13homolog is not only closely related at the amino acid sequence level, but also has the same ADAMTS13-specific intron/exon structure in its tail, which is unique among the ADAMTSgene family. This is in agreement with a unique function for ADAMTS13 as a protease cleaving von Willebrand factor, leading to abnormal blood clotting. Although the mouse ADAMTS13gene was not included in this analysis, it has been identified (Genbank accession number NM\_001001322). A second evolutionarily related group is comprised of ADAMTS1, -4, -5, -8 and -15and their single sister in Ciona. Vertebrate members of this group share unique intron positions and lack all of the intron positions held by other ADAMTSgenes and their invertebrate homologs (Figure [3](#F3){ref-type="fig"}). Three members of this group (ADAMTS1, -4, and -8) encode proteins with aggrecanase and angiogenesis-related functions, which suggests the examination of ADAMTS5 and -15 for similar biological activities. This putative \"angiogenesis/aggrecanase group\" appears most closely related to ADAMTS20and -9. Further, the unique intron positions shared by ADAMTS1, -4, -5, -8, and -15, and lack of invertebrate orthologs in this putative \"angiogenesis/aggrecanase group\" suggest that this group\'s progenitor arose within chordates viaa retrotransposition event from the common ancestor of the group comprised by ADAMTS20and -9(Figures [2](#F2){ref-type="fig"} and [4](#F4){ref-type="fig"}). The intron/exon structures of ADAMTS1, -4, -5, -8, and -15are similar to that of the mouse ADAMTS1gene \[[@B63]\], and our analysis shows four ADAMTSgenes with this characteristic gene structure in the genome of F. rubripes. Therefore, retrotransposition of an ancestor of the angiogenesis/aggrecanase subfamily of genes, its acquisition of new introns, and subsequent gene duplications that produced five related genes occurred prior to the divergence of human, mouse and pufferfish lineages. In at least one case (intron 17 in ADAMTS8) we see evidence of acquisition of a new intron following the process of duplication, but prior to the divergence of mammals and pufferfish. ADAMTS20and -9are most closely related and, along with the members of the angiogenesis/aggrecanase clade, are most closely related to invertebrate gon-1(Caenorhabditis) and CG6107 (Drosophila). The finding that ADAMTS9and -20together as a group have a single sister gene in Cionaindicates that they evolved by gene duplication in the vertebrate lineage, after their divergence from urochordates. However, the results of our phylogenetic analyses demonstrate that neither ADAMTS9nor ADAMTS20can alone be rightfully dubbed as being orthologous to gon-1, as has been recently proposed \[[@B64],[@B65]\]. In fact, our analyses suggest that while gon-1and CG6107 are likely orthologs, the chordate ortholog of these genes was the common ancestor of Ciona ADAMTS9and -15, i.e.also the common ancestor of the later-duplicated vertebrate ADAMTS genes ADAMTS9, -20, -15, -5, -8, -4, and -1(Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}). Only one invertebrate sequence (DrosophilaCG4096) was found that grouped with the remainder of the human ADAMTShomologs. The placement of this gene with or within a group of these remaining ADAMTSgenes would suggest the number of gene duplication events in this gene family that occurred prior to the divergence of vertebrates and invertebrates from a common ancestor. The Kishino-Hasegawa test revealed that the likelihood of DrosophilaCG4096 being most closely related to an ancestor of the group of all remaining mammalian ADAMTS proteins, or of the various groups nested within it, was not significantly different from the likelihood that DrosophilaCG4096 is most closely related to ADAMTS 7and -12(data not shown). The most parsimonious explanation of this result is that a single gene duplication of an ancient ADAMTS homolog occurred early during the evolution of animals, prior to the divergence of chordates from invertebrates, followed by lineage-specific gene loss and gene duplications (Figure [4](#F4){ref-type="fig"}). If this scenario is correct, the Caenorhabditisortholog of DrosophilaCG4096 has been subsequently lost, but the vertebrate ortholog has been retained and underwent several gene duplications within and among chromosomes in the vertebrate lineage (ADAMTS2, -3, -6, -7, -10, 12--14, 16--19). The exons comprising the TSR tail each contain a single variant TSR, with the C(S/T)XCG motif 5\' and the W4XW motif at the 3\' end. This exon structure may facilitate the formation of alternately spliced isoforms, such as we describe here, but it would also lend itself to duplication or loss of individual repeats. However, the number of variant TSRs in the tails of these proteins has been conserved for the gene pairs ADAMTS17and -19, ADAMTS6and -10, and ADAMTS18and -16(Figure [4](#F4){ref-type="fig"}). While this may suggest a series of relatively recent gene duplications, a more likely explanation is that each TSR has an important and non-redundant role, or that the presence of a specific number of TSRs is critical for each protein\'s function. The apparent absence of any ortholog of ADAMTS17or -19in the pufferfish genome (Figure [2](#F2){ref-type="fig"}), but their presence in Musand Rattus\[[@B51]\] suggests that this gene duplication either occurred in mammals after they diverged from fish, or that ADAMTS17or -19evolved earlier than the mammal/fish divergence but were lost in Fugu. Gene duplication is a common way by which new genes with similar functions may evolve. In fact, duplication of large segments of chromosomes has been a common occurrence during animal evolution \[[@B66],[@B67]\]. Both the phylogenetic trees and the intron/exon structures of ADAMTSgenes show a history of such duplications. In addition to the similarity in intron positions of ADAMTS-1, -4, -5, -8, and -15in both mouse and pufferfish, human ADAMTS1and -5are located in tandem on chromosome 21, and human ADAMTS8and -15, on chromosome 11 (Figure [3](#F3){ref-type="fig"}). The remaining genes have additional introns at conserved locations, and both proteins in each set of pairs have the same intron/exon structure. Thus, by combining the phylogenetic analysis and intron/exon structure determinations, we are able to propose the following series of events leading to the ADAMTS protein family (Figure [4](#F4){ref-type="fig"}): (i) The ancestral ADAMTSgene duplicated prior to the divergence of the ecdysozoan and chordate lineages, approximately 673 million years ago \[[@B68]\]. (ii) In the following approximately 250 million years prior to divergence of fish and mammals \[[@B69]\], multiple gene duplications occurred. (iii) A retrotransposition of the common ancestor of the ADAMTS9and -20gene pair resulted in an intronless gene that proceeded to gain multiple introns, giving rise to the angiogenesis/aggrecanase clade. (iv) This gene was involved in a duplicative chromosomal inversion, and later a duplication of the chromosomal segment containing both ADAMTSgenes. (v) Another intron was gained, in ADAMTS8, prior to the divergence of the pufferfish and mammalian lineages. In the other branch of the ADAMTSfamily, we see a remarkable frequency of genes located on chromosome 5, suggesting it as the location of the ancestor of these genes. We can speculate that a similar scenario of within-chromosome duplication followed by duplication of chromosomal segments took place, although none are in as close physical proximity as the ADAMTS1-subfamily genes. Conclusions =========== This comprehensive bioinformatic survey of the human genome affirms the widely held belief, derived from experimental work, that the nineteen known human ADAMTS proteins constitute the complete gene family. By examining both the amino acid sequences using rigorous phylogenetic methods and comparing the exon structure of these proteins, we were able to draw conclusions about the evolutionary history of this family of proteins which, in turn, provides a framework for further analysis of the functions of these clinically-relevant genes. Methods ======= Gene discovery -------------- Sequences of ADAMTShomolog sequences presented here were identified from 2001 to 2004 using genomic sequences cataloged in the NCBI, JGI (Fuguand Ciona\[[@B52],[@B70]\]) and Celera genome databases using BLASTp, BLASTn, BLASTx, tBLASTn and tBLASTx searches \[[@B71]\]. We searched databases of Expressed Sequence Tags (ESTs) for all human genes and, with the exception of ADAMTS15, found ESTs that corresponded to those genes, confirming their transcription. Splice site predictions were made by neural network viathe Berkeley Drosophila Genome Project and also by eye using Sequencher 4.2 (Genecodes), with reference to protein multiple sequence alignments. We used the same method to identify Caenorhabditis elegans, Drosophila melanogaster, and Fugu rubripeshomologs of ADAMTSgenes, and their genomic structure. Multiple sequence alignments of the inferred amino acid sequences were prepared using Multalin \[[@B72]\] and ClustalX1.81 \[[@B73]\] and manually refined and annotated within BioEdit \[[@B74]\] and MacClade4.06 \[[@B75]\]. Phylogenetic analysis --------------------- Initial phylogenetic analyses were conducted including all of the human ADAMTS proteins along with seven invertebrate homologs, three from Drosophila melanogasterand four from Caenorhabditis elegans(Figure [1A](#F1){ref-type="fig"}), and then these analyses were repeated with the most divergent invertebrate sequences removed (Figure [1B](#F1){ref-type="fig"}). The datasets for these analyses were comprised of 359 and 543 unambiguously aligned amino acid sites, respectively. The alignments were analyzed using parsimony and distance methods. Parsimony analyses used a heuristic search with random stepwise addition of data and tree-bisection-reconnection in PAUP\4.0b10 for 1000 bootstrap replicates \[[@B76]\]. Distance matrices were inferred using the Jones, Taylor, Thornton (JTT) substitution model with PROTDIST in PHYLIP 3.6a3 \[[@B77],[@B78]\]. Neighbor-joining trees were constructed with the input order jumbled for 1000 bootstrap replicates using NEIGHBOR, SEQBOOT and CONSENSE in PHYLIP. Using Tree-Puzzle 5.0 \[[@B79]\] we generated maximum-likelihood distance matrices in which among-site substitution rate heterogeneity was corrected using an invariable and eight gamma-distributed substitution rate categories and the JTT model. 10,000 quartet-puzzling steps were also used (Tree-Puzzle) to assess branch support. To better resolve the evolutionary relationships of the vertebrate ADAMTSsubfamily members and to provide information on their presence in other chordate lineages, sequences from Mus, Fuguand Cionawere identified and added to the alignment and phylogenetic analysis. An annotated ADAMTS homolog from the honeybee Apis melliferawas also included in the analysis as another representative invertebrate. 571 unambiguously aligned amino acid sites of ADAMTS-homologous sequences encoded by Homo, Mus, Fugu, Ciona, Apis, Drosophilaand Caenorhabditiswere analyzed (Figure [2](#F2){ref-type="fig"}). MrBayes3.0 \[[@B80]\] was used to construct a maximum likelihood (ML) phylogenetic tree from this protein alignment. MrBayes was run for 1000000 generations, with four incrementally heated Markov chains, and a sampling frequency of 1000 generations. The temperature setting was increased to 0.5. Among-site substitution rate heterogeneity was corrected using an invariable and eight gamma-distributed substitution rate categories and the WAG model for amino acid substitutions \[[@B81]\], abbreviated herein as WAG+I+8Γ. The consensus ML tree topology, the arithmetic mean log-likelihood (lnL) for this topology, and branch support were estimated from the set of sampled trees with the best posterior probabilities. Means and 95% confidence intervals for the gamma distribution shape parameter α and the proportion of invariable sites (pI) were also estimated. List of abbreviations ===================== ADAMTS (ADisintegrin-like and Metalloprotease with Thrombospondin motifs) ADAM (ADisintegrin-like and Metalloprotease) TSR (Thrombospondin type 1 Sequence Repeat) MMP (matrix metalloprotease) ECM (extracellular matrix) SAGE (serial analysis of gene expression) TTP (thrombotic thrombocytopenic purpura) IL (interleukin) TGF (transforming growth factor) TNF (tumor necrosis factor) BLAST (Basic local alignment search tool) Authors\' contributions ======================= ACN and EGVM initiated the project and all authors were involved in the design phases. ACN, SBM and JML inferred sequences of previously un-annotated ADAMTS genes from Genbank, Celera, and JGI, and determined intron positions. SBM performed the phylogenetic analyses. ACN and SBM drafted the manuscript and JML and EGVM contributed to writing the paper and advised throughout. Supplementary Material ====================== ::: {.caption} ###### Additional File 2 Alignment used for phylogenetic analyses of animal ADAMTS homologs.Unambiguously aligned amino acid sites, indicated by the \"mask\" line in the alignment, were used for phylogenetic analyses (Figure [2](#F2){ref-type="fig"}). Accession (GI) numbers for sequences are provided. Intron positions in the corresponding genomic sequence are indicated, with color-coding for intron phases (Figure [3](#F3){ref-type="fig"}). ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 1 Alignment used for phylogenetic analyses of human ADAMTS proteins and invertebrate homologs.Unambiguously aligned amino acid sites, indicated by the \"mask\" line in the alignment, were used for phylogenetic analyses (figure [1](#F1){ref-type="fig"}). Intron positions in the corresponding genomic sequence are indicated, with color-coding for intron phases (Figures [3](#F3){ref-type="fig"} and [4](#F4){ref-type="fig"}). ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ ACN was supported by NIH training grant T32-NS07480. SBM was supported by an Emory Graduate Fellowship, an Avis Cone Fellowship (Iowa), and JML\'s grant. JML was supported by a grant from the NSF (MCB-0216702). EGVM was supported by grants from the NIH (CA86335 and CA87830). Fugu rubripesand Ciona intestinalis*data are from the Joint Genome Institute databases.	PubMed Central	2024-06-05T03:55:52.886835	2005-2-4	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549037/", "journal": "BMC Evol Biol. 2005 Feb 4; 5:11", "authors": [ { "first": "Ainsley C", "last": "Nicholson" }, { "first": "Shehre-Banoo", "last": "Malik" }, { "first": "John M", "last": "Logsdon" }, { "first": "Erwin G", "last": "Van Meir" } ] }
PMC549038	Background ========== The proliferation of astrocytes is stimulated by polypeptide growth factors such as PDGF, EGF, bFGF and IGF-1 acting on cellular signaling pathways which involve tyrosine kinases, protein kinase C, and the Ras-Raf-MAP kinase pathway \[[@B1],[@B2]\]. Astroglial proliferation is also stimulated by neurotransmitters such as acetylcholine and glutamate \[[@B3],[@B4]\], by direct stimulation of protein kinase C with phorbol ester \[[@B5],[@B6]\], and by peptides such as endothelin and prolactin \[[@B7],[@B8]\]. Astroglial proliferation is prominently inhibited by ethanol both in vivo and in vitro \[[@B9]-[@B11]\], and this interference likely contributes to the development of the fetal alcohol syndrome (alcoholic embryopathy) (reviewed in \[[@B12]\]). Ethanol has been shown to potently antagonize proliferative effects of several individual astroglial mitogens including PDGF, IGF-1, acetylcholine and prolactin \[[@B8],[@B13]-[@B15]\]. The molecular target of ethanol\'s antimitogenic actions in astroyctes is not known with certainty, but inhibitory interactions of ethanol with lipid signaling pathways have been implicated \[[@B15]\]. Our group has recently reported strong evidence that the growth-inhibitory effect of ethanol in astrocytes is caused by the disruption of the phospholipase D (PLD) signaling pathway \[[@B16],[@B17]\]. Under physiological conditions, PLD catalyzes the hydrolysis of phosphatidylcholine (PC) to yield phosphatidic acid (PA) and choline. In the presence of ethanol, however, PLD forms phosphatidylethanol (PEth), a non-physiological phospholipid, at the expense of PA. This PLD-specific phenomenon of transphosphatidylation is the reason why downstream events mediated by PLD activation and PA formation are dose-dependently inhibited in the presence of ethanol (or other primary alcohols such as 1-butanol). In our previous work, we have found that astroglial PLD is activated by mitogenic factors including fetal calf serum (FCS), PDGF, and phorbol ester, and we observed that ethanol reduced both astroglial proliferation and PA formation in a parallel manner. 1-butanol reduced PA formation and DNA synthesis with the same potency while t-butanol was inactive for both effects \[[@B16]\]. More recently, we demonstrated that exogenous PLD as well as PA, when introduced into the cytosol by transient permeabilization, stimulated astroglial cell proliferation. Importantly, the action of PLD was suppressed in the presence of ethanol (0.3 %, v/v) while the mitogenic effect of PA was not affected \[[@B17]\]. Thus, disruption of the PLD signaling pathway by ethanol is sufficient to suppress astroglial cell proliferation. Recent findings from other groups are also compatible with a central role for the PLD signaling pathway in ethanol toxicity in astrocytes. First, several mitogenic factors including those that are known to be particularly sensitive to ethanol activate PLD activity in astrocytes. This holds true for PDGF \[[@B16]\], acetylcholine \[[@B5],[@B18]\], glutamate \[[@B19]\], phorbol esters \[[@B5],[@B6]\], endothelin \[[@B20],[@B21]\], and prolactin \[[@B22]\]. In fact, disruption of PLD signaling by ethanol was recently found to be responsible for ethanol\'s inhibitory effect on astroglial DNA synthesis induced by muscarinic agonist \[[@B23]\]. Second, PLD is activated via the mitogenic Ras-Ral pathway in many cell types \[[@B24]\], and PA, the immediate product of PLD activity, interacts with and activates proteins such as Raf kinase, protein kinase Cζ, and mTOR which are known to be central to mitogenic signaling (reviewed in \[[@B25],[@B26]\]). In addition, PA is a precursor of diacylglycerol (DAG), the endogenous activator of classical PKC\'s, and of lyso-PA, a potent mitogen in many cell types \[[@B25],[@B26]\]. Taken together, current evidence suggests that intact PLD signaling is a prerequisite for the proliferative effects of several mitogens, and that disruption of the PLD pathway by ethanol may be a common theme in ethanol-induced inhibition of astroglial proliferation. The present study in fetal astrocytes was motivated by recent reports that ethanol induces apoptosis in astrocytes, an effect that was accompanied by activation of the sphingomyelinase pathway and formation of ceramide \[[@B27],[@B28]\]. Apoptosis denotes an active cellular program causing cellular death upon contact with toxicants. Apoptotic cell death in the CNS has been under intensive study in recent years and involves several intracellular reaction cascades linked by the activation of caspases (reviewed in \[[@B29]\]). Apoptosis is almost universally accompanied by the formation of ceramide which may occur through de novo-synthesis, inhibition of ceramide breakdown, or activation of (acidic and/or neutral) sphingomyelinase (SMase), an enzyme which catalyzes the hydrolysis of sphingomyelin to ceramide and phosphocholine (see \[[@B30]\] for review). Ceramide has emerged as a second messenger for apoptotic pathways targeting kinases and phosphatases which are required for the execution of apoptotic cell death (reviewed in \[[@B31],[@B32]\]). In cerebellar astrocytes and in glioma cells, ceramide levels were found to be reciprocally related to cell proliferation \[[@B33],[@B34]\]. For the present study, we developed the hypothesis that the formation of ceramide may be secondary to the inhibition of PLD signaling which we had described earlier (see above). We now report that ethanol-induced ceramide formation in astrocytes is mimicked by 1-butanol, but not by t-butanol, and that PA, the product of PLD activity, antagonizes ethanol-induced formation of ceramide. We also found that ceramide is a potent inhibitor of stimulated PLD activity. Thus, we obtained evidence of a cross-talk between PA and ceramide, two lipid messengers with opposite effects on cellular proliferation. Results ======= Markers of apoptosis -------------------- When primary astrocyte cultures were exposed to ethanol (0.3--1%, v/v), staining of the cells with Hoechst 33258, a dye intercalating into DNA, revealed condensation and fragmentation of the nuclei which was visible after 16 hrs; the maximum effect was observed after 21 hours (Fig. [1A](#F1){ref-type="fig"}). Higher magnification demonstrated the presence of \"apoptotic bodies\" in the nuclei (Fig. [1B](#F1){ref-type="fig"}). A similar effect was observed after treatment of the cells with the well-known apoptogen, staurosporine (1 μM), or with C~2~-ceramide (50 μM) but not with t-butanol (1%, v/v) (not illustrated). In parallel experiments, ethanol caused fragmentation of nuclear DNA in serum-starved astrocytes which is reflected by \"DNA laddering\" on agarose electrophoresis (Fig. [2](#F2){ref-type="fig"}). Serum withdrawal alone was not effective while incubation with C~2~-ceramide (50 μM) mimicked the effect of ethanol. Ethanol at 0.3% (v/v) was almost as effective as 1 % (Fig. [2](#F2){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### [Nuclear fragmentation of astrocytes after treatment with ethanol.]{.underline} In this experiment, astrocytes were incubated with ethanol (1 %, v/v) for 21 hours, fixed in methanol/acetic acid (3:1) and stained with bisbenzimide (Hoechst 33258, 1 μg/ml). The characteristic condensation and fragmentation of nuclei indicates apoptosis. Enlargement: left picture, 400 fold; right picture, 1,000 fold. The experiment was repeated three times with similar results. ::: ![](1471-2210-5-3-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### [DNA fragmentation in astrocytes after treatment with ethanol.]{.underline} Astrocytes were incubated with the compounds given: lane 1, control (serum-free medium); lane 2, serum-containing medium; lane 3, C~2~-ceramide (50 μM) in serum-free medium; lanes 4--7, ethanol in serum-free medium (concentrations and times as given); lane 8, size markers. After incubation, cells were lysed, DNA was purified, separated on a 3 % agarose gel and stained with ethidium bromide. The experiment was repeated three times with identical results. ::: ![](1471-2210-5-3-2) ::: Effects of ethanol on sphingomyelin hydrolysis ---------------------------------------------- Formation of \[^3^H\]-ceramide was measured after labeling sphingomyelin with \[^3^H\]serine. Under basal conditions, the ratio of \[^3^H\]-ceramide to \[^3^H\]-sphingomyelin (\"C/S ratio\") was approximately 1:30. This ratio was not significantly changed during serum withdrawal (Figs. [3](#F3){ref-type="fig"} and [4](#F4){ref-type="fig"}). The incubation of astrocytes with ethanol (1 %, v/v) in serum-free medium caused an increase of \[^3^H\]-ceramide (cpm per dish) but did not significantly change the total labeling of the large pool of \[^3^H\]-sphingomyelin by \[^3^H\]-serine (data not shown). As the total incorporation of \[^3^H\]-serine into \[^3^H\]-sphingomyelin was somewhat variable between individual preparations, we used the C/S ratio to calculate ethanol-induced changes. As shown in Figs. [3](#F3){ref-type="fig"} and [4](#F4){ref-type="fig"}, ethanol caused a significant increase of the astroglial C/S ratio in a biphasic and dose-dependent manner. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### [Formation of ceramide in ethanol-treated astrocytes: time course.]{.underline} Astrocytes were labeled with \[^3^H\]-serine for 72 hours, washed and treated with ethanol (0.3 %, v/v) in serum-free medium. At the indicated time points, the cells were extracted with methanol/chloroform (2:1), lipid extracts were separated by TLC, and radioactivity associated with \[^3^H\]-ceramide and \[^3^H\]-sphingomyelin was determined by liquid scintillation counting. Data (N = 3--7) are means ± S.E.M. and are expressed as \[%\] ceramide/sphingomyelin. Statistics: ANOVA, F~1,53~= 2.28, p = 0.02. \, p \< 0.05 vs. control at time zero (Dunnett\'s post test). ::: ![](1471-2210-5-3-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### [Formation of ceramide in ethanol-treated astrocytes: concentration dependence.]{.underline} Astrocytes were labeled with ^3^H-serine for 72 hours, washed and treated with ethanol (0.1--1 %, v/v). After (A)1 hour and (B)18 hours, the cells were extracted with methanol/chloroform (2:1), phospholipids were separated by TLC, and the radioactivity associated with ceramide and sphingomyelin was determined by liquid scintillation counting. Data (N = 5--6) are means ± S.E.M. and are expressed as \[%\] ceramide/sphingomyelin. Statistics: one-way ANOVA for repeated measurements, (A) F~3,19~= 3.98, p = 0.03; (B) F~3,23~= 4.88, p = 0.02. \, p \< 0.05 vs. controls (Dunnett\'s post test). ::: ![](1471-2210-5-3-4) ::: The rapid and transient phase of ceramide formation occurred within 15 min and reached a maximum at 1 hour after addition of ethanol (1 h value without ethanol: 2.93 ± 0.35%; 1 h value with 0.3% ethanol: 3.92 ± 0.60%; p = 0.02). A second increase gradually developed after 4 hours and reached a maximum at 18 hours of ethanol exposure (18 h value without ethanol: 3.46 ± 0.41 %; 18 h value with ethanol: 4.18 ± 0.51 %; p = 0.005). At this later time point, staurosporine (1 μM) caused an increase of the C/S ratio to 7.97 ± 1.78 % (p \< 0.01; not illustrated). Inhibition of ceramide formation by PLD activity and phosphatidic acid ---------------------------------------------------------------------- To investigate whether ceramide formation is secondary to a disruption of PLD signaling, we used the isomeric alcohols, 1-butanol and t-butanol. 1-Butanol -- but not t-butanol -- is a substrate of PLD for transphosphatidylation and leads to the formation of phosphatidyl-1-butanol at the expense of PA. The correlations between butanol exposures and disruption of PLD signaling (i.e., suppression of PA formation) in astrocytes were documented in detail in our previous work \[[@B16]\]. In the present experiments we measured the differential effects of 1- and t-butanol on the formation of ceramide (C/S ratio) in astrocytes. As shown in Fig. [5](#F5){ref-type="fig"}, there was a tendency for an increased level of the C/S ratio after addition of 1-butanol at 0.1% while highly significant increases were observed with 0.3%. In contrast, t-butanol (0.1 and 0.3%) had no effect. This pattern was identical at the 1 hour and 18 hours time points (Fig. [5A](#F5){ref-type="fig"} and [5B](#F5){ref-type="fig"}). This pattern suggested a change of the C/S ratio that was secondary to the disruption of the PLD pathway. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### [Effects of isomeric butanols on ceramide formation in astrocytes.]{.underline} Astrocytes were prelabeled with \[^3^H\]-serine for 72 hours, washed and treated with 1-butanol (\"1-But\") or t-butanol (\"t-But\"). After (A)1 hour and (B)18 hours, the cells were extracted with methanol/chloroform (2:1), phospholipids were separated by TLC, and the radioactivity associated with ceramide and sphingomyelin was determined by liquid scintillation counting. Data (N = 8--10) are means ± S.E.M. and are expressed as \[%\] ceramide/sphingomyelin. Statistics: Repeated measures ANOVA, (A) F~4,49~= 7.2, p = 0.0002; (B) F~4,39~= 25.0, p \< 0.0001. \, p \< 0.05; \\, p \< 0.01 vs. controls (\"Ctr\"). \#, p \< 0.05; \#\#, p \< 0.01 vs. effect of 1-butanol (Tukey-Kramer multiple comparisons test). ::: ![](1471-2210-5-3-5) ::: To obtain more direct evidence for this hypothesis, we tested the influence of exogenous PA, the product of PLD, on the C/S ratio. For this purpose, we used a permeabilization procedure which makes use of an oxygen-insensitive mutant (C530A) of streptolysin-O (SL-O) to introduce the membrane-impermeable PA into the astroglial cytosol \[[@B17]\]. In preliminary experiments, we found that transient permeabilization with SL-O for 15 min by itself did not affect \[^3^H\]-ceramide levels (data not shown). Basal ceramide levels were also unchanged if exogenous PA was added to the astrocytes in the absence (data not shown) or presence of SL-O (Fig. [6](#F6){ref-type="fig"}). However, the ceramide formation evoked by ethanol (0.3 %) was significantly reduced in the presence of PA (Fig. [6](#F6){ref-type="fig"}). At the 1 h timepoint, the ethanol-induced effect (C/S ratio: 5.14 ± 0.53 %) was reduced by PA pretreatment to 3.98 ± 0.33 %, a relative reduction by 71 percent (Fig. [6A](#F6){ref-type="fig"}). At the 18 h timepoint, PA pretreatment reduced ethanol-induced ceramide formation by 61 percent (Fig. [6B](#F6){ref-type="fig"}). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### [Effects of phosphatidic acid on ceramide formation in astrocytes.]{.underline} Astrocytes were prelabeled with \[^3^H\]-serine for 72 hours, washed and treated with PA (200 μM) or ethanol (EtOH, 0.3 % v/v) during transient permeabilization with streptolysin-O (144 ng/ml) in calcium-free medium. After 15 min, the cultures were washed and re-exposed to calcium-containing medium to initiate pore repair. After (A)1 hour and (B)18 hours, the cells were extracted with methanol/chloroform (2:1), phospholipids were separated by TLC, and the radioactivity associated with ceramide and sphingomyelin was determined by liquid scintillation counting. During the experiments, PA was only present for 15 min during cell permeabilization whereas ethanol was present throughout the incubation period. Data (N = 9) are means ± S.E.M. and are expressed as \[%\] ceramide/sphingomyelin. Statistics: Repeated measures ANOVA, (A) F~3,35~= 5.52, p = 0.005; (B) F~3,35~= 14.5, p \< 0.0001. \\, p \< 0.01 vs. controls (\"Ctr\"). \#, p \< 0.05; \#\#, p \< 0.01 vs. effect of ethanol (Tukey-Kramer multiple comparisons test). ::: ![](1471-2210-5-3-6) ::: Inhibition of phospholipase D activity by ceramide -------------------------------------------------- As the previous experiments indicated an inhibitory effect of the PLD pathway on ceramide formation, the following experiment tested a possible effect of ceramide on PLD activity measured by the transphosphatidylation assay. In these experiments, the membrane-permeable C~2~-ceramide (50 μM) slightly but non-significantly reduced basal PLD activity by 30% (Fig. [7](#F7){ref-type="fig"}). However, when PLD activity was stimulated by addition of serum (Fig. [7A](#F7){ref-type="fig"}) or by PDB, a phorbol ester and stimulator of protein kinase C (Fig. [7B](#F7){ref-type="fig"}), C~2~-ceramide at both 10 and 50 μM strongly and significantly reduced PLD activity. Interestingly, serum-induced PLD stimulation was more sensitive to ceramide than PDB-induced PLD activity; the relative inhibitions for 10 and 50 μM C~2~-ceramide were (for serum stimulation) 84 and 93 % and (for PDB stimulation) 53 and 64 %, respectively. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### [Inhibition of phospholipase D activity by ceramide.]{.underline} PLD activity in serum-starved, \[^3^H\]-glycerol-labeled astrocytes was determined by the transphosphatidylation assay; in the presence of ethanol, PLD converts \[^3^H\]-phosphatidylcholine (PC) into \[^3^H\]-phosphatidylethanol (PEth) which reflects PLD activity. In (A), PLD activity was stimulated by addition of medium containing 10% fetal calf serum (FCS) for 5 min. In (B), PLD activity was stimulated by 4β-phorbol-12β,13α-dibutyrate (PDB; 1 μM) for 30 min. C~2~-ceramide (\"C~2~\") was added to the cultures in concentrations of 10 and 50 μM 45 min before the addition of FCS and PDB, respectively. Data are means ± S.E.M. of 7--8 experiments and are expressed as \[%\] PEth/PC. Statistics: ANOVA, (A) F~4,36~= 14.0, p \< 0.0001; (B) F~4,37~= 75.2, p \< 0.0001. \\, p \< 0.01 vs. controls. \#\#, p \< 0.01 vs. stimulated PLD activity (Tukey-Kramer multiple comparisons test). ::: ![](1471-2210-5-3-7) ::: Discussion ========== We have previously documented that ethanol suppresses signaling through the mitogenic phospholipase D (PLD) pathway, and we and others have provided evidence that this effect may be responsible for the antiproliferative actions of ethanol in astrocytes \[[@B16],[@B17],[@B23]\]. The present study was motivated by findings that ethanol induces astroglial apoptosis via activation of the sphingomyelinase pathway \[[@B27],[@B28]\]. We confirm these earlier reports by showing the induction of apoptotic markers and ceramide formation in ethanol-treated astrocytes. The novel finding of our study is that ethanol-induced formation of ceramide is reciprocally regulated by phosphatidic acid (PA) and the phospholipase D pathway which is itself inhibited by ceramide. We used three different approaches to demonstrate that apoptotic cell death in astrocytes can be induced by exposure to ethanol. First, we report that ethanol can induce nuclear condensation and degradation (Fig. [1](#F1){ref-type="fig"}). Second, application of ethanol to serum-starved astroglial cultures caused \"DNA laddering\", a typical hallmark of apoptotic degradation of nuclear DNA (Fig. [2](#F2){ref-type="fig"}). The effect of ethanol was mimicked by a cell-permeable ceramide, C~2~-ceramide (Fig. [2](#F2){ref-type="fig"}), and by staurosporine (not illustrated). Third, ethanol induced an increase of ceramide in astroglial cultures (Figs. [3](#F3){ref-type="fig"} and [4](#F4){ref-type="fig"}). Our findings clearly confirm that ethanol can induce apoptosis and ceramide formation in astrocytes, a finding which is in agreement with some \[[@B28]\] but not all \[[@B35]\] previous studies. We performed a time course of \[^3^H\]-ceramide formation after ethanol exposure and found that it was maximal at 1 and 18 hours. Biphasic formations of ceramide such as those observed here have been described previously in a range of peripheral cell types although their significance is unclear; they may reflect different modes of ceramide formation, or different pools of ceramide \[[@B36]\]. At both time points of maximum ceramide formation (1 hour and 18 hours), we observed the same ethanol-evoked enhancement of ceramide formation. Importantly, apoptotic cell death and ceramide formation were induced by ethanol levels as low as 65 mM which corresponds to blood alcohol levels (0.3%) which are found in heavy drinkers. It should be noted that the present experiments do not unequivocally identify the mechanism of ceramide formation. We used \[^3^H\]-serine to pre-label sphingomyelin for 72 hours, removed the precursor, and measured formation of ceramide as an increase of the \[^3^H\]-ceramide/ \[^3^H\]-sphingomyelin ratio during incubations with ethanol. This ratio most likely reflects the activity of sphingomyelinase(s), and sphingomyelinase activity was actually shown to be responsible for ethanol-induced ceramide formation in a recent study \[[@B28]\]. However, our present data do not exclude alternative pathways of increased ceramide formation such as de novo-synthesis of ceramide or inhibition of ceramidase. The important findings of this study relate to the interaction between lipid second messenger pathways. It was known from previous work (see Introduction) that PA, the product of phospholipase D (PLD), mediates mitogenic stimulation in astrocytes whereas formation of ceramide by sphingomyelinase activation accompanies apoptosis. We now tested the hypothesis that ethanol causes astroglial apoptosis by inhibiting PLD and, as a consequence, stimulates the sphingomyelinase pathway. The results shown in Figs. [5](#F5){ref-type="fig"} and [6](#F6){ref-type="fig"} are evidence of a direct inhibitory influence of the PLD pathway on ceramide formation. First, we observed that the addition of 1-butanol, a primary alcohol which suppresses PLD signaling, caused an increase of ceramide levels (Fig. [5](#F5){ref-type="fig"}). This effect was not seen with the inactive isomer t-butanol which does not interfere with PLD signaling in astrocytes (see our previous study \[[@B16]\]). Second, we used transient permeabilization of astrocytes by streptolysin-O to introduce PA, the product of PLD, into the astroglial cytosol \[[@B17],[@B37]\]. We found that exogenous PA almost completely prevented the ethanol-induced increase of ceramide at early (1 hr) and delayed (18 hrs) phases of ceramide formation (Fig. [6](#F6){ref-type="fig"}). The fact that ethanol and 1-butanol, but not t-butanol increase ceramide formation, whereas PA antagonized this effect, gives strong evidence that PLD-mediated formation of PA keeps ceramide levels low under basal conditions (Fig. [5](#F5){ref-type="fig"}), and that PLD activity antagonizes ceramide formation under the influence of toxicants (Fig. [6](#F6){ref-type="fig"}). Unfortunately, we could not determine the effect of exogenous PA on astroglial apoptosis because the permeabilization procedure was found to induce a delayed apopototic response in astrocytes (not shown). We also probed the reciprocal effects of ceramide signaling on PLD activity. The results shown in Fig. [7](#F7){ref-type="fig"} demonstrate that C~2~-ceramide inhibits PLD activity at a concentration as low as 10 μM. Basal PLD activity was only slightly inhibited, but the increases of PLD activity induced by addition of serum or phorbol ester were strongly antagonized. This finding in astrocytes corroborates previous reports that ceramide can inhibit PLD signaling in peripheral cell types \[[@B38],[@B39]\]. It remains a matter of speculation why serum-induced PLD was somewhat more susceptible to inhibition by ceramide than phorbol ester-stimulated activity. Growth factors in serum and phorbol ester may affect different signaling pathways leading to PLD activation, and we previously presented inhibition data with bacterial toxins which supported this idea for astroglial PLD \[[@B40]\]. Interestingly, ethanol was also observed to inhibit serum- and growth factor-mediated astroglial proliferation more effectively than phorbol ester-induced proliferation \[[@B6],[@B16]\]. At this time, we cannot distinguish which isoform of PLD is responsible for PA formation; previous attempts to selectively down-regulate astroglial PLDs failed due to the long biological half-lives of the proteins \[[@B41]\]. The molecular target of ceramide for inhibiting PLD also remains to be identified; previous work has implicated direct inhibition of PLD by ceramide as well as upstream molecules such as protein kinase C which activate PLD \[[@B42],[@B43]\]. Conclusions =========== In summary, the present results give evidence of a cross-talk between lipid-signaling pathways in astrocytes such that the product of PLD, namely PA, inhibits ceramide formation whereas ceramide inhibits PLD activation (Fig. [8](#F8){ref-type="fig"}). The experimental evidence suggests that the ratio \"PA:ceramide\" contributes to the decision whether astrocytes proliferate or undergo apoptosis. Our data suggest that ethanol induces astroglial apoptosis during brain development by disrupting PLD signaling, thereby reducing PA and increasing ceramide formation. This effect likely contributes to the microencephaly and delay of brain development observed in fetal alcohol syndrome. ::: {#F8 .fig} Figure 8 ::: {.caption} ###### [Hypothetical cross-talk between phospholipase D and sphingomyelin pathways in astrocytes, and effect of ethanol.]{.underline} Phosphatidic acid (PA), the product of phosphatidylcholine (PC) hydrolysis by phospholipase D (PLD), inhibits hydrolysis of sphingomyelin (SM) by sphingomyelinase (SMase). Vice versa, ceramide (Cer), the product of SM hydrolysis by SMase, inhibits activation of PLD. Ethanol induces apoptosis by disrupting the mitogenic PLD signaling pathway thereby decreasing the PA:Cer ratio and disinhibiting the pro-apoptotic SMase pathway. ::: ![](1471-2210-5-3-8) ::: Methods ======= Materials --------- \[^3^H\]-Serine and \[^3^H\]-glycerol were from Biotrend (Köln, Germany). Ceramide (from bovine brain), C~2~-ceramide (N-acetyl-D-erythro*-sphingosine), and staurosporine were from Alexis (Lausen, Switzerland). Hoechst 33258, L-α-phosphatidic acid (sodium salt, from egg yolk) and 4β-phorbol-12β,13α-dibutyrate (PDB) were from Sigma (Deisenhofen, Germany); most other chemicals and TLC plates were obtained from Merck (Darmstadt, Germany) or Roth (Karlsruhe, Germany) at the highest purity available. Fetal calf serum (South America) was from Invitrogen, cell culture materials were from Sarstedt (Nürnbrecht, Germany). Phosphatidylethanol (PEth) standard was synthesized as described \[[@B16]\]. Recombinant, oxygen-insensitive streptolysin-O (C530A) was prepared as described \[[@B44]\]. Cell culture ------------ Astrocyte-rich cultures were prepared from newborn rat pups. Cerebral hemispheres were collected, meninges and blood vessels were removed, the brain tissue was dissociated by trituration, passed through a 50 μm nylon mesh, and the cells were seeded onto plastic culture dishes (14,000 cells per cm^2^). The growth medium was DMEM containing 10% fetal calf serum (FCS), 2 g/l NaHCO~3~, 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were incubated at 37°C in a 95/5% mixture of air and carbon dioxide. For the experiments, astrocytes were grown for two weeks in culture and were used when they reached confluency. As judged by GFAP immunostaining, these cultures contained \>90% astrocytes. Fluorescence microscopy ----------------------- Cells were seeded on microplates (5,000 cells per 200 μl) and incubated with different apoptogens (ethanol 0.3 and 1 %, C~2~-ceramide 50 μM or staurosporine 1 μM) for 24 hrs in serum-free medium. Subsequently, cells were washed and fixed with ice-cold methanol/acetic acid (3:1). Dried and re-hydrated cells were stained with bisbenzimide (Hoechst 33258, 1 μg/ml) solution for 10 min, washed and sealed in gelatin. Photographs were obatined using a Leica Leitz DMRB fluorescence microscope and a Nikon Digital Camera DXM. DNA fragmentation ----------------- The test was carried out as described \[[@B45]\] with some modifications. Briefly, cells were incubated with apoptogens (Ethanol 0,3%, 1%; C2-Ceramide 50 μM) in serum-free medium. Then, the cells were transferred and resuspended in Tris-EDTA buffer containing 0.5% Igepal CA 630. Further lysis was performed in buffer containing RNAse A (100 μg/μl), proteinase K (0,5 μg/ml) and SDS (1.2 %). After 5 minutes, the clear solution was mixed with 3 M CsCl in acetate buffer. Precipitated debris and chromosomal DNA was removed by centrifugation, and the supernatant was loaded onto a QIAprep column (twice), centrifuged and eluted by hypotonic Tris-EDTA buffer. The eluate was analyzed by electrophoresis on a 3 % agarose gel and visualized with ethidium bromide. Measurement of \[^3^H\]-ceramide formation ------------------------------------------ Phospholipids were labeled by addition of \[^3^H\]-serine (1 μCi/ml) to astrocytes kept in growth medium (DMEM plus FCS) for 72 h. After washing, cells were incubated in serum-free medium with ethanol, butanol, C~2~-ceramide or staurosporine (see Results). C~2~-ceramide or staurosporine were added in DMSO; the final DMSO concentration was \< 0.1 %. At the end of the incubation, cells were fixed with methanol, transferred and extracted first with with chloroform: methanol (1:1), then with chloroform: methanol: water (10:10:9) to separate water and lipid phases. After addition of ceramide and sphingomyelin standards, the lower (lipid) phase was evaporated, taken up in chloroform:methanol (3:2) and separated by thin-layer chromatography (TLC). For the determination of \[^3^H\]-ceramide, we used one-dimensional TLC (HP-TLC plates Merck 11845; eluent: chloroform/acetic acid 9:1). For the determination of \[^3^H\]-sphingomyelin, we used two-dimensional TLC (TLC plates Merck 1.05721); solvent I was chloroform/methanol/25% aqueous ammonia (13:7:1), solvent II was chloroform/ methanol/water/acetic acid (30:30:2:5). ^3^H-lipids were visualized by using iodine vapor, spots were scraped and suspended in scintillation cocktail (Lumasafe Plus), and radioactivity was measured by liquid scintillation counting (Packard 1600 CA). Formation of \[^3^H\]-ceramide was calculated as percentage of radioactivity found in \[^3^H\]-sphingomyelin. Cell permeabilization --------------------- For the introduction of PA, astrocytes were transiently permeabilized with streptolysin-O \[[@B37]\]. Briefly, astrocytes were washed and exposed to an oxygen-insensitive mutant of streptolysin-O (C530A) in calcium-free HBSS buffer (prepared by dissolving 8 g NaCl, 0.4 g KCl, 60 mg KH~2~PO~4~, 60 mg Na~2~HPO~4~× 2 H~2~O, 100 mg glucose in 1 L of sterile water). PA (sodium salt; 200 μM) was added as a suspension in buffer prepared by sonication. After 15 min, the cells were washed and incubated in serum-free, calcium-containing DMEM. Formation of \[^3^H\]-ceramide was determined as described above. Ethanol, if present, was present throughout the incubation period. Using 144 ng/ml of streptolysin-O, this procedure yielded transient permeabilization of \> 80% of astrocytes followed by repair of the pore in \> 80% of permeabilized cells. The procedure was previously found to allow the entry of approx. 10^6^molecules of PA per cell \[[@B17]\]. Determination of phospholipase D activity ----------------------------------------- Phospholipase D activity was determined using the transphosphatidylation assay \[[@B46]\]. For this purpose, astrocytes were kept in serum-free medium containing \[^3^H\]-glycerol (1 μCi/ml) for 24 h in order to label phospholipids. More than 60% of the phospholipid label was associated with phosphatidylcholine (not illustrated). Subsequently, the cells were washed and re-exposed to medium containing ethanol (2 %) and PDB (1.0 μM) or FCS (10 %, v/v) as stimulators. PDB and C~2~-ceramide, when used, were dissolved in DMSO (end concentration of DMSO \< 0.1 %). After 30 min of reaction time, the cells were washed in cold phosphate-buffered saline and extracted as described above for ceramide determinations. After addition of phosphatidylethanol (PEth) and PA standards, aliquots of the lipid phase were separated by two-dimensional TLC (TLC plate Merck 1.05721) using chloroform/methanol/25 % aqueous ammonia (13:7:1) for the first run and the upper phase of ethylacetate/isooctane/ acetic acid/water (13:2:3:10) for the second run. Individual phospholipids were stained by iodine, and the spots corresponding to PEth, PA and phosphatidylcholine (PC) were isolated and counted for radioactivity in a scintillation counter. To determine PLD activity, formation of \[^3^H\]- PEth was calculated as percentage of \[^3^H\]-PC. Statistics ---------- Data are shown as means ± SEM of N experiments whereby N refers to the number of different astroglial preparations from different animals. Results were obtained from two replicate dishes which were pooled to represent a single experiment. Statistical calculations were performed by GraphPad InStat 3.0 program package, using analysis of variance (ANOVA) of paired or unpaired data as indicated in text and figure legends. Abbreviations ============= Cer, ceramide; FAS, fetal alcohol syndrome; FCS, fetal calf serum; PA, phosphatidic acid; PC, phosphatidylcholine; PDB, 4β-phorbol-12β,13α-dibutyrate; PEth, phosphatidylethanol; PKC, protein kinase C; PLD, phospholipase D; SL-O, streptolysin-O; SM, sphingomyelin. Author\'s contributions ======================= B.S. carried out the cell culture experiments and phospholipid measurements and participated in the experiments concerning apoptotic markers. S.J. contributed substantially to the apoptotic marker experiments. K.L. participated in the design of the study and the final draft of the manuscript. J.K. conceived and supervised the study, performed the statistical analyses and drafted the manuscript. Acknowledgements ================ The authors are grateful to Dr. Ivan Walev, Department of Medical Microbiology and Hygiene, Johannes Gutenberg University of Mainz, for supplying oxygen-insensitive streptolysin-O, and to the Deutsche Forschungsgemeinschaft for financial support (Kl 598/5--3).	PubMed Central	2024-06-05T03:55:52.889743	2005-2-4	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549038/", "journal": "BMC Pharmacol. 2005 Feb 4; 5:3", "authors": [ { "first": "Beate", "last": "Schatter" }, { "first": "Shenchu", "last": "Jin" }, { "first": "Konrad", "last": "Löffelholz" }, { "first": "Jochen", "last": "Klein" } ] }
PMC549039	Background ========== Maternal morbidityrefers to complications that have arisen during the pregnancy, delivery or postpartum period. Every year an estimated 50 million women are affected by maternal morbidity. Defining, interpreting and measuring maternal morbidity, however, is recognised to be difficult and the prevalence of such morbidity (both general and specific) has been poorly described \[[@B1],[@B2]\]. Over the past decade, the nature and extent of postpartum maternal morbidity has received increasing interest in both developed and developing countries with a range of research methods of varying sophistication being used to identify long and short-term and acute and chronic morbidity following childbirth \[[@B1],[@B3]-[@B8]\] The WHO (1998) \[[@B1]\] defines the postpartum period, or puerperium, as beginning one hour after the delivery of the placenta and continuing until 6 weeks (42 days) after the birth of the infant. As the woman recovers from labour, adapts to her new role and reverts physically to her non-pregnant state, it is a special but critical time for both the mother and her infant \[[@B9]\]. Many of the complications leading to postpartum maternal morbidity arise during labour and delivery and in the first 1--2 weeks following delivery; for at least 18 million women these morbidities become long-term and are often debilitating \[[@B1]\]. Major acute obstetric morbidities include haemorrhage, sepsis and pregnancy-related hypertension. Longer-term morbidities include uterine prolapse, vesicovaginal fistulae (VVF), incontinence, dyspareunia and infertility \[[@B10]\]. Fortney & Smith \[[@B2]\] have described 6 dimensions to maternal morbidity: aetiology, severity, duration, time of onset, accumulation and sequelae. However, in many developing countries health services data on postpartum morbidity remains extremely limited. In many settings, data from hospital-based studies is hard to interpret because of the small proportion of women that have access to supervised deliveries and medical care. But, as Fortney and Smith suggest, in areas with good access to and uptake of health care the measurement of the type and incidence of postpartum complications severe enough to require hospitalisation may provide useful baseline information on the acute and severe direct morbidity women experience in the early weeks following childbirth. Zambia\'s capital city, Lusaka, offers such a context. This paper describes the application of a pragmatic and inexpensive approach to the baseline assessment of the burden of moderate-to-severe morbidity in the postpartum period in this urban African setting, with a view to monitoring trends and identifying preventable risk factors. Methods ======= A network of 23 public sector clinics and a single referral hospital, the University Teaching Hospital (UTH), comprise the public health care system for Lusaka\'s population of approximately 1.5 million. There is also a small private sector (2% of deliveries) \[[@B11]\] and there are traditional practitioners. An estimated 10.5% of deliveries occur at home. All public clinics provide antenatal care and a postnatal care service at 6 weeks after delivery. In addition, 10 of the clinics provide 24-hour care for labour and delivery and a 1-week postnatal care service. In Lusaka urban district, a recent systematically sampled community survey suggests that there is a relatively high coverage of antenatal, delivery and postnatal services (Table [1](#T1){ref-type="table"}). Women interviewees also reported good access to medical treatment for serious problems during pregnancy and in the first month postpartum. Hospital admission data, if used as a proxy for moderate-to-severe morbidity, may therefore be considered pragmatically representative of the population health needs in this setting. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Uptake of maternal health care services, Lusaka Urban community survey data \[11\] ::: Uptake of maternal health care services % of women reporting (n = 946) ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------ \% of women with 5 or more antenatal check-ups 73% Proportion of deliveries with a professional attendant 89.5% \% of women attending a postnatal check-up in the 6 weeks postpartum 84% \% of women reporting a \"serious problem\" in the antenatal or postpartum period who said that they had been able to get medical attention \"as soon as they felt they had needed it\" 85% ::: At the University Teaching Hospital, which acts as the district referral hospital for the city, admission and discharge registers are kept in each ward and department. In an earlier retrospective study, analysing referrals for all pregnancy-related complications, undertaken at UTH \[[@B12],[@B13]\], 4% of these cases were identified as referrals in the postpartum period (95/2,892 over a two- month study period). In this study, the hospital registers were used to identify all cases of postpartum morbidity presenting to UTH, and from these to estimate the incidence of, and identify the nature of postpartum morbidity severe enough to require admission for hospital-level treatment. Data collection was carried out by LV between July and September 2000. Ethical clearance for the study was obtained from the University of Zambia Research Ethics Committee. For the purpose of this study, the WHO definition of the postpartum period (from delivery until 6 weeks after delivery) was used as the time period inclusion criteria \[[@B1]\]. All women identified as having been admitted to UTH for in-patient treatment for morbidity during this period were included for the purpose of the review, whether or not their morbidity was explicitly \"obstetric\" in origin. Women who were admitted to hospital to accompany and nurse their babies that had neonatal problems were excluded. Retrospective data collection ----------------------------- Relevant admission and discharge registers at UTH were reviewed for the six-month period July-December 1999. Dependent on the type, timing and severity of a postpartum problem, women may be referred or may self-refer to one of three different units within the hospital: (i) Women with early postpartum complications, defined as problems occurring within 24 hours of delivery, are admitted through the labour ward admission room; (ii) Women with postpartum problems occurring more than 24 hours after delivery are referred or may present themselves to the gynaecology filter clinic, from where they may then be referred on to the emergency admission ward; (iii) Cases of breast abscess are generally admitted though the surgical unit. From any of the units women may be then admitted onto a longer stay gynaecology ward for further management and treatment. Women admitted to hospital irrespective of the length of stay were included in the data capture. Table [2](#T2){ref-type="table"} outlines the identification process and inclusion criteria that were used in each of the wards. The figures are likely to be an underestimate of the total number of postpartum admissions because as is often found in studies of this nature, diagnosis was frequently poorly recorded in the registers. Only women who could be positively identified as postpartum morbidity admissions were included in the final analysis. Any women admitted to medical wards with non-obstetric conditions -- such as malaria -- in the later part of the puerperium would have been missed. Using admissions registers to identify postpartum cases also excludes any women who were admitted to hospital prior to the puerperium (for example antenatally, or in labour) and subsequently developed postpartum problems requiring prolonged in patient care. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Identification of postpartum cases and inclusion criteria ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Emergency Admission Ward Gynaecology Wards Labour Ward Surgical Unit --------------------------------------------------------------- ---------------------------------------------------- ------------------------------------------------------------------------ ----------------------------------- -------------------------------------- Source of identification of cases Admission Register Discharge Register Admission Register Discharge Register Inclusion criteria to be defined as postpartum admissions All cases recorded as postpartum.\ All cases recorded as postpartum.\ All cases recorded as postpartum. All cases of breast abscess/mastitis All cases with infected caesarean section wound.\ All cases with infected caesarean section wound.\ All cases with infected episiotomy/perineal tears. All cases with infected episiotomy/perineal tears.\ Any non-obstetric conditions e.g. malaria, PTB, pneumonia, meningitis. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ::: Cases were crosschecked by name and age between the emergency admission and gynaecology wards and between the short and longer stay surgical wards, to prevent double counting. Cases of breast abscess were also crosschecked between the surgical unit and the gynaecology department to ensure cases had not been referred. Data was entered on Epi. Inf. 6.04 for analysis. Routine health service statistics for the same six-month study period (July-December 1999) were also collated. There were 19,691 deliveries within UTH and the satellite clinics (5,511 and 14,180 deliveries respectively), of which 1,021 were by caesarean section (an average of 39 per week). Women delivering either at UTH or the clinics were instructed to attend the local satellite clinic for postnatal follow-up visits at week 1 after delivery and again at 6 weeks. Some women were seen at UTH for a postnatal visit at week 1 to follow-up on some complication while those who had a caesarean section were reviewed at 6 weeks. Recorded postnatal check-up at the clinics at 1-week was 40% and 21% at 6 weeks; however, this data was incomplete for some clinics. At UTH, an average of 42 women per week attend the postnatal clinic -- most of them after a caesarean section. During the same six-month period, there were 93 maternal deaths at UTH. Of these maternal deaths 8 (8.5%) were known to be due to puerperal sepsis. A much larger number of the women who died had sepsis and stigmata of AIDS (Y Ahmed, personal communication). Prospectively identified cases ------------------------------ Due to the limitations of the routine data sources used in the retrospective review, a small cross sectional study was also conducted using prospective identification of cases in order to verify the findings. Over a 4-week period, from 14 August to 10 September 2000, all early postpartum admissions to the maternity unit at UTH were identified through the labour ward admission register. Obstetric case notes were sought and reviewed. Late postpartum cases (\>24 hours and up to 6 weeks after delivery) for the same time-period were also identified and recorded by the same means as described for the retrospective study. On the gynaecology ward, cases were identified through daily review of the ward round books, to identify new admissions, and through consultation with the senior ward sister and the ward clerk. Results ======= Retrospective data ------------------ After crosschecking of data to prevent double counting, 365 maternal postpartum admissions to the hospital were positively identified for the 6-month study period July-December 1999. Cases of retained placenta (n = 55), removed in theatre, were not included in the final analysis as it was not possible to differentiate between clinic referrals and UTH deliveries. ### Referral source and admission status Of the 365 admissions, 236 (65%) cases were identified through the emergency admission ward, 120 were identified on the gynaecology ward and the remaining 9 cases were identified on the surgical wards. More than half of the emergency admission ward cases were referred from the satellite MCH clinics (Table [3](#T3){ref-type="table"}). Two-thirds of all cases were admitted to either the gynaecology transit ward or one of the longer gynaecology wards (97 \[41%\] and 61 \[26%\] respectively). A quarter of women were discharged home later the same day after a period of treatment or observation. Six women (2.5%) were admitted to either the special observation unit or medical intensive care unit; five women (2%) were referred to the general adult \"filter clinic\". ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Sources of postpartum referrals to the emergency admission ward, University Teaching Hospital ::: Referral source Number (%) (n = 236) ---------------------------------------- -------------------------- Satellite clinics 135 (57%) Self-referral 28 (12%) Out-patient department within hospital 12 (5%) Not documented 61 (26%) ::: ### Age range The age range for all cases was 15--48 years. Of all postpartum admissions (365 women), more than half were aged between 20--29 years, a reflection of the fact that the largest number of births takes place within this age group. ### Nature of morbidities requiring hospital admission There were 39 different recorded diagnoses across the 365 cases (Table [4](#T4){ref-type="table"}). Puerperal sepsis was the most frequent diagnosis, accounting for one-third (34.8%) of all postpartum hospital admissions over the 6-month period. Infection of the reproductive tract, including infected tears and episiotomy, infected caesarean section wound and puerperal sepsis accounted for 47% of all admissions (170/365). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Identified postpartum admission by diagnosis, University Teaching Hospital, July--December 1999 ::: Diagnosis Cases (n = 365) Percent of all admissions -------------------------------------------------- --------------------- ------------------------------- Puerperal sepsis 127 34.8 Malaria 53 14.5 Infected tears/episiotomy 26 7.1 Hypertension 24 6.6 Pneumonia 22 6.0 Infected caesarean section 17 4.7 Anaemia 11 3.0 Breast Abscess 10 2.7 Symphisiotomy 9 2.5 Eclampsia 9 2.5 Puerperal Psychosis 7 1.9 \"After pains\" 6 1.6 Pulmonary Tuberculosis 5 1.4 Pyrexia (not linked to diagnosis) 5 1.4 Postpartum Haemorrhage 4 1.1 Pre-eclampsia 3 0.8 Retained products of conception 3 0.8 Urinary tract infection 3 0.8 Other (gastroenteritis, meningitis, measles etc) 21 5.8 ::: Malaria was the second most common diagnosis accounting for 14.5% of all cases. Hypertensive disorders including hypertension, pre-eclampsia and eclampsia accounted for 10.9%. ### Estimating the population burden of moderate-to-severe postpartum morbidity Using MacKeith et al.\'s \[[@B14]\] estimates for public sector coverage of deliveries (87.5%) and the UTH and clinic figures for the period (19,691 deliveries), we estimate that there were approximately 22,000 births in Lusaka during the 6-month period July-December 1999, and 365 hospital admissions for postpartum morbidity were positively identified for the same period. If hospital admission can be taken as a practical proxy measure for the moderate-to-severe end of morbidities, then the burden of moderate-to-severe postpartum morbidity in this urban African population may therefore be estimated to be at least1.7% (365/22,000 births). Because this incidence refers only to women admitted live to hospital in the postpartum period, the actual incidence will be somewhat higher, with the addition of postpartum morbidities that occurred in women who were already hospital in-patients, any maternal deaths that occurred outside of the hospital, and any missed late postpartum admissions to medical wards. Cross-sectional study of prospectively identified cases ------------------------------------------------------- Over the one-month period August-September 2000, 64 admissions to the hospital for postpartum maternal morbidity were identified. The routine hospital procedures differentiate between \"early\" (up to 24 hours postpartum) and \"late\" (after 24 hours) postpartum admissions. The former are admitted through the labour ward admissions and the latter through the emergency gynaecology ward. Twenty women (31%) were \"early\" referrals and 44 (69%) were \"late\". This categorisation of the data therefore concerns time elapsed between delivery and admission to hospital, and not between time of delivery and onset of the condition. ### Early-postpartum referrals Just over one-third (7/20) of the maternal referrals in the first 24 hours after delivery were for retained placenta and just over one-third were for pregnancy- related hypertension or eclampsia (7/20). Three referrals were for postpartum haemorrhage. There were no maternal deaths in this group. ### Late-postpartum referrals Referrals from the satellite clinics accounted for 73% of all \"late\" (\>24 hours after delivery) postpartum admissions to UTH; the remaining cases were mainly self-referral. The diagnoses of the 44 cases of late-postpartum admissions in the one-month period are shown in Table [5](#T5){ref-type="table"}. ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Late postpartum referrals (Cross-sectional study of prospectively identified cases) ::: Diagnosis Number (%) (N = 44) ---------------------------------- ------------------------- Puerperal sepsis 5 (11%) Malaria 5 (11%) Pregnancy-related hypertension 5 (11%) Infected tears/episiotomy 4 (9%) Infected caesarean section 3 (7%) Symphisiotomy 3 (7%) Puerperal psychosis 2 (4.5%) Secondary postpartum haemorrhage 2 (4.5%) Meningitis 2 (4.5%) Retained products 1 (2%) Eclampsia 1 (2%) Breast abscess 1 (2%) Other 10 (23%) ::: Twenty-one cases (47%) were admitted to the longer stay gynaecology ward. More than half of all cases (52%) were in the 20--29 years age group. Among the 44 women admitted to hospital at more than 24 hours postpartum, 4 died; 2 of these deaths occurred on the longer stay ward. Of the 4 deaths, one could be directly linked to obstetric events (anaemia); the others were related to non-obstetric causes (pneumonia, cryptococcal meningitis, and encephalitis). Discussion ========== This hospital study used time-period based inclusion criteria in its identification of cases of maternal morbidity sufficiently severe to require hospital admission. The majority of morbidity thus identified was directly linked to obstetric causes e.g. puerperal sepsis, infected wounds, and pregnancy-induced hypertension. However, non-obstetric conditions, including malaria and pneumonia were found to have accounted for at least one-fifth of all postpartum admissions in the retrospective review. This mirrors the increased role that indirect causes have been found to be playing in maternal mortality rates in countries such as Zambia \[[@B14]\]. Data accuracy ------------- We have already outlined some of the practical difficulties with using routine hospital data sources such as admission registers. Admission rates estimated from the small prospective part of the study do not suggest, however, that many cases were lost in the identification process in the larger retrospective part. The former identified an average of 11 late-postpartum maternal admissions to hospital per week in the month observed, and the latter, an average of 14 late-postpartum maternal admissions per week over the six months reviewed. In both parts of the study, puerperal sepsis and malaria were identified as leading causes of postpartum morbidity requiring hospital admission. In the prospective identification of postpartum morbidity requiring hospital admission, puerperal sepsis, malaria and hypertensive disease each accounted for the same number of admission (5 in each) but the numbers are too small to draw any conclusions. Some inaccuracy in the classification of certain morbidities may be expected due to the use of admission diagnoses. It is also a limitation in the design of this element of our study that data collection did not extend to the medical wards. It is therefore not possible for us to ascertain whether, or to what extent, there are medical ward admissions of women in the late puerperium with non-obstetric conditions such as malaria. Puerperal sepsis ---------------- Puerperal sepsis was the leading cause of postpartum hospital admissions in this population, accounting for 34.8% of all identified postpartum cases in the retrospective part of this study. Other hospital-based studies as well as surveys of women\'s self-reports of postpartum morbidity report puerperal sepsis as a leading cause of postpartum morbidity in developing countries \[[@B5],[@B6],[@B15]-[@B18]\]. Puerperal sepsis cases identified through the retrospective data collection part of the study accounted for more than twice as many cases as the second commonest postpartum morbidity requiring hospital admission, malaria. For the retrospective study the overall rate of puerperal sepsis cases requiring hospital-level care and admission was 0.64% of all supervised deliveries in the public sector services, a rate that falls in between those estimates from earlier hospital-based studies in Niger: 0.22% \[[@B5]\]; and in Nigeria: 1.7% \[[@B15]\]. However, it should again be remembered that in this study, women who delivered in hospital and developed septic complications before discharge would be excluded from this figure. The overall figure can therefore be expected to be higher. Other obstetric postpartum morbidity ------------------------------------ A number of other obstetric-related postpartum morbidities including anaemia, breast abscess, symphisiotomy, puerperal psychosis, after-pains, urinary tract infection, secondary postpartum haemorrhage and retained products of conception were also identified as late-postpartum referrals through the retrospective study. Anaemia in the postpartum period is not an uncommon health problem \[[@B1]\]. Surveys of women\'s self-reported morbidity frequently cite symptoms in the postpartum period that could be suggestive of or lead to anaemia, including chronic fatigue \[[@B19]\] and excessive bleeding \[[@B15],[@B20],[@B21]\]. Non-obstetric postpartum morbidity ---------------------------------- Malaria and pneumonia together accounted for one-fifth of all the postpartum hospital admissions that we identified. This finding suggests the usefulness of an approach that employs a \"time-period\" definition to identify cases rather than simply a set of purely obstetrically-related diagnostic categories. In all, 14.5% of identified postpartum maternal admissions to hospital were due to malaria. While it is widely recognised that the severity and frequency of malaria is greater in pregnant, compared to non-pregnant women, until recently it was generally thought that the importance of pregnancy-related malaria ends with delivery \[[@B22]\] and malaria is rarely mentioned as an important postpartum morbidity in the obstetric literature. Diagne et al.\'s study from Senegal \[[@B22]\], however, was one of the first to suggest that the increased susceptibility to malaria in pregnancy persists up to 60 days after delivery. They found that compared to the non-pregnant state, the incidence of episodes of malaria increased significantly during the second and third trimesters of pregnancy and reached a maximum during the first 60 days after delivery. A number of factors may modify susceptibility to malaria in the postpartum period. The age at which partial immunity to malaria is acquired is critically dependent on transmission intensity \[[@B23],[@B24]\]. Wide variations are seen in levels of immunity to malaria among Zambian women secondary to geographical and other factors affecting transmission \[[@B23]\]. Many of the malaria cases identified in the study may be the result of recrudescence rather than new infection particularly because the study took place during the transition between dry and wet seasons and study participants were primarily from urban and peri-urban communities. Susceptibility may also be dependent on haematological and nutritional factors as well as HIV status \[[@B25]\]. The contribution of HIV/AIDS to maternal postpartum morbidity and mortality --------------------------------------------------------------------------- Pneumonia and Pulmonary TB were important causes of postnatal morbidity in this study and were likely related to HIV/AIDS. The HIV serostatus was rarely available of the index postpartum cases, though unlinked anonymous testing of HIV in the antenatal population in 4 sentinel sites in Lusaka during 1998 showed a high HIV prevalence of 27.4% \[[@B26]\]. HIV positive women are more prone to postpartum infections including urinary tract infections, chest, episiotomy and caesarean section wound infections \[[@B27],[@B28]\]. Furthermore, in this study, causes of postpartum morbidity included puerperal psychosis, cerebral malaria and HIV related cerebral complications -- all of which can be difficult to diagnose with certainty in a malaria and HIV endemic area \[[@B29]\]. Of the 93 cases of maternal mortality during the six-month retrospective study period in 1999, almost a third of the cases were attributed to a presumptive diagnosis of HIV/AIDS and had no other direct or indirect cause of maternal mortality (personal communication, Y Ahmed, 2003). Hospital admissions in the first 24 hours following delivery ------------------------------------------------------------ Review of registers on the labour ward, for both the retrospective and prospective aspects of this study suggest that the majority of referrals in the early-postnatal period (first 24 hours) were for infant rather than maternal indications. Of the early postpartum referrals for maternal indications, retained placenta is the leading reason for referral from clinics to the hospital. This reflects the urban context of the study and the relative ease of transportation that permits a district policy of removal of placenta at hospital level rather than by the practitioner with essential obstetric care skills at the delivery clinic. Conclusion ========== The high public sector maternity care usage in this community permits the low-cost review of routine data to be reasonably meaningful. The caveats are those associated with extraction of data from health facility admission registers, which are not always complete, and which cannot take account of changes in diagnosis or subsequently arising complications. In the absence of more robust data, such reviews, if carried out meticulously, do offer the opportunity to identify the extent of moderate-to-severe postpartum morbidity and the principle causes. They thus may provide the groundwork for detailed condition-specific research to take place exploring aetiology, duration, time of onset and outcome, and the implications for health care provision. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= LV: Conceived of the study, conducted the field data collection and data analysis and contributed to the paper. SFM: Wrote the first draft of the paper, participated in the study design and advised on the data collection and analysis. YA: Contributed to the study design, advised on the data collection and analysis and contributed to the paper. All authors read and approved the final manuscript. The views expressed in this article are those of the authors and not of their institutions. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2393/5/1/prepub> Acknowledgements ================ The authors would like to thank the following for their support and input at different points during the study period: Nancy MacKeith, project officer of the \"Women Friendly Services\" Project, Lusaka District, Zambia; Dr Christine Kaseba, Head of Department, Obstetrics & Gynaecology, UTH; midwifery and nursing staff of the Obstetric & Gynaecology Department, UTH; Professor Krikor and the nursing staff from the Department of Surgery, UTH; Dr R. Kumwenda-Phiri, Director, Lusaka Urban District Management Team; Department for International Development (DFID) for its funding of the Women Friendly Services Project (Project SCF219), through which this collaborative piece of work was facilitated. However DFID can accept no responsibility for any information or views expressed.	PubMed Central	2024-06-05T03:55:52.892558	2005-2-1	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549039/", "journal": "BMC Pregnancy Childbirth. 2005 Feb 1; 5:1", "authors": [ { "first": "Lisa", "last": "Vallely" }, { "first": "Yusuf", "last": "Ahmed" }, { "first": "Susan F", "last": "Murray" } ] }
PMC549040	Background ========== Endemic HIV infection, in sub-Saharan Africa, where in many countries more than 20% of pregnant women are HIV seropositive leads to a diagnostic problem in the evaluation of their infants. Without intervention, more than 25% of infants born to seropositive women will acquire HIV infection in the first year of life. HIV testing with enzyme immunoassay-based rapid tests have expanded capacity to identify seropositive women and provide interventions, but even with single dose Nevirapine and other antiretrovirals, infection of infants still exceeds 10% in the first year of life. Serologic tests for HIV do not accurately identify those infants who have acquired infection within the first 18 months of life because of transplacentally acquired maternal IgG antibodies. As antimicrobial and antiviral interventions are developed to reduce morbidity and mortality, among infants born to seropositive women, the early diagnosis of infection is increasingly important. The gold standard for diagnosis of HIV-1 infection in infants under the age of 2 years is DNA polymerase chain reaction (PCR) or reverse transcriptase (RT)-PCR \[[@B1]-[@B3]\]. With the increasing availability of single dose nevirapine for prevention of mother-to-child transmission (MTCT) of HIV \[[@B4]\] and generic antiretroviral drugs for treatment of AIDS in resource-poor countries, there is an urgent need to develop alternative laboratory methods for early diagnosis of infant HIV-1 infection as well as identifying infants who meet the criteria for commencing cotrimoxazole prophylaxis and/or initiation of ARV therapy. The World Health Organization (WHO) recommends initiation of highly active antiretroviral therapy (HAART) in HIV-seropositive infants under the age of 18 months, who have WHO Pediatric Stage III disease and CD4 percentage (%CD4) \<20% in resource-poor countries where %CD4 are available but virologic tests (DNA PCR, RT-PCR or immune-complex dissociated p24 antigen) for confirmation of HIV infection are not available \[[@B5]\]. Thus, in infants under the age of 18 months, a single laboratory test that can identify both HIV-infection and provide %CD4, two important parameters for decision-making in initiating HAART, may be useful. Infants who are positively identified as HIV-infected and meet clinical criteria are likely to benefit from HAART. Immunological changes in HIV-1 infection include a decrease in CD4^+^cells, a transient increase in CD8^+^cells, total lymphocytes and inversion of the CD4/CD8 ratio \[[@B6],[@B7]\]. As HIV infection progresses, the CD4^+^cells decline, while the CD8^+^cells which may remain at high levels for long periods, eventually decrease but not to baseline levels. Since in healthy children the CD4^+^and CD8^+^cells account for 60% and 30% of the T lymphocytes respectively, a normal CD4/CD8 ratio should always be \>1.0. Thus, in HIV-1 infection where there is a decrease in CD4^+^cells and an increase in CD8^+^cells, the reversal of the CD4/CD8 to \<1.0 should in theory be useful for diagnosis of HIV-1 infection. Certain flow cytometers when used in tandem with a haematological analyzer can provide absolute CD4^+^cell counts, their percentages as well as the CD4/CD8 ratio. The use of the ratio in combination with %CD4, may lead to a timely identification of infected infants, who meet the WHO criteria for initiation of CTX prophylaxis and/or ARV therapy. While the infrastructure needed to conduct flow-cytometric analyses of HIV infection are still largely confined to a few centers, an increasing number of point of care diagnostic testing systems, inexpensive methods to measure CD4 cells are currently in development. These include the development and evaluation of simplified volumetric flow cytometric methods using a low cost flow cytometer that can be powered from a car battery or by solar panels (Cyflow SL, Partec, Munster, Germany) by Cassens and colleagues \[[@B8],[@B9]\] and modification of a commercially available 4-parameter flow cytometer, Luminex 100 (Luminex, Austin Texas, USA) to a compact portable prototype instrument that can operate with a 12-volt rechargeable battery \[[@B10]\]. Furthermore, use of generic CD4, CD8 and CD45 fluorescence-conjugated monoclonal antibodies can reduce the cost of determining T cell subset profile even when employing standard flow cytometers \[[@B10],[@B11]\]. The main objective of the current study was to evaluate the CD4/CD8 ratio for diagnosis of subtype C HIV-1 infection in infants under the age of 2 years among infants where DNA PCR was performed to diagnose HIV infection. Materials and Methods ===================== Study cohort ------------ The infant specimens used in this study were obtained from two independent prospective studies; the short course zidovudine (AZT) and Pediatric AIDS Clinical definition (PACD). The Medical Research Council and the Institutional Ethics Committees approved both studies and present study. The infants included in the study were under the age of 2 years. All the infants were breastfeeding at the time of specimen collection. Infants who were followed in the short course AZT study aimed at preventing MTCT were enrolled between May 2001 and June 2002. In the AZT study, mothers received short course AZT starting at 36 weeks gestation and throughout labor. Their infants received AZT for 7 days. In the PACD study, hospitalized children aged between 2 months and 18 months were prospectively enrolled into the study, between July 2002 and July 2003, following informed consent from their mothers. The mothers of these children did not receive antiretroviral therapy for prevention of MTCT nor for HIV disease. The PACD study is a hospital based analytical cross-sectional survey. Laboratory specimens were obtained once from each subject. Children presenting in the moribund state, requiring immediate resuscitation or those with known HIV status or those whose mothers/guardians refused to sign the informed consent were excluded from the study. The objective of the PACD study is to identify clinical symptoms, associated with HIV-1 infection in infants under the age of 18 months, which may be used in the absence of laboratory tests, for HIV-1 diagnosis. Blood collection and processing ------------------------------- A total volume of 2 ml whole blood was collected in ethylenediamine tetraacetic acid from each of the 137 infants in the short course AZT and the PACD studies. The whole blood was then aliquoted into two tubes (500 microlitres in each) for determination of T cell subset profiles, and the second for DNA PCR and the rest centrifuged at 200 g to obtain plasma, which was stored at -80°C. The laboratory tests described below were conducted in a blind fashion. Flow cytometry analysis ----------------------- T cell subset profiles were determined by flow cytometry using a Coulter Epics XL equipped with System II software (Beckman Coulter, Miami, Florida, USA) within 4 hours of blood collection. This flow cytometer was run, in a double platform setting where the absolute counts for both white blood cells and lymphocytes were obtained on a Celldyn 3500R haematological analyzer (Abbott, GmbH, Germany). Then from the combined results, the absolute CD4^+^and CD8^+^cell counts, CD4/CD8 ratios, as well as the %CD4 and %CD8 values among lymphocytes were automatically calculated. DNA PCR analysis ---------------- DNA PCR Roche amplification assay version 1.5 (Roche Diagnostics, Branchburg, NJ, USA) was employed as the reference standard for diagnosis of infant HIV-1 infection status. DNA extraction, amplification and detection were performed and results interpreted following the manufacturer\'s instructions (Roche Diagnostics, Branchburg, NJ, USA) as we previously described \[[@B12],[@B13]\]. Infant grouping --------------- Reference ranges of T cell subset profiles for infants and children are usually stratified by age as \<12 months, 1 to 5 years and 6--12 years. In the current study, in addition to overall evaluation of samples obtained from all the infants aged 0 to 18 months, the evaluated parameters were also compared based on infant age groups 0--11, and 12--18 months. Statistical Analysis -------------------- Diagnostic tests (sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and test efficiency (TE) with 95% confidence interval) were used to assess the assay under evaluation with DNA PCR as the reference standard. A MTCT prevalence rate of 30.7% (based on the current rate in Zimbabwe) was used in the calculations of PPV and NPV. Sensitivity was defined as the percentage of reference standard HIV positive samples found reactive by assay under evaluation. Specificity was defined as the percentage of reference standard HIV-negative samples that were negative by the assay under evaluation. TE refers to the overall ability of a test to correctly identify all positives and negatives (the absence of false positives and false negatives). It is a combination of the sensitivity and the specificity of an assay and gives an idea of the total effectiveness of the assay. PPV was defined as the probability that a specimen had CD4:CD8 ratio less than 1 when the test was DNA PCR positive. NPV was defined as the probability that a specimen does not have a CD4:CD8 ratio ≥ 1 when the test was DNA PCR negative. Comparisons of T cells between infected and uninfected infants were done using non-parametic equivalent of the T-test (Kruskal-Wallis test). Pvalues less than 0.05 were considered statistically significant. Results ======= Of the 137 infant specimens tested using DNA PCR, 76 were HIV-1 positive and 61 were HIV-1 negative. The 76 PCR positive infants included 9 infants who had evidence of in uterotransmission as determined by serial DNA PCR of birth and subsequent samples tested in longitudinal short course AZT MTCT studies (Zijenah et al, unpublished data). T cell subset of infected and uninfected infants ------------------------------------------------ T lymphocyte subset profiles were performed for the 137 infants who had whole blood specimens for flow cytometry. The median age of these infants at specimen collection was 5.5 months (Interquartile Range \[IQR\]: 3--13) and 8.0 months (IQR: 4--14) for HIV infected and uninfected respectively (p = 0.08). As expected, the median CD4^+^cell counts of PCR negative infants were significantly higher than those of the PCR positive infants, p \< 0.001(Table [1](#T1){ref-type="table"}). Inversely, the median CD8^+^cell counts were significantly higher among PCR positive infants than PCR negative infants, p \< 0.001(Table [1](#T1){ref-type="table"}). The median CD4/CD8 ratio of the PCR positive infants (0.4, IQR: 0.3--0.6) was significantly lower than that of the PCR negative infants (1.8, IQR: 1.4--2.3), p \< 0.001. The median %CD4 of PCR positive infants was also significantly lower than that of PCR negative infants, p = \< 0.01(Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### T cell subset profile of HIV-1 infected and uninfected infants ::: PCR Positive (n = 76) PCR Negative (n = 61) **Pvalue^a^ ----------------------------- --------------------------- --------------------------- ----------------- Median age (months) 5.5 (IQR: 3--13) 8 (IQR: 4--14) 0.08 Median CD4^+^(cells/μL) 521.5 (IQR: 323--805) 1356 (IQR: 916--1769) \<0.001 Median CD8^+^(cells/μL) 1302.5 (IQR: 829--2054) 799 (IQR: 471--1020) \<0.001 Median CD4/CD8 ratio 0.4 (IQR: 0.3--0.6) 1.8 (IQR: 1.4--2.3) \<0.001 Median %CD4 13.9 (IQR: 9.2--19.1) 29.9 (IQR: 25.3--34.5) \<0.001 Median %CD8** 31.3 (IQR: 22.3--42.9) 18.4 (IQR: 14.2--21.5) \<0.001 Abbreviations: PCR, polymerase chain reaction; n, number tested; Pvalue^a^for statistical significance between group medians was estimated using the Kruskal-Wallis test. ::: DNA PCR versus CD4/CD8 ratio ---------------------------- Seventy-five infants of the 76 PCR positive group (98.7%) had CD4/CD8 ratio \<1 while 60 infants of the 61 who were PCR negative had CD4/CD8 ratio ≥ 1. All the 9 infants infected in utero had a CD4:CD8 ratio less than 1. Their median CD4/CD8 ratio was similar to that of the rest of the PCR positive babies (0.4; IQR: 0.3--0.6), albeit the numbers are too small for statistical significance considerations. The infant (aged 11 months at specimen collection) who was DNA PCR positive with a CD4/CD8 ratio of 1.1 was from the PACD study. The PCR negative infant (aged 4 months at specimen collection) with a CD4/CD8 ratio of 0.3 was also from the PACD cohort. The overall sensitivity and specificity of the CD4/CD8 ratio were 98.7% (95% CI: 96.1--100 and 98.3% (95% Confidence Interval (CI): 95.1--100) respectively with PPV and NPV of 96.3% and 99.4% respectively and a test efficiency of 98.5% (95% CI: 96.5--100) (Table [2](#T2){ref-type="table"}). Comparison of all the evaluated parameters between the 0--11 and 12--18 months infant age groups showed that the 95% CI overlap between the groups which implies no statistically significant difference in these two age groups. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Evaluated parameters for CD4/CD8 ratio for the three infant age groups using DNA PCR as reference standard ::: 0--18 months infant age group (n = 137) 0--11 months infant age group (n = 95) 12--18 months infant age group (n = 42) --------------- --------------------------------------------- -------------------------------------------- --------------------------------------------- \%Sensitivity 98.7 (CI: 96.1--100) 98.2 (CI: 94.7--100) 100 (CI: 100--100) \%Specificity 98.4 (CI: 95.2--100) 97.5 (CI: 92.7--100) 100 (CI: 100--100) \%PPV 96.4 94.6 100 \%NPV 99.4 99.2 100 \%TE 98.5 (CI: 96.5--100) 97.9 (CI: 95.0--100) 100 (CI: 100--100) Abbreviations: n, number tested; NPV, negative predictive value; PPV, positive predictive value; TE, test efficiency. ::: Discussion ========== In resource-poor countries, the major constraint in the use of DNA PCR for diagnosis of HIV-1 infection in infants under the age of 18 months is the cost of the equipment and the reagents. In addition, highly trained laboratory personnel and stringent quality assurance measures are needed to run this assay for routine diagnosis of HIV infection in infants. In Zimbabwe, enzyme linked immunosorbent assay (ELISA) is routinely performed in both public and private laboratories for diagnosis of various infections including HIV-1. However, because of the transplacental transfer of maternal IgG antibodies, which may persist in infants for up to 18 months, ELISA is not suitable for diagnosis of HIV-1 infection in these infants. Therefore alternative methods are needed for this purpose. In addition, both public and private laboratories have flow cytometers or FACSCount machines for enumeration of T cell subset profile and automated haematological analysers for routine full blood counts with differential. With this equipment available in the country we evaluated the CD4/CD8 ratio as an alternative diagnostic test for infant HIV-1 infection. In our investigation we used Epics XL Coulter flow cytometer equipped with System II software (Beckman Coulter, Miami Florida, USA) for the following reasons. With this instrument, CD4/CD8 ratios can be conveniently obtained. In addition, when used in double platform setting tandem with a haematological analyzer, the results also show absolute CD4^+^cell counts and both %CD4 and %CD8 values among lymphocytes. The %CD4 value among lymphocytes is the recommended parameter for analyzing pediatric samples, as absolute counts for infants are age sensitive and variable. A simpler single platform system such as the FACScount (Becton-Dickinson Immunocytometry Systems, San Jose, CA, USA), is not fully suited for pediatric work as it provides CD4^+^, CD8^+^, CD3^+^T lymphocyte counts and CD4/CD8 ratio but %CD4 are not available. Of note, %CD4 expressed among CD3^+^T lymphocytes, is a different parameter from the customary %CD4 expressed among lymphocytes. The %CD4 expressed among lymphocytes and not the %CD4 expressed among CD3^+^T lymphocytes is recommended for decision making to initiate ARV therapy in children under the age of 18 months \[[@B5]\]. In Zimbabwe, CD4^+^cell count, is less expensive than PCR and may provide additional information of value to the clinician with respect to prognosis, and the need for prophylaxis and treatment. Optimal flow cytometry for determination of T cell subset profile, offers the added advantage that CD4/CD8 ratio will determine the infection status of the infant while the % CD4 will provide information on decision-making for commencement of HAART. Overall, the CD4/CD8 ratio had a ≥ 98% sensitivity for diagnosis of infant HIV-1 infection and a specificity ≥ 98%. Both sensitivity and specificity were 100% for infants in the 12--18 months age group. Interestingly, in parallel studies performed in 250 HIV-1 seropositive adults, 249 had a CD4/CD8 ratio of \<1. The CD4/CD8 ratio of the one patient was 1 at enrollment and has remained so for over one year. When interpreting our data, it is important to note that normal T cell subset values among African children differ from those of other populations \[[@B14]-[@B16]\]. A study in Guinea Bissau \[[@B16]\], reported that Guinean children under the age of 2 years had lower %CD4 and CD4/CD8 ratios and higher %CD8 when compared to their counterparts from developed countries. Interestingly, girls had higher CD4/CD8 ratios and lower %CD8 than boys. In our study, there were no statistically significant differences in absolute T cells, or percentages or CD4/CD8 ratio between boys and girls (Table 3 \"refer to [Additional file 1](#S1){ref-type="supplementary-material"}\"). A very few studies in Africa have compared T cell subset profiles between HIV-1 infected and uninfected infants under the age of 2 years \[[@B14],[@B17]\]. Moodley and colleagues in South Africa reported that the most marked changes in lymphocyte subset of HIV-1 infected children aged between 3 and 15 months were a lower %CD4 and higher %CD8 relative to uninfected infants \[[@B17]\]. In addition, CD4/CD8 ratio was a good predictor of poor clinical outcome at 3 months. The authors concluded that CD4/CD8 ratio and %CD4 among lymphocytes are reliable markers of HIV-1 infection in an African pediatric population. Furthermore a raised CD8^+^cell count rather than a CD4^+^cell count was a more specific prognostic marker of disease progression in HIV infected children. Embree and colleagues, in Kenya, also reported that HIV-1 infected children had lower %CD4 and higher %CD8 by 3 months when compared to uninfected children \[[@B13]\]. The authors concluded that %CD4 and %CD8 among lymphocytes could be useful as an adjunct in HIV-1 diagnosis. The two African studies mentioned above, have documented the clinical value of %CD4, CD4/CD8 ratio and CD8 counts in HIV-1 infection in infants. In summary, the CD4/CD8 ratio may be useful in identifying infected infants while %CD4 will identify infants who may benefit from cotrimoxazole prophylaxis and/or initiation of HAART, and for management of HIV-infected infants in developing countries in general. We thus propose use of flow cytometry, where available, as a point of care diagnostic tool for ill infants admitted to hospitals with clinical symptoms suggestive of HIV infection and/or AIDS. Competing interest ================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= LSZ designed the study, analyzed the data and drafted the manuscript. DAK, KJN, AB and GJ participated in the design of the study and preparation of the manuscript. SR conducted the statistical analysis and participated in the preparation of the manuscript. OT performed DNA PCR and analysed the data. AB and CG conducted flow cytometry and analysed the data, with the guidance of GJ. MN and PM participated in the collection of infant specimens and demographic data as well as preparation of the manuscript. All authors read and approved the final manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Table 3. Gender-based comparison of T cell subset profiles of HIV infected and uninfected babies. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ This work was financially supported in part by a grant from the Doris Duke Charitable Foundation.	PubMed Central	2024-06-05T03:55:52.895358	2005-2-1	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549040/", "journal": "J Transl Med. 2005 Feb 1; 3:6", "authors": [ { "first": "Lynn S", "last": "Zijenah" }, { "first": "David A", "last": "Katzenstein" }, { "first": "Kusum J", "last": "Nathoo" }, { "first": "Simbarashe", "last": "Rusakaniko" }, { "first": "Ocean", "last": "Tobaiwa" }, { "first": "Christine", "last": "Gwanzura" }, { "first": "Arsene", "last": "Bikoue" }, { "first": "Margaret", "last": "Nhembe" }, { "first": "Petronella", "last": "Matibe" }, { "first": "George", "last": "Janossy" } ] }
PMC549041	Background ========== Humans are routinely exposed to mutagenic and carcinogenic aromatic amines via smoking, well-cooked food and other sources \[[@B1]\]. These chemicals can form DNA adducts in vivoand thus lead to DNA damage \[[@B2]\]. The integrity of most of the so-damaged DNAs is typically restored as a consequence of the action of certain DNA-repairing enzymes, the normal function of which is important for maintaining genomic integrity and preventing cellular neoplastic transformation \[[@B3]\]. Since the genetic polymorphisms of DNA-repair enzymes might be able to influence DNA adduct levels \[[@B4]-[@B6]\], the particular degree of DNA-repair capacity has often been associated with the risk of human cancers \[[@B7]-[@B11]\]. Amongst the known genetic polymorphisms of the DNA-repair genes \[[@B12]\], the xeroderma pigmentosum group D (XPD, also known as ERCC2) and x-ray repair cross-complementing groups 1 and 3 (XRCC1 and XRCC3) have been studied most commonly \[[@B13]\]. The XPDgene encodes a helicase that is a component of the transcription factor TFIIH \[[@B14]\], this factor being an essential member of the nucleotide-excision repair (NER) pathway that is responsible for effecting repairs to bulky adducts and UV-induced DNA damage \[[@B15]\]. In 2002, Qiao et al. \[[@B16]\] reported that individuals featuring XPD751Gln/Gln did demonstrate suboptimal DNA-repair capacity (DRC) in regard to its ability to remove UV photoproducts when compared to the XPD751Lys/Lys and Lys/Gln genotypes. The XRCC1 protein is a scaffolding protein directly associated with polymerase beta, DNA ligase III and poly (ADP-ribose) polymerase (PARP) and functions in a complex to facilitate the base-excision repair (BER) and single-strand break-repair processes \[[@B17]-[@B19]\]. In 1999, Lunn et al. noted that individuals harboring the XRCC1399Gln allele were associated, more significantly, with higher levels of both aflatoxin B1-DNA adducts and glycophorin A variants when compared to individuals who exhibited the Arg/Arg genotype \[[@B4]\]. XRCC3 participates in DNA double-strand break repair and is a member of an emerging family of Rad-51-related proteins that likely participate in homologous recombinational repair (HRR) in order to maintain chromosome stability \[[@B20]\]. To the best of our knowledge, studies pertaining to these DNA-repair genes focusing on colorectal cancer risk would appear to be limited and controversial. In 2000, Abdel-Rahman et al. \[[@B21]\] observed that the XRCC1399Gln allele, compared to the XRCC1399Arg/Arg genotype, was associated with an increased risk for developing colorectal cancer, especially amongst young urban residents, although in 2003, Mort et al. \[[@B22]\] failed to reveal any significant associations between colorectal cancer risk and any polymorphisms of four of the NER genes (XPD, XPF, XPG, ERCC1) or XRCC1. In the present paper, we conducted a hospital-based case-controlled study to examine the role of genetic polymorphisms of three DNA-repair genes (XRCC1, XRCC3and XPD) in the context of colorectal cancer risk for the Taiwanese population. Methods ======= Subjects -------- Detailed descriptions of the specific characteristics of the study participants have been published previously \[[@B23]\]. In brief, participants were recruited from the Chang Gung Memorial Hospital between January 1995 and January 1999 inclusively. The colorectal adenocarcinoma cancer cases (n= 776) participating in this study were newly diagnosed and histologically confirmed. Patients suffering from familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer, or inflammatory bowel disease and other related malignancies were excluded from study participation. Seven hundred and twenty-seven of the original 776 cases (94%) were finally included in this study. Seven hundred and forty-seven age (same age) and sex-matched controls were recruited from the Physical Check-Up Department during the same period. All the participating controls had received comprehensive health examinations including colonoscopies. After excluding individuals diagnosed with other colorectal diseases, a history of other cancers or the existence of a family history of colorectal cancer, 736 controls (98%, 736/747) were finally included in this study. With informed consent, the socio-demographic characteristics of study participants were ascertained by means of the application of a structured questionnaire, at which time 10 ml of venous blood was collected. Genotyping ---------- Genomic DNA isolated from 10 ml whole blood was used to genotype XRCC1Arg399Gln, XRCC3Thr241Met and XPDLys751Gln by means of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. All of the PCR reactions were carried out by a Mastercycler gradient thermocycler (Eppendorf, Hamburg, Germany) in a final volume of 25 ul containing 200 ng of each primer, 50 ng genomic DNA, 1.5 mM MgCl~2~, 200 ul dNTPs and 1.0 unit of Taq DNA Polymerase in the buffer provided by the manufacturer. In addition, all laboratory genotyping personnel were blind to the case-control status of the samples. The 615 bp XRCC1PCR products were amplified with the primers 5\'-TTGTGCTTTCTCTGTGTCCA-3\' (sense) and 5\'-TCCTCCAGCCTTTTCTGATA-3\' (antisense) and digested with MspI (New England BioLabs, Beverly, MA, USA). The Arg allele revealed 374 and 221 bp fragments following digestion and polyacrylamide gel electrophoresis, whilst the Gln allele was not digested by MspI \[[@B4]\]. The 136 bp XRCC3PCR products were amplified with the primers 5\'-GCCTGGTGGTCATCGACTC-3\' (sense) and 5\'-ACAGGGCTCTGGAAGGCACTGCTCAGCTCACGCACC-3\' (antisense) and digested with NcoI (New England BioLabs). The Met allele revealed 97 and 39 bp fragments following digestion and polyacrylamide gel electrophoresis, while the Thr allele was not digested by NcoI \[[@B24]\]. The 734 bp XPDPCR products were amplified with the primers 5\'-CCTCTCCCTTTCCTCTGTTC-3\' (sense) and 5\'-CAGGTGAGGGGGACATCT-3\' (antisense) and digested with PstI (Takara, Japan). The Gln allele revealed 646 and 88 bp fragments following digestion and polyacrylamide gel electrophoresis, whilst the Lys allele was not able to be digested by PstI \[[@B25]\]. Statistical analysis -------------------- Tests for Hardy-Weinberg equilibrium amongst controls were conducted using observed genotype frequencies and a chi-square test featuring one degree of freedom. Baseline sociodemographic characteristics between cases and controls were analyzed using the chi-square test and two-sample Students\' t-test. Multivariate unconditional logistic regressions were used to examine the association between the XRCC1, XRCC3and XPDgenotypes and the risk for colorectal cancer. Since the differences in the estimated risks between conditional logistical regression and unconditional logistical regression were small, unconditional logistical regression was used to estimate odds ratio (OR) and 95% confidence interval (CI) with the matching factors (age and gender) included in the model for estimation \[[@B26]\]. Other potential confounders (such as physical activity, cigarette smoking, alcohol use, coffee intake, and consumption of staple, meat, vegetable/fruit and fish/shrimp) would be also included if they have \>10% effect on the gene main effects. However, none of these factors have met the inclusion criteria. Based on the multiplicative scale, the likelihood ratio test was further used to evaluate the interaction between XRCC1, XRCC3and XPDgenes on the risk for the colorectal cancer. Since the associations between polymorphisms of DNA-repair gene, cancer susceptibility and DNA repair capacity are inconsistent \[[@B13]\], we defined the risk allele as the allele with OR\>1 observed in the present study. Stratified analyses were also conducted to evaluate the differences between specific tumor sites (colon and rectum) and age groups (\< = 60 years old and \> 60 years old). All analyses were performed using the SAS statistical package (version 8.1 for windows; SAS Institute, Inc., Cary, NC, USA) and all tests were two-sided. Results ======= More men (56%) than women participated in this study (Table [1](#T1){ref-type="table"}). The mean age for both groups was 60 years. From the cancer cases, 352 patients suffered from colon cancer (48%) and 375 patients from rectal cancer (52%), with most such cases being deemed to be at stage II or stage III (both were 33%). Detailed analyses of the sociodemographic characteristics and potential risk factors associated with colorectal cancer amongst the study population have been published previously \[[@B23]\]. The genotypic distributions of the three DNA-repair genes for both cancer cases and controls are shown in Table [2](#T2){ref-type="table"}. The frequencies for the XRCC1399Gln, XRCC3241Met and XPD751Gln allele amongst the controls were, respectively, 0.27, 0.05 and 0.07, these genotype frequencies being in Hardy-Weinberg equilibrium. Due to the relatively low frequencies of variant alleles for these genes in the present study, any genotype featuring one or more variant alleles was combined for further analyses. The risk for colorectal cancer was not significantly different for individuals featuring the XRCC1399Arg/Arg genotype (OR = 1.18; 95% CI, 0.96--1.45), the XRCC3241Thr/Thr genotype (OR = 1.25; 95% CI, 0.88--1.79) or the XPD751Gln allele (OR = 1.20; 95% CI, 0.90--1.61). As revealed in Table [3](#T3){ref-type="table"}, those individuals exhibiting a greater number of risk genotypes (genotype with OR greater than 1) faced a greater risk for colorectal cancer when compared to those individuals who did not display any risk genotypes (Trend test, P= 0.03). Subjects who demonstrated two or three of the putative risk genotypes did reveal a significantly greater risk for colorectal cancer (OR = 1.95; 95% CI, 1.08--3.52 and OR = 2.43; 95% CI, 1.21--4.90, respectively) as compared to those individuals who did not feature any putative risk genotypes, although it appears that no gene-gene interactions arose amongst these three genes (all Plevels for interaction were \>0.21). When stratified by tumor site and age at diagnosis, these combined gene effects upon cancer risk were observed for individuals who revealed that their tumor was located in the rectum (Trend test P= 0.03) and those individuals for whom their tumor was diagnosed prior to their being 60 years of age (Trend test P= 0.004; Table [4](#T4){ref-type="table"}). The ORs for subjects with three putative risk genotypes were 3.18 (95% CI, 1.29--7.82) for rectal cancer and 4.90 (95% CI, 1.72--14.0) for those individuals diagnosed prior to 60 years of age, respectively. Discussion ========== To the best of our knowledge, few studies have investigated the role of polymorphisms in DNA-repair genes for patients suffering colorectal cancer \[[@B21],[@B22]\]. In this hospital-based case-control study of colorectal cancer-suffering patients in Taiwan, we found polymorphisms in three DNA-repair genes associated with an elevated risk of colorectal cancer. In addition, a gene-dosage effect was found for rectal cancer and younger patients. These findings suggest that those genes involved in different DNA-repair pathways may act simultaneously in the process of carcinogenesis for colorectal cancer. Although we didn\'t find any significant independent associations between these DNA-repair genes and colorectal cancer risk, the risk appeared to be slightly increased for individuals who featured the XRCC1399Arg/Arg, XRCC3241Thr/Thr genotypes and the XPD751Gln allele. Our results do not appear to be entirely consistent with the results of some previous reports \[[@B21],[@B22]\], the former group reporting that the XRCC1399Gln allele significantly increased the risk of colorectal cancer (OR = 3.98, 95% CI = 1.50--10.6). In 2003, Mort et al. \[[@B22]\] noted that the risk of suffering colorectal cancer was significantly heightened for individuals who featured the XRCC3241Thr allele (OR = 1.52, 95% CI = 1.04--2.22) and only slightly increased for those individuals who revealed the XRCC1399Gln and XPD751Gln alleles. In 2001, Park et al. \[[@B27]\] reported that advanced colorectal cancer-suffering patients who revealed the XPD751Gln/Gln genotype featured a poorer (positive) response rate to chemotherapy and also a shorter survival period compared with colorectal cancer-suffering individuals who belonged to either the 751Lys/Lys or the 751Lys/Gln group. The apparent divergence between these studies and ours might be due to one of two reasons. Firstly, it may simply be that ethnic differences in allele frequency for the polymorphism might explain the controversial findings. The frequencies for the XRCC1399Gln, XRCC3241Met and XPD751Gln alleles amongst the healthy controls in this study (0.27, 0.05 and 0.07, respectively) were similar to the results for other studies conducted in Taiwan \[[@B28],[@B29]\] and China \[[@B30],[@B31]\], but appeared to be much lower than those reported in 2003 by Mort et al. \[[@B22]\] (0.42 for XRCC1399Gln, 0.45 for XRCC3241Met and 0.36 for XPD751Gln) for a British population. On the other hand, the XRCC1399Gln allele frequency observed in the present study is greater than that observed by Abdel-Rahman et al. in 2000 (0.14 for XRCC1399Gln) \[[@B21]\]. Abdel-Rahman et al. also found that urban residents have 9.97-fold increased risk of early-onset colorectal carcinoma than rural residents with the XRCC1399Gln allele \[[@B21]\]. Therefore, it is possible that the divergence in results from different studies might be related to different levels of carcinogen exposure for different populations. Moreover, inadequate study design such as a too-small sample size and/or the inadequate controlling for certain confounders (such as age and gender) should also be considered as constituting the underpinning for such differing results. Combined effects of polymorphisms of the XRCC1Arg399Gln, XRCC3Thr241Met and XPDLys751Gln genes in regard to colorectal cancer risk were observed in the present study. With the complexity of detail of environmental exposures to various carcinogens, it is plausible that the effective repair of DNA damaged by chemical mixtures necessitates multiple DNA-repair pathways (including BER, HRR and NER pathways). The failure of, or the presence of deficient DNA repair capacity for each specific DNA-repair pathway may contribute to an increase in cancer risk. Additive or multiplicative effects of combined genetic variants for different DNA-repair pathways have been previously reported for lung cancer \[[@B32]\], melanoma \[[@B33]\] and breast cancer \[[@B34]\]. In 2003, Zhou et al. \[[@B35]\] found that the risk of lung cancer amongst nonsmokers increased progressively with the increase in the number of high-risk alleles of XRCC1and XPDgenes. Hu et al. \[[@B36]\] also observed that prolonged cell-cycle delay was significantly associated with the number of variant alleles of the APE1and XRCC1genes that were present. It would therefore appear reasonable to hypothesize that genetic polymorphism(s) for DNA-repair genes may simultaneously contribute to colorectal cancer susceptibility. In this study, we found that the combined effect of multiple DNA-repair genes upon colorectal cancer risk was significant for our younger age group, but not so for the older age group. This finding appears to be similar to the results of most of the previous studies pertaining to colorectal cancer \[[@B21]\], basal-cell carcinoma \[[@B37]\], head-and-neck cancer \[[@B38]\], hepatocellular carcinoma \[[@B39]\], and lung cancer \[[@B6],[@B32],[@B35],[@B40]\] that we reviewed, although we did note that one study reported that such an elevated risk was also observed for old-aged head-and-neck cancer patients \[[@B41]\]. In addition, few studies that we reviewed failed to find a difference between age groups in regard to for cancer risk \[[@B31],[@B42]-[@B46]\]. In 2000, Duell et al. \[[@B47]\] noted that old healthy subjects who featured the XRCC1399Gln allele appeared to be significantly associated with detectable DNA adducts in their blood mononuclear cells when compared to younger subjects who featured the 399Arg/Arg genotype. In 2001, Hemminki et al. \[[@B48]\] also found that old subjects who revealed the XPD751Gln/Gln genotype exhibited a decreased DNA-repair rate for, specifically, UV-specific cyclobutane pyrimidine dimers in the skin. Therefore, it is possible that individuals who demonstrate a less-efficient DNA-repair capacity might develop tumors at a younger age than individuals who reveal an efficient DNA-repair capacity. In our study, we also found that the combined genetic effect of these three DNA-repair genes in regard to cancer risk was more pronounced for the rectum than for the colon. This difference may reflect certain etiological differences between colon and rectal carcinogenesis. In fact, different epidemiological characteristics, etiology, pathogenesis and clinical behavior have been reported for different anatomical sites of colorectal cancer \[[@B49],[@B50]\], this latter group suggesting that carcinogenesis within the distal colon was associated with bulky-adduct-forming (BAF) agents and that these DNA lesions were repaired through NER pathways. In 2001, Hong et al. \[[@B51]\] also found that much more DNA-repair and apoptosis activity occurred in the distal rather than the proximal colon for the rat azoxymethane carcinogenesis model. Taken together, these observations support the notion that insufficient DNA-repair capacity could contribute to the risk of cancer associated with exogenous carcinogen exposure. To the best of our knowledge, this study is the first to report on XRCC1, XRCC3and XPDpolymorphisms in relation to the risk of colorectal cancer for the Taiwanese population. Our results suggest that genetic polymorphisms of the XRCC1, XRCC3and XPDgenes, particularly in combination, may be associated with an individual\'s susceptibility to colorectal cancer. Acknowledging the relatively limited sample size in the subgroups for the low allelic frequencies, further studies incorporating a larger sample size and/or another ethnic population are needed to confirm the genetic role of DNA-repair mechanisms as regards colorectal cancer susceptibility. Conclusions =========== Our results suggest that DNA-repair pathways may simultaneously modulate the risk of colorectal cancer for the Taiwanese population, and, particularly for rectal cancer and younger patients. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= CCY carried out the genotyping analysis, performed the statistical analysis and drafted the manuscript. FCS participated in the design of the study. RT and CRCC participated in the design of the study and provided clinical biospecimens. LLH conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/5/12/prepub> Acknowledgments =============== This study was supported by Grant number NSC 89-2314-B-002-373, NSC 90-2320-B-002-123 and NSC 91-2320-B-002-121 provided by the National Science Council and Grant number DOH 85-HR-516, DOH 86-HR-516, and DOH 87-HR-516 provided by the National Health Research Institute, Department of Health, The Executive Yuan, Republic of China. Figures and Tables ================== ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristic details for the cases and controls ::: Variables Cases, n (%) (N = 727) Control, n (%) (N = 736) p ---------------------- ------------------------ -------------------------- --------- Gender 0.75^a^ Male 410 (0.56) 409 (0.56) Female 317 (0.44) 327 (0.44) Mean age (SD), years 60.3 (12.8) 60.7 (13.0) 0.64^b^ Colon/rectum, N 352/375 \- Stage 0 9 (1.2) \- I 84 (11.6) \- II 242 (33.3) \- III 241 (33.2) \- IV 151 (20.8) \- a: Chi-square test b: t test ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Distribution of three DNA repair genes for cases and controls ::: Polymorphism Cases, n (%) Control, n (%) OR (95% CI) ^a^ ----------------- -------------- ---------------- ------------------- XRCC1 Arg399Gln Arg/Arg 407 (56.7) 384 (52.7) 1.00 Arg/Gln 260 (36.2) 291 (39.9) 0.84 (0.67--1.07) Gln/Gln 51 (7.1) 54 (7.4) 0.82 (0.53--1.25) Missing 9 7 With Gln^b^ 311 (43.3) 345 (47.3) 1.00 Arg/Arg 407 (56.7) 384 (52.7) 1.18 (0.96--1.45) XRCC3 Thr241Met Thr/Thr 660 (91.5) 658 (89.7) 1.00 Thr/Met 60 (8.3) 74 (10.1) 0.83 (0.56--1.21) Met/Met 1 (0.1) 2 (0.3) 0.50 (0.05--5.51) Missing 6 2 With Met^c^ 61 (8.5) 76 (10.4) 1.00 Thr/Thr 660 (91.5) 658 (89.7) 1.25 (0.88--1.79) XPD Lys751Gln Lys/Lys 602 (84.0) 631 (86.3) 1.00 Lys/Gln 112 (15.6) 96 (13.1) 1.29 (0.94--1.76) Gln/Gln 3 (0.4) 4 (0.6) 0.81 (0.18--3.62) Missing 10 5 Lys/Lys 602 (84.0) 631 (86.3) 1.00 With Gln^d^ 115 (16.0) 100 (13.7) 1.20 (0.90--1.61) a: ORs and 95% CIs were estimated from unconditional logistic regressions, controlling for age and gender b: Arg/Gln+ Gln/Gln c: Thr/Met+ Met/Met d: Lys/Gln+ Gln/Gln ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Combined effect of XRCC1, XRCC3and XPDgenes upon colorectal cancer risk ::: XRCC1 Arg399Gln XRCC3 Thr241Met XPD Lys751Gln Cases Controls OR (95% CI)^a^ OR (95% CI)^a^ ----------------- ----------------- --------------- ------- ---------- ------------------- --------------------------- With Gln With Met Lys/Lys 18 34 1.00 1.00 Arg/Arg With Met Lys/Lys 30 30 1.91 (0.89--4.10) With Gln Thr/Thr Lys/Lys 239 257 1.77 (0.97--3.22) 1.79 (0.99--3.24) With Gln With Met With Gln 6 6 1.90 (0.54--6.76) Arg/Arg Thr/Thr Lys/Lys 312 307 1.93 (1.07--3.50) Arg/Arg With Met With Gln 7 5 2.65 (0.74--9.56) 1.95 (1.08--3.52) With Gln Thr/Thr With Gln 48 46 1.99 (0.99--4.01) Arg/Arg Thr/Thr With Gln 54 42 2.43 (1.21--4.90) 2.43 (1.21--4.90) 0.03^b^ a: ORs and 95% CIs were estimated from unconditional logistic regressions, controlling for age and gender b: Trend test ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Number of risk-genotypes of three DNA repair genes and colorectal cancer risk by age at diagnosis and by tumor site ::: Number of risk-genotypes\* ------------------ ---------------------------- -------------- -------------- -------------- ------- Age at diagnosis \< = 60 years Cases/Controls 7/20 114/126 169/155 29/17 OR 1.0 2.58 3.12 4.90 0.004 (95% CI)^a^ (reference) (1.05--6.33) (1.28--7.58) (1.72--14.0) \>60 years Cases/Controls 11/14 161/167 198/203 25/25 OR 1.0 1.24 1.25 1.28 0.74 (95% CI)^a^ (reference) (0.55--2.82) (0.55--2.83) (0.49--3.35) Tumor site Colon Cases/Controls 10/34 132/293 177/358 23/42 OR 1.0 1.51 1.65 1.86 0.16 (95% CI)^a^ (reference) (0.72--3.14) (0.80--3.42) (0.78--4.44) Rectum Cases/Controls 8/34 143/293 190/358 31/42 OR 1.0 2.13 2.32 3.18 0.03 (95% CI)^a^ (reference) (0.96--4.73) (1.05--5.11) (1.29--7.82) \*Risk-genotype was defined as genotype with OR\>1 as shown in Table 3. a: ORs and 95% CIs were estimated from unconditional logistic regressions, controlling for age and gender b: p for trend test :::	PubMed Central	2024-06-05T03:55:52.897394	2005-1-28	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549041/", "journal": "BMC Cancer. 2005 Jan 28; 5:12", "authors": [ { "first": "Chih-Ching", "last": "Yeh" }, { "first": "Fung-Chang", "last": "Sung" }, { "first": "Reiping", "last": "Tang" }, { "first": "Chung Rong", "last": "Chang-Chieh" }, { "first": "Ling-Ling", "last": "Hsieh" } ] }
PMC549042	Although more than 20 years have passed since the identification of HIV as the cause of AIDS, several essential questions about its pathogenicity remain as yet unanswered. In particular, a central, still unresolved issue is the mechanism underlying the progressive development of immunodeficiency. It is now well established that HIV infection determines a rapid turnover of infected CD4 cells \[[@B1],[@B2]\]; consistent with this finding, multiple molecular pathways triggered by different HIV proteins are known to lead to cell apoptosis \[[@B3],[@B4]\]. However, the capacity of the immune system to regenerate its cells by far exceeds the number of dying HIV infected cells. Thus, the extension of the apoptotic message to neighboring, bystander cells has long been recognized as a potential mechanism sustaining the immunodeficiency that accompanies HIV disease progression \[[@B5]\]. In this context, the finding that the virus-encoded Tat protein is released by the infected cells and can be taken up by neighboring, uninfected cells via an endocytic mechanism \[[@B6],[@B7]\] has long suggested the possibility that some of the bystander apoptotic effects exerted by HIV might be mediated by this protein. Over ten years ago different investigators did indeed show that extracellular Tat can trigger apoptosis in T-cell lines and primary T-cells \[[@B8],[@B9]\]. The classical apoptotic pathway, involving the cell\'s mitochondria, is regulated by the Bcl-2 family of proteins. This family contains both anti-apoptotic (Bcl-2, Bcl-XL) and pro-apotpotic (Bax, Bid, Bim) members that exert their function primarily at the mitochondrion by either preventing or inducing mitochondrial dysfunction. Upon receiving a death signal, the pro-apoptotic proteins translocate from the cytoplasm to the outer mitochondrial membrane, where they interact with their pro-apoptotic partners. This occurrence is followed by mitochondrial dysfunction, release of pro-apoptotic proteins out of the mitochondrion (among which, a prominent role can be ascribed to cytochrome c), and subsequent caspase activation \[[@B10]\]. One of the cellular events that trigger the mitochondrial pathway of apoptosis is the disturbance of the dynamic formation of microtubules in the cell. This event can be triggered by a variety of microtubule-targeted, tubulin-polymerizing agents (MTPAs), which include paclitaxel (Taxol) and several other anticancer drugs \[[@B11]\]. Following intracellular uptake, MPTAs bind β-tubulin and promote tubulin polymerization, which interferes with the function of the mitotic spindle resulting in mitotic arrest at the metaphase-anaphase transition and subsequent induction of the mitochondrial pathway of apoptosis. A link between microtubule polymerization and the pro-apoptotic effect of Tat has first been suggested a few years ago in the observation that Tat directly interacts with the αβ-tubulin dimers and polymerized microtubules in the cytoplasm of the cell \[[@B12]\]. The functional consequence of this interaction, which requires the integrity of four amino acids in the conserved Tat core domain, is the stabilization of microtubules and the consequent prevention of microtubule depolymerization. This disturbance in the microtubular network is a powerful inducer of the mitochondrial pathway of cellular apoptosis, an event that is transduced by the pro-apoptotic Bcl-2 relative Bim. These findings supported previous observations that had already shown that Tat causes changes in mitochondrial membrane permeability \[[@B13],[@B14]\] and that it interferes with the polymerization of microtubules \[[@B15]\]. Two papers now published in Retrovirologyextend the link between the microtubule network, the mitochondrial pathway of apoptosis, and Tat. De Mareuil and coworkers show that Tat enhances tubulin polymerization into microtubules, an effect similar to that exerted by the MTPAs, and physically associates with the polymerized microtubuli \[[@B16]\]. As opposed to paclitaxel, however, Tat only increases the rate of tubulin polymerization while it does not permanently affect the organization of the microtubule network, nor does it blocks cell cycle progression. Most notably, the ability of different Tat variants to induce tubulin polymerization correlates with their capacity to induce apoptosis. Similar to paclitaxel and other microtubuli damaging agents, the pro-apoptotic effect of Tat parallels the induction of cyctochrome c release from the mitochondria, a critical event triggering apoptosis. The accompanying manuscript by Epie and coworkers describes the identification of a microtubule-associated protein, LIS1, which specifically binds Tat \[[@B17]\]. In the course of a biochemical project entailing the fractionation of T-cell extracts searching for Tat-associated kinases that phosphorylate the C-terminal domain of RNA polymerase II -- a known biochemical activity associated to Tat -, these authors found that LIS1 co-purifies with a complex of proteins including one of the CTD kinases, CDK7, its cyclin partner, cyclin H and the MAT1 co-factor. Of note, out of the four purified proteins, only LIS1 directly bound Tat, as shown by GST-pulldown and co-immunoprecipitation experiments, and by the yeast two hybrid assay. LIS1 is known to regulate microtubule dynamics by interacting with dynein and additional components of the dynein motor \[[@B18]\]. What might be the relevance of these findings in the context of HIV-1 infection? They clearly provide a mechanism for CD4 T-cell apoptosis and for the extension of the apoptotic effect to bystander, uninfected cells in the lymph node. Moreover, the interaction of Tat with the microtubular network might explain the occurrence of neuropathogenesis accompanying the progression of HIV disease, since many human neurodegenerative conditions are elicited by a reorganization of the neuronal cytoskeleton \[[@B19]\]. Thus, the disturbance of the microtubular network induced by Tat adds to other potentially pro-apoptotic mechanisms induced by the protein, such as the upregulation of FasL \[[@B9]\], TRAIL \[[@B20]\], Bax \[[@B21]\] and caspase 8 \[[@B22]\] and the downregulation of Bcl2 \[[@B21]\]. As commonly happens in biology, the findings reported in these manuscripts raise more questions than answers. First, the Tat domains involved in the described interactions are different, a surprising finding given the very small size of Tat. This observation might possibly suggest that Tat is part of a large multi-molecular complex associated with the tubular network, making multiple contacts with different proteins. This issue can be experimentally addressed biochemically, or even within the cell, by taking advantage of the biophysical techniques available to investigate protein-protein interactions in vivo \[[@B23]\]. Secondly, the role of LIS1, if any, in the Tat-triggered mitochondrial pathway of apoptosis or in the functions of CDK7 and its partners, with which it unexpectedly co-purifies is unclear. Third, and most importantly, it remains to be seen whether the concentration at which Tat binds tubulin and exerts its pro-apoptotic effects is compatible with the concentration at which the protein is expressed in the infected cells and diffuses to neighboring cells. As a matter of fact, the measurement of the extracellular concentration of Tat still remains a holy grail in the HIV research field \[[@B24]\], partly due to the weak avidity of the currently available anti-Tat antibodies, partly because of the biological property of extracellular Tat that is sequestered by extracellular matrix proteoglycans \[[@B25]\]. Until more reliable methods are developed to determine the levels of extracellular Tat in vivo, the full biological implications of Tat-induced apoptosis cannot be entirely appreciated. Abbreviations ============= MTPAs: microtubule-targeted, tubulin-polymerizing agents CTD: carboxy-terminal domain Competing interests =================== The author(s) declare that they have no competing interests. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### To form microtubules, α- and β-tubulin molecules join to form a heterodimer. These dimers then attach to other dimers forming oligomers that elongate into protofilaments; eventually, the oligomers will join to give rise to a ringed microtubule. Microtubules or unpolymerized tubulin bind microtubule-associated proteins (MAPs), which regulate polymerization, facilitate assembly, stabilize the microtubules and regulate microtubular transport of macromolecules and vesicles. The HIV-1 Tat protein binds to αβ-tubulin dimers and microtubules thus enhancing microtubule polymerization, and to the microtubule-associated protein LIS1, which is also known to facilitate assembly of microtubules. Disturbance of the dynamics of microtubular network formation activates the intrinsic mitochondrial apoptotic pathway. Pro-apoptotic Bcl2 family members -- in particular, Bim -- are recruited to the mitochondrion; as a consequence, the mitochondrial membrane potential collapses, and pro-apoptotic factors are released into the cytoplasm. These include reactive oxygen intermediates (ROIs), apoptosis-inducing factor (AIF), and cytochrome c, among others. Release of cytochrome c is a point of no return as it leads to autoactivation of caspase 9, which in turn proceeds to cleave the downstream effector caspases (caspase 3, 6, etc.). ::: ![](1742-4690-2-7-1) :::	PubMed Central	2024-06-05T03:55:52.899813	2005-2-7	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549042/", "journal": "Retrovirology. 2005 Feb 7; 2:7", "authors": [ { "first": "Mauro", "last": "Giacca" } ] }
PMC549043	Background ========== Surveys in many countries have found that mental disorders have a high prevalence and are a major cause of disability in the population \[[@B1]-[@B3]\]. For example, the Australian National Survey of Mental Health and Wellbeing found that close to one in five adults met the criteria for a mental disorder at some time during the 12 months before the survey \[[@B3]\]. The most common mental disorders were anxiety (10%), depressive (6%) and substance use disorders (8%). These disorders are so prevalent that everyone in the community can expect to have close contact with someone experiencing a mental disorder. How people initially respond to others with a mental disorder may influence their recovery. For example, it is known that many people with mental disorders get no professional help \[[@B4]\] and that help-seeking is more likely if relatives or friends suggest it \[[@B5]\]. It is also known that recovery can be assisted if family members are supportive and not critical \[[@B6]\]. This type of initial help from a person\'s social network can be defined as mental health first aid. There has been no previous research on mental health first aid knowledge in the population. Previous mental health literacy surveys have assessed knowledge and beliefs about mental disorders and their treatment \[[@B5]\], but these surveys have not assessed the public\'s intentions for mental health first aid responses to hypothetical cases of persons with mental disorders. Thus it is not known which mental health first aid responses are currently adequate and which need improving. Hence a relevant question was asked in a recent national survey of mental health literacy in Australia. Methods ======= The Australian survey --------------------- In 2003--2004 a household survey was carried out of Australian adults aged 18 or over by the company AC Nielsen. Households were sampled from 250 census districts covering all states and territories and metropolitan and rural areas. Up to 5 call backs were made to metropolitan selections and 3 to non-metropolitan selections. To achieve a target sample of 4,000 interviews with adults aged 18 years or over, visits were made to 28,947 households. The outcome of these visits was: no contact after repeated visits 14,630; vacant house or lot 306; refused 7,815; person sampled within household temporarily unavailable 1,132; no suitable respondent in household 287; did not speak English 383; incapable of responding 213; and unavailable for the duration of the survey 181. The achieved sample was 3998 persons, with 1001 receiving the depression vignette, 999 the depression with suicidal thoughts vignette, 997 the early schizophrenia vignette, and 1001 the chronic schizophrenia vignette. Interview content ----------------- The interview was based on a vignette of a person with a mental disorder. On a random basis, respondents were shown one of four vignettes: a person with major depression, one with major depression together with suicidal thoughts, a person with early schizophrenia, and one with chronic schizophrenia. All vignettes were written to satisfy the diagnostic criteria for either major depression or schizophrenia according to DSM-IV and ICD-10. The vignette with depression and the one with early schizophrenia were written to satisfy these diagnostic criteria at a minimal level, so that we could ascertain the public\'s reaction to cases of a developing disorder which had reached the point where intervention was needed. The vignette of the person with depression together with suicidal thoughts was identical to the depression vignette in all respects except the suicidal thoughts and was designed to assess how this symptom affected the public\'s response. The chronic schizophrenia vignette was designed to assess the response to someone with a severe long-standing disorder, where acceptance seemed less likely. Respondents were also randomly assigned to receive either male (\"John\") or female (\"Mary\") versions of the vignette. These vignettes (John version) are shown in Table [1](#T1){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Case vignettes used in the survey ::: Disorder Vignette ----------------------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Depression John is 30 years old. He has been feeling unusually sad and miserable for the last few weeks. Even though he is tired all the time, he has trouble sleeping nearly every night. John doesn\'t feel like eating and has lost weight. He can\'t keep his mind on his work and puts off making decisions. Even day-to-day tasks seem too much for him. This has come to the attention of his boss, who is concerned about John\'s lowered productivity. Depression with suicidal thoughts John is 30 years old. He has been feeling unusually sad and miserable for the last few weeks. Even though he is tired all the time, he has trouble sleeping nearly every night. John doesn\'t feel like eating and has lost weight. He can\'t keep his mind on his work and puts off making any decisions. Even day-to-day tasks seem too much for him. This has come to the attention of John\'s boss who is concerned about his lowered productivity. John feels he will never be happy again and believes his family would be better off without him. John has been so desperate, he has been thinking of ways to end his life. Early schizophrenia John is 24 and lives at home with his parents. He has had a few temporary jobs since finishing school but is now unemployed. Over the last six months he has stopped seeing his friends and has begun locking himself in his bedroom and refusing to eat with the family or to have a bath. His parents also hear him walking about his bedroom at night while they are in bed. Even though they know he is alone, they have heard him shouting and arguing as if someone else is there. When they try to encourage him to do more things, he whispers that he won\'t leave home because he is being spied upon by the neighbour. They realize he is not taking drugs because he never sees anyone or goes anywhere. Chronic schizophrenia John is 44 years old. He is living in a boarding house in an industrial area. He has not worked for years. He wears the same clothes in all weathers and has left his hair to grow long and untidy. He is always on his own and is often seen sitting in the park talking to himself. At times he stands and moves his hands as if to communicate to someone in nearby trees. He rarely drinks alcohol. He speaks carefully using uncommon and sometimes made-up words. He is polite but avoids talking with other people. At times he accuses shopkeepers of giving information about him to other people. He has asked his landlord to put extra locks on his door and to remove the television set from his room. He says spies are trying to keep him under observation because he has secret information about international computer systems which control people through television transmitters. His landlord complains that he will not let him clean the room which is increasingly dirty and filled with glass objects. John says he is using these \"to receive messages from space\". ::: After being presented with the vignette, respondents were asked a series of questions to assess their recognition of the disorder in the vignette, their beliefs about treatment and long-term outcomes, beliefs about causes and risk factors, stigmatizing attitudes, awareness of mental disorders in the media, contact with people like those in the vignette, and the health and sociodemographic characteristics of the respondent. The questions relevant to the present paper are described below. To assess recognition of the problem in the vignette, respondents were asked: \"What would you say, if anything, is wrong with John?\". Responses of \"depression\" were counted as correct for the first two vignettes above, and responses of \"schizophrenia\" or \"psychosis\" for the second two. To assess mental health first aid responses, respondents were asked the open-ended question: \"Imagine John is someone you have known for a long time and care about. You want to help him. What would you do?\". Answers were recorded verbatim by the interviewer. To assess contact with people like those in the vignette, respondents were asked: \"Has anyone in your family or close circle of friends ever had problems similar to John\'s?\"; \"Have they received any professional help or treatment for these problems?\'; \"Have you ever had problems similar to John\'s?\"; \"Have you received any professional help or treatment for these problems?\"; and \"Have you ever had a job that involved providing treatment or services to a person with a problem like John\'s?\". Those that said \"yes\" to these questions were respectively labelled in the analyses reported below as \"carers\", \"consumers\" or \"professionals\". To assess stigma, respondents were asked a series of nine questions designed to elicit their attitudes towards the person in the vignette (personal stigma) and nine items concerning what they thought others in the community would believe about the person in the vignette (perceived stigma) \[[@B7]\]. Personal stigma items were of the form: \"Please indicate how strongly you agree or disagree with each statement. People with a problem like John\'s could snap out of it if they wanted. Strongly agree, Agree, Neither agree nor disagree, Disagree, Strongly disagree\". Perceived stigma items were of the form: \"Now we would like you to tell us what you think most [other]{.underline} people believe. Please indicate how strongly you agree or disagree with the following statements. Most people believe that people with a problem like John\'s could snap out of it if they wanted. Strongly agree, Agree, Neither agree nor disagree, Disagree, Strongly disagree\". Sociodemographic characteristics recorded included age group (coded as 18--39, 40--59 and 60+ years), gender, and education (dichotomized as bachelor\'s degree or higher versus lower-level qualifications). Content analysis of responses to open-ended question ---------------------------------------------------- Responses were coded according to the categories identified from an earlier study where the same question was administered as part of a randomized controlled trial of Mental Health First Aid \[[@B8]\]. Responses were coded with a \"yes\" or \"no\" in each category, such that multiple categories were possible. The categories were: A. Encourage professional help-seeking B. Listen to / talk to / support person C. Listen to / talk to / support family D. Assess problem / assess risk of harm E. Give or seek information F. Encourage self-help Responses coded into category A. Encourage professional help-seeking, were subcoded into multiple categories to identify the type of professional help recommended. These categories were: A1. GP / doctor unspecified A2. Counsellor A3. Psychiatrist A4. Psychologist A5. Mental health team / services A6. Other mental health professionals A7. Unspecified professionals and other professionals A8. Accompany person (eg. Offer to go with him/her) A9. Contact help on their behalf Examples of responses coded into category B included \"support, understanding and caring, someone to talk to him\", \"talk to her about it\", \"listen\", \"be there for him\". Responses coded into category C were the same for category B, but referred to giving support, listening to, or talking to the sufferer\'s family. For example, \"talk to her family\", \"contact relatives\", \"ask advice of parents\", \"support his parents\". Responses coded into category D included \"keep an eye on her, make sure she is safe\", \"make a contract with her so if she wants to harm herself she rings me first\", \"find out what is the real problem behind the behaviour, focus on the problem\". Responses coded into category E included \"ring the local health authority to get advice\", \"talk to other people who have been in the situation\", \"speak to health professionals and get best advice\", \"get some brochures from community health and give them to him\". Responses coded into category F included \"I suggest that he have a holiday/exercise/change jobs\", \"try and help him get into something he is interested in\", \"support groups\", \"do something for herself to get out of the situation\". Inter-rater reliability of the coding was assessed by a second rater who independently coded 100 responses which were randomly selected using the SPSS Select Cases procedure \[[@B9]\]. Statistical analysis -------------------- Inter-rater reliability of the content coding was assessed using kappa. Kappa values were interpreted according to Altman \[[@B10]\] as follows: 0.8--1.0 very good; 0.6--0.8 good; 0.4--0.6 moderate; 0.2--0.4 fair; and \<0.2 poor. The frequency of codings was analysed by pooling across male and female versions of each vignette and percent frequencies calculated. Percentages were calculated applying survey weights to give better population estimates. Standard errors of these percentages were estimated using the Complex Samples procedure in SPSS 12.0 \[[@B9]\]. This procedure takes account of sampling weights and geographic clustering in the sample. For simplicity of exposition, the standard errors are not reported for each estimate. However, they were always \<2%. Such a standard error implies that a difference of 4% between vignettes was always statistically significant at the P \< 0.05 level. Multiple logistic regressions were then conducted to examine the levels of association between participants suggesting particular treatment options and their sociodemographic and mental health experience attributes. The following predictor variables were included: age group; consumer, carer and professional status, including whether or not professional help was obtained; and levels of perceived stigma and personal stigma. Two vignette measures -- type of vignette provided and whether respondents correctly identified the problem portrayed in that vignette -- were also included in the analyses. Each logistic regression was also adjusted to take into account of sampling weights and clustering method applied in this survey. These analyses were undertaken using STATA 8 \[[@B11]\]. Results ======= Reliability of coding open-ended responses ------------------------------------------ Inter-rater reliability was assessed for a randomly chosen 100 responses. Kappa was very good or good for encourage professional help-seeking (0.89), listen/talk/support person (0.70), listen/talk/support family (1.00), encourage seeing doctor (0.98), encourage seeing counsellor (0.93), encourage seeing psychiatrist (0.94), encourage seeing psychologist (0.88), and accompanying the person to a professional (0.95). It was moderate for give or seek information (0.48), encourage seeing unspecified and other professionals (0.56), and contact professional on their behalf (0.56). Kappa was fair for encourage self-help (0.34) and poor for assess problem/risk of harm (0.15). Kappa could not be calculated because of zero frequencies from the first rater for the categories of mental health team/services and other mental health professional. To better understand the reasons for the fair and poor agreement with two of the codes, positive and negative agreement were examined separately \[[@B12]\]. In both cases, negative agreement was high (0.95 for both), but positive agreement was low (0.38 and 0.15 respectively). The low positive agreement resulted because the second rater used these codes much less frequently than the first rater. However, despite this difference, neither rater used these two codes frequently, suggesting a low frequency of these responses in the population. Frequencies of mental health first aid responses ------------------------------------------------ Table [2](#T2){ref-type="table"} shows the percentage frequency of each category of response. The most common responses for all vignettes were to encourage professional help-seeking and to listen/talk/support the person. The differences between the vignettes were comparatively small. For the chronic schizophrenia vignette, there was a greater frequency of encouraging professional help-seeking and giving or seeking information, and a lesser frequency of listen/talk/support the person. Assessing the problem/risk of harm was more common for the depression vignettes than for the schizophrenia vignettes. Listen/talk/support family was more common for the early schizophrenia vignette, which was the only one to specifically mention family members. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Percentage of respondents who mentioned various first aid responses ::: Response Depression Vignette Depression/Suicidal Vignette Early Schizophrenia Vignette Chronic Schizophrenia Vignette ------------------------------------- ------------------------- ---------------------------------- ---------------------------------- ------------------------------------ Encourage professional help-seeking 58.6 62.4 57.5 66.0 Listen/ talk/ support person 69.1 73.4 70.3 65.6 Listen/ talk/ support family 2.2 2.8 8.5 2.4 Assess problem/ risk of harm 17.3 14.8 9.7 6.8 Give or seek information 5.5 7.1 9.1 12.9 Encourage self-help 12.0 10.8 12.3 10.2 ::: Table [3](#T3){ref-type="table"} shows types of professionals mentioned by respondents who encouraged professional help-seeking. The most commonly mentioned was GP/doctor unspecified. This recommendation was more common for the depression vignettes than for the schizophrenia vignettes. Conversely, psychiatrists were mentioned more for the schizophrenia vignettes. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Percentage of respondents who mentioned encouraging help-seeking with various types of professionals ::: Type of Professional Depression Vignette Depression/ Suicidal Vignette Early Schizophrenia Vignette Chronic Schizophrenia Vignette -------------------------------------------------- ------------------------- ----------------------------------- ---------------------------------- ------------------------------------ GP/ doctor unspecified 40.1 35.9 26.9 27.4 Counsellor 8.1 8.3 7.6 7.3 Psychiatrist 2.9 3.3 6.4 7.3 Psychologist 2.2 2.5 2.5 3.1 Mental health team/services 0.1 0.4 1.5 2.3 Other mental health professional 0.2 0.2 1.0 1.1 Unspecified professionals or other professionals 14.1 21.1 20.9 29.9 ::: Table [4](#T4){ref-type="table"} gives the percentage frequency of ways of encouraging professional help-seeking. Accompanying the person was more common for the chronic schizophrenia vignette, while contacting the professional on the person\'s behalf was more common for both the schizophrenia vignettes. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Percentage of respondents who mentioned ways of encouraging professional help-seeking ::: Type of Professional Depression Vignette Depression/ Suicidal Vignette Early Schizophrenia Vignette Chronic Schizophrenia Vignette -------------------------------------- ------------------------- ----------------------------------- ---------------------------------- ------------------------------------ Accompany person 8.7 11.0 9.7 16.7 Contact professional on their behalf 3.0 4.1 12.8 15.9 ::: Predictors of responses ----------------------- Table [5](#T5){ref-type="table"} shows the predictors of first aid responses from the multiple logistic regressions. Taking predictors with P \<0.01, encouraging professional help-seeking was more likely in response to the chronic schizophrenia vignette, from women, and from those who correctly recognized the problem in the vignette. It was less likely from consumers who had not sought help and respondents high on personal stigma. Listening/talking/supporting the person was more likely from consumers who had sought help. Listening/talking/supporting the family was more likely in response to the chronic schizophrenia vignette, and less likely from those high on personal stigma. Assessing the problem/ risk of harm was less likely in response to either schizophrenia vignette and from people aged 60+. Giving or seeking information was more likely in response to either schizophrenia vignette and from those who perceived stigma in others, but less likely from those high in personal stigma. Encouraging self-help was more likely from respondents high in personal stigma and less likely from those who correctly recognized the problem in the vignette. ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Odds ratios (and P-values) from multiple logistic regression analyses predicting first aid responses ::: Predictor Encourage professional help-seeking Listen/ talk/ support person Listen /talk/ support family Assess problem/ risk of harm Give or seek information Encourage self-help --------------------------------------------- ----------------------------------------- ---------------------------------- ---------------------------------- ---------------------------------- ------------------------------ ------------------------- Type of vignette Depression 1.00^1^ 1.00^1^ 1.00^1^ 1.00^1^ 1.00^1^ 1.00^1^ Depression/ suicidal 1.13 (0.284) 1.19 (0.149) 1.28 (0.433) 0.82 (0.154) 1.21 (0.360) 0.87 (0.385) Early schizophrenia 0.95 (0.636) 1.13 (0.298) 4.67 (0.000) 0.46 (0.000) 1.83 (0.002) 1.07 (0.671) Chronic schizophrenia 1.66 (0.000) 0.88 (0.266) 1.00 (0.997) 0.32 (0.000) 2.83 (0.000) 0.75 (0.100) Sociodemographic characteristics Age 18--39 1.00^1^ 1.00^1^ 1.00^1^ 1.00^1^ 1.00^1^ 1.00^1^ Age 40--59 1.04 (0.646) 0.89 (0.138) 0.91 (0.645) 0.94 (0.628) 0.87 (0.341) 1.04 (0.759) Age 60+ 0.97 (0.753) 0.79 (0.010) 0.85 (0.515) 0.64 (0.002) 0.66 (0.031) 1.04 (0.809) Female gender 1.32 (0.000) 1.20 (0.024) 0.92 (0.663) 0.74 (0.015) 0.99 (0.937) 0.87 (0.247) University degree 1.20 (0.065) 1.07 (0.428) 0.92 (0.660) 1.19 (0.218) 1.01 (0.934) 1.06 (0.676) Experience with mental disorders Consumer -- not sought help 0.50 (0.000) 1.47 (0.061) 1.56 (0.291) 1.54 (0.047) 0.61 (0.212) 1.77 (0.010) Consumer -- sought help 0.88 (0.314) 1.49 (0.000) 0.47 (0.024) 1.02 (0.920) 0.78 (0.247) 1.12 (0.514) Carer -- not sought help 0.78 (0.149) 1.08 (0.666) 0.48 (0.195) 1.11 (0.658) 1.01 (0.965) 1.17 (0.503) Carer -- sought help 1.22 (0.032) 1.02 (0.841) 1.09 (0.668) 0.83 (0.187) 1.05 (0.741) 0.98 (0.900) Professional 0.92 (0.403) 1.20 (0.068) 1.19 (0.400) 0.97 (0.853) 0.96 (0.801) 1.06 (0.679) Stigma Personal stigma 0.95 (0.000) 0.99 (0.494) 0.93 (0.001) 1.01 (0.612) 0.95 (0.000) 1.06 (0.000) Perceived stigma 1.01 (0.112) 0.99 (0.300) 1.00 (0.883) 1.00 (0.876) 1.04 (0.003) 0.99 (0.580) Correct recognition of disorder in vignette 1.60 (0.000) 1.05 (0.550) 1.22 (0.320) 1.27 (0.095) 1.32 (0.060) 0.70 (0.007) ^1^Reference category ::: Table [6](#T6){ref-type="table"} shows predictors of encouraging help-seeking with various types of health professionals. Encouraging the person to see a GP or doctor was more likely in response to either schizophrenia vignette, from women and from those who correctly recognized the problem in the vignette, while it was less likely in those high on personal stigma. Encouraging help-seeking from a counsellor was less likely from those aged 60+. Encouraging help-seeking from a psychiatrist was more likely in response to either schizophrenia vignette, while encouraging help-seeking from a psychologist was more likely from the university educated. Encouraging help-seeking from a mental health team/services was more likely in response to the chronic schizophrenia vignette and from those with professional experience in the area of mental health. Encouraging help-seeking from other mental health professionals was less likely in respondents high on personal stigma, while encouraging it from unspecified professionals was more likely in response to the depression/suicidal vignette, to either schizophrenia vignette, and from respondents who perceive stigma in others, while it was less likely in those high on personal stigma. ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Odds ratios (and P-values) from multiple logistic regression analyses predicting encouragement of help-seeking from various types of professionals ::: Predictor GP/ doctor unspecified Counsellor Psychiatrist Psychologist Mental health team/ services Other mental health professionals Unspecified professionals or other professionals --------------------------------------------- ---------------------------- ---------------- ------------------ ------------------ ---------------------------------- --------------------------------------- ------------------------------------------------------ Type of vignette Depression 1.00^1^ 1.00^1^ 1.00^1^ 1.00^1^ 1.00^1^ 1.00^1^ 1.00^1^ Depression/ suicidal 0.77 (0.012) 1.05 (0.797) 1.04 (0.895) 1.13 (0.688) 1.92 (0.597) 1.28 (0.784) 1.76 (0.000) Early schizophrenia 0.55 (0.000) 0.92 (0.697) 2.23 (0.002) 1.42 (0.281) 13.79 (0.013) 3.68 (0.102) 1.72 (0.000) Chronic schizophrenia 0.60 (0.000) 1.16 (0.461) 2.66 (0.000) 1.76 (0.062) 20.34 (0.004) 7.12 (0.024) 2.96 (0.000) Sociodemographic characteristics Age 18--39 1.00^1^ 1.00^1^ 1.00^1^ 1.00^1^ 1.00^1^ 1.00^1^ 1.00^1^ Age 40--59 1.19 (0.055) 0.88 (0.366) 1.35 (0.096) 0.96 (0.887) 0.50 (0.112) 1.03 (0.949) 0.93 (0.476) Age 60+ 1.35 (0.010) 0.49 (0.001) 1.55 (0.044) 1.24 (0.505) 1.00 (1.00) 0.54 (0.447) 0.74 (0.017) Female gender 1.34 (0.000) 1.29 (0.08) 0.74 (0.061) 1.06 (0.823) 1.00 (1.00) 1.22 (0.676) 1.10 (0.294) University degree 0.96 (0.638) 1.34 (0.054) 0.87 (0.473) 2.29 (0.001) 1.80 (0.132) 1.89 (0.148) 1.12 (0.264) Experience with mental disorders Consumer -- not sought help 0.69 (0.074) 0.63 (0.237) 0.38 (0.126) 0.86 (0.813) -^2^ -^2^ 0.61 (0.056) Consumer -- sought help 0.91 (0.365) 1.23 (0.227) 0.82 (0.457) 1.64 (0.060) 1.02 (0.967) 1.22 (0.735) 0.88 (0.334) Carer -- not sought help 0.87 (0.477) 1.10 (0.759) 1.53 (0.255) 0.96 (0.938) 0.88 (0.907) 0.78 (0.817) 0.97 (0.904) Carer -- sought help 1.08 (0.389) 1.32 (0.074) 1.46 (0.025) 1.51 (0.110) 1.05 (0.908) 1.08 (0.842) 1.01 (0.916) Professional 1.00 (0.985) 0.95 (0.751) 0.891 (0.570) 1.17 (0.570) 2.42 (0.008) 1.01 (0.990) 0.98 (0.828) Stigma Personal stigma 0.97 (0.005) 1.00 (0.969) 1.00 (0.835) 1.00 (0.917) 0.92 (0.120) 0.89 (0.007) 0.95 (0.000) Perceived stigma 0.99 (0.079) 1.01 (0.468) 1.00 (0.969) 0.98 (0.439) 1.04 (0.188) 1.05 (0.083) 1.03 (0.000) Correct recognition of disorder in vignette 1.43 (0.000) 1.49 (0.019) 1.17 (0.379) 0.74 (0.171) 1.60 (0.255) 3.00 (0.072) 1.24 (0.031) ^1^Reference category ^2^Could not be calculated ::: Table [7](#T7){ref-type="table"} shows predictors of ways of encouraging professional help-seeking. Accompanying the person to professional help was more likely in response to the chronic schizophrenia vignette and from women. Contacting the professional on the person\'s behalf was more likely in response to either schizophrenia vignette and from respondents who perceived stigma in others, while it was less likely from those high on personal stigma. ::: {#T7 .table-wrap} Table 7 ::: {.caption} ###### Odds ratios (and P-values) from multiple logistic regression analyses predicting ways of encouraging professional help-seeking ::: Predictor Accompany person Contact professional on their behalf --------------------------------------------- ---------------------- ------------------------------------------ Type of vignette Depression 1.00^1^ 1.00^1^ Depression/ suicidal 1.21 (0.285) 1.68 (0.071) Early schizophrenia 1.08 (0.646) 5.27 (0.000) Chronic schizophrenia 2.26 (0.000) 8.01 (0.000) Sociodemographic characteristics Age 18--39 1.00^1^ 1.00^1^ Age 40--59 1.07 (0.549) 0.97 (0.828) Age 60+ 0.90 (0.485) 0.63 (0.017) Female gender 1.43 (0.003) 1.06 (0.683) University degree 0.86 (0.220) 1.31 (0.046) Experience with mental disorders Consumer -- not sought help 0.51 (0.060) 0.33 (0.068) Consumer -- sought help 1.19 (0.228) 0.71 (0.136) Carer -- not sought help 0.83 (0.491) 1.16 (0.620) Carer -- sought help 0.98 (0.893) 1.12 (0.424) Professional 1.21 (0.109) 1.12 (0.427) Stigma Personal stigma 0.99 (0.659) 0.95 (0.000) Perceived stigma 0.99 (0.283) 1.03 (0.008) Correct recognition of disorder in vignette 1.16 (0.280) 1.37 (0.028) ^1^Reference category ::: Discussion ========== The most common first aid responses were found to be encouraging professional help-seeking and listening/talking/supporting the person. Nevertheless, these responses were far from universal, with 32--44% not mentioning professional help and 27--34% not mentioning listening/talking/supporting. Given the likely helpfulness of these first aid responses, they need greater promotion in the community. Other first aid responses were mentioned only by a minority. Of particular concern is the low percentage assessing risk of harm for the person in the depression/suicidal vignette. Asking about suicidal intentions is often recommended as a response \[[@B13],[@B14]\], although there is no evidence on whether this actually helps prevent suicide. Previous research has investigated first aid responses of young people to suicidal intent in their peers, finding that many would not tell a responsible adult about it \[[@B15]\]. However, we are unaware on any previous research on adults\' responses to suicidal intent in someone they know. Encouraging self-help was another minority response, but was associated with stigma and lack of recognition of the mental disorder in the vignette. Respondents appear to have suggested self-help as an alternative to professional help, rather than as a complement to it. We have previously reviewed the evidence on self-help interventions for depression and anxiety disorders and found that some have support \[[@B16],[@B17]\]. Such interventions need to more widely promoted, but not as a substitute for professional help. When correlates of first aid responses were examined, most variables had at least one significant association. However, the variables that most often predicted first aid responses were female gender, low personal stigma and correct recognition of the disorder in the vignette. The latter two predictors indicate potential barriers to providing first aid. Respondents who saw the person in the vignette as having negative attributes were less likely to respond by encouraging professional help-seeking or providing personal support. Efforts to reduce stigma in the community may therefore facilitate greater first aid. People who did not recognize the disorder showed a similar pattern of responses. These people lack knowledge of mental disorders, at least to the extent of being able to apply a psychiatric label. Therefore community education about how to recognize these disorders may also facilitate helpful first aid responses. One approach to improving public responses to people with a mental disorder is an individual training course in mental health first aid \[[@B18]\]. Such a course has been developed and teaches an action plan with five steps of mental health first aid: (1) Assess the risk of suicide or harm; (2) Listen non- judgementally; (3) Give information and encouragement; (4) Encourage person to get appropriate professional help; and (5) Encourage self-help strategies. \[[@B19]\]. Two randomised controlled trials of this course, one in a work place environment \[[@B20]\] and the other with members of the public in a rural area \[[@B8]\], have shown benefits of the training: better recognition of disorders, changes in beliefs about treatment to be more like those of professionals, decreased social distance, increased confidence in providing help, and increase in actual help provided. The workplace trial also found improved mental health in course participants \[[@B20]\]. The study has two limitations which must be acknowledged. The major one is that the study has assessed intended first aid to a hypothetical person in a case vignette. Whether these intentions would be implemented in practice is unknown. Intentions might be seen as placing an upper limit on responses, such that if a respondent fails to state an intention, this is unlikely to be seen in practice. An alternative approach would have been to ask the respondent how they had treated actual people they knew with mental health problems. However, the disadvantage of this alternative would have been the lack of standard situations. A second limitation is that the inter-rater reliability of coding some of the first aid responses was low. Conclusions about these responses must be viewed with caution. On the other hand, the strengths of the study are the large representative sample, the open-ended responses which did not constrain the respondents to particular alternatives, and the ability to compare responses to a series of standard scenarios. Conclusions =========== There is room for improving the range of mental health first aid responses in the community. Lack of knowledge of mental health and stigmatizing attitudes are important barriers to effective first aid. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= AFJ was involved in securing funding for the survey, had a major role in the design of the survey and the interview questionnaire, carried out the descriptive statistical analysis and had a major role in writing the manuscript. KAB provided research assistance with the survey, coded all the responses, and wrote the method relevant to this coding. KMG was involved in the design of the study, developed the stigma scales and wrote some of the manuscript. BAK developed the coding scheme, was the second rater for inter-rater reliability, and wrote some of the manuscript. RAP did the regression analyses and wrote the section of the Method describing this. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-244X/5/9/prepub> Acknowledgements ================ This study is part of the Australia-Japan Partnership, which is an agreement between the governments of the two countries for joint projects in areas of health. Funding for the survey was provided by the Australian Department of Health and Ageing, a National Health and Medical Research Council Program Grant, and \"beyondblue: the national depression initiative\". We thank Helen Christensen for her role in the study.	PubMed Central	2024-06-05T03:55:52.901300	2005-2-6	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549043/", "journal": "BMC Psychiatry. 2005 Feb 6; 5:9", "authors": [ { "first": "Anthony F", "last": "Jorm" }, { "first": "Kelly A", "last": "Blewitt" }, { "first": "Kathleen M", "last": "Griffiths" }, { "first": "Betty A", "last": "Kitchener" }, { "first": "Ruth A", "last": "Parslow" } ] }
PMC549044	Introduction ============ Respiratory syncytial virus (RSV) is an important respiratory pathogen that produces an annual worldwide epidemic of respiratory illness primarily in children, but also in the elderly \[[@B1],[@B2]\]. In the USA alone, RSV infection of children causes about 100,000 hospitalizations and 4,500 deaths annually (MMWR, 1996). RSV commonly precipitates bronchiolitis and exacerbates asthma but is also associated with severe life threatening respiratory infections in individuals with coronary artery disease or who are immunocompromised \[[@B3]-[@B6]\]. At the molecular level, RSV infection up-regulates the expression of several cytokines and chemokines, such as IL-1β, IL-6, IL-8, TNF-α, MIP1α, RANTES, and the adhesion molecule ICAM-1, ET-1, LTB4 and LTC4/D4/E4 \[[@B7]-[@B13]\]. Furthermore, elevated levels of cytokines and chemokines have been found in the nasal secretions of naturally RSV-infected children and of artificially-infected adults \[[@B14]-[@B17]\]. Defects in IL-12 and IFN-α production have been associated with severe RSV disease \[[@B18]\]. Despite progress in our understanding of immunopathology, the lack of a suitable animal model, with pathophysiology similar to humans, allowing appropriate virology, immunology, pathology and toxicology testing, has hindered the development of prophylactic and therapeutic interventions against RSV infection \[[@B19]\]. The pathology of RSV infection has been examined in a number of animals including primates, cotton rats, mice, calves, guinea pigs, ferrets and hamsters \[[@B19]\]. The choice of an experimental model is governed by the specific manifestation of the disease. The development of multiple animal models reflects the multifaceted nature of human RSV disease, in which clinical manifestations and sequelae depend upon age, genetic makeup, immunologic status and concurrent disease status of the individual \[[@B19]\]. Currently there is no single animal model that duplicates all forms of RSV disease. While cotton rats provide a good model for toxicologic evaluations, mice are considered advantageous for immunology and vaccine development. Furthermore, in mice the importance of IFN-γ, IL-6, IL-10 and IL-13 has been described \[[@B20]-[@B24]\]. The mouse provides an excellent model for human RSV infection because of the following: (a) the mouse is the best-characterized animal model, and experiments can be performed in this model in a cost- and time-effective manner, (b) a wide array of immunological reagents is available for studies in this model, and (c) the A2 strain of human RSV administered intranasally readily infects lungs of mice, and exhibits a time course of infection, pathology and resolution similar to that seen in humans \[[@B25]\]. Treatment of mice with the anti-RSV compound ribavirin decreases RSV titers in the lungs \[[@B26]\]. Depending upon the amount of RSV administered, the illness in micemay range from mild pneumonitis of the lung to weight loss \[[@B27]\]. Healthy BALB/c mice are semi-permissive to RSV and develop only limited inflammation and airway reactivity. Based on the reports that deficiency in IL-12 and IFN production increases severity of RSV disease, we hypothesized that rendering mice immunocompromised, will improve permissiveness to RSV and provide a better model for RSV infection. To test this hypothesis, BALB/c were treated with cyclophosphamide, infected with RSV, and characterized in terms of viral infectivity and pathology, immunology, and immunohistology. The results show that cyclophosphamide temporarily decreases IL-12 production and thus augments viral replication and the immunopathology of RSV disease. Materials and Methods ===================== Animals ------- Female six-week old BALB/c mice were purchased from Jackson Laboratory (Bar Harbor, ME) and maintained in a pathogen-free environment. All procedures were reviewed and approved by the University of South Florida Committee on Animal Research. Cyclophosphamide treatment -------------------------- Cyclophosphamide (CYP; Sigma, St. Louis, MO) was administered to mice intraperitoneally (i.p.) at a single dose of 100 mg per kg five days prior to RSV infection. RSV infection, weight determination and tissue collection --------------------------------------------------------- The A2 strain of human RSV (American Type Culture Collection, Manassas, VA) was propagated in Hep-2 cells (ATCC) in a monolayer culture as previously described (Behera et al., 1998). Mice were infected intranasally with 5 × 10^5^PFU of RSV in a volume of 50 μl five days after treatment with CYP. One set of animals was monitored for weight loss at days 5, 10, 15 and 22 following CYP treatment (0, 5, 10 and 17 days after RSV infection). A second set of animals was sacrificed five days after infection and their lungs were removed for determination of RSV titers, cytokine levels and histopathology. ### RSV plaque assay HEp-2 cells (5 × 10^5^/well) in 6-well plates were infected with 5 × 10^5^pfu RSV per well for 2 hours at 37°C. The RSV was removed and the wells were overlaid with 1.5 ml of growth medium containing 0.8% methylcellulose. The cells were then incubated at 37°C for 72 hours, after which the overlay was removed. Following incubation, the cells were fixed in cold 80% methanol for 3 hours, blocked with 1 % horse serum in PBS at 37°C for 30 min, then incubated with anti-RSV monoclonal antibody (NCL-RSV 3, Vector Laboratories, Burlingame, CA) diluted 1:400 for 1 hour at 37°C. Secondary antibody staining and substrate reactions were performed using the Vectastain ABC Kit (Vector Laboratories) and diaminobenzidine in H~2~O~2~(Pierce, Rockford, IL) was used as a chromagen. The plaques were enumerated by microscopy and the results were expressed as mean ± standard error of the mean. Determination of airway hyperresponsiveness (AHR) ------------------------------------------------- AHR was measured in unrestrained mice using a whole body plethysmograph (Buxco, Troy, NY), as previously described (Schwarze et al., 1997) and expressed as enhanced pause (Penh). Groups of mice (n = 4) were exposed for 5 min to nebulized PBS and subsequently to increasing concentrations (6, 12, 25 and 50 mg/ml) of nebulized methacholine (MCh; Sigma, St, Louis, MO) in PBS using an ultrasonic nebulizer. After nebulization, recordings were taken for 5 minutes. Penh values were averaged and expressed as a percentage of baseline Penh values obtained following PBS exposure. Immunohistochemical analysis ---------------------------- Mouse lungs were rinsed with intratracheal injections of PBS then perfused with 10 % neutral buffered formalin. Lungs were removed, paraffin-embedded, sectioned at 20 μm and stained with hematoxylin and eosin (H & E). A semi-quantitative evaluation of inflammatory cells in the lung sections was performed as previously described (Kumar et al., 1999). Whole lung homogenates were prepared using a TissueMizer and assayed for cytokines IL-10, IL-12 and IFN-γ by ELISA (R & D Systems, Minneapolis MN), following the manufacturer\'s directions. The results are expressed as cytokine amount in picograms per gram of lung (pg/g). Detection of RSV and cytokines in the lungs by RT-PCR ----------------------------------------------------- Total cellular RNA was isolated from lung tissue using TRIZOL reagent (Life Technologies, Gaithersburg, MD). Forward and reverse primers used were as follows: RSV-N forward: 5\'-GCG ATG TCT AGG TTA GGA AGA A-3\'; reverse: 5\'-GCT ATG TCC TTG GGT AGT AAG CCT-3\'; mouse IFN-γ Forward: 5\'-GCT CTG AGA CAA TGA ACG CT-3\'; reverse: 5\'-AAA GAG ATA ATC TGG CTG TGC-3\'; mouse IL-10 forward: 5\'-GGA CTT TAA GGG TTA CTT GGG TTG CC-3\'; reverse: 5\'-CAT TTT GAT CAT CAT GTA TGC TTC T-3\'; mouse IL-12 forward: 5\'-CAG TAC ACC TGC CAC AAA GGA -3\'; reverse: 5\'-GTG TGA CCT TCT CTG CAG ACA -3\' and β-actin forward: 5\'-GAC ATG GAG AAG ATC TGG CAC-3\'; reverse: 5\'-TCC AGA CGC AGG ATG GCG TGA -3\'. All PCRs were denatured at 95°C for 1 min, annealed at 56°C for 30 sec, and extended at 72°C for 1 min for 25--35 cycles. All amplifications were done in triplicate and repeated three times. The PCR products were separated by agarose gel electrophoresis and quantified using Advanced Quantifier Software (BioImage, Ann Arbor, MI). Cell enumeration of bronchoalveolar lavage fluid ------------------------------------------------ Bronchoalveolar lavage (BAL) fluid was collected and differential cell counts were performed as previously described (Kumar et al., 1999). Briefly, BAL was centrifuged and the cell pellet was suspended in 200 μl of PBS and counted using a hemocytometer. The cell suspensions were then centrifuged onto glass slides using a cytospin centrifuge at 1000 rpm for 5 min at room temperature. Cytocentrifuged cells were air dried and stained with a modified Wright\'s stain (Leukostat, Fisher Scientific, Atlanta, GA) which allows differential counting of monocytes and lymphocytes. At least 300 cells per sample were counted by direct microscopic observation. Statistical analysis -------------------- Values for all measurements were expressed as mean ± SD or SEM. The data were analyzed by ANOVA. Paired and unpaired results were compared by a Wilcoxon rank sum test or Mann-Whitney test respectively. Differences between groups were considered significant at p\< 0.05. Results ======= Cyclophosphamide treatment augments RSV infection in mice --------------------------------------------------------- Groups of mice were injected i.p. with a single dose of CYP or PBS and five days later infected with RSV. Five days after infection, RSV titers in one group of mice were measured by plaque assay of lung homogenates (Fig. [1A](#F1){ref-type="fig"}). The mice pretreated with CYP produced significantly more (p\< 0.01) RSV plaques compared to the PBS control group. Weight loss is a clinical correlate of RSV infection, therefore weights were measured in a parallel group of mice on day 5, 10, 15, and 22 after CYP treatment (day 0, 5, 10 and 17 after RSV infection) (Fig. [1B](#F1){ref-type="fig"}). CYP treatment alone resulted in a weight loss or reduced weight gain compared to PBS, but the RSV-infected, CYP-treated mice lost significantly more weight than those exposed to RSV alone p \< 0.05; †.p \< 0.01(vs. RSV) or PBS (p \< 0.01 vs. Control). Pretreatment with CYP resulted in increased weight loss in the RSV-infected mice through day 15 and reduced weight gain at day 22 indicating that cyclophosphamide treatment exacerbated the pathology of RSV infection. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### (A) CYP increases RSV titer in the lungs of BALB/c mice.Mice were treated with CYP (100 mg/kg, i.p.) or PBS and 5 days later infected with RSV (50 μl i.n. twice, 10^6^PFU/ mouse). Animals were sacrificed on day 4 and RSV titers were measured in whole lung homogenates by RSV plaque assay. (n = 4 for each group; § P \< 0.01 vs PBS group). (B) Cyclophosphamide affects body weight.Mice (n = 4) were infected with RSV alone or were treated with CYP (100 mg/kg i.p.) prior to infection. Body weights were measured on day 1, 5, 10, 15, and 22 after treatment. Bars represent means ± SEM. (\* P \< 0.05; †. P \< 0.01 vs RSV); ‡ P \< 0.05; § P \< 0.01 vs control). ::: ![](1743-422X-2-3-1) ::: Cyclophosphamide pretreatment increases RSV-inducible lung inflammation ----------------------------------------------------------------------- To examine whether CYP treatment increases inflammatory effects in the lungs of RSV-infected mice, we determined airway hyperresponsiveness (AHR), cellular infiltration into the lung and lung histopathology. Groups of BALB/c mice either infected with RSV alone or treated with cyclophosphamide (CYP) prior to infection were lavaged and cells in the fluid were centrifuged onto slides. BAL cells were stained with Leukostat. Cells were counted from 4 different slides from each group in a blinded fashion. Cell counts were plotted as percentage of total cells (Fig. [2A](#F2){ref-type="fig"}). There was a decrease in the number of macrophages and increases in lymphocyte and neutrophil numbers following RSV infection that was enhanced by prior treatment with CYP. To analyze the extent of lung pathology, lungs were paraffin embedded, sectioned and stained with hemotoxylin-eosin (HE). The lung sections from RSV-infected, CYP-treated mice (Fig. [2C](#F2){ref-type="fig"}, a & b) showed significantly greater inflammation than lungs from mice given RSV alone (Fig. [2C](#F2){ref-type="fig"}, c & d). The RSV-infected groups showed greater inflammation than uninfected control mice (Fig. [2C](#F2){ref-type="fig"} e & f). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### (A) BAL cell differential of RSV-infected mice. Mice were treated with cyclophosphamide (CYP) or vehicle 5 days before infection with RSV. Animals were sacrificed on day 4 postinfection and BAL was performed. Following cytocentrifugation, BAL cells were stained with Leukostat and counted from 4 different slides from each group in a blinded fashion. Cell counts as percentage of total were plotted. (B) Measurement of airway hyperrresponsiveness (AHR). Mice treated as above were tested for AHR by methacholine challenge in a plethysmograph. AHR is expressed as PENH, percent of control. (C) Lung histopathology.Mice were infected with RSV alone (C and D)or treated with cyclophosphamide (A and B)prior to RSV infection. The third group of mice was not exposed to RSV (E and F).Animals were sacrificed on day 5 and their lungs removed and sectioned. Paraffin-embedded lung sections were stained with hematoxylin-eosin. ::: ![](1743-422X-2-3-2) ::: Increased RSV infection in CYP-treated BALB/c mice is associated with increased production of immunoregulatory cytokines ------------------------------------------------------------------------------------------------------------------------ To examine the cytokine profile in lungs from RSV-infected mice with or without CYP treatment, the gene expression of IL-10, IFN-γ and IL-12 was measured by RT-PCR. Gel profiles and densitometric analyses are shown in Fig. [3A](#F3){ref-type="fig"} and [3B](#F3){ref-type="fig"}. The results show that mice infected with RSV after CYP treatment have increased mRNA expression for all of three cytokines compared to control mice or mice infected with RSV alone. To determine whether mice treated with CYP and infected with RSV do produce more of these proteins, cytokines were measured by ELISA on homogenates prepared from whole lungs. IL-10 (p \< 0.05 vs. PBS), IL-12 (p \< 0.05 vs. RSV) and IFN-γ (p \< 0.05 vs. PBS) levels were significantly higher in the CYP-treated group than the control group (Fig. [4A--C](#F4){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Detection of RSV and cytokines in the lungs of BALB/c mice. (A)RSV-N and IL-10, IL-12, IFN-γ and β-actin were checked by RT-PCR. Mice were infected with RSV alone or treated with CYP prior to infection. The third group was uninfected (PBS) as control. Animals were sacrificed on day 5, their lungs removed and RNA was isolated and used in RT-PCR assay. (B) Densitometric analysis of the band densities from part A.Relative intensity refers to the ratio of the intensity of each cDNA product to that of β-actin. ::: ![](1743-422X-2-3-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### CYP-treated BALB/c mice produce higher levels of Th1 and Th2 cytokines. Mice were infected with RSV alone or were treated with cyclophosphamide prior to infection. The third group of mice received no virus (PBS only) as control. Animals were sacrificed on day 5 and their lungs removed. Whole lung homogenates were prepared and cyrtokines were measured by ELISA. Results are given as mean ± SEM (n = 4 for each group). (A)CYP-pretreated mice produce higher IL-10 in the lungs. (‡ P \< 0.05 vs. PBS). (B)IL-12 was higher in CYP-treated mice. (§ P \< 0.01 vs. PBS. \* P \< 0.05 vs. RSV). (C)IFN-γ was higher in CYP-treated mice. (\* P \< 0.05 vs. RSV; ‡ P \< 0.05 vs. PBS). ::: ![](1743-422X-2-3-4) ::: Cyclophosphamide treatment causes a transient reduction in IL-12 and IFN-γ expression ------------------------------------------------------------------------------------- To examine the mechanism underlying the increased RSV infection in CYP-treated animals, the levels of two cytokines that exert antiviral activity, IL-12 and IFN-γ was measured after treatment with CYP (Fig. [5](#F5){ref-type="fig"}). Mice were sacrificed on days 1, 2, 4, 6 after treatment and IL-12 and IFN-γ protein levels were measured in whole lung homogenates by ELISA. Untreated BALB/c mice were used as controls. Treatment with CYP gradually decreased both IL-12 (p \< 0.05) and IFN-γ (p \< 0.01 vs. Control) until day 4. These results show that decreased production of IL-12 and IFN-γ may play a role in the observed increase in RSV infection in CYP-treated mice. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Changes in IL-12 and IFN-γ levels over time in CYP-treated mice.Mice were treated with cyclophosphamide at 100 mg/kg i.p. Animals were sacrificed on day 1, 2, 4, 6 after treatment and their lungs removed. Whole lung homogenates were prepared and IL-12 (A)and IFN-γ (B)were measured by ELISA. Untreated mice were used as control. Results are shown as mean ± SEM (n = 2; ‡ P \< 0.05; § P \< 0.01 vs. control). ::: ![](1743-422X-2-3-5) ::: Discussion ========== The main focus of this study has been to establish and characterize an immunocompromised mouse model for studying RSV infection. Mice are only semi-permissive to RSV infection, yet can serve as a useful model for immunological studies. Compared to the traditional BALB/c mouse model, the use of cyclophosphamide to create an immunocompromised condition provides an effective means of augmenting RSV replication and disease. Pretreatment of BALB/c mice with CYP results in RSV titers in the lungs of these immunocompromised mice that are increased significantly compared to the group infected with RSV without cyclophosphamide treatment. These results are consistent with an earlier study in the cotton rat model \[[@B28]\]. In another study, a high titer RSV inoculum (10^7^PFU/ml) was administered intranasally to old mice and resulted in clinical illness and appreciable pathology in the lung \[[@B25]\], while mice inoculated with 10^6^PFU/ml, or less, did not exhibit symptoms of illness. Most studies using mice as models employ lower doses of RSV because RSV infection in humans, which induces a pneumonia-like pulmonary inflammation, occurs typically at sub-clinical RSV doses. The loss of body weight in CYP-treated mice following RSV infection compared to untreated control mice confirmed that CYP-treatment increased the susceptibility to RSV infection at lower inocula. This finding that permissiveness to RSV can be augmented by rendering mice immunocompromised is significant, as it increases the utility of the mouse model for RSV infection. Consistent with increased RSV infection, the cellular population in BAL fluid was altered. Especially significant are the increases in lymphocytes and neutrophils in CYP-treated mice compared to controls. This data is in agreement with previous reports showing that RSV infection increased lymphocyte infiltration in the lung \[[@B2]\]. Along with increased cellular infiltration, lung pathology, particularly epithelial denudation and goblet cell hyperplasia, is also markedly increased. Expression of IL-10, IL-12 and IFN-γ was examined to determine if RSV-induced changes in the levels of these cytokines in the lungs of CYP-treated mice played a role in the increased lung immunopathology. RSV-infected CYP-treated mice exhibited significantly increased expression of these cytokines at the protein and mRNA level in agreement with previous observations that RSV infection induced enhanced expression of Th1 and Th2 cytokines \[[@B22],[@B29]\]. Other studies have shown that IFN-γ can induce production of IL-12 in a self-activating loop, by activating macrophages which produce IL-12 \[[@B30]\]. Although CYP treatment is known to induce an immunosuppressed condition, the mechanism is unclear. The results of cytokine analysis on days 1 to 5 after cyclophosphamide treatment indicated that IL-12 and IFN-γ were reduced and the reduction was highest on days four and two, respectively. These results suggest that the ability of cells to produce these cytokines at the time of RSV infection is an important determinant of the magnitude of infection in terms of increased RSV replication and titer. Impairment in IL-12 and IFN-γ production at the key moment of acute infection leads to rapid viral replication and subsequent pathology. In a previous report we demonstrated the importance of IFN-γ by artificially increasing IFN-γ levels and showing that viral titers were decreased because of the induction of 2\'-5\'oligoadenylate synthetase which activates RNase L to degrade viral RNA \[[@B31]\]. Also, studies in humans have suggested that individuals lacking IFN-α or IL-12 are at a higher risk of severe RSV disease. Thus, down-regulation of the production of these cytokines is a likely factor underlying the observed enhancement of RSV infection by cyclophosphamide treatment. In conclusion, the results of this study demonstrate that cyclophosphamide treatment of BALB/c mice renders them more susceptible to RSV infection as revealed by increased RSV titers in the lung and decreased body weight. The mechanism of this increase in infection involves transient down regulation of IFN-γ and IL-12 induced by cyclophosphamide treatment. Competing Interests =================== The author(s) declare that they have no competing interests. Authors\' Contributions ======================= XK, BAL and cell enumeration; GH, data analysis; GP, AHR, RT-PCR; MK, RSV infection and assay, tissue collection, RT-PCR; AB, cyclophosphamide treatment, tissue collection, ELISAs; TSR, immunohistochemistry; JZ, cell culture and virus preparation; RFL, experimental design and analysis; SSM, project design, experimental analysis and data interpretation. Acknowledgements ================ This study was supported by a VA Merit Review Award and by an American Heart Association (Florida Affiliate) research grant to SSM, and by the Joy McCann Culverhouse Endowment to the Division of Allergy and Immunology Airway Disease Research Center.	PubMed Central	2024-06-05T03:55:52.905035	2005-2-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549044/", "journal": "Virol J. 2005 Feb 8; 2:3", "authors": [ { "first": "Xiaoyuan", "last": "Kong" }, { "first": "Gary R", "last": "Hellermann" }, { "first": "Geoff", "last": "Patton" }, { "first": "Mukesh", "last": "Kumar" }, { "first": "Aruna", "last": "Behera" }, { "first": "Timothy S", "last": "Randall" }, { "first": "Jian", "last": "Zhang" }, { "first": "Richard F", "last": "Lockey" }, { "first": "Shyam S", "last": "Mohapatra" } ] }
PMC549045	Background ========== Maintenance of blood brain barrier (BBB) integrity is critical to prevent the passage of potentially harmful factors, such as pathogens or toxins into the brain. During the progression of central nervous system (CNS) infectious disease, pathogens might gain access to the brain by compromising the integrity of the BBB. In the course of AIDS, HIV enters the brain at early stages, disrupting the components of the BBB resulting in a chronic state of inflammation known as HIV encephalitis (HIVE) \[[@B1],[@B2]\]. HIVE is characterized by the presence of HIV-infected microglia and / or macrophages in the brain, the formation of multinucleated giant cells and microglial nodules, astrogliosis and myelin pallor, the combined effects of which could result in cognitive impairment \[[@B3]\]. Because endothelial cells of the BBB provide the first point of contact between blood-borne viral products and the brain, they provide the front line of defence against viral entry into the CNS. Alterations in signalling between components of the BBB with either HIV proteins or factors produced in response to HIV infection, such as cytokines and chemokines, may disrupt BBB integrity, resulting in a compromise that could promote transmigration of activated monocytes or HIV infected cells into the brain. Toxic viral products released by HIV-infected cells such as, gp120, Tat or Nef, together with cytokines and chemokines from activated monocytes, can act to increase BBB permeability \[[@B4]-[@B8]\]. Cell-free gp120 is found in the serum of HIV infected patients and crosses the BBB by absorptive endocytosis \[[@B9]\] and has been detected in the perivascular regions of the brain \[[@B10]\]. Gp120 is toxic to uninfected cells such as cerebral endothelial cells \[[@B8]\], and induces numerous signalling alterations in glial cells leading to indirect neuronal dysfunction and death \[[@B11],[@B12]\]. Huang et al. have shown that gp120 promotes apoptosis in human umbilical vascular endothelial cells (HUVEC) by acting through CXCR4 and CCR5 chemokine receptors to increase activation of protein kinase (PKC) \[[@B13],[@B14]\]. Furthermore, these studies show that the toxic effects of gp120 were blocked by PKC antagonists, sphingosine, phorbol esters and fibroblast growth factor 2 (FGF2) \[[@B13]\]. While viral products and inflammatory response proteins may damage components of the BBB, other factors, such as growth factors, may work to preserve BBB integrity through maintaining endothelial cell fitness. In this context, FGF2 is of particular interest for several reasons. First, FGF2 is produced primarily by astrocytes that are in proximity to cerebral endothelial cells in the blood brain barrier \[[@B15]\]. Among the known astrocyte-derived growth factors, only FGF2 mimics the signalling actions of astrocytes to the BBB \[[@B15],[@B16]\]. Second, of the four FGF receptors (FGFR), FGFR1 is mainly expressed on neurons and endothelial cells while FGFR2 and FGFR3 are found on glial cells \[[@B17]-[@B19]\]. FGF2, which binds to FGFR1, exhibits a wide range of angiotrophic effects \[[@B15],[@B16]\] and promotes the survival of cortical and hippocampal neurons \[[@B15],[@B16],[@B20]-[@B22]\]. Third, FGF2 signals through FGFR1 and activates phosphoinositol 3 kinase (PI3K), protein kinase C (PKC), extracellular regulated kinase (ERK), and p38 pathways \[[@B23]-[@B25]\]. Both ERK and p38 belong to the mitogen-activated protein kinase (MAPK) signalling pathways and have been shown to be involved in regulating endothelial cell survival \[[@B15],[@B16]\]. FGF2 protection of HUVEC from gp120 is proposed to occur by preventing the gp120-mediated increase in PKC activity \[[@B13]\], however, protective signalling mechanisms directly induced by FGF2 have not been addressed. Therefore, we investigated the signalling pathways involved in FGF2-mediated protection against gp120 toxicity in HUVEC. Our studies indicate that FGF2 protects endothelial cells from gp120-mediated toxicity by crosstalk among several signalling pathways downstream of the tyrosine kinase FGFR. These pathways include, the ERK, PI3K/AKT and PKC signalling cascades. Likewise, other studies have suggested that signalling pathways that inhibit cell death (e.g., p38, MAPK/ERK) and survival pathways (e.g., AKT/PKB) may represent the next investigational step in inhibition of HIV-related CNS toxicity \[[@B26]\]. In this context, FGF2-mediated signalling may play an important role in maintaining BBB integrity during HIV trafficking into the brain and/or cell-free gp120 interactions with cerebral endothelial cells. Results ======= FGF2 protects endothelial cells from gp120-mediated toxicity ------------------------------------------------------------ Consistent with previous reports \[[@B13],[@B14],[@B27],[@B28]\], our results showed that gp120 (25 ng/ml) increased cell death of HUVEC above control by approximately 27.5% (average of results from all viability assays) after 24 h exposure (Fig. [1](#F1){ref-type="fig"}) as determined by Trypan Blue Exclusion, TUNEL, and FA/PI staining (Fig. [1E, J, O](#F1){ref-type="fig"}, respectively). However, cells pre-treated with FGF2 (20 ng/ml) for 24 h and then exposed to gp120 displayed essentially the same percentage of cell death as untreated control cells (Fig. [1D, E, I, J, N, O](#F1){ref-type="fig"}). Although FGF2 treatment of HUVEC most likely improved overall cell fitness \[[@B15],[@B16]\], no significant differences in the total numbers of cells (Fig. [2A](#F2){ref-type="fig"}) or in cell viability were observed between control and FGF2 treated cultures (Fig. [1E, J, O](#F1){ref-type="fig"} and [2B](#F2){ref-type="fig"}). Furthermore, time course experiments indicated that simultaneous treatment (data not shown) or pre-treatment with FGF2 up to 24 h was effective at protecting cells from gp120 toxicity (Fig. [2B](#F2){ref-type="fig"}). These results indicate that FGF2 is protective against gp120-mediated toxicity in HUVEC. FGF2 activates ERK in HUVEC --------------------------- To explore mechanisms involved in the angio-protective effects of FGF2 against gp120, we first investigated FGF2-stimulated signalling mechanisms that are involved in cell survival pathways. The binding of FGF2 to its receptor (FGFR1) induces several signalling cascades, such as MAPK-mediated ERK activation and AKT-mediated GSK3β inactivation, both of which regulate cell survival. We first determined the effects of FGF2 stimulation on phosphorylation of ERK and GSK3β in time course experiments in HUVEC (Fig. [3](#F3){ref-type="fig"}, lanes 1--3). Western blot analysis showed that HUVEC treated with FGF2 (20 ng/ml) resulted in maximum ERK phosphorylation 5--10 min after stimulation (Fig. [3A](#F3){ref-type="fig"}, lanes 1--3), followed by a progressive decrease reaching undetectable levels at 60 min (data not shown), with no effect on levels of total ERK (Fig. [3B](#F3){ref-type="fig"}, lanes 1--3). Neither GSK3β (Fig. [3C](#F3){ref-type="fig"}) nor PKC (data not shown) phosphorylation was affected by FGF2 treatment. To test the specificity of FGF2 on signalling, HUVEC were exposed to pharmacological inhibitors for PI3K (LY294002), ERK (U0126), and PKC (Bis I and Gö6983) for 30 min prior to FGF2 treatment (Fig. [3A](#F3){ref-type="fig"}, lanes 4--7, respectively). ERK phosphorylation was inhibited by blocking ERK and PKC (Fig. [3](#F3){ref-type="fig"}, lanes 5--7). Interestingly, blocking the PI3K/AKT/GSK3β pathway resulted in a dramatic increase in ERK phosphorylation (Fig. [3A](#F3){ref-type="fig"}, lane 4). Neither FGF2 nor inhibitors affected levels of total ERK (Fig. [3B](#F3){ref-type="fig"}). With regard to GSK3β, blocking PI3K with LY294002 and PKC with Bis I or Gö6983 also inhibited GSK3β phosphorylation (Fig. [3C](#F3){ref-type="fig"}, lanes 4, 6, 7), albeit to a lesser degree. Treatment with the ERK inhibitor U0126 increased GSK3β phosphorylation (Fig. [3C](#F3){ref-type="fig"}, lane 5). Neither FGF2 nor inhibitors affected total levels of PI3K or GSK3β (Fig. [3D, E](#F3){ref-type="fig"}). These inhibitor studies suggest that FGF2 signalling involves crosstalk between PI3K/AKT/GSK3β and ERK that is possibly mediated by PKC (Fig. [3A](#F3){ref-type="fig"}, lane 4 and [3C](#F3){ref-type="fig"}, lane 5). To further confirm that these changes in kinase signalling are mediated by FGF2, immuno-complex kinase assays were performed (Fig. [4A, B](#F4){ref-type="fig"}). As indicated by an astrisk (\) in Fig. [4A](#F4){ref-type="fig"}, lane 2, FGF2 treatment increased ERK activity significantly above levels observed in un-treated control cells (Fig. [4A](#F4){ref-type="fig"}). Likewise, and as shown in Figure [3](#F3){ref-type="fig"}, FGF2-mediated ERK activity was significantly greater than control in the presence of the PI3K inhibitor LY294002 (Fig. [4A](#F4){ref-type="fig"}). The ERK inhibitors PD and U0126, and the PKC inhibitors Bis I and Gö6983 significantly blocked FGF2-mediated ERK activity (Fig. [4A, D](#F4){ref-type="fig"}) as shown in Figure [3](#F3){ref-type="fig"}. Conversely, FGF2 alone or in the presence of the inhibitors LY294002, Bis I and Gö6983 had minimal effects on GSK3β activity (Fig [4B, D](#F4){ref-type="fig"}). However, the ERK inhibitor U0126 significantly decreased GSK3β activity (Fig. [4B](#F4){ref-type="fig"}). PD98059 also decreased GSK3β activity although at statistically insignificant levels (Fig. [4B](#F4){ref-type="fig"}). Cell viability was not significantly affected by FGF2 or inhibitor treatments (Fig. [4C](#F4){ref-type="fig"}) ensuring that effects of inhibitors on kinase activity were not due to cell death. Taken together these data show that FGF2 activates ERK signalling in HUVEC but has little effect on GSK3β activity unless FGF2-mediated ERK phosphorylation is blocked. Furthermore, independently of FGF2, PI3K/AKT and PKC signalling is necessary for GSK3β phosphorylation. However, once GSK3β is phosphorylated, the kinase activity of GSK3β is independent of PI3K/AKT and PKC downstream signalling. On the other hand, GSK3β phosphorylation is influenced, to some degree, by FGF2-mediated ERK phosphorylation since blocking ERK phosphorylation results in a significant increase in the phosphorylation of GSK3β. Likewise, the kinase activity of GSK3β also appears to require ERK phosphorylation for maximal activation. In summary, the FGF2-mediated kinase activity of ERK and GSK3β appears to involve crosstalk between these pathways and possibly PKC. The potential roles of ERK and GSK3β phosphorylation and activity in FGF2-mediated protection from gp120 were investigated. FGF2 angioprotection in HUVEC against gp120 toxicity is mediated, in part, by ERK signalling -------------------------------------------------------------------------------------------- To investigate the potential role of ERK and PI3K/AKT/GSK3β signalling in FGF2-mediated angioprotection against gp120, HUVEC were treated with LY294002, U0126, Bis I, or Gö6983 for 30 min prior to FGF2 and gp120 exposure (Fig. [5](#F5){ref-type="fig"}). Results from cell toxicity assays determined by Trypan blue exclusion (Fig. [5A](#F5){ref-type="fig"}), support our previous data (Fig. [1](#F1){ref-type="fig"}) showing that exposure to gp120 alone significantly increased cell death above control and FGF2 treated cells; whereas, cells pre-treated with FGF2 before exposure to gp120 were protected (Fig. [5](#F5){ref-type="fig"}). The protective effects of FGF2 against gp120 were significantly blocked by U0126, which inhibits MEK to block ERK phosphorylation, (Fig. [5A](#F5){ref-type="fig"}). Blocking PI3K with LY 294002 partially blocked FGF2 protection, although at levels insignificant from control. FGF2 protection from gp120 was not affected by blocking PKC with Bis I or Gö6983 (Fig. [5A](#F5){ref-type="fig"}). Treating cells with U0126 to block ERK phosphorylation, and gp120 in the absence of FGF2 resulted in significant cell death compared to untreated cells (Fig. [5B](#F5){ref-type="fig"}). Moreover, pre-incubation of FGF2 with anti-FGF2 antibody completely neutralized FGF2-mediated angioprotection against gp120 (Fig. [5B](#F5){ref-type="fig"}). These results indicate that ERK phosphorylation is significantly involved in FGF2-mediated angioprotection from gp120. PI3K/AKT/GSK3β signalling is partially involved in FGF2 protection from gp120; whereas, PKC signalling in the presence of FGF2 is not necessary for protection from gp120. These results suggest that FGF2 protects endothelial cells from gp120 largely by ERK stimulation with a partial contribution by GSK3β phosphorylation. To further confirm the contribution of these signalling pathways in FGF2 protection against gp120, HUVEC infected with caERK or caAKT were exposed to gp120 and assayed for cell viability. As expected, endothelial cells infected with caERK and exposed to gp120 were significantly protected from gp120 toxicity (Fig. [6](#F6){ref-type="fig"}). caAKT conveyed only partial protection from gp120 toxicity, less than either caERK or FGF2 treatment (Fig. [6](#F6){ref-type="fig"}). In control experiments where HUVEC were infected with GFP adenovirus, no protective effects against gp120 were observed (Fig. [6](#F6){ref-type="fig"}). Furthermore, none of the adenoviral constructs alone promoted significant cell toxicity (Fig. [6](#F6){ref-type="fig"}). In agreement with our previous data, these results suggest that ERK activation plays a significant role in protection of endothelial cells from gp120, and AKT/GSK3β is also be involved. To confirm that the gene transfer approach resulted in ERK and AKT phosphorylation and kinase activation, Western blot (Fig. [7A--D](#F7){ref-type="fig"}) and immuno-complex assays (Fig. [7E, F](#F7){ref-type="fig"}) were performed. ERK kinase activity was detected using an antibody that recognizes only the phosphorylated form of ERK1/2. Consistent with our previous experiments (Fig. [3A](#F3){ref-type="fig"}), FGF2 stimulation resulted in an increase of both ERK1 (44 kDa) and ERK2 (42 kDa) phosphorylation (Fig. [7A](#F7){ref-type="fig"}). Levels of FGF2-mediated phosphorylation of ERK2 were greater than ERK1 (Fig. [7A](#F7){ref-type="fig"}, lane 2). Infection with the GFP adenoviral construct alone had no effect on ERK1/2 phosphorylation (Fig. [7A](#F7){ref-type="fig"}, lane 3). In contrast, infection with caERK resulted in a significant increase in ERK1 phosphorylation with no effect on ERK2 (Fig. [7A](#F7){ref-type="fig"}, lane 5). FGF2 treatment in combination with caERK induced high levels of ERK1 phosphorylation with only moderate increases in ERK2 phosphorylation (Fig. [7A](#F7){ref-type="fig"}, lane 6). These results indicate that FGF2 stimulation results in phosphorylation of mainly ERK2; whereas gene transfer of caERK or the combination of FGF2 and caERK mainly increased ERK1 phosphorylation. Importantly, total ERK activity levels were similar in caERK with or without FGF2 (Fig. [7E](#F7){ref-type="fig"} and [7F](#F7){ref-type="fig"}). Moreover, the level of protection conveyed by FGF2 alone was similar to protection by caERK or caERK plus FGF2. On the other hand, caAKT alone had no effect on ERK1/2 phosphorylation (Fig. [7A](#F7){ref-type="fig"}, lane 7), whereas, FGF2 treatment in combination with caAKT (Fig. [7A](#F7){ref-type="fig"}, lane 8) had similar effects on ERK1/2 phosphorylation as observed with FGF2 (lane 2) alone or with GFP and FGF2 (lane 4). Levels of total ERK were not affected by FGF2, GFP, caERK or caAKT (Fig. [7B](#F7){ref-type="fig"}). Infection of HUVEC with caAKT resulted in a slight increase in baseline levels of AKT phosphorylation (Fig. [7C](#F7){ref-type="fig"}, lane 7). Levels of total AKT were not affected by FGF2, GFP, caERK, or caAKT (Fig. [7D](#F7){ref-type="fig"}). Consistent with Western blot analyses, immunocomplex assays show that caERK and/or FGF2 increased levels of ERK activity (Fig. [7E](#F7){ref-type="fig"}, lanes 2--4, 6 and [7F](#F7){ref-type="fig"}), whereas neither caAKT nor GFP resulted in increased ERK activity in the absence of FGF2 (Fig. [7E](#F7){ref-type="fig"} lanes 5, 7 and Fig. [7F](#F7){ref-type="fig"}). Results from inhibitor studies (Fig. [5](#F5){ref-type="fig"}) and gene transfer experiments (Fig. [6](#F6){ref-type="fig"}) suggest that both ERK and PI3K/AKT (albeit to a lesser degree) are involved in FGF2-mediated protection against gp120 toxicity. Furthermore, blocking the ERK-mediated pathway results in an increase in GSK3β phosphorylation and vice versa: blocking the AKT/GSK3β pathway after FGF2 stimulation results in an increase in ERK phosphorylation. These results suggest that when endothelial cells are exposed to gp120, FGF2 may mediate protection that involves crosstalk between the ERK and PI3K pathways (Fig. [3A](#F3){ref-type="fig"} and [3C](#F3){ref-type="fig"}, and Fig. [6](#F6){ref-type="fig"}). Moreover, inhibitor studies suggest PKC may be involved in this signalling convergence, but a direct role of PKC in FGF2 protection against gp120 is unclear. PKC may be involved in crosstalk between ERK and AKT signalling pathways during FGF2 protection from gp120 ---------------------------------------------------------------------------------------------------------- Our studies using pharmacological inhibitors suggest that PKC may be involved in a crosstalk mechanism observed between the ERK and AKT/GSK3β pathways in FGF2 signalling. For example, when HUVEC were exposed to PKC inhibitors Bis I and Gö6983 prior to FGF2 treatment, ERK phosphorylation was inhibited to below baseline levels, showing that FGF2-mediated ERK phosphorylation is at least in part influenced by PKC phosphorylation (Fig. [3A](#F3){ref-type="fig"}, lanes 6 and 7). Likewise, PKC inhibitors partially inhibited GSK3β phosphorylation after FGF2 stimulation (Fig. [3C](#F3){ref-type="fig"}, lanes 6 and 7). Furthermore, since Huang et al. have shown that total PKC phosphorylation increases with gp120 treatment in HUVEC and that FGF2 is protective \[[@B13]\], we explored the possibility that similar crosstalk might be involved in the FGF2-mediated protection from gp120. To address these signalling events, we determined which signalling pathways were initiated by FGF2 and which were initiated by gp120. To differentiate the effects of gp120 on ERK, GSK3β and PKC phosphorylation from those obtained in Fig. [3](#F3){ref-type="fig"} where FGF2 alone was utilized, we treated endothelial cells with 1) gp120 alone (Fig. [8A, C](#F8){ref-type="fig"}), [2](#F2){ref-type="fig"}) gp120 in combination with inhibitors (Fig. [8A, C](#F8){ref-type="fig"}), and [3](#F3){ref-type="fig"}) inhibitors, FGF2 and gp120 (Fig. [8B, C](#F8){ref-type="fig"}). Treatment of endothelial cells with gp120 alone (Fig. [8A](#F8){ref-type="fig"}, lane 2, [8C](#F8){ref-type="fig"}) or with inhibitors alone (data not shown) did not change levels of ERK phosphorylation. However, when endothelial cells were treated with LY204002 and then exposed to gp120 for 30 min, a significant increase in ERK phosphorylation was observed (Fig. [8A](#F8){ref-type="fig"}, lane 3, [8C](#F8){ref-type="fig"}). Furthermore, in the presence of both FGF2 and gp120 and the inhibitor LY294002, ERK phosphorylation also increased (Fig. [8B](#F8){ref-type="fig"}, lane 3, [8C](#F8){ref-type="fig"}). Interestingly, in the presence of the PKC inhibitor that includes inhibition of the ζ isoform (Gö6983), ERK phosphorylation is returned to approximately control levels (Fig. [8A--B](#F8){ref-type="fig"}, lane 5, [8C](#F8){ref-type="fig"}). On the other hand, inhibition of the classic isoforms of PKC, α, β and γ with Bis I almost completely blocks ERK phosphorylation in the presence of FGF2 and gp120 (Fig. [8B](#F8){ref-type="fig"}, lane 4), as does inhibition of ERK phosphorylation with U0126 (Fig. [8B](#F8){ref-type="fig"}, lane 6). These results suggest that PKC signalling may be involved in FGF2-stimulated ERK phosphorylation that protects against gp120. Treatment of HUVEC with gp120 alone, or with gp120 and inhibitors to block ERK, or PI3K/AKT/GSK3β had little effect on GSK3β phosphorylation (Fig. [8A](#F8){ref-type="fig"}, lanes 1--3, 6, [8C](#F8){ref-type="fig"}); whereas, blocking PKC decreased levels of GSK3β phosphorylation (Fig. [8A](#F8){ref-type="fig"}, lanes 4 and 5). Likewise, treatment of HUVEC with FGF2 alone or with FGF2, gp120 and inhibitors to block PI3K/AKT/GSK3β or ERK had little effect on GSK3β phosphorylation (Fig. [8B](#F8){ref-type="fig"}, lanes 1--3, 6, [8C](#F8){ref-type="fig"}); whereas, blocking PKC decreased levels of GSK3β phosphorylation (Fig. [8B](#F8){ref-type="fig"}, lanes 4 and 5, [8C](#F8){ref-type="fig"}). In summary, in the presence of FGF2 and inhibitors for FGFR and PI3K/AKT/GSK3β, ERK phosphorylation increases (Fig. [3A](#F3){ref-type="fig"}). However, in the presence of FGF2 or FGF2 and inhibitors for PKC or ERK, ERK phosphorylation decreases (Fig [8A, B, C](#F8){ref-type="fig"}). Likewise, PKC inhibitors almost completely abolish GSK3β phosphorylation in the presence of gp120, independently of FGF2 stimulation (Fig. [8B, C](#F8){ref-type="fig"}). Together, these findings point to PKC involvement with FGF2 stimulated signalling in HUVEC during challenge with gp120, however further experimentation is needed to confirm any role of PKC in FGF2-mediated protection from gp120. Discussion ========== The present study is the first to show that FGF2 protects HUVEC against the toxic effects of gp120 via crosstalk of the ERK-PI3K/AKT pathways (Fig. [9](#F9){ref-type="fig"}). Consistent with these finding, FGF2 has been shown to protect endothelial cells from oxidative stress \[[@B29]\] and radiation \[[@B30],[@B31]\]. These studies suggest that PKC is involved in protection against ultra-violet radiation, since blocking PKC abrogates FGF2-mediated protection \[[@B31]\]. Similarly, a recent study showed that FGF2 also protected endothelial cells from gp120-mediated toxicity that was induced by dysregulation of PKC activity to promote apoptosis \[[@B13],[@B28]\]; however, the pathways by which FGF2 protected endothelial cells from gp120 remained unclear and may be represented by independent mechanisms. Therefore, our study focused on signalling pathways involved in angioprotection upon exposure to gp120. gp120 has been reported to dysregulate PKC signalling but also to induce ERK phosphorylation in several systems by different pathways \[[@B32]-[@B34]\]. Likewise, our studies suggest that gp120 and FGF2 signalling in HUVEC may, in some aspects, overlap and involve primarily ERK and to a lesser extent AKT/GSK3β signalling. In this context, when HUVEC were treated with the ERK inhibitor U0126, then exposed to gp120, a significant increase in cell death above control was observed; however, the amount of cell death observed under these conditions was less than that observed in cells treated with gp120 alone. In HUVEC, PKC phosphorylation does not change when stimulated with FGF2 and PKC does not appear to be directly involved in FGF2-mediated protection from gp120 since inhibitors of this pathway had no effect on angioprotection. However, previous studies have shown that PCK may play a role in the MAPK signalling cascade, through upstream crosstalk with Ras (Figure [9](#F9){ref-type="fig"}) \[[@B35],[@B36]\]. Moreover, in the presence of gp120 with or without FGF2, both ERK and PKC inhibitors completely block ERK phosphorylation, suggesting that while PKC is involved in ERK phosphorylation, the protective properties of ERK are not dependent on PKC. In support of these conclusions, the current study shows that inhibition of ERK, and to a lesser degree PI3K/AKT, blocks FGF2-mediated protection from gp120. Our data suggest that FGF2 signalling via ERK-PI3K/AKT crosstalk is responsible for protection of endothelial cells from gp120. Other mechanisms that could contribute to FGF2-mediated protection against gp120 may include, but are not limited to, interaction of FGF2 with heparin sulfate receptors and/or stimulation of alternative pathways not involving ERK \[[@B37]\]. Consistent with these findings, FGF2 protects cardiac myocytes from inducible nitric oxide synthetase induced apoptosis by the ERK signalling pathway \[[@B38]\], and in neuronal cells FGF2-mediated ERK activation is essential for survival signalling \[[@B39]\]. Our studies provide evidence for the first time that FGF2-mediated protection of endothelial cells against gp120 toxicity largely occurs through an ERK-dependent pathway. Our data also suggest crosstalk between the PI3K/AKT and ERK pathways, since blocking PI3K resulted in a significant increase in ERK phosphorylation in FGF2 treated endothelial cells. Likewise, blocking ERK caused an increase in phosphorylation of GSK3β, which is directly downstream of PI3K/AKT signalling. In this context, it is possible that upon stimulation by growth factors such as FGF2, endothelial cells utilize several signalling cascades that are capable of crosstalk to promote cell fitness and survival, as suggested by studies involving vascular endothelial growth factor (VEGF) signalling in the presence or absence of serum \[[@B40]\]. In these studies, it was shown that crosstalk between the AKT and p38 pathways may regulate cell survival during serum withdrawal and VEGF stimulation \[[@B40]\]. Our studies also point toward signalling crosstalk during FGF2 protection from gp120. Crosstalk between PI3K and p38 was shown to be mediated by MAPK kinase kinase (MEKK3) in VEGF signalling \[[@B40]\]. Likewise, in FGF2 signalling, crosstalk between PI3K/AKT and ERK might be mediated by PKC \[[@B41]\]. This is consistent with previous studies showing that in VEGF-stimulated endothelial cells, inhibition of PI3K resulted in an increase in the phosphorylation of ERK1/2 and p38 phosphorylation \[[@B42]\]. Together with the findings in this study, these reports emphasize the importance of different signalling pathways communicating to regulate intracellular signal transduction in endothelial cell survival \[[@B43],[@B44]\]. The observations reported in this study have potential importance to the maintenance of BBB integrity in host response during HIV infection. FGF2 is produced by astrocytes in close proximity to endothelial cells of the BBB and functions to improve cell fitness and barrier integrity. In in vitromodels of the BBB, FGF2 treatment of endothelial cells mimics the effects of astrocyte co-culture by improving tight junction integrity \[[@B15]\]. Numerous studies have shown that disruption of this key component in the BBB is central to HIV infection of the CNS and is a hallmark of HIVE \[[@B45]\]. This is particularly important during HIV trafficking into the CNS because endothelial cells of the BBB are the first neural cells to come in contact with HIV infected cells or HIV products. Regulation among signalling crosstalk in endothelial cells by FGF2 is important since these are the cells of the BBB that first encounter HIV-infected and/or activated cells and HIV products such as gp120. Migration of HIV-infected and/or activated cells into the brain is largely regulated by endothelial cell integrity. During the progression of HIVE, activated and HIV-infected monocytes produce cytokines and chemokines and release HIV products that act in concert to compromise the integrity of the BBB \[[@B46]\]. This triggers a series of signalling events that may result in the alteration of tight junction proteins, such as zona occludins, thereby promoting migration of HIV-infected cells into the brain parenchyma \[[@B45],[@B47],[@B48]\]. Alternatively, astroglial cells that are also an important component of the BBB might produce trophic factors such as FGF2 in response to endothelial cell distress in attempts to maintain BBB integrity. Among them, factors produced by damaged endothelial cells, including tissue factor, can induce the early growth response-1 gene (Egr-1) transcription factor in astrocytes that in turn directs expression of FGF2 \[[@B49]\]. Conclusions =========== In summary, the present study shows that FGF2 is protective against gp120 toxicity via crosstalk of ERK-PI3K/AKT signalling pathways during compensatory signalling. This finding is important for understanding the pathogenesis of HIVE because factors produced by components of the BBB, such as FGF2 by astrocytes, in response to toxins such as HIV-gp120 may be responsible in part for angio-protection of endothelial cells of brain microvasculature. Methods ======= Cell culture ------------ HUVEC (Clonetics^®^, BioWhittaker, Inc., Walkersville, MD) were grown in complete media (endothelial basal medium \[EBM\] supplemented with bovine brain extract (12 μg/ml), human epithelial growth factor (10 ng/ml), hydrocortisone (1 μg/ml), GA-1000 (Gentamicin and Amphotericin B, 1 μg/ml) (Clonetics) and 20% fetal bovine serum (Irvine Scientific, Irvine, CA). Complete growth media were changed to minimal media (EBM, GA-1000, 1% serum, Clonetics) for 24 h prior to treatments. HUVEC were chosen because previous studies have characterized this cell line with regard to FGF2 mediated signaling responses and much of the work conducted in the present study complements and builds on data from these studies \[[@B13],[@B27]\]. Furthermore, HUVEC mimic numerous characteristics of cerebral endothelial cells. Both short-term signaling events and long-term viability of HUVEC were addressed after treatment with a combination of inhibitors, FGF2, and gp120, or with each component alone, as described below. HUVEC treatments to determine viability --------------------------------------- For viability assays, HUVEC were treated with either 20 ng/ml FGF2, (Calbiochem, La Jolla, CA) or full-length recombinant HIV-1~BaL~gp120 (25 ng/ml) NIH Research and Reagent Program, Rockville, MD and Bartels-Mardx, Carlsbad, CA) for 30 min, 1 h, 6 h, 12 h and 24 h. Recombinant HIV-1~BaL~used in these experiments is a macrophage trophic virus and binds to CD4 and signals via CCR5. For protection assays, HUVEC were treated either simultaneously with FGF2 and gp120 or pre-treated with FGF2 for 30 min, 1 h, 6 h, 12 h and 24 h before the addition of 25 ng/ml gp120. HUVEC were harvested 24 h after the addition of gp120 for viability assays. Viability assays ---------------- For trypan blue exclusion assays, HUVEC were rinsed with warm PBS, harvested, collected by gentle centrifugation, resuspended in a PBS/ trypan blue solution (1:1, vol:vol) and counted as previously described \[[@B50]\]. Terminal dUTP end labeling (TUNEL) staining was carried out essentially as described previously \[[@B51],[@B52]\]. Cells were grown on coverslips, rinsed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. After rinsing with PBS, cells were permeabilized with 1% H~2~O~2~in 1× PBS-Tween-20 for 10 min at room temperature, rinsed twice with PBS and air-dried for 2 min. TUNEL was conducted according to the manufacturer\'s instructions for staining (Roche Diagnostics, Indianapolis, IN) and counterstained with Eosin Y. TUNEL positive cells were detected with 3, 3\' diaminobenzidine (DAB) (Sigma) and counted with a computer-aided analysis system (Quantinet 570C, Leica, Bannockburn, IL). Cell death was also assayed by fluorescent staining with fluorescein diacetate (FA) and propidium iodide (PI) as previously described \[[@B27]\]. The FA (Sigma) working solution was prepared by adding 10 μl of stock FA (50 mg FA in 10 ml of acetone) to 2.5 ml PBS. The FA/PI (Sigma) cocktail was prepared by adding 1 μl of FA working solution to 300 μl of PI (1 mg PI in 50 ml PBS). After rinsing once in warm PBS, 20 μl of the FA/PI cocktail was added to cells on coverslips and incubated 15 min in the dark. Coverslips were placed cell-side up on SuperFrost slides (Fisher Scientific, Pittsburgh, PA) under anti-fading media (Vector Laboratories, Inc., Burlingame, CA) and immediately imaged with laser scanning confocal microscope (LSCM, MRC1024, Bio-RAD, Hercules, CA). HUVEC treatments for signalling events -------------------------------------- Signalling events mediated by FGF2 and/or gp120 were determined via Western Blot (WB) analyses. Cells were treated with either 20 ng/ml FGF2 (Calbiochem, La Jolla, CA) or gp120 for 30 min, 1 h, 6 h, 12 h and 24 h and analyzed by WB. Additionally, HUVEC were treated with inhibitors alone. To test the effects of FGF2 stimulation or gp120 exposure on downstream signalling, prior to FGF2 treatment, cells were pre-treated with inhibitors targeting different steps in the MAPK, PKC or AKT/glycogen synthase kinase 3-beta (GSK3β) pathways. For these experiments, cells were incubated for 30 min with the: (i) 10 μM PI3K inhibitor LY294002 (Calbiochem), (ii) 10 μM PKC inhibitors Gö6983 (Calbiochem), 2 μM Bisindolymaleimide I (Calbiochem), (iii) 10 μM MEK inhibitors U0126 or 20 μm PD98059 (Calbiochem). To test the specificity of FGF2-mediated protection against gp120, HUVEC were incubated with 20-fold excess anti-FGF2 neutralizing antibody prior to the addition of 20 ng/ml FGF2. Cells were incubated in the presence of anti-FGF2 antibody and FGF2 for 24 h, then exposed to 25 ng/ml gp120 for 24 h and assayed for viability, ERK phosphorylation and kinase activity. To determine the signalling events caused by gp120, with or without FGF2 and inhibitors, the following conditions were utilized: 1) cells were treated with inhibitors for 30 min as previously described, and then exposed to 25 ng/ml for 30 min, 2) inhibitors for 30 min then gp120 for 30, 3) inhibitors for 30 min, FGF2 for 10 min and gp120 for 30 min. After treatments cells were immediately harvested for Western analyses. Western blot analysis in HUVEC ------------------------------ Briefly, after treatments, cell monolayers were harvested and solubilized in HEPES homogenization buffer (1 mM HEPES, 5 mM Benzamidine, 2 mM 2-Mercaptoethanol, 3 mM EDTA, 0.5 mM Magnesium Sulfate, 0.05% Sodium Azide, Protease Inhibitor Cocktail III and Phosphatase Inhibitor Cocktail I) (Calbiochem). Protein concentration was determined by the method of Lowry and between 10--15 μg of protein were separated by electrophoresis on 10% Bis-Tris NuPage Gels (Invitrogen, Carlsbad, CA). Samples were then electroblotted onto Immunobilon P nitrocellulose membranes (Millipore, Bedford, MA). Proteins were immunolabeled with primary antibodies against phosphoERK1/2 (1:2500) (Thr202/tyr204, mouse monoclonal phosphoERK antibody) (Cell Signalling Technology), total ERK1/2 (1:2500) (anti-mouse monoclonal ERK1/2 antibody) (Pharmigen), phosphoGSK3β (1:2500) (Ser 9, anti-rabbit polyclonal phospho-GSK3β antibody) (Cell Signalling Technology), total GSK3β (1:2500) (anti-mouse monoclonal GSK3β antibody) (Transduction Laboratories, Lexington, KY), phosphoAkt (Thr308, anti-rabbit polyclonal phosphoAKT antibody) (Calbiochem), total AKT (1:2500) (anti-rabbit polyclonal AKT antibody), (Calbiochem), anti-mouse monoclonal PI3K antibody(1:1000) (Transduction Labs), anti-rabbit phospho-PKC (pan) that detects phosphorylation of PKC isoforms α, β, δ, ε, and η (Cell Signalling Technology, Beverly, MA) and anti-rabbit actin antibody (1:1000) (Chemicon, San Diego, CA). Blots were incubated with the HRP-tagged secondary antibody, detected with the ECL reagent (DuPont NEN, Boston, MA), followed by autoradiography. As a control, HUVEC were pre-treated with one of the following pharmacological inhibitors: MTA, LY294002, Gö6983, Bisindolymaleimide I, U0126 or PD98059 for 30 min and then FGF2 and gp120 were added simultaneously. Cell viability was assayed 24 h later. Adenoviral constructs and transfection -------------------------------------- Recombinant adenoviral constructs encoding constitutively active (ca) forms of ERK and AKT were prepared as previously described \[[@B53],[@B54]\] (kindly provided by Dr. Kazuhiko Namikawa, Asahikawa Medical College, Asahikawa, Japan and Dr. Kenneth Walsh, Tufts University, Boston, MA, respectively). Adenovirus encoding the green fluorescent protein (GFP-Ad) as previously described \[[@B55]\] was used as a control to account for any effects that may be due to adenoviral infection. Briefly, for ca-ERK, cDNA fragments containing the entire coding regions for human MAP/ERK kinase 1 (MEK1) were isolated from human embryonic kidney cells (HEK293) by PCR. ca-ERK lacks the nuclear export signal (amino acids 32--51) and has glutamic acid substitutions for two phosphorylation sites, Ser^218^and Ser^222^, was prepared by site-directed mutagenesis and fused to the hemagglutinin tag sequence, as previously described \[[@B56]\]. ca-AKT, has the c-srcmyristoylation sequence fused in frame to the N-terminus of the FLAG-AKT coding sequence \[[@B54]\]. High titer recombinant viral stocks (10^11^plaque forming units) were generated in HEK293 cells and stored at -80°C. Endothelial cells were plated at approximately 50% confluency in complete media (20% serum) and grown for 24 h at 37°C, 5% CO~2~. HUVEC were changed to minimal media (1% serum) for 6 h and then half of the media was removed from each sample, pooled and stored at 37°C, 5% CO~2~. HUVEC were infected at a multiplicity of infection of 50 in pre-conditioned minimal media for 4 h, achieving a 40--50% transduction efficiency (data not shown). Minimal medium containing adenovirus was replaced with pooled pre-conditioned minimal media and cell cultures were further incubated for 48 h at 37°C and 5% CO~2~. After 48 h, cells were treated with FGF2 (10 ng/ml) for 10 min, harvested in lysis buffer, stored at -20°C, and later used for ERK and AKT kinase assays. For immunocytochemistry, cells on coverslips were blocked overnight at 4°C in 10% horse serum and 5% BSA. Coverslips for ca-ERK were then labelled overnight at 4°C with primary anti-Hemagglutinin (1:150) (Roche Diagnostics) and for ca-AKT with primary anti-FLAG (1:50) (Sigma) followed by incubation with secondary biotinylated IgG (Vector Laboratories) (1:200) for 1 h at room temperature. Hemagglutinin and FLAG proteins were detected with DAB (Sigma) and visualized by light microscopy to access HA production. Experiments were conducted at least three times to ensure reproducibility. Immunocomplex kinase assays --------------------------- ERK and AKT Assays were performed essentially as previously described with some modifications \[[@B57]\]. Briefly, cells were rinsed twice with cold phosphate-buffered saline and incubated for 20 min on ice in lysis buffer (1% Triton X-100, 10 % glycerol, 50 mM HEPES, pH 7.4, 140 mM NaCl, 1 mM EDTA, 1 mM Na~3~VO~4~, 1 mM phenyl-methylsulfonylfluoride, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 1 mM dithiothreitol). The cell lysates were then centrifuged for 10 min at 14,000 rpm and protein concentration was determined using the BCA reagent (Pierce, Rockford, IL). Two hundred microliters of the supernatant were pre-absorbed with a protein G-sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) for 1 h at 4°C. The pre-cleared lysates were incubated with 1 μg/sample of anti-ERK monoclonal antibody (1:50) (Pharmingen, San Diego, CA) or polyclonal anti-human AKT antibody (anti-PKB 88--100) (Calbiochem) overnight at 4°C, followed by incubation with protein G-sepharose for 2 h at 4°C. After washing twice with the lysis buffer and twice with a kinase buffer (20 mM HEPES, pH 7.2, 0.1 mM Na~3~VO~4~, 10 mM glycerophosphate, 10 mM MgCl~2~, 1 mM dithiothreitol, 0.1 mM EGTA), the immune complexes were incubated in 30 μl of the kinase buffer containing 20 μg myelin basic protein for ERK (Sigma) or 1 μg of GSK3β fusion protein (Cell Signalling, Beverly, MA) for AKT and 10 μCi of \[γ-^32^P\] ATP (6000 Ci/mmol, PerkinElmer, Boston, MA) for 30 min at 30 °C. Reactions were terminated by the addition of 5 μl of 500 mM EDTA and 5 mM ATP. After adding 4× Laemmili SDS sample buffer and boiling 5 min, samples were separated by 15% SDS-PAGE, followed by autoradiography. Quantification was performed with the PhosphorImager using the Image Quant software (Molecular Dynamics, Sunnyvale, CA). Statistical analysis -------------------- All experiments were performed in a blind code fashion. After results were obtained, the code was broken and analysis was performed by utilizing one-way analysis of variance (ANOVA) with post hoc Dunnett\'s or Tukey-Kramer. Authors\' contributions ======================= DL designed and conducted FGF2, gp120 and inhibitors experiments, Western analyses, viability assays and composed the manuscript. RH assisted in design of, and conducted FGF2, gp120 and inhibitors experiments, Western analyses and viability assays. MH designed and conducted activity assays and gene transfer experiments. MD obtained adenoviral constructs, designed experimental methods for infection and analysed output measures. EM performed light microscopy and immunocytochemical experiments and composed with DL the first draft of the manuscript. Acknowledgements ================ We wish to thank Dr. Aline Grigorian for helpful comments and Dr. Kenneth Walsh and Dr. Kazuhiko Namikawa for their generous gifts of the AKT and ERK constructs, respectively. This work was supported by NIH Grants MH62962, MH59745, MH45294, MH58164, and DA12065 to EM and MH071206 and an HNRC pilot to DL. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Cell viability assays for FGF2 protection of HUVEC against gp120-mediated toxicity(A-D) Phase contrast, (F-I) TUNEL staining, and (K-O) fluorescent staining of HUVEC. Panels A, F, and K show images of untreated HUVEC control; B, G, and L show HUVEC treated for 24 h with FGF2 (20 ng/ml); panels C, H, and M show HUVEC treated for 24 h with gp120 (25 ng/ml) and panels D, I, and N show HUVEC pre-treated with FGF2 for 24 h before a 24 h exposure to gp120. Panels E, J and O show the percentage of cell death as determined by Trypan blue exclusion, percentage of DNA fragmentation by TUNEL and FA/PI staining, respectively. Results shown in panels E, J and O represent the average of three separate experiments performed in triplicate. \ = P \< 0.05 by One-Way ANOVA with post-hoc Dunnett\'s when compared to control. Bar = 20 microns. ::: ![](1471-2202-6-8-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Effects of FGF2 and gp120 on cell proliferation and viabilityA) Density of proliferating HUVEC exposed to FGF2 and/or gp120. B) Cell viability of cells exposed to gp120 after pre-treating HUVEC with FGF2 for different lengths of time. Cell viability was measured 24 h after gp120 addition. \* = P \< 0.05 by One-Way ANOVA with post-hoc Dunnett\'s when compared to control. ::: ![](1471-2202-6-8-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Effects of inhibitors on FGF2-mediated ERK activationA)Western blot (WB) reacted with anti-phospho-ERK after treatment with FGF2 for 0, 5, 10 min (lanes 1--3) and after inhibitors LY294002 (to block PI3K), U0126 (to block MEK), and Bis I and Gö6983 (to block PKC) (lanes 4--7, respectively). B)WB reacted with anti-total ERK antibody. (C)WB reacted with anti-phospho-GSK3β. D)WB reacted with anti-GSK3β. E)WB reacted with anti-PI3K antibody. The same WB was used in each experiment after stripping, reblocking and incubating with new antibodies for the given protein. ::: ![](1471-2202-6-8-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Effects of inhibitors on FGF2-stimulated ERK and GSK3β activityImmunocomplex kinase assays for (A)ERK and (B)GSK3β activity without FGF2 treatment, or after inhibition with PD98059, U0126, LY29004, Bisindolymaleimide I or Gö6983 followed by FGF2 treatment. (C)Inhibitor treatment alone. (D) Summary of changes in phosphorylation (P) versus changes in activity (Act) of ERK and GSK3β. ⇑ Indicates an increase, ⇓ indicates a decrease, -- indicates no change. \* = P \< 0.05 by One-Way ANOVA with post-hoc Dunnett\'s when compared to control. ::: ![](1471-2202-6-8-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Viability assays indicate that ERK activation is required for FGF2 protection against gp120(A) Trypan Blue Exclusion assay for cell survival after 30 min of treatment with inhibitors LY294002, U0126, Bis I, or Gö6983 followed by treatment with FGF2 and/or gp120. (B)Trypan Blue Exclusion assay for cell survival after 30 min of treatment with the MEK inhibitor U0126 alone, and followed by exposure to gp120 and/or FGF2 for 24 h. In cells treated with FGF2 and anti-FGF2, HUVEC were exposed to anti-FGF2 antibody for 1 h followed by treatment with FGF2 alone or in combination with gp120. \* Indicates a significant difference from control. \* = P \< 0.05 by One-Way ANOVA with post-hoc Dunnett\'s when compared to control. ::: ![](1471-2202-6-8-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Gene transfer of constitutively active ERK and AKT protect cells from gp120 toxicity(A)After gene transfer of constitutively active (ca) ERK or ca AKT, HUVEC were exposed to gp120, FGF2 or a combination of both and cell viability was assayed via Trypan Blue Exclusion. Controls consisted of ca ERK, ca AKT and GFP gene transfer without further treatment. \* indicates a significant difference from control. \\ indicates a significant difference from \. \ = P \< 0.05 by One-Way ANOVA with post-hoc Dunnett\'s when compared to control. \\ = P \< 0.05 by One-Way ANOVA with post-hoc Tukey-Kramer when compared between experimental groups. ::: ![](1471-2202-6-8-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Effects of gene transfer of constitutively active ERK, AKT or GFP phosphorylation(A-D)Western Blot of HUVEC after treatment with FGF2, infected with the GFP, ca ERK or ca AKT adenoviral constructs, or treated with FGF2 and infected with adenoviral constructs. The same Western blot was used for all antibodies after stripping and rehybridizing. A) Reacted with anti-phospho ERK antibody (B)reacted with anti-total ERK antibody. (C)Reacted with anti-phospho AKT antibody. (D)Reacted with anti-total AKT antibody. (E)Immuno-complex assay showing changes in ERK activity with or without FGF2 treatment in HUVEC with caERK, caAKT or GFP. (F)Quantification of ERK activity levels in HUVEC +/- FGF2 treatment with caERK, caAKT or GFP with the PhosphorImager as described in the Materials and Methods. \* indicates a significant difference from control. \* = P \< 0.05 by One-Way ANOVA with post-hoc Dunnett\'s when compared to control. ::: ![](1471-2202-6-8-7) ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Effects of FGF2, gp120, and inhibitors on ERK and GSK3β phosphorylation(A, B)Western blots showing ERK and GSK3β phosphorylation with (A)gp120 alone (lane 2), and with inhibitors (lanes 3--6), (B)FGF2 and gp120 (lane 2), and FGF2 with inhibitors and gp120 (lanes 3--6). (C)Table summarizing data from western blots (Aand B)showing changes in phosphorylation of ERK and GSK3β. ⇑ Indicates and increase, ⇓ indicates a decrease, -- indicates no change. ::: ![](1471-2202-6-8-8) ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Diagrammatic representation of signalling pathways that may be involved in FGF2-mediated protection from gp120As indicated by the diagram and as described by the data, it is possible that the crosstalk between the PI3K/AKT/GSK3β and ERK pathways is mediated in part by PKC. PKC signalling also is reported to occur directly with PI3K and upstream of Ras. Furthermore, direct interaction between MEK and PKC downstream of Ras is reported. Likewise, ERK2 (p42) is reported to signal to Raf-1 via a positive feedback mechanism. The points of action of the inhibitors LY290042, U0126, Bisindolymaleimide I, and Gö6983 are also shown. ::: ![](1471-2202-6-8-9) :::	PubMed Central	2024-06-05T03:55:52.906678	2005-2-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549045/", "journal": "BMC Neurosci. 2005 Feb 2; 6:8", "authors": [ { "first": "Dianne", "last": "Langford" }, { "first": "Rosemary", "last": "Hurford" }, { "first": "Makoto", "last": "Hashimoto" }, { "first": "Murat", "last": "Digicaylioglu" }, { "first": "Eliezer", "last": "Masliah" } ] }
PMC549046	Background ========== A great challenge in pharmacology and toxicology is to understand the molecular mechanisms behind how mixtures of compounds affect living organisms. This study focuses on two classes of substances, imidazoles and xenoestrogens, and how these chemicals alone and in combination affect hepatic drug-metabolizing hepatic cytochrome P450 (CYP) enzymes -- specifically, CYP1A and CYP3A enzymes, in juvenile Atlantic cod (Gadus morhua). Imidazoles and triazoles are used as fungicides both clinically as well as in horticulture and agriculture, posing a potential threat to wildlife. The triazole propiconazole has been detected in the aquatic environment \[[@B1]\]. The azole antifungal effect resides in inhibition of CYP51 mediated ergosterol biosynthesis \[[@B2]\]. In addition to disrupting key enzymes in fungus, azoles such as the imidazoles clotrimazole, ketoconazole, miconazole and prochloraz also cause endocrine disruption in vertebrates by inhibition of key enzymes in steroid homeostasis \[[@B3]-[@B7]\]. Moreover, these fungicides inhibit drug-metabolizing CYP forms, including members of the CYP1, CYP2 and CYP3 gene families in vertebrates \[[@B5],[@B8]-[@B13]\]. Effects on CYP forms may have adverse effects on metabolic clearance of endobiotics and xenobiotics. For example, in a study in fish, pre-exposure to clotrimazole resulted in increased bioaccumulation of the pro-carcinogen benzo \[a\]pyrene in gizzard shad (Dorosoma cepedianum) \[[@B14]\]. Xenoestrogens comprise a wide variety of structurally diverse chemicals such as o,p-DDT, ethynylestradiol, alkylphenols and bisphenol A. These substances are well-known or supposed to be endocrine disrupting substances in vertebrates and share in common that they activate the estrogen receptor (ER) and thereby elicit estrogenic responses \[[@B15]-[@B17]\]. In addition to being estrogenic, these xenoestrogens interact with drug-metabolizing CYP forms, including members of the CYP1A and CYP3A subfamilies in vertebrates \[[@B18]-[@B22]\]. Xenoestrogens are continuously released into the environment as a result of various anthropogenic activities. Induction of vitellogenesis in fish is a biomarker routinely used to assess the presence of estrogenic substances in the aquatic environment \[[@B23],[@B24]\]. Induction of CYP1A-mediated ethoxyresorufin-O-deethylase (EROD) activity is another established biomarker used to assess exposure to aromatic hydrocarbons. This response proceeds through activation of the aryl hydrocarbon receptor (AHR) by aromatic hydrocarbons including polyaromatic hydrocarbons, and planar polychlorinated biphenyls and dioxins \[[@B25]\]. Some AHR agonists have been shown to be anti-estrogenic and cross-talk between AHR and ER has been suggested in vertebrates \[[@B26]-[@B33]\]. In addition to activation of the ER, xenoestrogens also affect other steroid receptors. Nonylphenol up-regulated CYP3A1 gene expression in rat, through activation of the pregnane X receptor (PXR) \[[@B34],[@B35]\]. We previously reported induction of CYP3A and CYP1A protein levels in Atlantic cod exposed to alkylphenols \[[@B22]\]. Azole fungicides induce expression of multiple vertebrate CYP genes including members of the CYP1A, CYP2B and CYP3A subfamilies \[[@B8],[@B9],[@B13],[@B36]-[@B38]\]. Clotrimazole activates the ligand-binding domain of the PXR, involved in CYP3A signalling, in vitrofrom several mammalian species and zebra fish (Danio rerio) \[[@B39]\]. Both imidazoles and xenoestrogens inhibit drug-metabolizing enzymes, including members of the CYP1A and CYP3A subfamilies in vertebrates \[[@B8]-[@B13],[@B18],[@B20],[@B22]\]. Thus, xenoestrogens and imidazoles conceivably share common routes for biotransformation. However, there is a lack of data regarding effects of combined exposure of imidazoles and xenoestrogens on these CYP forms in wildlife. Living organisms usually are exposed to mixtures of different classes of xenobiotics. Conceivably, exposure to mixtures may be more of a health threat than exposure to single compounds, as a result of interactions. Anthropogenic compounds may enter the environment through industrial activities and through the use of pharmaceuticals \[[@B40]\]. Atlantic cod is an economically important species for fishery and a growing aquaculture industry, in addition to its ecological relevancy. Its distribution in the Northern Atlantic and the North Sea makes it vulnerable to effluents from on-shore and off-shore industries and from run-off entering the waters near highly industrialized and urbanized areas. The rationale of the present study was to identify possible sites of interactions between imidazoles and xenoestrogens. We hypothesise that combined exposure to these compounds may compromise the metabolic clearance not only of these xenobiotics themselves, but also of endobiotics such as circulating steroid hormones that share common routes of metabolism through hepatic CYP1A and CYP3A. Such endocrine disrupting effects may adversely affect the stability of wildlife populations. The specific aim of our study was to examine interactions between two classes of compounds in livers of Atlantic cod. Thus, we investigated the effects of the model imidazole ketoconazole and of two types of xenoestrogens (nonylphenol and ethynylestradiol), as well as of a mixed exposure to ketoconazole and nonylphenol, on hepatic CYP1A and CYP3A protein expression and catalytic activities, and also on vitellogenesis and plasma levels of sex steroid hormones. Results ======= In vivoeffects on CYP1A ------------------------- Exposure to ketoconazole (12 mg/kg b.w.) and/or a combination of ketoconazole and nonylphenol (12 mg/kg b.w. + 25 mg/kg b.w.) resulted, respectively, in 159 and 172% average induced increases in CYP1A-mediated EROD activities (Fig. [1A](#F1){ref-type="fig"}), and in 133 and 193% increases in CYP1A protein levels in Atlantic cod (Fig. [1B](#F1){ref-type="fig"}). Treatment with nonylphenol (25 mg/kg b.w.) resulted in 41% reduction and ethynylestradiol (5 mg/kg b.w.) resulted in 72% reduction, respectively, of CYP1A activities compared to vehicle treated fish (Fig. [1A](#F1){ref-type="fig"}). However, when compared to fish exposed to the combination of ketoconazole and nonylphenol, exposure to nonylphenol alone and ethynylestradiol resulted in 65% and 84% decrease in CYP1A activity (Fig. [1A](#F1){ref-type="fig"}). Exposure to nonylphenol and ethynylestradiol had no effect on CYP1A protein expression (Fig. [1B](#F1){ref-type="fig"}). The CYP1A protein levels were elevated by 93% in fish exposed to a mixture of ketoconazole and nonylphenol (Fig. [1B](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### *A) In vivoCYP1A enzyme activities (A) and in vivoCYP1A protein expression (B). CYP1A enzyme activities and protein expression in juvenile Atlantic cod exposed in vivoto vehicle (5 ml peanut oil/kg fish), ketoconazole (12 mg/kg fish), nonylphenol (25 mg/kg fish), ethynylestradiol (5 mg/kg fish) and ketoconazole + nonylphenol (12 + 25 mg/kg fish). A) EROD activities. B) CYP1A protein levels analyzed using PAb against rainbow trout CYP1A. Each bar represents mean values of eight to nine fish ± SD; ^a^Significantly different from vehicle treated fish; ^b^Significantly different from ketoconazole+nonylphenol treated fish; P\< 0.05. ::: ![](1476-5926-4-2-1) ::: In vivoeffects on CYP3A ------------------------- Fish exposure to ketoconazole, ethynylestradiol and nonylphenol resulted in decreased CYP3A-mediated benzyloxy-4-\[trifluoromethyl\]-coumarin-O-debenzyloxylase (BFCOD) activities, when compared to vehicle treated fish (Fig. [2A](#F2){ref-type="fig"}). Furthermore, mixed exposure to ketoconazole and nonylphenol resulted in a 98% decrease in CYP3A activity, which was greater than the additive effects of these two compounds administrated alone (Fig. [2A](#F2){ref-type="fig"}). Fish exposed to the ketoconazole and nonylphenol mixture displayed significantly reduced CYP3A activities when compared all other treatment groups (Fig. [2A](#F2){ref-type="fig"}). No effect on CYP3A protein expression was observed in fish treated with ketoconazole and nonylphenol, either alone or in combination (Fig. [2B](#F2){ref-type="fig"}). However, ethynylestradiol treatment resulted in 22% decrease in CYP3A protein levels (Fig. [2B](#F2){ref-type="fig"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### *In vivoCYP3A enzyme activities (A) and in vivoCYP3A protein expression (B)*. CYP3A enzyme activities and protein expression in juvenile Atlantic cod exposed in vivoto vehicle (5 ml peanut oil/kg fish), ketoconazole (12 mg/kg fish), nonylphenol (25 mg/kg fish), ethynylestradiol (5 mg/kg fish) and ketoconazole + nonylphenol (12 + 25 mg/kg fish). A) BFCOD activities. B) CYP3A protein levels analyzed using PAb against rainbow trout CYP3A. Each bar represents mean values of eight to nine fish ± SD; ^a^Significantly different from vehicle treated fish; ^b^Significantly different from ketoconazole+nonylphenol treated fish; P\< 0.05. ::: ![](1476-5926-4-2-2) ::: Western blot analyses of CYP3A proteins using PAb against rainbow trout CYP3A revealed the presence of one CYP3A immunoreactive protein band in liver microsomes, with an apparent molecular size above 50 kD, in Atlantic cod (Fig. [3A](#F3){ref-type="fig"}). By using 2D gel electrophoresis followed by immunoblotting, two immunoreactive CYP3A protein spots were detected above 50 kD, with pI values around 4.8 and 5.1, respectively (Fig. [3B](#F3){ref-type="fig"}). The most basic isoprotein appears to be inducible by treatment with ketoconazole (Fig. [3B](#F3){ref-type="fig"}). Ethynylestradiol and nonylphenol treatment did not induce expression of the more basic isoform. Present data does not elucidate whether those two protein spots are different gene products, or if they result from post-translational modifications such as phosphorylation. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### CYP3A Western blot (A) and CYP3A 2D-immunoblots (B). A) Western blot of hepatic microsomal CYP3A proteins in juvenile Atlantic cod treated with vehicle (5 ml peanut oil/kg fish) and ketoconazole (12 mg/kg fish) detected using PAb against rainbow trout CYP3A. B) 2D-gel electrophoresis followed by immunoblotting using PAb against rainbow trout CYP3A. Each blot represent pooled liver microsomes of eight to nine fish for each treatment; vehicle (5 ml peanut oil/kg fish), ketoconazole (12 mg/kg fish), nonylphenol (25 mg/kg fish), ethynylestradiol (5 mg/kg fish), ketoconazole + nonylphenol (12 + 25 mg/kg fish). ::: ![](1476-5926-4-2-3) ::: In vitroinhibition studies ---------------------------- In vitroinhibition studies using pooled Atlantic cod liver microsomes showed that ketoconazole, nonylphenol, ethynylestradiol and the ketoconazole:nonylphenol (1:5) mixture inhibited CYP1A (EROD) activity, with IC~50~values (inhibitor concentration required to achieve a 50% inhibition) ranging from 0.6 to 20 μM. The CYP3A-mediated BFCOD activity also was inhibited by ketoconazole (IC~50~= 0.3 μM), ethynylestradiol (IC~50~= 40 μM) and the ketoconazole:nonylphenol (1:5) mixture (IC~50~= 5:25 μM). Nonylphenol alone was an insignificant inhibitor of microsomal CYP3A activities in Atlantic cod (IC~50~= 160 μM). For comparison, IC~50~values for nonylphenol and ethynylestradiol also were determined in cDNA expressed human CYP3A4 baculovirus supersomes, compared to the prototypical CYP3A4 inhibitor ketoconazole (IC~50~= 0.4 μM). In contrast to Atlantic cod liver microsomes, nonylphenol inhibited the human CYP3A4 mediated BFCOD activity (IC~50~= 35 μM) and ethynylestradiol was a weak inhibitor (IC~50~= 50 μM) of this activity. The IC~50~values are summarized in Table [1](#T1){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### IC~50~values and inhibition constants (K~i~) for ketoconazole and xenoestrogens on CYP1A- and CYP3A activities assayed in vitro. ::: Compound(s)* IC~50~(μM)^1,a^ K~i~(μM)^1,a^ IC~50~(μM)^2,b^ K~i~(μM)^2,b^ IC~50~(μM)^3,b^ ------------------- --------------------- -------------------- ---------------------- ------------------- --------------------- Ketoconazole (KC) 0.6 (0.0)^c^ 0.04 -- low \[S\] 0.3 (0.1)^c^ 0.2 0.4^c^ 0.2 -- high \[S\] Nonylphenol (NP) 5.2 (1.1) 3.5 160 (40) Not analysed 35 Ethynylestradiol 20 (1.2) 5.4 -- low \[S\] 40 (7.1) 54 -- low \[S\] 50 10.3 -- high \[S\] 95 -- high \[S\] KC:NP (1:5) 1.3 (0.2):6.2 (1.0) Not analysed 5.3 (1.1):25.0 (5.3) Not analysed Not analysed ^1^Hepatic Microsomal CYP1A activity; ^2^Hepatic Microsomal CYP3A activity; ^3^cDNA Expressed Human CYP3A4; ^a^Substrate \[S\] = 7-Ethoxyresorufin; ^b^\[S\] = 7-Benzyloxy-4-\[trifluoromethyl\]-coumarin; ^c^Published in \[22\]. Each IC~50~value represents the mean from 2--4 separate assays, followed by the SD, in brackets. The K~i~values are estimated from one representative Dixon plot. ::: The inhibitory effects of these compounds were further investigated on hepatic microsomal CYP1A and CYP3A enzyme kinetics. The K~i~values were determined in Dixon plots (Figs. [4](#F4){ref-type="fig"} and [5](#F5){ref-type="fig"}) and summarized in Table [1](#T1){ref-type="table"}. Ketoconazole was a potent non-competitive inhibitor of both CYP1A and CYP3A activities with K~i~values in the sub-μM range (Fig. [4](#F4){ref-type="fig"}; Table [1](#T1){ref-type="table"}). Ethynylestradiol was a non-competitive inhibitor of CYP1A with K~i~from 5.4 to 10.3 μM and an uncompetitive inhibitor of CYP3A with K~i~from 54 to 95 μM (Fig. [5](#F5){ref-type="fig"}; Table [1](#T1){ref-type="table"}). Nonylphenol was a non-competitive inhibitor of CYP1A activity with K~i~around 3.5 μM (Table [1](#T1){ref-type="table"}). There were no effects of pre-incubation either with ketoconazole or ethynylestradiol on hepatic microsomal CYP3A protein levels in this study (Fig. [6](#F6){ref-type="fig"}). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Non-competitive inhibition of CYP1A by ketoconazole (A) and non-competitive inhibition of CYP3A by ketoconazole (B). Dixon plots for ketoconazole on A) EROD activity (diamonds represent 8.2; squares represent 25 and triangles represent 677 pM ethoxyresorufin). B) BFCOD activity (diamonds represent 48; squares represent 84 and triangles represent 200 μM BFC). ::: ![](1476-5926-4-2-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Non-competitive inhibition of CYP1A by ethynylestradiol (A) and uncompetitive inhibition of CYP3A by ethynylestradiol (B). Dixon plots for ethynylestradiol on A) EROD activity (diamonds represent 8.2; squares represent 25 and triangles represents 677 pM ethoxyresorufin). B) BFCOD activity (diamonds represent 200; squares represent 267 and triangles represents 356 μM BFC). ::: ![](1476-5926-4-2-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### *CYP3A Western blot after in vivoincubation. Western blot of CYP3A proteins in pooled liver microsomes from Atlantic cod detected using PAb against rainbow trout CYP3A. The blot illustrates representative samples after in vitroincubation with 1.0 μM ketoconazole and 50 μM ethynylestradiol for 30 or 60 min. ::: ![](1476-5926-4-2-6) ::: Plasma vitellogenin- and sex steroid hormone levels --------------------------------------------------- Treatment with nonylphenol, ethynylestradiol and the combination of ketoconazole and nonylphenol resulted in induction of vitellogenin, whereas these treatments had no statistically significant effect on 17β-estradiol, testosterone and 11-keto-testosterone plasma levels compared to either vehicle treated fish or fish treated with each test compound alone. The results are summarized in Table [2](#T2){ref-type="table"}. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Plasma levels of vitellogenin and sex steroid hormones in juvenile Atlantic cod exposed in vivoto ketoconazole and xenoestrogens. ::: Treatment* Vitellogenin 17β-Estradiol Testosterone 11-Keto-Testosterone ----------------------------------- ------------------- ------------------- ------------------ -------------------------- (μg/ml plasma) (pg/ml plasma) (pg/ml plasma) (pg/ml plasma) n = 7--8 n = 8 n = 7--8 n = 6--8 Vehicle (5 ml peanut oil/kg b.w.) 0.6 (1.0) 62 (56) 86 (68) 33 (38) Ketoconazole (KC) (12 mg/kg b.w.) 0.6 (0.9) 60 (26) 120 (78) 71 (46) Nonylphenol (NP) (25 mg/kg b.w.) 266 (199) ^a^ 76 (52) 75 (33) 42 (67) Ethynylestradiol (5 mg/kg b.w.) 4,350 (1,463) ^a^ 100 (69) 90 (40) 35 (23) KC + NP (12 + 25 mg/kg b.w.) 268 (205) ^a^ 36 (28) 61 (51) 36 (38) ^a^Significantly different from vehicle treated fish (P\< 0.001; Kruskal-Wallis ANOVA, followed by Mann-Whitney U-test). Each value represents the mean from 6--8 fish, followed by the SD, in brackets. ::: Discussion ========== Effects on CYP1A ---------------- For data evaluation we must bear in mind that western blot analysis of CYP1A protein levels is less sensitive than the EROD assay \[[@B41]\] and so densitometry analysis of western blot data fails to detect minor changes. Treatment of juvenile Atlantic cod with ketoconazole resulted in elevated EROD activities. Mixed exposure to ketoconazole and nonylphenol resulted in induced EROD activities and CYP1A protein levels. Induction of hepatic CYP1A gene expression by exposure to imidazoles and/or triazoles also has been reported in rat, bobwhite quail (Colinus virginianus) and rainbow trout (Oncorhynchus mykiss) \[[@B8],[@B13],[@B37],[@B38]\]. However, it is possible that induction of EROD activity, partly or completely, is masked by CYP1A inhibition caused by ketoconazole present in the tissue. Inhibition of CYP1A is supported in the present study, showing that ketoconazole was a potent non-competitive inhibitor of EROD activity in vitro. Ketoconazole and other imidazoles also have been shown to be potent inhibitors of EROD activities in other vertebrates \[[@B9],[@B13],[@B14],[@B42]\]. Treatment of Atlantic cod with nonylphenol and ethynylestradiol resulted in decreased EROD activities, whereas no effects of these substances were observed on CYP1A protein levels. This decrease in EROD activity is probably caused by nonylphenol or ethynylestradiol present in the liver microsome fraction. Nonetheless, chemical data are required, in the future, to confirm this. In vitroinhibition studies in liver microsomes confirmed that nonylphenol and ethynylestradiol acted as non-competitive inhibitors of the EROD activity. Hence, ketoconazole, nonylphenol, and ethynylestradiol interact with CYP1A enzymes, indicating a possible site for interaction of these different classes of xenobiotics. In addition, ketoconazole treatment induces CYP1A expression, which further may affect this interaction. Effects on CYP3A ---------------- Atlantic cod exposed to nonylphenol, ethynylestradiol and ketoconazole displayed reduced hepatic CYP3A (BFCOD) activities. The CYP3A inhibitory effect by ketoconazole is well known and ketoconazole is the most established diagnostic inhibitor, used to assess human in vitroCYP3A4 activity \[[@B12],[@B43]\]. Studies in fish demonstrate that ketoconazole is a potent inhibitor of hepatic BFCOD activities in killifish (Fundulus heteroclitus), rainbow trout and Atlantic cod with IC~50~values at 0.01, 0.1 and 0.3 μM, respectively \[[@B13],[@B22]\]. In rainbow trout, exposure to ketoconazole resulted in elevated hepatic and intestinal CYP3A protein levels \[[@B13]\]. In the present study, 2D gel electrophoresis revealed the presence of two CYP3A immunoreactive spots in Atlantic cod liver microsomes with pI values around 4.8 and 5.1, respectively. The more basic isoform (pI 5.1) appeared to be responsive to ketoconazole treatment. The existence of multiple CYP3A genes has been shown in several vertebrate species, including teleosts \[[@B44]\]. It is conceivable that there are two different CYP3A genes in Atlantic cod and that these genes respond differently to ketoconazole treatment. Protein isoforms revealed on 2D gel electrophoresis may also be due to post-translational modifications such as phosphorylation \[[@B45]\]. Phosphorylation of several members of the CYP2 gene family, through phosphokinase A, resulted in immediate loss in catalytic activity \[[@B46]\]. The shift to a more basic form in this report could imply a dephosphorylation of CYP3A upon ketoconazole treatment. However, as these spots were not detected directly on the 2D gels by using either Coomassie blue or silver staining, no spots could be selected for sequencing to investigate whether these two immunoreactive spots represent different gene products. In juvenile Atlantic salmon (Salmo salar), multiple hepatic CYP3A proteins also were seen \[[@B19]\]. The two proteins responded differently to nonylphenol treatment. High doses of nonylphenol (125 mg/kg b.w.) suppressed the high-molecular weight CYP3A protein band, whereas lower doses of nonylphenol (25 mg/kg b.w.) resulted in induction of this isoform \[[@B19]\]. In the present study, exposure to nonylphenol resulted in reduced CYP3A activities in juvenile Atlantic cod liver. Nevertheless, nonylphenol did not inhibit microsomal BFCOD activities in vitro, whereas nonylphenol was a weak inhibitor of that activity using recombinant human CYP3A4. The Atlantic cod we exposed to a mixture of ketoconazole and nonylphenol displayed in vivoCYP3A activities that were lower than the additive effect of each compound administered alone. The mechanism for this possible interaction still is not known. In mammals, more than one substrate can simultaneously bind to the active site of CYP3A4 \[[@B11]\]. Thus, in Atlantic cod, conceivably both ketoconazole and nonylphenol might bind to CYP3A enzyme and prevent access of the diagnostic BFC substrate. The CYP3A protein levels remained unchanged in these fish suggesting that combined exposure of ketoconazole and nonylphenol selectively inhibits in vivoCYP3A activity. Ethynylestradiol has been shown to act as a mechanistic inactivator (i.e.\"suicide\" substrate) of the CYP3A4 enzyme, resulting in loss of CYP3A4 protein levels \[[@B47],[@B48]\]. In Atlantic cod, a possible mechanism-based inactivation of CYP3A by ethynylestradiol was suspected. Thus, exposure to ethynylestradiol resulted in significantly reduced CYP3A levels and ethynylestradiol acted as an uncompetitive inhibitor of microsomal CYP3A activities. However, pre-incubation of hepatic microsomes with ethynylestradiol for up to 60 min did not result in any significant loss of CYP3A protein content, which implies that, in Atlantic cod, ethynylestradiol is not acting as mechanism-based inhibitor of CYP3A. Nonetheless, further studies are needed, as for example 2D gel electrophoresis of pre-incubated liver microsomes followed by immunoblotting. Vitellogenesis and sex steroid hormones --------------------------------------- In humans, prolonged ketoconazole therapy results in decreased clearance of 17β-estradiol, which may cause gynecomastia, presumably through inhibition of hepatic CYP3A4 \[[@B5]\]. In the present study, nonylphenol dependent induction of vitellogenesis was not significantly affected by treatment with a single dose of ketoconazole. In the present study, exposure to xenoestrogens and ketoconazole alone had no statistically significant effect on sex steroid levels compared to control fish. In another study in first spawning Atlantic cod, exposure to alkylphenols resulted in decreased plasma levels of 17β-estradiol (in females) and testosterone and 11-keto-testosterone (in males) \[[@B49]\]. Additional data, including plasma levels of ethynylestradiol, and increasing the sample sizes are required to definitely elucidate whether, in Atlantic cod, exposure to xenoestrogen and ketoconazole alone or in combination may affect sex steroid homeostasis. Conclusions =========== This study identifies, in Atlantic cod, interactions between ketoconazole and two different types of xenoestrogens on CYP1A and CYP3A. Ketoconazole acted as a non-competitive inhibitor of CYP1A and CYP3A activities and ketoconazole treatment also induced CYP1A protein expression. Ethynylestradiol acted as a non-competitive inhibitor of CYP1A and an uncompetitive inhibitor of CYP3A activities. In vitrostudies revealed that nonylphenol was a non-competitive inhibitor of CYP1A; but it did not inhibit CYP3A. However, in vivo, nonylphenol synergistically impaired the ketoconazole-mediated inhibition of CYP3A activity, without affecting CYP3A protein levels. The study further illustrates that induction of CYP1A- and CYP3A gene expression can be partly or completely masked by inhibition of catalytic activities or vice versa. Taken together, the results indicate that CYP1A and CYP3A represent sites of interactions between those classes of xenobiotics. In future risk-assessment of, e.g., municipal effluents or produced water from oil platforms, that have been shown to contain xenoestrogens, it should be considered to identify other classes of substances, for example azoles that also interact with CYP1A and CYP3A. Our data may warn for ecotoxicological implications, as induction of EROD activity as well as plasma vitellogenin routinely are used as biomarkers to assess exposure to AHR and ER agonists in various biomonitoring programs in the aquatic environment. Methods ======= Chemicals --------- The 4-nonylphenol and the 17α-ethynylestradiol, for the in vivoexposure experiment, were obtained from Fluka Chemie AG (Buchs, Switzerland). The 4-nonylphenol for the in vitroinhibition studies was from Berol Nobel (Stenungsund, Sweden). Dimethylsulphoxide (DMSO), 7-ethoxyresorufin, horseradish peroxidase- (HRP) conjugated goat-anti-mouse IgG, iodoacetamide, ketoconazole, ponceau-S, resorufin and tween-20 were obtained from Sigma Aldrich (Stockholm, Sweden). Reduced nicotinamide-adenine-dinucleotide-phosphate (NADPH) was from Roche Diagnostics (St Louis, MO, USA and Bromma, Sweden). Ready gels (12% continuous acrylamide in Tris:HCl), 3-\[(3-cholamidopropyl)-dimethylammonio\]-1-propanesulfonate (CHAPS), precision protein standards (low range) and supported nitrocellulose membrane (0.45 μm) were purchased from BioRad (Sundbyberg, Sweden). The 17β-estradiol and testosterone enzyme immuno assay (EIA) kits were purchased from Cayman Chemical (Ann Arbor, MI, USA). The 11-keto-testosterone EIA kit and the Atlantic cod vitellogenin Enzyme Linked ImmunoSorbent Assay (ELISA) kit were obtained from Biosense Laboratories AS (Bergen, Norway). HRP-conjugated donkey-anti-rabbit IgG, the ECL™ Western blotting detection reagents and Immobiline™ DryStrip 7 cm ranging from pH 4 to 7 were from Amersham Biosciences (Uppsala, Sweden). Ampholytes for isoelectric focusing (Servalyt^®^Carrier ampholyt 3--10) was purchased from Serva Feinbiochemica (Heidelberg, Germany). Dithiothreitol (DTT), Kodak X-Omat AR-ray film, X-ray developer and fix were from VWR International (Stockholm, Sweden). The 7-benzyloxy-4-\[trifluoromethyl\] coumarin (BFC), 7-hydroxy-4-\[trifluoromethyl\] coumarin (HFC) and the CYP3A4 inhibition kit were from BD Biosciences Company, Gentest™ (Woburn, MA, USA). All other chemicals used were of the purest grade available in Sweden or Norway, from Sigma-Aldrich, BioRad and VWR international. Animals and sampling -------------------- Hatchery reared juvenile Atlantic cod of both sexes with an average body weight (b.w.) around 400 g were supplied by Sekkingstad, Preserving AS, Hordaland, Norway. The fish were kept in 0.5 m^3^indoor glass fibre tanks, at Industrial Laboratory (ILAB), Bergen High Technology Centre (Bergen, Norway), provided with continuously flowing seawater at a temperature of 8 ± 0.5°C and a salinity of 3.4%. Throughout the experimental period, the fish were subjected to continuous 24 h artificial light (the regime the farm used for optimal fish growth). The fish were acclimated to these conditions for five days prior to the experimental period. During the experimental period the fish were starved and i.p. injected with either 12 mg ketoconazole/kg b.w. resuspended in peanut oil (2.5 mg/ml); 25 mg nonylphenol/kg b.w. dissolved in peanut oil (5.0 mg/ml); 5 mg ethynylestradiol/kg b.w. dissolved in peanut oil (1.0 mg/ml) or a mixture of ketoconazole and nonylphenol (12 mg ketoconazole + 25 mg nonylphenol/kg b.w. in peanut oil). Control fish were injected with 5 ml peanut oil/kg b.w. (vehicle). There were eight to nine fish in each treatment group. When designing the experiment, we could only test one combination due to limited fish numbers. We selected nonylphenol to combine with ketoconazole because a previous study showed that, in Atlantic cod, alkylphenols affect CYP1A/3A more strongly than the natural estrogen 17β-estradiol \[[@B22]\]. The ketoconazole dose (12 mg/kg) was selected based on the results on CYP1A and CYP3A protein levels and enzyme activities from a previous dose-response study in rainbow trout \[[@B13]\]. The nonylphenol dose (25 mg/kg) was selected as this dose is known to induce vitellogenesis in a number of fish species. In addition, in a previous study on the Atlantic salmon, this dose of nonylphenol also had effects on CYP1A and CYP3A \[[@B19]\]. After five days exposure, the fish were sacrificed by a sharp blow to the head. Blood samples were collected from the dorsal vein using a heparinized syringe and the liver was quickly dissected out and placed in ice-cold homogenization buffer (0.1 M sodium phosphate buffer pH 7.4, containing 0.15 M KCl, 1 mM EDTA and 1 mM DTT). Liver microsomes were prepared according to the published protocol by Goksøyr \[[@B50]\], and stored at -80°C. Total microsomal protein content was measured according to a published method by Bradford \[[@B51]\], using bovine serum albumin as standard, and a SpectraFluor spectrophotometer from Tecan (Grödig/Salzburg, Austria). Blood plasma was isolated by centrifugation at 5,000 g for 10 min at room temperature and stored at -80°C. Ethical approval licence number of ILAB Bergen: 119. Experiment no. 0204. For in vitroinhibition studies, feral Atlantic cod of both sexes were caught off the West coast of Sweden and placed in concrete basins provided with recirculating aerated seawater at 10 ± 2°C and a salinity of 3.0% and alternative light/dark photoperiods of 12 hours. Prior to sampling, the animals were starved and acclimated to these conditions for three weeks. Eight fish were injected i.p. with β-naphthoflavone (BNF), 50 mg/kg b.w. dissolved in peanut oil (5.0 mg/ml). The fish were placed in a 100 l glass aquarium provided with aerated seawater (above) and 30% of the water volume was replaced each day. To eliminate visual stress, the sides of the aquaria were covered with black plastic sheets. After 3 days exposure, the fish were sacrificed. Livers were quickly dissected out and placed in ice-cold homogenization buffer. Livers were pooled from twenty untreated Atlantic cod and from eight BNF treated Atlantic cod, respectively. Microsomal fractions were isolated (above) and stored in aliquots at -80°C. Ethical approval from the Ethical committee of Göteborg license number (99--2003). The duration of exposure was decided according to results from previous time-course studies showing maximal CYP1A protein and EROD activities in rainbow trout and in the marine viviparous blenny (Zoarces viviparous), 3 days post-injection with either the prototypical CYP1A inducers BNF or 3-methylcholanthrene \[[@B52]-[@B54]\]. CYP1A- and CYP3A protein blot analyses -------------------------------------- Western blot analyses of 40 μg hepatic microsomal protein were carried out using enhanced chemoluminescence (ECL), based on the protocol previously described \[[@B55]\] and PAb raised against rainbow trout CYP1A and CYP3A \[[@B41],[@B55],[@B56]\]. The intensity of each protein band was determined by densitometry on scanned fluorograms using Labview 7.0 from National Instruments (Austin, TX, USA). The 2D gel electrophoresis was performed using immobilised pH gradient gels with linear gradient from pH 4 to 7. The samples were concentrated by acetone precipitation and pellets dissolved in rehydration buffer (8 M urea, 2 M thiourea, 20 mM DTT, 4% CHAPS, 0.5% Triton X-100, 0.5% ampholyte 3--10 and \<0.02% bromophenolblue) to a final protein concentration of 20 μg/μl or 80 μg/μl. The samples were rehydrated overnight followed by isoelectric focusing for 2.5 h. The rehydration was passive and carried out overnight in an Immobiline Dry Strip reswelling tray (Amersham Biosciences). First-dimension isoelectric focusing (IEF) was performed on a Multiphor II unit (Amersham Biosciences) at 20°C using a MultiDrive XL power supply (Pharmacia LKB). Settings for IEF were 30 min at 100 V and 3 h at 3500 V for a total of 10,520 Vh. Amperage and wattage were set to 2 mA and 5 W. The proteins were resolved on 9% continuous acrylamide gel in Tris:HCl, including sodium dodecyl sulphate polyacrylamide using a mini-gel apparatus from BioRad at 200 V for 45 min. Each sample consisted of pooled liver microsomes from eight to nine fish from each treatment group. Gels loaded with 25 μg microsomal protein were initially stained with 0.1 % (w/v) Coomassie brilliant blue, and then destained, followed by silver staining. The latter was performed according to Heukeshoven and Dernick \[[@B57]\]. Stained gels were scanned and analyzed using the PDQUEST 7.1 software (BioRad). Gels loaded with 100 μg microsomal proteins were electrotransferred to nitrocellulose membrane and immunoblotted for CYP3A, as described above. Catalytic assays ---------------- The CYP1A activity was determined as EROD activity, using resorufin as standard in a SpectraFluor plate reader according to the protocol provided by Nilsen et al.\[[@B58]\]. The CYP3A catalytic activity was measured as BFCOD activity, using HFC as standard. The BFC assay was performed based on a published protocol by Miller et al.\[[@B59]\] and optimized for rainbow trout liver microsomes (T. Hegelund and M. Celander, unpublished data). The reaction mixture consisted of 200 μM BFC, bovine serum albumin (1.6 mg/ml), 2 μM NADPH and 10 μl liver microsomes in a total volume of 200 μl in 0.2 M potassium phosphate buffer pH 7.4 in a 96-multiwell plate using a VICTOR™ 1420 Multilabel Counter from Wallac Sverige AB (Upplands Väsby, Sweden). In vitroinhibition of CYP1A and CYP3A --------------------------------------- In vitroinhibition studies were carried out in 96-multiwell plates using a VICTOR™ 1420 Multilabel Counter. The IC~50~values were determined for nonylphenol, ethynylestradiol, ketoconazole and the ketoconazole:nonylphenol (1:5) mixture on CYP1A and CYP3A activities. The substances were dissolved in DMSO and diluted with ethanol. The final concentrations never exceeded 0.01% (v/v) DMSO and 0.001% ethanol (v/v). For CYP1A and CYP3A inhibition studies, pooled liver microsomes from BNF-treated and from untreated Atlantic cod, respectively, were used. For comparison, the IC~50~values for ketoconazole, nonylphenol and ethynylestradiol were determined in cDNA expressed human CYP3A4 baculovirus supersomes using the CYP3A4 inhibition kit from BD Gentest. In vitroincubation studies ---------------------------- Pooled liver microsomes from untreated Atlantic cod were pre-incubated for 10, 30 and 60 min, at room temperature, with ethynylestradiol and ketoconazole following CYP3A western blot analysis. The reaction mixture consisted of microsomes (2.5 or 5.0 mg protein/ml) and various concentrations of ethynylestradiol (35, 50 and 100 μM) or ketoconazole (0.3 and 1.0 μM) ± 3 μM NADPH in a total volume of 50 μl in homogenization buffer, containing 20% (v/v) glycerol. The CYP3A western blot analysis was performed as described above. Ethynylestradiol and ketoconazole were dissolved in acetonitrile (vehicle) and the final acetonitrile concentration in the reaction mixture was 0.02% (v/v). Plasma vitellogenin analysis ---------------------------- Plasma levels of vitellogenin protein were determined using a non-competitive sandwich ELISA kit and employing rabbit PAb against Atlantic cod vitellogenin from Biosense Laboratories AS (Bergen, Norway) \[[@B58]\]. Each plasma sample was diluted (1:20, 1:15,000 and 1:50,000) and 100 μl was analyzed and compared to purified Atlantic cod vitellogenin protein standards (ranging between 0.12 and 2,000 ng/ml). The signal was detected at A~492~after 15 min incubation with substrate solution, using a VICTOR™ 1420 Multilabel Counter. Plasma sex steroid hormone analyses ----------------------------------- Plasma levels of 17β-estradiol and testosterone were determined using competitive EIA kits from Cayman Chemical (Ann Arbor, MI, USA). Plasma levels of 11-keto-testosterone were analyzed using a competitive EIA kit, from Biosense Laboratories AS (Bergen, Norway). Plasma from each fish was concentrated (2:1) by extraction once with six volumes diethyl ether and 50 μl was analyzed and compared to purified standard substances. The signals were detected at A~405~after 40 min (17β-estradiol), 60 min (testosterone) or 80 min (11-keto-testosterone) incubation with substrate solution, using a VICTOR™ 1420 Multilabel Counter. Statistics ---------- Data were tested for homogeneity of variances using the Levene\'s test. When there was homogeneity of variances we used a parametric one-way ANOVA, followed by Scheffé post hoc test. When there was no homogeneity of variances we used the non-parametric Kruskal-Wallis ANOVA, followed by the two-tailed Mann-Whitney U test. No values were log transformed. Data are presented as means (n = 6--9 fish) accompanied with the standard deviations (SD). The significance level (α) was set at 0.05. The statistical analyses were performed using SPSS 11.0 software, from SPSS Sweden AB (Sundbyberg, Sweden). Authors\' contributions ======================= LH performed most of the analyses, participated in fish exposure, sampling, experimental design and drafted the manuscript. BEG assisted with experimental design, 2D-analysis and writing. AG participated in experimental design and writing. MCC rose funding, coordinated, participated in fish exposure, sampling, experimental design and writing. Acknowledgements ================ We thank Åsa Berglund and Susan Westerberg, Department of Zoophysiology, Göteborg University and Christina Tolfsen Department of Molecular Biology, University of Bergen for excellent technical assistance. The Improving Human Potential Programme from the European Union, through Contract No HPRI-CT-2002-00188 to LH and MC, has funded access to installations from the University of Bergen. Financially supported by the Faculty of Science, Göteborg University, and grants from Swedish EPA (ReproSafe), C.F. Lundström Foundation and His Swedish Royal Majesty Carl XVI Gustaf\'s 50-Anniversary Foundation to MC.	PubMed Central	2024-06-05T03:55:52.910316	2005-2-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549046/", "journal": "Comp Hepatol. 2005 Feb 8; 4:2", "authors": [ { "first": "Linda", "last": "Hasselberg" }, { "first": "Bjørn E", "last": "Grøsvik" }, { "first": "Anders", "last": "Goksøyr" }, { "first": "Malin C", "last": "Celander" } ] }
PMC549047	Background ========== Agnogenic myeloid metaplasia (AMM), polycythemia vera (PV), essential thrombocythemia (ET) and chronic myelogenous leukemia (CML) are characterized as myeloproliferative disorders (MPDs) arising due to exponential amplification of a haematopoietic stem cell. A typical feature of these disorders is the presence of high number of circulating progenitor cells and their cytokine independent growth in culture. So far, the molecular basis for the disorder has been recognized only for CML and is attributed to the Bcr/Abl or Philadelphia (Ph) chromosome arising due to a translocation event. The aetiology for the other three disorders referred to as Ph negative MPDs is unknown. AMM, one of the Ph negative MPD is diagnosed by hyperplasia of atypical megakaryocytes, hepatosplenomegaly, extramedullary haematopoiesis and bone marrow fibrosis. Fibrosis is considered to be a secondary consequence of enhanced levels of fibrogenic growth factors such as TGF β1, bFGF and PDGF produced by enhanced numbers of megakaryocytes (MKs), while the primary cause is considered to be the enhanced proliferation of a defective stem cell. What leads to generation of such a defective cell with enhanced proliferation is unknown. We have earlier demonstrated \[[@B1]\], which has later been confirmed \[[@B2]\] that thrombopoietin (TPO) is one of the growth factors whose level is enhanced in patients with AMM. TPO is a haematopoietic growth factor that is essential for megakaryocytopoiesis and thrombocytopoiesis \[[@B3]\]. TPO binds to its receptor Mpl (Myeloproliferative leukaemia), gets internalised \[[@B4]\], and initiates a STAT5 signalling cascade \[[@B5]\], which results in thrombopoiesis. Studies with TPOknockout mice have shown reduced numbers of platelets and myeloid progenitor cells confirming the role of TPO. Enhanced expression of TPOin mouse by Retroviral/Adenoviral gene transfers and in mice transgenic for TPO, the animals developed extramedullary haematopoiesis and splenomegaly due to MK and granulocytic hyperplasia. The severity of the conditions was related to the expression levels of TPO, resulting in myelofibrosis under very high expression level \[[@B6]-[@B8]\]. These results of TPOover expression are similar to the symptoms of AMM and hence indicate that high levels of TPO found in AMM patients could be responsible for the clinical features of AMM. In the mouse knockouts for Mplreceptor, there is about 85% reduction in the number of platelets, MKs and other haematopoietic cell types \[[@B9]\] and Mplover expression leads to myeloproliferation \[[@B10]\]. In vitro, Mplantisense oligonucleotides inhibited megakaryocytopoiesis \[[@B11]\]. These reports augment the role of Mpl in megakaryopoiesis. But in contrast to TPO, Mpl protein level is reduced in AMM patients \[[@B12]\]. This abnormality in the TPO/Mpl pathway may depict the clinical features, but is it the cause for MK hyperplasia and the disease? Studies so far have not been conclusive. According to Taksin et al. \[[@B13]\] defects in the TPO/Mpl pathway are unlikely to be primarily responsible for AMM. Autonomously growing MKs in AMM did not have enhanced TPOtranscripts and they were not inhibited by TPO antibody. Also no mutations were detected in the Mplgene in these patients, to account for its reduced protein level. The observation of Li et al. \[[@B14]\] that Mplanti-sense oligonucleotide treatment could inhibit the autonomous growth of MKs from ET/PV/AMM in vitro suggests that expression of Mpl is essential for MK autonomy in these disorders. However Moliterno et al. \[[@B12],[@B15]\] have reported reduced Mpl protein level with glycosylation defects in the platelets of patients with these disorders. Although this could be responsible for the impaired TPO-mediated platelet protein tyrosine phosphorylation seen in PV and AMM patients \[[@B16]\], it does not confer with MK autonomy/hypersensitivity for growth in vitro. This reduced expression of Mplin a disorder characterized by MK hyperplasia whose development and differentiation is dependent on this cytokine is paradoxical and needs further investigation. Hence to better understand the role of TPO/Mpl in AMM, we have extended the study of this growth factor and its receptor gene in these patients. Our studies reveal enhanced plasma TPO level, reduced endogenous level of Mpl protein in the platelets of patients with AMM and absence of any co-relation between TPO level and Mpl expression. We have cloned MplcDNA from 15 AMM patients and have found that it has full potential for expression similar to controls both in vitro and in the in vivo heterologous system of 293 T cells. We also assayed for the level of eIF4E, the translation initiation factor that under normal limiting level favours translation of strong mRNAs such as those of house keeping genes and at higher level, enhances the translation of weak mRNAs such as Mpl. AMM patients were found to have elevated eIF4E level. Results ======= TPO ELISA --------- Undiluted platelet poor plasma (PPP) samples from 20 AMM patients and 10 controls were assayed for their TPO levels. Although some patients exhibited low TPO levels, overall, TPO level was enhanced in AMM patients as compared to controls \[0--684 pg/ml in AMM patients (with a mean of 229 pg/ml) as compared to 0--71 pg/ml in controls (with a mean of 22.8 pg/ml)\]. This increase in TPO was significant (P \< 0.001) as determined by t-test (Fig. [1](#F1){ref-type="fig"}). Minimum detectable dose with the TPO ELISA kit is 2.78--18.5 pg/ml according to the manufacturer. The Optical Density (OD) values for samples were within the range obtained with the highest standard (2000 pg/ml) used in the ELISA. Some samples from controls and patients as well failed to register any OD probably due to very low TPO levels. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### TPO is elevated in AMM patients.TPO level in the undiluted Platelet Poor Plasma samples of 10 controls and 20 AMM patients was tested by ELISA. While TPO level was in the range of 0--71 pg/ml in controls (Mean 22.8 pg/ml), it ranged between 0--684 pg/ml in AMM patients (Mean 229 pg/ml). This elevation of TPO level in patients was significant (P \< 0.001) compared to controls. ::: ![](1478-811X-3-4-1) ::: Mpl Expression is reduced in platelets of patients with AMM ----------------------------------------------------------- Platelets obtained from 12 AMM patients and 12 healthy controls were lysed in 1X Laemelli buffer and analysed on SDS PAGE. Mpl antibody that was raised against the full length Mpl protein was used for western blot analysis. This antibody recognizes both glycosylated and non-glycosylated forms of Mpl. The blots were re-probed with β Actin antibody for normalization. The western blots on chemiluminescence detection showed an overall reduction in the expression of Mpl in the platelets of patients with AMM and there was no co-relation (r^2^= 0.0038) between TPO level and Mpl levels (Fig. [2](#F2){ref-type="fig"}). While there was high expression of glycosylated form of Mpl in the controls, the expression of Mpl in the platelets of patients with AMM varied from none to having reduced amounts of both glycosylated and faster moving non-glycosylated forms. A representative blot with results from 4 controls and 7 patients is shown in Fig. [3](#F3){ref-type="fig"}. The developed blots were analysed using the Image J program to quantitate the intensity of the bands and the values for Mpl expression after normalization against β Actin expression are represented in Fig. [4](#F4){ref-type="fig"}. Statistical analysis of the values by t-test revealed the reduced Mpl expression to be significant (P \< 0.05). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### TPO level is not co-related to platelet Mpl protein level.Plasma TPO levels and platelet Mpl protein levels from 12 AMM patients were compared for co-relation. Statistical analysis revealed absence of any co-relation between the levels of these two proteins (r^2^= 0.0038). ::: ![](1478-811X-3-4-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Mpl protein is reduced in AMM patients.Platelet lysates from 12 controls and 12 AMM patients in 1X Laemelli buffer were analysed by 6% SDS PAGE. Mpl antibody raised against the full-length protein was used for western blot analysis. The blots were re-probed with β Actin Antibody for normalization. This representative autoradiogram shows the results for 4 controls and 7 AMM patients. ::: ![](1478-811X-3-4-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Densitometric scanning results of Mpl expression in platelets.The intensity of the bands on the Mpl western blot autoradiograph was quantitated using Image J program. The values represent Mpl expression after normalization with β Actin expression assayed for 12 controls and 12 AMM patients. The reduction in Mpl levels in the platelets of AMM patients was statistically significant (P \< 0.05). ::: ![](1478-811X-3-4-4) ::: Mpl RNA level is similar in controls and AMM patients ----------------------------------------------------- Since Mpl protein level was reduced in AMM patients, to know if it was due to reduced transcription of the Mplgene, we compared the levels of MplRNA in AMM patients and controls by Real-Time RT-PCR. Total RNA from the platelets of 12 controls and 12 AMM patients were used as templates for PCR with gene specific primers. The values were normalized against GAPDHexpression and are represented in Fig. [5](#F5){ref-type="fig"}. On statistical analysis (t-test), the difference in Mplexpression was found not to be significant (P \> 0.05), indicating absence of any transcriptional inhibition of Mplin AMM patients. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Transcription of Mpl is similar in AMM and controls.Total RNA isolated from platelets of 12 AMM patients and 12 controls were subjected to Real-Time RT-PCR using Mplgene specific primers. The values for Mplexpression after normalization against GAPDHvalues were found not to be significant (P \> 0.05) between the two groups. ::: ![](1478-811X-3-4-5) ::: AMM patients have high eIF4E levels ----------------------------------- We assayed for the expression of eIF4E in the platelets of 12 AMM patients to see if reduced Mpl protein level could be due to its reduced translation owing to limiting levels of eIF4E, a translation initiation factor that could favour translation of weaker mRNA such as Mplat higher amounts. At limiting levels it favours translation of stronger mRNA over the weaker mRNA. The western blots on chemiluminescence detection showed an overall increase in the expression of eIF4E in the platelets of patients with AMM. A representative blot shown in Fig. [6](#F6){ref-type="fig"} includes 5 controls and 12 AMM patients (Platelet sample from Patient \# 7 has inadequate protein). The developed blots were analysed using the Image J program to quantitate the intensity of the bands and the values for eIF4E expression after normalization against β Actin expression are represented in Fig. [7](#F7){ref-type="fig"}. Statistical analysis of the values by t-test revealed the elevated eIF4E expression to be significant (P \< 0.0001) and absence of any significant co-relation between the expression levels of Mpl and eIF4E (data not shown). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### AMM patients have high levels of eIF4E.Platelet lysates from 12 controls and 12 AMM patients in 1X Laemelli buffer were analysed by 10% SDS PAGE. Antibody for eIF4E was used at a dilution of 1:1000 for western blot analysis. The blots were re-probed with β Actin Antibody for normalization. A representative autoradiogram including 5 controls and all 12 patient samples is shown in this figure. Platelet sample from patient \# 7 has inadequate protein. ::: ![](1478-811X-3-4-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Densitometric scanning results of eIF4E expression in platelets.The intensity of the bands on the eIF4E western blot autoradiograph was quantitated using Image J program. The values represent eIF4E expression after normalization with β Actin expression for the 12 controls and 11 patient samples. The increase in eIF4E levels in the platelets of AMM patients was statistically significant (P \< 0.0001). ::: ![](1478-811X-3-4-7) ::: The coding region of Mplfrom AMM patients has no mutations ------------------------------------------------------------ MplcDNA was amplified from the platelets of 15 AMM patients along with those from 15 normal controls by RT-PCR, cloned into the Blue Script vector and the recombinant clones sequenced completely. Base changes were detected in some patients and controls as well in the exons 2,3, 4, 6 and 12 but not at the same nucleotide (data not shown). The same changes were however not observed on re-cloning the cDNA of the samples with mutations and sequencing the corresponding genomic region. These base changes were therefore considered to be due to PCR errors and not genuine mutations. Hence Mplgene abnormalities were not detected in AMM patients. Mplfrom AMM patients is able to transcribe and translate in vitro ------------------------------------------------------------------- About 1 μg of the MplcDNA of 15 controls and 15 AMM patients in the pcDNA3 vector were subjected to in vitro transcription/translation employing the rabbit reticulolysates to see if they were capable of translation in vitro. Mplfrom AMM patients were able to translate as efficiently as the cDNA from controls revealing their full potential for translation. Also, in the presence of canine microsomal membranes, the translated protein was glycosylated similar to those from the controls. Results from 2 controls and 4 AMM patients are represented in Fig. [8](#F8){ref-type="fig"}. The developed autoradiographs were analysed using the Image J program to quantitate the intensity of the bands and the values are represented in Fig. [9](#F9){ref-type="fig"}. Statistical analysis of the values by t-test revealed absence of any difference (P \> 0.05) between cDNA from controls and AMM either in the amount of the translation product or its glycosylation. ::: {#F8 .fig} Figure 8 ::: {.caption} ###### **Mplfrom AMM patients can be translated in vitro similar to controls.**MplcDNA clones of 15 controls and 15 Patients with AMM in pCDNA3 vector were subjected to in vitro transcription/translation using Promega\'s TNT Quick Coupled Transcription/Translation Systems with ^35^S Methionine in the absence (-) or presence (+) of canine microsomal membranes. The entire reaction product was loaded onto 6% SDS PAGE and electrophoresed. The dried gel was exposed to X-ray film and a representative autoradiograph with 2 control and 4 patient samples is shown. ::: ![](1478-811X-3-4-8) ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### *Densitometric scanning results of Mplin vitro transcription/translation.The intensity of the bands on the autoradiograph of Mplin vitro transcription/translation was quantitated using Image J program. The values represent Mpl band intensity for 15 samples each of controls and patients. There was no significant (P \> 0.05) difference between AMM and controls in the intensity of the non-glycosylated/glycosylated bands. ::: ![](1478-811X-3-4-9) ::: Mplfrom AMM patients is able to transcribe and translate in vivo ------------------------------------------------------------------ MplcDNA was tested for in vivo expression in the 293 T human embryonic kidney cells. Equal amount of MplcDNA from 15 AMM/15 controls was transfected into the 293 T cells and Mpl expression analysed by western blot as described for the platelets. The expression level of Mpl appeared to be similar between controls and AMM patients. Fig. [10](#F10){ref-type="fig"} is a representative blot depicting results of 5 controls and 5 AMM patients. Two bands were identified. The developed blots were analysed using the Image J program to quantitate the intensity of the bands and the values for Mpl expression are shown in Fig. [11](#F11){ref-type="fig"}. Statistical analysis of the values by t-test showed no significant difference (P \> 0.05) in the level of Mpl expression. ::: {#F10 .fig} Figure 10 ::: {.caption} ###### *Mplfrom patients with AMM can be translated in vivo.*Western blot analysis of lysates of 293 T cells transfected with pcDNA3/Mplconstructs from 15 AMM patients and 15 controls. Transfection lysates were analysed by 6% SDS PAGE. Blots were probed with Mpl antibody raised against the full length Protein. A representative blot with data for 5 controls and 5 patients is shown in this figure. The second band probably corresponds to non-glycosylated Mpl. ::: ![](1478-811X-3-4-10) ::: ::: {#F11 .fig} Figure 11 ::: {.caption} ###### Densitometric scanning results of western blot of Mpl in vivo expression.The intensity of the bands for Mpl western blot with in vivo transfection lysates was quantitated using Image J program. No significant (P \> 0.05) difference in the intensity of the Mpl bands between AMM and control\'s cDNA transfection lysates was observed. ::: ![](1478-811X-3-4-11) ::: Discussion ========== AMM is one of the MPDs for which the molecular defect is unknown. A characteristic feature of the disorder is the presence of atypical megakaryocytes in high numbers and their autonomy or hypersensitivity to growth factors in vitro. Since the haematopoietic growth factor TPO that is essential for the development of megakaryocytes is present at high level in AMM patients, it is of interest to study the role of TPO and its receptor in AMM. We have earlier reported that AMM patients have elevated plasma TPO level and this is not associated with its enhanced transcription in the bone marrow cells \[[@B1],[@B17]\]. To determine if this enhanced TPO level is due to its receptor abnormalities, we have analysed 12 AMM patients for the presence of Mpl protein in their platelets and found it to be reduced in AMM relative to controls. However, this reduced level was not due to its reduced transcription since Real-Time RT-PCR revealed similar levels of transcripts from AMM patients and controls. To analyse if it could be due to its reduced translation owing to its G-C rich 5\' UTR, we estimated the expression of the translation initiation factor eIF4E. The promoter of Mpldisplays characteristics of weak mRNA that are not translated very efficiently due to G-C rich 5\' UTR \[[@B18]\]. The translation initiation factor eIF4E, that is present at low level under normal conditions favours the translation of strong mRNA at these limiting levels. At higher levels, although the translation of strong mRNA is not increased greatly, the translation of weak mRNAs is highly enhanced and this is usually associated with hyper proliferation of cells as in cancers \[[@B19]\]. Hence we assayed for the expression of eIF4E in AMM to see if reduced Mpl protein level could be due to its reduced translation owing to limiting eIF4E level compared to controls. However eIF4E level was rather significantly higher than in controls as seen in other diseases with hyper cellular proliferation. Although this is not direct evidence, at these levels of eIF4E, normal translation of Mplcan be expected. Since there was no significant difference in the MplRNA level of AMM patients as shown by RT-PCR, we cloned and sequenced Mplgene from these patients to over-rule any structural defects that could prevent translation. Although base changes were observed for some patients and controls as well, on sequencing the corresponding regions of the genomic DNA, the same base changes were not observed. Hence these were not considered to be genuine mutations. Taksin et al. \[[@B13]\] also have reported absence of Mplgene mutations in AMM patients. Hence to date, there are no reports describing Mpl gene mutations in AMM patients although an activating mutation in this gene has been reported by Ding J et al. \[[@B20]\] in a case of familial ET, a rare hereditary MPD. Moliterno et al. \[[@B21]\] have recently reported a single base change polymorphism in the Mplgene of African American patients with MPD that leads to substitution of the 39^th^amino acid Lysine, with Asparagine in the extra cellular domain of the protein. Patients with this polymorphism exhibited high platelet counts and low platelet Mpl protein. African Americans were however not represented amongst our patients tested. We also performed in vitro transcription/translation with the cloned cDNA from the AMM patients. Similar levels of proteins were formed irrespective of the source of the cDNA, indicating that the cDNA from AMM patients had no structural defects preventing its translation. There was no difference in the extent of glycosylation of the protein either when the reaction was performed in the presence of microsomal membranes capable of post-translational modification in vitro. To further confirm this in vivo, these cDNAs were transfected into 293 T human embryonic kidney cells and the cell lysates analysed by western blots for Mpl expression. Irrespective of the source of the cDNA, similar level of expression of Mpl protein was observed. Two bands were however detected. Since the antibody used can react with both glycosylated and non-glycosylated forms of Mpl, it is likely that the second band corresponds to non-glycosylated form. As the in vivo system is saturated with the over-expressed protein, the system for modification of the protein could be exhausted. Overall, Mplgene from AMM patients does not seem to have any impaired structural features to prevent its transcription/translation. Our present study shows elevated TPO level; reduced Mpl platelet protein level; absence of Mpltranscriptional inhibition and absence of any structural defects in Mplcoding region in AMM patients. However, there was no co-relation between TPO level and Mpl expression, similar to the observation of Harrison CN et al. \[[@B22]\] in ET patients. Plasma TPO level is regulated by platelet counts and MK mass \[[@B23]\]. TPO binds to its receptor Mpl on platelets/MKs and gets internalised. This receptor mediated endocytosis of TPO leads to its catabolism by the lysozymes, without any recycling of the receptors to the surface \[[@B24]\]. In AMM patients there is initial MK hyperplasia and thrombocytosis that leads to enhanced release of growth factors such as Platelet Derived Growth Factor, bFibroblast Growth Factor and Transforming Growth Factor β1 \[[@B25]-[@B27]\]. This may lead to increase in TPOtranscription, since these growth factors have been shown to stimulate the transcription of TPOfrom the bone marrow stromal cells in cultures \[[@B28],[@B29]\]. Our earlier observation that elevated plasma TPO level is not associated with its enhanced transcription in the bone marrow cells \[[@B17]\] could be due to the fact that our cultures predominantly consisted of fibroblasts and not stromal cells. Enhanced expression of TPO and absence of an accompanying increase in Mpltranscription or re-cycling of the internalised receptor may be responsible for persistence of high plasma TPO level in AMM patients. Reduced Mpl level seen in AMM patients may be due to exhaustion of the Mpl pool by binding to the enhanced TPO and its subsequent catabolism. This gains support from an earlier observation in thrombocythemic mice wherein very high and persistent level of TPO due to its induced over expression were found to be associated with reduced platelet Mpl protein without any reduction in Mpltranscription \[[@B30]\]. Also in other MPDs such as ET/PV, high plasma TPO level is associated with low Mpl protein level \[[@B16],[@B31]\]. In vivo Mpl post-translational defect may also be responsible for impaired Mpl protein forms and levels in these patients as suggested by Moliterno et al. \[[@B15]\]. Cloning and sequencing of TPO5\' UTR from these patients may provide additional valuable information on elevated TPO levels since mutations in 5\' UTR of TPOhave been associated with elevated TPO levels without any increase in its transcription in patients with thrombocytosis \[[@B32],[@B33]\]. Conclusions =========== Our studies with TPO/Mpl show elevated TPO and reduced Mpl level in AMM patients, without any significant co-relation in their expression levels. MplcDNAs from AMM patients had full potential for transcription/translation including glycosylation, in vitro and in vivo, similar to those of normal controls. Reduced Mpl protein level may not be due to defects in its transcription/translation but could be due to increased internalisation or intrinsic defects in translation and glycosylation. Methods ======= Patients -------- Peripheral blood was obtained from patients with AMM after informed consent and peripheral blood was obtained from normal volunteers to serve as controls. Five volumes of peripheral blood was mixed with 1 volume of ACD buffer pH 4.5--5.5 (2.2% W/V Sodium Citrate; 0.8% Citric Acid; 2.2% Glucose; 50 ng/ml PGE-1) and centrifuged at 250 g for 10 minutes. The cell pellet was diluted 5X with Hank\'s buffer and subjected to Ficoll/Hypaque density gradient centrifugation. The ring of mononuclear cells was washed 2X with Hank\'s buffer and processed for isolation of genomic DNA using the Wizard Genomic DNA Purification kit from Promega, Madison, WI, following their instructions. The Platelet Rich Plasma (PRP) supernatant was centrifuged at 800 g for 10\' to obtain the platelets. The Platelet Poor Plasma (PPP) was stored at -80°C, and the platelet pellet washed 2X with the Citrate buffer, pH 6.2 (0.05 mmol/L Sodium Citrate; 0.1 mol/L NaCl; 0.14 mol/L Glucose). They were then processed for RNA isolation using the Totally RNA isolation kit from Ambion, Texas, following their instructions or were suspended in 1X PBS and lysed with equal amount of 2X Laemelli buffer (0.125 M Tris HCl pH 6.8; 4% SDS; 0.56 M β Mercaptoethanol; 0.02% Bromophenol Blue; 20% Glycerol) for western blot analysis. TPO ELISA --------- TPO level in the blood plasma was estimated using the Human thrombopoietin Quantikine ELISA kit from R & D Systems, Minneapolis, MN. The assay is based on the two-sided sandwich principle. Standards and undiluted PPP samples from 10 normal controls and 20 AMM patients were incubated in micro-titer Plates whose wells are pre-coated with monoclonal TPO antibody. After washing off the unbound substances, the wells were incubated with polyclonal TPO antibody conjugated with HRP enzyme. After subsequent washes to remove any unbound antibody-enzyme, a substrate solution for HRP was added to the wells. The color developed that is proportional to the amount of TPO from plasma bound to the plate was quantitated using the Opsys MR plate reader. Real-Time RT-PCR ---------------- Real-Time RT-PCR was performed to determine the expression of Mplin platelets of 12 controls and 12 AMM patients, using the Geneamp 5700 SDS Instrument from Applied Bio-systems, Foster City, CA. Reagent for the assay was the Quantitect RT-PCR kit (Qiagen, Valencia, CA). MplmRNA in the platelets was reverse transcribed and PCR amplified from 10 ng of total RNA with gene specific primers and a florescent reporter probe that hybridises to the gene in between the two primers. The sequences of the primers were: MPL FP 5\'-AAGTCCTCAGAGAGGACTCCTTTG-3 MPL RP 5\'-CAGGCAAGAAGGCTGCAATC-3\' MPL PROBE 5\'-FAM CCTCCCAGGCCCAGATGGACTAC-3\' BHQ1 The reaction conditions were reverse transcription at 50°C for 30\'; 40 cycles of amplification with de-naturation at 94°C for 15\", annealing and extension at 60°C for 10\". Sequence Analysis of Mpl* -------------------------- Total RNA extracted from platelets obtained from 15 patients with AMM and 15 normal controls was used to clone the full length MplcDNA. First strand cDNA was synthesized using the SuperScript First Strand Synthesis System for RT-PCR from Invitrogen, Carlsbad, CA, following their specifications. About 3 μg of total RNA was mixed with 50 ng of random hexamers, and 1 mM dNTPs in 10 μl reaction volume and incubated at 65°C for 5\' and left on ice for 1\'. A cocktail mix of RT buffer, MgCl~2~and DTT was added to a final concentration of 1X, 5 mM and 10 mM respectively along with 40 Units of RNase OUT Ribonuclease Inhibitor and incubated at 25°C for 2\'. 50 Units of SuperScript II Reverse Transcriptase enzyme was added and incubated at 25°C for 10\' and 42°C for 50\'. The reaction was terminated by heating at 70°C for 15\' and chilled on ice. 2 Units of RNase H was added and incubated at 37°C for 20\' to remove the RNA from cDNA: RNA hybrids to increase the sensitivity of PCR from cDNA. Mplwas amplified using the Expand High Fidelity PCR kit from Roche, Indianapolis, IN, with gene specific primers containing EcoR 1 (FP) and Xho1 (RP) Restriction Enzyme sites to enable directional cloning. The Restriction Enzyme sites are indicated in lower case in the Primer sequences given below. FP 5\'-CGgaattcGAAGGGAGGATGGGCTAAGGC-3\' RP 5\'-CCGctcgagAGTTTAGCTCTGTCCAGGGAA-3\' The Products were purified using the Wizard PCR Preps DNA Purification System from Promega, Madison, WI, and digested with the Restriction Enzymes, EcoR 1 and Xho1. The digested products were then cloned into Blue Script vector. The recombinants were checked by restriction analysis and the selected positive clones were sequenced completely at Northwoods DNA Inc., Becida, MN. MplcDNA were re-cloned from samples that had mutations and sequenced completely. The putative mutations were confirmed by sequencing the corresponding region of the Mplgene from respective samples. For this, the genomic DNA isolated from the peripheral blood samples were PCR amplified with gene specific primers and the PCR products were directly sequenced after purification using the Wizard PCR Preps DNA Purification System from Promega, Madison, WI. In vitro transcription/translation of Mpl ------------------------------------------- The MplcDNA clones in the Blue Script vector were digested with EcoR 1 and Xho1 and the released MplcDNA was cloned into pcDNA 3 vector to obtain cDNA with 3\' translational signals. These constructs were then subjected to in vitro transcription/translation using the TNT Quick Coupled Transcription/Translation Systems from Promega. Madison, WI, in the presence of ^35^S Methionine from Amersham Biosciences. About 0.5 μg of the constructs were incubated with the TNT Quick-Coupled Master Mix and ^35^S Methionine in the absence or presence of 2 μl of Canine microsomal membranes provided with the TNT Quick Coupled kit, at 30°C for 2 hours. The reactions were terminated by adding 100 μl of 1X SDS loading buffer (50 mM Tris. HCl pH 6.8; 100 mM DTT; 2% SDS; 0.1% Bromophenol Blue; 10% Glycerol) and stored at -20°C. Appropriately translated and processed products were then separated on a 6% polyacrylamide gel. The gel was dried using Model 583 Gel dryer from BIO-RAD, Hercules, CA. The products of translation were visualized by autoradiography of the dried gel. The bands were quantitated using the Image J program. Transfections ------------- 293 T human embryonic kidney cells were used for the transient transfection studies with pcDNA3/Mplconstructs of controls and patients used in the in vitro transcription/translation studies. The cells were seeded onto a 6 well plate at a density of 5 × 10^4^cells/well a day before transfection with DMEM medium containing 10% FBS. On the day of the transfection, the cells were washed with DMEM medium without serum and covered with 800 μl of the same. In an eppendorf tube, 1 μg of the DNA was mixed with 100 μl of serum free DMEM medium and 100 μl of DMEM containing 2 μl of Lipofectamine transfection reagent (Invitrogen, Carlsbad, CA) was added to this DNA. The mixture was incubated for 30\' at room temperature and then added to the 800 μl of medium covering the cells. After incubating for 5 hours at 37°C the transfection medium was removed and replaced with DMEM medium with 10% FBS. The cells were harvested 48 hours post-transfection. They were washed twice with 1X PBS and the cells dislodged by gentle re-suspension. The cells were collected and centrifuged at 1000 RPM for 10\'. The cell pellets were re-suspended in 100 μl of 1 X PBS each and lysed with 100 μl of 2X Laemelli buffer. Western Blot Analysis --------------------- The Transfection cell lysates/platelet cell lysates in 1X Laemelli buffer were boiled for 5\', clarified by centrifugation at 14 000 RPM for 5\', loaded onto a 6% SDS PAGE, and transferred onto immobilon nylon membrane. The membrane after transfer was rinsed with 1X PBS and incubated in the blocking solution (5% milk in 1 X PBS; 0.1 % Tween 20) for 1 hr., at room temperature. The membrane was then incubated with 1:10 000 dilutions of Rabbit anti Mpl antibody from Upstate USA Inc. VA, over night at 4°C in the blocking solution. The membrane was then washed 4 X with 1X PBST (1X PBS; 0.1% Tween 20) for 15\' each and incubated with 1:5000 dilutions of HRP conjugated anti-rabbit secondary antibodies for 1 hr., at room temperature. Washes with 1X PBST were repeated and the membrane processed for detection using the Western Lightning Chemiluminescence Reagent Kit from Perkin Elmer, Boston, MA, following their recommendations. The bands were quantitated using the Image J program. Similar procedure was followed for the detection of eIF4E protein in the platelets except that a 10% SDS PAGE was used for separation of the proteins. The eIF4E antibody from Transduction Laboratories, CA, was used at 1:1000 dilutions. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= KCH performed most of the experiments and was responsible for acquisition, analysis and interpretation of the data in addition to preparation of the manuscript. JCW conceived, designed the study, provided patient samples for the study and critically reviewed the data. KS performed eIF4E western blot. GH performed the early work and helped in preparation of application for grant support. ADN was involved in critical revision of the article, in obtaining patients for the study and obtaining grant support. All authors read and approved the final manuscript. Acknowledgements ================ This work was supported by a research grant from \'Aplastic Anemia and MDS Foundation\' and \'Maimonides Research & Development Foundation\'.	PubMed Central	2024-06-05T03:55:52.913100	2005-2-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549047/", "journal": "Cell Commun Signal. 2005 Feb 3; 3:4", "authors": [ { "first": "Kirugaval C", "last": "Hemavathy" }, { "first": "Kathir", "last": "Suppiah" }, { "first": "Gazala", "last": "Hashmi" }, { "first": "Allan D", "last": "Novetsky" }, { "first": "Jen C", "last": "Wang" } ] }
PMC549048	Background ========== There is broad agreement between existing guidelines for asthma \[[@B1]-[@B4]\] regarding diagnostic procedures, patient education and medical treatment \[[@B5]\]. The statements for chronic obstructive pulmonary disease (COPD) \[[@B6]-[@B8]\] are also consistent, despite the lack of evidence for some recommendations \[[@B9]\]. However, it has been shown previously that the management of these diseases in practice was not fully consistent with the guidelines \[[@B10]-[@B12]\], which may be related to the variety of views on the treatment of these conditions \[[@B13],[@B14]\]. Furthermore, the knowledge about the diseases is changing rapidly and the management has become more complex \[[@B15],[@B16]\]. Many patients with asthma or COPD are managed in primary care and it is important to optimise the treatment given the prevalence and impact \[[@B17]\]. However, it remains difficult to draw a clear picture of management of obstructive lung diseases in primary care since the diagnoses of asthma and COPD often have not been separated in many surveys \[[@B7]\]. This could be due to some diagnostic and therapeutic overlap between these both diseases. Especially in primary care it is difficult to distinguish both entities when the symptoms are presented in an early stage \[[@B18]\]. In particular the treatment response in this stage often seem to be not strongly related to the stated diagnoses \[[@B19],[@B20]\], and also the treatment response of COPD in the long term course is disappointing \[[@B21]\]. Furthermore, systematic registration of diseases in primary care are often hampered by missing of recorded diagnoses \[[@B22]\] or discrepancies between diagnoses and prescribed medications \[[@B23]\]. A specific problem for health services research and performance assessment in Germany might be that diagnoses saved in medical records are used for reimbursement. For that reason it is unclear to what respect ICD-10 codes reflect the real problems of patients in general practice. It may be possible that physicians enter diagnoses in their records to get reimbursement, but use other information such as results of diagnostic tests for treatment. Empirical evidence on this phenomenon is limited, but its existence would complicate the assessment of practice patterns. The aim of this study was to assess the appropriateness of the recorded diagnoses and to determine what diagnostic information is used to guide medical treatment. For this purpose we analysed associations between recorded diagnoses, diagnostic procedures and medical treatment regarding asthma and COPD in general practice. Methods ======= Study design ------------ A cross-sectional observational study was performed in six computerised general practices with a total of eleven GPs in the Rhine-Neckar region in Southern Germany. Setting and study subjects -------------------------- All patients who had attended their physicians with asthma, COPD or other lower airway diseases from January until June 2003 were included in the study. Ethical approval ---------------- The study was approved by the Medical Ethics Committee of the University of Heidelberg. Procedure for selection of subjects ----------------------------------- Patients were identified by searching the electronic files for documented diagnoses concerning lower airway diseases within the time frame. The data were evaluated systematically according to the international classification of diseases (ICD-10). In detail, the practice software was checked for the ICD-10-codes of the chapter \"chronic lower airway disease\": J40 (bronchitis, not specified as acute or chronic), J41 (simple and mucopurulent chronic bronchitis), J42 (unspecified chronic bronchitis), J43 (emphysema), J44 (chronic obstructive pulmonary disease), J45 (asthma), J46 (status asthmaticus), J47 (bronchiectasis). Additionally, the ICD-10-codes J98 (diseases of bronchus, not elsewhere classified) and R05 (cough / bronchial hyperreactivity), and the repeated documentation of ICD-10-codes belonging to the chapter \"acute lower airway disease\" were extracted in order to detect potentially existing but not detected asthma or COPD. The chapter \"acute lower airway disease\" includes the diagnoses J20 (acute bronchitis), J21 (acute bronchiolitis) and J22 (unspecified acute lower respiratory infection). The performed diagnostic procedures and the actual medication for each identified patient were extracted and documented manually by two independent scientific fellows (L.G., I.M.) of the department. The variables of the used protocol for documentation are listed in table [2](#T2){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Baseline characteristics ::: ---------------------------------------------------------------------------------------------------------------------------- Overall (n = 857) Asthma (n = 255) COPD (n = 112) Asthma + COPD (n = 25) Other (n = 465) ----- ----------------------- ---------------------- -------------------- ---------------------------- --------------------- age 41.84 ± 22.18\ 36.48 + 21.00\ 56.28 + 18.38\ 46.23 + 19.16\ 41.06 + 22.32\ (min 1; max 97) (min 1; max 88) (min 21; max 92) (min 26; max 71) (min 1; max 97) sex m 385; f 472 m 121; f 134 m 55; f 57 m 15; f 10 m 194; f 271 ---------------------------------------------------------------------------------------------------------------------------- ::: Analysis -------- Every ICD10-code specified above was extracted and written down, at maximum up to three ICD-10-Codes per patient were found. The diagnoses were clustered into three groups: asthma, COPD, and other lower airway diseases. If more than one diagnosis was documented, asthma (J45) or COPD (J44) were extracted as the leading diagnosis. The ICD-10 group J43 (emphysema) was included into the ICD-10 group J 44 (COPD). The ICD-10 groups other than J45 (asthma), J44 or J43 were merged into the group \"other lower airway diseases\". The performed diagnostic procedures were extracted manually. Four ways of making diagnoses have been found in the records: 1. taking medical history only, 2. taking medical history plus trial of medication, 3. taking medical history and performing spirometry, 4. taking medical history plus single measurement of PEF. The additional performance of bronchial challenge testing and chest X-ray was documented, too. Baseline data were evaluated descriptively. The t-test was used to detect differences of age. Linear logistic regression was used to test relationships between diagnosing asthma or COPD and the performed diagnostics. The relation between prescribing patterns and performed diagnostics was also examined with logistic regression analysis. Results ======= Overall, 8765 patients attended the practices within the time frame. 857 (9,8%) patients had lower airways diseases (385 male, 472 female). A total of 255 (2.9% of 8765) had the recorded diagnosis of asthma and 112 (1.3% of 8765) had COPD (107 with J44 = COPD, 5 with J43 = emphysema). A combination of asthma and COPD was documented in 25 (0.2% of 8765) cases. The 465 (5.3% of 8765) patients with \"other lower airway diseases\" included seven cases with acute bronchitis (J20), none with acute bronchiolitis (J21), one case with unspecified acute lower respiratory infection (J22), 251 (2.9% of 8765) cases with not specified bronchitis (J40), 38 with simple and mucopurulent chronic bronchitis (J41), 57 with unspecific chronic bronchitis (J42), one with bronchiectasis (J47), seven with diseases of bronchus, not classified (J98), 103 (1.2% of 8765) with cough / bronchial hyperreactivity (R05), Patients with COPD were significantly older than those with asthma or other lower airway diseases. There was no case of twofold documentation of \"acute lower airway disease\" (J20 - J22) within the time frame. The details regarding the patient population are described in table [1](#T1){ref-type="table"}. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Performed diagnostics and documented diagnoses ::: Asthma (n = 255) COPD (n = 112) Other (n = 465) ----------------------------------------- ---------------------- -------------------- --------------------- ------------- ------- ------- ---------- ------------- ------- ------- ---------- ------------- Performed diagnostics n % OR 95% CI n % OR 95% CI n % OR 95% CI Medical history only 42 16.5 0.11 0.07--0.15 33 29.3 0.31 0.20--0.46 338 72.7 10.9 7.89--15.02 Medical history and trial of medication 57 22.4 8.11 4.68--14.08 6 5.4 0.51 0.23--1.13 12 2.6 0.14 0.07--0.26 Medical history and spirometry 77 30.2 1.30 0.99--1.72 65 58.1 4.38 2.99--6.40 90 19.4 0.37 0.27--0.50 Medical history and PEF-measurement 79 31.0 7.57 4.93--11.60 8 7.1 0.81 0.47--1.40 25 5.4 0.18 0.11--0.28 Additional bronchial challenge test 18 3.9 1.35 0.73--2.48 24 21.4 6.74 3.66--12.42 1 0 0.16 0.08--0.35 Additional chest X-ray 34 13.3 0.88 0.58--1.31 42 37.5 4.06 2.67--6.19 48 10.3 0.44 0.30--0.65 ::: Diagnostics ----------- Table [2](#T2){ref-type="table"} shows the performed diagnostic procedures. Spirometry was performed in 30% of the patients with recorded asthma. A high amount was diagnosed with \'medical history and single measurement of peak expiratory flow\' (31%), and 22% of cases fell into the category of medical history taking plus trial of medication (= diagnosing by therapy). This implies that the physician gives sympathomimetics in suspected asthma. If the patient feels better, the diagnosis \'asthma\' is made. In about 17% the diagnosis asthma was made only on the basis of medical history Spirometry was performed in 58% of the cases with recorded COPD. In 29% the diagnosis was made only by medical history. A small percentage (7%) was diagnosed by history taking and PEF-measurement and 5% by diagnosing by therapy. Concerning the other lower airway diseases, spirometry was done in 19% in order to exclude asthma or COPD. These diagnoses were made mainly on the basis of medical history (73%). The associations between recorded diagnoses and diagnostic procedures are calculated by binary logistic regression (also in table [2](#T2){ref-type="table"}). The higher the odds ratio (OR) the higher is the positive association between the variables. Patients with the combined diagnosis of asthma and COPD were not included into the analysis. An odds ratio smaller than one means that there is a negative association between those variables. Significant results are bold. The logistic regression showed that the diagnosis asthma was often based only on single measurement of PEF (OR 7.6) or \'trial of medication\' (OR 8.1). The use of spirometry was not significantly associated with the recorded diagnosis asthma. Concerning the recorded diagnosis of COPD, \'trial of medication\' was not associated significantly. Instead, the recorded diagnosis was based on spirometry (OR 4.4). Bronchial challenge testing was used to exclude asthma (OR 6.7). Also chest X-ray showed a significant association with the recorded diagnosis of COPD (OR 4.1). The recording of other lower airway diseases was mainly based on the medical history (OR 10.9), the other diagnostic procedures are used only in few cases (OR \< 1). Prescriptions ------------- The documentation of the prescribed medication showed that 66% of patients with asthma and 42% with COPD received sympathomimetics (table [3](#T3){ref-type="table"}). 38% of the asthma patients received inhaled corticosteroids, whereas 46% of the patients with COPD were treated with inhaled corticosteroids. Cromoglycate was given in 36% of patients with asthma and 14% of COPD. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Relationship between performed diagnostis. actual treatment and documented diagnoses ::: Performed diagnostics and actual treatment Asthma (n = 255) COPD (n = 112) Other (n = 465) ------------------------------------------------ ---------------------- --------------------- --------------------- ------------- ------- ------- ---------- ----------------- ------- ------- ----------- ------------- n % OR 95% CI n % OR 95% CI n % OR 95% CI Sympathomimetics (n = 167; 65.5%) (n = 47; 42.0%) (n = 36; 7.7%) Medical history only 9 3.5 0.22 0.10--0.51 7 6.3 1.35 0.56--3.26 10 2.2 4.97 2.05--12.02 Medical history and trial of medication 52 20.4 7.99 3.06--20.85 4 3.6 2.93 0.51--16.71 8 1.7 31.48 8.95--111.2 Medical history and spirometry 60 23.5 2.28 1.24--4.18 36 32.1 4.06 1.77--9.35 14 3.0 3.10 1.51--6.36 Medical history and PEF-measurement 46 18.0 0.67 0.43--1.07 0 0 0 0-0 4 1.0 2.39 0.77--7.36 Additional bronchial challenge test 12 4.7 3.51 0.77--16.04 9 8.0 1.16 0.44--3.08 0 0 0.01 0--10^9^ Additional chest X-ray 22 8.6 1.02 0.48--2.17 22 19.6 1.98 0.91--4.31 6 1.0 1.91 0.75--4.86 Corticosteroids (n = 96; 37.6%) (n = 52; 46.4%) (n = 28; 6.0%) Medical history only 4 1.6 0.23 0.08--0.66 9 8.0 2.22 0.85--5.78 5 1.0 2.37 0.78--7.23 Medical history and trial of medication 20 7.8 0.91 0.49--1.68 3 2.7 1.16 0.22--6.03 5 1.05 13.99 4.11--47.64 Medical history and spirometry 49 19.2 5.17 2.91--9.19 40 35.7 4.67 2.05--10.64 15 3.2 6.05 2.72--13.45 Medical history and PEF-measurement 23 9.0 0.58 0.33--1.02 0 0 0 0--14 × 10^4^ 3 1.0 2.26 0.63--8.04 Additional bronchial challenge test 9 3.5 3.30 1.07--10.17 16 14.3 6.22 1.93--20.11 0 0 0.00 0-0 Additional chest X-ray 16 6.3 1.63 0.79--3.37 29 25.9 4.56 2.00--10.38 10 2.2 6.19 2.65--14.47 Cromoglycate (n = 91; 35.7%) (n = 16; 14.3%) (n = 39; 8.4%) Medical history only 9 3.5 0.16 0.07--0.37 1 1.0 0.23 0.05--1.02 21 4.5 12.02 4.93--29.29 Medical history and trial of medication 24 9.4 1.52 0.83--2.79 0 0 0.00 0--1.6 × 10^18^ 4 1.0 6.16 1.77--21.51 Medical history and spirometry 23 9.0 0.74 0.42--1.32 10 8.9 1.07 0.40--2.86 10 2.2 1.70 0.89--3.25 Medical history and PEF-measurement 35 13.7 1.74 1.01--3.00 5 4.5 14.1 2.96--67.18 4 1.0 2.17 0.71--6.65 Additional bronchial challenge test 5 2.0 1.06 0.34--3.26 4 3.6 1.67 0.48--5.83 1 0 1.62 0.19--13.54 Additional chest X-ray 9 3.5 0.65 0.29--1.46 5 4.5 0.73 0.23--2.25 8 1.7 2.58 1.11--6.01 ::: Only a small amount of patients with \'other lower airway diseases\' received anti-obstructive medicine (6--8%). The logistic regression showed that sympathomimetics were given in asthma when having shown efficacy (trial of medication) (OR 8.0) or when spirometry was performed (OR 2.3). Therapy with corticosteroids was significantly associated with spirometry (OR 5.2) and bronchial challenge testing (OR 3.3). Treatment of COPD with sympathomimetics was significantly associated with the use of spirometry (OR 4.1). Inhaled corticosteroids were also given more often when spirometry (OR 4.7) and Chest X-ray (OR 4.6) had been performed. Bronchial challenge testing was used to exclude asthma (OR 6.2). In asthma as well as in COPD corticosteroids are not prescribed only by trial of medication and are not given after making the diagnosis only by medical history. Prescribing of cromoglycate was associated with PEF measurement in both asthma and COPD (OR 1.7 respectively 14.1) Treatment of the other lower airway diseases with sympathomimetics respectively corticosteroids was mainly associated with testing the efficacy of therapy (OR 31.5 respectively 14.0). The prescription is lower associated with spirometry (OR 3.1 respectively 6.1) or x-ray (OR 1.9 respectively 6.2). Cromoglycates were given mostly on the basis of \'taking medical history only\' (OR 12.0) and trial of medication (OR 6.2). Discussion ========== Although guidelines recommend the use of spirometry, this was used in only in 30% of the patients with a recorded diagnosis of asthma and in 58% of the patients with a recorded diagnosis of COPD. A high number of recorded diagnoses of asthma and COPD were insufficiently diagnosed by PEF-measurement, medical history and diagnosing by therapy. On the first sight, patients with asthma seem to be treated insufficiently with anti-inflammatory therapy and patients with COPD seemed to be over-treated with steroids. However, further analysis showed a high association between prescribed corticosteroids and sympathomimetics as long term medication and performed diagnostic procedures. This improvement of diagnostic investigation when potential harmful drugs are given demonstrates that treatment of asthma and COPD by the GPs may be more consistent with guidelines than other studies have found \[[@B10],[@B13]\]. This hypothesis is facilitated by the fact, that cromoglycates seem to be administered if there was diagnostic uncertainty, as there was no significant association between performing spirometry and making diagnoses in those cases. According to this, our study shows that a deeper look insight with analysis of diagnoses together with performed diagnostics and medication could prove a better care for people with obstructive airway diseases than a single analysis of the documented diagnoses. As a conclusion, ICD-10 codes seem not to be able to reflect the work in primary care, in particular if the difficulties of the diagnostic and therapeutic differentiation between asthma and COPD in earlier stages are taken into account \[[@B18],[@B20]\]. Therefore it seems that the documented diagnosis due to ICD-10 often serves as a \'working hypothesis\'. The needs of family physicians for practice and research would be best met by the International Classification of Primary Care (ICPC-2) \[[@B24]\], but the general practitioners in Germany are politically obliged to record ICD-10 diagnoses to get reimbursement. As the documentation of the reason of encounter is restricted in these terms of ICD-codes there could be some necessity to assign diagnoses although they do not match with the real status of the patient. This makes the recorded diagnoses unreliable, sometimes resulting in apparently poor coherence between medication and diagnoses, and this leads to difficulties in drawing valid conclusions on the quality of primary care. Another critical point is, that the \'trial of medication\' as a \'fact of life in primary care\' could implicate a patients\' perception of improvement which could lead the doctor to perpetuate the therapy, thus resulting in over-prescribing accompanied by an avoidable risk of side-effects. Especially this \'trial of medication\' may reflect diagnostic uncertainty in general practice, which must normally have consequences for the classification and labelling. In absence of a reasonable classification system the GPs are forced to hide this uncertainty by documenting diagnoses in ICD-10-codes. As a result, GPs could feel the uncertainty nevertheless, but start seeing it as a personal shortcoming rather than a reality of medicine. Therefore this exaggeration of \'labelling with ICD-10\' could also constrain a systematic quality improvement in diagnostic performance in primary care. The strength of the study was the detailed analysis with manual extraction and documentation of performed diagnostics and actual medication of each identified patient. As the ICD-10 classification made it difficult to identify patients with obstructive airway diseases in junction with performed diagnostics and therapy we searched all records manually. Because of this complexity the study had the limitation that the number of GPs was small, which reduces the generalisability of the descriptive findings, although the estimates of associations are less sensitive for this problem. The prevalence of asthma and COPD in this German sample of general practice seems to be low compared to the prevalence of about 5% in studies of other countries \[[@B25]-[@B27]\]. One explanation might be the high doctor-patient contact rate in Germany that decreases the prevalence of severe diseases in general practice. Conclusions =========== The conclusion of this study is that performance assessment should take the reliability problems of recorded diagnoses into account, especially if the ICD-10 classification is used. A more detailed analysis may be necessary for adequate evaluation of primary care, consequential pointing out to the fact, that treatment of obstructive airways diseases by the GP\'s may be more consistent with guidelines than other studies have found. Despite these findings improvement of diagnostic performance in general practice is necessary, because a routine use of spirometry is of growing importance against the background of the increasing prevalence of obstructive airway diseases. Competing interests =================== The author(s) declare that they have no competing interests. Authors contributions ===================== AS conceived and designed the study, wrote the report. LG and IM checked the practice software and documented data. MB helped with interpreting the data and writing the report. MW helped with interpreting the data and writing the report. JS helped with revising the report. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6963/5/11/prepub> Acknowledgements ================ We are grateful to the general practitioners, namely Jörg Barlet, Stefan Bilger, Vera Datzer, Richard Michel, Berthold Musselmann and Andreas Wichmann for collaborating in this study. And we are indebted to the reviewer for his very helpful comments to improve this manuscript, thus leading to a higher precision in presentation and discussion of our findings.	PubMed Central	2024-06-05T03:55:52.915951	2005-2-1	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549048/", "journal": "BMC Health Serv Res. 2005 Feb 1; 5:11", "authors": [ { "first": "Antonius", "last": "Schneider" }, { "first": "Lutz", "last": "Gantner" }, { "first": "Inko", "last": "Maag" }, { "first": "Mathias M", "last": "Borst" }, { "first": "Michel", "last": "Wensing" }, { "first": "Joachim", "last": "Szecsenyi" } ] }
PMC549049	If only it were all so simple. If only there were evil people somewhere insidiously committing evil deeds, and it were necessary only to separate them from the rest of us and destroy them. But the line dividing good and evil cuts through the heart of every human being, and who is willing to destroy his own heart?Alexander Solzhenitsyn, The Gulag Archipelago The notion that the immune system may be manipulated into recognizing and eradicating neoplasia is not new. Heroic efforts to develop a cancer vaccine can be traced as far back as 1777 when the surgeon to the Duke of Kent injected himself with malignant tissue as a prophylaxis against development of cancer. In 1808, another attempt was made to develop a cancer vaccine by the doctor to Louis XVII who inoculated himself with breast cancer in hope of reversing a soft-tissue sarcoma, although no therapeutic effect was observed. However, it was not until 1891 that the first report of successful immunotherapy was published by William Coley, a clinician at the Memorial Sloan Kettering Cancer Institute in New York. Using heat-killed endotoxin-containing bacteria (streptococci and Serratia marcescens), Coley was able to achieve a cure rate of 10% in soft-tissue sarcoma \[[@B1],[@B2]\]. Nevertheless, despite the numerous attempts over the past centuries to use the immune system in the eradication of cancer, the success rate of cellular immunotherapy remains abysmally low. In light of the successes in the development of vaccines targeting pathogenic agents, this review suggests that lessons learned from the immunology of infectious disease may be applicable to the treatment of neoplasia. The immunology of infectious disease teaches that the clearance of a pathogenic challenge requires the initiation of an immune response of the appropriate quality and quantity. Clinical experience demonstrates the perils of an inappropriate immune reaction. Pathogens may be of an intracellular (viral, some bacterial strains) or extracellular (bacterial, parasitic) nature; accordingly, the specific immune response is bifurcated into a cell-mediated branch, which offers protection against intracellular pathogens, and an antibody-mediated branch, which offers protection against extracellular pathogens. Following a challenge with Mycobacterium leprae, the leprosy inducing bacterium, a cell-mediated immune response will result in protection whereas an antibody-mediated immune response will result in cachexia and disease progression \[[@B3]\]. The ability of the immune system to act as a double-edged sword implies that in any given condition the initiation of an immune response may result in either protection or destruction of healthy tissue. An appreciation of the duality of the immune reaction is imperative in the design of immunotherapeutic approaches that attempt to attain a therapeutic benefit through the manipulation of the immune system. Two distinct theories aim to define the role of the immune system in cancer. The theory of immunosurveillance postulates that the role of the immune response in cancer is one of protection -- the organism is patrolled for incipient tumor cells by the effector cells of the immune system. In contrast, the theory of immunostimulation postulates that, while in experimental systems highly immunogenic tumors may be eradicated by the immune response, the role of the immune response in spontaneous neoplasia is not one of protection, but rather of tumor promotion. This review gives a historical overview of these theories and highlights recent data supporting the validity of both immunosurveillance and immunostimulation. To explain the conflicting roles of the immune response in neoplasia it must be noted that the immune system is not a single entity, but a complex system of constituents. While these theories have historically been considered to be mutually exclusive, it is proposed that these theories describe the activation of different constituents of the immune system and hence illustrate that an appropriate immune reaction will result in protection whereas an inappropriate immune reaction will result in tumor promotion. The theory of immunosurveillance ================================ The rise of the theory of immunosurveillance -------------------------------------------- As early as 1909 Paul Ehrlich postulated that cancer occurs spontaneously in vivoand that the immune system is able to both recognize and protect against it \[[@B4]\]. In the late 1950s Lewis Thomas \[[@B5]\] introduced the theory of immunosurveillance, which was subsequently developed by Sir MacFarlane Burnet \[[@B6]\]. The theory postulates that effector cells of the immune system actively patrol the body to identify and eradicate incipient tumor cells. Following the identification of T cells in the 1970s, these became the effector cells postulated to mediate immunosurveillance. The concept of immunosurveillance initially had much intellectual appeal. First, it could explain clinical observations of spontaneous remission. Second, the most potent chemical carcinogens, dimethlybenzanthracene \[[@B7]\], urethan \[[@B8]\] and other polycyclic hydrocarbons \[[@B9]\], are also powerful immunosuppressors, their suppressive effects being apparent even after a single exposure \[[@B7]\]. Third, specific immune responses had been observed in the transplantation of chemically induced tumors. Finally, the theory of immunosurveillance was developed at a time when the only known function of T cells was to reject foreign grafts; thus, there was a clear need to further determine the biological function of these cells. Discrepancies in the theory of immunosurveillance ------------------------------------------------- A number of discrepancies were noted in the theory of immunosurveillance as initially described by Thomas and Burnet, where T-cells function as the effector cells mediating immunosurveillance. First, T-cell deficient (athymic nude) mice did not develop significantly more cancers than control mice \[[@B10]\] Second, a corollary of the theory of immunosurveillance is that patients who lack an immune response would have an increased incidence of neoplasia. While this has been reported in immunosuppressed patients who have undergone renal transplant, most of these tumors (as many as 60%) were reported to be hematological malignancies or neoplasms with viral etiology. It would be reasonable to expect the immune system to protect against viral carcinogenesis, as the protective role of the immune system against viral pathogens is well established. One interpretation of these results is that the predisposition toward hematological malignancy is due to the viral etiology of certain lymphomas (which are thought to be caused by the Epstein-Barr virus). The only non-lymphoid tumors that increase significantly upon immunosuppression are non-melanoma skin cancers, cervical carcinoma and Kaposi\'s sarcoma (the latter two having a viral etiology, specifically the human papilloma virus and human herpes virus 8, respectively). In these early studies, the incidence of most tumor types was not increased by immunosuppression \[[@B11]\]. In fact, the incidence of mammary carcinomas actually decreases in immunosuppressed individuals \[[@B12]\]. A third discrepancy is that diseases such as leprosy, sarcoidosis and uremia, which are characterized by immunosuppression, are not accompanied by an increased incidence of tumors \[[@B13],[@B14]\]. Finally, the theory of immunosurveillance predicts that sites that are excluded from the immune system -- that is, sites of immunological privilege such as the anterior chamber of the eye and the brain -- should have an increased incidence of cancer. However, this is not the case. Furthermore, several studies suggest that incipient tumors may not be immunogenic. A correlation between the dose of carcinogen administrated and the immunogenicity of the subsequent tumor has been reported in mouse models \[[@B15]-[@B17]\]. Since human tumors most likely occur as a result of low exposure to carcinogens, incipient tumors would be expected to be poorly immunogenic. In addition, since incipient tumors would contain few cells, the question arises of whether the antigenic load would be sufficient to induce a response \[[@B18]\]. This phenomenon may be observed in the immunology of infectious diseases where naïve C57Bl/6 mice challenged with Taenia taeniaeformiswill reject a large inoculum of the cestode larva yet will succumb to infection by minute inoculations \[[@B19]\]. Finally, it is only in recently years that the tumor specific- and not tumor associated antigens have been identified \[[@B20]\]. The revivification of immunosurveillance ---------------------------------------- Over the past two decades, data have emerged suggesting that constituents of the immune system such as natural killer (NK) cells and cytokine networks may be able to guard against cancer. Furthermore, recent studies of the incidence of neoplasia in immunosuppressed patients after organ transplantation, in contrast to earlier studies, reported that these patients are more susceptible to a wide range of cancers, including epithelial cancers \[[@B21],[@B22]\]. ### Natural killer cells Several studies suggest that NK cells are able to protect against tumors. Beige, natural-killer-cell defective, mice have an increased incidence of spontaneous tumors \[[@B23]\] and cancer metastases \[[@B24]-[@B28]\]. This is consistent with the pattern seen in patients with Chediak Higashi syndrome, an autosomal recessive disorder characterised by abnormal NK cytotoxic function. These patients also have a 200-fold increased risk of developing malignancy \[[@B29]\]. Furthermore, several studies show that cancer cells secrete soluble factors that suppress NK cells in vitro\[[@B30]-[@B32]\] and that patients with a variety of tumor types have suppressed NK cell activity \[[@B33]-[@B38]\]. NK cell activity is also a positive prognostic indicator in several tumor types \[[@B39]-[@B43]\]. These data suggest that NK cells are involved, directly or indirectly, in the surveillance of incipient tumors and micrometastases. This theory is further substantiated by data showing that oncogene-transfected fibroblasts can be selectively lysed by NK cells while sparing untransfected controls \[[@B44]\]. The precise mechanism though which NK cells mediated immunosurveillance is not yet understood. It is likely that the role of NK cells, in addition to their direct cytotoxic effects, is to activate other cells of the immune system through providing cytokine support \[[@B45]\]. Mature NK cells do not produce T-helper 2 (Th2) cytokines, but rather the T-helper1 (Th1) cytokines tumor necrosis factor (TNF)-α, interferon (IFN)-γ and GM-CSF \[[@B46]\]. In fact, secretion of IFN-γ by NK cells can influence the development of a Th1 type immune response both against pathogenic agents and against MCA-induced tumors \[[@B47]\]. ### Cytokine networks The initiation of an immune response requires cytokine support provided by T-helper cells. The nuance of cytokines produced by these cells determines the type of immune response initiated. The initiation of a cell-mediated immune response requires the support of Th1 cytokines, while the initiation of an antibody-mediated immune response requires the support of Th2 cytokines. T-helper cells that have differentiated into Th1 cells secrete IFN-γ and to a lesser extent interleukin (IL)-2 and IL-12, whereas Th2 cells secrete IL-10, IL-4 and to a lesser extent IL-5. The cell-mediated immune response is generally regarded as possessing tumor-inhibitory activities both clinically and in animal models \[[@B48]-[@B51]\]. Accordingly, a number of studies suggest that the expression of Th1 cytokines is associated with a favorable clinical outcome, while the expression of Th2 cytokines is associated with an unfavorable clinical outcome. In renal-cell carcinoma, the presence of an IL-4 receptor polymorphism that resulted in an increase in IL-4 signaling and an increased likelihood of a Th2 response was an independent indicator of adverse prognosis \[[@B52]\]. In other studies of patients with renal cancer, an elevated level of IL-10 was an adverse prognostic indicator \[[@B53]\], while the serum levels of IFN-γ were negatively correlated with tumor mass \[[@B54]\]. In malignant melanoma, patients that had an early relapse had lower serum levels of IL-2 and IL-12. Furthermore, decreases in the serum concentrations of IL-2 and IL-12 and increases in IL-10 were observed at least one month before relapse \[[@B55]\]. In B-cell diffuse large cell lymphoma, patients who achieved complete remission had a higher ratio of Th1 to Th2 cells \[[@B56]\]. Finally, in a range of advanced solid cancers, the serum IL-10 level was an independent prognostic indicator of overall survival and time to treatment failure \[[@B57]\]. These data suggest that the Th1-Th2 paradigm is relevant to cancer, although it should be noted that the Th1/Th2 paradigm is a generalization and that these data show a correlation, and as such are not evidence of a causal relationship. Since many tumors secrete IL-10, it is possible, for example, that the relationship between expression of IL-10 and tumor prognosis reflects the poor prognosis of patients with a larger tumor burden. This is plausible given that the role of IL-10 in tumor immunology is complex (reviewed in \[[@B58]\]). IL-10 appears to be a pleotropic cytokine whose reported effect is a function of the nuances of the experimental system. While several reports suggest that IL-10 is immunosuppressive, several groups cite IL-10 to have stimulatory effects on T cells and NK cells \[[@B59]\]. In addition, IL-10 transfected into murine carcinoma models decreases tumorigenicity and sensitizes tumor cells to immune mediated lysis by either NK cells or NK cells and cytotoxic T-cells \[[@B60]-[@B62]\]. Clinical data further corroborates a protective role for IL-10 in tumor immunology by showing that metastatic melanoma patients who responded to an immunotherapeutic regimen had tumors with significantly higher expression of IL-10 mRNA as compared to patients that did not respond \[[@B63],[@B64]\]. Furthermore, tumors have a number of specific and non-specific ways of evading a Th1 response. Tumors secrete a number of agents, including transforming growth factor (TGF)-β, IL-10 and prostaglandin E-2, which have been shown to promote a Th2 immune response while suppressing the Th1 immune response. Indeed, it has been shown that the cytokine networks of some cancer patients are skewed toward Th2 \[[@B55],[@B65],[@B66]\]. That is, these patients exhibit enhanced expression of Th2 cytokines or decreased expression of Th1 cytokines systemically or in the local tumor microenvironment. The observation that tumors have developed numerous methods of evading the Th-1 response is consistent with the notion of immunosurveillance. Nowell\'s clonal-evolution hypothesis postulates that cancer is a Darwinian process: mutations that provide a growth or survival advantage will be selected for in the population \[[@B67]\]. The fact that malignancies have many ways of evading the Th-1 response suggests that the ability to evade this response confers a survival advantage on malignant cells, which further suggests that the Th-1 response poses a threat to the neoplasm. ### Interferon-γ The Th-1 cytokine IFN-γ has both direct and indirect antitumor properties. A series of experiments have demonstrated the importance of IFN-γ in eradicating incipient tumors by showing an increase in the efficacy of carcinogenesis in the absence of IFN-γ. These experiments were conducted either with neutralizing antibodies to IFN-γ or in animal models deficient in IFN-γ, the IFN-γ receptor or downstream signaling mechanisms of IFN γ, specifically, the signal transducer and activator of transcription-1 protein. In all of these models, an increase in the incidence of MCA-induced tumors and a decrease in the latency period of the tumors were observed \[[@B68]-[@B71]\]. Furthermore, an increase in the number of spontaneous lymphomas and lung tumors was observed in IFN-γ deficient mice compared with genetically matched wild-type controls \[[@B72]\]. IFN-γ may inhibit tumor growth by affecting proliferation, apoptosis and angiogenesis. IFN-γ has been shown to have a direct anti-proliferative effect on certain tumor models \[[@B73]-[@B75]\]. It is thought that this effect is mediated through p21 (WAF1/CIP1) and p27 (kip1) as IFN-γ activates these tumor suppressors \[[@B76],[@B77]\]. Furthermore, IFN-γ has been reported to modulate apoptosis in certain models \[[@B78],[@B79]\] by inducing expression of caspase 1 and Fas/FasL \[[@B80],[@B81]\]. With respect to angiogenesis, IFN-γ induces expression of three angiostatic non-ELR (non-Glu-Leu-Arg) chemokines: interferon-gamma-inducible protein 10 (IP-10), monokine induced by gamma interferon and interferon-inducible T-cell alpha chemoattractant \[[@B82]-[@B84]\]. The effects of these chemokines on angiogenesis will be discussed. IFN-γ may also have indirect antitumor effects by stimulating an effective antitumor immune response. In addition to influencing the Th1-Th2 cytokine balance, IFN-γ can activate cytotoxic macrophages, NK cells and NK T cells \[[@B85]\]. The theory of immunostimulation =============================== The proof of the principle that an inappropriate type of immune response will enhance tumor growth was demonstrated as early as 1907 by Flexner and Jobling, who showed that injection of dead autologous tumor cells enhanced the growth of pre-existing tumors \[[@B86]\]. In general, Th2-driven antibody responses to tumors are non-protective and may contribute to tumor progression by inhibiting the Th1 cell-mediated immune response \[[@B87]\]. However, the notion that the antibody-mediated immune response may be detrimental in cancer was suggested long before Mossman and Coffman demonstrated the Th1-Th2 paradigm in 1986 \[[@B88]\]. In the 1950s Kaliss popularized the term \"immunological enhancement\" to describe the enhancement of tumor growth by non-cytotoxic antibodies \[[@B89]\]. It was theorized that these antibodies bind to tumor cells, masking their epitopes and thus preventing a cell-mediated immune response, although this has never been demonstrated experimentally. In 1972, Richmond Prehn formulated the theory of immunostimulation of tumor growth \[[@B90]\]. This theory states that, in contrast to the strong immune response generated by transplantable tumors, a quantitatively mild immune response, such as that generated by spontaneous tumors, is stimulatory to the growth of neoplasia. Several experimental observations support the hypothesis that such a weak immune response to cancer may stimulate tumor growth. The co-injection of lymphocytes (spleen cells) from syngeneic mice that had been growing tumors for 10--20 days with tumor cells from MCA-induced sarcomas into thymectomized irradiated syngeneic mice at a range of doses accelerated tumor growth when the ratio of lymphocytes to tumor cells was low \[[@B90]\]. However, when the ratio of lymphocytes to tumor cells was high, lymphocytes from specifically immunized mice inhibited growth compared with naïve lymphocytes that continued to augment tumor growth. This suggests the existence of a biphasic dose response whereas a \"weak\" immune response results in stimulation of tumor growth while a strong immune response results in protection. This premise is also demonstrated in another study where MCA induction of tumors occurred more rapidly in irradiated thymectomized mice that had received increasing doses of lymphocytes (spleen cells) to partially restore their immune system than in mice that had either fully restored or unrestored immune systems \[[@B91]\]. Comparable results have been observed in mice that have had their immune systems compromised to various degrees by irradiation \[[@B92]\] and in T-cell deficient nude mice that have had their immune systems partially restored by the injection of various quantities of thymic or spleen cells \[[@B93]\]. These studies support the notion that a weak antitumor immune response will not confer protection and may exacerbate tumor growth. Furthermore, in transplantation immunology, an immune response against a graft generally promotes graft rejection. However, rabbits that received skin grafts of similar but not identical major histocompatibility complex launched an immune response that resulted not in rejection but in enhanced growth of the tissue, resulting in hyperplasia \[[@B94]\]. This is consistent with the notion that a quantitatively \"weak\" immune response against a tumor may enhance tumor growth. Collectively, these data support the essence of the theory of immunostimulation: specifically, that spontaneous tumors may not stimulate an appropriate immune response but rather stimulate non-protective immune responses that quantitatively are not adequate for tumor eradication. These resulting \"weak\" immune responses are not merely non-protective but actually facilitate tumor growth. Specifically, a \"weak\" immune response is a state in which immunological recognition of the tumor occurs but resolution is not achieved. As this theory was developed in an era when our understanding of the composition of the immune system was limited, the exact mechanism by which a \"weak\" immune response stimulates tumor growth has yet to be defined in the terminology of contemporary immunology. Recent data, however, suggest numerous mechanisms by which the immune system can facilitate tumor growth and progression. Inappropriate immune reactions exacerbate cancer ------------------------------------------------ Based on the observation that a tumor is in a state of chronic inflammation, Dvorak compared cancer to a wound that never heals \[[@B95]\]. Numerous tumor types constitutively produce cytokines and chemokines; this results in the migration of leukocytes into tumors. Unfortunately, these immune infiltrates often do not offer protection but actually facilitate tumor growth. Tumor-infiltrating leukocytes can facilitate tumor progression by secreting growth factors, reactive oxygen and nitrogen species, proteases, prostaglandins and angiogenic growth factors. Reactive oxygen and nitrogen species may directly promote tumor progression by inducing DNA damage, and hence the acquisition of additional mutations. In addition, these cells may skew the cytokine milieu to favor the generation of a non-protective Th2 immune response. Mast cells and macrophages are examples of inflammatory cells that are often recruited to tumor sites through chemotactic gradients. The mechanisms by which mast cells, macrophages and chemokines themselves facilitate tumor growth and progression are discussed below. ### Mast cells Mast cells are leukocytes that contain inflammatory agents, such as proteases, histamine and heparin, and mediate hypersensitivity reactions. The possibility that mast cells are involved in cancer has been considered since the late 1800s. In 1877, Paul Ehrlich described a mast cell for the first time in his doctoral thesis, after identifying it using histological staining. In subsequent investigations, he and others observed that mast cells localize around tumor tissues \[[@B96]\]. Interestingly, mast cells were more likely to be localized in the periphery than in the centre of tumor sections. Many types of human tumors contain mast-cell infiltrates \[[@B97]-[@B100]\]. The accumulation of mast cells in neoplastic tissue results from the secretion of growth factors such as vascular endothelial growth factor (VEGF) \[[@B101]\], epidermal growth factor \[[@B102]\], basic fibroblast growth factor (bFGF) \[[@B103],[@B104]\], platelet derived growth factor \[[@B101]\] and stem cell factor (SCF) \[[@B105]\] by the tumor. These cytokines act as chemotactic agents to induce the migration of mast cells. A number of studies show a correlation between mast-cell infiltration and tumor progression. For example, co-injection of mast cells with an inoculum of rat sarcoma tumors results in enhanced tumor growth, while pharmacologically decreasing the quantity of mast cells slows tumor growth \[[@B106]\]. Furthermore, genetic evidence supports the role of mast cells in tumor growth and progression. SCF is important for mast-cell development, proliferation, migration and degranulation. W/W^v^mice (which express a mutation in the SCF receptor) do not develop functional mast cells. In W/W^v^mice, tumors do not metastasize as readily and they vascularize at a slower rate than in wild-type mice. Tumor metastasis and vascularization return to wild-type levels following restoration of mast cells \[[@B107]\]. Furthermore, administering antisense targeting SCF in a rat mammary-tumor model results in a decrease in mast-cell degranulation, microvascular density and tumor growth \[[@B108]\]. These studies suggest that the presence of mast cells in tumors is not indicative of a protective immune reaction, as these cells actually facilitate tumor progression. The mechanisms by which mast cells facilitate tumor progression appear to involve the factors contained within mast-cell granules. Experimental evidence suggests that degranulation is critical in the ability of mast cells to enhance tumor growth. Inhibition of mast-cell degranulation using disodium cromoglycate impedes tumor growth \[[@B109]\]. One of the functions of degranulation in tumor growth may be to facilitate angiogenesis. Addition of mast cells or isolated mast-cell granules induces vascularization in the chorioallantoic membrane model, while no effect of adding previously degranulated mast cells was observed \[[@B110]\]. This effect appears to be partially mediated through bFGF and VEGF, as addition of anti-bFGF and anti-VEGF antibodies significantly decreased the degree of vascularization. Indeed, mast cells secrete a number of growth factors that regulate angiogenesis and endothelial-cell survival, including TNF-α \[[@B111]\], IL-8 \[[@B111]\], bFGF \[[@B112]\] and VEGF \[[@B113],[@B114]\]. Moreover, mast-cell granules contain the proteolytic enzyme tryptase, while some mast-cell subsets also contain chymase. These enzymes have both direct and indirect effects on the integrity of the extracellular matrix (ECM). Directly, these enzymes induce degradation of ECM components \[[@B115]\]. Indirectly, they can induce degradation of the ECM by activating latent forms of matrix metalloproteinases \[[@B116]\]. Furthermore, it has been reported that these proteinases may induce proliferation of vascular endothelial cells \[[@B117]\]. As is often the case in networks, activation of one component induces the activation or suppression of interacting components. Activated mast cells secrete a number of cytokines that induce the chemotactic migration of macrophages into the tumor, including IL-1, IL-6, TNF-α, IL-8, monocyte chemotactic protein-1, macrophage inflammatory protein-1α and macrophage inflammatory protein-1β \[[@B118],[@B119]\]. The effect of macrophages on tumor growth and progression is discussed in the following section. In addition, activation of mast cells induces the synthesis of the cylooxygenase enzyme, which is involved in the metabolism of arachidonic acid \[[@B120]-[@B122]\] and prostaglandins. Pharmacological inhibition of cylooxygenase significantly impedes metastasis in several animal models \[[@B123]-[@B125]\]. ### Macrophages Macrophage infiltration of tumor sites has been observed in a range of tumor types. Monocytes are recruited to the tumor by constitutive tumor-cell expression of chemoattractant cytokines. In fact, the level of expression of monocyte chemotactic protein-1, macrophage colony stimulating factor (M-CSF) and VEGF by tumor cells correlates well with the extent of macrophage infiltration \[[@B126],[@B127]\]. Within the tumor microenvironment, monocytes differentiate into macrophages. The role of the macrophage in the tumor microenvironment appears to be specific to the tumor type. In breast \[[@B128]\], cervical \[[@B129]\] and bladder \[[@B130]\] cancers macrophage infiltration is an adverse prognostic indicator. However, in prostate \[[@B131],[@B132]\], lung \[[@B133],[@B134]\] and brain \[[@B135],[@B136]\] cancers the prognostic significance of tumor associated macrophages, TAMs, depends on the method of assessing macrophage infiltration, the endpoints of the study and the specifics of the patient cohort. This discrepancy highlights the chameleon-like nature of the macrophage. Depending on their microenvironment, macrophages may either exhibit antitumor cytotoxic activity or facilitate tumor growth and progression while reinforcing a Th2 biased immune response \[[@B137]\]. Following exposure to IFN-γ or bacterial lipopolysaccharide, macrophages can exhibit direct or indirect tumor cytotoxicity. Macrophages may participate in antibody-dependent cellular cytotoxicity responses if an IgG2a antibody binds to a tumor surface antigen. In such a scenario, the macrophage would bind to the Fc portion of the antibody, releasing cytotoxic mediators such as proteinases and TNF-α. Macrophages may also mediate cytotoxicity independently of antibodies through the secretion of reactive oxygen and nitrogen species, proteinases and TNF-α. Furthermore, macrophages of this \"cytotoxic\" phenotype affect the cytokine profile of the microenvironment because they secrete IL-12. IL-12 favors the differentiation of naïve T-helper cells to Th1 cells, which will subsequently secrete IFN-γ and TNF-β. The role of these cytokines in the promotion of a cell-mediated immune response has already been discussed. Finally, cytotoxic macrophages produce matrix metalloproteinase-12 \[[@B138]\]. Matrix metalloproteinase-12 has been shown to convert plasminogen to angiostatin \[[@B139]\]. Angiostatin is important in the inhibition of angiogenesis \[[@B140]\]. In contrast to macrophages that are activated by classical mechanisms and are capable of cytotoxic activity, TAMs, for the most part, do not exhibit cytotoxic activity, are Th1 immunosuppressive and, in fact, facilitate tumor growth, vascularization and metastasis. Tumors secrete a number of cytokines including IL-4, IL-10, TGF-β, prostaglandin E-2 and VEGF. These factors modulate the macrophage phenotype, changing them from cytotoxic macrophages to suppressive macrophages \[[@B141]-[@B143]\]. Furthermore, TAMs appear to have functional defects; for example, in contrast to other macrophages, TAMs are poor antigen-presenting cells \[[@B141]\] and have reduced cytotoxicity owing to impaired production of TNF-α \[[@B144]\] and nitric oxide \[[@B145]\]. However, these impairments in the function of TAMs are consistent with macrophages that are not activated rather than defective. A number of studies suggest that TAMs facilitate tumor growth, vascularization and metastases. As previously stated, numerous studies have shown the presence of TAMs to be an adverse prognostic indicator. In addition, genetic evidence suggests that macrophages are important in vascularization and metastasis of experimental tumors. To assess the role of macrophages in carcinogenesis, the M-CSF deficient osteoporotic (op/op) mouse was crossed with the polyoma virus middle T transgenic mouse, which develops spontaneous mammary tumors. While no differences were observed in the early stages of tumor growth, the resulting offspring of these tumor-bearing mice had a reduced rate of progression to invasive carcinoma and contained fewer pulmonary metastases than control mice \[[@B146]\]. Furthermore, induced M-CSF expression in the mammary tissue of these mice resulted in restored metastatic spread of experimental tumors. In addition, transplantation of the Lewis lung carcinoma in the op/op mouse resulted in a decrease in its growth and vascularization compared with wild-type littermates \[[@B147]\]. This attenuation could be reversed by administration of M-CSF. These data suggest that macrophage infiltration is important in angiogenesis and tumor progression. Interestingly, TAMs are not found ubiquitously within tumor tissue. Data suggest that TAMs localize to poorly vascularized regions -- that is, regions characterized by hypoxia, low pH and tissue necrosis \[[@B127],[@B148]-[@B150]\]. In response to hypoxia, macrophages express a number of hypoxia-regulated gene-products, including VEGF, hypoxia inducible factor 1α and hypoxia inducible factor 2α \[[@B149],[@B151],[@B152]\]. Furthermore, hypoxic conditions impair the chemotactic migration of macrophages \[[@B150],[@B153]\]. This suggests that macrophages could be detained in regions of low oxygen tension. While the precise nature of the relationship between macrophages and hypoxia requires further investigation (macrophages are phagocytic cells, so their physiological role may well be to engulf necrotic tissues), it is possible that macrophage recruitment to these sites facilitates angiogenesis. One mechanism by which TAMs may augment angiogenesis is through the production of a plethora of cytokines and proteinases. As mentioned above, hypoxia induces production of VEGF. The angiogenic properties of VEGF have been extensively discussed by others \[[@B154]\]. In addition to VEGF, the repertoire of cytokines produced by TAMs includes granulocyte macrophage colony-stimulating factor, TGF-α, TGF-β, IL-1, IL-6, IL-8 and prostaglandin E-2. These cytokines are involved in the regulation of angiogenesis. Furthermore, TAMs secrete urokinase-type plasminogen activator and matrix metalloproteinase-9 \[[@B155],[@B156]\]. These proteinases are involved in the induction of angiogenesis and metastasis through degradation of the ECM. Finally, TAMs have numerous indirect immunological effects. TAMs produce copious amounts of IL-10 but low amounts of IL-12 \[[@B157]\]. This shift in the cytokine profile reinforces the Th2 imbalance that is often present in cancer models. More significantly, IL-10 prevents T-cell activation by inducing a state of anergy (a state of T-cell non-responsiveness associated with tolerance induction). IL-10 can induce anergy both in T cells that have been activated in its presence and in T-cells activated by antigen-presenting cells that were previously exposed to IL-10 \[[@B158]\]. These data suggest that TAMs may also facilitate tumor growth by inducing tolerance to the tumor and hence suppressing the antitumor immune response. ### Chemokines Chemokines are a subclass of cytokines that direct the migration of leukocytes to sites of inflammation. It has recently been observed that tumors express chemokine receptors \[[@B159]-[@B161]\]. Thus, it should not come as a surprise that, aside from their role in mediating the recruitment of tumor-infiltrating leukocytes to tumor sites, chemokines may also affect neoplastic proliferation, neovascularization and metastasis. Specific chemokines, such as growth-related oncogene (GRO)-α, GRO-β, GRO-γ and IL-8, directly induce the proliferation of melanoma cells \[[@B162]\], while ligands of the chemokine receptor CXCR-2, such as IL-8, induce proliferation of lung \[[@B163],[@B164]\], ovarian \[[@B165]\], pancreatic \[[@B166]\] and head and neck \[[@B167]\] tumors. Chemokines may both augment and inhibit angiogenesis. In general, chemokines with an ELR motif promote angiogenesis and facilitate the chemotactic migration of endothelial cells \[[@B168]\]. Nevertheless, IFN-γ induces the expression of three non-ELR chemokines that have been reported to have angiostatic properties. For example, expression of IP-10 is negatively correlated with human lung-cancer tumor growth \[[@B169]\], while administering recombinant IP-10 slows tumor growth \[[@B170]\]. Furthermore, administration of IL-12 inhibits bFGF-mediated in vivoneovascularization of matrigel \[[@B171]\]. The angiostatic effects of IL-12 appear to be mediated through the induction of IP-10 and of monokine induced by gamma interferon, as neutralizing antibodies to these chemokines negated the angiostatic effects of IL-12 \[[@B172],[@B173]\]. Finally, in some systems IL-8 activates the transcription of matrix metalloproteinase and is associated with an increased degree of invasion and metastasis. Furthermore, chemokines may be involved in the attraction of circulating cancer cells to sites of metastasis \[[@B174]-[@B177]\]. Strategic research directions ============================= Based on our knowledge of tumor immune evasion and immune contribution to growth of tumors, several therapeutic implications arise including: a) Use of adjuvant therapies to decrease tumor immune suppression while vaccination, b) Extracorporeal elimination of immune suppressive molecules; and c) Gene silencing of tumors to generate immunity. The armamentarium of clinically useful drugs for inhibition of tumor immunity is rapidly growing. The challenge is to identify drugs that intrinsically possess antitumor activity, while at the same time can reverse immune stimulation. One promising candidate that fits these criteria is the clinically used anti-VEGF antibody bevacizumab (Avastin). Currently approved by the FDA for treatment of advanced colon cancer patients, this antibody is believed to mediate its effects primarily by inhibition of angiogenesis \[[@B178]\]. From the tumor immunotherapy perspective, VEGF plays an important role for tumor suppression of immune responses. In fact, administration of anti-VEGF antibody increases efficacy of immunotherapy in mouse models \[[@B179]\]. Mechanisms by which VEGF inhibits antitumor immunity include suppression of NF-kB activation in DC resulting in an immature phenotype \[[@B180]\], as well as suppression of T cell activation \[[@B181]\]. Suppression of immune inhibitory molecules could also be accomplished by immunization. For example, the pregnancy associated molecule human chorionic gonadotropin (hCG) is associated with inhibition of Th1 generation in vitroas well as in vivo\[[@B182]\]. Vaccination with the carboxy-terminal peptide of hCG has been shown to break tolerance to this self-molecule and induce a marginal anticancer response \[[@B183]\]. Combining vaccination against immune suppressive molecules with vaccination against tumor-specific antigens will likely improve efficacy. Another method of reversing tumor associated immune suppression would involve extracorporeal removal of inhibitory molecules. Although plasmapheresis techniques have been attempted with mediocre success \[[@B184]\], a novel and promising method involves ultrapheresis. Lentz et al described a pilot study of 16 metastatic patients in which the \<100,000 kDa fraction was removed through membrane ultrapheresis, Six of the 16 patients had reduction of the sum of mean cross-sectional diameters of measureable lesions by 50% or more. Additionally, tumor infiltrating lymphocytes were observed in many of the lesions after 2 months of treatment \[[@B185]\]. Follow-up studies demonstrated that the immune suppressive component being removed through the ultrapheresis procedure was soluble TNF-R alpha \[[@B186]\]. Recent developments in hollow-fiber technologies allow for specific removal of plasma bourne components such as HIV gp120 and various toxins \[[@B187]\]. Applications of these techniques to immune therapy could yield valuable new approaches that are currently not studied. The introduction of RNA interference (RNAi) as a potent method of gene-specific silencing has opened new territories for gene therapy. Successful use of RNAi for immune modulation was first reported by Hill et al \[[@B188]\]. Subsequently, it was demonstrated that silencing of the inhibitory cytokine IL-10 led to the generation of dendritic cells with potent Th1 priming abilities \[[@B189]\]. More recently, siRNA modified DC were used for induction of antitumor immunity \[[@B190]\]. The fact that naked siRNA can be directly endocytosed by target cells \[[@B191]\], or administered using polyethylenimine-complexe \[[@B192]\], suggests the possibility of direct intratumoral injection of siRNA targeting immune suppressive molecules. Additionally, tumor-targeting immunoliposomes could be used for systemic delivery of siRNA into cancer cells. Suitable targets would include the wide variety of immune suppressive molecules mentioned above. Conclusions =========== The debate on the role of the immune system in cancer has been one of the most controversial areas of science with opinions vacillating from optimistic highs to skeptic nadirs on a cyclical basis. These oscillations in opinion that are reflective of the relative progress in the immunotherapy of cancer, can be explained by the complexity of the relationship of the immune system and cancer -- an interaction that can result in anti-tumor protection, tumor promotion, or in no net effect. The apparent schizoid role of the immune system in cancer may in fact be a variation on a theme borrowed from the immunology of infectious disease. That is, the mere generation of an immune response is not sufficient to attain protection against a pathogen: it is only an immune response of the proper type and the proper magnitude that will result in protection. An immune response against an agent that is not of the proper type and magnitude will be deleterious to the host. A proper type of immune response against cancer will result in protection. Evidence exists that in set conditions components of the immune system can recognize and eradicate incipient cancer cells. It is likely that some sort of immune surveillance exists in the healthy individual -- though the exact mechanisms have yet to be elucidated. The genetic evidence discussed herein suggests that NK cells and IFN-γ are likely to be involved in this protection. Nevertheless, the clinical presentation of cancer suggests that neoplasia can evade these putative mechanisms of immunosurveillance. This further suggests that the ensuing immune response is not protective, or not adequately protective, to control the nascent tumor. However, this lack of a protective response is not the whole extent of the role of the immune system in cancer. Several examples have been presented in which the immune response actually facilitates tumor growth, neovascularization and progression. These observations suggest a mechanism that may explain the theory of immunostimulation, though undoubtedly, many other mechanisms may exist. This duality of the immune system in cancer arises in part because of the decentralized nature of the immune system. The non-protective immune reaction that arises resembles a bureaucratic organization in which a lack of orchestration between departments results in redundancy and counter-productivity: the individual entities that constitute the immune system react to the presence of the tumor yet they are not orchestrated to achieve the end point of tumor eradication. Secondly, in advanced tumors, the duality of the immune system in cancer may also arise from the bilateral nature of the interaction between the tumor and the immune mechanisms of the host. Not only can the immune system affect the tumor, but the tumor may also affect the immune system. The skewed cytokine milieu of a tumor, which results in the recruitment of inflammatory cells through the secretion of chemokines and growth factors and which further prevents the cytotoxic activation of these cells, is an example of how the tumor influences the immune system. This effect of the tumor on the immune system may be explained by the theory of immunoediting, which describes the iterative process of selection for cells that can evade the natural immunosurveillance mechanisms \[[@B193]\]. This selection process may generate tumors that not only escape detection by the immune system, but that have actually generated mechanisms of depressing those branches of the immune response that would offer anti-tumor protection and/or enhancing those branches that would elicit tumor promotion. Akin to the successful development of vaccines against infectious agents, the development of effective immunotherapy against cancer will require recognition of the duality of the immune response in cancer, and the stimulation of the proper type of immune response. The challenges ahead, especially in late-stage patients who have undergone immunoediting, will be to overcome the tendencies of the tumor to elicit an inappropriate immune response, one used by the tumor to promote its own growth. It may be the case that in subsets of patients the sort of immunotherapy that is desirable is not that which stimulates the immune system but that which suppresses an inappropriate, tumor promoting, immune reaction \[[@B194],[@B195]\]. Chemotherapy, may in fact be an example of \'immunotherapy\'. Chemotherapy both directly and indirectly affects the immune system; however, given the multiplicity of the functions of the immune system in cancer, the net effect of this treatment modality remains to be defined. While the direct effect of chemotherapy may be myelosuppression, this may not be an undesirable consequence given that cells of the myeloid lineage, such as mast cells and macrophages, often promote the growth, vascularization and invasion of tumors. Furthermore, myelosuppression may actually stimulate a protective, Th1-type, cell mediated immune response because mast cells and macrophages secrete factors that promote a Th2 type of immune response. \"If only it were all so simple.\" If only the immune system were all good and it were necessary only to stimulate it to achieve the eradication of tumors. But the potential for good and evil is entwined within the heart of the immune reaction, and who is willing to destroy their own heart? Acknowledgements ================ I thank Carlo V. Hojilla, Richard G. Miller, Richmond T. Prehn (University of Washington, Seattle, WA), A. Michael Rauth, David E. Spaner, Richard A. Wells and Minna Woo for reading of the manuscript and helpful suggestions.	PubMed Central	2024-06-05T03:55:52.919340	2005-2-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549049/", "journal": "J Transl Med. 2005 Feb 8; 3:8", "authors": [ { "first": "Christine V", "last": "Ichim" } ] }
PMC549050	Background ========== Human T-lymphotropic virus type 1 (HTLV-1) is endemic in southern Japan, intertropical Africa, Melanesia, Latin America, and the Caribbean basin \[[@B1]\]. HTLV-1 is the etiological agent of adult T-cell leukemia \[[@B2]\] and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), an inflammatory disease of the central nervous system \[[@B3],[@B4]\], and has also been implicated in several other inflammatory disorders, such as polymyositis \[[@B5]\], uveitis \[[@B6]\], Sjögren\'s syndrome \[[@B7]\], alveolitis \[[@B8]\], and infective dermatitis \[[@B9]\]. The possibility that HTLV-1 may cause joint disease was initially raised by reports of arthralgia and polyarthritis in patients with adult T-cell leukemia \[[@B10],[@B11]\]. Polyarthritis has also been observed in some patients with HAM/TSP \[[@B12]\]. Nishioka et al. \[[@B13]\] described the association of a polyarthritis syndrome with HTLV-1 infection in the absence of clinical ATL or HAM/TSP, and proposed the term HTLV-1-associated arthritis (HAA). Cases of HTLV-1-infected patients with mixed connective tissue disease have also been described \[[@B14]\], although an association between HTLV-1 infection and systemic lupus erythematosus has not been established \[[@B15]\]. Apart from the possibility of neurological signs, the clinical features of HAA are similar to those of idiopathic rheumatoid arthritis (RA) \[[@B16]-[@B18]\]. Epidemiological studies have demonstrated that HTLV-1 seropositivity is a risk factor for RA in Japan \[[@B19],[@B20]\], but a recent study conducted in South Africa, another HTLV-1 endemic area, failed to detect any association between HTLV-1 and RA \[[@B21]\]. This discrepancy might be due to differences in genetic background, although the possibility cannot be excluded that HAA in Japan results from the coincidental coexistence of two relatively common diseases. Interestingly, a recent prospective study demonstrated an increased incidence of arthritis in cohorts of former US blood donors infected with HTLV-1 or HTLV-2 \[[@B22]\]. Several findings support the hypothesis of an etiopathogenic role for HTLV-1 in HAA: ATL-like T lymphocytes have been identified in the synovial fluid and synovial tissue \[[@B17],[@B18],[@B23]\]; high titers of IgM antibodies against HTLV-1 have been found in the synovial fluid \[[@B23]\]; HTLV-1 proviral DNA has been detected in synovial fluid cells and synovial tissue cells \[[@B23]\], cultured adherent synovial stromal cells \[[@B24]\], and synovial macrophage cells \[[@B25]\]; and Tax mRNA and protein have been detected in synovial stromal cells \[[@B26]\]. HTLV-1 tropism for synovial cells has been confirmed in vitro \[[@B27]\]. Moreover, mice transgenic for Tax develop an inflammatory arthropathy resembling RA in humans \[[@B28]\]. The development and progression of RA is dependent on the migration of T lymphocytes into the synovial compartment \[[@B29],[@B30]\]. Similarly, the tissue damage in HAM/TSP is thought to be caused by T cells that have infiltrated the central nervous system \[[@B31],[@B32]\]. T lymphocytes, especially CD4^+^T cells, are the main target of HTLV-1 in vivo and carry the majority of the HTLV-1 proviral load \[[@B33]\]. The HTLV-1 proviral load in peripheral blood mononuclear cells (PBMCs) is higher in patients with HAM/TSP than in asymptomatic HTLV-1 carriers \[[@B34]\] and the equilibrium set point of the proviral load is suspected to determine the development of the disease \[[@B35]\]. We postulated that HTLV-1 proviral load might also influence the initiation and course of HAA, and measured this marker in PBMCs from a previously described cohort \[[@B16]\] of HTLV-1-infected patients with RA and in a group of HTLV-1-infected patients with connective tissue disease. Results ======= The HTLV-1 proviral load was measured in the peripheral blood of HTLV-1-infected patients with RA or connective tissue disease and in matched asymptomatic and HAM/TSP controls (Figure [1](#F1){ref-type="fig"}). The number of copies of HTLV-1 proviral DNA per 10^6^PBMCs ranged from 14,600 to 373,000 in the patients with RA (Table [1](#T1){ref-type="table"}) and from 1,500 to 411,200 in patients with connective tissue disease (Table [2](#T2){ref-type="table"}), the corresponding ranges in the HTLV-1 asymptomatic carriers and in patients with HAM/TSP being 50 to 97,700 and 2,100 to 392,000, respectively. The mean ± SD and median proviral loads were 133,800 ± 134,600 and 75,800 in the HTLV-1-infected patients with RA or connective tissue disease combined, the values for the RA subgroup being 114,400 ± 112,200 and 67,400 and those for the connective tissue disease subgroup 172,500 ± 176,700 and 120,800, while the corresponding values in the asymptomatic carriers were 18,800 ± 26,400 and 10,100 and those in patients with HAM/TSP 86,800 ± 90,600 and 62,400. The HTLV-1 proviral load was significantly higher in the HTLV-1-infected group with RA or connective tissue disease than in the matched asymptomatic HTLV-1 carriers (P= 0.0012 in Wilcoxon\'s test, P= 0.0002 in the paired t-test after log-transformation), and the difference remained significant when the analysis focused on the RA subgroup (P= 0.0022 in Wilcoxon\'s test, P= 0.0002 in the paired t-test). No differences were observed between the HTLV-1-infected group with RA or connective tissue disease and the matched HAM/TSP controls (P\> 0.05 in both Wilcoxon\'s test and the paired t-test). As expected, the difference between the asymptomatic carriers and the HAM/TSP group was significant (P= 0.0001 in Wilcoxon\'s test, P\< 0.0001 in the paired t-test). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### HTLV-1 proviral load in the peripheral blood from HTLV-1-infected patients with RA or connective tissue disease and from HAM/TSP or asymptomatic controls.The 10^th^and 90^th^percentiles are shown as the lower and upper horizontal bars on the vertical line, while the 25^th^and 75^th^percentiles are shown as the lower and upper edges of the box; the median is shown within the box. The results shown as dots fall outside the 10^th^and 90^th^percentiles. ::: ![](1742-4690-2-4-1) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Clinical and biological features of HTLV-1-infected patients with RA ::: Patient Sex Age Associated Diseases Disease duration (year) Sharp score ESR (mm) CRP (mg/l) RF (IU/ml) Treatment HTLV-1 proviral load (copies / 10^6^PBMCs) --------- ----- ----- --------------------- ------------------------- ------------- ---------- ------------ ------------ ------------------------- -------------------------------------------- 1 F 75 \- 7 65 111 313 128 Corticosteroids, DMARDs 373,300 2 F 63 HAM/TSP 2 10 70 5 32 Corticosteroids, DMARDs 39,800 3 F 62 \- 9 3 41 6 \- NSAIDs 126,500 4 F 58 \- 7 46 5 3 64 Corticosteroids, DMARDs 55,000 5 F 72 \- 7 109 56 16 \- NSAIDs, DMARDs 38,500 6 F 59 \- 10 5 38 5 \- Corticosteroids, DMARDs 28,900 7 F 64 \- 2 8 25 12 128 Corticosteroids, DMARDs 79,800 8 F 69 \- 16 126 21 30 16 Corticosteroids, DMARDs 230,000 9 F 57 HAM/TSP 13 6 29 13 \- Corticosteroids 247,400 10 F 58 HAM/TSP 2 17 35 9 32 Corticosteroids, DMARDs 106,500 11 F 42 \- 18 3 22 5 \- Analgesics 32,200 12 F 77 HAM/TSP 7 110 88 41 \- Corticosteroids, DMARDs 14,600 Sharp score : joint space narrowing score and erosion score; CRP: C-reactive protein; ESR, erythrocyte sedimentation rate; RF: rheumatoid factor; NSAIDs: non-steroidal anti-inflammatory drugs; DMARDs: disease-modifying anti-rheumatic drugs (hydroxychloroquine, leflunomide, methotrexate, etc.) ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Clinical and biological features of HTLV-1-infected patients with connective tissue disease ::: Patient Sex Age Connective tissue disease Associated diseases Disease duration (year) ESR (mm) CRP (mg/l) ANA Treatment HTLV-1 proviral load (copies / 10^6^PBMCs) --------- ----- ----- --------------------------- --------------------- ------------------------- ---------- ------------ ----- --------------------- -------------------------------------------- 13 F 38 SLE uveitis 13 38 6 \+ Corticosteroids, HC 18,400 14 F 68 PM \- 1 17 1 \+ Corticosteroids 411,200 15 F 60 SS \- 2 10 1 \+ NSAIDs, HC 1,500 16 F 56 PM HAM/TSP 10 17 12 \+ Corticosteroids 71,700 17 F 63 SS \- 3 22 15 \+ NSAIDs, HC 169,900 18 F 65 SS HAM/TSP, alveolitis 5 21 1 \- Corticosteroids 362,300 SS, Sjögren\'s syndrome, SLE : Systemic lupus erythematosus, PM : Polymyositis, ANA: anti-nuclear antibodies; HC : hydroxychloroquine ::: Of the 12 HTLV-1-infected patients with RA, 4 (patients 2, 9, 10, and 12) had HAM/TSP (Table [1](#T1){ref-type="table"}); their respective HTLV-1 proviral loads were 39,800, 247,400, 106,500, and 14,600 copies per 10^6^PBMCs. Two patients with connective tissue disease (Table [2](#T2){ref-type="table"}, patients 16 and 18) had HAM/TSP, which was associated with alveolitis in patient 18, with proviral loads of 71,600 and 362,300. These samples did not account for the higher proviral load in the arthritic group compared to the asymptomatic carrier group. Indeed, after excluding the patients with co-existing HAM/TSP, the difference between the asymptomatic controls and the group with RA or connective tissue disease remained significant (P= 0.0121, Wilcoxon\'s test; P= 0.0027, paired t-test), even when the analysis was restricted to the subgroup with RA alone (P= 0.0117, Wilcoxon\'s test; P= 0.0003, paired t-test). The patient with connective tissue disease and uveitis (patient 13) had 18,420 copies per 10^6^PBMCs. For one of the patients with RA and HAM/TSP (patient 10), 4 consecutive frozen dry pellets of PBMCs from 1996 to 2002 were available; in these, the proviral load was relatively stable at 92,100, 73,600, 143,300, and 106,500 copies per 10^6^cells, respectively. No correlation was found between HTLV-1 proviral load and the age of the patient, the duration of illness, or the Ritchie\'s index score (P\> 0.05, Spearman test). HTLV-1 proviral load did not correlate with erythrocyte sedimentation rate or C-reactive protein level (P\> 0.05, Spearman test). No significant difference in HTLV-1 proviral load was seen in patients positive or negative for rheumatoid factor or antinuclear antibody (P\> 0.05, Mann-Whitney test) or receiving or not receiving either specific treatments for RA or corticotherapy (P\> 0.05, Mann-Whitney test). The proviral load was measured in two sets of PBMCs and synovial fluid or synovial tissue cells obtained from one HTLV-1-infected patient with RA at an interval of one year (Table [1](#T1){ref-type="table"}, patient 5). In the first set of samples, the HTLV-1 proviral load in the synovial fluid cells and paired PBMCs was 845,200 and 125,300 copies per 10^6^cells, respectively, while, in the second set of samples from a year later, the proviral load in the synovial tissue cells and paired PBMCs was 666,700 and 38,500 per 10^6^cells, respectively. Lymphocytes subsets and activation status were examined in the peripheral blood of HTLV-1-infected patients with RA or connective tissue disease. When correlations were examined between HTLV-1 proviral load and the percentage of T lymphocytes expressing CD45RO, CD45RA, or HLA-DR in the HTLV-1 infected patients with RA or connective tissue disease (Figure [2](#F2){ref-type="fig"}), HTLV-1 proviral load correlated positively with the percentage of CD4+ T cells expressing CD45RO and negatively with that of CD4+ T cells expressing CD45RA (P= 0.039 and P= 0.021, Spearman test). A positive correlation was found between HTLV-1 proviral load and the percentage of CD4+ T cells expressing HLA-DR (P= 0.008), while the correlation did not reach significance for CD8+ HLA-DR T cells. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Correlation between HTLV-1 proviral load, and memory or activated T lymphocytes subsets in the peripheral blood from HTLV-1-infected patients with RA or connective tissue disease. ::: ![](1742-4690-2-4-2) ::: Lymphocyte distribution and activation were compared in the peripheral blood and synovial fluid of one HTLV-1-infected patient with RA (patient 5). The percentage of CD4+ T cells expressing CD45RO was dramatically increased in the synovial fluid (98%) compared to the peripheral blood (48%), as was the percentage of CD4+ T lymphocytes expressing HLA-DR (94% compared to 18%). Discussion ========== The etiology of autoimmune diseases, such as RA or mixed connective tissue disease, has yet to be established, but appears to result from complex interactions between host genetic and environmental factors \[[@B36]\], and retroviruses have been considered as possible causative factors \[[@B37]\]. HTLV-1 has been suggested to be implicated in the pathogenesis of RA in Japan, where this retrovirus is endemic \[[@B13],[@B17],[@B18]\]. Epidemiological studies have shown an association between HTLV-1 infection and RA in Japan \[[@B19],[@B20]\], and between HTLV-1/2 infection and arthritis in the United States \[[@B22]\]. HTLV-1-seropositive cases with connective tissue disease have also been described, although the authors suggested a geographical, rather than an etiological, link \[[@B14]\]. Another study failed to demonstrate any association between HTLV-1 infection and systemic lupus erythematosus in Jamaica \[[@B15]\]. Considering the age, sex ratio, and HTLV-1 prevalence (4.6%) in our RA cohort, a fortuitous coincidence cannot be excluded. The prevalence of HTLV-1 infection in Martinique is estimated to be around 1.5% and increases with age, particularly among women \[[@B38]\]. An epidemiological study is therefore required to determine whether there is no, or a weak, association between HTLV-1 and arthritis in Martinique. Nevertheless, the present study provides biological data suggesting a contribution of HTLV-1 to the development of some cases of RA or mixed connective tissue disease. We found that: (1) The circulating HTLV-1 proviral load was higher in HTLV-1-seropositive patients with RA or connective tissue disease than in asymptomatic HTLV-1 carriers and similar to that in patients with HAM/TSP; (2) In the peripheral blood of patients with arthritis, HTLV-1 proviral load correlated with the percentage of memory and activated T CD4^+^cells; (3) A high HTLV-1 proviral load was found in the synovial fluid and tissue cells in a patient with RA; (4) In this patient, the percentage of memory and activated CD4^+^T cells was higher in the synovial compartment than in the peripheral blood. The HTLV-1 proviral load is thought to be a major determinant of HTLV-1-associated diseases. The HTLV-1 proviral load is higher in the peripheral blood from patients with HAM/TSP than in blood from asymptomatic carriers \[[@B34]\]. It is also higher in the peripheral blood of patients with HTLV-1-associated uveitis than in asymptomatic carriers \[[@B39],[@B40]\]. Similarly, we observed a significantly higher proviral load in HTLV-1-infected patients with either RA or connective tissue disease than in HTLV-1 asymptomatic carriers. Moreover, the proviral load was higher in the synovial fluid and tissue from one HTLV-1-infected patient with RA than in the peripheral blood. Assuming an average of one HTLV-I provirus per infected cell, the proviral load values in these synovial samples suggest that the majority of infiltrated cells are infected. HTLV-1 proviral load is known to be higher in the spinal fluid than in paired blood samples from HAM/TSP \[[@B41],[@B42]\], but not asymptomatic carriers \[[@B43]\]. Interestingly, a higher HTLV-1 proviral load than in the peripheral blood has also been reported in bronchoalveolar lavage fluid in patients with HTLV-1-associated alveolitis \[[@B44]\] and in the labial salivary glands in patients with HTLV-1-associated Sjögren\'s syndrome \[[@B45],[@B46]\]. Thus, a high proviral load might be involved in the pathogenesis of several other HTLV-1-associated inflammatory disorders in addition to HAM/TSP. In HAM/TSP, the HTLV-1 proviral load reaches an equilibrium set-point that is correlated with progression of motor disability, and fluctuates by no more than 2- to 4-fold over a decade \[[@B35]\]. In one HTLV-1-infected patient with RA, the proviral load was found to be stable over a 6 year period. In HTLV-1-associated uveitis, the proviral load has been shown to correlate with disease activity \[[@B40]\]. However, in our cohort, HTLV-1 proviral load in the peripheral blood did not correlate with disease activity and was not influenced by treatment of the rheumatological disease. This suggests that HTLV-1 proviral load reaches a set-point determining the onset of the rheumatological disease, but the intensity of the symptoms might be influenced by subsequent in situ events. The unusually high proviral loads in HTLV-1 infection results mainly from the Tax-driven activation and expansion of infected cells \[[@B47]\]. The HTLV-1 targets are mainly CD45RO-expressing CD4^+^T lymphocytes and the proviral load is reported to correlate with the number of memory T cells \[[@B48]\]. In our HTLV-1-infected cohort with RA or connective tissue disease, HTLV-1 proviral load correlated positively with the percentage of CD4^+^T cells expressing CD45RO and negatively with that of CD4^+^T cells expressing CD45RA. The HTLV-1 proviral load also correlated positively with the percentage of HLA-DR-expressing T cells. Migration of HTLV-1-infected CD4^+^T cells and HTLV-1-specific CD8^+^cytotoxic T lymphocytes (CTL) into the central nervous system is a critical step in the pathogenesis of HAM/TSP \[[@B31],[@B32]\]. Similarly, infiltration of T cells plays a central role in the initiation and perpetuation of RA \[[@B29],[@B30]\]. Thus, our finding of an increase in memory (CD45RO) and activated (HLA-DR) CD4^+^T cells in the joint fluid of an HTLV-1 carrier with RA supports the hypothesis of a pathogenic involvement of HTLV-1-infected T lymphocytes. Several mechanisms are potentially involved in the occurrence of rheumatological disorders during HTLV-1 infection. Firstly, HTLV-1 infection upregulates the expression of adhesion molecules potentially involved in the migration of lymphocytes into the spinal and joint compartments \[[@B17]\]. Secondly, HTLV-1 might be transmitted from the infiltrated T lymphocytes to the synoviocytes \[[@B23]-[@B26]\], and subsequent Tax expression might induce proliferation of these cells. Extracellular Tax protein has also been reported to stimulate the proliferation of synoviocytes \[[@B49]\]. Finally, Tax expression stimulates the production of a variety of cytokines, including IL-15 and its receptor. IL-15 might represent a cornerstone between HAM/TSP and HTLV-1-associated rheumatological diseases. IL-15 favors T cell migration into the target tissue compartment \[[@B50]\]. Moreover, IL-15 inhibits IL2-mediated activation-induced cell death, and is suspected to both facilitate the persistence of MHC I restricted memory CD8+ T cells involved in the pathogenesis of HAM/TSP, and enhance the survival of self-reactive T cells, leading to the development of autoimmune disease \[[@B51]\]. Recent data argue for a possible autoimmune mechanism of tissue damage in HAM/TSP \[[@B52]\]. Moreover, IL-15 can also induce TNF-α synthesis by macrophages, which, in turn, stimulates a cascade of proinflammatory cytokines, including IL-1β, IL-6, and GM-CSF \[[@B50],[@B53]\], which induce synoviocyte proliferation and are thought to be deleterious for the central nervous system \[[@B31]\]. Thus, the coexistence of HAM/TSP in one-third of the HTLV-1-infected patients with RA might be explained by the shared features of a high proviral load and common downstream pathways. Alternatively, autoimmune arthritis or its etiological factors might secondarily enhance HTLV-1 proviral load through cell activation, with subsequent migration of HTLV-1-infected cells into the joint or CNS. The accumulation of HTLV-1-infected lymphocytes in the synovium could result from selective infiltration and/or from oligoclonal expansion once the PBMCs have infiltrated the joint. Whether the increase in HTLV-1-infected cells in the peripheral blood and the even greater increase in the synovial compartment are the cause or an effect of the associated arthritis remains uncertain. A role of the anti-inflammatory and anti-rheumatic drugs can also not be excluded. In conclusion, our data in HTLV-1-infected patients with RA or connective tissue disease are consistent with a role of the proviral load in the development of these rheumatological disorders, although the direction of causality in this interaction remains open to question. HTLV-1 might cause a systemic immune-mediated inflammatory disease potentially involving tissues other than the central nervous system, HAM/TSP being only the major syndrome. The clinical expression of this disease might be determined by the amount of HTLV-1-infected T lymphocytes, their level of activation, and their capacity to accumulate in different body compartments. Further research is needed to increase our knowledge of the molecules involved in the homing of HTLV-1-infected CD4+ T lymphocytes and of anti-HTLV-1-specific CD8+ CTL to different target tissues. Materials and Methods ===================== Patients -------- The study was performed in Martinique, an island in the lesser Antilles archipelago, with a population of 400,000. Between 1988 and 2001, 280 patients with RA, defined according to the American Rheumatology Association (ARA) criteria, and 335 patients with connective tissue disease were followed on an inpatient or outpatient basis at the Rheumatology Department of the Regional Teaching Hospital. Thirteen (4.6%) of the 280 patients with RA (1 male and 12 female) were found to be HTLV-1 seropositive, confirmed by Western blotting (antibodies recognizing at least rgp21, p19, and p24) and peripheral blood was obtained from 12 of these. In addition, 6 patients with connective tissue disease (three with Sjögren\'s syndrome, two with inflammatory myopathy, and one with systemic lupus), who were seropositive for HTLV-1, were included in the study. Samples were collected between September 2001 and June 2002. For one patient, sequential samples cryopreserved since 1996 were available. The mean age at the time of sampling was 63 years for the HTLV-1-seropositive RA patients compared to 60 years for the total RA cohort, while the mean age of the HTLV-1-seropositive patients with connective tissue disease was 58 years. The mean time interval between onset of the auto-immune disease and sampling for HTLV-1 proviral load determination were 8 years and 6 years in the patients with RA and connective tissue disease, respectively. Of the 12 HTLV-1-seropositive RA patients, 4 had HAM/TSP, defined according to the WHO guidelines \[[@B54]\]. Two of the patients with connective tissue disease also presented HAM/TSP. The clinical and biological features of the HTLV-1-infected patients with either RA or connective tissue disease are summarized in Tables [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}, respectively. Each HTLV-1-infected patient with RA or connective tissue disease was matched for age (± 5 years) and sex with 2 patients with HAM/TSP and 2 asymptomatic HTLV-1 carriers. Measurement of HTLV-1 proviral load ----------------------------------- PBMCs were isolated from EDTA blood by density gradient centrifugation. Synovial fluid samples were obtained by arthrocentesis and synovial tissue was obtained during arthroscopy. The synovial tissue was minced into small pieces, washed with phosphate-buffered saline, and passed through a wire mesh to collect synovial tissue cells. Cells were cryopreserved until use. DNA was extracted from 10^6^cells using a phenol/chloroform procedure. The HTLV-1 proviral load was quantified using a real-time TaqMan PCR method \[[@B55]\]. SK110/SK111 primers were used to amplify a 186 bp fragment of the polgene and the dual-labeled TaqMan probe (5\' FAM and 3\' TAMRA) was located at 4829--4858 bp of the HTLV-1 reference sequence (HTLV~ATK~). Albumin DNA was quantified in parallel to determine the input cell number and was used as an endogenous reference to normalize variations due to differences in the PBMC count or DNA extraction. For both HTLV-1 and the albumin gene, amplifications were performed on 10 μl of DNA extract using the TaqMan PCR Core Reagent kit, data being acquired with the ABI Prism 7700 Sequence Detector System (Perkin Elmer, Foster City, California, USA). Standard curves were generated using ten-fold serial dilutions of a double standard plasmid (pcHTLV-ALB) containing one copy of the target regions of both the HTLV-1 polgene and the cellular albumin gene. The HTLV-1-infected human lymphocyte line MT2 (ECACC 93121518) was used as a control for quantification, the limit for an acceptable result being taken as 2.4--3.3 copies of the HTLV-1 pol gene per cell and the variation between series being normalized on the basis of three copies per MT2 cell. All standard dilutions and control and patient samples were run in duplicate for both HTLV-1 and albumin DNA quantification. Standard curves for HTLV-1 and albumin were accepted when the slope was between -3.322 and -3.743 (corresponding to amplification efficiencies of 100% to 85%) and the correlation coefficient, r^2^, was \>0.992. If the variation between duplicate values of HTLV-1 or albumin DNA copy numbers was greater than 30%, the analysis was repeated. The normalized value for the HTLV-1 proviral load was reported as the HTLV-1 average copy number/albumin average copy number ratio × 10^6^and expressed as the number of HTLV-1 copies per 10^6^PBMCs. Determination of CD4- and CD8-positive lymphocyte counts -------------------------------------------------------- Lymphocyte subsets in PBMCs were characterized using a panel of labeled anti-human monoclonal antibodies, consisting of fluorescein isothiocyanate-conjugated anti-CD3 (clone SK7, mouse IgG~1~), allophycocyanin-conjugated anti-CD4 (clone SK3, mouse IgG~1~), peridinin chlorophyll protein-conjugated anti-CD8 (clone SK1, mouse IgG~1~), phycoerythrin (PE)-conjugated anti-CD45RO (clone UCHL-1, mouse IgG~2a~), and PE-conjugated anti-HLA-DR (clone L243, mouse IgG~2a~) (all from BD Biosciences Immunocytometry Systems, San Jose, CA), and PE-conjugated anti-CD45RA (Hl100 mouse IgG~2b~, from BD Biosciences Pharmingen). The analyses were performed on a FACSCalibur (BD Biosciences Immunocytometry Systems). Statistical analysis -------------------- Mann-Whitney\'s Utest, Wilcoxon\'s signed rank test, paired Student\'s t-test, and Spearman\'s rank correlation were used, as appropriate. A Pvalue \< 0.05 was considered to be statistically significant. List of Abbreviations ===================== HTLV, human T-lymphotropic virus; HAM/TSP, HTLV-1-associated myelopathy/tropical spastic paraparesis; RA, rheumatoid arthritis; PBMCs, peripheral blood mononuclear cells. Competing Interests =================== The author(s) declare that they have no competing interests. Authors\' Contributions ======================= MY carried out most of the clinical and experimental work. AL was involved in the molecular biology work and contributed to the design of the study. FD and GL performed the flow cytometry analysis. SO and GJB participated in the neurological and rheumatologic evaluations, respectively. SA supervised the design and the course of the clinical study. RC conceived the study and drafted the manuscript. All authors read and approved the final manuscript. Acknowledgments =============== We thank P. Numeric and F. Dubreuil for their contribution to the clinical part of the study, and R. Marlin for experimental help. This research was supported by the Université des Antilles et de la Guyane (EA 2434), the Délégation Régionale de l\'Institut National de la Santé et de la Recherche Médicale (INSERM) and the Fonds National de la Science du Ministère de l\'Education Nationale, de l\'Enseignement et de la Recherche.	PubMed Central	2024-06-05T03:55:52.923090	2005-2-1	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549050/", "journal": "Retrovirology. 2005 Feb 1; 2:4", "authors": [ { "first": "Maria", "last": "Yakova" }, { "first": "Agnès", "last": "Lézin" }, { "first": "Fabienne", "last": "Dantin" }, { "first": "Gisèle", "last": "Lagathu" }, { "first": "Stéphane", "last": "Olindo" }, { "first": "Georges", "last": "Jean-Baptiste" }, { "first": "Serge", "last": "Arfi" }, { "first": "Raymond", "last": "Césaire" } ] }
PMC549051	Background ========== Early in 1970s, the concept of suppressor T cells was developed and it was envisioned that this subset of lymphocytes was responsible for the active control, and ultimately the termination, of immune responses \[[@B1]\]. But the characters of this subset had not been well studied mainly because its distinct phenotype was not identified. In 1990s, Sakaguchi et al found that a subset of CD4+ lymphocytes in peripheral blood of normal mice expressed the IL-2R-α (CD25) and it down-regulated the immune response to self and non-self antigens \[[@B2]\]. Soon the CD4+CD25+ lymphocytes were verified as one group of suppressor T cell and termed as thymic derived \"naturally occurring\" regulatory T cells (T~R~). T~R~represents a minor (5--10%) component of peripheral CD4+ T cells but plays an important role in controlling immune responses \[[@B3]\]. Accumulating evidences show that T~R~cells possess potent suppressive activity both in vivo and in vitro and are involved in autoimmune diseases, transplantation tolerance and tumor immunity \[[@B2]-[@B5]\]. The transfer of CD4+CD25- cells into nude mice resulted in autoimmune diseases; reconstitution of CD4+CD25+ cells after transfer of CD4+CD25- cells prevented the development of autoimmunity \[[@B2]\]. Similarly, depletion of these cells induced gastritis and late-onset diabetes \[[@B6]\], impaired development or dysfunction of these cells increased susceptibility to experimental autoimmune encephalomyelitis \[[@B7]\], multiple sclerosis \[[@B8]\] and other autoimmune diseases \[[@B9],[@B10]\]. Conversely, an increased percentage of CD4+CD25+ T~R~cells in total CD4+ T cells was found in peripheral blood of cancer patients \[[@B11]-[@B14]\] and depletion of CD25+ cells alone or combination with other strategies might cause tumor regression \[[@B4],[@B15],[@B16]\]. All these studies indicated the importance of T~R~cells in controlling immune response. The mechanism of how the T~R~cells control immune response is still unclear. Previous studies show that activated T~R~cells strongly inhibit proliferative responses of CD4+ or CD8+ T cells in vitro \[[@B17],[@B18]\], moreover, it down-regulates co-stimulatory molecules on dendritic cells (DC) \[[@B19]\], inhibit the maturation and antigen-presenting function of DC \[[@B20]\], and suppress activated and matured DC driven responses \[[@B21]\]. The important role of T~R~cells in immunoregulation makes it be recognized as an attractive therapeutic target for immune-related diseases. In our animal experiments of antitumor immunotherapy that targeting CD4+CD25+ T~R~cells, to our surprise, we did not find an increase of CD4+CD25+/CD4+ in peripheral blood of tumor bearing BALB/c or C57BL/6 mice, this is not in accordance with the increase of the proportion in cancer patients as reported by Wolf et al \[[@B11]\]. In order to find a way to evaluate the CD4+CD25+ T~R~cells in tumor-bearing mice, we analyzed CD4+CD25+ subset in peripheral blood and spleen lymphocytes from normal or C26 colon-carcinoma-bearing mice by flow cytometry. Methods ======= Mice and tumor model -------------------- 6 to 8 weeks BALB/c mice were purchased from the Laboratory Animal Center of Sun Yet-sen University. Mouse C26 colon carcinoma cell line was a gift from Prof. Li-Jian Xian (Cancer Center, Sun Yet-sen University). The C26 Cells were cultured in RPMI 1640 medium (Gibco Invitorogen Corporation) supplemented with 10% fetal calf serum (FCS; Gibco Invitorogen Corporation, Carlsbad, CA), 100 U/ml of penicillin G and 100 μg/ml of streptomycin, and the medium was renewed every 2 to 3 days. After growing to confluency, the cells were detached with trypsin-EDTA, resuspended in serum-free RPMI 1640 medium and inoculated subcutaneously at right axilla with 1 × 10^5^to 1 × 10^7^live tumor cells per mouse. Reagents -------- PE-conjugated anti-mouse CD4, Cychrome-conjugated anti-mouse CD25 antibodies were purchased from eBioscience. Red blood cell lysis buffer is composed of 0.155 M ammonium chloride, 0.01 M potassium bicarbonate, and 0.1 mM EDTA. Fixation solution contains 1% paraformaldehyde in PBS. Samples preparation and flow cytometry -------------------------------------- Mouse peripheral blood was collected from orbital plexus and anticoagulated with 20 U/ml sodium heparin. Single-cell suspensions of splenocytes were prepared by grinding the spleen with the plunger of a disposable syringe, passing the ground spleen through nylon mesh, and suspending the cells in PBS. Mouse peripheral blood or spleen single-cell suspensions were stained with PE-conjugated anti-mouse CD4 and Cychrome-conjugated anti-mouse CD25 antibodies at 4°C for 30 minutes. Then, erythrocytes were lysed by red blood cell lysis buffer. After wash with PBS, the samples were fixed with fixation solution and analyzed on a FACScalibur™ flow cytometer (BD Biosciences) with CELLQuest™ software. Statistical Analysis -------------------- The data are summarized as the mean ± standard error. Statistical analysis was performed using the Student ttest, statistical significance was accepted at the P\< 0.05 level. Results ======= CD4+CD25+/CD4+ in peripheral blood and spleens from normal BALB/c mice ---------------------------------------------------------------------- To evaluate the normal proportion of CD4+CD25+/CD4+ in mice, 6 to 8 weeks normal BALB/c mice (n = 10) were sacrificed to test the proportion in spleen and peripheral blood lymphocytes by flow cytometry using anti-mouse CD4 and CD25 antibodies. The total CD4+ lymphocytes, CD4+CD25+ subset, and CD4+CD25+/CD4+ in spleen or peripheral blood lymphocytes were shown in Table [1](#T1){ref-type="table"}. In normal mice, the CD4+CD25+ T~R~cells appear in spleen or peripheral blood in a relative stable percentage manner. The proportion of CD4+CD25+/CD4+ in peripheral blood was 6.19 ± 0.86%, which is in accordance with the results reported by others \[[@B3]\]. Otherwise, the CD4+CD25+/CD4+ proportion in spleen was higher than that in peripheral blood (10.23 ± 1.88% vs 6.19 ± 0.86%, P\< 0.001). And, the higher level of the proportion in spleen is due to a lower level of the total CD4+ lymphocytes (CD4+CD25+ plus CD4+CD25-) in spleen than that in peripheral blood (37.06 ± 5.76 vs 56.80 ± 6.38, P\< 0.001). The representable figures of peripheral blood and spleen lymphocytes double stained with CD4 and CD25 antibodies were shown in Figure [1](#F1){ref-type="fig"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### The percentages of CD4+CD25+ and CD4+, and the proportions of CD4+CD25+/CD4+ in peripheral blood and spleen lymphocytes from normal BALB/c mice. ::: CD4+ CD4+CD25+ CD4+CD25+/CD4+ total lymphocyte --------------------------- -------------- ------------- ---------------- ----------------------- peripheral blood (n = 10) 56.80 ± 6.38 3.50 ± 0.45 6.19 ± 0.86 6.73 ± 0.84 (10^9^/L) spleen (n = 10) 37.06 ± 5.76 3.79 ± 0.93 10.23 ± 1.88 1.54 ± 0.23 (× 10^8^) Pvalue \<0.001 0.38 \<0.001 \- ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The proportions of CD4+CD25+ subset in peripheral blood and spleen lymphocytes from normal BALB/c mice. Mouse peripheral blood (A) or spleen single-cell suspensions (B) were collected or prepared, and stained with PE-conjugated anti-mouse CD4 and Cychrome-conjugated anti-mouse CD25 antibodies, after the lysis of erythrocytes, the samples were analyzed by flow cytometry. ::: ![](1479-5876-3-5-1) ::: CD4+CD25+/CD4+ in peripheral blood and spleens from C26 tumor-bearing BALB/c mice --------------------------------------------------------------------------------- To investigate the possible changes of the proportion in tumor bearing mice, 1 × 10^5^to 1 × 10^7^live C26 colon carcinoma cells were inoculated subcutaneously at right axilla of BALB/c mice respectively (n = 12). 20 days later, tumor nodules were formed at different sizes from 7 to 40 mm in diameters. The mice were sacrificed and peripheral blood and spleen lymphocytes were prepared for double staining with anti-mouse CD4 and CD25 antibodies. In peripheral blood, we did not find an increase in CD4+CD25+/CD4+ in tumor bearing mice, compared with that in normal mice. Otherwise, an increased proportion of CD4+CD25+/CD4+ in spleen lymphocytes was observed in tumor bearing mice, moreover, the proportion increased in accordance with the increase in tumor sizes, as shown in Figure [2A](#F2){ref-type="fig"}. The representable double staining figure of peripheral blood or spleen lymphocytes from tumor bearing mice were shown in Figure [2B--C](#F2){ref-type="fig"}. Considering the short tumor bearing duration, we prolonged the observation to 50 to 60 days, the increase in the proportion was not yet observed in peripheral blood (data not shown). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### The relationship between tumor sizes and the CD4+CD25+/CD4+ proportions in peripheral blood or spleen lymphocytes in tumor bearing mice. (A) 1 × 10^5^to 1 × 10^7^C26 colon carcinoma cells were inoculated subcutaneously at right axilla of BALB/c mice (n = 12). 20 days later, after tumor sizes were measured, the mice were sacrificed and peripheral blood lymphocytes (○) and spleen (■) lymphocytes were stained with anti-mouse CD4 and CD25 antibodies. x-axis represents the diameters of tumors; y-axis represents the proportion of CD4+CD25+/CD4+. The absolute total lymphocyte counts were 9.85 ± 2.34 (× 10^9^/L) in peripheral blood and 2.37 ± 0.77 (× 10^8^) in spleen. The representative figures of CD4+CD25+ subset in peripheral blood (B) or spleen (C) lymphocytes from tumor bearing mice were also shown. ::: ![](1479-5876-3-5-2) ::: The changes of the percentages of CD4+CD25+, CD4+CD25- and total CD4+ cells in spleen lymphocytes from tumor bearing mice ------------------------------------------------------------------------------------------------------------------------- The proportion of CD4+CD25+/CD4+ was determined by two factors: CD4+CD25+ in lymphocytes (numerator) and total CD4+ in lymphocytes (denominator). The increase of the proportion may be due to the increase of CD4+CD25+ subset or decrease of CD4+ subsets, or both. To investigate the possible reason that the proportion increased in spleen lymphocytes of tumor-bearing mice, we analyzed the CD4+CD25+ and total CD4+ cells in spleen lymphocytes. We found there was no obvious change in CD4+CD25+ in spleen lymphocytes from tumor bearing mice, otherwise, a decrease in total CD4+ lymphocytes was found with the increase of the tumor sizes, and the decrease was mainly due to the decrease of CD4+CD25- subset, as shown in Figure [3](#F3){ref-type="fig"} and Figure [2C](#F2){ref-type="fig"}. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### The relationship between tumor sizes and the percentages of total CD4+ (■), CD4+CD25- (□) or CD4+CD25+ (○) cells in spleen lymphocytes in tumor bearing mice. Samples were prepared and analyzed as in Figure 2, x-axis represents the diameter of tumors; y-axis represents the percentages in spleen lymphocytes. ::: ![](1479-5876-3-5-3) ::: Discussion ========== The identification of CD4+CD25+ as the phenotype of regulatory T lymphocytes is one of the highlights of recent immunological progress. These cells are proven to be involved in autoimmune diseases, transplantation tolerance and tumor immunity, etc \[[@B3]\]. The relationship between cancer and immune system has been studied and debated for a long time, now we know that immunodeficient or immunosuppressed humans or animals show greater incidences of cancer \[[@B22]\]; at the same time, immune function in cancer patients are often compromised by tumor itself or related treatment, and this often leads patients to disadvantageous situation. To restore the immune function in cancer patients is an important element in cancer treatment. The identification of CD4+CD25+ T~R~cells provided a new way to study relationship between tumor development and immune suppression. A higher proportion of CD4+CD25+ T~R~cells was found in peripheral blood of cancer patients and to be related to poor prognosis of the diseases \[[@B11],[@B12]\]. Depletion of CD4+CD25+ T~R~cells using anti-CD25 mAb could promote anti-tumor immunity \[[@B4],[@B15],[@B16]\]. All these indicated that CD4+CD25+ T~R~cells maybe an attractive target to restore or improve immune function in cancer treatment. In our animal experiments of antitumor immunotherapy, we did not find an increase of CD4+CD25+/CD4+ in peripheral blood in tumor bearing BALB/c mice, this is not in accordance with the results in cancer patients reported previously \[[@B11]\]. To find a way to evaluate the CD4+CD25+/CD4+ in antitumor immunotherapy targeting CD4+CD25+ T~R~cells, we analyzed the proportion in peripheral blood and spleen lymphocytes in normal or C26 colon-carcinoma-bearing mice by flow cytometry. In present study, the proportion of CD4+CD25+/CD4+ in peripheral blood of normal mice was about 6.19%, which was compatible with the results reported previously (5--10%). But in spleen lymphocytes from normal mice, we found a higher proportion of CD4+CD25+/CD4+ (around 10%), and the higher proportion is due to a lower level of total CD4+ lymphocytes in spleen, compared with that in peripheral blood, whereas the percentages of the CD4+CD25+ cells are similar. In C26-colon-carcinoma bearing BALB/c mice, we found an increase of CD4+CD25+/CD4+ in spleen but not in peripheral blood, furthermore, the proportion in spleen lymphocytes increased with the increase of tumor sizes. The phenomenon that the increase of the proportion in spleen separates with that in peripheral blood may be due to: 1). Spleen is a professional immune organ, which maybe more sensitive to the changes of immune situation than peripheral blood; 2). In this study, what we used is artificial tumor model, not spontaneous tumor model, and the tumor grew so quickly to cause mice moribund or dead that the increase of the proportion did not appear in peripheral blood. To observe the increase of the proportion in peripheral blood of tumor bearing mice may need a longer observation duration, or had better use spontaneous tumor models. In our experiments, we found the increase of CD4+CD25+/CD4+ is due to the decrease of CD4+ in lymphocytes, which is the result of decreased CD4+CD25- subset in lymphocytes. Our results support the observations reported by Sasada \[[@B12]\], in which the relative increase in the proportion of CD4+CD25+ T cells in patients with gastrointestinal malignancies are due to a selective reduction in the number of CD4+CD25- T cells. A possible explanation for this is that CD4+CD25- subset is more sensitive to clonal deletion or apoptosis than CD4+CD25+ T cells \[[@B12],[@B23],[@B24]\]. Furthermore, it is possible that some factors, such as tumor-derived antigens or molecules, can induce apoptosis selectively in the CD4+CD25- subset but not in the CD4+CD25+ subset \[[@B12]\]. The relationship between cancer and immune system has been debated for a long time. Our results provided direct evidence that the tumor might compromise the immune function, since our tumor model was established on BALB/c mice with normal immune function. It is known that tumor cells secrete immunosuppressive cytokines such as IL-10 and TGF-β \[[@B25]-[@B27]\], and the cytokines may induce CD4+CD25- lymphocytes to convert to CD4+CD25+ T~R~cells \[[@B28],[@B29]\]. These all support the theory that tumor may compromise the immune function. Conclusions =========== In normal BALB/c mice, CD4+CD25+/CD4+ proportion in spleen lymphocytes is higher than that in peripheral blood lymphocytes. In C26-colon-carcinoma bearing mice, no difference was found in the proportion in peripheral blood lymphocytes compared with normal mice; Otherwise, the proportion in spleen lymphocytes obviously increased, moreover, the proportion increased in accordance with the increase of tumor sizes. The increase of the proportion is due to the decrease of total CD4+ in lymphocytes, which is resulted from decreased CD4+CD25- subset in lymphocytes. Our observation suggest the CD4+CD25+/CD4+ proportion in spleen lymphocytes might be a sensitive index to evaluate the T~R~in tumor mouse models rather than that in peripheral blood lymphocytes, and our results provide some information on strategies of antitumor immunotherapy targeting CD4+CD25+ regulatory T lymphocytes. Acknowledgements ================ This work was supported partly by the China Postdoctoral Science Foundation (No. 2004035180).	PubMed Central	2024-06-05T03:55:52.925598	2005-1-28	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549051/", "journal": "J Transl Med. 2005 Jan 28; 3:5", "authors": [ { "first": "Ji-Yan", "last": "Liu" }, { "first": "Xiao-Shi", "last": "Zhang" }, { "first": "Ya", "last": "Ding" }, { "first": "Rui-Qing", "last": "Peng" }, { "first": "Xia", "last": "Cheng" }, { "first": "Nian-Hua", "last": "Zhang" }, { "first": "Jian-Chuan", "last": "Xia" }, { "first": "Yi-Xin", "last": "Zeng" } ] }
PMC549061	Background ========== Telomeres are the nucleoprotein ends of linear eukaryotic chromosomes. In most organisms, telomeric DNA consists of a simple, repeated sequence with a G-rich strand running 5\' to 3\' towards the chromosome end, and terminates with a short, single-stranded 3\' overhang (reviewed in \[[@B1],[@B2]\]). The length of the duplex repeated region varies, from 20 base-pairs (bp) in hypotrichous ciliated protozoa to around 300 bp in yeast and several kilobases (kb) in mammalian cells. These DNA repeats recruit telomeric proteins to form the telosome, a structure that resists nucleolytic degradation and prevents chromosome ends from eliciting recombination and end-joining pathways for repairing double-strand DNA breaks \[[@B3]\]. Telomeres are also essential for the complete replication of chromosomes, because conventional DNA polymerases do not copy the extreme ends of linear DNA molecules. In the absence of a mechanism to compensate for this \'end-replication problem\', progressive telomere shortening leads to replicative senescence, which in yeast is characterized by chromosome instability and low cell viability \[[@B4],[@B5]\]. Replicative senescence in mammals is characterized by growth arrest and altered gene expression \[[@B6]\]. The end-replication problem is managed in most eukaryotes by the enzyme telomerase, which adds telomeric DNA sequences to the 3\' end of chromosomes through the action of its catalytic subunit and RNA template (reviewed in \[[@B7]\]). DNA polymerase then forms duplex DNA by synthesizing the complementary C-rich strand of the telomere \[[@B8]\]. In fission yeast, the catalytic subunit of telomerase is encoded by the gene trt1^+^\[[@B9]\]. In some cases, cells can endure the loss of telomerase and give rise to a population of survivors. In the budding yeast Saccharomyces cerevisiae, survivors maintain long, heterogeneous telomeres on linear chromosomes using a RAD52-dependent homologous-recombination pathway \[[@B10]\]. Global gene-expression profiles of budding yeast lacking telomerase revealed the induction of a DNA damage response when telomeres were short and a sustained stress response in survivors \[[@B11]\]. Human alternative lengthening of telomeres (ALT) cells are cancerous cells lacking detectable telomerase activity that maintain long, heterogeneous telomeres using what is believed to be a strand invasion mechanism \[[@B12],[@B13]\]. S. pombecells without telomerase cease dividing after about 120 generations, and can give rise to a subpopulation of survivors \[[@B14]\]. Interestingly, these survivors have either circular chromosomes or linear chromosomes with long, heterogeneous amplified telomeres (presumably maintained through recombination) that resemble their budding yeast and human ALT-cell counterparts. While survivors with circular chromosomes arise more frequently, those with linear chromosomes grow faster \[[@B14]\]. Circular chromosomes in S. pombeare believed to form as a result of the genomic instability due to loss of telomeres, which normally prevent end-joining and suppress recombination. Interchromosomal fusions yield unstable dicentric chromosomes, while intrachromosomal fusions produce circular chromosomes. S. pombe, with only three chromosomes, is more likely than other organisms with larger numbers of chromosomes to successfully form exclusively intrachromosomal fusions \[[@B14],[@B15]\]. S. pombestrains with circular chromosomes also result after concurrent deletion of rad3^+^and tel1^+^, two genes with sequence similarity to human ATM(ataxia telangiectasia mutated) \[[@B15]\]. Although S. pombesurvivors with linear chromosomes grow remarkably well and have a morphology similar to wild-type cells, survivors with circular chromosomes display obvious growth defects such as slower growth rates and larger sizes \[[@B14]\]. Survivors with circular chromosomes presumably cope with impaired DNA segregation, and perhaps DNA breakage and rearrangement. We hypothesized that cells would show altered expression of genes necessary for coping with the loss of telomerase and concomitant changes in chromosome structure. In this study, we determined the S. pombeglobal gene-expression response to loss of trt1^+^to investigate changes in expression of genes during senescence, and to compare survivors with circular or linear chromosomes. We report that survivors with circular chromosomes maintain an extended stress response not observed in survivors with linear chromosomes. Furthermore, we present evidence for regulation of a telomeric gene by the RNAi machinery. Results ======= Wild-type reference strains --------------------------- Wild-type isogenic reference strains WT 3 and WT 5 were used to determine relative gene-expression changes in trt1^-^samples. Before averaging the expression values from the two reference strains, the similarity of their expression profiles was assessed. The dye ratios measured by microarray for each strain were plotted against each other (Figure [1a](#F1){ref-type="fig"}). All genes had expression values that varied less than twofold between the two samples, indicating that the samples were highly similar. The wild-type values used in this paper are thus the average expression values of strains WT 3 and WT 5. To learn whether changes in gene expression would result from subjecting cells to the continuous growth program for 15 days, gene-expression values from strain WT 5 on day 1 of the growth curve were compared with those of the same strain harvested on day 15 (Figure [1b](#F1){ref-type="fig"}). Only three genes (SPBC354.08c, atp8^+^and cox1^+^) changed their expression values by more than twofold, and they were only slightly greater; thus, the vast majority of genes do not have altered expression as a result of long-term growth in culture, provided that expression is measured while the cells are in early log phase (see Materials and methods). These three genes also had expression changes of more than twofold in one or more conditions measured for trt1^-^cells, but given their variable expression in wild-type cells, these changes were most probably unrelated to the absence of telomerase. Watching cells pass through crisis and characterizing survivors --------------------------------------------------------------- Diploid S. pombecells that were heterozygous for trt1^+^and able to maintain full-length telomeres were sporulated, and the resulting trt1^+^and trt1^-^cells propagated through a 15-day growth curve (Figure [2a](#F2){ref-type="fig"}). Cells lacking telomerase gave rise to survivors after day 8 concomitant with heterogeneous amplified telomeric repeats and telomere-associated sequence (TAS) (Figures [2b-d](#F2){ref-type="fig"}), indicative of linear chromosomes \[[@B14]\]. By day 15, the culture was dominated by faster-growing cells with linear chromosomes. The linear structure of these chromosomes was confirmed by their ability to enter a pulsed-field gel (Figure [3b](#F3){ref-type="fig"}, lane g), and the existence of terminal chromosome fragments C, I, L and M after digestion of chromosomes with NotI (Figure [3a-d](#F3){ref-type="fig"}, lane e) \[[@B14],[@B15]\]. Cells passing through crisis (days 7 and 9) also had weak hybridization signals for the C+M and I+L fragments (Figure [3d](#F3){ref-type="fig"}, lanes c-d), suggesting a mix of cells with either linear or circular chromosomes, or perhaps cells containing both linear and circular chromosomes. The inability to detect intact chromosomal DNA at day 7 (Figure [3b](#F3){ref-type="fig"}, lane e) may have resulted from the presence of cells with circularized chromosomes (Figure [3d](#F3){ref-type="fig"}, lane c) that do not enter pulsed-field gels. Strains C1 and C5 had circular chromosomes as evidenced by lack of telomeric repeats (data not shown), lack of TAS2 sequence (data not shown), the inability of chromosomes to enter a pulsed-field gel (Figure [3b](#F3){ref-type="fig"}, lanes b-c), the lack of terminal chromosome fragments C, I, L and M (Figures [3c,d](#F3){ref-type="fig"}, lanes g-h) \[[@B14],[@B15]\], and hybridization signals to fragments C+M and I+L (Figure [3d](#F3){ref-type="fig"}, lanes g-h). Two waves of expression are observed in the growth curve -------------------------------------------------------- Two waves of altered gene expression were seen during the growth curve (Figure [4a](#F4){ref-type="fig"}), the first with a peak at day 7, consisting of around 110 genes with expression upregulated twofold or more, and the second with a peak at day 9, consisting of three microarray signals that appear to represent a single ORF (see below) (Figure [4a](#F4){ref-type="fig"}). The peak of the first wave (day 7) was nearly coincident with crisis in the cell population (day 8) (Figure [2a](#F2){ref-type="fig"}) and the time when telomeres were shortest (near day 7) (Figure [2c,d](#F2){ref-type="fig"}). The second peak of gene expression at day 9 was coincident with the emergence of survivors (Figure [2a-d](#F2){ref-type="fig"}). The vast majority of expression changes involved upregulation, and only seven genes had downregulated expression of twofold or greater on two or more days of the growth curve. Notably, there were three cases of reduction in expression greater than tenfold: trt1^+^(intentionally knocked out), SPAC2E1P3.04 (a predicted copper amine oxidase) and SPAC2E1P3.05c (unknown function). Hybridizations of genomic DNA to microarrays (data not shown) revealed that genes SPAC2E1P3.04 and SPAC2E1P3.05c were deleted from the genome in all strains except WT 3, WT 5 and C1. Interestingly, these two genes are within about 4 kb of transposable element SPAC167.08 (Tf2-2), suggesting a hotspot for DNA excision. In no case was gene amplification detected by genomic hybridization (data not shown), so the observed increases in expression were most probably due to transcriptional or post-transcriptional regulation, as opposed to changes in gene copy number. Gene-expression changes in trt1^-^cells ----------------------------------------- Because a relatively large number of trt1^-^strains were studied, the identification of genes with consistently altered expression was facilitated by selecting those genes with expression changes of twofold or more in two or more days of the growth curve or, alternatively, in both strains C1 and C5. This criterion was met by 123 genes, of which 54 (44%) overlapped between the growth curve and survivors with circularized chromosomes. In addition, of the 67 genes that had their expression changed twofold or more exclusively in the growth curve, many displayed altered expression just below the cutoff in survivors with circularized chromosomes. Two genes - SPBC1683.06c (a predicted uridine ribohydrolase) and SPBC1198.01 (a predicted formaldehyde dehydrogenase) - had expression changes of twofold or more in both strains C1 and C5, but no significant changes during the growth curve. As a measure of confidence, 84 of the 123 genes (approximately 68%) met a more stringent criterion requiring a gene to change its expression in three or more of the 17 conditions. Additional confidence that expression changes scored as significant were not false positives came from the remarkably continuous manner in which gene expression changed throughout the growth curve (Figure [4a](#F4){ref-type="fig"}). The 123 genes with altered expression encompass a broad range of functions, but were especially enriched in genes associated with energy production and carbohydrate metabolism (Table [1](#T1){ref-type="table"}). There were seven pseudogenes and 29 predicted genes that did not have assigned functions at the time of writing. For nearly all the gene-type categories, there was a larger number of genes with altered expression in the growth curve than in the survivors with circular chromosomes (Table [1](#T1){ref-type="table"}). This difference may be attributable to the fact that cells in the growth curve were experiencing crisis whereas strains C1 and C5 were survivors, presumably with established mechanisms to cope with the absence of or the loss of telomeres. The telomerase-deletion response had a large overlap with genes that changed expression in response to environmental stresses. Fission yeast stress-response genes can be separated into a CESR, in which genes changed expression in all or most of the stresses studied (oxidative stress, heavy metals, heat shock, osmotic stress and DNA damage), and into more specific stress responses \[[@B16]\]. Of the 123 genes with altered expression in trt1^-^cells, 48 (about 39%) also had upregulated expression among a conservative list of CESR genes (P\~ 10^-77^) \[[@B16]\], and two genes had downregulated expression in the CESR and in this study. Of the 110 genes with expression upregulated twofold or more on day 7 of the growth curve, 44% overlapped with the CESR. Comparison with a less conservative list of CESR genes \[[@B16]\] suggested that 54% of the 123 genes with altered expression in trt1^-^cells had overlap with the CESR (P\~ 10^-81^). With respect to specific stress responses \[[@B16]\], there were 17/123 genes in common with the oxidative stress response (P\~ 10^-32^), and 11/123 genes in common with the heat stress response (P\~ 10^-24^). The stress response study found that the DNA damage response and the oxidative stress response have substantial overlap \[[@B16]\]. Therefore, the genes with altered expression in this study that overlap with the oxidative-stress response may represent a DNA damage response to short telomeres. Chromosome structure and gene expression ---------------------------------------- Comparisons of all the gene-expression profiles in this study revealed striking differences between the profiles of survivors with linear chromosomes versus those with circular chromosomes. Survivors with linear chromosomes (days 12-15 of the growth curve) had gene-expression patterns similar to those of cells with native telomeres in the first two days of the growth curve. To illustrate, by day 12 of the growth curve, the gene-expression profiles of survivors became relatively constant and remained so through day 15. The profiles of days 12-15 appear most similar to days 1 and 2 of the growth curve, immediately after cells lost telomerase and were experiencing shortening telomeres (Figure [4b](#F4){ref-type="fig"}). This observation was confirmed by hierarchical clustering (Figure [4c](#F4){ref-type="fig"}). Conversely, survivors with circular chromosomes had gene-expression profiles that most resembled those of cells in crisis during days 5-8 of the growth curve (Figure [4b,c](#F4){ref-type="fig"}). Sustained stress response in survivors with circular chromosomes ---------------------------------------------------------------- There were 54 genes with clearly altered expression (twofold or more) mainly during crisis in the growth curve that also had altered expression in the survivors with circular chromosomes (Table [2](#T2){ref-type="table"}, Figure [5](#F5){ref-type="fig"}). The expression of all but three of these 54 genes was not altered in survivors with linear chromosomes (growth curve days 12-15) (Table [2](#T2){ref-type="table"}). Of the 54 genes, 30 (56%) overlapped with the conservative list of CESR genes (P\~ 10^-46^), and eight genes (15%) overlapped with the oxidative stress response (P\~ 10^-14^). There were 8/54 genes (15%) that overlapped with the heat stress response (P\~ 10^-17^). Because of the extensive overlap of the 54 genes with the CESR, we conclude that survivors with circular chromosomes had a sustained stress response. Of the 54 genes, 51 represent a gene-expression signature that differentiates survivors with circular chromosomes from those with linear chromosomes. As an independent test of whether these 51 genes can serve as a signature for cells with circularized chromosomes, two additional cultures (strains H1 and H2, see Materials and methods) with circularized chromosomes were grown and analyzed by microarray. Both strains clearly displayed altered expression of the 51 genes whereas survivors with linear chromosomes did not (Figure [5](#F5){ref-type="fig"}), thus validating this gene signature. No altered expression of genes encoding recombination and telomere factors -------------------------------------------------------------------------- One feature of microarray studies is that genes not previously recognized to be under the control of a common regulator can often be associated by similar expression patterns \[[@B17]\]. On the basis of this hypothesis, a list of genes known to be involved in telomere maintenance and recombination was inspected. However, the expression patterns of all these genes were not substantially changed throughout the course of the study (data not shown). Genes investigated included pku70^+^and lig4^+^, which encode components of the non-homologous end-joining pathway \[[@B18]\]; taz1^+^\[[@B19]\] and pot1^+^\[[@B20]\] encoding telomere DNA-binding proteins; telomerase component est1^+^\[[@B21]\]; homologous recombination-related genes rad22^+^\[[@B22]\], rhp54^+^\[[@B23]\], rad32^+^\[[@B24]\] and rhp51^+^\[[@B25]\]; RecQ helicase gene rqh1^+^\[[@B26]\]; silencing component clr4^+^\[[@B27]\]; and telomere maintenance components pof3^+^\[[@B28]\] and rad3^+^\[[@B15]\]. Interestingly, even though pof3^+^and clr4^+^expression did not change, the genes with altered expression in this study had a statistically significant overlap with the lists of genes with induced expression in pof3mutants (P\< 10^-45^) \[[@B28]\] and clr4mutants (P\< 10^-45^) \[[@B29]\]; a significant correlation was also observed with genes that changed expression in the RNA interference (RNAi)-machinery mutants dcr1^+^, ago1^+^and rdp1^+^(P\~ 10^-22^) \[[@B29]\]. These genes with altered expression may act in common pathways downstream of trt1^+^, clr4^+^, pof3^+^and the RNAi machinery. A second wave of expression represents sub-telomeric ORF with homology to RecQ helicases and dhrepeats -------------------------------------------------------------------------------------------------------- The second wave of gene-expression changes during the growth curve (Figure [4a](#F4){ref-type="fig"}) consisted of three microarray signals: SPAC212.11 (largest magnitude), SPAC212.06 (second largest magnitude) and the reverse transcript of centromeric dhrepeats \[[@B30]\]. Inspection of the sequences revealed that the microarray signals from SPAC212.06 and centromeric dhrepeats most probably resulted from cross-hybridization with the SPAC212.11 transcript (see Materials and methods). A BLAST search of the SPAC212.11 predicted protein sequence found that the ORF has the most similarity to RecQ DNA helicases of superfamily II (Figure [6](#F6){ref-type="fig"}) (reviewed in \[[@B31]\]). We report a role for the helicase in cells passing through crisis in a separate study (J.G.M., K.J. Goodrich, J.B. and T.R.C., unpublished work) and investigate its transcriptional regulation here. SPAC212.11 is the last sequenced ORF on the left arm of chromosome I. The sub-telomeric regions of chromosomes I and II have significant similarity \[[@B32]\]. A BLAST search performed with the SPAC212.11 DNA sequence (5.6 kb) revealed a paralog, SPBCPT2R1.08c (6.3 kb), located on the right arm of chromosome II (the microarray had no probe for SPBCPT2R1.08c), and partial homology on the right arm of chromosome I. The annotated sequence of SPBCPT2R1.08c includes the entirety of the SPAC212.11 sequence with only a single base change. The SPAC212.11 sequence does not contain a stop codon because the ORF is located at the end of the sequencing contig, which ended before a stop codon was reached. Comparison with the annotated SPBCPT2R1.08c sequence suggests that SPAC212.11 has an additional 95 bp before the stop codon. Both SPBCPT2R1.08c and SPAC212.11 are the last predicted genes on their respective sub-telomeric sequencing contigs. Analysis of contig pT2R1 revealed that the 3\' end of SPBCPT2R1.08c is approximately 2.8 kb upstream from the start of TAS3 (Figure [2b](#F2){ref-type="fig"}). Since TAS3 is around 7 kb from the chromosome end, the 3\' end of SPBCPT2R1.08c is approximately 10 kb from the telomeric repeats. It is not known which of the paralogs contributed to the SPAC212.11 microarray signal. For the sake of simplicity, further references in the text to \'the putative helicase\' are meant to include SPAC212.11, SPBCPT2R1.08c and any paralogs, collectively. The nucleotide BLAST search performed with the SPAC212.11 sequence also revealed that the ORF contains regions of homology to dhrepeats (Figure [6](#F6){ref-type="fig"}), which are targeted for heterochromatin formation via an RNAi-mediated mechanism in S. pombe\[[@B33],[@B34]\]. These repeats are typically located at centromeres and the Kregion of the mating-type locus \[[@B30],[@B33],[@B35]-[@B37]\]. RNAi machinery implicated in controlling expression of the putative helicase ---------------------------------------------------------------------------- Centromeric repeats, previously thought to be transcriptionally silent, are transcribed in both the forward and reverse directions, leading to formation of double-stranded RNA (dsRNA). However, these transcripts do not accumulate in wild-type cells. Reverse-strand centromeric transcripts are synthesized and rapidly processed by the RNAi machinery, while forward-strand synthesis is silenced transcriptionally. RNA-dependent RNA polymerase (Rdp1) associates with centromeric repeat DNA and may use siRNAs corresponding to centromeric transcripts \[[@B38]\] to prime forward transcription from reverse-strand templates, thus resulting in dsRNA formation and maintenance of the heterochromatic state. In the RNAi mutants dcr1^-^, ago1^-^and rdp1^-^, centromeric silencing is abolished and accumulation of both forward and reverse centromeric transcripts is observed \[[@B33]\]. Microarray, northern blot and reverse transcription (RT)-PCR analysis indicated that the putative helicase gene was robustly expressed in cells emerging from crisis, but was weakly (or not at all) expressed in wild-type cells, strains C1 and C5 and survivors with linear chromosomes (Figures [4a,b](#F4){ref-type="fig"}, [7a](#F7){ref-type="fig"}, and data not shown). As the putative helicase transcript was not detectable by northern blot in wild-type cells (data not shown), we hypothesized that this ORF could be silenced by its dhrepeats, but that this silencing may have been disrupted in trt1^-^cells as a result of genomic instability. Arguing against this hypothesis, however, Southern analysis with probe P~5\'~(Figure [6](#F6){ref-type="fig"}), which is specific for the helicase, did not reveal any DNA rearrangements during crisis close to the helicase that might have contributed to loss of silencing (data not shown). Nevertheless, the loss of silencing observed might lead to expression of both strands of the putative helicase, as was found for centromeric dhrepeats in RNAi mutants. To test for the presence of both strands, strand-specific RT-PCR was used with primers spanning the dhrepeats of the putative helicase (region P~dh~in Figure [6](#F6){ref-type="fig"}). The forward strand was expressed at levels higher than in wild type in cells from days 7, 9 and 15 of the growth curve. These results were consistent with microarray analysis that detected the 3\' end of the forward transcript (Figure [7a](#F7){ref-type="fig"}). The reverse strand was weakly detectable in cells from days 7 and 9 of the growth curve (Figure [7a](#F7){ref-type="fig"}). dsRNA arising from the repeats presumably could have formed on days 7 and 9 of the growth curve, but why such RNA was not all processed by the RNAi machinery is not clear. On days 7 and 9 of the growth curve, the RNAi machinery was not apparently affected by the mutation of telomerase as centromeric dhrepeat transcripts were not detected by RT-PCR (Figure [7a](#F7){ref-type="fig"}). We next hypothesized that if the RNAi machinery were involved in transcriptional silencing of the putative helicase in wild-type cells, transcript should accumulate in mutant RNAi strains. Strikingly, both ago1^-^and dcr1^-^strains displayed significant accumulation of the forward transcript of the putative helicase, and the rdp1^-^strain showed slightly increased accumulation with respect to wild-type (Figure [7b](#F7){ref-type="fig"}). The reverse strand did not accumulate in these three strains. Thus, transcriptional silencing of the putative helicase appeared to be relieved in RNAi mutants, implicating RNAi in the control of expression of this ORF. Discussion ========== Correlation of chromosome structure and gene expression ------------------------------------------------------- The genome-wide survey of expressed genes in this study provided an opportunity to investigate the cellular response to loss of the gene for the telomerase catalytic subunit Trt1. A major finding was the tight correlation between the structures of chromosomes in survivors and gene expression profiles. Survivors with linear chromosomes had expression profiles remarkably similar to cells with canonical - yet shortened - telomeres, whereas cells with circular chromosomes maintained the upregulated expression of a significant number of genes that also had upregulated expression during senescence. The stress response in survivors with circular chromosomes had significant overlaps with the S. pombeCESR and with the heat and oxidative stress responses. The CESR consists of genes that had upregulated expression in all or most responses to oxidative stress, heavy metal stress, heat shock, osmotic stress and DNA damage \[[@B16]\]. The stress response may persist in survivors with circularized chromosomes because of impaired DNA segregation and DNA breakage and rearrangement. Indeed, compared with wild-type cells, survivors with circular chromosomes are larger and have slower growth rates, indicating that functions related to cell division are impaired \[[@B14]\]. Telomeric repeats contribute to recruiting the molecular components collectively involved in the protective capping of chromosome ends \[[@B20],[@B39],[@B40]\]. These repeats are maintained in the absence of telomerase in cells from diverse organisms that normally use telomerase (reviewed in \[[@B3]\]). Interestingly, the survivors with linear chromosomes abated their stress response concomitant with the appearance of amplified telomeric and TAS repeats as rare survivors took over the population, suggesting that the repeats helped to ameliorate the stress response. Neither cells in the growth curve that experienced shortened telomeres nor survivors with long telomeres displayed upregulation of telomeric gene expression, supporting the notion that telomeric length changes alone do not affect gene expression in S. pombe\[[@B19]\]. In addition, in survivors with circular chromosomes, only eight microarray signals, corresponding to as few as two genes (due to cross-hybridization) near former telomeres had altered expression, although such changes might have been expected as a result of the large alterations in chromosome structure at these sites. Comparison with the budding yeast response to loss of telomerase ---------------------------------------------------------------- As in fission yeast, genes with changed expression in the budding yeast response to loss of telomerase had significant overlaps with genes whose expression was altered by environmental stresses such as heat shock, osmotic shock, dithiothreitol (DTT),nitrogen starvation and peroxide (\[[@B11],[@B41]\] see also \[[@B42]\]). A difference in the stress responses between the two yeasts was that in budding yeast a large but specific subset of the environmental stress-response genes persisted in survivors with linear chromosomes four days after crisis, whereas in fission yeast survivors with linear chromosomes, the stress response mostly abated by the fourth day after crisis (Figure [4b](#F4){ref-type="fig"}, day 12). The different yeast responses may be due to a fission yeast telomere structure that was not as strongly recognized as aberrant, perhaps mitigating a DNA-damage response. It is also possible that had budding yeast survivors been followed longer, providing a period for adaptation, the stress response would have subsided. In fission yeast, the expression of a number of mitochondrial ATP synthase genes was upregulated (Table [1](#T1){ref-type="table"}) with orthologs similarly induced in budding yeast. In both cases, the changes did not overlap with the DNA-damage responses of the yeasts, further supporting a link between short telomeres and alterations in the metabolic program suggested by Nautiyal et al. \[[@B11]\]. Significance of putative RecQ helicase -------------------------------------- RecQ helicases have recently been implicated in telomerase-independent telomere maintenance in both S. cerevisiaeand human ALT cells. BLM and WRN, human RecQ helicases associated with cancer and disease \[[@B31]\], have both been shown to associate with duplex telomere repeat binding protein TRF2 in vivo, and BLM co-localizes to telomeric foci exclusively in ALT cells \[[@B43]-[@B45]\]. The S. cerevisiaeortholog of human WRN and BLM, Sgs1, was also shown to be required for telomere elongation of type II survivors in the absence of telomerase \[[@B46]-[@B48]\]. The long, heterogeneous telomeres of S. pombesurvivors with linear chromosomes are similar to those of S. cerevisiaesurvivors and human ALT cells, suggesting a role for RecQ helicases in fission yeast telomerase-independent telomere maintenance. dhrepeats and RNAi at the telomere ------------------------------------ This is the first report to our knowledge of naturally occurring dhrepeats outside of the centromeric and mating-type regions in fission yeast. We have presented several results that suggest that sub-telomeric dhrepeats promote heterochromatin formation at the helicase locus. First, transcript from this ORF was only weakly expressed in wild-type cells as determined by RT-PCR (Figure [7a](#F7){ref-type="fig"}) (and was not detectable at all by northern hybridization, data not shown), consistent with transcriptional regulation of this ORF by heterochromatin. Second, expression of the putative helicase was robust in ago1^-^and dcr1^-^mutants, which would be expected if RNAi has a role in transcriptionally silencing this ORF. In trt1^-^mutants experiencing genomic instability, we detected both forward and reverse transcripts of sub-telomeric dhrepeats (Figure [7a](#F7){ref-type="fig"}). The presence of these complementary transcripts suggests the existence of dsRNA that had not been processed by the RNAi machinery, consistent with a lack of silencing at this locus. Intriguingly, after maximal expression of both strands on day 9 of the growth curve, subsequent downregulation was observed by day 15 (Figure [7a](#F7){ref-type="fig"}), consistent with restoration of silencing. While the finding of homology with dhrepeats at the sub-telomere was unexpected, dhrepeats have been shown to function in silencing at sites outside of centromeres and the mating-type locus. Reporter genes fused to centromeric repeat fragments as short as 580 bp were silenced when integrated at ectopic locations in the genome \[[@B49],[@B50]\] and this silencing required the RNAi machinery \[[@B51],[@B52]\]. The longest (nearly continuous) stretch of sequence with homology to dhrepeats found in the helicase ORF was about 600 bp (Figure [6](#F6){ref-type="fig"}), presumably long enough to promote heterochromatin formation. In addition, RNAi-mediated silencing triggered by both a synthetic hairpin RNA and transposon long terminal repeats have been shown to induce heterochromatin formation away from centromeres and the mating-type locus \[[@B53]\]. In a separate study, telomeric silencing of a reporter gene and binding of Swi6 at the telomere were not affected in dcr1^-^, ago1^-^and rdp1^-^mutants \[[@B54]\]. The lack of an observed effect may have been due to the ability of telomeric repeats to recruit silencing factors. Indeed, telomeric heterochromatin is largely promoted by telomeric repeats. However, the study by Hall and co-workers \[[@B54]\] did report defective mitotic and meiotic telomere clustering in RNAi mutants, supporting a role for RNAi at telomeres. Given the correlation between disruption of telomeric heterochromatin and expression of the helicase ORF, events other than telomere erosion that disrupt heterochromatin might also induce helicase expression. Materials and methods ===================== Strain construction ------------------- The trt1^+^and trt1^-^cells used in this study were generated by sporulating S. pombediploid strain G4 (h^-^/h^+^ade6-M210/ade6-M216 trt1^+^/trt1^-^) on ME plates \[[@B18]\]. The parent diploid strain was made heterozygous for trt1^+^by using a standard two-step integration procedure \[[@B55]\] with a linearized plasmid containing about 1 kb each of the 5\' and 3\' flanking regions of the trt1^+^ORF separated by HSV1-tkand KanMX4\[[@B56]\]. The plasmid was linearized in the middle of the 3\' flanking region with FseI and transformed using the lithium acetate method \[[@B57]\] into a diploid strain created by crossing PP68 (h^-^ade6-M210) and PP69 (h^+^ade6-M216). Cells were re-streaked twice on yeast extract low adenine (YEA) + geneticin plates \[[@B18]\] to select for stable genomic integrants, which were subsequently confirmed by Southern hybridization to a uniquely sized EcoRI restriction fragment 3\' of trt1^+^which was present only in integrants. Cells were then plated on YEA + 50 μM 5-fluorodeoxyuridine (5-FUdR) plates to select for those that had excised HSV1-tk, KanMX4 and the XbaI-XhoI fragment (around 5 kb) of trt1^+^from their genomes. Random surviving colonies were screened for heterozygous diploids by Southern hybridization to the 3\' region of the trt1^+^KpnI restriction fragment. The heterozygous state was evidenced by hybridization signals to both full-length trt1^+^and a shortened, nonfunctional version. Loss of markers was confirmed by lack of a Southern hybridization signal to HSV1-tk, and by lack of growth on YEA + geneticin plates. Selection of strains -------------------- After germination of G4 and growth of spores at 32°C for three days on YEA plates \[[@B18]\], plates were stored at 4°C while the genotypes of random colonies were determined. A portion of single colonies was used for crossing and visual inspection to identify those that had an h^-^ade6-M210genotype, which were further screened by Southern hybridization for the presence or absence of trt1^+^(performed as described in \'Strain construction\' below). Colonies were subsequently used as described in \'Growth curve\', or alternatively used to create strains C1, C5, H1 and H2. Strains C1, C5, H1 and H2 were created from four separate trt1^-^colonies that were each successively re-streaked on YEA plates 15 times (with growth for 2 to 3 days at 32°C between re-streaks), to permit colonies to form without competition from faster-growing survivors with linear chromosomes. During this time cells were presumed to senesce and give rise to survivors. After the last re-streak, a single colony from each strain was randomly selected and used to prepare freeze stocks. Growth curve ------------ Three strains were grown: two wild-type isolates (h^-^ade6-M210 trt1^+^) designated WT 3 and WT 5, and a single mutant isolate (h^-^ade6-M210 trt1^-^) designated as \'GC Day X\', where X represents the day of the growth curve that cells were collected. Single colonies were used to inoculate 5-ml starter cultures in yeast extract full supplements (YES) medium \[[@B18]\] and grown for 24 h with shaking at 32°C. Cells were counted and used to inoculate 200-ml YES cultures in 500-ml Erlenmeyer flasks at 2.5 × 10^4^cells/ml, and were grown in an incubator (Innova 4430, New Brunswick Scientific) with continuous shaking at 200 rpm at 32°C. Cell density was monitored by periodic counting, and a portion of the cells was harvested for microarray analysis and Southern hybridization when the density reached 3-5 × 10^6^cells/ml (early log phase). Cells harvested at this point were referred to as day 1 of the growth curve. The unharvested cells were permitted to continue growing until 24 h from the time of inoculation, at which time cells were counted and used to inoculate a fresh 200-ml YES culture at 2.5 × 10^4^cells/ml, and the process repeated for 15 days. To harvest cells for microarray analysis, a volume of culture containing approximately 1.6 × 10^8^cells was gently centrifuged at room temperature (2,000 rpm for 2 min), the supernatant removed, and the cell pellet snap-frozen in liquid N~2~. For Southern hybridization, approximately 2 × 10^8^cells were collected by centrifugation, washed twice in H~2~O, and snap-frozen in liquid N~2~. A portion of cells for pulsed-field gel analysis was also collected in the same manner as for Southern hybridization at the end of each 24 h period. trt1^-^cells were collected daily, WT 3 and WT 5 on day 1, and WT 5 on day 15. Growth and collection of strains C1, C5, H1 and H2 -------------------------------------------------- Cells were streaked onto YEA plates from freeze stocks, grown for 3 days at 32°C, and single colonies used to inoculate 5-ml starter cultures in YES medium. After 24 h, cells were counted and 200-ml YES cultures were inoculated at 2.5 × 10^4^cells/ml, and cultures grown with constant shaking at 200 rpm at 32°C. When the cell density reached around 3 × 10^6^cells/ml (early log phase), cells were collected as described in \'Growth curve\' for microarray analysis, Southern hybridization and pulsed-field gel electrophoresis. Strains H1 and H2 are trt1^-^isolates with circular chromosomes, as evidenced by pulsed-field gel electrophoresis (data not shown) Genomic DNA preparation and Southern hybridization -------------------------------------------------- DNA from approximately 2 × 10^8^S. pombecells was prepared as described \[[@B18]\]. After digestion with either EcoRI or HindIII, the DNA was subjected to electrophoresis on a 1% agarose gel in 1 × TBE (90 mM Tris, 90 mM borate, 2 mM EDTA pH 8.3). DNA was denatured by sodium hydroxide treatment and transferred to a nylon membrane (Hybond-N+ membrane, Amersham) by capillary transfer in 10 × SSC (1.5 M NaCl, 0.15 M sodium citrate). DNA was immobilized on the membrane by irradiation with 120 mJ/cm^2^at 254 nm in a UVStratalinker1800 (Stratagene). For molecular weight markers, the 1 kb DNA ladder (New England Biolabs) was labeled by filling in 5\' overhangs with \[α-^32^P\]dATP using DNA polymerase I Klenow fragment. Probes for pol1^+^, act1^+^, the putative helicase and the C, I, L and M chromosome fragments were generated by PCR amplification from a genomic DNA template and were gel purified. Probes were labeled by random-primed transcription of PCR products with the use of \[α-^32^P\]dCTP and High Prime Mix (Boehringer Mannheim). Probes specific for the telomeric and telomere-associated sequences were created with the use of gel-purified fragments of pNSU70 \[[@B58]\]. Pulsed-field gel electrophoresis -------------------------------- Cells (approximately 1 × 10^8^) were collected as described above. Plug preparation, chromosome digestion and electrophoresis were performed exactly as described \[[@B18]\]. DNA was visualized by staining with ethidium bromide (1 μg/ml) for 30 min. The gel was then irradiated with 120 mJ/cm^2^at 254 nm in a UVStratalinker1800 to nick the DNA, treated with HCl, NaOH and neutralization buffer, and processed as described in \'Southern hybridization\'. RT-PCR ------ RNA was prepared as for microarray analysis and used for RT-PCR (OneStep RT-PCR kit, Qiagen). First-strand cDNA synthesis was performed using primers complementary to either the forward or reverse strands. Both primers were present in subsequent cycles of PCR amplification after heat inactivation of reverse transcriptase at 95°C for 15 min. The control reaction lacking reverse transcriptase (act1^+^sense, -RT) was not subjected to first-strand cDNA synthesis, but was otherwise treated identically. Probes and PCR primers ---------------------- The PCR primers used to generate probes C, I, L, and M have been published previously \[[@B14]\]. The PCR primers spanning the regions described in Figure [6](#F6){ref-type="fig"} were: P~5\'~: 5\'-CTTCAAAAACTGCTAGAGATATCGCCGG-3\' and 5\'-GTACTGGTAGTCCTCTGATGTATGGG-3\' P~3\'~: 5\'-ATGCCCCGTACGCTTATCTA-3\' and 5\'-TTTGCCTTTCTAGCCCATGA-3\' P~dh~: 5\'-CAACACCAATACTGACGATGATG-3\' and 5\'-GCAATAGAACCAGCGGTTTG-3\' Primers for centromeric dhrepeats have been published previously \[[@B33]\]. RNA preparation and reference pool for microarrays -------------------------------------------------- Whole-cell RNA was isolated from S. pombecell pellets (\~1.6 × 10^8^cells) by hot-phenol extraction and purification with RNeasy columns (Qiagen) following a published protocol \[[@B59]\]. Aliquots (10 μg) were made (henceforth referred to as \'sample RNA\') and RNA quality was assessed by UV absorbance, by agarose gel electrophoresis to confirm intact rRNA bands, and by northern hybridization to act1^+^. A reference pool consisting of RNA from each sample was made, comprising 76% trt1^-^cells and 24% trt1^+^cells. This pool was divided into 10 μg aliquots (henceforth referred to as \'reference RNA\') and used as the reference RNA in all hybridization experiments reported here. A single large batch of YES medium was made at the start of the study and used to culture all cells analyzed by microarrays to prevent batch-to-batch medium variations that might yield artifactual microarray results. Microarray cDNA labeling, hybridization and data acquisition ------------------------------------------------------------ The procedures performed and the S. pombemicroarrays used have been described previously \[[@B59]\]. Whole-cell RNA (10 μg) was labeled by directly incorporating either Cy3-dCTP (reference RNA) or Cy5-dCTP (sample RNA) through reverse transcription. The resulting cDNA was hybridized onto DNA microarrays containing spotted PCR products for over 5,269 different genes and genomic elements printed in duplicate on glass slides representing 99.9% of all known and predicted fission yeast genes. Microarrays were scanned using a GenePix 4000B laser scanner (Axon Instruments) and analyzed with GenePix Pro software. Low-quality signals were filtered out, and data were normalized using a customized Perl script (local adjustment of median of ratios to one within running windows of 1,000 spots). Data evaluation and gene classification --------------------------------------- Normalized data (Cy5/Cy3 ratios) were evaluated using GeneSpring (Silicon Genetics). All gene-expression values were normalized to the average of two trt1^+^biological replicates (strains WT 3 and WT 5) collected on day 1 of the growth curve. Experiments and genes were clustered in GeneSpring using the Pearson correlation around zero (termed the Standard correlation in GeneSpring) with a minimum distance of 0.001 and a separation ratio of 1. Gene annotations were taken from GeneDB at the Wellcome Trust Sanger Institute \[[@B60]\]. Lists of genes whose expression changed in the fission yeast stress response \[[@B16]\] were taken from the authors\' website \[[@B61]\]. BLAST searches were performed using the NCBI BLAST server \[[@B62]\]. The density of genes with changed regulation along the chromosome was determined by using a running window of 20 consecutive genes along each chromosome \[[@B63]\]. For each window, the probability of obtaining the observed results by chance was calculated using the hypergeometric distribution. There were two microarray signals - SPAC212.06 (a pseudogene) and the reverse transcript of centromeric dhrepeats - that we believe were due to cross-hybridization with the SPAC212.11 transcript (or transcripts from identical ORFs, see text). Cross-hybridization becomes apparent with array element sequence identities higher than about 70% \[[@B59]\]. The SPAC212.11 transcript is capable of hybridizing to the entire SPAC212.06 microarray probe (99% sequence identity), but the SPAC212.06 transcript does not contain the sequence required to hybridize to the SPAC212.11 microarray probe. The SPAC212.11 transcript also has a high degree of homology with the dhrepeat microarray probe sequence (83% sequence identity), and both the SPAC212.11 and dhrepeat transcripts are expected to be capable of hybridizing to each other\'s microarray probes. Significant levels of forward and reverse centromeric dhrepeat transcripts could not be detected using RT-PCR with RNA from days 1, 7, 9 and 15 of the growth curve (Figure [7a](#F7){ref-type="fig"}) (indicating they could not hybridize to the microarray), although helicase RNA was detected by both RT-PCR and northern hybridization (Figure [7a](#F7){ref-type="fig"} and data not shown). Twenty-one microarrays were used in this study, representing two wild-type biological repeats, 15 days of the growth curve, and four strains with circularized chromosomes. The complete raw and normalized data sets are available from ArrayExpress \[[@B64]\] (Accession number: E-MEXP-201). Acknowledgements ================ We thank Peter Baumann, Valerie Wood, Juan Mata, Gavin Burns and Karen Goodrich for helpful discussions and assistance, Christopher Penkett for his help in making the data public, and Peter Baumann for critically reading the manuscript. J.G.M. was supported by a postdoctoral fellowship from the Damon Runyon Cancer Research Foundation, DRG 1617. J.B. is funded by Cancer Research UK. R.A.M. was supported by NIH grant R01GM067014. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Stability of wild-type strain gene expression profiles. (a)Microarray expression data for two wild-type biological replicates, WT 3 and WT 5, on day 1 of the growth curve are plotted against each other. The expression data plotted are the normalized ratio of dyes Cy5- and Cy3-dCTP representing sample and reference pool, respectively. Lines showing limits of twofold change are drawn on both sides of the line of identity (identical values between datasets). The axes are log scale. Every gene for which there is data is shown (filled circles). All genes fall within the lines of twofold change. (b)As in (a), except WT 5 from day 1 of the growth curve is compared with WT 5 from day 15. Only three out of 5,050 genes, marked with arrows, changed expression by more than twofold. These genes are SPBC354.08c, encoding a hypothetical protein (2.15-fold); atp8^+^, F~0~-ATP synthase subunit 8 (2.15-fold); and cox1^+^, cytochrome coxidase subunit I (2.98-fold). ::: ![](gb-2004-6-1-r1-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Senescence and emergence of survivors in trt1^-^cells. (a)Growth curves. YES cultures (200 ml) were inoculated at 2.5 × 10^4^cells/ml with either trt1^+^or trt1^-^cells. Cell density is shown for trt1^+^cells (open circles) and trt1^-^cells (filled squares) at the end of each 24-h period, after which a new culture was inoculated at 2.5 × 10^4^cells/ml. When cells were counted on day 1, they had already undergone about 45 generations after germination. Note that when the culture density reached 3-5 × 10^6^cells/ml, a portion of the cells was harvested for microarray analysis and Southern hybridization. Cells appeared enlarged near day 8 and were morphologically normal by day 11. (b)Restriction-enzyme sites in the TAS of one chromosome arm cloned into the plasmid pNSU70 \[58\]. Locations of the probes used for Southern hybridization are indicated by the bottom bars. These probes hybridize to multiple chromosome arms because the TASs are found on the four arms of chromosomes I and II and, depending upon the strain background, on one or both arms of chromosome III. (c)Telomere length in wild-type and trt1^-^strains from the growth curve. DNA (\~15 μg) was digested with EcoRI, subjected to electrophoresis, transferred to a nylon membrane and probed with the ^32^P-labeled telomere fragment shown in (b) that was expected to report the state of the telomere end. As a loading control, a probe for the single-copy gene pol1^+^was included. Signals arising from the telomeres are labeled. (d)As in (c), but DNA was digested with HindIII and the blot probed with TAS2 and a fragment of pol1^+^. The TAS2 probe was expected to hybridize to sequences at least 2 kb, and up to 6 kb, from the telomere end. ::: ![](gb-2004-6-1-r1-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Chromosome structures of trt1^-^survivors. (a)The 13 NotI restriction sites in S. pombechromosomes I and II \[65\] are indicated by vertical lines. Chromosome III does not have a NotI site. Terminal fragments are labeled according to convention and highlighted in black. (b)Pulsed-field gel analysis of intact chromosomes visualized by staining with ethidium bromide. Lanes d-g correspond to days 1, 7, 9 and 15 of the growth curve, respectively. (c)Pulsed-field gel of NotI-digested chromosomes visualized with ethidium bromide. Days 1,7, 9 and 15 correspond to days of the growth curve. Lanes a and f were repositioned from the original gel image. (d)The gels from (c) were transferred to a nylon membrane and probed with a mixture of ^32^P-labeled probes to internal regions of the C, I, L and M fragments, identified in (a). The terminal fragments of linear chromosomes are labeled on the left, and fragments C+M and I+L resulting from circularized chromosomes are shown on the right. ::: ![](gb-2004-6-1-r1-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Gene-expression profiles of cells experiencing senescence and survivors. (a)Graph of expression for all genes showing fold-change relative to wild type for each day of the growth curve. Each gene is represented as a line with discontinuities resulting from missing data. For clarity, three genes (missing from the genome, see text) with expression reduced tenfold or more are not shown: trt1^+^, SPAC2E1P3.04 and SPAC2E1P3.05c. (b)Hierarchical clustering of the 123 genes whose expression changed by twofold or more relative to wild-type in two or more days of the growth curve (see text for details). Samples d1-d15 are days of the growth curve. Each column represents expression of all 123 genes for a unique condition. Each row represents the expression pattern of a single gene throughout all conditions. Genes shown in red had upregulated expression and those in green had downregulated expression. Values of fold-change less than 1.2 are in black, and gray areas indicate missing data. Brackets labeled with letters a-b along the right-hand side denote sets of genes with similar expression patterns for one or more conditions. Band \'a\' consists of genes with downregulated expression: SPAC2E1P3.05c, SPAC2E1P3.04, trt1^+^and SPBC359.02; and band \'b\' represents the second wave of gene expression in the growth curve. The wild-type sample was an average of biological replicates WT 3 and WT 5. (c)Dendrogram of the experimental conditions and strains shown in (b). Experiments were hierarchically clustered on the basis of the similarity of expression ratios of the 123 genes shown in (b). ::: ![](gb-2004-6-1-r1-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Expression signatures of cells with circular chromosomes. For each condition, the 51 genes from Table 2 that had expression changes of twofold or more in both strains C1 and C5, but not in survivors with linear chromosomes, are graphed in clusters of vertical bars. The height of each bar represents fold-change in expression relative to wild type. Survivors with linear or circular chromosomes are labeled. Strains H1 and H2 have circular chromosomes as evidenced by their inability to enter into a pulsed-field gel (data not shown). Strains H1 and H2 were not used to derive the expression signature and are shown as an independent verification of it. ::: ![](gb-2004-6-1-r1-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Homology of the putative helicase with RecQ helicases and dhrepeats. The 5.6 kb sequence of SPAC212.11 is represented as a rectangle. Horizontal lines above the gene indicate the regions spanned by primers used in this study. P~3\'~was the fragment of SPAC212.11 on the microarray (180 bases), and P~5\'~was used in Southern hybridizations (642 bases). Region P~dh~was amplified in RT-PCR experiments (Figure 7) to detect dhrepeat forward and reverse strands. Solid black rectangles are regions of homology with dhrepeats found at centromeres and in the Kregion of the mating-type locus. The predicted amino-acid sequence of the region marked with cross-hatching has homology with the RecQ helicase family. The BLAST expect (E) value is shown, with the exception that the approximately 70 bp region of homology to dhrepeats 3\' of the putative RecQ helicase domain has an E value of 2 × 10^-8^. ::: ![](gb-2004-6-1-r1-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Expression of dhrepeats at the sub-telomere. (a)Expression of sub-telomeric dhrepeats in trt1^-^mutants. Strand-specific RT-PCR using primers spanning the region of dhrepeats in the putative helicase (P~dh~in Figure 6) was used to detect the expression of both forward (For) and reverse (Rev) transcripts. We define the forward transcript to be homologous to the DNA strand running towards the chromosome end in the 5\' to 3\' direction (this is also the strand with the longest ORF). Strand-specific control reactions were also performed using primers specific for centromeric (Cen) dhrepeats \[33\], as well as act1^+^sense and act1^+^antisense transcripts (a control lacking reverse transcriptase is labeled -RT). Strains WT 5 and days 1, 7, 9 and 15 of the growth curve are shown. (b)Expression of sub-telomeric dhrepeats in RNAi mutants. RNA was isolated from trt1^+^RNAi mutant strains ago1^-^, dcr1^-^and rdp1^-^\[33\], and subjected to strand-specific RT-PCR using the same primers described in (a). A different wild-type strain from that in (a) was used. ::: ![](gb-2004-6-1-r1-7) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Genes with significantly altered expression in trt1^-^cells ::: Category Examples GC Circ -------------------------------------------- ---------------------------------------------------------------------------- ------ ------ Acetyltransferase (2) ppr1^+^, SPBC1271.07c\* 2-0 1-0 Alcohol metabolism (2) SPCC24B10.20\, SPAPB24D3.08c\ 2-0 0-0 Amino acid and derivative metabolism (6) SPBC119.03\, SPBPB21E7.04c\, SPAC139.05\* 5-1 3-1 Carbohydrate metabolism (14) eno102^+^, tms1^+^, fbp1^+^, SPCC663.08c\* 13-1 8-0 Cell organization (3) eng1^+^, SPBC8E4.10c\, SPAC11D3.01c\ 2-1 0-0 Cofactor metabolism (2) SPAC513.07\, SPAC2E1P3.04\ ^§^ 1-1 0-0 DNA maintenance and recombination (3) SPAC212.11\, trt1^+^ 2-1 0-1 Energy production (5) SPBC23G7.10c\, SPAC513.02\, SPBC1773.06c\ 5-0 2-0 Ion homeostasis (2) zym1^+^, SPBC947.05c\* 2-0 1-0 Meiosis and sporulation^‡^(5) mfm2^+†^, meu3RC^+^\^†^, meu8^+^\^†^, meu27^+^, SPBC354.08c\^†^ 4-1 0-0 Methyltransferase (1) SPAC1B3.06c\ 1-0 0-0 Mitochondrial energy and proteins (10) cox1^+^, cox3^+^, cob^+^, atp6^+^, atp8^+^, atp9^+^ 10-0 0-0 Nucleotide metabolism (2) SPBC1683.06c\, SPCC965.14c\ 1-0 1-0 Proteolysis (6) isp6^+^, SPBC1685.05\, SPCC338.12\ 6-0 1-0 Pseudogene (7) SPBC16E9.16c\, SPBPB21E7.08\ 7-0 3-0 RNA binding and regulation (3) SPCC74.09\, SPAC4G8.03c\ 3-0 1-0 Non-coding RNA (1) meu3RC 1-0 0-0 Signal transduction (2) hri1^+^, SPBC725.06c\* 2-0 1-0 Stress response (8) hsp16^+^, cta1^+^, hsp9^+^, ish1^+^, pyp2^+^ 8-0 3-0 Sulfur metabolism (2) gst2^+^, SPBC1198.01\* 1-0 2-0 Transcription (3) aes1^+^, SPAC30.02c\, SPBC1105.14\ 3-0 1-0 Transporter (6) cta3^+^, SPCC1840.12\* 5-1 2-0 Unknown function/hypothetical protein (29) SPAC25H1.01c\* 29-0 24-0 The total number of genes in each category is indicated in parenthesis. For each category, the number listed before the hyphen is the number of genes with at least two instances of upregulated expression, and the number after the hyphen is the number of genes with at least two cases of downregulated expression. GC, growth curve; Circ, strains C1 and C5, where numbers represent changes that occurred in both strains. \Putative function. ^†^Meiosis-associated genes with changed expression in the CESR \[16\]. ^‡^This category contains genes that may also appear in other categories. All other categories are nonredundant. ^§^SPAC2E1P3.04 appears to have been deleted from the genome in all strains except WT 3, WT 5 and C1. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Maintained expression in strains C1 and C5 ::: Gene name Category Gene name Category --------------- -------------------------------------- --------------- ---------------------- SPBC1271.07c Acetyltransferase\ aes1^+^ Transcription SPBPB21E7.04c Amino acid/derivative metabolism\* SPCC1840.12 Transporter\* SPBC119.03 Amino acid/derivative metabolism\* cta3^+^ Transporter SPAC139.05 Amino acid metabolism\* SPBP4G3.03 Unknown/hypothetical SPBC359.02 Amino acid metabolism\* SPBC660.05 Unknown/hypothetical SPACUNK4.17 Carbohydrate metabolism\* SPAC25H1.01c Unknown/hypothetical SPBC24C6.09c Carbohydrate metabolism\* SPAC29A4.12c Unknown/hypothetical SPAC3G9.11c Carbohydrate metabolism\* SPBC19C7.04c Unknown/hypothetical SPAC4H3.03c Carbohydrate metabolism\* SPAC15E1.02c Unknown/hypothetical SPCC1739.08c Carbohydrate metabolism\* SPBC1348.03 Unknown/hypothetical SPCC663.08c Carbohydrate metabolism\* SPAC23C11.06c Unknown/hypothetical SPAC513.02 Carbohydrate metabolism\* SPAC637.03 Unknown/hypothetical SPCC663.06c Carbohydrate metabolism\* SPCC584.16c Unknown/hypothetical tms1^+^ Carbohydrate metabolism SPBC21C3.19 Unknown/hypothetical trt1^+^ DNA maintenance SPBC56F2.06 Unknown/hypothetical SPAC19G12.09 Energy\* SPCC16A11.15c Unknown/hypothetical zym1^+^ Ion homeostasis SPCC338.18 Unknown/hypothetical SPCC338.12 Protease inhibitor\* SPAPB24D3.07c Unknown/hypothetical SPBC16E9.16c Pseudogene SPCC70.04c Unknown/hypothetical SPCC18B5.02c Pseudogene SPCC757.03c Unknown/hypothetical SPBPB21E7.08 Pseudogene SPBC1271.08c Unknown/hypothetical SPCC70.08c rRNA methyltransferase\* SPCC191.01 Unknown/hypothetical SPBC725.06c Signal transduction\* SPAC27D7.10c Unknown/hypothetical hsp16^+^ Stress response SPBC725.10 Unknown/hypothetical cta1^+^ Stress response SPCC737.04 Unknown/hypothetical gst2^+^ Stress (sulfur metabolism) SPAC27D7.09c Unknown/hypothetical SPAC4H3.08 Stress response (lipid metabolism)\* SPBC725.03 Unknown/hypothetical Fifty-four genes with maintained expression changes twofold or more in both of strains C1 and C5 that also had changed expression of twofold or more during 2 or more days in the growth curve. All but three genes (trt1^+^, cta3^+^and SPBC359.02) are without changed expression in survivors with linear chromosomes (days 12-15 of growth curve). \*Putative function. :::	PubMed Central	2024-06-05T03:55:52.927439	2004-12-15	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549061/", "journal": "Genome Biol. 2005 Dec 15; 6(1):R1", "authors": [ { "first": "Jeffrey G", "last": "Mandell" }, { "first": "Jürg", "last": "Bähler" }, { "first": "Thomas A", "last": "Volpe" }, { "first": "Robert A", "last": "Martienssen" }, { "first": "Thomas R", "last": "Cech" } ] }
PMC549062	Background ========== Population genomics, the study of genome-wide patterns of genetic variation in a large number of organisms, is emerging as a vigorous new field of study \[[@B1]-[@B3]\]. Rapid, accurate and inexpensive resequencing could enable a variety of potential applications and studies. For the biowarfare (BW) pathogen, Bacillus anthracis, genomic sequences from multiple strains and non-pathogenic close relatives could aid studies that definitively identify B. anthracisin environmental and clinical samples, determine forensic attribution and phylogenetic relationships of strains, and uncover the genetic basis of phenotypic variation in traits such as mammalian virulence. Moreover, first recognizing the presence of a novel pathogen, and then attempting the difficult task of discerning between novel naturally occurring pathogenic organisms (for instance Bacillus cereusG9241 \[[@B4]\]) and artificially enhanced bacterial pathogens, requires a thorough knowledge of extant patterns and levels of genetic variation in natural populations. Unusual patterns of genetic variation may serve as evidence aiding the detection of these unusual types of pathogens. The current technological model for genome sequencing employs high-throughput shotgun sequencing at large centers. This highly successful enterprise has completed about 200 bacterial genomes with more than 500 ongoing as of July 2004 \[[@B5]\]. The genome sequences of the B. anthracisAmes chromosome (5.2 Mb, NC\_003997) and plasmids pXO1 (181.6 kilobases (kb), NC\_001496) and pXO2 (96.2 kb, NC\_002146) have been determined \[[@B6]-[@B8]\], as have the genomes of three near neighbors, B. cereusATCC 14579 \[[@B9]\], B. cereusATCC 10987 \[[@B10]\] and B. cereusG9241 \[[@B4]\]. A strain of B. anthracisAmes strain isolated from a victim of the autumn 2001 bioterror attack in Florida was also sequenced to a high level of coverage using the random shotgun method and compared to the Ames sequence to identify 60 new markers that included single nucleotide polymorphisms (SNPs), inserted or deleted sequences, and tandem repeats \[[@B11]\]. The success of this effort has led to an extensive phylogeny-based whole-genome shotgun resequencing effort in B. anthracis(reported by \[[@B12]\]). Whole-genome shotgun studies are increasingly being used to explore variation among more closely related bacterial strains \[[@B13]-[@B15]\]. However, the relatively high costs of these efforts have limited the extent of their application. Numerous molecular methods for genotyping B. anthracisand near neighbors of the Bacillus cereus sensu latogroup \[[@B16]\] have been developed and successfully employed in a wide variety of studies. These include DNA sequence surveys from one or a few number of loci \[[@B17]-[@B21]\], repetitive element polymorphism-PCR \[[@B22],[@B23]\] and amplified fragment length polymorphisms (AFLP) \[[@B24]-[@B27]\]. However, because of the relative paucity of genetic variation between isolates \[[@B28]\], the most effective method for subtyping B. anthracishas employed multiple locus variable number of tandem repeats analysis (MLVA) \[[@B29]-[@B31]\]. Similar to the mammalian short tandem repeat methodology, MLVA determines strain phylogenetic relationships based on a relatively few, highly variable genomic repeat regions. While being relatively rapid and inexpensive, a key limitiation of MLVA lies in its exclusive focus on loci with common alleles that are differentiated by size. Because of the relatively rapid mutational process generating variation at these loci, similarly sized markers may have different evolutionary origins. Clearly, a method for rapid, inexpensive genome resequencing of bacterial strains would be of great benefit for genotyping, forensics and studies of the genetic basis of strain phenotypic variation. Developing DNA-based biodetection assays depends upon prior knowledge of patterns of genetic variation within and between bacterial species. It would be ideal to enable technologies that could combine the high information content of whole-genome resequencing of strains while also being rapid and inexpensive like MLVA, AFLP and multi-locus sequence typing (MLST). Furthermore, while conventional strain typing methodologies have focused on the utility of common variants, rare variants may prove to be especially informative for forensic applications. High-density oligonucleotide resequencing microarrays are a highly parallel technology that can enable the rapid identification of DNA sequence variants with minimal laboratory effort and infrastructure \[[@B32],[@B33]\]. Previous applications of microarrays on bacterial genomes \[[@B34],[@B35]\] or small eukaryotic genomes like yeast \[[@B36],[@B37]\], focused on methods that scanned specific genes or a genomic region for genetic variants. Initial high-throughput microarray applications in the human genome for SNP discovery \[[@B38]-[@B40]\] were successful, but also reported that between 12% and 45% of the detected variants were false. Subsequent experimental improvements and the development of the ABACUS algorithm/software package \[[@B32]\] significantly reduced SNP false-positive ascertainment, radically improved genotype calling and automatically assigned quality scores to each genotype call. These fundamental advances enabled rapid resequencing of 40 human genomic regions \[[@B32],[@B41]\] and ABACUS is now the standard application for microarray-based resequencing. Here we present the first microarray-based high-throughput resequencing of a large collection of B. anthracisisolates. Our study first reaffirms, and then directly demonstrates that the quality of microarray-generated DNA sequence data is directly comparable to that produced by conventional shotgun sequencing. We then estimate the levels of genetic variation in the annotated genomic regions we resequenced, characterize the frequency spectrum of DNA sequence variants we observe, and finally explore patterns of linkage disequilibrium and recombination among those variants. Because of the scalability and minimal effort associated with microarray-based resequencing, our work demonstrates the possibility of a rapid and cost-effective method of genome resequencing that could be applied to both environmental, and ultimately clinical specimens. Results ======= Resequencing B. anthraciswith microarrays ------------------------------------------- A panel of 56 B. anthracisstrains from the Biological Defense Research Directorate\'s strain collection (see Additional data file 1) was resequenced using Affymetrix resequencing arrays (RAs) and base calls determined using the ABACUS software package \[[@B32]\]. Each RA was capable of resequencing 29,212 base-pairs (bp) or about 0.5% of the B. anthracisgenome from a single isolate sample (see Additional data file 2). Long PCR sample preparation and chip processing was conducted for 118 RAs. Analysis of these 118 RAs with the ABACUS software package shows that 115 are successful (97.5%). Experimental failure occurs when less than 60% of the total possible bases fail to achieve quality scores exceeding the ABACUS user-defined threshold. For this study, the total threshold was set at 31 and a strand minimum of -2 \[[@B32]\], as determined from analysis of a replication experiment described below. The 115 successful RAs call 92.6% of the possible bases (3,109,539 bp out of a total possible of 3,359,380 bp). Figure [1](#F1){ref-type="fig"} shows the distribution of quality scores across all 3,359,380 base calls. Amplicon failure, typically arising from long PCR (LPCR) failure, accounts for 1.1% of the uncalled bases. The remaining base-calling failure (6.3%) consists of features on the RAs that fail to generate quality scores exceeding the experimental threshold. Previous results demonstrated that base-calling failure was concentrated among RA oligonucleotide probes containing multiple purines. Purine-rich probes were observed to have lower hybridization intensities at identical positions across multiple RAs. Guanine-rich probes, in particular, showed the greatest reduction in hybridization intensity (see Figure 6 in \[[@B32]\]). Consequently, total quality scores at these sites frequently failed to exceed the quality-score threshold and they remained uncalled. To determine if probe sequence composition, specifically purine and guanine content, contributed to the 6.3% of bases not called, the sequence composition of the purine-rich oligonucleotide probes at 4,209 sites successfully called on all 115 RAs (484,035 total sites) was compared to that at the 886 sites that failed to be called on any RA (101,890 total sites). These failed sites account for 3.0% of the total base calling failure in the experiment. Uncalled sites are composed of oligonucleotide probes with a significantly higher purine composition (P\< 10^-22^). A similar pattern is detected if we limit our analysis to guanine-rich probes (P\< 10^-9^). This latter result is surprising given that the B. anthracisgenomic sequences we examined have a low G+C content (\~34%). Nevertheless, these analyses demonstrate that both purine-rich and guanine-rich oligonucleotide probes are significantly more likely to fail to generate quality scores exceeding the experimental threshold. Assessing microarray resequencing data quality ---------------------------------------------- Building on the recognition of the importance of automated algorithms to assess data quality \[[@B42],[@B43]\], we used two methods to assess the quality of microarray resequencing data \[[@B32]\]. The first consisted of a replicate experiment where 51 samples were independently hybridized on 102 RAs. A parameter search that optimized the percentage of called bases, while minimizing the number of discrepancies between replicates was then performed. A total of 1,489,812 bases could have been called in each replicate experiment. At the optimal parameter values (total threshold of 31, strand minimum of -2 see Cutler et al.\[[@B32]\]), 90.6% (1,349,178) of sites are called in both replicates. Other parameter values provide similar levels of base calling and discrepancy rates. The optimal parameter values are similar to those previously used by Cutler et al.\[[@B32]\]. Of the bases called in both replicates, 1,349,177 are called identically. Only one site is called differently. This corresponds to a replication discrepancy rate of 7.4 × 10^-7^(Table [1](#T1){ref-type="table"}). If repeatability could be related to accuracy, then this level of repeatability would correspond to a phred score of at least 61 \[[@B42],[@B43]\]. This calculation assumes that the discrepancy rate corresponds to a binomial error probability of P, where phred = -10 log~10~P. These replication levels and discrepancy rates are consistent with those previously reported \[[@B32]\], providing further evidence for the ability of RAs analyzed with ABACUS to produce highly replicable data. While RA data is highly replicable, repeated systematic errors would not be detected in a replicate experiment. To obtain an independent estimate of RA sequence accuracy, we compared the sequence data from 30 RAs where the same B. anthracisstrain had been sequenced using the random shotgun approach and deposited in GenBank (B. anthracis: strain Ames, NC\_003997 \[[@B8]\], Vollum, NZ\_AAEP00000000, 4 June 2004 update, strain Australia 94, NZ\_AAES00000000, 7 June 2004 update, strain Kruger B NZ\_AAEQ00000000, 7 June 2004 update (J Ravel, DA Rasko, MF Shumway, L Jiang, RZ Cer, NB Federova, M Wilson, S Stanley, S Decker, TD Read, et al., unpublished work). In a comparison of 398,467 bp of RA- and shotgun-generated sequence, we observed 15 discrepancies occurring at six sites. This corresponds to a discrepancy rate of 3.8 × 10^-5^. If we make the conservative assumption that all discrepancies lie in the RA-generated sequence, this level of accuracy would correspond to a phred score of at least 44. To determine if this conservative assumption is warranted, we examined in greater detail the nature of the RA/shotgun sequence discrepancies. Five of the discrepant sites, accounting for 10 discrepancies total (twofold RA replication at each site), were found in Kruger B strain sequences. The one remaining site, accounting for five discrepancies (fivefold RA replication at this site), was found in Vollum strain sequences. At all 15 discrepancies, the RA called a base identical to the Ames reference sequence \[[@B8]\], while the Kruger B/Vollum shotgun sequence called a new SNP. The fact that the shotgun sequence called a SNP at every discrepancy was surprising, leading us to examine more closely the level of shotgun coverage and assembly at each discrepant site. A comparison of the latest shotgun assembly of the Kruger B strain (J Ravel, et al., unpublished work) with the RA Kruger B strain base calls agreed with the RA base calls. The latest Vollum shotgun assembly (J Ravel, et al., unpublished work) still disagreed at the one site (five discrepancies total), but this discrepancy was based on a single shotgun sequencing read with a phred score of 7 at the discrepant base. Clearly, the shotgun coverage lacks sufficient depth at this site to make a reliable base call and it seems far more likely that the fivefold RA base call is correct. Hence, the RA sequence data has less than one discrepancy per 398,467 bases called, or a discrepancy rate of \< 2.5 × 10^-6^(Table [1](#T1){ref-type="table"}). This observed level of sequencing accuracy corresponds to a phred score of 56. These data demonstrate that our conservative assumption is not warranted. Resequencing array data quality from a single experiment matches, and in some cases perhaps exceeds, that obtained by multiple DNA sequencing reads using conventional DNA sequencing technologies \[[@B42],[@B43]\]. Patterns and levels of genetic variation in B. anthracis ---------------------------------------------------------- We identify 37 SNPs among 56 B. anthracisstrains. The SNP location, base-call, and position relative to the respective GenBank reference sequences \[[@B6]-[@B8]\] are contained in Additional data file 3. Twenty-four of the 37 SNPs, including two singletons, were independently confirmed in identical strains where whole-genome random shotgun sequence was available (A0039, A4088 and A0442 in Additional data file 1 (J Ravel, et al., unpublished work)). Of the remaining 13 SNPs not independently verified by The Institute of Genomic Research (TIGR), 11 were seen only once in our collection of strains and two SNPs were seen three times. Population genetic inference typically assumes that study samples are selected without prior knowledge of their patterns of genetic variation. For this study, we selected diverse strains from widely distant geographic regions in an attempt to sample the full extent of genetic variation in B. anthracis. The number of SNPs identified, the amount of sequence generated and the nucleotide diversity \[[@B44]\] of the 56 strains is contained in Table [2](#T2){ref-type="table"}. We performed analyses for sequences comprising the total dataset, for each genomic region separately, and for the total dataset with each resequenced base assigned into an annotated SNP class. We report three main findings. First, the total average level of DNA sequence variation in B. anthracisis very low. This finding is in agreement with previous studies \[[@B11],[@B28]\]. This level of genetic variation is much lower than that seen in commonly studied bacterial species \[[@B14]\], roughly half of that observed in the human genome and 25-fold lower than that observed in D. melanogaster\[[@B38],[@B39],[@B45]-[@B48]\]. Second, the B. anthracischromosome appears less variable than either the pXO1 or pXO2 plasmids, although this difference is not statistically significant. Third, the patterns of genetic variation by SNP class (see Table [2](#T2){ref-type="table"} and Additional data file 4) are similar to that seen in other well studied bacterial \[[@B14]\] and eukaryotic genomes \[[@B45]\]. Silent sites, those sites that when mutated do not alter the protein primary structure, are significantly more variable than are amino acid altering replacement sites (P= 0.0011). Intergenic regions are observed to have intermediate levels of genetic variation, whereas replacement sites, those sites that when mutated alter the protein primary structure, are the least variable. Replacement sites are marginally significantly less variable than intergenic sites (P= 0.039) whereas silent sites are not significantly more variable than intergenic sites (P= 0.22). The neutral theory of molecular evolution predicts a characteristic frequency spectrum of SNPs, or segregating sites, for populations at equilibrium \[[@B49]\]. Deviations from this expected distribution are observed when an experimental population sample contains an excess of low frequency, rare SNPs, or an excess of high frequency, common SNPs, relative to the neutral expectation. These deviations can arise as a consequence of demographic history and/or the action of natural selection \[[@B50]\]. Figure [2](#F2){ref-type="fig"} compares the observed and expected percent of SNPs in four allele-frequency classes. The data suggest an observed excess of rare SNPs as compared to that expected under the neutral theory. For example, while the neutral theory predicts that approximately 60% of SNPs should have minor allele frequencies less than or equal to 0.25, we observe that more that 92% of the B. anthracisSNPs we discovered have minor allele frequencies that fall into this class, a statistically significant difference (Figure [2](#F2){ref-type="fig"}). We used the Tajima\'s D statistic \[[@B50]\] to further assess this pattern for the entire dataset, for SNPs from each genomic region and for each SNP class (Table [2](#T2){ref-type="table"}). Tajima\'s D is a summary statistic for the site (or SNP) frequency spectrum, whose value is negative when there is an excess of rare variants and positive when there is an excess of common variants, relative to the neutral expectation. The test statistic is calculated from two different estimates of levels of genetic variation, the number of segregating sites \[[@B44]\] and the average number of nucleotide differences estimated from pairwise comparisons \[[@B50]\]. We observe that Tajima\'s D is negative for SNPs comprising the total dataset, each genomic region and each SNP class. While none of the individual test statistics is statistically significant, they collectively suggest an excess of rare variants in B. anthracis. If we scale our variation estimates drawn from the 0.5% resequenced in 56 B. anthracisgenomes, we can estimate a range around the total number of SNPs that one would detect upon sequencing two random B. anthracisisolates, sampled in the same fashion as isolates in this study were chosen. Our results indicate that we should expect to find, on average, between 944 (standard deviation (SD) 454) \[[@B50]\] and 1,586 SNPs (SD 762) \[[@B44]\]. A substantial proportion of these SNPs, probably more than expected under the neutral theory, would be rare. Using multiple sequence alignments of 17 genes from B. anthracis(NC\_003997, Ames) and B. cereus(NC\_004722, ATCC 14579 \[[@B9]\] and NC\_003909, ATCC 10987 \[[@B10]\]) the patterns of genetic polymorphism and divergence at silent and replacement sites was assessed. The raw counts are presented in Table [3](#T3){ref-type="table"}. It is striking that two B. cereusstrains exhibit more polymorphism at silent and replacement sites than divergence from B. anthracis. This result confirms, at the DNA sequence level, previous results suggesting that the B. cereusspecies group is diverse and polyphyletic in origin. B. anthracisthen appears to be a clonal lineage derived from, and nested within, a diverse species. In other words, the species names do not encompass or reflect the evolutionary history of the species \[[@B10],[@B51],[@B52]\]. No evidence for recombination in B. anthracischromosome --------------------------------------------------------- The 37 SNPs discovered on the B. anthracischromosome and plasmids pXO1 and pXO2, possess in total, 636 pairs of sites where two alleles are observed. In principle, the alleles at each pair of sites could form four distinct haplotypes. Plasmid transfer between different B. anthracisstrains would affect physically unlinked site pairs resulting in four distinct haplotypes. Homologous recombination or gene conversion between physically linked site pairs is also expected to produce all four haplotypes. The straightforward counting of the number of haplotypes that one detects in a large population sample, such as the one used in this study, is often referred to as the four-gamete test \[[@B53]\]. Among the 636 site pairs in our sample, we observe 26 pairs of sites with two haplotypes, 610 pairs of sites with three haplotypes, and no pairs of sites with four haplotypes. This striking result implies that the value of D\', the standardized measure of linkage disequilibrium (LD) \[[@B54]\], is equal to 1, its maximum value, for all site pairs that we observe. Among the 137 site pairs where we could have detected statistically significant LD at P\< 10^-3^, we observe that 52 site pairs exhibit statistically significant LD. Four of the six site pairs showing significant LD on the B. anthracismain chromosome are over 500 kb apart. Correlation of RA resequencing data with MLVA typing ---------------------------------------------------- Because of the low level of genetic variation in B. anthracis(\[[@B28],[@B29]\] and this study), determining the phylogenetic relationship among B. anthracisstrains has proven difficult. Twenty-four B. anthracisstrains characterized with a single fluorescent AFLP primer combination were reported to be monomorphic \[[@B27]\]. One recent MLST study sequenced seven housekeeping genes (approximately 3 kb total) in 5 B. anthracisstrains and reported that the strains were monomorphic at the sites examined. Another recent MLST study sequenced seven genes (approximately 3 kb total) in 11 diverse B. anthracisstrains finding three polymorphic nucleotides \[[@B55]\]. Neither the AFLP nor the MLST studies discover and genotype sufficient genetic variation to distinguish between B. anthracisstrains. The most successful marker-based approach used to date, MLVA, determined the genotypes at eight VNTR loci in 426 B. anthracisisolates, enabling the construction of a phylogenetic tree of B. anthracisstrains \[[@B29]\]. We sought to determine if our resequencing of 0.5% of each of 56 B. anthracisgenomes is capable of confirming the major phylogenetic groupings determined by MLVA. To test this, we concatenated the 37 variant positions for all strains in this study, calculated a distance matrix using a simple Kimura substitution model, and generated an Unweighted Pair Group Method Arithmetic Mean (UPGMA) tree (see methods \[[@B56]\]; Figure [3](#F3){ref-type="fig"}). The strains group together in a manner broadly similar to that found by Keim et al.\[[@B29]\] with B strains forming an outgroup and most A strains being found together in the same subgroups (Figure [3](#F3){ref-type="fig"}). There are exceptions: one group in Figure [3](#F3){ref-type="fig"} contains a mix of A3a, A1a, A1b and A2 strains. This anomaly is probably due to the relatively few SNPs that effectively distinguish these groups when only 0.5% of the genome is sampled. All B. anthracisAmes strains but ASC394 correctly cluster in an A3b group. B. anthracisASC394 may be a case of an originally mistyped or mislabeled strain. Nevertheless, our data suggest that limited, random resequencing of 0.5% of the 56 B. anthracisgenomes discovers and genotypes sufficient genetic variation to determine the major phylogenetic relationships among B. anthracisstrains. Discussion ========== Population genomics requires the random sampling of genome-wide patterns of DNA sequence variation in a large number of organisms. Such studies require high-throughput, highly accurate, cost-effective resequencing technologies. While the conventional industrial-scale shotgun-sequencing model is clearly the best technology available for de novogeneration of genomic sequence, it may not be the best approach for resequencing large numbers of strains. RAs, as originally applied for human genome resequencing \[[@B32]\], offer one competing technology that can rapidly produce very high-quality data with limited personnel and infrastructure requirements. Our application of RAs to resequence multiple genomic regions in the biowarfare pathogen, Bacillus anthracis, further supports this perspective. Studies of DNA sequence variation are most informative when both rare and common variants are identified. While the limited ascertainment of selected common variants can be employed to identify broad evolutionary relationships among bacterial genomes, and in fact underlies most bacterial strain typing methodologies, the ultimate forensic application of resequencing lies in the ascertainment of rare, presumably newly arising variants, that may allow more precise determination of a strain\'s origin. Rare variants may be particularly informative since they are likely to be restricted to specific strains (substrains/isolates). Strain genotyping of common variants provides an incomplete description of genomic patterns of DNA sequence variation, while obtaining most or all of the genomic sequence from multiple strains allows a maximally informative analyses of DNA sequence variation, its function, and ultimately, the evolutionary history of the organisms. The ability to rapidly, accurately and inexpensively resequence entire bacterial genomes should also contribute to an understanding of a variety of important phenotypic traits in B. anthracisand other bacterial pathogens \[[@B57]-[@B62]\]. Our study demonstrates that microarray-based resequencing is technologically robust and generates highly replicable and accurate data when compared to alternative sequence technologies (Table [1](#T1){ref-type="table"}). In this experiment, 115 RAs, or 97.5% of the total attempted, were processed successfully obtaining an average high-quality base-calling rate of 92.6%. Called bases are shown to be highly replicable (discrepancy rate of 7.4 × 10^-7^) and accurate when compared to conventional shotgun sequence (discrepancy rate of \< 2.5 × 10^-6^). Clearly, RA-generated resequencing data from a single experiment is comparable, in terms of data quality, to DNA sequence generated from multiple shotgun reads by a DNA sequencing center. The major technical challenge facing RA-based resequencing is to increase overall call rates while not compromising data quality. Modifications of RA synthesis, experimental protocols and the ABACUS software algorithm could all contribute to improved base-calling rates. While it is possible to increase call rates while sacrificing data quality, there is a need to focus on generating very high-quality data at virtually all sites. If this is absent, the second-best outcome is to call all bases in an environment in which we understand the nature of probable errors. In diverse fields where RAs might be widely used as a first-stage screening tool, such as BW agent identification or human clinical testing, the imperative is to use highly sensitive technologies that minimize the false-negative rate. False-positive findings could be confirmed later in a second-stage screen with an alternative technology such as conventional dideoxy chain termination sequencing. Microarray-based resequencing identifies and genotypes SNPs in a single experiment. No prior knowledge of the variability of a site is required - only a reference genomic sequence. Microarray design and applications are flexible. It is, however, important to note that the use of RAs in this study is not as a SNP typing technology. Thus, problems in interpreting the inferred phylogenetic relationships between strains that arise from SNP typing schema are avoided \[[@B63]\]. RA-based resequencing resembles MLST methodology used for bacterial strains \[[@B52],[@B55],[@B64]\]. MLST attempts to choose the most informative genomic regions to resequence, largely because of the costs associated and technological limitations in generating enough DNA sequence data on a large collection of variant strains. While a typical MLST approach might resequence between 3 and 4 kb, in organisms like B. anthracisthat have low levels of genetic variation (\[[@B28],[@B51],[@B55]\] and this study), this amount of generated sequence is insufficient. Clearly, RAs, such as those used in this study that can resequence approximately 29 kb, could rapidly increase this amount and be used for MLST studies. Furthermore, manufacturing improvements that reduce RA feature sizes enable the resequencing of greater quantities of genomic sequence per microarray. Ongoing work at NMRC/BDRD is evaluating RAs that can resequence 300 kb per chip. At that RA feature density, when combined with whole-genome amplification protocols, a single technician in two days could resequence the entire B. anthracisgenome on approximately 15 RAs. Our data provides the first population genetic estimation of the levels and patterns of DNA sequence variation in B. anthracis. We report three main findings. First, among B. anthracisisolates sampled in the same fashion as in this study we would expect two randomly selected B. anthracisstrains to differ, on average, at between 944 (SD 454) and 1,586 SNPs (SD 762). The variance surrounding these expectations is large, and any two isolates may differ from the expectation. Closely related, nonrandomly sampled isolates, such as those sequenced in \[[@B11]\], will have far fewer SNPs than that expected for samples drawn from a worldwide collection. Nevertheless, our data suggest that were it possible to rapidly resequence entire B. anthracisgenomes, sufficient genetic variation is likely to be found to make very fine-level discrimination of strain collections. Resequencing offers the best chance to identify newly arising, rare, strain-specific variants that will discriminate between very closely related strains, since we expect identical genotypes at the known common genetic variants \[[@B11]\]. We also observe, that as seen in eukaryotic genomes \[[@B45]\], the amount of silent variation per site within genes is much higher than that seen at replacement sites. Intergenic regions are seen to have intermediate levels of polymorphism. This pattern is expected to arise if noncoding intergenic regions possess variants visible to natural selection. If SNPs in intergenic regions were purely neutral, then we would expect to see levels of polymorphism similar to that at silent sites, which are undoubtedly under less stringent selective forces. Second, the neutral theory of molecular evolution predicts that in a population at equilibrium, a significant proportion of the observed genetic variation will consist of rare genetic variants \[[@B49]\]. We observe a significant excess of rare SNPs as compared to that expected under the neutral theory (Table [2](#T2){ref-type="table"}). This pattern of variation classically has at least two possible causes. The first consists of a recent population expansion from a small founder population. The second consists of the action of natural selection on genetic variants \[[@B65]-[@B67]\]. Resequencing technologies will be of particular use in populations of organisms exhibiting this pattern of genetic variation. Finally, we see no evidence for plasmid exchange or recombination altering the patterns of DNA sequence variation among B. anthracisstrains in the regions that we resequenced. Some of the regions that we resequenced contain genes whose function influences B. anthracispathogenicity or surrounded the bacterial origin of replication. In other bacterial species, these types of regions are the most likely to exhibit recombination \[[@B14]\]. The fact that we observe no evidence of plasmid exchange or recombination among physically linked markers in the regions that we resequenced, is striking. The simplest interpretation of this observation is that the B. anthracisstrains that we examined are ultimately derived from a single clonal ancestor and that the exchange of plasmids and recombination between strains during the course of their evolution is either very rare or nonexistent. While models of natural selection could also account for the patterns that we see, we think a simple demographic model of recent, rapid clonal expansion is parsimonious and best supported by our data. Hence, our findings suggest that B. anthracispopulations consist of multiple closely related clones whose life histories prevent the opportunity for homologous recombination between different strains. We note, however, that while we resequenced 0.5% of the B. anthracisgenome, including regions where we expected to detect recombination, further data collection from multiple genomic regions, or the entire genome, would allow a more thorough analysis of this pattern. Sequencing a larger percentage of the genome in a similar-sized or larger sample of isolates would provide greater power to detect rare recombination events. We are undertaking such a project to test the validity of our inference and to better determine if recombination is rare or absent among B. anthracisstrains. The absence of recombination in B. anthracis, a potential biowarfare agent, suggests a novel approach to identifying a newly arising or a genetically engineered strain. A recombination event could arise through rare natural genetic exchange or as a consequence of genetic engineering. Irrespective of the cause, the discovery of a B. anthracisstrain possessing evidence of genetic recombination would warrant close examination and probably demand immediate further phenotypic and genomic characterization. Taken together, the findings of a low number of differences between strains, a preponderance of rare variants, and an absence of recombination all point to a scenario where the current world population of B. anthracishas expanded recently from a single clone derived from, and nested within a diverse species, B. cereus. Other bacterial pathogens, such as the potential biowarfare agent Yersinia pestis, possess a similar recent pattern of rapid expansion \[[@B15]\]. However, the patterns of genetic variation in Y. pestisare quite different from that seen in B. anthracis, for instance in the much more active role of insertion sequences in Yersinia. We speculate that the B. anthracishistory of clonal expansion could arise as a consequence of the life history of a highly pathogenic sporulating mammalian pathogen. Exploring the population biology of less virulent members of the B. cereusgroup could directly test this. These population genomics studies could determine if clonal clusters of B. cereusstrains exhibit similar population dynamics and patterns of genetic variation, or whether the picture of B. anthracisemerging from studies such as this is as unusual as the level of pathogenicity of the species itself. Conclusions =========== Microarray-based resequencing can rapidly generate very high quality data, enabling population genomics studies in bacteria. We find no evidence for plasmid exchange or recombination altering the patterns of DNA sequence variation among B. anthracisstrains in the regions that we resequenced The patterns of genetic variation in the B. anthracisregions resequenced are consistent with that expected for a bacterial species that has undergone a rapid, historically recent expansion from a single clone. Detecting plasmid exchange or recombination between B. anthracisgenetic variants could act as an indicator of a newly emerging or genetically engineered strain. Materials and methods ===================== B. anthracisstrains surveyed ------------------------------ We selected a geographically diverse panel of 56 B. anthracisstrains from the Biological Defense Research Directorate collection (see Additional data file 1). Twenty-four of the strains originated from the Louisiana State University collection \[[@B29]\]. These have been typed by MLVA \[[@B29]\] and in order to sample diversity, we chose a group that had representatives of the A1a, A1b, A2, A3a, A3b, A3d, A4, B1 and B2 lineages. The remaining 35 strains originate from a UK collection and were chosen to represent geographical variation as well as unusual phenotypes such as gamma phage and penicillin resistance. Six of the UK strains were reisolates of the Ames strain \[[@B11]\], which allowed us to test the reproducibility of resequencing. Resequencing array design ------------------------- Unique genomic sequences were identified using Miropeats \[[@B68]\] at the default thresholds from among the B. anthracisAmes chromosome (5.2 megabase-pair (Mb), NC\_003997) and plasmids pXO1 (181.6 kb, NC\_001496) and pXO2 (96.2 kb, NC\_002146). The genomic regions that we resequenced included at least one gene of interest (pXO1: toxin lethal factor precursor lef, toxin moiety, protective antigen pagA; pXO2: encapsulation protein gene CapC; Ames chromosome: vrrA, DNA-directed RNA polymerase rpoB, yfhpprotein), but also included many surrounding loci (see Additional data file 4 for complete listing). The total chip design consisted of 6,191 bp from pXO1, 6,725 bp from pXO2, and 16,584 bp from the Ames chromosome (total submitted sequence 29,500 bp). From these unique sequences, a single 20 × 25 μm RA design capable of resequencing 29,212 bp or 0.5% of the B. anthracisgenome was fabricated by Affymetrix (see Additional data file 3). The final sequences submitted for RA design are contained in Additional data file 5. B. anthracisstrain genomic DNA isolation ------------------------------------------ Five milliliters of brain heart infusion (BHI) was inoculated and grown 12-16 h at 37°C. One-ml aliquots of cells were centrifuged for 10 min at 5,000-7,500g. Pellets were resuspended in 720 μl enzymatic lysis buffer (20 mM Tris-Cl pH 8.0, 2 mM EDTA, 1.2% Triton X-100, 20 mg/ml lysozyme) and incubated at 37°C for 1 h. After incubation 100 ml of Proteinase K was added along with 800 ml of Qiagen buffer AL, and incubated at 70°C for an additional 30 min. Then, 800 ml of 100% ethanol was added and this was split onto four of the Qiagen DNAeasy tissue kit. The DNA was then washed and eluted according to the Qiagen protocol. After the DNA was eluted, it was passed through a 0.22 mm filter. Sterility was confirmed by plating 10% DNA preparation directly on SBA plates with a second 10% inoculated into a 5 ml broth culture. The plate and the broth were allowed to incubate for 7 days. Two hundred milliliters of the broth culture was subcultured onto SBA at day 4. If there was no growth on any of these cultures the DNA was considered sterile and removed from the BSL-3 lab for subsequence analyses. Sample preparation and RA hybridization --------------------------------------- Genomic DNA was amplified using Long PCR (LPCR) protocols described in Cutler et al. \[[@B32]\]. The primers that amplified each RA fragment are shown in Additional data file 3. The primer sequences were: ant8 AAAAAGACGAGATGCGTCAACATCCCGTCCCA, ant9 TCAACTAAATCCGCACCTAGGGTTGCTGTAAG, ant10 ATTACTTTGAGTGGTCCCGTCTTTATCCCCCT, ant11 ACATTAGCAGGCAAGGACAGTGGTGTTGGAGA, ant14 ATTCACGCTCTCCCACCCAGATATTCCTACAT, ant15 GTCCTAATATCGGTGAGCAACGCAGGGTAGTT, ant20 GAGAAGAACCCCTACTACACGCATTGATACTG, ant21 TTTAGTAGCGAGGGTACAGGCGCGTTTATACC, ant26 TGGAAGCAGGCTTCGTAAGTGTAGGCGACGTT, ant27 GTTGCATGTTCGCTCCCATAAGTGCGCGGTTA, ant 32 AATGGGTGTATAGGGGTGATCTGTTGTGATGG, ant33 TCCATGTTCGGCCATCTGATTCCGTCACTACT. Long PCR product concentration was determined by using Pico-Green (Molecular Probes, Inc.) with lambda DNA standards (Invitrogen). The LPCR products were then pooled, DNAse digested, biotin endlabelled and hybridized to individual RAs overnight following established protocols contained in \[[@B32]\]. Subsequent washes and stains were carried out as described in Cutler et al.\[[@B32]\] and were only washed and not antibody stained. RAs were scanned at 570 nm, with a pixel size of 3 μm/pixel averaged over two scans. Automated grid alignment and base calling was performed for the .DAT files on a Mac G5 computer with the ABACUS software suite. RA sequence determination ------------------------- An ABACUS parameter search was employed to determine those parameters that called the maximal number of bases while minimizing discrepancies \[[@B32]\]. This total experiment consisted of 118 RAs, of which three failed (\< 60% base calling). Of the remaining 115 RAs, 8 were used to sequence individual strains once. Of the remaining 107 RAs, 96 were used to replicate hybridize 48 B. anthracisstrains, while the remaining 11 RAs were used as additional multiple replicates of these same strains. In total, sequence data was generated from 56 unique B. anthracisstrains (see Additional data file 1 for strain listing). In order to obtain the most complete data possible, for those strains with replicate RA sequences, a single composite strain sequence was generated for subsequent population genetic analyses. The current version of ABACUS algorithm is not designed to detect insertion/deletion variation. The effect of oligonucleotide probe composition was determined by choosing for each base, the probe with the most purines or the most guanines. The number of times that a given base was called was tabulated across all 115 successful RAs. The mean purine and guanine composition was determined for the classes that were called in all 115 RAs and uncalled in all 115 RAs. A Student\'s ttest with unequal variances was used to test for difference in mean sequence composition (purines/guanines) between the always called and never called classes. The DNA sequence files for the 115 RAs and the original RA image files (.DAT files) are available from the authors and will be made available through the NCBI Trace Archive. Population genetic analyses --------------------------- All population genetic analyses were calculated using the popgen\_fasta2.0.c code (Cutler DJ, unpublished work) on the collection of 56 sets of B. anthracisfasta files. The fasta files were analyzed in total and separately for the main chromosome and plasmids pXO1 and pXO2. The identification of genes was taken from publicly available annotation contained in the relevant GenBank refseq files (B. anthracisstr. Ames NC\_003997; pXO1, NC\_001496; pXO2 NC\_002146). The statistical significance of linkage disequilibrium between site pairs was performed by using the Fisher\'s Exact Test at P\< 10^-3^\[[@B69]\]. Estimating levels of genetic variation -------------------------------------- To account for missing data, θ is estimated by \[Σ~n~(S~n~/a~n~)\]/L, where S~n~is the number of observed segregating sites at positions with exactly n alleles sequenced (n is a maximum of 56, fewer with missing data), a~n~= Σ~i\ =\ 1..n-1~1/i, and L is the total length of the sequence examined. Var{θ} is estimated by \[Σ~n~(L~n~θ/a~n~+ (L~n~)^2^b~n~θ^2^/(a~n~)^2^\]/L^2^, where L~n~is the number of sites with data from exactly n alleles, and b~n~= Σ~i\ =\ 1..n-1~1/i^2^. With missing data π is estimated by \[Σ~i~2p~i~q~i~n~i~/(n~i~- 1)\]/L, where the sum is taken over all sites i, p~i~and q~i~are the allele frequencies at site i, and n~i~is the number of alleles sequenced at site i. To determine if the estimates of theta between SNP types (silent, replacement, intergenic) are significantly different, we used the number of samples sequenced, the number of segregating sites, and the length of the region to find a maximum-likelihood estimate of theta per site for each SNP type using equations 11 and 12 in Hudson \[[@B70]\]. We compared all possible SNP types against each other (silent vs replacement, silent vs intergenic, replacement vs. intergenic). For a given pair of SNP types, we first determined the maximum-likelihood estimator of theta for each type individually. We then determined the maximum-likelihood estimator of theta, assuming both types had identical theta per site. We ask whether the model with different thetas for each type fits significantly better than the model with a single theta through a likelihood ratio test. Reported significances are the p-values from the likelihood ratio test. Site frequency spectrum ----------------------- Comparing the observed site frequency spectrum with that expected under the neutral theory is a powerful approach to detect unusual patterns of genetic diversity. We employed two different approaches for this analysis. First, we calculated the expected number of sites with minor allele frequency i as Σ~n~θL~n~\[1/i + 1/(n - i)\] and from this determine the expected percent of sites under the neutral expectation. This is directly compared with the observed percent of SNPs in Figure [2](#F2){ref-type="fig"}. Confidence intervals for the sample proportion of each SNP minor allele frequency classes as ![](gb-2004-6-1-r10-i1.gif) where N is the number of SNPs observed for each class, ![](gb-2004-6-1-r10-i2.gif) is their observed frequency, and ![](gb-2004-6-1-r10-i3.gif). As a second method, we employed Tajima\'s D statistic \[[@B50]\], estimated as (π - θ)/Var(π - θ). Under the neutral model, π and θ have the same expectation, hence Tajima\'s D is expected to be 0. Since π is a function of site heterozygosities and θ is a function of the total number of segregating sites, Tajima\'s D is negative (positive) with an excess (deficit) of rare sites. We use our estimated values of π \[[@B51]\] and θ \[[@B45]\], multiplied by the total genome B. anthracisgenome length (5,505,178), to determine the expected number of SNPs that we would expect to observe among two B. anthracisstrains sampled in same random fashion as isolates in this study were chosen. Using Equations 6-9 in \[[@B51]\], we calculated the variance of ![](gb-2004-6-1-r10-i4.gif) and θ estimators. The one standard deviation (SD) that we report is the square root of this variance. Phylogenetic tree inference --------------------------- The 37 variable positions identified in this study were concatenated together to create artificial sequence types. A DNA distance matrix was created using DNADIST, plotted as a UPGMA tree using NEIGHBOR and the tree plotted using DRAWGRAM \[[@B57]\]. Additional data files ===================== The following additional data are available with the online version of this article. Additional data file [1](#s1){ref-type="supplementary-material"} lists B. anthracisstrains from the Biological Defense Research Directorate (BDRD) strain collection resequenced in this study. Additional data file [2](#s2){ref-type="supplementary-material"} lists the BDRD-01 RA fragment names, the GenBank reference sequence from which they are derived, the length of the unique genomic sequences submitted to RA design, the length of the unique genomic sequences capable of being queried, and the LPCR primer pairs used to amplify the RA fragments. Additional data file [3](#s3){ref-type="supplementary-material"} lists the B. anthracisSNPs identified in this study. The data include the BDRD SNP ID, the GenBank reference sequence and RA fragment containing the SNP, the SNP position relative to the GenBank reference sequence and the RA sequence, the SNP frequency, and the listing of the base calls in all strains at sites harboring SNPs. Additional data file [4](#s4){ref-type="supplementary-material"} lists the 31 B. anthracisgenes partially or wholly resequenced in this study. The observed number SNPs by SNP type (silent vs replacement) for each gene are provided. Finally, Additional data file [5](#s5){ref-type="supplementary-material"} shows the genomic sequences submitted to RA design for BDRD-01. Supplementary Material ====================== ::: {.caption} ###### Additional data file 1 B. anthracisstrains from the Biological Defense Research Directorate (BDRD) strain collection resequenced in this study ::: ::: {.caption} ###### Click here for additional data file ::: ::: {.caption} ###### Additional data file 2 The BDRD-01 RA fragment names, the GenBank reference sequence from which they are derived, the length of the unique genomic sequences submitted to RA design, the length of the unique genomic sequences capable of being queried, and the LPCR primer pairs used to amplify the RA fragments ::: ::: {.caption} ###### Click here for additional data file ::: ::: {.caption} ###### Additional data file 3 The B. anthracisSNPs identified in this study. The data include the BDRD SNP ID, the GenBank reference sequence and RA fragment containing the SNP, the SNP position relative to the GenBank reference sequence and the RA sequence, the SNP frequency, and the listing of the base calls in all strains at sites harboring SNPs ::: ::: {.caption} ###### Click here for additional data file ::: ::: {.caption} ###### Additional data file 4 The 31 B. anthracisgenes partially or wholly resequenced in this study ::: ::: {.caption} ###### Click here for additional data file ::: ::: {.caption} ###### Additional data file 5 The genomic sequences submitted to RA design for BDRD-01 ::: ::: {.caption} ###### Click here for additional data file ::: Acknowledgements ================ Funding from the Defense Threat Reduction Agency (DTRA) was used to support this study. The authors would like to thank Peter Turnbull for aid in B. anthracisstrain selection, Michael Chute for B. anthracisgenomic DNA isolation and David Rasko for comments on the manuscript. The views expressed in this paper are those of the authors and do not reflect the official policy or position of the Department of Navy, Department of Defense or US Government. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### ABACUS quality scores for base calls in B. anthracis. A quality score measures the difference, in log~10~units, between the likelihood support level for the best base-call model minus that for the second-best model \[32\]. Of the bases, 92.6% possess quality scores that exceeded the threshold (31) used for this study. ::: ![](gb-2004-6-1-r10-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### B. anthracisSNP frequency spectrum. An excess of rare SNPs are observed in our sample. Ninety-two percent of the SNPs that we discovered have a minor allele frequency less than or equal to 0.25. This finding (92%) is significantly different from the neutral theory expectation (60%). This excess can arise as a consequence of rapid, population expansion from a small founder population and/or the action of natural selection. ::: ![](gb-2004-6-1-r10-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Radial tree showing inferred phylogenetic relationships of B. anthracisstrains from this study. The 37 variable positions identified in this study were concatenated together to create artificial sequence types. Groups of strains with identical sequence types were A0488 and ASC006; A0039, ASC025, ASC031, ASC070, ASC074 and ASC394; ASC074 and ASC054; A0328, ASC061 and ASC073; A0034, ASC159, ASC165 and ASC398. A DNA distance matrix was created using DNADIST, plotted as a UPGMA tree using NEIGHBOR and the tree plotted using DRAWGRAM \[56\]. The B1 strain A0465 was used as an outgroup. ::: ![](gb-2004-6-1-r10-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Assessing microarray resequencing data quality ::: ------------------------------------------------- ----------- Replication experiment Total number of bases called in replicate 1 1,383,229 Total number of bases called in replicate 2 1,373,905 Total number of bases called in both replicates 1,349,177 Total number of bases called differently 1 Replication experiment discrepancy rate 7.4E-07 Accuracy estimation experiment Total number of bases called identically 398,452 Total number of bases called differently 15 Accuracy experiment discrepancy rate 3.8E-05 ------------------------------------------------- ----------- ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Observed genetic variation in B. anthracis ::: Observed number of SNPs Total amount resequenced (bp) Nucleotide diversity (× 10^4^) ± 2 SEs Tajima\'s D ---------------------- ------------------------- ------------------------------- ---------------------------------------- ------------- Total 37 1,544,913 2.9 ± 1.3 -0.93 Genomic location Chromosome 18 874,564 2.5 ± 1.4 -0.95 pXO1 9 325,397 3.3 ± 2.4 -0.54 pXO2 10 344,952 3.5 ± 2.5 -0.73 SNP class Silent 15 243,481 7.5 ± 4.3 -0.55 Replacement 9 898,837 1.2 ± 0.80 -0.64 Intergenic 13 402,595 3.8 ± 2.3 -1.09 ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Observed patterns of polymorphism/divergence between B. anthracis(Ames) and B. cereus(ATCC 14579, ATCC 10987) ::: Silent sites Replacement sites ------------------------------------------------------- -------------- ------------------- Polymorphic sites within B. cereusstrains 660 136 Divergent sites between B. anthracisand B. cereus 646 125 Polymorphic sites within B. anthracisstrains 11 3 :::	PubMed Central	2024-06-05T03:55:52.932328	2004-12-17	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549062/", "journal": "Genome Biol. 2005 Dec 17; 6(1):R10", "authors": [ { "first": "Michael E", "last": "Zwick" }, { "first": "Farrell", "last": "Mcafee" }, { "first": "David J", "last": "Cutler" }, { "first": "Timothy D", "last": "Read" }, { "first": "Jacques", "last": "Ravel" }, { "first": "Gregory R", "last": "Bowman" }, { "first": "Darrell R", "last": "Galloway" }, { "first": "Alfred", "last": "Mateczun" } ] }
PMC549063	Background ========== The human genome is a blueprint, but for what machinery? One approach to understanding the complex processes encoded by the human genome is to assign its enzyme products to biochemical pathways that define regulated sequences of biochemical transformations. Pathway and interaction assignments place genes in their larger biological context, and enable causal inferences about the likely effects of mutations, drug interventions and changes in gene regulation. They are a first step toward quantitative modeling of metabolism. Assignment of genes to pathways also permits a validation of the human genome annotation because patterns of pathway assignments spotlight likely false-positive and false-negative genome annotations. For example, false-negative assignments appear as pathway holes: missing enzymes within a pathway that are likely to be hiding in the genome. SRI\'s Bioinformatics Research Group has developed a pathway-bioinformatics technology called a pathway/genome database (PGDB), which describes the genome, the proteome, the reactome and the metabolome of an organism. A PGDB describes the replicons of an organism (chromosome(s) or plasmid(s)), its genes, the product of each gene, the biochemical reaction(s), if any, catalyzed by each gene product, the substrates of each reaction, and the organization of those reactions into pathways. Pathway Tools is a reusable software environment for constructing and managing PGDBs \[[@B1]\]. It supports many operations on PGDBs including PGDB creation, querying and visualization, analysis, interactive editing, web publishing, and prediction of the metabolic-pathway complement of an organism. The power of Pathway Tools is derived from both its database schema, and its software components. Both were originally developed for the EcoCyc project \[[@B2],[@B3]\]. A PGDB can be thought of as a symbolic computational theory of a species\' metabolic functions and genetic interactions \[[@B4]\], encoding knowledge in a manner suitable for computational analysis. Indeed, once an organism\'s genome and biochemical network are encoded within the schema of a PGDB, new possibilities for symbolic computational analysis arise, because many important semantic relationships are described in a computable fashion. PathoLogic is one of the Pathway Tools software components. Its primary function is to generate a new PGDB from an organism\'s annotated genome. PathoLogic predicts the metabolic pathways of the organism, providing new global insights about its biochemistry, and generates reports that summarize the evidence for the presence of each predicted metabolic pathway. We used PathoLogic to generate HumanCyc, a PGDB for Homo sapiens, from the annotated human genome. The genome data used as input to PathoLogic combined data from the Ensembl database \[[@B5]\], the LocusLink database \[[@B6]\] and GenBank \[[@B7]\]. Our analysis assigns 2,709 human enzymes to 135 predicted metabolic pathways. It provides a genome-based view of human nutrition that associates the essential dietary requirements of humans that were previously derived mainly from animal and tissue extract studies to a set of metabolic pathways whose existence is derived from the human genome. The analysis also identifies probable omissions in the human-genome annotation in the form of pathway holes (missing enzymes within the predicted pathways); we have identified putative genes to fill some of those pathway holes. This paper describes the generation of HumanCyc, and presents an analysis of the human metabolic map. The computationally predicted pathways are consistent with known human dietary requirements. We compare the predicted human metabolic pathway complement to the pathways of Escherichia coliand Arabidopsis thalianaand identify 35 pathways that are shared among all three organisms, and therefore define an upper bound on a potential set of universally occurring metabolic pathways. Results ======= Prediction of human metabolic pathways -------------------------------------- We applied PathoLogic to the input files containing the H. sapiensannotated genome, as described in Materials and methods, generating HumanCyc. Table [1](#T1){ref-type="table"} shows the results of PathoLogic\'s enzyme matching during the PGDB automated build. This computational matching process found more than 2,300 matches between gene products in the annotated genome and reactions in MetaCyc. Both the ambiguous matches (row 3 in Table [1](#T1){ref-type="table"}) and the proteins labeled as \'probable enzymes\' by PathoLogic (row 5) were examined manually; about half of them were manually matched to enzymes, as explained in Materials and methods. Sometimes one gene product is matched to more than one reaction, as happens with multifunctional enzymes (for example, the gene product shown in Figure [1](#F1){ref-type="fig"} would be matched to two different reactions). So the number of matches is higher than the number of proteins matched. The \'Unmatched\' row includes human proteins that are not enzymes. Table [2](#T2){ref-type="table"} shows statistics from version 7.5 of HumanCyc (released in August 2003), after manual refinement of the PGDB was completed. The 2,742 enzyme genes in HumanCyc correspond to 9.5% of the human genome, and can be subdivided into 1,653 metabolic enzymes, plus 1,089 nonmetabolic enzymes (including enzymes whose substrates are macromolecules, such as protein kinases and DNA polymerases). Our best estimate of the total number of human metabolic enzymes is the sum of the 1,653 known enzymes plus the 203 pathway holes, for a total of approximately 6.5% of the human genome allocated to small-molecule metabolism (compared to 16% of the E. coligenome). Of the 1,653 metabolic enzymes, 622 are assigned to a pathway in HumanCyc, and the remainder are not assigned to any pathway; we expect that in the future some of the latter group of enzymes will be assigned to some known human pathways not yet in HumanCyc, and to some human pathways that remain to be discovered. Of the metabolic enzymes, 343 are multifunctional. The number of enzymes is less than the number of enzyme genes because, in many cases, the products of multiple genes are required to form one active enzyme complex. Table [3](#T3){ref-type="table"} shows all pathways present in HumanCyc, arranged according to the MetaCyc pathway taxonomy. Only the top two levels in the taxonomy are shown for the sake of brevity. The 135 metabolic pathways in HumanCyc is a lower bound on the total number of human metabolic pathways; this number excludes the 10 HumanCyc superpathways that are defined as linked clusters of pathways. The average length of HumanCyc pathways is 5.4 reaction steps. Example HumanCyc pathways are shown in Figures [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}. All HumanCyc pathways can be accessed online from the HumanCyc Pathways page \[[@B8]\]. HumanCyc 7.5 contains 1,093 biochemical reactions, 896 of which have been assigned to one or more of the 2,709 enzymes in HumanCyc. There are more enzymes than reactions because of the existence of isozymes in the human genome. This leaves 203 reactions that have no assigned enzyme. These reactions correspond to the above-mentioned pathway holes for the HumanCyc pathways. Of the 896 reactions that have assigned enzymes, 428 have multiple isozymes assigned. Filling holes in HumanCyc pathways ---------------------------------- The PathoLogic-based analysis of the annotated human genome inferred 135 metabolic pathways. A total of 203 pathway holes (missing enzymes) were present across 99 of these pathways; that is, 38 pathways were complete. Using our hole-filling algorithm \[[@B9]\], no candidate enzymes were found for 115 of the 203 pathway holes. For the remaining 88 pathway holes, candidates were obtained and evaluated. In 25 of these 88 cases putative enzymes were identified with sufficiently strong support that the enzyme and pathway annotations within HumanCyc have been updated to reflect these findings. See the HumanCyc release note history \[[@B10]\] for a list of these 25 hole fillers added to HumanCyc version 7.6. The original annotations of the human proteins that were identified as candidate hole fillers fell into several classes: A description of each class is presented below, with examples included for some. ### Open reading frames (ORFs) with no assigned function (6 candidates) Putative enzymes were identified, for example, for the N-acetylneuraminate lyase (LocusLink ID 80896), aldose 1-epimerase (LocusLink ID 130589) and imidazolonepropionase (LocusLink ID 144193) reactions. In each of these cases, the function of the protein was previously unknown. ### Proteins assigned a nonspecific function (7 candidates) The pathway hole filler assigned an enzyme previously annotated with a general function. For example, \'amine oxidase (flavin-containing) B\' (LocusLink ID 4129), was assigned to a more specific reaction, putrescine oxidase. A \'fatty acid synthase\' (LocusLink ID 54995) was identified to fill the 3-oxoacyl-ACP synthase reaction. ### Proteins assigned a single function but which our analysis indicates are multifunctional (9 candidates) In these cases the program is postulating an additional function for a gene that already has an assigned function. The pathway hole filler identified the enoyl-CoA hydratase enzyme (LocusLink ID 1892) as a potential hole filler for the 3-hydroxybutyryl-CoA dehydratase reaction in the lysine degradation and tryptophan degradation pathways. The dihydrofolate synthase hole in formylTHF biosynthesis was filled by the enzyme (LocusLink ID 2356) catalyzing the folylpolyglutamate synthase reaction. ### Proteins that may have been assigned an incorrect specific function Although our analyses of other pathway/genome databases have revealed examples we consider to have been assigned an incorrect function in the original annotation, our analysis of the 25 HumanCyc pathway holes that we filled revealed no candidates in this category. The pathway hole filler not only identifies candidate proteins for each pathway hole, but also determines the probability that each candidate has the desired function. Table [4](#T4){ref-type="table"} displays the homology-based features used by the pathway hole filler to compute this probability. The table shows three example reactions, each with two candidate enzymes and the data gathered for each. The columns in the table display the computed probability that the candidate has the desired function; the number of query sequences that hit the candidate (number of hits); the E-value for the best alignment between the candidate and a query sequence (best E-value); the average rank of the candidate in the lists of BLAST hits; and the average percentage of each query sequence that aligns with the candidate. In the first example, 28 imidazolonepropionase sequences from other organisms were retrieved from Swiss-Prot and the Protein Information Resource (PIR). Using BLAST, each sequence was used to query the human genome for candidate enzymes. Protein A was found in all of the 28 lists of BLAST hits. From the numbers in the table, it is fairly obvious that protein A is more likely to catalyze the imidazolonepropionase reaction than is protein B. In the second example, given the best E-value (1e-110) it is again not surprising that the computed probability that protein C has N-acetylglucosamine-6-phosphate deacetylase activity approaches 1.0. In the last example, both proteins have excellent BLAST E-values; in fact, the E-value for protein F indicates a better match with the query sequences than the E-value for protein E. In this case, protein E is found in 19 lists of BLAST hits versus four for protein F, and on average aligns with a much larger fraction of each query sequence. When examined in more detail, we discover that the four query sequences that identified candidate F in their BLAST output are multifunctional proteins with both aldose-1-epimerase activity and UDP-glucose 4-epimerase activity. Protein F aligns with the amino-terminal region of each of the four query sequences, and has no detected similarity in the carboxy-terminal regions. The UDP-glucose 4-epimerase activity lies in the amino-terminal region of each multifunctional query protein. Nutritional analysis of the human metabolic network --------------------------------------------------- Nutritional requirements and their genetic and biochemical basis are thought to have evolved principally in prokaryotes, over billions of years \[[@B11]\]. Specific nutritional challenges have driven the evolution of metabolic pathways and the functional capabilities mediated by them. Indeed, eukaryotic life acquired the basic building blocks of metabolism, that is, sets of genes encoding enzymes that mediate specific metabolic pathways, from prokaryotic ancestors. One may define a metabolic pathway as a conserved set of genes that endow an organism with specific nutritional/metabolic capabilities, for example, the ability to grow in the absence of phenylalanine because of the ability to synthesize phenylalanine. Current knowledge of human nutrition based on metabolic pathways is derived from various sources. One is clinical observation of inherited human metabolic diseases and nutrient deficiency states. For some pathways, like oxidative phosphorylation and the TCA cycle, direct studies of human tissues, such as human muscle biopsies, have been made. Nuclear magnetic resonance (NMR) has been used directly on humans to study aspects of carbohydrate and energy metabolism. Stable isotopes have been used to trace human metabolism, from which inferences about nutrition have been made. Dietary studies have been made in experimental mammals such as rats and mice and metabolic pathways experimentally elucidated in model organisms. Here we compare previously accepted human nutritional requirements with pathways derived from the human genome to evaluate their agreement. For example, biosynthetic pathways for essential human nutrients, that is, substances that must be provided in the diet such as the essential amino acids and vitamins, would not be expected to occur in the human genome. Integration of human genome data with clinical, biochemical, physiological and other data obtained both directly from humans and indirectly from model organisms should, over time, lead to a deeper understanding of human metabolism and its nutritional implications in health and disease. When the genome sequences of individuals are available, it may be possible to address questions about the variation in optimal nutrition from person to person. Explicit identification of specific areas of inconsistency will serve to focus ongoing experimental efforts to elucidate the molecular basis of human nutrition and metabolism. For all of the nine amino acids essential for humans, PathoLogic did not predict the presence of a corresponding biosynthetic pathway (see Table [5](#T5){ref-type="table"}) \[[@B12]\]. And for all of the 11 nonessential amino acids, PathoLogic did predict the presence of a corresponding biosynthetic pathway. For 12 of 13 essential human vitamins, PathoLogic did not predict the presence of a corresponding metabolic pathway (note that PathoLogic could not have predicted such a pathway for six of those vitamins because MetaCyc does not contain such a pathway). PathoLogic did predict the presence of a pathway called \'pantothenate and coenzyme A biosynthesis pathway\', which is not expected given that pantothenate is an essential human nutrient. However, examination of the predicted pathway reveals that no enzymes in the first part of the pathway (biosynthesis of pantothenate) are present; all enzymes are in the portion of the pathway that synthesizes coenzyme A from pantothenate. Thus, this false-positive prediction can be attributed to the fact that MetaCyc does not draw a boundary between what should probably be considered two distinct pathways. No hard-and-fast rules are generally accepted as to how to draw boundaries between metabolic pathways; therefore the PathoLogic method cannot produce objective and well accepted pathway boundaries (nor can any other known algorithm). Comparative analysis of the metabolic networks of human, E. coliand Arabidopsis ----------------------------------------------------------------------------------- Table [6](#T6){ref-type="table"} indicates whether or not each HumanCyc pathway is present in the EcoCyc E. coliPGDB and in the AraCyc PGDB for A. thaliana\[[@B13]\]. More precisely, we say a pathway is shared among multiple PGDBs if the same MetaCyc pathway has been predicted to be present in each PGDB; that is, if the pathway has exactly the same set of reactions in the PGDBs (the unique identifier of the MetaCyc pathway is reused in any PGDB to which the pathway is copied). The comparison does not consider how many pathway holes are in the PGDBs, but relies on the PathoLogic prediction (plus subsequent manual review) that the pathway is present; that is, if PathoLogic determines that the pathway is present despite its holes, the comparison considers it to be present. Note that we do not count the presence of related pathway variants; that is, if organism A contains pathway P and organism B contains a variant of P, we do not score this case as a shared pathway. Some shared pathways will include pathway holes. Figure [4](#F4){ref-type="fig"} shows how the three metabolic networks intersect by means of a Venn diagram, depicting each PGDB\'s pathway complement as a circle. The number within a given intersecting area denotes the number of pathways shared by the corresponding combination of PGDBs. For example, HumanCyc has 55 pathways in common with EcoCyc, as well as 67 with AraCyc, while EcoCyc and AraCyc share 69 pathways. Thirty-five pathways are common to all three databases, and are shown in Table [6](#T6){ref-type="table"}. The 35 pathways include significant numbers of pathways from all the pathway classes (biosynthesis, catabolism and energy metabolism), and constitute a significant fraction of the pathway complements of both E. coli(20.1% of the 174 pathways in EcoCyc) and H. sapiens(25.7% of the 135 pathways in HumanCyc). Those 35 pathways therefore constitute a likely upper bound on the number of universally and exactly conserved metabolic pathways. It is an upper bound in the sense that as more organisms are considered, the list of universal pathways cannot grow larger. We propose that the cofactor biosynthesis pathways shared among all three organisms have been conserved because first, they produce complex molecules that are not available from the environments of these organisms; second, these molecules are used as cofactors in so many reactions within the metabolic networks that the requirement for them is absolute; and third, no other pathway to accomplish the synthesis of that molecule has evolved. A study by Ouzounis and Karp surveyed global properties of the E. colimetabolic network, including the most frequently used substrates and cofactors \[[@B14]\]. Together, the two pyridine nucleotides NAD and NADP are the third most common substrate in the E. colimetabolic network (after water and ATP): removing the ability to synthesize NAD would disable so many reactions as to be insurmountable. Pyridoxal-5\'-phosphate is the second most common cofactor (after Mg^2+^). Coenzyme A and acetyl-CoA together constitute the seventh most common substrate in E. coli, formylTHF constitutes the 23rd most common substrate, and thioredoxin and glutathione constitute the 40th and 41st most common substrates. Discussion ========== Pathway variants ---------------- The level of metabolic pathway variation in the biosphere remains to be determined. Metabolic pathways have been experimentally elucidated in a small number of model prokaryotic and eukaryotic organisms. Despite the relatively small number of carefully studied organisms, significant pathway variation has been observed both between distinct organisms and within a given organism. For example, at least four variants of the \'glycolytic pathway\' have been described \[[@B15]\]. Sets of variant pathways for glycolysis \[[@B16]\], leucine degradation \[[@B17]\], and NAD biosynthesis \[[@B18]\] can be viewed through the MetaCyc website. In MetaCyc, variant pathways are named with roman numerals; for example, \'NAD biosynthesis I\' and \'NAD biosynthesis II\'. Metabolic pathways appear to have diverged in a manner analogous to the divergence of biological sequences. The demonstrated existence of pathway variants and ongoing uncertainty as to the full extent of such variation has significant implications for ongoing efforts to predict biochemical pathways from incomplete genomic data. First, it means that the precision with which we can infer pathways in one organism from another solely on the basis of genomic data remains to be determined, because when genomic evidence is found in organism O~k~for the presence of a pathway, P~j~, that was experimentally elucidated in an organism O~j~, this alone does not constitute conclusive evidence for the presence of P~j~in O~k~, since a variant of P~j~(P~k~) may be present in O~k~(note that any such P~k~variant may not even have been experimentally characterized). Second, for those pathways with known closely related variants (for example, two pathways differing by only a single step with one step differing from the other only at the level of co-reactants/products, such as one using NADP/NADPH the other using NAD/NADH as cosubstrates/products) it is often impossible to choose among these variants on the sole basis of genomic data because of the limited resolution of sequence analysis. We have developed a general approach to representing the presence of pathway variants. MetaCyc pathways (approximately 500) have been grouped using a function-based classification system. Each grouping defines a \'pathway family\', each member of which (a pathway variant) endows an organism with one or more specific functional capabilities, for example, the ability to grow in the absence of phenylalanine. Within a given pathway family, variants may be clustered into one or more subfamilies. Subfamilies are groups of pathways that show significant overlap/similarity in terms of individual reaction steps (reactants and products of each reaction); enzymatic activities that catalyze these steps; and genes encoding the enzymes that mediate these activities The similarity between variants within a given subfamily suggests they evolved from a common ancestor pathway. For example, at least four variants of the glycolytic pathway have been described; all enable the conversion of glucose to pyruvate and show significant overlap/similarity in their component reactions. Other pathway variants have been observed that show little similarity to each other (for example, some of the amino-acid degradation pathways) and these are therefore believed to have evolved from distinct ancestor pathways. The existence of pathway subfamilies indicates that multiple pathways have coevolved to meet common nutritional/metabolic challenges. For the purposes of this paper, genomic evidence for the presence of a specific biochemical pathway, P~1~, in humans is taken as evidence that P~1~and/or other members of the pathway family to which P~1~belongs are likely to be present in humans (including those not yet included in MetaCyc and/or experimentally elucidated). Indeed, PathoLogic sometimes inferred the presence of multiple variant pathways in humans. This occurred because when evidence was found in the genome for the presence of one member of a pathway family/subfamily, this evidence often also supported the presence of other members of this family/subfamily. In these cases, all inferred variants were included in HumanCyc. Of course, the specific members of a given pathway family actually present in humans may include one or more of those inferred from MetaCyc or other members of this pathway family not yet described in MetaCyc and/or not yet experimentally elucidated from any organism. It is attractive to think that multiple variant pathways might refer to metabolically differentiated tissues in the body, or to different regulatory states available to the same tissue. An example of the latter would be the liver; at different times of day it either synthesizes glycogen, taking glucose from the blood, or it degrades glycogen to maintain the blood glucose level. HumanCyc as a tool ------------------ The HumanCyc PGDB is freely available for use by the scientific community from the SRI website \[[@B19]\]. Basic queries to HumanCyc can be issued through the BioCyc Query Page \[[@B20]\]. This page supports a number of query types. For text searches through the DB, for example, enter \'tryptophan\' next to the \'Query All (by name)\' box, and then click \'submit\' to retrieve a list of all enzymes, pathways, compounds, and reactions whose name includes that word. Click on \'Choose from a list of pathways\' to generate a list of all pathways within HumanCyc. Click \'submit\' near \'Browse Ontology\' to browse one of several possible classification hierarchies, such as the ontology that classifies metabolic pathways according to their physiological role. When viewing a HumanCyc pathway display, be aware that the software omits enzyme names for pathway holes. That is, when no human protein has been identified that catalyzes a reaction within a pathway, no enzyme name is drawn next to that reaction. The cellular overview \[[@B21]\], a full human metabolic pathway map, provides a tool for analysis of high-throughput datasets. The Omics Viewer \[[@B22]\] allows the user to visualize gene-expression data, protein-expression data, metabolomics measurements, or reaction flux data in a pathway context by painting reaction steps in the metabolic overview with colors that represent the expression levels of the genes coding for enzymes that catalyze those steps. The PathoLogic summary page for H. sapiensincludes a report that lists the evidence for each predicted pathway in HumanCyc, with pathways sorted according to the MetaCyc pathway ontology \[[@B23]\]. HumanCyc is currently available for local installation at your institution \[[@B24]\] as a set of flat files, and as part of an executable program running on Windows/Intel, Linux/Intel and Solaris/Sun. The latter configuration can function as both a desktop application, and as a local mirror of the HumanCyc website on the user\'s intranet. Related work ------------ Related databases of human metabolic pathways include the GenMAPP \[[@B25]\] collection of 14 drawings of human metabolic pathways that are available through the web; the KEGG \[[@B26]\] metabolic pathway website, which allows coloring of a set of reference pathway maps to indicate which enzymatic steps have corresponding human enzymes; and the Reactome (Cold Spring Harbor Laboratory, European Bioinformatics Institute (EBI) and the Gene Ontology (GO) Consortium) project, which has curated human metabolic pathways in a web-accessible database \[[@B27]\]. PathoLogic was the first program for automated inference of genome-scale pathway reconstructions from genome data \[[@B28]\]. Additional methods for prediction of metabolic pathways include methods that infer \'extreme pathways\' and \'elementary modes\' from flux-balance models of reaction networks \[[@B29]\], which have been applied to the human mitochondrion \[[@B30]\]. Rather than recognizing known, historically defined pathways in a large reaction network, as does PathoLogic, these methods infer pathways from the stoichiometric matrix representing a biochemical network by convex analysis. The biological utility and meaning of these pathways, and their correspondence to known metabolic pathways that have been established through experimental studies, remains to be demonstrated. One benefit of pathway analysis by projecting previously known pathways onto a genome using PathoLogic is the fact that projection of known pathways tells us what reactions to expect to occur in another organism, leading to the identification of pathway holes, and the directed search for their fillers. In fact, the occurrence of hundreds of pathway holes in every genome we have analyzed raises the question of how the extreme pathways method can work given that the reaction networks it relies on must omit hundreds of reactions (the pathway holes) as a result of the incompleteness of genome annotations. The following additional pathway prediction methods are related to this work, but have not been applied on a genome scale and have received little validation. Zien and colleagues use gene-expression data to score pathways that are enumerated combinatorially from a reaction database. Van Helden and colleagues use clustering of gene-expression data to generate seed reactions that are used to combinatorially enumerate pathways \[[@B31],[@B32]\]. McShan et al. infer novel biotransformation rules for xenobiotics from the molecular graphs of compounds \[[@B33]\]. A recent review discusses the potential for reconstruction of metabolic networks using metabolomics technology \[[@B34]\]. Limitations of HumanCyc and future work --------------------------------------- The user of HumanCyc should be aware of several potential limitations that influence the interpretation of the DB contents. First, HumanCyc is incomplete in the sense that some known (and unknown) human metabolic pathways are not present in it. It will require a significant database curation effort to enter all known pathways. Second, HumanCyc probably contains false-positive pathway predictions. Our approach is to err on the side of being more inclusive in our pathway predictions so that all potential pathways are brought to the attention of the scientific community for evaluation. For example, HumanCyc sometimes contains multiple pathway variants that we currently lack the evidence to choose between, and in other cases the actual human pathway may be a variant of the pathway present in HumanCyc. Third, HumanCyc does not encode information about the location (compartment(s), cell type(s) or tissue(s)) in which a pathway occurs. Each pathway is defined using all identified human enzymes, meaning that location-specific versions of a pathway are not specifically identified. Furthermore, the presence of a pathway could be incorrectly inferred because the enzymes that make up a pathway are found in different locations and are never expressed in a common location, meaning that the pathway can never occur in its entirety. Fourth, HumanCyc does not contain nucleotide or amino-acid sequences. Our future work will address the first three of these limitations, and will include a significant effort to manually refine and update HumanCyc with pathway and enzyme information from the biomedical literature. To date, three pathways have been added to HumanCyc through manual curation. Experimentally elucidated pathways in HumanCyc will be annotated as such with evidence codes; pathways predicted by PathoLogic are annotated as computationally inferred. HumanCyc can be used to develop an agenda for experimental refinement of the human metabolic map. Predicted pathways should be experimentally verified, with particular attention to choosing among multiple pathway variants. Candidate \'fillers\' for the 178 unresolved pathway holes identified herein should be identified. Conclusions =========== PGDBs endow genomic information with an extended dimension that allows researchers to analyze an organism\'s genome with respect to the causal relationships inherent to a metabolic network. In this sense, HumanCyc provides the opportunity to look at human metabolic processes within the context of the annotated human genome, and vice versa. The computationally predicted metabolic network provided a rational framework for understanding the genetic basis of some well-characterized human dietary requirements, that is, 11 nonessential amino acids and 22 essential nutrients (9 essential amino-acids and 13 essential vitamins). PathoLogic\'s pathway prediction process provides a reasonably accurate picture of the metabolic network, and Pathway Tools provides the user with extensive capabilities for refining the DB to reflect improvements in our understanding of the human metabolic network. The query and visualization capabilities of the Pathway Tools software (such as the visualization of gene-expression data superimposed on the metabolic map) will facilitate novel approaches for analyzing the complexity of functional relationships within the human genome. Materials and methods ===================== Data gathering and preparation ------------------------------ The PathoLogic program generates a new PGDB starting from the annotated genome of an organism, meaning a complete genome sequence (closed or gapped) for which gene prediction and sequence analyses have already identified the locations of likely coding regions, and have predicted the functions of these genes. We used Ensembl Build 31 as our main data source for the annotated human genome, and complemented that information with data from LocusLink, the National Center of Biotechnology Information (NCBI) database of genetic loci. We used GenBank as our source for the mitochondrion genome, as this information was not included in Ensembl. LocusLink mitochondrial loci were used to complement this information when applicable. The information from these different sources required special preprocessing to make it available to PathoLogic. That preprocessing addressed three needs: first, to convert the disparate data formats used by these sources into a format parsable by PathoLogic; second, to extract information useful to PathoLogic; and third, to remove redundancy among the sources. In the case of Ensembl, the standard Ensembl flat files included just a subset of the information needed for the PGDB generation process. We therefore used the EnsMart facility provided by Ensembl \[[@B5]\] to generate files with the required data. EnsMart allows the user to select subsets of the genome (from small regions to entire chromosomes) and output desired data about each gene in different tabular formats. Additional data file 1 lists the genetic element files generated using EnsMart. One file was generated for each human chromosome and contig, with the file names corresponding to the chromosome and contig names in Ensembl. When the corresponding chromosome for a given contig was known, the contig\'s file name would be constructed by prepending the chromosome\'s name to the contig\'s name, otherwise \'Un\', for unknown, was prepended to the contig\'s name. We selected LocusLink file LL3\_030319 (Version from 19 March, 2003), which contained all data required by PathoLogic (the LocusLink organism-specific files, like the Ensembl files, did not include all the needed database fields). Table [7](#T7){ref-type="table"} shows the types of information extracted from each source when available. For example, we included a large number of comments extracted from LocusLink in HumanCyc; each such comment in HumanCyc ends with a citation to LocusLink to properly attribute its source. Function descriptions for gene products are very important for PathoLogic, as they will be matched against enzyme activity names stored in MetaCyc, a multi-organism database of experimentally determined metabolic pathways and enzymes \[[@B15]\]. In Ensembl, such functional information had to be parsed from the \'function\' field in EnsMart data. This field sometimes includes long and complex descriptions of the gene product\'s function, mostly extracted by Ensembl from SwissProt \[[@B35]\]. These descriptions could include synonyms, EC numbers, and multiple functions (see Figure [1](#F1){ref-type="fig"}). All that information had to be extracted and presented to PathoLogic in a structured form. When generating the input files for PathoLogic, information from Ensembl and LocusLink was combined only when the Ensembl record for a gene included a cross reference to a LocusLink ID. In such a case, data from the LocusLink entry was merged with that of the Ensembl gene, meaning that when both databases provide an attribute such as a gene name, the Ensembl data is preferred. This approach created gene objects for HumanCyc that include information from both the Ensembl and the LocusLink entries. Those HumanCyc gene objects use the Ensembl ID as their unique identifier, and have database links to the corresponding LocusLink entries. For the mitochondrial data, the number of LocusLink mitochondrial loci was small enough so that they were easily checked for matches with corresponding genes in the GenBank files. LocusLink loci that had no direct counterpart in either the Ensembl data or the GenBank mitochondrial data (for example, LocusLink loci that were not directly referenced in any Ensembl gene record) were assigned their own record in the PathoLogic input files. The LocusLink ID was used as the unique identifier for these gene objects. Only loci corresponding to \'real\' genes (not models, phenotypes or pseudogenes) were included in HumanCyc. We must point out that records corresponding only to LocusLink loci lack gene position information, so they cannot be precisely placed on a chromosome map. We were aware that adding these LocusLink-only-based records to HumanCyc would produce some redundancy in the database, but this was accepted for the sake of completeness. Manual analysis of similar Ensembl and LocusLink-based gene objects after building HumanCyc led to the fusion of gene objects corresponding to the same gene. The number of LocusLink gene objects that were not merged to corresponding gene objects from Ensembl or GenBank is shown in the \'nonredundant\' column of Table [7](#T7){ref-type="table"}. It is readily apparent from Table [7](#T7){ref-type="table"} that HumanCyc, thanks to the combination of the Ensembl and LocusLink data sources, has excellent cross-reference coverage to many other biological databases (including Ensembl and LocusLink themselves). In addition to the databases mentioned in Table [7](#T7){ref-type="table"}, we added links to the GeneCards genomic database for those genes with known HUGO IDs. Seventy-six PathoLogic input files were generated from the preceding data sources: 24 for the human chromosomes, one for the mitochondrion, 50 for the different contigs not yet integrated to the chromosomal sequences, and one called \'unknown\' for all the loci that had no chromosome information. A replicon object was created in HumanCyc for each of these files. Prediction of human metabolic pathways using PathoLogic ------------------------------------------------------- This section summarizes the PathoLogic algorithm (for a more detailed description of the method see \[[@B36],[@B37]\]). For an evaluation of the accuracy of PathoLogic see \[[@B36]\]. After initializing the schema of the new PGDB, a database object is created for each replicon and contig, and for each gene and its corresponding gene product. PathoLogic then tries to determine the metabolic reaction catalyzed (if any) by each gene product in the organism by using its EC number, if provided in the annotation, and by matching the name of each gene product against the extensive dictionary of enzyme names within the MetaCyc DB \[[@B15]\]. Finally, the list of reactions now known to be catalyzed by the organism is matched against all the pathways in MetaCyc. For pathways with significant numbers of matches (see \[[@B36]\] for a detailed description of this algorithm), PathoLogic imports the pathway and its associated reactions and substrates from MetaCyc into the new PGDB. This method of pathway prediction is analogous to predicting the function of a protein based on sequence similarity to a protein of known function, in that both methods recognize the presence of something known (a known pathway versus a known protein function) based on a similarity between patterns (a pattern of enzymes present versus a sequence pattern). The two methods share similar limitations: just as sequence similarity cannot predict protein functions that are not in the sequence database, PathoLogic cannot predict pathways that are not in MetaCyc. As mentioned above, PathoLogic will assign reactions for those enzymes that have an exact EC number match or name match against MetaCyc. A gene product name may not exactly match that of any enzyme in MetaCyc. Some enzyme names will produce ambiguous matches or no match at all. PathoLogic assembles a list of \'probable enzymes\' that includes both ambiguous matches and nonmatched proteins whose names suggest enzymatic activity. This list is examined manually through a PathoLogic module that helps the user evaluate possible matching candidates within MetaCyc and assign probable enzymes to the correct reaction, if possible. An alternative to our strategy of using the existing EC number and function assignments from LocusLink and Ensembl would be for us to discard those assignments and to reanalyze the genome using sequence analysis methods to produce new assignments. We rejected this approach for two reasons: first, it would discard some experimentally derived function assignments in place of less reliable computational assignments; and second, we consider the Ensembl function predictions to be of high quality, and we are aware of no evidence that our group, or any other group, has a sequence analysis methodology that will produce function assignments that are substantially more accurate than those of Ensembl. Finally, PathoLogic generates reports summarizing the amount of evidence supporting each pathway in the new PGDB, and listing the pathway holes, that is, the enzymes missing from each predicted pathway. This information helps the user identify pathways that should be deleted from the PGDB, such as variant pathways and false-positive predictions made by PathoLogic. For example, MetaCyc includes eight variants of the TCA cycle. Several of these might appear in a newly predicted PGDB. False-positive pathways, some of which are predicted because they share reactions with other pathways in MetaCyc, should be removed from the PGDB. Once variant or false-positive pathways are eliminated by the user, PGDB generation has been completed. Filling holes in HumanCyc pathways ---------------------------------- To determine the function of a protein sequence, researchers typically use a single sequence to search for potential homologs in a large public database. To identify sequences to fill pathway holes, we have, in effect, reversed this search process. We search the genome for a sequence that will provide the enzymatic function needed to fill each pathway hole. Our method uses multiple isozyme sequences (retrieved via MetaCyc from Swiss-Prot and PIR) to search a genome for similar sequences (hole-filler candidates). We then evaluate each candidate to determine the probability that the sequence has the desired function based on homology and pathway-based data. A hole-filler tool that implements this pathway-driven gene-finding methodology has been developed \[[@B9]\]. Analysis of HumanCyc metabolic network -------------------------------------- Once HumanCyc was built and manually refined, as explained above, we examined the metabolic network within HumanCyc in order to make a preliminary assessment of the quality of PathoLogic\'s predictions and to check for pathways not previously thought to occur in humans. We also compared the metabolic network of HumanCyc to that of two of our curated PGDBs, corresponding to a bacterium and a plant. These PGDBs are EcoCyc (E. coli) and AraCyc (A. thaliana). Additional data files ===================== The following additional data are available with the online version of this article. Additional data file [1](#s1){ref-type="supplementary-material"} contains a table listing the data file names generated from EnsMart for each human chromosome or contig and provided as input to PathoLogic, thus indicating which contigs are associated with which chromosomes. Supplementary Material ====================== ::: {.caption} ###### Additional data file 1 A table listing the data file names generated from EnsMart for each human chromosome or contig and provided as input to PathoLogic, thus indicating which contigs are associated with which chromosomes ::: ::: {.caption} ###### Click here for additional data file ::: Acknowledgements ================ This work was supported by funds from a major pharmaceutical company, and by grants R01-HG02729-01 from the NIH National Human Genome Research Institute, R01-GM65466-01 from the NIH National Institute for General Medical Sciences, and DE-FG03-01ER63219 from the US Department of Energy. The contents of this article are solely the responsibility of the authors and do not necessarily represent the views of these sponsors. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### A typical description of a gene product\'s function in Ensembl. This example aims to communicate to the reader exactly what information was obtained from Ensembl; it shows multiple functions, synonyms and EC numbers, as well as a Swiss-Prot accession number, all in one line of text. A Perl script was developed to parse these descriptions and extract the relevant information. ::: ![](gb-2004-6-1-r2-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Predicted HumanCyc pathway for arginine degradation. The computer icon in the upper-right corner indicates this pathway was predicted computationally. Neither enzyme names nor gene names are drawn adjacent to the first three reactions of this pathway to indicate that these steps are pathway holes, meaning no enzyme has been identified for these steps in the human genome. The graphic at the bottom indicates the positions of genes within this pathways on the human chromosomes. Moving the mouse over a gene in the webpage for this diagram will identify the gene and the chromosome. ::: ![](gb-2004-6-1-r2-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Curated HumanCyc pathway for oxidative ethanol degradation. This pathway was not predicted by PathoLogic, but was entered into HumanCyc as part of our subsequent literature curation effort. The flask icon in the upper-right corner indicates this pathway is supported by experimental evidence. The complete comment for this pathway is available at \[38\] ::: ![](gb-2004-6-1-r2-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Numbers of pathways, including superpathways, shared by the three PGDBs HumanCyc (H. sapiens), EcoCyc (E. coli), and AraCyc (A. thaliana). The numbers outside the circles represent the total number of pathways in the corresponding PGDB. The numbers inside the intersecting areas represent the number of pathways that fall into each area. For example, there are 55 pathways in common between HumanCyc and EcoCyc (20 + 35). AraCyc contains 177 total pathways: 76 that are unique to A. thaliana, and 101 that are shared with other organisms. ::: ![](gb-2004-6-1-r2-4) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### The number of human proteins that were assigned enzyme activities (which caused them to become connected to reaction objects within HumanCyc), according to the mechanism of reaction matching ::: Type of match Number of proteins --------------------------------- -------------------- PathoLogic matched by EC number 2,057 PathoLogic matched by name 314 Ambiguous 27 Unmatched by PathoLogic 27,185 Probable enzymes 1,320 Manually matched 625 ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### HumanCyc statistics ::: PGDB objects Quantity -------------------------- ---------- Replicons 76 Genes 28,783 Protein genes 28,583 Enzyme genes 2,742 RNA genes 200 tRNAs 50 Compounds 661 Polypeptides 28,602 Protein complexes 22 Enzymes 2,709 Enzymatic Reactions 1,093 With enzyme in HumanCyc 896 Pathways 135 Database links 389,262 Citations 41,810 ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### The entire set of pathways in HumanCyc, grouped by classes using the MetaCyc pathway classification hierarchy ::: Class Subclass Pathway EcoCyc AraCyc ------------------- ------------------------------------------------- ------------------------------------------------------------------------------------------ -------- -------- Biosynthesis Polyamines Betaine biosynthesis \* \* Betaine biosynthesis II Spermine biosynthesis \* Polyamine biosynthesis II Ornithine spermine biosynthesis \* Polyamine biosynthesis \* \* UDP-N-acetylgalactosamine biosynthesis \* UDP-N-acetylglucosamine biosynthesis \* Nucleotides De novobiosynthesis of purine nucleotides \* Purine and pyrimidine metabolism Purine biosynthesis 2 De novobiosynthesis of pyrimidine ribonucleotides \* Salvage pathways of pyrimidine ribonucleotides \* De novobiosynthesis of pyrimidine deoxyribonucleotides \* Salvage pathways of pyrimidine deoxyribonucleotides \* Fatty acids and lipids Fatty acid elongation - saturated \* \* Fatty acid biosynthesis - initial steps \* \* Phospholipid biosynthesis \* \* Phospholipid biosynthesis II Mevalonate pathway \* Triacylglycerol biosynthesis \* Cofactors, prosthetic groups, electron carriers Heme biosynthesis II NAD biosynthesis II NAD biosynthesis III NAD phosphorylation and dephosphorylation \* Pyridine nucleotide biosynthesis \* \* Pyridine nucleotide cycling \* Glutathione-glutaredoxin redox reactions \* Glutathione biosynthesis \* \* Thioredoxin pathway \* \* Pantothenate and coenzyme A biosynthesis \* \* Pyridoxal 5\'-phosphate salvage pathway \* \* FormylTHF biosynthesis \* \* Polyisoprenoid biosynthesis \* \* Methyl-donor molecule biosynthesis \* Cell structures Colanic acid building blocks biosynthesis \* \* GDP-mannose metabolism \* \* Mannosyl-chito-dolichol biosynthesis \* UDP-N-acetylglucosamine biosynthesis \* Carbohydrates GDP-D-rhamnose biosynthesis Gluconeogenesis \* \* Mannosyl-chito-dolichol biosynthesis \* Trehalose degradation - low osmolarity \* \* Aminoacyl-tRNAs tRNA charging pathway \* \* Amino acid biosynthesis Alanine biosynthesis II \* Arginine biosynthesis 4 \* Citrulline biosynthesis Asparagine biosynthesis I Aspartate biosynthesis II Cysteine biosynthesis II Glutamate biosynthesis II \* Glutamine biosynthesis II Glycine cleavage \* Glycine biosynthesis I \* \* Methionine salvage pathway Proline biosynthesis I \* \* Serine biosynthesis \* \* Tyrosine biosynthesis II Degradation Sugars and polysaccharides Lactose degradation 4 \* Lactose degradation 2 \* Sucrose degradation III Galactose metabolism \* \* Glucose 1-phosphate metabolism \* \* Glycogen degradation \* \* Mannose degradation \* Non-phosphorylated glucose degradation \* UDP-glucose conversion \* Ribose degradation \* \* Trehalose degradation - low osmolarity \* \* Sugar derivatives Lactate oxidation Mannitol degradation \* Sorbitol degradation \* Glucosamine catabolism \* Other degradation Removal of superoxide radicals \* \* Methylglyoxal degradation Nucleosides and nucleotides (Deoxy)ribose phosphate metabolism \* \* Periplasmic NAD degradation Fatty acids Fatty acid oxidation pathway \* \* Triacylglycerol degradation \* Lipases pathway \* Carboxylates, other Propionate metabolism - methylmalonyl pathway \* 2-Oxobutyrate degradation Acetate degradation \* \* Pyruvate metabolism N-acetylneuraminate degradation C1 compounds Carbon monoxide dehydrogenase pathway \* Serine-isocitrate lyase pathway \* Amino acids, amines Alanine degradation 3 \* Arginine degradation III Arginase degradation pathway Arginine proline degradation \* Asparagine degradation 1 \* Aspartate degradation 1 Malate/aspartate shuttle pathway L-cysteine degradation IV \* L-cysteine degradation VI Cysteine degradation I Glutamate degradation I \* Glutamate degradation IV Glutamate degradation VII \* Glutamine degradation 1 Glutamine degradation II Glycine degradation II Glycine degradation I Histidine degradation III Histidine degradation I Homocysteine degradation I Isoleucine degradation I \* Isoleucine degradation III Leucine degradation II Leucine degradation I \* Lysine degradation I \* Methionine degradation 1 \* 4-Hydroxyproline degradation \* S-adenosylhomocysteine degradation Phenylalanine degradation I Proline degradation III Proline degradation II L-serine degradation \* \* Threonine degradation 2 Tryptophan degradation I Tryptophan degradation III \* Tryptophan kynurenine degradation Tyrosine degradation Valine degradation I \* Alcohols Aerobic glycerol degradation II \* Glycerol metabolism \* \* Glycerol degradation I \* Ethanol degradation \* Amines and polyamines, other Citrulline degradation N-acetylglucosamine, N-acetylmannosamine and N-acetylneuraminic acid dissimilation \* \* Glucosamine catabolism \* Energy metabolism Glycolysis 3 \* Glycolysis \* \* Glycolysis 2 Glyceraldehyde 3-phosphate degradation \* Non-oxidative branch of the pentose phosphate pathway \* \* Oxidative branch of the pentose phosphate pathway \* \* Aerobic respiration - electron donors reaction list \* Pyruvate dehydrogenase \* \* TCA cycle - aerobic respiration \* \* Entner-Doudoroff pathway \* More detailed subclasses were not included for brevity. An asterisk in one of the last two columns means that the pathway is also present in the EcoCyc (E. coli) and/or AraCyc (A. thaliana) databases, respectively. Note that pathway names are derived from the MetaCyc database, which explains why HumanCyc contains a pathway called \'Heme Biosynthesis II\' but not \'Heme Biosynthesis I.\' ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### A comparison of candidates for three missing enzymes ::: Candidate P (has-function) Number of hits Best E-value Average rank Percentage of query aligned -------------------------------------------------------------- ----------------------------------------------------------------------- ------------------ ---------------- -------------- -------------- ----------------------------- Reaction hole: imidazolonepropionase A ENSG00000139344-MONOMER Functional annotation: UNKNOWN 0.98 28 7.0e-69 1.0 91.9 B ENSG00000119125-MONOMER Functional annotation: Guanine deaminase 0.00018 6 3.0e-6 3.5 37.9 Reaction hole: N-acetylglucosamine-6-phosphate deacetylase C ENSG00000162066-MONOMER Functional annotation:CGI-14 protein 0.998 9 1e-110 1.0 94.6 D ENSG00000119125-MONOMER Functional annotation: Guanine deaminase 1.0e-5 4 0.85 4.0 19.9 Reaction hole: aldose 1-epimerase E ENSG00000143891-MONOMER Functional annotation:AMBIGUOUS 0.98 19 3e-74 1.58 81.9 F ENSG00000117308-MONOMER Functional annotation:UDP-glucose 4-epimerase 0.93 4 1e-100 1.0 58.3 ::: ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Comparison of known essential human nutrients with corresponding biosynthetic pathways in MetaCyc and in HumanCyc ::: Essential nutrient in humans Biosynthetic pathway in MetaCyc? Biosynthetic pathway inferred in humans? ------------------------------ ---------------------------------- ------------------------------------------ Amino acids Arginine Y N Histidine Y N Isoleucine Y N Leucine Y N Lysine Y N Methionine Y N Phenylalanine Y N Threonine Y N Valine Y N Vitamins Ascorbic acid (Vitamin C) Y N Biotin (Vitamin H) Y N Folic acid (Vitamin M) Y N Niacin (Vitamin B~3~) N N Pantothenic acid Y Y Pyridoxine (Vitamin B~6~) N N Riboflavin (Vitamin B~2~) Y N Thiamine (Vitamin B~1~) Y N Cobalamin (Vitamin B~12~) Y N Retinol (Vitamin A) N N Vitamin D N N Tocopherol (Vitamin E) N N Vitamin K N N Note that a pathway cannot be predicted in HumanCyc if it does not exist in MetaCyc. ::: ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Pathways (including superpathways) that are common to human, bacteria and plant PGDBs ::: Class Subclass Pathway ------------------- ------------------------------------------------- ------------------------------------------------------ Biosynthesis Polyamines Betaine biosynthesis Polyamine biosynthesis Fatty acids and lipids Phospholipid biosynthesis Fatty acid biosynthesis - initial steps Fatty acid elongation - saturated Cofactors, prosthetic groups, electron carriers Pyridine nucleotide biosynthesis Thioredoxin pathway Glutathione biosynthesis Pantothenate and coenzyme A biosynthesis Pyridoxal 5\'-phosphate salvage pathway Polyisoprenoid biosynthesis FormylTHF biosynthesis Cell structures Colanic acid building blocks biosynthesis GDP-mannose metabolism Carbohydrates Gluconeogenesis Trehalose degradation - low osmolarity Aminoacyl-tRNAs tRNA charging pathway Amino acid biosynthesis Proline biosynthesis I Glycine biosynthesis I Serine biosynthesis Degradation Sugars and polysaccharides Glucose 1-phosphate metabolism Galactose metabolism Trehalose degradation - low osmolarity Glycogen degradation Ribose degradation Other degradation Removal of superoxide radicals Nucleosides and nucleotides (Deoxy)ribose phosphate metabolism Fatty acids Fatty acid oxidation pathway Carboxylates, other Acetate degradation Amino acids, amines L-serine degradation Alcohols Glycerol metabolism Energy metabolism Pyruvate dehydrogenase TCA cycle - aerobic respiration Glycolysis Oxidative branch of the pentose phosphate pathway Nonoxidative branch of the pentose phosphate pathway HumanCyc, H. sapiens; EcoCyc, E. coli; AraCyc, A. thaliana. The pathways in the table are included in all three PGDBs. ::: ::: {#T7 .table-wrap} Table 7 ::: {.caption} ###### Information extracted from different data sources ::: Data source (version) Information extracted (for each gene or locus) Number of genes ------------------------------------ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ----------------- ------- Ensembl (Build 31) Gene name, chromosome or contig, start and end positions, strand (transcription direction), exons, gene-product (including function name(s) or description(s), synonyms and EC number(s)), cross references (IDs) to other databases (SwissProt, HUGO, PDB, GO, RefSeq, OMIM, Entrez, SPTREMBL, EMBL, LocusLink). 24,847 LocusLink (03/29/2003) Gene name, chromosome, gene product (function name or description), function synonyms, EC number(s), gene and protein comments, cross references (IDs) to other databases (Entrez, UCSC Genome, RefSeq, GO, OMIM, UniGene, PubMed) 18,880 3,936 GenBank NC\_001807 (mitochondrion) Gene name, start and end positions, transcription direction, gene product (function name or description) 35 Functional information in Ensembl had to be extensively parsed to extract multiple functions, EC numbers, and/or synonyms. The \'nonredundant\' column shows the number of genes from LocusLink that had no corresponding gene in the other two data sources (Ensembl and GenBank). :::	PubMed Central	2024-06-05T03:55:52.936959	2004-12-22	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549063/", "journal": "Genome Biol. 2005 Dec 22; 6(1):R2", "authors": [ { "first": "Pedro", "last": "Romero" }, { "first": "Jonathan", "last": "Wagg" }, { "first": "Michelle L", "last": "Green" }, { "first": "Dale", "last": "Kaiser" }, { "first": "Markus", "last": "Krummenacker" }, { "first": "Peter D", "last": "Karp" } ] }
PMC549064	Background ========== The unfolded protein response (UPR) regulates the protein-folding and secretory capacity of eukaryotic cells by monitoring conditions within the endoplasmic reticulum (ER) and regulating a downstream gene-expression program (reviewed in \[[@B1]-[@B3]\]). In yeast, about 5% of the genome is under the transcriptional control of the UPR \[[@B4],[@B5]\]. Induction of this vast set of genes is thought to lead to a restructuring of the secretory pathway to allow an increased protein flux to the cell surface and enable the cell to tolerate protein-folding stress. Hence, the UPR adjusts the secretory capacity of cells by feedback regulation. The identification and characterization of the UPR signaling components revealed a unique mechanism of signal transduction whose salient features are conserved among all eukaryotes. The UPR is initiated when the amino-terminal portion of the serine/threonine ER-transmembrane kinase Ire1 detects unfolded proteins within the ER lumen \[[@B6],[@B7]\]. Accumulation of unfolded proteins sequesters chaperones and thereby allows Ire1 molecules to oligomerize in the plane of the ER membrane. Oligomerization, in turn, results in trans-autophosphorylation of the cytosolic kinase domain, providing the means by which the signal is transmitted across the ER membrane. Activated Ire1 acts as a site-specific endoribonuclease, cleaving the mRNA encoding the transcription activator Hac1 at two discrete positions, and removing a 252-nucleotide nonclassical intron \[[@B8],[@B9]\]. A second enzyme, tRNA ligase (Rlg1), then joins the severed exons to produce a spliced version of HAC1mRNA, termed HAC1^i^mRNA (ifor UPR-induced) \[[@B10]\]. The HAC1mRNA splicing reaction mediated by Ire1 and Rlg1 is spliceosome-independent, and utilizes chemistry that closely resembles pre-tRNA splicing \[[@B9],[@B11]\]. As in pre-tRNA processing, 5\' and 3\' splice-site cleavage of HAC1mRNA occur independently. In contrast, spliceosome-mediated splicing is a series of two transesterification reactions that have to be strictly ordered: 3\' splice-site cleavage cannot occur before 5\' splice-site cleavage. Intriguingly, this Ire1-mediated splicing reaction happens on polyribosome-bound HAC1mRNA in the cytosol \[[@B12],[@B13]\]. In the unspliced HAC1mRNA, the intron forms a long-range base-pairing interaction with the 5\' untranslated region (UTR) that is responsible for preventing its translation \[[@B12],[@B13]\]. Splicing abolishes translational inhibition, allowing production of the Hac1 transcription factor and induction of UPR target genes. Hence, removal of the intron provides a key regulatory step in the signaling pathway. The amino-acid sequence of the nuclease region of Ire1 reveals significant similarities to that of RNase L, a mammalian endoribonuclease that is activated by interferon during viral infection \[[@B14],[@B15]\]. RNase L functions to eliminate infected cells by nonspecific degradation of cellular RNA. Mutagenesis analysis maps the endoribonuclease activity of both proteins to homologous carboxy-terminal domains \[[@B16],[@B17]\]. The nuclease domains in both proteins are preceded by kinase domains, and only the oligomerized forms of each protein appear to be active nucleases. Despite these similarities, the endoribonuclease activity of Ire1 is specific for the HAC1mRNA, whereas the nuclease activity of RNase L shows no sequence specificity. Previously, to demonstrate the sequence specificity of Ire1, we devised an in vitroassay which utilized a purified, recombinant Ire1 protein \[[@B9]\]. This protein, here referred to as Ire1\, is composed of a linker region (bridging between the membrane anchor of full-length Ire1 and the cytosolic kinase domain), the kinase domain itself, and the RNase domain. Ire1\ cleaves HAC1mRNA faithfully at both 5\' and 3\' exon-intron junctions and thus recapitulates the substrate specificity of full-length Ire1 in vivo. Other RNAs, including actin mRNA and poly(U) RNA, which has been demonstrated to be an RNase L substrate \[[@B18]\], are not cleaved by Ire1\* \[[@B9],[@B16]\]. In metazoans, multiple parallel pathways originate from the ER and contribute to the UPR. Two bona fideIre1 orthologs IRE1α and IRE1β \[[@B19],[@B20]\] are present in higher eukaryotes. In addition, a Hac1 ortholog (XBP1) is activated by a similar nonconventional splicing step that changes the protein\'s carboxy-terminal sequence by introducing a frameshift \[[@B21]-[@B23]\]. Another transmembrane kinase, PERK, shares structural similarity to Ire1 in its ER-luminal unfolded-protein-sensing domain and is also activated upon accumulation of unfolded proteins \[[@B24],[@B25]\]. Activated PERK phosphorylates the translation initiation factor eIF2α, thereby inactivating it. This branch of the pathway leads to a global repression of translation, which is thought to lessen the secretory burden on the ER. In cells with reduced eIF2α activity, mRNAs containing small open reading frames (ORFs) in their 5\' UTRs become preferentially translated. One such mRNA codes for the transcription factor ATF4, which collaborates with XBP1 to induce UPR target genes \[[@B26]\]. A third branch of the metazoan UPR activates the transcription factor ATF6, which is initially synthesized as an ER transmembrane protein \[[@B27],[@B28]\]. Accumulation of unfolded proteins allows ATF6 to leave the ER and move to the Golgi compartment, where it encounters proteases that release its cytosolic portion as a soluble protein \[[@B27],[@B29]-[@B32]\], which participates along with XBP1 and ATF4 in executing the transcriptional program of the UPR \[[@B33]\]. To date, HAC1mRNA is the only known RNA substrate for yeast Ire1, prompting the question of whether other substrate RNAs exist or whether Ire1 and HAC1mRNA function as a matched enzyme-substrate pair that interact exclusively with each other. Here we describe three independent genome-scale methods that address this question. Each approach successfully identifies HAC1mRNA as a substrate of Ire1; none of the approaches identifies any other mRNA as a qualified candidate. Two of these approaches represent novel applications of cDNA array technology and could be adapted to the study of other signal transduction pathways regulated by RNA processing. Results and discussion ====================== We first devised a molecular screen to identify mRNAs cleaved by the Ire1 nuclease (Figure [1](#F1){ref-type="fig"}). In brief, we isolated a total poly(A)^+^RNA fraction from cells and subjected it to in vitrocleavage reactions in the presence or absence of Ire1\. Fragments that lost their poly(A)^+^tails due to cleavage by Ire1\ were re-isolated, reverse transcribed, fluorescently labeled and hybridized to genomic cDNA microarrays to identify Ire1 substrates. For these experiments, we expressed and purified a recombinant Ire1\* tagged with glutathione-S-transferase (GST). The GST moiety was removed during the purification by protease cleavage. Upon incubation of Ire1\* with in vitrotranscribed HAC1mRNA, we observed efficient and accurate cleavage at both splice junctions as previously described \[[@B9],[@B11]\]. We optimized reaction conditions such that HAC1mRNA contained in a total poly(A)^+^RNA fraction from yeast would be efficiently cleaved, even in the presence of significant excess of other mRNAs. We incubated total poly(A)^+^RNA with Ire1\* and then fractionated the products using oligo(dT) cellulose (Figure [2](#F2){ref-type="fig"}). Input RNA and the bound and unbound fractions of the cleavage reaction were analyzed by Northern blotting using a HAC1-specific probe covering the 5\' exon (Figure [2](#F2){ref-type="fig"}, lanes 1-3). Note that HAC1mRNA present in the input fraction (Figure [2](#F2){ref-type="fig"}, lane 1) was efficiently converted to a faster migrating species corresponding to the released 5\' exon and intron, which, lacking poly(A) tails, were recovered exclusively in the unbound fraction after oligo(dT) chromatography (Figure [2](#F2){ref-type="fig"}, lane 3). Conversely, analyzing the same RNA fractions with a HAC1probe directed to the 3\' exon, the resulting 3\' exon (still containing its poly(A) tail) was recovered exclusively in the oligo(dT)-bound fraction (Figure [2](#F2){ref-type="fig"}, lane 5). No significant levels of uncleaved HAC1mRNA were detectable after Ire1\* cleavage (Figure [2](#F2){ref-type="fig"}, lanes 2, 3, 5 and 6). Using these reaction conditions, we isolated Ire1\-cleaved RNA fragments from total poly(A)^+^RNA, which were recovered in the unbound fraction after oligo(dT) chromatography. We prepared a mock-treated reference sample using reaction conditions which were identical except that Ire1\ was omitted. We reverse-transcribed both samples and fluorescently labeled the resulting cDNA with Cy3 (Ire1\-treated; green) or Cy5 (mock-treated; red), respectively. The two probes were then simultaneously hybridized to yeast microarrays. We expected that contaminating poly(A)^-^RNA or uncleaved poly(A)^+^RNA would be equally represented in the oligo(dT) unbound fractions of both Ire1\-treated and mock-treated reactions, thus leading to equal representation of both fluorescent probes in the mixture. Indeed, scatter plots generated by quantitating the fluorescence levels of both Cy3 and Cy5 (Figure [3a](#F3){ref-type="fig"}) showed that most spots appeared on a tight diagonal, having hybridized to roughly equal amounts of both the Cy3 and Cy5 probes. We expected those mRNAs that are specifically cleaved by Ire1\* to be enriched in the oligo(dT) unbound fraction of the Ire1\-treated reaction compared to the unbound fraction of the mock-treated reaction, resulting in a higher Cy3/Cy5 fluorescence ratio in the microarray hybridization. Depletion of particular mRNAs by Ire1\ digestion thus provides an enzymological tool to fractionate substrate from nonsubstrate mRNAs. We therefore plotted a histogram of the log~2~Cy3/Cy5 ratio for all mRNAs (Figure [3b](#F3){ref-type="fig"}, and see Additional data file 1). The histogram approximates a tight normal distribution (mean = 0.06; σ = 0.41) with only one significant outlier: At a log~2~Cy3/C5 ratio of 2.3, HAC1mRNA falls 5.6σ from the mean of the distribution, clearly identifying this mRNA as an Ire1\* substrate. Indeed, under these conditions, HAC1mRNA is the only substrate for cleavage by Ire1\* represented in the poly(A)^+^RNA fraction. As the Ire1 cleavage sites on other mRNAs could, in principle, be located within the 5\' or 3\' UTRs, we also hybridized the same probe to genomic microarrays containing yeast intergenic regions in addition to the ORFs. Our results were similar to those observed using ORF-only microarrays, with HAC1being the only significant outlier (data not shown). To exclude the possibility that other potential RNA substrates might be hiding in the scatter of the data, our second approach combined the microarray analysis with a bioinformatics approach to search the genome for potential Ire1 cleavage sites. We had previously defined a consensus stem-loop motif by comparing the two splice sites of HAC1mRNA and mutational analysis (Figure [4a](#F4){ref-type="fig"}) \[[@B11]\]. Both cleavages occur between the third and fourth nucleotide of a predicted seven-nucleotide loop bounded by a stem; the first nucleotide of both loops is a C; the third and sixth nucleotide of both loops is a G; and the first nucleotide of the 3\' leg of the stem is in both cases a G. The primary and secondary sequence information in this consensus is illustrated in Figure [4b](#F4){ref-type="fig"}. In vitro, Ire1\* can cleave short RNA substrates containing only the 5\' or 3\' stem-loop sequences \[[@B11]\]. Mutational studies at nucleotide resolution demonstrate that each of the shared primary and secondary sequence elements is essential for efficient cleavage. We computationally searched the yeast genome for the presence of sequences fitting the consensus stem-loop motif. In this analysis, we searched ORFs, as well as 1,000 nucleotides upstream and downstream to include potential stem-loop motifs in 5\' and 3\' UTRs. The search yielded a total of 52 hits (Additional data file 2). In the list of genes identified, only a single gene, HAC1, contains two predicted stem-loop structures, consistent with the notion that HAC1mRNA is the only Ire1-dependent splicing substrate in yeast cells. For the remaining 52 predicted stem-loop structures, the possibility remained open that Ire1 would cleave some substrates at only a single site, perhaps to downregulate particular mRNAs. However, in the light of the data obtained from the microarray analysis described in Figure [3](#F3){ref-type="fig"} we consider this possibility unlikely. We would have expected to see the 5\' fragments resulting from such cleavage events in the oligo(dT) unbound fraction of Ire1\-treated poly(A)^-^mRNA, resulting in high outlying Cy3/Cy5 ratios; however, none of the genes predicted to bear single HAC1splice consensus sequences fell more than 3σ from the mean of the distribution (that is, none fell outside a 99% confidence interval). Because previous in vitroanalyses have shown that stem-loop structures matching the consensus are sufficient for cleavage, we consider it most likely that the predicted stem-loop structures are either not included in the transcripts or do not fold as predicted in the context of the full mRNA sequences. In a third genome-scale approach, we exploited the phenotype of a mutant in tRNA ligase that is defective in UPR induction \[[@B10]\]. Characterization of this mutant, rlg1-100, previously showed that Ire1-dependent cleavage of HAC1mRNA occurs normally in the absence of ligation, but that the cleavage products are rapidly degraded. We assume that any other mRNA following this pathway should suffer the same fate, and that we could identify substrates of Ire1 by looking for mRNAs whose steady-state levels drop upon UPR induction in the rlg1-100mutant, but not in a wild-type cell. The Ire1-dependent selective disappearance of HAC1mRNA in rlg1-100cells thus provides us with a tool to assess the spectrum of mRNAs that utilize the Ire1-mediated splicing pathway. We treated rlg1-100cells with either tunicamycin (to induce the UPR by inhibition of N-linked glycosylation in the ER) or with no drug as a control, isolated total mRNA, reverse transcribed and fluorescently labeled the cDNA with Cy5 (for the tunicamycin-treated sample) and Cy3 (for the untreated sample) before simultaneous hybridization to genomic microarrays. mRNAs which are depleted during tunicamycin treatment should have Cy3/Cy5 ratios greater than 1 (log~2~Cy3/Cy5 ratios greater than 0). As shown by the data in Figure [5](#F5){ref-type="fig"}, the steady-state levels of most mRNAs remain unchanged upon tunicamycin treatment (Figure [5a](#F5){ref-type="fig"}, and Additional data file 3). As before, a histogram of the log~2~Cy3/Cy5 ratios followed a tight quasi-normal distribution (mean = 0.11, σ = 0.27) with a single outlier. HAC1, at a log~2~Cy3/5 ratio of 1.5, HAC1falls 5σ from the mean, and is thus successfully identified as a splicing target of the UPR. Once again, no other mRNA clearly satisfied the criteria for identification as an Ire1 substrate. The depletion of HAC1mRNA upon tunicamycin treatment was specific to rlg1-100cells. A similar analysis of wild-type cells showed the expected induction of UPR target genes and no depletion of HAC1mRNA relative to an untreated control (data not shown). Thus, HAC1mRNA again stands out as the unique substrate for Ire1-dependent cleavage in yeast. Conclusions =========== The experiments presented in this paper represent three genome-scale approaches for identifying mRNA substrates of the Ire1-dependent mRNA splicing pathway in yeast. Each approach successfully and selectively identifies the HAC1mRNA as a target of Ire1 nuclease. In vitrocleavage of mRNAs by Ire1\, followed by microarray detection of the fractionated mRNAs, identifies HAC1mRNA as significantly enriched in the population of mRNAs specifically cleaved by Ire1. A computational search for the experimentally determined Ire1 consensus cleavage sites identifies HAC1as the unique gene containing two such sequences. Finally, in vivoinduction of splicing in a cell containing wild-type tRNA ligase or the mutant rlg1-100, and subsequent microarray detection of \'genetically fractionated\' mRNAs, identifies HAC1as selectively depleted in the absence of the wild-type ligase. No other mRNA met any of these criteria for identification as an Ire1 substrate. We consider it reasonable to conclude that among the set of robustly expressed genes, HAC1mRNA is the lone substrate of Ire1. In principle, there are many reasons why each of the approaches presented here could have missed identifying an Ire1 substrate other than HAC1mRNA. For example, a poorly expressed substrate would exhibit a low signal-to-noise ratio in the microarray readout of the in vitrocleavage assay, or a substrate cleaved close to the 5\' end would still hybridize to cDNA arrays with an efficiency comparable to that of the uncleaved mRNA, and hence could have escaped detection. In our computational screen, we might have missed cleavage sites that are significantly divergent from the experimentally defined consensus. Finally, because tRNA ligase would not take part in a cleavage-only reaction on a single-site substrate, we would also not expect our third method to lead to a relative reduction of the abundance of such mRNAs in rlg1-100mutant cells as compared to the wild-type (though we would have expected such substrates to be identified in the in vitroIre1\* cleavage experiment). Thus rigorously, we cannot conclude that HAC1mRNA is the only Ire1 substrate. However, because the potential caveats are noncongruent and because HAC1stands out unambiguously in each of the three methods applied, we consider it highly unlikely that additional substrates exist. Previous studies in metazoans have suggested that other RNAs are degraded when Ire1 is activated \[[@B17],[@B34]\]. When overexpressed, wild-type IRE1α mRNA accumulated at levels that were greatly reduced compared to those in which an RNase-dead mutant was overexpressed, raising the possibility that Ire1 might downregulate its own mRNA by degradation. Similarly, overexpression of IRE1β appeared to cause fragmentation of 28S rRNA. In both cases, however, no Ire1 cleavage consensus stem-loop structures were found. It therefore remains questionable at this time whether these degradation events are directly due to cleavage by Ire1 itself or to indirect secondary effects. According to the current evidence, therefore, Ire1 has a single identified target in yeast and thus functions solely for the purpose of post-transcriptional regulation of HAC1. In contrast, other signaling pathways in the cell are commonly branched, using one enzyme to activate multiple different substrates. The utilization of common principles of protein modification, such as phosphorylation or ubiquitination, readily allows cross-talk among different signaling pathways that use common components. One phosphatase, for example, can dephosphorylate substrates that have been phosphorylated by several different kinases. In contrast, communication between the ER and nucleus through Ire1 appears to be a very private affair. Ire1-dependent splicing offers unique mechanistic advantages, such as the ability to quickly complete the synthesis of partially made Hac1 transcription factor by releasing the translational arrest of stalled polyribosomes. Given that the ER is a topologically distinct compartment of the cell with metabolic considerations quite distinct from those of the cytosol, a communication route that is wired differently and therefore more insulated from other informational \'chatter\' in the cell may be particularly beneficial in the secure passage of information from the ER to the nucleus. The linear connectivity (activation of Ire1 → production of Hac1) of the two key players of the yeast UPR is supported by genetic analysis. Both Δire1and Δhac1mutant cells display indistinguishable growth phenotypes and highly correlated gene-expression profiles \[[@B4]\]. In higher eukaryotes, signaling through the UPR is more complex, with multiple ER-proximal components each activating distinct downstream targets. Metazoan Ire1 exists as two isoforms (Ire1α and Ire1β) \[[@B20],[@B35]\]; in addition, the transmembrane kinase PERK is activated and the membrane-tethered transcription factors ATF6α and ATF6β are released when unfolded proteins accumulate in the ER \[[@B27],[@B36]\]. The diversity of these signaling components varies widely between different tissues, and the relative contribution of each of the parallel pathways to the induction of the downstream transcriptional program is currently the subject of intense study. Given this increased complexity, it is possible that there are other metazoan Ire1 splicing substrates in addition to the HAC1ortholog XBP1, and the methods developed here may prove useful in searching for such putative, additional Ire1 substrates in metazoans. Materials and methods ===================== RNA isolation ------------- RNA samples were prepared as described previously \[[@B6]\]. Briefly, yeast cells were grown to mid-log phase in selective media. rlg1-100cells were treated with 1 μg/ml tunicamycin (Calbiochem) for 40 min in the experiments described in Figure [5](#F5){ref-type="fig"}. Total RNA was isolated in SDS-high salt buffer by hot phenol extraction. For the microarray experiments described in Figures [3](#F3){ref-type="fig"} and [5](#F5){ref-type="fig"}, poly(A)^+^RNA was isolated from total RNA using the PolyA^+^Tract mRNA isolation system (Promega) according to the manufacturer\'s instructions. Poly(A)^+^RNA used for in vitroIre1\* nuclease reactions was prepared by two rounds of purification using the PolyA^+^Tract system. Northern blot analysis ---------------------- Total cellular RNA and RNA recovered from in vitronuclease reactions was analyzed by Northern blot hybridization as described previously \[[@B8]\] by separation on 1.5% agarose gels containing 6.7% formaldehyde. Hybridization probes were generated by random labeling of PCR fragments using \[^32^P\]α-dCTP according to the manufacturer\'s instructions (Amersham). All probes prepared from DNA fragments were generated by PCR amplification of yeast genomic DNA. In vitroIre1\* nuclease reaction ---------------------------------- In vitronuclease reactions were performed by incubating yeast poly(A)^+^RNA with recombinant Ire1\* (expressed and purified from Escherichia coli) as described previously \[[@B9],[@B11]\]. Following phenol/chloroform extraction, the reaction mixture was fractionated on oligo(dT) magnetic beads using the PolyA^+^Tract mRNA Isolation System (Promega) according to the manufacturer\'s instructions. Control nuclease reactions were performed and processed identically, except that Ire1\* was omitted from the reaction mixture. Microarray hybridization of amino-allyl coupled cDNA probe ---------------------------------------------------------- General protocols for microarray hybridization and for preparation of probe were as described previously \[[@B37]\]. A sample of oligo(dT) unbound RNA was reverse transcribed with StrataScript (Stratagene) using random primer A (5\' GGTTCCCAGTCACGATCNNNNNNNNN 3\', where N is any nucleotide) followed by addition of Sequenase to synthesize the second strand. The resulting double-stranded cDNA above was amplified in the polymerase chain reaction (PCR) (20 cycles) using primer B (5\' GGTTCCCAGTCACGATC 3\') complimentary to the specific sequence portion of primer A. This extra PCR amplification step was added because of the low abundance of Ire1\-cleaved RNA fragments. After ethanol precipitation, one fourth of these PCR reactions was used to re-PCR in the presence of fluorescently labeled nucleotides (either Cy3 or Cy5 dTTP). The resulting Cy3- and Cy5-labeled probes were combined. Unreacted fluorescent dye was quenched. Probes were cleaned up in a QIA-quick PCR purification spin column and hybridized at 50°C for 24 h to glass slide microarrays containing the entire yeast genome. Synthesis of the cDNA for poly(A)^+^RNA (2 μg) used for the experiment in Figure [5](#F5){ref-type="fig"} was carried out by reverse transcription in the presence of aminoallyl-dUTP at 42°C for 2 h. cDNA prepared from untreated cells was coupled with Cy3, and cDNA prepared from tunicamycin-treated cells was labeled with Cy5. Data analysis ------------- Hybridized microarrays were scanned with a GenePix 4000A microarray scanner (Axon Instruments). GenePix Pro was used to analyze and to display the data. All data points with absolute fluorescence intensity less than 250 in either channel were discarded. Cy3 and Cy5 fluorescence intensities were normalized against one another to adjust for differences in labeling efficiency and scanner gain. Quantitative analysis was performed in Microsoft Excel. Stem-loop consensus search -------------------------- Pattern searching was performed using the public-domain software scan\_for\_matches \[[@B38]\]. Sequence file input was a database of all yeast ORFs ± 1,000 nucleotides obtained from the SaccharomycesGenome Database \[[@B39]\]. Pattern file input was r1 = {au,ua,gc,cg,ug,gu} RNA base-pairing rules were used; allowed base-pairs are the fields of argument r1. p1 = 4..4 YCNGNNGNG \~p1 These parameters instructed the program to use RNA base-pairing rules to find a sequence of four nucleotides (one arm of stem), followed by a sequence matching YCNGNNGNG (where Y is pyrimidine; the seven-nucleotide loop flanked the conserved closing nucleotide pair), followed by the reverse complement of the four nucleotides at the beginning (the second arm of the stem). The output of the computational screen is listed in Additional data file 2. Additional data files ===================== The following additional data are available with the online version of this paper. Additional data file [1](#s1){ref-type="supplementary-material"} contains supplementary Table 1 which lists all the data displayed in Figure [3](#F3){ref-type="fig"}; Additional data file [2](#s2){ref-type="supplementary-material"} contains supplementary Table 2, which lists the complete output of the computational screen; Additional data file [3](#s3){ref-type="supplementary-material"} contains supplementary Table 3 which lists all data displayed in Figure [5](#F5){ref-type="fig"}. Additional data file [4](#s4){ref-type="supplementary-material"} is a Word file containing the captions and keys to the tables. Supplementary Material ====================== ::: {.caption} ###### Additional data file 1 Supplementary Table 1 which lists all the data displayed in Figure 3 ::: ::: {.caption} ###### Click here for additional data file ::: ::: {.caption} ###### Additional data file 2 Supplementary Table 2, which lists the complete output of the computational screen ::: ::: {.caption} ###### Click here for additional data file ::: ::: {.caption} ###### Additional data file 3 Supplementary Table 3 which lists all data displayed in Figure 5 ::: ::: {.caption} ###### Click here for additional data file ::: ::: {.caption} ###### Additional data file 4 The captions and keys to the supplementary tables ::: ::: {.caption} ###### Click here for additional data file ::: Acknowledgements ================ C.P. was supported by a Howard Hughes Medical Institute Predoctoral Fellowship, and by the Burroughs Wellcome Foundation Program in Quantitative Biology. This work was supported by grants from the National Institutes of Health to J.D. and P.W.; P.W. is an Investigator of the Howard Hughes Medical Institute. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Schematic of screen for RNA substrates of Ire1p endoribonuclease using an in vitronuclease reaction. Recombinant Ire1\ expressed and purified from E. coliis incubated with poly(A)^+^RNA isolated from wild-type S. cerevisiaeto cleave endogenous HAC1mRNA and other potential RNA substrates of Ire1p. Cleaved RNA (lacking the poly(A) tail) is separated from uncleaved RNA as the unbound, poly(A)^-^fraction from an oligo(dT) column and used to prepare fluorescent probe by reverse transcription followed by PCR amplification in the presence of Cy3-dTTP. A second control probe, using poly(A)^-^RNA from mock nuclease reactions (identical reactions except for the lack of Ire1\) is prepared similarly, except that PCR amplification was carried out in the presence of Cy5-dTTP. Equal amounts of these probes are mixed and used to probe the yeast DNA microarray. Because RNA fragments generated by Ire1\ cleavage are represented only in the Cy3 probe, microarray spots hybridizing to cleaved fragments should appear green, whereas microarray spots hybridizing to molecules common to both probes should appear yellow upon superimposition of green (Cy3) and red (Cy5) channels. ::: ![](gb-2004-6-1-r3-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Efficient cleavage of HAC1mRNA by Ire1\* in the presence of cellular mRNA. (a)Schematic diagram of the Ire1\-cleaved (poly(A)^-^) and uncleaved (poly(A)^+^) RNA fractions separated after an in vitronuclease reaction on yeast poly(A)^+^RNA. (b)Northern blot of the RNA fractions indicated in (a) probed with a PCR fragment encompassing either the 5\' exon of HAC1(lanes 1-3) or the 3\' exon (lanes 4-6). Lanes 1 and 4, yeast poly(A)^+^RNA before Ire1\ cleavage; lanes 2 and 5, bound uncleaved RNA fraction (b, poly(A)^+^); lanes 3 and 6, unbound cleaved RNA fraction (u, poly(A)^-^). Positions of uncleaved and cleaved HAC1mRNA are indicated. Note that the 5\' exon and 5\' exon plus intron, and the 3\' exon and 3\' exon plus intron RNA species, respectively, co-migrate on these agarose gels. ::: ![](gb-2004-6-1-r3-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### HAC1is a unique RNA cleaved by Ire1\. (a)Scatter plot of Cy3 and Cy5 signal intensities following hybridization to a yeast ORF microarray with both Cy3- and Cy5-labeled probes, prepared from the cleaved RNA fragments generated from either Ire1\ or mock treatment, respectively, as described in Figure 1. Each point on the plot represents a single yeast ORF. Points below the diagonal represent ORFs that hybridize predominantly to RNAs represented in the Cy3 probe. The position of the spot displaying the brightest Cy3 signals corresponds to HAC1. (b)Histogram representation of the log~2~Cy3/Cy5 ratio. Inset, HAC1is the only gene with a log~2~Cy3/Cy5 ratio near 2.3. All data displayed in Figure 3 are provided in Additional data file 1. ::: ![](gb-2004-6-1-r3-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### HAC1stem-loop motif used for the genome wide computational consensus search. (a)Predicted secondary structure and sequence flanking both 5\' and 3\' Ire1p cleavage sites in HAC1mRNA. Cleavage occurs after a G residue located at the third position of the seven-nucleotide loop. (b)Parameters used for the computer search. A four base-pair stem with a seven-nucleotide loop with C at the first position, and G both at third and sixth positions, are determined experimentally as described previously \[14\]. Possible base-pairs (N = N\') in the stem include AU, UA, GC, CG, UG, GU. The complete output of the computational screen is listed in Additional data file 2. ::: ![](gb-2004-6-1-r3-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### HAC1is the only transcript decreasing in rlg1-100cells following tunicamycin treatment. (a)Scatter plot of Cy3 and Cy5 signal intensities following hybridization to a yeast ORF microarray with both Cy3- and Cy5-labeled probes, prepared from RNA isolated from rlg1-100cells either untreated or treated with tunicamycin for 40 min, respectively. Points below the diagonal represent ORFs that hybridize predominantly to RNAs represented in the Cy3 probe, and which are therefore present at reduced levels upon tunicamycin treatment. The position of the spot displaying the brightest Cy3 signals (corresponding to the HAC1) is shown. (b)Histogram presentation of microarray data measuring log~2~of the relative mRNA abundance between tunicamycin treated (40 min) and untreated rlg1-100cells. Levels of most mRNAs were not altered, and the only mRNA significantly changed upon tunicamycin treatment is HAC1. Inset, HAC1is the only gene with a log~2~fold-change in abundance near 1.5. All data displayed in Figure 5 are provided in Additional data file 3. ::: ![](gb-2004-6-1-r3-5) :::	PubMed Central	2024-06-05T03:55:52.944304	2004-12-22	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549064/", "journal": "Genome Biol. 2005 Dec 22; 6(1):R3", "authors": [ { "first": "Maho", "last": "Niwa" }, { "first": "Christopher K", "last": "Patil" }, { "first": "Joe", "last": "DeRisi" }, { "first": "Peter", "last": "Walter" } ] }
PMC549065	Background ========== The flux of ions across excitable cellular membranes is a signaling mechanism that is extensively utilized by organisms from all the three major superkingdoms of life. This directional flow of ions across cellular membranes is mediated by a wide range of ion channels that may be gated by a variety of signals, such as voltage, mechanical forces or chemical first messengers \[[@B1]\]. Ion-dependent signaling is particularly critical for the functions of the animal nervous system, where propagation of signals along neuronal processes and the transmission of signals from neurons or receptor cells to their targets is mediated by the action of ion channels. The neuronal ligand- or neurotransmitter-gated ion channels (LGICs) combine the functionalities of a receptor and ion channel in a single protein, and mediate fast synaptic signaling \[[@B1]\]. The neurotransmitter released by the presynaptic cell, within a few microseconds binds to the extracellular ligand-binding module of the ion channel and causes the channel to open. This results in a selective flow of ions down their electrochemical gradients through the water-filled pore of the channel, and the excitation or inhibition of the train of action potentials in the postsynaptic cells. Furthermore, within a few milliseconds the neurotransmitter dissociates from the receptor and thereby terminates the synaptic signal. Thus, the LGICs act as molecular switches to provide a specific impulse of ion flux in response to a neuronal signal \[[@B1]\]. One of the most prominent superfamilies of the animal LGICs has as its prototype the acetylcholine-gated channels and includes the receptors for a variety of neurotransmitters in both vertebrates and invertebrates (\[[@B2]\], also see \[[@B3]\]). The known endogenous ligands bound by these receptors are acetylcholine, GABA, serotonin, glycine, histidine, glutamate and cationic zinc \[[@B4]-[@B8]\]. The receptors are also the targets of plant toxins such as nicotine and strychnine, conotoxins of snails, lophotoxins of corals, and many of the neurotoxins of elapid snakes \[[@B4]-[@B6]\]. This superfamily is commonly referred to as the Cys-loop superfamily (named after a conserved cystine bridge seen in the animal representatives of this superfamily) or the acetylcholine-receptor-type LGIC superfamily (ART-LGIC). All the known members of this superfamily possess stereotypic domain architectures, with an all-β amino-terminal ligand-binding domain (LBD) and a carboxy-terminal transmembrane domain comprised of four membrane-spanning helices (4-TM). The members of this superfamily exhibit a pentameric quaternary structure, with the second transmembrane helix from each monomer (helix M2) contributing to the wall of a transmembrane pore through which the ion passes. The animal ART-LGICs may exist as heteropentamers, containing up to four distinct paralogous monomers. The ligand is bound at the dimer interface of two adjacent LBDs, and residues from both subunits form a box-like cavity to accommodate the ligand \[[@B9],[@B10]\]. In the case of most animal neurotransmitter receptors in their open state, only two (or occasionally three) of the five subunit junctions in the pentameric receptor are occupied by the ligand \[[@B4]-[@B6]\]. The ART-LGICs characterized to date show ion selectivity. The excitatory channels, such as the acetylcholine and serotonin receptors, the mammalian Zn receptors and some invertebrate GABA receptors, allow the flow of cations, whereas the inhibitory receptors, such as those for glycine and GABA, invertebrate glutamate and histamine receptors, and some invertebrate serotonin receptors (such as Caenorhabditis elegansMOD-1), allow the flow of anions. Cation or anion selectivity of the channel is principally governed by the charge distribution in the linker between the transmembrane helices M1 and M2 \[[@B11],[@B12]\]. Several recent studies based on the X-ray structure of the recombinant homopentamer of the soluble acetylcholine-binding domain (ACHB) from the snail Lymnaea stagnalis\[[@B9]\] and the electron microscopic structure of the transmembrane domain \[[@B13]\] have thrown light on the possible mechanisms of ligand interaction and channel gating of the ART-LGICs. The current model for the mechanism of these channels posits that the binding of the ligand causes a preferential rotation of one of the β sheets of the LBD. The resultant conformational change is believed to be transmitted via interactions with the loop between helices M2-M3 to the hydrophobic constriction in the middle of the M2 helices that line the channel walls \[[@B13]\]. This causes a relaxation of the middle of the girdle and allows the flow of the ions. Despite intense studies, there remain several unresolved issues with respect to the mechanism by which the binding of the ligand to a segregated site transmits the conformational change to the rest of the LBDs to trigger the rotation. Furthermore, the extent of the applicability of the conclusions drawn from the acetylcholine receptor model for other members of the superfamily remains somewhat unclear. Thus far, the ART-LGIC superfamily is known only from multicellular animals (metazoans). Phylogenetic analysis suggests that the common ancestor of the bilateral animals already possessed multiple members belonging to two major families of the superfamily that correspond, respectively, to the excitatory cationic channels, including the acetylcholine and serotonin receptors, and the inhibitory anionic channels, including the GABA, glycine and invertebrate histamine and glutamate receptors \[[@B2],[@B14],[@B15]\]. This restricted phyletic pattern is in contrast with what has been previously observed for the voltage-gated potassium channels of the Shaker-type superfamily and the voltage-gated sodium channels. In both these cases, several representatives are known from both non-animal eukaryotes, as well as numerous prokaryotes, suggesting that they were employed in signaling in other contexts well before the origin of the animal nervous system \[[@B16]-[@B19]\]. This prompted us to investigate if distant representatives of the Cys-loop/ART-LGIC superfamily could be detected in organisms outside the animal lineage. We also sought to use these distant relatives in comparative sequence-structure and genomics studies to understand the most general functional and mechanistic features that typify this superfamily. We report here the identification of several prokaryotic members of the ART-LGIC superfamily and discuss the general implications of these proteins for the mechanisms and origin of the Cys-loop receptors of the animal nervous system. Results and discussion ====================== Identification of prokaryotic versions of the ART-LGIC superfamily ------------------------------------------------------------------ To investigate the origins of the animal ART-LGIC superfamily, we tried to obtain a complete picture of their phyletic spread in all organisms with currently available genomic sequence information. All bona fideanimal members of this superfamily (with the exception of snail ACHB) contain a globular, extracellular, amino-terminal LBD and a carboxy-terminal 4-TM domain. The membrane-spanning helices, being compositionally biased, tend to frequently recover false positives in iterative sequence profile searches. Accordingly, we only used the globular extracellular domains of the known ART-LGIC receptors, which are typically around 200-220 amino acids in length, for our iterative sequence profile searches with the PSI-BLAST program. Iterative searches from a number of starting queries, such as the human acetylcholine receptor α7 chain (gi: 2144875; region 24-230), C. elegansMOD-1 receptor (gi: 25154135; region 32-238) or the human GABA receptor α4 chain (gi: 1346079; 46-256) recovered a consistent set of receptors from diverse animals with significant expect (e)-values prior to convergence (run with inclusion threshold of 0.01). Interestingly, in addition to the animal sequences these searches also recovered sequences from different bacteria. For example a search initiated with the above-mentioned acetylcholine receptors as the seed recovered Gloeobacter violaceus, Crocosphaera watsonii(both cyanobacteria) in iteration 3 (e-values = 10^-5^-10^-7^) and Rhodopseudomonas palustris(α-proteobacteria) in iteration 6 (e-value = 10^-4^). However, no significant hits belonging to any of the other eukaryotic lineages, such as the fungi, Dictyostelium, plants, alveolates or Giardiawere detectable. To further investigate the occurrence of ART-LGIC homologs in bacteria, we constructed a PSI-BLAST profile of the LBDs recovered in the above searches and used it to systematically search all the bacterial genomes, which are available as whole-genome shotgun reads or as completely assembled chromosomes. As a result of these searches we recovered statistically significant hits to the ART-LGIC LBDs from several other phylogenetically diverse bacteria including Cytophaga hutchinsonii, α-proteobacteria like Bradyrhizobium japonicumand Magnetospirillum magnetotaticum, γ-proteobacteria, like Erwinia chrysanthemi, Microbulbifer degradansand Methylococcus capsulatus, several cyanobacteria and a single archaeal genus Methanosarcina. All these bacterial hits corresponded to the full length of the animal LBDs, which were used as seeds to build the sequence profiles. When signal peptides and the transmembrane helices were predicted for the bacterial proteins, all of them showed a general structure similar to the animal receptors; that is, an amino-terminal signal peptide and a LBD followed by a carboxy-terminal 4-TM domain. However, some of the bacterial proteins showed additional domains between the amino-terminal signal peptide and the ART-LGIC superfamily ligand-binding and channel domains (see below for further discussion). Reciprocal searches with either just the region corresponding to the LBD or the whole unit comprising both the LBD and the following 4-TM domain of the bacterial proteins recovered the animal Cys-loop proteins with significant e-values (0.001-10^-17^in iterations 1-3). For example, a search with the sequence of Chut0841 (gi: 23135736) from C. hutchinsoniirecovered the animal receptors with e-values in the range 10^-4^-10^-6^in the second iteration. The secondary structure was predicted for the region corresponding to the LBD in the bacterial proteins using the programs PHD \[[@B20]\] and JPRED2, using the combined information from the multiple alignment, a PSI-BLAST position-specific score matrix and a hidden Markov model derived from the alignment \[[@B21]\]. The predicted secondary structure of the bacterial proteins precisely corresponded to the secondary structure of the conserved core of the animal LBDs typified by the ACHB (PDB:1UV6), with an amino-terminal helix followed by nine β strands, which form a β sandwich \[[@B9]\]. Taken together, the above observations suggested that the bacterial proteins were bona fidehomologs of the animal neurotransmitter receptors of the ART-LGIC/Cys-loop superfamily. Mechanistic and functional implications of the comparative sequence-structure analysis of the bacterial and animal ART-LGIC receptors ------------------------------------------------------------------------------------------------------------------------------------- To obtain information regarding the potential functional and structural similarities and differences of the predicted bacterial ART-LGIC and the animal receptors we prepared a multiple alignment of the bacterial sequences with the representatives of all the major classes of animal Cys-loop proteins (Figure [1](#F1){ref-type="fig"}) using the T\_Coffee program \[[@B22]\]. The alignment was further refined on the basis of secondary structure predictions and comparisons with the available structure of the stand-alone animal LBD, ACHB. The multiple alignment shows that the majority of the highly conserved positions in the LBD are in the conserved strands, and when mapped onto the structure of ACHB, they correspond to the positions stabilizing the hydrophobic core of the β-sandwich (Figure [1](#F1){ref-type="fig"}, see also Additional data file 1). This observation strongly suggests that the bacterial versions would adopt a tertiary structure similar to the animal LBDs. The bacterial LBDs differ notably from the animal LBDs, however, in lacking the characteristic cysteine residues which form the disulfide bridge in practically all known animal receptor subunits (Figure [1](#F1){ref-type="fig"}). However, in place of the second cysteine the bacterial sequences possess a highly conserved hydrophobic position that is likely to be buried in the hydrophobic core of the sandwich and, thereby, similarly stabilize the region corresponding to the Cys-loop of the animal sequences (Figure [1](#F1){ref-type="fig"}). This absence of the cysteines in the bacterial versions of these family is reminiscent of what was previously observed in the bacterial homologs of several animal extracellular protein domains, such as the SCP1/PR1 domain, the immunoglobulin domains and the MAC-perforin domains \[[@B23]-[@B25]\]. Eukaryotic cells typically possess an extensive secretory compartment, with a strongly oxidizing environment, in the form of the endoplasmic reticulum, through which a protein passes before secretion \[[@B26]\]. In contrast, in bacteria most disulfide bond formation occurs after extrusion to the periplasmic compartment \[[@B27]\]. The presence of this extensive secretory compartment in eukaryotic cells might have allowed a greater role for stabilization through disulfide bonds, and thereby favored the emergence of interacting cysteines in eukaryotic versions of domains as opposed to the bacterial counterparts. Over and above the general conservation of hydrophobic residues in the 4-TM domain, there are some potential functionally relevant conserved positions shared by the bacterial and metazoan proteins. One of these is the helix-bending position in helix M1 (usually occupied by a proline (P), glycine (G) or serine (S), and corresponding to P221 in Torpedo californicaACHR α-chain), which is predicted to be critical for the flexibility of the structure to conformational change \[[@B13],[@B28]\]. Another position of interest is in the middle of helix M2, and is occupied by a small residue (corresponding to S252 in T. californicaACHR α-chain) that initiates a bend in the helix resulting in the hydrophobic constriction or girdle that forms the channel gate \[[@B13]\]. Glycine 275 of T. californicaACHR α-chain, in the loop between helices M2 and M3, has been implicated as one the residues that may be critical for the rotational freedom of the ACHR M2 helix during the gating process \[[@B13]\]. The strong conservation of a small residue at this position in both the bacterial and animal members of this family suggests that it is likely to support this function throughout the superfamily. Less obvious is the role of a polar residue just before the start of helix M4 that is highly conserved across both bacterial and animal members of this superfamily. From its position in the structure, it is possible that interactions of residue with solvent water might play a role in stabilizing one of the conformational states. One of the major determinants of ion selectivity is the sequence just amino-terminal to the helix M2 on the cytoplasmic side. The cation channels usually have a sequence motif of the form glutamate (E)-\[arginine (R)/lysine (K)\] with the glutamate playing a role in cation selection. The anion channels usually have a motif of the form alanine (A)-\[RK\] with the basic residue participating in anion selection \[[@B11],[@B12],[@B29]\]. A glutamate corresponding to that of the cation channels is seen in about eight of the bacterial sequences and a basic residue similar to the anion channels is seen in six of the bacterial sequences, suggesting that both selectivities are likely to be encountered in the bacterial sequences (Figure [1](#F1){ref-type="fig"}). In addition, like the animal sequences, the bacterial sequences contain poorly conserved polar or charged residues at the carboxyl terminus of the M2 helix, which might play a role in fine-tuning their selectivity \[[@B4],[@B6],[@B11],[@B13]\]. The long hydrophilic linker between helices M3 and M4 is highly variable in length and sequence in the animal proteins. It has been implicated in cytoplasmic interactions with functional partners such as the P2X family of ATP receptors \[[@B30]\] and the cytoskeletal receptor-clustering protein gephyrin \[[@B31]\]. In contrast to the animal members of the superfamily, all bacterial versions possess an abbreviated cytoplasmic M3-M4 loop and are unlikely to have functional interactions that are seen in the animal versions. The ligand-binding box in ACHB has been termed the aromatic box as it is bounded by multiple aromatic residues (Figures [1](#F1){ref-type="fig"}, [2](#F2){ref-type="fig"}). In several metazoan receptors the positively charged group on the ligands has been suggested to form cation-π interaction with the π-orbitals of different aromatic residues in the binding box \[[@B32]-[@B34]\]. An examination of the ACHB structure \[[@B9]\] revealed that the side chains of eight residues almost completely envelop the ligand, and are the principal constituents of the ligand-binding box (Figure [2](#F2){ref-type="fig"}). Of these, the dyad of two consecutive cysteines, which are amino-terminal to the final strand of the LBD is observed only in a subset of the animal cation channels, and does not represent a conserved interaction position. Of the remaining six positions, two are from one of the subunits while the remaining four are from the other subunit (Table [1](#T1){ref-type="table"}). The average number of aromatic residues in these positions in the bacterial proteins is 2.1, whereas in the animal sequences it is 2.6. Every sequence in our representative set, animal or bacterial, with the exception of the human Zn receptor \[[@B8]\], contains at least a single aromatic residue in one of these positions. This suggests that aromatic residues are critical for ligand interaction throughout this superfamily, though the exact position in the ligand-binding box that is occupied by an aromatic residue does not seem to be preserved. However, the smaller number of aromatic residues in the ligand-binding box of bacteria may indicate some differences in the type of ligand and the nature of the interactions. Furthermore, an interesting difference is noted in the aromaticity of the positions corresponding to leucine (L) 112 (subunit D) and tryptophan (W) 143 (subunit C) of the ACHB structure between the bacterial and animal sequences (Figure [2](#F2){ref-type="fig"}). The ratio of aromatic residues at these positions is anti-correlated, and this anti-correlation is strongly preserved in the individual sequences. This suggests that these two positions might represent mutually exclusive, but functionally equivalent, surfaces for ligand interaction. The presence of at least one aromatic residue in most of the predicted ligand-binding pockets could imply that cation-π interactions with the bound ligand are widespread in the entire superfamily. However, other explanations are also possible. For example, one or more aromatic residues could have a possible structural role in constraining the pocket to favor a particular ligand or ligand orientation. Alternatively, they could provide the requisite hydrophobic environment in the pocket or interact with the ligand through aromatic stacking. In addition to the residues discussed above, there are several other conserved residues in the LBD that may have a role in transmission of conformational changes. Among the most highly conserved features is the aPaD signature (where \'a\' is any aromatic residue, and D is aspartate) in the middle of the region corresponding to the Cys-loop (Figure [1](#F1){ref-type="fig"}) and these residues are essential for wild-type receptor function \[[@B5]\]. They lie far away from the ligand-binding region and close to another nearly universally conserved basic residue at the end of the terminal strand of the LBD (Figure [1](#F1){ref-type="fig"}). This basic residue is known to be mutated in the glycine receptor α1 subunit in the human genetic disease sporadic hyperekplexia \[[@B35]\]. The aspartate from the aPaD motif and the basic residue could potentially form a salt bridge to stabilize the \'outer sheet\' of the β sandwich and thereby regulate the preferential movement of the sheets after ligand binding. This proposal is consistent with recent studies that implicate some of these charged residues, especially the aspartate in the Cys-loop, in coupling ligand binding to further conformational changes leading to channel gating \[[@B36],[@B37]\]. The other highly conserved positions are a tryptophan at the end of strand 2 (W58 in ACHB) and an aromatic or hydrophobic position (W82 in ACHB) that are in hydrophobic interaction with each other (Figures [1](#F1){ref-type="fig"}, [2](#F2){ref-type="fig"}). These residues are at the center of a set of fairly conserved positions (including D61, P84, D108, G109 and isoleucine (I) 150 in ACHB) in both bacterial and eukaryotic proteins that form a chain on either side from the ligand-binding box to the surface of the \'inner sheet\' at the top of the LBD \[[@B9],[@B28]\]. It is likely that these residues form a conduit for the propagation of the conformational change from the ligand-binding box to the inner sheet (Figure [2](#F2){ref-type="fig"}). The conservation of certain key features in both the LBD and the 4-TM domains of the bacterial and eukaryotic receptors suggests that despite their extensive sequence divergence they are likely to share general functional and mechanistic properties. In the pentamer these residues appear to form a continuous ring passing through the top surface of the LBD, and undergo conformational changes in relation the presence of a bound ligand (Figure [2](#F2){ref-type="fig"}) \[[@B9]\]. Functional significance of domain architectures and gene neighborhoods of bacterial ART-LGICs --------------------------------------------------------------------------------------------- We analyzed the domain architectures and gene neighborhoods of the predicted bacterial ART-LGICs to glean further insights regarding their biological functions. Unlike the animal ART-LGICs, the bacterial receptors show a greater diversity in their domain architectures, while preserving the core module which comprises the extracellular LBD and 4-TM channel-forming domain (Figure [3](#F3){ref-type="fig"}). The representatives from cyanobacteria, Rhodopseudomonasand one of the three versions from C. hutchinsoniishow a simple architecture identical to the animal forms. Some versions, like those from the α-proteobacteria, M. magnetotacticumand B. japonicum, show a further amino-terminal fusion to a domain of the periplasmic binding protein type I (PBP-I) superfamily (Figure [3](#F3){ref-type="fig"}). The archetypal domains of the PBP-I superfamily are the bacterial proteins such as the lysine/arginine/ornithine-binding protein, that bind amino acids and other small molecules in the extracellular or periplasmic space and facilitate their uptake by ABC-family transporters \[[@B38]\]. Interestingly, PBP-I domains also form the LBDs of two distinct superfamilies of animal neuronal receptors. The NMDA-type receptors, which comprise a class of ligand-gated channels distinct from the ART-LGIC/Cys-loop superfamily, contain an amino-terminal PBP-I domain and a carboxy-terminal domain belonging to the second major superfamily of bacterial periplasmic binding proteins (the PBP-II superfamily, for example, HisJ) \[[@B39],[@B40]\]. The channel-forming transmembrane domain in these proteins is inserted into the carboxy-terminal PBP-II domain. The metabotropic glutamate receptor and vertebrate taste receptors, which are G-protein-coupled receptors, contain a PBP-I domain amino-terminal to their 7-TM domains \[[@B39],[@B40]\]. A third architectural theme in the bacterial ART-LGICs is a fusion of two additional amino-terminal domains to the core receptor module, namely the MCP-N (methyl-accepting chemotaxis protein-N domain) and Cache domains \[[@B41]\]. This version is seen in a number of phylogenetically distant prokaryotes, such as the archaeon Methanosarcinaand the bacteria Cytophagaand Microbulbifer(Figure [3](#F3){ref-type="fig"}). The MCP-N and Cache domains are prevalent prokaryotic sensor domains that bind a variety of extracellular or periplasmic ligands and regulate signal transduction via a variety of carboxy-terminal signaling domains. In an interesting parallel to the PBP-I/II domains, the MCP-N and Cache domains are found in a regulatory subunit (α2-δ) of the animal voltage-gated calcium channels, and appear to comprise the binding site for the drug GABApentin and possibly an as-yet unknown endogenous ligand \[[@B41]\]. Thus, these architectures suggest that many of the predicted bacterial receptors might possess multiple ligand-interaction domains and that an interplay of allosteric effects could regulate their function. Remarkably, the additional domains found with the bacterial ART-LGIC proteins are also encountered in animal neuronal receptors, suggesting that all these domains belong to a common network of ancient sensory modules that have been utilized in diverse contexts \[[@B42]\]. Contextual information in the form of conserved gene neighborhoods or predicted operons in prokaryotes often provides hints to identify gene products that functionally or physically interact or belong to the same pathways or signaling cascades \[[@B43],[@B44]\]. Accordingly, we examined the gene neighborhoods of all the predicted bacterial ART-LGICs to identify conserved neighborhoods or persistent patterns of genomic clustering of genes with similar functions. In some bacteria, the gene for the ART-LGIC was found in a conserved gene neighborhood along with a gene for a stand-alone version of the PBP-II superfamily (Figure [3](#F3){ref-type="fig"}). This is analogous to the above-noted fusion of the PBP-I domain to the ART-LGIC in other bacteria, and suggests that these stand-alone PBP-II domains probably functionally cooperate with the receptors. In one bacterium, namely Rhodopseudomonas, there is a similar predicted operon, but instead of a gene for a PBP-II superfamily protein, there is one for a stand-alone Cache domain. This situation parallels the fusion with the Cache domain in some of the receptor versions and these two independent proteins may similarly cooperate functionally. Taken together, these observations suggest that bacterial ART-LGICs may function as chemotaxis receptors. As most bacterial genomes in which they are present contain only a single member of the ART-LGIC superfamily, it is likely that, in contrast to many of the well studied metazoan receptors, they function as homopentamers. The PBP-I, PBP-II MCP-N and Cache domains that are either fused or operonic with many of the predicted bacterial receptors may help in a preliminary concentration or sensing of amino acids or other small-molecule ligands. These ligands may then bind to the channel\'s LBD domain and activate an ionic flux across the cell membrane that in turn regulates the motility of the bacterium in response to the ligand. This proposal is analogous to the recently reported activity of a voltage-gated Na^+^channel in the bacterium Bacillus pseudofirmusin chemotaxis, motility and the regulation of the Na^+^-cycle \[[@B16]\]. Interestingly, in at least one bacterium, Microbulbifer degradans, the ART-LGIC with a predicted cation selectivity is in a predicted operon with a Na^+^/H^+^symporter, suggesting possible interactions with the Na^+^cycle. Phyletic patterns and phylogenetic relationships of the bacterial and eukaryotic ART-LGICs ------------------------------------------------------------------------------------------ Comparative genomics of ART-LGICs suggests that they show a highly non-uniform phyletic patterns. Among the eukaryotes they are only seen in animals, and could not be detected in the currently available genomic sequences of other crown-group eukaryotes such as plants, fungi, Dictyostelium, Entamoeba, apicomplexans or earlier-branching eukaryotic taxa such as Giardiaand Trichomonas. Among the prokaryotes, too, they show a highly sporadic distribution: very distantly related taxa may possess similar receptors (for example, Cytophagaand the archaeon Methanosarcina, Figure [3](#F3){ref-type="fig"}), whereas closely related taxa may differ from each other in possessing or lacking a predicted ART-LGIC. These phyletic patterns are similar to those observed for several signaling receptors in prokaryotes and are suggestive of a high degree of mobility through lateral transfer, and frequent gene loss \[[@B45]\]. We constructed phylogenetic trees of the ART-LGICs by using an alignment that spanned the entire length of the LBD and the 4-TM channel domain, and included all bacterial members and representatives of all the major animal receptor groups. The trees constructed using several different methods - maximum likelihood, Bayesian inference, minimum evolution and neighbor-joining - produced congruent tree topologies (Figure [3](#F3){ref-type="fig"}). As expected, the tree showed a strongly supported monophyletic animal branch that in turn split up into the two major families corresponding to the great split between the classical acetylcholine-serotonin type (usually cationic) receptors and their relatives and the classical glycine-GABA type (usually anionic) receptors and their relatives \[[@B2],[@B7],[@B14],[@B15]\]. All the animal sequences are much closer to each other to the exclusion of all other prokaryotic sequences (Figure [3](#F3){ref-type="fig"}). They possess several unique sequence and structure features, including the characteristic cysteines of the Cys-loop and the extra large variable region between the transmembrane helices M3 and M4. Its absence in the bacterial forms suggests that they are \'simpler\' versions, which are closer to the primitive state. The mean intra-group distance of the metazoan versions, measured using the JTT substitution matrix on an alignment of 368 positions, is 1.7. This value is much lower than the intra-group distance of 3.01 that is observed for the bacterial forms (the overall mean distance being 2.8). The prokaryotic proteins also show greater diversity of architectures in comparison to the stereotypic architecture of all the animal members of this superfamily. These observations suggest that the diversification of the prokaryotic forms preceded the emergence of the eukaryotic forms and thus that the root of the tree is more likely to lie in the bacterial lineage than within the metazoan lineage. Certain bacterial versions (those from Crocosphaera, Gloeobacter, Erwiniaand Rhodopseudomonas) are markedly more similar in sequence to the eukaryotic forms (Figures [1](#F1){ref-type="fig"}, [3](#F3){ref-type="fig"}). Specifically, these similarities include the extension of strand 2 of the LBD, before the universally conserved W, and the hWxP motif (where h is a hydrophobic residue and x any residue) amino-terminal to strand 4 of the LBD. Constrained trees, where the animal branch was artificially grouped with the more distantly related bacterial sequences, were significantly worse (using the Kishino-Hasegawa and Bayesian posterior probability tests; data not shown) than the trees in which they were grouped with their closer bacterial homologs. This observation, taken together with the greater likelihood of the root being amongst the prokaryotes, suggests that the above features shared by some of the bacterial sequences and the animal versions are synapomorphies or derived characters. Taken together, the phyletic patterns and the specific relationship of the animal sequences to a subset of the bacterial forms suggests that the common ancestor of the animal ART-LGICs probably arose via an early lateral gene transfer from a bacterium to the ancestral lineage leading to the modern metazoans. Following this transfer, the ancestral eukaryotic version acquired the characteristic cysteines of the Cys-loop and duplicated and diverged to give rise to the two major metazoan Cys-loop families. By the time of the common ancestor of the bilateral animals the two major families appear to have diversified into about nine distinct lineages (Figure [3](#F3){ref-type="fig"}). The biased sampling of eukaryotic genome sequences and the high frequencies of gene loss in the eukaryotes could imply that the transfer of the ART-LGICs from bacteria to the eukaryotes might have occurred well before the emergence of the animal lineage, and has been lost repeatedly in the other eukaryotes. While this possibility cannot be ruled out until more representative eukaryotic sequences become available, it is likely that there was a single precursor for all the animal sequences, which was acquired at some point from a bacterial source, and the massive radiation of the Cys-loop receptors occurred only after the animals branched off from the rest of the crown group. In principle it is possible that the bacterial sequences emerged through a secondary transfer from the animals. However, the potentially greater antiquity of the prokaryotic lineages possessing these proteins, combined with their greater diversity, makes this direction less likely given the current data. In addition, as discussed below, the case of the ART-LGIC receptors seems to fit the general pattern, which is observed for many other eukaryotic signaling proteins that appear to have a bacterial provenance. It is of interest to note that several other animal neurotransmitter receptors show connections to bacterial signaling systems. In addition to the MCP-N and Cache domains shared by the metazoan voltage-gated Ca^2+^channels, and the PBP-I domains of various G-protein-linked and NMDA-type receptors, there are similar parallels in the receptors for the gaseous neurotransmitter nitric oxide (NO). The NO receptors of animals share two domains, namely the HNOB and HNOBA, which are involved in heme-dependent NO sensing with several bacterial signaling proteins \[[@B46]\]. Likewise, a recent analysis of the enzymes in the biosynthetic pathways of all common metazoan neurotransmitters suggested that many of them may have been laterally transferred from bacteria to eukaryotes at different points in eukaryotic evolution \[[@B47]\]. Some of these include some potentially late transfers, analogous to previous observations for the NO receptors and the present report on ART-LGICs. Furthermore, parallel instances of connections to bacterial sensory proteins have been noted in the case of plant receptors for cytokinin, ethylene and light (phytochromes), and certain small-molecule receptors of the cellular slime mold Dictyostelium(see \[[@B48]\] and references therein). Thus, the ART-LGICs appear to belong to a larger sensory network that probably first emerged in the bacterial signaling systems and was subsequently recruited by the eukaryotes in contexts unique to their own functional milieus. Conclusions =========== We report here the identification of several prokaryotic homologs of the animal acetylcholine receptor-type ligand gated ion channels (Cys-loop receptors). The pattern of the residues conserved in both the metazoan and bacterial receptors suggests that a common mechanism of channel-gating is likely to operate throughout this superfamily. Furthermore, the ligand-binding box appears to preserve at least one aromatic residue, although its exact position may not necessarily be conserved. The conservation pattern also suggests that a chain of positions leading out on either side from the ligand-binding box may mediate the transmission of the conformational change through the \'top\' of the LBD, which may then transmit through the rest of the structure. The charge interactions between the acidic residue in the middle of the Cys-loop region and a basic residue the extreme carboxyl terminus of the LBD, just before the transmembrane domain also appear be universal features that might be involved in the process of channel gating. On the basis of the domain architectures and operon organizations, we predict that the bacterial ART-LGICs are likely to function as chemoreceptors for low-molecular-weight solutes in the environment. Phyletic and phylogenetic analyses suggest that the ancestor of the animal lineage probably acquired a single progenitor from a bacterial source, and it subsequently radiated to give rise to all the Cys-loop receptor subunits of the extant metazoans. Materials and methods ===================== The nonredundant (NR) database of protein sequences (National Center for Biotechnology Information (NCBI)) was searched using the BLASTP program \[[@B49]\]. Unfinished microbial and eukaryotic genomes were searched using the TBLASTN program with protein queries \[[@B49]\]. Iterative database searches were conducted using the PSI-BLAST program with either a single sequence or an alignment used as the query, with a position-specific score matrix inclusion expectation (E) value threshold of 0.01 (unless specified otherwise); the searches were iterated until convergence \[[@B49]\]. For all searches with compositionally biased proteins, the statistical correction for this bias was used. Multiple alignments were constructed using the T\_Coffee \[[@B22]\] or PCMA programs \[[@B22]\], followed by manual correction based on the PSI-BLAST results and structural information. All large-scale sequence-analysis procedures were carried out using the SEALS package \[[@B50]\]. Transmembrane regions were predicted in individual proteins using TMPRED \[[@B51]\], TMHMM2.0 \[[@B52]\] and TopPred II \[[@B53]\] with default parameters. For TopPred, the organism parameter was set to \'prokaryote\' or \'eukaryote\' depending on the source of the protein. Signal peptides were predicted using the SIGNALP program \[[@B54]\]. Protein structure manipulations were performed using the Swiss-PDB viewer program \[[@B55]\]. Protein secondary structure was predicted using a multiple alignment as the input for PHD \[[@B20]\] or JPRED2 \[[@B21]\]. Similarity-based clustering of proteins was carried out using BLASTCLUST \[[@B56]\]. Phylogenetic analysis was carried out using the maximum-likelihood, neighbor-joining, Bayesian inference and minimum evolution (least squares) methods. The MrBayes program was used for the Bayesian inference of phylogeny \[[@B57]\]. The alignment for phylogenetic analysis was prepared by visually deleting all those columns that contained non-conserved residues from five or fewer sequences. Regions with substantial gaps, which are replaced by numbers in Figure [1](#F1){ref-type="fig"}, were also entirely deleted from the alignment. The resulting alignment with 49 sequences and 368 columns was used for all subsequent phylogenetic analysis. Maximum-likelihood distance matrices were constructed with the TreePuzzle 5 program \[[@B58]\] using 1,000 replicates generated from the input alignment and used as the input for construction of neighbor-joining trees with the Weighbor program \[[@B59]\]. Weighbor uses a weighted neighbor-joining tree construction procedure that has been shown to correct effectively for long-branch effects. The minimal evolution trees were constructed using the FITCH program \[[@B60]\] of the Phylip package on 1,000 bootstrap replicates prepared from the input sequence. For maximum-likelihood analysis two different procedures were used. In the first, a minimum evolution tree obtained using FITCH was provided as a input for the Protml program \[[@B61],[@B62]\], which then produced a maximum-likelihood tree using local rearrangements. The statistical significance of the internal nodes of this maximum-likelihood tree was assessed using the relative estimate of logarithmic likelihood bootstrap (Protml RELL-BP) \[[@B61],[@B62]\], with 10,000 replicates. In the second procedure an initial full maximum likelihood tree was constructed using the Proml program of the Phylip package \[[@B60]\]. A gamma distribution with one invariant and four variable sites with different rates was used for constructing this tree, which was then used as the guide tree to generate further full maximum-likelihood trees using the PhyML program with 100 bootstrap replicates generated from the input alignment \[[@B63]\]. The consensus of these 100 trees was derived using the Consense program of the Phylip package to obtain the bootstrapped full maximum-likelihood tree. Gene neighborhoods were determined by searching the NCBI PTT tables with a custom-written script. These tables can be accessed from the genomes division of the Entrez retrieval system. Additional data files ===================== The following additional data are available with the online version of this paper. Additional data file [1](#s1){ref-type="supplementary-material"} contains the conservation pattern of the ART-LGIC superfamily plotted onto the three-dimensional structure of the ACHB protein. Additional data file [2](#s2){ref-type="supplementary-material"} contains the alignment of the proteins in Figure [1](#F1){ref-type="fig"} in Word format. Supplementary Material ====================== ::: {.caption} ###### Additional data file 1 The conservation pattern of the ART-LGIC superfamily plotted onto the three-dimensional structure of the ACHB protein ::: ::: {.caption} ###### Click here for additional data file ::: ::: {.caption} ###### Additional data file 2 The alignment of the proteins in Figure [1](#F1){ref-type="fig"} ::: ::: {.caption} ###### Click here for additional data file ::: Acknowledgements ================ E.J. and A.T. are funded by grant from the Research Board of the University of Illinois at Urbana-Champaign and NSF grant 0235792 to E.J. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### A multiple alignment of the ART-LGIC/Cys-loop superfamily (see also Additional data file 2 for alignment; an alignment of metazoan members only may also be obtained from PFAM: PF02931 LBDs; PF02932: TM domain). Proteins are denoted by their gene names, species abbreviations and gi. The secondary-structure assignments, based on the available crystal structures of the acetylcholine receptor pore (pdb: 1OED) and Achbp protein (pdb: 1UV6), are shown above the alignment where E denotes extended or strand, and H, helix. The coloring reflects the composition of the amino acids at 90% consensus. The coloring scheme and the consensus abbreviations are as follows: h, hydrophobic (ACFILMVWY), l, aliphatic residues (ILV), and a, aromatic residues (FHWY) are shaded yellow; s, small (AGSVCDNPT) and u, tiny residues (GAS) are colored green; c, charged (DEHKR), +, basic (HKR), -, acidic (DE), p, polar (CDEHKNQRST) are colored magenta. The conservation pattern as plotted onto the three-dimensional structure of the ACHB is shown in Additional data file 1. Also shown below the alignment are the key residues described in the text. \# and @ represent residues of adjacent chains (PDB id: 1UV6, chain C and chain D respectively) involved in ligand binding (shaded gray). Residues predicted to be potentially involved in the transmission of conformational change are marked by an asterisk (\) at the bottom of the column and are colored violet and shaded gray. The highly conserved positions - the acidic residue in the middle of the Cys-loop and the basic residue at the carboxyl terminus of the LBD - are shown in inverse blue shading. The arginine residue involved in ion selectivity in anionic channels is shaded green and the glutamate residue involved in ion selectivity in cationic channels is shaded purple. Species abbreviations are as follows: Bjap, Bradyrhizobium japonicum; Ce, Caenorhabditis elegans; Crwa, Crocosphaera watsonii; Cyhu, Cytophaga hutchinsonii; Dm, Drosophila melanogaster; Echr, Erwinia chrysanthemi; Glvi, Gloeobacter violaceus; Hs, Homo sapiens; Lst, Lymnaea stagnalis; Mba, Methanosarcina barkeri; Mcap, Methylococcus capsulatus; Mdeg, Microbulbifer degradans; Meac, Methanosarcina acetivorans; Mmag, Magnetospirillum magnetotacticum; Npun, Nostoc punctiforme; Rpal, Rhodopseudomonas palustris; Syn, Synechococcussp.; Toma, Torpedo marmorata. ::: ![](gb-2004-6-1-r4-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Cartoon representations of ACHB. (a)The ligand-binding dimer of ACHB; (b)the pentamer of ACHB. The representations were derived from the crystal structure of the snail acetylcholine-binding protein (PDB 1UV6). Residues forming the ligand-binding box are shaded orange. The chain of residues that could potentially act as a conduit for transmission of conformational changes is colored green and the prominent conserved ones among them have been labeled. ::: ![](gb-2004-6-1-r4-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### A phylogenetic tree of proteins containing the ART-LGIC domain with relevant domain architectures and gene neighborhoods. Bacterial branches are colored blue and animal branches magenta. Nodes with maximum-likelihood RELL bootstrap support of more than 70% are shown as yellow circles. Selected gene neighborhoods that provide contextual functional information are shown as box arrows. The globular domains in the domain architectures are drawn approximately to scale. LBD, the ligand-binding domain of the ART-LGIC domain. Species abbreviations are as in Figure 1. ::: ![](gb-2004-6-1-r4-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Character of residues in the conserved positions of the ligand-binding box ::: Ratio of aromatic residues Overall aromaticity ---------------------------- --------------------- --------- --------- --------- --------- --------- ----- Position\ R104(D) L112(D) W143(C) T144(C) Y185(C) Y192(C) Bacteria 0.2 0.53 0.2 0.0 0.6 0.53 2.1 Animals 0.14 0.22 0.82 0.0 0.54 0.89 2.6 \*The positions correspond to the D and C chains of ACHB (PDB: 1UV6) :::	PubMed Central	2024-06-05T03:55:52.946809	2004-12-20	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549065/", "journal": "Genome Biol. 2005 Dec 20; 6(1):R4", "authors": [ { "first": "Asba", "last": "Tasneem" }, { "first": "Lakshminarayan M", "last": "Iyer" }, { "first": "Eric", "last": "Jakobsson" }, { "first": "L", "last": "Aravind" } ] }
PMC549066	Background ========== Much is known about the sequence of cell and tissue behavior that leads to repair of a mammalian skin wound \[[@B1],[@B2]\] but we still have a rather incomplete knowledge of the portfolio of genes that drives these events. From late fetal stages onwards, tissue repair is always accompanied by a robust inflammatory response and this intimate association between wound healing and inflammation has made it difficult to dissect out the key elements of the repair process from those that are simply a consequence of inflammation and not necessary for healing. For this reason, and because adult skin healing is a complex process drawn out over several days to weeks, no systematic microarray analysis has yet been undertaken to encompass all those episodes from initial injury to the final sealing of the wound. The compelling argument for performing such a study comes from microarray investigations of genes upregulated in fibroblasts in response to serum exposure. Cluster analysis of these results hints at the roles of hundreds of genes by the similarity of their temporal profile with genes whose function as part of the serum response cascade is well characterized \[[@B3],[@B4]\]. To overcome the problems of extended wound repair time course and to distinguish repair genes from those involved in, or a consequence of, inflammation, we have developed an incisional wound model in neonatal mice where healing is rapid and largely complete by 24 hours and we have used this model to compare wound-expressed genes in wild-type mice versus PU.1null sibs which are genetically incapable of raising an inflammatory response because they lack key leukocyte lineages. PU.1is an ETS family transcription factor that is crucial for several lineage decisions in hematopoetic cells; consequently, PU.1null mice lack a number of hematopoetic cell types \[[@B5]\]. They are born with no macrophages or osteoclasts, and there is a late onset of neutrophil and T-cell development \[[@B5]\]. However, although there are no neutrophils or macrophages for recruitment to sites of tissue damage, neonatal PU.1null mice can efficiently heal skin wounds \[[@B6]\]. Indeed, repair in the PU.1null mice results in less indication of fibrosis and an altered cytokine and growth-factor profile compared to wild-type. For example, interleukin 6 (IL6) mRNA, which is robustly expressed at wild-type wound sites, is almost undetectable in PU.1null wounds, and TGFβ1 mRNA, previously implicated in several fibrosis scenarios, is significantly reduced in PU.1null wounds, as revealed by RNase protection analyses \[[@B6]\]. In this study we use Affymetrix GeneChip analysis of mRNAs collected at various time points after wounding of wild-type versus PU.1null skin, to distinguish those key transcriptional events that are part of the tissue repair process but independent of whether or not there is an accompanying inflammatory response, from those genes that are \'inflammation dependent\'. The latter are expressed either by inflammatory cells recruited to the wound, or upregulated by fibroblasts, endothelial cells, muscle cells and others at the wound site, as a consequence of inflammation. Using cluster analysis we have grouped more than 1,000 wound-induced genes according to their temporal profiles, with each cluster having a unique temporal profile of expression that correlates with a clear physiological episode during the repair process. For a small sample of genes from each of these clusters, we show in situhybridization data that also reveals spatial resolution. Results and discussion ====================== For our wound model we chose neonatal mouse back skin which raises a robust inflammatory response to wounding that is not dissimilar to that seen at sites of tissue damage in adult skin, but which heals rapidly, such that incisional lesions are generally fully re-epithelialized by 24 hours. This compression of the repair process reduces the temporal \'noise\' and thus the potential loss of gene-expression synchrony between wild-type and PU.1null animals, which naturally will increase with time after the initial wound insult. Wounding of neonatal back skin results in rapid healing with or without an associated inflammatory response ----------------------------------------------------------------------------------------------------------- Resin histology of healing incisional wounds in neonatal mouse skin reveals closure of the wound commencing within 3 hours of the lesion; by 24 hours, the epidermal wound edges have generally met and fused along much, if not all, of the length of the wound. This is true for both wild-type neonates and for PU.1null sibs, with the only obvious differences apparent in the histology being an absence of inflammatory cells in the PU.1null wounds (Figure [1c-j](#F1){ref-type="fig"}). As previously described, in situhybridization studies using a c-fmsmacrophage-specific probe reveal large numbers of these cells drawn to the wound connective tissue just beneath the epidermal fusion seam at 24 hours after wounding wild-type skin, but their complete absence in PU.1null equivalent wound sections (Figure [1k,l](#F1){ref-type="fig"}). The same difference, although with an earlier temporal profile, is observed if wounds of wild-type and PU.1null skin are probed with histochemical stains that reveal neutrophil influx (data not shown). These wound dynamics lead us to believe that the neonatal mouse skin wound model provides a good opportunity to analyze the transcriptional events that regulate the various tissue-repair episodes from initial activation steps through to the \'stopping\' signals that occur when the tissue defect has been filled in, both in the presence and absence of an inflammatory response. More than 1,000 genes are differentially expressed post-wounding ---------------------------------------------------------------- For microarray comparison, a consistent series of horizontal and vertical incisional (criss-cross) wounds were made to the back skin of 2-day-old neonatal PU.1null mice and their wild-type littermates. Each time-matched pair (PU.1null and wild-type) were chosen from the same litter to reduce the possibility of differential expression \'noise\' due to environmental differences. Wound tissues were harvested at either 30 minutes to identify immediate early genes, 3 hours for early tissue repair effector genes, and 12 or 24 hours to reveal later tissue repair effectors as well as inflammatory genes. Total RNA was then extracted and hybridized to Affymetrix GeneChips, and differentially expressed genes were identified by comparison of expression levels for each time point with unwounded skin samples that served as baseline controls. Genes were selected if transcript levels exceeded a twofold increase over either the unwounded baseline, or between time points, or between the wild-type and PU.1null wounds. On the basis of these criteria, 1,001 genes were identified as wound-induced (see Additional data file 1 for an annotated database of all these genes together with full details of expression levels at all time points). Cluster analysis to group these genes reveals temporal profiles that correlate with distinct physiological episodes in the repair process ----------------------------------------------------------------------------------------------------------------------------------------- Cluster analysis with Spotfire Array Explorer 3.0 software was used to organize the 1,001 wound-induced genes into groups according to the cosine coefficient similarity measurement; this includes within a group all those genes that have a similarly shaped temporal profiles, regardless of the levels of gene expression. Nine clusters were identified in this way, and of these, seven correlated with clear episodes in the repair process. The other two had profiles that, as far as we can tell, do not correspond to any currently understood step in the repair process and so were discarded for further analysis, although they appear in our supplementary data (see Additional data file 2 for median graphs of these clusters). Of the seven clusters associated with known repair episodes, five contain one or more known genes with good functional associations to that repair episode, and this encourages us to name each cluster according to that physiological episode. This does not provide definitive proof of function for any gene in that cluster, but it gives the best opportunity to predict function, particularly for expressed sequence tags (ESTs) with no further sequence information. Four clusters have profiles that are independent of an inflammatory response ---------------------------------------------------------------------------- Four clusters of genes have profiles that are largely independent of inflammation. Genes in these clusters are expressed with similar profile whether wounds are in wild-type skin, where there is an influx of inflammatory cells, or in PU.1null skin, where there is none. In both these situations there is full and complete repair, and so we propose that these four clusters represent the basic repertoire of repair genes that are activated during the repair response. Figure [2](#F2){ref-type="fig"} shows line graphs that display the temporal profile of the median expression levels at each time point to give a representation of that cluster and these have been termed the \'activation\' (Figure [2a](#F2){ref-type="fig"}), \'early effector\' (Figure [2b](#F2){ref-type="fig"}), \'late effector\' (Figure [2c](#F2){ref-type="fig"}) and \'stop\' (Figure [2d](#F2){ref-type="fig"}) clusters. The number of genes found in each cluster is displayed on each graph. Three gene clusters correlate with various phases of the inflammatory response ------------------------------------------------------------------------------ Three further clusters of genes represent expression profiles that correlate with the onset of inflammation and thus we consider them inflammation-associated genes. In neonatal animals, the inflammatory response is generally induced by 12 hours and is well established by 24 hours post-wounding. Two of these inflammation-associated gene clusters contain genes that are not expressed in unwounded skin or at early stages of repair; rather, they are upregulated in wild-type skin directly coincident with the onset of the wound inflammatory response but are generally not upregulated in the PU.1null wound site at any stage. We have called these two groups of genes, the \'early inflammatory\' cluster (Figure [2e](#F2){ref-type="fig"}) and the \'late inflammatory\' cluster (Figure [2f](#F2){ref-type="fig"}). A third cluster does not display the standard inflammatory response profile as typified by the early and late inflammatory clusters. Rather, this cluster contains genes that are expressed at early stages of repair in both PU.1null and wild-type mice but, whereas expression appears to increase in the wild-type wound coincident with the inflammatory response, the same genes are downregulated in the PU.1null wound, where there is no inflammatory response; we have called this group of genes the \'inflammation-maintained\' cluster (Figure [2g](#F2){ref-type="fig"}). Nearly 100 genes are expressed with an immediate early gene profile at the wound site ------------------------------------------------------------------------------------- One of the most clear-cut clusters of genes is of those whose temporal expression profiles are suggestive of a transient, immediate early response to wounding. These genes show almost identical profiles, whether in the wild-type or PU.1null situation, and thus are independent of an inflammatory response. We have named this group the activation cluster, as many will be kick-start activators of the various cell behaviors that together comprise the wound-healing process. This cluster is dominated by transcription factors and contains several well known immediate early genes, such as Egr1(Krox 24), JunB, Myc, and I-Kappa-Bα(Nfkbia). We present a heatmap for the 90 genes in this cluster arranged with the most highly expressed at the top of the map (Figure [3a](#F3){ref-type="fig"}). Heatmaps provide a visual representation of temporal profiles only, and so for a small sample of these genes we also include in situhybridization data on wounded skin sections to illustrate which cells and tissues express that particular gene. This spatial expression profile reveals expression in the in vivosetting, giving clues to the function of that gene during repair. Krox24(Figure [4a](#F4){ref-type="fig"}) has previously been shown to be transiently induced in both embryonic and adult mouse wounds \[[@B7]\]. In situhybridization reveals Krox24to be expressed by those epidermal cells extending back 10-12 cell diameters from the cut edge of neonatal wounds and all the associated hair follicles within this zone also (Figure [4b](#F4){ref-type="fig"}). MKP-1 (Figure [4c](#F4){ref-type="fig"}) is a dual-specificity phosphatase with close homology to Drosophilapuckered, which has been shown genetically to be key in braking the Jun N-terminal kinase (JNK) cascade activated during morphogenetic episodes such as dorsal closure in the fly embryo \[[@B8]\]. In situhybridization shows that the front few rows of wound epidermis express MKP-1, although expression extends less far back from the wound edge than for Krox24(Figure [4d](#F4){ref-type="fig"}). By analogy to Drosophilamorphogenetic episodes, it may be that MKP-1 operates as suppressor of MAP kinase (MAPK) signaling by phosphorylation of extracellular-regulated kinases 1 and 2 (ERK1 and 2), and so may actually function as a brake on the earliest tissue movements activated at the wound site. Expression of Fos-like antigen 1 (Fosl1, Fra1), has previously been associated with epithelial migrations during tumorigenesis but has not been analyzed in a wound-repair model \[[@B9],[@B10]\]. Fosl1has a classic activator temporal profile (Figure [4e](#F4){ref-type="fig"}) and a similar spatial profile to Krox24, with high levels of expression in wound-margin epidermal cells but somewhat weaker expression in damaged hair follicles at the wound site (Figure [4f](#F4){ref-type="fig"}). Its close relative c-foshas previously been shown to be upregulated during repair of embryonic skin wounds \[[@B11]\], and in vitrostudies show that blocking wound-induced fosinduction may hinder cell migration \[[@B12]\]. Because cluster analysis allows us to group genes together that are likely to have similar functions \[[@B13]\], the temporal profiles of, as yet, uncharacterized ESTs in the activation cluster implicates them as having an immediate-early activator function during repair. A good example of such a gene is EST GenBank accession number AI853531 (Figure [4g](#F4){ref-type="fig"}), which is weakly homologous to human Mitogen-Inducible-Gene-6(Mig-6, Gene 33). The exact function of Mig-6 remains elusive but it has been shown to interact with Cdc42, a member of the Rho family of GTPases, via the activation of stress-activated protein kinases (SAPKs) \[[@B14]\]. In situhybridization reveals clear expression of this gene in wound fibroblasts (Figure [4h](#F4){ref-type="fig"}); together with its potential Cdc42 effector status and its induction in quiescent fibroblasts upon mitogenic stimulation and expression in many human cancer cell lines \[[@B15]\], this suggests that Mig-6 may mediate a fibroblast migration signal. The remaining genes in the activation cluster all have very similar temporal profiles, suggesting that they too may have important roles in activating or modulating early cell behavior at the wound edge. A further 200 genes are also expressed independently of inflammation, but with later onset and a less transient time course --------------------------------------------------------------------------------------------------------------------------- Two further clusters of genes have increased expression levels post-wounding in a manner that is also inflammation-independent but where expression occurs at a later time than with the activation genes. The profiles of these two clusters are temporally distinct from one another and so we have called them the early effector and late effector clusters. Between them they contain 184 genes that fit the expected profile of genes that might direct re-epithelialization and granulation tissue assembly events. The temporal profiles of all these genes can be seen by heatmap in Figure [3b](#F3){ref-type="fig"} (early effector cluster) and Figure [3c](#F3){ref-type="fig"} (late effector cluster). These two clusters contain varied types of tissue repair effectors such as tissue remodelers, genes encoding extracellular matrix (ECM) proteins, those involved in the signaling machinery and structural genes required for cell migration. Again, we provide here several examples of genes within these clusters with accompanying in situhybridization data to provide an insight into the spatial localization of some genes in these clusters. Map4k4, a member of the serine/threonine protein kinase family that activates the JNK and MAPK signaling pathways in response to stress signals, cytokines and growth factors \[[@B16]\], is a member of the early effector cluster. The temporal profile (Figure [4i](#F4){ref-type="fig"}) and expression of Map4k4, in both keratinocytes up to 10-12 cell diameters from the wound edge and a subset of dermal fibroblasts extending a similar distance back from the wound edge (Figure [4j](#F4){ref-type="fig"}), confirms the activation of this intracellular signaling cascade at sites of tissue repair. The JNK pathway has recently been shown to have a role in Paxillin regulation during fibroblast migrations triggered by in vitroscratch wounds \[[@B17]\], and so expression of Map4k4is also suggestive of a cell migratory regulatory role for this signaling pathway in keratinocytes and fibroblasts during in vivorepair. Also in the early effector cluster, retinol binding protein-1 (Rbp1), a Fabp/p2/Crbp/Crabp family retinol transporter is expressed in wound epidermal cells approximately 15 cell diameters back from the wound site (Figure [4k](#F4){ref-type="fig"} and [4l](#F4){ref-type="fig"}). This suggests a role for retinoids in re-epithelialization of the wound, and indeed, there is some evidence that these molecules can trigger epidermal proliferation via heparin-binding epidermal growth factor (HB-EGF) expression in suprabasal epidermal cells \[[@B18]\]. Typifying the late effector profile is Keratin 6(K6), a classic wound-induced gene \[[@B19]\] (Figure [4m](#F4){ref-type="fig"}). K6encodes a nonconventional keratin which is thought to facilitate the packaging up of other intermediate filaments in activated keratinocytes, so that these cells can migrate forward to re-epithelialize the wound \[[@B19]\]. High levels of expression of K6by the front 10-12 rows of wound-edge keratinocytes were confirmed by in situhybridization (Figure [4n](#F4){ref-type="fig"}). Interestingly, another member of the late effector cluster, the intracellular Ca^2+^-binding protein MRP8 (S100A8) is expressed in a similar temporal and spatial pattern to K6(Figure [4o](#F4){ref-type="fig"} and [4p](#F4){ref-type="fig"}). MRP8 binds to keratin filaments as an MRP8/14 heterodimer in a Ca^2+^dependent manner \[[@B20],[@B21]\] and is postulated to interact with these keratin filaments and guide cytoskeletal rearrangements during tissue repair \[[@B22]\]. The temporal and spatial coexpression of K6with MRP8may highlight a relationship between them and as such reveals another advantage of cluster analysis - the ability to identify potential interactions between genes and genetic pathways within the same cluster. Not all functionally related genes cluster together, however. The heterophilic binding partner of MRP8 is MRP14, which does not appear in the same cluster but rather is expressed within the early inflammation cluster (see later), since, in addition to keratinocyte expression, it is expressed at high levels by wound leukocytes. As both the MRP8/MRP14 heterodimer and a homodimer, MRP8 is a potent chemoattractant \[[@B22],[@B23]\] and, interestingly, the MRP8/14 heterodimer also has an entirely different role, operating as a wound antimicrobial factor, although the MRP14 subunit seems to be responsible for this activity \[[@B24]\]. The pleiotropic activities of MRP8/MRP14 may reflect different functions of monomeric versus complexed subunits. A final cluster of inflammation-independent genes may indicate players in the \'contact inhibition\' stopping process --------------------------------------------------------------------------------------------------------------------- At the end of the repair process many of the cell behaviors that drive repair - such as migration and proliferation - clearly need to cease as tissues re-establish approximately their pre-wound state. This will be a gradual process and yet we might expect to see such genes depressed during the repair period and becoming upregulated as wound edges meet and closure is finishing. We see a cluster of genes with exactly this profile, suggesting that some of these genes are re-expressed to control the later stages of repair. We have loosely termed this the stop cluster. Because of their known biology, several genes in this cluster make ideal candidates for players in the processes of contact inhibition and epithelial fusion that occurs as cells from the two epidermal wound fronts confront one another. The Eph receptors and their ligands, the ephrins, have features that might make them ideal for sensing and responding to stop cues. In vitrostudies show that both ligand- and receptor-bearing cells become activated upon cell-cell contact \[[@B25],[@B26]\], and this interaction leads to a repulsive response by receptor-expressing growth cones during the developmental wiring of the nervous system \[[@B27]\]. Further evidence for ephrin-mediated control of epithelial sheet movement and fusion comes from studies in Caenorhabditis elegans, where Eph receptor mutants display defects in the movement of epidermal cells over neuroblasts, and in Eph knockout mice, where various morphogenetic epithelial fusions fail, leading, for example, to cleft palate and hypospadius \[[@B28],[@B29]\]. All these results suggest that the transcriptional regulation of EphB1 revealed in the heatmaps for our stop cluster (Figure [5a](#F5){ref-type="fig"}) may reflect a functional role in the stopping or final fusion episodes of wound re-epithelialization. Similarly, the expression levels of the receptor Notch also dip and rise during the repair period, and in situhybridization studies reveal that this transcriptional regulation is also occurring within leading wound-edge epidermal cells (Figure [5b-e](#F5){ref-type="fig"}). Notch has exceptionally complex biology with several ligands, including Delta and Serrate, and is a widely used as a signaling cassette at various stages of embryogenesis, and has been shown to be downregulated in several invasive tumors \[[@B30]\]. In Drosophila, Notch signaling has been implicated in the contact inhibition and fusion events that occur during dorsal closure at the end of embryogenesis (A. Martinez-Arias, personal communication), and during gut cell migratory episodes, which are also dependent on transcriptional activation of the short stopgene \[[@B31]\], the mammalian orthologue of which, Actin crosslinking family 7 (ACF7), is another member of our wound stop cluster. Several other genes within the stop cluster have characteristics that indicate they may be involved in sensing contact-inhibition cues or be downstream of these signals and operate to adhere epidermal fronts together. They include genes for Plexin 3 (Plxn3), a member of the plexin family of semaphorin receptors \[[@B32]\], Desmocollin 3 (Dsc3), which is a cadherin component of intercellular desmosomal junctions \[[@B33]\] and ACF7, a cytoskeletal linker protein \[[@B34]\]. As with the other clusters, suggestive biology is no proof of function, and it is worth noting that several other genes with this temporal profile do not have biology suggestive of a role in these late stages of wound healing. We feel that this cluster, more than any other, can only hint at function, and definitive function testing using knockout or knockdown assays will be necessary to investigate any speculative roles in the repair process. Expression of 200 genes at the wound site is dependent on the inflammatory response ----------------------------------------------------------------------------------- A comparison of those genes expressed during the repair process in wild-type versus PU.1null mice reveals most clearly genes that are dependent on the presence of an inflammatory response at the wound site. The heatmaps for early and late inflammatory gene clusters strikingly reveal robust expression in wild-type wounds, but little or no expression in the PU.1null mice for these genes (Figure [6](#F6){ref-type="fig"}). Together, the early and late inflammatory clusters comprise 169 genes that are not expressed in unwounded wild-type skin or at early stages of repair but appear to be upregulated in the wild-type wound directly, coincident with the onset of the inflammatory response. The early inflammatory cluster typically contains genes whose expression is upregulated rapidly in the wild-type, often reaching a peak by 12 hours (Figure [6a](#F6){ref-type="fig"}), coincident with the influx of neutrophils to the wound site. In the late inflammatory cluster, expression typically peaks a little later, at 24 hours post-wounding (Figure [6b](#F6){ref-type="fig"}), more suggestive of a link to the later influx of macrophages. A further 17 genes are initially expressed at both the wild-type and PU.1wound site, but are maintained at high level only in the wild-type wound, where there is an influx of leukocytes. In PU.1null wounds, where there is no such influx, these genes are only transiently expressed. We assume that expression of these inflammation-maintained genes (Figure [6c](#F6){ref-type="fig"}) is directly or indirectly dependent on signals released by inflammatory cells. Inflammation-dependent genes may be expressed by leukocytes or by host cells as a \'response signature\' to inflammatory signals -------------------------------------------------------------------------------------------------------------------------------- Genes that are expressed only in wild-type wounds and whose temporal expression patterns are coincident with the influx of neutrophils and/or macrophages will include those genes that are constitutively expressed by one or both of these lineages, or genes that are upregulated as part of the leukocyte activation state, or may be expressed by cells other than the invading leukocytes as a downstream consequence of host fibroblast, endothelial and muscle cells being exposed to signals from these leukocytes. We present a selection of in situhybridization studies to illustrate each of these scenarios as revealed by very distinct classes of spatial expression pattern. ### Early inflammatory cluster L-plastin (Lcp1) is a pan-leukocyte, calcium-dependent, actin-bundling protein that has previously been implicated in macrophage activation and migration, although it is also overexpressed in many types of malignant human tumors \[[@B35]\]. It is first expressed in the wild-type wound coincident with the early stages of the wound inflammatory response, with a peak of expression at 12 hours post-wounding; our temporal data indicate no expression at any stage in PU.1null wounds (Figure [7Aa](#F7){ref-type="fig"}). In situhybridization studies reveal intense expression by leukocytes clustered within the wild-type wound site but no expression in surrounding skin (Figure [7Ab](#F7){ref-type="fig"}), and they confirm the absence of expression in PU.1null wounds (Figure [7Ac](#F7){ref-type="fig"}). The wound-restricted expression pattern of L-plastinsuggests that expression of this gene is limited to activated leukocytes only. Also expressed by leukocytes in the early inflammatory cluster are C3, a key component of the classical and alternative complement pathways, and its receptor, C3R. C3is expressed at similar levels in unwounded PU.1null and wild-type skin, but whereas expression is rapidly upregulated by 30 minutes post-wounding and continues until 24 hours in wild-type wounds, upregulation of C3is delayed and much weaker in the PU.1null wound (Figure [7Ad-f](#F7){ref-type="fig"}). This delay in C3expression suggests that inflammation has a significant role in raising and maintaining a rapid complement response at the wound site. Onzinalso appears as a member of the early inflammatory cluster; it encodes a leukaemia-inhibitory factor-regulated protein that has previously been identified in a screen for genes controlling inflammatory dermatitis \[[@B36]\]. Unwounded wild-type skin expresses Onzinat low levels but it is completely absent in PU.1null, unwounded skin and remains so until 12 hours post-wounding, when it is upregulated, but to a much lesser extent than in wild-type (Figure [7Ag](#F7){ref-type="fig"}). In situhybridization studies reveal a rather similar expression pattern in both wild-type and PU.1null wounds (Figure [7Ah,i](#F7){ref-type="fig"}). This suggests that Onzinmight be expressed in wild-type skin by resident inflammatory cells and in the PU.1null wound, either by inflammatory cells whose development is delayed, such as T cells, or that there may be an alternative or compensatory mechanism of gene regulation in non-inflammatory cells at the wound site. As discussed previously, both the genes for MRP8 and its binding partner MRP14 are upregulated by wound-edge keratinocytes. Both are also expressed by leukocytes, and in the case of MRP14this expression predominates and leads to cluster separation of the two genes, with MRP14categorized as part of the early inflammatory cluster. In the wild-type wound, it is expressed from 3 hours, with expression peaking at 12 hours post-wounding, whereas in the PU.1null, expression does not begin until 12 hours post-wounding and levels are much reduced compared with wild-type (Figure [7Aj](#F7){ref-type="fig"}). In situhybridization clearly shows MRP14to be expressed, in addition to expression in keratinocytes, in the region of the wound populated by inflammatory cells in the wild-type only (Figure [7Ak,l](#F7){ref-type="fig"}), and indeed, previous experiments suggest that both neutrophils and macrophages express MRP8and MRP14\[[@B22]\]. It may be that genes expressed by host connective-tissue cells at the wound site as a consequence of inflammatory signals are detrimental to healing and lead to some of the imperfect aspects of repair seen in adult healing such as fibrosis and scarring. One candidate for such a gene is Osteopontin(Spp1, minopontin), encoding a glycoprotein that can mediate cell-matrix interactions via the engagement of a number of adhesive receptors (reviewed in \[[@B37]\]). Previous wound-healing studies on Spp1null mice report differences from wild-type in that repair is characterized by abnormal macrophage debridement and abnormal maturation of collagen bundles \[[@B38]\]. Osteopontinhas a clear inflammation-related profile (Figure [7Am](#F7){ref-type="fig"}) and in situhybridization reveals an unusual pattern of expression at the wild-type wound site, with some expression by a subset of leukocytes but with most positive cells located in what appears to be the deep dermal or muscle layers of the wound region (Figure [7An,o](#F7){ref-type="fig"}). Both the early and late inflammatory clusters contain chemokine and growth factor receptors unique to leukocytes, and presumably used by these cells to detect various chemotactic cues that will guide them to the wound site. For example, the gene for chemokine receptor 1 (CCr1), a receptor for several chemokines including MIP-1α, CCL5 and Scya7, is expressed as early as 3 hours post-wounding, with expression levels peaking by 12 hours. There is no expression at the PU.1null wound site (Figure [7Ap](#F7){ref-type="fig"}). In situstudies show CCr1to be expressed in the wild-type wound by leukocytes recruited to the wound site (Figure [7Aq,r](#F7){ref-type="fig"}). As well as chemokine receptors, chemokines themselves are found in these clusters. CXCL10(IP-10) encodes an α-chemokine that functions as a potent chemoattractant for macrophages and T cells, and is upregulated by 12 hours in wild-type wounds but is absent in PU.1null wounds (Figure [7As](#F7){ref-type="fig"}). In situstudies reveal intense staining by what could be either leukocytes or host fibroblasts at the wild-type wound site (Figure [7At,u](#F7){ref-type="fig"}). Either this chemokine is an amplifying chemotactic signal expressed by leukocytes to draw in further leukocytes, or its expression is triggered in fibroblasts, but only if they receive signals from the first influx of neutrophils. ### Late inflammatory cluster Cathepsin Sis a typical gene of the late inflammatory cluster, being highly upregulated at 24 hours post-wounding in the wild-type, but with no expression in the PU.1null wound (Figure [7Ba](#F7){ref-type="fig"}). Cathepsin S is one of a large family of leukocytic proteases - this one largely macrophage-specific - that catalyze the remodelling of ECM proteins. In situhybridization studies in the wild-type wound show Cathepsin Sto be expressed by macrophages clustered around the wound site, but also by cells in the dermis at skin sites well away from the wound (data not shown), suggesting that it is constitutively expressed by cells of the monocyte lineage, rather than being part of the macrophage activation profile. No expression of Cathepsin Sis seen in wounded or unwounded skin of the macrophageless PU.1null mouse (Figure [7Bb,c](#F7){ref-type="fig"}). Repetinis an epidermal differentiation gene and a member of the fused gene subgroup of the S100 family that encodes multifunctional epidermal matrix proteins \[[@B39]\]. This temporal profile at the wound site implicates Repetinas being responsive to inflammatory signals (Figure [7Bd](#F7){ref-type="fig"}), and yet in situhybridization studies reveal it is not expressed by inflammatory cells, but rather by leading-edge keratinocytes in both wild-type and PU.1null wounds (Figure [7Be,f](#F7){ref-type="fig"}). While not absolutely dependent on inflammatory signals, it appears that Repetinexpression by wound keratinocytes is significantly enhanced by inflammatory cues. As several studies have shown somewhat enhanced rates of re-epithelialization where one or more components of the inflammatory response are reduced during healing \[[@B6],[@B40],[@B41]\], it is tempting to speculate that genes like Repetin, which are upregulated in the wound epidermis in response to inflammatory signals, may in some way retard the re-epithelialization process. As with the early inflammatory cluster, there are several genes in the late inflammatory cluster that may directly or indirectly, via their effects on signaling pathways, be responsible for wound fibrosis. The angiotensin II receptor has previously been implicated in mediating the fibrotic response in several tissue injury situations, such as myocardial infarction \[[@B42]-[@B45]\]; its gene is also a member of the late inflammatory cluster but is expressed at both wild-type and PU.1null wounds. Expression is clear in both wild-type and PU.1null wounds but significantly higher in the wild-type (Figure [7Bg](#F7){ref-type="fig"}). The spatial expression pattern of Angiotensin II receptoris reminiscent of Osteopontinin the early inflammatory cluster, with the brightest staining in the deep dermal or muscle layer of the wild-type wound and only very faint expression seen at the PU.1null wound site (Figure [7Bh,i](#F7){ref-type="fig"}). Presumably, a subset of genes found in these inflammatory clusters, which are upregulated by host granulation tissue lineages rather than by leukocytes, may turn out to be markers, or direct regulators, of the fibrotic response that is routinely activated in adult wound granulation tissue. Clearly, therapeutic reduction of the products of these genes at the wound site might result in the reduction of wound fibrosis. ### Inflammation-maintained cluster A final cluster of genes appears to be regulated by the inflammatory response in that they are generally expressed at early stages post-repair in both PU.1null and wild-type mice, but, whereas their expression subsequently diminishes in the PU.1null mouse, expression is maintained, or increases, coincident with the inflammatory response in wild-type wounds. This temporal expression profile is most clearly visualized from heatmap data (Figure [6c](#F6){ref-type="fig"}). Some of the genes in this cluster implicate mast cells in the recruitment of other leukocyte lineages which then amplify the inflammatory signal. For example, Mast Cell Protease 5 (Mcpt5) is a serine chymase stored in the secretory granules of mast cells and acts as a potent chemoattractant \[[@B46]\]. Mcpt5is rapidly and transiently upregulated immediately post-wounding and by 12 hours is back to near basal levels in the wild-type wound. However, it is secondarily upregulated at 24 hours. Expression is also clear at the PU.1null wound site as an immediate response but levels remain low and there is no second peak of expression (Figure [7Ca](#F7){ref-type="fig"}). In situhybridization studies show expression by scattered cells within the wild-type wound, with low levels of expression detected at the PU.1null wound site also (Figure [7Cb,c](#F7){ref-type="fig"}). These data suggest that Mcpt5is initially expressed independently of signals from macrophages and neutrophils, but that leukocytes are subsequently responsible for a secondary expression, either directly by expressing Mcp5themselves, or indirectly by triggering expression in another cell type, possibly supplying cues that reinforce expression by mast cells or prevent their dispersal from the wound site. Chemokines are also represented in this inflammation-maintained cluster. CCL2 and CCL7 are C-C chemokines with roles in directing the cellular composition of the inflammatory response. They are upregulated at 3 hours with expression tailing off by 24 hours post-wounding in the wild-type. In the PU.1null wound, CCL2and CCL7are also upregulated at 3 hours but to a lesser degree than in the wild-type, and unlike in the wild-type, expression is immediately downregulated, so that by 12 hours post-wounding there is a complete absence of expression (Figure [7Cd,g](#F7){ref-type="fig"}). This suggests that expression is enhanced and maintained in the wild-type by the presence of macrophages and neutrophils, whereas in the PU.1null wound, initial expression is independent of these leukocytes but without them expression cannot be amplified and maintained. Our in situstudies suggest that these chemokines are expressed by host wound connective-tissue cells rather than leukocytes at both the wild-type and PU.1null wound sites (Figure [7Ce,f](#F7){ref-type="fig"}, and [Ch,i](#F7){ref-type="fig"}). Conclusions =========== Here we report an Affymetrix GeneChip microarray study of in vivowound healing using a neonatal mouse wound model where all phases of the repair process are compressed into a 24-hour period. Cluster analysis of wild-type wounds versus those of PU.1null mice that are genetically incapable of raising an inflammatory response allow us to distinguish repair genes from those involved in, and a consequence of, wound inflammation. Several previous studies have modeled wound healing in vitroby exposure of fibroblasts to serum, as tissue damage to blood vessels in vivoleads to exposure of connective-tissue cells to blood serum \[[@B3]\]. Our results show that for the earliest phases of the repair process, this model does indeed mirror the in vivorepair response. When we consider only those genes present on both experimental microarrays, we see that more than 40% of the genes that are upregulated with an immediate early profile at the wound site have previously been shown to have similar temporal profiles after in vitroactivation of fibroblasts. Other in vitromodels of repair also turn out to be rather good predictors of the in vivoresponse. For example, exposure of keratinocytes to keratinocyte growth factor (KGF) in vitroreveals many of the early and late effector genes that are expressed by keratinocytes at the wound edge \[[@B22]\]. Other aspects of the repair process, in particular the stopping phase where migratory and proliferative behaviors cease as wound edges confront one another, have not yet been successfully modeled in vitro, but our study shows that even in the complexity of in vivohealing, many hints as to the genes responsible for these episodes can be gleaned by microarray surveys. Clearly, the most novel aspect of our study is its capacity to highlight those gene responses that are specifically associated with, or a consequence of, the wound-activated inflammatory response. Those genes expressed at the wound site by virtue of their being expressed by the invading leukocytes provide clues as to the migratory machinery of leukocytes in vivo, informing us, for example, which chemokines might be key attractive cues by revealing which of the chemokine receptors are expressed by these cells. Of most therapeutic significance are those genes expressed as a consequence of inflammation by host wound fibroblasts, endothelial and muscle cells. These genes are clearly not absolutely essential for repair, or PU.1skin wound not heal. Rather, they will include genes that contribute to the negative side effects of inflammation at the wound site including retarded re-epithelialization and fibrosis. Dissecting out exactly which genes from the inflammation-associated clusters might sit in such a category will be a major goal of our future studies. How full a survey of the wound healing process is revealed by our microarray study? The Affymetrix GeneChip we used covered approximately half of the mouse genome and so we cannot claim this to be a saturation screen. Moreover, it has not escaped our attention that several well established wound players that we know to be represented on the chips are apparently absent from any of the wound clusters. This is true for several growth factors, most notably TGFβ1, which we have previously shown to be differentially expressed in wild-type versus PU.1null wounds in RNase protection assays \[[@B6]\], and the same may be true for several other classes of genes expressed at low copy number. A similar observation was made in a recent serial analysis of gene expression (SAGE) study of Drosophilagenes expressed downstream of JNK signaling, which highlighted many such genes but revealed barely any change in expression of the TGFβ family member dpp, for which there is excellent genetic evidence for downstream activation by JNK signaling \[[@B47]\]. For these reasons it is clear that our screen underestimates the numbers of genes associated with each of the repair episodes and is perhaps somewhat biased towards genes expressed at higher copy number. As we have highlighted throughout this paper, revealing a temporal expression profile that coincides with one of the physiological episodes of the repair process in no way proves function for a wound-expressed gene. Although limited to a small sample of genes, our in situhybridization studies add spatial resolution to this dataset, revealing whether a gene is expressed by the wound epidermis or connective tissue cells, or by inflammatory cells, but given the vast array of genes expressed at the wound site, how can one dissect each of their roles during the repair process? Using recent developments in Cre driver lines, it will be possible to knockout genes specifically in appropriate cell lineages within the mouse, so that the requirement for a particular chemokine receptor in the recruitment of inflammatory cells can be assessed, or the link between expression of a candidate \'fibrosis\' gene by wound fibroblasts and subsequent scarring, can be tested. Complementary studies in which mRNAs for candidate inflammation/fibrosis genes are simply knocked down by local delivery to the wound site of antisense oligodeoxynucleotides (AS ODNs) \[[@B48]\] will provide a testbed for whether such approaches may be of therapeutic benefit to improve the repair process. While mammalian models may remain the best guide for potential clinical application, it may be faster and more efficient to turn to simpler, more genetically tractable organisms to trawl through the vast numbers of candidate repair and inflammation genes revealed by microarray studies. Indeed, we and others \[[@B49]-[@B51]\] have begun to use Drosophilaas a testbed to dissect the genetics of some aspects of the repair process and to determine by mutant analysis precisely which genes are required for which repair episodes. In summary, we present here a portfolio of genes expressed during the in vivowound healing process and categorized according to the physiological episodes that best correlate with their temporal expression profile. Such a classified listing provides good clues to the genetic regulation of all the cell behaviors that contribute to healing, and supplies us with a pool of genes whose modulation may prove to be therapeutically beneficial to the repair process. Materials and methods ===================== Mice and wounding ----------------- Generation and PCR genotyping of PU.1mice has been described previously \[[@B6]\]. Tail-tip blood smears were stained with Giesma (Sigma) for rapid identification of null individuals by the absence of neutrophils. Two-day pups received local anesthetic and full-thickness incisional wounds were made to a 1 cm × 0.5 cm area of the back skin. For the microarray study, a criss-cross network of 10 × 8 incisional wounds were made with a scalpel (Figure [1a](#F1){ref-type="fig"}) such that all cells within the rectangle of skin were adjacent to a cut. For in situhybridization studies, three incisional wounds were made along the long axis of this patch of skin (Figure [1b](#F1){ref-type="fig"}). Subsequently, PU.1null mice and control sibs were maintained with daily antibiotic injections until sacrifice at appropriate times. For microarray experiments, full-thickness back skin was dissected and immediately transferred, without fixation, into liquid nitrogen for subsequent RNA extraction. Wound harvesting and cryostat tissue sectioning for immunohistochemistry and in situhybridization were performed after perfusion fixation with 4% paraformaldehyde in PBS as described \[[@B6]\] with tissue sections cut at 14 μm. RNA extraction and preparation for hybridization ------------------------------------------------ Total RNA was isolated from all skin samples using RNAzol (Biogenesis) according to the manufacturer\'s instructions with a secondary clean-up stage using an RNeasy kit (Qiagen). RNA was amplified and labeled with biotin as described previously \[[@B52]\]. Array hybridization and scanning -------------------------------- Double-stranded cDNA was generated from 10 μg total RNA using Superscript Choice kit (Life Technologies) with a T7-poly(T) primer. Approximately 1 μg cDNA was used to generate biotinylated cRNA by in vitrotranscription using Bioarray High Yield RNA Transcript Labelling kit (Enzo Diagnostics Inc). Fragmented cRNA (10 μg) was hybridized in 100 mM β-mercaptoethanol, 1 M NaCl, 20 mM EDTA, 0.01% Tween20, 0.1 mg/ml herring sperm DNA, 0.5 mg/ml acetylated BSA, 50 pM control oligonucleotide and eukaryotic hybridization controls, to Affymetrix MGU74A arrays at 45°C for 16 h. Arrays were washed using Affymetrix protocols in nonstringent buffer (6x SSPE buffer, 0.01% Tween20, 0.005% antifoam) at 25°C and stringent wash buffer (100 mM MES buffer, 0.1 M NaCl, 0.01%Tween20) at 50°C and stained with streptavidin phycoerythrin (10 μg/ml) including an antibody amplification step. Arrays were scanned using an Affymetrix confocal scanner. Analysis of GeneChip data ------------------------- The data were analyzed using Microarray Analysis Suite version 4.0 (Affymetrix). The data were scaled to a target intensity of 300. Values representing genes not expressed are unreliable and are given an absent call. All genes were subjected to a filter to identify genes with at least one present call across the full eight time points and that had on one or more occasion a greater than twofold change in gene expression levels between time points or between the wild-type and PU.1null. The remaining genes were sorted into nine clusters using Spotfire Array Explorer 3.0 software, of which seven correlated with clear physiological episodes of repair or inflammation and we have loosely named those clusters according to these criteria. Resin histology and c-fmsin situ hybridization ------------------------------------------------ Wound tissues were processed for resin histology with sections cut at 5 μm and stained with Toluidine Blue as previously described \[[@B6]\]. To visualize macrophages we carried out in situhybridization studies using the macrophage-specific c-fmsprobe, using the protocol outlined in \[[@B6]\] and see below. In situhybridization ---------------------- Probe details are available in Additional data file 3. In situhybridization on frozen sections was performed as previously described \[[@B53]\], with probe hybridization carried out in a humidity chamber at 55°C for 16 h. Expression was visualized by BCIP/NBT precipitation (Roche Biochemicals) and sections viewed on a Zeiss Axiphot microscope after mounting in Citifluor (UKC). This spatial expression information gave us a good indication of which were the expressing cell lineages in the wound site, particularly for epidermal keratinocytes (most superficial cell layer), and leukocytes (scattered individual cells in the wound granulation tissue), but without double staining with lineage-specific antibodies we cannot be definite, particularly in distinguishing specific leukocyte lineages from subpopulations of wound fibroblasts and so forth. Additional data files ===================== The following additional data are available with the online version of this paper. Additional data file [1](#s1){ref-type="supplementary-material"}, wound gene bioinformatics, is an Excel file containing an annotated database for the 1,001 differentially expressed genes in the nine temporal gene clusters: activation, early effector, late effector, stop, early inflammatory, late inflammatory; inflammation-maintained, and excluded clusters 1 and 2. For each gene this database provides an Affymetrix ID and GenBank description, together with absolute fluorescence levels and absence/presence calls for each time point, and known functional information gained from GenBank and Swiss-Prot databases. Additional data file [2](#s2){ref-type="supplementary-material"}, excluded clusters, contains line graphs displaying the temporal profile of the median expression levels at each time point to give a representation of excluded clusters 1 and 2. Additional data file [3](#s3){ref-type="supplementary-material"}, in situhybridization probe source, is a Word document listing the origins of the various RNA probes used in the in situhybridization studies. Additional data file [4](#s4){ref-type="supplementary-material"}, full array expression data, is an Excel file containing the raw data - absolute fluorescence levels and absence/presence calls - for all genes on the Affymetrix MGU74 arrays at all experimental time points. Supplementary Material ====================== ::: {.caption} ###### Additional data file 1 An Excel file containing an annotated database for the 1,001 differentially expressed genes in the nine temporal gene clusters: activation, early effector, late effector, stop, early inflammatory, late inflammatory; inflammation-maintained, and excluded clusters 1 and 2. For each gene this database provides an Affymetrix ID and GenBank description, together with absolute fluorescence levels and absence/presence calls for each time point, and known functional information gained from GenBank and Swiss-Prot databases ::: ::: {.caption} ###### Click here for additional data file ::: ::: {.caption} ###### Additional data file 2 Line graphs displaying the temporal profile of the median expression levels at each time point to give a representation of excluded clusters 1 and 2 ::: ::: {.caption} ###### Click here for additional data file ::: ::: {.caption} ###### Additional data file 3 A Word document listing the origins of the various RNA probes used in the in situ hybridization studies ::: ::: {.caption} ###### Click here for additional data file ::: ::: {.caption} ###### Additional data file 4 An Excel file containing the raw data - absolute fluorescence levels and absence/presence calls - for all genes on the Affymetrix MGU74 arrays at all experimental time points ::: ::: {.caption} ###### Click here for additional data file ::: Acknowledgements ================ L.C. was funded by a Pfizer Prize studentship. We are also grateful to Marine Mione for excellent tips on successful in situhybridization with difficult probes, and to Kate Nobes and Scott McKercher for comments on the manuscript. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Wound histology. The location of skin wounds on the back of a neonatal mouse is shown. (a)For the array studies a series of criss-cross wounds were made so that all the skin cells were as close as possible to a wound edge for collection of wound RNA. (b)For in situhybridization studies and immunohistochemistry we made a series of three incisional wounds, so that transverse sections (broken line) contained the profiles of several wounds. Resin histology through wild-type (left-hand column) and PU.1null wounds (right-hand column) at (c,d)0.5 h, (e,f)3 h, (g,h)12 h and (i,j)24 h post-wounding. At all stages, arrows mark the epidermal wound edges, which are seen to have met and fused in both genotypes by 24 h. An asterisk (\) marks the migrating epithelial edge. (k,l)*In situhybridization using a macrophage-specific C-fmsprobe reveals large numbers of macrophages recruited to the granulation tissue in frozen sections through 24 h wounds in wild-type skin (k), while none are present in equivalent tissues of the PU.1null mouse (l). Scale bars = (c-j) 400 μM; (k,l) 250 μM. ::: ![](gb-2004-6-1-r5-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Median temporal profile graphs of identified repair and inflammation clusters. Line graphs displaying the median level of absolute mRNA expression (y-axis) at each time point: 0, 0.5, 3, 12 and 24 h (x-axis), for genes within each of the four repair clusters and the three inflammation clusters, giving representative temporal profiles for the cluster. Pink lines represent the temporal profiles of expression for the PU.1null wound site, blue lines those for the wild-type wound site. (a-d)The inflammation-independent gene clusters: (a) activation; (b) early effector; (c) late effector; (d) stop. (e-g)The inflammation-dependent clusters: (e) early inflammatory; (f) late inflammatory; (g) inflammation-maintained. The scale of absolute expression levels along the y-axis varies according to the maximum levels of expression in each cluster. ::: ![](gb-2004-6-1-r5-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Heatmaps for activation and effector clusters. Color depiction of the temporal profiles of mRNA intensity during the 24 h repair period for genes in (a)the activation cluster, (b)the early effector cluster and (c)the late effector cluster. Higher levels of expression are indicated by progressively brighter shades of red, and lower expression levels by increasingly brighter shades of green. The scale bar indicates absolute expression as a measure of fluorescence units. Genes are ordered with the most highly expressed first. Gene names are shown to the right of the maps and further bioinformatics data for each can be found in Additional data file 1. Expression levels for the PU.1null wound site at 0, 0.5, 3, 12 and 24 h are shown on the right and the equivalent expression levels for the wild-type (WT) wounds on the left. ::: ![](gb-2004-6-1-r5-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Temporal and spatial expression profiles of sample genes from the activation and effector clusters. Temporal and spatial profiles of the (a-h)activation and (i-p)effector clusters. The line graphs display temporal expression: absolute expression levels (y-axis) at each time point (x-axis) with both PU.1null (pink) and wild-type profiles (blue). The y-axis range varies depending on the expression levels for each gene. The photomicrographs show in situhybridization on 3 h (b,d,f,h) and 12 or 24 h (j,l,n,p) frozen wild-type wounds. (a,c,e,g) Temporal profiles of each of the activation genes show a rapidly induced but transient expression peak at 3 h in both PU.1null and wild-type wounds. (b) Krox24is expressed by wound margin epidermal cells extending back 10-12 cell diameters from the wound edge and also by associated hair follicles (arrows). (d) MKP-1is expressed by the first 5-8 front-row keratinocytes and a subset of dermal fibroblasts (arrows). (f) High levels of Fosl1expression in wound margin epidermal cells and weaker expression in damaged hair follicles (arrows). (h) EST GenBank accession number AI853531 appears to be expressed by wound fibroblasts (arrows). (i,k,m,o) The early and late effector gene samples all exhibit expression profiles with upregulation either at 12 h (i,k), or 24 h (m,o), whether in PU.1null or wild-type wound tissues. (j) Map4k4is expressed up to 10-12 cell diameters from the wound edge and in dermal fibroblasts (arrows). (l) Rbp1is expressed in epidermal cells approximately 15 cell diameters from the wound site. (n) K6expression is restricted to 10-12 rows of wound edge keratinocytes. (p) MRP8has a rather similar keratinocyte expression to K6, but is also expressed to a lesser extent in leukocytes in wild-type wounds (arrows). Scale bar = 400 μm. ::: ![](gb-2004-6-1-r5-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Heatmap and in situhybridization data for genes in the stop cluster. (a)The temporal expression profiles of genes of the cluster are represented by a heatmap. The highest levels of expression are indicated by the brightest shades of red, while lower expression levels are represented by progressively brighter shades of green, as indicated by the scale bar. Genes are ordered with the most highly expressed first, and gene names are shown to the right of the maps. (b-e)A temporal series of in situstudies revealing expression of one gene in this class, Notch, at 0.5 h (b), 3 h (c), 12 h (d) and 24 h (e), showing how mRNA levels in the leading-edge keratinocytes appear to dip during the period of re-epithelialization and then increase again coincident with the time at which epidermal fronts contact one another. Arrows highlight region of gene expression. Scale bar = 100 μm. ::: ![](gb-2004-6-1-r5-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Heatmaps for inflammation-dependent genes. (a-c)Heatmaps of the temporal profiles of mRNA intensity during the 24 h repair period for inflammation-dependent genes in wild-type and PU.1null wounds. (a) The early inflammatory cluster corresponds to the earliest onset of the inflammatory response with a temporally later induction seen in the late inflammatory cluster (b). (c) The inflammation-maintained cluster also appears to be regulated by the inflammatory response. Highest levels of expression are indicated by progressively brighter shades of red and lower expression levels represented by progressively brighter shades of green, as shown by the scale bar. Genes are ordered, for each cluster, with the most highly expressed first, and gene names are shown to the right of the maps. ::: ![](gb-2004-6-1-r5-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Temporal and spatial expression profiles of sample genes from the three inflammation-dependent clusters. Temporal and spatial profiles of the (A)early inflammatory, (B)late inflammatory and (C)inflammation-maintained clusters. Line graphs display absolute temporal expression levels (y-axis) at each time point (x-axis) for both PU.1null (pink) and wild-type (blue) wounds. y-axis expression levels vary according to individual gene expression levels. In situhybridization studies of (A) 12 h, (B) 24 h or (C) 3 h frozen sections illustrate the contrasting expression patterns of each of these classes of genes in wild-type (WT) versus PU.1null wounds. (Aa,d,g,j,m,p,s)In the early inflammatory cluster, expression in wild-type wounds peaks at 12 h but is absent or significantly reduced in PU.1null wounds. (Ab,c)In situstudies show L-plastinto be expressed by activated leukocytes in the wild-type only (arrow). (Ae,f)Faint expression of C3is seen in both genotypes (see arrows). (Ah,i)Onzinexpression appears to be in the same cells within the connective tissue in both genotypes. (Ak,l)Both keratinocytes and leukocytes (arrows) express MRP14in the wild-type but only keratinocyte expression (arrow) is seen in the PU.1null wound. (An,o)Osteopontindisplays a possible \'fibrosis\' gene spatial profile with expression in deep dermal cell layers (arrow) in the wild-type only. (Aq,r)CCr1is expressed only in the wild-type wound, in cells whose clustered location suggests they are one of the leukocyte lineages.(At,u)Expression of CXC10is broad and throughout the wound connective tissue of wild-type wounds (arrow) suggesting that expressing cells are wound fibroblasts. (B) In the late inflammatory cluster, expression in wild-type wounds appears to peak beyond 12 h in wild-type wounds and is absent or reduced in PU.1null wounds. (Bb,c)Expression of Cathepsin Sis seen in activated leukocytes in the wild-type only (arrow). (Bd)Repetinis expressed by both genotypes but to a lower level in the PU.1null. (Be,f)Repetinis only upregulated by keratinocytes but is not restricted only to wild-type wounds (arrows). (Bh,i)Expression of the potential fibrosis gene Angiotensin II Receptor 1is seen in deep dermal cell layers of wild-type and, to a significantly reduced level, PU.1null wounds (arrows). (C) In the inflammation maintained cluster, the expression profiles suggest that while these genes may be initially expressed in PU.1null wounds, persistent expression requires the presence of an inflammatory response as in the wild-type wound situation. (Cb,c)Expression of Mcpt5is seen at both wound sites (arrows) in scattered cells throughout the wound connective tissue. (Ce,f)CCL2appears to be expressed by host wound cells at both wound sites (see arrows). (Ch,i)CCL7is expressed in an almost identical temporal and spatial profile to CCL2. Scale bars = 400 μm (Aa-o, Ba-f, C) and 250 μm (Ap-u and Bh,i). ::: ![](gb-2004-6-1-r5-7) :::	PubMed Central	2024-06-05T03:55:52.951285	2004-12-23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549066/", "journal": "Genome Biol. 2005 Dec 23; 6(1):R5", "authors": [ { "first": "Lisa", "last": "Cooper" }, { "first": "Claire", "last": "Johnson" }, { "first": "Frank", "last": "Burslem" }, { "first": "Paul", "last": "Martin" } ] }
PMC549067	Background ========== Large-scale genomic and cDNA sequencing projects have revealed thousands of new genes whose open reading frames (ORFs) are highly conserved during vertebrate evolution, but whose precise cellular functions remain unclear. Although functional analysis by gene disruption is possible after transfection of murine embryonic stem cells and the breeding of knockout mice \[[@B1]\], these whole-animal studies are laborious and expensive. If the mutant phenotype can be distinguished in cell culture, the chicken B-cell line DT40 is a valid alternative to murine knockouts because of its high ratio of targeted gene integration \[[@B2]-[@B4]\]. Additional advantages of DT40 are tightly regulated conditional gene-expression systems for the analysis of essential genes \[[@B5]-[@B7]\] and the ability to study genetic interactions by the stepwise modification of multiple loci \[[@B8]\] and marker recycling \[[@B7]\]. The recent release of the chicken genome sequence \[[@B9]\] greatly benefits the DT40 research community. For the first time, the entire genome can be searched for sequences that are conserved during vertebrate evolution and whose function might be clarified after genetic modification in DT40. However, in silicogene structure prediction methods have a high error rate and often do not correctly annotate the intron-exon structure of genes. Only full-length cDNAs unambiguously define the boundaries of the transcription units within whole-genome assemblies and cloned full-length cDNAs are also of immense practical value to complement mutant phenotypes and artificially express the encoded protein \[[@B10]\]. For these reasons, many genome sequencing projects in higher eukaryotes have been complemented by large-scale efforts to obtain a maximum number of full-length cDNAs \[[@B11],[@B12]\]. Although relatively large expressed sequence tag (EST) databases from bursal lymphocytes \[[@B13]\] and other tissues have been described \[[@B14]\], relatively few chicken cDNA sequences had been deposited in the public databases. Here we describe a project to sequence and characterize a large number of full-length cDNAs from bursal lymphocytes. The corresponding genes are likely to be expressed in DT40 and this should facilitate their analysis by targeted gene modifications. In combination with the recently released cDNAs from other tissues \[[@B15]\], the bursal cDNAs will be a valuable resource for many laboratories working with the chicken as a model organism. Results and discussion ====================== Generation of bursal cDNA sequences ----------------------------------- The overall strategy for producing the greatest possible number of new full-length cDNAs expressed in bursal lymphocytes is outlined in Figure [1](#F1){ref-type="fig"}. We previously described a cDNA library of bursal lymphocytes, but it contained only a low number of full-length cDNA clones \[[@B13]\]. It was therefore decided to synthesize a new cDNA library, called \'riken1\', using the biotinylated cap trapper method which is optimized to generate a large percentage of full-length cDNA inserts \[[@B16]\]. To assess the quality of the library and guide the selection of clones for full insert sequencing, the 5\' ends of over 14,000 clone inserts were sequenced. BLAST \[[@B17]\] searches against the public protein databases indicated that about 80% of the 11,116 high-quality ESTs obtained showed significant homology to existing entries and more than 80% of these extended further upstream than the methionine start codon of their homologs in the databases. This indicated that the riken1 library indeed contains an extraordinary high percentage of full-length cDNA inserts. Only clones whose ESTs showed significant BLAST matches against the public protein databases and covered the methionine start codon of their homolog were considered for full-length sequencing, as evolutionarily conserved genes are of highest interest for the DT40 research community. The remaining ESTs were clustered to remove duplicates corresponding to the same gene. In addition, ESTs corresponding to already known chicken genes in the public databases were removed. The plasmids corresponding to the remaining 2,796 ESTs were chosen for full insert sequencing by bidirectional primer walks. Once the end of the walks had been reached, the sequences of the full-length cDNA inserts were assembled. From the BLAST search results the most likely methionine start codon was assigned to each sequence. About 15% of the cDNA sequences showed evidence for premature frameshifts in the form of short ORFs and stretches of conserved sequence in a different frame further 3\'. If overlapping ESTs were found in the public databases, the cDNA sequences were edited to correct the likely reverse transcription error, otherwise these sequences were discarded. Length distribution and GC content ---------------------------------- A total of 2,272 high-quality chicken full-length cDNA clones were sequenced and assembled, manually annotated with respect to their likely translation start codon and deposited both at The Bursal Transcript Database website \[[@B18]\] and in the public databases. The lengths of the proteins encoded by the annotated ORFs were compared with the lengths of UniProt \[[@B19]\] database entries and the lengths of the untranslated region (UTR) sequences were compared with the lengths of known vertebrate UTRs available from the UtrDB collection \[[@B20]\] (Figure [2](#F2){ref-type="fig"}). The distributions obtained for the bursal cDNAs closely resemble those calculated for known sequences. Most of the 5\' UTRs have lengths in the range of 100 base-pairs (bp) \[[@B21]\], a value conserved in diverse taxonomic classes. The length distribution of 3\' UTRs is much broader, with a significant number of long sequences exceeding 1 kilobase (kb). The similarity between the length distributions observed for the collection presented here and those sequences stored in public databases suggests that most of our sequences are full-length cDNAs with correctly annotated start codon positions. The most remarkable feature noted in the analysis of 5\' UTRs of the bursal cDNAs is a very high GC content (67%). This supports the observation that the GC content of 5\' UTRs is particularly high in warm-blooded species \[[@B22]\]. On the other hand, the percentage of GC base-pairs in 3\' UTRs of the bursal cDNAs (41%) is close to the value observed for database sequences (42%). The ORFs of the bursal full-length cDNAs contain 49% GC base-pairs. Analysis of start codon context ------------------------------- The accurate prediction of the translation start codon remains difficult and in some cases our annotations remain tentative. Sequences surrounding the translation start codons are not random and in mammals match the consensus GCCRCCaugG (where aug is the start codon and R is either A or G) \[[@B23]\]. The most conserved nucleotides in the consensus are a purine, usually A, at position-3 and G at position 4. It has also been observed that a large fraction of 5\' UTRs contain AUG codons upstream of the translation start site, but these codons are unlikely to be flanked by the consensus sequence \[[@B21]\]. A detailed analysis shows that the riken1 collection of cDNA sequences contains 4,406 AUG codons upstream of the annotated translation start codons in 2,218 of the bursal cDNAs. Nine hundred one of these alternative start codons were in the same reading frame as the annotated ORF. An in-frame stop codon within the 5\' UTR region was present downstream of 501 of these 901 alternative start codons. The total number of ORFs present in 5\' UTR regions of riken1 cDNAs was 1,289. We have checked whether the context of the annotated AUG start codons differs from the context of the alternative upstream AUG sequences of the bursal cDNAs. We therefore extracted 10-bp long sequences surrounding the annotated start codons and the alternative upstream AUGs and visualized sequence variability using the sequence logo software \[[@B24]\] (Figure [3](#F3){ref-type="fig"}). The annotated start codons closely match the consensus, but the alternative upstream AUG codons do not exhibit flanking nucleotide preferences. This provides further evidence that the ORFs in our collection are correctly annotated. Similarity to predicted Ensembl transcripts and UniProt protein sequences ------------------------------------------------------------------------- All full-length cDNAs were compared to the collection of transcripts predicted from the chicken genome sequence by the Ensembl system \[[@B25]\]. The transcripts were downloaded before the Ensembl team used our collection of full-length bursal cDNA sequences to improve transcript predictions. Distribution of the percent identity and coverage of the best BLASTN alignments are shown in Figure [4a](#F4){ref-type="fig"}. Only 494 of the chicken full-length transcripts matched predicted mRNAs with a length coverage greater than 90%. This is not surprising taking into account that computational prediction of untranslated regions, based on the genome sequence alone, is very difficult, if not impossible. However, there were also significant differences between sequenced and predicted cDNAs within ORF regions. There are 1,463 sequences in which either the 5\' or the 3\' end of the ORF was not covered by predicted transcripts. In most cases (1,106), the discrepancy concerned the 5\' end. The statistics presented above and summarized in Table [1](#T1){ref-type="table"} indicate that our collection of full-length cDNA sequences may be used to significantly improve the annotations of more than 1,400 chicken genes. This analysis is further supported by the mapping of bursal serial analysis of gene expression (SAGE) tags to Ensembl transcripts and the genome sequence \[[@B26]\]. Figure [4b](#F4){ref-type="fig"} shows the distribution of the percent identity and coverage statistics of the BLASTP comparison of the proteins encoded by the bursal cDNAs to the UniProt collection of protein sequences. In most cases (1,524), the proteins encoded by riken1 cDNAs were almost fully covered in the alignments (more than 90% coverage) and showed a high percentage identity (greater than 70%) to known protein sequences. When compared to available chicken ESTs or cDNAs in the public databases, some of the bursal cDNAs showed significant structural differences most likely due to differential transcript processing. In addition, the bursal cDNA collection has been used to define a large number of intragenic single-nucleotide polymorphisms (SNPs) \[[@B27]\]. Functional domain assignment ---------------------------- All full-length cDNAs were compared to the Pfam database \[[@B28]\], which stores sequence profiles representing functional domains and the 10 most frequently occurring domains are shown in Table [2](#T2){ref-type="table"}. Subsequently, we have used the Gene Ontology (GO) \[[@B29]\] annotation of Pfam domains provided by the InterPro \[[@B30]\] database to assign functional descriptors to the domains detected in our sequences. It is important to note that the assignment of a GO term to a given cDNA sequence indicates only the presence of a functional domain rather than an orthologous relationship to other genes annotated by the term. Determination of orthologous relationships is best done at the level of whole-genome comparisons and is therefore beyond the scope of this study. This classification will be valuable for the selection of candidate genes for further analysis in DT40, but it is unlikely to be representative for the whole chicken genome because only a selected subset of cDNAs expressed in bursal cells were chosen for sequencing. Tables [3](#T3){ref-type="table"} and [4](#T4){ref-type="table"} list the assignments of GO molecular function and biological process descriptors to functional domains detected in riken1 full-length cDNAs. The most frequent molecular function associated with a domain is \'ATP binding\' (166 cDNAs) assigned to the protein kinase domain and other domains such as the AAA ATPase family, ABC transporters and others. The 64 DNA-binding proteins represented in our collection include RNA polymerase II, C5-cytosine-specific DNA methylase and 19 proteins exhibiting transcription factor activity according to biological process annotation. GTP-binding proteins are involved in translation initiation (eIF-2-gamma), cell-cycle regulation (Septin 5) and regulation of transport from the endoplasmatic reticulum to the Golgi apparatus. There are 22 full-length cDNAs in our collection containing Pfam domains annotated by the GO term \'molecular function unknown\'. Experimental information concerning the molecular mechanisms of action is very sparse or nonexistent for proteins sharing these evolutionarily conserved domains. Highly similar human proteins exist for the chicken proteins, an example being the human protein BM02. Taking into account the ease of targeted genome modification and availability of numerous functional assays, the DT40 cell line is an attractive model system to provide first insights into the functions of the evolutionarily conserved domains described above. Bursal Transcript database -------------------------- All the full-length cDNA sequences are stored within the Bursal Transcript database \[[@B18]\]. This database links the previously published EST data with the new cDNAs and can be searched by keyword or by using BLAST. Browsing of functional categories is also available as dynamically generated web pages link the bursal cDNAs to Ensembl, UniProt, Pfam and to GO data. To highlight gene expression differences between DT40 and bursal cells, the bursal cDNAs are also linked to SAGE data from both of these types of cells \[[@B26]\]. Conclusions =========== The cDNAs from bursal lymphocytes represent one of the largest full-length cDNA collections in the chicken, comprising about one third of all currently available, experimentally verified transcripts and will be of general interest to researchers using the chicken as an experimental model as well as to the poultry industry. The resource has already been integrated with the chicken genome sequences to build a unigene catalog \[[@B9]\], to define the nature and frequency of intragenic chicken strain polymorphisms \[[@B27]\] and to develop a chicken gene microarray for gene-expression profiling (B. Wong, T. Makeev and C. Davies, unpublished data). However, the main beneficiary of the full-length cDNAs is the DT40 research community. Although the release of the genome sequence has greatly simplified the identification of candidate genes for disruption and the design of the knockout constructs, it is still not a trivial task to predict the ORFs as well as 3\' and 5\' UTRs without cDNA sequences. Other uses are the expression of the cDNAs in vitroor for complementation of mutant DT40 phenotypes with the added convenience that the cDNA sequences are not only known, but also available as cloned pieces of fully sequenced DNA. Materials and methods ===================== Construction of the riken1 cDNA library and 5\' EST sequencing -------------------------------------------------------------- The riken1 library was synthesized from mRNA of 2-week-old CB strain bursal lymphocytes using the biotinylated cap trapper method \[[@B16],[@B31]\]. The resulting phage library was converted into pKS-derived plasmids and individual clones were then selected on ampicillin-containing agarose plates. About 45,000 colonies were picked and transferred into 384-well microtiter plates to prepare a permanent clone stock. Plasmids from 14,976 of the arrayed clones were sequenced on an Applied Biosystems automated sequencer using a primer that anneals to the plasmid backbone upstream of the 5\' end of the cDNA inserts (see \[[@B18]\] for details of the cloning vector sequence). The ABI sequencing files were processed as described previously \[[@B13]\]. About 5% of the riken1 clones contained an insert sequence which was 100% identical to the GenBank entry AJ277662, annotated as a human genomic fragment including the LMO1locus. This sequence was present as a stuffer of the lambda vector used for the library construction and the clones containing it were removed from further analysis. In total, the 5\' single-pass sequencing of 14,976 clones yielded 11,116 high-quality ESTs of the riken1 library. Selection of clones for full-length insert sequencing ----------------------------------------------------- BLAST searches against the \'All non-redundant GenBank CDS\' database showed that approximately 80% of the 5\' EST sequences matched GenBank entries with a score of at least 50. This score threshold was chosen because it allowed us in most cases to align the putative start codon of the query sequence to the EST. These sequences were chosen and clustered \[[@B32]\] to remove duplicates. In addition, all sequences matching chicken entries in the public databases with a score of over 300 were not considered further. The BLAST results of all remaining sequences were manually inspected and only those sequences which covered the methionine start codon of their closest match in the public databases were retained. In the end, the cDNA inserts corresponding to 2,796 ESTs were chosen for full insert sequencing. Full-length insert sequencing ----------------------------- Sufficient plasmid template for numerous sequencing reactions was prepared from the clones corresponding to the selected ESTs. All plasmids were then sequenced with a primer complementary to a plasmid sequence 3\' of the cDNA insertion site. Subsequently, custom-made 20-mer primers based on available sequences were used for sequencing until the 3\' and 5\' ends of the cDNA inserts were reached. All sequences were processed as described previously \[[@B13]\], except that a routine of manual proofreading and editing of the chromatograms within the Staden pregap program was implemented to increase the quality of the base calling and to decrease the failure rate of the next primer walk. The FOUNTAIN software \[[@B32]\] was extended to automatically design primers in 96-well format suitable for these walks. The primers were positioned to give an average of a 70-bp overlap between sequences. Once both ends of the insert were reached by the primer walks, the Staden gap4 program \[[@B33]\] was used to produce a double-stranded consensus of the cDNA insert. A total of 2,565 high-quality cDNA contigs were assembled for further analysis. Quality check and correction of frameshifts in the cDNA sequences ----------------------------------------------------------------- The integrity of the conserved ORF within each assembled cDNA sequence was manually examined by inspecting BLAST search results against the public protein and EST databases. To facilitate this task, a new EstSet module was added to FOUNTAIN \[[@B32]\]. The user interface displays the sequence of the cDNA insert together with its three possible translations and its BLAST search results against the public protein and EST databases. On the basis of this information a likely methionine start codon can be assigned to the cDNA. Around 15% of the cDNA sequences showed evidence of an artificial frameshift in the form of suspicious BLAST matches in two or more ORFs, presumably due to errors in the reverse transcription process. These sequences were compared to other Gallus gallusESTs from the public databases. If the short ORF could be corrected by adopting the sequence of an overlapping EST, the cDNA sequence was edited. The type of editing was recorded and the corresponding riken1 clone was annotated as likely to be defective. In total, 293 cDNAs were removed because either a likely artificial frameshift could not be corrected by using sequences of overlapping ESTs or they contained multiple stop codons in all three reading frames or they showed evidence for unspliced introns. All cDNA clones are freely available upon request to the corresponding author and their sequences have been submitted to the EMBL public database (accession numbers AJ719267-AJ721138 and AJ851370-AJ851825). Analysis of the start codon context ----------------------------------- The sequences surrounding the annotated start codons (10 bp upstream and downstream) were exported and submitted for information content visualization by the WebLogo software \[[@B24]\]. Subsequently, we have exported the ± 10 bp context of every ATG codon located upstream of the annotated start of the coding sequence. These sequences were also submitted to analysis by WebLogo software. Sequence-similarity searches and functional class annotation ------------------------------------------------------------ The riken1 cDNAs were compared with the collection of predicted chicken transcripts downloaded from the Ensembl ftp site \[[@B34]\] using the BLASTN program. BLASTP software was used to compare translated ORFs with the protein sequences stored in the UniProt database. Functional domains were assigned by comparing riken1 cDNAs with sequence profiles representing Pfam domains. This comparison was performed with RPSBLAST software (e-value cut-off of 10^-6^) run on the binary database files downloaded from the National Center for Biotechnology Information (NCBI). Functional classes were assigned according to Pfam to GO mapping provided by the InterPro database. The XML information exchange standard was used to interface the BLAST program outputs with the FOUNTAIN package. Acknowledgements ================ This work was supported by the EU grants \'Chicken IMAGE\', \'Genetics in a cell line\' (QLK3-2000-00785) and \'Mechanisms of gene integration\' (LSHG-CT-2003-503303). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Outline of full-length bursal cDNA production. ::: ![](gb-2004-6-1-r6-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Comparison of the length distributions of bursal cDNAs and public database sequences. The length distributions of 5\' UTRs from (a)bursal cDNAs and (d)5\' UTR sequences present in UtrDB. The length distribution of annotated ORFs from (b)bursal cDNAs and (e)UniProt database sequences. The length distributions of 3\' UTRs from (c)bursal cDNAs and (f)3\' UTR sequences present in UtrDB. Lengths of UTR sequences are given in nucleotides; lengths of ORFs in amino acids. ::: ![](gb-2004-6-1-r6-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### ATG codon context in the riken1 cDNA sequences. (a)Sequence logo of annotated start codon context. (b)Sequence logo of the context of ATGs found upstream of the annotated start codons. Figure created using WebLogo \[35\]. ::: ![](gb-2004-6-1-r6-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Statistics of BLAST searches. (a)BLASTN search of full-length cDNAs against chicken transcripts predicted by Ensembl. Note that only 494 cDNAs are identical or nearly identical with predicted transcripts. Therefore, the remaining 1,777 experimentally determined full-length cDNAs may be used to improve the gene annotation of chicken genomic DNA. (b)BLASTP searches of annotated ORFs against the UniProt database of protein sequences. ::: ![](gb-2004-6-1-r6-4) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Comparison of riken1 cDNAs and cDNAs predicted by Ensembl ::: Number of riken1 cDNAs Percentage of the total number of riken1 cDNAs --------------------------------------------------- ------------------------ ------------------------------------------------ More than 90% identity and more than 90% coverage 494 22 More than 90% identity and more than 70% coverage 808 36 More than 90% identity and more than 50% coverage 1258 55 Less than 90% identity 19 0.8 3\' or 5\' UTR of riken1 cDNA uncovered 1,463 64 5\' UTR of riken1 cDNA uncovered 1,106 49 ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### The 10 most frequently occurring Pfam domains in chicken full-length cDNAs ::: Pfam ID Description Number of occurrences --------- ---------------------------------------------------------------- ----------------------- PF00069 Protein kinase domain 66 PF00076 RRM\_1: RNA recognition motif 46 PF00071 Ras family 32 PF00025 Arf: ADP-ribosylation factor family 23 PF00169 PH: pleckstrin homology domain. 22 PF00271 Helicase\_C, Helicase conserved C-terminal domain 20 PF00018 SH3 (Src homology 3) domain 18 PF00651 BTB or POZ domain present in some of zinc finger proteins 18 PF00270 DEAD, DEAD/DEAH box helicase 18 PF00004 AAA, ATPase family associated with various cellular activities 14 ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### The 10 molecular function GO terms most frequently assigned to chicken cDNAs ::: GO ID Description Number of occurrences\* ------------ ------------------------------- ------------------------- GO:0005524 ATP binding 166 GO:0004672 Protein kinase activity 66 GO:0003677 DNA binding 64 GO:0005525 GTP binding 57 GO:0005515 Protein binding 37 GO:0016491 Oxidoreductase activity 27 GO:0005554 Molecular function unknown 22 GO:0003700 Transcription factor activity 19 GO:0008565 Protein transporter activity 18 GO:0008270 Zinc ion binding 17 \Number of cDNAs containing domain annotated by the GO term. ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### The 10 biological process GO terms most frequently assigned to chicken cDNAs ::: GO ID Description Number of occurrences\ ------------ -------------------------------------------- ------------------------- GO:0006468 Protein amino acid phosphorylation 66 GO:0006355 Regulation of transcription, DNA-dependent 39 GO:0006508 Proteolysis and peptidolysis 38 GO:0007264 Small GTPase mediated signal transduction 33 GO:0006412 Protein biosynthesis 29 GO:0006118 Electron transport 28 GO:0006886 Intracellular protein transport 26 GO:0007165 Signal transduction 18 GO:0005975 Carbohydrate metabolism 17 GO:0006457 Protein folding 11 \*Number of cDNAs containing domain annotated by the GO term. :::	PubMed Central	2024-06-05T03:55:52.956700	2004-12-23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549067/", "journal": "Genome Biol. 2005 Dec 23; 6(1):R6", "authors": [ { "first": "Randolph B", "last": "Caldwell" }, { "first": "Andrzej M", "last": "Kierzek" }, { "first": "Hiroshi", "last": "Arakawa" }, { "first": "Yuri", "last": "Bezzubov" }, { "first": "Jolanta", "last": "Zaim" }, { "first": "Petra", "last": "Fiedler" }, { "first": "Stefan", "last": "Kutter" }, { "first": "Artem", "last": "Blagodatski" }, { "first": "Diyana", "last": "Kostovska" }, { "first": "Marek", "last": "Koter" }, { "first": "Jiri", "last": "Plachy" }, { "first": "Piero", "last": "Carninci" }, { "first": "Yoshihide", "last": "Hayashizaki" }, { "first": "Jean-Marie", "last": "Buerstedde" } ] }
PMC549068	Background ========== Mammalian phenotypes are complex and the term itself is imprecise. Generally, we use the word phenotype in referring to the appearance or manifestation of a set of traits in an individual that result from the combined action and interaction of genotype and environment. Because mouse is the premier model organism for the study of human biology and disease, the goal of comparative phenotyping and building new animal models through genetic engineering holds great promise. The mouse has distinct advantages for studies that translate to humans. It is a small, short-lived mammal with a fully sequenced genome in which all life stages can be accessed, and for which myriad tools are available for precisely experimentally manipulating its genome. Further, the large collection of inbred strains of mice and the controlled environment in which the animals live provides the ability to confirm phenotype observations and to systematically perturb environmental factors and genetic input to measure effects under defined conditions. Current international efforts to \'make a mutation\' for every gene through mutagenesis \[[@B1]\] and genetic engineering \[[@B2],[@B3]\] make it imperative for phenotype data to be represented in standard descriptive formats to enable computational analysis and comparison. Mammalian phenotypes are frequently genetically complex. Mutation of even a single gene almost always produces pleiotropic effects. Conversely, non-allelic mutations can produce indistinguishable phenotypes. Modifier genes and epistatic interactions can markedly alter the phenotype. Combining different allelic combinations of different genes can produce unique phenotypes not found in the single-gene mutation genotype. Imprinting of genes can dramatically affect phenotype. Mutations expressed in different inbred strains of mice can manifest as an increase or decrease of severity or penetrance of the corresponding phenotype. Quantitative trait loci (QTL) can contribute in complex nonlinear ways to the phenotype. In addition, mutations that are \'genomic\' in nature, either disrupting or deleting multiple genes or occurring in intergenic regions, can produce distinct phenotypes and challenge us to think beyond gene effects to genomic effects. The outcome of these complex interactions can be dissected and reproducibly examined by characterizing inbred strains that represent the combined phenotype of the \'whole-genome\' genotype in its environmental context. The Mouse Genome Database (MGD) at the Mouse Genome Informatics website \[[@B4],[@B5]\] serves as the model organism database for mouse, representing the genetics, genomics and biology of the mouse and as a community resource for mammalian studies. Significant reorganization and modeling of phenotypes is now underway to support these data robustly, to represent phenotypes in ways that are computationally accessible, and to provide human interfaces to these data that will enable knowledge building and hypothesis generation. One component of this work is the development of the Mammalian Phenotype (MP) Ontology, a structured vocabulary that will aid in standardizing annotations and, with its concepts definitions, unambiguously describe phenotypic observations. Results and discussion ====================== The problems of text -------------------- Written descriptions of phenotypes in higher organisms reflect the complexity of the subject, the richness of language, and the phenomenal diversity that these data represent. While text descriptions are commonly used in publications describing phenotype, and have been the basis of electronically accessible phenotypic descriptions (for example, Online Mendelian Inheritance in Man (OMIM) \[[@B6]\] and the Mouse Locus Catalog (MLC) \[[@B7]\], text is unreliable for searching, either manually or computationally. From the user\'s perspective, even the best full-text search including Boolean operators will miss appropriate records (false negatives) and return unwanted records (false positives). Consider the example in Table [1](#T1){ref-type="table"} where searches were done to find spontaneous mutations in which mice were entirely or partially devoid of hair/fur. To obtain a complete result, the user would need to use a number of search terms and synonyms. The wording within the text depends upon the author of the record and his/her particular word usage and editorial style. A minimum of four search terms is needed to recover the 27 relevant mutations displayed in this table and it cannot be ascertained if this is a complete set of mutations for this phenotype. Conversely, the user is returned with 23 irrelevant results. Irrelevant results can be returned for many reasons including, but not limited to, the following: the author of the record is contrasting the phenotype of a mutation in one gene with a mutation in another gene; the author is making a statement that includes the negation of the trait; the match is based on gene name rather than phenotype; the mutant was used as a linkage marker to genetically map another gene. A further detriment to database text records is their difficulty to update and maintain. As new information is learned about a phenotypic mutant, the record must be continually rewritten. Although this practice might be sustained for a small number of records, it does not scale when thousands of mutant records are considered. The alternative of simply adding on another paragraph to existing text records becomes confusing, with potentially conflicting information and different writing styles appearing in one textual description, and unwieldy, with more and more text that may no longer represent a logical synthesis. Nomenclatures, vocabularies and ontologies ------------------------------------------ Formal nomenclatures for genes, mutant alleles and inbred strains of mice have existed since the 1940s \[[@B8],[@B9]\]. The MGD \[[@B4]\] serves as the authoritative source for the names and symbols associated with mouse genes, alleles and strains. The advantage of applying such nomenclatures has been increasingly recognized as genomes become better defined and the realized power of comparative genomics allows homologous and orthologous gene relationships to be explicitly defined. At present, human, mouse and rat gene nomenclatures operate in parallel, using coordinated symbols for all three species\' genes. In addition, mouse and rat strain nomenclatures were merged to one standard strain nomenclature recently, making strain identity and nomenclature conventions consistent. Nomenclature guides for mouse and rat genes, mutant alleles, and strains are available online and regularly revised based on international nomenclature committees\' reviews \[[@B10]\]. Beyond nomenclatures, which are key to object identities and relationships, are vocabularies that can be used to describe broader concepts and categorizations. Vocabularies can take many forms, including simple lists of controlled terms, such as the cytogenetic band designations used to name the bands defined by chromosome staining or the classes of genetic markers, such as gene, pseudogene, expressed sequence tag (EST), and so forth. The annotation of complex biological data and concepts requires more than lists and simple vocabularies. Ontologies, or \'descriptions of what there is\', contain both concepts, with precise meanings, and relationships among those concepts. As such ontologies are able to support descriptions of complex biology and are useful in making these data more amenable to computational analyses. The first widely used ontology developed and adopted in the biological domain is the Gene Ontology (GO) \[[@B11]-[@B13]\] which contains concepts of molecular function, cellular localization and biological process for annotating the functional aspects of genes. The GO is structured as a directed acyclic graph (DAG), where each vocabulary term (node) may have both multiple parent term and multiple child term relationships. MGD uses GO extensively for gene annotation \[[@B14]\]. In addition, MGD has adopted the Mouse Embryo Anatomy Nomenclature Database \[[@B15]\] and the Anatomical Dictionary for the Adult Mouse \[[@B16]\] for annotating data that include anatomical attributes, such as tissue sources for clones and phenotypes. The Gene Expression Database (GXD) \[[@B17]\], integrated with MGD through the Mouse Genome Informatics (MGI) system \[[@B4]\], applies these anatomical ontologies as a central concept in the description of expression data. Mammalian Phenotype Ontology ---------------------------- Although the need for vocabularies as key components to consistent phenotype annotations for mammals has been recognized for some time \[[@B18]\], and many smaller controlled vocabularies have been implemented to describe various aspects of phenotype in MGD (for example, class of mutation, embryonic stem (ES) cell lines used for generating targeted mutations, type of inheritance), much of the data has remained in text form. Over the past two years, the Mammalian Phenotype (MP) Ontology has emerged to more precisely describe phenotypes, and to allow easier access to phenotype-sequence interactions. Our goal is to describe the richness of phenotypes as precisely as they are known, recognizing that phenotype data are by nature complex and usually incomplete. Taking advantage of structural properties of a DAG, we have the ability to annotate phenotypes to the level of data resolution available, whether general or very specific and the ability to query with a high-level term, returning all phenotypes containing annotations to that term or to terms more specific than the query term. Thus, one can query for \'respiratory signs/symptoms\' and retrieve all phenotypes annotated to this term and its hierarchical \'children\' (abnormal breathing, abnormal respiratory sounds, anoxia, apnea, dyspnea, hypercapnia, and so on), or specifically request annotations to any of these sub-terms. The top level terms of the MP Ontology include physiological systems, behavior, developmental phenotypes and survival/aging. Physiological systems branch into morphological and physiological phenotypes at the level immediately below. A browser to view the ontology is available at \[[@B19]\] (Figure [1](#F1){ref-type="fig"}). In this browser the DAG structure is flattened into a hierarchy, with multiple hierarchies representing unique paths to a term displayed sequentially. MP terms and synonyms can be searched or users can browse the ontology starting from the high-level terms and open levels continuously to increasingly granular terms. Each MP ontology term has a unique identifier, a definition and synonyms. In the term detail pages, these data and the number of hierarchical paths of the vocabulary where the terms appear are displayed. A plus sign following the term indicates that children of this term exist. In this figure, displayed next to the term, is a link indicating the number of annotation instances in MGD using this term or children of this term. This feature, due to be publicly available in early 2005, will greatly improve phenotype-centric searching in MGD. Developing the MP vocabulary ---------------------------- To initiate the vocabulary, we first developed a high-level categorization of phenotypes consisting of approximately 100 terms, such as heart/cardiovascular dysmorphology and skeletal axial defects. As we used this list for annotations, terms were refined and general organizing principles for the MP vocabulary were developed. An important component of our approach has been to address two practical implementation questions. From the biologist\'s perspective, the question is what term would be used to describe a specific phenotypic trait. From the curation perspective, we ask what terms reflect biological reality and maximize curator productivity. From a purely ontological perspective, every trait could be broken down into a core object, such as \'cornea\' or \'gastrulation\', defined by anatomical, behavioral or physiological terms, and a series of attribute vocabularies that describe the quality, quantity and character of a trait. For the practical reason of needing robust terms to describe phenotypes up-front to speed curation and the problem of losing biological meaning, particularly for clinical or dysmorphology terms, when terms are completely deconstructed (that is, the sum of the parts is less than the term itself), we have chosen to use compound terms in the MP Ontology. A few examples of terms where it is difficult to preserve the full biological meaning once they are deconstructed are shown in Table [2](#T2){ref-type="table"}. In addition, it should be noted that each of these terms requires multiple annotations to recover all aspects that the single term provides. Use of complex terms in the MP Ontology, however, does not preclude also storing the decomposed version should this later prove desirable (see PATO model discussed in \[[@B20]\]). More important, the MP Ontology can currently hold, for each term, database cross-references to other ontologies. This is a common practice in GO when compound terms are developed. For the MP Ontology, these cross-references include anatomical terms from the Mouse Anatomy ontologies \[[@B15],[@B16]\] and the GO process terms \[[@B21]\]. Three major strategies are being pursued to further develop the vocabulary itself. First and most important is through the ongoing process of curating phenotype data. As new phenotypic traits are described and published, the need for new terms is recognized. New terms added in this way may be a simple addition to an existing hierarchical path or may result in the addition of entire new branches in the hierarchy. Second, collaborative efforts between the MGD phenotype curators, the mouse mutagenesis centers and the rat genetics community identify new specific terms and suggest improved organization of terms within particular hierarchical branches. Third, we are recruiting individuals with expertise in specific biological domains to review and evaluate sections of the vocabulary for accuracy, completeness and systematic arrangement. The MP Ontology is a work in progress and remains incomplete in some areas. We welcome the participation of the mammalian research community so that the most useful, definitive and universally applicable terms will be included. Information can be obtained by sending e-mail to <pheno@informatics.jax.org>. While common pathological and clinical terms are used in the MP Ontology, considerations for term placement within the structure and for precise terminology is often derived from comparison with other open biological ontologies (OBO) \[[@B22]\]. Recently, a cell-type ontology has become available \[[@B23]\] and a comparison of terminology to this ontology has not yet been completed. We are working with the mutant mouse pathology database Pathbase \[[@B24],[@B25]\] to map and cross-reference terms from their Pathology Ontology. Vocabulary tools ---------------- The MP Ontology was built as a DAG using the DAG-Edit software written by John Richter and Suzanna Lewis \[[@B26]\]. The MP Ontology is updated daily and can be browsed or searched online at \[[@B19]\]. MP files also are available in flat file format and OBO format from our ftp site \[[@B27]\] and are posted at the OBO site \[[@B28]\]. Phenotype data annotation ------------------------- Phenotypes are described in the MGD relative to the genotype of the individual. Genotype objects specifically consist of one or more allele pairs describing mutations or QTLs and the genetic background strain(s) where the phenotype was observed. Each phenotype annotation associates a MP Ontology term with a genotype/strain and the reference or data source supporting this assertion. Additional modifying text may be annotated to describe detail that is not easily standardized. Examples include experimental conditions, age of onset and incidence, and trait penetrance, among others. The annotation note may also include specifics of the phenotype where such details are deemed to be too case specific to be a MP term. In addition, genotypes are associated with OMIM where a particular mouse genotype is a model for human diseases and syndromes. Figure [2](#F2){ref-type="fig"} shows the portion of one phenotype record that uses the MP Ontology. Conclusions =========== The MP Ontology and annotation schema was designed to minimize curatorial time, yet remain precise enough to describe phenotypic data. It supports robust phenotypic annotations and querying capabilities for mouse phenotype data. While this vocabulary is far from complete, we have designed strategies for its continued development as a collaborative effort for supporting the representation of existing mutations and those that continue to be created. As of 1 November 2004, over 11,150 phenotypic alleles representing mutations in 5,214 unique genes had been catalogued in MGD. For these alleles, 9,696 genotype records exist, with 21,556 phenotypic annotation instances. The MP Ontology is also used in phenotypic data annotations at the RGD \[[@B29]\]. As our database groups continue to accumulate annotations, it will be possible to mine these data to ask interesting questions about similarities and differences in comparable allele effects between the species, as well as within species. Comparative phenotype data will potentially uncover new modifier effects and point to new pathway relationships and genetic networks tied to disease processes. The MP Ontology will be critical for enabling computational analyses and providing a framework for improved web views and other human-comprehensible displays for the research community. Acknowledgements ================ We thank Martin Ringwald and Susan Bello for helpful comments on the manuscript, and acknowledge members of the MGI and RGD teams, Neuromice consortium and Richard S. Smith for contributions to the MP Ontology development. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Screen shot of the Mammalian Phenotype (MP) Browser in which the term \'lethality-embryonic/perinatal\' was selected. At the top (yellow shading) the term, its synonym and MP unique identifier appear. The number of paths to term (in this case, one) indicates how many paths through the DAG structure can be traversed to reach the term. The main body of the browser page shows the selected term highlighted and within the context of the hierarchical path(s) of the MP Ontology. In this example page, three levels of the hierarchy are visible (Phenotype Ontology, its 34 sub-terms, and the two sub-terms that fall beneath \'lethality-embryonic/perinatal\'. The plus sign, appearing for many of the terms on this page, indicates that these terms have additional sub-terms that can be viewed by clicking on the term to expand the view of that portion of the ontology. The number of genotypes and annotations following the term \'lethality-embryonic/perinatal\' is a hypertext link to those data. This latter feature will be available in early 2005. ::: ![](gb-2004-6-1-r7-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Example showing a portion of the MGI web display of the phenotype annotations for homozygotes for the Ccm1^tm1Dmar^genetically engineered allele (the first targeted mutation from the Douglas Marchuk laboratory in the cerebral cavernous malformations 1 gene). Homozygotes are embryonic lethal, showing developmental and cardiac abnormalities. Note the organization of annotations under the high level phenotype categories and the link to OMIM where the mouse and human show similar phenotypic characteristics. See \[30\] to view the complete record for the Ccm1^tm1Dmar^phenotypic allele. Searches for phenotypes at MGI can be done via the Alleles and Phenotypes Query Form \[31\]. ::: ![](gb-2004-6-1-r7-2) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Text search for mutations with hair/fur loss defects in the Mouse Locus Catalog ::: --------------------------------------------------------------------------------------------------------------------------- Hairless Nude Bald Hair loss Loss of hair Furloss Loss of fur Composite \'true\' result ----------------- -------------- ----------- ----------- -------------- --------- ------------- --------------------------- Al Al Aldoc\* alp alp ao ao Bda Bda Bda Blai2^†^ Bmp6^†^ Btk^†^ Ctsl Ctsl dep dep Dh^†^ Dsg3 Dsg3 Dsg3 Ebp Ebp Eda Eda Eda Edar Edar Edaradd Edaradd exf exf Fgf7^†^ Foxj1\* Foxn1 Foxn1 Foxn1 Foxn1 Frl Frl Frl hl hl hl Hoxb8^†^ Hr Hr Hr Hr Htr2b^†^ Il7^†^ Itgam^†^ Itgav^†^ jb jb jd jd jd Krt2-6g Krt2-6g ma ma Ngef^†^ Nras^†^ Ny Ny Ny Ny Otc Otc Otc Pdcd8 Pdcd8 Rbpsuh^‡^ Rbpsuh-ps1^‡^ Rbpsuh-ps2^‡^ Rbpsuh-rs3^‡^ Scd1 Scd1 Shc1^†^ Slc30a4 Slc30a4 St^§^ tf tf Tgfb1^†^ Tgfbi^†^ Tnfrsf6^†^ wal wal 15 18 10 14 2 2 2 27 true results\ 23 irrelevant --------------------------------------------------------------------------------------------------------------------------- The term heading each column was used to search the Mouse Locus Catalog \[7\]. The gene symbols listed in the column are those returned in response to the search. Because this is a text search, any matching text within the written description of the phenotypes for mutants known for that gene will be counted as a match. The 23 irrelevant results were returned due to: \match based on mapping this gene close to the mutant displaying this phenotype; ^†^experimental result of using mice displaying this phenotype in relation to the gene in question; ^‡^match based on gene name, not phenotype; ^§^match based on another mutation which is stated as arising in a stock carrying this phenotype. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### MP Ontology terms and their decomposed forms ::: MP Ontology term\ Object Body location (anatomy/cells) Attribute Modifier Value ---------------------------------- --------------------- ------------------------------- ------------------ ------------------ ----------- Hydrocephaly^†^ Cerebrospinal fluid Brain cerebral ventricles Amount Relative Excessive Brain Size, mass Increased Trauma Brain Qualitative Observed Dystrophic cardiac calcinosis^‡^ Calcium salts Heart Deposition Observed Inflammation Heart Qualitative Observed Lesions Heart Qualitative Artherosclerotic Observed Lenticonus^§^ Eye lens capsule Shape Conical Bulge Eye cortex Shape Conical Bulge Osteopetrosis^¶^ Trabecular bone Amount Dense Excessive Cartilage Amount Calcified Excessive Erythrocytes Relative\_number Decreased Hematopoiesis Location Extramedullary Ectopic \*Definitions from the MP Ontology. ^†^Hydrocephaly, excessive accumulation of cerebrospinal fluid in the brain, especially the cerebral ventricles, often leading to increased brain size and other brain trauma. ^‡^Dystrophic cardiac calcinosis, a condition characterized by the localized deposition of calcium salts in the heart; often occurring in association with inflammation or atherosclerotic lesions and other pathological states. ^§^Lenticonus, a conical bulging of the lens capsule and the underlying cortex of the eye. ^¶^Osteopetrosis, excessive formation of dense trabecular bone and excessive calcified cartilage formation; may lead to anemia and extramedullary hematopoiesis. :::	PubMed Central	2024-06-05T03:55:52.959037	2004-12-15	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549068/", "journal": "Genome Biol. 2005 Dec 15; 6(1):R7", "authors": [ { "first": "Cynthia L", "last": "Smith" }, { "first": "Carroll-Ann W", "last": "Goldsmith" }, { "first": "Janan T", "last": "Eppig" } ] }
PMC549069	Background ========== Mutant mice are the premier genetic models for human diseases. An increasing number of laboratories and companies worldwide are now carrying out detailed analyses of mouse phenotypes that have been generated from large-scale mutagenesis of the mouse genome. Description of mouse phenotypes has not traditionally adhered to predefined rules or been recorded in databases. However, the sheer volume of data from high-throughput screens (such as N-ethyl-N-nitrosourea (ENU) mutagenesis \[[@B1]\]) is now driving the need to manage information about mutants in a paperless environment and to build databases that will allow this data to be shared between laboratories and used to formulate hypotheses about gene function. The key to satisfying this need is the ability to describe different phenotypes in a consistent and structured way. There is a need for consistency in the way different communities of biologists attempt to present this kind of data since consistent representation of phenotypes across different domains (such as pathology and anatomy) and species is crucial for the semantic interpretation and the efficient use of this complex information in different kinds of study, such as comparison of gene functions between species. Ontologies have been an important tool for structuring biological information since the time of Linnaeus. With the advent of the Gene Ontology (GO) in 2000 \[[@B2]\] these techniques for strictly specifying the semantic relationships between terms have become a standard to support knowledge representation in the field of genomics. Hierarchical ontologies hold information about the structure of a particular domain of knowledge at varying degrees of detail (granularity), thus permitting us to integrate concepts and descriptions at different levels of resolution. This approach is forming the basis of new methods for mining biological data \[[@B3],[@B4]\]. In this article, we describe developments in describing mouse phenotypes using ontologies. Ontologies and knowledge bases ------------------------------ The term ontology is derived from the Greek and is used in philosophy to mean \'a description of what exists\'. There are many definitions of the word, however, and for the purpose of this article, an ontology is \'a specification of entities and their relationships\' \[[@B5]\]. The key word \'specification\' implies a formal organization. Thus, an ontology is a formalism to describe entities and the relationships between them. Ontologies for computing applications are schemas for metadata \[[@B6]\]. They provide a controlled organization of terms and their relationships that has explicitly defined and machine-processable semantics \[[@B7]\]. The controlled semantic portrayal of entities and their relationships allows the description of a domain of knowledge. For our purposes ontologies mainly attempt to replace free-text descriptions of phenotypes with equivalent computable descriptions that can be used to draw inferences about these data. An ontology together with a set of individual instances of the kinds of entities it specifies constitutes a knowledge base \[[@B8]\]. It may be difficult to distinguish between the knowledge contained in an ontology and the knowledge contained in a knowledge base \[[@B9]\]. In phenotype ontologies the distinction between the ontology and the knowledge base must be clear. The ontology should capture the general conceptual structures necessary to describe the domain, whereas the knowledge base should provide the individual instances that are described using the ontology. So, in the ontology one can first define the entity (class) of \'pain perception\' and further, assign to this entity the attribute \'relative sensitivity\' and specify for this attribute a range of allowed values using concepts such as \'sensitive\' or \'insensitive\', and so on, thereby allowing us to describe pain-perception phenotypes. The knowledge base, however, holds data about particular instances \[[@B10]\], for example a particular mouse with a particular genotype, under defined handling conditions and of a certain age, that has a particular level of sensitivity to pain according to a particular assay. In other words, the ontology constitutes a general theory (how to describe phenotypes), whereas the knowledge base describes particular circumstances, in our case particular instances of phenotype. Why use ontologies? ------------------- An important question here is why do we need to use ontologies; why not simply use a series of unconnected, standard terms such as provided by a controlled vocabulary? The advantages of using ontologies have been argued extensively, but the main reason is that ontologies are attempting to capture the precise meaning of terms. Furthermore, ontologies can be used for reasoning and inference (for example, consistency checking or drawing conclusions from the knowledge). The most important factor from our perspective is the need to combine information from different phenotypes or from different protocols (assays). For example, if a mutant mouse has six digits in each forelimb we will wish to use this information in a variety of ways (for example, to group mice with limb pattern defects, or with affected forelimbs, or with abnormal numbers of digits in any limb). For this, we need not just a controlled vocabulary of terms, but also information about how these terms relate to one another (for example, that forelimb is an instance of \'limb\', that the normal number of digits in the forelimb is five, that the number of digits is an instance of \'pattern\', and so on). Current approaches to the description of mouse phenotypes --------------------------------------------------------- Traditionally, the main source of information for most scientists is the peer-reviewed journal literature. Electronic versions of published information have opened the road to accessing and retrieving information in a much easier and more cost-effective manner. The growth and wider availability of the world-wide web has led to a significant growth in the amount of readily available electronically stored information \[[@B11]\]. With this surge of readily available information the location and retrieval of relevant information has become a major (commercial) activity \[[@B12]\]. One of the most important issues in information retrieval is constructing effective indexing methods that are required for the sophisticated querying of the stored data. Free-text searching forms the basis of information retrieval but is extremely limited because of the inherent lack of accuracy and specificity. Complex free-text descriptions, such as are used for phenotypes, are almost impossible to index and retrieve in a useful way directly from the biomedical literature. The potential power of complex searches against information from multiple experiments requires the annotation of free text into structured representations that can be understood and where the power of computational algorithms can maximize the potential of the information to be compared and contrasted. The most comprehensive attempt to annotate mammalian phenotypic data so far, the Mammalian Phenotype Ontology (MP) \[[@B13]\], is currently under development by the Jackson Laboratory \[[@B14],[@B15]\]. The current structure of the ontology is generated using DAG-Edit \[[@B16]\], the current GO standard, and allows a hierarchical display of terms and their definitions. These terms include a combination of entities and values, for example, id MP:0001509 corresponds to \'abnormal body position\', which at a high level provides a sufficient description of phenotypic data. This approach allows high-level access to the knowledge held in the ontology, but also has certain limitations similar to the GO paradigm. If one attempts to create too much specificity within an ontology of this type it can expand to unmanageable proportions and parentage relationships can be overlooked as their number grows. For example, merely creating new terms by prepending the two qualifiers, \'increased\' and \'decreased\', everywhere that is applicable, will massively increase the size of the ontology. To allow a systematic approach to the model, combinations would also have to be instantiated that might never be used. Because there is a practical limit to the number of values that can be managed, such an approach is limited. Inevitably, decisions have to be made as to which individual combination describes a particular phenotypic entity best. We note here though that the development of MP is being developed pragmatically, with instances being added as needed to annotate mouse phenotypes, following the paradigm used by GO developers. MP is a cross-product ontology that includes mouse anatomy ontology, GO and other controlled terms as part of the construction of MP terms. Although the cross-reference IDs are not visible, they are part of the design of MP. Some of the work described here reflects insights gained during extensive discussions about the representation of phenotypes at the Phenotype Consortium meeting held in Bar Harbor, ME in September 2003. The developers of the MP ontology are part of this consortium and have intentionally created their ontology in such a way that it can be easily extended to form instances of the compositional approach discussed in the next section. With the objective of capturing information about phenotypes in any organism, Ashburner proposed the Phenotype And Trait Ontology (PATO) \[[@B17]\] in 2002. PATO is a schema according to which, \"phenotypic data can be represented as qualifications of descriptive nouns or nounal phrases\" (M. Ashburner, unpublished work). Each noun represents an observable characteristic and for each noun there will be a set of attributes, for each of which is defined a set of appropriate values. In addition to these three semantic classes (namely observable entities together with the associated attributes and values), the concepts that are needed to describe phenotypes include the assays by means of which the phenotypes were determined and the environmental and genetic conditions (Microarray Gene Expression Data Society \[[@B18]\]) under which these assays were performed. Taken together, the semantic concepts and relationships defined for PATO, assays, genetic and environmental conditions, will form the basis for the systematic description of phenotypes. Results ======= A proposal for describing mouse phenotypes ------------------------------------------ The description of mutant phenotypes must provide a practical way to capture the biologically relevant information about the phenotype in machine-readable form \[[@B19]\]. It should allow us to compare, combine and analyze different phenotypes. For this, the ontology must first be consistent, and second be able to generate statements that have a logically well-formed structure in order to support reasoning from descriptions of different phenotypes. To provide these functionalities we propose a compositional method of describing phenotypes \[[@B19]\]. By this we mean that the description of the phenotype combines terms from different standard ontologies, each of which supports a particular domain of knowledge. A list of ontologies that should be included in such a phenotype ontology is given in Table [1](#T1){ref-type="table"}. These ontologies are combined in a specified formula or schema that provides the logical structure of the whole. The schema itself can be considered as a meta-ontology that describes how other ontologies relate to one another. Figure [1](#F1){ref-type="fig"} illustrates such a schema. According to the schema in Figure [1](#F1){ref-type="fig"}, the whole organism has certain attributes, such as genotype, identity number, and exists under certain handling conditions (Table [2](#T2){ref-type="table"}). The organism also has a set of core components including its anatomy, development, physiology and behavior. Each of these core components is represented by a separate ontology and each has a set of attributes, again represented by an ontology. For example, the organism may have an anatomical component \'left eye\' which is a term from the anatomy ontology. The left eye, in turn, may have attributes of \'color\', \'size\', and so on, taken from the attributes ontology. This combination of core entity and attribute constitutes a phenotypic character - something that can be measured. Phenotypic characters, in turn, link to \'assays\', which return a variety of \'values\', again represented by an ontology, which may be applied to the phenotypic character in question. When this schema is used to describe actual phenotypes, instances of single phenotypic characters are linked together to provide a full phenotypic description of an individual organism. Each character can be represented by a line in a table where the table represents the full phenotype. Figure [2](#F2){ref-type="fig"} presents this schematically. According to the schema in Figure [1](#F1){ref-type="fig"}, five classes of ontology (in circles), namely organism, entity, attribute, assay and value, are required to express a phenotypic instance. ### Organism This class holds the information (organism attributes) of an organism in which the phenotypic characters are observed (see Table [2](#T2){ref-type="table"}). ### Entity Entities will be formed by importing ontologies discussed in Table [1](#T1){ref-type="table"}: behavior, anatomy, and so on. Each entity may be associated with a set of attributes, for example, color and size, that may also be shared with other entities. ### Attribute Attributes will be provided by PATO \[[@B17]\]. PATO should hold general attributes that can be applied through different phenotypic ontologies. This has the advantage of economy and also enables cross-referencing between domains. New attributes should be assigned to classes only when they cannot be modeled with existing options. ### Assay Assays will have a hierarchical structure and will define a range of values that correspond to a particular combination of entity and attribute (that is, phenotypic character). They hold multiple relations to values, qualifiers and free text as well as their own metadata. The slot for free text is included to capture knowledge that cannot be expressed through the ontology as yet. ### Values Splitting PATO into two different ontologies, PATO attributes (above) and PATO values, allows the PATO ontology to be incorporated into the schema \[[@B19]\]. Values can thus be either specific values provided by the assay or common values, provided by PATO. A possible relationship between these sets of values would be \'interpretation\_of\'. Although values provided directly by the assay are usually the objective recordings of a test for a specific phenotypic character, there can be an interpretation of these recordings in terms of a higher level phenotypic character. For example, in an assay of memory in the mouse that uses a water test, the values returned by the test may be that a mouse completed the task in a certain time and manner, but these results may be interpreted to indicate a value corresponding to the phenotypic character comprised by the entity \'memory\' that was assayed for the attribute of \'short-term recall\' and returned the interpretative value \'loss of memory\'. By introducing the \'interpretation\_of\' relationship, we could make this distinction in a machine-understandable manner and allow the possibility, if required, of expressing the original objective values of the test, thus avoiding information loss. This aspect of the schema remains under study. A central idea in this schema is that of the \'phenotypic character\', which we can define as any feature of the organism that is observed or \'assayed\'. An example for the mouse is tail length. A phenotypic character is a compound composed of an entity, in this case an anatomical entity \'tail\', and an attribute of tail, here \'length\'. Similarly the physiological entity \'hearing\' (GO:0007605) has the attributes \'sensitivity\', \'range\', and so on. Thus, \'hearing range\' and \'hearing sensitivity\' are distinct phenotypic characters. The ideal phenotypic character is one that can be measured independently of others. In practice, however, phenotypic characters are rarely independent. Furthermore, the observations from any particular assay will most probably depend on several different phenotypic characters. For example, the results returned by the click-box test for hearing sensitivity in the mouse actually depend, not only on hearing, but also on the mouse\'s ability to make a detectable locomotor response (the Preyer reflex \[[@B20]\]). These multiple dependencies are captured in the schema, enabling the ontology to support the appropriate possible groupings of phenotypes. This will allow us, for example, to group all mutants that have (by direct assay), or may have (for example, those failing the click-box test), an effect on the locomotor system. Conversely, different assays may provide information about a single phenotypic character. For example, an acoustic brain-stem response (ABR, a sound-evoked potential within the acoustic nerve) \[[@B21]\] can be measured to assay basic hearing ability as well as to give a threshold-response curve for differing frequencies. Linking assays with characters in this way will support machine reasoning, enabling us, for example, to make the hypothesis that a particular mouse has a locomotor rather than hearing defect. Indeed, the need to capture this network of relationships between assays and phenotype is a strong indication of the need for an ontology rather than merely a controlled vocabulary of unrelated terms. The expressivity of representation languages such as DAML+OIL \[[@B22]\], OWL \[[@B23]\] and OBO \[[@B17]\] could also dynamically account for the possibility of a cross product or dependence required for representing a phenotype. For example, if a cross product between ontologies does not exist (that is, one of the required terms is not to be found in an ontology), one can assign an \'anonymous class\' that is dynamically defined as being both a class in one case and an instance in another. As an example, one might want to refer to the term cocaine dependence, but that cross product may not exist. An \'anonymous class\' can be dynamically defined as being both \'cocaine\' (coming from a chemical ontology) and \'dependence\' (coming from the behavior ontology) to generate this cross product. Finally, we note here that it should be possible to link current high-level structures (such as the current MP ontology), which are necessary in many cases for annotation purposes, to the more expressive form we propose here, so that it can also be explored computationally. Example ------- In this section we describe an example of the application of the compositional schema. We chose a phenotype example at random from the MP database: \'nest building\' \[MP:0001447\]. Several descriptions of nest-building patterns can be found in the corresponding reference \[[@B24]\]. For example, the authors comment: \"Note the fluffy well formed nests built in the +/+ cages and the huddling of mice in these nests, in contrast to the poorly formed nests in -/- cages with random sleeping patterns.\" and later: \"In addition, +/+ mice built nests from nestlet material that averaged 50 mm in depth, while -/- mice built significantly shallower nests (Figure 4D), with depths that averaged less than 20 mm \[t(10) = 3.754, p\< 0.004\].\" The authors also describe the assays used to record these observations: \"Nesting Patterns: six cages of wild-type and six cages of mutant mice (N= 4 mice per cage) were used to evaluate nesting patterns. A 5 × 5 cm piece of cotton nesting material (Ancare, Bellmore, NY) was placed in each cage. After 45 min, photographs were taken of each nest and the nest depth was measured. Nest height data were analyzed using the Student\'s ttest.\" For some users/applications the compound term \'abnormal nest building\' might be a sufficient description of this particular phenotypic instance, but this would result in information loss. A human would have to retrieve and read the reference to extract further information. Our schema allows the expression of this information in a machine and human readable manner. In Table [3](#T3){ref-type="table"} we provide the relevant part of our ontology modeled according to the schema. One can easily express these phenotypic instances. In order to describe fluffy, well formed nests or poorly formed nests one would use the following combination: Nest building{has\_attribute} attribute:quality{characterized\_by} defined\_quality\_assay(described in Nesting Patterns \[[@B24]\]) {returns\_value} well-formed Nest building{has\_attribute} attribute:quality{characterized\_by} defined\_quality\_assay(described in Nesting Patterns \[[@B24]\]) {returns\_value} poorly-formed Nest building{has\_attribute} attribute:quality{characterized\_by} defined\_quality\_assay(described in Nesting Patterns \[[@B24]\]) {returns\_value} fluffy We note here that had the value \'fluffy\' not been included in the standard values for a quality assay, it could be captured in the free-text field provided by the schema. To express a nest of 50 mm depth or significantly shallower: Nest building{has\_attribute} attribute:absolute\_depth{characterized\_by} undefined\_ absolute\_depth\_assay{returns\_value} 50 mm Nest building{has\_attribute} attribute: relative\_depth{characterized\_by} undefined\_ relative\_depth\_assay{returns\_value} shallow{has\_qualifier} significant With this information one could go back to a higher level and still be able to express a more general characterization of this phenotype as \'abnormal nest building\' but obviously the opposite is not possible. An important unresolved issue concerning the use of ontologies to describe phenotypes arises from the fact that all the ontological structures developed so far are designed to describe individual mice. Mutagenesis experiments usually characterize a number of mutant mice to take into account variable penetrance of the mutation and other stochastic effects. A strategy will therefore need to be developed to describe the generalized phenotypic properties of a cohort of mice. This may involve the use of more sophisticated relations such as {usually characterized by} or even quantitative relations such as {80% characterized by}. Discussion ========== Importance of the assay ----------------------- The assay plays a central role in our schema (Figure [1](#F1){ref-type="fig"}). Assays are the means of making observations and as they determine what can be observed they are a necessary complement to the attribute ontology. Generally, they are recorded as protocols or even as standard operating procedures (SOPs). However, even a visual observation is a form of assay and this needs to be reported when one expresses a phenotypic instance, for example: eye{has\_attribute} attribute:color{characterized\_by} visual inspection{returned\_value} pink On a practical level, assays can add specificity and functionality to the relationship between entities, their attributes and the corresponding values. Most important, an assay vocabulary allows the entire schema to be dynamic by including new assays and capturing explicit differences between assays in different laboratories. The assay will also allow standardization and definition of values for a given phenotypic character, for example, how abnormal is defined in relation to body position. Implementation -------------- Our schema can be expressed using a variety of modeling tools and knowledge representation (KR) languages \[[@B25]\]. We chose DAG-Edit \[[@B16]\] (version 1.408) and Protégé-2000 \[[@B26]\] (version 1.9) which is Java-based, well supported and incorporates multiple inheritance, relation hierarchies, meta-classes, constraint axioms and F-Logic \[[@B27]\]. Although the complexity of our current models can be described with existing tools, in the future more complex phenotype domains may require migration to a finer-grained conceptualization. Populating the Mouse Phenotype Ontology --------------------------------------- The schema was designed to be easily populated using extant core ontologies, such as anatomy, and defining attributes related to each entity. The assay vocabulary can be constructed as required. Permitted values are defined in the range of different assay attributes in part devised in the form of a general scheme and in part built from the output of particular assays. Although we include for demonstration purposes three core ontologies, namely behavior, anatomy, and developmental anatomy (Figures [3](#F3){ref-type="fig"} and [4](#F4){ref-type="fig"}), we have tested the schema only on behavior. We also include a possible structure for PATO attributes and a separate ontology for common values. We note, however, that the structure of PATO has not been finalized. Figure [3](#F3){ref-type="fig"} shows the implementation of the schema in DAG-edit. Figure [4](#F4){ref-type="fig"} shows a typical implementation of the Schema in Protégé 2000. Options for providing a definition, definition reference, documentation, associated annotations, synonyms, and so on, are offered in our schema. Similar options can be used for attributes using the metaslot options. Since most of the ontologies we are planning to use were generated using the DAG-edit \[[@B16]\] format, we had to convert them to the Protégé-2000 format using the tools and methodology described by Yeh et al. \[[@B27]\], with minor modifications. This task, however, should no longer be necessary as the latest version of DAG-edit allows the export of ontologies in OWL format. Modeling issues --------------- Decisions will inevitably have to be made to combine a core ontology with its attributes and then define facets of that relationship, for example, cardinality, attribute value type and attribute range. In our schema, the class hierarchy of all ontologies employed represents an \'is-a\' relation. So, mouse social behavior \'is-a\' mouse behavior, or mouse social behavior is a \'kind-of\' mouse behavior and so forth. All other relationships, including PATO and \'part-of\' relationships, are modeled as attributes. However, we note here that efforts are currently being made by the GO consortium to define and formalize the \'part-of\' relationship, which is considered vital and special in bio-ontologies, especially anatomy \[[@B28]\]. Because our phenotype ontology and PATO need to be the result of a collaborative effort within the communities, we feel that it is important to set out the basic modeling concepts that need to be applied upon allocating attributes to the core ontologies. Deciding whether to introduce a new attribute or represent this functionality through an entity is often quite difficult. Several things need to be considered in order to make the best decision, although it should be noted that there are no clear distinction as to what is a right or wrong decision. The first thing to take into account is that subclasses of a class inherit all properties of the parent and could have additional properties and different restrictions from the latter. PATO should remain as general as possible, and, when possible, care should be taken to avoid making PATO domain specific. For example, in the behavior ontology there is a class named \'reflexes\' that contains children such as \'blinking reflex\', \'Preyer reflex\' and \'righting reflex\'. It might be worth considering having one \'attribute of reflex\' available in PATO rather than creating a separate attribute \'of\' for each individual reflex, such as \'attribute of blinking reflex\', \'attribute of Preyer reflex\', and so on. Then again, if one wishes to assign different functionalities to these properties, creating separate attributes might be useful. As a rule though, one should consider that PATO needs to be consistent, usable and interoperable if it is to be applied to the general domain of phenotypes. Repetition between core ontologies and PATO should be avoided where possible. What is also often not clear is whether one should add a new class to represent functionality or assign attributes to already existent classes. For example, think of the entity \'body position\'. There are several ways to model this entity in the mouse behavior phenotype ontology. One could declare \'body position\' as a child of a class called \'posture\'. An \'attribute of body position\' could then be assigned to this class with a range of values that might be specific to an assay, for example SHIRPA \[[@B29]\] allows the value \'lying on its left side\' among other values to an assay for body position. Alternatively, a more general \'attribute of position\' could be assigned to this class. The choice depends on the functionality of the ontology and the range of phenotypes we wish to express. If the entity requires more specific attribute values to represent specific functionalities important to the domain of knowledge, we assign more specific attributes. If this functionality is not important for the domain, we assign specific attribute values \[[@B8]\]. \'Body position\' could also be split into an entity of \'body\' and an attribute of \'position\'. Again, a new class \'body position\' should be assigned, if one considers the objects with different attributes as different kind of object and this distinction important in the domain. As a general rule, before assigning new classes and attributes one should consider the functionality and their role in the domain, creating more distinctions as the depth of knowledge that is required to be expressed in the ontology increases. Classes in the hierarchy should not necessarily have to introduce new properties \[[@B8]\]. Although, in many cases these entities could be represented as attributes, it is not necessary for the functionality of the domain. If the expert thinks that this distinction is significant for the class hierarchy and the logical representation of his knowledge of the domain, then these entities should be represented as classes \[[@B8]\]. An important additional consideration is whether creating new terms in an ontology results in terms that cannot be consistently distinguished experimentally (\'resolution\'). Conclusions =========== We have presented here an approach to the use of ontologies in describing mouse phenotypes that could provide a platform for the consistent representation of mouse phenotypic data. We have also described in detail a possible methodology to construct applications of this schema across different domains. We have dealt with modeling issues and provide guidelines to deal with semantic and practical problems. We maintain that such modeling efforts in any domain should be done in a collaborative fashion in the community. Repetition between different parts of the mouse phenotype ontologies is unavoidable. However, the use of consistent IDs, synonyms and records for associated annotations could allow seamless integration of ontology products. The nature of the schema proposed, as well as its components, is extremely dynamic; therefore coordination of efforts is vital. The structure allows extensibility and interoperability. Although an ontology should not cover all possible information about a domain, the main idea behind this concept is to allow the phenotype ontology to cope with novel and unpredictable phenotypes and account for new assays, serving scientific autonomy and information validity and integrity. We have built a software system \[[@B30]\] which includes a browser that allows searching and viewing the knowledge captured though the complex relations described here and databases that allow the dynamic update of different parts of the core ontologies, including PATO, without the loss of applied facets. Acknowledgements ================ This project is funded by the European Commission under contract number QLG2-CT-2002-00930. We thank Michael Ashburner, Suzie Lewis, Judith Blake, Pat Nolan and the Phenotype Consortium for helpful discussions. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Proposed schema for constructing phenotype ontologies (modified from \[13\]). ::: ![](gb-2004-6-1-r8-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Schematic of phenotype description as the sum of the results of assaying different characters. PC, phenotypic character. ::: ![](gb-2004-6-1-r8-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Two snapshots of the ontology visualized using DAG-edit. ::: ![](gb-2004-6-1-r8-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### A snapshot of the ontology using Protégé-2000. ::: ![](gb-2004-6-1-r8-4) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Ontologies to be incorporated in a combinatorial phenotype ontology ::: Ontology Description URL ----------------------- ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- -------- Adult anatomy The Anatomical Dictionary for the Adult Mouse \[17\] has been developed by Terry Hayamizu, Mary Mangan, John Corradi and Martin Ringwald, as part of the Gene Expression Database (GXD) \[31\] Project, Mouse Genome Informatics (MGI), The Jackson, Laboratory, Bar Harbor, ME \[14\] \[17\] Developmental anatomy The Anatomical Dictionary for Mouse Development has been developed at the Department of Anatomy, University of Edinburgh, Scotland (Jonathan Bard) and the MRC Human Genetics Unit, Edinburgh (Duncan Davidson and Richard Baldock) as part of the Edinburgh Mouse Atlas project (EMAP), in collaboration with the Gene Expression (GXD) project at MGI, The Jackson Laboratory, Bar Harbor, ME \[31,32\] \[17\] Behavior Parts of behavior have been expressed in a consistent manner \[13,17\] \[13\] Pathology The Pathbase mouse pathology (Paul Schofield) ontology provides a description of mutant and transgenic mouse pathology phenotypes and incorporates 425 known mouse pathologies hierarchically organized as \'instances of\' pathological processes \[33\] \[17\] Gene Ontology GO describes the roles of gene products and allows genomes to be annotated with a consistent terminology (The Gene Ontology consortium 2002) \[2\] \[17\] Others ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Organism attributes ::: ---- ----------------------------------------------------------------------------------------------- id Identifier for individual (n) T Species (for example, NCBI taxonomy browser \[34\]) G Genotype I: Strain (for example, StrainID from MGI \[14\]) S: Genotypic sex A: Alleles at named loci (for example, MGI \[14\]) E Handling conditions (see EUMORPHIA \[35\]) D Age/stage of development (Theiler \[36\] and other staging criteria, for example EMAP \[37\]) ---- ----------------------------------------------------------------------------------------------- ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Nesting behavior ::: Entity Attribute Assay Value ------------------- --------------------------------------------------- --------------------------------------- ------------------------------------- Social behavior 1\. Attribute:qualitative Undefined\_qualitative\_assay 1\. Abnormal Huddling behavior 1\. Inherited attribute of class Social Behavior 1. 2\. Attribute:huddling\_frequency Undefined\_huddling\_frequency\_assay 2. 2a. Attribute:relative\_huddling\_frequency 2a 2b. Attribute:absolute\_huddling\_frequency 2b Nesting behavior 1\. Inherited attribute of class Social behavior 1. Nest building 1\. Inherited attribute of class Nesting behavior 1\. Attribute:duration Undefined\_duration\_assay 1. 1a. Attribute:relative\_duration 1a. Slow, fast 1b. Attribute:absolute\_duration 1b. 45 min 2\. Attribute:height Undefined\_height\_assay 2. 2a. Attribute:relative\_height 2a. short\_height, tall 2b. Attribute:absolute\_height 2b. 20 mm 3\. Attribute:weight Undefined\_weight\_assay 3. 3a. Attribute:relative\_weight 3a. heavy. light 3b. Attribute:absolute\_weight 3b. 10 g 4\. Attribute:quality Undefined\_quality\_assay 4\. Good, well-formed, poor, fluffy 4a. Attribute:shattering 4a. 4b. Attribute:threshability 4b. 5\. Attribute:depth 5. 5a. Attribute:relative\_depth Undefined\_relative\_depth\_assay 5a. Shallow 5b. Attribute:absolute\_depth Undefined\_absolute\_depth\_assay 5b. 50 mm :::	PubMed Central	2024-06-05T03:55:52.962209	2004-12-20	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549069/", "journal": "Genome Biol. 2005 Dec 20; 6(1):R8", "authors": [ { "first": "Georgios V", "last": "Gkoutos" }, { "first": "Eain CJ", "last": "Green" }, { "first": "Ann-Marie", "last": "Mallon" }, { "first": "John M", "last": "Hancock" }, { "first": "Duncan", "last": "Davidson" } ] }
PMC549070	Background ========== The recent definition of the complete nucleotide sequence of the human genome \[[@B1],[@B2]\] has motivated the full annotation of the sequence. The true promise of the human genome project, to become the foundation for medical and biological research benefiting human health and quality of life \[[@B3]\], can only be realized if the coding sequences are conclusively identified, intron/exon structures are accurately described and the potential protein products from each gene in different tissues and cellular states are determined. Current methods for gene-prediction provide useful information but are still limited \[[@B4]\]. It is not presently possible to predict all features of the genome from its sequence alone. Therefore, the value of the human genome sequence can be enhanced through the collection of different types of experimental data and its integration and validation in a genomic context \[[@B5]\]. Current use of expressed sequence tags (EST) and full coding DNA (cDNA) sequences is extremely helpful in achieving complete genome annotation \[[@B6]-[@B9]\]. However, these data are not sufficient to unequivocally predict which proteins (and with what covalent structure) are expressed in a given tissue. The complete characterization of all proteins across disease states, tissues and stages of development can now be addressed through experimental protein identifications generated by proteomic methods. Experiments carried out over the past years have illustrated that peptides resulting from proteolytic digests of complex protein mixtures can be identified in a high-throughput mode using a combination of liquid chromatography (LC) and tandem mass spectrometry (MS/MS) (LC-MS/MS) \[[@B10]-[@B15]\]. Peptides are thus useful as the currency of MS/MS-based protein identification \[[@B16]\]. By combining a large number of experiments sampling different cell and tissue types, the observed peptides can be mapped onto the genome covering a significant part of its chromosomes. Results and discussion ====================== To begin annotating the human genome with protein-level information, we have built PeptideAtlas. The generally applicable procedure to annotate eukaryotic genomes with peptide sequences can be applied when datasets are acquired using different experimental protocols. In each case, sample proteins were first proteolytically cleaved into peptides using the enzyme trypsin. The resulting peptide mixture was then subjected to chromatographic separation by strong cation exchange and reverse-phase capillary chromatography. In addition, those experiments using the ICAT (isotope-coded affinity tag) reagent for quantification included an avidin affinity-purification step to select peptides containing biotinylated, stable-isotope-tagged cysteines \[[@B16]\]. The resulting peptide pools were then analyzed by electrospray ionization (ESI)-MS/MS. The database search program SEQUEST \[[@B17]\] was used to assign the resulting MS/MS spectra to a peptide sequence. The confidence of these peptide assignments was evaluated using PeptideProphet \[[@B18]\]. All of the experimental data products, including PeptideProphet probability scores, are loaded into SBEAMS - Proteomics, a proteomics analysis database built as a module under the Systems Biology Experiment Analysis Management System (SBEAMS) framework. All of the identifications above a certain probability threshold within a specific set of experiments are extracted from the main database tables into another set of tables containing the attributes of each distinct peptide. Resulting from this, 26,840 distinct peptide sequences were identified from 224,973 spectra with identifications of a high probability (P≥ 0.9) of being correct. Each peptide is given a unique and stable identifier with an eight-digit number in the form PAp00000001. We then attempted to map the 26,840 distinct peptides to the human genome sequence using the analysis pipeline shown schematically in Figure [1](#F1){ref-type="fig"}. An example of visualizing the result in a genome browser is shown in Figure [2](#F2){ref-type="fig"} (see Materials and methods for details). The mapping results are summarized in Table [1](#T1){ref-type="table"}. The result of this process is stored as a freely available public resource, the human PeptideAtlas database \[[@B19]\]. The current build of PeptideAtlas contains peptide sequences identified in 52 proteomic experiments in which proteins were extracted from a particular cell or tissue type, digested with trypsin and analyzed with a mass spectrometer. The 52 proteomic experiments comprised 14 published as well as 38 unpublished human datasets from various cell types such as T cells, B cells, lymphocytes, lymphoblasts, hepatocytes, intestinal cells, hepatoma cells and others. The 14 published datasets contain 47% of the distinct peptides in PeptideAtlas. A full listing of all the experiments and samples currently in PeptideAtlas can be found at the project website \[[@B19]\]. The raw data for all published datasets is also provided in a repository there. The cumulative number of distinct peptides as a function of the addition of identified spectra (with P≥ 0.9) in the atlas is shown in Figure [3](#F3){ref-type="fig"}. As most of the observable peptides in the proteome are matched with genes, the curve is expected to saturate and adding additional data will yield few new matched genes. However, the current behavior is still completely linear, with approximately 1 in 10 identified spectra contributing a previously uncataloged peptide. Each data point represents an added experiment; the experiments are presented approximately in chronological order of data collection. Among the 52 experiments, there is clearly great variability in the total number of identified spectra contributed as well as new distinct peptides contributed. A repeated, complex-sample experiment might yield many new spectra but few new distinct peptides, while a new sample of a type not previously analyzed might yield relatively few spectra but most of these might contribute a new distinct peptide. Applying our pipeline described in Materials and methods, 25,754 of the 26,840 distinct peptides in PeptideAtlas were mapped to 9,747 (28.6 %) of the 34,091 human Ensembl proteins (version 22.34d.1, 2004-06-02). These proteins represent unique proteins or splice forms from 6,423 genes (27%) of human genes in Ensembl. Some peptides have indistinguishable, perfect protein sequence matches to multiple proteins. These proteins are typically paralogs (protein families), protein isoforms or repeated protein domains in the human genome. We identified 3,718 proteins unambiguously by one or more \'discrete peptides\' - peptides that map uniquely to a single protein - in the current build of PeptideAtlas. Those peptides are marked in the genome browser as \'discrete peptide\'. \'Degenerate peptides\' that map to several protein isoforms are also used to identify proteins. It would thus be more accurate to state that a product of a certain gene, rather than a certain protein, has been identified \[[@B20]-[@B22]\]. Moreover, the experimental data from those degenerate peptides generally do not allow differentiation between the sequence alternatives that exist in Ensembl. In fact, not all splicing variants that are in Swiss-Prot are also present in Ensembl and, therefore, it is impossible to ascertain the number of unambiguous identifications at the moment. This limitation underscores the requirement for mapping large-scale proteomic data to the human genome, such as presented in this report to aid in the generation of unambiguous sequence databases. A significant number of distinct peptides (1,086), assigned by SEQUEST/ProteinProphet from over 5,000 MS/MS spectra, could not be mapped to Ensembl database version 22.34d.1. These peptides were identified by SEQUEST searches against the IPI database \[[@B23]\] or ABCC non-redundant protein database (NCI) \[[@B24]\]. These peptides are of special interest as they often document interesting biological phenomena such as single-nucleotide polymorphisms (SNPs) and novel splice variants, demonstrating the need for annotating the human genome sequence with high-quality experimental data obtained from expressed proteins. The existence of these sequences also illustrates the flux in the genome annotation and sequence databases. For example, in Ensembl version 18.34.1, only 92% of genes from the previous build were transferred across to the new build. The missing 8% were predominantly inappropriate protein-coding genes coming from large-scale cDNA projects, which have a number of artifactual errors, or from chimeric cDNA clones from cancer cell lines. Experimentally observed, unmapped peptides are an ideal source of information for refining genome assembly and gene prediction. The absence of Ensembl matches does raise the question of whether these peptides are false positives or whether real proteins are missing in the Ensembl database. When these peptides were investigated in more detail it was found that nearly 100 were identified 10 or more times in several different experiments, and that many had protein sequence matches for Swiss-Prot entries. They are therefore likely to be true peptide attributions. For example, peptide PAp00000363 (AGKPVICATQMLESMIK) was identified 626 times at different charge states and with different mass modifications in 22 distinct experiments and mapped to KPY1\_HUMAN, a pyruvate kinase M1 isozyme. Interestingly, the protein appears to have a likely SNP, which mutates the valine present in the Ensembl genome sequence to the isoleucine observed in PAp00000363. The 9,747 mapped proteins represent 28.6% of the predicted human proteome in Ensembl version 22.34d.1. The distribution of peptide matches to these proteins (Figure [4](#F4){ref-type="fig"}) revealed coverage of all chromosomes. Void areas were observed in the centromere region of chromosome 1 and the telomere regions of chromosomes 13, 14 and 15. These missing regions represent the unsequenced parts of human chromosomal heterochromatin structures and are therefore expected to be devoid of peptide matches. Very few peptides were observed mapped to chromosome Y. The development of PeptideAtlas and a method for mapping observed peptides to the genome allows us to determine the distribution of multiple peptide hits to specific proteins and the distribution of peptide sequences that are present in multiple proteins. Also, in some cases splice junctions and gene boundaries could be confirmed. Our method allows us also to identify peptides corresponding to abundant proteins such as actin, elongation factor and glyceraldehyde-3-phosphate dehydrogenase, which are commonly identified in high-throughput LC-MS/MS experiments. These proteins are products of housekeeping genes, which are expressed most of the time in almost every tissue \[[@B25]\], or are structural proteins which are also known to be abundant in cells. The identification of proteins that are specific to a given cell, tissue or disease state allows for the selection of marker proteins. The knowledge of a single marker, or a set of marker proteins, is crucial for the development of new strategies for rapid protein analysis and quantitative proteome profiling \[[@B16],[@B26]\]. In PeptideAtlas we identify proteins to which two or more peptides map. In fact, for some proteins, 100 or more peptide matches were determined. These proteins were often unusually large in size and contained many exons. Examples of such proteins include the 1,462-amino-acid alpha-2-macroglobulin precursor (ENSP00000323929), which was matched by 161 peptides, or the 4,126-amino-acid DNA-dependent protein kinase catalytic subunit (ENSP00000313420) matched by 90 peptides (Figure [5](#F5){ref-type="fig"}), the 2,472-amino-acid spectrin alpha-chain protein (ENSP00000238302) with 102 peptides, and cytoplasmic 2 actin (ENSP00000331514) with 127 peptides. We also identified peptides whose amino-acid sequence is shared by members of protein families or shared domains among proteins in the genome. Peptides were matched to all identical sequences in all proteins. Multiple hits were possible and the resulting peptides were called degenerate peptides \[[@B22]\], in contrast to discrete peptides that matched one protein uniquely. For example, peptide PAp00001228 (CNGVLEGIR) matched to 26 proteins in the myosin family and peptide PAp00025728 (HCQLAIR) mapped to 23 proteins. Furthermore, our method was able to confirm intron/exon boundaries by identifying peptides that spanned these regions in a gene. We identified 4,800 intron/exon boundary-spanning peptides, corresponding to 2% of the splice junctions in the human Ensembl database, experimentally confirming specific intron/exon junctions. In most cases, these boundaries were already known to exist from cDNA information. However, using peptide information we were able to specifically confirm those boundaries on the level of expressed proteins. In one case (Figure [6](#F6){ref-type="fig"}) we observed a peptide confirming a skipped exon. This event was previously proposed to occur during expression of the A-type lamins in the lung adenocarcinoma cell line GLC-A1 \[[@B27]\]. The presence of some lamin A10 isoforms can easily be overlooked owing to their relatively low abundance. This new peptide information confirms the existence of this splice variant and shows that low-abundance proteins can be detected through the proteomics technologies described in this paper. The need for public proteomics data repositories is recognized \[[@B28]\] and we intend PeptideAtlas to become a growing database and public resource. We have structured the system in a way that allows scientists to submit their own MS data for incorporation into PeptideAtlas, thus increasing the number of experiments and identified peptides. Naturally, to be useful for the project, inclusion of third-party data is dependent upon data compatibility and consistent data quality. Consequently, only data with accurate statistical measures of confidence computed by, for example, PeptideProphet, or another published and tested statistical algorithm, will be included. Datasets for which such statistical analyses have been performed can be submitted for incorporation following the procedure detailed at the PeptideAtlas website. Alternatively, data contributors can submit raw MS/MS data directly. This information should preferably be formatted into mzXML \[[@B29]\] or mzData (HUPO Proteomics Standards Initiative) which are open file formats for the representation of MS data. Other traditionally used data formats are accepted as well. This data will then be searched by the PeptideAtlas curators using SEQUEST to correlate MS/MS spectra of peptides with amino-acid sequences using protein databases such as IPI, and the results will be further analyzed with PeptideProphet. An effort to add support for additional search engines is underway. This procedure will ensure the highest degree of consistency for the data in PeptideAtlas. In the future, the pipeline in general and the data submission process in particular, can be further improved and make compliant with the community accepted statistical data-validation standards and data file formats when such standards emerge \[[@B30]\]. Please see the submission section on the PeptideAtlas web-site for the most up-to-date submission methods and curator contact information. With an increasing number of included peptides, the utility of the resource will improve, as increasing numbers of genes, exons, transcripts and variant transcripts in many tissues and developmental stages will be verified on the protein level. All MS/MS spectra are stored in the SBEAMS - Proteomics database, from which PeptideAtlas is derived. While at present it is not possible easily to access the MS/MS spectra starting from the public PeptideAtlas interface, this possibility could be added in the future. All spectra for published experiments are available in the mzXML files in the repository. Access to raw spectra can be beneficial for many applications not related to the main purpose of PeptideAtlas. Furthermore, because peptide modifications (for example, phosphorylation) are stored, this information could be displayed as well. It is well understood and discussed in the literature \[[@B21]\] that all large-scale datasets obtained using high-throughput methods inherently contain a certain fraction of false-positive data. Thus, estimation of false-positive error rates is a very important but often challenging task. One significant advantage of the high-throughput pipeline implemented in this work is that computed peptide probabilities (here produced by PeptideProphet) allow estimation of the upper bound (most conservative estimate) of the false-positive identification error rates for any dataset submitted to PeptideAtlas. As the main purpose of PeptideAtlas is to map peptide identifications to the genome, the most relevant estimate of the false-positive error rates is the one at the level of distinct peptide assignments that have a defined mapping to Ensembl. Initial datasets of peptide assignments to MS/MS spectra, obtained by searching acquired MS/MS spectra using the database search program SEQUEST, were statistically validated using the computational tool PeptideProphet. For each peptide assignment to an MS/MS spectrum, PeptideProphet computes a probability of its being correct, based on its database search scores, difference between the measured and theoretical peptide mass, the number of termini consistent with the type of enzymatic cleavage used, the number of missed cleavage sites and other factors. Probabilities computed by PeptideProphet have been shown to be accurate in the entire probability range and, therefore, can be used to compute the false-positive identification error rate (fraction of all identifications passing the filter that are incorrect) resulting from filtering each dataset using any minimum computed peptide probability threshold \[[@B18]\]. The false-positive identification error rates for the combined dataset of peptide assignments (all 52 experiments) filtered using minimum probability thresholds 0.7, 0.9, 0.95 and 0.99 are shown in Table [2](#T2){ref-type="table"}. To assess the effect of using a particular probability threshold on the number of peptides in the atlas, we ran the PeptideAtlas pipeline using probability thresholds P≥ 0.7, 0.9, 0.95 and 0.99. Decreasing the probability threshold increases the number of peptides, both correctly and incorrectly identified, and the corresponding proteins (Table [2](#T2){ref-type="table"}). The most stringent threshold of P ≥ 0.99 produced 21,030 peptides with protein sequence matches (4,845 protein identifications), almost 8,400 fewer than the lowest threshold of 0.7 (2,252 fewer protein identifications). The P ≥ 0.9 threshold yielded 25,754 peptides with protein sequence matches at an estimated false-positive rate of less than 7%, and we selected this as an acceptable level for the default PeptideAtlas. The number of false-positive identifications could be reduced by selecting a higher threshold; however, a significant number of correct peptides and proteins would then also be eliminated. The additional peptides resulting from the low-probability threshold were valuable for adding additional peptide evidence in combination with higher-probability peptides corresponding to the same protein (peptides corresponding to proteins to which other peptides correspond are more likely to be correct than their probability value indicates \[[@B22]\]). We provide at our website the option for users to browse or download versions of the Atlas generated with the other Pthresholds, which might be useful for some applications. To validate our approach for general use in eukaryote genomes, we have extended our methods to peptides obtained from Drosophila melanogasterLC-MS/MS experiments. We collected data obtained from cytoplasmic, nuclear and membrane fractions derived from a DrosophilaS2 Schneider cell line. The resulting 4,406 different peptides with P \> 0.9 were compared to the 18,289 proteins (Ensembl fly database version 18.3a.1, 2003-07-01) using the same pipeline as described for human. From the fly, 3,107 proteins could be validated, representing 1,876 (14%) of the fly\'s genes. These results show that our method could easily be adapted to other organisms, thus opening up the way for comparative proteome-level evaluations of eukaryotic organisms. Conclusions =========== We have annotated the human genome with protein evidence for nearly 10,000 proteins. Although this number only represents a fraction of the genome and still contains some erroneous identifications, it is a first step towards the final goal: to fully annotate eukaryotic genomes via validation of expressed proteins. PeptideAtlas provides a method and a framework to accommodate proteome information generated by high-throughput proteomics technologies and is able to efficiently disseminate experimental data in the public domain. Its significance continues to grow as more data are submitted. Moreover, PeptideAtlas also allows one to address the important question of how big the human proteome is. Due to the technical limitations of current proteomics technologies, it is not possible yet to determine the complete proteome in one experiment. However, if the data from diverse experiments, using different cellular compartments and enrichment methods were combined, the determination of the complete proteome could eventually be achieved. PeptideAtlas offers the framework to answer this question accurately and to determine the size of the complete human proteome using pooled experimental data. Furthermore, PeptideAtlas provides a resource for the development of new avenues of research. The dataset will provide a rich source of data for computational scientists to develop and test new algorithms for proteomic analysis, gene discovery and splice-variant prediction. The methods described here, combined with the ever-increasing power of proteomics and bioinformatics technologies, will facilitate the determination or characterization of protein-coding genes, their features, and their processing and expression in relationship to the sequence of the human genome, thus contributing significantly to our understanding of genome structure. Materials and methods ===================== Pipeline -------- The assembly of experimentally derived distinct peptides is mapped to the human genome in the following way. First, we use BLAST \[[@B31]\] to match the peptides to the Ensembl human protein database. The Ensembl database project \[[@B32]\] provides a bioinformatics framework to organize biology around the sequences of large genomes and, furthermore, extensive resources and visualization options as well as remote access to the underlying relational databases \[[@B33]\]. The human genome sequence (release 22.34d.1, 2004-06-02) contains 23,758 genes and 34,091 gene transcripts. Second, complete matches, spanning each peptide\'s complete length, were used to determine human chromosomal coordinates. The method for retrieving chromosomal coordinates within the human genome accounts for splice junctions; in cases where a peptide maps onto a splice junction, it is projected to both parts of the chromosome, generating multiple sets of coordinates. Third, the results are loaded into a relational database. This database schema (available at the project website \[[@B19]\]) is able to accommodate data for different PeptideAtlas builds, for different organisms or different reference protein sequence sets as starting material and is thus extremely versatile. Fourth, visualization of the results was achieved using the Distributed Annotation System (DAS) (Figure [2](#F2){ref-type="fig"}) in conjunction with the Ensembl database. DAS allows sequence annotations to be decentralized among multiple third-party annotators and integrated on an as-needed basis by the Ensembl genome browser \[[@B34]\]. Data collection --------------- LC-MS/MS analysis was performed on LCQ, Ion-trap (Thermo Finnigan LCQ) and Q-Tof (Micromass Waters) instruments. To estimate the false-positive error rate on the level of distinct peptide identifications, we first note that there is an almost 10-fold difference between the number of peptide assignments to MS/MS spectra and the number of resulting distinct peptide identifications. This can be explained by the fact that many peptides were sequenced multiple times, with some of the most abundant peptides sequenced more than 1,000 times (for example, peptides PAp00004784, PAp00003568, PAp00026910). While many correct peptide assignments to MS/MS spectra represent the same peptide sequence, the majority of incorrect peptide assignments are expected to be single identifications. As a result, the false-positive error rate on the level of distinct peptides is higher than that on the level of peptide assignments to MS/MS spectra. Second, it should also be taken into account that a considerable fraction of all distinct peptides did not match any Ensembl entry. This is due to the fact that MS/MS spectra were searched against larger databases, such as human IPI, which contained a number of protein sequences not present in Ensembl. The fraction of all distinct peptide identifications that did not map to any Ensembl entry can be estimated using information provided in Table [2](#T2){ref-type="table"}. Among peptides with probability of being correct of 0.99 or greater, only 2.6% of all distinct peptides did not map to any Ensembl sequence. The fraction of unmapped distinct peptides increases to 8.3% among peptides in the 0.95-0.99 probability range, 12.9% in the 0.9-0.95 range and 18.2% in the 0.7-0.9 range, reflecting the increase in the number of incorrect peptide identifications among peptides with lower probabilities. Thus, one can estimate that at least 18.2% of all incorrectly identified peptides did not map to any entry in Ensembl. The false-positive error rate among distinct peptides that mapped to Ensembl (peptides with protein sequence match) can then be estimated to be not higher than the maximum possible number of distinct incorrect peptide identifications that have protein sequence matches (computed by multiplying the total number of peptide assignments to MS/MS spectra by the corresponding false-positive error rate and applying an 18.2% correction to account for peptides with no mapping to Ensembl) divided by the total number of peptides with protein sequence matches. The corresponding estimates are 16%, 6%, 3% and 0.8% in the case of minimum-probability thresholds 0.7, 9, 0.95 and 0.99, respectively (Table [2](#T2){ref-type="table"}). It should be noted that these are conservative (upper bound) estimates and the actual error rate may be significantly smaller. Population of the database -------------------------- The PeptideAtlas pipeline begins with the download of the Ensembl human protein database from \[[@B35]\]. Release 22.34d.1 (2004-06-02) was used here. PeptideAtlas peptides were then searched against the human proteins using BLAST with the following parameters adapted for searching small peptides \[[@B31]\]: -E 1 -W 2 -M PAM30 -G 9 -e 10 -K 50 -b 50 -F F. The BLAST results were then filtered for identical matches and mapped into chromosomal coordinates using Bio::EnsEMBL and Bioperl \[[@B36]\] Perl modules. The results are uploaded into the PeptideAtlas database and then the Ensembl genome browser. The PeptideAtlas database can handle different PeptideAtlas builds, different organisms and different versions of underlying genome data for maximum flexibility. Acknowledgements ================ We thank Mike Carlson, B. Brett Finlay, Philip R. Hardwidge, Stephen D. Hauschka, Charis L. Himeda, and Priska von Haller for making the raw data from their published results available for this study. This project has been funded in part with federal funds from the National Heart, Lung, and Blood Institute, National Institutes of Health, under contract No. N01-HV-28179, and from the National Institute on Drug Abuse under contract P30DA015625. Some unpublished data contributing to this work was supported in part by a grant from the NIH to J.W. (RO1-AI-51344-01). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Analysis pipeline for the annotation of the human genome with high-quality peptide sequences derived from high-throughput MS analysis of biological samples. ::: ![](gb-2004-6-1-r9-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Visualization of PeptideAtlas peptide entries in the Ensembl DAS browser as a separate track at the top called PeptideAtlas, displayed as light blue rectangles. The Ensembl genome browser, here showing 10 kilobases (kb) on chromosome 12, can be used to zoom into the genome down to the nucleotide level. A light blue line connects peptides that map on intro/exon boundaries. Details about the peptide, including its unique identifier, peptide sequence, best PeptideProphet probability \[22\] (marked SCORE) and PeptideAtlas hyperlink are displayed. ::: ![](gb-2004-6-1-r9-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Cumulative number of distinct peptides as a function of the addition of more good spectra (identified with P≥ 0.9). Eventually the pattern is expected to show saturation, as most observable peptides will have been cataloged. However, at present there is no evidence of saturation and around 100 new peptides are still cataloged per 1,000 identified spectra added. ::: ![](gb-2004-6-1-r9-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Distribution of PeptideAtlas peptides on the human genome. Each chromosome is described by three columns. The left-most column shows a chromosome\'s standard banding. The right-most column presents a histogram of the mapping of peptides to chromosomal regions; a line\'s length represents the number of peptides mapped to a chromosomal region. The central column indicates the over/under representation of peptides in a given region. Green regions represent more mapped peptides than expected at uniform random; red regions indicate fewer mapped peptides than expected at uniform random. ::: ![](gb-2004-6-1-r9-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### View of the DNA-dependent protein kinase catalytic subunit PRKDC gene (ENSG00000121031), which is matched by 90 distinct peptides in PeptideAtlas. ::: ![](gb-2004-6-1-r9-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Example of peptides confirming a case of alternative splicing of the lamin A/C gene (LMNA). PAp00038023 was identified as part of protein ENSP00000310687 from the SiHa human cell line experiment. PAp00042742 was identified as part of protein ENSP00000292304 from a human B-cell experiment. ::: ![](gb-2004-6-1-r9-6) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Summary of PeptideAtlas results ::: Human Drosophila -------------------------------------- --------------------------- -------------------------------- Ensembl version 22.34d.1 2004-06-02 19.3a.2, 2003-07-01 Ensembl gene predictions 23758 13525 from Release 3.1 FlyBase Ensembl gene transcripts 34091 18289 PeptideAtlas version FullHumanEns22APD0704P0.9 Fly 2 PeptideAtlas peptides 26840 4406 Number of experiments 52 3 PeptideProphet probability threshold 0.9 0.9 PeptideAtlas mapped peptides 25754 4406 PeptideAtlas mapped proteins 9747 3107 PeptideAtlas mapped genes 6423 1876 Percentage of the genome 27 % 14 % ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Comparison of different probability thresholds that were applied to the MS results ::: ------------------------------------------------------- -------- -------- -------- -------- Probability ≥ 0.70 ≥ 0.90 ≥ 0.95 ≥ 0.99 Total number of passing spectra 245724 224793 211674 179410 Peptides 31290 26840 25022 21598 Distinct peptides with protein sequence matches 29393 25754 24172 21030 Number of mapped proteins 11612 9747 9016 8134 Number of simple reduced proteins 7097 5826 5383 4845 False-positive estimate MS/MS spectra 2.4% 0.9% 0.05% 0.01% False-positive estimate with protein sequence matches \<16% \<6% \<3% \<0.8% ------------------------------------------------------- -------- -------- -------- -------- :::	PubMed Central	2024-06-05T03:55:52.966358	2004-12-10	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549070/", "journal": "Genome Biol. 2005 Dec 10; 6(1):R9", "authors": [ { "first": "Frank", "last": "Desiere" }, { "first": "Eric W", "last": "Deutsch" }, { "first": "Alexey I", "last": "Nesvizhskii" }, { "first": "Parag", "last": "Mallick" }, { "first": "Nichole L", "last": "King" }, { "first": "Jimmy K", "last": "Eng" }, { "first": "Alan", "last": "Aderem" }, { "first": "Rose", "last": "Boyle" }, { "first": "Erich", "last": "Brunner" }, { "first": "Samuel", "last": "Donohoe" }, { "first": "Nelson", "last": "Fausto" }, { "first": "Ernst", "last": "Hafen" }, { "first": "Lee", "last": "Hood" }, { "first": "Michael G", "last": "Katze" }, { "first": "Kathleen A", "last": "Kennedy" }, { "first": "Floyd", "last": "Kregenow" }, { "first": "Hookeun", "last": "Lee" }, { "first": "Biaoyang", "last": "Lin" }, { "first": "Dan", "last": "Martin" }, { "first": "Jeffrey A", "last": "Ranish" }, { "first": "David J", "last": "Rawlings" }, { "first": "Lawrence E", "last": "Samelson" }, { "first": "Yuzuru", "last": "Shiio" }, { "first": "Julian D", "last": "Watts" }, { "first": "Bernd", "last": "Wollscheid" }, { "first": "Michael E", "last": "Wright" }, { "first": "Wei", "last": "Yan" }, { "first": "Lihong", "last": "Yang" }, { "first": "Eugene C", "last": "Yi" }, { "first": "Hui", "last": "Zhang" }, { "first": "Ruedi", "last": "Aebersold" } ] }
PMC549072	Tribute ======= I arrived in Chicago last November for the ASC meeting, and reflexively reached for the phone to let Dr. Wied know that I had arrived, just as I had for each of countless trips to Chicago over the past 25 years. Then I remembered that he was no longer here, having passed away in Salzburg, Austria, on July 25th. But as the meeting unfolded, I realized that George\'s mark was deeply imprinted on the entire scientific program, and that his legacy was carried by most of the people in attendance. George Papanicolaou is attributed with the discovery of the cytologic method for detecting epithelial tumors. His laboratory at Cornell Medical Center in New York was the culture medium for the discipline, instructing such notable cytologists as Leopold Koss and George Wied. The medical specialty itself, however, owes its success to George Wied, for without him, it is doubtful whether it would have survived the skepticism of most other medical specialists. Dr. Wied had faith in the importance of cytology that was unwavering and his conviction was transmitted to all who followed him around the globe. A survivor of Nazi Germany, he was a fierce defender of individual freedoms, and he translated that zeal into inclusion of all peoples in his vision. He was an instinctual teacher and taught those around him to convey the criteria of those strange cellular samples via the Tutorials of Cytology. His attention to detail was impeccable, a trait that he insisted his faculty emulate. The TOC faculty quickly became internationally recognized experts, and the Tutorials were a successful enterprise for over 40 years. Until cytopathology became a mandatory part of the curriculum of U.S. pathology training programs, the Tutorials were among the few opportunities for physicians to become facile with the discipline. Repeat enrollment was common, as the specialty became more complex and its importance became apparent to the medical profession. For many nations TOC remained the sole source of first-hand and first-rate cytology education. Judging by the multinational backgrounds of presenters at the ASC meeting, and the excellence of the papers, Dr. Wied\'s goal has most assuredly been achieved. Setting and maintaining a level of excellence was a responsibility that Dr. George Wied eagerly assumed, and expected the rest of us to uphold. It was easy to follow his example, for his charisma infused us with commitment to those eager to learn. In order to validate the professionals involved in the practice, he established the examinations of the International Academy of Cytology. He personally traveled to every venue where the exam was held, usually following either a Tutorial or International Congress of the IAC. He savored each opportunity to bring another disciple into the fold of cytology. But where Dr. Wied had the most fun was in the realm of cytology automation. As I listened Sunday to the presentations of experiences with the \"new\" imaging systems and their integration into the clinical cytology laboratory, I recalled the numerous conferences devoted to development of computerized scanners. As early as 1951, Dr. Wied recognized that computers would become an essential part of our daily lives, for data management and communication. He also realized that the task of screening slides was work intensive, subjective, and fraught with opportunities for error. If Dr. Wied didn\'t have the knowledge himself, he immediately reached out to others who did, and drafted them to the cause of automating his specialty. One of those recruits was Peter Bartels, a gifted optical scientist and incredibly creative problem solver. Together they built a team of researchers at the University of Chicago that attacked each obstacle to success like an army of dragon slayers. Rather than seek the glory of discovery alone, Dr. Wied\'s generous spirit inspired him to organize meetings to discuss problems common to automation, bringing scientists from a variety of disciplines and numerous countries. If a potentially important contributor to the meeting did not have funding to attend, Dr. Wied would offer to support them, often personally. He was most supportive of young scientists and mentored them through their careers. These were usually pleasant meetings, as Dr. Wied\'s gentle and considerate nature didn\'t allow personal bickering. But he loved scientific controversy and he encouraged us to challenge each other to the next level of achievement. He fully believed that what was good for one research group was good for the entire profession. He was most distressed when the commercialization of automated scanners led to open fighting among the companies trusted to develop the fruits of so many years of collegial research. He was a frequent member of NIH study sections that reviewed research proposals in automation. He was also known to drop in unexpectedly on a research group thousands of miles from his home, just to see what was happening. He was free with his advice without being condescending. He would never knowingly offend anyone. His constant encouragement and validation of the importance of scientific discovery for this young specialty was manifested through the two journals he founded and edited, ACTA Cytologicain 1957 and Analytical and Quantitative Cytology and Histologyin 1979. Recognition for his accomplishments came frequently and from various sources, including an Outstanding Investigator Award from the NIH. But if you were to reach into the heart of George Wied, I wager that he felt that his greatest accomplishment was being father to Kazutaka (George) Wied, son of his wife, Kay. An unlikely parent, Dr. Wied soon became a master at constructing dioramas out of shoe boxes, holding flash cards with German vocabulary words, and generally encouraging young George to treasure his education. Being a musician himself, Dr. Wied patiently tolerated the squawks and squeaks of his young son\'s efforts to be a violinist. As young George became more accomplished, after dinner performances soon became an expected treat whenever the occasion allowed. Dr. Wied and Kay continued to travel the world even though Dr. Wied\'s health was becoming increasingly fragile. His final trip could have been written by a romantic novelist. He accepted a speaking engagement in Japan, but instead of a simple round trip to Tokyo, he insisted on an around-the-world ticket, \"because it\'s cheaper!\" For one of the stops, he chose to go to Vienna, a city that he loved, to once more savor the delicious schnitzel at the Intercontinental Hotel, site of the Vienna Tutorials. Kay realized that the Salzburg Music Festival was nearby and would be a good place to relax and hear the music that Dr. Wied loved so dearly. Young George, having just graduated from Stanford University, was with them, not always the case. They spent the day at numerous concerts in Salzburg, all dedicated to Czech composers, an uncanny coincidence since Dr. Wied was born in what was then Czechoslovakia. After the concerts, Kay, young George and Dr. Wied spent the evening together, remarking on the beauty of the music. Dr. Wied died in the night, with his beloved family nearby, and across the street from the home of his most admired composer, Wolfgang Amadeus Mozart. There is an old adage, that when a loved one dies, you mourn each time for all the roles they played in your life. If it is a parent or sibling, you mourn once. However, if the person has been multiple influences in your life, you will mourn for each role. For Dr. Wied, many of us will mourn multiple times. For me, he was a teacher, mentor, advisor, friend, and role model. I will mourn many times, but will be comforted by having known him. I have already and will continue to repay his influence by transmitting his ideals to those who follow. His is truly a legacy that deserves to be perpetuated. George Wied was a man of rare breadth and depth, the kind of professional and human for which mankind will eternally thirst. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Dr. Wied at the University of Chicago Cytopathology Laboratory, circa 1990. ::: ![](1742-6413-2-2-1) :::	PubMed Central	2024-06-05T03:55:52.969104	2005-2-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549072/", "journal": "Cytojournal. 2005 Feb 8; 2:2", "authors": [ { "first": "Dorothy L", "last": "Rosenthal" } ] }
PMC549073	Introduction ============ The deleterious effects of exposure to ETS on the respiratory system of adults and children is well documented \[[@B1]-[@B4]\]. Exposure to ETS not only influences respiratory health among those affected but also leads to increased health care utilization and costs because of respiratory problems \[[@B5],[@B6]\]. For example, a recent study investigating more than 10,000 children in Germany showed that the risk of emergency department visits and hospitalizations from asthma was more than double for children exposed to 10 or more cigarettes/day compared to less exposed children \[[@B6]\]. ETS exposure was found to be associated with respiratory symptoms, abnormal lung functions, and increased bronchial responsiveness in children and adults \[[@B1]-[@B4],[@B7],[@B8]\]. Of special significance for developing countries, lower respiratory infection, the single most important cause of death for children below the age of 5 years, has been found to be associated with exposure to ETS \[[@B1],[@B9]-[@B12]\]. However, with most studies of ETS exposure and respiratory health being done in developed countries, local evidence to promote clean air policies and to enforce existing policies are lacking in most of the developing world. The situation with exposure to ETS in developing countries is likely to be aggravated by the widespread of smoking, lack of restrictions regarding indoor smoking, overcrowded housing conditions, and failure of health services \[[@B13]-[@B15]\]. Cigarette smoking in Aleppo is affecting some 70% of men and 20% of women aged 30--45 years, with an average of 1.2 cigarette smoker per household \[[@B16]\]. Moreover, Syria as well as other countries in the Eastern Mediterranean region (EMR) are experiencing an alarming increase in the popularity of waterpipe smoking \[[@B17],[@B18]\]. Although this form of smoking is generally considered an outdoor social practice, research done at the Syrian Center for Tobacco Studies (SCTS) shows that more serious smokers demonstrate a predominantly individual use pattern (home, and alone) \[[@B19]\]. As such, waterpipe smoking may be an important source of ETS due to its emissions and length of smoking bouts \[[@B18],[@B20]\]. Assessment of exposure to ETS, therefore, needs to encompass all information relevant to the studied setting and the smoking patterns of the target population. Despite these troubling facts, there is a dearth of research examining the relationship between ETS exposure and respiratory health in developing countries, with the few available studies limited by poor outcome definition, lack of systematic exposure assessment, and inadequate control of confounding \[[@B21],[@B22]\]. A recent review of this subject identifies a major limitation of available data being restricted to industrialized nations \[[@B3]\]. Generally, the use of different methodologies and markers of exposure and outcome precludes arriving at a clear picture of the relationship between ETS exposure and respiratory health. For example, not all studies have found a relationship between exposure to ETS and lung function impairment \[[@B23],[@B24]\], some did not find a dose-response relationship \[[@B25]\], while others demonstrated gender-specific effects \[[@B24],[@B26]\]. The reliance on single or historic indicators of exposure (spouse\'s smoking, maternal smoking during pregnancy) can lead to an imprecise estimation of exposure or recall problems \[[@B27],[@B28]\]. Previous quantitative and qualitative research done in Aleppo has identified ETS as a potentially major health hazard in the indoor environment \[[@B16],[@B29]\]. The current study, which is the first to assess respiratory health of adults and its relation to ETS exposure in Syria, is based on a population-based household survey (Aleppo Household Survey, AHS) done in Aleppo in 2004 utilizing multiple self-reported indicators of exposure and outcome as well as expired breath CO and spirometry. Methods ======= Population and sampling ----------------------- The target population consisted of adults 18--65 years of age residing in the greater city of Aleppo (around 2,500,000 inhabitants). Detailed description of the sampling design and procedures of the AHS is reported elsewhere and illustrated in Figure [1](#F1){ref-type="fig"}\[[@B16]\]. Briefly, stratified cluster sampling was used where residential neighborhoods of the city were stratified into two strata: formal and informal; according to the official description of the municipal registry (Figure [1](#F1){ref-type="fig"}). From each stratum, residential neighborhoods were randomly selected with probability proportional to size (PPS). Within each neighborhood, households were selected with equal probably and an adult was randomly selected from each. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The overall sampling scheme of the Aleppo Household Survey. In the 1^st^step the target population was divided into two strata, formal and informal zones (where residential areas are build illegally or on a land not designated for housing). In the next step residential neighborhoods were selected with PPS, and within selected neighborhoods households and one adult within each were selected with equal probability. ::: ![](1465-9921-6-13-1) ::: The survey was conducted between May-August 2004, and the protocol and the informed consent documents were approved by the Institutional Review Boards at the University of Memphis and SCTS. Design and procedures --------------------- This interviewer-administered survey involved six, 2-person, mixed gender teams of surveyors equipped with notebook computers programmed to record questionnaire responses and measurements using a custom data entry program (Delphi programming language and SQL server DBMS). The questionnaire included 8 main sections; socio-demographics, general health and disability, chronic disease, respiratory health, household members\' health, environmental health, smoking, and ETS exposure. For the assessment of respiratory health and risks, the questionnaire was developed based on relevant instruments (especially the European Community Respiratory Health Survey-ECRHS, the International Study of Asthma and Allergy in Children-ISAAC, and ETS exposure assessment instrument developed by Eisner and colleagues), as well surveys done in Syria \[[@B29]-[@B35]\]. Next, the survey instrument and strategy were modified in terms of format, content, language, response categories, and recall period based on formative work conducted with key informants involved in the provision of health care as well as with residents, in addition to piloting in 20 randomly selected households \[[@B16],[@B29]\]. After being randomly selected, all study participants underwent the detailed study interview and objective measurements; height using a sliding wall meter (Seca, Germany), body weight using digital scales (Camry, China), expired breath carbon monoxide (CO) using breath CO monitors (Vitalograph, US), and lung function tests using portable spirometers (Micro-plus, UK) according to a standard protocol (16). Exposure -------- Data from self-reported non-smokers (both cigarettes and waterpipe), validated by breath CO levels ≤ 10 ppm (Table [1](#T1){ref-type="table"}) \[[@B36],[@B37]\], were analyzed for this report. ETS exposure assessment utilized responses to multiple inquiries about short- vs. long-term, indoor vs. outdoor, and cigarette vs. waterpipe exposures, as well as sensory irritation due to ETS exposure (a marker of intensity of exposure) (Table [2](#T2){ref-type="table"}) \[[@B29]-[@B35]\]. Spouse\'s and parental smoking assessment included inquiries about length, intensity, and type of smoking (cigarette, waterpipe). Occupational exposure to respirable pollutants other than ETS was assessed by asking those involved in paid work whether they are exposed to dust, foams, smoke or other respirable particles at their work categorized as no exposure, mild exposure (a day or less weekly), moderate exposure (more than a day per week but not daily), and severe exposure (almost daily). Parental allergy was assessed by asking whether the respondent\'s parents ever suffered from respiratory or nose allergy with responses categorized into none, father, mother, or both. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Basic indicators of Aleppo Household Survey (AHS) participants (n = 2038), and non-smokers satisfying criteria for inclusion in the analysis (n = 1118) ::: All participants n (%) Non-smokers with breath CO ≤10 ppm n (%) ----------------------------------------- ------------------------ ------------------------------------------ Age 18--29 years 736 (36.1) 450 (40.3) 30--45 years 874 (42.9) 418 (37.4) 46--65 years 428 (21.0) 250 (22.4) Gender Men 921 (45.2) 303 (27.1) Women 1117 (54.8) 815 (72.9) Religion Muslim 1938 (95.3) 1073 (96.1) Christian 82 (4.0) 37 (3.3) Other 13 (0.6) 6 (0.5) Race Arabs 1625 (79.9) 912 (81.6) Non-Arabs 409 (20.1) 205 (18.4) Education Illiterate 425 (20.9) 257 (23.0) ≤ 9 years 1131 (55.5) 578 (51.7) \> 9 years 482 (23.7) 283 (25.3) mean ± SD mean ± SD Total number of people in the household 6.5 ± 3.3 6.7 ± 3.2 Adults 3.3 ± 1.8 3.5 ± 1.9 Children 3.2 ± 2.5 3.2 ± 2.5 Household density (household/rooms) 2.2 ± 1.3 2.2 ± 1.3 Total SES score 4.3 ± 2.0 4.0 ± 2.0 ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Various indicators of exposure to ETS among adults non-smokers (n = 1118) in Aleppo, Syria. ::: Non-smokers with CO≤10 ppm n (%) ------------------------------------------------ ---------------------------------- Spouse\'s smoking (cigarettes and waterpipe) 351 (43.7)\* Parental smoking None 374 (33.5) Father 591 (52.9) Mother 36 (3.2) Both 117 (10.5) Number of household smokers Cigarettes ≥ 1 smoker 543 (48.6) Waterpipe ≥ 1 smoker 47 (4.2) Both ≥ 1 smoker 108 (9.7) Past year regular exposure to other\'s smoke 769 (68.8) Past week sensory irritation from ETS exposure Sometimes 188 (16.8) Many times 103 (9.2) Past week hours spent daily with smokers At home ≤ 1 hour/day 651 (58.2) \>1 hour/day 467 (41.8) At other places ≤ 1 hour/day 795 (71.1) \>1 hour/day 323 (28.9) Exposure to smoking at workplace Yes/well ventilated 205 (58.9)\* Yes/poor ventilated 32 (9.2)\* Average cigarettes smoked daily in the house 1--10 cig/day 438 (39.2) \> 10 cig/day 281 (25.1) Average waterpipes smoked daily in the house 1--2 waterpipe/day 33 (3.0) \> 2 waterpipe/day 8 (0.7) House policy regarding smoking Smoking is not allowed at all 40 (3.6) Smoking is allowed for only few guests 137 (12.3) Smoking is allowed only in special places 106 (9.5) Smoking is not restricted at all 812 (72.6) Differs between cigarettes and waterpipes 23 (2.1) \* Calculated from the number of non-smokers who are currently married (n = 803), or employed (n = 348) ::: Outcomes -------- Past year recurrent cough and recurrent shortness of breath were defined as having 3 or more recognizable episodes of these symptoms. Those reporting recurrent cough or shortness of breath were asked to select, from a provided list, one or more options that best described their symptoms (Table [3](#T3){ref-type="table"}). The main asthma symptom (past year wheezing/whistling in the chest) and asthma diagnosis were inquired about from all participants, while other asthma symptoms (recurrent cough accompanied by wheezing, recurrent nocturnal cough unrelated to colds, and recurrent episodic shortness of breath accompanied by wheezing) were inquired about among those reporting recurrent cough or shortness of breath. Items related to physician-diagnosed conditions included ever having a diagnosis of (asthma, chronic bronchitis, or emphysema), or the occurrence of a diagnosed condition (sinusitis, acute bronchitis, pneumonia) during the past year. Hay fever was defined conservatively based on positive responses to two questions about past year nasal allergic symptoms (episodes of sneezing, runny or blocked nose when not experiencing a cold), and the co-occurrence of itchy and watery eyes \[[@B38]\]. Severity of respiratory complaints was based on the number of wheezing/whistling episodes for asthma (≤ 12 and \> 12), reporting more than one episode of sinusitis or acute lower respiratory tract infection, and medical care (medication use, hospital or clinic visits) for respiratory problems (Table [4](#T4){ref-type="table"}). Medication or health facility use because of respiratory problems was broken down further by condition (asthma, pneumonia, bronchitis, etc.), but because none of these outcomes were associated with exposure to ETS in our study we used only the parent general question. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Relation between different levels of exposure to ETS and respiratory symptoms/diagnosis among adult non-smokers in Aleppo-Syria (n = 1118) ::: ETS score\* -------------------------------------------------------------------------------------- ----------------- ---------------- --------- General respiratory symptoms Past year recurrent cough (≥ 3 recognizable episodes) 1.3 (0.8--1.9) 1.9 (1.2--2.9) 0.004 Past year recurrent shortness of breath (≥ 3 recognizable episodes) 1.6 (1.1--2.3) 1.7 (1.1--2.6) 0.001 Past year recurrent exertional shortness of breath that disappears after rest 1.8 (1.2--2.7) 2.0 (1.3--3.2) \<0.001 Past year recurrent shortness of breath almost all the time 3.0 (1.3--6.8) 2.6 (1.1--6.3) 0.02 Symptoms/diagnosis suggestive of asthma Past year wheezing/whistling in the chest 1.4 (0.8--2.2) 1.7 (1.0--2.8) 0.05 Past year recurrent episodic dry cough accompanied by wheezing/whistling 1.9 (1.0--3.7) 1.9 (0.9--3.9) 0.05 Past year recurrent nocturnal cough, not related to colds, that wakes the subject up 1.2 (0.7--1.9) 1.9 (1.1--3.2) 0.02 Past year recurrent episodic shortness of breath accompanied by wheezing 2.2 (1.1--4.4) 1.6 (0.7--3.7) 0.06 Ever diagnosed asthma 1.8 (1.2--2.8) 1.4 (0.9--2.4) 0.1 Hay fever (nasal allergy symptoms with eye itching and watering) 0.9 (0.6--1.3) 1.5 (0.9--2.3) 0.01 Symptoms/diagnosis suggestive of chronic bronchitis Productive cough that lasts most of the winter 1.2 (0.7--2.2) 1.6 (0.8--2.9) 0.2 Recurrent shortness of breath accompanied by cough and phlegm 2.2 (1.1--4.7) 2.5 (1.1--5.6) 0.02 Ever diagnosed chronic bronchitis/emphysema 1.1 (0.5--2.2) 1.2 (0.5--2.7) 0.6 Symptoms/diagnosis suggestive of respiratory infection Past year recurrent cough accompanying upper respiratory infections (cold, flue) 1.0 (0.7--1.6) 1.5 (0.9--2.4) 0.1 Past year recurrent cough with bloody phlegm 1.1 (0.5--2.6) 1.4 (0.6--3.4) 0.7 Past year sinusitis 1.0 (0.6--1.7) 1.7 (1.0--2.9) 0.09 Past year diagnosed acute lower respiratory infection (bronchitis, pneumonia) 1.3 (0.7--2.3) 1.9 (1.1--3.6) 0.03 \* Odds ratio and 95% confidence interval for the relation between ETS score tertiles (lower being referent) and respiratory symptoms/diagnosis according to multivariate logistic regression adjusted for age, gender, SES score, hay fever, parental allergy, pack-years (for ex-daily smokers), occupational exposure to respirable pollutants other than ETS ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Relation between different levels of exposure to ETS and severity of respiratory problems of adult non-smokers in Aleppo-Syria ::: ETS score\* ----------------------------------------------------------------------------------------- ----------------- ---------------- ----- Number of wheezing attacks in the past year (≤12 vs. \>12) 0.6 (0.2--2.2) 1.2 (0.3--4.5) 0.6 Number of episodes of sinusitis (once vs. more than once) 0.5 (0.2--1.4) 1.2 (0.4--4.1) 0.3 Number of episodes of acute lower respiratory tract infection (once vs. more than once) 1.4 (0.4--4.6) 0.7 (0.2--2.3) 0.3 Past year doctor\'s or hospital visit because of respiratory problems 1.4 (0.9--2.1) 1.2 (0.7--2.0) 0.4 Past month medication use for respiratory problems 1.0 (0.5--1.7) 1.0 (0.5--1.9) 0.7 \* Odds ratio and 95% confidence interval for the relation between ETS score tertiles (lower being referent) and respiratory symptoms/diagnosis according to multivariate logistic regression adjusted for age, gender, SES score, hay fever, parental allergy, pack-years (for ex-daily smokers), occupational exposure to respirable pollutants other than ETS ::: Forced Expiratory Volume in the 1^st^second (FEV~1~) and Forced Vital Capacity (FVC) were measured for all participants according to standard guidelines \[[@B39]\]. We used hand-held spirometer (Micro-plus, Micro Medical, Rochester, UK), which have been shown to have good precision and reproducibility \[[@B40]\]. We used newly calibrated spirometers and tested them weekly by team members with known lung functions (allowing for no more than 5% variation between different spirometers). Multiple maneuvers were performed until three satisfactory ones were recorded. The best effort that did not exceed the next best by more than 5% was included in the analysis \[[@B41]\] (Table [5](#T5){ref-type="table"}). ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Relation between exposure to ETS and lung functions among men and women non-smokers (n = 623) in Aleppo-Syria ::: ETS score ---------------- ------------------------- ------------------------ ----- Men FEV~1~(ml) -46.8 (-215.4 to 121.7) 34.3 (-140.5 to 209.1) 0.3 FVC (ml) 63.9 (-167.5 to 295.3) 147.9 (-89.1 to 385.0) 0.5 FEV~1~/FVC (%) -1.2 (-3.7 to 1.6) -1.0 (-3.5 to 1.6) 0.5 Women FEV1 (ml) -87.8 (-164.8 to -10.7) -58.7 (-136.5 to 19.1) 0.3 FVC (ml) 3.1 (-103.8 to 110.1) -72.2 (-182.4 to 38.1) 0.2 FEV~1~/FVC (%) -2.0 (-3.8 to -0.3) -1.0 (-2.9 to 0.8) 0.1 \* Unstandardized linear regression coefficient and 95% confidence interval for the relation between ETS score tertiles (lower being referent) and lung function tests adjusted for age, BMI, SES score, hay fever, pack years (for ex-cigarette smokers), occupational exposure, parental allergy, and interaction terms of age with height and age with weight ::: Analysis -------- Descriptive statistics were calculated for the overall study population and for measures of ETS exposure among non-smokers (Tables [1](#T1){ref-type="table"},[2](#T2){ref-type="table"}). Composite scores for socioeconomic status (SES score) and self-reported ETS exposure indices were constructed for the analysis (as illustrated in the additional file, Appendix 1). Spearman correlation coefficients were calculated to assess the relation between FEV~1~, FVC, FEV~1~/FVC and ETS score. Logistic regression was used to estimate the odds ratio (OR) and the 95% confidence interval for the relation between ETS score and respiratory symptoms adjusting for age, sex, SES score, parental allergy, occupational exposure to other respiratory pollutants, and pack years (for ex-daily smokers). Linear regression analysis was used to assess the relationship between ETS score and lung function (FEV~1~, FVC, and FEV~1~/FVC) adjusting for age, BMI, SES score, and occupational exposure to other respiratory irritants, as well as interaction terms of age with height and weight. This analysis was performed separately for men and women, since lung development and response to ETS has been shown to differ by gender \[[@B26],[@B28]\]. In both multivariate models (logistic, linear), ETS score was first entered as a categorical variable (low; bottom tertile, middle; middle tertile, high; top tertile) for the calculation of OR for different gradients of exposure, then as continuous variable for the calculation of pfor dose-response relationship. Because of the concern that ex-smokers may avoid ETS exposure and have respiratory problems (giving us a group with potentially most respiratory problems but least exposure), we repeated the analysis including only never smokers, but this did not affect the results considerably (analysis not shown). All analyses were done by SPSS 11. Results ======= From a total of 2038 valid survey responses (45.2% men, mean age 35.3 ± 12.1, response rate 86%), 1118 (27.1% men) satisfied the inclusion criteria for the exposure-symptoms analysis (Table [1](#T1){ref-type="table"}), and 623 (30% men) for the exposure-lung functions analysis (Table [5](#T5){ref-type="table"}). According to ETS score (mean ± SD 8.8 ± 3.6, median 9), the vast majority of non-smokers in our population were exposed to ETS, whereby only 3.6% had levels ≤ 2 and 21.1% had levels ≤ 5 (Table [2](#T2){ref-type="table"}). Logistic regression analysis of the relation between ETS score and self-reported symptoms/diagnosis generally shows a dose-response association with symptoms and diagnosed acute lower respiratory tract infection (acute bronchitis, pneumonia). General respiratory symptoms associated with exposure to ETS were past year recurrent cough (ORs for comparison between middle, high, with the low exposure group were 1.3 and 1.9, respectively, with pfor dose response 0.004), past year recurrent shortness of breath (ORs 1.6 and 1.7, p = 0.001), past year recurrent exertional shortness of breath that disappears after rest (ORs 1.8 and 2, p \< 0.001), past year recurrent shortness of breath almost all of the time (ORs 3 and 2.6, p = 0.02). Additionally, several symptoms suggestive of asthma/allergy were related to ETS exposure, including past year recurrent wheezing/whistling in the chest (ORs 1.4 and 1.7, p = 0.05), past year recurrent episodic dry cough accompanied by wheezing (ORs 1.9 and 1.9, p = 0.05), past year recurrent episodic shortness of breath accompanied by wheezing (ORs 2.2 and 1.6, p = 0.06), past year recurrent nocturnal cough not related to cold that wakes the subject up (ORs 1.2 and 1.9, p = 0.02), and past year hay fever symptoms (ORs 0.9 and 1.5, p = 0.01). Among symptoms suggestive of chronic bronchitis, ETS exposure was associated with recurrent shortness of breath accompanied by cough and phlegm (ORs 2.2 and 2.5, p = 0.02). And finally, past year episodes of acute lower respiratory infection (bronchitis, pneumonia) were associated with exposure to ETS (OR 1.3 and 1.9, p = 0.03) (Table [3](#T3){ref-type="table"}). In contrast, exposure to ETS was not related to severity of respiratory complaints judged by the number of episodes (wheezing, sinusitis, acute bronchitis, pneumonia), medical care utilization for respiratory problems in general (Table [4](#T4){ref-type="table"}), as well as medical care utilization for a specific problem (e.g. asthma, pneumonia, analysis not shown). In the univariate analysis, there was a weak inverse correlation between ETS score and FEV~1~(coefficient -0.1, p \< 0.001), FVC (-0.1, p = 0.002), and %FEV1/FVC (-0.6, p = 0.1). Linear regression analysis between ETS exposure score and lung functions showed significant inverse associations with indices of airflow limitation (FEV~1~and FEV~1~/FVC) only in women. Women in the middle category of ETS exposure had on average 88 ml deficit in FEV~1~and 2% in FEV~1~/FVC in comparison to those in the low exposure category. This association did not show a dose-response relationship (Table [5](#T5){ref-type="table"}). Discussion ========== This study shows that exposure to ETS is universal among non-smoking adults in Aleppo-Syria. This exposure is associated with respiratory complaints of both infectious and non-infectious etiology in a dose-response fashion, suggesting a causal relationship. Unlike data from developed countries, however, exposure to ETS was not related to increased severity of asthma or other respiratory conditions judged by symptoms frequency and medical care utilization because of respiratory problems. ETS exposure was associated with decrements in lung functions suggestive of airflow limitation (FEV~1~and FEV~1~/FVC) among women only. Error and bias in ascertainment are always a concern in cross-sectional studies, due to imprecise or differential recall. We tried to minimize such problems by not highlighting tobacco or ETS exposure when introducing the study to participants \[[@B42]\], by using symptoms/diagnosis descriptors that are native to the target population, and by assessing multiple indices of both exposures and outcomes. We have some indicators that such bias was limited, including lack of associations between a diagnosis of asthma or chronic bronchitis and ETS score. Remarkably, recurrent cough with bloody phlegm, one of the potentially most startling respiratory symptoms, was not associated with ETS exposure in this study, giving further support of minimal recall bias. Our reliance on self-reported exposure is also a potential limitation, but studies have repeatedly shown that self-report is a valid measure of ETS exposure that correlates with other objective markers such as cotinine \[[@B43]-[@B46]\]. Understandably, the composite ETS score is a crude quantitative measure of ETS exposure. We opted for its use to incorporate various sources of information about exposure to ETS in order to differentiate between meaningful gradients of exposure for the purpose of the analysis. On the other hand, the diversity of information relevant to ETS exposure considered in this study, in addition to the verification of non-smoking status by breath CO measurement, and the use of multiple subjective and objective outcomes, helped delineate the exposed group and conduct a robust analysis. The widespread exposure to ETS among adults in Aleppo, suggests that it is rather hard to avoid such exposure in this environment. The vast majority of non-smokers in Aleppo are exposed to ETS both at home and outside. This exposure is sufficiently intense to cause sensory (eye and nose) irritation for a quarter of nonsmokers. In comparison, less than a quarter of non-smokers in a national sample of 43,732 adults in the US report exposure to ETS \[[@B47]\]. Interestingly, such spread of exposure is occurring in the face of enacted laws banning smoking in public buildings, worksites, and transportation in Syria since the nineties \[[@B48]\]. Our results indicate that these laws are not enforced, as about two thirds of working non-smokers report exposure to others\' smoke at work. Accordingly, these results should provide solid ground for public health advocates and authorities to push for the application of policies and measures to protect non-smokers from this hazardous exposure. Although not in the realm of laws and regulations, the widespread liberal attitude towards smoking in the house suggests a general lack of awareness, or dismissal, of the health damaging effects of ETS exposure to household members. Remarkably, about three quarters of studied households do not restrict indoor smoking whatsoever, and only a small minority (3.6%) has total restriction. This shows that in societies where smoking is rather a norm, it becomes hard to employ smoking restrictions even in one\'s own house. Increasing public awareness of this health hazard is thus an area where public health advocacy can make a difference. In general, this study shows stronger and dose-dependent relationships of ETS exposure with general respiratory symptoms (i.e. recurrent cough or shortness of breath) than with symptoms characteristic of specific respiratory problems (asthma, bronchitis). Arguably, general symptoms are more easily identifiable as well as shared among many respiratory problems. The magnitude of difference in self-reported asthma symptoms according to exposure level is generally in the range of a twofold increase. This effect magnitude is similar to that reported in adults from 16 European countries (European Community Respiratory Health Survey, ECRHS), but lower than that reported from the Swiss Study on Air Pollution and Lung Diseases in Adults (SAPALDIA) \[[@B49],[@B50]\]. Similar to studies from developed countries, we found a dose-response relationship between exposure to ETS and respiratory symptoms, implying a causal relationship \[[@B28],[@B49]-[@B52]\]. On the other hand, unlike data from European nations (ECRHS), we found a 50% increase in hay fever symptoms for the high exposure group compared to those with low exposure. We defined hay fever according to symptom report, however, while the ECRHS inquired about suffering from allergic rhinitis or hay fever \[[@B49]\]. Also, among symptoms indicative of chronic bronchitis, only recurrent shortness of breath with cough and phlegm was associated with ETS exposure in our study, while in the ECRHS ETS exposure during childhood was associated with increased reporting of recurrent cough and phlegm in adulthood \[[@B28]\]. Since cough and phlegm are common symptoms of infectious respiratory problems, likely to be widespread in our population due to overcrowding and poor housing conditions, such symptoms can be non-specific indicators of chronic bronchitis in our setting, while shortness of breath can be more specific marker of this condition. Level of exposure to ETS in our population was not associated with severity of asthma, sinusitis, or lower respiratory tract infection. In contrast, in a study of 349 adults with asthma in the US, Eisner and colleagues found that exposure to ETS at baseline was associated with more symptom severity and emergency/hospital admissions because of asthma at 18 month followup \[[@B53]\]. Medical care utilization for respiratory problems is likely to be an inadequate indicator of severity in a low-income country such as Syria, where the lack of medical insurance and limited public health services render seeking private health care the last resort for most adults in this country. On the other hand, as it will be discussed later, the lack of a comparison group of non-exposed individuals may have contributed to the absence of association between ETS exposure and symptoms severity in this study. Studies of the relation between exposure to ETS and lung function have generally shown a detrimental effect of such exposure. This effect, however, was not consistent and of low magnitude generally (50--100 ml) \[[@B3]\]. For example, in a population-based sample of adults from the NHANES III survey in the US, exposure to ETS was associated with decreased lung functions (FEV~1~, FVC, FEV~1~/FVC) in women but not men \[[@B26]\]. Data from the ECRHS involving 18,922 adults from 17 European countries show that exposure to parental smoking in childhood was associated with impaired lung function \[[@B28]\]. The effect on lung function differed, however, according to participant\'s gender, parental smoking (mother, father, both), and period of exposure (during pregnancy, childhood) \[[@B28]\]. Our study shows that exposure to ETS is associated with decreased lung function indicative of airflow limitation (FEV~1~, and FEV~1~/FVC) in female but not male non-smokers. The reduction for the middle compared to low exposure category was in the magnitude of 88 ml for FEV~1~and 2% for FEV~1~/FVC. Although other studies have reported similar gender-specific vulnerability of women \[[@B26]\], it is important to emphasize the small number of male non-smokers in our sample (less than one third). We also could not elicit a dose-response in the relation between ETS exposure and airflow limitation among women, although ETS score was weakly correlated to FVE~1~in the univariate analysis. It is possible that we are dealing in our setting with levels of exposure that exceed those occurring in western societies. Indeed, because of the low magnitude of the effect of ETS exposure on lung function, studies have relied on comparisons of exposed vs. non-exposed individuals to assess this relationship \[[@B49],[@B53]-[@B55]\]. In our sample, however, we had very few subjects with no or little exposure, which could have reduced the sensitivity of our analysis. Another possibility is that the relatively crude measure of ETS (ETS score) we used may better differentiate between gradients of exposure at its lower stratum than higher. Conclusions =========== This study shows that exposure to ETS is rampant among adult non-smokers in Syria, where it is hard to escape it due to a high prevalence of smoking, household over-crowding, and lack of smoking restrictions. This exposure is leading to increased respiratory symptoms/disease of both infectious and non infectious etiologies, and is likely to have deleterious effects on respiratory function among women. In addition, the dose-response association found between exposure to ETS and respiratory symptoms point towards causal relationship. These results send a clear message to health advocates and policy makers about the spread and harmful effects of exposure to ETS in Syria and on the importance of collective efforts to educate both the public and authorities about this major health hazard and ways to effectively protect non-smokers from it. In addition to giving further support to the health hazards of ETS exposure, this study can provide guidance for future research on this issue in other developing countries. List of abbreviations ===================== • AC- air condition • AHS- Aleppo Household Survey • ECRHS- European Community Respiratory Health Survey • ETS- environmental tobacco smoke • FEV~1~- forced expiratory volume in the 1^st^second • FVC- forced vital capacity • NHANES III- third National Health and Nutrition Examination Survey • OR- odds ratio • PC- personal computer. • ppm- part per million • PPS- probability proportionate to size • SAPALDIA Swiss Study on Air Pollution and Lung Diseases in Adults • SD-standard deviation • TV- television Authors\' contribution ====================== W Maziak, designed the study, conducted the analysis and wrote the 1^st^draft of the manuscript. KD Ward, T Eissenberg, participated in the study design and co-authored the manuscript. S Rastam and F Mzayek participated in the data management, analysis, and co-authored the manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Appendix 1 : Composite scores for SES and ETS used in the study with the total score categorized around tertile cut off points. ::: ::: {.caption} ###### Click here for file ::: Acknowledgment ============== This work is supported by USPHS grants R21 TW006545 and R01 TW05962. We especially thank our surveyors for the excellent performance of the challenging task of data collection for this study.	PubMed Central	2024-06-05T03:55:52.970145	2005-2-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549073/", "journal": "Respir Res. 2005 Feb 8; 6(1):13", "authors": [ { "first": "Wasim", "last": "Maziak" }, { "first": "Kenneth D", "last": "Ward" }, { "first": "Samer", "last": "Rastam" }, { "first": "Fawaz", "last": "Mzayek" }, { "first": "Thomas", "last": "Eissenberg" } ] }
PMC549074	Background ========== Ovarian cancer is the most lethal gynecological malignancy. Indeed, epithelial ovarian cancer is detected at a late clinical stage in as much as 75% of patients, in whom the overall survival rate is a dismal 14--30% \[[@B1]\]. Hindering the development of effective treatments for the cancer is the fact that the molecular events responsible for the biological behavior of ovarian cancer are poorly understood. Implicated in both ovarian tumorigenesis and physiological follicular proliferation are the insulin-like growth factor I (IGF-I) and IGF-II systems. IGFs are regulated by at least six members of the IGF binding protein (IGFBP) family. High levels of IGFBP2 were detected in the serum or cystic fluid from patients with ovarian cancer compared with those with benign and borderline tumors \[[@B2]-[@B4]\]. A recent study further showed that IGFBP2 mRNA was overexpressed to a greater extent in advanced-stage serous ovarian cancer than normal ovarian tissue \[[@B5]\]. In addition, the increase in IGFBP2 expression was found to correlate positively with the levels of the serum tumor marker CA125 \[[@B4]\]. The progression-free interval and overall survival have also proved to be significantly shorter in patients with a high serum level of IGFBP2 at diagnosis than in those with lower levels \[[@B6]\]. Taken together, these data suggest an important role for IGFBP2 in the biology of ovarian cancer. IGFBP2 is also overexpressed in a wide spectrum of other cancers, including glioma, prostate cancer, synovial sarcoma, neuroblastoma, colon cancer, adrenocortical cancer, lung cancer, Wilms\' tumor, and hepatoblastoma \[[@B7]-[@B17]\]. The overexpression of IGFBP2 also correlates with the aggressiveness of some tumors, including prostate cancer, hepatoblastoma and glioma \[[@B10],[@B17],[@B18]\], suggesting that IGFBP2 possesses a carcinogenic property. The observation that IGFBP2 has an RGD motif suggests that IGFBP2 modulates the integrin/cytoskeleton system. Indeed, IGFBP2 was recently found to interact with the alpha v beta 3 and alpha 5 beta 1 integrins \[[@B19],[@B20]\]. In addition, IGFBP2 has been found to stimulate the growth of prostate cancer cells, an effect that can be blocked by MAP-kinase and PI3-kinase inhibitors \[[@B21]\]. IGFBP2 was also found to be co-expressed with the vascular endothelial growth factor in pseudopalisading glioma cells surrounding tumor necrosis \[[@B22]\]. Further, IGFBP2 enhances glioma cell invasion by increasing invasion-related genes including MMP2 \[[@B23]\]. These findings collectively suggest that IGFBP2 plays a key role in human cancer development. In this study, we found that overexpression of IGFBP2 enhanced the invasiveness of ovarian cancer cells. Further, attenuation of IGFBP2 expression by siRNA reduced the invasiveness of ovarian cancer cells. Results and Discussion ====================== IGFBP2 is associated with invasive epithelial ovarian carcinoma --------------------------------------------------------------- We first performed western blotting analysis using frozen tissue specimens, which consisted of four paired normal and carcinomatous ovarian tissues, one normal ovarian tissue, and three unpaired ovarian carcinoma tissues. This result showed that IGFBP2 was frequently overexpressed in ovarian cancer tissues compared with normal ovarian tissues (Figure [1A](#F1){ref-type="fig"}). We then compared the expression of IGFBP2 in normal ovaries, borderline serous ovarian tumors, and invasive serous ovarian carcinomas using a tissue microarray, which showed that IGFBP2 was expressed at significantly different levels between normal tissues and borderline tumors (p= 0.03) and between borderline tumors and invasive carcinomas (p= 0.03) (Figure [1B](#F1){ref-type="fig"}, Table [1](#T1){ref-type="table"}). Because borderline ovarian tumors have no obvious stromal invasion or infiltrative growth in contrast with invasive ovarian carcinoma \[[@B24]\], this finding suggests that IGFBP2 overexpression could induce invasion nature of ovarian cancer. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Overexpression of IGFBP2 in ovarian carcinoma. (A) Western blotting analysis in paired normal and cancer tissues (N1 to T4), one normal ovarian (N5) and 3 unpaired ovarian cancer tissues (T6 to T8) showed frequent overexpression of IGFP2 in ovarian cancer tissues compared with normal ovarian tissues. (N: normal ovarian tissue, T: ovarian cancer tissue). (B) Expression of IGFBP2 in normal ovary (100×), borderline ovarian tumor (100×), and invasive ovarian carcinoma (200×) using tissue microarray. IGFBP2 was expressed at greater level in invasive ovarian carcinoma than normal and borderline ovarian tumors. ::: ![](1476-4598-4-7-1) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Increasing expression of IGFBP2 with progression of ovarian serous tumors\. ::: Tissue IGFBP2 expression (Scores) Pvalue ----------------------------- ---------------------------- ---------- ------- ------- ---------- --------- Normal Ovaries ◇ ◇◇◇◇◇ 0.03^\#^ Borderline Ovarian Tumors ◇◇◇◇ ◇ ◇◇ ◇◇◇◇◇ ◇◇◇◇◇ Invasive Ovarian Carcinomas ◇◇ 0.03^§^ ◇◇◇◇◇ ◇◇◇◇◇ ◇◇◇◇ ◇◇◇◇ ◇◇◇◇◇ ◇◇◇◇◇ ◇◇◇◇◇ ◇◇◇◇◇ \Each diamond represents one case (Mann-Whitney U test). ^\#^Pvalue between normal ovaries and borderline ovarian tumors ^§^Pvalue between borderline ovarian tumors and invasive ovarian carcinomas ::: IGFBP2 enhances the invasiveness of ovarian cancer cells -------------------------------------------------------- Thus far, the biological or pathophysiological role of IGFBP2 in ovarian cancer is unknown. To elucidate the role of increased IGFBP2 in ovarian cancer, we therefore generate IGFBP2-overexpressing cells. To obtain suitable cells for this study, we first examined the endogenous expression of IGFBP2 in six ovarian cancer cell lines: NIH:OVCAR3, SKOV3, PA-1, OV-90, TOV-112D, and TOV-21G. IGFBP2 was expressed at different levels in all six cell lines (Figure [2A](#F2){ref-type="fig"}). Both SKOV3 and OV-90 cells showed very low levels of endogenous IGFBP2; NIH:OVCAR3, PA-1, and TOV-112D cells expressed IGFBP2 at high levels; and TOV-21G cells expressed IGFBP2 at a relatively moderate level. We thus selected SKOV3 cell line and generated two vector transfected clones and three IGFBP2-overexpressing clones by transfection (Figure [2B](#F2){ref-type="fig"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### IGFBP2 overexpressing promotes ovarian cancer cell invasion. (A) IGFBP2 expression of six ovarian cancer cell lines. The western blotting analysis shows that the expression level of IGFBP2 is heterogeneous in cell lines. SKOV3 and OV-90 ovarian cancer cell lines have very low endogenous IGFBP2. NIH:OVCAR3, PA-1 and TOV-112D have high levels of IGFBP2 expression whereas TOV-21G has relatively moderate expression of IGFBP2. (B) Two vector transfected clones and three IGFBP2 stable clones with different expression level were obtained. The expression of IGFBP2 was determined by western blotting analysis (p: parental SKOV3 cell line, v: vector transfected cell lines, b: IGFBP2 stable cell lines). (C) The invasion capacity of stable clones showed that IGFBP2 overexpressing cells have invasion potential as 1.84 -- 2.89 fold as parental and vector transfected cells. (\*p\< 0.05) ::: ![](1476-4598-4-7-2) ::: We studied the cellular phenotype of IGFBP2-overexpressing cell lines. Similar to observations in gliomas \[[@B23]\], we did not observe differences in cell proliferation (data not shown). This is contrary to the findings in prostate cancer, adenocortical cancer and neuroblastoma, in which IGFBP2 has been found to stimulate cell proliferation \[[@B21],[@B25],[@B26]\]. We then assessed the invasive capacity by a Matrigel in vitro invasion assay. The invasiveness of the IGFBP2-overexpressing cells was 1.8- to 2.9-fold greater than that of the vector-alone-transfected cells (p\< 0.05; Figure [2C](#F2){ref-type="fig"}). Our tissue microarray findings were consistent with this finding. This suggests that acquisition of IGFBP2 is a very important step in the penetration of the extracellular matrix by ovarian cancer cells, which they may need to do before they can move to adjacent tissue and the lymphovascular space. This provides a site for occult progression or the formation of recurrent ovarian cancers. Indeed, ovarian cancer frequently spreads through the lymphatic system \[[@B27]\]. In fact, in one study, 20% of patients with early-stage invasive ovarian carcinoma whose tumors appeared to be confined to the ovary (stage I disease) were found to have lymph node metastasis \[[@B28]\]. The mortality rate in such patients is higher than that in patients without lymph node metastasis \[[@B29]\]. Our study therefore also provides strong evidence that IGFBP2 could be a factor indicating a poor prognosis. Disruption of IGFBP2 inhibits ovarian cancer cell invasion ---------------------------------------------------------- The observation that IGFBP2 increased the invasive capacity of ovarian cancer cells and those of previous studies prompted us to determine whether IGFBP2 could serve as a target of therapy for ovarian cancer. We used PA-1 ovarian cancer cells for this experiment because they express a high level of endogenous IGFBP2 and show a relatively high invasive capacity compared with other ovarian cancer cell lines. We designed siRNAs for four different target sites of IGFBP2 mRNA because not all siRNAs were expected to effectively attenuate IGFBP2 expression. Western blotting analysis showed that the IGFBP2 level after siRNA-3 transfection was comparable to the levels after the transfection of Lamin A/C and negative control siRNA in OVCAR3 and PA-1 ovarian cancer cell lines, suggesting that the siRNA-3 was not effective (Figure [3A](#F3){ref-type="fig"}). Considering that not all siRNA molecules work, this was not surprising. Therefore, we used siRNA-3-transfected cells as a negative control. siRNA-1 and siRNA-4 attenuated IGFBP2 expression in the PA-1 cells more than did either siRNA-2 or siRNA-3 (Figure [3B](#F3){ref-type="fig"}). Likewise, in the invasion assay, the invasive capacity of the cells transfected with siRNA-1 or siRNA-4 was significantly decreased compared with that of the cells transfected with siRNA-2 or siRNA-3 (p\< 0.05; Figure [3C](#F3){ref-type="fig"}). These findings therefore support to the notion that IGFBP2 is a viable target of therapy for ovarian cancer. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Attenuation of IGFBP2 inhibits ovarian cancer cell invasion. (A) Western blotting analysis of IGFBP2 after transfection of siRNA in OVCAR3 and PA-1. 4 siRNAs inhibit the IGFBP2 with various levels. IGFBP2 levels of siRNA-3 transfected cells have similar to those of Lamin A/C and negative control transfected cells. (B) Four different siRNAs were transfected to PA-1 ovarian cancer cells which has high endogenous IGFBP2. Different inhibiting levels of IGFBP2 were determined by western blotting analysis. siRNA-1 and -4 were working better than siRNA-2 and -3. (C) The invasion activity after 72 hours of siRNA transfection was significantly decreased in siRNA-1 and -4 treated cells comparing with siRNA-2 and -3 treated cells (\*p\< 0.05). ::: ![](1476-4598-4-7-3) ::: Conclusions =========== In this study, we showed that IGFBP2 was significantly overexpressed in invasive ovarian carcinomas compared with borderline ovarian tumors as well as normal ovarian tissues and that IGFBP2 increases invasion capability of ovarian cancer cells. These results provide evidence that IGFBP2 could promote ovarian cancer progression by augmenting the invasion potential. Although further investigations of molecular mechanisms are required, our findings using siRNA study support IGFBP2 as a novel target for the treatment of ovarian cancer. Methods ======= Ovarian tissues and cell lines ------------------------------ Ovarian cancer and normal control tissues were obtained from The University of Texas M. D. Anderson Cancer Center tumor tissue bank with the approval of the Institutional Review Board. The ovarian cancer cell lines NIH:OVCAR3, SKOV3, OV-90, TOV-112D, TOV-21G, and PA-1 were purchased from the American Type Culture Collection (Manassas, VA). NIH:OVCAR3 cells were maintained in RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS); SKOV3 and PA-1 cell were maintained in McCoy\'s 5a medium and Dulbecco\'s modified Eagle medium (DMEM)/F12, respectively, supplemented with 10% FBS; OV-90, TOV-112D, and TOV-21G cells were maintained in a 1:1 mixture of MCDB 105 medium and medium 199, supplemented with 15% FBS. All cells were kept at 37°C in a humidified atmosphere with 5% CO~2~. Media were routinely changed every 3 days. Construction of progression tissue microarray for ovarian serous tumors ----------------------------------------------------------------------- Formalin-fixed, paraffin-embedded archival tissue blocks from ovarian cancer patients who had undergone surgery at The University of Texas M. D. Anderson Cancer Center between 1990 and 2001 were used to construct progression tissue microarrays according to previously described methods \[[@B30]\]. The progression tissue microarray consisted of normal ovarian surface epithelium from 6 individuals, serous borderline tumors from 17 patients, and invasive serous carcinomas from 40 patients. Tissue cores with a diameter of 1.0 mm were obtained from each sample and assembled into two separate tissue array blocks. Immunohistochemistry studies ---------------------------- Polyclonal antibody against IGFBP2 (c-18; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used in the immunohistochemistry studies. This antibody is specific and does not cross-react with other isoforms of IGFBP. A standard indirect immunoperoxidase procedure (ABC-Elite; Vector Laboratories, Burlingame, CA) was used for all stains. In brief, antigen retrieval was performed by first placing unstained slides in a steamer for 25 min. The antibody against IGFBP2 (in a 1:1000 dilution) was overlaid on the tissue sections of tissue arrays, and incubation was performed at 4°C overnight. Secondary antibody incubation was performed at room temperature for 60 min. Mayer\'s hematoxylin nuclear stain was used as a counterstain. Staining intensity was graded on a 0--3 scale, where 0 = no staining as assessed by staining with anti-goat secondary antibody alone, 1 = weak (\<10%), 2 = moderate (10--50%), and 3 = strong (50--100%). The results of the immunohistochemistry studies were statistically analyzed using a Mann-Whitney nonparametric U test. Stable clone establishment -------------------------- To establish stable cell lines that overexpressed IGFBP2, we transfected SKOV3 ovarian cancer cell lines with a pcDNA3.1 expression vector encoding IGFBP2 cDNA using FuGENE6 reagent (Roche Diagnostics Corporation, Indianapolis, IN). Transfected cells were subsequently selected in the presence of G418 (300 μg/ml) for 5 weeks. The expression of IGFBP2 clones was determined from western blots of cell extracts with anti-IGFBP2 antibody (C-18). Two vector-transfected cell lines and three IGFBP2 stable cell lines were used in this study. Established stable cells were maintained without antibiotics. Western blot analysis --------------------- Equal amounts of proteins from the total cell lysates was separated by 10% SDS-PAGE and transferred electrophoretically to a Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Chicago, IL). The membrane was blocked in 5% skim milk in 1× PBS and probed with a 1:1000 dilution of a goat polyclonal anti human IGFBP2 (C-18) overnight at 4°C. An enhanced chemiluminescence kit (ECL; Amersham Pharmacia Biotech, Piscataway, NJ) was used to visualize the proteins. In vitro chemoinvasion assay ---------------------------- We used 24-well BioCoat Matrigel invasion chambers (Becton Dickinson Labware, Bedford, MA) with an 8-μm pore polycarbonate filter coated with Matrigel to measure chemoinvasion. The lower compartment contained 0.75 ml of medium with 0.5% FBS as a chemoattractant. In the upper compartment, 5 × 10^4^to 2 × 10^5^cells/well were placed in triplicate wells and incubated for 22 h at 37°C in a humidified incubator with 5% CO~2~. After incubation, the cells that had passed through the filter into the lower wells were stained with Giemsa (Fisher Scientific, Orangeburg, NY) and counted by analyzing images under a microscope using software program as described previously \[[@B31]\]. All assays were repeated at least three times. Student\'s t-test was used to analyze the differences in the invasion rates between control cell lines and stable cell lines. A pvalue of \<0.05 was considered statistically significant. siRNA transfection ------------------ Four different siRNA molecules designed for IGFBP2 mRNA and siRNA molecules for Lamin A/C and negative control were synthesized and purified (Qiagen, Valencia, CA). siRNA that targets Lamin A/C and siRNA that bears no homology with relevant human genes was used as a negative control. AATGGCGATGACCACTCAGAA was the target sequence for siRNA1; AAGGGTGGCAAGCATCACCTT was the target sequence for siRNA2; AAGCGCCGGGACGCCGAGTAT was the target sequence for siRNA3; AACCTCAAACAGTGCAAGATG was the target sequence for siRNA4; AACTGGACTTCCAGAAGAACA was the target sequence for Lamin A/C; and AATTCTCCGAACGTGTCACGT was the target sequence for the negative control. siRNAs were dissolved in siRNA suspension buffer to a final concentration of 20 μM, and the mixture was heated to 90°C for 1 min and incubated at 37°C for 60 min. PA-1 ovarian cancer cells (2 × 10^5^) were plated to a 6-well plate and allowed to adhere for 24 h; the confluency of the cell monolayer at the time of transfection was 40--60%. 5 μg of siRNA and 15 μl of RNAiFect Transfection Reagent (Qiagen, Valencia, CA) was used. The cells were incubated under normal cell culture conditions. All assays were performed 72 h after treatment. Authors\' contributions ======================= EL carried out Western blotting analysis, establishing stable cells, invasion assay and siRNA transfection, participated in the analysis of all data and drafted the manuscript. CM and IS contributed to the statistical analysis. HW and JL provided frozen tissues and carried out tissue microarray and its analysis. AN performed the analysis of invasive assay. JK participated in its design of the study. JL and WZ participated in its design and coordination and helped draft the manuscript. All authors read and approved of the final manuscript. Acknowledgements ================ We thank Hua Wang, Woonyoung Choi and Seung-Hoon Lee for helpful suggestions and comments. We thank Daniel Rosen for the assistance in tissue microarray construction. This study was partially supported by SRC from the Korea Science and Engineering Foundation. The Cancer Genomics Core Laboratory is supported by a Cancer Center Support Grant from NCI/NIH, the Tobacco Settlement Fund to M. D. Anderson Cancer Center as appropriated by the Texas Legislature, and donations from the Kadoorie Foundation and the Goodwin Foundation.	PubMed Central	2024-06-05T03:55:52.974003	2005-2-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549074/", "journal": "Mol Cancer. 2005 Feb 2; 4:7", "authors": [ { "first": "Eun-Ju", "last": "Lee" }, { "first": "Cristian", "last": "Mircean" }, { "first": "Ilya", "last": "Shmulevich" }, { "first": "Huamin", "last": "Wang" }, { "first": "Jinsong", "last": "Liu" }, { "first": "Antti", "last": "Niemistö" }, { "first": "John J", "last": "Kavanagh" }, { "first": "Je-Ho", "last": "Lee" }, { "first": "Wei", "last": "Zhang" } ] }
PMC549075	Introduction ============ AIDS is due to human immunodeficiency virus (HIV-1) infection of CD4 T cells and is characterized by the cell death of HIV-infected lymphocytes and uninfected bystander cells \[[@B1]-[@B4]\]. Recent discoveries have shown that this could be related to the extra-cellular effects of Tat, which is a toxic protein secreted early by HIV-infected cells \[[@B5],[@B6]\]. Tat was first identified as a regulatory protein essential in the HIV viral cycle due to its ability to dramatically increase HIV gene expression \[[@B7]\]. The size of Tat varies and exists in forms between 86 and 101 residues. Yet 101 residues now appear to be the dominant size in the field \[[@B6]\]. Interest in this protein was raised with the discovery that Tat was secreted from HIV-infected cells and that it could have extra-cellular functions related to AIDS pathogenesis such as Kaposi\'s sarcoma \[[@B8]\]. The extra-cellular roles of Tat are suspected to be the major reason for the maintenance of HIV-infected cells (or reservoir cells) \[[@B5]\], and could explain the failure of current antiviral therapies to eradicate HIV \[[@B9]\]. Tat induces apoptosis in different cell lines such as macrophages and cytotoxic T-lymphocytes (CTL) \[[@B10]\], which are essential for the cellular response of the immune system to eliminate virus-infected cells \[[@B6]\]. Different mechanisms by which Tat induces apoptosis in T cells were proposed: (i) The up-regulation of Fas ligand \[[@B10]\]; (ii) The up or down regulation of cellular genes encoding for cytokines \[[@B11]\], for cell survival factors such as Bcl-2 \[[@B12]-[@B14]\], for superoxide-dismutase \[[@B15]\], and for p53 \[[@B16]\]; (iii) The inhibition of the expression of manganese-dependant superoxide dismutase \[[@B17]\]; (iv) The activation of cyclin dependant kinases \[[@B18]\]. Another mechanism was proposed that involves microtubules to induce Tat-mediated apoptosis \[[@B19]\]. We showed recently, using two short Tat variants of 86 residues, that Tat is able to form a complex with tubulin \[[@B20]\]. Microtubules are tubulin polymers necessary for the change and preservation of cellular morphology, intracellular organelle distribution, chromosome migration during mitosis, cell differentiation, as well as intracellular transport and signalization \[[@B21]\]. Microtubule damaging agents (MDAs) are classified in microtubule-stabilizing agents such as Taxanes and microtubule depolymerizing agents such as Vinca.alkaloids. The MDAs inhibit microtubule dynamics in living cells and lead to apoptosis after cell cycle disturbance \[[@B21]-[@B24]\]. They activate the intrinsic mitochondrial apoptotic pathway as shown by the mitochondrial membrane potential collapse and the opening of the permeability transition pore \[[@B25],[@B26]\]. The subsequent release of pro-apoptotic factors, such as cytochrome c, leads to the caspase cascade activation and thus to apoptosis \[[@B27]\]. Moreover, MDAs can also directly affect mitochondria, as shown in vitroby the release of cytochrome cfrom isolated mitochondria \[[@B28]\]. The aim of this study was to evaluate the consequence of Tat binding to microtubules and the correlation with the mitochondria-mediated T cell apoptosis. We used three Tat variants with sequences from 99 to 101 that have the size of the main Tat variants found in the field \[[@B6]\]. The HIV-1 HxB2 and HIV-1 Eli are representative of rapid progressor (RP) patients in respectively Euro-American strains \[[@B29]\] and African strains \[[@B30]\]. HIV-1 Oyi strain was isolated from a highly exposed persistently sero-negative (HEPS) individual during an epidemiological study in Gabon \[[@B31]\]. In order to identify the regions of Tat essential for the interaction with microtubules, we used peptides corresponding to different part of the Tat sequence. We compared the effect of these Tat variants and Tat peptides with paclitaxel, a well-known anti-cancer drug agent that strongly increases tubulin polymerization and stabilizes microtubules \[[@B32]\]. Results ======= Tat acts on tubulin in vitro ------------------------------ The minimal concentration of tubulin (Cr) necessary to obtain tubulin polymerization without drug was 7 μM in our buffer conditions \[[@B33]\]. We measured the polymerization effect of various concentrations of Tat and paclitaxel with 15 μM tubulin. We did observe the enhancement of tubulin into microtubules in the presence of Tat and paclitaxel (Fig. [1A](#F1){ref-type="fig"}). In comparison with tubulin alone (line 1), the rate of assembly as well as the final extent of assembly was enhanced by Tat. Furthermore, the lag time to start polymerization is shorter with Tat (lines 2 and 4) as compared with tubulin alone (line 1). With 4 μM (line 2) and 8 μM Tat (line 4), turbidity reached a plateau at 0.55 and 1.20 respectively, which corresponds to a 1.7 and 3.7 fold increase compared with the control plateau at 0.32 obtained with tubulin alone (line 1) (Fig. [1A](#F1){ref-type="fig"}). With higher concentrations of Tat (16 μM and 20 μM) the turbidity plateaus were superior to 5. The lowest effective concentration of Tat that induced an increase in polymerization was 0.5 μM (data not shown). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Turbidity time course of the in vitro microtubule assembly. (A) Tubulin in presence of various ligands. Samples with 15 μM tubulin are maintained at 4°C. After addition of 8 μM Tat (line 4), 4 μM Tat (line 2) from HIV-1 HxB2 strain or 15 μM paclitaxel (line 3), the assembly reaction was started by warming the samples at 37°C (time 0, arrow) and compared to tubulin alone (line1). The ΔOD350 nm is measured every 30 sec. After 30 min the temperature was lowered to 10°C (arrow). (B) Electron microscopy of microtubules formed in the presence of Tat. Aliquot from samples reaching the ΔDO350 nm plateau at 37°C were adsorbed on coated Formvar films on copper grids. Electron micrographs of microtubules formed without Tat (Control) or with indicated concentrations of Tat and paclitaxel are presented with 4000-fold magnification. Microtubules formed with Tat at 8 μM are also presented at 40000-fold magnification. (C) Production of microtubules in the presence of Tat. Samples of 15 μM tubulin alone (Control) and with 8 μM Tat (Tat 8), 16 μM Tat (Tat 16) or 15 μM paclitaxel (Paclitaxel) at the time they reach the plateau at 37°C were ultracentrifuged and supernatant (S) and pellets (P) were analyzed on SDS-PAGE. (Tat 8) × 5 indicates a fivefold increase in the quantity of the sample loaded. The mass of tubulin (Tub 55 kDa) and Tat (Tat 10 kDa) are indicated. ::: ![](1742-4690-2-5-1) ::: When the temperature of the samples was decreased to 10°C, we observed a complete depolymerization for tubulin alone (line 1) and tubulin with 4 μM and 8 μM Tat (lines 2 and 4 respectively). Even at the highest concentrations of Tat (16 μM and 20 μM), at 10°C the turbidity decreased to the original values (data not shown). By contrast, in the same conditions, we did not observe any depolymerization in the presence of paclitaxel (line 3). However, full depolymerization in the presence of paclitaxel was obtained with a longer incubation at 4°C (data not shown). The reversibility of following an incubation at 10°C or 4°C strongly suggests that turbidity enhancement is not related to precipitation or aggregation but to microtubule formation. We also observed that when Tat concentration is increased to stoichiometric quantities with tubulin, the turbidity values increased dramatically to an absorbance greater than 2. These values are superior to the absorbance obtained in the same conditions using the stoichiometric quantity (15 μM) of paclitaxel, which induces effective tubulin polymerization. Turbidity is known to be a function of the total weight concentration of scattering particles only when the particles have small diameters compared with the wavelengths of the incident light \[[@B34],[@B35]\]. Thus, it seemed Tat would be able to act quantitatively and qualitatively on tubulin polymerization. In order to address these possibilities, our first control was to verify the shape of microtubules formed in the presence of Tat. Examination with electron microscopy confirmed the formation of microtubules in the presence of Tat, that were similar in shape to the controls without Tat (Fig. [1B](#F1){ref-type="fig"}). However, microtubules in the presence of Tat and paclitaxel were slightly packed and shorter than non-treated microtubules. These phenomena were in part responsible for the variation in turbidity. To determine whether a part of the enhancement of turbidity was due to an increase in tubulin polymerization, we did evaluate the amount of polymerized tubulin on gel after ultra-centrifugation. Results in figure [1C](#F1){ref-type="fig"} show that with Tat or paclitaxel the band intensity of pellets (P) are enhanced and the band intensity of supernatants (S) are decreased compared with the control tubulin alone. Increasing the concentration of Tat to 8 μM (Tat 8) and 16 μM (Tat 16) indeed enhanced the proportion of tubulin in the pellets (P) and decreased the proportion of tubulin in the supernatant (S) (Fig. [1C](#F1){ref-type="fig"}). Thus, although the enhanced turbidity is due in part to microtubule aggregation, these results show that the increase expansion in the turbidity plateau is also due to an increasing amount of microtubules. Interestingly, analysis of the samples by gel electrophoresis, using a five-fold amount of the tubulin samples loaded on gels showed that Tat was only detectable in the pellet fraction (Fig. [1C](#F1){ref-type="fig"}). To eliminate non-specific binding, we used a glycerol cushion during the ultra-centrifugation step \[[@B33]\]. This confirmed that Tat was associated with the microtubules. We evaluated the ability of different Tat variants to modify tubulin polymerization. In our conditions, when 15 μM of tubulin are incubated at 37°C with 8 μM Tat HxB2, Eli or Oyi, the turbidity plateau values at 350 nm are 1.01, 1.61 and 0.80 respectively (Table [1](#T1){ref-type="table"}). These values are higher than the turbidity value of 0.30 obtained with 15 μM tubulin alone (Table [1](#T1){ref-type="table"}). Thus Tat from different HIV subtypes are able to enhance tubulin polymerization. However, Tat Eli and Tat HxB2 are more effective in this enhancement than Tat Oyi (Table [1](#T1){ref-type="table"}, column O.D. ratio). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Enhancement of tubulin polymerization by different Tat and derived peptides ::: Compounds Concentration ΔOD~350\ nm~ ΔOD ratio ---------------- --------------- -------------- ----------- none 0.31 1 Tat HxB2 8 μM 1.01 3.31 Tat OYI 8 μM 0.80 2.63 Tat ELI 8 μM 1.61 5.27 Paclitaxel 15 μM 0.55 1.79 Pep38--72 OYI 8 μM 0.78 2.56 Pep38--72 ELI 8 μM 1.59 5.20 Pep73--101 OYI 8 μM 0.31 1.01 Pep73--99 ELI 8 μM 0.30 1 15 μM tubulin is incubated at 37°C in buffer, the increase of ΔOD~350\ nm~is followed and the value of the plateau is indicated in the column ΔOD~350\ nm~. Lane 1 is the control with tubulin alone (none), lanes 2--4 are Tat variants, lane 5 is the paclitaxel control, lanes 6--9 are the Tat peptides. The ΔOD~350\ nm~ratio is the value that the ΔOD~350\ nm~plateau reaches in presence of the indicated product divided by the value of the ΔOD~350\ nm~plateau reached with tubulin alone. ::: To identify the sequence(s) region(s) implicated in tubulin, peptides overlapping the sequence of Tat HxB2 and Tat Oyi were synthesized. The sequences of peptides 38--72 and 73--99 derived from both Eli and Oyi are indicated in figure [2](#F2){ref-type="fig"}. Using 8 μM of peptide 38--72 from Eli (line Pep38--72 ELI) and Oyi (line Pep38--72 OYI) in the presence of 15 μM tubulin, we obtained a turbidity plateau value of 1.59 for Eli and 0.78 for Oyi which correspond to an increased ratio of 5.20 and 2.56 (Table [1](#T1){ref-type="table"}). These effects were comparable with the effect of the entire parental Tat proteins. Turbidity with the peptide 73--99 derived from Eli (Pep73--99 ELI) and Oyi (Pep73--101 OYI) were at the same level as the control, indicating that they were not active in tubulin polymerization (last lines, Table [1](#T1){ref-type="table"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Sequences of HIV-1 Tat strains. These three Tat variants and the peptides were obtained by solid phase synthesis with a procedure previously described \[43, 50\]. Sequences correspond to the viral strains HIV-1 Eli \[30\], HIV-1 HxB2 \[29\] and HIV-1 Oyi \[30\]. ::: ![](1742-4690-2-5-2) ::: Tat-mediated apoptosis in T-cells correlates with the effect on microtubules ---------------------------------------------------------------------------- Drugs that are able to bind to tubulin and/or microtubules are known to affect the microtubule network organization and block the cell cycle in mitosis, leading to cell death. We first evaluated the toxicity of the different Tat variants on Jurkat lymphocyte cells, using 4\'6-diamidino-2-phenylindole (DAPI) staining. This method allows the quantification of apoptotic cells by the observation of the characteristic nuclear fragmentation. After 20 hours treatment, the percentage of apoptotic cells in the presence of 10 μM Tat HxB2 (11 %) and 10 μM Tat Eli (12.5 %) were higher than those obtained with 10 μM Tat Oyi (3.5 %) (Fig. [3A](#F3){ref-type="fig"}). These differences in apoptosis were significant (p= 0.037) using one-sided ANOVA. The percentages of apoptotic cells in the presence of 1 μM Tat Oyi were similar to the control cells (0.1 %) (Fig. [3](#F3){ref-type="fig"}). The percentages of apoptotic cells in the presence of 1 μM Tat HxB2 (2.7 %), 1 μM Tat Eli (2 %) and 10 μM Tat Oyi (3.5 %) was weak and comparable to the basal level of the non-treated control cells (Fig. [3A](#F3){ref-type="fig"}). Thus, at 10 μM Tat HxB2 and Tat Eli induced apoptosis inversely to 10 μM Tat Oyi. These results indicate that Tat-mediated apoptosis in T-cells could be correlate with the effect on microtubules since the differences in toxicity between the different Tat variants correspond to the same differences in tubulin polymerization. These differences were also observed using trypan blue exclusion (data not shown). Tat toxicity was then confirmed by flow cytometry analysis after propidium iodide (Pi) staining. Figure [3B](#F3){ref-type="fig"} shows that 1 μM and 10 μM Tat HxB2 induces 13% and 27% apoptosis respectively, revealed by the proportion of cells with hypodiploid DNA content (Fig. [3B](#F3){ref-type="fig"}, H values). A high proportion of apoptotic cells (31 %) was also obtained with 10 μM of Tat Eli. Furthermore, the percentage of hypodiploid cells obtained with 10 μM Tat Oyi is low (10.4 %) and is the same as the control without Tat (9.9 %). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Effect of different Tat variants on cell cycle progression, apoptosis and microtubule network in lymphocytes. (A) Table showing data from fluorescence microscopy after 20 hours treatment with indicated concentration of Tat or paclitaxel. Percentage of apoptotic cells are determined after DAPI staining and the differences in apoptosis between treated and untreated cells used as control obtained in three independent experiments are presented. (B) Jurkat cells were treated with indicated concentrations of paclitaxel and various Tat or were untreated (control). After 20 hours, cells were stained with PI and analysed by flow cytometry. Percentage of cells in G2/M (G2 / M) or apoptotic cells (H) with hypodiploid DNA are indicated in the upper corner of each cell (C) Jurkat cells were treated with 10 μM Tat Eli or 1 μM paclitaxel and processed for immunofluorescence labeling with anti-alpha tubulin antibody as described in materials and methods. Control corresponds to untreated cells. ::: ![](1742-4690-2-5-3) ::: Tat does not affect microtubule network organization nor cell cycle progression ------------------------------------------------------------------------------- Paclitaxel stabilizes the microtubule network by inducing the formation of pseudoasters in mitotic cells and the formation of bundles, structures that correspond to strongly associated microtubules, in interphasic cells \[[@B21]\]. Considering that Tat increased microtubule polymerization, we investigated if Tat was able to induce bundle formation or another disturbance in the microtubule network organization in the treated cells. The effects of Tat and paclitaxel cytotoxic concentrations (10 μM and 1 μM respectively) were evaluated by immunofluorescence microscopy on Jurkat cells. After 6 hours treatment with paclitaxel, strong modifications of the microtubule network (that form bundles) were observed (Fig. [3C](#F3){ref-type="fig"}). However, in the presence of 10 μM Tat, we did not observe bundles or any other modification of the microtubule network. By disturbing microtubule functions, MDAs generally lead to a mitotic block of treated cells. Although study have implicated the transactivation effect of Tat on cyclin and other gene implicated in cell cycle progression, we examined if Tat induced a mitotic block that could be due in part to the tubulin polymerization of Tat. Following treatment, the percentage of cells in various phases was obtained by flow cytometry analysis after propidium iodide incorporation. After 20 hours treatment, 1 μM paclitaxel blocks almost 50 % of the cells in the G2/M phase (Fig. [3B](#F3){ref-type="fig"}, G2/M values). This strong blockage in G2/M with paclitaxel attests that we were in the appropriate time conditions to evaluate the effects of Tat on cell cycle regulation in these Jurkat cells. A longer incubation time (i.e.48 h) in the presence of paclitaxel leads to 90 % of cell death (data not shown). After 20 hours treatment, the percentage of the G2/M cells in the presence of 1 μM Tat HxB2 or 10 μM Tat Oyi were 28 % and 32 % respectively and similar to the 33 % observed in the untreated cells (Fig. [3B](#F3){ref-type="fig"}). In the presence of 10 μM Tat HxB2 or 10 μM Tat Eli the percentage of G2/M cells (22 % and 24 % respectively), were lower than the control cells (33 %). This decrease of the G2/M is not correlated with an increase in G1, and seems to be related to the strong cell death (30 %) rather than to a specific block in another cell cycle phase. In parallel, DAPI results confirmed that the percentages of metaphasic cells were similar between untreated cells and Tat-treated cells (1 %), in contrast to paclitaxel-treated cells that were blocked in metaphase (50 %). Thus, contrary to paclitaxel, the different Tat variants did not lead to a G2/M block before inducing apoptosis. Tat induces the release of cytochrome c from isolated mitochondria ------------------------------------------------------------------ MDAs induce apoptosis through the mitochondrial apoptosis pathway. Our previous work on paclitaxel showed that this agent is able to induce cytochrome crelease from purified mitochondria \[[@B28]\]. To investigate whether Tat could directly target mitochondria, we isolated these organelles and incubated them with growing concentrations of Tat Eli (0.2 μM, 2 μM and 10 μM). Using Western Blot analysis, we detected cytochrome cin the supernatant of mitochondria incubated with Tat Eli (Fig. [4A](#F4){ref-type="fig"}, Cyt c). Enhanced amounts of cytochome cwere observed with 2 μM and 10 μM Tat Eli (lane T10 and T2) whereas the level of cytochrome cwith 0.2 μM of Tat Eli (lane T0.2) was comparable to the untreated mitochondria (lane T-). In parallel, cytochrome clevels decreased slightly in mitochondria treated with Tat, as revealed by Western Blot of the pellets (Fig. [4B](#F4){ref-type="fig"}, Cyt c). Levels of VDAC, mitochondrial outer membrane porin, were systematically measured to ensure that equal amounts of mitochondria were present in the pellets (Fig. [4B](#F4){ref-type="fig"}, VDAC), and that no mitochondria remained in the supernatants (Fig. [4A](#F4){ref-type="fig"}, VDAC). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Tat induces cytochrome crelease from isolated mitochondria. After 2 hour treatment with 10 μM (P10) of peptide 73--99 or 0.2 μM (T0.2), 2 μM (T2) and 10 μM (T10) of Tat Eli or without peptide (T-), samples were centrifuged and supernatants and pellets were separated. (A). Western blots of the supernatant with antibodies against cytochrome c(Cyt C) and with antibodies against VDAC. (B) Western blots of Mitochondria Pellets. Data are representative of four independent experiments. ::: ![](1742-4690-2-5-4) ::: Discussion ========== Our in vitrostudy clearly shows that Tat directly interacts with microtubules and is able to enhance their formation. Turbidimetry tests showed that Tat strongly enhanced pure tubulin polymerization. The Tat concentration inducing tubulin polymerization is higher compared to the concentration of Tat in the plasma and it is possible that Tat concentration increases in the cell due to an active uptake similar to what is observed with paclitaxel. Furthermore, it is also possible that Tat concentration in the plasma is higher nearby HIV infected cells. We found that Tat acts qualitatively on microtubules making them shorter, such as paclitaxel, and also induces microtubule packing. These effects on microtubule formation are more pronounced than with paclitaxel as observed by turbidimetry and electron microscopy. The high effect of Tat on the self-association of tubulin is of interest for studies on the mechanism of microtubule formation and could be used in the design of new agents targeting microtubules. We also showed that the full-length Tat protein is not necessary to obtain tubulin polymerization enhancement as peptides derived from its central region harbored the same properties as the full-length protein. These peptides contain the glutamine-rich region of Tat and confirms that the glutamine-rich region of Tat is involved in Tat mediated apoptosis \[[@B20]\]. It also contains the basic region (sequence 49--60) that is essential for Tat to cross membranes \[[@B36]\] and the adjacent region (sequence 38--48) that has been previously shown to be involved in the Tat-tubulin interaction \[[@B19]\]. In this study, we demonstrate that the N-terminus and the C-terminus of Tat are not necessary for the interaction with tubulin. We showed that the more toxic Tat variants were also those that were more active in the polymerization of tubulin in vitro, suggesting that the toxicity of extra-cellular Tat is mediated by a mechanism involving microtubules. MDAs such as paclitaxel, which affect tubulin polymerization, generally disturb the microtubule network functions and inhibit cell proliferation. Contrary to paclitaxel, in the presence of high concentrations of Tat, we did not observe cell cycle arrest in G2/M. Moreover, by immunofluorescence microscopy, we did not detect any early modification of the microtubule network organization, even with high Tat concentrations. MDAs are able to induce apoptosis at lower doses than those required to induce bundles \[[@B21]\]. Interestingly, low concentrations of MDAs can suppress microtubule dynamics and disturb their functions, without modifying microtubule network morphology \[[@B21],[@B27],[@B37]\]. Thus, low cytoplasmic concentrations of Tat could modulate microtubule dynamics participating in the apoptotic signaling pathway induction in lymphocytes. The effect of Tat on microtubule dynamic needs to take in account that Tat could also modify the activity of proteins acting on microtubule dynamic such as LIS1 \[[@B38]\]. We hypothesize that Tat is able to induce the release of pro-apoptotic factors from mitochondria as we have previously shown this property for microtubule damaging agents \[[@B28]\]. We show here for the first time that Tat is able to directly affect isolated mitochondria and trigger cytochrome crelease, a key event in the mitochondrial apoptotic pathway activation. This result is in agreement with those from Macho et al.that show that, in lymphocyte cultures under low serum conditions, Tat accumulates at the mitochondria. Moreover, this localization correlated with disruption of the mitochondrial membrane potential \[[@B39]\], a process that leads to the release of pro-apoptotic factors such as cytochrome c. Thus, we show in this study that secreted Tat can both act on tubulin polymerization and on the mitochondria in vitro. Interestingly, although our study involves only three Tat variants, our results show that, in comparison with Tat Oyi, the Tat HxB2 and Tat Eli derived from RP are more potent in tubulin polymerization and apoptosis induction in T-cell. This open the possibility that these properties of Tat could be associated with more severe disease phenotype. Apoptosis of bystander cells has been demonstrated to be very important in AIDS \[[@B40]\] and the rate of lymphocyte apoptosis has been correlated with progression rates \[[@B41]\]. It now appears that extra-cellular Tat secreted from HIV-infected cells is involved in the apoptosis of non-infected T cells. The elucidation of the mechanism responsible for Tat-mediated inhibition of the immune response therefore should have a tremendous impact for AIDS therapy. Methods ======= Protein synthesis, Purification and Biochemical Characterization ---------------------------------------------------------------- Tat variants and their derived peptides were synthesized with an ABI 433A peptide synthesizer (Perkin Elmer, Applied Biosystem Inc.) with FASTMoc chemistry according to the method of Barany and Merrifield \[[@B42]\] on 4-hydroxymethyl-phenoxy-methyl-copolystyrene-1% divinylbenzene preloaded resin (HMP; 0.50--0.65 mmol; Perkin Elmer, Applied Biosystem Inc., Foster City, CA), as previously described \[[@B43],[@B44]\]. Purity and integrity of proteins were confirmed by amino acid composition (6300 Beckman analyzer), partial sequence analyses (473A Protein Sequencer, Applied Biosystem) and by MALDI-TOF mass spectrometry (Perspective Biosystems, Voyager DE-RP). Purified Lamb Brain Tubulin --------------------------- Lamb brain tubulin was purified by ammonium sulfate fractionation and ion-exchange chromatography, stored in liquid nitrogen and prepared for use as described \[[@B45],[@B46]\]. During this purification particular caution was taken to remove microtubule-associated proteins (MAPs). Protein concentrations were determined spectrophotometrically with a Perkin-Elmer spectrophotometer Lambda 800. Tubulin extinction coefficient is ε~275\ nm~= 1.07 L·g^-1^·cm^-1^in 0.5% SDS in neutral aqueous buffer or ε~275\ nm~= 1.09 L·g^-1^·cm^-1^in 6 M guanidine hydrochloride. Tubulin polymerization ---------------------- In vitrostudies have been derived from experiments that show that the variation of temperature permits the disassembly/reassembly cycles of tubulin in the presence of GTP and Mg^2+^\[[@B47],[@B48]\]. Microtubule assembly was performed in 20 mM sodium phosphate buffer, 1 mM EGTA, 10 mM MgCl~2~, and 3.4 M glycerol, pH 6.5. The various concentrations of Tat or paclitaxel were mixed with tubulin at 4°C. The reaction was started by warming the samples to 37°C in a 0.2 × 1 cm cells, and the mass of polymer formed was monitored by turbidimetry at 350 nm using a Beckman DU7400 spectrophotometer thermostated. Depolymerization occurs when the temperature is lowered to 10°C. The data showed in fig [1a](#F1){ref-type="fig"} were representative of three independent experiments. ΔOD~350\ nm~values correspond to values without the buffer. The microtubule mass was determined by centrifugation assay. Samples were taken at the turbidity plateau at 37°C. They were ultra-centrifuged at 50,000 rpm for 15 min at 37°C using fixed angle rotor TLA 100.2 in a TL100 Beckman apparatus. In these conditions, microtubules precipitate in the pellet and non- polymerised tubulin remains in the supernatant. Pellet and supernatant were loaded separately on 10 % acrylamide SDS-PAGE, colored with Coomassie blue after migration (Fig. [1C](#F1){ref-type="fig"}). Electron microscopy ------------------- Small aliquots of tubulin associated with Tat were adsorbed on coated Formvar films on copper grids. They were negatively stained for 1 min in 2 % uranyl acetate and observed with a Jeol 1220 electron microscope. Cell Culture ------------ The lymhoblastoid Jurkat cell line was cultured in RPMI 1640 supplemented with 2 mM ultraglutamine (BioWhittaker, Verviers, Belgium), penicillin (100 U/ml), streptomycin (100 μg/ml), and 10 % heat-inactivated fetal calf serum (BioWhittaker, Verviers, Belgium). Human neuroblastoma SK-N-SH cells were routinely maintained at 37°C and 5 % CO~2~, in standard culture RPMI-1640 medium (BioWhittaker, Verviers, Belgium) containing 10 % fetal bovine serum (BioWhittaker), 2 mM glutamine (BioWhittaker, Verviers, Belgium), 1 % penicillin and streptomycin (BioWhittaker, Verviers, Belgium). Exponentially growing cells (10^5^cells/ml) were seeded 3 days before paclitaxel or Tat treatment. Drugs and Antibodies -------------------- Stock solution of paclitaxel (Sigma) was prepared in DMSO. The final concentration of DMSO used in cell culture was less than 0.05 %. For Western blotting, anti-VDAC antibody (anti-porin, 31 HL France Biochem, Meudon, France) was used at 1/500 and anti- cytochrome cantibody (7 h82C12; PharMingen, San Diego, CA) was used at 1/1000. Secondary antibody was goat antimouse monoclonal antibody conjugated with peroxidase (Jackson ImmunoResearch Laboratories, USA). The antibodies used for immunofluorescence microscopy were anti-α-tubulin (clone DM1A Sigma, Saint Louis, USA) and FITC mouse anti-goat IgG (Jackson ImmunoResearch Laboratories, USA). Cell death analysis ------------------- Jurkat cells at a concentration of 5 × 10^5^cells/ml were incubated in the absence or presence of Tat or paclitaxel at different concentrations (see Fig. [3](#F3){ref-type="fig"}). After 20 hours incubation at 37°C, they were counted under optic microscope and cell death was estimated by trypan blue exclusion. Cells were then permeabilized with glacial alcohol, stained with propidium iodide and DNA content was measured by flow cytometry (Facs Calibur, Becton Dickinson, Mississauga, Canada). Cytogram analysis was performed with Cell Quest Pro^®^software (Becton Dickinson, Mississauga, Canada). The proportion of hypodiploid cells was used as an estimate of apoptosis. Cell death was further analysed using fluorescence microscopy of cells stained with DAPI. 5 × 10^5^. Jurkat cells/ml were incubated at 37°C for 20 hours with various Tat or paclitaxel in Lab-Tek chamber slides. (Nalge Nunc International., USA). After centrifugation for 10 min at 3500 rpm, spin cells were fixed with 3.7 % formaldehyde, permeabilized with 0.1 % saponin and treated with PBS containing 10 μg/ml 4\'6-diamidino-2-phenylindole (DAPI) (Sigma). The morphology of the cell nuclei was observed with a fluorescence microscope using an excitation wavelength of 350 nm. Nuclei were considered to have the normal phenotype when glowing bright and homogenously. Specific well drawn line up chromosome were seen in equatorial plates for cells in metaphase. Apoptotic nuclei were identified by the condensed chromatin gathering at the periphery of the nuclear membrane or a total fragmented morphology of nuclear bodies. Cells were counted and the percentage of normal., mitotic and apoptotic nuclei determined. Data indicated in the figure [3](#F3){ref-type="fig"} are representative of three independent experiments. The apoptosis data obtained from the three independent experiments in the presence of paclitaxel and various Tat were compared using one-sided ANOVA following baseline subtraction using the values of untreated control cells. Indirect immunofluorescence staining of alpha-tubulin ----------------------------------------------------- 10^6^Jurkat cells/ml were treated for 6 hours with 10 μM Tat HxB2 or 1 μM paclitaxel and were processed for tubulin indirect immunofluorescence staining as previously described \[[@B49]\]. After incubation with paclitaxel or Tat for 6 hr in lab-tek chamber slides (Nalge Nunc International, USA), the cells were centrifuged for 10 min at 3500 rpm. They were then fixed with 3.7 % formaldehyde and permeabilised with 0.1% saponin. Immunofluorescence microscopy of the microtubule network was performed using anti- α-tubulin antibody (Amersham, USA) and an FITC-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, USA). Effect on mitochondria ---------------------- Mitochondria were isolated by cell fractionation from the neuroblastoma SK-N-SH cell line as previously described \[[@B28]\]. 9 × 10^7^cells were suspended in a sucrose buffer (250 mM sucrose, 1 mM dithiothreitol, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1.5 mM MgCl~2~, phenylmethylsulfonyl fluoride, protease inhibitors, 20 mM Hepes, pH 7.4) at 4°C, homogenized with 50 strokes in a glass homogenizer (Kimble Kontes, Vineland, NJ), and centrifuged twice at 800 g for 10 min. The supernatants were then centrifuged at 15,000 g for 10 min at 4°C. Mitochondrial pellets were immediately washed three times in the sucrose buffer. Isolated mitochondria were aliquoted and incubated for 2 hr at 37°C with various Tat concentrations. After centrifugation (15,000 g for 10 min at 4°C), pellets and supernatants were carefully separated. Mitochondrial pellets were lysed in lysis buffer (62.5 mM Tris.HCl; pH 6.8, 0.5% SDS, 5% mercaptoethanol, 10% glycerol) and loaded in parallel with the supernatants on a reducing 15 % SDS-PAGE gel. The gel was processed for immunoblotting and revealed using antibodies against VDAC and cytochrome c. Anti-VDAC antibody (Calbiochem, USA) was used to ensure that equal amounts of mitochondrial proteins were loaded on the gels and that no mitochondria or lyzed mitochondria were present in the supernatant. Secondary antibodies were added and visualization was carried out using an enhanced chemiluminescence detection kit (ECL, Amersham, Aylesbury, United Kingdom). The results presented are representative of four independent experiments. List of abbreviations ===================== HIV, human immunodeficiency virus MDA, microtubule damaging agent VDAC, voltage-dependent anion selective channel. Competing interest ================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= JdM carried out most of the experimental work, performed the experimental design and drafted the manuscript. MC performed part of the cell, mitochondrial and immunofluorescence study directed by DB. PB performed tubulin purification, part of tubulin experiments on polymerization, quantification of microtubules and electron microscopy directed by VP. GRC, SL, SO, DE and JW synthezised the peptides and characterized them biochemically and biophysically. CP performed FACS studies. EPL participated in the design of the study, the redaction of the manuscript and funded the studies. Acknowledgements ================ This work was supported by Agence Nationale pour la Valorisation de la Recherche (ANVAR), Conseil régional Provence Alpes Côtes-d\'Azur, Conseil Général des Bouches-du-Rhones, Ville de Marseille, Association Faire Face au SIDA, Centre National pour la Recherche Scientifique (CNRS) and the Université d\'Aix-Marseille. We thank Eddy Paquier for his help. We thank Alain Renaud for technical assistance. SO and JW have scholarships from the Conseil Régional Provence Alpes Côtes-d\'Azur and SynProsis. GC has a scholarship from the Entente Cordiale program between the UK and France.	PubMed Central	2024-06-05T03:55:52.975936	2005-2-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549075/", "journal": "Retrovirology. 2005 Feb 3; 2:5", "authors": [ { "first": "Jean", "last": "de Mareuil" }, { "first": "Manon", "last": "Carre" }, { "first": "Pascale", "last": "Barbier" }, { "first": "Grant R", "last": "Campbell" }, { "first": "Sophie", "last": "Lancelot" }, { "first": "Sandrine", "last": "Opi" }, { "first": "Didier", "last": "Esquieu" }, { "first": "Jennifer D", "last": "Watkins" }, { "first": "Charles", "last": "Prevot" }, { "first": "Diane", "last": "Braguer" }, { "first": "Vincent", "last": "Peyrot" }, { "first": "Erwann P", "last": "Loret" } ] }
PMC549076	Introduction ============ According to the United States National Institute of Mental Health (NIMH), Bipolar Disorder (BPD), also known as manic-depressive illness, is a serious medical illness that causes shifts in a person\'s mood, energy, and ability to function. Different from the normal ups and downs that everyone goes through, the symptoms of bipolar disorder are severe. Bipolar disorder is a complex, chronic condition associated with considerable morbidity and mortality, including a high rate of suicide. Bipolar disorder causes dramatic mood swings from overly \"high\" and/or irritable to sad and hopeless, and then back again, often with periods of normal mood in between. Severe changes in energy and behavior go along with these changes in mood. The periods of highs and lows are called episodes of mania and depression. Most people with bipolar disorder can achieve substantial stabilization of their mood swings and related symptoms over time with proper treatment. A strategy that combines medication and psychosocial treatment is optimal for managing the disorder over time. Background ========== Omega-3 fatty acids (0-3FA) may have a beneficial effect on irritable mood. Low O-3FA levels in red blood cell membranes of depressed patients hint that O-3FA may be helpful in treating mood disorders \[[@B1]\]. A recent article has given an excellent review of O-3FA and studies showing their effectiveness in depression, bipolar disorder and aggression \[[@B2]\]. In this article, two published studies are discussed that have reported on similar therapeutic effects of O-3FA \[[@B2]\]. One placebo-controlled study of 20 patients revealed that ethyl ester of eicosapentaenoic acid (E-EPA) was effective in stabilizing the moods of depressed patients \[[@B3]\]. Another report, a double-blind, placebo controlled study (N = 22/19), measured the effect of O-3FA docosahexaenoic acid (DHA) on the aggressive tendencies of college students. The O-3FA DHA group (1.5--1.8 g O-FA DHA/day) did not display any increase in aggressive tendencies when external stressors peaked, while the placebo group displayed a significant increase in their aggressive tendencies under similar circumstances \[[@B4]\]. In a recent study, 25% of 111 patients with bipolar-I disorder who met criteria for a DSM-IV major depressive episode also experienced substantial irritability in the absence of associated symptoms of mania. These findings suggest that abnormal irritability is not limited to mania or mixed states \[[@B5]\]. However, recent studies give caution that at a 6 gram per day average daily dose, as a single agent, omega 3 fatty acids may not be as effective as an antidepressant \[[@B6]-[@B9]\]. O-3FA may also help with the irritability component of different clinical conditions, such as depression, mania, schizophrenia, borderline personality disorder and other psychiatric conditions with a common presenting sign of irritability. Numerous other conditions have an irritability component, including Borderline Personality Disorder, Alzheimer\'s disease, Premenstrual Dysphoric Disorder, to name a few \[[@B10]-[@B12]\]. There is one report suggesting beneficial effect of Omega-3 Fatty acid treatment for Borderline Personality Disorder. This double-blind, placebo-controlled pilot study specifically showed that EPA may influence both aggression and depression \[[@B12]\]. Although attention-deficit/hyperactivity disorder (ADHD) also has an irritability component, recent publications bring doubt to the O3FA connection in ADHD \[[@B13],[@B14]\]. A recent, open ended, O-3FA add-on study has shown beneficial effect of O-3FA on irritability in 19 patients with mood disorders \[[@B15]\]. These patients had already been receiving different combinations of pharmacotherapy and talk therapy. Despite their treatment, the irritability component of their illness was still causing social, occupational and other life disturbances. Hence, they were chosen for the O-3FA add-on component of the study. In the nineteen-patient phase of the study, bipolar patients of every subtype, ages 18 to 65 years, with significant irritability were studied. All patients received a systematic assessment battery at entry and were treated by a psychiatrist, trained to deliver care and measure outcomes in patients with bipolar disorder, consistent with expert recommendations. At every follow-up visit, the treating psychiatrist completed a standardized assessment and assigns a clinical status based on DSM-IV criteria. Patients had independent evaluations at regular intervals throughout the study and remain under the care of the same treating psychiatrist while receiving variable medications and talk therapy, depending on their need \[[@B15]\]. In the 19-patient study, a paired sample t-test revealed a large decrease in the percent of days irritable after O-3FA was administered. Before treatment, the mean irritability percentage was 81.05 (SD = 23.31) and after treatment the mean irritability percentage dropped to 30.00 (SD = 36.67). Despite the small number of patients in the study (n = 19), the difference between means was statistically significant (t (18) 4.512, p \< .001). Using a paired sample t-test, a significant difference was also found between the highest irritability score (mean = 2.79; SD = 0.92) and the last recorded irritability (mean = 0.79; SD = 0.85) while taking O-3FA (t(18) = 8.270; p \< .001) \[[@B15]\]. Methods ======= This is a report on a 37-patient continuation phase of the open ended, O-3FA add-on study. Subjects consisted of the original 19 patients, in addition to the 18 new patients recruited and followed in the same fashion as the first nineteen \[[@B15]\]. Subjects carried a DSM-IV-TR \[[@B16]\] diagnosis of Bipolar Disorder and were visiting a Mood Disorder Clinic regularly throughout the length of the study. At each visit, patients\' clinical status was monitored using the Clinical Monitoring Form \[[@B17]\]. Subjects reported on the frequency and severity of irritability experienced during the preceding ten days; frequency was measured by way of percentage of days in which subjects experienced irritability, while severity of that irritability was rated on a Likert scale of 1 -- 4 (if present). The irritability component of Young Mania Rating Scale \[[@B18]\] (YMRS) was also recorded quarterly on 13 of the 39 patients consistently. The patients were asked about general dietary omega-3 intake before the fish oil was added on, and basic nutritional guidance was given to subjects at the clinic. Patients in general were not heavy fish/product consumers. Dosage ------ Starting dose and last maintenance dose were available for 37 subjects (Table [1](#T1){ref-type="table"}). Subjects self-medicated, and therefore, the last maintenance dose of O-3FA was chosen by each subject. The mean starting dose was 1824.32 mg (SD 1075.07), and the mean for the last maintenance dose was considerably higher at 2878.38 mg (SD 2011.79). The increase was statistically significant using a paired sample t-test (t = -3.44, 36df, p = .001). Statistical Results =================== Percentage of Irritable (Days) ------------------------------ The initial mean was 63.51 (SD 34.17), indicating that on average, subjects were irritable for about six of the previous ten days. The mean for the last recorded percentage was less than half of the initial score: 30.27 (SD 34.03). The decrease was found to be statistically significant using a paired sample t-test (t = 4.36, 36 df, p \< .001). The difference between the distributions was examined using the non-parametric sign test. The number of negative differences (25) significantly exceeded positive differences (7); there were five ties, and the pre/post distributions were significantly different (p \< .003). YMRS Irritability Sub-score --------------------------- Thirty four subjects had initial and last recorded YMRS irritability sub-scores. As with the above means there was a sizable decrease. The initial mean score was 3.18 (SD 1.09). The mean for the last recorded percentage was 1.68 (SD 1.89). The decrease was found to be statistically significant using a paired sample t-test (t = 4.21, 33 df, p \< .001). YMRS Total Score ---------------- Starting and last recorded YMRS scores were available for 34 subjects. The mean starting score 10.71 (SD 6.77), and the mean for the last recorded score was 4.85 (SD 5.63). The decrease found to be statistically significant using a paired sample t-test (t = 4.14, 33 df, p \< .001). Severity -------- Thirty six subjects had initial and last recorded severity scores on the ADE. Again, a decrease was found. The initial mean score was 2.14 (SD 1.22). The mean for the last recorded score was 0.94 (SD 0.92). This decrease was found to be statistically significant using a paired sample t-test (t = 5.23, 35 df, p \< .001). Composite: Severity and Irritability ------------------------------------ As an exploratory measure, a composite score was created by multiplying the ADE severity score, which has a maximum of 4 points, by the percentage of the ten days prior to measurement which the patient was rated as irritable. The initial mean on this composite was 159.72. As with other measures, there was wide variation: SD = 122.92. The mean for this measure on the last recorded scores was percentage was about one-fourth of the initial score: 43.89 (SD 64.38). The decrease was found to be statistically significant using a paired sample t-test (t = 5.00, 35 df, p \< .001). Last Recorded Maintenance Dose and Percentage of Irritability After ------------------------------------------------------------------- Because of apparent wide variation on these two measures and a concern that outliers may have affected some results, the last recorded irritability scores were plotted against the maintenance dose. This revealed a rather bimodal pattern, in which relatively lower irritability measures (≤ 50%) clustered in the quadrant with lower dosage levels (≤ 4,000 mg). Duration and YMRS Total ----------------------- In response to a similar observation regarding wide variation in the last recorded values (84 days to 5.5 years) the values were also plotted. A clearly bimodal pattern appeared in which 11 subjects (about one-third of study participants) clustered in the quadrant representing short duration (\<500 days) and higher YMRS totals (\>7). The remaining two-thirds of subjects clustered in the quadrant representing short duration and lower YMRS totals (\<6). Subject Weight -------------- The mean start weight was 176.97 lbs (SD 43.13), and the mean for the last weight recorded was slightly higher at 178.59 lbs (SD 43.24). The increase was not statistically significant. Follow-up Subjects ================== Follow-up information, recorded after the collection of the \"last\" scores for most of the above variables, was available for 13 of the 37 subjects. Final YMRS total or scale scores were not available for this sub-group. Omega 3 Duration ---------------- The final date recorded for the duration of O3 was derived based from an O3 start date and a \"final\" date recorded for O3. The time period ranged 84 days to 1995 days (5.46 years). The mean duration of O3 for this group was 439.62 days (SD = 487.46). Dosage ------ For these subjects, the mean starting dose was 1807.69 mg (SD 990.34), and the mean for the last maintenance dose was higher at 2615.38 mg (SD 1894.66). The increase was not significant. Percentage Irritable (Days) --------------------------- The initial mean was 82.31 (SD 20.88). The mean for the last recorded percentage was dramatically lower: 25.38 (SD 32.04). The decrease was found to be statistically significant using a paired sample t-test (t = 6.52 12 df, p \< .001). The difference between the distributions was examined using a sign test. The number of negative differences (12) significantly exceeded positive differences (0); there was one tie, and the pre/post distributions were significantly different (p \< .001). Severity -------- The initial mean score for the 13 subjects with final scores was 2.69 (SD 0.95). The mean for the final score was 0.77 (SD 0.83). This decrease was found to be statistically significant using a paired sample t-test (t = 6.22, 12 df, p \< .001). Composite: Severity and Irritability ------------------------------------ An exploratory composite score, described above, was also created for the subjects with final scores. For these subjects, the initial mean was higher than that of the total group, 223.08. Again, there was wide variation: SD = 104.19. The mean for this measure on the last recorded scores was percentage was much lower that the initial score: 33.08 (SD 39.87). The decrease was found to be statistically significant using a paired sample t-test (t = 6.70, 12 df, p \< .001). Weight ------ For these 13 subjects, the mean start weight was 166.23 lbs (SD 35.68), and the mean for the final weight recorded was also slightly higher at 168.23 lbs (33.62). As with the previous finding regarding weight, the increase was not statistically significant. Results ======= Omega-3 Fatty Acids added onto the existing treatment helped with the irritability component of a significant percentage of patients suffering from bipolar disorder with a persistent sign of irritability. Discussion ========== As seen from the standard deviations of several of the variables discussed here, measures ranged widely. This creates difficulty in using descriptive data, such as means, to adequately portray subject attributes and performance. Using data reduction techniques or grouping subjects according to high and low scores on various attributes may be one way to increase the descriptiveness, which would be possible and more reasonable with a larger pool of subjects. A potential limitation or interpretive consideration merits discussion. For many of the variables discussed above, noticeable differences in measures were observed between the \"starting\" versus \"last recorded\" group (n = 37) and the \"starting\" versus \"final\" measures group (n = 13). Given these differences and the smaller number of subjects in the second set of comparisons, \"starting\" versus \"final\" comparisons should be interpreted with caution until differences inherent in this \"final\" subgroup (n = 13) are more clearly understood. This is clearly seen in the results of sign tests, in which the apparent magnitude of the \"final\" effects is pronounced. Statistically significant within-subjects differences were found in several independent variables. This is especially notable given the small number of subjects. The preliminary findings suggest that a rigorously designed study tailored especially to the examination of the effects of O-3FA is warranted. The majority of data were collected within an ongoing \"best-practice, outpatient bipolar disorder study\" that involved medications and talk therapy which we have not reported or discussed herein. Results must, therefore, be interpreted with caution. There are several mechanisms through which O-3FA are theorized to help with mood, irritability, aggression etc. Suggested theories of mechanism converge on the theory of nerve cell membrane stabilization. A recent study has come closest to showing physical proof of effectiveness of O-3FA through indirect demonstration of greater membrane fluidity, as detected by reductions in Tesla-2 (T2) values in MRI scans \[[@B19]\]. The overlapping beneficial effects of antipsychotics, antidepressants, anticonvulsants, O-3FA, and nonpsychoactive cannabinoids, as they relate to pain, stroke, schizophrenia, psychoneuroimmunology, Alzheimer\'s disease, and stress, may be because of their common effects at protein kinases, thus affecting the structure and function of the cell membrane and the cell \[[@B20]\]. These changes should help the cell operate within an optimal level of excitation, which may be related to emerging evidence that these therapeutic agents have neuroprotective value \[[@B20]\]. A recent randomized placebo controlled double blind intervention study suggests an adaptogenic role for O-3FA in stress \[[@B21]\]. We would like to discuss briefly the issue of daily dosing of O-3FA for nutrition and medicinal purpose: Recent studies give caution that at a 6 gram per day average daily dose, as a single agent, omega 3 fatty acids may not be as effective as an antidepressant \[[@B6]-[@B9]\]. However, these studies may have given too high of a dose of O-3FA, above 6 grams daily, with possibly beyond a therapeutic window of effectiveness for O-3FA. Our scatter plots indicate that the optimum effective dose for irritability is at 1--2 gram of EPA plus DHA per day, which would be the dosing we suggest. A recent exploratory dose study of O-3FA for schizophrenic patients showed that 2 g/day EPA-treated patients had lower symptom scores, and needed less medication greatest. In this study, there was a positive relationship between improvement on rating scales and rise in red blood cell arachidonic acid concentration as well \[[@B22]\]. The United States (US) accounts for more than 51% of the 430.3 billion dollar expended on pharmaceutical products worldwide each year \[[@B23]\]. World healthcare society first needs access to low-cost, nontoxic, non-expert-dependent interventions to ensure basic health outcomes. Food may represent the most cost-effective means of promoting public health \[[@B23]\]. The American Heart Association recommends consumption of two servings of fish per week for persons with no history of coronary heart disease and at least one serving of fish daily for those with known coronary heart disease \[[@B24]\]. Approximately 1 g per day of EPA acid plus DHA acid is recommended for cardioprotection \[[@B24]\]. Higher dosages of omega-3 fatty acids are required to reduce elevated triglyceride levels (2 to 4 g per day) and to reduce morning stiffness and the number of tender joints in patients with rheumatoid arthritis (at least 3 g per day) \[[@B24]\]. We conclude that it is beneficial in many ways to establish a regular intake of 1--2 g per day EPA acid plus DHA, similar to daily intake of vitamins with minerals. Dietary interventions to remedy omega-3 deficiency is necessary \[[@B23]\]. It is time for more aggressive funding for research into medicinal foods, such as omega-three fatty acids \[[@B23]\]. Figures and Tables ================== ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Initial, Last and Final Omega 3 Dosages (mg). summarizes dosage under three conditions. Figures for the Initial Dose include two subjects (n = 39) for whom no corresponding follow-up data were available. ::: Initial, Last Recorded and Final Omega 3 Dosages (mg) ------------------------------------------------------- --------------- --------------- ----------- Initial Last Recorded Final^T1^ n 39 37 13 Mean 1833.33 2878.38 2615.38 Mode 1000^T2,\ T3^ 1000 2000 Median 2000 2000 2000 SD 1071.91 2011.79 1894.66 T1 = Final group results (n = 13) are discussed below. T2 = Multiple modes exist. The smallest value is shown. T3 = One gram (1,000 miligram) of fish oil; of which about 180 milligrams is (eicosapentaenoic acid) EPA and 120 milligrams is DHA (docosahexaenoic acid), (for a total of 300 milligrams of omega 3\'s) in each clear capsule. :::	PubMed Central	2024-06-05T03:55:52.978992	2005-2-9	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549076/", "journal": "Nutr J. 2005 Feb 9; 4:6", "authors": [ { "first": "Kemal", "last": "Sagduyu" }, { "first": "Mehmet E", "last": "Dokucu" }, { "first": "Bruce A", "last": "Eddy" }, { "first": "Gerald", "last": "Craigen" }, { "first": "Claudia F", "last": "Baldassano" }, { "first": "Ayşegül", "last": "Yıldız" } ] }
PMC549077	Background ========== Wegener granulomatosis (WG) is a systemic inflammatory disease of unknown aetiology characterized by granulomata of the respiratory tract and systemic necrotizing vasculitis \[[@B1]\]. There is a strong and specific association with presence of anti-neutrophil cytoplasmatic antibodies to a defined target antigen, proteinase 3 (PR3-ANCA), which is present within primary azurophil granules of neutrophils (PMN) and lysozymes of monocytes \[[@B2]\]. Upon cytokine priming of PMNs, this enzyme translocates to the cell surface, where PR3-ANCAs can interact with their antigens and activate PMNs \[[@B3]\]. It has been shown that PMNs from patients with active WG expressing PR3 on their surfaces produce respiratory burst and release proteolytic enzymes after activation with PR3-ANCA \[[@B4]\]. The consequence is a self-sustaining chronifying inflammatory process. WG appears as a multifactorial disease, and environmental influences still remain elusive. Several factors, such as bacterial infections, have been proposed as probable initiators of the disease \[[@B5]\]. It has been reported that chronic carrier status of Staphylococcus aureusis a risk factor for disease exacerbation in WG \[[@B6]\]. Recently a strong association of WG with distinct HLA-DPB1alleles or rather an extended haplotype, respectively, in the MHC class II region has been reported \[[@B7]\]. In addition, analyses of candidate genes revealed several associations, e.g.with α(1)-antitrypsin, and proteinase 3 \[\[[@B8]\] and \[[@B9]\]\]. A primary candidate gene, PLCγ-2, was proposed on the basis of a novel animal model system. A mutation in the abnormal limb mutant 5 (ALI5) mouse \[[@B10]\] causes a phenotype comparable to human autoimmunity disease like WG: inflammations, granulomatosis, affected organs (lung, kidney with glomerulonephritis, eye and skin) and ANCAs were detected in the ALI5 mouse initially. Yet, this result has to be confirmed in further studies. The ALI5 mutation is located in the genomic region coding for the hydrophobic ridge loop 3. In mouse PLCγ-2mutation leads to acute and chronic inflammations with a phenotype comparable to WG. Therefore, human PLCγ-2is a good candidate gene for seeking predisposing genetic factors for WG. The human gene is a member of the PLC family comprising 12 closely related molecules involved in signal transduction from numerous receptors \[[@B11]\]. The protein is activated by cytoplasmatic tyrosine kinases (Lyn, Syk, and Btk) which are induced by engagement of B-cell receptors. In turn, the PLCγ-2 activation leads to the generation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphat (IP3). While DAG activates protein kinase C (PKC, \[[@B12]\]), IP3 mediates Ca^2+^mobilization, which is required for activation of B-cells \[[@B13]\]. PLCγ-2 itself is expressed mainly in B-cell, hematopoetic cells, macrophages, granulocytes, testis, sperm, skin and brain \[[@B14]\]. The protein consists of two catalytic domains, which are separated by two SH2 and one SH3 domains \[[@B15]\]. It was established that PLCγ-2 mediates the coupling of G-protein-coupled receptors (GPCRs) and Ca^2+^entry in cell lines \[[@B16]\]. The human PLCγ-2gene is localized on chromosome 16 (16q24.1) spanning 179 kb of genomic DNA. The 4.2 kb mRNA consists of 33 Exon coding for a M~r~140,000 protein with 1265 amino acids \[[@B17]\]. PLCγ-2is highly polymorphic \[[@B18]\]. Here, we report on an indirect association screen by microsatellite analysis and pooling of DNA in a case-control study design. Furthermore, we screened exons 11, 12 and 13, partly coding for the hydrophobic ridge loop 1 and 2 of PLCγ-2 protein, by single stranded conformation polymorphism (SSCP), sequencing and the PCR/RFLP method. As the ALI5 mutation is located in the region corresponding to the human exon 27, this exon was also screened by SSCP. Results ======= Analyses of microsatellites --------------------------- In the present study we analysed 4 microsatellites intra- or juxta-genic of the PLCγ-2gene with pooled DNAs from WG patients compared with those from controls (table [1](#T1){ref-type="table"}; figure [1](#F1){ref-type="fig"}). All markers exhibited at least 3 alleles and did not show any intra-subgroup differences. The microsatellite analyses did not reveal significantly different allele distributions between WG patient and control pools (figure [1](#F1){ref-type="fig"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Primer sequences and information about microsatellites used in study ::: Primer sequence --- ---------------------- ----------------------- ---------------- 1 CGCACATGTATCCAGAACT AGAGGTGGACCCATGCTTA \di (AT) 2 CAAAGAAGATAAGGGCAGGC CCTAGGCGACTCAGTGAGACT \tetra (TTTA) 3 AGGAGTTCGAGAAGAGCCTG TGCCACTACACCCAGATGAT \di (AC) 4 TGATCTGTGTCTGGGCTTTC AGTTGTGACCCTAACATTGCA \di (AC) ^1^The fluorescence labelled tail (5-Fam-CATCGCTGATTCGCACAT) was added to the 5\'end of each sense primer ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Schematic representation of the human PLCγ-2 gene with relative localization of exons and investigated microsatellites. P values were generated by contingency tables (for details see \"materials and methods\"). Vertical lines: exons; No1-6: investigated microsatellites 1-6; Ex: exon. ::: ![](1477-5751-4-1-1) ::: SSCP, sequencing and PCR/RFLP analyses -------------------------------------- SSCP analysis was chosen to identify mutations or polymorphisms, respectively, in exons 11, 12, 13 and 27 of the PLCγ-2gene. DNA\'s of patients and controls with altered migration behaviour were sequenced. Identified variations were genotyped individually by SSCP and/or PCR/RFLP in individual patient and control samples (see table [2](#T2){ref-type="table"}). Exon 12 revealed a low frequent SNP (1122G\>A), which represents the first base pair of the exon. In exon 13 two previously identified SNPs (1293T\>C and 1296T\>C) were detected \[[@B18]\]. Both SNPs showed similar frequencies as reported \[[@B18]\]. All variations were found not significantly different in WG patients compared to the control group, and they were not associated with amino acid substitutions. In one patient our analyses revealed a single base substitution (3030G\>A) in exon 27. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Summary of found variations in the human PLCγ-2gene in exons 11, 12, 13 and 27 ::: variation frequency of alleles ---- ------------- ---------------------- ---------- ---------- ------ ------- 11 \- \- \- \- \- \- 12 1122G\>A T329T 3/262 3/162 0.55 BanI 13 1293T\>C^2^ F382F 7/350 4/324 0.68 PfIFI 1296T\>C^2^ D383D 143/262 96/186 0.78 TaqI 27 3030G\>A T961T 1/166^3^ 0/180^3^ 0.51 \- ^1^Numbering according to BC007565 (UCSC); ^2^Previously reported SNPs; ^3^Frequencies determined by SSCP analyses ::: Discussion ========== As recently reported a mutation in the PLCγ-2 gene in the ALI5 mouse leads to acute and chronic inflammation and granulomatosis. In addition, the ALI5 mice show similar symptoms as WG patients. For this reason screening of PLCγ-2 appears as a logical consequence in seeking candidate genes for WG. In the present study PLCγ-2 was analysed by an indirect microsatellite approach using pooled DNA from WG patients with a defined PR3-ANCA^+^status and a matched control cohort as reported before \[[@B7]\]. Furthermore, exons partly representing the catalytic domains of the PLCγ-2 protein, HRL1, 2 and 3, were analysed by SSCP, sequencing and by the PCR/RFLP method. We did not find any hints of an involvement of the PLCγ-2 gene in the pathophysiology of WG. Hence, we exclude at this time that the PLCγ-2 gene predisposes for WG in our cohort. Our study revealed 4 single base substitutions, 2 of which were reported before \[[@B18]\]. A further silent and low frequent SNP was detected in exon 12 which did not differ significantly between patient and control cohorts after PCR/RFLP analyses. As this SNP is the first basepair (bp) after the 3\'-splice site one might hypothesise an influence on splicing but to present knowledge this SNP does not change a consensus sequence required for splicing. The ALI5 mutation is located in the HRL3 domain partly corresponding to the human exon 27 of PLCγ-2. Our analysis revealed a SNP (G\>A) at position 3030 in one WG patient. The indirect microsatellite based approach did not reveal any association of PLCγ-2 with WG. Altogether 4 microsatellites spread in the PLCγ-2 gene were analysed. The approach using pooled DNA and ad hocdesigned markers intragenic or in the immediate vicinity of a distinct gene has proven to be a reliable and efficient method to detected predisposing loci in WG before \[[@B7]\]. Here, none of the markers did show a significant allele distribution between the patient and control group. Conclusion ========== In conclusion, our analysis of the human PLCγ-2 gene did not reveal an association of PLCγ-2 with WG. In contrast to ALI5 mice, where a single mutation leads to distinct symptoms of inflammatory autoimmunity, human WG depends on a more complex genetic background. Further analysis of all exons of PLCγ-2 might yield an association with WG but our microsatellite analysis strongly suggests that predisposition for WG is not due to variations in the PLCγ-2 gene. Material and Methods ==================== Patients and controls --------------------- 175 well-characterized patients of German origin with a clinical diagnosis of WG and a defined PR3-ANCA^+^status were included in present study. Diagnosis of WG was established according international standards \[\[[@B19]\] and \[[@B20]\]\]. All patients were biopsy-proven. Biopsies were seen in German reference centre for vasculitis (Department of Pathology, University of Schleswig-Holstein Campus Luebeck, Germany) by 2 different observers. A group of 165 healthy individuals of German origin were used as controls. All persons gave informed consent. Microsatellite analysis ----------------------- Pooling of DNA was performed as reported before \[[@B7]\]. Patient (n = 150) and healthy control (n = 100) individuals from the abovementioned groups were divided into 3 and 2 sub-pools, respectively, containing 50 persons each. In this study 3 intragenic microsatellites as well as one in the immediate vicinity of the gene were included (table [1](#T1){ref-type="table"}; see also UCSC Database, June 2002 Freeze; URL). Oligonucleotides were designed by Primer Express 2.0 software (ABI) adjusted to an annealing temperature of 55°C. For PCR we used the \'tailed primer PCR\' as described before \[[@B7]\]. For amplification three oligonucleotides were used: 1. tailed sense primer (tailed F), 2. anti-sense primer and 3. labeled primer (labeled F) corresponding to the 5\'-tail sequence of tailed F. PCR conditions were as follows: 1 × PCR buffer (Qiagen), 1.5 pmol labeled F, 0.2 mM each dNTP, 3 mM MgCl~2~, 0.2 pmol tailed F, 1.5 pmol reverse primer, 0.25 U Qiagen Hot Start Taq (Qiagen) and 50 ng DNA. Electrophoreses were run using ABI standard protocols. Raw data were analyzed by the Genotyper software (ABI) producing a marker-specific allele image profile (AIP, see \[[@B21]\] and \[[@B7]\]). AIP consists of series of peaks with different heights reflecting the allele frequency within each analyzed DNA pool. Statistics for comparisons of allele frequencies of patients and controls was performed as described before \[\[[@B21]\] and \[[@B7]\]\]. Case and control distributions were compared statistically by means of contingency tables. SSCP, sequencing and PCR/RFLP analyses -------------------------------------- Exons 11, 12, 13 and 27 were analysed by the SSCP method. PCR was performed using oligonucleotides reported before (\[[@B18]\], exon 27 corresponding to exon 26). Heat-denaturated fragments were then separated by polyacrylamide gel electrophoresis under non-denaturating conditions yielding specific band patterns for each of the alleles. Results were visualized by autoradiography. Alleles of representative probes were determined by direct DNA sequence analysis on a 377 ABI automatic sequencer (ABI). Afterwards, variations were genotyped individually by PCR/RFLP method with restriction enzymes specific for the respective change (for details see table [2](#T2){ref-type="table"}). The variation in exon 27 was individually genotyped by the SSCP method. Authors\' contribution ====================== PJ and SW carried out the molecular genetic studies, performed the statistical analysis and drafted the manuscript. PY participated in study design and helped to draft the manuscript. EC and WLG provided the samples and performed diagnostics of the patient group. JTE conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.	PubMed Central	2024-06-05T03:55:52.981231	2005-2-9	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549077/", "journal": "J Negat Results Biomed. 2005 Feb 9; 4:1", "authors": [ { "first": "Peter", "last": "Jagiello" }, { "first": "Stefan", "last": "Wieczorek" }, { "first": "Philipp", "last": "Yu" }, { "first": "Elena", "last": "Csernok" }, { "first": "Wolfgang L", "last": "Gross" }, { "first": "Joerg T", "last": "Epplen" } ] }
PMC549078	Introduction ============ CytoJournalis published by BioMed Central, an independent publisher committed to ensuring peer-reviewed biomedical research is Open Access -- it is universally and freely available online to everyone, its authors retain copyright, and it is archived in at least one internationally recognised free repository \[[@B1]\]. CytoJournalhowever, has taken this further, by making all its content Open Access. Since its launch in August 2004, BioMed Central has graciously supported the processing of all the articles published during CytoJournal\'s first 6 months. However, as we move forward it is crucial that CytoJournaldevelops its own financial viability, at least to support publication expenses. Thus, from 1^st^March, 2005, authors will be asked by the publisher to pay a flat £330 (approximately US \$600/€500) article-processing charge (APC) \[[@B2]\]. As explained below, a significant proportion of authors may not have to pay the fee directly. This is because the APC can be covered/waived under a variety of different mechanisms such as institutional and society memberships with BioMed Central \[[@B2]\]. Please note that authors submitting any article online to CytoJournal before 1^st^March, 2005 will not have to pay an APC, even if it is accepted for publication after peer review. Problems with the traditional publishing model ============================================== Traditionally, readers pay to access articles, either through subscriptions or by paying a fee each time they download an article. Escalating journal subscriptions have resulted in libraries subscribing to fewer journals \[[@B3]\], and the range of articles available to readers is therefore limited. Although traditional journals publish authors\' work for free (unless there are page or colour charges), having to pay to access articles limits how many people can read, use and cite them. This compromises the scientific impact of the publication and overall visibility of the researchers work. Definition of Open Access ========================= CytoJournal\'s Open Access policy changes the way in which articles are published. First, all articles become freely and universally accessible online, and so an author\'s work can be read by anyone at no cost. Second, the authors hold copyright for their work and grant anyone the right to reproduce and disseminate the article, provided that it is correctly cited and no errors are introduced \[[@B1]\]. Third, a copy of the full text of each article is permanently archived in an online repository separate from the journal. CytoJournal\'s articles are archived in PubMed Central \[[@B4]\], the US National Library of Medicine\'s full-text repository of life science literature, and also in repositories at the University of Potsdam \[[@B5]\] in Germany, at INIST \[[@B6]\] in France, and in e-Depot \[[@B7]\]- the National Library of the Netherlands\' digital archive of all electronic publications. Benefits of Open Access ======================= Open Access has four broad benefits for science and the general public. First, authors are assured that their work is disseminated to the widest possible audience, given that there are no barriers to access it. This is accentuated by the authors being free to reproduce and distribute their work, for example by placing it on their institution\'s website. They can still use the published material for any future plans including book chapters, monograms, and presentation after due acknowledgement about its original publication. It has been shown that free online articles are more highly cited because of their easier availability \[[@B8]\]. Second, the information available to researchers will not be limited by what their library can afford, and the widespread availability of articles will enhance literature searching \[[@B9]\]. Third, the results of publicly funded research will be accessible to all taxpayers and not just those with access to a library with a subscription. Note that this public accessibility is becoming a legal requirement for many countries and organizations \[[@B10]\]. Fourth, a country\'s economy will not influence its scientists\' ability to access articles because resource-poor countries (and institutions) will be able to read the same material as wealthier ones (although creating access to the internet is another matter \[[@B11]\]). Open Access to original peer-reviewed scientific information to both the general public and experts alike has many benefits. It can affect and shape the public opinion. In the long run the availability of such information in the field of cytopathology will translate into further progress in cytopathology and maximize its benefits to various fields of life sciences. Description of APC payment ========================== APCs will allow continued Open Access to all of CytoJournal\'s articles. Authors are asked to pay a flat payment of £330, during 2005, if their article is accepted for publication \[[@B2]\]. Waiver requests will be considered on a case-by-case basis, by the Editor-in-Chief. Authors can circumvent the charge by getting their institution to become a \'member\' of BioMed Central, whereby the annual membership fee covers the APCs for all authors at that institution for that year \[[@B12]\]. Current members include numerous institutions in USA, NHS England, the World Health Organization, the US National Institutes of Health, and all UK universities \[[@B12]\]. Biomed Central has also opened a new avenue which allows membership to various societies such as different cytology, cytopathology, and pathology societies all over the world. The members of these societies could publish in CytoJournalwithout incurring an APC. We strongly recommend readers to approach their respective societies to become members under this provision. No charge is made for articles that are rejected after peer review. Many funding agencies have also realized the importance of Open Access publishing and have specified that their grants may be used directly to pay APCs \[[@B13]\]. As another avenue, Cytopathology-Foundation, Inc <http://www.cytopathology-foundation.org>, a non-profit organization, is looking for help from appropriate organisations to support the publication cost in CytoJournal, so that ultimately our journal will be an Open Access journal free of any financial burden for all CytoJournalauthors. We appreciate any help in this matter to achieve this goal. You may contact by e-mail: <cytojournal@mcw.edu> to extend the support. What the APC covers =================== The APC pays for the manuscript publication process. It allows the article to be freely and universally accessible in various formats online, and for the processes required for inclusion in PubMed and archiving in PubMed Central, e-Depot, Potsdam and INIST. Although some authors may consider £330 expensive, it must be remembered that CytoJournaldoes not levy additional page or color charges on top of this fee, which can easily exceed £330 with conventional journals. As the entire manuscript with color PDF file is freely available on-line, authors do not have to stock reprints and spend mailing charges to share the publication with their peers. The PDF files can be directly downloaded by the interested colleague/researcher or the author to share the PDF file through e-mail at the click of the mouse! With reference to conventional journals it is usually stated in instructions to authors that- \'this journal does not reproduce color illustrations unless the cost of such reproduction is subsidized by the author and agreed upon in advance\'. Restricting the number of images published in a manuscript or publishing them as black & white would significantly compromise the publication standard we hope to maintain for a morphology based discipline such as cytopathology. With CytoJournal, the article being online only, any number of color figures and photographs can be included at no extra cost. In addition, CytoJournalbeing a web based publication, a small video clip could also be included as part of publication without extra cost. This provision to include video clip(s) is an excellent opportunity to improve the clarity of the details to the readers about dynamic processes such as procedures and methodologies \[[@B14]\]. Free versus Open Access ======================= Although several journals now offer free access to their articles online, this is different from Open Access (as defined by the Bethesda Statement \[[@B15]\]). These journals often delay free access for 6-12 months, and even when the full text is available, readers are not allowed to reproduce and/or disseminate the work because of restrictions imposed by the copyright policy. That said, CytoJournalis not alone in the move to Open Access funded by APCs. The British Medical Journal has recently announced that it cannot continue to provide free access to its website \[[@B16]\] and is considering various sources of revenue, including APCs \[[@B17]\]. Also, the Public Library of Science has set up new Open Access journals, and have elected to set APCs of US\$1500 for each accepted article \[[@B18]\]. Given that the Public Library of Science has used television advertising to promote journals \[[@B10]\], the high profile of these journals will raise awareness of Open Access and encourage researchers in all disciplines to understand and accept Open Access, with APCs as an acceptable method to fund it. Conclusion ========== By providing a forum for Open Access, APCs will enable CytoJournalto serve the pathology, cytopathology, and entire medical community. We believe this change will benefit and aid scientific research in general. We hope you will support this progress by submitting your next article to CytoJournal. Abbreviations ============= APC = article-processing charge. Competing interests =================== At CytoJournal, the work of the Editors-in-Chief, the Editorial Board, and of all invited outside peer reviewers is entirely voluntary, without tangible remuneration of any kind. Our goal is publication and dissemination of the highest quality literature and research in cytopathology and related areas. Our (intangible) rewards are in the achievement of these goals. Any decisions about manuscripts are based entirely on the quality of the scientific content, and not on the ability of authors to pay article-processing charges.	PubMed Central	2024-06-05T03:55:52.982561	2005-2-10	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549078/", "journal": "Cytojournal. 2005 Feb 10; 2:3", "authors": [ { "first": "Vinod B", "last": "Shidham" }, { "first": "Anthony F", "last": "Cafaro" }, { "first": "Barbara F", "last": "Atkinson" } ] }
PMC549079	Background ========== Quantitative real-time PCR (QPCR) has become the favoured tool in mRNA expression analysis and also in virus diagnostics \[[@B1]\]. Real-time PCR has outperformed classical and semi-quantitative PCR methods in terms of accuracy, reproducibility, safety and convenience for the precise monitoring of viral load in clinical material, as well as for the investigation of the expression of cellular genes in response to virus infection. However, the most prominent problem in quantitative mRNA expression analysis is the selection of an appropriate control gene. For years, the glyceraldehyde 3-phosphate dehydrogenase (GAP) gene and the β-actin (Act) gene were used as control genes in classical molecular methods for RNA detection. Recently, evidence accumulated that especially these two genes, GAP and Act, are unsuitable controls in quantitative mRNA expression analysis due to setting dependent variations in expression \[[@B2]-[@B4]\]. Recently, we have confirmed these results by investigating the expressional stability of 13 potential reference genes in 16 different tissues and presented more suitable genes like the RNA polymerase II gene \[[@B5]\]. However, an evaluation of reference genes in virus infected cells has not been performed so far. Therefore, the selection of the 10 most promising reference genes, GAP, Act, peptidyl prolyl isomerase A (PPI), glucose 6-phosphate dehydrogenase (G6P), TATA-Box binding protein (TBP), β2-microglobulin (β2M), α-tubulin (Tub), ribosomal protein L13 (L13), phospholipase A2 (PLA) and RNA polymerase II (RPII) were evaluated in cell lines infected with members of different virus families: coronavirus (SARS-coronavirus), flavivirus (yellow fever virus, (YF)), herpesvirus (Human herpesvirus-6 (HHV-6) and cytomegalovirus (CMV)) and orthopoxvirus camelpox (CAMP), covering also DNA and RNA viruses. Quantification of viral RNA was performed to proof and monitor infection. Thereafter the candidate reference genes were evaluated by the BestKeeper tool \[[@B6]\], the GeNorm tool \[[@B7]\] and the algorithm we described previously \[[@B5]\]. Results ======= An efficient infection could be evidenced by a significant increase of viral RNA or DNA for all 5 viruses over time (table [1](#T1){ref-type="table"}). Despite progressing viral replication, the expression of some of the reference genes remained constant, while other genes were varying in expression according to accumulation of infected cells. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Cell culture conditions and results of virus kinetics ::: CMV HHV-6 CAMP SARS YF ------------------------------ ----------------- ------------------- --------------- ------------- --------------- cell line MRC-5 CCRF-HSB-2 HepG2 Huh-7D12 HepG2 multiplicity of infection 2.0 0.5 0.5 1.0 0.5 time to maximal infection /h 72 120 24 72 96 max. infected cells % 100 \>70 \>90 \>70 \>80 measuring point /h 0,6,12,24,48,72 0,24,48,72,96,120 0,1,3,6,12,24 0,2,4,22,42 0,24,48,72,96 ::: The experimentally obtained data for each virus and each gene were analysed using three different methods. The reference gene evaluation of the BestKeeper tool is shown in table [2](#T2){ref-type="table"}. A low standard deviation (SD) of the C~T~values should be expected for useful reference genes and a high SD for genes that are susceptible to virus replication. Corresponding to the recent estimation the SD of the C~T~value was highest for Act in 4 of 5 viruses, indicating that Act is no reliable reference gene in this setting. In contrast, TBP and PPI displayed the highest expressional stability for 4 of 5 viruses. To find a general conclusion, the total of all SD values from all virus experiments (sum~V~) was calculated for each reference gene. As shown in table [2](#T2){ref-type="table"}, TBP and PPI seemed to be the least regulated genes in this analysis (sum~v~= 2.29 for both), followed by GAP (sum~v~= 3.49) and β2M (sum~v~= 3.96). All other genes showed moderate total SD values (sum~v~\> 4.58), except Act (sum~v~= 11.28), confirming to be the most inappropriate reference gene. It is remarkable that the obtained BestKeeper index values are low, despite the inclusion of Act in the calculation. Calculating BestKeeper vs. each reference gene using Pearson correlationdisplayed very inconsistent results (table [3](#T3){ref-type="table"}). Act showed the highest SD values in all virus infections, but a significantly high correlation. In contrast TBP displayed low correlation that was statistically not significant in most cases. When summing up the SD values of all reference genes for each virus infection (sum~HRG~), it seems that CAMP infection caused the highest variations in reference gene expression. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Results from BestKeeper analysis, SD \[±C~T~\] ::: RPII Act β2M L13 PLA TBP GAP PPI G6P Tub BK sum~RGC~ ------------ ------ ------- ------ ------ ------ ------ ------ ------ ------ ------ ------ -------------- CMV 0.59 2.70 0.51 0.36 0.72 0.41 0.66 0.43 0.71 0.69 0.56 7.78 HHV-6 2.77 1.09 0.50 0.87 0.88 0.35 0.59 0.26 0.92 0.78 0.63 9.02 CAMP 1.84 2.70 1.46 2.34 1.72 0.49 0.61 0.70 1.47 1.36 1.10 14.70 SARS 0.39 1.72 0.41 0.53 0.58 0.32 0.56 0.34 0.81 0.55 0.40 6.21 YF 1.36 3.06 1.07 0.67 1.64 0.71 1.08 0.56 0.80 1.19 0.98 12.16 sum~V~ 6.95 11.28 3.96 4.77 5.55 2.29 3.49 2.29 4.71 4.58 ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Results from BestKeeper analysis, Bestkeeper vs. Reference gene candidate ::: Coeff. of corr. \[r\] (p-Value) RPII Act β2M L13 PLA TBP GAP PPI G6P Tub ----------------------------------- --------- --------- --------- --------- --------- --------- --------- --------- --------- --------- CMV 0.75 0.79 0.76 0.13 0.89 0.10 0.92 0.91 0.75 0.95 (0.005) (0.002) (0.005) (0.698) (0.001) (0.763) (0.001) (0.001) (0.005) (0.001) HHV-6 0.79 0.73 0.54 0.30 0.93 0.79 0.94 0.75 0.82 0.97 (0.002) (0.007) (0.069) (0.350) (0.001) (0.002) (0.001) (0.005) (0.001) (0.001) CAMP 0.91 0.18 0.98 0.95 0.99 0.78 0.63 0.99 0.45 0.59 (0.002) (0.662) (0.001) (0.001) (0.001) (0.022) (0.092) (0.001) (0.268) (0.127) SARS 0.48 0.77 0.41 0.27 0.85 0.88 0.46 0.36 0.73 0.84 (0.162) (0.010) (0.236) (0.452) (0.002) (0.001) (0.177) (0.307) (0.017) (0.002) YF 0.90 0.91 0.96 0.25 0.98 0.94 0.99 0.92 0.93 0.92 (0.001) (0.001) (0.001) (0.492) (0.001) (0.001) (0.001) (0.001) (0.001) (0.001) Abbreviations: SD \[± C~T~\]: the standard deviation of the C~T~; BK: BestKeeper; Sum~V~: Sum of viral infection SD values; Sum~RGC~: Sum of reference gene SD values ::: Analysing the expression data with the GeNorm tool showed slightly deviant results (table [4](#T4){ref-type="table"}). First, the value sum~V~, representing the SD of a reference gene over all viruses, was lowest for PPI (sum~V~= 6.08) confirming the results obtained by the Bestkeeper tool. However, β2M (sum~V~= 6.11), GAP (sum~V~= 6.19) and TBP (sum~V~= 6.29) turned out to be comparably reliable as reference genes. Second, also the GeNorm tool showed that Act is by far the worst reference gene (sum~V~= 14.20). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Results from GeNorm analysis (M ≤ 0.5) ::: RPII Act β2M L13 PLA TBP GAP PPI G6P Tub sum~RGC~ ------------ ------ ------- ------ ------ ------ ------ ------ ------ ------- ------ -------------- CMV 1.41 3.41 1.42 1.63 1.45 1.69 1.38 1.37 4.79 1.54 20.09 HHV-6 2.82 1.38 1.15 1.55 1.19 1.03 0.95 1.08 1.15 0.96 13.27 CAMP 1.70 3.84 1.40 1.94 1.49 1.57 1.66 1.40 2.04 1.92 18.95 SARS 0.83 1.88 0.82 1.06 0.87 0.70 0.89 0.84 1.04 0.80 9.73 YF 1.65 3.69 1.32 1.87 2.02 1.30 1.31 1.39 1.31 1.48 17.34 sum~V~ 8.41 14.20 6.11 8.05 7.03 6.29 6.19 6.08 10.33 6.70 Abbreviations: Sum~V~: Sum of viral infection GeNorm values; sum~RGC~: sum of reference gene GeNorm values ::: Applying the calculation mode presented previously \[[@B5]\], that is based on the calculation of ΔΔC~T~values (table [5](#T5){ref-type="table"}), Act was most susceptible to virus infection for 3 of 5 viruses and displayed the highest ΔΔC~T~value over all viruses (sum~V~= 45.23). The two genes with the lowest ΔΔC~T~value were TBP (sum~V~= 9.82) and PPI (sum~V~= 10.04), corresponding to the results of the Bestkeeper and the GeNorm tool. ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Results from ΔΔC~T~analysis ::: RPII Act β2M L13 PLA TBP GAP PPI G6P Tub sum~RGC~ ------------ ------- ------- ------- ------- ------- ------ ------- ------- ------- ------- -------------- CMV 2.10 11.55 3.03 2.18 3.95 2.36 2.90 2.54 12.51 2.39 45.49 HHV-6 5.98 3.54 3.35 2.89 4.99 0.88 2.27 1.25 3.35 2.30 30.78 CAMP 3.59 14.19 3.94 3.17 2.71 1.23 3.19 1.78 2.22 3.33 39.33 SARS 1.19 1.71 2.14 1.93 2.52 1.11 2.75 1.34 4.14 1.78 20.58 YF 9.01 14.25 5.78 2.90 9.62 4.24 6.35 3.14 5.42 7.48 68.17 sum~V~ 21.87 45.23 18.22 13.07 23.78 9.82 17.45 10.04 27.62 17.27 Abbreviations: sum~V~: sum of viral infection values; sum~RGC~: sum of reference gene values ::: Discussion ========== To date, it is generally accepted, that the selection of the ideal reference gene in gene expression analysis has to be done for each individual experimental setting by evaluating several genes and using the best two or three of these genes as reference. Obviously there is no \"one good gene for all experiments\" recommendation. However, it is helpful to find putative candidates that can be shortlisted when setting up a new experimental design. Therefore, we determined the expression of previously tested reference genes in a setting of virus infected human cell lines. Capable reference genes were evaluated using three independent methods: Bestkeeper, GeNorm and the ΔΔC~T~method, and their results were compared. All three tools ranked actin at the last position, indicating that it is an unsuitable reference gene in virus infected cells. The actin gene shows significant variations with increasing degree of infection. The best genes obtained from all three calculation tools were TBP and PPI. TBP seems to be a relative stable expressed gene during the course of virus replication of different viruses in different cells. However, as previously shown \[[@B5]\] TBP is not expressed in all tissues and therefore its use may be limited. Interestingly, classical reference genes like β2M and GAP were also acceptable regarding to a stable expression in virus infected cells. All other genes showed moderate expression stability. The analysis of our data set according to the Bestkeeper tool revealed very good BestKeeper indices; even actin was included into our gene panel. These findings demonstrate the usefulness of analysing a wide variety of reference gene candidates. The inconsistent data regarding to the Bestkeeper calculation of the coefficient of correlation and the corresponding p-values may be a result of the Pearson correlation. As described by Pfaffl et al. its use is limited to groups without heterogeneous variances, but the tested reference genes have very different expression levels resulting in significant variances. Paffl et al. also described that new versions of Bestkeeper should circumvent these problems by use of Spermanand Kendall Tau correlation. However, one problem still remains to be solved; both tools, the BestKeeper and the GeNorm, can not compare paired probes. This is the great advantage of the ΔΔC~T~method, or any other method which directly compares paired samples. From this point of view the use of a method like the ΔΔC~T~should be applied first before considering additional tools for further elucidation of the acquired data. Conclusions =========== In summary, TBP and PPI turned out to be the best reference genes in virus infected cells. These genes are a good point to start reference gene selection in gene expression studies in virus infection experiments. Material and Methods ==================== Virus culture and virus detection by real-time PCR -------------------------------------------------- Camelpox strain CP-19, CMV strain AD169, HHV-6 strain U1102, SARS coronavirus strain 6109 and YFV strain 17D were propagated according to standard procedures \[[@B8]-[@B10]\]. The respective MOI and time of cell culture are shown in table [1](#T1){ref-type="table"} and were chosen to allow maximal infection as determined by immunofluorescence and real-time PCR \[[@B8]-[@B11]\]. For kinetic studies, cells were harvested at several time points (table [1](#T1){ref-type="table"}) and RNA was extracted. The RNA transcription level of putative reference genes was determined by quantitative real-time PCR as described below. Extraction of RNA ----------------- Total RNA from 1 × 10^6^cells was prepared using the QIAamp RNA Blood Mini Kit and RNase-free DNase set (Qiagen, Hilden, Germany) according to the manufacturer\'s recommendations for cultured cells. RNA solution was treated with DNA-free(Ambion, Huntingdon, United Kingdom). cDNA synthesis -------------- cDNA was produced using the Superscript III RT-PCR System (Invitrogen, Karlsruhe, Germany) according to the manufacturer\'s recommendations for oligo(dT)~20~primed cDNA-synthesis. cDNA synthesis was performed using 1 μg of RNA, at 50°C. Finally, cDNA was diluted 1:5 before use in QPCR. Quantitative TaqMan PCR ----------------------- Primers, TaqMan probes and QPCR conditions for reference gene analysis were used as previously described \[[@B5]\]. PCR was performed in a Perkin Elmer 7700 Sequence Detection System in 96-well microtiter plates using a final volume of 25 μl. Calculations ------------ Analysis was performed with the BestKeeper \[[@B6]\] and GeNorm \[[@B7]\] tools. The ΔΔC~T~value was calculated as follows: First the ΔC~T~for each time point of probe assessment between virus and Mock infected cells was calculated. In a second step the maximal differences between the time points were calculated as ΔΔC~T~. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= AR conceived the study, carried out the HHV-6 experiments and real-time PCR assays and drafted the manuscript. ST carried out the CMV experiments. HB carried out the YF experiments. MM carried out the SARS experiments. WS participated in the design of the study. AN carried out the CAMP experiments, participated in design and coordination of the study and helped to draft the manuscript. All authors read and approved the final manuscript. Acknowledgements ================ We gratefully acknowledge the excellent technical assistance of Delia Barz and Jung-Won Sim-Bandenburg. The authors are grateful to Andreas Kurth for critical reading of the manuscript.	PubMed Central	2024-06-05T03:55:52.983600	2005-2-10	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549079/", "journal": "Virol J. 2005 Feb 10; 2:7", "authors": [ { "first": "Aleksandar", "last": "Radonić" }, { "first": "Stefanie", "last": "Thulke" }, { "first": "Hi-Gung", "last": "Bae" }, { "first": "Marcel A", "last": "Müller" }, { "first": "Wolfgang", "last": "Siegert" }, { "first": "Andreas", "last": "Nitsche" } ] }
PMC549080	Background ========== Mimivirus is the largest known virus, both in particle size (\>0.4 μm in diameter) and genome length, recently discovered in amoeba, following the inspection of a hospital cooling tower prompted by a pneumonia outbreak \[[@B1]\]. Recently, its entire 1.2-Mbp genome sequence was determined \[[@B2]\]. Extensive phylogenetic studies and gene content analyses defined Mimivirus as a new family of nucleocytoplasmic large DNA viruses (NCLDV) besides Poxviridae, Iridoviridae, Phycodnaviridaeand Asfarviridae, and suggested its early origin, probably before the individualization of the three domains of life \[[@B2]\]. While analyzing Mimivirus genome sequence, we noticed the unusual length of its putative DNA polymerase. A detailed analysis identified an intein in this gene. After the recent discovery of an intein in Chiloiridescent virus \[[@B3]\], an insect-infecting NCLDV of Iridoviridae, this is the second report of an intein sequence in a eukaryote-infecting virus. Inteins are \"protein introns\" that catalyze self-splicing at the protein level. The splicing is defined by the self-catalytic excision of an intervening sequence (\"intein\") from a precursor host protein where it is located, and the concomitant ligation of the flanking amino- and carboxy-terminal fragments (\"exteins\") of the precursor. Inteins often possess a homing endonuclease domain, and are considered as mobile elements. Since their first discovery in 1990 \[[@B4],[@B5]\], inteins have been identified in a wide variety of organisms, including bacteria, archaea, and unicellular eukaryotes, albeit with sporadic distribution (see <http://bioinformatics.weizmann.ac.il/~pietro/inteins/> for a comprehensive list). For instance, they are relatively abundant in some hyperthermophilic archaea species (such as Methanococcus jannaschiipossessing nineteen inteins), but absent in closely related species such as Methanococcus maripaludis\[[@B6]\]. Similarly, they are observed in many unrelated bacterial clades, but appear often limited to several species within each clade. It was suggested that viruses were potential \"vectors\" of inteins across species and responsible for the sporadic distribution of inteins \[[@B3]\]. Accordingly, inteins have been identified in many bacteriophages and prophages \[[@B7]-[@B10]\]. To our knowledge, the sole published account of eukaryote-infecting viruses harboring an intein concerns iridoviruses \[[@B3]\]. Results ======= Eukaryotic Polδ-like Mimivirus PolB ----------------------------------- Mimivirus genome sequence exhibits a putative ORF (R322, 1740 amino acid long) corresponding to a family B DNA polymerase PolB. This ORF R322 exhibits high scoring sequence homology (BLAST E-value\<10^-24^) against eukaryotic PolBs in the public database. However, this Mimivirus PolB is much larger than its eukaryotic and viral homologues (about 1000 aa), and its optimal alignment with the other PolB sequences reveals four unmatched extraneous segments (Fig. [1A](#F1){ref-type="fig"}, Fig. [S1](#S1){ref-type="supplementary-material"}). Focusing on these extra segments, we identified a 351-aa intein (position 1053 to 1403) in the Mimivirus PolB sequence. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### (A)Locations of inteins found in different DNA polymerases of the family B (PolB) (I, II, III; filled triangles) and other extra segments identified in the Mimivirus PolB (i1, i2, i3; open triangles). Nanoarchaeum equitansPolI is encoded in two pieces of genes (NEQ068, NEQ528), the break point of which corresponds to the position III intein integration site. Full intein motifs are comprised of the C-terminal part of NEQ068 and N-terminal part of NEQ528. (B)A phylogenetic tree of the family B DNA polymerases (PolBs) from diverse organisms, including Mimivirus (R322; GenBank AY653733), Paramecium bursaria Chlorella virus 1 (PBCV), Ectocarpus siliculosus virus (ESV), Invertebrate iridescent virus 6 (IIV), Lymphocystis disease virus 1 (LDV), Amsacta moorei entomopoxvirus (AME), Variola virus, Asfarvirus, eukaryotic DNA polymerase α and δ catalytic subunits, and archaeal DNA polymerase I. Intein containing genes are indicated by bold letters in the figure. Numbers in parentheses on the right of species name designate the numbering of paralogs. Sequences corresponding to inteins or Mimivirus extra segments (i1, i2, i3) were removed for the tree reconstruction. N. equitansPolI split genes were concatenated. (C)A phylogenetic tree based on the intein sequences found in PolBs. Numbers (I, II, and III) in parentheses on the right of species names indicate the intein integration sites. In (B) and (C), trees were built using a neighbor joining method, and rooted by the mid-point method. Bootstrap values larger than 70% are indicated along the branches. ::: ![](1743-422X-2-8-1) ::: After removing those four Mimivirus specific insertions, the Mimivirus PolB sequence exhibited the highest BLAST scores (E-value = 10^-125^, 32% identity) against a soybean DNA polymerase Polδ (SWISS-PROT: O48901) with an alignment covering both the entire Mimivirus and the target sequence. Near equivalent matches are observed with a variety of eukaryotic (from yeast to human) family B DNA polymerase sequences. The best viral homologues were found in phycodnaviruses (E-value = 10^-116^). Conserved carboxylate residues (aspartate and glutamate) at the exonuclease and polymerase active sites \[[@B11],[@B12]\] were all identified in the Mimivirus PolB (Fig. [S1](#S1){ref-type="supplementary-material"}). There was no other ORF encoding a putative PolB in the genome. These suggest that R322 encodes a functional PolB. Consistent with the homology search result, a phylogenetic analysis places the Mimivirus PolB near the root of eukaryotic Polδs (Fig. [1B](#F1){ref-type="fig"}). A similar branching position is obtained for the seven universally conserved Mimivirus genes \[[@B2]\]. Despite low bootstrap values for some of the deep branches in the Fig. [1B](#F1){ref-type="fig"}, this tree clearly indicates the lack of any specific affinity between the Mimivirus PolB and the archaeal PolB sequences containing inteins (bold letters in the Fig. [1B](#F1){ref-type="fig"}). It should also be noted that several other large DNA viruses are known to possess PolBs with a similar phylogenetic pattern \[[@B13]\]. Canonical/archaeal type Mimivirus intein ---------------------------------------- The Mimivirus intein sequence (351 aa) exhibits significant sequence similarities to several known inteins (E-value\<10^-4^), all of which are from thermophilic/halophilic archaea. The best matching intein (E-value = 3 × 10^-8^) is the second intein of the Thermococcus sp. PolB (InBase: Tsp-GE8 Pol-2) with 24% amino acid sequence identity. The Mimivirus sequence exhibits all the expected features required for an active intein (Fig. [2](#F2){ref-type="fig"}). Sequence motifs \[[@B14]\] characterizing the splicing domain (N1-4, C2, C1) and the dodecapeptide LAGLIDADG homing-endonuclease domain (EN1-4) were all identified in the Mimivirus sequence except N4 motif. N4 motif is occasionally absent in the previously characterized active inteins \[[@B14]\]. Amino acid residues providing nucleophilic groups in self-splicing reactions are all present: the first serine and the last asparagine residues of the intein, and the first threonine residue of the downstream extein. Accordingly the Mimivirus intein is a canonical \"asparagine-type\" intein, of which the close homologues have previously been observed only in archaea species. In contrast, the previously reported Chiloiridescent virus intein is a non-canonical \"glutamine-type\" exhibiting a glutamine residue at the C-terminus \[[@B3],[@B15]\]. The threonine and histidine residues in the N3 motif assisting in the initial acyl rearrangement at the N-terminal splice junction are also conserved. Thus, we predict that the Mimivirus intein is an active intein capable of self-splicing. The presence of a homing endonuclease domain suggests that this intein also retained its capacity to spread to other sites of the genome or to other organisms. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### The Mimivirus DNA polymerase PolB intein. The 351 amino acid residues intein sequence is shown with, respectively, the last and the first three amino acid residues of the N-extein and the C-extein. Bold letters represent amino acid residues essential for protein splicing. Conserved intein sequence motifs are indicated by underlines (N1, N2, N3, EN1, EN2, EN3, EN4, C2 and C1). The sequence part matching to the Pfam LAGLIDADG endonuclease domain (PF00961, E-value = 0.16) is indicated by italic letters. The intein/extein boundaries are shown by \'\\|\'. ::: ![](1743-422X-2-8-2) ::: Other three inserts that we identified in the Mimivirus PolB are rather short. Those inserts are unique to Mimivirus, being not found in other PolB sequences. One of the extra segments of 197 aa found at the position \'i3\' (Fig. [1A](#F1){ref-type="fig"}) exhibits a marginal sequence similarity to an intein within the replication factor C of Methanococcus jannaschii(E-value = 0.002, Fig. [S2](#S2){ref-type="supplementary-material"}). However, it also exhibits a comparable level of sequence similarities to several unrelated database sequences, apparently containing low complexity sequences. The i3-insert lacks sequence features required for an active intein. The remaining two extra segments (88 and 121 aa at the position \'i1\' and \'i2\', respectively) did not exhibit any significant similarity to known protein sequences. The biological properties of those three Mimivirus specific inserts remain to be characterized. Mimivirus intein belongs to a specific allele type -------------------------------------------------- Inteins have been identified in different types of DNA polymerases \[[@B16]\]. DNA polymerase catalytic subunits known to contain inteins are archaeal PolI, archaeal DNA polymerase II (PolII), bacterial DNA polymerase III α subunit (DnaE) and bacteriophage DNA polymerase I. Among these, archaeal PolI belongs to the family B DNA polymerase. Archaeal PolI contains up to three intein alleles, the insertion of which always occurs at one of three strictly conserved positions (I, II and III in Fig. [1A](#F1){ref-type="fig"}). Interestingly, the location of the bipartite inteins that separate the two PolI gene pieces of Nanoarchaeum equitans\[[@B17]\] coincides with position III. Remarkably, Mimivirus intein is exactly located at the position III (Fig. [1A](#F1){ref-type="fig"}). The sequence around the insertion site is highly conserved among different PolBs from evolutionary distant organisms such as Escherichia coliand human (Fig. [3](#F3){ref-type="fig"}). The crystal structure of Pyrococcus kodakaraensisPolI \[[@B11]\] reveals that those three distinct sites are in close spatial proximity, in the middle of the DNA binding domain and active site. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Sequence alignment of Family B DNA polymerases from the Archaea, Bacteria and Eukarya domains. The Mimivirus PolB sequence was used without its intein sequence. Only the region of the alignment around Mimivirus intein insertion site (\"YGD\\|TDS\") is shown. The insertion site precisely coincides with the most conserved positions in the sequences, as indicated by bold letters. This is the sole region in the entire sequence exhibiting 6 consecutive identical residues among PolB of the Archaea, Bacteria and Eukarya domains. SWISS-PROT/TrEMBL IDs are DPOL\_ARCFU (Archaeoglobus fulgidus), Q8TWJ5 (Methanopyrus kandleri), DPO2\_ECOLI (Escherichia coli), Q87NC2 (Vibrio parahaemolyticus), Q8SQP5 (Encephalitozoon cuniculi), and DPOD\_HUMAN (Human). ::: ![](1743-422X-2-8-3) ::: Perler et al. observed that inteins present in the same location within homologous genes (\"intein alleles\") tend to be more similar with each other than with inteins in different locations of the same gene or in different genes \[[@B18]\]. This phenomenon appears not only the simple consequence of regular vertical transmission of inteins, but also the result of lateral acquisitions through \"homing\" \[[@B19]\] at the same site of highly similar genes (i.e. \"alleles\") by the mechanism involving gene conversion \[[@B18]\]. Remarkably, the Mimivirus PolB intein holds this rule. The Mimivirus intein exhibits higher sequence homology scores to inteins at the position III of archaeal PolI (designated as \"pol-c allele\") than to inteins in the other PolI locations (I, II) or inteins in other genes. A phylogenetic analysis of the Mimivirus intein and other PolI inteins also supports the classification of the Mimivirus intein in this specific \"intein allele\"-type (Fig. [1C](#F1){ref-type="fig"}). This underlines the presence of intein subclasses (\"intein alleles\") each exhibiting its own preference of harboring site, even in such distantly related homologous genes such as Mimivirus PolB and archaeal PolI. It is implausible that the intein homing mechanism involving gene conversion have led to the direct transfer of an intein between such distantly related homologous genes. Nucleotide sequences (18 bp) around the pol-c allele insertion site do not exhibit unexpectedly high level of sequence similarities between Mimivirus (TATGGAGAC/ACGGACTCA for the amino acid sequence YGD/TDS) and archaeal sequences. For instance, the sequences from M. jannaschiiand Pyrococcus horikoshiiexhibit 7-missmaches (TAT[ATT]{.underline}GAC/AC[T]{.underline}GA[TGG]{.underline}A; MJ0885) and 5 mismatches (TAT[AT]{.underline}AGAC/ACGGA[TGG]{.underline}A; PH1947), respectively. To the best of our knowledge, no evidence has been reported for a homing endonuclease recognizing such different sequences, although homing endonucleases are known to be rather tolerant of single-base-pair changes in their lengthy DNA recognition sequences \[[@B19]\]. A similar observation has been reported for DnaB inteins of Rhodothermus marinusand Synechocystissp. PCC6803 \[[@B20]\]. A shift in the base compositions between intein and extein coding sequences is considered as indicating a recent acquisition of inteins \[[@B20]\]. Mimivirus PolB extein/intein DNA sequence compositions do not show a significant difference. Both exhibit similar G+C-contents (29%) and codon usages. In contrast, Thermococcus fumicolansPolI coding DNA (GenBank: Z69882) exhibits a G+C-content of 57% for the extein regions, compared to G+C-contents of 47% and 49% for its two inteins. Discussion ========== Archaeal PolI inteins have been described only in extremophiles, growing under conditions of temperature over 80°C (hyperthermophiles) or of high salinity (10 times that of sea water; halophiles). Mimivirus is mesophilic, growing in amoeba under the temprature of 37°C. The association of an archaeal-seqeunce-like intein with a eukaryotic-like PolB in Mimivirus thus suggests an indirect interaction between mesophilic eukaryotic viruses and extremophilic archaeabacteria. Mesophilic euryarchaea species similar to the methanogens associated with rumen \[[@B21],[@B22]\] or related species found in human beings \[[@B23]\] might have mediated the transition of inteins between extreme environment and moderate one in the course of evolution. However, no data are available yet on the presence of inteins in the PolB genes of such mesophilic archaebacteria. Lateral transfer (homing) might be responsible for the phylogenetic incongruence between inteins and exteins, and the same intein locations within homologues of distantly related organisms such as Mimivirus and archaea. However, given the specificity of homing endonucleases to long recognition sequences (12--40 bp) and the low level DNA sequence similarity between viral and archeal PolB homologues, a single recent homing event appears quite unlikely. The spread of inteins is better explained by a series of transfers, where inteins progressively accommodated small changes in their homing recognition sequences while retaining their gene position specificity. Such a cascade of transfers could have been mediated by DNA viruses \[[@B3]\]. Consistent results now start to accumulate including recent identification of several inteins in different iridoviruses (S. Pietrokovski pers. comm.), and an intein in a golden brown alga-infecting virus HaV of the Phycodnaviridae\[[@B24]\]. Given the similar base compositions of Mimivirus intein and extein, the low level of intein homology between Mimivirus and archaea, and the likely early origin of the Mimivirus/NCLDV lineage \[[@B2]\], it is tempting to speculate that these DNA viruses might have acquired inteins very early on, and acted as their central reservoir disseminating inteins across different domains of life in the long course of evolution. Conclusions =========== We have characterized a new viral intein found in the eukaryotic-type putative DNA polymerase PolB of Mimivirus by binformatics methods. The conservation of the active site motifs for splicing as well as its insertion at a catalytically important site of the PolB sequence suggests that the intein is most likely to be functional. Our phylogenetic analyses revealed that the intein sequence is closest to extremophilic archaeal inteins. The intriguing association of an extremophilic archaeal-type intein with a mesophilic eukaryotic-like PolB in Mimivirus is consistent with the hypothesis that DNA viruses might have been the central reservoir of inteins throughout the course of evolution. Methods ======= Sequence homology searches were carried out with the use of the BLAST programs \[[@B25]\] against the SWISS-PROT/TrEMBL database \[[@B26]\] and the New England Biolabs Intein Database \[InBase, <http://www.neb.com/neb/inteins.html>; \[Perler, 2002 \#1380\]\]. Pfam \[[@B27]\] searches were carried out with the use of its web site <http://www.sanger.ac.uk/Software/Pfam/>. Multiple sequence alignments were generated with the use of T-Coffee \[[@B28]\]. Intein sequence motifs were identified through the inspection of a multiple intein sequence alignment. Neighbor joining tree analyses were conducted with the use of MEGA version 2.1 \[[@B29]\]. All the gap containing columns in multiple sequence alignments were removed before phylogenetic tree analyses. The gamma distance was applied to compute evolutionary distances. The gamma shape parameter (alpha) was estimated using the GZ-GAMMA program \[[@B30]\]. The sequence and annotation data for the Mimivirus PolB and intein was deposited to GenBank (accession number: AY606804). The complete genome sequence of Mimivirus is also available at GenBank (accession number: NC\_006450). For a comprehensive description of the Mimivirus complete genome sequence and preliminary characterizations of the viral particle, see \[[@B2]\]. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contribution ====================== HO carried out most of the sequence analysis, contributed to the interpretation of the results, and drafted the manuscript. DR contributed to the interpretation of the results. JMC contributed to the construction of the sequence alignment, participated in the interpretation of the results and finalized the manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Supplementary figure S1Sequence alignment of Mimivirus PolB and eukaryotic Polδs. The Mimivirus intein sequence is removed, and its insertion site is highlighted by amino acid residues in red corresponding to the left three and right three resides around the insertion site. Three Mimivirus specific inserts (i1, i2 i3) were highlighted by blue letters. Conserved carboxylate residues in the exonuclease and polymerase active sites are highlighted by green background. Eukaryotic sequences were Encephalitozoon cuniculi(TrEMBL/SWISS-PROT: Q8SQP5), Schizosaccharomyces pombe(P30316) and Glycine max(soybean, O48901). Sequence alignment was obtained with the use of T-Coffee. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 Supplementary figure S2Sequence alignment of Mimivirus insert i3 and known intein sequences. Intein sequences are from Methanococcus jannaschiireplication factor C (Mja RFC-3) and Pyrococcus abyssireplication factor C (Pab RFC-2). ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ The authors wish to thank Dr. Shmuel Pietrokovski for his precious comments, Dr. Keizo Nagasaki for the information about their recent finding of a HaV intein, and Dr. Deborah Burn and Dr. Guillaume Blanc for their critical reading of the manuscript.	PubMed Central	2024-06-05T03:55:52.985533	2005-2-11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549080/", "journal": "Virol J. 2005 Feb 11; 2:8", "authors": [ { "first": "Hiroyuki", "last": "Ogata" }, { "first": "Didier", "last": "Raoult" }, { "first": "Jean-Michel", "last": "Claverie" } ] }
PMC549081	Background ========== Ulcerative colitis (UC) is a chronic, relapsing mucosal disorder that extends in continuous fashion proximally from the rectum and is limited to the colon. The aetiology of UC includes a genetic component possibly involving an abnormal cell-mediated immune response to commensal enteric bacteria in the large intestine. The relapse/remission pattern of the disorder and substrate driven nature of microbial metabolism in the large bowel implicate environmental factors such as diet. Apart from nutritional repletion, dietary measures do not play a role in the management of UC. Nonetheless, attempts to link the cause of UC with specific foods date back at least 50 years\[[@B1]\]. Many foods or food groups have been related to UC (table 1 -- see [additional file 1](#S1){ref-type="supplementary-material"}) \[[@B2]-[@B13]\] including sugar, eggs, soft drinks, fruit and vegetables, protein, carbohydrate and fat. However none have been proven to be of significant benefit or to contribute to the cause of UC. This may partly be because both the assessment of disease activity in UC and dietary intake are difficult to measure, or because the actual dietary component that is key to this relationship has not been measured. It has been proposed that sulfide, produced in the large bowel from either amino acid fermentation or sulfate reduction, may be a triggering factor in the inflammatory process of UC \[[@B14]-[@B16]\]. Recently, in a prospective dietary study where foods rich in sulfur compounds were quantitated, evidence that sulfur compounds may increase the likelihood of subsequent relapse in UC was found\[[@B17]\]. The main source of inorganic sulfur, predominantly sulfate, in the diet are the S (IV) family of additives; the sulfiting agents. Sulfites have been used as food preservatives since the 17th century and are amongst the most widely accepted and versatile of additives. Sulfiting agents, denoted by E220--228 in Europe and generally recognized as safe (GRAS) substances in the USA, include sulfur dioxide, hydrogen sulfites, sulfites and metabisulfites. Sulfiting agents are cheap, easy to use and extremely effective at preventing microbial growth and reducing spoilage\[[@B18]\]. They serve as antioxidants, inhibit enzymatic and non-enzymatic browning reactions and act as a texture modifier in biscuit dough. Sulfites improve color extraction from, and stabilization of grape must in winemaking and preserve lobsters and shrimps from discoloration during iced storage. However, there are some problems with sulfite use\[[@B19],[@B20]\]. In the early 1980s ingestion or inhalation of sulfites was shown to cause bronchospasm in about 5 % of asthmatics. Sulfite sensitivity can pose a particular threat in the workplace where sulfiting agents are used, but may also occur with ingestion of sulfited foods such as potato products and wine. In addition, skin sensitivity has been reported and there are anti-nutritional effects particularly to thiamin which is readily cleaved by the sulfite ion\[[@B21]\]. The mechanism involves an initial nucleophilic attack to the methylene carbon activated by the positive charge on nitrogen, the reaction rate peaking between pH 5 and 6\[[@B18]\]. As a result of this anti-nutritional effect the GRAS status for sulfites was reviewed in the USA and in 1986 the use of sulfites in fresh and frozen fruit and vegetables revoked and a declaration on the label required\[[@B22],[@B23]\]. Earlier (in the USA) their use in meat had been prohibited, because these foods are an important source of thiamin. A study of diet and disease activity in UC using a 7 d dietary diary, a full assessment of disease activity and a method of dietary data analysis that allows trends in food consumption not apparent using customary dietary software was therefore undertaken. Methods ======= Subjects -------- Eighty-one UC patients were recruited and informed consent obtained. Ethical permission was granted by Tayside Committee on Medical Ethics, Dundee, UK (ref 007/00). As it was important to have a range of disease activities present, recruitment included patients at all stages of the disease. Patients were excluded if clinical examination or histology indicated Crohn\'s disease or indeterminate colitis, if there was a positive stool culture for pathogens or if the patient had antibiotic treatment within 3 months preceding the start of the study. Dietary Assessment ------------------ All the UC patients were asked to complete a 7 d diet diary\[[@B24]\]. The diet diary used has been validated for use in the European Prospective Investigation into Cancer study (EPIC). Following completion of the diet diary, subjects attended the research clinic and a full clinical assessment (see below) was carried out. The time interval between the first day of the diary and the clinical visit was on average 28 d. Thus the dietary data is prospective. 7d diet diaries were coded and analyzed using Tinuviel, WISP v3.0 nutritional analysis software (Warrington, UK). Due to the variation in the sulfiting protocols and widespread use of sulfiting agents, current tables of food composition do not contain inorganic sulfur values and cannot be used to quantify intake. Instead of quantitating the intake of particular dietary components, foods and food groups were assessed in their entirety using the method described in the dietary data analysis section (below). Clinical Assessment ------------------- Clinical assessment included history, physical examination and global clinical grading, plus full blood count, liver function tests and inflammatory markers. Patients were examined by rigid sigmoidoscopy or flexisigmoidoscopy and graded on a scale 0--6 (integers and half integers used) according to the macroscopic appearances of the rectal mucosa at a distance 5--10 cm from the anal verge\[[@B25]\]. The clinical assessment of disease activity was confirmed in each case by histological examination, by a single histopathologist blinded to the clinical details, of a rectal biopsy taken from the posterior rectal wall 5--10 cm from the anal verge\[[@B26]\]. A simple clinical colitis score was assigned to patients on each visit following Walmsley\'s scoring system\[[@B27]\], together with blood parameters of disease severity (Hb, plasma viscosity, CRP, serum albumin). Dietary Data Analysis --------------------- Patterns of dietary intake associated with disease activity became apparent through the study of the dietary diaries, e.g. high intakes of sulfite containing foods coupled with a modern processed, convenience diet was associated with a high sigmoidoscopy score. Traditional dietary coding (WISP) did not show any such clear associations between micro or macro nutrient intake and sigmoidoscopy score. Traditional dietary analysis was therefore thought to be missing important patterns in dietary data and a new method of dietary assessment was subsequently developed. This new method used the following procedure. To calculate the association of a particular food with clinical score, each food or food group consumed was given a food sigmoidoscopy score (FSS) calculated by summing the products of food weight and sigmoidoscopy score for each occurrence of the food or food group and dividing by the total weight of the food or food group contained in all diaries. In order for each diary to make equal contributions to the FSSs, the weight of each food was adjusted using the calorific intake for each person. This procedure was carried out separately for every food item recorded in the 7 d diet diaries but is explained below using the example of red wine. Red wine score = (Σv(i)s(i))/Σv(i) for i = 1 to 81 equation 1. Where: - i is the 7 d dietary diary number (n = 81). v(i) is the volume (divided by calorific intake for patient (i) of red wine recorded in 7 d dietary diary i. s(i) is the sigmoidoscopy score associated with 7 d dietary diary (i). Thus foods eaten in large quantities by patients with high levels of disease activity will have high scores and vice versa. The denominator in the above equation is the total volume of the food in question from all diaries (corrected for calorific intakes) so the food scores can be equated with the effect of a typical portion of the food in question on the sigmoidoscopy scores of the patients. This procedure is repeated for every food item. Foods or food groups were excluded from the analysis if 10 or fewer people consumed them or if they made up less than 1 kg of the total intake of the entire population. The decision as to where food group boundaries lay was made depending on the size of the group and whether the differences between the foods were considered important for this study. Statistics and Data Handling ---------------------------- Dietary data was exported from WISP to Microsoft EXCEL 98 (Macintosh version, 1998). A worksheet containing the core headings; Patient ID, food description, weight and patient sigmoidoscopy score was completed. The data was then sorted by food description and each food copied to a separate EXCEL file. Equation 1 was then used to calculate food sigmoidoscopy scores for each food in a manner similar to the example in table 2 (see [additional file 2](#S2){ref-type="supplementary-material"}). Correlation values for scatter plots were obtained using the linear regression function in EXCEL. The equation t = r √((n-2)/(1-r^2^)) combined with t tables provided corresponding significance levels. Results ======= Of the 81 patients recruited 43 were male and 38 female. The average age (range) of the males and females were respectively 53 (26--78) y and 47 (19--74). The distribution of sigmoidoscopy scores is shown in fig [1](#F1){ref-type="fig"}. One third of the patients had sigmoidoscopy scores of 0, 0.5 or 1. The mean sigmoidoscopy score for all 81 patients was 2.09. The correlation between the clinical activity indexes and sigmoidoscopy scores was r^2^= 0.25 (n = 81). Table 3 (see [Additional file 3](#S3){ref-type="supplementary-material"}) shows the foods and food groups with associated sigmoidoscopy scores and average portion sizes. In total 75 foods (or food groups) were given FSS scores. The higher the FSS value the greater the association with disease activity and vice versa. The total weight of foods in all diaries was 1,681 kg. The average food sigmoidoscopy score (i.e. a food sigmoidoscopy score calculated for the entire dietary intake data set was 2.127). Foods excluded from the FSS table (Table 3), by virtue of contributing \<1 kg or being consumed by \<10 people, made up 8 % of the total weight of all foods and had a score slightly lower (2.001) than that of an average food (2.127). Standard errors are not quoted for the food scores as the data used to generate them (weight \* sigmoidoscopy score) was not normally distributed due to the number of sigmoidoscopy scores of 0. The dietary diaries were assessed for completeness by comparing calorific intakes with expected values for the sexes. Expected (calculated from dietary reference tables using age and sex)\[[@B28]\] versus actual values for men and women were respectively 2481 kcal/d versus 2326 kcal/d and 1925 kcal/d versus 1887 kcal/d. Foods for which regulations exist in the EU permitting sulfite addition are shown in table 4 (see [Additional file 4](#S4){ref-type="supplementary-material"}) \[[@B29]\]. Typically a manufacturer will add sulfite up to the maximum permitted level in order to achieve the longest shelf life for the product. A report on sulfite usage in the UK was produced in 2001\[[@B30]\]. Sweet wines, langoustines (prawns), dehydrated potatoes and dried fruit were not given FSS scores because their data quantity fell below the \<10 people or \<1 kg rule. Soft drinks were split into those known to contain sulfite (drinks made from fruit squash concentrates and lucozade) and the rest. In terms of intake (portion size\sulfite concentration), for this population, the major sources of sulfite (FSS, FSS table position) were bitter beer (3.91, 75), white wine (2.87, 73), burgers (2.84, 72), soft drink concentrates (2.79, 70), sausages (2.68, 68), lager (2.47, 64) and red wine (2.00, 29). A Mann-Whitney test on the FSS positions of these foods gave a significance of p \< 0.001. The sulfite-containing, alcoholic beverages; wines and beers, were associated with increased UC disease activity, but spirits were not, which suggests a role for sulfite rather than alcohol in the disease process. A plot of alcohol consumption from wine and beer against sigmoidoscopy score revealed a significant positive correlation (n = 81, r^2^= 0.07, p \< 0.02). Decaffeinated coffee appeared better for the UC patient than the caffeine-containing counterpart. Decaffeinated tea is not shown on table 3 because it was only drunk by 9 people but had a FSS of 1.71 versus 2.01 for the caffeine-containing product. Whole fruit consumption appeared better than the corresponding juice (e.g. fruit juice scored 2.43 compared to citrus fruits at 1.96 and apples at 1.67). An average thiamin concentration (mg / 100 g) for each food or food group is also shown in table 3. There is a significant correlation (p \< 0.005) between this thiamin value and the food\'s sigmoidoscopy score. Discussion ========== Ulcerative colitis is considered to have a genetic component. Twin studies\[[@B31]\] have shown a 10% concordance of UC in monozygotic and 3% in dizygotic twins suggesting about 90% environmental and 10% genetic contributions. The pool of genetically susceptible individuals is therefore at least 10 times greater than those diagnosed with the condition. A failure to date in identifying the gene(s) responsible points to a complicated genetic component featuring multiple polymorphisms. The first acute episode of UC must disrupt either, the ecology of, or the sensitivity and selectivity of the immune system to, the commensal enteric microflora sufficiently to cause the chronic condition. More extreme versions of the environmental conditions that lead to subsequent relapses could conceivably lead to the first acute episode. Of all the dietary components studied in relation to UC risk and disease severity, milk has probably received the most attention. Andreson\[[@B1]\] was the first to postulate that food allergy was the cause of UC in two-thirds of his patients, and by the use of elimination diets claimed to identify the offending food and remove it. In Andreson\'s experience, the most common provoking antigen was cow\'s milk. His views were confirmed by Rowe\[[@B32]\] and later by Truelove\[[@B33]\]. They all postulated that milk protein sensitivity was an aggravating cause of disease in up to 5% of colitic patients, who benefited from a milk-free diet. While able to demonstrate circulating antibodies to milk proteins more frequently and in higher titer than in matched controls, they were unable to correlate the occurrence and titer of these antibodies with the extent, severity, or duration of colitis, or with the response to a milk-free diet. Mishkin\[[@B34]\] concluded, in a review of the subject, that IBD patients avoid dairy products to a much greater extent than the prevalence of lactose malabsorption and/or milk intolerance in this population group would justify. This observation was probably due to the incorrect perceptions of patients and arbitrary advice of physicians and authors of popular diet books. In order to ascertain whether dietary antigens may sustain the mucosal inflammatory response, two prospective controlled trials have investigated the effectiveness of bowel rest and total parenteral nutrition as primary therapy in the management of acute UC\[[@B35],[@B36]\]. Neither study found any benefit over conventional corticosteroid treatment alone and so the possibility of a dietary antigen driving the chronicity of the disease seems unlikely. These results are in agreement with work demonstrating\[[@B37]\] that a split ileostomy is of little benefit in the management of UC, but the latter observations may have been confounded by the development of diversion colitis\[[@B38]\]. The dietary analysis procedure proposed here has the potential to highlight trends in dietary data that would not be apparent using traditional dietary analysis software and could be useful in the study of other diseases with dietary associations. This system would highlight any possible dietary factors both positive and negative, not just sulfite. The proposed method is less reductionist than traditional coding as it assesses the risk of each food item or group rather than the risk from the foods\' (quantitated) constituents. Part of the power of this study derives from the availability of a sigmoidoscopic grading (0--6) of the severity and extent of the disease. This grading provides the statistical variable that is normally obtained from a non-UC control group. Other alternative systems for analysis of disease risk for dietary components are; the use of disease occurrence odds ratios between the top and bottom quartiles of intakes, and assessing the correlation coefficients between disease activity and intakes. The odds ratio method loses data and data accuracy by characterizing intakes as high, high middle, low middle and low and then discarding the middle two quartiles. The correlation method is dependent on spread. The proposed system has neither of these disadvantages. The food sigmoidoscopy score calculation does rely on the assumption that the sigmoidoscopy score is an approximately linear scale, i.e. a sigmoidoscopy score of 6 is caused by the consumption of a double portion of a harmful food item of sigmoidoscopy score 3. This could be argued to be reasonable. Both the sigmoidoscopy grading and dietary analysis method are validated methodologies. The food sigmoidoscopy score is simply a mathematical function of these two variables. As all data is transformed according to the same simple rules any statistical treatment of the results is as valid as statistical treatment of the raw data. Whilst clinical activity indices were used to generate analogous scores to the food sigmoidoscopy scores, the results from these measurements are not included in this paper. Clinical activity index involves subjective measurements such as a feeling of well being. Thus, the food orders generated by these measurements were not thought to be as accurate as those generated by the sigmoidoscopy scores. The consensus of previous studies on diet and UC pointed to the modern, processed, highly refined, Western diets as being damaging. The results presented here linking diet with disease activity are broadly in agreement with this. Additionally they propose a new risk factor for UC, namely intake of sulfited foods. The involvement of diet in UC is controversial. Differences in dietary intake between patients and controls could be a result of changes in diet brought on by the symptoms of the disease process\[[@B4]\]. While this explanation is possible it does not seem likely that patients would increase their beer and wine intake as a consequence of feeling unwell. The relationship between sulfite intake and sigmoidoscopy score in this study was extremely strong and therefore an explanation for why sulfite should be a risk factor for UC is required. Sulfite has a number of effects that may be relevant to this discussion. Sulfite may be important because it is a precursor of sulfate. Sulfate can potentially be reduced to sulfide by sulfate reducing bacteria in the colon. Sulfide is a plausible metabolic toxin in UC. Supplementing patients with sulfate decreases the microbial incorporation of hydrogen into methane (as measured by breath methane) and increases the in vitro sulfide production rate of feces\[[@B39]\]. The end metabolic product of both sulfite and protein is sulfate. Sulfate from both sources can be reduced to sulfide in the gut. The absence of a significant relationship between protein intake and disease activity in this study does not support a mechanism for UC that involves a common pathway for sulfite and protein. Alternatively, the relevance of sulfite to UC may be because of its ability to degrade thiamin (particularly at colonic pH). Thiamin deficiency manifests itself in the nervous and cardiovascular systems. It is unlikely that it is the status of the patient that is important, but rather the amount of thiamin available to the gut microflora. An example of the importance of thiamin to the gut microflora is the requirement of the probiotic bacteria, lactobacilli, for thiamin. Thiamin status is influenced by a number of factors. Firstly, thiamin intake; in foods such as pork, fortified cereals and legumes which are good sources of thiamin, intakes were associated with improved clinical state. Traditional dietary analysis did not reveal a significant correlation between thiamin intake and sigmoidoscopy scores though no allowance is made in dietary coding software for the reduction in thiamin content caused by sulfite usage. Secondly, carbohydrate intake; Elmadfa et al*. demonstrated that the thiamin status of adult humans depends on carbohydrate intake\[[@B40]\]. Carbohydrate (and sugar) intakes have previously been associated with UC relapse (table 1). Finally, thiamin status can be affected by caffeine\'s anti-thiaminergic properties. For both coffee and tea intake, the decaffeinated version was associated with better clinical state. However, there was a sub group (n = 8) of this population who recorded an intake of either vitamin B complex or multivitamins. This sub group did not have a mean sigmoidoscopy score significantly lower that the general UC population. It is likely that vitamin B1 is a factor in the disease process but not the only nutritional one. An additional possible interpretation for the experimentally determined food order is the carbohydrate nature and content of the foods. Carbohydrates, such as the α-amylase resistant starch (RS) and prebiotics, escape digestion in the small intestine and provide an energy substrate for the colonic microflora. Both prebiotics (found in chicory, legumes, artichokes alliums, and in small amounts in cereals) and resistant starch (potatoes, bananas, lentils and legumes) have been hypothesised to improve the colonic health of the host. For RS, resistance to digestion is a function of the morphology of the starch granules and their crystalline organisation, which is determined by the botanical source of the starch and the processing it has undergone before being eaten\[[@B41]\]. Prebiotics are non-digestible carbohydrates that selectively stimulate the growth of lactobacilli and bifidobacteria with benefit to health. Prebiotics are mainly fructose and galactose polymers with a degree polymerisation of between 2 and 60. Of the prebiotic sources; chicory and artichokes were not found in typical diets, legumes and cereals were seen to have probable benefits in this study and alliums were not. This study therefore provides only limited support for the use of prebiotics in UC. The foods containing RS were all found to be of benefit in this study and therefore the role of RS in UC is strongly supported. Any dietary advice provided to ulcerative colitis patients should be based on the FSS table. The table is of course imperfect because of experimental error, natural variation and the associations between foods. For example, milk and cereal are coded separately but are often consumed together. Thus the magnitude of the difference in the FSSs for these two foods is less than if they\'d been independent variables. Suggestions have been made in this discussion as to the factors responsible for the FSS order and to distill these factors into the advice given in Table 5 (see [Additional file 5](#S5){ref-type="supplementary-material"}). This table is speculation, as this diet has not been formally tested in the UC population. It does however represent the only comprehensive dietary advice available to ulcerative colitis patients at this time. The list of dietary risk factors for colon cancer\[[@B42]\] bears a similarity to the dietary risk factors presented here for UC. UC patients have an increased risk of colorectal cancer and it is probable that factors responsible for inflammation in UC patients are also responsible for neoplasia in the colon cancer population. Conclusion ========== A dietary analysis method is described that provides a new tool for establishing relationships between diet and disease. This method has been applied to the study of ulcerative colitis and points to sulfite and caffeine as being harmful, with thiamin and resistant starch being potentially therapeutic. For the first time, dietary guidelines for ulcerative colitis patients, including food portion sizes have been developed. Abbreviations ============= Ulcerative colitis (UC); Food sigmoidoscopy score (FSS); European Union (EU); Odds ratio (OD) and Confidence interval (CI). Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= JHC, EAM and LME contributed to the study design and the writing of the manuscript. EAM was the co-ordinator of the study and along with ST had responsibility for managing the patients and coding the diaries. LME developed the dietary data analysis protocol assisted by RC. CK and JHC performed the sigmoidoscopic examinations of the patients. All authors read and approved the final manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Review of studies of diet and ulcerative colitis (UC). ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 Food sigmoidoscopy score (FSS) calculation example for red wine (NB incomplete data set used). ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 3 Foods consumed in order of food sigmoidoscopy scores (FSS)\[[@B43]\]. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 4 Permitted levels of sulfite in the UK. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 5 Proposed dietary advice for ulcerative colitis patients\[[@B44]\]. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ Core funding for this study was provided by the National Association of Colitis and Crohn\'s Disease. Additional funding was provided by the Sir Halley Stewart Trust, Chief Scientist Office, Scotland and the Anonymous Trust, Dundee. We are very grateful to all these organisations. Thanks go to all those who volunteered for this study. Their enthusiasm and humour throughout the study was greatly appreciated. Thanks go to the endoscopy staff; Shirley McLeod, Marion Winters, Shona Pryde and Sheena McWha and to our secretary Helen Cowper without whom the study would not have run so smoothly. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Frequency distribution of sigmoidoscopy scores (n = 81) for ulcerative colitis patients recruited at all stages of disease. ::: ![](1475-2891-4-7-1) :::	PubMed Central	2024-06-05T03:55:52.987215	2005-2-10	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549081/", "journal": "Nutr J. 2005 Feb 10; 4:7", "authors": [ { "first": "Elizabeth A", "last": "Magee" }, { "first": "Laurie M", "last": "Edmond" }, { "first": "Shiona M", "last": "Tasker" }, { "first": "San Choon", "last": "Kong" }, { "first": "Richard", "last": "Curno" }, { "first": "John H", "last": "Cummings" } ] }
PMC549082	Innumerable animal species produce noises of different kind and many of them sound musical to us -- human beings -- eliciting a variety of emotional feelings, as the frogs\' chants in a rainy night, or the wild birds\' songs in a sunny morning perhaps mixed with our finch\'s joyful trills from its small cage, or the deep thrilling whales\' hissing who very few have had the chance to listen (as I once did in Colombia). Most likely, they all are just communication ways, perhaps to let the other guy know that the male is there, waiting for the female to come and meet it, because usually only males sing. Is all that really music? I do not think so, for music is unique to man and woman and only we, human beings, get together either in small or large groups just to listen to music or to make music. Music conveys a message, which only the human being can decipher in his/her own fashion; as in animals, it is no doubt a communication means. The isolated man or woman, the Robinson Crusoe, is essentially non-existent. Music is a natural human need, almost at the level of the basic biological ones (self and species preservation). The current literature on music and the human being is enormous, from artistic to technical to psychological to physiological aspects, or what have you. Music is so much embedded in us that never many studies are or will be enough. This little book, little in size but not in scope, authored by Dorita S. Berger (an accomplished concert pianist and certified music therapist, with superb academic background and solid experience), seems to cover a paucity because it falls within the reach of those who, without the requisite of being specialists are not fully outsiders; it also serves as an introduction to those who may like to proceed further in any of the several paths that converge and diverge to and from this fascinating area of human adaptation. The book consists of a foreword, a preface, 11 chapters and 2 appendices. Its foreword is unique and deep in content, indeed. Written by Donna Williams, herself a diagnosed autistic, captivating and exemplary woman, author of several publications (among them a book entitled Autism: An Inside-Out Approach, 1996, Jessica Kingsley Publishers), its sentences must be read over and over until one really apprehends its profound meaning, as her initial paragraph, where she states: \"Mind and consciousness are like a gradual sunrise that takes time to come up. In the meantime, we are not stagnant, waiting for life to happen.\" She refers to \"the music of life, felt with our bodies, long before we identify mind with self \... \... some of us stay here longer than others.\" And a little further down, \"music in its broader sense is everywhere, and it is our first language \... it has the most important of all places in the lives of those who find the realm of the mind a place of rusty cogs and heavy effort.\" Donna declares that music was an exceptionally important part of her life and almost naïvely and charmingly confesses: \"I could not understand three sentences in a row until I was nine\". These deep thoughts and experiences remind of James Joyce\'s agenbite of inwit, or the wound of in-looking, the hurt of self-analysis, the painful knowing of oneself, introduced in his famous, conflicting and monumental Ulysses. Man at large is the prey of inner suffering. The preface is already in the hands of the author, where the book is introduced along with some general concepts, such as: \"Entering the brain through the auditory system is a key to obtaining automatic brain attendance and response\" and \"physiologic changes resulting from music intervention (or stimulation) can be a major factor in bringing about a feeling of well-being\". Dorita aims the text at music therapists, parents, teachers and health clinicians. However, in my opinion and as anticipated above, its audience should not be restricted to such relatively small group but rather can be much broader, as there are, for example, students or even researchers developing specific projects in the area. Already ending the preface, the author underlines quite sensibly that the book is not a recipe since music therapy is an approach to solving a physiologic or psychological problem. Chapter 1 (Introduction: Who Defines \"Appropriate\"?, pp17--25) is a dareful good beginning that encourages the reader to proceed. For the human animal, the expectation is to \"follow the rules\" of the pack and behave accordingly, and Dorita very wisely asks who defines \"appropriateness\" and for whose benefit the rules are designed. What is normal and typical? Are the two words interchangeable? What determines \"right\" and \"wrong\" responses? Are these not \"labels\" somehow imposed by society? The concepts of \"right\" and \"wrong\" are basically determined by socio-cultural standards rather than scientific givens. All this set of questions go far beyond the intended objective of the book and scrape deep in the human self, both as an individual and as a member of society. Allow me to bring about the painful, senseless, dire and cruel Irak War (April 2003): Is the Bush administration not imposing its own pack rules, denying rights as illegal for other countries (other societies or packs) while retaining those very same rights as legal for itself? Is it bad behavior for the others to have weapons while it is good behavior for the USA to have them? No doubt, Dorita touches, perhaps without realizing, a huge and essentially unsolvable problem that only true generosity and unabashed love, mostly and so far unsuccessfully called for by the core of our unfulfilled religions, might one distant and dreamt of day bring forth. Yes, excellent and well-posed question: Who defines \"appropriate\"? In Chapter 2 (Aspects of Autism, pp26--34) the author says that autism is a neurologically atypicalmanner of function, with genetic and sensory-motor implications; what appears in childhood, remains throughout life. She introduces the concept of a different world the child is immersed in and, as such, cannot possibly get connected to this \"real\" one, with other channels and incomprehensible rules. His/her desire for sameness is broken at worst or disturbed at best. Another good point: By and large, most of the people avoid anything bringing change, anxiety, confusion, insecurity and the like. Sameness, conservation of what we know, appears as safer. A trip to a foreign country or place carries with its sole organization an element of tension and we often think \"oh, we had better call it all off\". And children are particularly susceptible and sensitive to these changes (such as a new school or neighborhood). Chapters 3 (Aspects of Sensory Integration, pp35--48), 4 (Functional Adaptation Defined, pp 49--60), 5 (Understanding Basic Sensory Systems, pp 61--77) and 6 (Are You Listening? Part One: About Hearing and Listening, pp78--90) deal mainly with physiological aspects, perhaps a little in excess because it tends to overload the reader removing him/her from the central subject, which is music, as stimulus or intervention, akin in a sense to electrical or mechanical or chemical or thermic or magnetic stimulation. However, it is fine and useful. As an interested reader and somewhat also as an actor within the discipline, the comment may be taken simply as an expression of desire. One paragraph called my attention almost at the end of chapter 3: \"Music does not require semantic interpretation. It simply provides an environment -- a sound blanket -- wrapping itself around the body and providing a sense of safety and security.\" Good attractive concept, but I would emphasize what I say previously: Music carries a message, it may not be semantic or absolute, but the listener somehow decodes it to suit his/her own cenesthetic state. The basic driving forces of man and woman are clearly stated in Chapter 4, two are purely biological (as anticipated above); other two pertain only to the human being, self-determinationor freedom of choiceand spiritual fulfillment. Let me add that one essential tool for their satisfaction is communication, which is a need, and music appears as a phenomenal way to accomplish it. The messages left by Johann Sebastian Bach, Wolfgang Amadeus Mozart or Ludwig van Beethoven are there and will be there forever, hence demonstrating unparalleled self-determination and spiritual fulfillment, even after death. Finally, accommodation, as discussed here, is a good classical physiology concept that fits very well this new frame of music and human adaptation. An appealing idea advanced in Chapter 5 refers to a possible release of chemical neurotransmitters (such as dopamine, endorphines, encephalines or others) elicited by music stimulation. The whole thing may mark a difficult but attractive research avenue. Chapter 6 is in my modest opinion and with due respects the weakest of the book: auditory scanning is definitely obscure, the section titled \"sound coding tango\" (what does the word \"tango\" do here?) does not show a relationship with the text that follows and the remaining sections are at best confusing. The last paragraph in page 89 sounds very appealing, at least to me, and I tend to agree with it \... but it lacks so far solid scientific support. In Chapter 7 (Are You Listening? Part Two: Dimensional Hearing and Erroneous Assumptions, pp 91--111) Dorita starts with the case of a four year old boy, Jason. No musical instrument really moved him, until one day he spotted a gong. It captured his love and reacted as fully unfazed by its intense volume. Quite an amazing response considering that most people (the pack) get annoyed by a gong sound, especially if it is played within a small room. Thereafter, she briefly discusses some common erroneous assumptions, as for example, \"music is always fun\" and \"the content of a recognizable tune is being perceived by another in the same format in which it is being presented by the therapist\". The latter agrees with the previous contention: each listener to suit his/her cenesthetic needs decodes the musical message. Such assumptions deserve to be taken into consideration in other environments, too. Example: A person complains that his neighbor plays piano every day after midnight and, thus, disturbs his sleep. The player argues back saying that he plays beautiful classic music loved by everybody. Who is right? The author brings also forward an interesting and novel possibility: Is there such a thing as auditory dyslexia? There seems to be no term for a phenomenon in which sounds are retrieved in incorrect order (as in reading or writing dyslexia). Researchers may have in this another subject to investigate. After finishing this chapter and reviewing the variety of concepts and subjects touched in it, I could not but wondering that the human being enjoys with the eyes the landscapehe or she moves about, simultaneously the complex auditory tract perceives the soundscapethat surely accompanies the former, probably also seasoned by an odorscape, tastescapeand even a touchscape; but everything is slowly evolving in the timescape, as a multimensional function of time. The whole configures the complex scenery we live in creating what Donna calls the music of life. Chapter 8 (Elements of Music for Sensory Adaptation, pp 112--129) goes, with some tardiness in the development of the book (at least to my particular taste), into music itself, and I would like to use the term music stimulation, characterized by six properties, as Dorita explains: rhythm, melody, harmony, dynamics, timbre, and form. This is not the place to give details about these elements for the author clearly fathoms in them solidly and without a single doubt. My only comment, and just as a curiosity recalling my days as cardiovascular physiologist, refers to one teacher actually using pulsed hand-clapping (a pacemaker) to call her entire class of children to order, to subdue chaotic impulses (page 114). A fibrillating heart has its fibers in a dynamic state of dyssynchrony (as the children shouting in the classroom). No effective contraction to eject blood takes place. One or may be two electric shocks usually stop the disorder and the myocardial mass resumes its rhythmic synchronic activity. This is called a defibrillating maneuver. Are the similarities of nature not nice? As Albert Einstein used to say, the most incomprehensible thing of the world is that the world is comprehensible. Chapter 9 (Music Therapy in the Realm of Sensory Integration, pp 130--151) is more practical as it discusses the different types of musical instruments from the viewpoint of their suitability to specific therapeutic objectives. In Chapter 10 (Formulating Music Therapy Treatment for Sensory Adaptation Goals, pp 152--165), instead, several aspects are discussed, some of them somewhat superficially and not meeting the expectations developed by the subtitles, as for example \"adaptive responses to environment of auditory and visual stimuli\" or \"auditory integration and differentiation\". The last Chapter 11 (Conclusions, pp 166--175) is some kind of summary. Let me quote a wise paragraph, a caveat emptoror let the buyer beware: \"Music therapy does not lend itself readily to rigorous scientific explanation. Findings are anecdotal at best, and based on observations, insights and informed assumptions. Music therapy does not replace other interventions, nor does it need to copy the goals of other therapies in order to be effective\". This is why the field is fully open for serious research. I think the book meets the intent of its author (page 173): \[It\] \"is an introduction to physiologic information and perspectives that can help the music therapist assess more astutely the behaviors and presenting problems of an atypically functioning patient.\" Now, and to finish this review by being a little prickly but honestly (and perhaps naïvely) constructive, let me point out a few minor aspects that might improve another edition of the book: The title does not properly reflect the content of the book. It should be named The Autistic Child, Sensory Integration and Music Therapy, for this is really the order followed in it. The physiology section could be reduced. There are also some unnecessary repetitions. The bibliography is given in three groups: specific references to each chapter, recommended references, and a general list at the end of the book. It is not practical. I would rather have a single list at the end properly referred to in the course of the text. Appendix A is the reproduction of an article by Schneck and Berger. It adds something to the book but not to its core material. Thus, it might be deleted in the future. Appendix B is a collection of four case histories. By far is too long containing unnecessary information. It should be greatly reduced and limited to the pertinent information. The music portion could be enhanced. Having said more than enough, I recommend the book and congratulate the author for her dedication, effort and beautiful activity by combining art with benefit to the many time forgotten children.	PubMed Central	2024-06-05T03:55:52.990095	2005-2-10	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC549082/", "journal": "Biomed Eng Online. 2005 Feb 10; 4:9", "authors": [ { "first": "Max E", "last": "Valentinuzzi" } ] }

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