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although systemic chemotherapy is the mainstay of treatment for distant metastasis of cancer , there are several cancers known to have survival benefit from pulmonary metastasectomy , which include colorectal carcinoma , renal cell carcinoma , and osteosarcoma .
systemic chemotherapy remains the standard treatment for metastatic urothelial cancer since cisplatin - based combination chemotherapy introduced in the 1980s . despite relatively high response rate of chemotherapy ( range , 50% to 70% ) , patients frequently experiences recurrence and have poor prognosis .
surgical resections of lung metastases from urothelial carcinoma are not common , and there are limited data for role of metastasectomy on long - term survival . in this study , we retrospectively reviewed our experience to identify the feasibility and benefits of pulmonary metastsectomy from urothelial cancer .
between 1998 and 2010 , 16 patients of urothelial carcinoma with lung metastasis underwent metastasectomy at samsung medical center .
eligible criteria for surgical resection are as follows : 1 ) the pulmonary lesion must be completely resectable ; 2 ) metastasis was limited to the lung ; 3 ) primary tumor had been or could be completely controlled ; and 4 ) patient was medically operable and had sufficient expected pulmonary reserve after resection .
after surgery for primary tumor , adjuvant chemotherapy was administered according to the guidelines of national comprehensive national comprehensive cancer network , unless pathologic stage was t2 or less and there was no nodal involvement or lymphovascular invasion .
palliative chemotherapy was administered when there was evidence of disease progression like distant metastasis or unresectable loco - regional recurrence .
regimens for adjuvant chemotherapy were generally 3 cycles of gemcitabine and cisplatin ( gp ) .
palliative regimens were mostly 4 to 6 cycles of gp , but sometimes methotrexate , vinblastine , doxorubicin , and cisplatin were used .
after initial surgery for primary tumor , chest computed tomography ( ct ) for the surveillance of pulmonary metastasis was performed by 6 month interval generally . since 2004 ,
positron emission tomography ( pet ) was routinely performed 1 year after surgery for primary tumor .
after pulmonary metastasectomy , chest ct was performed at every 3 months for the first year , every sixth months for the next 2 years , and annually thereafter .
surgery was generally targeted to limited resection such as wedge resection and segmentectomy . however , in case that tumor was located centrally or could not be differentiated from primary lung cancer by intraoperative frozen section biopsy , lobectomy or more extended resection was considered .
descriptive statistics for categorical variables are reported as frequency and percentage , and continuous variables are reported as mean or median as appropriate .
chicago , il , usa ) . this study was reviewed and approved by institutional review boards in samsung medical center .
sublobar resection was performed in 10 patients ( 8 wedge resections and 2 segmentectomies ) .
lobectomy was performed in 6 patients . in 3 patients , intraoperative frozen section of nodule
could not differentiate it from primary lung cancer due to very poor differentiation of tumor , and therefore , lobectomy was performed with mediastinal lymph node dissection . in the other 3 patients ,
the median hospital stay was 5 days ( range , 2 to 10 days ) .
the 5-year overall survival and disease free survival were 65.3% and 37.5% , respectively ( fig .
site of recurrence included lung ( n=5 ) , loco - regional relapse in bladder with lung metastasis ( n=1 ) , liver ( n=1 ) , and multiple mediastinal and cervival lymph nodes ( n=1 ) . in all the 5 patients with pulmonary recurrence ,
newly detected pulmonary nodules were found to be resectable again , and repeated metastasectomy was performed , which included 4 wedge resections and 1 lobecctomies .
median disease free interval between the first metastasectomy and repeated resection was 10 months ( range , 1 to 56 months ) .
one patient with 1 nodule after 3 months from second metastasectomy underwent wedge resection , and the other with 6 nodules after 4 months from second metastasectomy underwent bilateral wedge resection .
patient with loco - regional relapse in bladder with lung metastasis underwent transurthral resection of the bladder tumor and metastasectomy simulataneously but spine metastasis relapsed 7 months later and radiation therapy applied .
total 3 patients expired after initial pulmonary metastasectomy , one patient was who recurred at cervical and mediastinal lymph nodes , another was recurred at liver and last one was not known because of follow - up loss but confirmed by telephone interview .
all five patients with recurrence limited to lung without extrathoracic metastasis who underwent repeated metastasectomy are alive for median survival length of 31 months ( range , 5 to 39 months ) .
despite distant metastasis is regarded as systemic disease , there are some malignancies such as colorectal cancers , renal cell carcinoma , or various types of sarcoma in which pulmonary metastasectomy contributes to prolong survival .
furthermore , in these tumors , even repeated metastasectomy has been shown to have favorable outcomes .
urothelial cell carcinoma is known to be relatively chemosensitive , and standard treatment for systemic metastasis from urothelial carcinoma has been palliative combination chemotherapy .
however , despite that systemic chemotherapy promises high response rate ( range , 50% to 70% ) in metastatic urothelial carcinoma , its effect lasts transiently and the disease eventually progresses with poor prognosis .
several authors reported favorable outcomes regarding pulmonary metastasectomy in combination with chemotherapy for urothelial carcinoma . in 2004 ,
siefker - radtke et al . reported result of 31 patients who underwent metastasectomy , which included 24 metastasis to the lung , 4 to distant lymph nodes , and 2 to the brain .
five year overall survival after metastasectomy was about 33% and median overall survival time was 23 months .
in 2007 , abe et al . reported experience of multimodal treatment of 48 patients with metastatic urothelial cancer .
they found median survival time was significantly different ( 42 vs. 10 months ) in metastasectomy group and the number of chemotherapy cycle , sites of metastasis , and resection of metastasis were independent predictors on survival .
they reported 5-year overall survival rate of 50% and size of metastasis greater than 3 cm was a significant poor prognostic factor . in 2010 , kanzaki et al .
reviewed data from 18 patients who underwent pulmonary metastasectomy from transitional cell carcinoma and reported cumulative 3 and 5-year survival rate of 59.8% and 46.5% .
they also found solitary metastasis is associated with good prognosis . in our series , we also observed favorable survival in after metastasectomy from urothelical cell carcinoma .
those 5 patients in our series had recurrent pulmonary metastasis but not in other organs .
all of them underwent repeated resection and two of them underwent third resection for recurrent disease .
second , it was very difficult to compare the results of surgery with those of non - surgical treatment because of differences in patient characteristics between the two groups .
patients without metastasectomy usually had much advanced disease with multiple metastases to the lung or various organs , or their physical performance or pulmonary reserves are not sufficient for additional surgery .
third , many patients in samsung medical center who could have been potentially candidates of pulmonary metastasectomy might not have referred to surgery due to lack of agreements for pulmonary surveillance and treatment strategy for pulmonary metastasis after surgery of urothelial cell carcinoma , and these can lead to selection bias .
although there is still limitation , pulmonary metastasectomy can be applied in patients who have pulmonary metastasis from urothelial carcinoma with favorable outcomes .
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backgroundtreatment of pulmonary metastasis from urothelial cell carcinoma has been mostly palliative chemotherapy and the role of pulmonary metastasectomy has not been investigated much.materials and methodsthis study is a retrospective interim review of pulmonary metastasectomy from urothelial carcinoma at single institution between 1998 and 2010 .
overall 16 patients underwent pulmonary metastasectomies.resultsthere was no postoperative complication or hospital mortality .
mean hospital stay was 6 days .
overall and disease - free 5-year survival were 65.3% and 37.5% , respectively.conclusionin selected patients with pulmonary metastasis from urothelial carcinoma , surgical treatment is feasible and could contribute to long - term survival in selected patients .
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overweight and obesity prevalence rates are increasing worldwide . about 1.4 billon adults were overweight in 2008 , of whom 200 million men and 300 million women were considered to be obese . in the netherlands , prevalence rates of overweight ( and obesity ) are 60% ( 14% ) in men and 44% ( 13% ) in women .
high prevalence rates have been found specifically among ethnic minorities and low socioeconomic status ( ses ) groups [ 1 , 3 ] .
these health risks include cancer , cardiovascular diseases like hypertension and diabetes , and problems with mobility [ 57 ] .
the who recommends adults aged 1864 to engage in moderate - intensity aerobic physical activities such as walking , cycling , or doing household chores for at least 150 minutes a week . active transport ( i.e. , walking or cycling from a to b ) can thus help achieve sufficient levels of physical activity [ 9 , 10 ] .
this review of reviews showed that age and overweight were inversely correlated with physical activity , whereas the male gender and ethnic origin ( white ) were positively correlated with being active .
generally , these correlates were also found for active transport among low - income populations . in the past decade
particularly , studies from the usa and australia have found positive relations between physical activity and neighbourhood walkability [ 13 , 14 ] .
most of these studies , however , have been performed among high - ses populations who tend to live in neighbourhoods with relatively high walkability . moreover , high - ses populations have shown higher levels of physical activity and lower prevalence rates of overweight compared to low - ses populations .
it is not yet clear whether the findings from these studies are also valid for low - ses populations living in deprived neighbourhoods .
detailed information on the relation between neighbourhood characteristics and physical activity among subgroups within this population is currently lacking .
it is hypothesized that more favourable neighbourhood characteristics are associated with higher levels of activity .
past studies that focused on the effect of neighbourhood walkability on active transport have shown that associations are reduced after controlling individual demographic characteristics . however , in addition to the confounding role of demographic characteristics , knowledge about interactions between demographic variables and neighbourhood characteristics is required in order to distinguish subgroups that are more vulnerable or responsive to environmental features than others .
inhabitants of dutch deprived neighbourhoods vary in ethnic background and are often characterized by a low socioeconomic status . as a result ,
the prevalence rate of overweight is higher , and level of physical activity is lower compared to those of the average dutch population [ 17 , 18 ] .
this makes inhabitants of dutch deprived districts an important target group for physical activity interventions .
furthermore , previous research has shown that it is difficult to address ethnic minorities , and guidelines for this specific group are lacking .
more insight into the personal and neighbourhood factors that are associated with active transport could contribute to the development of effective interventions targeting ethnic minorities .
therefore , this study investigates which personal and neighbourhood environmental characteristics are associated with active transport among inhabitants of dutch deprived neighbourhoods . in addition , interactions between personal and neighbourhood environmental characteristics will be tested to explore differential associations of neighbourhood walkability with active transport within subgroups .
briefly , urban40 is a longitudinal study that evaluates the health impact of area - based interventions to improve housing , employment , education , social integration , and safety in the 40 most severely deprived neighbourhoods in the netherlands , also known as the dutch district approach . in 2007 ,
the dutch government made a list of the 140 most deprived neighbourhoods in the netherlands , based on scores on eighteen different registry - based indicators of physical and socioeconomic deprivation as well as physical and social problems reported by residents . from this list
, twenty neighbourhoods were selected that belonged to the 140 most deprived dutch neighbourhoods . in each district , 250 randomly selected adults aged 18 and older received a letter in which they were invited to participate , including a reply card that they could return free of charge . trained interviewers contacted the residents who sent a reply card and arranged home visits for the interview . up to two reminder letters were sent when people did not respond via the reply card . in addition , trained interviewers from an interview agency specialized in research among multicultural populations recruited participants by unplanned home visits ( ringing doorbell at the addresses on the original list ) .
the trained interviewers were tailored to gender and ethnicity ( spoken language , e.g. , dutch , english , turkish , classical arabic , or arab - berber ) of the residents .
financial constraints limited recruitment of respondents by unplanned home visits in all districts , favouring larger districts .
respondents filled out the questionnaire by themselves , but they could also ask the assistant that brought the questionnaire to help them , if needed in their own language ( dutch , english , turkish , classical arabic , or arab - berber ) .
in particular the participants that were of non - dutch origin made use of this option ; they were orally interviewed and the assistant filled in the answers .
a total of 5000 respondents were invited to participate . of the 374 participants that returned the reply card , 299 filled out the questionnaire .
this resulted in a total study population of 740 adults that were recruited between may 2010 and november 2011 .
after deletion of cases without data on key variables ( n = 118 ) , a final sample of 622 participants remained .
active transport was defined as the minutes per week spent on walking or cycling from a to b. the validated squash questionnaire for physical activity measured active transport by asking respondents to think about a regular week in the past months .
respondents then indicated how many days per week they engaged in several forms of physical activity and how many minutes they engaged in them .
subsequently , they could fill in the days , hours , and minutes spent on active transport .
total minutes of walking for active transport per day were calculated by multiplying the number of hours reported with sixty and adding them to the minutes reported .
subsequently , minutes of active walking per week were calculated by multiplying the minutes per day with the reported number of days per week .
this number of minutes of walking for active transport per week was used in the analyses .
minutes of cycling for active transport per week were calculated according to the same steps .
neighbourhood environmental characteristics were assessed by use of the neighbourhood walkability scale ( news ) .
three scales with twelve questions in total investigated the access to services ( three items ) ( = 0.71 ) ( e.g. , shops are within easy walking distance from my home ) , neighbourhood surroundings ( four items ) ( = 0.66 ) ( e.g. , the natural surroundings in my neighbourhood are beautiful ) , and safety from crime ( five items ) ( = 0.66 ) ( e.g. , because of criminality it is unsafe to walk in my neighbourhood during the day ) . three items of the safety from traffic scale were included separately in the analyses because of the low reliability of the scale ( < 0.6 ) .
the items were there is much traffic on nearby streets which make walking in the neighbourhood difficult or unpleasant ; the speed of traffic in nearby streets is normally low ( 30 km / h or less ) ; and most car drivers exceed maximum speed when driving through the neighbourhood .
the answers were given on a four - point likert scale ( completely disagree
a higher score corresponded with better access to services , better neighbourhood surroundings , and more safety from crime and traffic .
personal characteristics that were taken into account were age , sex , ethnicity , and body mass index ( bmi ) .
ethnicity of the respondent was operationalized by assessing the country of birth of both parents .
if a respondent had at least one parent who was born outside the netherlands , he or she was considered to be a migrant .
neighbourhood status was included as a confounder , to correct variations in degree of deprivation among neighbourhoods .
neighbourhood status was determined using the neighbourhood status score ( nss ) of the dutch social cultural planning office ( scp ) .
this score is based on the income , education level , and employment status of the inhabitants and indicates the degree of social deprivation per neighbourhood .
lower scores correspond with a larger degree of social deprivation , whereas high scores indicate a more favourable socioeconomic status . characteristics of the target population were investigated by means of descriptive analyses .
independent samples t - tests were performed to investigate the difference in scores on the news and in minutes of active transport per week between the following subgroups : respondents equal to or younger than the median age of 43 years and respondents older than 43 years : men and women ; native dutch respondents and migrants ; and respondents with a bmi lower than 25 and respondents with a bmi higher than 25 .
bivariate correlations were calculated to explore the associations between personal characteristics , and minutes per week spent in active transport and the associations between news scales and minutes spent engaging in active transport . in order to gain more insight into these correlations and to adjust for the difference in effect between baseline characteristics multivariate
linear regression analyses were performed . the full main effects model consisted of nss , the personal characteristics ( age , sex , ethnicity , and bmi ) , and the neighbourhood characteristics .
interactions between personal characteristics and neighbourhood variables were investigated by adding each interaction term separately to the full main effects model .
most of the participants were dutch , the migrants were mainly non - western ( 87.45% ; not tabulated ) .
the nss of the twenty participating districts varied between 3.64 ( highly deprived ) and 0.15 ( moderately deprived ) with a mean of 1.82 and a sd of 1.03 .
table 2 shows the scores for the neighbourhood characteristics and the minutes of active transport per week for subgroups of the population . on average ,
residents of deprived neighbourhoods walked 33 minutes and cycled for 32 minutes per week for active transport .
the younger participants were found to be more active in terms of walking as well as cycling than the older participants .
migrants were more active in terms of walking , whereas the dutch biked more often .
participants with a bmi below 25 were more active than those overweight , with a significant difference in activity levels for cycling .
respondents older than 43 years perceived the neighbourhood surroundings as more positive than the younger respondents . women experienced better access to services than men .
native dutch participants perceived better access to services , better neighbourhood surroundings , and felt safer from crime than migrants .
furthermore , they experienced less traffic on nearby streets that made walking difficult than migrants .
participants who lived in a neighbourhood with a relatively higher status within this population perceived all neighbourhood characteristics as more favourable than the people from districts with a lower nss .
multivariate regression analyses showed that minutes of walking decreased significantly by one minute for each year increase in age .
nss was also found to be a significant associate of walking ; respondents from higher status neighbourhoods walked less than respondents from lower status neighbourhoods .
perceived speed of traffic was the only neighbourhood variable found to be a statistically significant associate of walking .
interactions between ethnicity and the perception of traffic exceeding maximum speed ( : 0.40 ; p < 0.001 ) ; sex and the perception of traffic exceeding maximum speed ( : 0.26 ; p < 0.05 ) ; and between age and speed of traffic ( : 0.53 ; p < 0.01 ) were statistically significant .
subgroup analyses indicated that migrants and women reported more walking for transport when cars were perceived to not exceed maximum speed ( : 0.18 ; p < 0.01 and : 0.13 ; p < 0.05 , resp . ) , whereas this perceived environmental factor was unrelated to walking of native dutch and male respondents ( : 0.06 ; ns and : 0.04 ; ns , resp . ) .
stratification for age showed that low perceived speed of traffic was significantly associated with more walking in respondents of 43 years or younger ( : 0.18 ; p < 0.01 ) but not in respondents older than 43 ( : 0.06 ; ns ) .
every year of increase in age was associated with a decrease of one minute of cycling per week ( table 3 ) .
the native dutch cycled more compared with the migrants ( : 0.11 ; p 0.01 ) .
the interaction between ethnicity and neighbourhood surroundings was found to be significant ( : 0.50 ; p 0.01 ) .
more attractive neighbourhood surroundings were significantly associated with more cycling per week among the migrants ( : 0.23 ; p 0.001 ) , but not in the dutch respondents ( : 0.02 ; ns ) .
this study investigated which personal and neighbourhood environmental characteristics were associated with active transport among inhabitants of dutch deprived neighbourhoods .
this finding is supported by a study of ogilvie et al . and could be a result of differences in fitness between the younger and the older respondents .
people tend to perceive more barriers for physical activity as they age , such as physical disabilities and poorer perceived health .
migrants walked more minutes per week compared with the dutch , probably because walking is considered to be an easy and cheap mode of transportation , which is less sensitive to cultural habits compared to cycling .
the dutch cycled more than the migrants , probably because they are more used to cycling and have more access to this mode of transportation .
higher scores on bmi correlated with lower levels of active transport , which confirms findings of pucher et al . .
perceived speed of traffic was the only neighbourhood environmental characteristic that was associated with walking for transport .
when the average speed of traffic was perceived as low , people reported more walking for active transport .
a review of previous studies reported mixed evidence with regard to safety and active transport , which illustrates a need for clear definitions of safety in this field of research .
our measurements confirm this need for proper measurements of safety from traffic , since we were forced to include the relevant questions separately in the analyses as a result of low reliability of the scale .
interactions showed that with regard to ethnicity , migrants appeared to be more responsive to neighbourhood walkability than the dutch respondents .
speed of traffic ( walking ) and aesthetics ( cycling ) was found to be more strongly associated with active transport among migrants than among native dutch .
this could be a result of cultural differences ; migrants seem to be more in need of a safe , attractive , and stimulating environment to induce active transport .
an investigation of transportation among dutch migrants showed that migrants cycled more when high quality cycle infrastructure was available and if a strong cycle culture was present in the neighbourhood .
in addition , female and younger respondents were subgroups that appeared to be somewhat more responsive to traffic safety than male and older respondents . a dutch study on traffic safety found that higher levels of neighbourhood traffic safety correlated with increased odds of being active , especially in women and people aged 35 to 59 , which partly confirms our findings .
personal characteristics have been shown to be stronger associates of active transport than neighbourhood characteristics , a finding which is supported by multiple other studies [ 33 , 34 ] .
nevertheless , there were also studies that did find associations between neighbourhood walkability and active transport [ 13 , 14 ] .
this could be explained by differences in levels of transportation facilities in the countries under study .
most of these studies were conducted in the us or australia , countries that sometimes lack proper walking or cycling trails .
however , the netherlands especially is already well - equipped for facilitation of walking and cycling , so relative differences in neighbourhood characteristics may only have a minimal impact on active transport .
this implies that results of these studies are likely to be only valid for the population and country in which it was conducted , as was suggested in previous studies [ 12 , 34 ] .
the low variability of neighbourhood characteristics within the deprived districts in our study may have decreased the possibility to find strong environmental relationships even more .
it is also possible that the characteristics investigated in this study did not cover all environmental aspects that might be relevant in active transport .
nevertheless , interaction effects indicated that within some groups of the population under study , neighbourhood characteristics are important and need to be considered in efforts to promote physical activity .
since the data collected for this study was cross - sectional , statements about causality can not be made .
second , the overall response rate was low , although it was equivalent to those of comparable studies of the past years [ 10 , 12 , 35 ] .
third , self - reported measures of active transport were used , which could have been prone to recall bias and socially desirable answers .
fourth , an abbreviated version of the news questionnaire was used , which may be the reason for the one scale on safety from traffic being considered unreliable . despite some limitations , this study adds valuable information to current evidence in particular because of the focus on a low - ses population , on the understudied cycling behaviour , and the ethnic composition of the study population .
participants were hard to reach and are therefore often neglected in research , among others because of possible cultural and language differences between the interviewer and respondents .
intensive recruitment strategies such as matching of interviewer and participant and executing the study in the respondents ' mother tongue were used to overcome this difficulty .
the setting of deprived neighbourhoods was also a valuable characteristic , since they are the focus of governmental policies and interventions .
insight into the associations between neighbourhood characteristics and patterns of physical activity and their determinants in these districts could provide more information about the contents and implementation of such policies and interventions .
results of the current study may be used to design experimental research in order to test causality of the findings .
eventually , this may lead to evidence for effective intervention development for the subgroups most in need of interventions to increase active transport and subsequently levels of physical activity .
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background .
active modes of transport like walking and cycling have been shown to be valuable contributions to daily physical activity . the current study investigates associations between personal and neighbourhood environmental characteristics and active transport among inhabitants of dutch deprived districts
. method .
questionnaires about health , neighbourhoods , and physical activity behaviour were completed by 742 adults .
data was analysed by means of multivariate linear regression analyses . results .
being younger , female , and migrant and having a normal weight were associated with more walking for active transport .
being younger , male , and native dutch and having a normal weight were associated with more cycling for active transport .
neighbourhood characteristics were generally not correlated with active transport .
stratified analyses , based on significant person - environment interactions , showed that migrants and women walked more when cars did not exceed maximum speed in nearby streets and that younger people walked more when speed of traffic in nearby streets was perceived as low . among migrants , more cycling was associated with the perceived attractiveness of the neighbourhood surroundings .
discussion and conclusion .
results indicated that among inhabitants of dutch deprived districts , personal characteristics were associated with active transport , whereas neighbourhood environmental characteristics were generally not associated with active transport .
nevertheless , interaction effects showed differences among subgroups that should be considered in intervention development .
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this summary of the 2008 era edta registry report includes the data on renal replacement therapy ( rrt ) from 55 national and regional registries in 30 countries in europe and bordering the mediterranean sea ( figure 1 ) .
the renal registries provided datasets with individual patient data ( 36 registries ) or in aggregated form ( 19 registries ) .
the data from all registries were used to present incidence and prevalence of rrt data as well as transplant rates .
more detailed data than those presented in this paper can be found in the 2008 era edta registry report which is available at www.era-edta-reg.org .
incidence of rrt per million population ( pmp ) at day 1 , 2008 b&h , bosnia herzegovina ; fyrom , former yugoslav republic of macedonia .
incidence is the number of new cases of a condition during a specific period of time which , in this annual report , is a year ( 2008 ) . in 2008 ,
the overall incidence rate of rrt for end - stage renal disease ( esrd ) among all registries reporting to the era edta registry was 122 per million population ( pmp ) .
figure 2 shows that the highest incidence rates at day 1 were reported by turkey ( 264 pmp ) , portugal ( 232 pmp ) and greece ( 199 pmp ) , whereas incidence rates below 100 pmp where reported by ukraine ( 15 pmp ) , montenegro ( 30 pmp ) , russia ( 37 pmp ) , estonia ( 66 pmp ) , iceland ( 73 pmp ) , latvia ( 95 pmp ) , finland ( 95 pmp ) , romania ( 96 pmp ) and spain ( castile - la mancha ) ( 98 pmp ) .
the incidence rate of rrt for diabetic esrd was highest in turkey ( 80 pmp ) , israel ( 77 pmp ) and slovakia ( 71 pmp ) , while the registries of iceland , romania , ukraine , montenegro , russia , estonia and latvia reported incidence rates for esrd due to diabetes mellitus below 20 pmp ( figure 2 ) .
table 1 shows the incidence rate of rrt over the period 200408 for countries and regions providing individual patient data , adjusted for age and gender distribution .
incidence of rrt per million population ( pmp ) at day 1 in 2008 , for all patients and for patients with diabetes mellitus as primary renal disease , unadjusted .
figures include data from renal registries providing individual patient data ( left figure ) and aggregated data ( right figure ) .
incidence of rrt over the period 200408 per million population ( pmp ) at day 1 , adjusted for age and gender distribution for the age group 019 years at the start of rrt , the data were available for 14 registries of 11 countries including those of austria , denmark , finland , greece , iceland , norway , romania , spain ( andalusia ) , spain ( basque country ) , spain ( catalonia ) , spain ( valencian region ) , sweden , the netherlands and the uk ( scotland ) .
as numbers of children starting rrt were low we present averages for 2007 - 2008 ( table 2 ) .
for an overview of paediatric rrt data collected by the espn / era edta registry , please visit www.espn-reg.org .
incidence of rrt over the period 2007 - 08 per million age - related population ( pmarp ) per year at day 1 , by age group , unadjusted figure 3 shows that the mean age of patients starting rrt in 2008 ranged from 44 years in ukraine to 69 years in dutch - speaking belgium .
figures include data from renal registries providing individual patient data ( left figure ) and aggregated data ( right figure ) .
the incidence of the different treatment modalities was measured as number of patients pmp on treatment modality at day 91 of rrt ( table 3 ) .
whereas incidence rates of haemodialysis were highest in greece ( 160 pmp ) , israel ( 157 pmp ) , and in dutch- and french - speaking belgium ( 150 pmp and 153 pmp respectively ) , the highest incidence rates for peritoneal dialysis were reported from sweden ( 37 pmp ) , denmark ( 34 pmp ) and finland and iceland ( both 28 pmp ) .
the highest incidence rates of patients living on a functioning graft at day 91 of rrt were seen in norway ( 15 pmp ) and the netherlands ( 11 pmp ) .
incidence of rrt per million population ( pmp ) at day 91 in 2008 , by treatment modality , unadjusted categories may not add up because of rounding off .
hd , haemodialysis ; pd , peritoneal dialysis ; tx , transplantation ; unkn , unknown .
prevalence is the number of people in a given population with a particular disease at a given time , in this annual report at december 31 , 2008 .
the overall prevalence among all registries reporting to the era edta registry was 644 pmp .
figure 4 shows that the prevalence of rrt pmp at 31 december 2008 was highest in portugal ( 1408 pmp ) , belgium ( french - speaking ) ( 1153 pmp ) and spain ( catalonia ) ( 1124 pmp ) .
the lowest prevalence was reported by ukraine ( 89 pmp ) and russia ( 165 pmp ) .
table 4 shows the overall prevalence of rrt , adjusted for age and gender distribution .
prevalence of rrt per million population ( pmp ) on 31 december 2008 , for all patients and for patients with diabetes mellitus as primary renal disease , unadjusted .
figures include data from renal registries providing individual patient data ( left figure ) and aggregated data ( right figure ) .
data from czech republic , israel , italy ( 16 of 20 regions ) slovakia , and tunisia include dialysis patients only . in italy ( 13 of 20 regions )
, the percentage of missing prevalent rrt patients is estimated at 11% , due to an estimated 2530% underreporting of patients living on a functioning graft .
prevalence of rrt on december 31 over the period 2004 - 08 per million population ( pmp ) , adjusted for age and gender distribution only a limited number of registries provided complete data for prevalent patients in the age group 0 - 19 years in 2008 , including those of austria , denmark , finland , greece , iceland , norway , romania , spain ( andalusia ) , spain ( basque country ) , spain ( catalonia ) , spain ( valencian region ) , sweden , the netherlands and the uk ( scotland ) .
prevalence of rrt per million age - related population ( pmarp ) on 31 december 2008 , by age group , unadjusted the mean age of patients on rrt on 31 december 2008 ranged from 44 years ( ukraine ) to 64 years ( dutch - speaking belgium and italy ) for registries providing data on both dialysis and transplant patients ( figure 5 ) . mean age ( years ) of prevalent patients on rrt on 31 december 2008 .
figures include data from renal registries providing individual patient data ( left figure ) and aggregated data ( right figure ) .
data from czech republic , israel , italy ( 16 of 20 regions ) , slovakia and tunisia include dialysis patients only .
the prevalence of haemodialysis on 31 december 2008 was highest in portugal ( 875 pmp ) , greece ( 753 pmp ) and italy ( 16 of 20 regions ) ( 739 pmp ) , and the lowest in ukraine ( 66 pmp ) , russia ( 118 pmp ) and iceland ( 154 pmp ) ( table 6 ) .
the prevalence of peritoneal dialysis was highest in denmark ( 115 pmp ) , the uk , wales ( 102 pmp ) and sweden ( 92 pmp ) , whereas the lowest prevalence of this treatment was reported in montenegro ( 8 pmp ) , ukraine ( 11 pmp ) and russia ( 12 pmp ) .
in norway , 70% of the patients on rrt were living with a functioning graft ( 572 pmp ) .
prevalence of rrt per million population ( pmp ) on 31 december 2008 , by treatment modality , unadjusted categories may not add up because of rounding off .
hd , haemodialysis ; pd , peritoneal dialysis ; tx , transplantation ; unkn , unknown .
table 7 shows that the highest transplant rates were reported from spain ( catalonia ) ( 64 pmp ) , norway ( 58 pmp ) and spain ( basque country ) ( 55 pmp ) .
countries with the highest transplant rates with living - donor kidneys included the netherlands ( 25 pmp ) , norway ( 21 pmp ) and montenegro ( 19 pmp ) .
renal transplants performed per million population ( pmp ) in 2008 , by donor type , unadjusted categories may not add up because of rounding off .
for survival analysis data was used from 19 registries in 12 countries that provided individual patient records for the period 19992003 [ i.e. austria , belgium ( dutch - speaking ) , belgium ( french - speaking ) , denmark , finland , greece , iceland , italy ( calabria ) , norway , spain ( andalusia ) , spain ( asturias ) , spain ( basque country ) , spain ( cantabria ) , spain ( catalonia ) , spain ( valencian region ) , sweden , the netherlands , the uk ( england / wales ) and the uk ( scotland ) ] .
three spanish regions [ i.e. spain ( castile and leon ) , spain ( castile - la mancha ) and spain ( extremadura ) ] were also included in the analyses based on cohort 200206 because for these registries , complete data were available from 2002 .
the data are presented for all countries and regions together ( table 8 and figures 6 , 7 and 8) .
comparisons of survival by treatment modality were all adjusted for fixed values of age , gender and distribution of prd .
similar adjustments have been applied to survival comparisons by prd ( for fixed values and further methodology , consult the appendix section ) .
one- , 2- and 5-years survival probabilities , unadjusted for analysis methods , see the appendix section .
survival of incident dialysis patients and of patients receiving a first transplant between 1999 and 2003 , by treatment modality , adjusted for age , gender and primary renal disease . survival of incident haemodialysis patients in 19992003 , from day 91 , by primary renal disease , adjusted for age and gender .
survival of incident peritoneal dialysis patients in 19992003 , from day 91 , by primary renal disease , adjusted for age and gender .
austria : r kramar ; belgium , dutch - speaking : h augustijn , b de moor , and j de meester ; belgium , french - speaking : jm des grottes and f collart ; bosnia - herzegovina : h resi , b jakovljevi , and e mei ; croatia : croatian national renal registry , croatian regional renal registries , and croatian society of nephrology ; czech republic : i rychlk , j potucek , and f lopot ; denmark : j heaf ; estonia : m luman , m rosenberg , and pechter ; finland : p finne and c grnhagen - riska ; france : m lassalle , c couchoud , gm london , and c combe ; fyr of macedonia : o stojceva - taneva , g selim , and a sikole ; germany : f schaefer ; greece : ga ioannidis ; iceland : r palsson : israel : e golan and m shreibman ; italy : a rustici , m nichelatti , a limido , a molino , m salomone , g cappelli , e arosio , f antonucci , a santoro , e mancini , a rosati , gm frasc , g gaffi , m standoli , m bonomini , l diliberato , s digiulio , a dinapoli , d torres , f casino , c zoccali , m postorino , am pinna , p ravani , and e verrina ; latvia : h cernevskis , and v kuzema ; montenegro : m ratkovic , and s ivanovic ; norway : t leivestad ; poland : b rutkowski , g korejwo , and p jagodzinski ; portugal : f macrio , r filipe , and f nolasco ; romania : g mircescu , l garneata , and e podgoreanu ; russia : na tomilina and bt bikbov ; slovakia : v spustov and j fekete ; slovenia : j buturovi - ponikvar , j gubensek , and a kandus ; spain , andalusia : p castro de la nuez and ma prez valdivia ; spain , asturias : r alonso de la torre , roces , and e snchez ; spain , basque country : magaz , j aranzabal , i lampreabe , and j arrieta ; spain , cantabria : j gonzlez cotorruelo , and o garca ruz ; spain , castile and leon : am olmos and r gonzlez ; spain , castile - la mancha : g gutirrez vila and i moreno ; spain , catalonia : e arcos , j comas , r deulofeu , and j twose ; spain , extremadura : jm ramos aceitero and ma garca bazaga ; spain , valencian region : o zurriaga llorens and m ferrer alamar ; spain : r saracho ; sweden : kg prtz , l bckman , s schn , a seebergen , and b rippe ; the netherlands : a hoitsma , a hemke , am van den broek , r cornet , fw dekker , a kramer , m noordzij and kj van stralen ; tunisia : c mahjoubi , h trimech , and f jarraya ; turkey : k serdengeti and g sleymanlar ; ukraine : m kolesnyk , g vladzijevskaya , and j samuseva ; the uk , england / northern ireland / wales : d ansell and c tomson ; the uk , scotland : w metcalfe and k simpson .
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background . this study provides a summary of the 2008 era edta registry report ( this report is available at www.era-edta-reg.org).methods .
the data on renal replacement therapy ( rrt ) were available from 55 national and regional registries in 30 countries in europe and bordering the mediterranean sea .
datasets with individual patient data were received from 36 registries , whereas 19 registries contributed data in aggregated form .
we presented incidence and prevalence of rrt , and transplant rates .
survival analysis was solely based on individual patient records.results . in 2008 ,
the overall incidence rate of rrt for end - stage renal disease ( esrd ) among all registries reporting to the era edta registry was 122 per million population ( pmp ) , and the prevalence was 644 pmp .
incidence rates varied from 264 pmp in turkey to 15 pmp in ukraine . the mean age of patients starting rrt in 2008 ranged from 69 years in dutch - speaking belgium to 44 years in ukraine .
the highest prevalence of rrt for esrd was reported by portugal ( 1408 pmp ) and the lowest by ukraine ( 89 pmp ) .
the prevalence of haemodialysis on 31 december 2008 ranged from 66 pmp ( ukraine ) to 875 pmp ( portugal ) and the prevalence of peritoneal dialysis from 8 pmp ( montenegro ) to 115 pmp ( denmark ) . in norway , 70% of the patients on rrt on 31 december 2008 were living with a functioning graft ( 572 pmp ) . in 2008 , the number of transplants performed pmp was highest in spain ( catalonia ) ( 64 pmp ) , whereas the highest transplant rates with living - donor kidneys were reported from the netherlands ( 25 pmp ) and norway ( 21 pmp ) . in the cohort 19992003 , the unadjusted 1- , 2- and 5-year survival of patients on rrt was 80.8% ( 95% ci : 80.681.0 ) , 69.1% ( 95% ci : 68.969.3 ) and 46.1% ( 95% ci : 45.946.3 ) , respectively .
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differentiated thyroid carcinoma ( dtc ) accounts for 90% of all thyroid cancers , including papillary thyroid carcinoma ( ptc ) , follicular thyroid carcinoma ( ftc ) and hrthle carcinoma .
surgery , selective postoperative radioiodine and thyroid stimulating hormone ( tsh ) suppressive therapy are the primary treatment modalities for dtc .
although the overall treatment outcome regarding dtc is excellent and the 10-year survival rate is aboubt 90% , the rate of persistent or recurrent cases is 2330% ; this indicates that the risk of recurrence can not be underestimated . in 2009 , the american thyroid association ( ata ) published management guidelines for patients with thyroid nodules and dtc for the improved assessment of recurrence and mortality , and graded dtc patients into three categories ( low , intermediate and high ) in terms of the risk of recurrence . shortly afterward , tuttle et al .
confirmed that this recurrence staging system could effectively predict the risk of recurrence and persistent disease .
this recurrence risk stratification system can provide promising guidance concerning the initial postoperative management of dtc , but it can also give rise to controversy .
the prognostic factors described in this system include incomplete tumor resection , aggressive histology subtypes , tumor invasion , cervical lymph node metastasis , distant metastasis and i-131 uptake outside the thyroid bed on whole - body scans ( wbss ) undertaken after thyroid remnant ablation .
apart from the aforementioned points , the discrepancy between the actual thyroglobulin ( tg ) level and the posttreatment imaging findings has also been mentioned as a weighing factor for high recurrence risk in the ata guidelines , based on its predictive value for both ablation success and prognosis . nevertheless , the specific value of postoperative tg in indicating a high recurrence risk remains to be established .
in addition , some researchers have argued that the preablative stimulated tg ( ps - tg ) level may be influenced by postoperative thyroid tissue remnants , and that it would take at least 1-year for tg to become undetectable .
thus , the specific value and the significance of ps - tg regarding decision - making require further investigation . at present in china , surgical techniques such as cervical lymph node dissection are incongruently assigned to patients , and the time interval between surgery and treatment using nuclear medicine varies from a few days to a few years .
therefore , a marker that could reflect ongoing disease status is urgently needed , rather than static pathological findings during surgery . there is evidence to indicate that ps - tg measured just before radioiodine treatment may have a role as such a marker . to date , very few data regarding the relationship between ps - tg and the three recurrence risk categories of ata have been reported . in this study
, we introduced the ps - tg level into ongoing postoperative reassessment to explore the correlation between ps - tg level and ata recurrence risk stratification ; the objective was to provide evidence - based support for chinese patients for the role of ps - tg in postoperative reassessment and radioiodine treatment decision - making .
the study was approved by the ethics committee of peking union medical college hospital . in this retrospective study , 985 patients with dtc received total thyroidectomy followed by radioiodine treatment from 2007 to 2013 . a total 278 of these patients were excluded from the study ; 177 had high tg antibody ( tgab ) levels ( > 46 iu / ml ) and the other 101 had missing data regarding ps - tg or tgab levels .
therefore , 707 patients were finally enrolled in the study , including 482 females and 225 males with a mean age of 42.7 ( range , 477 ) years .
all patients underwent total thyroidectomy performed by experienced surgeons , with no macroscopic thyroid remnants remaining ; seven of these patients with papillary thyroid microcarcinoma ( tumor size 1 cm ) did not undergo cervical lymph node dissection .
patients received i-131 at a dose that varied from 30 mci ( 1.1 gbq ) to 200 mci ( 7.4 gbq ) according to their ata recurrence risk stratification within 3 months after surgery ; they underwent levothyroxine ( lt4 ) withdrawal or no replacement treatment and a low - iodine diet for at least 26 weeks when serum tsh levels were > 30 iu / ml . levels of ps - tg , tgab and tsh were measured before the first i-131 remnant ablation after thyroxine hormone withdrawal , and the tsh level had risen ( tsh > 30 iu / ml ) .
tg and tgab levels were determined using electrochemiluminescence immunoassay ( roche diagnostics gmbh , mannheim , germany ) , and the tsh level was determined using chemiluminescence immunoassay ( siemens healthcare diagnostics inc . ,
patient characteristics were compiled including age , sex , histology subtypes , tumor invasion , cervical lymph node metastasis , distant metastasis , remnant uptake on i-131-wbs , tsh , and ps - tg and tgab levels .
patients were divided into three groups according to the ata recurrence risk stratification : low - risk ( l ; n = 90 ) , intermediate - risk ( i ; n = 283 ) and high - risk ( h ; n = 334 ) . the h group was further subdivided into two subgroups , one with distant metastasis ( m1 ; n = 117 ) and the other without distant metastasis ( m0 ; n = 217 ) . additionally ,
while assigning patients to h group , we did not take the ps - tg level into account , because the predictive cut - off value was not specified in the ata guidelines .
patients with at least one of the following characteristics , according to ata and the national comprehensive cancer network guidelines , were considered for subsequent radioiodine therapy : male ; age > 45 years ; tumor size > 1 cm ; multiple lesions ( > 1 lesion ) ; and molecular characteristics such as the braf mutation . because increased tsh stimulates ps - tg during the lt4 withdraw period , indicating that tg release is tsh - dependent , we introduced the parameter ps - tg / tsh .
the associations between ps - tg , ps - tg / tsh and three recurrence risk groups were analyzed .
the m1 group was further analyzed in terms of metastatic evidence such as chest computed tomography , bone scan , postradioiodine - therapy wbs ( rxwbs ) combined with a suspicious high ps - tg level .
rank sum and f tests were used for the comparison of ps - tg and ps - tg / tsh values .
the kruskal - wallis and f tests were used for comparisons among the three groups ; the mann - whitney u and f tests were used to compare any two groups .
the most sensitive and specific ps - tg values for distinguishing each of the two group comparisons were obtained using receiver operating characteristic ( roc ) curves .
gender ( female or male ) , age ( < 45 years or 45 years ) , tumor size ( maximum diameter 1 cm or > 1 cm ) , multifocality ( single lesion or multiple lesions ) and ps - tg level ( < corresponding cut - off value or corresponding cut - off value ) were involved as independent variables of recurrence and distant metastasis .
these factors were further analyzed using logistic regression analysis to identify if ps - tg was an independent predictive factor for discerning different recurrence risk groups , and if distant metastases existed .
all of these statistical analyses were performed using spss software ( version 17.0 , inc .
in this retrospective study , 985 patients with dtc received total thyroidectomy followed by radioiodine treatment from 2007 to 2013 .
a total 278 of these patients were excluded from the study ; 177 had high tg antibody ( tgab ) levels ( > 46 iu / ml ) and the other 101 had missing data regarding ps - tg or tgab levels .
therefore , 707 patients were finally enrolled in the study , including 482 females and 225 males with a mean age of 42.7 ( range , 477 ) years .
all patients underwent total thyroidectomy performed by experienced surgeons , with no macroscopic thyroid remnants remaining ; seven of these patients with papillary thyroid microcarcinoma ( tumor size 1 cm ) did not undergo cervical lymph node dissection .
patients received i-131 at a dose that varied from 30 mci ( 1.1 gbq ) to 200 mci ( 7.4 gbq ) according to their ata recurrence risk stratification within 3 months after surgery ; they underwent levothyroxine ( lt4 ) withdrawal or no replacement treatment and a low - iodine diet for at least 26 weeks when serum tsh levels were > 30 iu / ml .
levels of ps - tg , tgab and tsh were measured before the first i-131 remnant ablation after thyroxine hormone withdrawal , and the tsh level had risen ( tsh > 30 iu / ml ) .
tg and tgab levels were determined using electrochemiluminescence immunoassay ( roche diagnostics gmbh , mannheim , germany ) , and the tsh level was determined using chemiluminescence immunoassay ( siemens healthcare diagnostics inc . , new york , ny , usa ) in the same laboratory .
patient characteristics were compiled including age , sex , histology subtypes , tumor invasion , cervical lymph node metastasis , distant metastasis , remnant uptake on i-131-wbs , tsh , and ps - tg and tgab levels .
patients were divided into three groups according to the ata recurrence risk stratification : low - risk ( l ; n = 90 ) , intermediate - risk ( i ; n = 283 ) and high - risk ( h ; n = 334 ) . the h group was further subdivided into two subgroups , one with distant metastasis ( m1 ; n = 117 ) and the other without distant metastasis ( m0 ; n = 217 ) . additionally , while assigning patients to h group , we did not take the ps - tg level into account , because the predictive cut - off value was not specified in the ata guidelines .
patients with at least one of the following characteristics , according to ata and the national comprehensive cancer network guidelines , were considered for subsequent radioiodine therapy : male ; age > 45 years ; tumor size > 1 cm ; multiple lesions ( > 1 lesion ) ; and molecular characteristics such as the braf mutation .
because increased tsh stimulates ps - tg during the lt4 withdraw period , indicating that tg release is tsh - dependent , we introduced the parameter ps - tg / tsh .
the associations between ps - tg , ps - tg / tsh and three recurrence risk groups were analyzed .
the m1 group was further analyzed in terms of metastatic evidence such as chest computed tomography , bone scan , postradioiodine - therapy wbs ( rxwbs ) combined with a suspicious high ps - tg level .
rank sum and f tests were used for the comparison of ps - tg and ps - tg / tsh values .
the kruskal - wallis and f tests were used for comparisons among the three groups ; the mann - whitney u and f tests were used to compare any two groups .
the most sensitive and specific ps - tg values for distinguishing each of the two group comparisons were obtained using receiver operating characteristic ( roc ) curves .
gender ( female or male ) , age ( < 45 years or 45 years ) , tumor size ( maximum diameter 1 cm or > 1 cm ) , multifocality ( single lesion or multiple lesions ) and ps - tg level ( < corresponding cut - off value or corresponding cut - off value ) were involved as independent variables of recurrence and distant metastasis .
these factors were further analyzed using logistic regression analysis to identify if ps - tg was an independent predictive factor for discerning different recurrence risk groups , and if distant metastases existed .
all of these statistical analyses were performed using spss software ( version 17.0 , inc . ,
the ratio of females to males was 2.14:1 , 96.89% of patients had ptc and the preablative tsh level was 91.36 35.57 u / ml .
analysis of the three risk classification groups of l , i and h revealed that the more advanced the stratification , the greater the likelihood of a higher ps - tg level .
this trend could also be seen when comparing the l , i and h groups with the m0 and m1 groups [ figure 1 ] . especially , in the case of the h group , it was found that the subgroup with distant metastasis was accompanied by the highest ps - tg level [ figure 1 ] .
characteristics of study subjects n : number of patients ; sd : standard deviation ; tsh : thyroid stimulating hormone .
( a ) scatter plot of ps - tg in three recurrence risk groups of low - risk(l ) , intermediate - risk(i ) and high - risk(h ) .
( b ) scatter plot of ps - tg in four groups of l , i , h without distant metastasis(m1 ) and m1 . the mean ps - tg level for the l , i and h groups was 5.278 , 11.588 and 159.939 ng / ml , respectively .
the corresponding standard deviation varied greatly between the three recurrence risk groups [ table 2 ] . the median ps - tg level for the l , i and h groups was 1.7 , 4.4 and 14.7 ng / ml , respectively , and the corresponding 2575% quartile was 0.35.4 ng / ml , 1.213.3 ng / ml and 2.2137.2 ng / ml , respectively [ table 2 ] .
comparison of ps - tg and ps - tg / tsh among different recurrence risk groups * p<0.001 , p values were all < 0.05 in further pairwise comparisons of ps - tg and ps - tg / tsh values among the three risk groups using the f test ; p<0.001 , p values were all < 0.05 in further pairwise comparisons of ps - tg and ps - tg / tsh values among the three risk groups using the mann - whitney u - test .
sd : standard deviation ; ps - tg : preablative stimulated thyroglobulin ; ps - tg / tsh : preablative stimulated thyroglobulin / thyroid stimulating hormone .
when comparing the ps - tg level among the three recurrence risk groups , significant differences could be found both using the f test ( p < 0.001 ; f = 48.254 ) and the kruskal - wallis test ( p < 0.001 ; = 61.388 ) [ table 2 ] .
further comparisons between groups also revealed a similar trend using both the f test ( l vs. i , p = 0.014 , f = 6.107 ; h vs. i , p < 0.001 , f = 24.905 ; h vs. l , p
whitney u - test ( l vs. i , p < 0.001 , z = 3.986 ; h vs. i , p < 0.001 , z = 7.367 ; h vs. l , p < 0.001 , z = 7.645 ) [ table 2 ] . receiver operating characteristic curves used for evaluating the most sensitive and specific ps - tg values for distinguishing each of the two group comparisons are shown in figure 2 .
the area under the roc curve for the ps - tg level used for distinguishing the groups was 0.631
( l vs. i ) , 0.668 ( i vs. h ) , 0.913 ( m0 vs. m1 in the h group ) and 0.931 ( m0 vs. m1 in all patients ) .
two definite cut - off values of ps - tg , namely 2.95 ng / ml and 29.5 ng / ml , were obtained for differentiating the l from the i group as well as the i from the h group ; these values might be used as indicators to distinguish each recurrence risk group [ figure 2 ] .
in addition , when only comparing patients in the h group , the cut - off ps - tg value in discriminating m1 from m0 was 47.1 ng / ml ( sensitivity : 79.5% ; specificity : 88.9% ) .
in addition , when dividing all patients into two groups according to whether or not distant metastasis was present , the specificity of the cut - off ps - tg value ( 47.1 ng / ml ; sensitivity : 79.5% ; specificity : 93.7% ) became even higher than that in high - risk patients [ figure 2 ] .
( a ) roc in distinguishing low from intermediate recurrence risk groups ; ( b ) roc in distinguishing intermediate from high recurrence risk groups ; ( c ) roc in distinguishing m1 from m0 in the high recurrence risk group ; ( d ) roc in distinguishing m1 from m0 in all patients .
roc : receiver operating characteristic curve ; auc : area under the curve ; ps - tg : preablative stimulated thyroglobulin ; ps - tg / tsh : preablative stimulated thyroglobulin / thyroid stimulating hormone ; m1 : distant metastasis ; m0 : no distant metastasis .
gender ( female or male ) , age ( < 45 years , or 45 years ) , tumor size ( maximum diameter 1 cm or > 1 cm ) , multifocality ( single lesion or multiple lesions ) and ps - tg level ( < corresponding cut - off value or corresponding cut - off value ) were analyzed as independent variables using logistic regression analysis .
in univariate logistic regression analysis , the role of ps - tg was significant in distinguishing between i and l groups ( odds ratio [ or ] : 2.757 ; 95% confidence interval [ ci ] : 1.6874.506 ; p < 0.001 ) , as well as between h and i groups ( or : 9.490 ; 95% ci : 5.73315.707 ; p < 0.001 ) .
even higher or values could be obtained in comparing both the m1 and m0 subgroups in the h group ( or : 31.161 ; 95% ci : 16.80557.784 ; p < 0.001 ) and between the m1 and m0 subgroup in all patients ( or : 57.916 ; 95% ci : 33.125101.260 ; p < 0.001 ) , which further confirmed the high accuracy and predictive value of ps - tg [ table 3 ] . logistic regression analysis of recurrence risk and distant metastasis status according to clinicopathologic factors * the cut - off values for ps - tg based status dichotomy were 2.95 ng / ml , 29.5 ng / ml , 47.1 ng / ml and 47.1 ng / ml while distinguishing between low - risk and intermediate - risk , intermediate - risk and high - risk , without and with distant metastasis in the high - risk group , and without and with distant metastasis in all patients ; the dichotomy values were age 45 years , 1 cm and one lesion for age , tumor size and multifocality , respectively .
m0 : no distant metastasis ; m1 : distant metastasis ; ci : confidence interval ; or : odds ratio ; ps - tg : preablative stimulated thyroglobulin . in further multivariate
logistic regression analyses , except for the interference of other factors , ps - tg was confirmed to be an independent prediction factor for differentiating the various groups ( h vs. i , or : 8.021 , 95% ci : 4.77113.486 , p < 0.001 ; m1 vs. m0 in the h group , or : 31.977 , 95% ci : 16.57561.688 , p <
0.001 ; m1 vs. m0 in all study patients , or : 52.141 , 95% ci : 28.58195.122 , p < 0.001 ) .
in spite of relative low efficacy ( area under the curve , 0.631 ) of ps - tg in discerning the l and i groups using roc analysis , the or ( or : 2.541 , 95% ci : 1.5294.223 , p <
0.001 ) was high enough to validate that ps - tg could be defined as an independent predictive factor [ table 3 ] .
the parameter ps - tg / tsh displayed similar trends to the ps - tg in value comparisons and roc analysis [ table 2 and figure 2 ] , implying that both the thyroid remnant and tsh had little influence on the ps - tg level in this study .
as stated above , the cut - off ps - tg value of 47.1 ng / ml was found to be a good predictive index in discriminating the m1 from m0 subgroup . utilizing this cut - off value ,
10.26% of patients could be identified before radioiodine treatment as m1 patients in this study , without any other evidence of distant metastasis ; however , they were finally confirmed to harbor distant metastases using imaging involving rxwbs after radioiodine treatment [ table 4 ] .
hence , the ps - tg level could be the only evidence available for those patients who would benefit from modified high - dose radioiodine treatment based upon ongoing preablative reassessment .
evidence of distant metastasis in patients in the m1 subgroup ( n = 117 ) * finding of distant metastatic lesions is defined as ( + ) , and finding of no distant metastatic lesions is defined as ( ) in the first and the third column ; ps - tg 47.1 ng / ml is defined as ( + ) , and ps - tg < 47.1 ng / ml is defined as ( ) in the second column ; three patients suffered from both pulmonary and bone metastases confirmed by both rxwbs and bone scintigraphy .
m1 : distant metastasis ; ct : computed tomography ; rxwbs : postradioiodine - therapy whole - body scanning ; ps - tg : preablative stimulated thyroglobulin .
the ratio of females to males was 2.14:1 , 96.89% of patients had ptc and the preablative tsh level was 91.36 35.57 u / ml .
analysis of the three risk classification groups of l , i and h revealed that the more advanced the stratification , the greater the likelihood of a higher ps - tg level .
this trend could also be seen when comparing the l , i and h groups with the m0 and m1 groups [ figure 1 ] . especially , in the case of the h group , it was found that the subgroup with distant metastasis was accompanied by the highest ps - tg level [ figure 1 ] .
characteristics of study subjects n : number of patients ; sd : standard deviation ; tsh : thyroid stimulating hormone .
( a ) scatter plot of ps - tg in three recurrence risk groups of low - risk(l ) , intermediate - risk(i ) and high - risk(h ) .
( b ) scatter plot of ps - tg in four groups of l , i , h without distant metastasis(m1 ) and m1 . the mean ps - tg level for the l , i and h groups was 5.278 , 11.588 and 159.939 ng / ml , respectively .
the corresponding standard deviation varied greatly between the three recurrence risk groups [ table 2 ] . the median ps - tg level for the l , i and h groups was 1.7 , 4.4 and 14.7 ng / ml , respectively , and the corresponding 2575% quartile was 0.35.4 ng / ml , 1.213.3 ng / ml and 2.2137.2 ng / ml , respectively [ table 2 ] .
comparison of ps - tg and ps - tg / tsh among different recurrence risk groups * p<0.001 , p values were all < 0.05 in further pairwise comparisons of ps - tg and ps - tg / tsh values among the three risk groups using the f test ; p<0.001 , p values were all < 0.05 in further pairwise comparisons of ps - tg and ps - tg / tsh values among the three risk groups using the mann - whitney u - test .
sd : standard deviation ; ps - tg : preablative stimulated thyroglobulin ; ps - tg / tsh : preablative stimulated thyroglobulin / thyroid stimulating hormone .
when comparing the ps - tg level among the three recurrence risk groups , significant differences could be found both using the f test ( p < 0.001 ; f = 48.254 ) and the kruskal - wallis test ( p < 0.001 ; = 61.388 ) [ table 2 ] .
further comparisons between groups also revealed a similar trend using both the f test ( l vs. i , p = 0.014 , f = 6.107 ; h vs. i , p < 0.001 , f = 24.905 ; h vs. l , p
whitney u - test ( l vs. i , p < 0.001 , z = 3.986 ; h vs. i , p < 0.001 , z = 7.367 ; h vs. l , p < 0.001 , z = 7.645 ) [ table 2 ] .
receiver operating characteristic curves used for evaluating the most sensitive and specific ps - tg values for distinguishing each of the two group comparisons are shown in figure 2 .
the area under the roc curve for the ps - tg level used for distinguishing the groups was 0.631
( l vs. i ) , 0.668 ( i vs. h ) , 0.913 ( m0 vs. m1 in the h group ) and 0.931 ( m0 vs. m1 in all patients ) .
two definite cut - off values of ps - tg , namely 2.95 ng / ml and 29.5 ng / ml , were obtained for differentiating the l from the i group as well as the i from the h group ; these values might be used as indicators to distinguish each recurrence risk group [ figure 2 ] .
in addition , when only comparing patients in the h group , the cut - off ps - tg value in discriminating m1 from m0 was 47.1 ng / ml ( sensitivity : 79.5% ; specificity : 88.9% ) .
in addition , when dividing all patients into two groups according to whether or not distant metastasis was present , the specificity of the cut - off ps - tg value ( 47.1 ng / ml ; sensitivity : 79.5% ; specificity : 93.7% ) became even higher than that in high - risk patients [ figure 2 ] .
( a ) roc in distinguishing low from intermediate recurrence risk groups ; ( b ) roc in distinguishing intermediate from high recurrence risk groups ; ( c ) roc in distinguishing m1 from m0 in the high recurrence risk group ; ( d ) roc in distinguishing m1 from m0 in all patients .
roc : receiver operating characteristic curve ; auc : area under the curve ; ps - tg : preablative stimulated thyroglobulin ; ps - tg / tsh : preablative stimulated thyroglobulin / thyroid stimulating hormone ; m1 : distant metastasis ; m0 : no distant metastasis .
gender ( female or male ) , age ( < 45 years , or 45 years ) , tumor size ( maximum diameter 1 cm or > 1 cm ) , multifocality ( single lesion or multiple lesions ) and ps - tg level ( < corresponding cut - off value or corresponding cut - off value ) were analyzed as independent variables using logistic regression analysis . in univariate logistic regression analysis ,
the role of ps - tg was significant in distinguishing between i and l groups ( odds ratio [ or ] : 2.757 ; 95% confidence interval [ ci ] : 1.6874.506 ; p < 0.001 ) , as well as between h and i groups ( or : 9.490 ; 95% ci : 5.73315.707 ; p < 0.001 ) .
even higher or values could be obtained in comparing both the m1 and m0 subgroups in the h group ( or : 31.161 ; 95% ci : 16.80557.784 ; p < 0.001 ) and between the m1 and m0 subgroup in all patients ( or : 57.916 ; 95% ci : 33.125101.260 ; p < 0.001 ) , which further confirmed the high accuracy and predictive value of ps - tg [ table 3 ] .
logistic regression analysis of recurrence risk and distant metastasis status according to clinicopathologic factors * the cut - off values for ps - tg based status dichotomy were 2.95 ng / ml , 29.5 ng / ml , 47.1 ng / ml and 47.1 ng / ml while distinguishing between low - risk and intermediate - risk , intermediate - risk and high - risk , without and with distant metastasis in the high - risk group , and without and with distant metastasis in all patients ; the dichotomy values were age 45 years , 1 cm and one lesion for age , tumor size and multifocality , respectively .
m0 : no distant metastasis ; m1 : distant metastasis ; ci : confidence interval ; or : odds ratio ; ps - tg : preablative stimulated thyroglobulin . in further multivariate
logistic regression analyses , except for the interference of other factors , ps - tg was confirmed to be an independent prediction factor for differentiating the various groups ( h vs. i , or : 8.021 , 95% ci : 4.77113.486 , p < 0.001 ; m1 vs. m0 in the h group , or : 31.977 , 95% ci : 16.57561.688 , p < 0.001 ; m1 vs. m0 in all study patients , or : 52.141 , 95% ci : 28.58195.122 , p < 0.001 ) . in spite of relative low efficacy ( area under the curve , 0.631 ) of ps - tg in discerning the l and i groups using roc analysis , the or ( or : 2.541 , 95% ci : 1.5294.223 , p <
0.001 ) was high enough to validate that ps - tg could be defined as an independent predictive factor [ table 3 ] .
the parameter ps - tg / tsh displayed similar trends to the ps - tg in value comparisons and roc analysis [ table 2 and figure 2 ] , implying that both the thyroid remnant and tsh had little influence on the ps - tg level in this study .
as stated above , the cut - off ps - tg value of 47.1 ng / ml was found to be a good predictive index in discriminating the m1 from m0 subgroup . utilizing this cut - off value ,
10.26% of patients could be identified before radioiodine treatment as m1 patients in this study , without any other evidence of distant metastasis ; however , they were finally confirmed to harbor distant metastases using imaging involving rxwbs after radioiodine treatment [ table 4 ] .
hence , the ps - tg level could be the only evidence available for those patients who would benefit from modified high - dose radioiodine treatment based upon ongoing preablative reassessment .
evidence of distant metastasis in patients in the m1 subgroup ( n = 117 ) * finding of distant metastatic lesions is defined as ( + ) , and finding of no distant metastatic lesions is defined as ( ) in the first and the third column ; ps - tg 47.1 ng / ml is defined as ( + ) , and ps - tg < 47.1 ng / ml is defined as ( ) in the second column ; three patients suffered from both pulmonary and bone metastases confirmed by both rxwbs and bone scintigraphy .
m1 : distant metastasis ; ct : computed tomography ; rxwbs : postradioiodine - therapy whole - body scanning ; ps - tg : preablative stimulated thyroglobulin .
previous studies have suggested that the predictive value of ps - tg regarding both the success of ablation and prognosis was highly informative .
one study showed that the 10-year disease - free survival rate of dtc patients was 100% when the ps - tg level was < 23 ng / ml , and decreased to 68.3% if the ps - tg level exceeded this limit .
a recent meta - analysis implied that ps - tg testing was a readily available and inexpensive tool with a high negative predictive value ( npv ) regarding future disease - free status .
a low ps - tg level could be considered as a favorable prognostic factor for patients with dtc .
kendler et al . reported that the level of ps - tg was the only independent predictor of ablation success ( p < 0.001 ) . recently
, considerable attention has been focused on ps - tg ; however , this parameter has not been incorporated into radioiodine decision - making , partly owing to its insensitivity and nonspecificity caused by the probable influence of residual thyroid after surgery .
some studies have utilized roc curves to evaluate the cut - off value of ps - tg as a predictor of the ablation success rate and prognosis .
a korean study reported that a cut - off value for the ps - tg level of 27.5 g / l was confirmed for predicting disease - free remission , with an npv as high as 98% .
another korean study found that the optimal cut - off value regarding the ps - tg level for successful ablation was 10 ng / ml with a sensitivity and specificity of 85.7% and 83.6% , respectively . in our previous studies
, we found that ps - tg might be considered as a predictive marker for distant metastasis from dtc ; these two studies mainly focused on the association between tg values and occult distant metastasis .
according to the above - mentioned findings , and the recently published chinese management guidelines for patients with thyroid nodules and differentiated thyroid cancer , a high ps - tg level was introduced as an important reassessment marker and regarded as a high recurrence risk index before radioiodine treatment . a high ps - tg level was recommended for high - dose i-131 treatment , although without a definite cut - off value .
this consideration is superior to the proposal that the ps - tg level is disproportionate to the rxwbs findings in high recurrence risk stratum , as stipulated in the ata guidelines and ignoring the predictive value of ps - tg in radioiodine dose decision - making . in the present study
, we considered ps - tg as one of the ongoing postoperative reassessment factors , correlating the ps - tg level with the newly developed recurrence risk staging system , and provided relevant data from chinese patients .
our findings suggested that a higher ps - tg level indicated a more advanced stage of recurrence risk .
significant differences in ps - tg levels have been found both among the three recurrence risk groups and between each two pairwise groups , indicating a remarkable difference in ps - tg levels in different recurrence risk groups .
roc analysis was conducted to define a certain cut - off value between each two pairwise groups .
logistic analysis was also used to determine the or values in verifying if ps - tg could be regarded as an independent predictive factor regarding the risk of recurrence .
two definite cut - off values for ps - tg level , 2.95 ng / ml and 29.5 ng / ml , were obtained for differentiating between the l and i and between the i and h groups , respectively ; these cut - off values might be candidate indicators for different recurrence risk stratifications . in addition
, in the h group , we found that the ps - tg cut - off value ( 47.1 ng / ml ) had a high sensitivity and specificity in distinguishing patients with distant metastasis from those with no metastasis .
in addition , a high specificity ( 93.7% ) and remarkably high or ( 42.492 ) regarding the ps - tg cut - off value ( 47.1 ng / ml ) have been identified while discriminating distant metastasis in all patients ; this finding might offer a specific cut - off value to further determine distant metastasis in high - risk patients in the absence of additional preablative metastatic evidence .
the l from the i and the i from the h groups relative to that achieved in differentiating the m1 from m0 subgroup , it still has a meaningful predictive value with or values of 2.712 and 31.801 , respectively .
all of these results indicated that the ps - tg level might be an independent postoperative assessment factor and provide ongoing serologic evidence for the recurrence risk stratification system .
in addition , from our findings , it is noteworthy that ps - tg might be the only indicator available for the identification of distant metastases , and thus if measured prior to radioiodine therapy would prevent 10.26% of patients with m1 from undertreatment .
therefore , the ps - tg level could be used in conjunction with other ata recurrence risk stratification indexes to improve the prediction of recurrence risk and dictate the most appropriate therapy . as it is well - established
, tsh can stimulate tg release from the residual thyroid tissue or metastatic carcinoma tissue , indicating that tg release is tsh - dependent .
consequently , we took tsh as a correction factor , and the ps - tg / tsh parameter was used in the present study to exclude the influence of postsurgical residual thyroid tissue .
similar results were obtained using ps - tg / tsh and ps - tg , both in the comparison of different recurrence risk groups and in the roc analyses ; this reflected the fact that tsh had no significant influence on ps - tg in the current study and also suggested that the patients had undergone complete thyroid resection .
a limitation of our study was that the follow - up outcomes were not available .
further studies are warranted to follow - up patients in the three risk recurrence categories after total thyroidectomy and radioiodine treatment .
in addition , therapy response would need to be evaluated by means of physical examination , neck ultrasound scans , imaging and tg serial values . in summary ,
the ps - tg level was found to be related to the risk of recurrence , and the corresponding cut - off value was obtained for discriminating different risks of recurrence with meaningful ors and relatively high specificity .
ps - tg could be considered as a convenient and reliable ongoing marker in indicating different risks of recurrence .
it might be the only indicator that can be used to identify distant metastases , and thus if measured before radioiodine therapy would prevent more than 10% of patients with m1 from undertreatment .
in addition , it could provide incremental value for both recurrence risk stratification and radioiodine decision - making .
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background : postoperative preablative stimulated thyroglobulin ( ps - tg ) has been evaluated in predicting prognosis and success of ablation regarding differentiated thyroid cancer ( dtc ) ; however , its relationship with recurrence risk and radioiodine decision - making remains uncertain , especially in chinese dtc patients .
we aimed to evaluate the association between ps - tg and recurrence risk stratification in dtc , to provide incremental values for ps - tg in postoperative assessment and radioiodine management.methods:seven hundred and seven patients with dtc were included ; low - risk ( l ; n = 90 ) , intermediate - risk ( i ; n = 283 ) , and high - risk ( h ; n = 334 , 117 with distant metastasis [ m1 ] ) patients were divided according to recurrence risk stratification .
the m1 group was further analyzed regarding evidence of metastasis . cut - off values of ps - tg were obtained using receiver operating characteristic analysis.results:patients with more advanced disease at initial risk stratification were more likely to have higher ps - tg levels ( i vs. l : p < 0.05 ; h vs. i : p < 0.001 ; h vs. l : p < 0.001 ) . the corresponding cut - off value of ps - tg for distinguishing sensitivity and specificity in each of the two groups was 2.95 ng / ml ( i vs. l : 61.5% , 63.3% ) , 29.5 ng / ml ( h vs. i : 41.9% , 92.6% ) , 47.1 ng / ml ( m1 vs. m0 in the h group : 79.5% , 88.9% ) and 47.1 ng / ml ( m1 vs. m0 in all patients : 79.5% , 93.7% ) . with the cut - off value at
47.1 ng / ml , ps - tg was the only factor that could be used to identify distant metastases , and consequently if measured before radioiodine therapy would prevent 10.26% of patients with m1 from undertreatment.conclusions:ps-tg , as an ongoing reassessment marker , favors differential recurrence risk grading and provides incremental values for radioiodine treatment decision - making .
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the ewing s sarcoma ( es ) family of tumours includes es and primitive neuroectodermal tumour ( pnet ) , which are primary malignant small round cell tumours of bone and soft tissue .
es and pnet have a similar neuronal phenotype and , because they share an identical chromosome translocation , they should be viewed as two variants of the same tumour that differ only in their degree of neuronal differentiation .
tumours that demonstrate neuronal differentiation are labeled pnets , and those that are undifferentiated are diagnosed as es .
however , characteristics and outcomes differ among patients with extraskeletal es / pnet in comparison with those with skeletal es / pnet ( table 1 ) [ 24 ] .
this neoplasm has been reported in various locations , but to the best of our knowledge , the case in question is the first case of es / pnet located in the hilus of liver and as a synchronous neoplasm .
it occurs two to six times more often in women and its rate increases with age . about 90% of cases
the most frequent type of the tumour is adenocarcinoma that accounts for more than 80% of gallbladder cancers .
the simultaneous occurrence of gallbladder adenocarcinoma and other malignancies has been reported only in a few cases involving the coexistence of adenocarcinoma of pancreas , lymphoma of mucosa associated lymphoid tissue of the gallbladder , hepatocellular carcinoma , uterine endometrioid adenocarcinoma and ovarian endometrioid carcinoma [ 69 ] .
laparoscopic simple cholecystectomy was performed in a 70-year old female due to the chronic cholelithiasis .
one month later , a hypoechogenic mass of 54 52 43 mm located in the hilus of the liver was found on control ultrasonography ( usg ) and computed tomography ( ct ) of the abdomen ( fig . 1a ) . computed
the patient underwent 2 cycles of intravenous chemotherapy according to the protocol of british columbia cancer agency ( bcca ) for es / pnet saime ( etoposide 100 mg / m , mesna 720 mg / m , ifosfamide 1800 mg / m ) given for 5 consecutive days every 3 weeks alternating with savac ( vincristine 1.5 mg / m , doxorubicin 75 mg / m , cyclophosphamide 1200 mg / m ) given over one day every 3 weeks .
control ct scans after two cycles showed a 57% tumour mass reduction in the biggest dimension ( fig .
thereafter , the patient was operated and resection of the hilus of the liver , hepato - duodenal ligament and local lymph nodes was performed .
two years later , a mass of 46 25 mm between inferior vena cava , left liver lobe and the head of the pancreas , and a second mass of 17 14 mm in the common bile duct were revealed on the control ct ( fig .
she underwent ercp ( endoscopic retrograde cholangio - pancreatography ) with insertion of the stent to the common bile duct .
the patient underwent palliative intravenous chemotherapy of cisplatin ( 25 mg / m ) and gemcitabine ( 1000 mg / m ) given both on the first and eighth day of the cycle repeated every 3 weeks .
partial regression according to recist ( response evaluation criteria in solid tumours ) 1.1 of the lesions located in the hilus of the liver and left liver lobe was observed on control ct scan after 2 cycles of chemotherapy and thus chemotherapy has been continued till 6 cycles .
one month later the patient s ps was 4 and she presented again with jaundice .
a ) hepatic tumor infiltrating head of the pancreas , vena cava inferior and portal vein .
c ) tumor infiltrating head of the pancreas , vena cava inferior , portal vein and hepatic artery
biopsy materials were examined with usage of formalin fixed , paraffin wax embedded tissue stained routinely with hematoxilin and eosine ( he ) .
immunohistochemistry included antibodies to cytokeratine 7 ( ck7 ) , cytokeratine 18 ( ck18 ) , leucocyte common antigen ( lca ) , epithelial membrane antigen ( ema ) , chromogranin , synaptophysin , vimentin , desmin , neurone specific enolase ( nse ) , thyroid transcription factor ( ttf ) , cluster differentiation 117 ( cd117 ) , s-100 protein , ki67 and myod1 .
a wide panel of immunohistochemical stainings was performed to exclude the diseases within the scope of the differential diagnosis of ewing s sarcoma .
briefly , a representative he section was selected for immunohistochemical investigation and 4 m sections were prepared .
the immunohistochemistry was performed by standard method : the slides were de - waxed , rehydrated and incubated in 3% peroxide solution for 10 minutes to block the endogenous peroxidase activity .
antigen retrieval was carried out by microwaving in citrate buffer ( ph = 6 ) or edta ( ph = 8) for 5 minutes at 700 w , then for 5 minutes at 600 w. the lab - vision detection system was used .
removed gallbladder , length 11 cm , with thickened wall , filled with gallstones was sent for the histological examination .
this revealed the presence of adenocarcinoma with infiltration of muscularis propria , t1bnxm0 ( fig .
an operative margin was free of cancer but a small group of cancer cells was visible in the lumen of the gallbladder at the resection margin .
most probably this finding was irrelevant and the cancer cells were placed there when cutting the material . a ) gallbladder wall with exophytic part of adenocarcinoma ; he , magnification 5. b ) histological picture of pnet localised within hilus of the liver : sheets of small , uniform , polygonal cells with scanty cytoplasm ; he , magnification 20 ; c ) immunohistochemistry for cd99 ; membranous staining in pnet cells , magnification 20 ; d ) immunohistochemistry for synaptophysin : positive , cytoplasmic , granular reaction in pnet cells , magnification 20 microscopic examination of the lesion located in the hilus of the liver , diagnosed as es / pnet showed a small cell neoplasm with membranous expression of cd99 , nse , vimentin and synaptophysin .
proliferative activity defined as the percentage of cells positive in ki67 was higher than 90% . staining for ck7 , ck18 , lca , ema , chromogranin , ttf1 , cd117 and s-100 protein were negative .
expression of myod1 was weak , cytoplasmatic and not characteristic for neoplasms of muscular origin ( fig .
examination of the specimens removed within surgery has revealed inflammatory cell infiltration and necrosis with no presence of neoplastic cells .
the histological picture together with the results of immunohistochemical reactions were consistent with es / pnet , which is a highly cellular neoplasm consisting of sheets and nests of small , uniform , round or polygonal cells with scanty cytoplasm and usually high mitotic activity .
presented case report highlights an extremely rare type of soft tissue sarcoma within atypical location .
unusual is also elderly age of the patient and existence of gbc in the same region as a synchronous neoplasm .
additionally , this case documents pathological diagnosis of es / pnet , which is based on both histology and immunohistochemistry ( the gold standard ) and may be problematic due to the rarity of these tumours and no typical location .
core biopsy , as it has been shown , is usually sufficient for making diagnosis .
immunohistochemistry is essential as the family of small round cell tumours is large and includes a lot of different neoplasms , for instance , non - hodgkin lymphoma , neuroblastoma , alveolar rhabdomyosarcoma , mesenchymal chondrosarcoma , retinoblastoma and desmoplastic small round cell tumour .
membranous expression of cd99 coexisting with positive expression of nse or synaptophysin are sufficient for the diagnosis of es / pnet .
other negative immunohistochemical reactions ( for instance lca , myod1 ) are helpful in eliminating neoplasms from the small round cell tumours family [ 1 , 10 ] .
genetic studies are useful because translocation t(11;22)(q24;q12 ) and expression of a fusion gene ews - fli1 are specific for es / pnets but they are not always available .
it should be borne in mind that up to 15% of es / pnets can be negative for this translocation , which is connected with presence of other rare mutations .
it is also known that the presence of this translocation is not likely to be considered diagnostic of es and pnet in the absence of supporting histological evidence .
treatment of ees / pnet usually includes multimodal chemotherapy combined with aggressive surgical treatment or radiotherapy .
multi - agent chemotherapy is superior to single agent one , includes cyclophosphamide , ifosfamide , etoposide , doxorubicin , vincristine , and should be given 1224 weeks before operation .
surgery should be performed after neoadjuvant chemotherapy because the probability of achieving complete resection with microscopic free margins is increased . in our case chemotherapy
resulted in the regression on ct imaging and finally , necrosis of the neoplasm , which was a sign of good response to the treatment and could be considered as a good prognostic factor .
the recommended operation is a wide resection encompassing a rim of 23 cm of normal tissue . when resection is either incomplete or impossible , radiotherapy is an alternative with the same dosage as in es / pnet .
therapy for refractory or relapsed disease consists of participation in clinical trials , chemotherapy with / without radiotherapy and surgery , if possible .
new strategies for the treatment of es / pnet include stem cell transplantation , antiangiogenic agents ( bevacizumab , vinblastine , celecoxib ) , mtor pathway inhibition ( sirolimus ) , bisphosphonates , anti - insulin - like growth factor receptor 1 ( igfr1 ) agents ( cixutumumab , r1507 ) and anti - cd99 agents . at our patient
no recurrence of pnet / ees has been reported within 42 months after diagnosis till her death , which was caused by the relapse of the gbc .
complete resection is the only potential curative treatment of gbc , however only 10% of patients are considered candidates for surgical treatment .
data concerning treatment of t1b stage disease are scant , however national comprehensive cancer network recommends in this case radical cholecystectomy . even for a patient undergoing aggressive surgery ,
several retrospective reports suggest beneficial effects of both adjuvant and neoadjuvant ( chemo)radiotherapy . in relapsed disease , palliative chemotherapy should be considered .
combination of cisplatin and gemcitabine has been used as a standard treatment because it has been shown to increase survival rate ( 11.7 vs. 8.1 months ) comparing with gemcitabine as the only agent . summing up
unfortunately , the overall survival of patients with gbc remains poor and the recurrence rate is still high .
that is why this major challenge in oncology requires the initiation of new clinical efforts and trials as well as the integration of the ones conducted previously .
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ewing s sarcoma ( es ) and primitive neuroectodermal tumour ( pnet ) are now considered to be the same tumour and usually occur in long bones .
extraskeletal ewing s sarcoma is an extremely rare neoplasm , accounting for 1% of soft tissue sarcomas , with most common location in the thorax . gallbladder cancer ( gbc ) represents the most common type among the biliary tract cancers with a poor prognosis even among patients undergoing aggressive therapy.we present study of extraskeletal es / pnet found in the hilus of the liver of an elderly , diagnosed one month prior with gbc woman .
the patient underwent two cycles of chemotherapy saime / savac for es and thereafter was operated . during three - year follow - up no recurrence of es / pnet has been reported .
however , two years after chemotherapy the patient suffered a relapse of adenocarcinoma of the gallbladder and thus received palliative chemotherapy of gemcitabine and cisplatin .
after 16 months of recurrence she died . to the best of our knowledge
, this is the first case of es / pnet located in the hilus of the liver and as a synchronous neoplasm .
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mostly chronic pain patients ( lower back pain , fibromyalgia , arthritis ) participated in these studies .
these studies assessed the effect of yoga over extended periods of time and applied regular yoga sessions once or twice per week . as a general trend
, the findings from these studies suggest that yoga has a beneficial effect on chronic pain .
a recent study by do rosario et al . showed immediate effects of modified yoga positions on musculoskeletal pain relief in participants with musculoskeletal pain .
it is likely that the benefits of yoga to sufferers of chronic pain are complex and manifold .
potentially involved mechanisms are hormonal changes , changes in neurotransmitters , reduction in muscular tonus , better posture , changes in attitude , and beliefs , and the lowering of anxiety .
responses to pain are also influenced by expectations and beliefs about pain and the emotional significance attributed to it .
furthermore , in the realm of sports medicine , there is the notion of exercise having hypoalgesic effects .
specifically , a recent meta - analysis showed that aerobic , isometric , and dynamic resistance exercise reduced the perception of experimentally induced pain in healthy participants .
yoga can be considered a mind body intervention that includes low impact exercise and , as such , yoga can be expected to have an immediate hypoalgesic effect .
its mindful , meditative way of combining breathing with stretching exercises ( asanas ) lowers the activity of the stress system ; therefore , sensations are perceived in a neutral way , as they are not intensified by emotions .
the results indicate that zen meditators have lower pain sensitivity and experience analgesic effects during mindful states . in this theoretical context ,
our primary hypothesis was that after a single yoga session a reduced pain perception could be objectified by algometry measurement . as a secondary hypothesis
, we assumed that generally the participant 's subjective expectation before intervention was to perceive less pain after a single yoga session .
thirdly , we wanted to quantify the potential correlation of the participant 's subjective expectations on the actual pain perception measurement .
ninety yoga students were recruited from five renowned hatha yoga schools ( krishna yoga , sapta yoga international , yoga langnau , iyengar yoga bern , unisport bern ) around bern , switzerland , between may and july 2011 .
each school had about 30 attendees of whom about two - thirds volunteered to participate in this study . to be included , volunteers had to be 18 years or older .
the second author ( s.t . ) observed the course and schedule of all yoga sessions , assessed all participants , and measured their pain perception before and after every yoga class .
all the participating yoga schools were teaching hatha yoga with mindful awareness , asanas , pranayama , meditation , and also a relaxation part .
all participants were asked about their general expectation as to whether a single yoga session would reduce or increase or have no effect on their measured pain perception .
pain perception was measured with a standardized pain provocation test ( algopeg , annette kocher , inselspital bern ) a polypropylene peg , calibrated to a clamping force of exactly 10 newton at an extension of 5 mm .
the use of this standardized pain provocation test has already been demonstrated in earlier studies .
the standardized and calibrated peg was clipped on the middle finger , ear lobes , and second toe of both body sides for 10 seconds each .
the participant was asked to rate pain immediately after the end of the 10 seconds on a numeric rating scale from 010 , where 0 signifies no pain and 10 the worst pain imaginable .
all data were analyzed using statistical package for the social sciences ( spss ) 18 .
we calculated the means of the algopeg measurements from the right and left for each of the three locations ; middle fingers , ear lobes , and toes .
a t - test was used to compare pain perception scores before and after the yoga session .
we further computed a general linear model to investigate whether cofactors such as yoga regularity and experience , as well as gender , age , and mood would influence outcomes .
we used the sign test to compare the actual change in pain scores to those that were expected by the participants .
expectation ( e ) was coded as follows : 0= aggravation ( i.e. more pain after the yoga session than before ) 2= improvement ( i.e. less pain after the yoga session than before ) we calculated the change scores ( cs ) as follows : pain scores before the yoga session minus pain scores after the yoga session .
if a participant experienced less pain after the yoga session , the cs would be positive .
we coded the cs as follows : 0 ( i.e. more pain after the yoga session compared with before ) 1 ( i.e. the same pain before as after the yoga session ) 2 ( i.e. less pain after the yoga session compared with before )
ninety yoga students were recruited from five renowned hatha yoga schools ( krishna yoga , sapta yoga international , yoga langnau , iyengar yoga bern , unisport bern ) around bern , switzerland , between may and july 2011 .
each school had about 30 attendees of whom about two - thirds volunteered to participate in this study . to be included , volunteers had to be 18 years or older .
observed the course and schedule of all yoga sessions , assessed all participants , and measured their pain perception before and after every yoga class .
all the participating yoga schools were teaching hatha yoga with mindful awareness , asanas , pranayama , meditation , and also a relaxation part .
before the experiment , all participants were asked about their general expectation as to whether a single yoga session would reduce or increase or have no effect on their measured pain perception .
pain perception was measured with a standardized pain provocation test ( algopeg , annette kocher , inselspital bern ) a polypropylene peg , calibrated to a clamping force of exactly 10 newton at an extension of 5 mm .
the use of this standardized pain provocation test has already been demonstrated in earlier studies .
the standardized and calibrated peg was clipped on the middle finger , ear lobes , and second toe of both body sides for 10 seconds each .
the participant was asked to rate pain immediately after the end of the 10 seconds on a numeric rating scale from 010 , where 0 signifies no pain and 10 the worst pain imaginable .
all data were analyzed using statistical package for the social sciences ( spss ) 18 .
we calculated the means of the algopeg measurements from the right and left for each of the three locations ; middle fingers , ear lobes , and toes .
a t - test was used to compare pain perception scores before and after the yoga session .
we further computed a general linear model to investigate whether cofactors such as yoga regularity and experience , as well as gender , age , and mood would influence outcomes .
we used the sign test to compare the actual change in pain scores to those that were expected by the participants .
expectation ( e ) was coded as follows : 0= aggravation ( i.e. more pain after the yoga session than before ) 2= improvement ( i.e. less pain after the yoga session than before ) we calculated the change scores ( cs ) as follows : pain scores before the yoga session minus pain scores after the yoga session .
if a participant experienced less pain after the yoga session , the cs would be positive .
we coded the cs as follows : 0 ( i.e. more pain after the yoga session compared with before ) 1 ( i.e. the same pain before as after the yoga session ) 2 ( i.e. less pain after the yoga session compared with before )
table 1 shows the characteristics of the participants per level of yoga regularity and experience .
participants characteristics the paired sample t - test comparing the means of the algopeg before and after the yoga ( on middle finger , ear lobe , and toe ) did not reveal any significant difference ( all p > 0.05 ) .
there was no significant difference in this outcome between the five different hatha yoga schools ( t - test ) .
moreover , in a general linear model , including experience , regularity , as well as gender , age , and mood as cofactors , no significant differences in the algopeg measurements before and after the yoga session were found ( data not shown in detail ) .
sixty out of 90 ( 66.7% ) yoga participants expected before testing to have a reduced pain perception after the yoga session [ table 1 ] only 3643% ( depending on the location ) of all the participants actually experienced less pain after one single yoga session [ table 2 ] .
the actual change in score of the pain measurements at the three locations regarding the 60 participants who expected less pain after the yoga session , [ table 3 ] shows the actual change scores ( cs ) of their pain perception .
subgroup of the participants with positive expectation ( less pain after a single yoga session ) among the subgroup of the 60 participants with positive expectation , 18 ( 30% ) experienced more pain after the yoga , 19 ( 32% ) experienced the same pain before and after the yoga , and23 ( 38% ) actually experienced less pain after the yoga session on their middle finger .
the results of the sign test show overall significant difference on finger ( p < 0.001 ) and ear ( p = 0.001 ) locations , and a trend toward statistical significance on the toe location ( p = 0.099 ) between the expectations of the participant 's pain and the actual measurements .
specifically , there were more participants who before the yoga session expected to have less pain after the yoga session , although there was actually no change in the measured pain perception between before and after the yoga .
to our knowledge , the effects of a single yoga session on pain perception have not previously been examined in subjects without a pain disorder . interestingly , before testing , two - thirds ( 66.7% ) of our yoga participants expected to have a lower pain reaction after the yoga session .
however , the application of a standardized pain provocation test did not uncover any significant difference in pain perception between before and after a single yoga session .
this means that the pain reducing effect in chronic pain patients through yoga interventions described in the literature can not be demonstrated through a single ( 60 minutes ) yoga session in healthy participants .
moreover , the previously reported immediate effects of sportive exercise on pain reduction could not be found following a single yoga session .
the pain - reducing effects of yoga in studies where participants practiced over a longer period of time might therefore have mechanisms that are different from those underlying an immediate analgetic effect . in all interventions aimed at reducing pain , the placebo effect of expectation should be considered . in order to include such a potential confounding factor through positive expectation , we assessed the participant 's expectation before testing .
as suspected in our second hypothesis , the majority of yoga practitioners ( 66.7% ) expected a pain - reducing effect of the yoga session .
however in our study , there was no correlation between these expectations and the actual change in pain perception .
this suggests that the general expectation did not have a strong influence on the actual algometric measurements in our study .
however , it should be acknowledged that there is literature to show that expectation is able to influence perception of experimentally induced pain .
also , our results might have been different if we had obtained expectation scores after the post reading of pain rather than before . for about one - third of the participants in our study who expected a positive effect of the yoga on pain perception
, we actually recorded increased pain perception within the scope of a single yoga session .
one reason for this could be that through positive expectations the total awareness of our body sensations and mental state could be increased .
therefore , shortly after a yoga session , a pressure sensation of an algopeg can even be perceived more intensively than before yoga .
whether the results of these single session interventions are generalizable , remains to be proven .
according to our results , however , we assume that the well - documented pain reducing effect of long - term yoga in literature can not be compared to the spectra of short - time mechanisms as demonstrated in this study .
regardless of the high positive expectations of the participants , this study showed that a single yoga session did not significantly affect provoked pain perception
. the positive impact of yoga on pain perception described in other yoga studies that were executed over a longer period of time must be due to other reasons than expectation or an immediate analgesic effect induced by exercise .
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background : several studies show yoga may benefit chronic pain management .
we investigated the effect of a single yoga session on the perception of pain , measured by a standardized pain provocation test in healthy yoga participants while also comparing pain perception to participants own expectations.materials and methods : ninety yoga participants were recruited at hatha yoga schools in switzerland .
pain perception was measured with a standardized algometric pain provocation test ; i.e. , a calibrated peg was applied for 10 seconds after which the participant rated pain intensity on a 010 numerical rating scale .
the test was applied to the middle finger , ear lobe , and second toe before and after a 60-minute yoga session.results:sixty out of 90 ( 66.7% ) yoga participants expected a reduced pain perception after the yoga session .
however , 36 ( 40% ) participants actually experienced less pain after compared to before the yoga session . but overall
, pain perception statistically did not significantly change from before to after the yoga session at any of the three body locations assessed .
the expectations and also the previous yoga experience did not significantly influence the participants pain perception.conclusions:regardless of the high positive expectations on the influence of yoga on pain , a single yoga session does not significantly influence pain perception induced by a pain provocation test .
hypoalgesic effects of yoga should be explained otherwise .
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globally , an estimated 185 million people have been infected with hepatitis c virus ( hcv ) as one of the major causes of cirrhosis and hepatocellular carcinoma ( 1 ) .
hcv genome consists of approximately 9.6 kilobases , positive - sense single - stranded rna , which encodes three structural ( c , e1 and e2 ) and 7 non - structural ( p7 , ns2 , ns3 , ns4a , ns4b , ns5a and ns5b ) proteins flanked by 5 and 3 untranslated regions ( utr ) ( 2 ) . e1 and e2 proteins are type i transmembrane proteins with both n - terminal ectodomain and a c - terminal domain ( 3 ) and contain 6 and 11 glycosylation sites , respectively ( 4 , 5 ) .
these proteins are involved in viral entry by interacting with cd81 and scavenger receptor class b member 1 ( srb1 ) ( 6 - 8 ) .
hcv glycosylation sites play an essential role in envelope proteins to ensure correct conformation for virus entry ( 5 , 9 ) and antigenic variation ( 10 ) .
hcv e2 glycosylation sites interact with cell surface receptors directly allowing the virus to enter the cell ( 11 , 12 ) .
glycosylation sites may mask important epitopes from host antibody responses ( 13 , 14 ) .
b - cell epitopes are essential in increasing the preferred immune responses ( 15 , 16 ) and number of epitopes and modulation of immune recognition of antigens can be influenced by deglycosylation of e1 proteins ( 17 ) .
the e1 derived peptide p35 ( amino acid ( aa ) 315323 ) ( 18 ) , e2-conserved synthetic peptides p37 ( aa 517531 ) and p38 ( aa 412419 ) have been reported to neutralize hcv particles , as important components of a candidate peptide vaccine ( 19 ) .
the molecular targets for current hcv direct - acting antiviral ( daa ) in development are mainly focused on non - structural proteins such as the ns3 protease , ns5a and the ns5b rdrp ( 20 ) .
recently , considerable progress has been made to understand hcv entry ( 21 , 22 ) and development of entry inhibitors ( 20 , 21 , 23 , 24 ) .
many patients do not respond to the current available therapy , therefore , there is an urgent need to develop effective hcv vaccines and specific therapeutic drugs . while both e1 and e2 are hypervariable in nature , it is difficult to design vaccines or therapeutic drugs against them .
genotype 5a accounts for over 50% of hcv infections in south africa ( 25 ) .
this study aimed to characterize genotype 5a e1 and e2 sequences to determine possible glycosylation sites , conserved b - cell epitopes and peptides in hcv that could be useful targets in the design of vaccine and entry inhibitors .
this study included 18 genotype 5a samples collected from treatment - naive hcv infected patients at dr .
george mukhari academic hospital ( dgmah ) , north - west of pretoria , south africa , from 2007 to 2011 .
patients demographics and genotyping based on 5utr were previously described in detail ( 25 ) .
six of 18 samples were sequenced as part of the genotype 5a near - full length analysis previously described ( 26 ) .
dgmah is an academic hospital serving a population of around 4 million from both rural and urban areas .
it is a referral hospital for patients from the north west , mpumalanga , limpopo and the northwest part of pretoria , gauteng .
viral rna was extracted from 140 l of serum using the qiaamp viral rna mini kit ( qiagen , hilden , germany ) according to the manufacturer s instructions .
hcv rna was converted into cdna using the enzyme revertaid tm rt - pcr ( fermentas , vilnius , lithuiana ) .
the cdna was amplified in three overlapping fragments ( table 1 ) covering complete e1 and e2 regions .
direct sequencing was performed with abi 3500xl ( inqaba biotechnological industry , pty , ltd , pretoria , south africa ) using second round pcr primers .
all sequences were aligned by mafft ( mafft.cbrc.jp/alignment/server/ ) and translated into amino acids using bioedit ( 27 ) .
the n glycosylation sites were predicted using the online prediction server netnglyc version 1.0 ( http://www.cbs.dtu.dk/services/netnglyc/ ) , which predicts n glycosylation sites in proteins by artificial neural networks that examine the sequence context of asn - xaa - ser / thr sequins
. the networks can identify 86% of the glycosylated and 61% of the non - glycosylated sequins , with an overall accuracy of 76% .
for identification of b - cell epitopes , 16-mer b - cell epitopes was predicted using the program abcpred ( http://www.imtech.res.in/raghava/abcpred/ ) at a 0.51 default threshold using a consensus sequence from 18 genotype 5a sequences created using bioedit .
abcpred server predicts b - cell epitopes using artificial neural network using fixed length patterns ( 28 ) .
antigenicity of all predicted epitopes was analyzed using vaxijen v2.0 online antigen prediction ( www.ddg-pharmfac.net/vaxijen/ ) .
vaxijen v2.0 allows antigen classification based on physicochemical properties of proteins without recourse to sequence alignment .
all predicted epitopes were analyzed for conservation using the iedb database ( http://tools.immuneepitope.org/tools/conservancy/iedb_input ) at a threshold of 100% conservation compared to 406 , 221 , 98 , 33 , 45 , 45 randomly selected sequences from each of the hcv genotypes 1a , 1b , 2 , 3 , 4 and 6 , respectively .
protparam computed different parameters including the molecular weight , theoretical pi , aa composition , atomic composition , extinction coefficient , instability index , aliphatic index and grand average of hydropathicity ( gravy ) . to check post - translational modifications ,
predicted peptides were predicted for n - linked glycosylation as described above and for n - linked phosphorylation using the netphos 2.0 ( 30 ) program .
the netphos 2.0 produces neural network predictions for serine , threonine and tyrosine phosphorylation sites in sequences .
this study included 18 genotype 5a samples collected from treatment - naive hcv infected patients at dr .
george mukhari academic hospital ( dgmah ) , north - west of pretoria , south africa , from 2007 to 2011 .
patients demographics and genotyping based on 5utr were previously described in detail ( 25 ) .
six of 18 samples were sequenced as part of the genotype 5a near - full length analysis previously described ( 26 ) .
dgmah is an academic hospital serving a population of around 4 million from both rural and urban areas .
it is a referral hospital for patients from the north west , mpumalanga , limpopo and the northwest part of pretoria , gauteng .
viral rna was extracted from 140 l of serum using the qiaamp viral rna mini kit ( qiagen , hilden , germany ) according to the manufacturer s instructions .
hcv rna was converted into cdna using the enzyme revertaid tm rt - pcr ( fermentas , vilnius , lithuiana ) .
the cdna was amplified in three overlapping fragments ( table 1 ) covering complete e1 and e2 regions .
direct sequencing was performed with abi 3500xl ( inqaba biotechnological industry , pty , ltd , pretoria , south africa ) using second round pcr primers .
all sequences were aligned by mafft ( mafft.cbrc.jp/alignment/server/ ) and translated into amino acids using bioedit ( 27 ) .
the n glycosylation sites were predicted using the online prediction server netnglyc version 1.0 ( http://www.cbs.dtu.dk/services/netnglyc/ ) , which predicts n glycosylation sites in proteins by artificial neural networks that examine the sequence context of asn - xaa - ser / thr sequins
. the networks can identify 86% of the glycosylated and 61% of the non - glycosylated sequins , with an overall accuracy of 76% .
for identification of b - cell epitopes , 16-mer b - cell epitopes was predicted using the program abcpred ( http://www.imtech.res.in/raghava/abcpred/ ) at a 0.51 default threshold using a consensus sequence from 18 genotype 5a sequences created using bioedit .
abcpred server predicts b - cell epitopes using artificial neural network using fixed length patterns ( 28 ) .
antigenicity of all predicted epitopes was analyzed using vaxijen v2.0 online antigen prediction ( www.ddg-pharmfac.net/vaxijen/ ) .
vaxijen v2.0 allows antigen classification based on physicochemical properties of proteins without recourse to sequence alignment .
all predicted epitopes were analyzed for conservation using the iedb database ( http://tools.immuneepitope.org/tools/conservancy/iedb_input ) at a threshold of 100% conservation compared to 406 , 221 , 98 , 33 , 45 , 45 randomly selected sequences from each of the hcv genotypes 1a , 1b , 2 , 3 , 4 and 6 , respectively .
protparam computed different parameters including the molecular weight , theoretical pi , aa composition , atomic composition , extinction coefficient , instability index , aliphatic index and grand average of hydropathicity ( gravy ) . to check post - translational modifications ,
predicted peptides were predicted for n - linked glycosylation as described above and for n - linked phosphorylation using the netphos 2.0 ( 30 ) program .
the netphos 2.0 produces neural network predictions for serine , threonine and tyrosine phosphorylation sites in sequences .
sequence alignment of 18 genotype 5a sequences with a reference sequence from the genbank showed that most regions in the genotype 5a e1 and e2 proteins were conserved except hypervariable 1 ( hvr1 ) , which was highly variable as expected .
comparison of genetic distances between sequences in this study showed intragroup genetic distances ranging from 8% to 17% , with an average distance of 13% ( table 2 ) .
the values range between 0 ( 0% ) and 1 ( 100% ) substitutions per nucleotide site .
the numbers 1 - 18 corresponds to the sequence number on the vertical side . e1 and
differences in the probability of glycosylation in e1 and e2 were observed in most sequences . whereas other studies reported five n - linked glycosylation sites in the e1 region , all strains in the current study showed three or four glycosylation sites , except for zadgm2088 , which showed 2 glycosylation sites , with n325 site not predicted as glycosylation sites from all sequences . in the e2 region , three sequences ( zadgm1104 , zadgm1707 and zadgm3013 ) showed nine glycosylation sites , while the remaining had variations in the number of glycosylation sites . in zadgm308 , position n430 was replaced by h , while in zadgm6544 , n448 was replaced by d. site n476 was found in only 6 of analyzed 18 sequences .
the e2 sites n423 and n576 were not predicted as glycosylation sites in all genotype 5a sequences in this study ( table 3 ) .
glycosylation probability is shown by + + + ( probability > 70% ) , + + ( probability between 60 and 70% ) , + ( probability between 50 and 60% ) , and - ( not predicted ) .
three conserved antigenic b - cell epitopes were predicted for genotype 5a sequences in the e2 region .
epitope e2(gpvycftpspvvvgtt ) had the highest antigenic score of 1.1613 , while e2(lpcsftptpalstgli ) and e2(lstglihlhqnivdtq ) had antigenic scores of 0.5340 and 0.6639 , respectively .
for conservancy analysis , epitope ewas highly conserved among other genotypes , while epitope eand ewere variable ( table 4 ) .
from the consensus sequences of genotype 5a e1 and e2 , eleven short peptides of 8 - 28 amino acids were designed from the highly conserved residues .
five peptides of 9 - 16 amino acids in length were derived in the e1 region , while six peptides of 8 - 26 amino acids were derived in the e2 .
three of the peptides had post - translation modification , which is the n - linked glycosylation , although at a low probability .
most peptides were found to be the best predicted peptides useful for designing entry inhibitors ( table 5 ) .
list of hydrophobic amino acids ( leu , val , ile , met , phe and trp ) .
glycosylation probability is shown by + + + ( probability > 70% ) , + + ( probability between 60 and 70% ) , + ( probability between 50 and 60% ) , and - ( not present ) .
sequence alignment of 18 genotype 5a sequences with a reference sequence from the genbank showed that most regions in the genotype 5a e1 and e2 proteins were conserved except hypervariable 1 ( hvr1 ) , which was highly variable as expected .
comparison of genetic distances between sequences in this study showed intragroup genetic distances ranging from 8% to 17% , with an average distance of 13% ( table 2 ) .
the values range between 0 ( 0% ) and 1 ( 100% ) substitutions per nucleotide site .
differences in the probability of glycosylation in e1 and e2 were observed in most sequences . whereas other studies reported five n - linked glycosylation sites in the e1 region , all strains in the current study showed three or four glycosylation sites , except for zadgm2088 , which showed 2 glycosylation sites , with n325 site not predicted as glycosylation sites from all sequences . in the e2 region ,
three sequences ( zadgm1104 , zadgm1707 and zadgm3013 ) showed nine glycosylation sites , while the remaining had variations in the number of glycosylation sites . in zadgm308 , position n430 was replaced by h , while in zadgm6544 , n448 was replaced by d. site n476 was found in only 6 of analyzed 18 sequences .
the e2 sites n423 and n576 were not predicted as glycosylation sites in all genotype 5a sequences in this study ( table 3 ) .
glycosylation probability is shown by + + + ( probability > 70% ) , + + ( probability between 60 and 70% ) , + ( probability between 50 and 60% ) , and - ( not predicted ) .
three conserved antigenic b - cell epitopes were predicted for genotype 5a sequences in the e2 region .
epitope e2(gpvycftpspvvvgtt ) had the highest antigenic score of 1.1613 , while e2(lpcsftptpalstgli ) and e2(lstglihlhqnivdtq ) had antigenic scores of 0.5340 and 0.6639 , respectively .
for conservancy analysis , epitope ewas highly conserved among other genotypes , while epitope eand ewere variable ( table 4 ) .
from the consensus sequences of genotype 5a e1 and e2 , eleven short peptides of 8 - 28 amino acids were designed from the highly conserved residues .
five peptides of 9 - 16 amino acids in length were derived in the e1 region , while six peptides of 8 - 26 amino acids were derived in the e2 .
three of the peptides had post - translation modification , which is the n - linked glycosylation , although at a low probability .
most peptides were found to be the best predicted peptides useful for designing entry inhibitors ( table 5 ) .
list of hydrophobic amino acids ( leu , val , ile , met , phe and trp ) .
glycosylation probability is shown by + + + ( probability > 70% ) , + + ( probability between 60 and 70% ) , + ( probability between 50 and 60% ) , and - ( not present ) .
genotype 5 is the most conserved hcv genotype classified into only one subtype ( 5a ) ( 26 ) .
this study was designed to identify conserved sequences of these proteins to predict antigenic epitopes and peptides that could serve as best targets for vaccine design and potential entry inhibitors . using different structural and sequence analyses tools helped with in - silico analysis for e1 and e2 regions .
hcv genotype 5a sequences were found to be conserved in most regions of e1 and e2 proteins .
the most variable region within the study sequences was the hvr1 and these hvr1 differed by up to 80% between hcv genotypes and subtypes ( 31 ) .
although highly variable , the hvr1 is the only region that contains neutralization determinant , which is the target for immune response ( 32 ) .
as expected due to hvr variability , comparison of genetic distances between sequences in this study showed high genetic distances ranging from 8% to 17% , with an average distance of 13% .
variability within the hvr1 is one of the reasons describing why human antibodies raised against hcv e2 epitopes do not provide protection against multiple viral infections ( 19 ) . in this study ,
analysis of n - linked glycosylation sites revealed that genotype 5a sequences were not conserved at glycosylation sites as compared to other genotypes .
site n476 with a level of 75% conservation among different genotypes was absent from the sequences of genotype 5a ( 5 ) and was found in six of the 18 analyzed sequences .
as reported previously , e2 sites n423 and n576 were absent in all genotype 5a sequences including the 18 sequences from this study , which is notable because these two sites were reported to be 99 - 100% conserved across all genotypes ( 5 ) .
the glycosylation sites were reported to be highly conserved among different genotypes ( 9 ) .
these sequence variations in genotype 5a glycosylation sites could be useful to design efficient vaccine to help host to produce good antibody response .
e2 is the main target for neutralizing antibody responses and variation of this region is thought to be related to maintenance of persistent infection by emerging escape variants and subsequent development of chronic infection ( 33 , 34 ) .
recently , a linear region of e2 encompassing amino acids 434 to 446 has been reported to elicit non - neutralizing antibodies that can inhibit neutralizing activity of antibodies targeting amino acids 412 to 423 ( 35 ) . however , a study by tarr et al . reported conflicting results showing that human antibodies that target the region encompassing amino acids 434 to 446 , are not inhibitory but capable of neutralizing hcvpp and hcvcc entry ( 36 ) .
all b - cell epitopes included in this study were found to be antigenic ally effective , and it can be implied that these epitopes may be important for inducing the desired immune response .
recently a study by ikram et al . reported conserved epitopes among genotype 3a that was also conserved among other genotypes ( 37 ) .
highly conserved epitopes might influence the immunogenic potential since variability within the epitopes can increase the chance of immune escape ( 38 ) .
short polypeptides derived from viral envelope sequences of other viruses have been used to investigate protein interactions involved in viral entry and some antiviral agents have been successfully developed ( 39 ) .
envelope protein peptide inhibitors for other viruses in the same family with hcv like dengue and west nile were shown to inhibit viral entry ( 40 , 41 ) . in hcv
, the post - binding entry step was prevented using peptides derived from the c terminal region of e2 , which plays an important role in the hcv entry process ( 42 ) .
for this study , conserved peptides were derived that can be used as targets for therapeutic purposes . in this study , only three peptides had glycosylation sites at low probability and no phosphorylation sites were predicted .
post translational modifications such as glycosylation and phosphorylation affect the stability of therapeutic peptides ( 43 ) .
using hcv glycoproteins in therapeutic strategies may offer protection against hcv infection ( 44 ) . in conclusion ,
the results showed that antigenic conserved predicted b - cell epitopes and stable peptides with few post - translational modifications .
these epitopes and peptides are potential candidates to design entry inhibitors and vaccines able to cover a global population , especially where genotype 5a is common .
further investigations would analyze these peptides to better understand their involvement in blocking hcv entry .
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background : hepatitis c virus ( hcv ) is one of the major causes of cirrhosis and hepatocellular carcinoma with an estimation of 185 million people with infection . the e2 is the main target for neutralizing antibody responses and the variation of this region is related to maintenance of persistent infection by emerging escape variants and subsequent development of chronic infection . while both e1 and e2 are hypervariable in nature , it is difficult to design vaccines or therapeutic drugs against them.objectives:the objective of this study was to characterize genotype 5a e1 and e2 sequences to determine possible glycosylation sites , conserved b - cell epitopes and peptides in hcv that could be useful targets in design of vaccine and entry inhibitors.patients and methods : this study was conducted through pcr amplification of e1 and e2 regions , sequencing , prediction of b - cell epitopes , analysis of n - linked glycosylation and peptide design in 18 samples of hcv genotype 5a from south african.results:differences in the probability of glycosylation in e1 and e2 regions were observed in this study .
three conserved antigenic b - cell epitopes were predicted in the e2 regions and also 11 short peptides were designed from the highly conserved residues.conclusions:this study provided conserved b - cell epitopes and peptides that can be useful for designing entry inhibitors and vaccines able to cover a global population , especially where genotype 5a is common .
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for the first time in 1817 , selenium was discovered by swedish chemist . since then , it has been recognized as an essential trace element with antioxidant , immunological , and anti - inflammatory properties .
dietary intakes of selenium vary between geographical regions depending on selenium content of crops used by animals . in most areas of north america and japan ,
intake is high ( 100200 /day ) while in australia , new zealand , and the majority of european countries , intake is only marginally adequate ( 3090 /day ) , but in some eastern european countries and certain regions of china , intake is very low ( 730 /day ) .
a previous study revealed that serum selenium levels in iranian healthy population is in normal range .
although there are some reports in iranian women , in which selenium status was in borderline status . selenium depletion has been reported in critical illness with systemic inflammatory response syndrome that correlates with an increased mortality and morbidity .
several studies have been shown that the supplementation of selenium either as monotherapy or in an antioxidant micronutrient combination was able to reduce illness severity , improve clinical outcome , reduce infectious complications , and decrease mortality . knowing the plasma levels of trace elements in a particular population for predicting possible nutritional deficiencies that might otherwise go unnoticed is important .
recently , several research projects investigate the effect of metals on health and their levels in body fluids . in this study
, we aimed to access the serum levels of selenium in septic patients early at the intensive care unit ( icu ) admission in one of referral hospitals , iran .
any probable selenium deficiency would help to consider the selenium supplementation during inflammatory cytokines and oxidative stress released during sepsis .
moreover , our evidences toward selenium administration are still inconclusive but have been supported in several studies .
this cross - sectional , descriptive analytic survey was conducted in the internal and surgical icu of rasool - e - akram hospital , affiliated to iran university of medical sciences , from august 2013 to the end of june 2014 .
the study protocol was approved by the local ethics committee of iran university of medical sciences .
patients or their legal surrogates provided written informed consent forms ( number : ir.26929 ) .
this study included 80 icu patients over the age of 18 years old diagnosed with sepsis consistent with the society of critical care medicine ( sccm ) guideline . according to sccm
, sepsis is defined as life - threatening organ dysfunction caused by a deregulated host response to infection ranging from infection and bacteremia to sepsis and septic shock , which can lead to multiple organ dysfunction syndrome and death .
the demographic data of patients such as age , gender , comorbidity , serum creatinine , and acute physiologic and chronic health evaluation ( apache ) ii score at the time of admission were collected in checklist .
we defined having comorbidity in the patients by suffering from one of chronic diseases such as diabetes , ischemic heart disease , chronic liver / lung , or kidney disease .
blood sampling was performed during 48 hours of the icu admission in patients with sepsis diagnosis .
fasting blood samples were drawn by venipuncture in the morning . trace element - free tubes containing ethylenediaminetetraacetic acid were used .
comparisons were done using the unpaired student 's t - test and mann whitney u - test for variables with normal and non - normal distributions , respectively .
chi - square and fisher 's exact tests were applied in the analyses of nominal variables in contingency tables . in all cases , a p < 0.05 was considered to be statistically significant .
in the present study , 80 icu - admitted patients , 49 males and 31 females were chosen .
the frequency of selenium plasma levels was depicted in figure 1 . as shown , the mean age of patients was 48.86 20.76 .
characteristic of participants and selenium plasma levels ( n=80 ) frequency of participants against plasma selenium levels the mean plasma levels of selenium in all of patients was 90.37 25.69 /l .
as shown in table 1 , the mean plasma levels of selenium in males and females was 98.14 23.52 and 78.1 24.46 /l , respectively ( p < 0.0001 ) .
although selenium plasma levels was higher in the icu male patients significantly , both had near normal range ( 80 /l ) .
there was no significant correlation between selenium plasma levels and apache ii score and comorbidity condition , respectively [ table 2 ] .
the benefits of selenium , an essential nutrient , and their function in human health are well established and have been extensively reviewed .
selenium status can be assessed by determining the selenium concentration of whole blood , plasma , serum , or erythrocytes .
a meta - analysis of 14 selenium supplementation / depletion studies confirmed that plasma or serum levels are the most commonly used and reasonably accurate biomarkers of selenium status , representing short - term changes in intake .
measurement is relatively easy , but trace element - free needles and collection tubes must be considered for any environmental contamination .
furthermore , in this study , we preferred to assess plasma selenium levels as it is feasible and available .
several previous studies revealed that serum selenium levels in iranian healthy population is in normal range .
for example , in study conducted in tehran , selenium serum levels is estimated 104.31 24.70
we have similar data 123 /l for adults in mazandaran although we have a report of selenium deficiency in women who are living in tehran .
the mean range of selenium plasma levels in our study was in similar manner with other reports .
selenium deficiency was defined as a levels < 80 g / l , which is a cutoff associated with increased mortality . by considering this cutoff , in this study , septic icu - admitted woman had normal or near normal selenium plasma levels and very near to normal , and septic icu - admitted men had normal range . a range of different recommendations to ensure adequate dietary intake of selenium for healthy populations has been estimated .
the united kingdom ( uk ) suggested the selenium intake of 75 mg / day for male and 60 mg / day for female via daily nutrition .
these amount has been determined as the intake believed to be necessary to maximize the activity of glutathione peroxidase in plasma , which occurs at a plasma selenium concentration of around 100 mg / l ( range 89114 mg /
the food and nutrition board has fixed a recommended daily allowance of 55 mg / day .
selenium deficiency is commonly reported in critically ill patients , particularly those suffering from sepsis , trauma , and burns .
it is reported that low plasma levels of selenium is associated with nosocomial infections ' risk and infusion of 1600 g selenium followed by initial bolus dose of 2000 g for 10 days categorized as a novel modality to increases selenium status , improves illness severity , and lowers the incidence of hospital - acquired pneumonia in the icu patients . however , randomized trials failed to support parenteral selenium supplementation in critically ill patients with sepsis .
currently , we do not have concrete evidences that support the administration of selenium in septic patients .
forceville et al . assessed selenium levels in 134 consecutive patients admitted to the icu .
mmol / l , which was significantly less than the reference population ( 1.000.15 mmol / l ; p < 0.0001 ) .
berger et al . investigated selenium levels in 11 trauma patients admitted to the icu .
intravenous trace element supplementation was started soon after admission including 62 mg of selenium / day for 7 days .
selenium levels were low on admission , and despite supplementation did not normalize for 7 days . in this study
, we have just found that in the iranian population at the beginning of admission to the icu , the selenium plasma levels is in reference range but deficiency probably will happen during critical ill condition . according to mentioned study , selenium plasma contents might have decline during icu hospitalization , critical ill condition and selenium urinary loss as well .
this alteration must be checked in well - designed controlled blinded clinical trials by controlling nutritional state and other confounding factors .
the first one is that the cross - sectional design of the study limits our conclusions and does not provide information about effects of nutritional status on disease progression and outcome of individuals .
we can not judge about other patients in different places of our country since they may be completely having different statues .
it was better to assess the changes in selenium plasma levels not only in the icu - admitted patients but also during icu hospitalization .
this study was funded by the school of medicine , iran university of medical sciences , tehran , iran .
this study was funded by the school of medicine , iran university of medical sciences , tehran , iran .
this study was funded by the school of medicine , iran university of medical sciences , tehran , iran .
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objective : selenium depletion has been reported in critical illness correlates with an increase in mortality and morbidity . in this study
, we aimed to access the selenium plasma levels of septic patients early at the intensive care unit ( icu ) admission in order to compare with reference range.methods:we conducted a cross - sectional study in a university affiliated hospital aiming to assess the early plasma level of selenium in icu admitted patients .
eighty patients diagnoses with sepsis were included and considered for characteristic evaluation , monitoring criteria assessment and also blood sampling .
all blood sampling was performed during 48 hours of the icu admission in order to determined the plasma selenium level by atomic absorption method.findings:the mean plasma levels of selenium in male and female was 98.14 23.52 and 78.1 24.46 /l , respectively . although selenium plasma levels was higher in the icu male patients significantly , both had near normal range ( 80 /l).conclusion : in this study we found that in early admitted iranian icu patients in tehran , selenium deficiency has not routinely seen but probably will happen during icu hospitalization .
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chryseobacterium indologenes ( previously classified as flavobacterium indologenes ) is a gramnegative rod organism found in soil and plants .
although this bacterium only rarely causes human disease , it is sometimes found in food and water sources , usually in hospitals as a nosocomial transinfection 1 , 2 , 3 .
understandably , an immunocompromised host may be affected by c. indologenes . in approximately half of
c. indologenes infection cases , the organism is present on an indwelling device , and severe illness is the result in half of cases 4 , 5 , 6 .
an even more troublesome fact is that c. indologenes presents a high rate of natural resistance against broadspectrum cefem compounds including carbapenem 7 .
worldwide reports of c. indologenes infections in humans have been increasing , although reports of stem cell transplantation ( sct ) recipients infected with c. indologenes are still rare . here
, we describe the first case of a patient with c. indologenes pneumonia after an sct for adult tcell leukemia / lymphoma ( atll ) , which illustrates the increasing risk of this emergent organism during chemotherapy for hematologic malignancies .
a 64yearold female was diagnosed with adult tcell leukemia / lymphoma ( atll ) , which became clinically overt as acute and lymphoma type 4 months prior to a bone marrow transplantation ( bmt ) .
she first received a modified lsg15 regimen 8 , with three intensified and alternating regimens of vcap ( vincristine , cyclophosphamide , doxorubicin , and prednisolone ) , amp ( adriamycin , mcnu , and prednisolone ) , and vecp ( vindesine , etoposide , carboplatin , and prednisolone ) , followed by anticcr4 antibody , which led to a good partial response .
the patient had undergone an unrelated bmt following a reducedintensity preparation regimen ( rist ) consisting of fludarabine 25 mg / m for 5 days and busulfan 0.8 mg / kg 4/day for 4 days , and 2 gy total body irradiation ( tbi ) . with combined tacrolimus and shortterm methotrexate ( mtx ) as graftversushost disease ( gvhd ) prophylaxis , she achieved engraftment uneventfully on day 19 . on day 50 , a routine cytomegalovirus ( cmv ) antigenemia testing converted to positive , and we initiated treatment with valganciclovir ( valgcv ) 180 mg / day orally .
this preemptive therapy 9 was effective , and the cmv antigenemia disappeared on day 70 . on day 61 ,
the patient 's course was complicated with hhv6 encephalopathy , with a suddenonset consciousness disturbance progressing over a 12h span .
the diagnosis of hhv6 encephalitis was made based on the detection of hhv6 dna 2.0 10 copies / ml in the patient 's cerebrospinal fluid ( csf ) the following day ( day 62 ) .
as soon as the encephalitis was diagnosed , we switched the valgcv to foscarnet 60 mg / kg , twice a day . on day 84 ,
a cranial mri scan showing diffuse high intensity on the limbic system confirmed the diagnosis of hhv6 encephalitis . despite the antiviral therapy , systemic convulsions impaired the patient 's pulmonary condition , and she required mechanical ventilator support on day 65 .
her neutrophil count was 6895/l at the onset of the febrile episode , indicating the fever was not neutropenic .
, a chest xray showed an infiltration shadow on bilateral lungs , and the patient 's condition deteriorated to extensive acute respiratory distress syndrome ( ards ) within 3 days ( day 85 ) ( fig .
sputum was suppurative ( miller & jones classification p3 ) , and geckler classification was 5 .
she underwent tazobactam / piperacillin as antimicrobial target therapy and meropenem as an empirical treatment , but died of pneumonia ( respiratory failure ) due to c. indologenes on day 91 .
the antimicrobial susceptibilities of the patient 's isolate are listed in table 1 . on day 85
, diffuse infiltration shadow in bilateral lungs was revealed by a chest xray of the patient , a 64yearold female .
antimicrobial susceptibilities of isolated chryseobacterium indologenes from the patient 's sputum culture mic , minimum inhibitory concentration ; s , sensitive ; i , intermediate ; r , resistant ; taz / pipc , tazobactam / piperacillin ; ctm , cefotiam ; caz , ceftazidime ; cpz / sbt , sulbactam / cefoperazone ; cpr , cefpirome ; azt , azactam ; mino , minocycline ; ipm / cs , imipenem / cilastatin ; mepm , meropenem ; cpfx , ciprofloxacin ; lvfx , levofloxacin ; amk , amikacin .
susceptibility was determined by disk diffusion , following the clsi recommendations ( clsi 2010 , performance standards for antimicrobial susceptibility testing ; twentieth informational supplement , m100s20 , jan .
the pathogenicity of c. indologenes has not been well defined , but some individuals infected with this bacterium were reported to have had an invasive infection with a fatal outcome .
the highly active protease of c. indologenes is in part responsible for its virulence 10 .
reports of c. indologenes infections in humans have been increasing worldwide , but reports of this infection in sct recipients remain rare .
some recent case reports recommend early detection of c. indologenes for an early determination of appropriate antimicrobials to eliminate it .
for example , the case of a critically ill patient with ventilatorassociated pneumonia ( vap ) was anecdotally described as having been diagnosed by a quantitative culture of the patient 's bronchoalveolar lavage 4 .
one case report of a patient with peritonitis caused by c. indologenes complicated by multiorgan failure describes treatment with an intraperitoneal injection of antibiotics 6 .
the proposed source of the chryseobacterium is the environment , including the air , water , or perhaps food .
although c. indologenes is an environmental organism , it is not a typical , hospitalacquired pneumonia pathogen . we propose that for sct recipients , careful monitoring for colonization and infection should be conducted by routine weekly culture . in a recent analysis of a vast number of isolates of c. indologenes collected worldwide , the majority of local hospital antibiograms for this organism remained susceptible to trimethoprim
interestingly , isolates from outside of asia were relatively sensitive to betalactams and fluoroquinolones , whereas isolates from the asia pacific region were not . with the development of broadspectrum cephalosporins ,
the majority of gramnegative rods ( other than stenotrophomonas maltophilia and multidrugresistant nosocomial pathogens ) affecting immunocompromised patients were eliminated within a few decades 12 .
c. indologenes , we believe , should be added to the list of emerging pathogens to watch for during sct . of note ,
the resistance of c. indologenes to broadspectrum cephalosporins is thought to be increasing rapidly in asia . given the resistance of such an organism to broadspectrum cephalosporin , we propose that a prudent selection of antimicrobials should be required as empirical therapy for sct recipients .
the emergence of highly resistant organisms is of particular concern in the especially vulnerable immunosuppressed population of patients after chemotherapy for hematologic malignancy and after stem cell transplantation .
major guidelines and local protocols may not yet take these lesscommon organisms into account in empiric antibiotic recommendations .
as the experts point out , selection of empiric antibiotics in such seriously ill patients may need to be reconsidered , as carbapenems may no longer be the
the broader problem of multidrugresistant gramnegative organisms such as acinetobacter and extended spectrum betalactamase ( esbl)producing enterobacteriaceae in the middle east and asia pacific regions may be influenced by local patterns of antibiotic use .
although the lack of management for these organisms makes the treatment outcome unfavorable , empiric therapy with carbapenem as a single agent for febrile neutropenia might no longer be the gold standard .
oi and mu : managed the patient 's case , contributed to the literature search , and wrote the manuscript .
mu : qualified the patient 's data , suggested important intellectual content , and reviewed the manuscript .
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key clinical messagea 64yearold female was diagnosed with adult tcell leukemia / lymphoma .
she then underwent an unrelated allogeneic bone marrow transplantation with a reducedintensity regimen .
she achieved engraftment followed by hhv6 encephalopathy .
this was complicated by chryseobacterium indologenes pneumonia .
chryseobacterium indologenes is now a possible emergent organism resistant to carbapenem after transplantation .
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calcium homeostasis , a very tightly regulated system in humans , includes the release of parathyroid hormone ( pth ) in response to hypocalcemia .
pseudohypoparathyroidism ( php ) , a rare genetic disorder of calcium metabolism , involves target organ resistance to the action of pth .
thus , despite high serum levels of pth , hypocalcemia occurs and can lead to seizures , which can be the presenting symptom in patients with php .
these patients can also develop tetany , convulsions , basal ganglia calcifications , and cataracts .
this form involves target organ resistance to other hormones in addition to pth , including the thyroid stimulating hormone ( tsh ) , growth hormone releasing hormone , and the gonadotropins ( lh , fsh ) .
php type ia is also associated with a constellation of unusual physical features , known as albright s hereditary osteodystrophy ( aho ) , and includes short metacarpals and metatarsals , short stature , and obesity .
we present a case of a 23-year - old somalian female with intractable epilepsy who was subsequently diagnosed with php .
however , in the months preceding her admission and diagnosis of php , she had loss of seizure control while remaining in the therapeutic range on phenytoin and phenobarbital .
the etiology of her recurrent seizures was multifactorial and included hypocalcemia from resistance to pth , extensive calcium deposition in the basal ganglia and cortex , and exacerbation of hypocalcemia due to vitamin d deficiency .
the latter was likely due to restricted exposure to direct sunlight and inhibition of hepatic 25-hydroxylation of vitamin d by certain antiepileptic agents .
a 23-year - old somalian female presented to the medical emergency room after her parents noted her to have a tonic
her medical history was significant for mild mental retardation and a seizure disorder , which was diagnosed in somalia at the age of 18 years . according to the patient s family
, the etiology of the seizures was never described , and the patient was started on phenytoin and phenobarbital .
she had remained seizure free for the first four years on these medications . despite therapeutic phenytoin levels ,
the patient began to have recurring seizures occurring twice monthly for 6 months before admission .
the patient had not sought medical attention in the united states prior to this episode due to financial instability .
however , the patient s family was able to provide further details . according to them ,
the patient had been in relatively good health prior to coming to the emergency room , and had not been hospitalized previously . over the past 2 years , the patient had complained of intermittent paresthesias in her fingers and toes , along with perioral numbness .
she had immigrated to the united states 3 years earlier and lived in central ohio , a region with limited sunny days during the winter months .
because of her poor functional status , the patient spent most of her days indoors under the supervision of her parents . her only home medications were phenytoin and phenobarbital , and she did not use alcohol , tobacco , or illicit drugs .
family history was significant for short stature , short metacarpals , and mild developmental delay in the patient s older brother and mother .
the patient also had two other siblings and a father who were of normal stature , had no brachydactyly , and had all completed school in somalia . on examination ,
she had short stature , a rounded face , and central adiposity , with height and weight measuring 139.7 cm and 64 kg , respectively .
examination of the extremities revealed brachydactyly of the fingers ( figures 1 and 1a ) and second , third , fourth , and fifth toes ( figures 2 and 2a ) .
the patient had no known personal or family history of congenital or acquired long qt syndrome . computed tomography of the head revealed diffuse bilateral calcifications throughout the basal ganglia and cerebral cortex ( figures 3 and 3a ) . given the clinical , radiographic , and laboratory findings , presumptive diagnoses of php type ia and mild primary hypothyroidism were made .
the patient was admitted to the medical wards , where her hypocalcemia was corrected using both intravenous and oral calcium , vitamin d , and calcitriol . with correction of the hypocalcemia
, there was complete resolution of the prolonged qt interval initially seen on electrocardiogram in the emergency room .
the patient was discharged on 112 mcg of levothyroxine daily , 1 g of calcium carbonate twice daily , 50,000 iu of ergocalciferol weekly , and 0.5 mcg of calcitriol twice daily .
phenytoin was discontinued and the patient was placed on levetiracetam for seizure prophylaxis as it does not interfere with hepatic conversion of vitamin d to 25-hydroxyvitamin d. phenobarbital was gradually tapered down to a lower dosage and eventually discontinued .
the patient was followed up at monthly intervals for a duration of 3 months and has remained seizure free since discharge . given the patient s diagnosis of php type ia , the patient s brother and mother , who exhibited the phenotypic appearance of aho , underwent laboratory evaluation for php type ia and pseudo - php .
laboratory results for the patient s brother revealed serum concentrations of calcium , phosphorus , and intact pth to be 7.2 mg / dl ( reference range : 8.910.3 mg / dl ) , 5.6 mg / dl ( 2.44.7 mg / dl ) , and 206 pg / dl ( 1288 pg / dl ) , respectively , suggesting the diagnosis of php type ia .
the patient s mother had laboratory testing done , which revealed serum concentrations of calcium , phosphorus , and intact pth to be 9.0 mg / dl , 2.8 mg / dl , and 14 pg / dl , respectively . due to normal laboratory results in the presence of features consistent with aho ,
when the serum calcium falls below normal levels , pth is released from the parathyroid glands .
pth regulates serum calcium through its effects on bone via bone resorption , the kidneys by promoting the reabsorption of calcium from the distal tubules , and by intestinal calcium absorption , following production of 1,25-dihydroxyvitamin d through pth - induced activation of the 1-alpha hydroxylase enzyme.1 php is a rare disease characterized by target organ resistance to the action of pth , resulting in hypocalcemia and hyperphosphatemia , despite high serum levels of pth .
few epidemiologic studies of php have been conducted due to the rarity of the disease .
one recent japanese study reported an estimated prevalence of 3.4 cases of php per million population.2 php exists as several distinct entities in the form of php type i , type ii , and pseudo - php ( table 2 ) .
type i is differentiated from type ii and pseudo - php based on the response to administration of synthetic pth , known as the ellsworth
howard test3 there are three subtypes of php type i. php ia , the most common type , is inherited in an autosomal dominant pattern and is caused by an inactivating mutation in the gnas gene that encodes the alpha subunit of the stimulatory g - protein ( gs).4 this signaling protein is involved in the actions of pth and several other hormones .
when pth binds to its receptor , gs activates adenyl cyclase , which is responsible for signal transduction , which produces the response to pth in target tissues .
the inactivating mutation in the gnas gene ultimately leads to failure of signal transduction , resulting in the inability of the target tissues to respond to pth.5 php ia not only involves end organ resistance to pth , but can also involve tissue resistance to other hormones that share the similar signaling pathway through gs - coupled receptors , including tsh , lh , fsh , and growth hormone releasing hormone.6,7 the occurrence of php involves a heterozygous loss of function mutation in the gnas gene and a phenomenon termed genomic imprinting , in which the gnas gene is expressed in tissues in a parent - specific manner.8 when an allele associated with the gnas gene is imprinted from one parent , this allele is inactivated or poorly expressed .
thus , the gene is expressed from the non - imprinted allele inherited from the other parent .
patients with php ia and pseudo - php exhibit about 50% activity of gs in unimprinted tissues .
however , in certain tissues , such as the renal proximal tubules , the thyroid , and the gonads , the paternal allele of the gnas gene is imprinted.9 thus , the gene is expressed from the maternal allele in these tissues.810 this explains why maternal inheritance of a heterozygous loss of function mutation in the gnas gene with the paternal allele being imprinted results in php ia , which involves end organ resistance to pth , tsh , lh , and fsh , while paternal inheritance of a heterozygous loss of function mutation in the gnas gene results in pseudo - php , which involves no associated hormonal resistance.611 thus , the patient and her brother inherited the gene from their mother and had php ia .
php ia and pseudo - php are both associated with aho , which is characterized by a constellation of unusual physical features , including short stature , obesity , round face , brachymetacarpals , brachymetatarsia , dental hypoplasia , and ectopic ossifications .
the aho phenotype in conjunction with end organ resistance to several hormones , including pth , constitutes the syndrome of php ia.11,12 the aho phenotype with no associated hormonal resistance constitutes the syndrome of pseudo - php .
resistance to pth in target tissue will cause hypocalcemia and hyperphosphatemia despite high serum levels of pth .
mild resistance to other hormones may be seen , including resistance to tsh , growth hormone releasing hormone , lh , and fsh .
a characteristic finding on computed tomography of the brain is calcification of the basal ganglia , but extensive intracranial calcifications may rarely be seen.13,14 our patient presented with intractable epilepsy , and during the work up to elucidate the etiology of her recurrent seizures , she was found to have php ia .
the causes included direct and indirect effects of php , as well as vitamin d deficiency .
it also contributed to the severe hypocalcemia , which was exacerbated by the vitamin d deficiency .
the latter was probably due to a combination of restricted exposure to direct sunlight and inhibition of hepatic 25-hydroxylation of vitamin d by the patient s antiepileptic medication .
calcification of the basal ganglia is a rare phenomenon , and approximately 70% to 80% of cases are caused by a disorder of calcium metabolism , such as hypoparathyroidism and php.13 other pathologic causes of symmetric bilateral basal ganglion calcification include fahr s syndrome , down syndrome , cockayne syndrome , and tuberous sclerosis , but none of these syndromes present with the laboratory findings or physical features of aho that are characteristic of php ia.15 the presence of a metabolic cause for bilateral basal ganglion calcifications , such as hypocalcemia due to php ia , effectively rules out the other aforementioned syndromes .
it has been suggested that vascular insufficiency in the extrapyramidal system causes this characteristic deposition of calcium in the basal ganglia , but there have been no data to definitely support this claim.13,16 approximately 50% of patients with php exhibit localized calcification of the basal ganglia.17 extensive calcification of the cerebral cortex extending beyond the basal ganglia , however , is a rare finding.17 when detected on imaging studies , these findings result in a diverse clinical presentation , including seizures , mental deterioration , and parkinsonism , although some patients may remain asymptomatic.13 our patient presented with extensive bilateral intracranial calcifications involving the basal ganglia and cerebral cortex , which may have contributed to intractable epilepsy .
paradoxically , another factor shown to contribute to loss of seizure control is long - term antiepileptic therapy , particularly phenytoin , phenobarbital , and carbamazepine .
these antiepileptic medications induce hepatic microsomal enzymes that convert 25-hydroxyvitamin d to inactive metabolites , resulting in low levels of vitamin d.18 this leads to low levels of 1,25-dihydroxyvitamin d , impaired absorption of calcium from the intestine , and reduced mobilization of calcium from bone , further exacerbating hypocalcemia.19 laboratory results in these patients will show low serum levels of 25-hydroxyvitamin d , 1,25-dihydroxyvitamin d , and calcium .
if a compliant patient on antiepileptic therapy presents with loss of seizure control , clinicians must consider the possibility of antiepileptic induced hypocalcemic seizures . in a patient with php ia who presents with loss of seizure control after previously being stable on antiepileptic therapy ,
an even further exacerbation of hypocalcemia may occur , resulting in intractable epilepsy . in these patients ,
hypocalcemia should be treated with supplementation of vitamin d and calcium , and efforts should be made to convert the patient to antiepileptic medications that do not interfere with vitamin d metabolism . finally , in patients with dark skin who have minimal exposure to sunlight , vitamin d deficiency may occur .
vitamin d is acquired mainly through direct exposure to sunlight , though diet may also influence levels to some degree.20 in order to optimize calcium absorption in the intestine , adequate levels of vitamin d are required.21 our patient from somalia with dark skin who had little exposure to direct sunlight during the winter months in the midwest region of the united states was deficient in vitamin d. this further exacerbated hypocalcemia , resulting in intractable epilepsy . though the etiology of intractable epilepsy in the setting of php ia is multifactorial , certain factors can be controlled and lead to improvement in seizure control
. calcium and vitamin d should be adequately replaced , and antiepileptic medications that interfere with vitamin d metabolism should be avoided .
calcium homeostasis is a tightly regulated system in humans . in patients with disorders of calcium metabolism ,
thus , physicians should be cognizant of disorders of calcium metabolism and medications that can exacerbate hypocalcemia , resulting in loss of seizure control .
php ia , a rare genetic disorder of calcium metabolism , may initially present as hypocalcemic seizures . in patients with disorders of calcium metabolism , such as php ia , who have a concomitant seizure disorder ,
caution should be placed on choice of antiepileptic therapy as certain medications may paradoxically exacerbate seizures by further lowering serum calcium levels .
life - long supplementation with calcium and vitamin d will be required in these patients to maintain sufficient calcium levels and prevent progression of the disease.22,23
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pseudohypoparathyroidism is a rare disorder of calcium metabolism that involves target organ resistance to the action of the parathyroid hormone . as a result
, calcium levels may become dangerously low , sometimes leading to seizures and other symptoms .
we present a case of a 23-year - old somalian female on antiepileptic therapy presenting with intractable epilepsy .
she was subsequently found to have pseudohypoparathyroidism type ia .
she had multiple reasons accounting for loss of seizure control , including worsening hypocalcemia from resistance to the parathyroid hormone ; vitamin d deficiency , which could have resulted from lack of exposure to direct sunlight and her antiepileptic medication ; and extensive calcium deposition in the brain due to pseudohypoparathyroidism .
the patient was stabilized with intravenous therapy and oral calcium , vitamin d , and calcitriol .
her antiepileptic therapy was changed to a medication that did not interfere with vitamin d metabolism or contribute to worsening hypocalcemia .
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asthma is one of the most common chronic diseases affecting children , especially in economically developed nations .
for example , in australia the prevalence of current asthma in children aged 015 years is approximately 11% .
childhood asthma is strongly linked to atopy , which in turn is characteristically associated with a th2-biased immunological response [ 24 ] . while this relationship is well documented
however , it is clear that both genetic predisposition and a variety of environmental factors contribute to the development of allergic asthma .
notable among the environmental factors that appear to be crucial in the induction of disease is respiratory viral infection , in particular with rhinovirus ( rv ) or respiratory syncytial virus ( rsv ) .
the association between childhood infections and asthma is complex , because at least in some settings , repeated early - life exposure to infectious agents may reduce the likelihood of developing allergic diseases . despite this
, epidemiological studies strongly suggest that lower respiratory viral infections associated with wheezing , occurring within a critical period of development in early childhood , play an important role in the subsequent development of asthma in children who are repeatedly exposed to inhaled allergens [ 610 ] .
another clearly defined risk factor for childhood allergic asthma is early - life exposure to airborne environmental irritants .
the importance of exposure to environmental tobacco smoke is well established [ 11 , 12 ] .
more recently , a number of large population - based studies , including prospective cohort studies , have clearly defined the increased risk of development of asthma in children exposed to traffic - related particulate pollutants [ 1315 ] .
the adverse respiratory effects of such pollutants , especially diesel exhaust particulates ( deps ) , are now recognised as a significant public health problem .
somewhat more contentious is the association between the use of paracetamol ( acetaminophen ) in infancy or childhood and the subsequent development of asthma [ 17 , 18 ] .
the evidence for an increased risk of asthma following exposures to other environmental chemicals is much less convincing .
fundamental questions remain unanswered about the underlying mechanisms by which environmental factors promote the development of childhood asthma .
in particular , if atopy and a th2-biased immunological response are indeed precursors to the development of childhood asthma , a key issue is how does injury by environmental factors drive an allergic response ? a possible final common pathway , for which there is now growing support ,
is based on the interaction between airway epithelial cells , dendritic cells , and cd4 + t - lymphocytes .
dendritic cells ( dcs ) have long been recognised as playing a crucial role in the induction of th2 polarisation during an immunological response .
as is increasingly being understood , the development of allergic immunological responses may be determined by innate host defence responses after initial exposure to pathogens , allergens , or other irritants [ 2123 ] .
these lead to local generation of cytokines that stimulate dc , with effects including upregulation of the expression of costimulatory molecules such as cd40 , cd80 , cd86 , jagged-1 , and ox40l , as well as the production of various chemokines ( figure 1 ) [ 2427 ] .
the subsequent maintenance of the th2 bias of the cd4 + t cells may be dependent on epigenetic changes [ 28 , 29 ] , which is now a focus of considerable interest in the study of the pathogenesis of asthma [ 30 , 31 ] .
key factors that may activate and/or drive the maturation of conventional or myeloid dc , to promote th2-biased differentiation of cd4 + t - lymphocytes , include the cytokines granulocyte - macrophage colony - stimulating factor ( gm - csf ) , thymic stromal lymphopoietin ( tslp ) , interleukin ( il)-25 and il-33 . because airway epithelial cells ( aec ) can secrete gm - csf , tslp , il-25 , and il-33 in response to injury , as well as chemoattractants for dc such as ccl20 , the airway epithelium appears likely to play a critical role in promoting recruitment / survival of dc and th2 polarisation of the immune response ( figure 1 ) [ 3335 ] .
accumulating evidence indicates that tslp activates dc to prime cd4 + t cells for inflammatory th2 differentiation . in the context of the induction of childhood asthma , a role for aec - derived tslp in the induction phase is strongly supported by in vitro studies .
following exposure to dep in vitro , human aec generated reactive oxygen species ( ros ) and secreted tslp , which caused dc precursors to enhance expression of ox40l and jagged-1 , which in turn promoted upregulation of th2 responses [ 37 , 38 ] .
similarly , aec exposed to double - stranded rna or infected with rv or rsv in vitro exhibited marked upregulation of expression of tslp [ 39 , 40 ] . in vivo ,
transgenic overexpression of tslp in the lungs promoted an antigen - driven allergic inflammatory response .
injury by proteases has also been shown to elicit generation of ros by epithelial cells , leading to oxidation of lipids , signalling through toll - like receptor ( tlr ) 4 , and the production of tslp that drives th2 responses following subcutaneous immunisation .
because many allergens exhibit endogenous protease activity , it is therefore also possible that inhaled allergens might themselves contribute to enhanced expression of tslp by aec .
to date , however , there are no reported in vitro studies using aec isolated from children nor have there been studies on the role of tslp in an animal model of childhood asthma .
further support for a role for tslp in the development of asthma comes from studies associating single nucleotide polymorphisms in the tslp gene or its promoter region with an increased risk of developing asthma in childhood [ 43 , 44 ] .
epithelial cell - derived il-25 is increasingly recognised as being important in the induction of allergic inflammation .
tslp - activated dc induce strong upregulation of the receptor for il-25 on th2 cells , thus linking these two cytokine pathways .
recent studies have identified novel populations of cells involved in the innate host response , which contribute to maintaining and enhancing the th2-biased response by secreting cytokines such as il-5 and il-13 in response to il-25 .
we have shown that il-25 produced by aec plays a key role in the induction of a th2-biased inflammatory response following respiratory viral infection .
furthermore , we have recently provided convincing evidence that il-25 is of crucial importance in the induction phase of childhood asthma . for the latter studies
, we used a novel animal model of neonatal infection with pneumonia virus of mice ( pvm ) , a species - specific paramyxovirus which simulates rsv infection in human infants .
following subsequent intranasal sensitisation with ovalbumin and long - term low - level challenge , these mice developed inflammation and remodelling typical of chronic asthma , together with a th2-biased immunological response . by themselves , neither infection nor allergen exposure led to the development of an asthmatic phenotype .
this model therefore simulates the interaction between early childhood infection with rsv and sensitisation to inhaled allergens during the development of childhood asthma . in this model
, we found that there was significant upregulation of expression of il-25 following neonatal infection with pvm .
therefore , we tested the effects of administration of a neutralising antibody to il-25 on the development of the asthmatic phenotype , in comparison to administration of an antibody to il-4 , which has long been recognised as having a crucial role in the induction of th2 responses .
anti - il-25 , administered either during chronic challenge or in early life alone , prevented key changes of airway remodelling such as subepithelial fibrosis and epithelial hypertrophy , and suppressed development of a th2 response .
anti - il-4 was more effective in inhibiting allergic inflammation , prevented goblet cell change but not other features of remodelling , and also suppressed development of a th2 response .
these novel findings suggest that blocking induction of a th2 response during the neonatal period or later in childhood could be effective for primary prevention of asthma , and that il-25 might play a crucial role in this process .
gm - csf clearly plays a key role in the development of dc and differentiated human respiratory epithelial cells release significant amounts of gm - csf when exposed to dep in vitro .
however , there is currently no direct evidence of a role for gm - csf produced by aec during the induction phase of childhood asthma .
there has been much speculation about the possible contribution of il-33 to the development of asthma , given the capacity of this cytokine to promote dc maturation towards a th2-inducing phenotype in vitro .
while il-33 is expressed by airway epithelium in asthmatics , there is once again a paucity of direct evidence for a role in the induction phase of allergic asthma .
there is now considerable evidence that an important mechanism by which environmental irritants cause injury to aec is via inducing the production of reactive oxygen species ( ros ) .
this has been well studied in vitro in response to injury by rsv [ 56 , 57 ] .
furthermore , activation of antioxidant defence mechanisms , through binding of the transcription factor nrf2 to antioxidant response elements ( ares ) , has been shown to be an important part of the host response to rsv infection in vivo .
rsv infection caused induction of various are - driven enzymes , and gene - targeted mice deficient in nrf2 had significantly lower levels of reduced glutathione in the lungs , higher levels of oxidative modification of lung proteins and lipids , and developed significantly more severe rsv disease , both in terms of inflammation and epithelial injury .
similarly , in vitro studies have demonstrated that dep induce oxidative stress , with greater effects on aec than on other target cells such as pulmonary macrophages .
there is clear evidence that paracetamol ( acetaminophen ) decreases intracellular levels of reduced glutathione and thus predisposes to injury by ros .
oxidative stress leads to activation of key intracellular signalling mechanisms , for example , the mitogen - activated protein kinase ( mapk ) and nf-b pathways , and these can be inhibited by administration of antioxidants [ 61 , 62 ] .
activation in turn leads to the generation of chemoattractants and proinflammatory cytokines . whether similar events occur in vivo during the induction phase of paediatric asthma has not been formally demonstrated
however , the concept that oxidant injury may be a key early event is supported by evidence that functional polymorphisms in oxidant defence genes increase the risk of developing asthma in childhood [ 63 , 64 ] . environmental irritants may also trigger aec via pattern recognition receptors , including the predominantly cell surface or endosomal tlrs and the cytoplasmic nod - like receptors ( nlrs ) .
these receptors are key components of host innate defences , capable of recognising conserved molecular patterns associated with pathogens or with cellular damage .
tlrs signal via adaptor proteins , notably myd88 , leading to activation of mapk and nf-b signalling pathways .
whether tlr - dependent signalling promotes the development of a th2-biased response following exposure to viral infection or environmental irritants is not altogether clear .
however , the potential importance of tlr - mediated responses is supported by evidence that polymorphisms in genes for tlr2 and tlr4 are related to increased prevalence of asthma from birth up to the age of 8 years
. members of the nlr family assemble into large multiprotein complexes , termed inflammasomes , which activate caspase-1 , a proteolytic enzyme that cleaves and thus activates cytokines such as il-1 and il-18 for secretion .
expression of nlrs has been demonstrated in the airway epithelium , but their role in the response to injury by environmental irritants , or in driving allergic inflammation , has not hitherto been investigated . another potentially important mechanism by which cytokine release from aec might be triggered is via the enzymatic activity of some allergens on protease - activated receptors .
for example , fungal antigens appear to be able to elicit secretion of tslp via par-2 activation .
an intriguing question , especially in relation to early - life viral infection , is whether environmental injury might lead to induction of a relatively stable epithelial phenotype that characterises and/or promotes the development of asthma .
there is no doubt that the airway epithelium of asthmatics is different to that of nonasthmatics , with evidence of abnormal proliferation / repair and enhanced production of proinflammatory cytokines [ 7173 ] .
notably , this includes enhanced expression of tslp and gm - csf by asthmatic aec [ 74 , 75 ] .
furthermore , impaired production of interferons has been related to the increased susceptibility of asthmatics to viral infections [ 76 , 77 ] .
however , what is not clear is whether this is a reason for development of asthma , or an effect of a predisposing factor or of asthma itself .
the issue is relevant to the ongoing debate about the relationship between viral infection and asthma .
interestingly , recent evidence suggests that in a th2-biased environment , which induces il-13-driven mucous cell change in the airway epithelium , the altered epithelial lining may be more susceptible to infection by rhinovirus .
thus , the mucous cell hyperplasia and other remodelling of the airway epithelium associated with the development of childhood asthma may predispose to progression of the asthmatic phenotype , by promoting a vicious cycle of viral and allergic inflammation .
injury to aec by environmental factors may have an important role in initiating a cascade of responses that can lead to the development of asthma in childhood .
oxidant stress by environmental injurious agents may activate a variety of intracellular signalling pathways , that in turn drive the synthesis and secretion of multiple cytokines able to promote the recruitment of t cells and their polarisation towards a th2 cytokine - secreting phenotype .
however , at present there are many gaps in our understanding of the pathogenetic sequence of events . further studies in animal models of childhood asthma may help to identify interventions that could reduce the risk of allergic asthma .
caution is warranted , however , because the mechanisms are likely to be complex and targeting single cytokines or regulatory pathways is rarely successful in asthma [ 81 , 82 ] .
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the pathogenesis of allergic asthma in childhood remains poorly understood .
environmental factors which appear to contribute to allergic sensitisation , with development of a th2-biased immunological response in genetically predisposed individuals , include wheezing lower respiratory viral infections in early life and exposure to airborne environmental pollutants .
these may activate pattern recognition receptors and/or cause oxidant injury to airway epithelial cells ( aecs ) . in turn , this may promote th2 polarisation via a final common pathway involving interaction between aec , dendritic cells , and cd4 + t lymphocytes .
potentially important cytokines produced by aec include thymic stromal lymphopoietin and interleukin-25 .
their role is supported by in vitro studies using human aec , as well as by experiments in animal models . to date , however , few investigations have employed models of the induction phase of childhood asthma . further research may help to identify interventions that could reduce the risk of allergic asthma .
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cardiac myxoma usually develops in the atria and presents in the left atrium in more than 75% of patients and in the right atrium in only 15 - 20% of patients.1 - 9 ) only 3% to 4% of myxomas are detected in the left and right ventricle each.10 - 12 ) myxomas of the heart valves are rare,1)2 ) and primary cardiac valve myxoma is unusual , especially aortic valve myxoma.2 )
a 72-year - old man was referred to our cardiovascular center with previous history of hypertension for 5 years and a 2-month history of worsening dyspnea on exertion that motivated him to seek medical attention .
atrial fibrillation was detected during a recent medical check up at a local clinic , and the patient was referred to our hospital and admitted for closer examination .
two - dimensional transthoracic echocardiography ( 2d tte ) showed 1.040.87 cm nodular mass attached to the aortic valve .
the echogenic mass originated from the ventricular side of the non - coronary cusp of the aortic valve ( fig .
because of his old age and atrial fibrillation , scheduled surgery was performed to avoid systemic embolization .
intra - operatory transesophageal echocardiography showed normal left ventricular outflow without regurgitation and no residual mass .
the margin of the mass was irregular but was easily distinguishable from the normal valve tissue .
the tumor consisted of plump gray purplish myxoid stroma and slender and long spindle stromal cells .
fibrocollagenous bands of aortic valve tissue were also noted in the myxoid stroma ( hematoxylin and eosin ; 200 ) ( fig .
the patient 's postoperative convalescence was uncomplicated and he has discharged on aspirin with regularly scheduled follow - up .
myxomas are one of the most common cardiac tumors but usually confined to the atria , more commonly the left atrium , or the ventricles
aortic valve tumors are rare and predominantly papillary fibroelastomas.13 ) the differential diagnosis of aortic valve myxoma includes vegetation , tumor , and degenerative changes ( lambl 's excrescence).14 ) 2d echocardiography may help differentiate thrombus , vegetation , and myxoma , because thrombus typically produces a layered appearance and in some myxomas an area of echolucency may be seen within the tumor . however , pathologic differentiation of cardiac tumor by image is very difficult .
therefore , the size , shape , location , mobility , and attachment site of a cardiac mass as well as the clinical presentation usually can differentiate these masses . the most important clue to the diagnosis is their location . by far
, the most common cardiac tumor of the heart is the myxoma , which is usually single and occurs in the atria . among tumors that affect
the valves ( tumors arising from the heart valves are rare ) , papillary fibroelastomas are by far the most common .
this tumor appears as a small mass attached to the mitral or aortic valve with motion independent from the normal valve and often locates on the downstream side of the valve by a small pedicle and are irregularly shaped with delicate frond - like surfaces . in case of our experience , a small spherical mass that showed a relatively round surface with central necrosis was attached to the cusp of non - coronary and was highly mobile without pedicle . unlike a typical fibroelastoma , according to tumor site , we speculated that the tumor was closer to fibroelastoma than other types of tumors .
although aortic valve myxomas are extremely rare in clinical practice , we should at least suspect them in patients with unusual tumor characteristics.15 ) nevertheless , the clinical diagnosis of cardiac myxoma is often challenging , and distinguishing myxoma from a vegetation or thrombus by echocardiography is often difficult .
our patient had an extremely rare aortic valve tumor as most aortic valve tumors are papillary fibroelastomas . a patient with atrial fibrillation and
an aortic valve tumor is at an increased risk of systemic embolic events from a cardiac source .
aortic valve myxomas are rare benign cardiac tumors , often discovered after embolization has occurred .
although long - term results from surgical treatment of cardiac myxomas are not completely understood due to its rarity , the rate of recurrence of aortic valve myxoma appears very low.16 ) some reports have suggested that recurrence may be due to incomplete removal.16)17 ) therefore , complete resection of the tumor was performed in this patient , with regular follow - up by echocardiography to monitor the remote chance of recurrence .
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myxoma of the aortic valve is an exceedingly uncommon condition . in this article ,
we report the case of a 72-year - old man with myxoma arising from the aortic valve . we extirpated the mass and repaired the aortic valve with the patient under cardiopulmonary bypass .
the postoperative course was uneventful .
histological examination confirmed that the mass was a myxoma .
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primary spontaneous pneumothorax ( psp ) is a common disorder for thin or tall young male and is characterized by its absence of underlying disease in the lung parenchyma and caused by the rupture of small blebs at the apex part of the lung .
based on the consensus of the american college of chest physicians , it suggests that patients with the second occurrence or persistent air leaks ( > 4 days ) undergo surgery for psp . and patients who are at risk ( scuba divers , divers , pilots , etc . )
the main purposes of surgical treatment are closure of the air leak and prevention of recurrence [ 4 - 6 ] .
video assisted thoracoscopic surgery is a generalized operative treatment of primary spontaneous pneumothorax which has the advantage of superiority in cosmetics , decrease of postoperative pain , curtailment of admission period , and quicker return to society [ 7 - 9 ] .
thoracoscopic bleb removal using automatic stapler is generalized as a standard method in the operative treatment of psp , but necessity of pleurodesis still remains controversial .
so we investigated methods of pleurodesis which are used in our hospital to compare the efficacy of pleurodesis for the risk of recurrence in patient with psp .
from january 2000 to july 2010 , 207 patients who had undergone thoracoscopic wedge resection and pleurodesis were enrolled in this study . the data including age , gender , smoking status , body mass index ( bmi ) , operative indications , method of pleurodesis , operative time , hospitalization , recurrence after surgery , and follow - up duration were reviewed retrospectively from medical records . the recurrence after surgery had been checked by reviewing medical record through follow - up in ambulatory care clinic until or calling the patients directly january 2011 .
the operation was performed by two surgeons in department of thoracic surgery of pusan national university hospital .
the indications of operative treatment were persistent air leak ( > 5 days ) in patients with first experience of pneumothorax , recurrence more than twice in ipsilateral side , and contralateral recurrence .
a 10-mm , 60 thoracoscope was introduced in the seventh intercostals space on the mid axillary line through a 1 cm skin incision . under visual control ,
two additional incisions were performed at the third intercostal space on the mid - axillary line , and at the useful site , so as to introduce endoscopic forceps , and stapling device .
after thorough inspection of the pleural cavity and the whole parenchymal surface , resection of bullae or blebs was performed by endoscopic stapling device .
pleurodesis was performed as two methods depend on surgeon 's preference : apical parietal pleurectomy ( group a ) and pleural abrasion ( group b ) .
pleurectomy was performed to remove all parietal pleura over the fifth intercostals space by using argon bovie except some portions of mediastinum region , and pleural abrasion was performed at the same location by brushing enough to cause petechia using a gauge .
one straight thoracic catheter ( 24fr ; mallinckrodt medical , athlone , ireland ) was placed through the incision of the seventh intercostals space on the mid - axillary line .
the chest tube was connected to a water seal system with 20 cm h2o suction .
chest x - ray was performed to confirm the position of the chest tube and expansion of lung .
postoperative pain was controlled by means of intra - venous patient controlled anesthesia associated with non - steroidal anti - inflammatory medications or opioid drugs .
chest tube removal was performed when completely expanded lung and absence of air leak and drainage less than 200 ml during 24 hours were obtained .
all patients were discharged the next day of the removal of chest tubes , if a chest x - ray was normal .
the statistics were evaluated by using spss ver . 12.0 ( spss inc . , chicago , il , usa ) , and the recurrence rate between two groups were compared by using kaplan - meier method and cox proportional hazard method .
the operation was performed by two surgeons in department of thoracic surgery of pusan national university hospital .
the indications of operative treatment were persistent air leak ( > 5 days ) in patients with first experience of pneumothorax , recurrence more than twice in ipsilateral side , and contralateral recurrence .
a 10-mm , 60 thoracoscope was introduced in the seventh intercostals space on the mid axillary line through a 1 cm skin incision . under visual control ,
two additional incisions were performed at the third intercostal space on the mid - axillary line , and at the useful site , so as to introduce endoscopic forceps , and stapling device .
after thorough inspection of the pleural cavity and the whole parenchymal surface , resection of bullae or blebs was performed by endoscopic stapling device .
pleurodesis was performed as two methods depend on surgeon 's preference : apical parietal pleurectomy ( group a ) and pleural abrasion ( group b ) .
pleurectomy was performed to remove all parietal pleura over the fifth intercostals space by using argon bovie except some portions of mediastinum region , and pleural abrasion was performed at the same location by brushing enough to cause petechia using a gauge .
one straight thoracic catheter ( 24fr ; mallinckrodt medical , athlone , ireland ) was placed through the incision of the seventh intercostals space on the mid - axillary line .
the chest tube was connected to a water seal system with 20 cm h2o suction .
chest x - ray was performed to confirm the position of the chest tube and expansion of lung .
postoperative pain was controlled by means of intra - venous patient controlled anesthesia associated with non - steroidal anti - inflammatory medications or opioid drugs .
chest tube removal was performed when completely expanded lung and absence of air leak and drainage less than 200 ml during 24 hours were obtained .
all patients were discharged the next day of the removal of chest tubes , if a chest x - ray was normal .
the statistics were evaluated by using spss ver . 12.0 ( spss inc . , chicago , il , usa ) , and the recurrence rate between two groups were compared by using kaplan - meier method and cox proportional hazard method .
of 207 patients , 188 patients were male and the age was 21.56.4 ( meanstandard deviation ) years .
mean duration of postoperative thoracostomy tube drainage and mean postoperative stay were 5.2 and 6.2 days , respectively .
all patients were divided into two groups according to the methods of pleurodesis : apical parietal pleurectomy ( group a , n=121 ) and pleural abrasion ( group b , n=86 ) .
the age of group a and b was 21.45.9 and 21.77.1 years and the gender ( male : female ) of group a and b was 112:9 and 76:10 .
the bmi of group a and b was 20.19.1 and 19.02.1 kg / m , respectively .
there was no significant difference in age , gender , and bmi between two groups .
the operative time of group a and b was 103.531 and 95.840.8 minutes , respectively .
the hospitalization after surgery of group a and b was 6.03.9 and 6.53.3 days , respectively ( table 1 ) .
the recurrence rate of group a and b was 9.1% and 12.8% , respectively and it showed a significant difference ( table 2 ) .
after the video assisted thoracoscopic surgery was generalized as a standard method in the operative treatment of primary spontaneous pneumothorax , the process of bleb removal using automatic stapler became common .
nevertheless , pleural procedure after wedge resection such as pleurodesis has been performed in some patients who need an extremely low recurrence rate .
pleurectomy is known to be highly effective to prevent a recurrence in psp . in the past
, it was performed through posterolateral thoracotomy or limited lateral thoracotomy and avoided due to cosmetic problem , chest pain , and nerve injury caused by thoracotomy . with the application of video - assisted thoracoscopic surgery , apical parietal pleurectomy was not operated frequently due to technical problem and long operative time . however , according to the development of thoracoscopic instrument and technique , more complicated operations have been performed by video - assisted thoracic surgery .
apical parietal pleurectomy with video assisted thoracoscopic surgery has also been more easy procedure . as other various methods of operating the pleurectomy
in contrast , pleural abrasion is preferred with video - assisted thoracoscopic surgery because of its simple skill , faster operative time , and being performed easily .
but pleural abrasion did not show same prevention effect for the recurrence compared to pleurectomy [ 14 - 16 ] .
surgical chemical pleurodesis using talc remains rarely performed , because difficult next thoracic surgery due to severe adhesion , the pain , and the possibility of carcinogenesis itself .
there was no significant difference of air leak in the postoperative complication between apical parietal pleurectomy ( group a ) and pleural abrasion ( group b ) .
the air leak means that lung is not completely expanded and the pleural adhesion is not properly induced .
therefore , it is important to make sure the complete closure of air leak , primarily . in 2010
, kim et al . reported that apical pleurectomy was no more advantageous than mechanical pleural abrasion in terms of operative time , postoperative course , and prevention of recurrent pneumothorax , so complete resection of bullae and existence of residual bullae are more important factors in reducing the incidence of recurrent pneumothorax than pleural symphysis .
however , results of our study present that the rate of recurrence of group a and b was 9.1% and 12.8% , respectively and there was a significant difference ( p=0.014 , hazard ratio=3.108 ) and suggest that the apical parietal pleurectomy could reduce the risk of recurrence .
but these two studies have different number of patients ( 87 vs. 207 ) and follow up duration ( 31.725.3 vs. 55.035.5 months ) .
this study suggests that the risk of recurrence after surgery in psp is significantly low in patients who underwent thoracoscopic wedge resection with parietal pleurectomy than pleural abrasion .
however , prospectively randomized clinical study will be required to clarify a clinical efficacy of apical parietal pleurectomy .
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backgroundthe standard operative treatment of primary spontaneous pneumothorax ( psp ) is thoracoscopic wedge resection , but necessity of pleurodesis still remains controversial .
nevertheless , pleural procedure after wedge resection such as pleurodesis has been performed in some patients who need an extremely low recurrence rate.materials and methodsfrom january 2000 to july 2010 , 207 patients who had undergone thoracoscopic wedge resection and pleurodesis were enrolled in this study .
all patients were divided into two groups according to the methods of pleurodesis ; apical parietal pleurectomy ( group a ) and pleural abrasion ( group b ) . the recurrence
after surgery had been checked by reviewing medical record through follow - up in ambulatory care clinic or calling to the patients , directly until january 2011.resultsof the 207 patients , the recurrence rate of group a and b was 9.1% and 12.8% , respectively and there was a significant difference ( p=0.01 , cox 's proportional hazard model ) . there was no significant difference in age , gender , smoking status , and body mass index between two groups.conclusionthis study suggests that the risk of recurrence after surgery in psp is significantly low in patients who underwent thoracoscopic wedge resection with parietal pleurectomy than pleural abrasion .
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we
demonstrate broadband tunability of light emission from dense ( 6,5 )
single - walled carbon nanotube thin films via efficient coupling to
periodic arrays of gold nanodisks that support surface lattice resonances
( slrs ) .
we thus eliminate the need to select single - walled carbon
nanotubes ( swnts ) with different chiralities to obtain narrow linewidth
emission at specific near - infrared wavelengths .
emission from these
hybrid films is spectrally narrow ( 2040 mev ) yet broadly tunable
( 10001500 nm ) and highly directional ( divergence < 1.5 ) .
in addition , slr scattering renders the emission highly polarized ,
even though the swnts are randomly distributed .
numerical simulations
are applied to correlate the increased local electric fields around
the nanodisks with the observed enhancement of directional emission .
the ability to control the emission properties of a single type of
near - infrared emitting swnts over a wide range of wavelengths will
enable application of carbon nanotubes in multifunctional photonic
devices .
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today the opiates are the most addictive substance used in the world wide and they are considered as one of the greatest neuropsychological disorder reasons [ 14 ] .
it has been shown that chronic consumption of opiates can significantly change the functions , structures , and morphology of neural systems [ 5 , 6 ] .
this kind of changes causes several different types of disorders such as drug addiction , carelessness , fluent speech disorders , memory and learning impairment , and psychological disorders [ 710 ] .
according to the studies , drug addiction is an abnormal form of learning and an adaptation of the memory system in certain regions of the brain , such as the hippocampus .
the learning processes are defined as a modification in some synaptic functions ( synaptic plasticity ) , changes in the pre- and postsynaptic ultrastructure , and the formation of new synapses [ 10 , 12 ] .
it is known that the synapses of hippocampus formation are more structurally affected by learning process [ 1216 ] .
there are previous researchers demonstrate that opiates have either direct or indirect effect on capacity of learning and memory , processes . concerning the role of the hippocampal synapses in learning , memory , and circuit reward process , it was concluded that morphine consumption as a main opiate substance can cause some functional , structural , and morphological changes in synapses of the hippocampus in the mammalians [ 17 , 18 ] .
however , many works support the link between synaptic plasticity changes and repeated morphine consumptions but only a few studies have recently start to examine the ultrastructural effects of morphine on synapses to explain the relationship between altered connectivity and opiate treatment [ 4 , 19 ] .
so to look into these issues with more details , the current experimental study employed transmission electron microscopy to compare the effects of morphine consumption on synaptic plasticity , postsynaptic density thickness , and synaptic curvatures in ca1 area of hippocampus formations . the electron microscopy studies could provide some important evidence of synaptic plasticity and the precise pattern of synaptic restructuring .
24 n - mary male rats , weighing 290300 g and 89 weeks of age , were used in this study .
they were housed 4 per cage in a climate - controlled room under a 12-hour alternating light / dark cycle at a controlled temperature of 23 1 .
then it was dissolved in tap water to produce solutions of 0.1 , 0.2 , 0.3 , and 0.4 mg / ml ( w / v ) .
we dissolved 3 gram of sucrose per 100 ml of solution to reach a sweet solution .
after 2 weeks of acclimation to the diet and the environment , 24 n - mary male rats were randomly divided into 3 groups , morphine ( n = 8) , placebo ( sucrose ) ( n = 8) , and control ( water ) ( n = 8) groups .
the rats in the morphine groups chronically consumed morphine solutions at doses of 0.1 , 0.2 , and 0.3 mg / ml for 48 hrs and 0.4 mg / ml up to 21 days .
sucrose ( 3 g/100 ml ) was added to drinking water to mask the bitter taste of morphine . in the placebo group sucrose ( 3 g per 100 ml ) was administrated in drinking water for the same duration of time . in the pilot study , the average water consumption during the administration of the highest dose ( 0.4 mg / ml ) was 50 mgkgday [ 20 , 21 ] . at the end of each drug treatment ,
all of the rats were deeply anesthetized with 100 mg / kg of pentobarbital and perfused transcardially with 4% paraformaldehyde/0.6% glutaraldehyde in 0.1 m phosphate , ph 7.4 .
then , their brains were separated by usual dissection methods and postfixed in 4% paraformaldehyde for 2 h. after fixation , the samples were washed with pbs , then postfixed with 1% osmium tetroxide for 1.5 h , again washed in pbs , dehydrated in an acetone series , and then embedded in epoxy resin [ 2224 ] . for ultrathin sections the block face was first trimmed and the semithin sections were provided at 500 nm thicknesses , stained with 1% toluidine blue , and were examined by light microscopy , for finding the desired area . after finding desired area ultrathin sections ( ~70 nm - thick )
were cut by ultramicrotome ( ultracut uct ) with glass knives ( leica emkmr2 ) at a thickness of silver - gold interface color and picked up on 100 mesh copper grids .
the sections were stained with a 2% aqueous solution of uranyl acetate for 3 min and then with a lead citrate ( 0.5% ) solution for 510 min .
ultimately , the samples were viewed on transmission electron microscope ( zeiss em900 ) and micrographs were taken at 30,000x magnification .
eight electron micrographs were taken from three separate regions of each brain ( 24 micrographs per brain ) at a magnification of 30,000x on a zeiss em900 transmission electron microscope at 80 kv ( three reference planes ) . here , the landmarks used to identify the correct location in each section typically consisted of cross sections of small myelinated fibers and clusters of mitochondria transversing the section .
the observer counted all of the synapses in the hippocampal sections and the physical dissector method was again used to calculate the synapse density , for which the number of synapses present in the reference section and not the look - up section was counted ( qsynapse ) .
the total number of synapses within an unbiased counting frame of a known area ( aframe ) was counted ( 1620 m per brain at 50,000x for the ultrathin sections ) .
again , the dissector volume of tissue ( vdis ) is calculated using the formula from above , where h is section thickness ( 70 nm ) multiplied by the number of sections .
synapse density , nvsynapse , was calculated using the following formula : nvsynapse = qsynaps / vdis ( using ultrathin sections ) .
synaptic ultrastructure , including thickness of postsynaptic density ( thickest part ) , and synaptic curvature were measured by transmission electromicroscopic methods .
analysis using the dissector methods showed a significant increase in the mean synaptic number per m in hippocampus of morphine treated groups ( 1.85 synapse per m ) compared to placebo ( 0.8 synapse per m ) and control ( 0.67 synapse per m ) groups ( p < 0.0001 ) .
it reflects a positive significant relationship between synaptic density increases and morphine consumption ( p < 0.0001 ) .
there was not any significant difference between placebo and control groups ( p < 0.585 ) . here , the 95% confidence intervals for morphine groups , placebo , and control groups were 2.09301.6069 , ( 0.92930.6706 ) , and ( 0.82090.5910 ) , respectively ( table 1 ) .
as shown in table 2 , the analysis of hippocampal synaptic curvatures in rats that received morphine revealed a significant increase in the convex form of synapse ( 87.5% ) compared to those of placebo ( 19.5% ) and control groups ( 12.5% ) ( p < 0.0070 ) . here
the concave form of synapses in morphine groups makes only 12.5% of all synapses compared to those of control ( 45% ) and placebo groups ( 39% ) .
the statistical analysis also revealed a significant difference between groups in terms of thickness of postsynaptic density .
as shown in the table below the minimum and maximum mean of thickness in morphine groups were 3 nm and 5.7 nm , respectively ( sd = 1.6 and f = 20.37 ) .
these results compared to those of placebo and control groups show significant increases in thicknesses of postsynaptic density ( p < 0.0001 ) . see table 3 and figures 1 and 2 . as shown in figure 1
, the synaptic contact zone curvature membrane is mostly convex in morphine groups ( see the arrows in figures 1 , 2(c ) , and 2(d ) ) relative to control ( see figures 1 and 2(a ) ) and placebo groups ( see figures 1 and 2(b ) ) .
m indicates the mitochondria . as shown in figure 2 , postsynaptic density ( psd ) is thick in morphine groups ( see arrows in figures 2(c ) and 2(d ) ) relative to control ( see figure 2(a ) ) and placebo groups ( see figure 2(b ) ) .
the aims of our present study were to determine the chronic effects of morphine consumption on synaptic number , the synaptic curvature , and synaptic thickness of the rat hippocampus .
analysis of our present data showed that chronic consumption of morphine has positive effects on the number of synapse in rat 's hippocampus .
in other words , the consumption of morphine for a long time causes a significant increase in the total number of synapses ( synaptic density ) in hippocampal ca1 area in rats .
these results are in agreement with those of kauer and malenka in 2007 , kelley in 2004 , and berke and hyman 2000 and vorel et al . 2001 findings [ 10 , 16 , 20 , 21 ] .
they reported that synaptic plasticity is essential for neuroadaptations that resulted from a wide range of environmental stimuli .
they also stated that use of the morphine , as environmental stimuli , for a long time can cause long - term changes in behavior by altering the synaptic structure , function , and number of synapses ( the synaptic plasticity ) in relevant brain circuits . and also alcantara et al . in 2011 and pattij et al . in 2009 showed that morphine causes behavioral changes in animals .
these morphine - induced behavioral changes are due to high sensitization of neuron to morphine consumption and changes in the number , function , and structure of synapse [ 4 , 25 ] .
it is thought that the structural and morphological changes of synapses , generation of new synapses , and synaptic plasticity have a main role in normal learning and memory process .
these findings suggested immediately that these processes are similar to associative learning and are essential in the early development of addiction . in this study ,
an overall increase in the synaptic contact zone curvature was observed in the ca1 area of hippocampus of morphine - treated animals . according to medvedev et al . in 2010 and connor et al . in 2006 ,
the synapses curvature was classified as follows : concave , a protrusion of presynaptic terminal into the postsynaptic button ; convex , a protrusion of postsynaptic button into the pre - synaptic terminal ; flat , no discernable curvature [ 26 , 27 ] .
the increase in synaptic contact zone curvature in our work was mostly convex , a protrusion of postsynaptic button into the pre - synaptic terminal , in morphine groups relative to placebo and control groups .
this finding is in agreement with agnihotri , suzuki reports [ 27 , 28 ] .
according to them , the curvature of synaptic membrane changes equally during normal development either as convex or concave , but this change in curvature of synaptic membrane in morphine treated groups was mostly convex .
these show that morphine dependence may cause unusual changes in cytoskeleton structure of postsynaptic contact zones of dendritic spines of neurons .
and also our data showed that the postsynaptic thicknesses change in morphine treated groups compared to other groups ( placebo and control groups ) .
it has been reported that the changes in postsynaptic density thicknesses have a close relationship to the alteration of protein profile , cytoskeletal , and morphology of postsynaptic site . according to the literatures , the postsynaptic density ( psd ) refers to the concentration of protein components in postsynaptic site , including signaling , receptors , and scaffold protein ; these proteins receive and transduce synaptic information [ 30 , 31 ] . according to the reports , it was concluded the postsynaptic density plays an important role in synaptic regulation and plasticity [ 3236 ] .
it also has been reported that the levels of these proteins are strongly influenced by several environmental factors , such as opiate drugs .
for example , the repeated administration of morphine induced significant changes in the levels of postsynaptic proteins [ 32 , 36 , 37 ] in one of the studies , morn et al .
2007 examined the levels of protein expression in synaptic membranes obtained from mouse hippocampus upon morphine administration using icat technology .
they found that chronic morphine exposure induced an increase in the levels of proteins in postsynaptic site . referring to the above results
, the changes of synaptic curvature ( scz ) between the three groups ( morphine , placebo , and control groups ) it is likely has been shown that chronic consumption of opiates can significantly change the functions , structures , and morphology of neural systems , so it is likely that the increase of postsynaptic protein levels may cause a significant increase in postsynaptic density thicknesses as indicated by transmission electron microscopy in our experimental study .
it is concluded that chronic morphine consumption plays a main role in the ultrastructural changes and synaptic plasticity in rats .
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it is well known that the synapses undergo some changes in the brain during the course of normal life and under certain pathological or experimental circumstances .
one of the main goals of numerous researchers has been to find the reasons for these structural changes . in the present study , we investigated the effects of chronic morphine consumption on synaptic plasticity , postsynaptic density thickness , and synaptic curvatures of hippocampus ca1 area of rats .
so for reaching these goals , 24 n - mary male rats were randomly divided into three groups , morphine ( n = 8) , placebo ( n = 8) , and control ( n = 8) groups . in the morphine group , complex of morphine ( 0.1 , 0.2 , 0.3 , and 0.4 ) mg / ml and in the placebo ( sucrose ) group complex of sucrose ( % 0.3 )
were used for 21 days .
after the end of drug treatment the animals were scarified and perfused intracardinally and finally the ca1 hippocampal samples were taken for ultrastructural studies , and then the obtained data were analyzed by spss and one - way analysis of variance .
our data indicated that synaptic numbers per nm3 change significantly in morphine group compared to the other two groups ( placebo and control ) ( p < 0.001 ) and also statistical analysis revealed a significant difference between groups in terms of thickness of postsynaptic density ( p < 0.001 ) and synaptic curvature ( p < 0.007 ) .
it seems that morphine dependence in rats plays a main role in the ultrastructural changes of hippocampus .
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although the incidence of end stage renal disease ( esrd ) has remained relatively stable in the united states , the prevalence has increased , such that 593,000 people are currently living on hemodialysis , on peritoneal dialysis , or with a functioning kidney transplant .
improvements in renal - replacement therapy , overall treatment , and access to care have increased patient survival in esrd . in the prevalent population
patients are living longer on renal replacement therapy and the population currently on dialysis is increasing in age .
the overall incidence of dialysis patients is 340 per million persons but since 2000 , the adjusted incidence rate of esrd has increased by 12.2% for those patients 75 years or older ( 1773 per million persons ) [ 1 , 2 ] .
because of the rising number of aging dialysis patients , there will likely be an increased need for use of hospital resources and cancer care in this population .
multiple studies have consistently showed increased complications and mortality in renal patients undergoing general , vascular , and cardiac surgical procedures [ 38 ] .
the question of whether patients with renal failure should undergo major cancer surgery has not been specifically addressed in the medical literature .
the complexity of the surgery and overall prognosis of the type of cancer involved will need to be considered in choosing options for these patients .
few studies address outcomes of pancreatic surgery in patients with renal dysfunction and all were single center studies consisting of small series of patients [ 911 ] .
the purpose of this study is to utilize a large , nationwide database to determine outcomes of pancreatic resection in patients with renal failure to assist surgeons and oncologists in selecting the optimal patients for these procedures .
data for this study was obtained from the american college of surgeons national surgical quality improvement program ( acs nsqip ) .
acs nsqip is a prospective , multiinstitutional , and clinical registry created by the veterans health administration in 1994 for quality improvement purposes .
over 130 preoperative variables through 30-day postoperative variables are collected on randomly assigned patients , including patient demographics , surgical profile , preoperative risk assessment , laboratory values , operative information , and 30-day morbidity and mortality rates . a highly trained surgical clinical reviewer ( scr )
all reviewers receive extensive initial training prior to starting data collection and ongoing training via continuing education .
acs nsqip monitors accrual rates and data sampling methodologies and conducts audits on a random basis , ensuring highly reliable data .
acs nsqip participant files for the years 20052011 were reviewed and current procedure terminology ( cpt ) codes were used to identify all patients who underwent pancreatic procedures ( 48100 - 48999 ) .
we excluded pancreatic biopsy and pancreatic debridement as these were presumably done for unresectable pancreatic malignancies or necrotizing pancreatitis . from the remaining pancreatic cases ,
two groups were created : patients who had pancreatic resection with renal failure ( group rf ) and those who underwent pancreatic resection with normal renal function ( group non - rf ) .
renal failure was defined as an increased creatinine above 3 mg / dl on laboratory studies within 24 hours prior to surgery and patients on dialysis .
dialysis dependent patients were those with acute or chronic renal failure requiring peritoneal dialysis , hemodialysis , hemofiltration , hemodiafiltration , or ultrafiltration within two weeks prior to surgery .
the comorbidities considered were diabetes , chronic obstructive pulmonary disease ( copd ) , congestive heart failure ( chf ) , hypertension requiring medications , disseminated cancer , and transfusions within 3 days prior to surgery .
postoperative complications of interest were superficial surgical site infection , deep incisional surgical site infection , organ space surgical site infection , wound disruption , pneumonia , urinary tract infections , unplanned intubation , pulmonary embolism , deep vein thrombosis , cardiac arrest requiring cardiopulmonary resuscitation , myocardial infarction , intraoperative or postoperative transfusions , sepsis , and septic shock .
finally other outcome measures reviewed included operative time , return to the operating room , hospital length of stay , 30-day mortality , and time from operation to death in those patients who expired .
associated risks were expressed as odds ratios ( or ) with a 95% confidence interval ( ci ) .
all reported p values are two - tailed , and for all tests , p < 0.05 was considered statistically significant . with nominal regression
, we used preoperative variables of sex , age > 65 years , history of diabetes , smoking , congestive heart failure , use of steroids , albumin < 3.0 gm / dl , bilirubin > 2.0 mg / dl , ast > 90 u / l , hematocrit < 30% , platelet count < 200 10/cc , and prothrombin time > 14 seconds and the presence of renal failure to determine if any of these factors were associated with 30-day mortality .
during the 7-year period , 20052011 , there were 18,533 patients who underwent pancreatic resection .
specific procedures included partial removal of pancreas ( 13 ) , pancreatectomy ( 6 ) , pancreatectomy with pancreatojejunotomy ( 3 ) , distal pancreatectomy ( 3 ) , and proximal pancreatectomy ( 3 ) .
rf patients were older and were more likely to have diabetes , myocardial infarction within the previous 6 months , hypertension requiring medications , use of steroids , bleeding disorder , disseminated cancer , and a blood transfusion prior to surgery .
rf patients had significantly worse initial bleeding parameters with higher protime and partial thromboplastin time , as well as a lower platelet count .
rf patients had similar bilirubin but higher ast , alkaline phosphatase , and lower albumin prior to pancreas surgery .
most notably , rf patients were more likely to need reintubation , have a cardiac arrest , and have bleeding that required blood transfusion .
although rf patients were not any more likely to have surgical site infections , wound infections , or sepsis , they were more likely to have septic shock .
of all patients who died in this study , those with rf expired in a mean of 7.8 days compared to 13.3 days in non - rf patients . using nominal regression ,
the factors predictive of 30 day mortality included age 65 or higher , presence of diabetes , history of chf , steroid use , albumin < 3.0 gm / dl , bilirubin > 2.0
mg / l , hct < 30% , protime > 14 seconds , and renal failure ( see table 5 ) . the presence of rf had the highest odds ratio at 6.13 .
advances in renal replacement therapy and better management of diabetes and cardiovascular problems have allowed patients with rf to live longer with their chronic illnesses , but they now may be living long enough to develop neoplasms that require surgical intervention . while previous large studies have addressed surgical procedures in dialysis patients , less
previous studies have demonstrated increased surgical morbidity and mortality in patients with renal failure who are dialysis dependent . in the largest study , gajdos et al . used nsqip to investigate the effect of long - term dialysis on general surgical procedures .
the 1506 dialysis patients had 12.7% mortality ( 30-day ) compared to 1.5% in the 164,094 patients with normal renal function .
dialysis patients also had a higher pulmonary complications , reoperations , cardiovascular complications , surgical site infections , and hospital length of stay .
while this study had a large number of dialysis patients , it included a variety of general surgical procedures with varying levels of complexity and is done for both benign and malignant reasons .
many factors contribute to this risk but the effect of renal dysfunction has been variably reported .
age has been the most commonly cited risk factor for outcome in pancreatic surgery [ 1316 ] .
diabetes , the reason for pancreatic resection ( benign versus malignant ) , hospital volume , preoperative biliary drainage , and resection of other organs have all been mentioned as contributing to prognosis [ 9 , 11 , 1719 ] .
renal failure and dysfunction have been shown to contribute to poor outcome in pancreatic head resections in 3 single center studies , but these studies had 300 cases or fewer in each series [ 911 ] .
two studies attempted to combine multiple risk factors in developing tools to assess patients ' risk for pancreatic surgery .
are et al . using the nationwide inpatient sample ( nis ) database developed a nomogram to predict outcome for patients undergoing pancreatic resection for malignancy .
on the other hand , parikh et al . using nsqip developed a risk calculator to predict outcome after pancreatic resection .
renal failure was not mentioned as a risk factor , but american society of anesthesiologists ( asa ) classification , functional health status , sepsis , surgical extent , age , dyspnea , body mass index , coronary heart disease , gender , and bleeding disorder were the most important .
two additional studies reviewed outcomes after pancreatic surgery utilizing the nis and discussed the effect of renal failure .
mcphee analyzed in - hospital mortality in patients who underwent pancreatectomy for neoplasm between 1998 and 2003 .
of 39,463 patients , 395 ( 1% ) had renal failure and mortality rate of rf patients was 36.1% compared to the overall mortality rate of 5.9% .
teh et al . evaluated the nis database for major pancreatic resections done for benign and malignant disease between the years of 1988 and 2003 . among the cohort of 103,222 patients , 1% had renal insufficiency and this patient subset
had increased in - hospital mortality ( or 6.3 ) , perioperative complications ( or 2.3 ) , and increased mortality following a major complication ( or 3.5 ) .
while these studies are helpful in stratifying renal failure as a risk , the clinical variables and specific outcomes in these rf patients are not described in detail .
our study clearly demonstrates that renal failure increased morbidity and mortality for patients undergoing pancreatic resection .
although only a cohort of 28 patients , 21% of these patients died in a mean of 7.8 days after surgery .
patients with renal failure also had a significantly higher rate of postoperative complications with an increased risk of unplanned reintubation , cardiac arrest , septic shock , and need for transfusion .
those patients who did survive remained hospitalized for almost twice as long as patients without rf .
this study did have limitations , as it utilized an administrative database and can only be as precise as the trained staff who enter the data .
although the number of pancreatic resections was large , the number of cases with rf was relative small and the data may have diminished accuracy especially in subcategories and with some of the laboratory values . in addition , nsqip is able to define a preoperative creatinine but can not tell us if creatinine is due to a chronic kidney disease or if this is an acute or relatively recent renal dysfunction .
one might assume that a patient with acute progressive renal failure would not be subjected to a major pancreatic resection , but unfortunately this study can not provide us with the exact information .
the grouping of the procedures by nsqip based on procedural codes was quite nonspecific and there was likely variability in coding by the staff .
it is likely that partial removal of pancreas may have included both distal pancreatic resections and pancreaticoduodenectomies with varying extents of each procedure .
a major limitation of the study is that nsqip database did not report the exact pathology of the resected specimen or the details of the operative procedure .
it is unclear if some of these cases represented benign or premalignant lesions such as cystic tumors or neuroendocrine tumors which may have significantly less risk and better short , and long - term outcome . finally ,
although nsqip can identify general complications , it is not a database that can delineate procedure - specific complications so important complications such as pancreatic leaks and fistulas could not be assessed .
in conclusion , this study establishes a significant negative effect of renal failure on outcome in pancreatic resections
. it will be difficult to acquire larger cohorts of patients with this particular problem and nsqip is really not able to assess risk based on specific diagnoses requiring pancreatic resection .
each rf patient should be assessed for overall risk and especially in the context of expected long - term survival based on the specific pancreatic diagnosis .
those patients who opt for pancreatic resection should be optimized in terms of bleeding parameters and should be monitored carefully for respiratory , bleeding , and septic complications .
strong consideration should be made for nonoperative therapies or no treatment in those patients who have prohibitive risk or limited long - term survival .
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background .
chronic kidney disease affects 20 million us patients , with nearly 600,000 on dialysis .
long - term survival is limited and the risk of complex pancreatic surgery in this group is questionable .
previous studies are limited to case reports and small case series and a large database may help determine the true risk of pancreatic surgery in this population .
methods .
the american college of surgeons national surgical quality improvement program database was queried ( 20052011 ) for patients who underwent pancreatic resection .
renal failure was defined as the clinical condition associated with rapid , steadily increasing azotemia ( rise in bun ) and increasing creatinine above 3 mg / dl .
operative trends and short - term outcomes were reviewed for those with and without renal failure ( rf ) .
results . in 18,533 patients ,
28 had rf .
there was no difference in wound infections , neurologic or cardiovascular complications .
compared to non - rf patients , those with rf had more unplanned intubation ( or 4.89 , 95% ci 1.8512.89 ) , bleeding requiring transfusion ( or 3.12 , 95% ci 1.3714.21 ) , septic shock ( or 8.86 , 95% ci 3.7520.91 ) , higher 30-day mortality ( 21.4% versus 2.3% , p < 0.001 ) and longer hospital stay ( 23 versus 12 days , p
< 0.001 ) . conclusions .
rf patients have much higher morbidity and mortality after pancreatic resections and surgeons should consider this before proceeding .
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since 1930 , investigators began to investigate the distant changes occur away from the original site(s ) of inflammatory process .
the discovery of c - reactive protein ( crp ) by tillett and francis opened this door wide .
these distant systemic changes have been referred to as acute phase response regardless whether they follow an acute or a chronic inflammatory process .
acute phase responses are divided according to the concentrations of many plasma proteins known as acute phase proteins .
acute phase proteins can be defined as those whose plasma protein concentration increase with inflammatory reaction ( positive acute phase proteins ) . however , negative acute phase proteins are those whose plasma protein concentrations decrease with inflammatory reaction .
interleukin- ( il- ) 6 is the major stimulator of the production of most acute - phase proteins .
cardiovascular diseases are accompanied by the elevation of several positive acute phase reactants such as crp , serum amyloid a ( saa ) , fibrinogen , white blood cell count , secretory nonpancreatic phospholipase 2-ii ( spla2-ii ) , ferritin , and ceruloplasmin .
cardiovascular diseases are also accompanied by the reduction of negative acute phase reactants such as albumin , transferrin , transthyretin , retinol - binding protein , antithrombin , and transcortin .
the concentration of acute phase reactants in plasma varies according to the severity of the cardiovascular disorder and also due to the differences of pattern of production of the individual protein .
this explains why an individual biomarker such as crp elevates in plasma while another under the same category ( such as saa ) does not .
in 1930 , tillett and francis described an acute phase reactant in the serum of patients with pneumonia that they called crp because of its precipitation with pneumococcal c - polysaccharide .
the gene for crp has been localized to chromosome 1 and codes for a mature , 206 amino acid polypeptide .
crp is a member of the pentraxin family of proteins and comprises five identical polypeptide chains in a pentameric structure .
crp has a half - life of approximately 19 hours and its plasma levels are determined predominantly by its rate of synthesis in the liver , although crp is also produced by other cells such as adipocytes .
crp is an acute phase protein [ 7 , 8 ] produced in the liver in response to interleukin- ( il- ) 6 which is stimulated , in turn , by tumour necrosis factor- ( tnf- ) and il-1 [ 8 , 9 ] .
recent studies suggest that crp plays a pivotal role in many aspects of atherogenesis including ldl uptake by macrophage , release of proinflammatory cytokines , expression of monocyte chemotactic protein-1 , intercellular adhesion molecule-1 , and vascular cellular adhesion molecule-1 [ 1012 ] .
activation of inflammation and the acute phase reaction appear to play an important role , not only in the pathogenesis of atherosclerosis , but also in the initiation of the acute coronary syndrome ( acs ) [ 13 , 14 ] .
cesari et al . suggested that the inflammatory markers crp , il-6 , and tnf- are independent predictors of cardiovascular events in older persons .
although crp is a nonspecific inflammatory marker which may not be a diagnostic marker for cardiac injury but it is a strong independent predictor for cardiovascular risk and events .
epidemiological studies and clinical trials have found that crp is a strong independent predictor of future cardiovascular risk .
several large epidemiological studies have suggested that crp measurement predicts the risk of future cv events [ 1619 ] although other investigators failed to identify crp as a significant independent risk factor [ 20 , 21 ] .
crp is also an early ischemic marker and elevated crp is predictive of future adverse events [ 22 , 23 ] .
high - sensitivity crp ( hs - crp ) rises acutely after tissue injury , including myocardial infarction ( mi ) .
intense cytokine production and inflammatory cell infiltration occur in the area of ischemia and necrosis .
this increase of hs - crp levels , in part , correlates with infarct size [ 24 , 25 ] and with a higher risk of cardiac rupture [ 26 , 27 ] . in short - term studies of patients with acs
, high crp concentrations have been shown to be predictive of death , but not recurrent ami [ 28 , 29 ] .
there are few studies assessing the long - term risks of an elevated crp in the acs population [ 3034 ] .
crp levels are influenced by additional factors such as aging , gender , ethnicity , obesity , diabetes , estrogen use , smoking , hypertension , and autoimmune diseases .
serum levels of crp can increase 1000-fold in febrile illnesses , various inflammatory states , and trauma .
ockene et al . indicated that crp is generally expressed at low levels ( < 1
mg / l ) in healthy adults and levels remain relatively stable in the absence of an acute inflammatory stimulus .
patients with unstable angina and crp > 3 mg / l at discharge are more likely to be readmitted for recurrent cardiovascular instability or mi within 1 year
. under acute conditions , concentrations of c - reactive protein increased during the first 6 to 8 hours and can reach peak levels approaching 300 mg / l after approximately 48 hours .
indicated that hs - crp measurement is the strongest correlative factor for future clinical events due to arterial inflammation , myocardial infarction , unstable angina , stroke , and peripheral vascular disease in both diseased and apparently healthy asymptomatic patients .
the crp plasma level also is the best risk assessment in patients with either stable or unstable angina , long term after myocardial infarction , and in patients undergoing revascularization therapies .
one study showed the only independent cardiovascular risk indicators using multivariate , age adjusted and traditional risk analysis were crp and total / hdl cholesterol ratio .
if crp , il-6 , and icam-1 levels are added to lipid levels , risk assessment can be improved over lipids alone .
a growing body of studies suggest that elevation of hs - crp levels predicts a poor cardiovascular prognosis .
the extent of the inflammatory response to injury appears to have prognostic significance , which is independent of the extent of myocardial injury .
hs - crp response after mi has been shown to predict future chd morbidity and mortality independent of infarct size .
, it adds a prognostic information on vascular risk at all levels of the metabolic syndrome . as a protein
it was shown that reference intervals for hs - crp determined by using fresh serum samples were identical to those obtained by using samples , from a similar population , that were stored for 4.5 yr . when crp was used primarily for measuring states of extremely active inflammation , such as sepsis or arthritis , values of 50100 mg /
, crp was measured using qualitative or semiquantitative laboratory technique , most commonly latex agglutination , which precluded its use as differential diagnostic test because any degree of inflammation produced positive results .
although most of the currently crp is measured by high sensitive methodologies ( hs - crp ) such as immunoturbidimetry and immunonephelometry , preanalytic and analytic variations still exist .
these methods still do not have the sensitivity needed for the use of crp in the assessment of future coronary risk .
the most recent hs - crp assays utilize either antibodies that are labelled with an enzyme ( elisa ) or a fluorescent compound or polystyrene beads - coated antibodies to achieve the desired sensitivity .
continuation in improvement of its sensitivity , precision , accuracy , and standardization is critical .
the interpretation , defining cut - off values , and integration into screening panel are essentials in guidance of clinical practice .
the american heart association and the centers for disease control and prevention ( aha / cdc ) issued guidelines for the utility of this marker in the primary prevention setting and in patients with acs .
the guideline recommended from the laboratory aspects the cut - off values for risk assessment : hs - crp concentration < 1 mg / l are considered low risk , 13 mg / l intermediate risk , and > 3 mg / l are in high risk .
saa is an acute phase reactant protein and is secreted by macrophages , vascular smooth muscle cells , and endothelial cells [ 49 , 50 ] .
saa is also expressed in adipose tissue and there is an emerging body of research investigating its role in adipose inflammation and insulin resistance and diabetes .
thus , the association of saa with cvds may be either direct from effects of saa on atherosclerosis , or indirect via effects of saa on clinical conditions known to confer increased risk for cvds .
saa is synthesized primarily in liver in response to stimulation by cytokines such as tnf - a , il-1 , and il-6 .
the saa family comprises acute - phase isoforms ( saa-1 and saa-2 ) and a constitutive isoform ( saa-4 ) .
saa-3 is an isoform that shares approximately 60% homology with the acute - phase isoforms but it is not expressed in humans and it does not appear to be a component of the acute - phase response .
saa promotes the chemotaxis for monocytes and neutrophils , stimulation of the production of other proinflammatory cytokines such as il-1b and tnf - a , and induction of the matrix metalloproteinases ( mmps ) [ 52 , 53 ] .
it was also confirmed that saa promotes thrombosis by increasing tissue factor [ 54 , 55 ] .
rct is well known to be promoted by hdl which plays a pivotal role in lipid metabolism .
( 1 ) saa is chemotactic for neutrophils and monocytes . ( 2 ) saa - carrying ldl may have greater proteoglycan - binding affinity leading to increased retention . ( 3 ) saa stimulates the synthesis of biglycan and increases its binding affinity for ldl .
( 4 ) saa can stimulate the production of other proinflammatory cytokines , exacerbating the vascular inflammation .
( 5 ) saa can induce matrix metalloproteinases ( mmps ) which can lead to the destabilization of the developing atherosclerotic plaque .
these proteins usually respond in parallel to a given stimulus ; however , the magnitude of the saa response has been found to be greater than that of crp [ 5658 ] .
some studies have shown that saa levels increase with higher degrees of inflammation , even in some noncardiovascular inflammatory conditions , whereas crp levels remain normal [ 59 , 60 ] .
thus , saa is considered more sensitive and useful than crp as a marker of acute inflammatory response [ 59 , 60 ] .
peak levels of saa were increased up to 1000-fold above the normal and those of crp about 100-fold of the normal . an excellent correlation between kidney allograft rejection and saa reaction
was found in this study and monitoring of saa concentrations in patients with kidney allograft was recommended as a valuable aid in the early diagnosis and prediction of acute allograft rejection .
indicated that patients with elevated saa levels had higher rates of adverse events ( death , myocardial infarction , or urgent target - vessel revascularization ) at 30 days , irrespective of whether crp was elevated .
in contrast , elevated crp levels with normal saa were not associated with adverse outcomes at 30 days .
the overall conclusion is that saa is more sensitive than crp in responding to cardiovascular and noncardiovascular events .
a study has been conducted by katayama et al . demonstrated a significant association between the saa level in the acute phase of ami and clinical prognosis .
this study also confirmed a significant positive correlation between the levels of saa in acute phase ami and the levels of hs - crp and peak ck .
have also demonstrated that elevation of crp and saa at the time of hospital admission predicts a poor outcome in patients with unstable angina .
have described the measurement of saa by latex - enhanced nephelometric immunoassay on a hitachi 7600 autoanalyzer ( hitachi , tokyo , japan ) .
have established a quantitative determination of saa by micro - elisa method with sensitivity of 0.1 mg/1 and precision expressed with cvs between 1.6 and 4.2% .
the association between the high concentration of fibrinogen and risk of cardiovascular disease is well established .
the relation was first reported in preliminary results from the northwick park heart study in 1980 .
fibrinogen is a symmetrical glycoprotein composed of six polypeptide chains of three types : two a , two b , and two .
the total molecular weight of high molecular weight ( hmw ) fibrinogen is 340,000 daltons .
first , it binds specifically to activated platelets via glycoprotein iib / iiia , contributing to platelet aggregation .
finally , it is an acute - phase reactant that is increased in inflammatory states .
occlusive thrombi are found in most cases of acute myocardial infarction ( mi ) , sudden cardiac ischemic death , and unstable angina pectoris [ 69 , 70 ] .
thrombosis is recognized as the central mechanism of these atherosclerotic complications [ 68 , 69 ] .
fogari et al . found that fibrinogen levels increased with the number of cigarettes smoked .
fibrinogen levels also quickly fall after smoking cessation , suggesting that this rapid fall in level may be a mechanism for the reduction in cardiovascular risk after smoking cessation .
association between the elevation in fibrinogen level and obesity increases the risk of cardiovascular disease .
a reduction in body mass index after a low - calorie diet for 6 months has been accompanied by a fall in fibrinogen level . the prospective cardiovascular munster ( procam ) study found that individuals who had ldl and fibrinogen levels in the highest tertile had a 6.1-fold increase in coronary risk compared with those in the lowest tertile .
the event rate was significantly lower when fibrinogen levels were in the lowest tertile even though ldl remained in the highest tertile .
association between diabetes and increased plasma fibrinogen levels was found to cause platelet hyperreactivity as fibrinogen is acting as a cross bridge between platelets .
this might explain why poor diabetic control is particularly associated with higher levels of fibrinogen .
gil et al . have determined that increased plasma levels of fibrinogen as an acute phase reactant marker and associated with unfavorable outcome of acute coronary syndrome ( acs ) .
shi et al . have demonstrated that elevated level of plasma fibrinogen is associated with a worse long - term prognosis in patients who have acs .
there are four main methods used to measure fibrinogen concentration : clotting rate assays ; clottable protein assays ; heat-/salt - precipitation and immunological assays .
the results obtained can be affected by the assays used , the fibrinogen heterogeneities described above , and other plasma constituents .
it was well established that elevated wbc count is associated with systemic bacterial infections and during inflammatory processes .
rana et al . indicated that coronary heart disease ( chd ) is associated with elevated total leukocyte count .
a clear and positive correlation between the leukocyte count and risk of chd has been established in several prospective studies conducted in chd - free subjects [ 7983 ] .
this correlation appears to persist even after adjustment for other risk factors [ 8486 ] . as smoking is a chd risk factor ,
madjid et al . confirmed elevated wbc count as an independent risk factor for chd . a correlation between baseline leukocyte count and the incidence of mi has been also described .
wbc count has been also counted as a prognostic indicator in subjects with stable chd after a previous mi within 3 or 6 months [ 88 , 89 ] .
the two conditions were associated with elevated total leukocyte count with an increased risk of re - infarction or death . in patients with acute mi ,
leukocytosis was an independent predictor of acute mi . in untreated patients with hypertension , elevated leukocyte count
was associated with subsequent cardiovascular morbidity independent of blood pressure levels , smoking , diabetes , lipid levels , and established markers of target organ damage including electrocardiographic left ventricular hypertrophy and glomerular filtration rate .
several cross - sectional studies have shown a positive relationship between systolic blood pressure ( sbp ) and inflammatory markers including crp and wbc count .
they demonstrated that increased sbp even within the normotensive range is associated with increased wbc count .
they indicated that this association persisted after adjustment for age , gender , smoking status , bmi , physical activity level , cholesterol / hdl - c ratio , and cholesterol - lowering medications .
an elevated wbc count may also be a marker for a state characterized by increased catecholamine levels or sympathetic nervous system activity which can raise blood pressure and may eventually result in sustained hypertension .
considering differential leukocyte cell count , a correlation was found between moderately elevated eosinophil count and increased risk of disease , as well as between neutrophil , eosinophil , and monocyte ( but not lymphocyte ) counts and the incidence of disease . in a case - control study ,
eosinophil counts were significantly higher in those who had severe vasospastic angina pectoris than in those who had mild vasospastic angina pectoris , stable angina , or no angina .
after treatment with antianginal drugs , however , the eosinophil counts decreased to control levels .
it was also reported by biasucci et al . that the number of neutrophils was much higher in the patients with acute mi than in those with ua or stable angina .
it was observed by rana et al . that granulocyte count is consistently associated with increased risk for chd .
rana et al . also revealed that there is no association of monocyte count or lymphocyte count and risk of chd and cvd .
granulocytes can release a variety of mediators of tissue injury that cause neutrophil stimulation giving rise to additional products with enhanced endothelial injury .
neutrophils can directly cause damage to coronary vascular endothelium by adherence - dependent mechanisms involving the early adhesion molecule p - selectin .
ros may influence vascular tone either indirectly by inactivating endothelium - derived relaxing factor and reducing the release of prostacyclin or directly by promoting smooth muscle cell contraction .
this may result in the loss of vasodilator , antithrombotic , and antiatherogenic properties of the vascular endothelium .
intense neutrophil activation in unstable angina or ami , as manifested by morphologic changes in neutrophils and elastase release , may relate to ongoing in vivo cellular activation .
another study has demonstrated that increased neutrophil platelet adhesion may contribute to neutrophil activation in unstable angina . on the other hand , schillaci et al . suggested a role for monocytes in atherogenesis and vascular thrombosis ( through an interaction with platelets ) by giving rise to foamy macrophages and ros .
one retrospective study of patients with coronary artery disease ( cad ) showed that five - year survival was significantly better for patients who had a normal as compared with a low relative lymphocyte count ( 92% versus 83% ) .
spla2-ii is a positive acute phase reactant with properties to catalyze the production of lipid mediators leading to impaired vasodilator function in patients with documented cad .
pla2 are ubiquitous enzymes that hydrolyse the sn-2-acyl bond of cell membrane phospholipids and lipoproteins and yield free fatty acids and lysophospholipids , precursors of various proinflammatory lipid mediators , including leukotrienes , prostaglandins , and platelet - activating factor [ 106 , 107 ] .
pla2 exist in many isoforms including the 14 kda secretory phospholipase a ii ( spla2 ) and the 85 kda cytosolic phospholipase a ii ( cpla2 ) .
spla2-i is described as pancreatic pla2 since it is expressed in high amounts in pancreas .
proinflammatory cytokines lead to an increase in expression and secretion of spla2-ii from different organs and tissues , including atherosclerotic plaques where spla2 was found to be highly expressed [ 109 , 110 ] .
extracellular spla2-ii is mainly localized at sites where it hydrolyses phospholipids from lipoproteins and lipid aggregates retained in the extracellular matrix of the arterial wall .
this may be a potential mechanism for in situ release of proinflammatory lipids , free fatty acids and lysophosphatidylcholine in regions of apo - lipoprotein b accumulation , which are abundant in atherosclerotic lesions .
additionally it was documented that spla2 induces the expression of chemokines and adhesion molecules in microvascular endothelium .
have demonstrated that elevated serum level of spla2-ii is an independent risk factor for cad .
they also indicated that the increase in spla2-ii serum levels is a predictor of developing clinical coronary events in patients with cad .
fichtlscherer et al . have referred this association to the fact that elevated serum spla2-ii causes impairment in systemic endothelial vasodilator function .
they also confirmed that spla2-ii and crp are sharing this function in terms of being independent predictors of vascular response to acetylcholine .
they concluded that both spla2-ii and crp can serve as mediators of endothelial dysfunction [ 106 , 114 ] .
the correlation with crp is much stronger than that with wbc count . in a large cohort study over 4 years on patients with chd ,
a single measurement of spla2-ii mass and activity at baseline was associated with recurrent events even after controlling other risk factors that might increase risk for cvd .
spla2-ii mass and activity were strongly correlated but the association of mass with secondary cvd event was even stronger .
ly315920 ( [ [ 3- ( aminooxoacetyl)-2-ethyl-1-(phenylmethyl)-1h - indol-4-yl ] oxy ] acetate ) , a pharmacological drug that functions as a selective stoichiometric inhibitor of the catalytic activity of spla2-ii . ly315920 has been administered orally and intravenously in transgenic mice expressing human spla2-ii .
some other drugs have several biological and pharmacological properties including the inhibition of spla2-ii serum activity such as nsaids ( alminoprofen ( a member of the phenylpropionic acid class of drugs ) , aspirin [ 115 , 116 ] , heparin , and indomethacin ) .
chemically modified tetracyclines , devoid of antimicrobial properties also inhibit the activity of serum spla2-ii .
ferritin , which is the major iron storage protein , plays a key role in iron metabolism .
serum ferritin senses the body iron stores and serves as the early sensitive marker for iron deficiency .
serum ferritin differs markedly from tissue ferritin in molecular weight , iron and carbohydrate content , subunit size , amino acid sequence , and possibly is encoded by genes distinct from those of tissue ferritin [ 121 , 122 ] .
ferritin maintains up to 4500 atoms of hydrolyzed and polymerized atoms in a soluble form within a protein shell with a hollow interior of approximately 90 [ 123 , 124 ] .
ferritin binds iron in a catalytically inactive manner ; accordingly , oxidative reactions can not be promoted by iron bound to ferritin but by free iron .
serum ferritin also increases with increasing bmi , possibly a result of higher il-6 levels in heavier subjects .
serum ferritin is inversely proportional to aspirin intake , as persons with higher aspirin use have lower serum ferritin [ 121 , 129 ] providing the property of aspirin as an anti - inflammatory agent .
many solid conclusions come to the finding that serum ferritin is a positive acute phase reactant and is strongly associated with inflammatory processes including heart diseases and diabetes .
the association between serum levels of ferritin and cardiovascular disease is still controversial and requires further investigation . from a study on finnish subjects ( a total of 1931 unselected men ) without symptoms of chd during an average followup for 3 years , it was reported that 2.2 times greater levels of cardiovascular disease were observed in the group with high serum iron ( indicative of elevated serum ferritin ) compared to the group with low serum iron [ 130 , 131 ] .
this oxidized ldl - cholesterol - induced inflammation in blood vessels , including the progression of atherosclerosis .
this role is thought to be due to prooxidant properties . in patients with serum ferritin concentrations
> 200 ng / ml , the risk of myocardial infarction was 2.2 times greater than the patients with serum ferritin levels < 200 ng / ml .
this indicated that serum ferritin indirectly enhances the role of ldl - cholesterol in the induction of cardiovascular diseases .
this role is further enhanced by the elevation of hscrp accordingly . in a prospective study performed in a french population ,
however , galan et al . failed to find a positive association between serum ferritin and ischemic heart disease .
these findings matched well with the suggestions of sempos et al . , 5 years earlier , as the results from the two studies did not support the hypothesis that positive body iron stores , as measured by serum ferritin , are associated with an increased risk of cvd , chd , or mi death .
dominguez - rodriguez et al . suggested even more extreme finding that major adverse cardiovascular events is associated with lower serum ferritin levels in a study on a total of 196 and 30 days followed - up patients with a first non - st elevation acs .
their observation was supported by an in vitro study that iron deficiency enhances atheroma inflammation through p38 mitogen activated protein kinase - nuclear factor-b - extracellular matrix metalloproteinase inducer / matrix metalloproteinase-9 pathway .
hp is an abundant plasma protein which binds with high affinity to hemoglobin [ 137 , 138 ] .
the binding of hp to hemoglobin can detoxify hemoglobin released into circulation during in vivo hemolysis .
the binding of hp to hemoglobin serves to decrease the ability of iron derived from hemoglobin from carrying out oxidative reactions providing that the heme iron in hemoglobin is a very potent oxidant . in other words ,
hp function is binding and scavenging of free hemoglobin through the liver or circulating monocytes [ 140 , 141 ] . in man ,
the protein product of the hp 2 allele is defective in its ability to block oxidative reactions mediated by iron - derived hemoglobin .
the hp 1-hb complex is relatively redox inert , while the hp 2-hb complex contains non - transferrin bound redox - active iron .
found that hp binding to hb is sufficient to prevent the generation of oxidant species from cell - free hb that would otherwise mediate hypertension and other adverse vascular outcomes .
interestingly , boretti et al . also showed that hp - bound hb has a very high oxygen affinity and the hp - hb complexes stimulate the production of the endogenous antioxidant no from nitrite [ 143 , 144 ] .
in addition , the heme - binding protein hemopexin has evolved to mop up the toxic porphyrin heme ring released from decomposing cell free hb , and heme - metabolizing enzymes , such as heme oxygenase-1 , provide a functional antioxidant effect that is protective to vascular health .
finally , plasma transferrin protein sequesters and safely transports elemental iron released from the heme ring , one of the most oxidative substances in the human body .
it was determined in many multiple independent longitudinal studies that the hp genotype is an independent determinant of the risk of incident cardiovascular disease in individuals with diabetes mellitus ( dm ) [ 146151 ] .
these studies have shown that dm individuals with the hp 2 - 2 genotype have a 25-fold increased risk of mi , stroke , and cardiovascular death as compared to hp 1 - 1 or hp 2 - 1 individuals .
it was also demonstrated that hp phenotype is predictive of development of microvascular complications in dm .
they found that patients who are homozygous for the hp 1 allele are at decreased risk for developing retinopathy and nephropathy .
this effect , at least for nephropathy , has been observed in both type 1 and type 2 dm .
furthermore , the hp phenotype may be predictive of development of macrovascular complications in dm .
hp can also bind to hdl ( most likely via an interaction with helix 6 of apoa1 ) and thereby serve to tether hemoglobin to hdl . in hp 2 - 2
dm individuals , there is impairment in the clearance of hp - hemoglobin due to a decreased uptake of the complex by the cd163 hp - hemoglobin scavenger receptor present on monocytes and kupfer cells .
hdl in these hp 2 - 2 dm individuals is dysfunctional in terms of its ability to promote reverse cholesterol transport which is believed to be the primary function of hdl .
it was also demonstrated that development of restenosis after percutaneous coronary angioplasty is significantly decreased in dm patients with the 1 - 1 hp phenotype . on contrary ,
found that hp 1 - 1 individuals are at elevated risk for chd mortality in a 10-year followup study .
they postulated that hp 2 - 2 might play a role in early atherogenesis , but hp 1 - 1 has a predominant influence in more advanced and life - threatening stages of the disease .
they also concluded that hp 1 - 1 is of greater prognostic importance for chd death than classical risk factors , such as smoking , obesity , and diabetes .
cp is a 132-kda monomer composed entirely of three 4245-kda domains with high amino acid sequence homology ( about 40% ) to each other .
cp is an abundant plasma protein that contains seven copper atoms per molecule and accounts for 95% of the total circulating copper in healthy adults [ 157 , 158 ] .
the physiological function activities of cp include copper transport , coagulation , angiogenesis , defense against oxidant stress , and iron homeostasis .
the ferroxidase activity of cp catalyzes oxidation of fe to fe and is thought to facilitate in vitro loading of iron into the iron transport and storage proteins transferrin and ferritin .
tang et al . measured serum cp levels in 3,569 consecutive stable patients undergoing elective coronary angiography with available leukocyte counts and hs - crp levels .
the role of cp in predicting incident major adverse cardiac events over 3 years of followup was evaluated . a strong positive correlation with hscrp was observed .
cp level ( quartiles 4 versus 1 ) was associated with almost 3-fold increase in risk of future major adverse cardiovascular events at 1 year . after adjusting for traditional risk factors including hscrp , and total leukocyte count , cp remained independently predictive of major adverse cardiovascular events at 1 year ( hr 2.51 , 95% ci 1.404.49 , p = 0.001 ) and at 3 years ( hr 1.48 , 95% ci 1.011.26 ) .
adelstein et al . and bustamante et al . observed substantial hypercupremia after mi [ 161 , 162 ] . since that report
, high serum cp levels were found in patients with multiple cardiovascular disorders including arteriosclerosis , abdominal aortic aneurysm , unstable angina , and vasculitis and peripheral arterial disease .
prospective studies also showed that serum cp may be an independent risk factor for cardiovascular disease .
a prospective finnish study verified that serum copper concentration was an independent risk factor for ischemic heart disease .
the association between cp and cardiovascular events is well explained by describing a role for cp in ldl oxidation by vascular cells ( free radical - mediated , transition metal - dependent mechanism ) . a model for ldl oxidation by cp in an atherosclerotic lesion
was described that shows that cp is synthesized by macrophages which are activated by tnf - a ( proinflammatory cytokine ) or by bacterial lipopolysaccharide ( cell wall product of infectious organism ) .
this secreted cp along with redox - active copper , are required for vascular cell - mediated ldl oxidation .
prognostic value of serum cp has been compared against that of crp and fibrinogen in 40 patients of unstable angina over 12 months period .
all acute phase proteins were measured in the total study group and in the subgroup with normal troponin t .
the levels of serum cp were found elevated when compared against those in stable angina patients .
serum cp increased significantly at 72 hours and reach peak levels at 7 days , both in the total study group and in the subgroup with normal troponin t . on the other hand , crp and fibrinogen serum levels elevated way earlier than those of cp and last to a way shorter period .
however , cp but not crp and fibrinogen levels showed a prognostic value because they were significantly higher in patients with complications in both groups , and logistic regression analysis showed that cp levels at 72 hours were the most important factor related to 12-month prognosis .
the role of serum copper in a 2-year progression of atherosclerotic carotid lesions ( measured by ultrasonography ) in eastern finnish men was examined by salonen et al . .
an accelerated progression of atherosclerosis was seen only in patients with both high serum ldl and high serum copper .
they thought that the prooxidant activity of the copper may be due to a synergism of copper and ldl .
mezzetti et al . also examined the importance of serum cp and ldl oxidation in lesion development in patients undergoing endarterectomy for internal carotid stenosis .
they reported that serum cp and ldl lipid peroxide levels measured 24 or 72 hours after surgery were highly correlated with the percentage renarrowing measured after 12 months .
taking together , these studies suggest that the contribution of cp to the risk of cvd is not independent but rather depends on the lipoprotein profile and possibly other factors .
lower levels of serum albumin within the normal range are associated with increased risk of all - cause and cardiovascular mortality , as well as with coronary heart disease ( chd ) [ 176 , 177 ] and stroke incidence .
these associations persist after adjustment for other known risk factors and preexisting disease and after exclusion of early mortality [ 175 , 177 ] .
serum albumin level accounts for about 5060% of the serum proteins with a range of 3550
g / l [ 179 , 180 ] . human serum albumin ( hsa ) , a protein of molecular weight 65 kda , consists of 585 amino acids and one free thiol ( cys 34 ) .
the disulphides are positioned in a repeating series of nine loop - link - loop structures centered around eight sequential cys - cys pairs to form heart - shaped .
plasma proteins have been suggested as the major extracellular antioxidants in extracellular fluid . among them , albumin , transferrin , and ceruloplasmin , can sequester transition metals , preventing them from acting as catalysts in highly reactive biochemical reactions .
several studies have suggested that albumin as a powerful oxidants scavenger in human plasma can inhibit hydroxyl radicals , peroxyl radicals , and hocl [ 179 , 181 ] .
substantial evidence support the significant inverse relation between serum albumin level and risk of coronary heart disease as well as cancer , other causes of death , and all - cause mortality [ 182184 ] .
hstmark reported that albumin as an extracellular antioxidant does have a cardioprotective role against lipid peroxidation . in patients with renal failure
, there is a pathophysiological link between cvd , malnutrition , inflammation and serum levels of crp [ 187189 ] .
additionally , a low level of serum albumin is also a strong predictor of morbidity and mortality in patients with kidney failure [ 190192 ] .
hypoalbuminemia of kidney failure , in part , may be a consequence of activation of the acute phase response and may represent a chronic inflammatory state [ 193195 ] .
( 1 ) cause - effect association : ( i ) albumin , atherogenic lipids and lipoproteins association .
a significant inverse relationship between serum albumin and lipoprotein ( a ) has been reported in patients with nephrotic syndrome and in dialysis patients [ 198 , 199 ] .
showed that an elevated rate of ldl - apo b production is highly correlated to the prevailing serum albumin levels in patients with nephrotic syndrome .
it was shown that by increasing serum albumin levels in continuous ambulatory peritoneal dialysis patients , serum lipoprotein ( a ) levels were decreased .
these findings have been confirmed these findings in humans and rats [ 202 , 203 ] .
fibrinogen levels correlate inversely with serum albumin levels in patients with nephrotic syndrome and in continuous ambulatory peritoneal dialysis patients .
. showed that addition of albumin abolished the free fatty - acid - associated increase in fibrinogen synthesis in mouse liver slices .
the intravenous infusion of albumin solution to seriously injured patients caused a significant fall in fibrinogen concentration .
there are several reports that platelet aggregation in patients with nephrotic syndrome and in patients on continuous ambulatory peritoneal dialysis , were inversely proportional to the serum albumin concentration [ 209211 ] .
several investigators reported that the addition of albumin in vivo or in vitro [ 210 , 213 , 214 ] corrected this defect .
showed that the addition of albumin to platelet - rich plasma from patients with nephrotic syndrome , or the intravenous infusion of albumin in quantities sufficient to correct hypoalbuminemia , also diminished the excessive production of prostaglandin metabolites by nephrotic platelets .
albumin is a lipooxygenase inhibitor in serum , which suggests that hypoalbuminemia increases synthesis of leukotrienes .
groth demonstrated that infusion of albumin or dextran 40 in 14 patients with various diseases decreased blood and plasma viscosity .
reported that albumin deficiency is accompanied by blood hyperviscosity , possibly by increasing red cell lysophosphatidylcholine .
continuous ambulatory peritoneal dialysis patients had higher whole - blood viscosity than control subjects when all samples were reconstituted to 70% hematocrit .
plasma viscosity was also higher in continuous ambulatory peritoneal dialysis patients than in hemodialysis patients and controls . in the study of gordge et al . , plasma viscosity correlated significantly with the degree of proteinuria in 21 diabetic patients with renal failure . ( 2 )
serum albumin was negatively correlated with c - reactive protein ( crp ) [ 222 , 223 ] .
however , infusion of albumin in patients with hypoalbuminemia did not have an effect on the level of crp .
these findings suggest that the correlation between albumin and crp may be secondary to inflammation rather than a direct effect of hypoalbuminemia .
as well , diabetes and cigarette smoking can cause hypoalbuminemia and atherosclerosis despite the fact that they are not major determinants of hypoalbuminemia in dialysis patients .
transthyretin ( ttr ) was formerly called prealbumin ( since it migrates closer to the anode compared to albumin on serum protein electrophoresis ) , but this term is misleading as ttr is not a precursor of albumin .
ttr is a globular , non - glycosylated protein with a molecular mass of 54.98 kda .
the total mass is approximately 76 kda with a complexed molecule of retinol - binding protein ( rbp ; 21 kda ) , which is still small enough to diffuse out of the vascular space as readily as albumin ( 66.3 kda ) or transferrin ( 79.6 kda ) ; slightly less than 50% of each of these proteins is normally intravascular as a result .
ttr is a negative acute phase protein synthesized primarily in the liver , except for tiny amounts produced by the choroid plexus and the retina [ 225 , 229 ] . in serum ,
the ttr protein circulates as a homotetramer , in which each monomer comprises 127 amino acids arranged as eight antiparallel pleated sheet domains .
the ttr gene is located on chromosome 18 and contains four exons . both wild type and mutated
ttr can form amyloid , which suggests that this highly structured protein is innately prone to form -pleated sheet fibrils [ 232 , 233 ] .
alterations in the primary structure of the ttr protein ( owing to ttr mutations ) can result in greatly accelerated amyloid formation and are the origin of all symptomatic cases of hereditary transthyretin - related amyloidosis ( attr ) which can consequently cause organ dysfunction .
ttr is associated with two distinct forms of cardiac amyloidoses : hereditary transthyretin - related amyloidosis ( attr ) which is caused by mutations in the ttr gene , which encodes ttr [ 225 , 235 ] , whereas systemic senile amyloidosis ( ssa ) is not inherited and is associated with wild type ttr . in patients with attr
, amyloid can infiltrate any or all of the cardiovascular structures , including the conduction system , the atrial and ventricular myocardium , valvular tissue , and the coronary and large arteries [ 236 , 237 ] .
branch bundle block leading to atrioventricular and sinoatrial block are the fate of the conduction system compromise .
myocardial infiltration results in a progressive increase in the thickness of the left and right ventricular walls and of the interatrial septum and is associated with worsening hemodynamic impairment . in a study of 38 patients with attr - related cardiomyopathy ,
only one patient ( 3% ) showed a dip plateau in the right ventricular pressure curve profile ; 13 patients ( 34% ) showed raised pulmonary capillary wedge pressures , and 14 ( 47% ) showed a doppler restrictive filling pattern .
interestingly , 11 ( 29% ) of the patients did not display any abnormalities in diastolic function at least when at rest .
amyloid infiltration of cardiac valves leads to the formation of nodules or diffuse thickening of the leaflets , accompanied by variable degrees ( generally mild ) of valvular regurgitation .
the clinical spectrum of cardiovascular involvement is wide , ranging from asymptomatic atrioventricular and bundle branch block to severe , rapidly progressive heart failure owing to restrictive cardiomyopathy .
diagnosis of systemic amyloidosis is based on monoclonal gammopathy by immunoelectrophoresis , immunofixation on serum and urine , or mutant transthyretin gene as potential risk factors for amyloid disease , and confirmed by positive congo red staining of any biopsy ( periumbilical fat aspiration , rectum , target organ ) .
fibril disruptersrecently , 4-deoxy-4-iododoxorubicin ( idox ) has been proved useful as a tool for disrupting ttr amyloid fibrils .
the amyloid disrupters might also prove useful for amyloidoses associated with other protein precursors , such as the l protein in alzheimer and the prion scrapie protein .
due to its cardiotoxicity , idox analogues able to disrupt amyloid fibrils are needed .
recently , 4-deoxy-4-iododoxorubicin ( idox ) has been proved useful as a tool for disrupting ttr amyloid fibrils .
the amyloid disrupters might also prove useful for amyloidoses associated with other protein precursors , such as the l protein in alzheimer and the prion scrapie protein .
due to its cardiotoxicity , idox analogues able to disrupt amyloid fibrils are needed .
ttr - diclofenac complex revealed that the ring with the acetate function is positioned at the innermost part of the binding site , allowing strong hydrogen bond interaction between the drug cooh group and the side chain of thr119 . in this case the two phenyl rings and the chlorine atoms provide extensive van der waals interactions with the protein residues that form the p1 and p3 pockets .
diflunisal , which is another nonsteroidal nsaid , has been found to stabilize the tetrameric structure of ttr , that is , reduces tetramer dissociation and subsequent monomer misfolding and aggregation into amyloid [ 236 , 242 ] .
ttr - diclofenac complex revealed that the ring with the acetate function is positioned at the innermost part of the binding site , allowing strong hydrogen bond interaction between the drug cooh group and the side chain of thr119 . in this case the two phenyl rings and the chlorine atoms provide extensive van der waals interactions with the protein residues that form the p1 and p3 pockets .
diflunisal , which is another nonsteroidal nsaid , has been found to stabilize the tetrameric structure of ttr , that is , reduces tetramer dissociation and subsequent monomer misfolding and aggregation into amyloid [ 236 , 242 ] .
the methods most commonly used at present are immunonephelometry ( in ) and immunoturbidimetry ( it ) .
of particular importance is body position ; individuals who have been standing or recumbent for long periods of time have higher or lower concentrations , respectively .
it is generally recommended that blood specimens for assay of plasma proteins be drawn after approximately 1520 min in the sitting position if possible .
the transferrin family ( trf ) constitutes the major iron transport and/or scavenging system in vertebrates and some invertebrates .
however , trf can also function as an iron chelator , which contributes to host defense by limiting iron availability for microbial pathogens .
trf is a soluble glycoprotein and a bilobal molecule , that is , it contains an n - terminal ( amino acids 1336 ) and a c- terminal ( amino acids 337679 ) globular domain .
each domain contains a metal - binding site and each lobe binds one iron atom .
amino acid sequences of trf from several species , including human , bovine , rabbit , and chicken , show a high degree of sequence similarity .
under normal conditions , these two high - affinity - iron binding sites prevent the existence of measurable amounts of unbound iron in the plasma .
the main source of iron for trf is catabolism of nonviable red blood cells and its main destination is the erythroid marrow .
serum trf transport iron from neutral biological fluids to the cytoplasm by receptor - mediated endocytosis .
free iron is capable of stimulating the production of free radicals which cause oxidative damage such as lipid peroxidation .
trf is one of the most important antioxidants and acts by sequestration of iron in a redox - inactive form .
apotransferrin , at physiological concentrations ( 2 - 3 mg / ml ) is known to inhibit lipid peroxidation in the liposome model by iron - binding .
however , at higher concentrations , apotransferrin does not inhibit lipid peroxidation any further , while at low concentrations , apotransferrin reduces antioxidant capacity in a concentration - dependent manner .
this finding was clinically relevant in diseases that are associated with lower plasma transferrin concentrations as a result of either a decreased synthesis or increased breakdown such as type 1 and type 2 diabetes mellitus .
it has been shown that oxidative damage to transferrin by neutrophil - derived superoxide increases ferrous iron release . in vitro
purified hemoglobin from diabetic patients compared to normal individuals contains more free iron , which increases progressively with the extent of the disease .
as trf is a negative acute phase protein , it is downregulated in inflammatory diseases such as diabetes . moreover , oxidative damage results in protein fragmentation and nephropathy which can lead to loss of transferrin in the urine of type 1 and type 2 diabetic patients .
interestingly , microtransferrinuria seems to be a more sensitive index for renal dysfunction than microalbuminuria [ 248 , 251255 ] .
the low concentration of transferrin often found in the disease also appears to reflect the increased iron stores , and the concentration returns to normal after iron - depletion therapy .
demonstrated that venesection therapy causes the rise of plasma transferrin due to the removal of iron overload and not to venesection per se .
another possible defect in hemochromatosis is the decreased ability of reticuloendothelial cells to retain iron .
the result of this defect is a return of greater amounts of iron from stores to plasma ferritin , increasing its iron saturation and thus causing more iron saturation and directing more iron into hepatocyte storage .
batey et al . have confirmed the existence of a nontransferrin - bound iron ( ntbi ) fraction in the sera of patients with untreated or inadequately treated primary hemochromatosis which may , in part explain the findings in primary hemochromatosis .
the presence and intensity of inflammation of cardiovascular disorders can be reflected by acute phase changes .
acute phase responses have long been used as a clinical guide to diagnosis , management , and prognosis .
not only this , but also they can be used to predict a new - onset cardiovascular disorder .
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acute phase reaction is a systemic response which usually follows a physiological condition that takes place in the beginning of an inflammatory process .
this physiological change usually lasts 1 - 2 days .
however , the systemic acute phase response usually lasts longer .
the aim of this systemic response is to restore homeostasis .
these events are accompanied by upregulation of some proteins ( positive acute phase reactants ) and downregulation of others ( negative acute phase reactants ) during inflammatory reactions .
cardiovascular diseases are accompanied by the elevation of several positive acute phase reactants such as c - reactive protein ( crp ) , serum amyloid a ( saa ) , fibrinogen , white blood cell count , secretory nonpancreatic phospholipase 2-ii ( spla2-ii ) , ferritin , and ceruloplasmin .
cardiovascular disease is also accompanied by the reduction of negative acute phase reactants such as albumin , transferrin , transthyretin , retinol - binding protein , antithrombin , and transcortin . in this paper
, we will be discussing the biological activity and diagnostic and prognostic values of acute phase reactants with cardiovascular importance .
the potential therapeutic targets of these reactants will be also discussed .
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interleukin-6 ( il-6 ) is a multifunctional cytokine that is produced by many different types of cells , including monocytes , lymphocytes , fibroblasts , endothelial cells , keratinocytes , mesangial cells , and endometrial cells [ 1 , 2 ] .
inflammation plays a key role in the pathogenesis of atherosclerosis [ 35 ] and contributes to plaque instability and thrombosis .
some studies have shown that elevated serum c - reactive protein ( crp ) levels were associated with coronary heart disease ( chd ) events [ 711 ] .
crp is the major acute - phase reactant in humans , which is derived mainly from hepatocytes in response to il-6 .
in addition , some epidemiological investigations have shown a direct association between circulating il-6 levels and risk or severity of chd [ 1115 ] .
a considerable number of studies have been performed on the correlation between helicobacter pylori ( h. pylori ) infection and chd .
some studies have shown a positive association between them [ 1622 ] , while others have shown no significant association [ 2327 ] ; a hypothesis was proposed that h. pylori infection might play an important role in altering systemic inflammation levels that could be connected to chd . however , only one study has investigated the association between h. pylori antibody titers and il-6 levels , which reported a significant correlation between them in 107 healthy subjects . to further elucidate the relationship between h. pylori infection and serum il-6 levels , we conducted a cross - sectional study to examine the association between serum anti - h .
pylori immunoglobulin g ( igg ) levels and serum il-6 levels in japanese h. pylori - infected adults .
subjects were japanese adults who visited an urban clinic in nagoya , japan , between december 2005 and october 2010 , for the purpose of being tested for h. pylori infection and for subsequent eradication .
they were apparently healthy individuals who were concerned about possible h. pylori infection . during this period
, we enrolled 158 h. pylori - infected individuals ( 54 men and 104 women ) aged 2378 years .
subjects with gastric cancer or idiopathic thrombocytopenia or those not infected with h. pylori or who had an unknown infection ( n = 109 ) were not included , nor were those with serum il-6 > 4 pg / ml
( n = 12 ) or with no data of h. pylori antibody levels ( n = 9 ) .
all subjects gave written informed consent to take part in this study , which was approved by the ethics committee of nagoya university school of medicine ( approval number 155 ) .
serum il-6 levels and pepsinogen ( pg ) isozymes i and ii were measured using a cleia ( chemiluminescence enzyme immunoassay ) .
the reference range for serum il-6 was 4 pg / ml for both sexes .
serological atrophic gastritis was defined as a serum pg i level of 70 ng / ml and a pg i / ii ratio of 3.0 .
subjects were classified into 3 groups according to serological levels of pepsinogens : strong positive ( pg i level of 30 ng / ml and a pg i / ii ratio of 2.0 ) , moderate positive ( pg i level of 50 ng / ml and a pg i / ii ratio of 3.0 ) , and positive ( pg i level of 70 ng / ml and a pg i / ii ratio of 3.0 but neither moderate nor strong positive ) [ 2933 ] . the rest of the subjects were regarded as serological atrophic gastritis negative .
h. pylori infection was determined using the c - urea breath test , with positive results defined as a value 2.5 , which is the cutoff value recommended in japan .
pylori igg level was assessed using an eia ( enzyme immunoassay ) . since the log10-transformed values of serum il-6 levels and the log10-transformed values of serum h. pylori igg levels showed a near - normal distribution , they were used in the analysis .
age was stratified in 10-year increments . the strength of association between the logarithm of serum il-6 levels and the logarithm of serum anti - h .
multivariate regression analysis was performed to assess the correlation of log10-transformed serum il-6 with log10-transformed serum anti - h .
pylori igg levels with adjustment for sex and age ; logarithm of serum anti - h .
dummy variables indicating levels of atrophic gastritis were used in the regression analysis ( no atrophic gastritis , positive , moderate positive , and strong positive ) .
additional multivariate regression analysis was performed to assess the influence of atrophic gastritis on log10-transformed serum anti - h .
all statistical analyses were performed using stata version 11.1 software ( stata corporation , college station , tx , usa ) .
the mean age was 56.9 years ( 58.4 years in men and 56.2 years in women ) and most were aged 6069 years for both sexes .
the geometric mean of serum h. pylori igg titer was 51.3 and the serum h. pylori igg level ranged from 6 to 280 .
the geometric mean serum il-6 level was 1.78 pg / ml for men , 1.57 pg / ml for women , and 1.64 pg / ml overall ; the level ranged from 1.0 to 2.0 pg / ml among 53.7% of men and 58.7% of women .
the logarithm of serum il-6 levels was positively correlated with the logarithm of serum h. pylori igg levels in men ( r = 0.27 , p = 0.0045 ) , in women ( r = 0.23 , p = 0.021 ) , and in both sexes combined ( r = 0.24 , p = 0.002 ) . in multiple linear regression analysis , adjusting for sex and age , a higher log10-transformed serum il-6 level was still significantly associated with a higher log10-transformed serum anti - h .
pylori igg level in all subjects ( = 0.18 , p = 0.012 ) .
the association was similarly observed in both men and women , though it did not reach statistical significance in either sex ; = 0.20 and p = 0.11 for men and = 0.17 and p = 0.055 for women .
when a cut - off value of 5% for the c - urea breath test , which is used worldwide , was applied as an h. pylori - positive definition , the results were not modified in any of the subjects ( = 0.14 , p = 0.045 ) . when stratified by status of atrophic gastritis ,
the association was significant among those without serological atrophic gastritis ( = 0.26 , p = 0.011 ) and not significant among those with positive ( = 0.046 , p = 0.75 ) , moderate positive ( = 0.20 , p = 0.254 ) , or strong positive ( = 0.22 , p = 0.93 ) serological atrophic gastritis .
after adjustment for sex and age , atrophic gastritis was not significantly associated with the logarithm of serum h. pylori igg levels ( = 0.0011 , p = 0.99 for positive , = 0.074 , p = 0.27 for moderate positive , and = 0.12 , p = 0.15 for strong positive ) . even in the multiple linear regression analysis adjusting for sex , age , and atrophic gastritis , a higher log10-transformed serum il-6 level was significantly associated with a higher log10-transformed serum anti - h .
pylori igg level in all subjects ( = 0.17 , p = 0.020 ) .
the present study showed that h. pylori - positive japanese adults with a higher anti - h .
the association in this study is consistent with the result of a previous study that involved 107 caucasians . over the years
, a growing body of evidence has demonstrated that higher levels of inflammatory markers such as serum il-6 and crp were associated with an increased risk of developing chd .
one systematic review showed that circulating il-6 was associated with an increased risk of cardiovascular disease .
in two prospective studies , evaluating associations with long - term average circulating il-6 levels provided an odds ratio for chd risk of 2.14 .
recently , a prospective cohort study showed that a one - sd increment in log - transformed serum il-6 was positively associated with an increased risk of cardiovascular mortality , with a hazard ratio ( hr ) of 2.04 ( 95% ci , 1.343.68 ) . in the fragmin and
fast revascularization during instability in coronary artery disease ii trial , elevated il-6 ( > 5 ng / l ) was associated with a higher 6- and 12-month mortality , independent of troponin or high - sensitivity crp .
a large prospective case - cohort study showed that increased concentrations of crp in men and il-6 in women were strong and independent predictors of chd risk even after adjustment for traditional cardiovascular risk factors .
crp has also been shown to be induced in response to il-6 . a systematic review and meta - analysis showed that the summary estimate of relative risk for incident chd was 1.58 ( 95% ci , 1.371.83 ) for crp levels greater than 3.0
mg / l , compared with levels of less than 1.0 mg / l . although our subjects had serum il-6 levels 4 pg / ml , which is within standard values , many cohort studies have shown a significant association between il-6 levels and the risk of chd when il-6 levels were within standard values [ 11 , 13 ] .
a prospective case - cohort study showed an association between il-6 concentrations and the incidence of chd , even when il-6 levels were within standard values ( 3.1 pg / ml in cases and 2.0 pg / ml in noncases ; p < 0.001 ) .
similarly , the risk of chd by fifths of baseline circulating il-6 levels in a pooled analysis of participants without known chd at baseline in the reykjavik study and british regional heart study has increased continuously with increasing fifths of circulating il-6 levels , even when they are within standard values .
pylori igg levels in the current study could play an important role epidemiologically and clinically .
established risk factors , such as genetics , diet , and exercise , play a more important role than h. pylori .
however , epidemiological studies have shown an association between chd and h. pylori infection when these risk factors are taken into consideration . moreover
, a number of cohort studies have shown that il-6 level is a risk factor of chd , independent of these factors [ 11 , 13 ] .
although controversial , a large number of studies have been published on the possible role of h. pylori infection in cardiovascular disease .
a previous study showed that the prevalence of h. pylori infection was significantly higher in patients than in controls ( 62% versus 40% ; p = 0.004 ) , with an odds ratio of 2.8 ( 95% ci : 1.37.4 ; p < 0.001 ) adjusted for age , sex , main cardiovascular risk factors , and social class . a case - control study on 1122 patients with acute myocardial infraction and 1122 age- and
sex - matched controls showed a higher prevalence of h. pylori seropositivity in acute myocardial infraction patients than in controls , even after adjustment for socioeconomic status ( or : 1.87 , 95% ci : 1.422.47 , and p < 0.0001 ) .
our findings suggest that a strong immune response to h. pylori enhanced the systemic inflammation , which was reflected in an increased level of serum il-6 . with regard to the biological mechanisms for this finding
, previous studies have shown that hp0175 , which is a secreted peptidyl prolyl cis - trans - isomerase of h. pylori , elicits il-6 gene expression and il-6 release from macrophages [ 34 , 35 ] .
a chronic low - grade inflammatory process leading to atherosclerosis could be one possible mechanism involved in the onset of h. pylori - induced ischemic heart disease .
in fact , a study found that h. pylori igg levels were higher for subjects who died of ischemic heart disease compared with those of survivors and higher for survivors compared with those of controls .
in addition , one study showed that patients with henoch - schnlein purpura had significantly higher levels of anti - h .
pylori igg compared with healthy controls ( 86 32 versus 32.5 23 u / ml ) .
pylori igg antibody titer may play a crucial role in other extradigestive diseases as well as cardiovascular disease .
pylori igg antibody titers were significantly correlated with the severity of inflammation in both the antrum and body of the stomach .
the igg h. pylori antibody absorbance index was significantly correlated with not only the density of antral h. pylori colonization but also the degree of gastritis of the antrum , as assessed using the whitehead score and activity , as well as the sydney system .
in contrast to this study , our findings showed that atrophic gastritis was not significantly associated with serum h. pylori igg levels after adjustment for sex and age .
furthermore , serum il-6 levels were significantly associated with h. pylori igg levels independent of sex , age , and atrophic gastritis stage .
thus , the possible elevation of serum il-6 due to h. pylori infection was not determined by the level of atrophic gastritis . in the current study
, there was no significant difference in serum il-6 levels between infected and uninfected subjects . in multiple linear regression analysis adjusted for sex and age , h. pylori infection status ( uninfected = 0 , infected = 1 )
was not significantly associated with il-6 levels in all of the subjects ( infected and uninfected ) ( = 0.047 , p = 0.411 ) .
therefore , uninfected ( n = 93 ) subjects and those with unknown infections ( n = 16 ) were excluded from this study .
however , we conducted this study to assess the correlation between serum anti - h .
therefore , this study evaluated the association of serum il-6 levels with the immune response to h. pylori infection as assessed by antibody levels .
in conclusion , our study revealed a significant positive association between serum il-6 levels and anti - h .
pylori igg levels in h. pylori - infected japanese adults . because this may imply that the immune response to h. pylori promotes systemic inflammation , further studies are needed to confirm this association .
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background . interleukin-6 ( il-6 ) is a multifunctional cytokine produced by many types of cells .
inflammation plays a key role in the pathogenesis of atherosclerosis that is an underlying cause of coronary heart disease ( chd ) . since the 1990s
, some studies have shown an association between h. pylori infection and chd , which may be mediated by inflammation .
therefore , this study aimed to evaluate the association between serum anti - h .
pylori igg levels and serum il-6 levels in h. pylori - infected adults . methods .
we enrolled 158 subjects who visited a clinic located in an urban area to be tested for h. pylori infection , using the 13c - urea breath test , and who were found to be infected and subsequently received eradication .
results .
the geometric mean serum il-6 level was 1.78 pg / ml for men , 1.57 pg / ml for women , and 1.64 pg / ml overall .
logarithms of serum il-6 levels were positively correlated with logarithms of serum h. pylori igg levels ( r = 0.24 , p = 0.002 ) . in multiple linear regression analysis adjusting for sex and age , the serum il-6 level was still significantly associated with the igg level in all subjects ( = 0.18 , p = 0.012 ) .
conclusion .
higher h. pylori igg levels were significantly associated with higher serum il-6 levels among h. pylori - infected individuals .
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osmotherapy is an option for the treatment of cerebral edema due to acute brain injury .
it is used to prevent progression of ce and lower intracerebral pressure ( icp ) .
mannitol and hypertonic saline ( hts ) are the drugs currently available for this purpose .
mannitol would be inappropriate in a patient with end - stage renal disease ( esrd ) due to its accumulation in a patient without excretory function .
hypertonic saline would be acceptable only as long as its accumulation and the excess extracellular volume generated by the resulting hypertonicity could be controlled . a patient with esrd
is described who developed an ich with subsequent ce and was placed on continuous venovenous hemodiafiltration ( cvvhdf ) using fluids specially designed to maintain a hypertonic state with goal serum sodium of approximately 150155 meq / l .
the patient is a 45-year - old male admitted after developing a headache , slurred speech , and left hemiparesis during his routine hemodialysis treatment .
a ct scan of the brain showed a 4.4 2.5 cm area of hyperattenuation in the right basal ganglia area , moderate edema associated with the ich , and mass effect with a 2 mm right - to - left midline shift .
the day after admission a repeat ct scan demonstrated that the edema and mass effect had increased and that the midline shift had increased to 10 mm .
the standard protocol has a dialysate sodium of 117 meq / l and a predilution replacement fluid sodium of 135 meq / l .
meq / l and a predilution replacement fluid sodium of 155 meq / l .
in addition , shortly after the initiation of cvvhdf , the patient was given a 250 cc bolus of 3% saline followed by an infusion at 100 cc / hr to quickly elevate his serum sodium .
the infusion was discontinued the next day , and the patient was maintained on hypertonic cvvhdf .
minor adjustments to the solutions were made to keep the serum sodium near the goal of approximately 155 meq / l .
after four days of cvvhdf , the decision was made to allow the serum sodium to slowly normalize .
dialysate sodium of 147 meq / l was used for one hemodialysis treatment and then 145 meq / l was used for the next treatment .
osmotherapy is an effective treatment option to control ce and reduce intracranial pressure in patients with acute brain injury .
mannitol has been reported to cause a variety of electrolyte disturbances and acute renal failure . in an esrd patient who is anuric
hts causes hypernatremia and extracellular volume expansion and can cause metabolic acidosis and hypokalemia among other side effects .
hts has been shown to be effective in patients resistant to mannitol therapy [ 4 , 5 ] .
hts may also have a beneficial effect on mortality in the treatment of raised icp when compared to mannitol .
it has been reported that serum tonicity as high as 365 mosm / l can be safely achieved with hts therapy whereas mannitol therapy is limited to a maximum of 320 mosm / l . when hypernatremia , whether or not a consequence of osmotic therapy , was evaluated in patients in a neurologic icu , it was an independent predictor of mortality only when the peak serum sodium exceeded 160 meq / l .
it has recently been suggested that continuous renal replacement therapy , due to greater cardiovascular and intracranial stability , is the preferred method to treat patients with acute brain injury and renal failure .
our pharmacy does not use commercially prepared solutions but rather uses an automatic compounder to custom mix the ordered solutions .
thus , it was straightforward for the pharmacy to mix the requested solutions for hypertonic cvvhdf .
this simplified the patient 's regimen as the need for a constant hypertonic saline drip was eliminated .
we observed no untoward effects from the hypertonic dialysis , and the serum sodium was readily maintained in the goal range .
we are aware of only one similar report in the literature , that of hofmann et al . using continuous venovenous hemofiltration ( cvvh ) and
we feel that hypertonic cvvhdf should be considered a reasonable treatment option for acute brain injury in patients with renal failure .
cvvhdf with solutions designed to induce hypernatremia was used to treat an esrd patient with ce due to an ich .
we were able to maintain the serum sodium at goal , and no adverse effects of the procedure were noted .
we suggest hypertonic cvvhdf be considered an option in the treatment of esrd patients with ce due to acute brain injury .
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continuous venovenous hemodiafiltration ( cvvhdf ) using solutions designed to maintain hypernatremia is described in an end - stage renal disease ( esrd ) patient with cerebral edema ( ce ) due to an intracerebral hemorrhage ( ich ) .
hypernatremia was readily achieved and maintained without complication .
cvvhdf should be considered as an alternative treatment option in esrd patients with cerebral edema who require hypertonic saline therapy .
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the criteria for the diagnosis of myocardial infarction have been redefined recently , as reported in a consensus document of the european society of cardiology ( esc ) and the american college of cardiology ( acc ) .
the increased risk associated with even minor amounts of myonecrosis has led to the concept that any amount of myocardial necrosis should be defined as a myocardial infarction .
this change in perspective will lead to an increase in the number of cases of ami that are recognized ( improved sensitivity ) .
presumably , fewer false - positive diagnoses will occur ( improved specificity ) owing to the improved performance of newer diagnostic technologies .
in contrast with the older world health organization criteria , the esc / acc criteria place a much greater emphasis on the role of biochemical cardiac markers in the diagnosis of ami .
however , the selection of the most optimal cardiac marker ( or combination of markers ) remains controversial .
h - fabp also elevated in heart failure and unstable angina patients ( uap ) .
the distinction between unstable angina and non - st - segment elevation is based on the absence ( unstable angina ) or presence ( non - st - segment elevation myocardial infarction ) of an elevated cardiac marker detectable in appropriately timed blood specimens .
although no marker meets all of the desired features of a biomarker , the more specific cardiac markers , troponin t ( ctnt ) and troponin i ( ctni ) , have a number of attractive characteristics .
in addition to being useful for diagnosis , the troponins also permit the estimation of prognosis and risk stratification of patients with acs .
cardiac tnt ( ctnt ) and cardiac tni ( ctni ) , therefore , have been accepted as the gold standard markers in the evaluation of patients with acs , but they have several limitations .
though very specific to cardiomyocytes necrosis , they are unable to differentiate ischemia from other mechanisms of injury and ctn release is often not detectable until 69 hours following injury , delaying diagnosis .
troponin levels are also reported to be elevated in patients with other medical conditions , such as congestive heart failure , myocarditis , or renal failure , making the test less suitable for diagnosis of acs .
these limitations have prompted us to evaluate the diagnostic properties of heart - type fatty acid binding protein in serum ( h - fabp ) .
it is a cardiomyocyte cytosolic protein and is small and quickly released into the circulation in myocardial injury .
h - fabp levels are detectable in blood 2 - 3 hours following initial injury , and they return to normal within 1224 hours . due to low normal levels in serum and high tissue content , a rapid rise above clinical cut - off level is warranted .
it is approximately 20-fold more specific than myoglobin and more sensitive than ctn for cardiac muscle in the early hours of chest pain .
we studied the diagnostic test properties of serum h - fabp as compared to cardiac tnt and tni in patients with chest pain admitted in iccu in a tertiary care hospital .
this is a case control study of 30 patients and 22 controls between december 2011 and may 2012 at the department of biochemistry and department of cardiology , nizam 's institute of medical sciences , hyderabad , a.p , india .
patients aged between 30 and 60 yrs . with first episode of chest pain within 68 hours
all these patients were admitted to iccu and diagnosed as ua or non - st - elevation acute coronary syndrome based on clinical and 12 lead ecg .
patients without chest pain but with other symptoms suggestive of an atypical presentation of acs by the assessing physician in the emergency department were also included .
the control group had 22 , age and sex matched apparently healthy voluntary blood donors .
patients with chest pain more than 8 hours duration , noncardiac chest pain , recent injuries , and renal failure were excluded .
informed written consent was obtained from all the subjects and the study was approved by the institutional ethics committee .
all patients underwent an initial clinical assessment that included a clinical history , physical examination , 12-lead electrocardiogram ( ecg ) , continuous ecg monitoring , standard blood pressure measurements , and chest radiography in the iccu .
ecg and systolic and diastolic blood pressures ( measured in the supine position ) were assessed under standardized conditions .
information with respect to smoking status ( smoker versus nonsmoker ) , alcoholism , and preexisting hypertension was obtained .
5 ml venous blood was drawn in plain tubes from patients within eight hours of symptom onset ; serum was separated within half an hour and stored at 70c until analysis and the samples were thawed only once .
the samples were processed for h - fabp by quantitative immunoturbidimetric method ( randox laboratories , ltd . ,
co. , antrim , united kingdom ) and cardiac troponin t and troponin i by time resolved immunofluorescence method ( aqt90 flex , radiometer , denmark ) .
sample size was calculated prospectively on the basis of obtaining estimates of sensitivity and specificity with adequate precision .
statistical software , medcalc statistical software version 12.7.7 ( medcalc software bvba , ostend , belgium ; http://www.medcalc.org/ ; 2013 ) was used for all data analysis .
descriptive statistics of normal data is reported as mean and sd and that of nonnormal data is reported by using five - numbered summary consisting of minimum ; 25th , 50th , and 75th percentile ; and maximum .
students t - test was used to test the significance of the difference between the means of normally distributed data and the mann - whitney u test was used to compare biomarker levels ( nonnormally distributed data ) between two independent groups ( e.g. , patients diagnosed as nste - acs and control subjects ) .
receiver operating characteristic ( roc ) curves were generated for each biomarker to assess their performance and were analyzed for the standard measures of test validity including sensitivity , specificity , predictive values , and likelihood ratios with 95% confidence intervals .
the optimal cut - off point for dichotomizing serum concentrations of h - fabp was selected to maximize the youden index .
the receiver operating characteristics curves were calculated to show the variability of sensitivity and specificity for cut - off points of different concentrations of h - fabp , ctnt , and ctni which were measured as continuous variables ( see table 3 ) .
the study population demographics of 30 patients , 20 male and 10 female , are shown in table 1 .
those admitted with an ami were older and had risk factors like smoking , alcohol , hypertension , and diabetes .
there was no significant difference between the mean levels of total cholesterol ( tc ) , low density lipoprotein cholesterol ( ldl - c ) , very low density lipoprotein cholesterol ( vldl - c ) , triglycerides ( tg ) , urea , and creatinine . significant difference was noted for serum high density lipoprotein cholesterol ( hdl - c ) , tc / hdl - c between cases , and controls .
table 2 compares the median , minimum , and maximum of each biomarker ( and the iqr ) in the patients .
median levels of ctni , ctnt , and h - fabp , ( p values : < 0.001 , < 0.0004 , and < 0.0001 , resp . ) are all significantly higher in patients when compared with the controls .
all the three levels are log transformed and presented as box and whiskers plots ( 25th percentile , median , 75th percentile ) and error bar for 95% ci graph comparing subgroups across different biomarkers in figure 1 .
the significance of the difference between the cardiac biomarkers was investigated using mann - whitney u test .
the serum concentration levels of ctnt and ctni in cases were significantly different from that of the levels in the controls , mann - whitney u = 192 , p = 0.008 and u = 180 , p = 0.003 , respectively .
the serum concentration levels of h - fabp in cases were significantly different from that of the levels in the controls , mann - whitney u = 138 , p = 0.0004 .
though all the three biomarkers were significantly different between cases and controls , h - fabp was highly significant .
data used : pretest probability 35% , cases 30 , and controls 22 . the diagnostic accuracy for nste - acs , as quantified using auc , is shown in figure 2 .
the auc for h - fabp ( 0.79 , 95% ci ( 0.66 0.89 ) ) was highest among the three .
at 100% specificity and 100% positive predictive value , ctni had the sensitivity of 40% ; at the serum concentration level above 0.009 g / l , ctnt at the serum concentration level above 9
g / l ; the sensitivity was 46.7% , specificity 100% , and positive predictive value of 100% .
h - fabp at the serum concentration above 6.5 ng / ml , the sensitivity was 56.7% , specificity 100% , and positive predictive value of 100% ( see figure 3 ) . at the optimal criteria for all the three biomarkers the negative predictive value was 55% , 58% , and 63% , respectively .
table 4 displaying the estimated specificity for a range of fixed and prespecified sensitivities of 80 , 90 , 95 and 97.5% as well as estimated sensitivity for a range of fixed and prespecified specificities with the corresponding criterion values with the confidence intervals .
this table can be used to calculate the positive predictive value and negative predictive value applicable in individual clinical settings when one knows the prior probability of disease ( pretest probability or prevalence of disease ) .
early diagnosis of ami facilitates rapid and appropriate triage of patients within the accident and emergency department , helping to prevent inadvertent discharge of patients with ami .
it also avoids delay in administering treatment for acute mi and reduces the possibility of patients without acute mi being given treatments from which they will not benefit , and which have the potential to cause significant harm .
the working diagnosis of nste - acs is a rule out diagnosis based on the ecg , that is , lack of persistent st elevation .
the 12-lead ecg is an important tool for early detection of acute mi , but it has significant limitations , and also the interpretation of the 12-lead ecg is dependent on the experience of the physician .
a test with high ability to rule out ( negative predictive value ) and correctly diagnose acs ( positive predictive value ) is of paramount interest .
nacb recommends for patients who present within 6 hrs . of the onset of symptoms ( level of evidence : b ( class ii b ) , an early marker of myocardial necrosis may be considered in addition to a cardiac troponin and had suggested that myoglobin is the most extensively studied marker for this purpose and secondly a rapid rule in protocol with frequent early sampling of markers of myocardial necrosis if tied to therapeutic strategies ( level of evidence : c ) .
a major drawback with cardiac troponins is that they are released relatively slowly from damaged myocytes and also have the drawback of biphasic release after tissue injury as first small amounts of cytoplasmic troponin is released before cytoskeletal troponin is released .
myoglobin is a smaller protein as compared to ck - mb and ctn and is released as early as 1 - 2 hours after symptom onset during ami .
myoglobin is no longer useful in the routine assessment of patients for possible acute myocardial infarction ( ami ) .
a number of studies have shown that myoglobin lacks both sensitivity and specificity for the cardiac myocyte [ 2023 ] myoglobin is also nonspecific due to low levels in heart tissue and high levels in skeletal muscle tissue .
the role of myoglobin will be relegated to that of total ck ( without mb ) , ldh , and sgot that is a class iii recommendation .
h - fabp is a sensitive biomarker for myocardial infarction [ 7 , 25 , 26 ] and one study showed increased sensitivity of 20.6% over troponin at 36 hours following chest pain onset similar to our study , where in h - fabp had higher sensitivity of 58% ( 95% ci 37.474.5 ) when compared with ctni 40% ( 95% ci 2359 ) and ctnt 47% ( 95% ci 2866 ) .
this sensitivity may be explained by the high concentration of h - fabp in myocardium compared to other tissues ; the stability and solubility of h - fabp ; its low molecular weight ; 15 kda compared to 18 , 80 , and 37 kda for myo , ck - mb , and ctnt , respectively [ 2830 ] ; its rapid release into plasma after myocardial injury60 minutes after an ischemic episode ; and its relative tissue specificity .
the effectiveness of using the combination of h - fabp with troponins to diagnose mi within 6 hours is well reported [ 9 , 32 , 33 ] .
these features of h - fabp make it an excellent potential candidate for the detection of acute myocardial injury .
body et al . evaluated the ability of 8 biomarkers to rapidly exclude ami at the point of presentation . in their
705 patients heart fatty acid binding protein ( h - fabp ) had an auc of 0.86 ( 95% ci 0.820.90 ) , which was significantly higher than any other biomarker including ctni . in our study
the discriminatory power for h - fabp was higher as indicated by auc 0.79 versus ctni 0.73 and ctnt 0.71 .
xu et al . investigated the effectiveness of h - fabp for diagnosis of ami in patients with different ethnic background and different time from symptom onset .
two hundred and eighty - nine patients admitted within 12 h after the onset of symptoms were recruited in the study .
it gave the highest sensitivity ( 96% ( 95% ci : 9198% ) ) and a comparable specificity ( 84% ( 95% ci : 7689% ) ) to ctni alone .
the range of time point for our patients was 3.77.9 hours and 100% specific and 100% .
ppv at the level of 6.5 ng / ml , the 63% npv of h - fabp was higher than that of ctni ( 55% ) and of ctnt ( 58% ) ( see table 3 and figure 4 ) . therefore , the proportion of patients with a negative test resulting in correctly diagnosed by h - fabp is 10% and 5% more than ctni and ctnt , respectively .
hall et al . from their study concluded that more patients were diagnosed with nste - acs and underwent coronary angiography after introducing the hs - ctnt assay . at the same time
there was an increase in the frequency of coronary angiograms without signs of coronary artery disease ( cad ) and fewer had significant dynamic ctnt concentration changes .
so far no study has reported over diagnosis of nste - acs with h - fabp .
the study of hafidh alhadi and keith fox highlights two important facts : first , h - fabp was more sensitive than myoglobin and second h - fabp had higher npv than myoglobin .
high sensitivity is essential for the early rule in of patients with ami , and high npv is important for the early rule out of ami , since more than 90% of patients who present with acute chest pain to an accident and emergency department do not have ami . in a study , out of the total number of patients who approached the emergency department with chest pain as the chief complaint , in the us , just 5 to 15% of them
were found to be suffering from heart attacks or other cardiac diseases . in countries like india ,
chest pain as the chief complaint . from the total sample of patients presenting with chest pain ,
rule out is more important for effective patient treatment . in our study , h - fabp with 100% specificity has a higher sensitivity ( 56.7% ) making it a better biomarker to rule out the disease .
the positive likelihood ratio of h - fabp at 100% specificity ( > 6.5 ng / ml ) is above 12.4 , which is clinically very significant showing strong evidence for the presence of the disease
. the prevalence rate of nste - acs in a tertiary care hospital is higher than that of a general hospital ; therefore , it becomes important to rule out the disease confidently .
the positive likelihood ratio of h - fabp ( cut off 6.5 ng / ml , + lr 12.5 , 95% ci : 1.886.8 ) is significantly higher than that of ctni ( cut off 0.009 g / l , + lr 1.71 , 95% ci : 1.03.0 ) and ctnt ( cut off 9.00 g / l , + lr 1.2 , 95% ci : 0.81.9 ) .
so the level of 6.5 ng / ml of h - fabp has much better certainty about a positive diagnosis .
in our study we have shown that serum levels of h - fabp can be used in the early hours of chest pain to rule out the existence of nste - acs in some and rule in in some and seems to be a preferred biomarker in the differential diagnosis of nste - acs .
we emphasize h - fabp testing as the main - line marker for triage of patients to assist in their optimal and efficient management .
though the number of cases is small , our observation might generate hypothesis for forthcoming studies .
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a prospective case control study was undertaken to evaluate the diagnostic performance of serum heart - type fatty acid binding protein ( hfabp ) in comparison to cardiac tnt and tni in 33 patients admitted with chest pain , diagnosed as nste - acs ( non st elevation acute coronary syndrome ) and 22 healthy controls .
area under the receiver operating curve ( auc ) was highest for h - fabp ( auc 0.79 ; 95% ci 0.660.89 ) versus ctni ( auc 0.73 ; 95% ci 0.590.84 ) and ctnt ( auc 0.71 ; 95% ci 0.570.83 ) .
the h - fabp level above 6.5 ng / ml showed 56.7% ( ci 37.474.5 ) sensitivity , 0.5 ( 95% ci 0.30.7 ) negative likelihood ratio ( lr ) , 100% ( ci 84.6100.0 ) specificity , and 100% ( ci 79.4100.0 ) positive predictive value ( ppv ) , 62.9% ( ci 44.978.5 ) negative predictive value ( npv ) .
ctni level above 0.009 g / l had 40% ( ci 22.759.4 ) sensitivity , 0.6 ( 95% ci 0.40.8 ) lr , 100% ( ci 84.6100.0 ) specificity , 100% ( ci 73.5100.0 ) ppv , and 55% ( ci 38.570.7 ) npv .
ctnt showed 46.7% ( ci 28.365.7 ) sensitivity , 0.5 ( 95% ci 0.40.7 ) lr , 100% ( ci 84.6100.0 ) specificity , 100% ( ci 76.8100.0 ) ppv , and 57.9% ( ci 40.873.7 ) npv at level above 9 g / l .
+ lr were 12.5 ( 95% ci 1.886.8 ) , 1.7 ( 95% ci 1.03.0 ) , and 1.2 ( 95% ci 0.81.9 ) for h - fabp , ctni , and ctnt respectively . in conclusion measurement of h - fabp is a valuable tool in the early diagnosis of patients with chest pain ( 68 hrs ) and seems to be a preferred biomarker in the differential diagnosis of nste - acs .
more studies are needed to determine whether serum h - fabp further improves diagnostic performance .
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lines of evidence have indicated that there are two types of immunologic responses in the human body and in animal models : t helper 1 ( th1)-based and t helper 2 ( th2)-based responses . in the th1 response , macrophages undergo m1-type activation , synthesizing chitotriosidases , protease , no , h2o2 , and other chemicals used to kill invaders , such as fungi , viruses , and bacteria .
the th1 mediated anti - cancer response , interferon- ( inf- ) , interleukin-12 ( il-12 ) , il-2 , and tumor necrosis factor- ( tnf- ) play major roles , .
however , the over - activation of th1 immunity sometimes causes certain types of autoimmune problems . in the th2 response ,
the mechanisms that control the immune response are not yet known ; however , dendritic cells , cd4cd25 regulatory t cells , and t helper 17 cells are involved . during the early stages of cancer growth
, some products from cancer cells and the tissue damage caused by invading cancer cells stimulate the th1 immune response , . because cancer cells are , on the whole , very similar to normal cells , cd4cd25 regulatory t cells , and possibly other types of regulatory cells ,
these regulatory cells secrete il-4 , il-10 , il-13 , and so on , to change the immune response from a th1 to a th2 response , . under the th2 response , the macrophages are of the m2-type and no longer produce chitotriosidases .
the job of m2 macrophages is to remove dead cells , remodel damaged tissue , and stimulate new blood vessel growth .
these actions actually help the cancer tissue to grow. as the number of cancer cells increases , the t regulatory system gets stronger quickly , and t regulatory cells not only occupy the tumor tissue but also spread into the neighboring lymph nodes and spleen .
because only activated m1 macrophages produce 50-kda chitotriosidases , we need to convert the th2 response to the th1 response in patients with cancer . to successfully induce the th1 response , three points need to be followed .
first , bretscher et al . , have noted that the amount of the stimulator used to stimulate the th1 response must be appropriately low . in their study of changing leishmania - susceptible balb / c mice to leishmania - resistant mice
, they showed that the amount of parasites used for the first inoculation must be less than 3.3 10 promastigotes per mouse for the mice to build up a th1 response .
balb / c mice injected with fewer than 3.3 10 promastigotes not only survived the inoculation but also became resistant to the next high - dose leishmania inoculation .
however , if the first inoculation used more than 3.3 10 promastigotes , the mice developed a typical th2 response , started to produce igg1 antibody and were still susceptible to the next inoculation .
further proved in culture that in the presence of dendritic cells or activated b cells , the concentration of antigen directly determined whether nave cd4 t cells would develop the th1 or th2 response . only within a special range of concentrations of the antigen
would nave t cells develop the th1 response and produce a large amount of ifn- and little il-4 .
when the antigen concentration was higher than that range , or sometimes when it was too low , naive t cells would develop the th2 response and produce a large amount of il-4 and little ifn-. a similar result occurred during the treatment of sarcoma-180-transplanted icr mice . when the mice were given subcutaneous injections of a mushroom component called lentinan with a dose of 1 mg / kg day for 10 days , their tumors completely regressed .
however , when the lentinan dose was 80 mg/(kg day ) for 5 days , no tumor regression occurred .
the second point is that even when using the right amount of stimulator , the total number of stimulations is also important .
virulizin is a product extracted from bovine bile that has been found to possess antitumor activity in a variety of human tumor xenografts in mice , .
when a suitably low dose of virulizin was injected intraperitoneally every day for 3 to 5 days , the serum levels of il-12 and il-12 increased by 2.3- and 2.6-fold , respectively .
the infiltration of macrophages and natural killer cells increased in the tumor tissue , and the tumor size decreased . when virulizin was given together with lps in the culture medium in vitro ,
activated macrophages produced more tnf-. however , when the mice were given continuous injections of the same dose of virulizin for 4 weeks , a typical th2 response developed : the expression of il-17 increased , a significant number of eosinophils were recruited to the tumor tissue , and the anticancer effectiveness decreased .
the third point is that at the time of th1 response stimulation , consuming large amounts of fruits , vegetables , and concentrated tea should be avoided .
it has been confirmed recently that compounds such as polyphenols , anthocyanins and falconoids are rich in fruits , vegetables , and concentrated tea and are strong inhibitors of m1 macrophage activation. although these compounds have been reported to be able to inhibit cancer cells by themselves , it is better to avoid taking a lot of them at the same time as th1 response stimulation .
when the th1 response is stimulated , the t regulatory system will also be augmented . to obtain a stronger th1 response , giving only the th1 response stimulator
cyclophosphamide ( cpa ) is a dna - alkylating agent that is actively against proliferating cells .
cpa has been used as an anticancer drug , but its anticancer effect is not very strong . lately , cpa has been found to be a good immune regulator , as it eliminates t regulatory cells or inhibits their activity . a single low dose of cpa can induce a cytokine profile shift from th2 to th1 in tumor - bearing animal models .
thus , it can be used to break cancer immune tolerance and enhance the function of th1 stimulators . in the past ,
some cytokines or interleukins , such as ifn- , il-2 , ii-12 and il-18 , have been used to directly stimulate the immune response against cancer . among these
injection of il-12 alone can induce complete regression of some small tumors , and the mechanism is directly related to ifn- production
first used the combination of a low dose of cpa and il-12 to treat animals bearing large tumors .
they found that if they first gave one intraperitoneal injection of 2.5 mg / mouse of cpa , and then , 4 or 5 days later , gave intraperitoneal injections of 0.5 g of il-12 once every other day for 5 or 9 days , large sarcomas were damaged .
furthermore , they noted that the effect of il-12 is based on the activation of macrophages . in ifn--knockout mice , il-12 showed little anticancer effect . in 2006 , lutsick et al . noted that a low dose of cpa actually inhibits cd4cd25 t regulatory cells .
this result suggested that the t regulatory system first needs to be inhibited and then , macrophage stimulators should be given .
if we administer macrophage stimulators prior to inhibiting the t regulatory system , the results would be different . in cpa treatment ,
t cells are essential for the eradication of established tumors , and cpa treatment seemed to be more effective on large tumors than on small tumors .
the studies outlined in this section are quite encouraging , but sometimes the elimination of cancer by interleukin injection can cause autoimmune problems .
in addition , the population of t regulatory cells in cancer patients comes back about 10 days after a single low dose of cpa is given , suggesting that the second to the fifth day after cpa injection is the best period of time for th1 stimulator administration .
it has been found recently that oral uptake of viable lactic acid bacteria will increase il-12 and ifn- levels in peripheral blood. it is possible that consuming yogurt can help cancer patients prevent recurrence of cancer. it might be beneficial to use a combination of cpa and lactic acid bacteria to raise the blood il-12 concentration , which would have a different result than the injection of il-12 .
the injection of il-12 will induce a high il-12 concentration peak in the blood , but orally taking lactic bacteria will not .
people have checked whether autoimmune induction occurs after orally consuming lactic acid bacteria in yogurt , and it does not .
in addition to lactic acid bacteria , some other edible bacteria , such as the bacteria used to make rice wine and vinegar , are also able to raise il-12 and ifn- levels in the blood and suppress colon cancer and b16 melanoma in mice .
in this paper , we have discussed the role of chitotriosidase in the anticancer function of macrophages ( figure 1 ) , but the exact molecular target of chitinase on cancer cells has not yet been identified .
increasing evidence indicates that a number of proteoglycans on cancer cells are different from those on normal cells and that tumor cells have their own types of surface mucin - type glycoproteins .
in addition to the stimulation of m1 macrophages in cancer treatment , recombinant human 50-kda chitotriosidase may be directly used to treat cancer patients .
if two suitable nanoparticles , one carrying recombinant human chitotriosidase and the other carrying a human protease , such as elastase , are prepared and intravenously injected into a cancer patient at the same time at a suitable ratio , both nanoparticles will penetrate into cancer tissue through the broken blood vessels , which are present in solid tumor tissues but not in normal tissues .
when the enzymes are released from the nanoparticles inside the cancer tissue , the local high concentrations of both enzymes will be able to kill cancer cells .
recently , xu et al . reported that a recombinant plant family 19 chitinase could kill cancer cells in culture directly .
because family 19 chitinases have a distinct structure and a catalytic mechanism different from that of family 18 chitinases , the recombinant plant family chitinase react directly and do not require protease , .
however , humans do not have family 19 chitinases , though plants have many of them . recently
, two laboratories have successfully prepared humanized plants , in their study of producing humanized monoclonal antibodies , through modifying the n - glycosylation pattern of the plant , .
thus , we have the opportunity to prepare suitable humanized family 19 plant chitinases from humanized plants and use them as a new type of biochemical drug to treat cancer patients .
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m1-type macrophages are capable of inducing lysis in various types of cancer cells , but the mechanism of action is unclear .
it has been noted that an unknown protein produced together with protease by activated macrophages is responsible for this action .
activated m1 macrophages have been recently reported to produce family 18 chitinases , all of which have been named chitotriosidase .
our experiments have demonstrated that family 18 chitinases work together with proteases and can damage various cancer cells both in vitro and in vivo .
thus , in this article , we suggest that the 50-kda chitotriosidase is the reported unknown protein .
in addition , we discuss how to properly stimulate activated m1 macrophages to produce 50-kda chitotriosidases and proteases for destroying cancer cells . because family 19 chitinase has recently been reported to kill cancer cells
, we also discuss the possibility of directly using human family 18 chitotriosidase and the humanized plant family 19 chitinase for cancer treatment .
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diagnostic criteria and treatment options for this condition are constantly evolving , but little focus has been directed towards pregnancy and childbirth in these patients . there is a scarcity of literature available addressing this important issue , and this paper aims to fill that gap , and provide sound evidence and guidance for mothers who are pregnant with eb babies .
milder forms of eb , such as eb simplex , often go undiagnosed : this could explain the relative lack of pregnancy cases reported .
on the other hand , very few reports in the literature detail pregnancy and childbirth experiences of mothers and infants with more severe forms of eb , including junctional eb ( jeb ) and recessive dystrophic eb ( rdeb ) .
it does not always follow that all patients with severe forms of eb will have difficult pregnancies ( price and katz , 1988 ) .
the bulk of the available literature is mainly on prenatal diagnosis of severe forms of eb ( ebs or jeb with pyloric atresia , herlitz jeb , and rdeb ) and its role in management decisions such as termination ( dalessio et al . , 2008 , marinkovich et al . , 1995 , norup 1999 , pfendner et al . , 2003 , yan et al . , 2007 ) .
a survey performed in denmark amongst obstetricians and pediatricians showed that in the case of newborns with severe eb , there was a strong consensus to withhold life - prolonging treatment , reflecting attitudes to eb ( norup 1999 ) . a patient with non - herlitz jeb
was reported who had two miscarriages prior to giving birth successfully via cesarean section under epidural anesthesia ( price and katz , 1988 ) . a patient with rdeb in germany had two vaginal deliveries resulting in healthy babies , with uncomplicated episiotomy wound healing , and no exacerbations of eb during her pregnancy ( bscher et al . , 1997 ) .
another patient with rdeb had preterm labor at 36 weeks and premature rupture of membranes , yet delivered a healthy baby via cesarean section ( bianca et al . , 2003 ) . in the french literature , there is a report of a patient with ebs who developed a herpetiform flare of ebs - dm during the first two months of her pregnancy ( diris et al . , 2003 ) .
more recent reports include that of a patient with kindler syndrome with vaginal stenosis who had a successful cesarean delivery ( hayashi et al . , 2007 ) .
the report most recently published is a case report of 11 pregnancies in three patients with recessive eb in australia .
one of the patients had non - herlitz jeb and had delivered two unaffected babies via nvd eight years apart .
one of them delivered three healthy unaffected babies via nvd , and the other delivered five unaffected babies via nvd .
they all had no complications or flare of their eb during their pregnancies and the peripartum period ( choi et al . , 2011 ) .
more recently , there has been a report of three more women , each with rdeb - intermediate ( rdeb - i ) , all of whom had successful vaginal deliveries without major cutaneous or mucosal complications ( hanafusa et al . ,
there is also an online patient information handout on pregnancy and childbirth in eb published by the dystrophic eb research association ( debra ) uk group in may 2006 which reports that women with eb have successfully had vaginal and cesarean deliveries ( pillay , 2006 ) .
labor and delivery practices include airway management strategies , the role of regional anesthesia , and the use of nonadhesive tape and padding ( i.e. mepitel , mepilex , mepitac , mepiform ) as minor trauma may lead to severe lesions ( pillay et al . , 2006 , price et al . , 1988 ) .
there are five reported cases that used either spinal or epidural anesthesia for cesarean section , and epidural anesthesia for vaginal delivery without any ensuing complications ( baloch et al . , 2008 , berryhill et al . , 1978 , broster et al . , 1987 )
in view of this limited information , we designed a survey looking at the experiences of a large group of obstetricians , unaffected mothers who delivered babies with eb , and eb patients themselves who have delivered babies .
we have developed a foundational database , and have developed recommendations on periobstetric advice in relation to eb .
this study was granted ethics approval by the south eastern sydney local health district human research ethics committee - southern sector on the 3rd of october , 2006 until july , 2012 .
questionnaires were sent out to three participant groups , namely obstetricians in australia , unaffected mothers who had given birth to eb babies , and eb females who had given birth .
the list of obstetricians was obtained from the royal australian and new zealand college of obstetricians and gynecologists ( ranzcog ) , whilst the list of eb patients and their parents was obtained from patients known to us , most of whom are in the australasian eb registry which is being maintained at st .
the appropriate questionnaires were mailed to these obstetricians and patients in the post with self - addressed envelopes .
some questionnaires were also handed out to member families of debra australia and new zealand , new patient referrals seen at st .
all participants had given signed informed consents to participate in the study and share their data .
a substudy was also performed that looked into the percentage and locations of blisters , if any , in babies born with eb to unaffected mothers and those diagnosed with eb this was achieved by sending out further questionnaires with body maps to both groups of respondents .
the data was then collated and summarized over a period of 4.8 years ( october 2006august 2011 ) .
statistical analysis was performed using chi - square tests , t - tests , and fisher exact tests .
peri - obstetric recommendations were made for eb patients and mothers giving birth to eb babies .
the questionnaires were sent out in one batch to 1346 obstetricians in australia , and 195 responded .
only 14 of the 195 obstetricians who responded had encountered mothers or babies born with eb .
six of the 14 obstetricians attempted a literature search on eb in pregnancy and childbirth , but only three were successful in finding any articles on pregnancy in eb .
however , one performed an elective cesarean section ( cs ) at the patient s request which resulted in poor wound healing and a post - operative wound infection .
furthermore , 111 of the 195 obstetricians indicated the need to have information about eb available in antenatal clinics .
we sent 122 survey questionnaires to eb - unaffected mothers who had given birth to at least one child with eb .
attempts were made to contact all non - responders , and they were re - sent the survey forms .
we received 75 completed questionnaires out of the 110 mailed out ( a 68% response rate or 75% response of those known to us ) .
these pertained to 176 pregnancies and 174 births , 84 ( 48% ) of whom were affected by various types of eb : ( 35 with ebs , 14 with jeb , 19 with ddeb , and 17 with rdeb ) .
the age of respondents ranged from 1978 ( mean age of 46.2 ) and they had given birth to between one and four children , with an average of about two children in each family .
a total of 43 out of the 75 mothers ( 57% ) had children under 18 years of age .
a summary of their characteristics is shown in table i. most mothers with eb had normal vaginal deliveries without any ensuing complications .
table ii shows the modes of delivery of both eb - affected and eb - unaffected children whilst table iii shows the list of complications during pregnancy and delivery of both groups of babies from mothers who were unaffected by eb themselves .
a chi - square analysis showed significantly more ( approximately two - fold ) complications in the pregnancies that delivered eb babies ( 22/84 ) as compared to eb - unaffected babies ( 11/90 ) ( p = .031 ) .
fisher s exact testing also revealed significantly more complications in eb babies delivered via cs ( including emergency cs ) as compared to nvd ( p < .001 ) .
five cases of full - term babies not known to have eb in advance due to a lack of family history of eb were delivered using vacuum suction and/or forceps , resulting in skin being eroded from the babies head , face and mouth areas ; these babies were subsequently diagnosed with severe forms of eb .
the first case was a baby with rdeb delivered via nvd and vacuum suction in which skin was removed from the baby s face and mouth .
the second case was a baby with rdeb delivered via nvd and forceps , where skin was removed from non - facial parts of the baby s head .
the third case was a baby with jeb delivered via nvd and forceps , which resulted in skin erosion from the baby s face .
the fourth case was a baby with jeb delivered via nvd and vacuum suction resulting in skin being eroded from the baby s feet .
the fifth case was a baby with jeb delivered via emergency cs using both vacuum suction and forceps , resulting in facial erosions and hematomas . the overall rate of emergency cs was 8% but for pregnancies with jeb offspring it was 21% , suggesting that labor in this group is more complicated .
out of the 84 babies with eb , 46 ( 55% ) had blisters at birth , most commonly in the severe types of eb ( jeb and rdeb ) and the others ( 45% ) developed blistering in the days or weeks subsequently .
most mothers surveyed were unaware that they were going to deliver a baby with eb .
some expressed the view that their obstetrician could have given more accurate information regarding the genetics and severity of the eb type that affected their babies .
other mothers , particularly with children affected by severe eb types recommended prenatal screening and informed decisions about termination options .
those whose babies had blistering due to birth trauma thought to be associated with vaginal delivery recommended delivery via cs .
out of 55 females with eb of childbearing age surveyed , 44/55 ( 80% ) returned completed questionnaires .
we tried to contact all 11 non - responders , and sent out new surveys forms .
the revised response rate of 44/52 or 85% of contactable female eb patients of childbearing age was very high .
for those known to have at least one child also affected with eb ( 32/36 ) , the response rate was 89% .
we received responses from 28 patients with ebs , 1 with jeb , 12 with ddeb and 3 with rdeb .
they had given birth to a total of 112 babies , 54 affected with dominant types of eb ( 37 with ebs and 17 with ddeb ) and to 58 babies unaffected by eb .
the respondents ' birth rates averaged about two per family , varying between one and eight children .
a summary of their characteristics is shown in table i. in general , the mothers ' eb conditions remained stable during pregnancy and the immediate peripartum period .
only two of the 44 patients , both with ebs , reported that it worsened .
two others ( 1 with ebs and 1 with rdeb ) reported improvement in their condition .
table iv compares the modes of delivery in this group of patients , and table v lists complications .
there were fewer complications during their pregnancies with eb - affected babies ( 8/54 ) , compared to their pregnancies with unaffected babies ( 15/58 ) .
however , a chi - square test showed no significant difference in the number of complications for these two groups ( p = .225 ) .
eb patients who delivered via nvd ( 91/112 deliveries , 81% ) all reported good healing of their episiotomy incisions and perineal tears , where occurring . during delivery , four ebs patients reported blistering at sites where adhesive tape was used to secure their epidural anesthesia .
post - delivery , 10 patients ( 8 with ebs , 1 with jeb , and 1 with rdeb ) reported nipple blistering while breastfeeding , which led them to switch to bottle - feeding .
the eb mothers who delivered via cs ( 21/112 or 19% ) , had good healing of their cs incision sites with just two reports of post - operative wound infections , which later healed well .
general advice from mothers with eb was to have genetic testing done in case a more serious type could be passed on . however , the general consensus was that the joy of having children was worth the discomfort and pain .
we sent out further questionnaires to the participants in group 3 ( eb females who had given birth ) to look at blistering at birth in eb - affected babies in relation to their mode of delivery .
a total of 32/44 group 3 mothers had given birth to babies affected with dominant forms of eb , and from this group , we received 13/32 responses .
this subgroup of respondents from group 3 had given birth to 19 babies with eb ( 14 with ebs and 5 with ddeb ) and 14 unaffected babies .
table vi shows the number of babies born with blisters at birth in relation to their mode of delivery . in this table , data were combined from groups 2 and 3 .
fifty - five percent ( 46/84 ) of eb babies born to eb - unaffected mothers ( group 2 ) had blisters at birth .
similarly , 58% ( 11/19 ) of eb babies born to mothers with eb ( group 3 ) had blisters at birth .
fisher s exact test showed that blistering in jeb and rdeb babies is significantly higher than in ebs babies ( p = .016 and p < .001 respectively ) . blistering in ddeb babies was not significantly greater than blistering in babies with ebs ( p = .769 ) .
fisher s exact test showed blistering at birth in rdeb babies is significantly more common than in ddeb babies ( p = .012 ) .
finally , blistering in jeb babies was not significantly different from blistering in rdeb babies ( p = .315 ) .
in addition , there were no significant differences ( p = .121 ) in blistering of rdeb babies delivered via nvd and planned cs .
overall , there was no significant difference in blistering at birth in all eb babies delivered via nvd versus cs ( p = .136 ) .
the questionnaires were sent out in one batch to 1346 obstetricians in australia , and 195 responded .
only 14 of the 195 obstetricians who responded had encountered mothers or babies born with eb .
six of the 14 obstetricians attempted a literature search on eb in pregnancy and childbirth , but only three were successful in finding any articles on pregnancy in eb .
however , one performed an elective cesarean section ( cs ) at the patient s request which resulted in poor wound healing and a post - operative wound infection .
furthermore , 111 of the 195 obstetricians indicated the need to have information about eb available in antenatal clinics .
we sent 122 survey questionnaires to eb - unaffected mothers who had given birth to at least one child with eb .
attempts were made to contact all non - responders , and they were re - sent the survey forms .
we received 75 completed questionnaires out of the 110 mailed out ( a 68% response rate or 75% response of those known to us ) .
these pertained to 176 pregnancies and 174 births , 84 ( 48% ) of whom were affected by various types of eb : ( 35 with ebs , 14 with jeb , 19 with ddeb , and 17 with rdeb ) .
the age of respondents ranged from 1978 ( mean age of 46.2 ) and they had given birth to between one and four children , with an average of about two children in each family .
a total of 43 out of the 75 mothers ( 57% ) had children under 18 years of age .
a summary of their characteristics is shown in table i. most mothers with eb had normal vaginal deliveries without any ensuing complications .
table ii shows the modes of delivery of both eb - affected and eb - unaffected children whilst table iii shows the list of complications during pregnancy and delivery of both groups of babies from mothers who were unaffected by eb themselves .
a chi - square analysis showed significantly more ( approximately two - fold ) complications in the pregnancies that delivered eb babies ( 22/84 ) as compared to eb - unaffected babies ( 11/90 ) ( p = .031 ) .
fisher s exact testing also revealed significantly more complications in eb babies delivered via cs ( including emergency cs ) as compared to nvd ( p < .001 ) .
five cases of full - term babies not known to have eb in advance due to a lack of family history of eb were delivered using vacuum suction and/or forceps , resulting in skin being eroded from the babies head , face and mouth areas ; these babies were subsequently diagnosed with severe forms of eb .
the first case was a baby with rdeb delivered via nvd and vacuum suction in which skin was removed from the baby s face and mouth .
the second case was a baby with rdeb delivered via nvd and forceps , where skin was removed from non - facial parts of the baby s head .
the third case was a baby with jeb delivered via nvd and forceps , which resulted in skin erosion from the baby s face .
the fourth case was a baby with jeb delivered via nvd and vacuum suction resulting in skin being eroded from the baby s feet .
the fifth case was a baby with jeb delivered via emergency cs using both vacuum suction and forceps , resulting in facial erosions and hematomas .
the overall rate of emergency cs was 8% but for pregnancies with jeb offspring it was 21% , suggesting that labor in this group is more complicated .
out of the 84 babies with eb , 46 ( 55% ) had blisters at birth , most commonly in the severe types of eb ( jeb and rdeb ) and the others ( 45% ) developed blistering in the days or weeks subsequently .
most mothers surveyed were unaware that they were going to deliver a baby with eb .
some expressed the view that their obstetrician could have given more accurate information regarding the genetics and severity of the eb type that affected their babies .
other mothers , particularly with children affected by severe eb types recommended prenatal screening and informed decisions about termination options .
those whose babies had blistering due to birth trauma thought to be associated with vaginal delivery recommended delivery via cs .
out of 55 females with eb of childbearing age surveyed , 44/55 ( 80% ) returned completed questionnaires .
we tried to contact all 11 non - responders , and sent out new surveys forms .
three were returned unanswered due to change of addresses . the revised response rate of 44/52 or 85% of contactable female eb patients of childbearing age was very high . for those known to have at least one child also affected with eb ( 32/36 ) , the response rate was 89% .
we received responses from 28 patients with ebs , 1 with jeb , 12 with ddeb and 3 with rdeb .
they had given birth to a total of 112 babies , 54 affected with dominant types of eb ( 37 with ebs and 17 with ddeb ) and to 58 babies unaffected by eb .
the respondents ' birth rates averaged about two per family , varying between one and eight children .
a summary of their characteristics is shown in table i. in general , the mothers ' eb conditions remained stable during pregnancy and the immediate peripartum period .
only two of the 44 patients , both with ebs , reported that it worsened .
two others ( 1 with ebs and 1 with rdeb ) reported improvement in their condition .
table iv compares the modes of delivery in this group of patients , and table v lists complications
. there were fewer complications during their pregnancies with eb - affected babies ( 8/54 ) , compared to their pregnancies with unaffected babies ( 15/58 ) .
however , a chi - square test showed no significant difference in the number of complications for these two groups ( p = .225 ) .
eb patients who delivered via nvd ( 91/112 deliveries , 81% ) all reported good healing of their episiotomy incisions and perineal tears , where occurring . during delivery , four ebs patients reported blistering at sites where adhesive tape was used to secure their epidural anesthesia .
post - delivery , 10 patients ( 8 with ebs , 1 with jeb , and 1 with rdeb ) reported nipple blistering while breastfeeding , which led them to switch to bottle - feeding .
the eb mothers who delivered via cs ( 21/112 or 19% ) , had good healing of their cs incision sites with just two reports of post - operative wound infections , which later healed well .
general advice from mothers with eb was to have genetic testing done in case a more serious type could be passed on
. however , the general consensus was that the joy of having children was worth the discomfort and pain .
we sent out further questionnaires to the participants in group 3 ( eb females who had given birth ) to look at blistering at birth in eb - affected babies in relation to their mode of delivery .
a total of 32/44 group 3 mothers had given birth to babies affected with dominant forms of eb , and from this group , we received 13/32 responses .
this subgroup of respondents from group 3 had given birth to 19 babies with eb ( 14 with ebs and 5 with ddeb ) and 14 unaffected babies .
table vi shows the number of babies born with blisters at birth in relation to their mode of delivery . in this table , data were combined from groups 2 and 3 .
fifty - five percent ( 46/84 ) of eb babies born to eb - unaffected mothers ( group 2 ) had blisters at birth .
similarly , 58% ( 11/19 ) of eb babies born to mothers with eb ( group 3 ) had blisters at birth .
fisher s exact test showed that blistering in jeb and rdeb babies is significantly higher than in ebs babies ( p = .016 and p < .001 respectively ) .
blistering in ddeb babies was not significantly greater than blistering in babies with ebs ( p = .769 ) .
fisher s exact test showed blistering at birth in rdeb babies is significantly more common than in ddeb babies ( p = .012 ) . finally , blistering in jeb babies was not significantly different from blistering in rdeb babies ( p = .315 ) .
in addition , there were no significant differences ( p = .121 ) in blistering of rdeb babies delivered via nvd and planned cs .
overall , there was no significant difference in blistering at birth in all eb babies delivered via nvd versus cs ( p = .136 ) .
an international expert consensus on delivery recommendations for patients with eb or for eb - unaffected mothers expecting infants with eb has yet to be established .
hence , this survey is quite timely . due to the rarity of this family of diseases , there was a relatively low response rate amongst obstetricians , most of whom felt that nvd should be the recommended mode of delivery for eb patients giving birth .
data from a larger prospective cohort study within the 2005 who global survey on maternal and perinatal health have shown that , overall , maternal morbidity and mortality were higher in the elective cs group ( 5.5% ) than the nvd group ( 1.8% ) .
furthermore , increased risk in nvd relates to maternal socio - demographic characteristics such as being single , young with a low level of education , gravidity , and primiparity . increased risk for maternal morbidity and mortality in the cs group related to women with previous complications in their pregnancies or perinatal outcomes ( villar et al . , 2007 ) .
this supports our data that nvd is still the recommended mode of delivery for most mothers carrying eb babies and for pregnant eb females . despite this recommendation
, there seems to be a growing preference for elective delivery by cs , particularly in western countries . in a recent structured survey performed to determine personal preferences of delivery method amongst obstetricians from australia and new zealand ( which had a 26% response rate ) , 11% of obstetricians chose elective cs in the absence of any clinical indication .
elective cs procedures were also the preferred method of childbirth in cases of predicted fecal incontinence ( 83.5% ) , urinary incontinence ( 81.5% ) , perineal damage ( 68.5% ) , and fear of damage to the baby ( 24% ) ( land et al . , 2001 ) .
the rates of cs in most developed countries are quite similar , with 23.3% of all births in australia , 21.3% in the uk and 26% in the us ( dodd et al . , 2007 ) performed by cs .
overall , nvd is still the most recommended mode of delivery worldwide and appears to be the safer method of childbirth .
it should be emphasized , however , that forceps delivery or vacuum suction should be avoided during nvd or cs , as our data have shown that babies with severe forms of eb had severe erosions on their head and feet .
the data also suggest that cesarean wounds heal well in mothers with eb , and that care during breastfeeding ( i.e. use of nipple shield ) or bottle - feeding , are recommended options if blistering is severe . as for applicability of data derived from the mothers of children with eb , our response rate of 75% from mothers of children with eb is significant , given
that the average response rate cited in the literature for mailed physician questionnaires is around 61% and this has remained quite stable over time ( cummings et al . , 2001 ) .
interestingly , the surveys of mothers who gave birth to babies with eb reveals that there were significantly more complications in deliveries by cs compared to the majority who delivered via nvd .
overall , either of the two modes of delivery seemed to be comparable for blistering rates .
blistering at birth in the different types of eb showed the more severe forms of eb ( jeb and rdeb ) had significantly more blistering than the milder ebs and ddeb forms , as might be expected .
together , this suggests that if it were known in advance that a mother was pregnant with a baby with eb , delivery via nvd would still be recommended as the preferred mode of delivery as long as it is safe to do so ; for example , providing that cephalopelvic disproportion is not a problem
. this would be the case for 50% of mothers with a dominant form of eb such as ebs and ddeb .
it would be more difficult , if not impossible , to predict complications in those with no known family history of a recessive form of eb .
genetic counseling and discussion of prenatal diagnostic options are recommended for all eb patients when contemplating pregnancy ( fassihi and mcgrath , 2010 , sybert , 2007 ) .
most patients with eb are capable of giving birth without increased risk of pregnancy - related complications .
unaffected mothers who have given birth to children with eb have had relatively normal pregnancies comparable to previous pregnancies yielding unaffected children .
however , when a mother is known to be carrying an eb pregnancy , delivery via normal vaginal delivery is no more likely to result in complications and blisters at birth in the eb - affected newborn .
hence , there appears to be no justification in performing a cesarean section to reduce complications for the mother with eb nor the infant with eb in order to avoid eb - related complications .
lastly , awareness of these data amongst obstetricians and dermatologists should lead to informed advice and improved quality of care for both eb mothers and eb babies alike .
1.normal vaginal delivery with regional anesthesia is generally safe , and episiotomy may reduce perineal tears.2.vacuum suction or forceps delivery is not recommended in mothers delivering babies with eb or where the eb status of the baby is unknown.3.in mothers expecting to deliver a baby with eb , normal vaginal delivery is still the preferred mode of delivery.4.only non - adhesive tape and dressings are to be used during anesthesia and surgery .
normal vaginal delivery with regional anesthesia is generally safe , and episiotomy may reduce perineal tears .
vacuum suction or forceps delivery is not recommended in mothers delivering babies with eb or where the eb status of the baby is unknown . in mothers expecting to deliver a baby with eb ,
normal vaginal delivery is still the preferred mode of delivery . only non - adhesive tape and dressings are to be used during anesthesia and surgery .
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backgroundpregnancy in epidermolysis bullosa ( eb ) has not been comprehensively studied.objectivewe aimed to develop a foundational database , which could provide peri - obstetric advice in eb.methodssurvey questionnaires were sent to obstetricians , unaffected mothers of eb babies , and mothers with eb .
results were analyzed using chi - square , fisher exact , and t-tests.resultsout of 1346 obstetricians surveyed , 195 responded , and only 14 had encountered eb .
all recommended normal vaginal delivery ( nvd ) , except for one elective caesarean section ( cs ) .
we received responses from 75 unaffected mothers who had delivered eb babies .
they had significantly more complications in their eb pregnancies compared to their non - eb pregnancies .
a further 44 women with various types of eb who had given birth responded .
most delivered via nvd and had no significant increase in complications in both their eb and non - eb pregnancies .
in both groups , there were no significant differences in blistering at birth in babies delivered via nvd and cs.conclusionin conclusion , most patients with eb who are capable of giving birth do not have an increased risk for pregnancy - related complications and nvd appears to be safe .
awareness of this data amongst obstetricians and dermatologists should lead to improved quality of care for mothers and babies affected with eb .
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prosthetic joint infection ( pji ) is a devastating complication with significant impact on patients and healthcare systems . despite recent advances
, the orthopaedic community recognizes that essential aspects are still controversial and further studies are needed to establish firm evidence - based guidelines .
one such controversial issue is whether to revise a chronically infected total joint in one or two stages .
some centres advocate to do it primarily in two - stages , others choose to do it one- or two - staged according to specific criteria and some believe it should ( almost ) always be performed in one single surgery .
the purpose of this paper is to report the results of a survey made to participants of the 34th european bone and joint infection society ( ebjis ) annual meeting , held in estoril - portugal in 10 - 12 september 2015 .
we aimed to draw a portrait of the actual standard of care around europe as well as understand the preferences and opinions of the participating experts in choosing between one- or two - stage revision surgery .
the results of this survey were already made public during the crossfire session held at the last day of the meeting .
this questionnaire was previously reviewed and approved by ebjis board members including its president at the time , dr heinz winkler .
the final format of the survey was then sent via e - mail to all 34th ebjis annual meeting participants one week before the event .
participants were explained the motives of the survey and asked to participate by anonymously answering the online survey .
the survey comprised 30 questions , divided in two groups : 10 multiple - choice questions [ personal , institutional and treatment choice information ] and 20 questions containing a series of factors with influence on pji revision procedures which were graduated by importance according with a 5-point likert scale .
categorical variable associations were dealt using pearson 's chi - squared test and likert scales using either student 's t - test or analysis of variance ( anova ) with post - hoc analysis , assuming both parametric tests as adequate according to the central limit theorem .
data processing was performed with ibm spss statistics version 20.0.0 , with the assumption of statistical significance for a p - value under 0.05 .
in total 502 delegates from 51 countries participated in the congress , of which 143 or 28% ( from 35 different countries ) responded this survey .
one can observe the vast majority of respondents are very skilful orthopaedic surgeons with a large experience in treating musculoskeletal infections .
they are evenly distributed among high and low volume hospitals . even in this highly specific group of experts
most of them work in institutions who treat less than 50 prosthetic joint infection cases yearly . a dedicated
bone and joint infection unit is part of the practice in about half of the cases and infectious diseases or microbiology experts are the most common medical specialty cooperating with orthopaedic surgeons ( table 2 ) .
predominantly two - stage is the most frequent choice for revision surgery of a chronically infected total joint arthroplasty .
about one - third answer they perform two- or one - stage according to the conditions of each specific case and only about 5% primarily perform one - stage .
it is noteworthy that among those selectively choosing two- or one - stage surgery the estimated proportion of one - stage is clearly under 50% . in respect to the degree of importance of different factors to choose a two - stage and not one - stage procedure , respondents pointed out as most relevant : 1 ) the presence of significant soft - tissue compromise ( median 4.0,interquartile range 2.0 ) ; 2 ) failure of previous ( single or staged ) revision surgery attempts ( median 4.0,interquartile range 1.5 ) ; 3 ) unclear infective microorganism(s ) preoperatively ( median 4.0,interquartile range 2.0 ) ; 4 ) highly resistant infective microorganism : methicilin - resistant s.aureus , vancomycin - resistant enterococci , esbl gram negative ( median 4.0,interquartile range 2.0 ) ; 5 ) infective microorganism : methicilin - resistant coagulase - negative staphylococci ( median 4.0,interquartile range 2.0 ) ; 6 ) patient presenting with concurrent sepsis / systemic infection ( median 4.0,interquartile range 2.0 ) ; 7 ) patients presenting with immunosuppression ( median 4.0,interquartile range 2.0 ) .
figures 2a and b explore the answer results according to the chosen philosophy for revision surgery .
those who advocate for one - stage systematically seem to give less importance to certain factors that are more consensual among the other two philosophies ( e.g soft tissue compromise , infection spreading to the neurovascular bundle , failure of previous revision surgery attempts , infection by methicillin - resistant coagulase - negative staphylococci or gram negatives ) . on a secondary and curious analysis
regarding the influence of several personal , local or institutional factors influence on chosen philosophy for revision surgery it was possible to find a significant trend towards more selective one- or two - stage approach in higher volume centres .
no other collected variables seem to influence the choice of philosophy including the number of pji cases treated yearly or the presence of a dedicated bone and joint infection unit ( table 5 ) .
prosthetic joint infection ( pji ) is one of the most challenging and frequent complications after total joint arthroplasty 1,2 .
a wide spectrum of uncertainties surrounding the best way to diagnose and treat this condition still persist despite the recent focus on this condition .
several attempts have been made lately to try and produce guidelines or even worldwide consensus 3,4 .
although these documents should be considered as a support for physicians managing pji cases according to the best known practices in the present , they do acknowledge that much is yet unclear .
one such ambiguous topic is whether revision surgery of the chronic infection should be made in one or two stages .
two - stage exchange consists of debridement of all non - viable tissues , resection of the infected implant with or without placement of a temporary antibiotic - impregnated cement spacer and delayed reimplantation of a new prosthesis in a separate surgery after infection is deemed to be eradicated . in a one stage exchange ,
although two - stage surgery is traditionally considered to be the safest option in regard to the chance for successful eradication of infection there are those who disagree and advocate similar success rates using a one - stage approach 5,6,7,8,9 .
in fact , there are several potential advantages of a successful one - stage revision .
it is better for both the patient ( only one surgery ; no disability period between stages and subsequent increased quality of life ; and perhaps improved final functional outcomes ) and for the healthcare systems ( reduced costs and workload ) . on the other hand , there are also potential disadvantages even if you assume a similar success rate in infection eradication . these disadvantages are chiefly related to the more aggressive bone and soft tissue debridement that is required and the technical alternatives for reconstruction necessary that may in some cases compromise a future re - revision procedure if it should become necessary . for these reasons
there is a third alternative philosophy that advocates a more selective approach to the problem 10,11,12 .
it is considered that some clinical scenarios may be addressed in one - stage and depending on different patient local and systemic factors , the infecting pathogen(s ) and its antibiotic susceptibility a two - stage approach may be indicated .
although this third way is gaining momentum , we are still a long way from a wide agreement on the specific indications for such an approach 3,4
. the results of this survey are in no way intended to serve as a review or consensus paper on this controversial and difficult topic but only as a contribution in helping us understand the current state of the art around europe .
the strength of these results is that they reflect the opinion of a sample of very experienced group of people ( including 28 heads of department ) with many years of dedication to bone and joint infections .
it is also relevant that even among the participants of a very specialized conference such as the european bone and joint infection society annual meeting , almost half work without the support of a multidisciplinary dedicated team .
one can only hypothesize that the vast majority of orthopaedic surgeons dealing with pji will face a similar problem .
two - stage approach continues to be the most commonly performed procedure around europe with only a small percentage of respondents acknowledging primarily one - stage as their chosen philosophy for revision .
there is nonetheless a significant proportion of people choosing to do a one- or two - stage approach according to the specific clinical scenario .
personal factors such as medical degree or even years of experience in dealing with musculoskeletal infections seem not to play a role in the chosen philosophy .
although a slight preference for a selective one- or two - stage approach could be found in centres preforming more total joint replacements per year , the same trend was not found in those treating more infection cases per year .
other institutional variables such as the type of institution you work on or even the presence or absence of support by a multidisciplinary team seem to have no influence .
host medical conditions , either local such as inadequate soft tissue envelope or systemic such as concurrent sepsis , immunosuppression or even uncontrolled diabetes seem to be much more important determinants . pathogen related issues such as unclear infective microorganism(s ) preoperatively or highly resistant microorganisms such as methicillin resistant s. aureus , vancomycin resistant enterococci or esbl - producing gram negative are also voted as very important factors .
there is a clear need of further research to help establish the relative importance of each host and pathogen factor . to determine the individual impact of each variable on the final outcome could help find out
such a standardized staging and prognosis system may ultimately enlighten the choice between one- or two - stage approaches .
a collective effort in conducting rigorous multicentre studies should be considered a priority as they would necessarily lead to improved patient outcomes .
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background : prosthetic joint infection ( pji ) is one of the most challenging problems in orthopaedic surgery and musculoskeletal infections specifically .
some very important controversies remain and strong evidence - based recommendations are still lacking in many clinical aspects .
therefore , an undisputed methodology of treatment does not exist yet and there are many different valid approaches.purposes : to draw a picture of the different practice patterns around europe and understand the motivations of the european bone & joint infection society ( ebjis ) members in choosing between one- or two - stage revision surgery in treating chronic pji.methods : the participants of the 34th ebjis annual meeting were surveyed through an online questionnaire .
the survey assessed the main philosophy in the treatment of chronic pji , personal and institutional information as well as the importance of different factors in choosing two - stage or one - stage procedures.results : one hundred and forty - three participants responded to the survey , including a significant group of skilful orthopaedic surgeons with large experience in treating musculoskeletal infections .
primarily two - stage was the most common philosophy regrading treatment of chronic pji ( 60.1% ) , followed by two - stage or one - stage accordingly ( 34,8% ) and primarily one - stage ( 5,1% ) .
significant soft tissue compromise , failure of previous revision surgery attempts , highly resistant or unclear infective microorganism(s ) preoperatively and patient presenting with sepsis or immunosuppression , were considered the more relevant factors in choosing two - stage instead one - stage procedures.interpretation : treatment of chronic pji is challenging and demanding .
an open dialogue to share the different experiences and a collective effort to plan a major multicentre research in order to establish standardized protocols are essential .
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members of the scientific community are increasingly expected to share data , and to do so in a standards - compliant manner .
this is evidenced by the recent mandates , announcements , and requests for information by the funding agencies15 and journals,6 and numerous essays and announcements by the scientific community,710 including pre - competitive initiatives by the life science industry.11 however , the scientific community is not necessarily well poised to comply.12 all stakeholders funders , journal editors , researchers and those supporting them , struggle to navigate the existing standards and make informed decisions.13 as an example , in 2009 one of our groups aimed to create a standards - compliant , integrated data repository for clinical and omics data , among other types .
through subsequent efforts to answer this question , three key points have become clear :
different groups and individuals have different definitions for what constitutes a
right standard across all cases ; rather , one must select a standard ( or even specific pieces of a standard ) based on one 's particular needs.integrated resources and registries are needed to help researchers navigate the fluid standards landscape and to choose and implement the right standard for their respective project .
right standard across all cases ; rather , one must select a standard ( or even specific pieces of a standard ) based on one 's particular needs . integrated resources and registries
are needed to help researchers navigate the fluid standards landscape and to choose and implement the right standard for their respective project .
the focus for that project was on omics data standards , but these points apply across the spectrum of biomedical data types . high - dimensional
big data equate to large numbers of parameters , which in turn require yet more data for sufficient statistical power .
importantly , this massive amount of data lends itself to many different analytic approaches , putting comprehensive analysis beyond the capabilities of any one researcher . the size and complexity of these data , combined with growing scarcity of research funding and the quest for personalized medicine ,
make it increasingly important to maximize the utility of research dollars through data sharing and re - use .
efforts to this end are demonstrated by a spate of new data sharing and aggregation initiatives by academics , private
public partnerships , and publishers , for example sage bionetworks,14 the pistoia alliance ( http://www.pistoiaalliance.org ) and dryad,15 among others.1618 at the national level in the usa , the data sharing trend is reflected in programs such as the national institutes of health 's ( nih ) recently announced big data to knowledge ( bd2k ) initiative,19 and the white house office of science and technology policy 's recent directive that the results of government - funded research be made publicly available.20 the innovative medicines initiative ( http://www.imi.europa.eu/ ) is europe s largest public private initiative that supports collaborative research projects and builds networks of industrial and academic experts in order to boost pharmaceutical innovation in europe .
internationally , the research data alliance ( https://rd-alliance.org/ ) has been established by an international steering group from funding agencies in the usa , eu and australia ; and recently the global alliance for genomic and clinical data sharing has brought together over 70 leading healthcare , research , and disease advocacy organizations , involving researchers from more than 40 countries , to enable secure sharing of genomic and clinical data.21 these types of initiatives , together with the evolving portfolio of grass - roots standards , have enhanced the need to maximize awareness and discoverability of standards .
there is a clear need for some level of coordination , without taking the form of a top - down authority .
how can we avoid requiring would - be standard adopters to spend considerable time and effort becoming well versed with a multitude of standards solely in order to rule most of them out ?
the international organization for standardization defines a standard as a document that provides requirements , specifications , guidelines or characteristics that can be used consistently to ensure that materials , products , processes and services are fit for their purpose.27 standards range from de jure , that is , ordained by some official organization such as the international organization for standardization or the american national standards institute , to de facto , that is , developed by grass - root initiatives and commonly adopted , but not prescribed by an official or specific authority .
the biosharing registry ( http://biosharing.org/ ) houses a fairly comprehensive , curated list of data standards ( primarily de facto ) in the life science , environmental , and biomedical space .
first , content standards take the form of reporting guidelines , for example , minimum information checklists .
these vary from general guidance to itemized prescriptions of the information that should be provided ( ie , curation guidelines ) , including both data and metadata .
the second category consists of syntax standards in the form of representations and formats that facilitate the exchange of information .
these fall broadly into two types : delimited text , or a markup language such as xml .
third are the semantic standards in the form of terminology artifacts , such as controlled vocabularies or ontologies .
these add an interpretive layer to the data by defining the concepts or terms in a domain , and in some cases the relationships between them .
other discussions of standards include the notion of a data model , which extends beyond terms and their definitions to describe the relationships between concepts in a domain.28 other groups also use additional terms such as conceptual model , conceptual schema , ontology , or domain analysis model,2932 but generally differ on what each of these terms means .
this is in fact part of the confusion even data standard experts do not agree on what constitutes a data standard .
nevertheless , focusing just within the context of transcriptomics , preliminary investigation yielded a list of 15 potentially relevant standards ( table 1 ) .
note that this list could grow depending on the type of sample and organism used , as many terminologies are species specific .
now imagine if a researcher has an associated dataset from a proteomics investigation , for example .
how is a mere mortal to sort through these ? a sampling of ( some of the ) standards related to microarray - based transcriptomics , generated by non - experts for evaluation of relevance to a project involving microarray - based transcriptomics data
in biomarker discovery , the phrase fit - for - purpose refers to the notion that the degree of rigor for assay validation should be tailored to the intended purpose of a given biomarker study.33 the same is true for data standards adoption .
while each individual project will inevitably have its own specific requirements , it can be useful to group projects across a spectrum of rigor . at the lowest level
, there is the use case of data sharing within a laboratory or between collaborators .
while minimum information guidelines should be followed , for the most part any documentation need only be human readable , and issues requiring clarification are merely a walk down the hall or an e - mail away ( at least until the student graduates or the postdoc moves on ) . data that are to be shared publicly , for example , accompanying a publication , require more rigor .
ideally , a prospective consumer of the data can both understand and reproduce those data without needing to contact the original author .
furthermore , much of the content of publications is now aggregated and curated by various online resources .
these value - added services can be much more efficient and effective at making content available via secondary sources when quality data standards are used .
minimally structured data can be very helpful for such purposes ; for example , the use of a unique identifier to describe a molecule or a standardized vocabulary term to denote the disease area under study .
the highest level of rigor is needed for contribution of data to a structured data repository . in this case
, additional effort is warranted in the form of structured fields and a standardized , machine - readable format .
such rigor enables querying across multiple datasets and integrative meta - analysis combining more than one set .
one key point in differentiating between these levels of rigor is that there are different flavors of annotation .
at every level , there is a difference between what needs to be documented , and what needs to be documented in a structured and queryable fashion .
while the option exists to select a standard that allows for maximum structure and adopt it only loosely , complexity can turn off would - be standards adopters , as well as waste time in development if such rigor will ultimately never be needed .
categories of criteria to be used in evaluating data standards for adoption include :
the standard itself
specification documentationease of implementation ( eg , level of documentation , requirement for programmer support)human and machine readabilityformal structureexpressivity the breadth of information that can be representedease of use , for example , minimal required fields , text - based interface familiarity to biologists.adoption and user community
broad adoption and implementation , outside the initial groupsupport supplied by the user communityuse by community databasessoftware development that supports the standard ( eg , for curating , submitting to databases)responsiveness to community requestsavailability of examples of userequirements of relevant authoritative bodies , for example , funders ( nih , national science foundation , centers for medicare & medicaid services ) , publishers , etc.additional factors
integration / compatibility with other standardsextensibility and flexibility to cover new domainsconversion and mapping , when applicablecost ( eg , open vs licensing fee ) . the standard itself
specification documentationease of implementation ( eg , level of documentation , requirement for programmer support)human and machine readabilityformal structureexpressivity the breadth of information that can be representedease of use , for example , minimal required fields , text - based interface familiarity to biologists .
specification documentation ease of implementation ( eg , level of documentation , requirement for programmer support ) human and machine readability expressivity
the breadth of information that can be represented ease of use , for example , minimal required fields , text - based interface familiarity to biologists .
adoption and user community
broad adoption and implementation , outside the initial groupsupport supplied by the user communityuse by community databasessoftware development that supports the standard ( eg , for curating , submitting to databases)responsiveness to community requestsavailability of examples of userequirements of relevant authoritative bodies , for example , funders ( nih , national science foundation , centers for medicare & medicaid services ) , publishers , etc . broad adoption and implementation , outside the initial group support supplied by the user community use by community databases software development that supports the standard ( eg , for curating , submitting to databases ) responsiveness to community requests availability of examples of use requirements of relevant authoritative bodies , for example , funders ( nih , national science foundation , centers for medicare & medicaid services ) , publishers , etc .
additional factors
integration / compatibility with other standardsextensibility and flexibility to cover new domainsconversion and mapping , when applicablecost ( eg , open vs licensing fee ) .
integration / compatibility with other standards extensibility and flexibility to cover new domains conversion and mapping , when applicable cost ( eg , open vs licensing fee ) .
of course , specific projects may have additional criteria to add , and different projects will place different weight on the different items .
unfortunately , standards adoption , when it happens , is often determined less by an objective criteria - based evaluation and more based on historical precedent ( my advisor used standard x ) , marketing ( i saw a press - release about standard x ) or sociopolitical circumstance ( i know someone on the standard x team ) .
what makes it even more difficult to select standards empirically , based on objective criteria , is that standards are often complex .
even well - documented standards can be dense and impenetrable to prospective users who were not involved in their development .
other factors include the desire for some level of control , or recognition for doing the work .
the recent data and informatics working group report to the advisory committee to the director of the nih included recommendations to establish a minimal metadata framework for data sharing , and to create catalogs and tools to facilitate data sharing.2 a truly minimal set of metadata elements is important if we are to have any hope of compliance because the activation energy required for data curation and annotation represents a significant hurdle in facilitating data sharing . the minimum information for biological and biomedical investigations ( mibbi ) project , part of the broader biosharing effort , worked with different research communities to coordinate their minimum information checklists,34 but each community has some unique requirements . also , data annotation presents an inherent tension : the easier we make it for investigators to annotate their datasets , the harder it will be to ensure discoverability .
conversely , the more discoverable we make the datasets , for example , through annotation using controlled terminologies , the more burden we put on the data generators .
biosharing is a great resource to register and discover standards , and has adopted the initial set of criteria described above , requiring the communities to do a self - appraisal and tag their entries accordingly . the standards development community also has an active role to play if they wish to maximize the use and uptake of their work .
reviewers of publications and associated adherence to data standards should include biocurators . in the absence of
widely agreed upon metrics to evaluate community standards , the decision about which is the right standard falls on the researcher . for reasons described above
table 2 lists some potential resources / functionalities to address this problem . for any of these resources , it is important to note that technology is dynamic , and therefore so are any associated standards .
potential resources to assist in the selection and adoption of appropriate standards ncbo , national center for biomedical ontology ( http://www.bioontology.org/ ) ; rdf , resource description framework .
while one can conjure up motivating scenarios from a regulatory or archiving standpoint , the value proposition behind adherence to standards only really makes sense if data are to be shared beyond the team that originally created them .
thanks in part to policies put in place by some funders and publishers,8 many high throughput datasets are made publicly available and , at some level , standards compliant .
some apply only to data generation through grants that exceed us$500 000.2 some require only a very low bar of compliance , and data are still difficult if not impossible to interpret . in many cases ,
the policies are simply not enforced,7 although the government and the nih have recently taken steps to rectify that fact.3
19
20 ideally , it should be noted , researchers themselves would be shielded from the complexity of data standards .
developers , informaticists , and curators are perhaps better equipped to delve into data standards than would be a clinician or bench scientist , but even they are typically not experts in specialized standards . in an ideal world , data generators would have access to user - friendly tools that enable the seamless use of relevant standards and can be customized to fit the different data and domain needs.9 the actual standards would be hidden from the data generators , and their use made automatic through intuitive , user - friendly tools . although we have described some tools for the discovery and evaluation of standards if one is so inclined , the real challenge is incentivizing researchers to go to the trouble .
this will probably need a combination of proverbial carrots and sticks . on the penalty side
, funders and publishers must continue to develop and publicize progressive data - sharing policies , and to enforce those policies through the delay of publication or future funding , if necessary . on the incentives side ,
a formal system for data citation must be developed , and those citations acknowledged and valued by funders , professional organizations , and university promotion and tenure committees .
recent activity in the realm of data publishing has been an important first step.35
36 only when obstacles are minimized and incentives are properly aligned will investigators be able to justify the effort required to do the right thing .
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in the era of big data , omic - scale technologies , and increasing calls for data sharing , it is generally agreed that the use of community - developed , open data standards is critical .
far less agreed upon is exactly which data standards should be used , the criteria by which one should choose a standard , or even what constitutes a data standard .
it is impossible simply to choose a domain and have it naturally follow which data standards should be used in all cases .
the right standards to use is often dependent on the use case scenarios for a given project .
potential downstream applications for the data , however , may not always be apparent at the time the data are generated . similarly , technology evolves , adding further complexity . would - be standards adopters must strike a balance between planning for the future and minimizing the burden of compliance .
better tools and resources are required to help guide this balancing act .
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by the term cardiac metastases , we define distant spread of a tumor to any of the structures composing the heart and also tumors affecting the heart cavities or producing actual intracavitary neoplastic thrombi .
any malignant neoplasm , in particularly melanoma , and lung and breast carcinoma can spread to the heart through four alternative paths : by direct extension , through the bloodstream , through the lymphatic system and by intracavitary diffusion through either the inferior vena cava or the pulmonary veins .
we report a case of an elderly man with a lung cancer discovered after a metastatic diffusion to heart and brain .
a 79-year - old male presented to the emergency department with a confusional state characterized by clouded consciousness , disorientation in time and space , difficulty in speech and gait and mouth deviation to the right .
he also showed dysarthria , right hemiparesis , hemi - hypoesthesia and babinski sign , bilateral grasping - sign and a positive epstein sign .
he was a former heavy smoker , hypertensive and diabetic patient with a previous history of ischemic cardiomyopathy and prior vascular and cardiac surgery due to aneurysm of the abdominal aorta and myocardial infarction , respectively .
a reduced breath sound on the right hemi - thorax was found at the chest examination while cardiac objectivity was unremarkable .
the electrocardiogram showed sinus rhythm with a heart rate of 65 beats / m , some premature ventricular complex and left atrial enlargement . chest x - ray revealed two nodular opacities on the right lung , the largest of which of 2.0 cm . owing to the neurological status , patient
was immediately submitted to a noncontrast - enhanced computed tomography ( ct ) head scan demonstrating a large , but nonspecific cerebral edema in the right frontal , temporal and parietal lobe and then , referred to the neurological department of our institution .
a brain magnetic resonance imaging ( mri ) , a chest ct scan and a transthoracic echocardiogram ( tte ) were quickly scheduled .
this last identified a large , round - shaped mass , in the left atrium ( la ) , mobile during the cardiac cycle suspected to arise from left superior pulmonary vein .
however , this approach failed to provide a full characterization of the lesion , comprehensive of size , morphology and spatial relationships with surrounding structures .
therefore , patient was immediately submitted to transesophageal echocardiogram ( tee ) . through this examination
we were able to :
confirm that the mass came from the left superior pulmonary vein [ figure 1],evaluate its size ( linear dimensions = 3.5 3.6 cm with a largest planimetric area of about 10 cm ) and morphology ( inhomogeneous texture for the presence of anechogenic areas in the context of a hyperechogenic tissue [ figure 1 ] andrule out any hemodynamic consequence into the left cavities by color doppler flow analysis .
confirm that the mass came from the left superior pulmonary vein [ figure 1 ] , evaluate its size ( linear dimensions = 3.5 3.6 cm with a largest planimetric area of about 10 cm ) and morphology ( inhomogeneous texture for the presence of anechogenic areas in the context of a hyperechogenic tissue [ figure 1 ] and rule out any hemodynamic consequence into the left cavities by color doppler flow analysis .
multiplanar transesophageal echocardiogram shows the mass ( a ) coming from the left superior pulmonary vein ( b and c ) and prolapsing into the left atrium ( d ) on the 2 day , after admission , the prearranged brain mri showed one heterogeneous , hypointense lesion , at the frontal right lobe , with perilesional vasogenic edema and partial compression of right lateral ventricle .
finger - like appearance in the right cerebellar hemisphere , compatible with neoplastic edema was also identified [ figure 2a and b ] .
( a and b ) brain magnetic resonance imaging t2-weighted axial images ( for explanations , please see the text ) .
( c ) axial unenhanced computed tomography scan shows a left inferior pulmonary lobe solid mass that infiltrates left pulmonary veins and left atrium ( white arrows ) due to the above - mentioned imaging findings from cerebral and cardiac evaluation and in relation to the presence of nodular opacities on chest x - ray , a metastatic lung cancer was suspected .
accordingly , patient underwent chest ct scan showing a solid mass in the left inferior pulmonary lobe , infiltrating the left pulmonary veins and left atrium [ figure 2c ] .
focal thickening of adjacent pleura was also seen and on parenchymal window , a pulmonary mass with maximum axial diameter of 57 mm was identified .
therefore , a starting diagnosis of lung cancer with brain metastases and intracavitary heart dissemination through the left superior pulmonary vein was made .
however , to achieve a definitive diagnosis from histology , patient was scheduled for a biopsy of pulmonary lesions but , unfortunately , he suddenly died before we can conclude the diagnostic procedures .
the heart can be metastasized by any malignant neoplasm able to spread to distant sites , in particularly melanoma , lung tumors , breast carcinoma and linfoma .
lung tumors are the leading metastatic cancers that spread to the heart through direct invasion , and rarely extend into the chambers of the left side of the heart via pulmonary veins .
neoplastic thrombi can also be produced in the la , after tumor embolism through the venous circulation of the lungs , mostly in patients with lung carcinoma .
large thrombi can even involve the mitral valve , and more commonly facilitate distant metastases .
although , extension from the pulmonary veins into the la is rare , this circumstance has been reported in limited case reports . in lung carcinomas , rate of metastasis
adenocarcinoma spread to the heart in 26% of cases , squamous cell carcinoma in 23.4% , and undifferentiated carcinoma in 21.2% .
intra - pulmonary veins and intra - atrial masses can induce widespread emboli and circulatory impairment via outflow obstruction .
in fact , sudden severe complications , including cerebral infarction , peripheral arterial occlusion and syncopal attacks have been reported in patients with this type of extension .
echocardiography , the first - choice modality , provides essential information with regard mass size , location , mobility , anatomical relationships , hemodynamic consequences and response to the treatment .
especially , tee adds important information , and it should always be considered when the transthoracic images are inappropriate , and in all the cases in which embolic sources are researched . particularly , echocardiography is helpful for la masses differential diagnosis , including primary or secondary cardiac tumors , infective endocarditis ( ie ) and thrombi . despite ,
ie may be excluded on the basis of medical history and clinical data , echocardiography is anyway an important tool providing unique diagnostic elements . in general ,
infective vegetation presents as an oscillating mass attached to a valvular structure , with a motion independent to that of the valve , however it may also present as non oscillating masses with atypical location .
tee enhances the sensitivity of tte to about 85 - 90% for the diagnosis of vegetations , while more than 90% specificity has been reported for both tte and tee .
on echocardiography , myxomas appear as mobile masses attached to the endocardial surface through a stalk , usually arising from the interatrial septum in the region of fossa ovalis .
however , 25% of myxomas may originate away from the atrial septum . in this case , the observed origin of the la mass from pulmonary vein , by tee , effectively allowed excluding a myxoma .
however , in case the stalk is not clearly visible on echo , the diagnosis can not be made by echocardiography and requires further imaging techniques , like mri or ct .
, the thrombus usually lies on the wall of la through a large base of implantation and can involve any segment , more often the la appendage in patients with atrial fibrillation ( af ) . in some cases ,
a diagnosis of thrombus should be highly suspected if an la mass is associated with af , dilated la , mitral stenosis , low ejection fraction , prosthetic mitral valves or spontaneous atrial contrast echoes .
nevertheless , as a general rule , nonseptal origin and pulmonary vein extension of the tumor strongly indicate the possibility of a secondary tumor .
briefly , tte and tee have been fundamental tools for differential diagnosis in this case .
however , it remains unclear whether the cerebral embolism presenting as an acute neurological syndrome was due to metastasis of the presumed primary lung cancer via bloodstream or to embolization of the la mass .
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metastatic cardiac malignancies are significantly more common than primary cardiac tumors .
primary lung cancers often invade the heart locally and they can also spread to the heart via the blood , lymphatic system or both ; in contrast , an extension to the left atrium from the pulmonary vein is very rare . here
, we report a case of a 79-year - old man with an acute neurological syndrome , in which an integrated multimodality imaging approach has allowed to discovery a lung cancer with cerebral embolism and intracavitary heart dissemination through the left superior pulmonary vein .
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multifocal intraocular lens ( iol ) implantation , introduced more than 20 years ago , is a popular procedure that achieves good visual acuity for both distance and near vision.1,2 generally speaking , there are 3 types of multifocal iols : refractive , diffractive , and a combination of diffractive and refractive.3,4 multifocal iols with a diffractive optic design have been proven to provide a significantly better near vision and reading performance than refractive multifocal iols and monofocal iols.5 with the addition of a + 3.75 d near power , good intermediate distance visions from diffractive multifocal iol models were also proven in previous studies.6,7 in this study , the visual and optical performances were evaluated and compared between two new - generation multifocal iol models , both with a near addition power of + 3.75 d but with different haptic designs .
this retrospective , single - center study comprised cataract patients who underwent cataract surgery with diffractive multifocal iol implantation from june 2012 to may 2013 at the shinagawa lasik center , tokyo , japan .
the inclusion criteria were patients with incipient or moderate cataracts resulting in a significant reduction of the visual quality .
the exclusion criteria were patients with active ocular diseases or significant amounts of corneal aberrations observed on the topography map .
eyes with serious postoperative complications ( such as posterior - capsular opacity [ pco ] and obvious capsule contraction ) were likewise excluded to achieve the accurate outcomes of visual and optical performance .
these included eyes were randomly divided into two groups and implanted with multifocal iols : acriva reviol bb mfm 611 ( vsy biotechnology , amsterdam , the netherlands ) for group 1 and acriva reviol bb mf 613 ( vsy biotechnology , amsterdam , the netherlands ) for group 2 . all patients read and signed the informed consent forms , which explained the surgical procedure , possible risks , and patient rights .
the study was carried out with approval from the institutional review board ( matsumoto clinic , tokyo , japan ) , and all patients consented .
preoperative examinations included uncorrected distance visual acuity ( udva ) , corrected distance visual acuity ( cdva ) , manifest refraction spherical equivalent ( mrse ) , uncorrected near visual acuity ( unva ) , corrected near visual acuity ( cnva ) , slit lamp biomicroscopy , funduscopy , intraocular pressure ( computerized tonometer ; topcon corp , tokyo , japan ) , tomography ( pentacam ; oculus optikgerte gmbh , wetzlar , germany ) , and corneal endothelial cell count ( noncon robo fa3609 ; konan medical inc , hyogo , japan).8 iolmaster ( carl zeiss meditec , jena , germany ) was used for the biometry and power calculation of the multifocal iols .
the diffractive multifocal iol with a plate haptic design used in this study was the acriva reviol bb mfm 611 iol ( figure 1 , left ) . according to the manufacturer , this diffractive multifocal iol has 3.75 d of addition power and ultradefinition aspheric optic design .
it has been verified to have smooth ridges at the diffractive ring transitions to increase retinal image quality .
it also has a 360 degree continuous square optic and haptic edge to reduce the pco formation.6 the acriva reviol bb mf 613 iol has the same ultradefinition aspheric optic design as the acriva reviol bb mfm 611 iol , but with a modified - c haptic design ( figure 1 , right ) .
sixty percent of the intraocular light was allotted for far focus , and 40% for near .
all surgeries were performed by the same surgeon ( mt ) , using femtosecond laser - assisted phacoemulsification.9 after topical anesthesia and adequate dilation , femtosecond laser ( catalys precision laser system ; optimedica corp , sunnyvale , ca , usa ) was used for the continuous curvilinear capsulorrhexis ( ccc ) and lens fragmentation of all cataracts .
viscoelastic material ( provisc ; alcon corp , fort worth , tx , usa ) was injected , and the cut capsule was removed .
the postoperative medications were given as follows : a 500 mg oral levofloxacin ( cravit ; daiichi sankyo , tokyo , japan ) once a day for 3 days and 0.1% diclofenac sodium eye drops ( diclofenac ophthalmic solution 0.1% ; nitto medic , toyama , japan ) , 0.1% dexamethasone metasulfobenzoate sodium ( dex ; nitto medic ) , and 0.5% moxifloxacin hydrochloride ( vegamox ; alcon corp ) five times a day for 1 week .
after 1 week , diclofenac use was reduced to 4 times a day for 1 month , and the latter drugs were shifted to topical 0.1% fluorometholone ophthalmic suspension ( fluorometholone ophthalmic suspension 0.1% t ; nitto medic ) and 0.3% ofloxin ophthalmic solution ( ofloxin ophthalmic solution 0.3% ; nitto medic ) , prescribed four times a day up to 1 month , after which their use was discontinued .
postoperatively , visual and refractive examinations were routinely performed during the 6 month follow - up , following the same investigational protocol . at 6 months
postoperatively , a defocus curve was obtained by spectacle defocus from + 2.00 d to 5.00 d in 0.50 d steps from the patient s manifest refraction .
the direct comparison method was used to compare the visual acuities between two groups at each defocus level.10 functional acuity contrast was tested using the takagi contrast glare tester cgt-1000 ( takagi seiko co ltd , nagano - ken , japan ) for different ambient light conditions ( with and without glare ) at luminance levels of 21 candelas ( cd)/m ( day testing ) and 11 cd / m ( night testing).11 wave - front aberration was tested using the wave - front analyzer kr-1w ( topcon corp ) 6 months postoperatively .
statistical analysis was performed with jmp 9 statistical package ( sas institute , inc , cary , nc , usa ) software .
descriptive statistical results were presented as mean and standard deviation . to compare the differences between the two groups , one - way analysis of variance and kruskal
this retrospective , single - center study comprised cataract patients who underwent cataract surgery with diffractive multifocal iol implantation from june 2012 to may 2013 at the shinagawa lasik center , tokyo , japan .
the inclusion criteria were patients with incipient or moderate cataracts resulting in a significant reduction of the visual quality .
the exclusion criteria were patients with active ocular diseases or significant amounts of corneal aberrations observed on the topography map .
eyes with serious postoperative complications ( such as posterior - capsular opacity [ pco ] and obvious capsule contraction ) were likewise excluded to achieve the accurate outcomes of visual and optical performance .
these included eyes were randomly divided into two groups and implanted with multifocal iols : acriva reviol bb mfm 611 ( vsy biotechnology , amsterdam , the netherlands ) for group 1 and acriva reviol bb mf 613 ( vsy biotechnology , amsterdam , the netherlands ) for group 2 . all patients read and signed the informed consent forms , which explained the surgical procedure , possible risks , and patient rights .
the study was carried out with approval from the institutional review board ( matsumoto clinic , tokyo , japan ) , and all patients consented .
preoperative examinations included uncorrected distance visual acuity ( udva ) , corrected distance visual acuity ( cdva ) , manifest refraction spherical equivalent ( mrse ) , uncorrected near visual acuity ( unva ) , corrected near visual acuity ( cnva ) , slit lamp biomicroscopy , funduscopy , intraocular pressure ( computerized tonometer ; topcon corp , tokyo , japan ) , tomography ( pentacam ; oculus optikgerte gmbh , wetzlar , germany ) , and corneal endothelial cell count ( noncon robo fa3609 ; konan medical inc , hyogo , japan).8 iolmaster ( carl zeiss meditec , jena , germany ) was used for the biometry and power calculation of the multifocal iols .
the diffractive multifocal iol with a plate haptic design used in this study was the acriva reviol bb mfm 611 iol ( figure 1 , left ) . according to the manufacturer , this diffractive multifocal
it has been verified to have smooth ridges at the diffractive ring transitions to increase retinal image quality .
it also has a 360 degree continuous square optic and haptic edge to reduce the pco formation.6 the acriva reviol bb mf 613 iol has the same ultradefinition aspheric optic design as the acriva reviol bb mfm 611 iol , but with a modified - c haptic design ( figure 1 , right ) .
sixty percent of the intraocular light was allotted for far focus , and 40% for near .
all surgeries were performed by the same surgeon ( mt ) , using femtosecond laser - assisted phacoemulsification.9 after topical anesthesia and adequate dilation , femtosecond laser ( catalys precision laser system ; optimedica corp , sunnyvale , ca , usa ) was used for the continuous curvilinear capsulorrhexis ( ccc ) and lens fragmentation of all cataracts .
viscoelastic material ( provisc ; alcon corp , fort worth , tx , usa ) was injected , and the cut capsule was removed .
in all cases , the postoperative medications were given as follows : a 500 mg oral levofloxacin ( cravit ; daiichi sankyo , tokyo , japan ) once a day for 3 days and 0.1% diclofenac sodium eye drops ( diclofenac ophthalmic solution 0.1% ; nitto medic , toyama , japan ) , 0.1% dexamethasone metasulfobenzoate sodium ( dex ; nitto medic ) , and 0.5% moxifloxacin hydrochloride ( vegamox ; alcon corp ) five times a day for 1 week .
after 1 week , diclofenac use was reduced to 4 times a day for 1 month , and the latter drugs were shifted to topical 0.1% fluorometholone ophthalmic suspension ( fluorometholone ophthalmic suspension 0.1% t ; nitto medic ) and 0.3% ofloxin ophthalmic solution ( ofloxin ophthalmic solution 0.3% ; nitto medic ) , prescribed four times a day up to 1 month , after which their use was discontinued .
postoperatively , visual and refractive examinations were routinely performed during the 6 month follow - up , following the same investigational protocol . at 6 months
postoperatively , a defocus curve was obtained by spectacle defocus from + 2.00 d to 5.00 d in 0.50 d steps from the patient s manifest refraction .
the direct comparison method was used to compare the visual acuities between two groups at each defocus level.10 functional acuity contrast was tested using the takagi contrast glare tester cgt-1000 ( takagi seiko co ltd , nagano - ken , japan ) for different ambient light conditions ( with and without glare ) at luminance levels of 21 candelas ( cd)/m ( day testing ) and 11 cd / m ( night testing).11 wave - front aberration was tested using the wave - front analyzer kr-1w ( topcon corp ) 6 months postoperatively .
statistical analysis was performed with jmp 9 statistical package ( sas institute , inc , cary , nc , usa ) software .
descriptive statistical results were presented as mean and standard deviation . to compare the differences between the two groups , one - way analysis of variance and kruskal
the mean age of all patients was 60.905.72 years ( range , 4576 years ) .
group 1 consisted of 89 eyes of 62 patients aged between 47 and 76 years ( mean age , 60.745.92 years ) ; group 2 included 69 eyes of 45 patients aged between 45 and 73 years ( mean age , 61.135.46 years ) .
table 1 shows the preoperative and 6 month postoperative outcomes in visual and refractive parameters of the two groups .
no statistically significant differences in terms of age , visual acuity , and refractive parameters were found between the two groups preoperatively ( p>0.05 ) . at 6 months
postoperatively , significant improvements in udva , cdva , mrse , unva , and cnva were found in both groups ( p<0.05 ) . comparing the two groups , plate haptic multifocal iols ( group 1 ) provided statistically better outcomes in udva , cdva , and cnva ( p<0.05 ) .
figure 2 summarizes the cumulative percentage of the udva in each group 6 months postoperatively .
eighty - five percent of the eyes in group 1 and 74% of the eyes in group 2 achieved a udva of 20/20 ( 0.00 logmar ) or better .
for unva , 69% of the eyes in group 1 and 54% of the eyes in group 2 achieved jaeger 3 ( 0.18 logmar ) or better at the end of the follow - up .
figure 4 shows the contrast sensitivities of two groups 6 months postoperatively . at any level of the target size ( visual angle , degree [ d ] ) ,
the mean values of contrast sensitivity were higher in group 1 than in group 2 .
these differences between the two groups were statistically significant both under photopic ( 6.3 d , p<0.0001 ; 4.0 d , p<0.0001 ; 2.5 d , p<0.0001 ; 1.6 d , p<0.0001 ; 1.0 d , p<0.0001 ; 0.7 d , p=0.01 ) and mesopic ( 6.3 d , p<0.0001 ; 4.0 d , p<0.0001 ; 2.5 d , p<0.0001 ; 1.6 d , p<0.0001 ; 1.0 d , p<0.0001 ; 0.7 d , p<0.0001 ) conditions . with a slight visual decline in the intermediate range ,
the defocus curves in figure 5 show good functional capacities for distant and near vision of eyes with either of the two multifocal iol models .
statistically significant differences were found between the two groups when the defocus addition was 1.00 d ( p=0.03 ) , 0.50 d ( p=0.00 ) , and 0.00 d ( p=0.02 ) .
the highest near - visual peaks were 0.070.10 logmar for the acriva reviol bb mfm 611 iol and 0.060.09 logmar for the acriva reviol bb mf 613 iol ; both peaks were present at 33 cm ( 3.00 d ) .
table 2 summarizes the comparison of postoperative higher - order aberrations ( hoas ) in the two groups .
no statistically significant difference in corneal hoas for either photopic vision ( 4 mm diameter ) or scotopic vision ( 6 mm diameter ) was found between two groups at the end of the follow - up ( p>0.05 ) .
however , the ocular hoas in group 2 were significantly greater than in group 1 ( p<0.05 ) .
both acriva reviol bb mfm 611 iol and acriva reviol bb mf 613 iol are new - generation diffractive multifocal iol models with a near addition of + 3.75 d and a 360 degree continuous square edge .
previous study has proven that a clear acriva reviol model with plate haptic design ( acriva reviol mfm 611 ) provides excellent visual acuity and contrast sensitivity.6 theoretically , both acriva reviol bb mf 613 and bb mfm 611 should have yielded the same visual and optical performances because they have the same optic design
. however , many factors can actually affect the visual and optical outcomes of multifocal iol implantations and can cause patient dissatisfaction , such as iol decentration or tilt.3,12 haptic deformation and flexion caused by anterior capsule contraction were thought to play major roles in these decentrations and tilts.13,14 the aim of the present study was to evaluate and compare the visual and optical performances of these two multifocal iol models with different haptic designs . before the cataract surgery
, no statistically significant differences were found in age , udva , cdva , mrse , unva , or cnva between the two groups . at 6 months
postoperatively , significant improvements in distance ( 8 lines increased in both groups ) and near ( 7 lines increased in group 1 , 8 lines in group 2 ) visual acuities were found , which confirmed the efficacy of the two multifocal iol models .
nevertheless , the plate haptic iol model ( acriva reviol bb mfm 611 , group 1 ) appeared to yield better visual and optical performances than the c haptic iol model ( acriva reviol bb mf 613 , group 2 ) .
similar outcomes can be found in eyes implanted with some other monofocal and refractive multifocal iol models.1318 for example , the plate haptic iol models of lentis mplus ( oculentis gmbh , berlin , germany ) can provide better refractive predictability and optical quality than the c haptic iol models.14 in the current study , because of the same optic design , the visual and optical differences between two diffractive multifocal iol models might be caused by their different positional stabilities within the capsular bags .
it was found that diffractive iols were much more sensitive to their own stability than monofocal and refractive multifocal iols.13 even a small misalignment of the central concentric rings can lead diffractive multifocal iols to provide significantly reduced results . in the current study ,
the different stabilities between the two models would have been because of the following reasons : first , the haptic designs of these models should have been the major reason for their different stabilities .
it has been found that three - piece c haptic iols had a greater tendency toward more decentration than one - piece iols;18 plate hap - tics provided greater stability for iols than the c haptics and can not be easily affected by the iol orientation.13,18,19 it was also proven that the iol tilt and decentration were correlated with coma and total aberrations.20 because our outcomes include both visual and optical performances , those eyes that could affect the visual and optical measurements ( such as pco and other serious postoperative complications ) were excluded from both groups .
this makes the iol decentration measurements unfeasible for statistically analyzing the stability performance of these iols .
even so , the visual and optical differences between two groups have indirectly reflected that a plate haptic design could provide multifocal iol models better stabilities .
second , the material and biocompatibility of iols have also played important roles in iol stability . for example
, capsule contraction syndrome ( including anterior capsule opacification and capsulorrhexis shrinkage ) was found in the eyes with silicone iols.21,22 in addition , hydrophilic multifocal iols were thought to be more sensitive to postoperative decentration.13 in this study , both multifocal iol models are produced from an ultrapure acrylate monomer that has a water content of 25% .
although this material makes iols much elastic and malleable , it could also render iols soft haptics .
thus , the modified c haptic of the acriva reviol bb mf 613 would be a little weak to stabilize the iol in some patients who have strong capsule contraction syndrome .
perfect ccc can give iols 360 degree support and excellent iol fixation.23 compared with a manual ccc , the femtosecond laser can produce a sharp and clean - edged capsulotomy without radial nicks and tears.2426 meanwhile , a properly sized , shaped , and centered ccc can be precisely created by a femtosecond laser for the ideal iol position.2729 in the present study , a new femtosecond laser device ( catalys precision laser system ) was used on all our cataract patients for the ccc incisions .
no serious intraoperative complication ( such as radial nicks and tears ) occurred during these surgeries .
in summary , both multifocal iol models provided excellent outcomes in distance and near visual acuities and refraction during our 6 month follow - up . compared with the acriva reviol bb mf 613 iol model , the acriva reviol bb mfm 611 iol model has a same optic design but appeared to result in better optical performance .
the plate haptic design seems to support a better stability for the diffractive multifocal iol than the c haptic design .
further studies should be performed to compare their longer - term visual and optic outcomes with other plate or c haptic multifocal iol models .
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purposeto evaluate and compare the visual acuity outcomes and optical performances of eyes implanted with two diffractive multifocal intraocular lens ( iol ) models with either a plate haptic design or a modified - c design.methodsthis retrospective study comprised cataract patients who were implanted with either a plate haptic multifocal iol model ( acrivaud reviol bb mfm 611 [ vsy biotechnology , amsterdam , the netherlands ] , group 1 ) or a modified - c haptic multifocal iol model ( acrivaud reviol bb mf 613 [ vsy biotechnology , amsterdam , the netherlands ] , group 2 ) between june 2012 and may 2013 .
the 6 month postoperative visual acuity , refraction , defocus curve , contrast sensitivity , and wave - front aberration were evaluated and compared between these eyes , using different iol models.resultsone hundred fifty - eight eyes of 107 patients were included in this study .
significant improvement in visual acuities and refraction was found in both groups after cataract surgery ( p<0.01 ) .
the visual acuity and contrast sensitivity were statistically better in group 1 than in group 2 ( p<0.01 ) .
no statistically significant difference in the corneal higher - order aberrations was found between the two groups ( p>0.05 ) .
however , the ocular higher - order aberrations in group 2 were significantly greater than in group 1 ( p<0.05).conclusionat 6 months postoperatively , both acrivaud reviol bb mfm 611 iol and acrivaud reviol bb mf 613 iol achieved excellent visual and refractive outcomes . the multifocal iol model with plate haptic design resulted in better optical performances than that with the modified - c haptic design .
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percutaneous nephrolithotomy ( pcnl ) , first reported in 1976 , is a minimally invasive surgery for the treatment of renal stones .
it has been the preferred procedure for the removal of large renal stones in iran since 1990 . during these years
we encountered an unknown and devastating complication during pcnl last year : a female patient became paraplegic following an uneventful pcnl .
we reviewed the medical literature , but we did not find reports of any similar complications . with consideration of the ethical issues surrounding this event , the urology and nephrology research center , which is affiliated with the shahid beheshti university of medical sciences , planned a widespread research project to study the possible etiologies of this event .
questionnaires consisting of a series of questions about the occurrence of neurologic complications following pcnl were sent to all iranian endourologists .
surprisingly , we encountered new cases of neurologic complications following pcnl that had not been reported as pcnl complications by endourologists because they had found no logical link between the complications and the pcnl .
there are several reports of nonparaplegic neurologic complications following pcnl owing to paradoxical air emboli [ 3 - 7 ] , but to the best of our knowledge , there are no reports of paraplegia following pcnl .
the cases of paraplegia in this study are therefore the first such cases to be presented .
we retrospectively reviewed the medical records for pcnl in 13 different endourologic centers and retrieved data related to neurologic complications , including coma , paraplegia , hemiplegia , and quadriplegia . with the exception of a few differences , all of the endourologists generally performed pcnl in a similar manner .
after the induction of general anesthesia , the patient was located in the lithotomy position and a 5- or 6-fr ureteral catheter , without a ureteropelvic junction occlusion balloon , was inserted through the ipsilateral ureter .
iodinated contrast materials were the most widely used means of opacifying the collecting system , but some endourologists preferred room air .
pcnl was usually performed through the antegrade percutaneous access ; once the targeted calyx was identified with fluoroscopy , an 18-gauge needle directed to the aforementioned calyx and a j - tip guidewire were passed .
the most widely used methods of tract dilatation were metal telescoping and semi - rigid amplatz dilatators . after tract dilatation
, a rigid nephroscope was inserted through an open , low - pressure system ; thereafter , lithotripsy was performed by use of ballistic lithotripters . in two centers ( yasooj and jahrom ) ,
pcnl was routinely performed under spinal anesthesia . in one center ( rasht ) , it was generally performed in the supine position ( complete supine pcnl ) . the amount of air injection for opacifying the collecting system often varied between 20 and 50 ml , but some endourologists used room air in amounts much greater than this range . with the exception of one center
, transverse rolls were located under the shoulders and anterior superior iliac spines of the patients who were in the prone position . in one center ,
a wedge foam pad was put under the thorax and abdomen in such a manner that the stone - containing side would be elevated at approximately 30 degrees . with the exception of one center in which half - saline solution was used as the irrigant solution ,
it is worth mentioning that the irrigant solution was provided at room temperature . in pediatrics , only normal
saline was used in all centers . to investigate the possible causes of serious neurologic complications
, we classified the cases into 4 groups ( a , b , c , and d ) according to their clinical manifestations and positive findings .
the total number of pcnl procedures in these 13 centers was 30,666 . among these procedures
, 11 cases were complicated by neurologic events , and 4 of these cases experienced paraplegia .
the pathogenic region in these four patients was determined to be in the spinal cord .
the data retrieved from the medical records of these cases are presented in tables 1 , 2 .
group a ( consisted of cases 1 , 2 , 4 , and 7 ) : in these cases , pcnl was performed with the patients under general anesthesia in the prone position .
the irrigant solution in cases 1 , 2 , and 4 was half - saline solution and that in case 7 was distilled water .
magnetic resonance imaging in cases 1 and 4 revealed ischemic regions in the thoracic cord .
group b ( consisted of cases 6 and 9 ) : these two patients were young and had no risk factors for cerebrovascular accident ( cva ) .
the irrigant solution was half - saline solution and distilled water in cases 6 and 9 , respectively .
case 6 became hemiplegic after the operation , which was a transient complication . in case 9 , after injection of 17 ml room air during the pyelography , cardiovascular collapse and arrhythmia occurred ; thus , the operation was terminated and the patient became conscious .
after several hours , the patient went into a coma , and following coma remission , he became aphasic for several days .
investigations in these two cases revealed a patent foramen oval in postoperative echocardiography , which was a predisposing factor for paradoxical air emboli .
group c ( consisted of cases 3 and 5 ) : in these two cases , in which cva occurred following pcnl , the procedure was performed with the patients under general anesthesia and in the prone position .
the irrigant solution was half - saline solution , and room air was used to opacify the collecting system .
in these two cases , a predisposing factor for cva was present . in case 3 , who was a known case of hypertension , a marked reduction in blood pressure following severe intraoperative hypertension led to an ischemic stroke . in case 5 ,
in whom a thromboembolic stroke occurred after pcnl , preoperative echocardiography revealed severe aortic valve insufficiency and the presence of a hypokinetic region in the left ventricular wall , which could be the cause of the cerebral thromboemboli in this patient .
group d ( consisted of cases 8 , 10 , and 11 ) : we could not categorize these cases in the above - mentioned groups . in these three cases ,
however , surprisingly , as in other the cases , pcnl in all of these patients was performed with the patients under general anesthesia in the prone position , and room air was used to opacify the collecting system .
although neurologic defects including paraplegia , hemiplegia , and quadriplegia are rare during or after pcnl , these events can occur and can result in devastation to the patient and noticeable financial and emotional pressures on the patient 's relatives . with consideration of the ethical issues related to this event , we designed a widespread study to recognize the possible etiologies of neurologic complications during or after pcnl .
we could not find any cases similar to group a in the medical literature following pcnl , but there are comparable case reports for other types of surgery that are performed in the prone position [ 8 - 10 ] .
a case series by bhardwaj et al . reported new neurologic deficits that were known to happen after spine surgery .
they presented four patients with cervical myeloradiculopathy who had undergone cervical laminectomy or fusion or both in the prone position , supported by chest rolls .
three cases were intubated and positioned while they were conscious , whereas the fourth case was positioned after induction .
the procedures were successfully concluded , except for temporary episodes of hypotension intraoperatively . in the recovery room , all cases had new neurologic deficits .
the authors concluded that the prone position in conjunction with compression on the abdomen can compromise the spinal cord perfusion and cause spinal cord ischemia .
they recommended using a frame that prevents abdominal compression and perioperative hypotension so as to maintain sufficient spinal cord perfusion during and after the surgery . in another case report , langmayr et al . presented a 33-year - old man undergoing surgery for a unilateral single - level disc herniation at l4 to l5 .
he developed quadriplegia and signs of occipital lobe dysfunction in a few hours after the procedure .
these authors concluded that a short - term mechanical occlusion of a vertebral artery can cause stasis , thrombi , and then embolism in the vertebrobasilar vessels ; therefore , surgeons should avoid excessive head rotation and neck extension in the prone position .
we reviewed the literature for case reports of air emboli ( group b ) during pcnl .
there were several reports of cases with suspicious air emboli during pcnl [ 3 - 7 ] .
manifestations and findings in these case reports were similar to those of group b. for example , in a case report by song et al .
, a 37-year - old man was referred for right calyceal diverticulum with milk of calcium .
after induction of general anesthesia , the patient was put in the lithotomy position . a 6-fr ureteral catheter with ureteropelvic junction occlusion balloon was inserted through the right ureter , and the case was rotated to the prone position .
the authors performed pyelography by injecting contrast media and a small volume of air into the right pelvocaliceal system .
six hours after the surgery , the patient complained of weakness of his right leg .
transesophageal echocardiography showed air bubbles in the left atrium revealing that the patient had a right to left shunt due to the patent foramen oval .
presented a similar case of paradoxical air emboli with an important difference : there was no report of air pyelography in that case .
the authors presumed that the prone position of the case produced a considerable gravitational gradient along the right side of the heart and the renal pelvis .
there were obvious predisposing factors related to the neurologic complications in the two cases in group c. probable neurologic complications in such cases were seen in all procedure types .
we have no hypothesis to explain these phenomena following the pcnl procedure in the cases of group d. overall , we assume that these rare but devastating complications have multiple etiologies . in all of these cases , pcnl was performed with the patient under general anesthesia and in the prone position and with the use of room air to opacify the collecting system .
therefore , each of these factors could be presumed to be a cause of these complications .
use of the prone position in conjunction with compression on the abdomen can compromise the spinal cord perfusion and cause spinal cord ischemia . on the other hand , excessive head rotation and neck extension in the prone position , especially in patients with atherosclerotic vessels ,
may lead to a short - term mechanical occlusion of the vertebral or carotid artery , which can cause stasis , thrombi , and then embolism in the cerebrovascular system .
the prone position produces a considerable gravitational gradient along the right side of the heart and the renal pelvis ; thus , negative pressure could have drawn air into open veins . in these circumstances ,
injection of air potentially makes the patient at risk of air emboli that may be concomitant with neurological complications in the presence of a patent foramen oval .
conversely , most of the pcnl procedures in iran are performed with the patient under general anesthesia in the prone position , and iodinated contrast materials are the most widely used means of opacifying the collecting system .
there were no reports of neurologic complications in cases with contrast agent instillation for opacifying the collecting system ; all of these complications happened after injection of room air .
there were also no reports of neurologic complications following pcnl procedures performed with the patient under spinal anesthesia or in the supine position .
consequently , it can be assumed that the use of room air to opacify the collecting system plays a major role in the occurrence of these complications and , likewise , that the prone position and general anesthesia may predispose to these events in the presence of air injection . this article is a descriptive study and
we do not claim that the aforementioned etiologies are the main causes of neurologic complications , because recognition of the original causes requires more comprehensive evaluation between advanced referral centers in different countries .
to avoid neurologic complications in pcnl , we recommend the following : 1 ) use of air during pcnl should be limited if not abandoned .
2 ) use of spinal anesthesia and the supine position in simple cases are recommended if possible .
3 ) use of a frame that prevents abdominal compression and perioperative hypotension to maintain sufficient spinal cord perfusion during and after the surgery is suggested .
4 ) excessive head rotation and neck extension in the prone position should be avoided .
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purposepercutaneous nephrolithotomy ( pcnl ) has been the preferred procedure for the removal of large renal stones in iran since 1990 .
recently , we encountered a series of devastating neurologic complications during pcnl , including paraplegia and hemiplegia .
there are several reports of neurologic complications following pcnl owing to paradoxical air emboli , but there are no reports of paraplegia following pcnl.materials and methodswe retrospectively reviewed the medical records of patients who had undergone pcnl in 13 different endourologic centers and retrieved data related to neurologic complications after pcnl , including coma , paraplegia , hemiplegia , and quadriplegia.resultsthe total number of pcnl procedures in these 13 centers was 30,666 . among these procedures , 11 cases were complicated by neurologic events , and four of these cases experienced paraplegia .
all events happened with the patient in the prone position with the use of general anesthesia and in the presence of air injection .
there were no reports of neurologic complications in pcnl procedures performed with the patient under general anesthesia and in the prone position and with contrast injection.conclusionsit can be assumed that using room air to opacify the collecting system played a major role in the occurrence of these complications .
likewise , the prone position and general anesthesia may predispose to these events in the presence of air injection .
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infectious diseases in type 2 diabetes can complicate diabetic ketoacidosis , derange hyperglycemia , or precipitate new onset diabetes . among infections ,
novel pandemic influenza virus h1n1 strain needs to be kept in mind when dealing with new onset diabetes with co - existing infectious agent , which , if present , is associated with high mortality .
a 63-year - old female , with no known chronic illness , was hospitalized in month of aug 2010 with fatigue , breathlessness on exertion , productive cough with scanty sputum , and high - grade fever of 04 days duration .
quick emergency assessment had shown temp 101f , tachycardia , tachypnea , p02 : 90% at room air , and normotensive .
further evaluation revealed nho in both lung fields on chest x - ray , hyperglycemia 325 mg / dl , detected for first time .
her symptoms and oxygen desaturation were out of proportion to clinical finding and chest x - ray findings . patient was managed with insulin infusion and empirical broad - spectrum antibiotic coverage in icu . as her condition worsened over next 12 hours , infection with novel influenza virus
was thought and empirically started on oseltamivir after taking throat swab for h1n1 test and later , the sample was tested positive for h1n1 influenza by rt - pcr [ figures 1 and 2 ] .
clinical course in the hospital was complicated by oxygen dependence requiring 10 - 12 ltr / hour of oxygen administration by nasal mask .
oseltamivir was continued for 02 weeks with escalation of antibiotic coverage for any methicillin - resistant staphylococcus aureus organisms .
appropriate prophylaxis against h1n1 was given to all nurses and paramedical staff and proper disposal of waste as per who guidelines .
she made an uneventful recovery and was discharged on biphasic insulin @ 35 units / day with well - controlled blood sugar levels .
chest x - ray showing pluffy opacities in both lung fields computed tomography scan chest : done at 48 hrs of admission , showing confluence of opacities with consolidation the first pandemic of 21 century , witnessed with detection of the first case in mexico 2009 , became evident that human - to - human transmission is caused by novel influenza virus , which is a re - assortment of 4 different strains of influenza virus genes causing novel h1n1 2009 pandemic strain .
though it behaved like seasonal flu virus , the morbidity and mortality were more with risk factors like diabetes , pregnancy , cardiovascular status , obesity ; chronic illness suffered more from this pandemic flu .
presently , we are in post - pandemic phase , and there are sporadic cases reported with mortality in high - risk cases [ figure 3 ] . the non - specific symptoms like fever ,
running nose , sore throat , body aches in flu make it difficult to identify and isolate cases initially [ table 1 ] .
who has listed diabetes as silent - epidemic , which is prevalent worldwide , taking toll of deaths in the age group of 35 - 65 years ; 1 out of 10 deaths are attributed to diabetes , a ratio that increases to 1 out of 4 in certain vulnerable populations .
people with diabetes and influenza are 3 times more likely to die from complications than without diabetes
. immunological research from the hospital clinico universitario de valladolid in spain and the university health network found high levels of a molecule called interleukin 17 in the blood of severe h1n1 patients and low levels in patients with the mild form of the disease .
two cases from iran with precipitation of diabetic ketoacidosis and h1n1 infection had fatal outcome . in a known diabetic ,
after h1n1 infection , hospitalization is known to triple and quadruple risk of icu admission once hospitalized .
our case was complicated by precipitation of new onset hyperglycemia and occurrence of pneumonia with rapid decline in general condition .
to best of our opinion , there were no reported cases of precipitation of diabetes with influenza a ( h1n1 ) pdm09 virus with positive outcome from india .
[ source : who influenza laboratory surveillance information by the global influenza surveillance and response system ( gisrs ) .
india , generated on 14/09/2012 06 : 14:47 utc ] distinguishing clinical features from common cold and flu this case lays emphasis on clinical acumen in recognition of a co - existing infectious process in a new onset diabetes mellitus , h1n1 infection , and prompt institution of oseltamivir to retard the progression of disease and decrease the mortality associated with it .
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infectious diseases in type 2 diabetes can complicate diabetic ketoacidosis , derange hyperglycemia , or precipitate new onset diabetes .
pulmonary tuberculosis being the most common .
high index of clinical suspicion is required for co - existing h1n1 virus , which if present has high mortality if not treated .
a 63-year - old female , with no known chronic illness , was hospitalized in month of aug 2010 with influenza - like symptoms and diabetes .
quick evaluation revealed tachycardia , tachypnea , p02 90% at room air , and normotensive .
clinical chest examination was normal .
further evaluation revealed nho in both lung fields on chest x - ray , hyperglycemia 325 mg / dl , detected for first time .
her signs and symptoms were out of proportion to clinical findings and chest x - ray findings .
patient was managed with insulin infusion and empirical broad - spectrum antibiotic coverage in icu .
as her condition worsened over next 12 hrs , infection with h1n1 was suspected and empirically started on oseltamavir after taking throat swab for h1n1 test and later , the sample was tested positive for h1n1 influenza by rt - pcr .
clinical course in the hospital was complicated by oxygen dependence requiring 10 - 12 ltr / hr by nasal mask .
she made an uneventful recovery . in a known diabetic , infection with h1n1 quadruples icu hospitalization , and only few cases of new onset
diabetes with h1n1 were reported .
two reported from iran had fatal outcome .
this case emphasis on clinical acumen in recognition , and prompt institution of therapy will reduce associated mortality .
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in the most recent reorganisation of the pleistocene period ( 2.58 ma0.01 ma ) , the ionian is defined as a geological stage between the bruhnes - matuyama boundary at 781 ka ( end of the calabrian stage 1.810.78 ma ) and the beginning of marine isotope stage 5 interglacial period at 126 ka ( beginning of the upper pleistocene 12611.7 ka ) . just prior to this
this is the transition from what is known as the 41 ka world to the 100 ka world , essentially a switch from 41 ka to 100 ka cyclicity in glacial cycles .
this led to major environmental changes in africa from about 1.0 ma to 700 ka .
the ionian is also a period when a broad group of potential modern human ancestors attributed to
archaic homo sapiens , ( or more specifically to homo heidelbergensis , homo helmei , or homo rhodesiensis ) evolved to become the first anatomically modern humans .
noonan et al . suggest that the split between neandertal and ancestral h. sapiens populations occurred at ~370 ka and that divergence from a last common ancestor occurred at ~706 ka . in europe
, h. heidelbergensis fossils such as those from sima de los huesos at 530 ka may represent fossils occurring soon after this initial split from a common ancestor .
in africa , ( figures 1 and 2 ) , the hominin record is more fragmentary and less well dated .
the kabwe ( broken hill 1 ) hominin remains from zambia may represent a similar post - divergence population in africa , as might the elandsfontein hominin remains from south africa ; however , the age of these fossils remains uncertain .
the fauna from kabwe is suggested to be broadly similar to bed iii - iv at olduvai ( > 1.07 ma or > 780 ka [ 5 , 6 ] ) although its association to the type specimen of homo rhodesiensis is questionable and some age estimates are as young as 125 ka .
the elandsfontein hominin remains are contemporary with a cornelian land mammal age faunal assemblages in southern africa , which date to between ~1.1 and 0.8 ma at other sites ( cornelia - uitzoek ; buffalo cave ; [ 9 , 10 ] ) and represent either homo erectus or h. rhodesiensis .
in contrast , the florisbad hominin skull ( the type specimen of homo helmei ) is associated with a florisian land mammal age
( see flma ; ~780~10 ka ) faunal assemblage and msa technology and is dated to ~259 ka [ 12 , 13 ] .
the oldest flma assemblage comes from gladysvale cave at between 780 and 578 ka .
currently , the earliest well - dated homo sapiens remains come from omo and herto in ethiopia between 198 and 147 ka [ 15 , 16 ] .
in southern africa , the oldest modern human remains come from border cave , klasies river mouth , and pinnacle point and are all dated to less than 184 ka [ 1720 ] .
the border cave 1 and 2 remains could be as old as 184143 ka based on esr and depending on their actual provenience , while bc5 is somewhere between 79 and 69 ka .
the pinnacle point remains are likely between 170 and 91 ka although again , their provenience is uncertain [ 17 , 18 ] .
the oldest klasies river mouth remains from layer lbs are likely around 115110 ka , while the younger sas layer fossils are likely around 9075 ka .
fish hoek man ( peers cave p4 ) , once thought to be an early modern human , has recently been radiocarbon dated to 7.87.6 kcal yr bp .
the ionian is also a period when we see the transition from the early to the middle stone age ( msa ) and the end of the acheulian industry in africa .
this is generally defined as a change from large cutting tools ( lcts ) such as hand axes and cleavers to the use of blades , points , and prepared core technology , specifically the levallois method .
an understanding of both the archaeological and hominin transitions in southern africa has been hampered by a lack of reliable dated sites in the region .
the current data suggest that the earliest msa in both eastern and southern africa is not associated with anatomically modern humans but with archaic homo sapiens also referred to as h. helmei at sites such as florisbad [ 13 , 23 ] and eyasi springs .
many researchers [ 2528 ] have suggested the occurrence of a number of transitional industries in southern africa , namely , the fauresmith and the sangoan , covering this transition , but they have generally been poorly defined , documented , and dated . the fauresmith is generally considered to have aspects of both msa and esa technology and is most often defined as a terminal phase of the acheulian .
the sangoan is characterised by a heavy - duty , less - elegant component of picks and steep , sometimes denticulated scrapers as well as lcts and msa elements .
both the sangoan and the fauresmith have long been considered to mark the beginning of regional cultural specialisations towards the end of the esa .
the fauresmith , with its msa - like features , perhaps holds the key to understanding this transition and the relationship between the archaeological and hominin record of this transitional time period between the esa and msa .
creating an all - encompassing term for the definitive acheulian artefacts , the hand axe , and the cleaver is in itself a problem .
alone does not encompass cleavers , while biface does not take account of size , and many cleavers and hand axes are unifaces , being made from a single large flake and unretouched on the dorsal face .
large cutting tools ( lcts ) is perhaps the best term and has been used elsewhere [ 30 , 31 ] .
this term is used throughout to denote hand axes and cleavers of acheulian character unless referring to a specific type of stone tool .
goodwin first split the palaeolithic of africa into three divisions , namely , the early , middle , and later stone age ( esa , msa , and lsa ) .
industries such as the still bay typified the msa , where the most prominent artefacts were triangular flakes with convergent dorsal scars and faceted butts ( platforms ) .
the esa was characterised by lcts as seen in the stellenbosch culture , first found at the type site of bosman 's crossing in stellenbosch ( western cape ) in 1899 .
these deposits were later resampled by hilary deacon and james brink , and there is now an attempt to date them by the latter and this author . the stellenbosch was officially recognised by the annual general meeting of the south african association for the advancement of science in 1926 .
the stellenbosch was later incorporated into the term chelles - acheul as used in eastern africa or simply hand axe culture .
the chellean was essentially an industry between the oldowan and acheulian which did not have cleavers , only hand axes .
this was later shown not to be the case , and the term acheulian was then used alone .
the formation of the msa was a splitting of a single entity ( the lsa ) into two with the lsa containing flakes with parallel dorsal scars and smooth butts and/or by microliths ( note that it was the msa style of tools that were originally classified as the lsa ) . while the type site for the stellenbosch were
the alluvial deposits of bosman 's crossing in stellenbosch , the vaal river gravel deposits ( northern cape ) have perhaps the greatest number of examples of what is now termed acheulian .
other well - established acheulian sites are cornelia - uitzoek [ 10 , 37 ] and several sites near vereeniging in the eastern free state as well as a number of caves sites discovered slightly later including sterkfontein ( gauteng ) , the cave of hearths , and olieboompoort ( limpopo ; mason and montague cave ( western cape ; ) .
goodwin included the fauresmith in the esa along with the stellenbosch and victoria west industries .
goodwin notes that these are all primarily core industries and that the chief instrument is the coup - de - poing , that is , the hand axe .
goodwin notes that raw material has a profound influence in the stellenbosch with bad material yielding bad implements , while more workable material yielded finer implements .
the victoria west industry was classified as having hand axes but with a large core that was made to produce a single large flake , the classic victoria west core .
there has been much debate regarding the relationship of the victoria west technology to other prepared core technologies ( pcts ) , notably levallois pcts [ 4143 ] .
the victoria west industry was later described as a technique within the final phases of the stellenbosch rather than an industry in its own right .
the fauresmith takes its name from the town of fauresmith in the south - western orange free state ( figure 1 ) . the first description of the fauresmith was given by goodwin when in a brief reference
he notes that it is difficult as yet to say how far this can be classed as an industry and how far it is only a local variation of the stellenbosch .
goodwin felt that the fauresmith was a specialised branch of the stellenbosch , while van riet lowe thought it was perhaps a more regional variant related to the presence of indurated shale or that it was cultural , due to the arrival of new people into south africa .
while the fauresmith may be a regional variant confined to the free state and northern cape regions , surface collections from the site of hopefield ( aka elandsfontein ) were also called cape fauresmith . the term southern rhodesian fauresmith has also been used , suggesting a wider occurrence of this industry . moreover ,
the site of van der elst donga , near vereeniging , is in gauteng and extends the fauresmith laterally across most of south africa .
the lack of fauresmith industries may also be simply related to deposits of that age not being found in other areas . in the first paper
devoted exclusively to the fauresmith , van riet lowe elaborated more on this culture .
he said it represents the culminating phase of the earlier stone age and , incidentally , the transition from core to flake treatment . with the greater use of flakes ,
we come in contact with an evolutionary process that shows marked improvement in technique , the introduction of less clumsy artefacts and the beginnings of a variety of attendant tools that were not in evidence .
in contrast , while earlier stellenbosh hand axes are also made on flakes , they are laterally struck .
prime examples of this can be seen in the early to middle acheulian hand axes of cornelia and the vaal river ( personal observation ) .
goodwin notes that in the fauresmith , the flake is always struck from a point situated , where the butt of the hand axe would eventually lie and that it is the beginning of a true flake industry . instead of flakes that are struck off during the making of the hand axe being made into unconventional implements ,
true flake cores make their appearance along with true flake implements with longitudinal trimming , including points . in this earlier description by goodwin , there is no mention of the size of the hand axes , something often taken to define the industry .
however , goodwin and van riet lowe later state that the hand axes are generally small and a neat almond , sometimes ovate shape , and that triangular hand axes are exceedingly rare .
fauresmith hand axes are also stated to have a straight edge , often with an s - shaped twist and differing from the zig - zag edge so often apparent in the stellenbosch .
they further state that the hand axes are far finer than the best seen in the stellenbosch and that the first beginnings of the levallois technique are potentially seen [ 26 , 48 ] . along with these are discs , scrapers , slightly trimmed flakes , and points .
moreover , fauremsith flakes are suggested to typically have a faceted platform , as in the msa . in comparison ,
the msa was characterised archaeologically in 1928 by the absence of lcts that characterise the esa , an increased prevalence of prepared core reduction strategies , the production of projectile points , and a variety of flake - based tools .
the oldest msa industry was considered to be the still bay with its lanceolate points , now known to date to ~72 ka .
goodwin also notes similarities between the fauresmith and some msa industries , for example , that the glen grey industry ( eastern cape ) directly evolved from the fauresmith .
so , here , goodwin is noting distinct similarities between the msa and the fauresmith but chooses to align the latter with the esa based on the occurrence of lcts .
sampson noted that the fauresmith has been primarily defined as an industry later than the stellenbosch and containing smaller , more refined hand axes .
however , in 1945 , van riet lowe suggested that the progress of man 's skill from stellenbosch to fauresmith times is thus not so much measured by a series of successive refinements in his hand axes and cleavers as by an abandonment of old methods and by an improved technical skill revealed in the greater refinement and variety of his waste products in the form of smaller and more elaborately prepared cores and flakes .
moreover , van riet lowe states that it is not the continued presence of hand axes so much as the integrally associated and now considerably improved [ fully developed ] levallois technique and while many of the earlier fauresmith hand axes are smaller and more refined than are their stellenbosch proto - types , this group of implements is generally neither so beautifully made nor so varied as a representative group from the final stellenbosch . here
van riet lowe sets out the differences between the stellenbosch and the fauresmith , which he [ 52 , 53 ] divided into three stages . in comparing the esa and msa in 1946 ,
goodwin states simply that the esa ( meaning the acheulian ) is defined by the hand axe , while the msa is defined by an accentuation of levallois ( notably used by tryon et al . to define the msa ) and
goodwin suggests that while we are dealing primarily with techniques , each of these techniques is foreshadowed in the previous phase .
we only reach each new age as the new technique becomes dominant and replaces previous modes .
it is not clear which dominates an interpretation in this scheme , the presence of lcts or the refinement of new techniques . as such
, this leans towards the idea of true intermediate periods as proposed by clark in 1965 . in 1954 , clark noted the similarity of the kalambo falls material to the fauresmith of south africa . by 1964 ,
clark had begun to see the fauresmith as essentially a southern african ( with pockets in eastern africa ) entity that was in essence the survival of earlier acheulian traditions , south of the limpopo . in central and central southern africa
( i.e. , zambia ) , the addition of heavy chopping tools and small denticulates was termed the sangoan .
it seems possible that the sangoan as described by clark at kalambo falls represents a transitional acheulian to msa industry that occurred in more forested areas , whereas the fauresmith occurred in more open savannah and thornvelt environments .
clark suggested that the sangoan / fauresmith represents an intermediate period between the final acheulian and the msa , which he terms the first intermediate period . at the 1955 panafrican congress , the term first intermediate period was adopted to describe this transition period between the esa and msa , but the term
was then dropped at the burg wartenstein symposium of 1965 . based on his work in the transvaal ,
mason suggested that all the hand axe cultures and developed oldowan be assumed under the term acheulian .
discontinues the use of the term fauresmith for the last known phase of the acheul sequence in the transvaal , for , like chellean ' it obscures the close relationship industries such as the cave of hearths earlier stone age series have with the acheul . in this statement ,
mason is setting out that the fauresmith holds greater similarity to the acheulian than the msa .
mason also notes that there is no factual stratigraphic support for the three stage division of the fauresmith .
he was in favour of simply using early ( sterkfontein , etc . ) , middle ( vaal river ) , and later ( cave of hearths ) acheulian and abandoning the term fauresmith for the final phase of the esa .
as inskeep notes in 1969 , the trend at that time was for a reduction of names referring to different regional variants of stone tool industries .
that is , incorporation of the stellenbosch into the acheulian and removal of the victoria west into a technique rather than an industry .
but he also notes that while hand axes and cleavers maybe characteristic they are by no means the only tools of the acheulian and not the most numerous .
going further , humphreys argues that all the tool types found within the fauresmith are also found in the acheulian .
humphreys notes that the distribution of fauresmith and stellenbosch sites raises two important points .
the first is that most of the stellenbosch sites are associated with the vaal river area , while all but two of the fauresmith sites are well away from that river , perhaps suggesting differences related to activities , raw materials , or even survival in different geological contexts .
humphreys also notes that the correlation of stellenbosch and fauresmith occurrences with the different types of raw materials suggested by the distribution of sites seems too clear to be accidental .
humphreys tries to take consideration of this and cites mason 's work at the cave of hearths as an example of a late acheulian industry contemporary with the fauresmith that suggests the latter is a regional or raw material - specific variation .
indeed mason originally called the assemblage fauresmith , in part based on the occurrence of small hand axes but later changed it to acheulian .
while the fauresmith occurrences have been shown to be clearly stratified above acheulian deposits , humpreys believes this is perhaps due to a lack of recovery or survival of artefacts in the gravel contexts compared to the sand contexts as at canteen kopjie .
excavations by gibbon and leader have shown that the small fraction is preserved in acheulian bearing gravel deposits in the vaal river , so this question can now begin to be tested with other recent fauresmith excavations at canteen kopjie , wonderwerk , and kathu pan , as will be discussed below . returning to van riet lowe , in 1945 , he suggested that throughout the fauresmith we have three principal types of cores , the majority with prepared striking platforms : ( a ) circular or tortoise cores , ( b ) triangular flake - cores , and ( c ) rectangular blade - cores .
the makers of fauresmith tools were not only masters of a fully developed levallois techniquebut specializedin flake tools of considerable variety . in the final stages
the hand - axe became less and less important and was gradually superseded and replaced by other types of tools until.we can not say whether we are at the end of the earlier or at the beginning of the middle stone age .
this final fauresmith is literally a period of transition and is distinguished only by the presence of hand axes ; the remaining tools and debitage being completely middle stone age in form and fineness . in 1952
, van riet lowe further suggested that the fauresmith showed the earliest local use of the mounted tool in the form of a spear , something most researchers would equate with the msa , not the esa .
here , van riet lowe appears to be stating that the only difference between the late fauresmith and the msa is the continued presence of lcts , and this could be used to suggest that the fauresmith should be included within the msa rather than the esa and that an industry should be defined on new technology rather than the continued use of old technology .
the idea that transitional industries such as the fauresmith and sangoan have more in common with the msa is a view since put forward by davies , van peer et al . , and
beaumont and vogel , the latter of which suggests the fauresmith should be termed early msa ( emsa ) and assemblages without lcts later msa ( lmsa ) . in van riet lowe 's 1937 definition of the 3 phases of the fauresmith , the 1st phase was described as having hand axes , cleavers , crude scrapers and cores that yielded long , narrow flake .
this is not dissimilar from the acheulian that we now know and likely represents the end phase of the acheulian .
the 2nd fauresmith phase was described as comprising beautifully finished hand axes on flakes , cleavers , trimmed points , end- and side - scrapers , typical concavo - convex side scrapers , typical levallois flakes and cores : long and slender blades cores , faceted polyhedral stones and gravers . and the
this is the typical mixture of esa and msa elements that is perhaps the best definition of the fauresmith as an industry or at least of clark 's first intermediate period . as will be described below , recent studies suggest that the so called fauresmith industry , and others , has a great deal in common with the msa and perhaps the occurrence of the first elements of the msa rather than the remaining elements of the acheulian within the fauresmith should be the most important factor in ( a ) defining it as its own transitional entity and ( b ) including it within the msa rather than the acheulian .
it is quite possible that some of the fauresmith being described is material that has since been defined as early msa .
only a chronological analysis of these sites to assess their contemporaneity would help resolve these issues .
to fully understand the context and difference of the fauresmith , an overview of the acheulian record in southern africa is first needed .
well - dated acheulian sites are still few and far between in southern africa mostly due to the difficulty of dating caves and river sequences , where much of this material is preserved .
acheulian sites often occur on deflated landscapes rather than in well - stratified and datable contexts and even then reliable means of dating such contexts were unavailable until recently .
the most extensive acheulian bearing deposits are the numerous palaeocave sites within the cradle of humankind world heritage area in gauteng ( cohk , e.g. , sterkfontein , swartkrans , and kromdraai [ 66 , 67 ] ) , donga sequences of the free state ( e.g. , vereeniging and cornelia [ 10 , 38 ] ) , the vaal river sites ( e.g. , pneil and canteen kopje ) , a series of pan deposits around kimberley ( e.g. , kathu pan and rooidam [ 69 , 70 ] ) , dune sequences of the western cape coast ( e.g. , elandsfontein and duinefontein ii ) , and a series of isolated caves occurring throughout the country ( e.g. , wonderwerk cave in northern cape , the cave of hearths in limpopo province , and montagu cave in western cape [ 40 , 47 , 72 ] ) .
other localities include amanzi springs in eastern cape and a series of sites in the mapangubwe national park along the limpopo river ( limpopo province ; ) .
have all suggested that the early acheulian first occurs in southern africa around 1.6 ma coeval with its occurrence in eastern africa . while a review of the dating makes this possible , the refinement of the dating is perhaps too coarse to currently make this statement .
kuman and clarke have argued for the acheulian from sterkfontein member 5c being as early as 1.71.4 ma based on faunal comparisons .
however , a recent comparison of fauna , palaeomagnetic , electron spin resonance ( esr ) , and uranium - lead ( u - pb ) age estimates [ 67 , 78 , 79 ] have suggested that the acheulian deposits are more likely dated to between 1.3 and 1.1 ma
. the robust nature of the esr ages is supported by identical age ranges for member 4 ( 2.62.0 ma ) based on independent esr , palaeomagnetism , and uranium - lead analyses [ 7981 ] as well as geochronological comparisons at other sites in the cradle ( see below ) .
moreover , the stw 53 infill ( aka member 5a ) at sterkfontein has been shown to be older than the member 5b and 5c deposits based on stratigraphy and geochronology [ 79 , 81 , 82 ] . the stw 53 infill has been dated to < 1.78 ma based on a u - pb age for speleothem that formed before the deposition of member 5a and the reversed magnetic signal of this deposit .
esr ages are again consistent with this and taken together an age between 1.8 and 1.5 ma is most likely [ 67 , 79 ] . given this data , there is no definitive reason based on the current data to discount the esr ages for the member 5c acheulian .
o'regan and reynolds suggest that the diversity of carnivore species in the deposit indicate that it is a palimpsest formed over a long period of time .
the spread of esr ages may support this suggestion . given this suggestion and taking the maximum age range for all the esr ages into account , rather than a weighted mean age as per , a maximum age range of 1.390.82 ma
is suggested for the acheulian , and so , it is not older than 1.4 ma ( the m5b deposit has a maximum esr range of 1.620.83 ma ( minus a single age with an extremely large error ) and suggests that the oldowan is not older than 1.6 ma at sterkfontein )
. the age for the acheulian is at the lower age range of kuman and clarke 's estimate .
while some fauna from sterkfontein member 5 suggests an older age , the fauna collections are likely quite mixed due to numerous years of excavation into the deposits ( see ) without understanding their complexity . vrba previously suggested the fauna from member 5 was likely around 1.5 ma , and a reanalysis of the carnivores from member 5c has suggested that it is likely younger than previously suggested [ 67 , 85 ] .
o'regan suggests that the only older element in member 5c , dinofelis barlowi , likely does not come from member 5 .
o'regan notes that a number of archaic carnivores ( megantereon and chasmaporthetes ) are present in swartkrans member 1 but not in sterkfontein member 5 despite the fact that they are generally considered contemporary .
. suggest that swartkrans member 1 maybe around 2.11.9 ma , and so , this may be a temporal difference .
have recently suggested that the hanging remanent at swartkrans is dated to sometime between 2.2 and 1.8 ma .
these temporal changes may explain some of the o'regan hypothesises that this might be related to taphonomic differences , different ages or different environments being sampled .
given that the caves are across the valley from each other the environment would likely be similar if of the same age .
taphonomic reasons can never be ruled out ; however , given the new younger esr ages for member 5b / c ( 1.41.1 ma ) and the suggested older age for swartkrans member 1 [ 67 , 86 ] , this difference , and differences noted by reynolds , is likely to be partly a reflection of age .
kuman and clarke 's age assessment is also in part due to the fact that the lcts from sterkfontein m5c are both limited in number and very unrefined . the earliest date for an assemblage assigned to the acheulian is often quoted as ~1.7 ma at locality kga of konso gardula in ethiopia .
however , the earliest acheulian deposits occur in deposits dated to somewhere between 1.69 and 1.41 ma ( 1.66 0.03 ma and 1.43 0.02 ma ; .
the first acheulian tools occur closer to 1.4 ma than 1.7 ma based on the stratigraphy and the occurrence of a stratigraphic break between the hand axe horizons and the basal age .
while the ks4 site from the nachukui formation ( west lake turkana , kenya ) is suggested to have an age slightly older than 1.65 ma , the majority of other early acheulian sites in eastern africa are generally dated to less than 1.5 ma including olduvai gorge , middle and upper bed ii ( 1.531.27 ma ; ) , to which many of the southern african early acheulian deposits are often compared . as such , there seems little typological basis for the sterkfontein member 5 acheulian being at the older part of the 1.71.4 ma age range as suggested by kuman [ 66 , 93 ] .
moreover , it is not unreasonable to assume that the acheulian may first occur later in southern africa than it does in eastern africa , as is the case for other parts of africa or the levant , where it does not occur until somewhere between 1.5 and 1.2 ma .
acheulian tools are also known from swartkrans member 2 and 3 dated to sometime between 1.65 and 0.6 ma ; however , swartkrans remains one of the least well - dated site in the cohk and many of the other sites have relatively undiagnostic stone tool assemblages .
many , including swartkrans member 1 , have been classified as developed oldowan or early acheulian based on the fact that many have no lcts , but a large flake size than the preceding oldowan [ 66 , 95 ] .
however , the recent dating suggests that parts of swartkrans member 1 are as old as 2.21.8 ma [ 67 , 86 ] , making an assessment of these stone tools as acheulian perhaps less likely .
that being said , swartkrans member 1 may be more complex than previously noted with multiple in - fills of more than one age .
have suggested that acheulian stone tools in the rietputs ( rietputs 15 ) formation of the vaal river are also dated to ~1.6 ma .
these deposits have maximum ages between 2.08 and 1.12 ma ( 1.89 0.19 ma and 1.34 0.22 ma ) and minimum ages between 1.881.08 ma ( 1.72 0.16 ma and 1.29 0.21 ma ) suggesting that the lca gravel deposits date to between ~2.1 and ~1.1 ma
. the lca gravel deposits are up to 4 m deep in some of the pits sampled and up to 7 m thick overall and appear to have accumulated over perhaps a million years .
the stone tools collected come from an undated pit and were collected out of context from mining debris piles and sporadically from an excavating conveyor belt over two days of 24 hour operation . as such , the rietputs acheulian described by gibbon et al .
the later collected acheulian assemblage described by leader comes from pit 5 , and so dates to somewhere between 1.63 and 1.11 ma ( 1.43 0.231.32 0.21 ma ) .
note that ~1.6 ma is at the extreme upper end of this age range , and a more likely age for this assemblage is perhaps 1.4 - 1.3 ma .
a further collection described by leader came from an in situ excavation with no current ages .
however , the stratigraphy of each of the pits and dating sample location compared to depth of the lca is never presented in either leader or gibbon et al . , making it difficult to confidently relate the ages to the artefacts collected beyond the age for the lca as a whole , 2.01.1 ma . in pit 2 ,
the top of the lca was dated to somewhere between 1.42 and 1.08 ma ( 1.27 0.151.22 0.14 ma ) , and the overlying ufa has been dated to somewhere between 1.45 and 1.01 ma ( 1.26 0.191.16 0.15 ma ) suggesting that the lca deposits are more likely older than ~1.2 ma . for some reason , gibbon et al .
and gibbon included the sample from the top of the lca [ 72 , table 1 ] within the overlying ufa when calculating the mean burial age for the lca deposit .
this will have had the effect of very slightly increasing the mean maximum age estimate for the lca deposits from 1.53 to 1.57 ma .
suggest the true age is likely closer to the maximum age estimate ; however , this is by no means certain , and an age between the maximum and minimum age estimates is also perfectly possible . as such
, some acheulian deposits from the vaal river could be as old as 1.6 ma or older , but the age of the collections described by leader is closer to ~1.4 - 1.3 ma .
cosmogenic burial ages for the base of the canteen kopjie acheulian bearing lower coarse alluvium ( stratum 2b ) range between 1.25 and 0.82 ma ( 1.06 0.19 ma and 1.00 0.18 ma ) in one pit and 1.61 and 1.23 ma ( 1.46 0.15 ma and 1.37 0.14 ) in a second pit .
gibbon notes that these ages suggest that deposition of the gravels was very complex on a local scale ( within 40 m ) , and as such , ages from one location may not represent the age of deposits on a locality scale . as such , for reliable dates of assemblages from the gravels , the levels need to be dated directly in each case , and as the collection described by gibbon et al
. comes from an undated pit , the age of the stone tools is much less certain .
chazan et al . also suggest an age of ~1.6 ma for the beginning of the acheulian at wonderwerk butagain this age is by far from secure .
the acheulian first occurs in sediments ( strata 11 ) that are dated to somewhere between 1.78 and 1.07 ma based on palaeomagnetism and a basal cosmogenic isotope burial age .
a number of depositional breaks appear to occur in the sequence , notably between the layers recording the palaeomagnetic transition at the end of the oldowan ( 1.78 ma ; strata 12 ) and the layers containing the acheulian ( strata 11 ) .
this may suggest the material is closer to 1.07 ma than 1.78 ma .
the vaal river and wonderwerk cave early acheulian may date to as old , or in theory older than 1.6 ma , but could be closer in age or contemporary with the acheulian from sterkfontein m5c at 1.41.1 ma . as such , there is little definitive evidence for the acheulian in south africa older than 1.4 ma .
hominin fossils are rare , but these earliest acheulian occurrences are normally associated with homo ergaster .
recent palaeomagnetic studies at the cornelia - uitzoek locality have shown that multiple levels of acheulian bearing deposits occur there between 1.07 and 0.78 ma .
the oldest acheulian layers at elandsfontein may also be of a similar age , and klein et al .
. however , klein et al . suggest that the site most likely dates closer to 600 ka based on the presence of rabaticeras and pelorovis antiquus , the hominin remains , and the typology of the acheulian artefacts .
klein et al . suggest that the extinct alcelaphine rabaticeras likely gave rise to the extant hartebeest genus , alcelaphus which has a first appearance date close to 600 ka , based on its occurrence at bodo and that the extinct long - horned buffalo pelorovis antiquus has a first appearance at or soon after 1 ma based on its evolution from pelorovis oldowayensis after the deposition of olduvai bed iv .
bodo is dated to sometimes between 690 and 520 ka ( 630 30 ka550 30 ka ) .
however , the bodo material shows a lot of affinities to material from olduvai bed iv at > 0.78>1.07 ma [ 6 , 96 ] .
moreover , rabaticeras itself actually occurs at buffalo cave and cornelia - uitzoek , both of which are dated to between 1.1 and 0.8 ma [ 9 , 10 ] .
the remainder of the fauna from elandsfontein is also similar to that from cornelia - uitzoek and buffalo cave , representing the cornelian land mammal age .
also note that the occurrence of sivathere and the dirk - toothed cat which would have to represent the youngest occurrences of these species by some margin .
this suggests that there is little evidence for elandsfontein being younger than at least 0.8 ma .
also , in western cape , the duinefontein ii fauna also belongs to the cornelian land mammal age ( brink , pers .
the site also contains an acheulian estimated to date to between 347 and 217 ka [ 71 , 97 ] based on a combination of thermoluminescence ( tl ) , optically stimulated luminescence ( osl ) , and infrared - stimulated luminescence ( irsl ) .
this is extremely young and of a similar age to some of the earliest msa industries in south africa ( see below ) .
moreover , the cornelian land mammal age is followed by the florisian land mammal age , which is currently dated back to sometime between 780 and 578 ka at gladysvale cave . this , along with the faunal similarities to elandsfontein , buffalo cave , and cornelia - uitzoek , may suggest that duinefontein ii shoul , in fact , be older than 800 ka , at odds with the luminescence age estimates . if the luminescence age estimates are correct , then this would indicate marked regional variation in the timing of the transition from the cornelian to the florisian land mammal age and the acheulian to the msa .
the acheulian certainly lacks elements of the msa as seen in fauremsith assemblages despite elandsfontein once being called cape fauresmith based on the small size and shape of some of its hand axes .
however , feathers outlines the problems with the dating samples from this site . based on the problems of variable dose rates and shifting dunes through time at this locality
, these age estimates maybe underestimations of the true age of the deposits and should perhaps be regarded as minimum ages .
these ages were also performed on feldspar whose luminescence signal is known to fade with time , causing younger age estimates and redating using post ir - irsl could be attempted .
however , given the faunal age estimates , the site is likely also beyond the limit of this method .
future potential for the dating of the west coast sites comes in the form of thermally transferred osl ( tt - osl ) , which can date back to at least 780 ka if the correct conditions occur .
gladysvale cave has also yielded a single hand axe dated to older than 780 ka [ 100 , 101 ] .
the age of the bed 13 cave of hearths acheulian is still a matter of debate , but it is certainly younger than 780 ka based on palaeomagnetism .
mcnabb suggests that the cave of hearths assemblage is lacking elements suggestive of the fauresmith or msa such as levallois and other prepared core technology points and blades . a direct esr date on the mandible from bed 3 suggests and age of 600400 ka however there are issues with the dosimetry for this sample .
given this esr age and based on the age of confirmed geomagnetic field events , a short geomagnetic reversal in the basal deposits [ 101 , 102 ] most likely dates to either the big lost event dated to between 580560 ka or the stage 17 excursion at ~670 ka .
as such , the bed 13 acheulian from the cave of hearths , including the hominin remains , may date to the period between 700 and 400 ka but most likely towards the older end of this age range .
the fossils associated with these sites are often referred to as h. rhodesiensis , h. heidelbergensis , h. erectus , or simply archaic h. sapiens .
taking h. erectus as an asian origin species and h. hedelbergensis as a european origin species , the use of h. rhodesiensis is perhaps most appropriate for much of the african hominin record ( as per [ 22 , 23 ] ) of this time period .
however , designation to a specific species should obviously be undertaken on a fossil by fossil basis , as there is always the possibility that species did not just leave africa but that h. erectus and h. heidelbergensis may have migrated into africa at various times .
in the older k3 deposits of the kapthurin formation ( gnjh-03 , gnjh-17 ; < 518 ka ) deino and mcbrearty document the use of lcts , levallois , and blades and a notable feature is the use of the levallois technique to make hand axes .
johnson and mcbrearty describe a further small set of blades from the base of the k3 deposit ( gnjh-42 and gnjh-50 ) at between 548 ( 545 3 ka ) and 500 ka ( 509 9 ka ) and suggest that the move to blade technology is a feature of the acheulian period . while the blades are standardised johnson and mcbrearty suggest the earliest blades are not made by the levallois method .
refinement of the k4 sequence by tyron and mcbrearty [ 108 , 109 ] indicates that > 272 ka ( 284 12 ka ) lcts ( cleavers ) and levallois technology occur at the same sites in the southern area , while lcts produced from levallois cores ( e.g. , at lha ) and points occur together in slightly older deposits in the northern area ( e.g. , at gnjh-17 ) .
by ~200 ka ( perhaps 250 ka ) the site of koimilot ( also in the kapthurin formation ) shows a range of levallois flake reduction strategies including convergent levallois flakes and points and an absence of lcts [ 109 , 110 ] . slightly further north between the turkana and baringo basins , much younger msa assemblages occur in the kapedo tuffs .
these sites illustrate that by 135120 ka ( 132 3 ka and 123 3 ka ) a wide range of levallois flake - based reduction techniques are being undertaken within the msa . as such , levallois blade and point technology characteristic of the msa
this sequence suggests the early occurrence of blades within the acheulian before 500 ka .
points begin to occur towards the end of the k3 deposits sometime between 518 and 272 ka . at sites within the base of the k4 deposits , lcts made from levallois flakes
soon after this centripetal levallois , flakes and cores occur at sites such as rorop lingop .
lcts in the form of cleavers are then last seen in the middle of the k4 deposits at some time before 272 ka .
after this ( ~250200 ka ) , at the site of koimilot a range of levallois flaking occurs including the presences of convergent levallois flakes and points [ 108 , 109 ] .
tryon uses this change in levallois flaking behaviours at koimilot as the beginning of the msa despite tools of msa - like character occurring earlier in the sequence .
mcbrearty suggests that this sequence indicates an independent evolution of blades , points , and levallois technology during the transition from the acheulian to the msa and that it does not occur as a package of behaviour evolving together . as such
, this causes a major issue in defining exactly what the msa is and when it might begin . as such
, the diversification in levallois technology is suggested as the defining features of the end of the esa and beginning of the msa around 250200 ka in the kapthurin formation .
malewa gorge in kenya is another site where msa ( kenyan still bay ) has been recovered from beneath a tuff that was originally dated to 240 ka .
clark suggested that if the still bay was older than 240 ka the tuff samples dated must have been contaminated .
an attempt at redating this tuff by morgan and renne gave an age of 102 16 ka , much younger than the original age .
pseudo - still bay artefacts were also dated to < 557 and > 440 ka at wetherill 's and cartwright sites and suggests that transitional industries could potentially occur here as early as half a million years .
morgan and renne describe the dating of msa and perhaps transitional esa / msa localities within the gademotta formation in southern - central ethiopia . at the type site unit 9 of laury and albritton lies beneath unit 10 which has been dated to sometime between 280 and 272 ka ( 276 4 ka ; g3 ; , and so , the earliest msa is older than 272 ka .
unit 10 at the nearby site of kulkuletti is dated to 280 8 ka , but this age is less precise . however , an age of 193173 ka ( 183 10 ka ) from here provides an age for younger msa deposits .
small acheulian hand axesare found at the base of unit 9 , while from the upper part of unit 9 and upwards , only msa artefacts are found .
these msa artefacts include medium to large retouched points and scrapers , some of which were made by the levallois method and many look quite advanced and like younger msa assemblages in the region .
this is suggested to be in part due to the use of obsidian at these sites when compared to others like kapthurin .
these artifacts display enormous variability in the sense of technology and retouched tool forms and neither the frequency of use of the levallois technique nor tool size showed consistent change within the sequence ( > 272173 ka ; [ 114 , 115 ] ) . despite the small hand axes in the base of unit 9 the gademotta and kulkuletti ,
msa is suggested not to have an underlying acheulian with characteristics of the fauresmith or sangoan .
clark et al . describe the remains of anatomically modern humans from the upper herto member of the bouri formation , middle awash in ethiopia dated to between 166 ka ( 163 3 ka ; ma9825 ) , and 147 ka ( 154 7 ka ; tg 120 ) that are associated with lcts .
along with the omo 1 fossil , these anatomically modern human remains represent some of the earliest in africa .
it should be noted that while 195 ka is the often cited date of the omo fossils , in reality , the fossils date to sometime between 198 and 97 ka ( 196 2 ka ; 104 7 ka ) although they are argued to lie closer to the older age .
millard suggests an alternative age range based on the data of 19198 ka but again stresses that the true age is likely closer to the older value .
this could make them roughly contemporary with the herto fossils rather than significantly older than them .
despite the young age , like the kapthurin formation , the herto assemblage includes some lcts being made from levallois flakes .
msa tools dated to around 125 7 ka have also been recovered in association with lcts and acheulian cores at the site of abdur in eritrea .
. suggest that this transitional industry indicates acheulian and msa technologies continued to coexist for much longer and that the acheulian maybe widespread on the red sea coast from egypt to djibouti at this period .
these two occurrences appear to represent the youngest occurrence of lcts in africa , but whether these stone tools are intrusive or re - used acheulian artefacts remains to be seen .
no lcts were recovered from in situ excavations at herto and all the lcts come from controlled surface collections .
the sangoan ( like the fauresmith ) has been classed as a final esa industry , possibly about 300 000 years old although some researchers would classify it as a transitional industry or even an early msa .
describes a transition from the acheulian , through sangoan to the lupemban - like msa at site 8-b-11 , sai island , sudan .
maximum age estimates for the sangoan deposits is based on osl dating of the underlying aeolian es sands between 242204 ka ( 223 19 ka ) .
minimum age estimates are provided by an osl age from above the middle sangoan levels at between 202142 ka ( 152 10 ka ) .
. suggest that this site , along with kalambo falls , strengthens the case for identifying the long - debated sangoan as a valid taxonomic entity for the early msa , with a geographical distribution extending far beyond the congo basin .
compared to other transitional sites , it is perhaps significantly younger at between 242 and 142 ka , when classical msa assemblages already occur in eastern and southern africa .
state that the osl ages below the lowest sangoan layer and above the acheulian layers were quite scattered . without publication of this data it is impossible to evaluate the reliability of these ages . in certain circumstances with high background radiation concentrations these ages could be considered as minimum age estimates due to saturation of the osl signal . in the lowest sangoan assemblage ,
hand axes are actually absent , but it also has few flake tools and has been designated primarily on the basis of heavy duty tools such as core axes .
the only hand axes that do occur come are two from the middle sangoan levels . rots and van peer suggest this is a reflection of core - axe manufacture at the site .
the core axes are suggested to have been hafted and represent a complex behavioural system suggestive of economic specialization .
rots and van peer suggest that the sangoan should be maintained as a taxonomic unit and that it represents the earliest stage of the msa , at least in that part of africa .
other sangoan sites in eastern africa include those from the lake eyasi beds in tanzania which has an interpreted age of > 132 7 ka based on uranium series ages from bones within the mumba rockshelter .
this age should be taken as a minimum age estimate and its stratigraphic correlation to the sangoan bearing deposits is also likely questionable .
younger simplistic flake industries from the northeast bay of lake eyasi , which have also been attributed to the msa and are dated to between 132 and 82 ka although high thorium concentrations in the samples make the dating less than ideal .
these msa artefacts are interestingly associated with an archaic homo sapiens skull despite this young age .
until recently , there was little evidence for the period between these acheulian deposits and the occurrence of widespread msa bearing caves along the coast of south africa in the last 120 ka .
the oldest msa sites were florisbad ( 279 47 ka [ 12 , 19 ] ) , klasies river mouth ( < 120 ka ; ~115107 ka ; ) , and border cave .
florisbad is associated with ( h. helmei ) and the other two sites with modern humans .
more recently a series of new potentially early msa sites have been identified in southern africa .
the new river terrace site of cafema , along the lower cunene river on the border of namibia and angola is noticeable in being in an area where little information was previously known .
however , current dating of the site is based on sand layers beneath the archaeology which provides a maximum age estimate of ~220 ka and as such the msa could be much younger . along the coast
the lack of older deposits was hypothesised to be due to the fact that the last interglacial high sea level stand at ~125 ka ( mis 5 ) would have eroded out any earlier deposits ( e.g. , mis 6 ) from these coastal cave sites , or they would have occurred on the now submerged coastal platform . however , there was little evidence of this .
recent confirmation of this has come in the form of msa deposits from the lc - msa lower horizon of cave 13b at pinnacle point at ~174153 ka .
these have survived as remnants along the wall of the cave and were not washed out by high sea levels due to the caves elevation and due to the cementation of the deposits by speleothem .
blades , points , and levallois flakes all occur in the lc - msa lower along with frequent bladlets .
blades are more common than points , which have similarities to the sequence described from the undated levels at the cave of hearths .
schoville suggests that edge damage is consistent with the use of points as knives rather than projectile points and as a whole the assemblage lacks significant retouch or formal tools .
brown et al . also suggest the presence of heat - treated silcrete lithics in these deposits although they are more numerous during the lc - msa middle deposits between 130 and 120 ka .
pinnacle point has also yielded humans remains ; however , their age is uncertain given that they were found out of context . an age of between 170 and 91 ka is a broad estimate for the age of the fossils based on the age of the deposits at the site .
all the h. sapiens fossil remains from border cave ( bc1 - 5 ) are older than the howieson 's poortlayers and are likely older than ~65 ka based on the dating of the howieson 's poort at numerous sites across s. africa [ 19 , 50 ] .
this is supported by the direct dating of the youngest bc5 fossil to between 79 and 69 ka using esr [ 19 , 127 ] .
perforated shells similar to the ones found in the burial are known from blombos and sibudu caves in association with the still bay around 7669 ka and in north africa with the aterian around ~82 ka .
the bc1 - 2 remains and the oldest msa lie somewhere between 179 and 80 ka depending on their provenience , while potentially older and relatively unexcavated msa layer , 6bs , is perhaps as old as 238217 ka .
as at sterkfontein ( see above ) , the reliability of the esr ages at border cave are high due to the very low levels of uranium in the samples , which simplifies uptake history modelling , as well as their broad agreement with the radiocarbon ages on younger deposits ( esr ages are very slightly younger than the c14 ages ) and amino acid age estimates [ 19 , 129 , 130 ] .
the cave of hearths has often been considered to be comparable to the older deposits at border cave , being termed part of the pietersberg industry , also suggested to occur at wonderwerk cave .
sinclair notes that hominins practiced pcts to make blades and convergent points with a small number of these flakes being modified further without clear evidence that they were being retouched to a clear design .
sinclair suggests that this minimal sense of patterning in the msa is consistent throughout the sequence .
however , in the earliest levels , blades generally tend to be much longer , while in the younger layers , retouched points tend to be more numerous . in both cases
beaumont and vogel suggest that bed 4 here represents a fauresmith industry due to the occurrence of two possible lcts from this layer .
however , mcnabb and sinclair note no evidence for a fauresmith like assemblage at the site .
florisbad contains a number of msa layer sand has also yielded remains of homo helmei , dated by esr to 259 35 ka ( 294225 ka [ 12 , 19 ] ) .
n - p ( peat 1 and brown sand ) which range in age between 327 and 208 ka based on a combination of esr and osl .
the esr and osl correlate quite well although with large error ranges ( esr : 259 35 ka / osl 279 47 ka and 281 73 ka ) .
however , there is a significant scatter in esr ages for some layers and inversion of ages for both esr and osl ages .
however , the esr ages are not completely inverted as stated by millard the depth scale is inverted in the grn et al . publication .
state that this is due to the complex history of the spring formation and intermixing of both fossils and older and younger quartz . as the osl dating was multigrain rather than the more modern single - grain dating method this is quite likely .
grn states that the main error is the uncertainty in the reconstruction of the radioactive environment .
most layers have a very low density of stone tools ( 15176 artefacts ) , the exceptions being unit f ( 1654 artefacts ) , dated to 121 6 ka ( 127115 ka ) using esr and 133 31 ka ( 164102 ka ) and 128 22 ka ( 150106 ka ) using osl . as such ,
this deposits has a large assemblage of expedient , lightly retouched msa tool types and utilized flakes with few formal tools and little variability .
florisbad unit m , has a relatively substantial occupation given the size of the trench at this depth ( 120 artefacts ) and is dated by osl to 157 32 ka ( 189125 ka [ 13 , 19 ] ) .
as such , this is an mis 6 occupation , a period when some researchers believe early modern humans were only located along the coast of south africa .
the florisbad dates and perhaps also those from border cave suggest this was not entirely the case , unless these msa assemblages are being made by remnant but soon to be extinct populations of archaic modern humans .
note that unit m has a proportion of heavily retouched artefacts , mostly sidescrapers and a number of retouched points and convergent flake blades .
the oldest msa artefacts are dated to between 327 and 208 ka , and kuman et al .
suggests that the oldest msa is on a wider range of raw materials and is generally quite informal when compared to the younger material .
there is less emphasis on prepared core techniques , and there are no heavily retouched pieces and only one point .
however , this is with the caveats that this is only the case if the material excavated by meiring ( see ; 75 artefacts plus kuman et al . )
were well provenance and representative . given the formation of the site the hominin skull likely dates to mis 7 ( ~145190 ka ) .
an upper age range for the msa at florisbad is probably the beginning of mis 8~310 ka .
the age of the oldest msa and the hominin remains from florisbad are therefore , roughly the same age estimates for the oldest msa in the kapthurin and gademotta formations ( see [ 106 , 113 , 115 ] 280250 ka ) and again older than the anatomically modern human remains from omo and herto 198147 ka [ 15 , 16 ] .
lincoln cave has yielded an msa assemblage dated by uranium series to somewhere between 278107 ka ( 252.6 35.6 and 115.3 7.7 ka ; ) .
note that acheulian like cores occur in lincoln cave and explain their occurrence as mixing from member 5c rather than the late occurrence of these core types .
contemporary msa bearing deposits ( post - member 6 ; ) from the main quarry at sterkfontein have also been dated to between 294210 ka using esr ( 252 42 ka ) .
significant mixing has taken place and homo ergaster fossils have also been recovered from lincoln cave .
given that the post - member 6 deposits are seemingly continuous with lincoln cave these esr ages may suggest that the lincoln cave msa is closer in age to 278 ka than 115 ka .
taken together , this suggests that msa tools are at least as old as 210 ka in the sterkfontein area .
younger msa deposits have also been recovered from member 4 deposits at swartkrans ( < 110.0 2.0 ka ) and between 90.361.6 ka ( 62.9 1.3 ka and 88.7 1.6 ka ) at plovers lake ( both dated by uranium series on speleothem ) . a direct single isochron esr date of 81.270.0 ka ( 75.6 5.6 ka ) for the plovers lake artefact horizon correlates well with the u - th age .
this data , along with that presented by herries et al . and herries and shaw further illustrates the robust nature of the esr dating of the cohk sites with esr correlating well with other geochronological methods over a time range of 2.6 ma to ~80 ka . as grn notes ,
such comparisons make it highly likely that esr ages for the esa bearing sterkfontein member 5 deposits are also reliable when discrepancies in the geological context are taken into account [ 75 , 85 ] .
these new dates for the cohk sites indicate a rich msa heritage that has until recently been overlooked due to a predominant interest in the early hominin and early stone age bearing layers of these sites .
this recent work suggests that msa deposits may occur in the cohk between ~250 and ~60 ka with the oldest being as old as those from east africa .
describe early msa technology from block a , and f at twin rivers in zambia . in block
a a flowstone at the base of the deposit as well as a piece within the deposits has been dated to > 400 ka and is an upper age limit for the deposit
. however , barham et al . suggest a hiatus occurs before deposition of the slurry flow which has seemingly eroded parts of the basal flowstone into the deposit .
the oldest msa deposits from twin rivers ( block a ) are suggested to date to between ~266 and ~172 ka , while those from f block are suggested to date to between ~200 and ~140 ka .
this a block assemblage includes backed tools that suggest early evidence for composite tool technology as well as bifacial projectile points and tanged points .
unifacial points were not recovered during the 1999 excavations but occurred in very low frequency during clark 's excavations during the 1950s .
the bifacial points include lanceolates characteristic of the early lupemban and flaking occurred using both an anvil technique and radial and prepared core technique .
twin rivers stands as some of the earliest potential evidence for the msa in southern africa and particularly the definitive use of ochre .
however , many researchers are sceptical over the association of the flowstone to the msa bearing deposits . in many instances ,
flowstone is sampled from the wall or edges of a cave cavity without definitive evidence for its association to the archaeology . at twin rivers , for example ,
the flowstone does not occur as a continuous layer capping the msa deposits but as remanents on the edge of the cavity .
therefore , the flowstone perhaps only provides a maximum age estimate ( [ 138 , figure 10.15 , 10.13 ] ) . the fact that younger speleothem dated to between 184172 ka ( 178 6 ka ) and 141137 ka ( 139 2 ka ) occurs under a speleothem dated to between 200190 ka ( 195 5 ka ; 131 ) in block a. all tl dates from g block are younger than 117 ka ( 101 16 ka ) and lend further suspicion to an extremely complicated stratigraphy and infill .
the speleothem dates to between 266 6 ka ( 272260 ka ) and 172 2 ka ( 174170 ka ) may also have been eroded out from earlier deposits when the msa in - filled the cavity .
again younger speleothem samples occur with depth with the 172 ka sample being deeper ( 3.2 m ) than the 192 2 ka sample at the top of a block at 2.2 m. all the msa in the top 1 m of a block is , therefore , younger than 174 ka , as the speleothem must have formed before it was eroded and incorporated into the breccia and so provides a maximum age .
a tl age from over 4 m depth gave an age of 132 31 ka and would suggest that all the deposits are younger than 163 ka , but the dose rate estimates for the deposits from which the sample came were not estimated , and a dose from higher in the sequence was used , making the date unreliable
. however , a u - th sample with a date of 160 3 ka also occurs at 2.4 m depth and is the youngest age from block a. this suggests that all the msa in block a may in fact be younger than 163 ka .
if the majority of speleothem represents material eroded into the deposit then the lupemban from block a would be younger than 141 ka , significantly younger than the 266170 ka cited by barham et al .
, the > 266 ka or > 230 ka age estimate that is often cited , or the ~300 ka age cited by barham .
there is little evidence that the msa deposits are older than 266 ka except for a small deposit at the very bottom of the excavation , and this does not apply to the assemblage as a whole .
the f block deposits are also potentially < 140 ka based on the u - th ages and g block is likely < 117 ka based on tl ages . an age range of 14148 ka could be estimated for the various msa deposits at twin rivers based on the uncertainty of the provenience of the speleothem samples ( excavators need to be extremely careful when relating fragments of flowstone to wider archaeological deposits in caves due to their complex depositional history . all publications should show a clear photograph of the association of the speleothem to the deposit been dated or even micromorphological analysis across the contact .
fauresmith sites have been noted at numerous sites on the vaal river ( e.g. , canteen koppie , nooitgedacht 2 , roseberry plain 1 , and powers site ) , the orange river , and the seacow valley as well as pan sites near kimberley ( kathu pan and rooidam ) and the cave site of wondwerk cave .
almost all these occur in eastern - central south africa , the exceptions being at and perhaps bushman 's rockshelter .
clarke notes that it is the smaller chert tools ( blades and unifacial points with faceted platforms and prepared cores ) in addition to the larger hand axes and cleavers that give the kalambo falls assemblages , particularly floor 4 , a fauresmith flavor , and make it seemingly distinct from the industries in the luangwa valley .
these floors were later termed archaeological horizons , but many are now not considered to be in situ accumulations but material redistributed by alluvial action . the locality is extensively described in , and four main sites have been defined [ a d ] .
the lowest horizons have been defined as upper to terminal acheulian in character and are referred to as the bwalya industry , and it has a very high percentage of lcts ( e.g. , 67% of the shaped tools in one excavated horizon ) .
many of the lcts are small , lanceolate in shape , and could be argued to represent bifacial points evolving in the direction of the later lupemban points .
small scrapers make up the majority of the light duty tools a few of which are convergent and resemble unifacial triangular points .
it has a much higher percentage of light duty tools , particularly scarpers , and it is suggested that this stage represents the beginning of the msa and disappearance of acheulian lcts . there is a trend towards more specialised flake forms , with blades .
the characteristic large tool is the core axe , which does occur in the lower acheulian horizons but is in much greater number in the sangoan levels .
clark states that the chipeta industry , though in some ways transitional , is the initial stages in the msa trend to increased specialisation .
while lanceolates are absent in the kalambo falls sangoan , clark notes that they do occur in other sangoan assemblages .
lanceolate bifaces are the characteristic tool of the lupemban levels of the msa along with an increase in lighter duty tools such as retouched unifacial points and blades .
potentially , utilised wood also come from the acheulian and sangoan levels and consist of possible clubs and pointed stakes and tools .
sheppard and kleindienst concluded that there is little change , at kalambo falls , in the basic techniques of blank production or the attributes of the blanks produced from the earliest acheulian layers to the later msa layers .
the only marked change to occur is the loss of lcts ( hand axes and cleavers ) and their replacement by heavy - duty forms of core axes and picks .
while lanceolates are absent in the kalambo falls sangoan , some of the lcts from the acheulian layers have a similar lanceolate character that suggests some continuity between these earliest levels and the lupemban
. the intermediate sangoan may represent an industry related to a particular climatic shift in the region , perhaps related to a warmer , more forested interglacial .
a range of radiocarbon ages exist for these early levels ( 60.332.6 ka bp ) , but they are considered to be infinite age estimates .
this was followed by an amino - acid racemization age on wood from the acheulian levels that suggested an age of ~110 ka .
a series of uranium series ages were then undertaken on wood , but because of the open system nature of the samples , they should be considered as minimum age estimates .
the acheulian layers were dated to 182 16 ka and 182 10 ka ( 198166 ka ) and the sangoan levels to 76 10 ka ( 8565 ka ) .
hopefully , osl dates will soon be forthcoming for at least the younger msa ( lupemban ) deposits at kalambo falls .
peter beaumont excavated a number of sites in the northern cape province throughout the 1970s and 1980s and it is these sites that now appear to hold the key to understanding the transition between the acheulian and the msa in the region .
kiberd recently described new excavations at the site of bundu farm in northern cape .
the site has yielded only one lbct from layer g6 but kiberd defines layers 46 as a terminal acheulian or transitional esa / msa industry ( i.e. , fauremsith ) .
layer g4 was dated by kiberd to 145.7 16.0 ka , providing a minimum age estimate for the g6 layer of > 129.7 ka .
unprovenienced teeth from the site gave esr age estimates of between 394 and 144 ka .
based on the occurrence and absence of fauna in layers of the site , kiberd suggests that this fauna most likely came from layers g4 and g5 .
this suggests that the g6 horizon containing the lcts is dated to > ~300400 ka based on a linear uptake model ( this model is the one that gives consistent ages for the cohk sites ) and ~200 ka based on an early uptake model .
prepared cores occur in all layers but are slightly more abundant in the younger layers that are < 161 ka . in contrast , flake blades ( this includes points , which are often referred to as convergent flake blades in south africa ) are generally more abundant in the oldest layer ( g6 ) , which also contains the one lbct
overall , the sequence shows the occurrence of levallois technology , blades , and points and in the oldest layer ( g6 ) a single lbct .
overall , the bundu farm sequence appears to contain all the components of the msa in layers that are most likely dated to sometime between 400 and 200 ka .
brink ( pers . comm . in kiberd ) notes that the connochaetes gnou fossils are of a similar size to those from florisbad ( 294225 ka ; ) and that the fossils reflect a period of increased rainfall . based on the early uptake model for
the esr ages the oldest deposits g46 perhaps dates to roughly mis 7 at ~240190 ka . if the linear uptake model is correct ( as it appears to be for many sites in south africa ) , the oldest layers likely date to mis9 between ~340310 ka .
it has a sequence of what are suggested to be almost continuous deposits dating back nearly 2 million years [ 47 , 71 ] .
beaumont and vogel suggest that msa assemblages are represented in major units ( mu ) 2 in excavations 2 , 3 , 5 , 6 and 7 and fauresmith assemblages in mu3 - 4 in excavations 1 and 2 .
note that no fauremsith like material is noted in their reanalysis of the excavation 1 material .
the excavation 1 material is , therefore , entirely acheulian in character , and its age has previously been discussed above .
chazan and horwitz further suggest that all material from excavation 6 is fauresmith in character with the co - occurrence of oversized blades , prepared core technology and lbct in contrast to beaumont and vogel 's interpretation of these deposits as being msa .
chazan and horwitz also note that in excavation 6 , there is no evidence for underlying earlier esa deposits as are found in excavation 1 at the front of the cave , the top of which dates to < 780 ka .
a single u / th date ( u-576 ) on a fragment of stalagmite recovered from within the fauresmith deposits of excavation 6 gave an age of 187 8 ka ( 195179
estimate for the age of the deposit and that it dates the fauresmith to between < 780 and > 179 ka .
in actual fact , if a fragment of speleothem occurs within a deposit it must have grown before the deposit was formed , not after it . as such , this is a maximum age estimate for the layer in which the speleothem fragment was found and suggests the fauresmith assemblage is < 195 ka , making it only slightly older than the pinnacle point msa at 170160 ka and perhaps similar to the lbct assemblage from herto .
if the speleothem capped the underlying deposit , then it would indeed suggest that the deposit below this were older than 179 ka . however , chazan and horwitz are not clear on the contextual issue of this sample , the age for which has come from the earlier publication by beaumont and vogel .
beaumont and vogel state that items of calcite , mainly in the form of small ( ~520 cm high ) stalagmites , were found sporadically in many strata .
they do not elaborate on the in situ or ex situ context of the material other than if it is a stalagmite of a stalactite . as stated above , if a fragment of speleothem occurs ex situ within a deposit , that deposit must be younger than the age of the speleothem , as the speleothem must have formed before being incorporated .
if , however , a speleothem caps a deposit by forming directly on it and is in situ , then the deposits under it are older than that speleothem and the deposits above it in theory younger .
if the speleothem is a flowstone , then it may also represent a false floor under which younger deposits may have been deposited .
as previously noted , it is very important to understand and state the relationship of speleothems to the deposits and archaeology being dated and present clear evidence of this in publications .
the other ages for fauresmith layers at wonderwerk come from the top of stratum 3 in excavation 2 and give ages of between 315 and 247 ka .
as these are soda straws that fell into the deposit after deposition of the fauremsith deposits , they provide little chronological evidence for the age of the majority of the deposit other than suggesting that the layers in which the straws were found are younger than 247 ka .
it does not , as beaumont and vogel suggest necessarily mean that the fauremsith layers date back to between 286 and 276 ka .
a stalagmite in the top part of the msa layers in excavation 2 had a basal age of 234206 ka and may suggest that the msa in this area is greater than 206 ka .
however , the exact relationship of the stalagmite to the deposits is again not stated .
there appears to be some evidence for the movement of material by water at the site which was once an active cave resurgence .
this can be seen by the mixed nature of artefacts in the rear mostly in - filled tunnel and the eroded surface in many sections which slope back towards the rear of the cave .
moreover , a stalagmite that is seemingly lower in the section is dated to between 182 and 154 ka .
this inversion of ages in the same excavation does not suggest the speleothems are in situ and would suggest that all of the msa deposits are in fact younger than 182 ka . until a better description of context is given for these samples all age assessments using , the data of beaumont and vogel should be regarded with extreme caution .
many of the speleothems from the top of the msa excavations in different trenches date to between 100 and 68 ka and without a context for the samples the deposits could in theory be not much older than that age or even in theory younger .
based on this re - analysis , there appears to be little current evidence for fauresmith deposits older than ~300200 ka .
previous dating of the site was based on elephant fossils that were more evolved than those from olduvai bed iv .
this simply gave the site an age of < 1.07 ma or < 780 ka based on the interpretation of the palaeomagnetic data . at kathu pan , the msa layers fall in the time range between 336 and 254 ka ( 291 45 ka ; , perhaps during mis9 ( 340310 ka ) .
the layer 4a fauresmith assemblage at kathu pan contains levallois cores , retouched points , blades and lcbts and has been dated , to somewhere between 511 and 435 ka based on a combination of osl ( 464 47 ka ) and esr ( 542 + 107/140 ka ) .
if so , the layer would date to between 682 and 435 ka ( based on esr alone ) .
an mis13 age ( 540470 ka ) might be a good estimate based on this data but certainly older than 417 ka .
this suggests that all the tool forms found in the msa are already in place by at least 417 ka .
the retouched points have facetted platforms and are in stark contrast to early msa assemblages that are suggested to lack formal tools and retouched points .
note that the extreme lateral convexity of the lithics distinguishes them from the norm for the levallois method , despite the fact that they typologically and technologically fit within the levallois .
the age of the acheulian 4b layers has not been determined other than being older than 4a .
note that lcts in the fauremsith horizons are made on a wide range of raw materials and are crude and irregular , while those from the acheulian are exclusively made on banded ironstone and are symmetrical and refined .
this may reflect the hominins developing new raw materials for lbct manufacture during the fauresmith as part of experimentation of new methods of stone tool manufacture .
the occurrence of a fauresmith industry at kathu pan , so close to wonderwerk cave , dated to > 417 ka may lend weight to the fauresmith at wonderwerk also being in this time range or at least older than 182 ka as suggested by chazan and horwitz unless it occurred for over 200 ka in the region and was being produced contemporarily with the msa .
describe a series of sites ( keratic koppie , hackthorne , and kudu koppie ) from the mapungubwe national park ( formerly vhembe dongola np ) in northern south africa on the border with botswana and zimbabwe .
suggest the site represents a charaman like industry as described from zimbabwe , which itself is perhaps a version of the sangoan .
these two industries were grouped together into clark 's first intermediate period along with the fauresmith .
although they show marked typological differences , the parallels between the sangoan and the fauresmith are striking .
suggest that after 200 ka classic msa technology occurs that is well developed , yet this does not reflect a change so much in the actual technology as a shift toward a lighter , more mobile toolkit which incorporates hafting .
note that the prepared core reduction strategies at kudu koppie suggest that both the late esa and msa toolmakers employed the levallois volumetric concept , but they often exploited a nodule 's natural convexities and form .
the msa toolmakers use a greater variety of prepared core methods and more intensively exploited cryptocrystalline and microcrystalline nodules , the scarcity of which may have resulted in a more formalized application of the levallois volumetric concept .
kempson suggests that the hackthorne site may represent mixing of esa and msa deposits , while keratic koppie preserves an assemblage with a mixture of acheulian and msa components .
kempson arguesd the assemblage is a post - acheulian industry with a major component of woodworking tools , suggestive of the sangoan industrial complex .
davies and kuman also suggest the occurrence of sangoan - like tools along the kwazulu - natal coast including coastal dunes near port edward , which if confirmed would truly throw out the idea that these are environmentally specific or regional entities .
this is close to the site of amanzi springs which deacon [ 73 , 151 ] suggested might be later acheulian .
rooidam 1 contained a rich stratum 9 , which sits 3.9 m below the surface and yielded 90% of the 19,000 artefacts recovered from the site [ 47 , 70 , 152 ] .
previously , the occurrence of small hand axes as well as a small amount of cleavers , blades and convergent points suggested this was a fauresmith layer .
beaumont and vogel later assigned this layer to the final acheulian , stating that while prepared cores and blades were present , there were no convincing levallois points . here ,
beaumont and vogel suggest that the late acheulian can be distinguished on the basis of true blades and that levallois points distinguish the fauresmith . here
the upper msa levels were also reclassified as fauremsith , which also occurs at rooidam 2 .
szabo and butzer conducted u - th dates on two calcretes sandwiched between the late acheulian and fauyremsith occupations and overlying the main stratum 9 occupation .
szabo and butzer 's unit c correlates with unit 9 of beaumont [ 47 , 70 ] . the u - th ages are inverted for the sequence .
the unit c deposit of szabo and butzer covering stratum 9 gave an age of 108 + 40/20 ka ( 14888 ka ) .
the stratigraphically higher unit g gave an age of 174 35 ka ( 209139 ka ) .
szabo and butzer argue that the younger lower age is due to the open system nature of the system in unit c and recrystallization .
they state that recrystallization does not appear to have occurred in unit g and so the age is accurate for that unit .
it seems likely that both ages are minimum age estimates due to the open nature of the system , and so , the age of the stratum 9 late acheulian depositsat the site can only be said to be older than 139 ka .
the upper fauresmith deposits in theory date to less than 209 ka and might suggest that the fauresmith occurs relatively late at the site ; however , if recrystalisation had also effected the lower sample to any degree , then these deposits might also be significantly older than this .
kuman describes a series of undated sites ( db3 and 4 ) near taung where lcts are associated with convergent points and prepared core technology and also with a higher representation of end struck rather than side struck flakes , diagnostic features of the fauresmith . at db3 kuman notes the occurrence of levallois and victoria west cores in the same context . hence , despite the dominance of simple flaking features , core types , faceted platforms and dorsal scar patterns together show a range of relatively advanced flaking techniques that are seen in the assumingly later msa of the region . at db3 , the majority of lct are side struck , while most small tools are end struck , which is out of character for lct of fauresmith type as described by goodwin .
the db3 acheulian assemblage contains three types of flaking technique ( radial , convergent , and parallel opposed ) , which are documented more consistently in the ensuing middle palaeolithic and msa .
bushman 's rockshelter was excavated by louw who describes a number of lcts or lct - like stone tools from layer 41 .
this has an uncalibrated c14 age of 47.5 bp and is no doubt an infinite age .
louw suggests that the hand axes may have been picked up by msa people and reused , but it is also possible that this is a transitional industry , perhaps the fauresmith , as the deposits do not seem to have sangoan elements like the limpopo sites .
if so , then this would suggest a greater regional spread of the fauresmith , but the deposit is as yet undated .
as outlined in the opening discussion , there are a number of theories concerning the terminology of the esa to msa transition and it is likely far from a simple transition that occured at exactly the same time or the same way in different areas of africa .
the sites of the kapthurin and gademotta formation in eastern africa indicate that the msa in its classical sense occurs at roughly the same time between 300 and 250 ka .
the kathu pan msa at 336254 ka is potentially older and suggests the earliest msa occurs synchronously in eastern and southern africa .
further potential evidence for this comes from florisbad and sterkfontein - lincoln cave sometime between 300 and 200 ka .
the sites of the kapthurin formation also indicate that many characteristic elements of the msa occur alongside elements of the esa including lcts from roughly 500 to 250 ka .
these have been termed msa , sangoan , fauresmith , and acheulian depending on the groups of tools found at different sites and has been used to suggest independent evolution of all the elements ( points , levallois , etc . )
that come together to form the msa as an entity and in essence augments previous acheulian technology .
blades come into use first around 545509 ka , followed by points , large prepared cores , and then centripetal levallois cores and flakes all the while occurring with lcts .
it is not until 250200 ka that convergent levallois flakes and cores occur in the absence of lcts that the msa is seen to start .
of course , lcts may occur much later ( < 160 ka ) in some contexts in etirea and ethiopia .
interestingly , the > 272 ka msa in the gademotta formation in ethiopia seems not to be underlain by such transitional industries .
potentially similar trends are seen at kalambo falls with a transition from the acheulian , through the sangoan and into the lupemban - msa , but this site remains unreliably dated . at kalambo
falls , there seems to be a definite trend through time but with the sangoan being more of a distinct entity , perhaps relating to a more forested , interglacial period .
the only reliably dated sangoan assemblage comes from sai island in the sudan at sometime between 242142 ka .
this is contemporary with the early msa assemblages in the kapthurin and gademotta formation as well as several sites in southern africa ( border cave , florisbad , and sterkfontein - lincoln cave ) . at kalambo
another age for the sangoan comes from tl dates ( 305203 ka ) that overlies sangoan deposits at the site of bt 1 in the ivory coast .
however , barham and mitchell note that the bt 1 site has a high percentage of lcts than other sangoan assemblages such as kalambo falls and may indicate mixing of this open deposit .
the sai island sangoan is also the same age as the lupemban - msa deposits at twin rivers in zambia if the dates of 266172 ka are correct .
this appears to show that the sangoan and early msa are contemporary in different areas of africa with lupemban - like msa at sai island also being later than seen in zambia .
it is possible that the sangoan here is of a similar age to that from sai island perhaps representing a spread from a more southern central area of africa with the kalambo and kapthurin formation sangoan being older .
however , if the twin rivers lupemban is younger than 160130 ka , as potentially seems the case based on the inverted speleothem ages , then it is of a similar age to the lupemban - like msa at sai island ( < 162 ka ) this is the same age as the pinnacle point 13b msa in south africa and material from border cave , wonderwerk cave , and florisbad but much younger than the earliest fauresmith from kathu pan at 417 ka .
if this is the case then there is perhaps little reason to see the sangoan at kalambo as much older than its 182 ka minimum age .
if it is related to a climate shift then it may be mis7 in age ( 240190 ka ) .
it has often been suggested that the sangoan is a woodworking specific industry in part due to its discovery in heavily forested areas such as zambia and the congo .
this is perhaps even more influenced by the association of wood at kalambo falls , one of the few sites to preserve such material .
a number of undated sangoan occurrences have also been described from south africa , both along the costal dune systems of kwazulu - natal and the very northern limpopo river region .
if confirmed this suggests a distribution , if sparse , across almost all of africa and appears to argue against either the sangoan being a regional entity , or perhaps an environmentally specific one .
moreover , rots and van peer suggest the core axes are being used as digging rather than wood working tools . based on known occurrences ,
the sangoan is , however , not widespread across most of south africa , where the related transitional industry , the fauresmith , has also been described .
this too has been suggested to be a regionally or environmentally specific industry , but again , a lack of dates has hampered its comparison to other industries such as the sangoan .
the majority of the fauresmith occurrences have been described from the river sequences of the northern cape and free state . however , a number of potential fauresmith sites ; namely , vereeniging and particularly bushman 's rock shelter occur at the very extremes of the fauresmith range [ 38 , 142 ] . only reanalysis and dating of these deposits and tools will help confirm if the sangoan and fauresmith are different regional entities or if the fauresmith is perhaps an earlier entity than the sangoan as the sai island dates suggest .
the acheulian levels at kalambo falls have some qualities distinctive of the fauresmith , in terms of lcts , although it do n't seem to include many of the light duty tools often associated with it . in south africa ,
the fauresmith industry has recently been dated in stratified deposits at kathu pan to at least 417 ka and perhaps as old as 647 ka and at wonderwerk cave to sometimes between 305 and 179 ka .
this is towards the older end of the kapthurin formation sequence . while the kapthurin formation is suggested to demonstrate independent evolution of all the traits that are used to classify the msa all these traits appear to occur at kathu pan during the fauresmith
of course , kathu pan is only one site and the kapthurin formation is many that indicate variation across both time and the landscape . given that kathu pan and wonderwerk are very close to each other the age of the fauresmith and msa at both sites suggests firstly that the fauresmith is a relatively long lived entity , perhaps 200 ka and secondly that fauremsith and msa assemblages may overlap in the time range between 300200 ka . either that or the msa assemblages from kathu pan represent the fauremsith but simply do not have the elements that would be used to classify it as such .
at wonderwerk , chazan and horwitz and beaumont and vogel certainly have different views on which layers are fauresmith and which are msa .
this in itself asks the question regarding the difference between the fauresmith and the msa and whether as some researchers have suggested that the fauresmith and other transitional industries should , in fact , represent the start of the msa and that the presence of lcts should be seen as merely a remnant of mode 2 technology in otherwise mode 3 dominant assemblages .
. argue that the acheulian biface persisted at abdur , possibly because it acquired a new , profitable use for the exploitation of aquatic resources by early modern humans .
if this is the case , then it provides further evidence that lcts are not distinctive only of the acheulian and their persistence in some assemblages should not be used to equate them with the acheulian but simply as a surviving esa element in an otherwise msa assemblage .
prepared core technology , particularly levallois , is inextricably linked to the msa , but later stages of the esa also document the use of prepared core reduction [ 30 , 47 , 58 , 154 , 157 ] .
good chronostratigraphic records are rare for this time period , but outside south africa , there is some evidence that prepared core reduction may even date back to almost 800 ka .
giant cores also referred to as levallois - like are known from the ii-6 archaeological unit from gesher benot ya'aqov in israel .
goren - inbar and saragusti suggest that biface production involved at least two well - established and different techniques : the levallois and the kombewa .
these techniques produced pre - determined , large - sized flakes that were modified into tools by a minimal amount of retouch .
a technological and morphometric comparison between tools manufactured by the different techniques does not demonstrate any bimodal patterning of the end products .
rink and schwarcz suggest the ii-6 unit dates to around 681623 ka based on a range of combined u - th / esr ages .
older ages have been suggested based on the location of the bruhnes - matuyama boundary at 780 ka in the lower unit ii-7 and depositional rate estimates .
however , given that the esr ages are undertaken on fossils within the ii-6 sand unit , these ages should perhaps be taken as the true age of the site unless there is significant uncertainty in the ages .
many researchers have seemingly dismissed the esr method as unreliable ( see ) . however , in the correct circumstances ( i.e. , low uranium concentrations as is the case of gby ) , the method is reliable when a linear uptake model is used based on comparisons with other geochronological methods ( see [ 19 , 79 ] ) .
the palaeomagentic reversal occurs in the base of clay unit ii-7 and as such the entire of ii-7 was deposited after the transition as well as a change of sedimentation and potential hiatus between units ii-7 and ii-6 . as such , the ii-6 unit could not be as old as ~780 ka as is most often quoted .
there is also a suggestion from the fauna that the site has a number of species not seen in europe until closer to 600 ka .
rink and schwarcz suggest that a hiatus in deposition may be responsible for this difference in palaeomagnetic and esr age estimates . however , rink and schwarcz 's study suffers from a common problem in esr studies ( see discussion in [ 67 , 79 ] for other examples ) , where all the ages are averaged together to create a mean value for different units or teeth with vastly different ages where the reasons and mechanisms for the differencing ages has not been established .
if the esr dates ( linear uptake , 40% moisture ) of rink and schwarcz are separated by units ; the lower ii-7 deposit has an optimal age of between 718 and 555 ka ( max age range 767534 ka ) and the upper ii-6 unit has an optimal age of 708559 ka ( max age range of 718492 ka ) .
the upper age range for the top of the ii-7 deposit , which contains the ~780 ka bruhnes - matuyama boundary in its base , is 767 ka , which is not much younger than 780 ka ( moreover , recent estimates put the boundary at 773 ka ) .
this suggests that the ii-6 archaeology most likely dates to sometime between ~700 and ~550 ka , although a slightly older age can not be ruled out .
these cores are not too dissimilar to both the large lct cores described for the kapthurin formation [ 106 , 109 ] between 509272 ka or the victoria west cores of south africa .
sharon and beaumont have suggested that victoria west cores were also developed as a prepared core technique within the acheulian as a means of producing ready - made blanks for lcts .
part of the problem in southern africa is that no victoria west core bearing sites have been accurately dated and few are well stratified .
victoria west technology has been recovered from stratified acheulian contexts in stratum 2a at canteen koppie in south africa .
the stratum 2a contains an acheulian assemblage that is overlain by fauresmith material in stratum 1 ( hutton sands ) , where it is then followed by an msa assemblage which has dates of 12080 ka . as such
, the victoria west technology appears to be associated with terminal acheulian artefacts and is older than the fauremsith .
while the victoria west layers at canteen kopjie are undated , if the ages for kathu pan are extrapolated , then they should be older than 540470 ka and perhaps not too dissimilar in age from those from gby at 700550 ka .
this points to a widespread use of this giant core technology around 700300 ka in africa and the levant in late acheulian and transitional industries . whether these early prepared core technologies for making large blanks for lcts are proto - levallois and evolved directly to msa levallois technology or para - levallois
is beyond the scope of this paper ( see [ 41 , 43 ] ) .
however , in the kapthurin formation , there seems to be a relative progression from these giant cores to centripetal and then convergent levallois cores and flakes .
lycett suggests that victoria west cores show independent development of prepared core technology from that of levallois technology , and as such it is para - levallois rather than proto - levallois .
however , the victoria west is chronologically older than the levallois technology of the fauresmith and similar progression as just described for the kapthurin formation could also be argued for south africa . blades also seem to be a component of either the late acheulian or the beginning of this transitional phase as shown by their first occurrence in the kapthurin formation between 545 and 509 ka and in the fauresmith at kathu pan .
large blades were one of the earliest indicators of the fauresmith and these are noted in the wonderwerk occurrence . as noted before , tryon et al .
and tryon and mcbrearty suggest that diversification of levallois technology as seen at koimilot by ~250 ka and the occurrence of convergent levallois points and cores stands as the marker of the early msa .
note that levallois technology for making lcts occurs by 350300 ka based on esr age estimates at the grotte des rhinocros and by osl at cap chatelier in north - west africa , and levallois - like cores make from lcts at kharga oasis ( egypt ) between 400300 ka based on u - series dating of tufa .
mcbrearty also suggests that the fundamental change from the esa to the msa is the end of lcts and a shift to projectile point technology .
of course , it should be noted that acheulian bearing hominins in europe were utilising an entirely wooden projectile technology for hunting as shown by the occurrence of the schningen spears at either ~400 ( mis 11 ) or ~310 ka ( mis 9d - e ; ) but were seemingly still disarticulating their kill with lcts . whether a similar wooden projectile technology was being used by hominins in africa is almost impossible to tell given the almost complete lack of preservation of such organic remains in most msa sites .
the exceptions are two wooden tools from floor 1 at kalambo falls in zambia [ 30 , 56 ] and one from florisbad in south africa .
other sites where large pieces of wood have been recovered include the acheulian sites of amanzi springs and gesher benot ya'aqov . despite the discovery of significant amounts of wood from these deposits ,
the kalambo falls tools are reminiscent in some ways of the european spears and are associated with large well - formed cleavers from the acheulian bearing floor 2 , below the sangoan . given their context
these wood tools might be older than those from europe and might point to a wooden projectile point technology in the late acheulian , complimenting the earlier lct technology . at most sites , the only clue would be in finding injury patterns on faunal remains indicative of such activities . in a similar vein
, the co - occurrence of lcts and projectile point technology in the sangoan and fauresmith may reflect similar activity patterns , or as mcbrearty suggests that the mix of technologies may , in fact , represent different hominins using different technologies at the same time in the same regions of africa .
all this has somewhat blurred the distinction between the terminal acheulian , the transitional industries , and the earliest msa and highlights the need to perhaps define a transitional phase as per clark or to perhaps redefine the msa as an entity as suggested by beaumont and vogel and van peer et al . .
certainly , the end of an industrial complex should not be defined on the last appearance of its classic tool form but by the introduction of new technology . however , in some views , this change should only be defined when this new form has become dominant , creating a rather arbitrary line . goodwin and van riet lowe defined the msa as being intermediate between the esa and lsa , both temporally and technologically .
it was in part defined by a lack of lcts at its upper age limit and lack of microliths at its younger age limit .
msa industries such as the howieson 's poort have shown that microlithic technology does occasionally occur within the later phases of the msa , perhaps in response to distinct climatic fluctiuations ( 6857 ka ) .
the msa was later recognized as being blade based , rather than flake based , and that it included the common use of the levallois technique [ 120 , 170 ] .
the sites described above show that all these markers of the msa , that is , blades , levallois technology begin to occur contemporaneously with lcts between 548 and 272 ka in both eastern and southern africa [ 107 , 108 , 148 ] .
kathu pan and bundu farm suggest that even at this early time period lcts seem to make up a much smaller component of the stone tool assemblages than at earlier acheulian sites .
klein notes that many researchers ( e.g. , [ 39 , 40 , 172 ] ) have moved beyond goodwin 's 1928 classification of the msa to include any industries which are characterized principally by retouched and unretouched flakes of various kinds and/or which are chronologically intermediate between the hand axe industries of the esa and the largely microlithic industries of the lsa .
klein notes that the prominence of triangular flakes with convergent dorsal scars and faceted butts is no longer an important criterion as they do not occur in all assemblages , many of which would be considered
if a similar age to material from the nearby caves at pinnacle point it could be of mis 6 age ( 200120 ka ) but there are definite differences , so perhaps it is even older or represents another part of mis 6 variation .
as most lithic specialists would concede goodwin emphasized in his early papers that the msa was not homogeneous in time and space and in this sense many researchers would see the classification of the fauresmith into the esa or msa a semantic one or an attempt at pigeonholing .
however , in archaeology , as with geology , classifications , frameworks , and the boundaries between these entities need to be established somewhere . with the ever increasing age for the first lsa industries based on the presence of microlithics and mode 4 and 5 technology and the retention of lcts ( particularly hand axes ) as the defining character of the acheulian
, the msa is beginning to be squeezed into an ever decreasing age range with little potential significance and no association with the hominin record .
cornelia - uitzoek and arguably elandsfontein and duinefontein ii are acheulian assemblages that date to around 1.10.8 ma , and all three do not contain prepared core technology or other technology reminiscent of the msa .
a such , they seem to represent what might be termed a middle acheulian period between the earliest acheulian found prior to 1.1 ma at sterkfontein and the oldest vaal river deposits and the final acheulian period that contains victoria west giant prepared core technology such as canteen kopjie .
it is clear that the msa first occurs before the advent of anatomically modern humans , and yet , there is also potential evidence that some of these modern human are also still utilising esa technology . the co - occurrence of msa technologies at florisbad with h. helmei at ~260 ka and h. sapiens with lcts at ~160 ka is the opposite of what might be expected if these industries were being made by different hominins based on the exclusive co - occurrence of the msa with h. sapiens fossils after 120 ka .
many researchers see the beginning of the acheulian as a clear change in hominin behaviour related to the appearance of h. ergaster and the middle acheulian site of elandsfontein , without prepared core technology is associated with h. erectus or very early h. rhodesiensis .
the first occurrence of msa - like characters in the fauresmith is a time period where archaic forms of homo sapiens begin to occur in africa in the form of homo rhodesiensis and then h. helmei .
unfortunately , the middle pleistocene hominin record of southern africa is very spartan and still not well dated . certainly , the traditional start of the msa between 300 and 200 ka associates it with h. helmei fossils such as the florisbad cranium and not h. sapiens .
it seems likely that the beginnings of msa style technology in the fauresmith began with h. rhodesiensis and gradually evolved through time to incorporate a greater range of technology , including diversity in projectile point , composite stone tool , and levallois technology by the time h. helmei is first seen soon after 300 ka . by the time the fauresmith is noted , it has all the features of the msa with small prepared cores and points that may very well represent the beginning of composite tool technology and also projectile points .
a number of the kathu pan tools certainly look like they were made as projectile points ( see [ 148 , figure 6 ] . the sangoan has certainly been suggested to contain some of the first hafted stone tools , and it is perhaps this major technological change along with the classic msa forms that occur in the fauresmith that makes these two entities either regional variants at the beginning of the msa or perhaps temporally distinct industries during a transitional phase between the esa and msa .
the switch is in essence one of scale with the period represented by lcts and prepared cores for making lcts representing the beginnings of a transitional period in both stone tool industries and hominin species .
as the acheulian is inextricably linked to h. ergaster and h. erectus , so too , the beginnings of msa technology appear to occur in the fauresmith with the advent of archaic h. sapiens ( h. rhodesiensis / h .
helmei ) and come to full flourishion before the advent of the first modern humans at ~200150 ka . only better dating of the fauresmith and sangoan sites will help answer what will most likely turn out to be an extremely complex process of transition from the esa to the msa .
|
an understanding of the age of the acheulian and the transition to
the middle stone age in southern africa has been hampered by a
lack of reliable dates for key sequences in the region .
a number
of researchers have hypothesised that the acheulian first occurred
simultaneously in southern and eastern africa at around
1.7 - 1.6 ma .
a chronological evaluation of the southern
african sites suggests that there is currently little firm
evidence for the acheulian occurring before 1.4 ma in
southern africa .
many researchers have also suggested the
occurrence of a transitional industry , the fauresmith , covering
the transition from the early to middle stone age , but again , the
fauresmith has been poorly defined , documented , and dated . despite
the occurrence of large cutting tools in these fauresmith
assemblages , they appear to include all the technological
components characteristic of the msa .
new data from stratified
fauresmith bearing sites in southern africa suggest this
transitional industry maybe as old as 511435 ka and
should represent the beginning of the msa as a broad entity rather
than the terminal phase of the acheulian .
the msa in this form is
a technology associated with archaic h. sapiens
and early modern humans in africa with a trend of greater
complexity through time .
|
the phosphatidylinositol 3-kinase ( pi3k)/akt pathway is frequently upregulated or activated in human cancer . the class i subgroup of pi3ks is thought to exclusively phosphorylate ptdins(4,5)p2 to generate ptdins(3,4,5)p3 ( pip3 ) in vivo .
pip3 induces the membrane localization and activation of the proto - oncogenic kinase akt / protein kinase b ( pkb ) . while akt mutations are less common , pi3k is often amplified in cancer .
oncogenic signaling via activated tyrosine kinase receptors and mutated ras will also lead to high constitutive activation of pi3k and hence also of akt .
furthermore , the counteractive tumor suppressor pip3 phosphatase pten ( phosphatase and tensin homolog deleted on chromosome 10 ) is frequently mutationally inactivated in many types of cancer .
downstream effector pathways of akt include the anti - apoptotic protein bad , inhibition of mdm2 , p27kip , and activation of mammalian target of rapamycin , mtor .
mtor regulates both cell growth and cell cycle progression through its ability to integrate signals from nutrient and growth factor stimuli .
in addition to contributing to tumorigenesis , the pi3k / akt pathway is strongly implicated in chemoresistance .
a number of investigators have reported that akt is a key factor in conferring cellular resistance to various chemotherapeutical agents in vitro , including cisplatin , oxaliplatin , and etoposide .
furthermore , akt promotes cell survival after exposure to such different death stimuli as uv irradiation , growth factor withdrawal , tgf - , anti - fas antibody , glutamate , and bile acids .
cellular responses to anticancer drugs are complex and may include apoptosis , necrosis , premature senescence , and mitotic catastrophe . in order to examine the role of akt specifically in apoptosis induced by chemotherapeutic drugs and to identify drugs which induce apoptosis independently of akt1 activity
, we have here screened a chemical library on an isogenic pair of hct116 cell lines , one of which expresses wild - type akt1 which by myristylation is made constitutively membrane - bound and active .
the mechanistic set was obtained from the developmental therapeutics program of the us national cancer institute ( http://www.dtp.nci.nih.gov ) .
akt antibody was from cell signaling ( beverly , ma ) , -tubulin antibody from sigma
aldrich ( st louis , mo ) , and antibody to phosphorylated gsk3 was a gift from katja pokrovskaja ( cancer center , karolinska ) .
hct116 colon carcinoma cells stably expressing constitutively active myristoylated akt ( myr - akt ) , and parental cells were generously provided by dr nissim hay ( university of illinois at chicago , il ) .
cells were maintained in mccoy s 5a modified medium supplemented with 10% fetal calf serum , l - glutamate , penicillin , and streptomycin ( and 10 g / ml puromycin for akt - transfected cells ) at 37 in 5% co2 .
hct116 cells were seeded in 96-well microtiter plates at 10,000 cells per well in 200 l medium and incubated overnight .
drugs were then added to a final concentration of 2.5 or 5 m from stocks in dmso ( to a final concentration of 0.5% ) .
after 24 h of incubation , np-40 was added directly to the tissue culture medium to a final concentration of 0.1% followed by mixing on a rotatory shaker for 5 min .
a 25-l aliquot of the content of each well was assayed for caspase - cleaved ck18 using the m30-cytodeath elisa assay ( a modification of the m30-apoptosense elisa intended for in vitro use ; peviva ab , bromma , sweden ) .
formation of the cleaved product depends on caspase activity and is inhibited by pan - caspase inhibitors such as zvad - fmk .
cells were seeded in the presence of transfection reagents ( 25 nm sirna , hiperfect transfection reagent ; dharmacon ) and incubated for 48 h. cells were then either drug treated for apoptosis assessment or lysed for western blotting .
cell extract proteins were resolved by nupage bis - tris gels ( invitrogen ) and transferred onto a polyvinylidene difluoride membrane .
membranes were probed with antibodies to akt ( dilution 1 : 2,000 ) , akt t473 ( dilution 1:1,000 ) , or -tubulin ( dilution 1:1,000 ) .
the web - based tool available at http://spheroid.ncifcrf.gov/spheroid/htmlnscnumberprojection.cfm was used to map the apoptotic compounds onto the self organizing maps ( soms ) .
this tool has been developed by the covell group at the national cancer institute at frederick and utilizes biological data ( tumor cell growth inhibition ( gi50 ) ) to generate soms .
the soms represent a neural network - based algorithm able to project high - dimensional data into lower dimensional space . for compounds that generated more than one map position ,
the mechanistic set was obtained from the developmental therapeutics program of the us national cancer institute ( http://www.dtp.nci.nih.gov ) .
akt antibody was from cell signaling ( beverly , ma ) , -tubulin antibody from sigma
aldrich ( st louis , mo ) , and antibody to phosphorylated gsk3 was a gift from katja pokrovskaja ( cancer center , karolinska ) .
hct116 colon carcinoma cells stably expressing constitutively active myristoylated akt ( myr - akt ) , and parental cells were generously provided by dr nissim hay ( university of illinois at chicago , il ) .
cells were maintained in mccoy s 5a modified medium supplemented with 10% fetal calf serum , l - glutamate , penicillin , and streptomycin ( and 10 g / ml puromycin for akt - transfected cells ) at 37 in 5% co2 .
hct116 cells were seeded in 96-well microtiter plates at 10,000 cells per well in 200 l medium and incubated overnight .
drugs were then added to a final concentration of 2.5 or 5 m from stocks in dmso ( to a final concentration of 0.5% ) .
after 24 h of incubation , np-40 was added directly to the tissue culture medium to a final concentration of 0.1% followed by mixing on a rotatory shaker for 5 min .
a 25-l aliquot of the content of each well was assayed for caspase - cleaved ck18 using the m30-cytodeath elisa assay ( a modification of the m30-apoptosense elisa intended for in vitro use ; peviva ab , bromma , sweden ) .
formation of the cleaved product depends on caspase activity and is inhibited by pan - caspase inhibitors such as zvad - fmk .
cells were seeded in the presence of transfection reagents ( 25 nm sirna , hiperfect transfection reagent ; dharmacon ) and incubated for 48 h. cells were then either drug treated for apoptosis assessment or lysed for western blotting . cell extract proteins were resolved by nupage bis - tris gels ( invitrogen ) and transferred onto a polyvinylidene difluoride membrane .
membranes were probed with antibodies to akt ( dilution 1 : 2,000 ) , akt t473 ( dilution 1:1,000 ) , or -tubulin ( dilution 1:1,000 ) .
the web - based tool available at http://spheroid.ncifcrf.gov/spheroid/htmlnscnumberprojection.cfm was used to map the apoptotic compounds onto the self organizing maps ( soms ) .
this tool has been developed by the covell group at the national cancer institute at frederick and utilizes biological data ( tumor cell growth inhibition ( gi50 ) ) to generate soms .
the soms represent a neural network - based algorithm able to project high - dimensional data into lower dimensional space . for compounds that generated more than one map position ,
characterization of an hct116 cell line expressing myristylated akt / pkb we here used an hct116 colon carcinoma cell line engineered to express a myristylated form of akt that is constitutively membrane - bound and active ( hct116-myr - akt , generously provided by dr .
western blot analysis confirmed higher levels of akt in hct116-myr - akt cells relative to parental cells ( fig .
furthermore , increased phosphorylation of the akt target gsk3 was observed in hct116-myr - akt cells ( fig .
in accordance with a requirement for glycolysis in the survival functions of akt , hct116-myr - akt cells showed an increased sensitivity to the glycolysis inhibitor 2-deoxyglucose ( 120 mm ) , as assessed in a survival assay over 48 h ( fig .
furthermore , hct116-myr - akt cells were less sensitive to apoptosis induced by cisplatin and camptothecin ( fig .
1phenotype of hct116-myr - akt cells . a akt expression in hct116 and hct116-myr - akt cells .
cell extracts were analyzed for akt expression by western blotting using an antibody to human akt .
cells were transfected with akt sirna where indicated ; b increased phosphorylation of akt at t473 in hct116-myr - akt cells .
akt phosphorylation was examined by western blotting using an antibody to the phosphorylated form of the protein .
gsk3 phosphorylation was examined by western blotting using an antibody to the phosphorylated form of the protein .
cells were treated with the indicated concentrations of 2-deoxyglucose and survival was examined using the srb assay ; * p < 0.05 ; * * p < 0.005 . e apoptosis induction by cisplatin and camptothecin in hct116-myr - akt cells .
cells were treated for 24 h , and apoptosis was quantified using the m30 cytodeath elisa .
f effect of akt sirna on apoptosis induced by one of the agents in the set ( nsc nsc632841 ) .
cells were transfected with sirna as indicated and treated with the drug for 24 h. caspase - cleaved ck18 was determined using the m30 cytodeath elisain order to confirm the role of myr - akt expression for the apoptotic response , we transfected hct116-myr - akt and control cells with akt sirna .
the transfection resulted in significantly decreased akt expression in both cell lines ( fig .
transfected cells were then treated with one of the apoptotic compounds from the nci mechanistic set ( 4-piperidinone , 1-(1-oxo-2-propenyl)-3,5-bis(phenyl - methylene ) ) , as shown in fig .
1f , untransfected control and myr - akt cells showed different responses to nsc632841 , whereas the apoptotic response was similar in both cell lines when transfected with akt sirna .
we conclude from these experiments that hct116-myr - akt cells show the expected phenotype with regard to signaling and drug sensitivity .
phenotype of hct116-myr - akt cells . a akt expression in hct116 and hct116-myr - akt cells .
cell extracts were analyzed for akt expression by western blotting using an antibody to human akt .
cells were transfected with akt sirna where indicated ; b increased phosphorylation of akt at t473 in hct116-myr - akt cells .
akt phosphorylation was examined by western blotting using an antibody to the phosphorylated form of the protein .
gsk3 phosphorylation was examined by western blotting using an antibody to the phosphorylated form of the protein .
cells were treated with the indicated concentrations of 2-deoxyglucose and survival was examined using the srb assay ; * p < 0.05 ; * * p < 0.005 . e apoptosis induction by cisplatin and camptothecin in hct116-myr - akt cells .
cells were treated for 24 h , and apoptosis was quantified using the m30 cytodeath elisa .
f effect of akt sirna on apoptosis induced by one of the agents in the set ( nsc nsc632841 ) .
cells were transfected with sirna as indicated and treated with the drug for 24 h. caspase - cleaved ck18 was determined using the m30 cytodeath elisa screening for compounds that induce myr - akt - insensitive apoptosis in order to identify agents that are effective in inducing apoptosis of hct116-myr - akt cells , we exposed the pair of cell lines to the nci mechanistic drug set at 2.5 or 5 m .
this drug collection contains 827 compounds selected from approximately 40,000 compounds on the basis on different mechanisms of action with regard to cell growth inhibition of the nci60 tumor cell line panel .
we initially screened the entire drug set for compounds effective in inducing apoptosis of hct116 then used a selection of apoptosis - inducing compounds on the cell pair .
apoptosis was measured by the m30 cytodeath elisa , an assay which is specific for a caspase - cleaved product of cytokeratin-18 formed in apoptotic cells .
the apoptosis product accumulates in cell cultures and was measured at a single time point ( 24 h ) .
the signals from untreated myr - akt and control cells were set to 1 , respectively .
for each drug , the induced levels of apoptosis in each cell line were then plotted against each other ( fig . 2 ) .
drugs that induced less than twofold the background apoptosis in parental cells are excluded from the figure .
most of the drugs shown generated a sufficient signal at a concentration of 2.5 m .
2 ) that most compounds induced stronger apoptotic responses in control cells compared to myr - akt cells ( the slope of the best - fit curve is 0.70 ) .
however , and importantly , a number of compounds induced similar levels of apoptosis regardless of cellular akt status .
2apoptotic responses of myr - akt and control hct1116 cells treated with 2.5 m of nci mechanistic set agents . with a minority of agents ,
caspase - cleaved ck18 was measured in extracts and medium after 24 h of treatment using the m30 cytodeath elisa .
only agents inducing caspase - cleaved ck18 of greater than twofold control are included in the figure .
the best - fit line of the dataset is showndrugs whose ratios of apoptosis induction in hct116-myr - akt to control hct116 cells were 0.9 were classified as akt - insensitive .
fifteen compounds were found to induce an apoptotic response in hct116-myr - akt cells that was < 50% of control cells and were classified as akt - sensitive ( table 2 ) .
table 1myr - akt - insensitive drugsnsc numbernamesom region(s)cancer active757colchicinemleukemiacarcinomasarcoma18268dactinomycinmleukemiacarcinoma24819peltatinmleukemiacarcinoma24818podophyllotoxinmleukemiasarcoma33410colchicine , n - mleukemiabenzoyl - deacetylmelanoma49842vinblastinemleukemiacarcinoma76022thaspinemsarcoma83265tritylcysteinenleukemiacarcinoma85236helenalinqleukemia219734mleukemiacarcinoma333856tetrocarcinmmelanoma345647chaetochrominmcarcinoma639828mnt647889pnt651079snt687850qnt705701alsterpaullonesntdrugs inducing similar levels of apoptosis in parental and myr - akt expressing hct116 cellsdrugs that induce increased survival and/or decreases in tumor mass according to testing performed at the national cancer institute ( http://www.dtp.nih.nci.gov ) were classified as cancer activent not testedtable 2myr - akt - sensitive drugsnsc numbernamesom region(s)285116siomycinp729618-adenosinev47147prodigiosins152731s123111mitomycin derivatives162907ellipticine , 6-(5-hexen-1-yl-)r335142s651084q687849q638645q636676q173904carbamic acid , esterq6299719-amino-20-camptothecins697923q1466045-fluorouridinesdrugs that induced more apoptosis in parental than in myr - akt expressing hct116 ( ratio > 2 ) apoptotic responses of myr - akt and control hct1116 cells treated with 2.5 m of nci mechanistic set agents . with a minority of agents ,
caspase - cleaved ck18 was measured in extracts and medium after 24 h of treatment using the m30 cytodeath elisa .
only agents inducing caspase - cleaved ck18 of greater than twofold control are included in the figure .
the best - fit line of the dataset is shown myr - akt - insensitive drugs drugs inducing similar levels of apoptosis in parental and myr - akt expressing hct116 cells drugs that induce increased survival and/or decreases in tumor mass according to testing performed at the national cancer institute ( http://www.dtp.nih.nci.gov ) were classified as cancer active myr - akt - sensitive drugs drugs that induced more apoptosis in parental than in myr - akt expressing hct116 ( ratio > 2 ) characteristics of compounds inducing similar levels of apoptosis in myr - akt and control hct116 cells based on responses in the nci60 cancer cell line panel , the profiles of cell growth inhibition of the drugs tested by the nci developmental therapeutics program have been assembled into soms , where the position of a particular drug reflects its main target process or mechanism of action . most of the agents currently in clinical use map to the m and s areas of these maps ( m = mitosis , s = nucleic acid synthesis ; fig . 3a ) .
we used a web - based tool to determine where the apoptotic compounds map on these soms ( see material and methods ) . approximately half ( 34/62 ) of the compounds identified by screening as inducing apoptosis in hct116 cells ( shown in fig .
2 ) mapped in the m and s areas and 15 mapped to the q region which is associated with metabolic stress .
3exploration of mechanisms of action of different sets of drugs using self - organizing maps ( 3d mind resource ; http://spheroid.ncifcrf.gov/spheroid ) .
som clustering of the nci60 gi50 data segregates compounds into nine major response categories : mitosis ( m ) ; nucleic acid metabolism ( s ) ; metabolic stress and cell survival ( q ) ; membrane function ( n ) ; kinases / phosphatases and oxidative stress ( p ) ; and four unexplored regions , r , f , j , and v .
a examples of drugs are in clinical use ( mostly mapping in the m and s areas ) ; b mapping of 62 drugs in the nci mechanistic set which induce strong apoptosis of hct116 cells ; c mapping of akt - sensitive drugs ( note the preferential location to the s and q regions ) ; d mapping of akt - insensitive drugs ( note the preferential location to the m region)akt - sensitive and akt - insensitive compounds showed different distributions on the maps ( fig .
akt - sensitive drugs mapped in the s region ( 6/15 ) , in the q region ( 6/15 ) , but not in the m region ( 0/15 ) . in contrast , 11/17 akt - insensitive drugs mapped in the m - region and only 2/17 in the s region . the difference in distribution of akt - sensitive and akt - insensitive drugs to the m area was statistically highly significant ( fisher exact test : p = 0.00011).as expected , the group of akt - insensitive drugs mapping to the m region included typical microtubuli - targeting drugs such as colchicine , vinblastine , and podophyllotoxin .
in addition , drugs such as actinomycin - d , tetrocarcin , and thaspine were found in this group . whereas actinomycin - d has been observed to inhibit tubulin polymerization in vitro ,
tetrocarcin a has been reported to inhibit bcl-2 function , while the mechanism of thaspine cytotoxicity is not well characterized.of the 17 akt - insensitive drugs , 12 have been tested for anti - tumor activity by the developmental therapeutics program ( http://dtp.nci.nih.gov ) .
all 12 were found to have antitumor activity in animal models ( table 1 ) .
three of the active compounds are well - known cytostatics , podophyllotoxin , vinblastine , and actinomycin - d . of the remaining
finally , thaspine ( nsc76022 ) is an alkaloid present in the cortex of the tree croton lechleri which grows in the upper amazon region of peru , ecuador , and colombia .
a red latex , dragon s blood ( sangre de drago ) , is extracted from the cortex of the tree and is extensively used by different tribes of the amazonian basin for medicinal purposes .
thaspine was previously reported to be cytotoxic and to have antitumor activity and may be an interesting anticancer drug .
helenalin ( nsc85236 ) is often used in vitro as an nfb inhibitor , but apoptosis induction by helenalin in the myr - akt cells is in line with its reported inhibitory effect on akt and its som location in the q region .
we have reported that helenalin induces apoptosis via camkii , ask1 , and jnk .based on the present apoptosis screening and further analysis using soms , we conclude that expression of constitutively active akt mainly affected apoptosis induced by dna - damaging drugs , whereas akt - insensitive apoptosis was associated mainly with drugs that interfere with the process of mitosis .
this conclusion is supported by the report that expression of constitutively active akt1 in a549 human non - small cell lung carcinoma cells resulted in increased survival in response to mitoxantrone and cisplatin but not to microtubuli - interacting agents such as paclitaxel .
that apoptosis induced by mitotic inhibitors is insensitive to akt overexpression is not altogether unexpected since apoptosis induced by mitotic inhibitors is likely to be secondary to mitotic catastrophe and not a primary signaling event .
in contrast to these results , other authors have reported that myr - akt confers resistance to microtubuli - interacting agents .
we suggest that this different result is due to the use of non - transformed il-3 dependent hematopoietic cells rather than transformed epithelial cells .
these cell types likely differ in signaling pathways that regulate survival energy metabolism and microtubule functions .
furthermore , due to the documented roles of akt in anti - apoptotic pathways , we have focused exclusively on acute apoptosis , whereas the other report is based on survival seen as levels of propidium iodide exclusion after 72-h treatment . over this time interval ,
microtubuli - interacting agents are expected to induce mitotic arrest followed by secondary apoptosis , and it is possible that this cell death is sensitive to blocking by constitutively active akt.cell-based assays have the advantage of reasonable experimental through - put while preserving disease - relevant molecular - pathway interactions .
this is particularly important in the field of oncology due to the complex mechanisms of action of cytotoxic drugs .
the conditional cell - based phenotypic screening approach used here promises be effective in defining drug mechanisms of that are insensitive to overexpression of various oncogenes , loss of tumor suppressors , or other phenotypic traits .
such studies are important in order to define the proper use of cancer drugs in clinical oncology .
exploration of mechanisms of action of different sets of drugs using self - organizing maps ( 3d mind resource ; http://spheroid.ncifcrf.gov/spheroid ) .
som clustering of the nci60 gi50 data segregates compounds into nine major response categories : mitosis ( m ) ; nucleic acid metabolism ( s ) ; metabolic stress and cell survival ( q ) ; membrane function ( n ) ; kinases / phosphatases and oxidative stress ( p ) ; and four unexplored regions , r , f , j , and v .
a examples of drugs are in clinical use ( mostly mapping in the m and s areas ) ; b mapping of 62 drugs in the nci mechanistic set which induce strong apoptosis of hct116 cells ; c mapping of akt - sensitive drugs ( note the preferential location to the s and q regions ) ; d mapping of akt - insensitive drugs ( note the preferential location to the m region )
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the phosphatidylinositol 3-kinase ( pi3k)/akt pathway is frequently upregulated in human cancer
. activation of this pathway has been reported to be associated with resistance to various chemotherapeutical agents .
we here used a chemical biology / chemical informatic approach to identify apoptotic mechanisms that are insensitive to activation of the pi3k / akt pathway .
the national cancer institute ( nci ) mechanistic set drug library was screened for agents that induce apoptosis in colon carcinoma cells expressing a constitutively active form of akt1 .
the cytotoxicity screening data available as self - organized maps at the developmental therapeutics program ( dtp ) of the nci was then used to classify the identified compounds according to mechanism of action .
the results showed that drugs that interfere with the mitotic process induce apoptosis which is comparatively insensitive to constitutive akt1 activity .
the conditional screening approach described here is expected to be useful for identifying relationships between the state of activation of signaling pathways and sensitivity to anticancer agents .
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all 3 isolates from italy were assigned to group b.ftnf00200 ( figure 1 , panel a ) .
although the sample size was small , these isolates were obtained in 3 different years ( table a1 ) , which suggests that this group is ecologically established in italy .
these results increase the known geographic distribution of this group , which appears to be the dominant clone in western europe ( figure 2 , panel a , purple shading ) .
all 42 isolates from austria , germany , hungary , and romania were assigned to group b.13 ( figure 1 , panel a ) , further demonstrating that b.13 is the most prevalent group of f. tularensis subsp .
holarctica in central and eastern europe ( figure 2 , panel a , red shading ) . within group b.13 ,
one isolate from hungary was assigned to subclade b.23/14/25 ( figure 1 , panel a ) ; isolates from finland , russia , and sweden were previously assigned to this subclade ( 6,8 ) ( figure 2 , panel b ) .
however , the other 41 isolates were assigned to subclade b.20/21 ( figure 1 , panel a ) .
terminal subgroups representing sequenced strains are shown as stars , and intervening nodes representing collapsed branches are indicated by circles .
isolates from austria , germany , hungary , italy , and romania ( n = 45 ) were assigned to existing subclades ( black arrows ) by using existing canonical snp assays ( 5,8 ) .
b ) maximum parsimony phylogeny constructed by using snps discovered from 6 f. tularensis whole - genome sequences , including 5 strains from group b.13 and an outgroup strain , osu18 ( not shown ) .
this phylogeny was rooted by using osu18 , and bootstrap values were based on 1,000 simulations by using a heuristic search . the newly sequenced hungarian strain ( tul07/2007 )
countries of origin for isolates assigned to select subclades within group b.13 are indicated by the letters a h .
red and purple shading indicates the known geographic distributions of groups b.13 and b.ftnf00200 , respectively , in this and previous studies ( 59 ) .
the country of georgia , which also contains isolates from group b.13 but is not depicted in the map , is indicated by red text and a red arrow pointing toward its location .
holarctica have been reported from some of these countries ( 5,8 ) , but most isolates from these countries are from groups b.13 and b.ftnf00200 .
b ) single nucleotide polymorphism based phylogeny of previously ( 5,6,8 ) and newly identified subclades within the b.13 group of f. tularensis subsp .
terminal subgroups representing sequenced strains are shown as stars , and intervening nodes representing collapsed branches are indicated by circles .
the countries of origin for isolates assigned to each subclade are indicated : aut , austria ; ce , central europe , unknown country ; cze , czech republic ; deu , germany ; fin , finland ; geo , georgia ; hun , hungary ; ita , italy ; rou , romania ; rus , russia ; swe , sweden ; ukr , ukraine ) . for mapping purposes ,
letters are assigned to a previously identified subclade that contains a new isolate from hungary now assigned to that subclade ( a ) and newly identified subclades ( b h ) .
the number of isolates listed for each subclade refers only to isolates examined directly in this study ( table a1 ) .
, san diego , ca , usa ) was used to sequence the genome of an isolate from hungary ( tul07/2007 , genbank accession no .
srx025133 ) assigned to subclade b.20/21 . putative single nucleotide polymorphisms ( snps ) were identified in the resulting sequence and the genomes of 4 other strains previously assigned to group b.13 ( lvs , am233362.1 ; fsc 200 , aasp00000000 ; rc503 , srx000104 ; georgia f0673 , srx025885 ) by using an existing bioinformatics pipeline ( 5 ) .
the more distantly related strain osu18 ( cp000437.1 ) genome was also included as an outgroup .
a maximum - parsimony tree was constructed by using the resulting 700 putative snps and paup 4.0b10 software ( sinauer associates , inc . ,
most of the putative snps separated osu18 from the b.13 strains ( data not shown ) , but the remaining putative snps provided resolution among the b.13 strains , including 20 putative snps specific to the branch leading to the strain from hungary ( figure 1 , panel b ) .
consistent with previous analyses ( figure 1 , panel a ) , the strain from hungary clustered as a sister taxon to strain fsc 200 ( figure 1 , panel b ) . to show additional phylogenetic structure within subclade b.20/21 , we designed genotyping assays targeting the 20 putative snps along the branch leading to the strain from hungary ( figure 1 , panel b ) and screened them across 64 isolates assigned to subclade b.20/21 .
this analysis included the 41 isolates from austria , germany , hungary , and romania , as well as 23 additional isolates from central europe , the czech republic , finland , russia , and sweden that were previously assigned to this subclade ( 6,8 ) ( table a1 ) .
the assays were constructed and performed as described ( 5 ) by using an annealing temperature of 60c .
all 20 snps were laboratory confirmed , and 52 of the isolates were assigned to 6 new subclades ( b.33/34 , b.34/35 , b.35/36 , b.36/37 , b.37/38 , and b.tul07/2007 ) ; the 12 other isolates remained in the basal subclade , now identified as b.20/21/33 ( figure 2 , panel b ; table a1 ) .
information about assays targeting canonical snps for the branches leading to the 6 new subclades are presented in the table .
our results are consistent with complex dispersal patterns within the b.13 group of f. tularensis subsp . holarctica .
several of the b.13 subclades identified in this study are broadly distributed throughout central and eastern europe ( figure 2 , panel a ) , including subclades b.20/21/33 , b.33/34 , and b.34/35 .
all of the new subclades containing > 1 isolate have representatives from multiple countries ( figure 2 , panel b ) .
other previously identified b.13 subclades , including b.27/28 , b.lvs , b.23/14/25 , and b.21/22 are also broadly distributed ( figure 2 , panel a ) .
holarctica in europe by placing isolates from multiple countries into the existing global phylogeographic framework . as a result
, the genetic background is becoming defined for each country ( i.e. , the specific subtypes reported from each country ) .
this information can be useful for identifying intentional ( e.g. , bioterrorism ) or unintentional movement of f. tularensis subsp .
holarctica between countries . for example , the isolate from romania examined in this study was actually isolated in italy from an infected hare that was shipped from romania for hunting .
genotyping results are consistent with a romanian origin for this isolate because it was assigned to the b.13 group that is widespread in central and eastern europe ( figure 2 , panel a ) and not to the b.ftnf00200 group , to which the isolates from italy were assigned ( figure 1 , panel a ) .
understanding global phylogeographic patterns is possible only if isolates from multiple geographic locations are placed within the same framework ( i.e. , examined with the same genomic signatures ) . because f. tularensis is genetically monomorphic and highly clonal , snps are preferred signatures for determining phylogenetic structure within this species ( 3 ) .
vogler et al . ( 5 ) conducted the first snp - based global phylogeographic analysis of f. tularensis .
subsequent studies ( 68 ) have used the snp signatures described by vogler et al .
( 5 ) and new snps discovered from new whole - genome sequences or multiple sequence typing data to further refine phylogeographic patterns within f. tularensis , particularly f. tularensis subsp .
these new signatures , when screened across diverse isolate collections , have identified new subclades within preexisting subclades .
this pattern will continue as whole - genome sequencing becomes less expensive and more widely available . as a result , the nomenclature of phylogenetic groups within f. tularensis and the particular subclade to which a given isolate is assigned are constantly changing and will continue to change , which makes comparison of results and findings across different studies difficult . to address this problem ,
holarctica snp - based phylogenetic groups within our phylogenetic trees ( figure 1 , panel a ; figure 2 , panel b ) , including those discovered by other researchers .
in addition , for the isolates analyzed in this study ( table a1 ) , where applicable , we have listed the phylogenetic groups to which they were assigned in previous studies .
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francisella tularensis subsp .
holarctica isolates from austria , germany , hungary , italy , and romania were placed into an existing phylogeographic framework .
isolates from italy were assigned to phylogenetic group b.ftnf00200 ; the other isolates , to group b.13 .
most f. tularensis subsp .
holarctica isolates from europe belong to these 2 geographically segregated groups .
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members of the arf family of small gtp - binding proteins , or gtpases , are activated by guanine nucleotide exchange factors ( gefs ) that catalyze gdp release from their substrate arf , allowing gtp to bind . in the secretory pathway ,
arf1 is first activated by gbf1 at the cis - golgi , then by big1 and big2 at the trans - golgi and trans - golgi network ( tgn ) . upon activation ,
arf1-gtp interacts with effectors such as coat complexes , and is able to recruit different coat complexes to different membrane sites in cells .
the copi coat is primarily recruited to cis - golgi membranes , whereas other coats , such as ap-1/clathrin , and gga / clathrin , are recruited to the trans - golgi and the tgn .
although arf1-gtp is required for stable association of these various coats to membranes , and is sufficient in vitro , other molecules , such as vesicle cargo and coat receptors on the membrane , contribute to specificity of coat recruitment in cells .
another mechanism to achieve specificity is interaction of effectors such as coats with the gef itself , which would increase the concentration of a given coat in proximity to the site where arf is activated , thus favoring its recruitment .
this interaction between a gef and an effector could also provide a mechanism for spatial organization of vesicle budding sites , similar to that described for cdc42-mediated establishment of polarity sites such as the emerging bud in yeast .
another factor affecting the amount of freely diffusible arf1-gtp in membranes is the gef(s ) themselves acting as effectors .
sec7p , the yeast homolog of mammalian big1 and big2 , and arno / cytohesin 2 , a pm - localized arf1 gef , both bind to arf1-gtp .
this binding to the products of the exchange reaction establishes a positive feedback loop for activation .
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elevated and refractory intracranial pressure ( icp ) resulting from acute hemorrhagic brain injury leads to tissue changes , cerebral ischemia , hernias , and damage to vital brain structures .
the use of osmotically active substances is a fundamental component of neurocritical care protocols and is recommended by current guidelines for the treatment of intracranial hypertension [ 14 ] .
mannitol and hypertonic saline solutions ( hss ) are most commonly used and have gained wide acceptance , despite the lack of high - quality clinical studies demonstrating their effectiveness . in the absence of definitive evidence of an optimal treatment , there is significant variation in both the choices and usage of such agents .
methods of hss administration vary considerably , including bolus infusion of a 2% or 3% solution and continuous or bolus infusion of 5% , 7.5% , or 23.4% solutions [ 811 ] .
results of a recent meta - analysis have suggested that hss ( 3% ) may be more effective than mannitol for controlling sharp increases in icp ; however , no previous studies have evaluated its long - term capacity to control icp . in our experience ,
continuous infusion of 3% hss is effective in reducing icp during the treatment of intracranial hypertension .
several possible mechanisms may account for the reduction of intracranial hypertension by treatment with hss [ 1315 ] .
accordingly , this study analyzed the effects of 3% saline solution infusion 1 hour after simulation of icp elevation in an animal model of intracranial hypertension .
twenty - nine hybrid pigs of the landrace , duroc , and pietrain breeds ( approximately 20 kg each ) were used in this study , under general anesthesia , duly assisted with ventilation and hemodynamic monitoring . of these , 1 animal was excluded because of anemia , with a hemoglobin level of 6.7 ( only animals with hemoglobin levels greater than 9.0 were used ) , and 1 animal was excluded because of hyponatremia , which may influence icp and therefore compromise our results .
the study procedures were approved by the animal research ethics committee ( number 0520/09 ) and by the department of neurology ( faculty of medicine ) at the university of so paulo .
all experiments were carried out according to the ethical principles for the use of laboratory animals adopted by this institution .
we attest that the animals were not subjected to conditions of suffering or pain in any step of the experiment .
the pigs were subjected to 12 h of fasting with free access to water until 1 h before the experiment .
prior to anesthesia induction , ketamine ( 5 mg / kg ; ketamin - s ; cristlia , itapira , brazil ) and midazolam ( 0.25 mg / kg ; dormire ; cristlia ) were administered intramuscularly .
after 15 min , the marginal ear vein was catheterized with a 20- or 22-gauge insyte vascular catheter ( bd , franklin lakes , new jersey , usa ) .
after venous access was established , anesthesia was induced intravenously ( i.v . ) using propofol ( 5 mg / kg ; provive 1% ; claris , ahmedabad , india ) .
anesthesia was maintained with propofol ( 3 mg / kg / h ) , and analgesia was maintained with fentanyl ( fentanest ; cristlia ) at a starting dose of 5 g / kg , followed by continuous i.v .
neuromuscular blockade was achieved by administration of pancuronium ( pancuron ; cristlia ) at a starting dose of 0.1 mg / kg i.v . , followed by continuous i.v .
infusion of saline solution ( 20 ml / kg , 0.9% nacl ) to compensate for volume loss due to fasting , and received supportive fluid therapy with 0.9% nacl at a rate of 5 ml / kg / h throughout the procedure .
after endotracheal intubation with a portex 6.0 mm tube ( smiths medical , rockland , massachusetts , usa ) , the animals received volume - cycled controlled mechanical ventilation ( dixtal 5010 ventilator ; dixtal biomdica , so paulo , brazil ) with a tidal volume of 10 ml / kg , fraction of inspired oxygen of 0.50 , and positive end - expiratory pressure of 5 cm h2o .
ventilation parameters were adjusted to maintain the partial pressure of co2 between 35 and 45 mm hg , the partial pressure of o2 between 100 and 150 mm hg , and the ph between 7.35 and 7.45 . in order to monitor and maintain sufficient ventilation ,
both the final pressure of expired co2 and peripheral hemoglobin saturation were continuously measured using pulse oximetry .
arterial blood gas analysis was performed using 0.3 ml samples collected at the beginning of the procedure in order to establish ventilation parameters and after the interventions described below .
hemodynamic data were collected using a multi - parameter monitor ( monitor portal dx 2020 ; dixtal ) .
a frontotemporal incision was performed , and bone trepanning was performed on the right hemisphere 1 cm lateral to the sagittal suture and 1 cm anterior to the coronal suture .
a neurovent - p intraparenchymal catheter with a multisensor that analyzes icp and brain temperature ( raumedic , helmbrechts , germany ) was inserted into the white matter of the right frontal lobe at a depth of approximately 10 mm .
right posterior temporal trepanning and insertion of a neurodur - p icp epidural monitoring catheter ( raumedic ) was also performed .
bone trepanning 1 cm lateral to the sagittal suture and 1 cm posterior to the coronal suture ( 3 mm in diameter ) allowed the insertion of an 8 french pediatric urethral catheter at a 20 lateral inclination and a depth of 2 cm , targeting the right frontal subcortical white matter .
balloon inflation was performed over a 15 min period using an infusion pump ( b braun medical , woburn , massachusetts , usa ) .
a hypertonic solution of 3% nacl ( 5.3 ml / kg ) was infused 30 min after the start of the final balloon inflation .
the effects on icp control was measured 60 minutes after saline solution infusion the 27 animals included in the study were divided into 3 groups ( n=9 per group ) . in group
a , the balloons were inflated to 4 ml in order to simulate mild to moderate hypertension . in group b , the balloons were initially inflated to 4 ml and were inflated by an additional 3 ml after 1 h , simulating expansion . in group c , an intracranial hematoma model with balloon with a volume of 7
the animals were sacrificed at the end of the experiment by means of an intravenous dose of propofol ( 20 mg / kg ) and fentanyl ( 10 mg / kg ) , followed by 40 ml of 19.1% potassium chloride solution .
after the experiment , the animals were placed in vivarium plastic bags with labels clearly identifying their origin and content and the researcher in charge .
we used a convenience sample ; and as this study was exploratory ( there s no study , as far as we know , that used this model to evaluate the use of hypertonic solutions ) , we defined a total of 30 pigs as being sufficient for a first analysis .
continuous variables were reported as means and standard deviations . to evaluate the icp following the interventions ( balloon inflation and hypertonic saline infusion ) we used a paired student s t test or wilcoxon rank - sum test according to normality of the data with significance at p=0.05 .
twenty - nine hybrid pigs of the landrace , duroc , and pietrain breeds ( approximately 20 kg each ) were used in this study , under general anesthesia , duly assisted with ventilation and hemodynamic monitoring . of these , 1 animal was excluded because of anemia , with a hemoglobin level of 6.7 ( only animals with hemoglobin levels greater than 9.0 were used ) , and 1 animal was excluded because of hyponatremia , which may influence icp and therefore compromise our results .
the study procedures were approved by the animal research ethics committee ( number 0520/09 ) and by the department of neurology ( faculty of medicine ) at the university of so paulo .
all experiments were carried out according to the ethical principles for the use of laboratory animals adopted by this institution .
we attest that the animals were not subjected to conditions of suffering or pain in any step of the experiment .
the pigs were subjected to 12 h of fasting with free access to water until 1 h before the experiment .
prior to anesthesia induction , ketamine ( 5 mg / kg ; ketamin - s ; cristlia , itapira , brazil ) and midazolam ( 0.25 mg / kg ; dormire ; cristlia ) were administered intramuscularly .
after 15 min , the marginal ear vein was catheterized with a 20- or 22-gauge insyte vascular catheter ( bd , franklin lakes , new jersey , usa ) .
after venous access was established , anesthesia was induced intravenously ( i.v . ) using propofol ( 5 mg / kg ; provive 1% ; claris , ahmedabad , india ) .
anesthesia was maintained with propofol ( 3 mg / kg / h ) , and analgesia was maintained with fentanyl ( fentanest ; cristlia ) at a starting dose of 5 g / kg , followed by continuous i.v .
neuromuscular blockade was achieved by administration of pancuronium ( pancuron ; cristlia ) at a starting dose of 0.1 mg / kg i.v . , followed by continuous i.v .
infusion of saline solution ( 20 ml / kg , 0.9% nacl ) to compensate for volume loss due to fasting , and received supportive fluid therapy with 0.9% nacl at a rate of 5 ml / kg / h throughout the procedure .
after endotracheal intubation with a portex 6.0 mm tube ( smiths medical , rockland , massachusetts , usa ) , the animals received volume - cycled controlled mechanical ventilation ( dixtal 5010 ventilator ; dixtal biomdica , so paulo , brazil ) with a tidal volume of 10 ml / kg , fraction of inspired oxygen of 0.50 , and positive end - expiratory pressure of 5 cm h2o .
ventilation parameters were adjusted to maintain the partial pressure of co2 between 35 and 45 mm hg , the partial pressure of o2 between 100 and 150 mm hg , and the ph between 7.35 and 7.45 . in order to monitor and maintain sufficient ventilation ,
both the final pressure of expired co2 and peripheral hemoglobin saturation were continuously measured using pulse oximetry .
arterial blood gas analysis was performed using 0.3 ml samples collected at the beginning of the procedure in order to establish ventilation parameters and after the interventions described below .
hemodynamic data were collected using a multi - parameter monitor ( monitor portal dx 2020 ; dixtal ) .
a frontotemporal incision was performed , and bone trepanning was performed on the right hemisphere 1 cm lateral to the sagittal suture and 1 cm anterior to the coronal suture .
a neurovent - p intraparenchymal catheter with a multisensor that analyzes icp and brain temperature ( raumedic , helmbrechts , germany ) was inserted into the white matter of the right frontal lobe at a depth of approximately 10 mm .
right posterior temporal trepanning and insertion of a neurodur - p icp epidural monitoring catheter ( raumedic ) was also performed .
bone trepanning 1 cm lateral to the sagittal suture and 1 cm posterior to the coronal suture ( 3 mm in diameter ) allowed the insertion of an 8 french pediatric urethral catheter at a 20 lateral inclination and a depth of 2 cm , targeting the right frontal subcortical white matter .
balloon inflation was performed over a 15 min period using an infusion pump ( b braun medical , woburn , massachusetts , usa ) .
this fluid system was tested prior to insertion . a hypertonic solution of 3% nacl ( 5.3 ml / kg )
the effects on icp control was measured 60 minutes after saline solution infusion the 27 animals included in the study were divided into 3 groups ( n=9 per group ) . in group
a , the balloons were inflated to 4 ml in order to simulate mild to moderate hypertension . in group b , the balloons were initially inflated to 4 ml and were inflated by an additional 3 ml after 1 h , simulating expansion . in group c , an intracranial hematoma model with balloon with a volume of 7 ml
the animals were sacrificed at the end of the experiment by means of an intravenous dose of propofol ( 20 mg / kg ) and fentanyl ( 10 mg / kg ) , followed by 40 ml of 19.1% potassium chloride solution .
after the experiment , the animals were placed in vivarium plastic bags with labels clearly identifying their origin and content and the researcher in charge .
we used a convenience sample ; and as this study was exploratory ( there s no study , as far as we know , that used this model to evaluate the use of hypertonic solutions ) , we defined a total of 30 pigs as being sufficient for a first analysis .
continuous variables were reported as means and standard deviations . to evaluate the icp following the interventions ( balloon inflation and hypertonic saline infusion ) we used a paired student s t test or wilcoxon rank - sum test according to normality of the data with significance at p=0.05 .
twenty - seven animals were included in the study ( 14 female and 13 male ) , with 9 in each experimental group . the mean weight of the animals was 19.741.12 kg , with no differences between groups .
icp measurements in each group confirmed the effectiveness of the intracranial hypertension model . in group
a , balloon inflation to a volume of 4 ml using an infusion pump generated a moderate increase in icp .
we also observed an increase in icp in group b , which represented an early reexpansion model of intracranial hematoma . in group c
, there was a marked increase in icp that remained higher when compared to the other two groups .
in the evaluation of increased icp , it was confirmed that the model was suitable to generate an increase in icp and that the increases in the mean icp between three groups were statistically significant ( p<0.001 ) ( figure 1 ) .
we observed no increases in the mean serum sodium concentration beyond the normal range ( p=0.09 ) .
in addition , no differences were identified in the serum sodium level measured during hss infusion within each group ( p=0.21 ) .
there was no effect of significant pressure reduction in any of the 3 groups ( figure 2 ) .
group c showed an increase in icp with saline infusion ; however , this increase was immediate but not statistically significant and therefore did not represent a rebound effect .
the saline solution used in our experiment was a 3% nacl solution , and the calculation for infusion was 5.3 ml / kg . in the present study , we did not observe reductions in intracranial pressure following hss infusion in any of the experimental groups .
animals in group c , in which extreme hypertension was simulated , showed an increase in icp following hss treatment , and the animals remained stable during this period .
cerebral perfusion pressure also did not change significantly with the administration of saline solution in any of the groups .
zornow described the effects of 3.2% hss infusion in a rabbit model of cryogenic brain injury , which included acute effects of pressure reduction with a continuous late increase in icp .
by contrast , qureshi et al . reported no beneficial effects of 3% hss on regional cerebral blood flow or cerebral metabolism rates in a canine model of intracranial hypertension .
it has been suggested as a treatment option by different studies and guidelines , but there is no clear consensus regarding its specific uses [ 1821 ] .
there are indications that the use of hss at higher concentrations ( for instance , 23.4% nacl at a dosage of 0.7 ml / kg ) may be a more appropriate strategy .
the present study analyzed only the acute effects of hss infusion , and no immediate complications were identified .
there are numerous theoretical adverse effects associated with the use of hss , although these are likely not of great clinical relevance .
electrolyte disturbances are among the most commonly observed findings , specifically hypernatremia and an associated hyperosmolar state .
the significance of these findings has yet to be clearly defined , as hss exerts its effects on icp reduction in hypernatremic patients suffering from hyperosmolar conditions .
acute renal failure is a known adverse effect of mannitol therapy ; however , with the exception of a few case reports , its importance for hss treatment is not well elucidated .
it was also demonstrated that hss might be effective when used in patients with renal failure .
we observed an increase in icp after 1 hour of hss infusion in group c , which modeled more severe hypertension ; however , this increase was not statistically significant .
nau suggests that the risk of developing rebound icp increases with repeated administration of hss , the degree of damage to the blood - brain barrier , and the position of the patient on the icp - volume curve .
the animals used in this study received a single dose of hss ; therefore , the effects of repeated hss infusions were not tested .
also , the use of 3% hypertonic saline as a continuous infusion may not be the ideal dose to cause a decrease of icp in our model .
additionally , the mechanism of action of hypertonic saline has been classically attributed to reduction of the water content of the interstitial space in a broken blood - brain barrier , a fact that could partially justify our results .
however , we believe that the area surrounding the balloon would cause rupture of axons and the blood - brain barrier , causing immediate inflammatory changes that contribute to icp increase and , consequently , are amenable to be treated with hypertonic saline . considering all these points together , the present results should be analyzed with caution .
however , this experimental study was done in a controlled environment where was possible to identify a consistent increase of icp over the different groups , showing , at least , that this model could be used to evaluate the different types of clinical and surgical interventions to reduce icp .
also , the use of 3% hypertonic saline as a continuous infusion may not be the ideal dose to cause a decrease of icp in our model . moreover
additionally , the mechanism of action of hypertonic saline has been classically attributed to reduction of the water content of the interstitial space in a broken blood - brain barrier , a fact that could partially justify our results .
however , we believe that the area surrounding the balloon would cause rupture of axons and the blood - brain barrier , causing immediate inflammatory changes that contribute to icp increase and , consequently , are amenable to be treated with hypertonic saline . considering all these points together , the present results should be analyzed with caution .
however , this experimental study was done in a controlled environment where was possible to identify a consistent increase of icp over the different groups , showing , at least , that this model could be used to evaluate the different types of clinical and surgical interventions to reduce icp .
this study identified a consistent increase of icp according to the different volumes of balloon inflation , showing that this model may be used to evaluate clinical and surgical treatments to decrease icp .
however , we did not see any effect of hss following balloon inflation in any of the three groups .
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backgroundcurrent clinical treatment methods for refractory intracranial hypertension include elevation of the decubitus , ventilation adjustment , and use of hypertonic solutions such as hypertonic saline and mannitol solutions .
previous studies have shown that hypertonic solutions are particularly effective .
although several concentrations of saline solution have been proposed , a 3% solution is the most widely used .
the aim of this study was to evaluate the maintained efficacy of a 3% hypertonic saline solution in an experimental model of intracranial hypertension.material/methodsa porcine model of reversible intracranial hypertension was created by inserting a balloon catheter into the brain parenchyma , which was inflated and deflated to simulate intracranial hypertension and its surgical correction .
the experiment included 3 groups of animals ( a , b , and c ) with different balloon inflation volumes .
in group b , balloons were inflated 2 times to simulate reexpansion .
a 20 ml / kg bolus of 3% saline solution was infused using a pump 90 minutes after the start of balloon inflation , and the effects of intracranial pressure were evaluated 60 minutes after infusion.resultsno increases outside of the normal range were observed in mean serum sodium concentrations ( p=0.09 ) . in addition , we identified no differences within each group in serum sodium levels measured during hypertonic saline infusion ( p=0.21 ) .
no significant reductions in intracranial pressure were observed in any of the 3 groups.conclusionsbolus infusion of 3% hypertonic saline solution with the aid of a pump does not significantly reduce intracranial pressure in an animal model of intracranial hypertension .
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the use of new prosthetic meshes has been an advance in incisional hernia repair and has become prevalent worldwide .
we should take into consideration that meshes shrink and can reach a maximum reduction of 25% to 30% in their cross - sectional area within about 6 to 12 months after implantation .
chronic pain too , may be a consequence of prosthesis retraction and of the method of fixation , both of which may produce algogenic tension on the affected tissues . when a synthetic - mesh repair is performed in a patient with wound infection , enteric fistula , or stoma , there is a high risk of mesh infection or mesh rejection that could lead to dramatic consequences .
that is why the use of polypropylene - mesh repair in such cases is not recommended .
more recently , biological prostheses , made of an acellular collagen network extracted from swine dermis , came into use : these meshes serve as a matrix to recolonize the collagen fibers of the recipient .
their main advantage is that they can be used in septic areas , but their greatest disadvantage is the high cost and the limited experience reported in the literature .
therefore , the surgeon is required to understand costs , applications , contraindications , and the incidence of complications for each prosthetic material that is available for the abdominal wall reconstruction .
surgeon s options in such cases include the use of biological meshes and flap surgery .
flap repair should be tailored to the following characteristics : defect type , defect location , availability of surrounding soft tissue , and in certain cases , reoperation . in this article
we present new flap reconstruction techniques , which were applied on swine models and the follow - up data that assess possible complications of the flaps repair versus mesh repair for a standard abdominal wall defect .
four swine models ( pic - fii-337 hybrid breed pigs ) , five months old , were acclimated and housed under standard conditions .
all animal care and operative procedures were performed under the supervision of the department of anesthesiology and reanimation , university of agricultural sciences and veterinary medicine .
the experimental study was conducted under the protocol approved by the ethics committee of the university of medicine and pharmacy resolution no .
281/2014 of the department of surgery of the university of agricultural sciences and veterinary medicine ) between november 2015 and december 2015 .
after the animals were anesthetized , a standard ellipse shaped abdominal wall defect ( 10 cm long 5 cm wide ) was created and repaired using flap procedures . performing the abdominal wall defect ,
the peritoneum was kept intact and all conventional flaps used into repair were de - epithelialized .
mindray dc 6 ultrasound machine equipped with variable frequency linear transducers ( 710 mhz ) was used preoperatively to locate perforating vessels , and also used to follow - up possible complications after the abdominal wall repair .
all swine models were examined on determined periods : three days , seven days , two weeks and one month .
ultrasound - guided diagnostic needle aspirations were performed to distinguish the nature of the collections : seromas or hematomas . in order to identify the pathogens which caused mesh infection , all the aspirated samples went for bacteriological examination . vascularized flaps provide healthy coverage without inducing foreign - body reaction at the closure site .
perforator flaps are especially required in cases where mobilization and rotation of the flaps is necessary to achieve a single - stage reconstruction .
the deep inferior epigastric artery perforator ( diep ) flap is one of the most commonly used perforator flaps for reconstruction .
a suprafascial dissection was performed under magnifying glass using the edge of the defect as the flap margin to avoid creating another incision that can compromise the intervening bridge of tissue .
once the dominant perforator was visualized , the remaining skin incisions were performed and the flap was elevated from lateral to medial direction .
the perforator was freed from the rectus abdominus fascia and surrounding connective tissue to gain further mobility and degree of rotation .
after the flap was harvested , it was rotated approximately 90 to cover the defect .
the flap was then inset without tension and the donor site area was closed primarily ( figure 1 ) .
the vessels that supply the perforator circulation to the flap are the deep inferior epigastric artery and vein , therefore diep is the acronym for the used perforator flap .
an incision at the base of the projection rib was done , which was continued through the skin and panniculus carnosus .
the subcostal perforator artery that supplies the flap was identified and isolated through careful dissection . during flap harvest
, perforator was meticulously dissected from the surrounding , but we preserved a small skin connection on the flap base .
the flap with its perforator and thin pedicle was placed over the abdominal defect and sutured with vicryl 3.0 ( figure 2 ) .
the fascia closure was tension - free , using a continuous suture and the skin was closed with intradermal suture using monocryl 3.0 .
these flaps are perfused through random or axial blood supplies , so understanding the vascular anatomy in terms of abdominal wall angiosomes and perforator location is critical to designing robust local flaps .
there are various flap transposition designs available including advancement , rotation / advancement , interpolation , v - y advancement , and bipedicled flaps .
, the flap is realized through a simple incision parallel to the long axis of the defect .
the flap is de - epithelized and advanced into defect ( e.g. figure 3 ) . in the open onlay mesh repair used on our model , the fascial closure was supplemented with a lightweight polypropylene - mesh , in the onlay position , extended beyond the line of the closure by 3 cm in all directions .
the mesh was fixed with a continuous suture around the periphery using a heavy gauge nonabsorbable suture ( e.g. figure 4 ) .
long sterile dressings ( size 9 x 30 ) were applied in order to prevent contamination . to support and fix the wound dressing a circular abdominal bandage ( 15.0 cm 10 cm )
vascularized flaps provide healthy coverage without inducing foreign - body reaction at the closure site .
perforator flaps are especially required in cases where mobilization and rotation of the flaps is necessary to achieve a single - stage reconstruction .
the deep inferior epigastric artery perforator ( diep ) flap is one of the most commonly used perforator flaps for reconstruction .
a suprafascial dissection was performed under magnifying glass using the edge of the defect as the flap margin to avoid creating another incision that can compromise the intervening bridge of tissue .
once the dominant perforator was visualized , the remaining skin incisions were performed and the flap was elevated from lateral to medial direction .
the perforator was freed from the rectus abdominus fascia and surrounding connective tissue to gain further mobility and degree of rotation .
after the flap was harvested , it was rotated approximately 90 to cover the defect .
the flap was then inset without tension and the donor site area was closed primarily ( figure 1 ) .
the vessels that supply the perforator circulation to the flap are the deep inferior epigastric artery and vein , therefore diep is the acronym for the used perforator flap .
an incision at the base of the projection rib was done , which was continued through the skin and panniculus carnosus .
the subcostal perforator artery that supplies the flap was identified and isolated through careful dissection . during flap harvest
, perforator was meticulously dissected from the surrounding , but we preserved a small skin connection on the flap base . the flap with its perforator and thin pedicle
was placed over the abdominal defect and sutured with vicryl 3.0 ( figure 2 ) .
the fascia closure was tension - free , using a continuous suture and the skin was closed with intradermal suture using monocryl 3.0 .
these flaps are perfused through random or axial blood supplies , so understanding the vascular anatomy in terms of abdominal wall angiosomes and perforator location is critical to designing robust local flaps .
there are various flap transposition designs available including advancement , rotation / advancement , interpolation , v - y advancement , and bipedicled flaps .
, the flap is realized through a simple incision parallel to the long axis of the defect .
the flap is de - epithelized and advanced into defect ( e.g. figure 3 ) . in the open onlay mesh repair used on our model , the fascial closure was supplemented with a lightweight polypropylene - mesh , in the onlay position , extended beyond the line of the closure by 3 cm in all directions .
the mesh was fixed with a continuous suture around the periphery using a heavy gauge nonabsorbable suture ( e.g. figure 4 ) .
long sterile dressings ( size 9 x 30 ) were applied in order to prevent contamination . to support and fix the wound dressing a circular abdominal bandage ( 15.0 cm 10 cm ) was used ( e.g. figure 5 ) .
the deep inferior epigastric artery perforator ( diep ) flap is one of the most commonly used perforator flaps for reconstruction .
a suprafascial dissection was performed under magnifying glass using the edge of the defect as the flap margin to avoid creating another incision that can compromise the intervening bridge of tissue .
once the dominant perforator was visualized , the remaining skin incisions were performed and the flap was elevated from lateral to medial direction .
the perforator was freed from the rectus abdominus fascia and surrounding connective tissue to gain further mobility and degree of rotation .
after the flap was harvested , it was rotated approximately 90 to cover the defect .
the flap was then inset without tension and the donor site area was closed primarily ( figure 1 ) .
the vessels that supply the perforator circulation to the flap are the deep inferior epigastric artery and vein , therefore diep is the acronym for the used perforator flap .
an incision at the base of the projection rib was done , which was continued through the skin and panniculus carnosus .
the subcostal perforator artery that supplies the flap was identified and isolated through careful dissection . during flap harvest
, perforator was meticulously dissected from the surrounding , but we preserved a small skin connection on the flap base . the flap with its perforator and thin pedicle
was placed over the abdominal defect and sutured with vicryl 3.0 ( figure 2 ) .
the fascia closure was tension - free , using a continuous suture and the skin was closed with intradermal suture using monocryl 3.0 .
these flaps are perfused through random or axial blood supplies , so understanding the vascular anatomy in terms of abdominal wall angiosomes and perforator location is critical to designing robust local flaps .
there are various flap transposition designs available including advancement , rotation / advancement , interpolation , v - y advancement , and bipedicled flaps .
, the flap is realized through a simple incision parallel to the long axis of the defect .
the flap is de - epithelized and advanced into defect ( e.g. figure 3 ) .
in the open onlay mesh repair used on our model , the fascial closure was supplemented with a lightweight polypropylene - mesh , in the onlay position , extended beyond the line of the closure by 3 cm in all directions .
the mesh was fixed with a continuous suture around the periphery using a heavy gauge nonabsorbable suture ( e.g. figure 4 ) .
long sterile dressings ( size 9 x 30 ) were applied in order to prevent contamination . to support and fix the wound dressing a circular abdominal bandage ( 15.0 cm 10 cm )
clinical and ultrasound evaluation of the swine models three days after surgery reported no infection and no recurrences .
seroma was found with two techniques : diep flap procedure and onlay technique , while hematoma and edema appeared almost with all techniques , except the advancement technique . from the aesthetic point of view , advancement flap procedure had the best esthetic result at three days clinical examination ( e.g. figure 6 ) .
seven days after surgery , hematoma and edema diminished , except in the perforator plus flap procedure where it was moderate . an interesting fact was that infection developed in one swine model ( the onlay technique ) , although special attention was given to the manipulation of the mesh and all sterile conditions were accomplished .
reaction that also affected the cosmetic image as we could see at the clinical examination ( e.g. figure 7 ) . all collections reported were solved over time by natural drainage and after two weeks examination no seromas or deep hematomas were reported .
this was the swine model with the most demanding technique , where high technical skills were required in the dissection of the perforator .
no recurrences were reported after one month , except superficial necrosis with the diep procedure and the onlay technique .
superficial dehiscence occurred in the case of the perforator plus flap , making the advancement flap technique the single procedure with the best cosmetic result ( e.g. figure 8) .
the paucity of reported data regarding incisional hernia outcomes in animal model left us with no alternative , therefore we compared our results with those from human clinical studies mentioned in literature .
one of the key points in survival of the flap is to avoid any tension above itself or its pedicle . once the pedicle is secured it is important to allow it to perfuse and to allow the spasm of the vessels to relax , in its original position , before the flap is placed into the defect .
topical vasodilators , such as papaverine or verapamil , can be instilled around the pedicle at this point . once the flap perfusion is satisfactory it is ready to be placed into the defect . with refinements of techniques ,
the availability of doppler examination , and efficient preoperative imaging modalities there has been a significant reduction in diep flap operative times .
the longest recorded surgery time was 5.4 hours for the subcostal perforating artery flap procedure and the shortest time recorded was 2.5 hours for the advancement flap technique .
dissections were performed by a general surgeon supervised by a senior plastic surgeon specialized in microsurgey . for a general surgeon who deals only occasionally with flaps , achieving
perforators dissection is more challenging than applying meshes , and our results show that the onlay technique lasted 3.1 hours .
although both techniques at three days post surgery generated seroma complications , dimensions were different 0.69 cm 3.45 cm in the onlay technique compared to the 0.42 cm 1.66 cm for the diep artery flap procedure .
the summation of these factors , inflammation cell death , and lymphatic leakage , may cause fluid to accumulate in the newly created space .
if , however , bacteria seed this sterile accumulation , it quickly can be converted into an infectious accumulation with possible communication with the mesh . in these cases , the removal of the mesh is recommended . at seven days after surgery ultrasonography identified a superficial hematoma , which evolved into a superficial necrosis .
persistence of such accumulation may be caused by continued irritation from the prosthetic material , and can diminish circulation to the area , from which necrosis occurs .
even though the surgery was carried out in sterile conditions there may be local and systemic factors that contribute to the mesh infection .
the most common infectious processes reported are related to local wound infections with common skin flora .
aspiration puncture was performed on our swine model , and the microbiological examination of the culture found staphylococcus spp . , bacillus and escherichia coli infection . the large - pore , monofilament , lightweight synthetic meshes are the current standard of practice , which is why we decided to use it in our study .
but whenever a prosthetic material is implanted into the body it triggers a cellular inflammatory reaction , which is why the risk of infection and other complications associated with the use of meshes are inevitable .
careful attention to technical detail in the closure of the abdominal incision should not be minimized .
slowly absorbable suture was used in all abdominal closures in our study , since no difference in hernia recurrence was found between slowly absorbable suture and non - absorbable one .
the costs of the various prostheses are established according to a classification based on their constitution and their properties . in terms of recurrences , contaminated abdominal wall defects , patients with infected wounds or systemic infections or other contraindications to the prosthetic materials , alternative repair consists of biological meshes or flap surgery .
the cost of the biomaterials should not be an important factor in such cases , but this issue of price can not be avoided especially when hospitals do not have enough funds ( poor or developing countries ) or have a limited number of meshes ( some developed countries ) or , even worse , the patient must support all the costs from his personal budget .
the most important fact is that the indications for bioprostheses have progressively increased , even though there have been only a few preclinical and clinical studies to assess their effectiveness compared with four times as many publications about implantation of these prostheses in uncontaminated fields .
another consideration is that this product usually is ordered on a per case basis because of the expense of having large numbers o matrix sheets available for intermittent use . and , practically , no cost - effectiveness studies are available for these bioprostheses .
the follow - up of these procedures is the key to make sure that the evolution of the flap used is going in the right direction .
mesh infection identified in our study could occur from a sterile accumulation that quickly converted into an infected one , or the properties of the mesh material affected the degree of local inflammatory response and fibrovascular tissue incorporation , which may have allowed for a late infection .
flap surgery is a simple , versatile , applicable method in repairing abdominal wall defects , with excellent short term results .
our low complications rate supports the theory in using this flap techniques for randomized trials .
the costs in performing flap surgery on living swine models are high but it is the best method to train on and to develop further flap surgical techniques especially for recurrent hernias .
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background and aimin the age of synthetic prostheses most of hernia studies include a careful examination of the various types of prosthesis , their characteristics and their repair indications .
biological prostheses are also beginning to draw attention .
but in terms of recurrence especially for poor or developing countries , the discussion is different , due to their high cost which makes them difficult to afford . in this article
we present new flap reconstruction techniques for the reconstruction of the abdominal wall versus mesh repair , applied on swine models , outline the results of each technique , and specify the indications for their use.methodsan experimental protocol using four swine models ( pic - fii-337 hybrid breed pigs ) , five months old , was conducted .
all animal care and operative procedures were studied following the protocol approved by the ethics committee of the university of medicine and pharmacy resolution no .
281/2014 of the department of surgery of the university of agricultural sciences and veterinary medicine ) ; the study was carried out between november 2015 and february 2016 .
the primary objective was to compare the effect of surgical strategies in the treatment of the abdominal wall defect using variable flaps versus mesh repair in a large - animal models . physical examination and ultrasound imaging of the abdominal wall repair were done on determined periods , during one month .
the complications occurring after the abdominal wall repair were edema , collections , superficial dehiscence an recurrences.resultsno recurrences were reported at one month results , all seromas reported were solved over time by natural drainage .
superficial necrosis appeared in two swine models and superficial dehiscence occurred in one model , the perforator plus flap .
mesh infection was detected in the onlay swine model.conclusionsin terms of recurrences , contaminated abdominal wall defects or other contraindications to the use of prosthetic materials , biological mesh repair or flap surgery are the only surgical options . based on our findings and considering the high cost reported by the biological meshes use
, flap surgery becomes the suitable treatment for such cases , allowing a good reconstruction of the abdominal wall .
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specialized analytical methods are required for identification of components of agricultural dusts such as those generated in harvesting , transportation , storage , and processing of cotton , corn , and soybeans .
the larger particles and trash components of the dusts can often be identified visually or with the aid of an optical microscope ( om ) .
the respirable portion of the dust , that which causes lung dysfunction , retains few structural features for identification .
electron microscopy and x - ray microanalysis , together with special optical microscopical techniques , can be used to characterize these microdusts .
combination studies with scanning electron microscopy ( sem ) and energy - dispersive x - ray ( edx ) analysis of cotton dusts have shown the presence of mineralogical particles probably of a soil origin and materials that can be associated with plant parts . even in screened and filtered
cotton dusts , fibrillar fragments are usually present due to their ability to penetrate openings the size of their diameters .
the corn and soybean dusts studied were different from the cotton dust in that the large fibrillar component of the cotton dust was absent in the screened grain dusts .
however , these dusts consisted of structurally unrecognizable particles that appeared similar to those found in cotton dust .
in addition they contained many spheroid particles identified as starch .
dusts from all three sources were found to agglomerate into larger particles , some of which were still less than 10 micron .
this agglomeration could confuse the instrumental measurement of dust particle size.imagesfigure 1.figure 2.figure 3 .
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dfigure 6.figure 7 .
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afigure 11 .
bfigure 11 .
cfigure 11 .
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in north america , opioid addiction to methadone , hydrocodone , and oxycodone has been described as an epidemic [ 1 , 2 ] . in canada , nearly 10% of women
self - report an addiction to some form of illicit substance ( e.g. , cannabis ; ) and nearly 2% abuse highly addictive opioid substances like oxycontin and heroin . many of these women are mothers of childbearing age and face greater challenges than nonusers with raising their children and meeting their children 's developmental , social , cognitive , and emotional needs .
substance abusing mothers are at increased risk of developing affective disorders , low self - esteem , anxiety , and depression and are often challenged by social isolation , reduced support networks , and exposure to violent relationships and all of these factors can influence a mother 's capacity to parent her children [ 8 , 9 ] . taken together , unmitigated maternal substance abuse exacts social and health care costs by increasing the need for child protective , mental health and criminal justice services . protecting children from exposure to maternal substance abuse is a public health priority [ 10 , 11 ] , particularly for nurses who work with childbearing families in the community .
children exposed to maternal substance abuse are at increased risk for developmental problems , such as cognitive deficits , language delays , emotional problems , behavioural disorders , and becoming substance abusers themselves [ 13 , 14 ] .
the increased likelihood of intergenerational transmission of substance abuse is linked to addicted mothers ' parenting behaviour , often characterized as neglectful .
notably , substance abuse is one of the top three stressors influencing children 's development , along with mental illness and family violence , and the presence of maternal substance abuse is a greater risk factor to children 's development than paternal substance abuse .
indeed , the american academy of pediatrics recently described maternal addictions as toxic to children 's development and called for preventative public health interventions to reduce the detrimental effect of maternal addictions .
given the increased risk for a variety of adverse health outcomes , opioid dependence is a growing public health concern , particularly for mothers responsible for the care of children .
nurses engaged in public health practice are at the forefront of ensuring these families receive appropriate support .
one public health approach that nurses use to support and refer patients to manage opioid addiction is methadone maintenance treatment ( mmt ) . compared to control groups and other forms of opioid addiction intervention
mmt has been associated with reduced opioid use [ 2225 ] , improved health outcomes and reduced mortality rates [ 2630 ] , enhanced mental health status [ 31 , 32 ] , and improved social and occupational functioning . in some cases mmt has led to reductions in criminal activity [ 23 , 33 ] and nonopioid drug use , but these have not been consistent findings across studies [ 34 , 35 ] .
furthermore , involvement in mmt has been found to positively influence retention in addiction - focused treatment .
conducted a meta - analysis of 52 studies on mmt and other forms of substance abuse intervention for opiate addiction and found that participants of mmt were less likely to prematurely leave treatment compared to clients who were receiving no addiction services , methadone detoxification , or buprenorphine maintenance .
although mmt appears to have meaningful benefits , less is known about mothers ' experiences and how to promote and maintain mothers ' engagement in treatment . given the impact of parental addiction on children , engagement in mmt may create opportunities for enhancing mothers ' parenting capacity through its positive impact on mothers ' emotional , behavioural , and physical health well - being .
however , little knowledge exists about the influences of mmt on mother 's perceptions of their parenting or on the types of parenting supports and resources women need to facilitate their long - term engagement in mmt .
insights from the field have identified numerous barriers for mothers that influence their decision or capacity to seek and enter substance abuse treatment , including social stigma , lack of gender - specific treatment to address women 's psychosocial needs , fear of losing custody of their children , lack of childcare during treatment , fear of partner retaliation or violence [ 22 , 28 , 39 ] , and living with an opioid - dependent partner .
nonetheless , for some mothers , being a parent may be a powerful incentive to seek professional help in addressing their addiction [ 16 , 29 ] . while mmt has the potential to enhance both a mother 's individual functioning and , thereby , her parenting capacity , mothers also experience challenges associated with being a parent that interfere with their engagement in mmt .
research from the center for substance abuse research has suggested that programs which bring together women and their children may be more effective at retaining women in treatment and have demonstrated long - term effects .
this conclusion has been reaffirmed in two recent systematic reviews by niccols and colleagues [ 42 , 43 ] , who found that substance abuse programs with integrated components focused on addiction , parenting , and child - related services lead to positive outcomes for both mothers and their children across a number of domains ( e.g. , parenting skills and child development ) . to date , however , researchers have not explored mothers ' perspectives of the supports and resources needed to assist them with their parenting or to successfully engage in long - term mmt .
consequently , the current research was designed to fill these gaps by accessing both experiential ( mothers ) and expert ( service providers ) knowledge of methods and means to promote mother engagement in mmt and to seek their perspectives on the characteristics of the desired parenting supports and resources .
specifically , the primary goal of the study was to explore the experience of mothers participating in an outpatient atlantic canadian mmt program and their service providers to gain an appreciation of the impact of this intervention on parenting and mother - child relationships .
the secondary goal was to learn from mothers about the types of supports and services that might assist them with their mother - child relationships and parenting to maintain engagement in mmt .
the atlantic canadian mmt program , which began operation in 2005 , is staffed by a multidisciplinary team of nurses , physicians , and social workers who advocate a harm reduction model of care .
referrals to the program come from a variety of sources including public health nurses , primary care physicians , psychologists , detoxification centre staff ( physicians , nurses , or psychologists ) , or self - referrals .
the program provides comprehensive , client - centred , community - based services to its participants in the form of access to prescribed methadone , individual counseling and group counseling , and educational programs associated with addiction , as well as facilitation of referrals to other relevant community resources as needed , such as mental health services and social services to assist with housing , financial , and parenting supports .
the model of intervention is based on health canada 's 2002 guidelines for the best practices in the delivery of mmt interventions .
participants are eligible for the program if they have an opioid addiction but can be polysubstance abusers .
individual and group - based interventions focus on themes of addiction , self - esteem , infectious disease , hope , wellness , and anxiety , as well as support for new mothers or expecting mothers .
long - term retention in this program is a desired outcome given the maintenance focus of treatment once a client has stabilized on methadone . a recent evaluation of the program reviewed all cases ( n = 458 ) admitted to this program between 2005 and 2010 .
the greatest gains were achieved during the first year of treatment , with self - reported improvements in opioid drug use , employment status , health , and mental wellness , as well as reduced involvement in criminal activity , including drug - related criminal activity .
after ethical approval was obtained , the study was performed in accordance with the ethical standards established in the 1964 declaration of helsinki and the 2010 canadian tri - council guidelines for ethical research .
mothers in outpatient treatment at the mmt program were recruited to participate if they met inclusion criteria .
eligible mothers ranged in age from 16 to 55 years and must have been actively parenting children between birth and 18 years of age .
no specific duration limit was set for involvement with the program , but all cases were past the 3-month stabilization phase of the program .
a combination of convenience , snowball , and word of mouth strategies were used to recruit the sample .
advertisements inviting mothers to participate were placed in strategic locations at the addiction treatment centre and presentations describing the study were made , individually , to mothers attending mmt .
mothers received a $ 30 grocery certificate to thank them for their participation in the study .
of approximately 90 mothers enrolled in mmt , , 12 mothers volunteered to participate , providing sufficient numbers for qualitative data saturation ( when no new themes emerged in the data ) .
table 1 contains a more detailed summary of the demographic characteristics of these 12 participants .
addiction service providers working with the atlantic canadian mmt program were recruited through consultation with the research project 's advisory committee that was formed specifically for this project and was composed of agency administrators , service providers ( nurses and social workers ) , and a client representative from the treatment centre .
a total of six service providers were recruited out of a potential pool of nine , including four social workers and two ancillary support providers who had administrative relationships with program clients ( program coordination ) .
mothers and service providers were interviewed using semistructured interview guides developed specifically for the study .
these interviews were designed to determine their perspectives on parenting resources and supports to engage in long - term mmt . although the interviews were structured slightly differently for mothers and service providers , the content tapped by the questions was consistent between the two interview protocols .
specifically , mothers and service providers were asked about ( 1 ) parenting support needs , ( 2 ) barriers to support faced by mothers , ( 3 ) knowledge and use of support resources , and ( 4 ) preferences for parenting support and resources to facilitate engagement in community - based addictions treatment .
all service provider interviews and most of the mother interviews were conducted at the addictions treatment centre .
one of two female graduate students conducted the interviews after receiving training in interviewing skills .
one mother interview was conducted in a private room in a public library closer to her home .
all interviews were audio recorded for later transcription and analysis using nvivo version 9 software .
demographic information ( age , gender , and ethnicity ) and family composition ( number of children and marital status ) were collected prior to the interview .
first , a category system ( coding framework ) of key themes was inductively created by the first two authors and an academic colleague to capture all data from a subsample of randomly chosen interviews .
second , two trained research assistants read and coded all interviews under the guidance of the first two authors .
additional codes were added to the coding framework as new themes emerged from the data .
after all data were coded , identified overarching themes and subthemes were identified that addressed the research questions .
the final step of the analysis involved triangulating data by source ( mothers in mmt / service providers ) to enhance the value and validity of the study 's findings .
the mothers described the onset of their addiction to opiates and other substances as occurring through a variety of circumstances .
the most common circumstance was from a medical issue , for example , chronic back pain .
others noted that they became addicted after being introduced to drugs through a boyfriend or partner , experimentation , or because drug use seemed glamorous .
( ) and not only that , my daughter was twelve going on thirteen and i thought
i do not want her growing up in a city either and maybe she 'll turn out like i am right now ( ) .
( ) and not only that , my daughter was twelve going on thirteen and i thought
i do not want her growing up in a city either and maybe she 'll turn out like i am right now ( ) .
( mmt 04 )
similarly , a number of service providers attributed mothers ' motivation to address their drug use to their desire to be a better parent .
as one service provider suggested , that 's number one , try and get cleaned up to be better moms , to be back in their ( children 's ) lives where they want to be .
( sp 01 )
when questioned about how mmt affected them as a person , most mothers thought it did so in a positive way .
after joining the mmt program , they felt better or stronger than before and were successful in getting and staying clean , and some thought the program saved their lives . according to one mother ,
it [ mmt ] has made me better ; ( ) so much that i do not want drugs , i do not think about them .
the only time i think about drugs is when i 'm here [ at treatment ] .
( mmt 04 ) it [ mmt ] has made me better ; ( ) so much that i do not want drugs , i do not think about them .
the only time i think about drugs is when i 'm here [ at treatment ] .
( mmt 04 ) when asked about the effect of mmt on mothers ' ability to parent , service providers described a host of improvements including making mothers more attentive to their children 's needs , teaching mothers useful life skills and parenting strategies , and providing structure and stability , as well as making mothers ' lives safer .
one service provider commented , they learn a lot of really good skills related to their drug use and related to their recovery ( ) .
they learn about their triggers and their cravings and what sets them off and makes them relapse .
( sp 01 ) they learn a lot of really good skills related to their drug use and related to their recovery ( ) .
they learn about their triggers and their cravings and what sets them off and makes them relapse .
( sp 01 ) when asked how the mmt program had affected their parenting , many women felt it had made them better mothers .
they felt more attentive to their children 's needs and better able to engage with them , as illustrated in the following statement : i'm not foggy anymore , i 'm more clear and i 'm just a better mother .
i play with [ my children ] all of the time , i do activities , i read to them , whereas before i just used to feel like everything i did was a chore .
( mmt 03 ) i 'm not foggy anymore , i 'm more clear and i 'm just a better mother .
i play with [ my children ] all of the time , i do activities , i read to them , whereas before i just used to feel like everything i did was a chore .
( mmt 03 )
most claimed their relationship with their children had improved after attending the mmt program .
mothers reported that they had more time and energy to devote to their children and were more emotionally available .
their relationships with their children were also described as more open , honest , and marked by a stronger bond . overcoming their addiction allowed some to get their relationships with their children back to normal after a period of relative neglect : before you are on methadone it just feels like you are lying all of the time , lying to yourself .
when i was on the pills i was n't active and wanting to do stuff like i should have , like going to school functions and stuff like that .
( but ) now that i 'm on methadone i feel better and i can do all of that stuff .
( mmt 02 ) before you are on methadone it just feels like you are lying all of the time , lying to yourself . when i was on the pills i was n't active and wanting to do stuff like i should have , like going to school functions and stuff like that .
( but ) now that i 'm on methadone i feel better and i can do all of that stuff .
( mmt 02 ) many mothers felt that the mmt program had a positive impact on their relationships with their families .
regarding their children 's personal development , however , the vast majority thought mmt had no significant impact at all . while describing both positive and negative changes in their children 's behaviour , mothers attributed these changes more to the removal of drugs in their lives rather than the mmt program itself .
some reported that their children were happier , while some children struggled with the frustration of having to attend sessions and take daily trips to the pharmacy with their mothers .
when discussing which of their support needs were successfully met by mmt , mothers indicated that the program was helpful by providing them someone with whom they could talk about their issues , access to classes on parenting skills , readily available social workers or counselors , and emotional support . according to one mother , just having somebody to talk to and being able to come in and give you different resources too , because i really did not have a clue , i did not ever think i would have kids .
( mmt 12 ) just having somebody to talk to and being able to come in and give you different resources too , because i really did not have a clue , i did not ever think i would have kids .
( mmt 12 ) while the methadone treatment facility is not designed specifically for mothers , it does offer access to external programs that address their unique needs , such as parenting groups and classes on parenting skills .
however , the mmt treatment group forum offers an opportunity for mothers in a similar situation to commiserate with each other about parenting concerns .
as one service provider stated , when moms learn from some of the other participants in a group about what they 've gone through , it might be a parenting issue , that 's where a lot of the moms get some of their support from peers , is , in groups or people who have gone through that before , and , they say my kids are just back in my care and i 'm having a real hard time saying no and maintaining boundaries .
has it happened with anyone else ? and then moms receive feedback that way .
( sp 01 ) when moms learn from some of the other participants in a group about what they 've gone through , it might be a parenting issue , that 's where a lot of the moms get some of their support from peers , is , in groups or people who have gone through that before , and , they say my kids are just back in my care and i 'm having a real hard time saying no and maintaining boundaries .
has it happened with anyone else ? and then moms receive feedback that way .
( sp 01 ) mothers discussed the support needs that were not met by mmt . the majority of complaints pertained to the absence of programs within the context of the mmt program itself that addressed the issue of children , such as services facilitating the bond between mothers and children .
as one mother suggested , they should have a program [ at mmt ] to watch your kids or anything while you go to group and stuff ( ) .
we 've even said it to them but they said they do not have [ a service ] to watch kids over there .
( mmt 05 ) they should have a program [ at mmt ] to watch your kids or anything while you go to group and stuff ( ) .
we 've even said it to them but they said they do not have [ a service ] to watch kids over there .
( mmt 05 )
they also encountered a number of barriers to support in their daily lives and in their attempts to access the mmt program .
the two most common barriers were a lack of daycare facilities and convenient , affordable transportation . transportation issues generally involved the complicated logistics of making regular trips to the centre , as few mothers had access to their own vehicles . according to one mother ,
my issue was when i first go in the program i was attending a meeting that i really enjoyed and we did not have a car at that point and they were giving us transportation to the meeting and then all of a sudden it stopped and i 'm like well how am i supposed to get there ? ( ) i think there should be transportation for people that need it .
( mmt 07 ) my issue was when i first go in the program i was attending a meeting that i really enjoyed and we did not have a car at that point and they were giving us transportation to the meeting and then all of a sudden it stopped and i 'm like well how am i supposed to get there ?
( mmt 07 )
compounding this problem was the distance between clients ' homes and the mmt centre , which some felt was too out of the way and was particularly difficult to use with children in tow .
one mother described her frustration with taking the bus to treatment , stating,it 's hard to get transportation out here sometimes .
when i first lived here , i was taking a bus with a newborn baby and [ my oldest child ] , with a four year old on a great big double stroller by myself ( ) .
when i first lived here , i was taking a bus with a newborn baby and [ my oldest child ] , with a four year old on a great big double stroller by myself ( ) .
( mmt 05 )
the cost of transportation was another prohibitive factor for some mothers , as cab fare and the cost of taking a bus surpassed their budget .
as one mother commented,having to come into town everyday was like a five dollar trip because coming in and going back out again , so that caused barriers .
( mmt 06 ) having to come into town everyday was like a five dollar trip because coming in and going back out again , so that caused barriers .
( mmt 06 )
they also lacked the financial means necessary to enroll their children in daycare , which limited their ability to attend sessions and was a significant barrier to their recovery .
as one mother suggested , too bad they did not have childcare here at [ mmt ] and then i think more people would come to the meetings because it would be easy for them .
it is hard finding sitters , ( ) what do they do ? ( mmt 07 ) too bad they did not have childcare here at [ mmt ] and then i think more people would come to the meetings because it would be easy for them .
it is hard finding sitters , ( ) what do they do ? ( mmt 07 ) wait time was an additional barrier to treatment for some , as were more personal issues , such as : lack of family support and embarrassment or fear .
another barrier identified by mothers was a lack of information about the availability of mmt , both within addictions services and the greater community .
( mmt 07 ) of all the potential support resources available outside of mmt , mothers ' parents were called upon most often and adopted the widest variety of supportive roles .
their parents provided basic needs , such as childcare , emotional support , and affirmational support .
in contrast , some mothers revealed that they were reluctant to leave their children with their own parents , due to troubled interpersonal relationship histories .
however , their parents were still a better option than other members of their social network , who were often drug abusers .
as the following passage illustrates , they often need their parents to care for their children during recovery, i was sick , like really , really sick because i could n't get my methadone .
i knew the outcome of it and i told her and i said , please come get [ child ] . it broke my heart to do that but i told her , i said come and get him because i can not sit here and be sick and take care of him and i know what i have to do to not be sick . ( mmt 05 )
i was sick , like really , really sick because i could n't get my methadone .
i knew the outcome of it and i told her and i said , please come get [ child ] .
it broke my heart to do that but i told her , i said come and get him because i can not sit here and be sick and take care of him and i know what i have to do to not be sick .
( mmt 05 )
mothers ' friends and siblings also helped by providing childcare .
one spoke about being able to talk to a friend about her urges to use , if she 's having a bad day or i 'm having a bad day and if she wants to use , she 'll tell me instead of going out and using .
it 's just better to talk about it ( ) than keeping it in because if you keep it all in and you end up just going and doing it then .
( mmt 05 ) if she 's having a bad day or i 'm having a bad day and if she wants to use , she 'll tell me instead of going out and using .
it 's just better to talk about it ( ) than keeping it in because if you keep it all in and you end up just going and doing it then .
( mmt 05 )
mothers also found support within the wider community , from counselors and social workers .
one mother stated , there 's a place over north though that gives you diapers and formula and stuff .
( ) i 'm on social assistance so they give you lots of help .
( mmt 12 ) there 's a place over north though that gives you diapers and formula and stuff .
( ) i 'm on social assistance so they give you lots of help .
( mmt 12 ) all twelve mothers were universally in favour of one - on - one support compared to group support . according to service providers , one - on - one support allowed providers to focus on the mother , addressing her needs specifically , rather than in general terms .
the private nature of sessions allowed mothers to share intimate details they might not be comfortable discussing in front of a group .
one service provider commented , especially the moms that have the kids , a lot of them have taken advantage of individual counselling , the one - on - one counselling , because lots of times their issues are so personal that do not really want to air them in a group .
( sp 04 ) especially the moms that have the kids , a lot of them have taken advantage of individual counselling , the one - on - one counselling , because lots of times their issues are so personal that do not really want to air them in a group .
( sp 04 )
group support , however , was also widely suggested by mothers , as it allowed them to support one another, they cover things in groups that you might not know how to deal with , like people give their advice , if you are craving what do you do ?
you might not think of all of these ideas by yourself but you get a lot of them at groups and just other people 's experiences because they 've been in the program longer ( mmt 07 ) they cover things in groups that you might not know how to deal with , like people give their advice , if you are craving what do you do ?
you might not think of all of these ideas by yourself but you get a lot of them at groups and just other people 's experiences because they 've been in the program longer ( mmt 07 ) for preferred method of contact , all twelve mothers favoured face - to - face interactions with service providers .
this was followed by telephone contact , which was favoured in part for its ability to provide instant , twenty - four hour access to service providers .
as one stated it would be nice for people to know that they could pick up the phone and have that instant kind of release and advice .
( mmt 11 ) service providers recommended face - to - face , telephone , and online treatment .
service providers preferred face - to - face contact given its ability to facilitate intimate communication .
a service provider stated , i prefer face - to - face , because that way you can gauge peoples cues , you can tell if someone is uncomfortable , you can tell if someone is really engaged , and it gives you just that opportunity to connect .
( ) when you sit with someone face to face and they 're letting you in on what 's going on and sharing that with you there 's an inherent trust there that 's very different than over the phone .
( sp 01 ) i prefer face - to - face , because that way you can gauge peoples cues , you can tell if someone is uncomfortable , you can tell if someone is really engaged , and it gives you just that opportunity to connect .
( ) when you sit with someone face to face and they 're letting you in on what 's going on and sharing that with you there 's an inherent trust there that 's very different than over the phone .
( sp 01 )
telephone support was considered an important resource , as it allowed mothers with hectic schedules to contact support staff should they have a crisis or need specific information .
as one provider suggested , i do not think telephone support exclusively , but i think telephone support is important .
for example , we have moms that telephone support is huge , they can call and do call often and we are available so that we can talk to them ( ) . we can not conduct long - term , significant therapeutic counselling on the phone , but we certainly can provide the affirmations , the support , the sort of assurances , the reassurances , the addressing the immediate perception or an immediate crisis , all of those sorts of things .
( sp 03 ) i do not think telephone support exclusively , but i think telephone support is important .
for example , we have moms that telephone support is huge , they can call and do call often and we are available so that we can talk to them ( ) .
we can not conduct long - term , significant therapeutic counselling on the phone , but we certainly can provide the affirmations , the support , the sort of assurances , the reassurances , the addressing the immediate perception or an immediate crisis , all of those sorts of things . ( sp 03 )
like telephone support , computer - based support was seen as a useful secondary resource , particularly for providing a convenient access point for information about addiction recovery .
a service provider commented , i think moms that have a hard time getting out , that might be a good way for them to be able to do that because they could go online .
( sp 05 ) i think moms that have a hard time getting out , that might be a good way for them to be able to do that because they could go online .
[ my concern is ] do they have computers ? ( sp 05 ) when discussing their preferred support provider for support groups , most mothers suggested a peer a former addict who had successfully gone through treatment .
they felt more comfortable talking to someone who knew what they were going through firsthand .
as one mother commented , because no one really understands unless you 've been through it yourself , really , they do not .
i mean they try to and god bless them but they do not , so i think that is the best person to talk to or someone who at least understands addiction .
( mmt 07 ) because no one really understands unless you 've been through it yourself , really , they do not .
i mean they try to and god bless them but they do not , so i think that is the best person to talk to or someone who at least understands addiction .
( mmt 07 )
some thought a professional facilitator would be the best , while others found a combination of the two to be ideal .
in contrast , all of the service providers felt that support groups should be facilitated by professionals , because of their specialized education and training , and some discussed the possibility of a combination of professional and peer facilitation .
for the ideal location , some mothers thought the program 's present community location was fine .
others thought it would have helped to have sessions closer to their homes , or else somewhere more generally accessible .
one mother suggested , it would have to be somewhere where everyone can get to , like maybe somewhere central .
( ) i think if it is just a walk - in thing it would work better because when you go in there on your terms .
( mmt 07 ) it would have to be somewhere where everyone can get to , like maybe somewhere central .
( ) i think if it is just a walk - in thing it would work better because when you go in there on your terms .
( mmt 07 ) when asked what topics the ideal treatment would cover , mothers suggested parenting and affirmational and emotional support . according to one mother , things are going to change in the household when someone starts getting well .
changes the whole dynamics of the household because when you 've got somebody who 's using ( ) the child becomes the parent .
all of a sudden , the child whose been probably getting away blue murder because he 's the parent is suddenly going to be the child again ( mmt 06 ) things are going to change in the household when someone starts getting well . changes the whole dynamics of the household because when you 've got somebody who 's using ( ) the child becomes the parent .
all of a sudden , the child whose been probably getting away blue murder because he 's the parent is suddenly going to be the child again ( mmt 06 )
for the ideal duration of mmt programs , mothers typically suggested between an hour and an hour and a half . for ideal frequency , mothers opted for once or twice a week .
lastly , mothers were asked to list potential areas of support they would find useful in an ideal mmt program .
an extension of this , group support , was also suggested.some people do not have family , some people do not have friends so it would be nice to have somewhere where they could go and talk to people if they needed to or even a place where moms could get together , ( ) i think a lot of parents get discouraged too once they lose their kids they think there is no hope .
( mmt 07 ) some people do not have family , some people do not have friends so it would be nice to have somewhere where they could go and talk to people if they needed to or even a place where moms could get together , ( ) i think a lot of parents get discouraged too
( mmt 07 )
in addition to emotional support , mothers felt information about methadone treatment would be important .
one suggested a fairly extensive program for children whose parents are undergoing treatment , teaching them about methadone and addiction as well as educating parents on practical aspects of parenthood , such as how to enroll children in school .
another suggested , when it comes to the children , i think they need education as well
( for example ) that the methadone is not another drug , that it is a process of helping to get well , because there 's a lot of information out there where people are saying , oh they 're just going from one drug to another and then the kids get fed up .
( mmt 06 ) when it comes to the children , i think they need education as well
( for example ) that the methadone is not another drug , that it is a process of helping to get well , because there 's a lot of information out there where people are saying , oh they 're just going from one drug to another and then the kids get fed up .
( mmt 06 )
two service providers also believed that treatment should extend beyond mothers and address the needs of children as well .
it is important , they felt , to treat children as individuals in need of counselling instead of roadblocks to their mothers ' recovery .
furthermore , while mothers and children must both be addressed in treatment , so too must the bond between them . facilitating stronger bonds between mother and child will help the family as a whole .
one service provider commented , what 's going on with women 's relationships with their children ?
because when women 's needs are being met , they 're better able to meet the needs of their children .
( sp 01 ) what 's going on with women 's relationships with their children ? with themselves ? are women 's needs being met ?
because when women 's needs are being met , they 're better able to meet the needs of their children .
mmt has been shown to be a useful treatment for opioid addiction ( e.g. , johansson et al . ) in targeted public health services . by participating in substance abuse treatment programs like mmt , mothers struggling with addiction not only stand to help themselves but
also may have the opportunity to minimize the risk of poor developmental , mental health , and behaviour outcomes for their children , reduce the likelihood of intergenerational transmission of addiction , and limit the social and health care costs associated with addiction .
extensive research has addressed the negative effect of substance abuse on mother - child relationships and child development ( e.g. , bowie ) , but very little research has examined mothering in the context of treatment for opioid addiction and general substance abuse .
existing research has suggested that motherhood status , residing with one 's children , and enhanced social services can each influence treatment success , as can integrating parent and child - focused interventions within substance abuse treatment for mothers .
however , mothers who decide to seek treatment still face significant challenges that can then interfere or complicate their engagement in substance abuse treatment , such as poverty , prior experiences of losing custody / access to one 's children , inadequate / unstable housing , parental stress , domestic violence victimization , and insufficient or absence social support .
thus , the current research examined mothers ' and service providers ' perspectives on their experience of being mothers in mmt , and on the parenting supports and resources that would assist mothers in successfully engaging in long - term mmt .
the information gained from the current research will be of value to public health nurses who deliver substance abuse treatment services , as well as to researchers , managers , and policy experts who develop best practice models for such public health interventions .
a significant strength of the study was that the perspectives of mothers and their service providers were largely in agreement .
many of the women in the current study chose mmt because they were often motivated by the desire to be better parents .
one of the major barriers for mothers seeking and/or maintaining treatment for substance abuse is the fear of losing custody of their children once professionals become aware of their addiction issues [ 38 , 49 ] .
similarly , hughes et al . found that mothers who were able to retain care of their children while being engaged in residential treatment programs have significantly better outcomes than mothers who did not retain their children .
positive outcomes also have been found for mothers who attended special mother - child service as part of their addiction intervention . in both of these latter two studies , retention and successful discharge from treatment
were enhanced by the presence of children in the residential care settings . in light of these types of findings
, child - centered policies have been criticized for focusing more on the child and limiting maternal rights rather than promoting mothers ' health , safely , and capacity to parent their children .
however , research into the role of children in mothers ' treatment success has been mixed . for opioid - dependent women , having a higher number of children has been associated with poorer treatment history ; that is , parenting responsibilities can limit treatment - seeking behaviours .
faupel and hanke also noted a significant negative relationship between motherhood and enrolment in community - based treatment .
thus , greater awareness of these barriers and how to overcome them within the context of a community - based substance abuse treatment program are required .
findings from this study agree with others ' observation that while mmt engagement leads to more positive outcomes for mothers , accessible and safe childcare and family responsibilities act as barriers to mmt even when this service is available within the community they reside .
for the mothers in the current study , attending treatment meant traveling long distances , often by means of public transportation that involved multiple bus transfers and reliance on third parties to provide a drive to the treatment site .
although not specific to mothers or women , burbridge reported that 42% of male and female clients in the same mmt program from which our mothers were drawn resided between 100 and 200 km away from the treatment centre and another 2% lived more than 200 km away . thus , providing onsite childcare and easier access to efficient transportation to attend mmt services would make treatment much more accessible for mothers .
these are public health issues that nurses engaged in mmt can address through patient advocacy and policy influence .
mothers ' perspectives in the current study echo those of lundgren and colleagues who advocated for childcare services for mothers in mmt and the need to develop specific parenting support services to promote engagement in mmt , such as skills training and parental counseling .
these services may be provided in typical primary health care settings by public health nurses .
the mothers in the current study specifically described a need for interventions that could help them rebuild relationships with their children that have been damaged or strained by the addiction .
the provision of parent and family - based interventions has been shown to reduce parental stress in parents engaged in mmt , while also reducing problem behaviour in their children and enhancing their family functioning [ 10 , 11 , 42 , 43 ] .
thus , multicomponent programs that address addiction issues , family dysfunction , and parenting have the potential to maximize treatment gains for both mothers and their children .
the need for this type of intervention is not unique to mothers in mmt , as experts have also noted a similar need for fathers .
further research is needed to establish ways that addiction services might address these parenting capacity needs through the provision of childcare , parenting skills , and family - based interventions .
given public health nurses ' frequent focus on childbearing families , better integration of services for mothers receiving mmt into the existing public health service network is an area worthy of exploration .
previous research has revealed that limited supportive treatment services are available for mothers who abuse substances relative to what is available for men and single women .
mothers in the current study revealed that they relied most heavily on their family and friends for support during treatment , followed by other community and service providers . according to the mothers in the current study
, mmt programs should focus on providing greater emotional support and encouragement to mothers , provide information to mothers about how to speak with their children about addiction and the recovery process or offer programs for children to attend on these topics , and provide services to better promote parenting skills . in general
, mothers believed that the formal adoption of these parent - child focused components to their mmt program would enable them to strengthen their relationships with their children and enhance their parenting capacity . the only program available directly through this particular mmt service that pertained to children was geared toward expectant and new mothers .
although these maternity focused interventions with mothers dependent on substances have been shown to have value for reducing risks to both the mother and her infant , they do not meet the needs of mothers who are raising older children and adolescents .
formal partnerships with community and public health programs and social service agencies that provide parenting enhancement focused interventions for mothers in substance abuse treatment appears to be a valuable component of addiction intervention for mothers , for both them and their children .
other addiction programs have recognized the value of going beyond treating the substance abuse by means of multidimensional need screenings , cross - agency case planning , and service partnerships , such as that used by the breaking the cycle program in the united states for criminal offenders and comprehensive outreach support services characteristic of such programs as the sheway project in canada , which assists high - risk women who are pregnant or caring for infants .
the sheway project has adopted a harm reduction approach to its work with mothers and emphasized a nonjudgmental and respectful decorum towards mothers amongst its staff and service delivery to promote engagement .
evaluations of the breaking the cycle and sheway project programs have demonstrated improvements in participants ' family functioning [ 47 , 57 ] .
poole further noted benefits of the sheway project with regard to stabilizing the housing situation of mothers , enhancing mother 's engagement in pre- and postnatal care , and the likelihood of mothers retaining custody of their children .
however , poole 's evaluation identified gaps with regard to services for the partners of the mother 's accessing addictions treatment and their children who were beyond the target age for the program of 18 months of age , parenting services , and other supports to provide safety and security to these families . in the current study
, the service providers who participated in our interviews appeared to appreciate mothers ' experiences and recognized the complexity of being a parent in treatment .
previous research has found that service providers sometimes hold stigma - related beliefs , stereotypes , and negative attitudes about mothers with addiction issues , and concerns for children of ten supercede concerns for mothers .
these views can translate into judgments and misrepresentation of mothers by staff , which then interferes with a mother 's willingness to engage in addiction treatment and support services for her children .
a qualitative analysis of service providers who provided health services to antenatal mothers with addiction issues revealed that staff tended to be more accepting and empathetic towards mothers with an addiction issues when these staff had a better understanding of the experience , challenges , and complexities of substance - dependent mothers .
thus , staff working with substance - abusing mothers and their families are likely in a better position to overcome barriers to patient engagement when they can use their empathy and understanding to realistically support and encourage mothers , rather than to judge or hold mothers to negative expectations .
in addition to parent - child focused supports , mothers in the current study also spoke about the nature of service delivery .
many felt that the provision of support through individual counseling would give them an opportunity to explore personal issues in a confidential setting that goes beyond the direct addiction issues .
these mothers also expressed value in group sessions as they viewed this medium of intervention as being able to provide them with the emotional support and words of encouragement they desired through the sharing of similar stories and experiences . while face - to - face support was by far the preferred mode of contact for these mothers with their service providers , they also recommended greater use of telephone contact because it would allow for 24-hour access to service provider support .
in addition , many mothers wanted mmt to incorporate supportive resources that were facilitated by a peer who has successfully gone through treatment .
although data saturation was obtained for the mothers ' data , this was not ensured in the service providers ' data . nonetheless , the themes identified in the 12 mothers ' and six service providers ' interviews shared a number of commonalities that lend credence .
thus , the current data likely reflects the experience of some women and service providers involved in this particular mmt program but may not necessarily fully generalize to the experience of mothers who chose not to participate or to mothers in other mmt programs .
fortunately , almost all of the staff employed with this mmt program volunteered to participate in the study .
a second major limitation of the current research was the lack of voice given to the children of the interviewed mothers .
inclusion of age - appropriate child - focused interviews and assessment methods would have allowed the research findings to be further contextualized from the perspective of children in terms of their needs for supports and services while their mothers are engaged in treatment .
mothers who struggle with addiction face significant barriers to engaging in substance abuse treatment , especially when treatment requires long - term commitments like mmt . in public health , mmt is the gold standard for helping individuals overcome addictions to opiates , but these interventions must be delivered in a manner that is sensitive to the needs of mothers and with an appreciation of the potential impact such programs have on parenting capacity and promotion of the well - being on the children of these clients .
inclusion of child and family - focused interventions into substance abuse treatment models that provide service to mothers is essential and indeed recommended .
mothers face great barriers to remaining engaged in community - based mmt due to challenges accessing safe and reliable childcare and accessing reasonable and reliable transportation to and from treatment sites .
public health nurses and other treatment providers and program managers must deliberately and creatively present solutions to these barriers to maximize the engagement of mothers in treatment
. however , it is important to acknowledge that research often falls behind innovative clinical practice , and many mmt programs in public health are likely already working to address these solutions .
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unmanaged maternal opioid addiction poses health and social risks to both mothers and children in their care .
methadone maintenance treatment ( mmt ) is a targeted public health service to which nurses and other allied health professionals may refer these high risk families for support .
mothers participating in mmt to manage their addiction and their service providers were interviewed to identify resources to maximize mothers ' engagement in treatment and enhance mothers ' parenting capacity .
twelve mothers and six service providers were recruited from an outpatient atlantic canadian methadone treatment program .
two major barriers to engagement in mmt were identified by both mothers and service providers including ( 1 ) the lack of available and consistent childcare while mothers attended outpatient programs and ( 2 ) challenges with transportation to the treatment facility .
all participants noted the potential benefits of adding supportive resources for the children of mothers involved in mmt and for mothers to learn how to communicate more effectively with their children and rebuild damaged mother - child relationships .
the public health benefits of integrating parent - child ancillary supports into mmt for mothers are discussed .
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patients suffering from any symptoms related to the anus frequently and often incorrectly assume that their symptoms are due to haemorrhoids 1,2,3,4 .
lockhart - mummery once wrote " nearly every lesion around the anus is liable to be called ' piles ' by the patient and not infrequently by the referring doctor also " 5 .
this practice still prevails : " almost everyone suffers from haemorrhoids at some time in their lives " 6 . "
haemorrhoids and their symptoms are one of the most common afflictions in the western world " 7 .
the cost of treating benign anal diseases ( bad ) in the united states exceeds 2 billion dollars annually 11.the german national insurance fund spends 38 million euros yearly for ointments and suppositories whose use is not supported by any scientific data 12 . history taking does not lead to accurate anal diagnoses 1,13 .
it is also unknown which examination position ( i.e. left - lateral , knee - chest , lithotomy or the upright standing - bented ) is the most reliable for determining the causes of anal bleeding , anal itch , anal pain or anal burning .
the sensitivity , specificity and predictive value of patients ' positioning in diagnosis of bad , concomitant anal findings ( caf ) , and multiple anal lesions ( mal ) with one individual also remain unknown 3,14,15 .
we investigated the types and frequencies of anal complaints with respect to anal findings at proctologic assessment using the knee - chest position in contrast to the widely used left lateral sims ' position to evaluate its pros and cons .
individuals were asked to complete a questionnaire that described their symptoms and signs ( table 1 ) .
proctologic assessment was performed in the knee - chest position 14 by inspection of the anal verge followed by digital examination of the anal canal , and anoscopy .
we used published definitions for bad such as haemorrhoids ( table 2 ) and pruritus ani ( table 3 ) .
concomitant anal findings ( caf ) such as skin tags were defined as anal verge anatomical findings of unknown importance for bad ( table 4 ) .
multiple anal lesions ( mal ) were defined as more than one bad found in one individual e.g. a fissure - in - ano with pruritus ani 14,15 .
definitions were tested in a two year pilot study , and adopted into routine use ten months before start of the study .
findings were entered into a personal computer immediately after proctologic assessment of each individual . means + /-
frequencies and percentages were calculated for categorical data such as the male to female ratio , history of symptoms , and anal lesions .
bivariate analyses were performed by using t - tests to compare independent groups and point - biserial correlations coefficients to analyse relationships between continuous and dichotomous variables .
binary logistic regression analysis was used to predict anal lesions based on answers of the patient questionnaire .
data were analysed using the statistical package for social sciences software ( spss , chicago , il ) version 15 .
individuals were asked to complete a questionnaire that described their symptoms and signs ( table 1 ) .
proctologic assessment was performed in the knee - chest position 14 by inspection of the anal verge followed by digital examination of the anal canal , and anoscopy .
we used published definitions for bad such as haemorrhoids ( table 2 ) and pruritus ani ( table 3 ) .
concomitant anal findings ( caf ) such as skin tags were defined as anal verge anatomical findings of unknown importance for bad ( table 4 ) .
multiple anal lesions ( mal ) were defined as more than one bad found in one individual e.g. a fissure - in - ano with pruritus ani 14,15 .
definitions were tested in a two year pilot study , and adopted into routine use ten months before start of the study .
frequencies and percentages were calculated for categorical data such as the male to female ratio , history of symptoms , and anal lesions .
bivariate analyses were performed by using t - tests to compare independent groups and point - biserial correlations coefficients to analyse relationships between continuous and dichotomous variables .
binary logistic regression analysis was used to predict anal lesions based on answers of the patient questionnaire .
data were analysed using the statistical package for social sciences software ( spss , chicago , il ) version 15 .
a total of 876 individuals of both genders aged 16 - 80 years old who consecutively entered our office from july 25 , 2005 until december 20 , 2005 were enrolled .
they were referred by general practitioners , physicians or gynaecologists in order to determine the causes of anal and/or abdominal complaints mostly without referral letters from their primary doctor .
we excluded 63 individuals because of tentative diagnoses of inflammatory bowel disease ( 20 ) , anal corticoid ointment harm ( 28 ) , condyloma acuminate ( 8) , anal abscess ( 4 ) , anal carcinoma ( 1 ) , m. bowen ( 1 ) , and hiv lesion ( 1 ) , leaving 807 patients for further calculation .
of these 807 individuals , 539 patients ( 66.8% ) were found to have bad , while 268 ( 33,2% ) participants did not have bad ( table 5 ) . of 539 patients with bad , 168 patients ( 31.2% ) presented with mal ( table 6 ) .
haemorrhoids and pruritus ani followed by anal fissures were found most frequently in patients with bad in contrast to patients with mal , who mostly presented with anal fissures , thrombosed external haemorrhoids , and pruritus ani ( table 6 ) .
stage 2 was by far the most frequently found stage in 317 patients presenting with pruritus ani .
only stage 1 pruritus ani as a single lesion presented more frequently ( table 7 ) .
the differences between the bad and the no bad group with regard to skin tags and a funnel - shaped anus were highly significant ( table 8) .
of 807 participants , 188 ( 34.9% ) with bad and 105 ( 39.5% ) without bad did not specify symptoms .
therefore we are only able to present the answers of the remaining 350 and 161 individuals with and without bad respectively ( table 9 ) . to determine whether certain symptoms could serve as predictors of bad , we used binary logistic regression analysis .
the database consisted of all 17 symptoms described in the questionnaire ( table 1 ) : haemorrhoids were predicted by anal bleeding ( p=0.032 ) , anal weeping ( p=0.017 ) , non - existence of diarrhoea ( p=0.008 ) , and anal pain ( p=0.005 ) . thrombosed external haemorrhoids
were predicted by anal lumps ( p<0.001 ) while anal bleeding ( p=0.010 ) was absent .
anal fissures were predicted by anal pain ( p<0.001 ) and anal bleeding ( p=0.006 ) in the absence of anal weeping ( p=0.033 ) .
pruritus ani was predicted by anal pain ( p<0.001 ) , anal itch ( p=0.001 ) , and anal soreness ( p=0.006 ) . for patients with mal , we were interested whether it would be possible to differentiate among existing bads using the symptoms described by the questionnaire .
we found that patients with haemorrhoids combined with pruritus ani stages 2 - 4 complained more often of anal itch ( p<0.001 ) , anal burning ( p<0.05 ) , anal soreness ( p=0.001 ) , and anal weeping ( p=0.001 ) than patients with haemorrhoids only .
individuals with and without bad suffered from symptoms and signs for 2 to 12 months ( 29.3% vs. 34.4% ) or more than 12 months ( 31,6% vs. 28,8% ) before seeking help from a doctor , in contrast to those who came after " up to one week " ( 11.6% vs. 7.5% ) or " within 2 to 4 weeks " ( 27.5% vs. 29.4% ) .
individuals without bad who tended to be younger ( table 5 ) , decided significantly more often to see their doctor immediately when symptoms appeared as compared to patients with bad who tended to be older .
the key to diagnoses of anorectal diseases remains the patient history , with confirmation by visual inspection , anoscopy , and rectoscopy 1,5,10,13,16 .
so far , diagnostics often exclude more serious causes of anal bleeding such as colorectal cancer 6,7 since patients with anal complaints but without colorectal cancer are neglected .
anal bleeding , anal itch , anal pain or burning rank among the most common symptoms of anal diseases seen in primary care practices 10,13,25,26,27,28 .
the utility of different examination positions for determination of the causes of anal symptoms is unknown 1,2,4,6,7,14,27 .
the knee - chest position may provide a better field of view than broadly used left lateral sims ' position , as the buttocks fall to each side , and finger tips of both hands of the investigator are free for gentle eversion of the anal skin with the help of a good lighting 5,14 .
a fundamental drawback might be that haemorrhoids could be found less frequently with the knee - chest position because of the sloping position of the patient : the large intestine is pulled down towards the patients ' head so that the haemorrhoids are unable to protrude .
the left lateral sims ' position is more comfortable and patients achieve it easily and quickly by themselves ; thus the investigating physician saves time by not having to position the patient .
proctologic patients often receive conflicting opinions from clinicians 3,13,14,24,26,27 since with different specialists , the labelling changes for various disorders .
as noted by alexander - williams 26 " perianal dermatitis is an umbrella term " .
pruritus ani was the second most frequent bad after haemorrhoids in our study ( table 6 ) , possibly because we used the knee - chest position with its clear view of the anal verge 14 .
our definitions of perianal dermatitis / pruritus ani stages are based on those reported in the literature 5,17,18,19,22,23 and are descriptive only , avoiding causative suggestions 3,14,24 .
the four stages illustrate transformation of the anal skin along a time course ( figures 6,7,14 ) from acute to chronic , according to our experience 3,14,24 , and those of others 10,17,18,22 .
stage 1 , defined as pain during palpation of the anal canal and/or anoscopy , is a well known phenomenon not always considered relevant when the physician 's finger or the anoscope touches the exquisitely sensitive squamous epithelium distal to the dentate line 6 .
mal presented in almost one third ( 31.2% ) of our patients with bad ( table 6 ) .
this is similar to other reports describing patients with three , four or five separate causes of anal itching 17,18,29 .
thus until all causes of patients complaints have been eliminated , the patients are unlikely to experience relief of symptoms 5,29 .
however since we found that patients with bad have highly significant more caf than those patients without caf ( p < 0,01 ) , it is possible that caf may play a role in the pathogenesis of bad ( table 8) . published symptoms of haemorrhoids are bleeding , prolapsing tissue , mucosal or faecal soiling , fullness after defecation , itching and pain 6,7 .
haemorrhoids themselves can not be painful or itchy , since there are no sensory nerve fibres above the dentate line where haemorrhoids are derived 3,6,7,13,24 . therefore it is understandable that our patients with mal differed in their spectra of symptoms compared to patients with only one bad .
the spectra of symptoms in these patients suggest that they have more than one bad to diagnose and to treat 3,12,24,29 .
interestingly our patients with bad and without bad did not differ much concerning their symptoms , with the exception of specific anal complaints like anal soreness ( p<0.05 ) , and anal weeping ( p<0.05 ) , both of which are suggestive of pruritus ani ( table 9 ) .
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background : the frequencies and types of anal symptoms were compared with the frequencies and types of benign anal diseases ( bad).methods : patients transferred from gps , physicians or gynaecologists for anal and/or abdominal complaints / signs were enrolled and asked to complete a questionnaire about their symptoms .
proctologic assessment was performed in the knee - chest position .
definitions of bad were tested in a two year pilot study .
findings were entered into a pc immediately after the assessment of each individual.results : eight hundred seven individuals , 539 ( 66.8% ) with and 268 without bad were analysed .
almost one third ( 31.2% ) of patients with bad had more than one bad .
concomitant anal findings such as skin tags were more frequently seen in patients with than without bad ( < 0.01 ) .
after haemorrhoids ( 401 patients ) , pruritus ani ( 317 patients ) was the second most frequently found bad .
the distribution of stages in 317 pruritus ani patients was : mild ( 91 ) , moderate ( 178 ) , severe ( 29 ) , and chronic ( 19 ) .
anal symptoms in patients with bad included : bleeding ( 58.6% ) , itch ( 53.7% ) , pain ( 33.7% ) , burning ( 32.9% ) , and soreness ( 26.6% ) .
anal lesions could be predicted according to patients ' answers in the questionnaire : haemorrhoids by anal bleeding ( p=0.032 ) , weeping ( p=0.017 ) , and non - existence of anal pain ( p=0.005 ) ; anal fissures by anal pain ( p=0.001 ) and anal bleeding ( p=0.006 ) ; pruritus ani by anal pain ( p=0.001 ) , itching ( p=0.001 ) , and soreness ( p=0.006).conclusions : the knee - chest position may allow for the accumulation of more detailed information about bad than the left lateral sims ' position , thus enabling physicians to make more reliable anal diagnoses and provide better differentiated therapies .
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the number of individuals who visit hospital emergency rooms ( ers ) due to oral and maxillofacial lacerations continues to increase with the diversification of industry , the exposure of individuals to expanded human activities , and an increasing number of traffic accidents1 .
the oral and maxillofacial regions are exposed outwardly , and therefore , carry a high risk of trauma .
if oral and maxillofacial lacerations are not managed adequately and immediately , they may lead not only to functional or esthetic disability , but also , at times , to more serious health concerns .
the types of oral and maxillofacial trauma can be classified into facial bone fractures , dento - alveolar trauma , and soft tissue injuries .
most related studies have been limited to hard tissue trauma , such as facial bone fracture and dento - alveolar trauma2 .
in contrast , soft tissue injuries , like facial lacerations , have neglected their studies3 . however , laceration is a frequent clinical problem arising from oral and maxillofacial trauma that brings patients to the er . according to a study by singer et al.4 , nearly 28% of all patients visiting the er with lacerations had facial lacerations .
the facial area is different from other body areas , as it has rich blood supply , which reduces the risk of postoperative infection .
however , due to the possibility of scarring , oral and maxillofacial lacerations can cause not only aesthetic problems , but also psychological issues5 .
therefore , oral and maxillofacial laceration treatment requires prompt and accurate evaluation , depending on the location and extent of the damaged area .
a study on the frequency and causes of facial laceration would allow for a better understanding of the types of lacerations and their development , and would enable faster and more accurate diagnosis . this study included oral and maxillofacial laceration patients who visited the er and investigate laceration types and trends to ensure effective treatment of future patients with facial lacerations .
this study included 1,742 patients with oral and maxillofacial lacerations with 2,014 different laceration locations , for which they visited the er of pusan national university hospital ( busan , korea ) over three years , from january 2011 to december 2013 . with the subjects ' basic reference data accessed from their clinical charts and radiographs , patients were classified by sex , age , visit day , etiology of injury , injury site , and the presence or absence of soft tissue and tooth damage .
exclusion criteria included patients with a scalp laceration , and those who returned home or passed away without treatment .
this study was approved by the institutional review board of pusan national university hospital ( pnuh e-2015045 ) .
a total of 1,742 patients with oral and maxillofacial lacerations visited the er of pusan national university hospital over three years . each year , the number of male and female patients gradually increased .
the ratio of male to female patients was 2.73:1 in 2011 , and 2.27:1 in 2013 , a slight decrease . across time , the total male to female ratio was 2.50:1 ; 1,244 of the 1,742 patients were male and 498 patients were female.(table 1 ) patients under 10 years old were most frequently seen , comprising 20.7% of all patients , followed by those in their 40s at 14.8% and individuals in their 50s at 14.7% .
individuals over 80 years old were the least frequently seen , comprising 2.0% of all patients.(fig .
1 ) the distribution of patient visit days was as follows : the most patients sought treatment on saturdays ( 21.0% ) , followed by sundays ( 20.2% ) .
wednesdays received the fewest number of visits by individuals with oral and maxillofacial lacerations ( 10.8%).(fig .
2 ) the distribution by trauma etiology showed that slip down was most common source of injury ( 43.9% ) , followed by collisions ( 19.7% ) and traffic accident ( 15.2% ) , while sports trauma was the least common etiology ( 5.1%).(fig .
3 ) during the study period , 817 patients had intra - oral lacerations exclusively , a number slightly higher than the 768 patients with extra - oral lacerations exclusively ; 157 patients had complex intra - oral and extra - oral lacerations.(fig .
4 ) complex laceration represents a case in which intra - oral and extra - oral lacerations occur simultaneously in a patient , and includes a penetrating wound .
when the intra - oral laceration locations were further broken down , the lips were the most common laceration location , found in 721 patients . among the extra - oral laceration cases ,
5 ) a total of 206 patients had a laceration accompanied by facial bone fractures , 250 experienced tooth injuries , and 57 patients experienced both .
a total of 1,742 patients with oral and maxillofacial lacerations visited the er of pusan national university hospital over three years . each year , the number of male and female patients gradually increased .
the ratio of male to female patients was 2.73:1 in 2011 , and 2.27:1 in 2013 , a slight decrease . across time , the total male to female ratio was 2.50:1 ; 1,244 of the 1,742 patients were male and 498 patients were female.(table 1 )
patients under 10 years old were most frequently seen , comprising 20.7% of all patients , followed by those in their 40s at 14.8% and individuals in their 50s at 14.7% .
individuals over 80 years old were the least frequently seen , comprising 2.0% of all patients.(fig .
the distribution of patient visit days was as follows : the most patients sought treatment on saturdays ( 21.0% ) , followed by sundays ( 20.2% ) .
wednesdays received the fewest number of visits by individuals with oral and maxillofacial lacerations ( 10.8%).(fig .
the distribution by trauma etiology showed that slip down was most common source of injury ( 43.9% ) , followed by collisions ( 19.7% ) and traffic accident ( 15.2% ) , while sports trauma was the least common etiology ( 5.1%).(fig .
during the study period , 817 patients had intra - oral lacerations exclusively , a number slightly higher than the 768 patients with extra - oral lacerations exclusively ; 157 patients had complex intra - oral and extra - oral lacerations.(fig .
4 ) complex laceration represents a case in which intra - oral and extra - oral lacerations occur simultaneously in a patient , and includes a penetrating wound .
when the intra - oral laceration locations were further broken down , the lips were the most common laceration location , found in 721 patients . among the extra - oral laceration cases ,
a total of 206 patients had a laceration accompanied by facial bone fractures , 250 experienced tooth injuries , and 57 patients experienced both .
this study investigated the relationship of oral and maxillofacial lacerations to age , etiology , distribution , and other related locations among 1,742 individuals who visited the er over a three - year period . a previous study noted that oral and maxillofacial area lacerations were the primary cause of er visits .
kim et al.6 noted that soft tissue damage , tooth damage , facial bone damage , tooth pain , bleeding , infection , and temporomandibular joint luxation were the sequential causes of er visits .
cho et al.7 reported the following were the sequential causes of er visits : oral and maxillofacial area laceration , facial bone fracture , tooth damage , abscess , and bleeding . according to a study report by jang et al.8 , laceration with soft tissue damage was a common cause for er visits , comprising 71% of er visits
compared to facial bone fractures related to the study of kim et al.9 , underwent the same period , in the sex distribution of oral and maxillofacial laceration , the male to female ratio was 2.50:1 , with 1,244 male subjects and 498 female subjects . over three years
, the male - to - female ratio decreased , due to the increase in the number of female patients . in the case of fracture patients ,
the male - to - female ratio was 4.31:1 , higher than the overall 2.50 .
it appears that men are exposed to more external injury than women , as fractures are caused by a greater external force than are lacerations .
slip down caused 43.9% of the lacerations and 33.7% of the fractures , the most common etiology for both injuries .
most of the laceration patients ( 20.7% ) were under 10 years old , and most of the fracture patients ( 22.2% ) were in their twenties .
the lacerations among those under 10 years old were often light injuries caused by parents or by the patients ' own carelessness . in a previous study by kim et al.10 exploring er visits for soft tissue injuries ,
patients under 10 years of age visited the er most frequently for such procedures , comprising 41.8% of patients seen ; patients in their 20s comprised 11.6% of the population , and patients in their 30s , 11.1% .
as for the causes of the injuries , 47.7% were from fall down , 19.1% were slip down , 9.4% were fist - related traumas , and 9.2% were traffic accidents .
likewise , jang et al.8 reported that most of the patients ( 56.6% ) were under 10 years of age ; 14.5% were between 10 and 20 years old , and 12.9% were in their 20s .
as for the causes of the lacerations , most of the patients ( 48.8% ) were slip down , and 25.5% were fist trauma . in this study ,
the age distribution of the etiology of injury and the highest frequency of injuries were among those under 10 year old was consistent with other studies ; those over 40 years old had a higher injury rate than in other studies .
this higher injury rate among those over 40 was attributed to the high frequency of slip down and collisions , as well environmental issues brought on by the large entertainment district near the hospital .
carelessness after drinking in the nearby entertainment district was believed to have resulted in more frequent injuries . in this study ,
47% of the lacerations were solely intraoral lacerations , and 44% were solely extra - oral lacerations ; only 9% were complex ( intra - oral and extra - oral ) lacerations . in the study by kim et al.10 ,
66.7% of the cases were intra - oral lacerations , 10.8% were extra - oral lacerations , and 19.4% , complex lacerations . from 1992 to 1996 ,
a study that examined individuals who visited the er of same hospital revealed that 42% of visits were for intra - oral lacerations with soft tissue damage , 17.8% for extra - oral lacerations , and 34.1% for complex lacerations .
these figures show a higher frequency of intra - oral lacerations than in the current study6 .
this discrepancy is thought to originate from differences in emergency department care systems and cooperation among other departments for maxillofacial trauma areas . in the aforementioned study ,
the extra - oral lacerations occurred mostly in the upper third of the oral and maxillofacial area and were affected by the protrusion of the forehead and the eyebrows1112 .
researchers found 221,190 lacerations on the forehead and eyebrows , and these lacerations showed a pattern similar to that in previous studies .
they also found a high frequency of injuries ( 260 cases ) in the chin area , which is in the lower third of oral and maxillofacial area .
these differences came from possibility of common location damages in the chin area in relation to mandible fracture and tooth trauma so it is thought that the lower third of laceration area works is entrusted to the department of oral and maxillofacial surgery .
this figure is considered very high , much higher than the 260 laceration cases in the chin area , where extra - oral lacerations occur most frequently .
because the lip area is a protruding structure and is near hard structures ( i.e. , teeth ) , it carries a high risk of injury and is the most easily perceived area of intra - oral lacerations at the time of the initial diagnosis in the er . understanding the causes , severity , and distribution of oral and maxillofacial trauma is known to be of great help in generating effective precautionary measures and treatments13 .
most previous er - related research focused on facial bone fracture patients or general patients who visited the er . in particular , research on soft tissue damage was very briefly mentioned or excluded from studies .
this study focused exclusively on oral and maxillofacial lacerations , which occur most frequently with soft tissue damage and could cause permanent complications .
it examined oral and maxillofacial lacerations in the context of diverse relevant factors . in this study , patients who were treated only in the department of oral and maxillofacial surgery were included , a potential limitation .
if a study focused on oral and maxillofacial laceration progress , including collaboration with other departments , it would be more comprehensive . in this study , because the medical chart information was not entered by a facial trauma specialist , some information , such as the size and depth of the laceration , was not included .
most lacerations patients were not hospitalized and did not receive regular follow up after stitches were removed .
thus , we were unable to include the frequency of postoperative complications , such as infection and scar formation
. therefore , it will be necessary to collect long - term data on oral and maxillofacial laceration patients . a future study on lacerations
should be performed to validate the results of this study , which would be of great help in the evaluation and treatment of oral and maxillofacial laceration patients seen in the er .
this study included 1,742 patients with oral and maxillofacial lacerations who visited the er of pusan national university hospital over three years , from january 2011 to december 2013 .
the overall male to female ratio was 2.50:1 ; 1,244 of the 1,742 patients were male and 498 were female .
most of the patients were under 10 years old , and most of the patients ' visits were on weekends .
most of the lacerations were caused by slip down , followed by collisions and traffic accidents , in that order ; a majority of the lacerations were either exclusively intra - oral or exclusively extra - oral lacerations rather than complex lacerations . among intra - oral lacerations ,
the lip area was the most vulnerable site ; among extra - oral lacerations , the chin area was more commonly injured .
therefore , it is necessary to continue collecting data from oral and maxillofacial laceration patients over time to establish optimal diagnosis and treatment strategies for oral and maxillofacial laceration patients who visit the er .
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objectivesthis study investigated patients with oral and maxillofacial lacerations who visited the emergency room over a three - year period in an effort to determine the optimal treatment for these injuries.materials and methodsthis study examined 1,742 patients with oral and maxillofacial lacerations with 2,014 different laceration locations who visited the emergency room of pusan national university hospital ( busan , korea ) over three years , from january 2011 to december 2013 .
patients were classified by sex , age , visit day , cause of injury , injury site , and the presence or absence of soft tissue and tooth injuries.resultsthe male to female ratio was 2.50:1 .
patients under 10 years old were seen most frequently .
most emergency room visits were on weekends . among intra - oral lacerations ,
the lip area was the most vulnerable site ; among extra - oral lacerations , the chin area was most frequently injured .
the most frequent etiology was a slip down .
most lacerations occurred without bone fracture or tooth damage.conclusionlaceration may differ in large part as compared with the fracture .
therefore , it is necessary to continue collecting data on oral and maxillofacial lacerations to establish optimal emergency room diagnosis and treatment strategies .
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the present paper is not a comprehensive review but it was written to illustrate findings that show the dynamic state of skeletal muscle fibers .
early studies on rabbit soleus muscle had shown that the maintenance of phenotypic properties depended on activity .
the application of this technique provided an opportunity to study the phenotypic adaptability of the terminally differentiated muscle fibre .
the purpose of this article is not to provide a review of all the observations made since but rather to show the evolution of ideas that have contributed to the notion of muscle plasticity .
the term plasticity of muscle has been coined during the preparation of an international symposium held at the university of konstanz in september 1979 .
the topics that were treated included the heterogeneity of muscle as a tissue , the programme of its ontogeny and differentiation , and the regulation of its properties by innervation , use , hormones and modulation of neural input .
the state of the art on these subjects was such that the time was ripe to bring together scientists from various disciplines to discuss these issues .
it has been known for a long time that mammalian muscle fibres are not all the same but differ in their various properties . the problem of understanding the significance of this diversity for the organization of motor control had been highlighted in the last century by studies of henneman and his colleagues who discovered that motoneurones supplying particular types of muscle fibres are specialized . those motoneurones that supply slow and fatigue resistant muscle fibres have a lower threshold and are more active , whereas motoneurones to muscle fibres that contract and relax fast have a high threshold and are therefore less active .
the relationship between the motoneurone and muscle fibres it supplies was most clearly illustrated by eccles and co - workers in the early 60th by their cross reinnervation experiments .
the mechanism as to how motoneurones control muscle properties was not clear at that time but has been attributed to a special trophic influence of the motor nerve on muscle .
an alternative proposal that could explain these observations , was that re - innervating the muscle with an alien nerve , had altered its activity pattern .
this interpretation was supported by tenotomy experiments on rabbit soleus muscle which changed the activity pattern of the muscle without altering its innervation .
returning the continuous activity of the soleus nerve to the silenced tenotomised muscle by electrical stimulation restored the slow time course of contraction .
it was concluded from these experiments that the continuous impulse activity normally present in postural soleus muscle , is responsible for the slow time course of contraction and relaxation of this muscle . due to the initiative of ernest gutmann and sidney hilton , gerta and dirk met at this stage , and decided to join forces to elucidate this programme .
the data reported here is mainly derived from that collaboration and should not be considered a comprehensive review as most of this has been covered by several reviews .
earlier studies on energy metabolism in functionally distinct muscles had led to the definition of specific and constant proportions in the enzyme activity patterns .
these findings supported the notion that energy supply is adjusted to specific functional demands of different muscles .
the analysis of enzyme activity patterns , therefore , represented an appropriate tool to investigate metabolic specialization of muscles and other tissues .
the application of this approach to cross re - innervated muscles in rabbit showed pronounced changes in the enzyme patterns matching the induced functional changes .
similar metabolic changes were described in cross re - innervated muscles of cat and rat . nevertheless , it was clear that these experiments did not provide an answer that distinguished between the hypotheses of trophic factors or activity regulating muscle properties .
the development of new implantable materials then allowed examining the hypothesis that activity is responsible for determining muscle properties by means of the chronic stimulation experiment .
electrodes were implanted either side of the peroneal nerve in one leg of anesthetized rabbits .
the electrodes were then connected to a stimulating device and impulses of supramaximal intensity at 10 hz were delivered to the nerve .
contractile properties of the stimulated tibialis anterior ( ta ) and extensor digitorum longus ( edl ) muscles were examined at various time intervals using conventional physiological methods . within two to four weeks
at this stage gerta and dirk started their collaboration and together they examined enzyme profiles of stimulated and control muscles . in these studies , a profound re - arrangement of enzyme activity and isozyme patterns confirmed that prolonged slow frequency ( 10 hz ) activity imposed on fast muscles that were normally getting their energy from breakdown of glycolytic pathways , induced their transformation towards a slow - oxidative muscle . at that time ,
the interpretation of our results was not readily accepted by the scientific community that favoured the idea of trophic factors released from the nerve being responsible for determining muscle properties even at that time this view was challenged by a.f .
huxley who suggested that the slow frequency activity of slow motoneurones induces a vibratory stress to fast muscles that elicits the change from fast to slow phenotype ( l.c .
the dramatic transformation of adult differentiated fast glycolytic muscle fibres into slow oxidative ones by altered activity inspired us to use this experimental model to study not only the potential of this tissue to adjust to activity but also to unravel the mechanisms by which such changes are induced .
in addition to establishing the physiological importance of activity as a decisive factor in determining muscle properties , chronic nerve stimulation of fast muscle at low frequency provided an excellent model for studying the time course of activity - induced changes .
in other words , the stimulation experiment allowed us to follow changes of cellular re - organization in a differentiated post - mitotic tissue during its transition from a relatively stable to an altered state induced by new functional demands .
it also enabled us to investigate both , the hierarchy and the sequence of changes at different levels of cellular organization and , establish the full range of adaptation during the transition from a fast to a slow muscle , and finally propose a mechanism that initiated this change .
another advantage of the stimulation experiment was that all motor units of the target muscle were simultaneously activated by the same pattern of activity .
systemic influences such as stress , changes in hormone homeostasis , etc . , would thus equally affect stimulated and contralateral control muscles .
analyses of time - dependent changes of chronic low - frequency stimulation ( clfs ) , demanded standardization of the experimental set - up so as to create reproducible conditions regarding patterns , intensity and duration of activity transmitted to the target muscle .
this was achieved by developing a method of tele - stimulation . in the following text ,
effects of clfs on selected properties of muscle fibres in response to this new type of activity are described .
apart from functional tests , our studies included energy metabolism , contractile and regulatory proteins of the myofibrillar apparatus and the sarcoplasmic reticulum .
these objects were chosen in the hope to elucidate both the hierarchy and sequence of changes that would help to explain the mechanisms underlying the adaptive potential of the muscle .
the metabolic transformation of rabbit fast - twitch muscles ( edl , ta ) exposed to low - frequency stimulation ( clfs ) was characterized by increases of enzyme activities involved in aerobic - oxidative metabolism and simultaneous decreases in glycogenolytic and glycolytic metabolic capacities .
twenty eight days after the onset of stimulation glycogenolytic and glycolytic capacities decreased to ~40% of their control values , while there was an almost 8-fold increase in mitochondrial volume density .
the relative activities of soluble and membrane - bound enzymes of aerobic oxidative metabolism ( the citric acid cycle , fatty acid oxidation ) remained unaltered although their absolute activity levels increased by 5 - 8-fold .
an even greater increase was seen in - hydroxybutyrate dehydrogenase , a structure - bound mitochondrial enzyme .
in contrast , glycerolphosphate oxidase , another mitochondrial structure - bound enzyme representing the mitochondrial part of the glycerolphosphate shuttle , markedly decreased and paralleled the changes in glycolytic capacity .
these results taken together , suggest that not only was there an increase in mitochondria but also a change of their composition .
the study of the time course of this increase compared to that of succinate dehydrogenase ( a marker for mitochondria ) revealed that the change in capillary density preceded that in aerobic - oxidative capacity . an improved oxygen supply provided by the capillaries
the altered time course of contraction after clfs had indicated a change in the contractile machinery of the muscle .
stimulated muscles displayed a greater proportion of fibres histochemically classified as slow ( type i ) .
subsequent studies showed alterations in the composition of myofibrillar proteins in whole muscle and in single fibre extracts .
the myosin isoform patterns , both at the levels of light ( mlc ) and heavy ( mhc ) chains were shifted towards slower isoforms .
the time course of these changes in rabbit muscle suggested a sequential exchange of fast with slow isoforms in the order of mhciid , mhciia , mhci . this sequence was confirmed in single fibre studies that also showed appearance of hybrid fibre types co - expressing several mlc and mhc isoforms .
expression patterns of mhc isoforms , both at the levels of mrna and protein in single fibres at different stages of transformation displayed a greater variability at the mrna than that at the protein level .
the coexistence of different mrna isoforms in the same fibre was not always seen at the protein level , indicating that the switch from transcription to translation is likely to be separately controlled .
studies of the time course of mhc isoform transitions performed on rat muscle provided similar results . however in rats , the fastest fibres in edl and ta muscles are type iib which contain mhciib . when exposed to clfs
considering the results from both , rat and rabbit muscle , the fast - to - slow transition isoforms occurs in an orderly manner so that mhciib - > mhciid - > mhciia - > mhci . during these fibre transitions , isoforms
initially present in the fibre were replaced by newly synthesised isoforms which resulted in the transitory appearance of hybrid fibres containing more than one mhc isoform .
as documented by single fibre analysis , the complete sequence of fast - to - slow transitions was as follows : mhciib - > mhciib / iid - > mhciid - > mhciid / iia - > mhciia - > mhciia / i - > mhci .
depending on the initial phenotype of the fibre , the clfs - induced fast - to - slow transitions differ between fibres .
therefore , discontinuing stimulation presents the possibility for the study of slow - to - fast transitions . as illustrated in studies on rat muscle ,
the corresponding half - lives amounted to approximately 3 days for the mrna and 11 d for the protein which indicated a rapid re - modelling of the myofibrillar apparatus in response to increased activity .
newly synthetized mhciid and mhciia replacing mhciib , were traced in rat ta muscle by measuring protein synthesis by s - methionine incorporation .
increases in synthesis of mhciid and mhciia occurred as soon as two days after stimulation onset .
this data supported our notion that changes in mhc isoform expression are early processes in response to the altered activity
fast - to - slow transformation included also exchanges of fast with slow isoforms of the regulatory proteins at the level of the thin filament . due to the greater number of isoforms , especially in the case of troponin with three fast and two slow isoforms ,
the fast - to - slow transitions proved to be more complex . nevertheless , the time course studies showed that the shift from fast to slow occurred in a manner coordinated with that of the mhc isoforms .
the major changes in the protein profile of the sarcoplasmic reticulum ( sr ) consisted of an exchange of the fast serca1 with the slow serca2 caatpase isoform .
this exchange paralleled the fast - to - slow transition at the stage from mhciia to mhci .
the switch in serca isoforms was preceded by a partial ( 40 - 50% ) inactivation of the atpase activity due to protein oxidation and nitration of tyrosyl groups of the enzyme . both processes
in addition to these alterations of ca - atpase , other proteins were also affected .
these included pronounced decreases ( ~60% ) in the levels of ryanodine receptor , dihydropyridine receptor , sarcalumenin , and triadin .
parvalbumin , a cytosolic ca - binding protein present only in fast - twitch fibres , was decreased rapidly during clfs .
its decay in rabbit muscle paralleled the first step ( mhciid to mhciia ) of the fast - to - slow transition in mhc isoforms .
these early changes of proteins involved in ca - uptake indicated alterations in intracellular ca - dynamics . these changes might induce alterations in gene expression related to ca - dependent signalling . indeed , measurements of ca transients in single fibres isolated from rat edl muscle exposed to clfs showed an instantaneous ~2.5-fold increase in resting ca which was maintained during the entire period of 10 days of stimulation .
this value was in the range characteristic of slow - twitch fibres from soleus muscle .
there was also a transitory increase of the integral ca per pulse with a maximum ( ~5-fold ) after 1 day .
steep decreases in the rate constant of ca - decay could be explained by an immediate impairment of the ca - uptake and by an additional loss of ca - binding capacity , most likely due to the decay in parvalbumin .
it has been shown that the expression of specific protein patterns in distinct muscle fibre types is under the control of ca - dependent signalling .
sustained increases of resting ca levels have been shown to activate transcription of slow fibre - specific proteins mediated by a calcineurin - dependent pathway .
an impairment of ca - uptake in the stimulated muscle by the early partial inactivation of the sr ca - atpase , and the decrease in cytosolic ca - binding capacity ( parvalbumin ) are likely to be responsible for the increased levels of resting ca and the activation of molecules controlling the slow phenotype .
the impairment of ca - uptake that preceded the serca1 to serca2 transition , may , however , not only result from the above mentioned mechanisms but may also be due to a compromised energy state of the stimulated muscle fibre .
in fact , measurements of the atp phosphorylation potential [ atp / adpfree ] in whole muscle and single fibres revealed an almost instantaneous drop after stimulation onset .
the atp phosphorylation potential remained for up to 50 days of stimulation at ~50% of that in unstimulated muscle .
the drop in the phosphorylation potential was accompanied by an almost instantaneous decrease in force production which was reduced to 28% of the control immediately after the onset of stimulation and gradually recovered during the following 10 days .
the recovery of force was most likely brought about by the metabolic transformation , i.e. the improved energy supply via aerobic - oxidative pathways .
consistent with this is the finding that lowering the cellular phosphorylation potential by other means , such as by administration of a creatine analogue , has also been shown to shift myosin isoform expression towards the slow phenotype , but these changes were less complete than those brought about by clfs .
the instantaneous effects of clfs thus affect at least two major elementary functions of the muscle fibre , its energy state and its ca homeostasis
. these may be the initial signals effecting signalling pathways controlling the expression of phenotypic properties in various compartments of the muscle fibre . in summary ,
these and other results of the multiple effects of clfs demonstrate the value of this experimental set - up for studying muscle plasticity and its regulatory mechanisms . in our opinion , this model has not been fully exploited and offers unique possibilities for studies of regulatory processes underlying muscle plasticity .
results of experiments on mutant mice suffering from severe muscle weakness ( c7bl dy/2j / dy2j ) , show that a particular pattern of activity imposed upon their muscles by stimulation of their nerve reduces the rate of deterioration of their muscles .
these mutant mice have a defect of laminin alpha 2 ( lama 2 ) gene .
clfs of their leg muscles lead to a reduction of the rate of loss of force and muscle bulk that is typical in these animals .
moreover , clfs reversed the changes in enzyme activities caused by the disease in these dystrophic mice .
the encouraging results obtained on animal models led to attempts to investigate the effects of clfs in patients suffering from duchenne muscular dystrophy .
consistent with the results on mice , the results showed that electrical stimulation reduces the rate of deterioration of ankle dorsi - flexors and quadriceps muscles , provided the stimulation was started before the muscles became excessively weak .
the frequency at which the muscles were stimulated was of critical importance for only stimulation at 10 hz or less had a beneficial effect , while frequencies of 30 hz or more enhanced the deterioration of the muscles . with the development of methods that attempt treatment of muscle diseases by trying to correct the genetic fault by exon skipping ( duchenne muscular dystrophy ) or by interfering with the splicing mechanism of the cell by antisense oligonucleotides ( sma )
it may be beneficial to induce prior to treatment a situation where the affected muscle is already undergoing changes in gene expression by clfs - induced alterations in transcriptional and/or epigenetic regulation .
finally , other effects of electrical stimulation such as increases of capillary density and blood flow , may allow better access of any substance to target muscles .
an additional change contributing to an altered responsiveness of the muscle , might relate to the increase in satellite cell activation under influence of low - frequency stimulation .
these few suggestions point out the possibilities of using stimulation induced changes either on their own or in combination with other therapeutic interventions for treatment of neuromuscular disorders .
it is surprising that these approaches have so far not been used , particularly in view of extensive evidence that electrical stimulation of human muscle is efficient as judged by biochemical and functional criteria , and has recently been so widely and successfully used for rehabilitation of various conditions .
these include procedures used for the maintenance of muscle function after spinal cord injury , denervation , and aging .
these important results also provided evidence that in human muscles electrical stimulation was able to reverse many of the degenerative changes that follow denervation .
as a consequence of these beneficial effects patients were able to use their muscles during locomotion and counteract the effects of aging and long term inactivity .
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studies carried out during the past 45 years on the effects of chronic low - frequency stimulation on skeletal muscle have revealed a multiplicity of adaptive changes of muscle fibres in response to increased activity .
as reflected by induced changes in the metabolic properties , protein profiles of the contractile machinery and elements of the ca2 + -regulatory system , all essential components of the muscle fibre undergo pronounced changes in their properties that ultimately lead to their reversible transformation from fast - to - slow phenotype .
the chronic low - frequency stimulation experiment thus allows exploring many aspects of the plasticity of mammalian skeletal muscle .
moreover it offers the possibility of elucidating molecular mechanisms that remodel phenotypic properties of a differentiated post - mitotic cell during adaptation to altered functional demands .
the understanding of the adaptive potential of muscle can be taken advantage of for repairing muscle damage in various muscle diseases .
in addition it can be used to prevent muscle wasting during inactivity and aging .
indeed , pioneering studies are still the sound grounds for the many current applications of functional electrical stimulation and for the related research activities that are still proposed and funded .
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kaposi s sarcoma ( ks ) was first described in the 1800s as a rare , fairly indolent tumor of specific populations .
this form of ks , now referred to as classic ks , usually presents on the skin of the lower extremities of elderly men of specific european regions and religious origins . in the middle of
the twentieth century ks became endemic in parts of africa and is currently one of the most common tumors in parts of central africa ( wabinga et al .
in the late twentieth century ks became one of the first aids defining illnesses and is the most common tumor of aids patients world - wide .
aids associated ks is generally far more aggressive than classic ks , arising on the skin in many parts of the body as well as in the oral cavity and can occur on internal organs where it is often fatal .
while they differ in aggressiveness , all forms of ks are relatively indistinguishable at the histological level . grossly , the tumors have a characteristic red to purple hue , indicative of the high vascularization of the tumor .
histologically , the tumor exhibits large vascular slits lined by flattened endothelium ; the slits are often , but not always , filled with blood cells
. there are discernable levels of extravasated red blood cells and infiltrating lymphocytes in the tumor . while a number of cell types are present , the tumor is predominantly made up of elongated spindle cells .
the spindle cells express endothelial cell markers on their surface including cd31 and cd34 , but express low levels of factor viiira ( russell jones et al . ,
recent expression data and array studies have found that spindle cells most closely resemble lymphatic endothelium , expressing vegf receptor 3 ( vegfr3 ) , a specific marker of lymphatic endothelial cells ( jussila et al . , 1998 ; skobe et al . , 1999
other lymphatic endothelial cell specific markers , including lyve-1 , podoplanin , and prox-1 , are also expressed by the spindle cells ( carroll et al .
based on the epidemiology and the multicentric nature of the tumor , ks was predicted to have an infectious cause ( beral et al . ,
, kshv was discovered associated with all ks tumors and is now considered to be the etiologic agent ( chang et al . , 1994 ) .
kshv was the eighth human herpesvirus discovered and is sub - classified as a gamma herpesvirus .
all of the greater than 90 viral genes are expressed , the virus replicates rapidly , produces infectious virions and ultimately causes cell death likely due to a combination of host cell shut off and virus production . during viral latency in endothelial cells ,
this locus includes lana , vcyc , vflip , a family of proteins from a repeat region called the kaposins , and 12 pre - micrornas encoding 17 or more mature mirnas . these latent genes
are responsible for the maintenance of the latent viral episome as well as the survival of latently infected cells . in later - stage ks tumors ,
, 1995 ; staskus et al . , 1997 ; dupin et al . , 1999
as expected the virus is predominantly found in the latent state in spindle cells where the limited number of latent genes and mirnas are expressed ( staskus et al . , 1997 ;
. however , approximately 15% of the spindle cells express lytic viral genes and produce infectious virus .
in addition to spindle cells , kshv is also found in other cell types in the ks lesion including monocytes ( blasig et al . ,
kshv can only sporadically be detected in the endothelium lining the vascular slits in the ks tumor ( dupin et al . , 1999 ) .
kshv is also associated with two lymphoproliferative diseases , primary effusion lymphoma ( pel ) a pleural cavity solid b - cell lymphoma , and plasmablastic multicentric castleman s disease , a lymph node b - cell growth ( cesarman et al .
because ks is an angioproliferative disease and the ks tumors are highly vascularized even at early stages , it has been proposed that kshv may directly induce angiogenesis .
endothelial cells of the vascular system are normally maintained in a quiescent , non - proliferating state .
however , during solid tumor formation , the secretion of pro - angiogenic cytokines by tumor cells can activate nearby endothelial cells to induce new blood vessel formation .
many of the vascular slits identified in histological sections of early stage ks lesions are lined by uninfected endothelium , suggesting they are formed by endothelial cells activated in a paracrine fashion ( dupin et al . , 1999 ) .
these uninfected cells may later become infected , as kshv has , in some cases , been detected in the cells surrounding the vascular spaces of later - stage nodular ks ( boshoff et al .
despite the evidence for paracrine activation of uninfected endothelial cells , kshv also likely activates infected endothelial cells in an autocrine or cell autonomous fashion .
because ks spindle cells are endothelial in origin , induction of the ks tumor cell is similar to the processes of angiogenesis .
many of the characteristics of activated endothelial cells and angiogenesis are also associated with oncogenesis , including proliferation , migration , and metalloprotease expression .
this review discusses the recent evidence that suggests that ( 1 ) kshv promotes continual neovascularization through paracrine factors and ( 2 ) kshv may drive tumor cell growth through autocrine and cell autonomous activation of angiogenic phenotypes .
the vascular endothelial growth factor ( vegf ) family of cytokines plays a prominent role in regulation of angiogenesis ( breen , 2007 ) .
vegf - a and its receptors are required for embryonic vascular development and are important for vascular permeability , proliferation , and survival of newly formed vasculature .
vegf - a expression is detected in spindle cells of ks lesions , and its secretion is known to be increased by inflammatory cytokines that are present in the ks lesions ( samaniego et al . , 1998 ) .
vegf - a is also expressed by kshv - infected pel cell lines and conditioned media from these cells is sufficient to induce capillary morphogenesis by endothelial cells ( liu et al .
, 2001 ; akula et al . , 2005 ; subramanian et al . , 2010 ) .
infection of endothelial cells with kshv directly leads to increased expression of vegf - a ( masood et al . ,
2002 ; sivakumar et al . , 2008 ; wang and damania , 2008 ) .
further , kshv conditioned media has been shown to regulate angiogenic phenotypes in endothelial cells ( sharma - walia et al . , 2010 ) .
therefore , kshv induction of vegf - a is likely to be critical for both the induction of angiogenesis as well as the activation of infected spindle cells .
although the mechanisms by which kshv induces vegf - a expression and secretion are still unclear , several potential pathways have been uncovered .
hypoxia induced factor ( hif)-1 is a transcription factor that has been shown to be important for upregulation of vegf - a ( sodhi et al . , 2000 ; shin et al . , 2008 ) .
however , during hypoxia , hif-1 is stabilized and can induce expression of genes through hypoxia responsive elements , including vegf - a .
interestingly , kshv infection of endothelial cells induces the expression of hif-1 during normoxia which leads to increased hif transcriptional activity ( carroll et al . , 2006 ) .
other studies have shown that kshv encodes proteins that can lead to increased stability of hif-1 .
the kshv latency associated nuclear protein ( lana-1 ) can stabilize hif-1 , through both degradation of its suppressors , von hippel lindau protein and p53 ( cai et al . , 2006 ) , and through direct interaction between hif-1 and lana-1 ( cai et al . , 2007 ) .
additionally , the virally encoded interferon response factor ( virf3 ) can , like lana-1 , interact with and stabilize hif-1 , leading to increased vegf - a expression ( shin et al . , 2008 ) .
the kshv viral g - protein coupled receptor ( vgpcr ) is able to increase the activity of hif-1 as a transcription factor through activation of the mapk and p38 signaling pathways and subsequent phosphorylation of hif-1 ( sodhi et al .
while induction of hif mrna expression by kshv infection has been shown , stabilization of hif directly by kshv infection of endothelial cells has yet to be clearly shown .
pro - angiogenic factors that are induced by kshv are indicated in red ovals , pro - lymphangiogenic factors induced by kshv are indicated in blue ovals .
white ovals indicate the activated pathway necessary for kshv induction of blood to lymphatic endothelial cell differentiation .
the upper left corner indicates phenotypes of angiogenesis that are activated in normal endothelium by kshv - infected cells and the gradient of cytokines secreted by kshv - infected cells is indicated by the red gradient .
the blue gradient in the upper right hand corner indicates the gradient of lymphangiogenic cytokines induced by kshv - infected cells that could induce lymphangiogenesis .
other host proteins have been shown to be involved in the induction of vegf - a during kshv infection of endothelial cells as well .
for example , emmprin is a membrane - associated glycoprotein that promotes matrix metalloproteinase expression .
its expression in kshv - infected cells promotes cell invasiveness through activation of the pi3k / akt and mapk pathways ( qin et al .
further work is ongoing in multiple labs to determine the cellular pathways essential for kshv induction of vegf - a .
several kshv genes expressed during lytic replication have been implicated in regulation of vegf - a expression ( table 1 ) . in bcbl-1 cells ( a pleural effusion lymphoma cell line ) , glycoprotein b ( gb ) and
k8.1 are required for enhanced vegf expression ( subramanian et al . , 2010 ) .
treatment of these cells with sirna or neutralizing antibodies to gb or k8.1 significantly reduced vegf - a production .
vgpcr is a constitutively active signaling receptor that has been linked to a variety of cell survival and pro - angiogenic signaling pathways ( arvanitakis et al .
when injected into mice , nih3t3 cells expressing vgpcr form highly vascularized tumors with some similarities to ks and this may be due , at least in part , to increased vegf - a secretion ( bais et al . , 1998 ; guo et al .
vgpcr upregulates vegf - a through activation of mapk and p38 , which , as described above , promotes hif-1 activity ( sodhi et al . , 2000 ) .
transgenic mice expressing vgpcr also form highly vascularized tumors that are reminiscent of ks tumors .
however , cell lines derived from these tumors expressed the lymphatic growth factor vegf - c , rather than vegf - a ( guo et al . , 2003 )
. increased vegf - a expression in cells expressing vgpcr is associated with constitutive activation of its receptor , vegfr2/kdr and downstream activation of pi3k / akt , contributing to endothelial cell survival ( montaner et al . , 2001 ; bais et al . , 2003 ) .
however , gb , k8.1 , and vgpcr have only been detected in cells supporting lytic kshv infection whereas the bulk of the tumor cells are latently infected .
the kshv glycoprotein k1 also induces increased vegf - a expression in endothelial cells and is capable of immortalizing primary endothelial cells ( wang et al .
while there is evidence that k1 is expressed at very low levels during latency , the majority of its expression occurs during lytic infection ( chandriani and ganem , 2010 ) . in summary ,
the lytic phase of kshv infection might play a role in the paracrine induction of angiogenesis through increased secretion of vegf - a into the tumor milieu .
in addition to vegf - a , kshv - infected endothelial cells also express other angiogenic cytokines . angiopoietin-1 and -2 are ligands for the receptor tyrosine kinase tie2 .
although less is known about the functions of angiopoietins and tie2 , their signaling is required for proper vascular development during embryogenesis ( dumont et al . , 1994 ) .
angiopoietin-1 is an agonist for the tie2 receptor , promoting endothelial cell survival and stability .
in contrast , ang2 acts as an antagonist for tie2 , although its role is context - dependent .
ang2 is upregulated during pathologic angiogenesis and this expression is thought to destabilize endothelial cells , allowing them to be activated by other pro - angiogenic stimuli , such as vegf , see figure 1 , circle 1 ( gale et al . , 2002 ) .
interestingly , ang2 is expressed in ks lesions , and is upregulated in endothelial cells infected with kshv ( brown et al . , 2000 ;
this induction can be activated by the kshv genes vgpcr and vil-6 , and can occur through a paracrine mechanism ( vart et al . , 2007 ) .
another study suggests that the mapk pathway activation of transcription factors ap-1 and ets-1 is involved ( ye et al . , 2007 ) .
in addition to ang2 , cells transfected with the vgpcr gene expressed increased levels of angiopoietin - like 4 , a member of the angiopoietin - like proteins that may play a role in vascular permeability and angiogenesis ( ma et al . , 2010 ) .
kshv induces a number of other cytokines known to be involved in angiogenesis in other systems .
these include interleukin 6 ( il-6 ) , monocyte chemoattractant protein-1 ( mcp-1 ) , pax-1 , and prostaglandin e2 ( schwarz and murphy , 2001 ; polson et al . , 2002 ; xie et al . , 2005 ; caselli et al . , 2007 ;
cyclooxygenase enzymes catalyze the rate limiting step in the conversion of arachidonic acid into prostaglandins .
prostaglandins signal through g - protein coupled receptors to regulate a variety of functions , including metabolic , neuronal , and immune functions .
cyclooxygenase-2 ( cox-2 ) expression is induced early during kshv infection of endothelial cells and plays a role in the establishment of latency ( naranatt et al . , 2004 ; sharma - walia et al . ,
this expression is associated with increased secretion of prostaglandin e2 , which promotes inflammatory cytokine expression , cell survival , and angiogenesis ( sharma - walia et al . , 2010 ) .
an additional cellular factor associated with angiogenesis , angiogenin , is induced in endothelial cells by the latent protein , lana-1 .
angiogenin was recently shown to aid in induction of angiogenesis by both vegf and basic fibroblast growth factor ( sadagopan et al . , 2009 ) .
kshv - induced angiogenin was able to promote endothelial cell migration and capillary morphogenesis ( sadagopan et al . , 2009 ) .
since angiogenin is internalized by both infected and uninfected cells , the authors suggested angiogenin may work in both paracrine and autocrine fashions .
in fact , all kshv - induced cytokines that act on endothelial cells have the potential to promote angiogenesis - like phenotypes on the endothelial - derived spindle cells .
kshv not only upregulates pro - angiogenic cytokines , it may also promote angiogenesis through repression of angiogenic inhibitors .
the kshv latent locus encodes for 17 mirnas which may play a role in downregulation of angiogenic gene expression ( cai et al . , 2005 ;
expression of 10 of these mirnas in 293 cells altered the expression of 81 genes ( samols et al . , 2007 ) .
thrombospondin-1 plays multiple roles in the repression of angiogenesis , however one of its main functions is activation of the anti - angiogenic growth factor transforming growth factor- ( tgf- ) .
this study found that thrombospondin-1 contains 34 putative mirna binding sites , and can be downregulated by multiple kshv mirnas ( samols et al . , 2007 ) .
therefore , downregulation of anti - angiogenic factors may be an important way by which kshv promotes continual neovascularization .
the kshv genome itself encodes for cytokine and chemokine - like factors that activate endothelial cells and stimulate angiogenesis ( table 1 ) . among these factors
are three genes with homology to the cellular chemokine macrophage inflammatory protein , the vmips i iii ( boshoff et al .
in addition to having chemoattractant properties , these proteins promoted neovascularization in the chick chorio - allantoic membrane angiogenesis assay ( boshoff et al . , 1997 ; stine et al . , 2000 ) .
kshv also encodes a viral homolog of interleukin 6 ( il-6 ) , a pro - inflammatory and pro - angiogenic cytokine .
this cytokine , when expressed in nih3t3 cells , promoted secretion of vegf - a ( aoki et al .
furthermore , when these cells were injected into mice , they gave rise to tumors more quickly than control cells and the tumors were more highly vascularized ( aoki et al .
the viral il-6 ( vil-6 ) is mostly detected in endothelial cells and spindle cells undergoing lytic replication but like k1 it has been shown to be expressed at very low levels in latently infected pel cells and to be induced during latency only under specific conditions ( chatterjee et al . , 2002 ; chandriani et al . , 2010 ) .
in summary , conditioned media from kshv - infected cells can induce angiogenic phenotypes in uninfected endothelial cells as indicated by the red gradient in figure 1 .
kshv infection of endothelial cells induces expression of a number of cytokines that are capable of inducing angiogenesis in a paracrine fashion .
paramount among these is vegf - a , an angiogenic cytokine that is induced by kshv infection of endothelial cells .
while the predominant viral mechanism of vegf - a induction is unknown , a number of lytic kshv genes are sufficient to induce vegf - a when overexpressed .
kshv - infected cells also produce a number of other angiogenic cytokines of cellular and viral origin that likely play a role in the induction of angiogenesis . taken together ,
all of the cytokines and induced pathways likely create a milieu that is beneficial to the induction of new blood vessels and play a significant role in the high vascularization of ks tumors .
therefore ks tumor formation is likely to include increased angiogenic capacity of the spindle cells .
there is growing evidence demonstrating the manipulation of host cell phenotypes by kshv and the role of these changes in the promotion of angiogenesis related phenotypes .
these infected cell phenotypes include increased stability of tubules formed by macrovascular endothelial cells , induction of capillary morphogenesis in low growth factor conditions , and enhanced migration and invasion ( qian et al . , 2007 ; sadagopan et al . , 2007 , 2009 ; wang and damania , 2008 ; couty et al . , 2009 ; dimaio et al . ,
additionally , kshv induces the expression of vegf receptors on the surface of infected endothelial cells as discussed below .
breakdown of these junctions is necessary for initiation of angiogenesis , immune cell extravasation , and tumor cell metastasis . interestingly
, several studies have evaluated the adherens junctions of kshv - infected endothelial cells and found them to be perturbed ( mansouri et al . , 2008 ; qian et al . , 2008 ; guilluy et al . ,
, 2008 ; qian et al . , 2008 ) as well as disruption of ve - cadherin
therefore , kshv infection can directly initiate a key angiogenic step , the breakdown of cell cell adherence .
while the direct mechanism of kshv - induced disruption of adherens junctions during latency is not known , there are a number of candidate kshv genes that could be involved ( table 1 ) .
the kshv - encoded ubiquitin ligase protein , k5 , targets ve - cadherin for degradation ( mansouri et al . , 2008 ) .
overexpression of the kshv vgpcr induces endothelial cell permeability and downregulation of cell surface ve - cadherin as well ( dwyer et al . , 2011 ) .
k5 also targets other cellular proteins , including platelet / endothelial cell adhesion molecule-1 ( pecam-1 , cd31 ) , a transmembrane protein important for endothelial cell cell communication , which could contribute to barrier dysfunction and increased permeability ( tomescu et al . , 2003 ; mansouri et al . ,
k1 , a primarily lytic protein that may also be expressed at low levels during latency was also shown to initiate signaling similar to that required for disruption of cadherin signaling ( guilluy et al . , 2011 ) .
while the exact viral mechanism of disruption of adherens junctions by kshv infection is not known , the virus encodes multiple genes capable of altering cadherin . during angiogenesis
, endothelial cells migrate from the pre - existing vasculature toward the site of angiogenic stimulus .
endothelial cells exhibit enhanced migration and invasion following latent kshv infection ( dimaio et al .
kshv - infected cells also express increased levels of the matrix metalloproteinases mmp-1 , -2 , and -9 ( qian et al . , 2007 ) .
mmp proteins break down the extracellular matrix supporting stable vasculature to allow for invasion and migration of endothelial cells during angiogenesis ( figure 1 , circle 2 ) .
expression of mmp proteins induced by kshv allows for increased invasion of both infected and uninfected endothelial cells ( wang et al .
while these processes constitute one component of angiogenesis , they are also known to play roles in oncogenesis ( gialeli et al . ,
2011 ) indicating that kshv activation of angiogenic phenotypes in endothelial cells may lead to enhanced oncogenesis as well .
endothelial cells grown in three - dimensional culture will migrate and organize into capillary - like structures .
this activity is dependent , at least in part , on growth factors and cytokines present in the matrix or growth media .
kshv - infected cells are able to undergo capillary morphogenesis in low growth factor conditions to a greater extent than uninfected cells ( wang and damania , 2008 )
. this could be partially due to increased cytokine secretion from kshv - infected cells .
in fact , when endothelial cells are cultured in the presence of conditioned media from kshv - infected bcbl-1 cells , their ability to organize into capillary - like structures is increased ( wang and damania , 2008 ) .
however , the effect of bcbl-1 conditioned media was greater on kshv - infected endothelial cells than on mock - infected cells , suggesting that infected cells are more receptive to angiogenic growth factors .
in addition , this same study found that capillary - like structures formed by kshv - infected endothelial cells are more persistent than mock - infected cells , indicative of the promotion of cell survival and continual angiogenesis by kshv ( wang and damania , 2008 and our unpublished results ) .
kshv latent infection of endothelial cells also induces vegf receptor expression , which may allow infected cells to respond more robustly to vegfs .
vegf receptors 1 and 2 play roles in angiogenesis while 2 and 3 play a role in lymphangiogenesis ( described below ) .
while kshv infection has not been reported to alter the expression levels of vegfr2 ( kdr ) , vegfr1 expression is significantly increased following kshv endothelial cell infection ( carroll et al . , 2004 ) .
drugs that inhibited hif-1 activation and signaling also inhibited vegfr1 upregulation ( carroll et al . , 2006 ) .
vegfr1 has been described as both a positive and negative regulator of angiogenesis depending on the context .
vegfr1 mouse knockouts have higher levels of angiogenesis ( fong et al . , 1995 ) .
however , in cell culture models , vegfr1 has been shown to potentiate angiogenesis ( cao , 2009 ) .
more studies will be needed to determine the importance of increased vegfr1 expression in kshv infection and ks tumor formation .
interestingly , expression of vegfr3 , the main receptor for vegf - c and d is also significantly increased by kshv infection ( carroll et al . , 2004 ; hong et al . , 2004 )
vegfr3 , a receptor specific to lymphatic endothelium and critical for lymphangiogenesis will be discussed below .
importantly , endothelial tip cells at the leading edge of new vascular protrusions are the only main adult cell type known to express both the blood endothelial cell receptor , vegfr1 , and the lymphatic endothelial cell receptor , vegfr3 ( tammela et al . , 2008 ) : kshv infection of endothelial cells directly induces expression of both of these receptors . the mechanisms by which kshv induces angiogenic phenotypes in latently infected cells are largely unknown . a number of angiogenic phenotypes are likely to be a direct result of the cytokine milieu of the infected cells .
as described above , kshv - infected cells secrete both viral and host cytokines that are sufficient to induce angiogenic phenotypes .
however , it is also apparent that some of the angiogenic effects seen in latently infected cells are cell autonomous , independent of either paracrine or autocrine factors . as described above , conditioned media from pel cells had stronger effects on tubule formation of kshv - infected endothelial cells ( akula et al . , 2005 ) .
we have also recently found that kshv infection induces the pro - angiogenic integrin , integrin 3 , during latent infection ( dimaio et al . , 2011 ) .
induction of integrin 3 leads to increased cell surface expression of the v3 integrin heterodimer .
we have further shown that latently infected endothelial cells become more adherent to the integrin ligands fibronectin and vitronectin , and are more migratory than mock - infected cells .
although both uninfected and infected cells organize in three - dimensional cultures in complete media , infected cells are more sensitive to inhibitors of integrin 3 and its downstream signaling molecules , such as src kinase ( dimaio et al . , 2011 ) .
this suggests that during latent kshv infection there is a shift in endothelial cell signaling that results in a more angiogenic phenotype dependent on v3 expression on the surface of the cell ( figure 1 , center ) .
therefore , kshv alteration of endothelial cell signaling pathways can dramatically affect how the cell responds to intra- and extra - cellular signals .
these changes that lead to alterations in angiogenic properties are likely to play a role in the growth and cell cell interactions of infected cells , thereby playing a role in ks tumor formation .
during development of the vascular system , a subset of endothelial cells in the cardinal vein begin to express markers of lymphatic differentiation , including the master regulatory gene , prox-1 .
these cells then bud from the cardinal vein , differentiate into lymphatic endothelial cells , and form the lymphatic vascular system ( wigle and oliver , 1999 ) .
immunohistochemistry of ks tumors showed that spindle cells express markers of lymphatic endothelium , suggesting these cells may arise from primary infection of lymphatic endothelial cells , rather than blood endothelial cells ( jussila et al .
, 1998 ; skobe et al . , 1999 ; weninger et al . , 1999 ; pyakurel et al . , 2006 ) .
an alternative hypothesis is that kshv infection of blood endothelial cells drives differentiation toward a more lymphatic phenotype .
this idea is supported by evidence that kshv infection of blood endothelial cells promotes expression of lymphatic - specific genes , including prox-1 , vegfr3 , podoplanin , and lyve-1 , effectively driving the reprogramming of blood endothelial cells to become lymphatic endothelium ( carroll et al .
microarray analysis comparing kshv - infected blood endothelial cells to blood and lymphatic endothelial cells indicate that kshv - infected blood endothelial cells have gene expression profiles that align more closely to lymphatic endothelial cells than that of blood endothelial cells ( carroll et al . , 2004 ;
the kshv latent gene kaposin b can directly promote the stability of prox-1 mrna ( yoo et al .
, 2010 ) leading to increased expression of prox-1 . however , this effect was not sufficient to induce prox-1 expression in blood endothelial cells .
we recently found that induction of blood to lymphatic endothelial cell reprogramming requires signaling through the cellular receptor gp130 .
endothelial cells that are latently infected with kshv have increased expression and signaling of gp130 ( morris et al . , 2008 ) .
this leads to activation of the jak / stat3 pathway and the pi3k / akt pathway leading to expression of lymphatic - specific genes starting with prox-1 .
inhibition of this pathway by sirnas that target gp130 or akt or pharmacological inhibitors that block pi3 kinase or jak2/stat3 signaling is sufficient to block lymphatic differentiation ( see figure 1 , center ) .
kshv vil-6 is sufficient to induce gp130 activation and we recently found that vil-6 is sufficient to induce lymphatic differentiation ( morris et al . , 2012 ) .
however , kshv lacking vil-6 is still able to cause blood to lymphatic endothelial cell differentiation , indicating that kshv has evolved multiple strategies to activate gp130 and induce blood to lymphatic endothelial cell differentiation ( morris et al . , 2008 ) .
induction of lymphatic differentiation by kshv is only part of the story , however . despite the expression of lymphatic - specific genes , blood endothelial cells infected with kshv
retain expression of some blood specific markers ( wang et al . , 2004a ) .
additionally , infection of lymphatic endothelial cells with kshv induces expression of blood specific markers ( wang et al . , 2004a ) .
kshv mirnas were found to target the transcription factor maf ( hansen et al . , 2010 ) .
downregulation of maf in lymphatic endothelial cells by sirna restored expression of blood endothelial markers , such as vegfr1 and cxcr4 .
thus , infection of blood or lymphatic endothelial cells by kshv alters host gene expression to an intermediate state between the two cell types .
as described above , this intermediate phenotype with both vegfr1 and r3 expression is only present in the leading tip of endothelial cells involved in active neo - angiogenesis . in the ks lesions only lana+ cells expressed prox-1 , indicating that this effect requires kshv gene expression ( hong et al . , 2004 ) .
this suggests that differentiation toward lymphatic endothelial cells may specifically allow the spindle cells to respond to lymphangiogenic growth factors .
in fact , kshv infection of endothelial cells induces both vegf - a and vegf - c ( sivakumar et al . , 2008 ) .
therefore , induction of both vegfr1 and r3 allow kshv - infected cells to respond to key angiogenic and lymphangiogenic factors in the tumor environment .
the direct role of kshv reprogramming of blood endothelial cells to lymphatic in induction of angiogenic and lymphangiogenic phenotypes is still under investigation .
the highly vascular nature of ks tumors and the large amounts of neo - angiogenesis in the tumor led to the proposal that the etiologic agent of the tumor might directly induce angiogenesis . in accordance with this hypothesis kshv infection of endothelial cells ,
in particular , kshv induces the expression of vegf - a and -c and other cytokines as well as encoding angiogenic cytokines from its own genome ( boshoff et al . , 1997 ; aoki et al . , 1999
; brown et al . , 2000 ; stine et al . , 2000 ; schwarz and murphy , 2001 ; masood et al . , 2002 ; polson et al . , 2002 ; wang et al . , 2004b ; xie et al . , 2005 ; caselli et al . , 2007 ; vart et al . , 2007 ; ye et al . , 2007
sivakumar et al . , 2008 ; wang and damania , 2008 ; sadagopan et al .
additionally , because the tumor cell is endothelial in nature , induction of angiogenic cytokines may also activate the tumor cells and aid in the growth of ks tumors .
kshv also induces angiogenic phenotypes directly in latently infected cells in a cell autonomous fashion , indicating that angiogenic activation of the infected endothelial cell may directly play a role in tumor formation .
while kshv activates many growth - signaling properties and in general the induction of angiogenic phenotypes supports endothelial cell proliferation , in most cultures kshv does not induce increases in endothelial cell proliferation .
it is possible that the cell culture milieu simply does not match the tumor milieu .
ks spindle cells are not fully transformed ex vivo and , except in very rare cases , have a limited life span indicating that factors in the tumor environment that come from other cells types could be necessary to maintain ks spindle cell growth . the increase in growth
could also be masked by the fact that endothelial cells in culture are rapidly dividing and therefore do not need additional growth signals . along those lines ,
that being said , the endothelial cell transforming potential of kshv in culture can be unmasked given specific conditions .
dermal microvascular endothelial cells that were immortalized with the e6 and e7 genes from papillomavirus are readily transformed by kshv , including increased proliferation ( moses et al . , 1999 ) .
therefore , kshv activation of endothelial cells can induce a proliferative advantage in the correct genetic environment .
however , it is unknown if viral induction of angiogenic phenotypes is necessary for the growth in the e6/e7 immortalized endothelial cells . in general
, viruses do not evolve to cause cancer , as it is likely a dead end for transmission .
kshv likely evolved to activate the cell where it is maintained to ensure survival and spread of the virus .
a major side effect of this activation may be providing an ideal environment for angiogenesis leading to increased vascularization of small tumor growths and expansion of ks tumors . while the study of viral induction of angiogenesis can lead to a better understanding of how kshv causes endothelial cell tumors , information gleaned from the study of viral mechanisms of induction of angiogenesis and lymphangiogenesis will also lead to a better understanding of endothelial cell activation and tumor angiogenesis in general .
thus , the study of kshv infection of endothelial cells provides a controlled system for analyzing the regulation and induction of angiogenic phenotypes that will likely shed light on the field of tumor angiogenesis .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
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kaposi s sarcoma ( ks ) is a highly vascularized tumor supporting large amounts of neo - angiogenesis .
the major cell type in ks tumors is the spindle cell , a cell that expresses markers of lymphatic endothelium .
kshv , the etiologic agent of ks , is found in the spindle cells of all ks tumors .
considering the extreme extent of angiogenesis in ks tumors at all stages it has been proposed that kshv directly induces angiogenesis in a paracrine fashion . in accordance with this theory , kshv infection of endothelial cells in culture induces a number of host pathways involved in activation of angiogenesis and a number of kshv genes themselves can induce pathways involved in angiogenesis .
spindle cells are phenotypically endothelial in nature , and therefore , activation through the induction of angiogenic and/or lymphangiogenic phenotypes by the virus may also be directly involved in spindle cell growth and tumor induction .
accordingly , kshv infection of endothelial cells induces cell autonomous angiogenic phenotypes to activate host cells .
kshv infection can also reprogram blood endothelial cells to lymphatic endothelium .
however , kshv induces some blood endothelial specific genes upon infection of lymphatic endothelial cells creating a phenotypic intermediate between blood and lymphatic endothelium .
induction of pathways involved in angiogenesis and lymphangiogenesis are likely to be critical for tumor cell growth and spread .
thus , induction of both cell autonomous and non - autonomous changes in angiogenic and lymphangiogenic pathways by kshv likely plays a key role in the formation of ks tumors .
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after stent graft implantation became a standard procedure for infra - renal abdominal aortic aneurysm , the number of cases of thoracic endovascular aortic repair ( tevar ) in korea has been rapidly increasing [ 1 - 3 ] , substantiated by the reports from korean health insurance review & assessment service ( fig .
1 ) . stent graft , which consists of prosthetic material , such as dacron or gore - tex patches , embedded in the cylindrical metallic frame , is devised to separate aortic aneurysm from true aortic lumen , and thus interrupt the blood flow into the aneurysm . because tevar eliminates the basic elements of surgical intervention ( i.e. thoracotomy , left heart bypass , aortic cross - clamping and deep hypothermic circulatory arrest ) , this new therapeutic modality is believed to lower the risks of morbidity and mortality in descending thoracic aortic aneurysm , traumatic aortic rupture and type b aortic dissection .
furterhmore , when the application of tevar for aortic arch aneurysm is difficult because proximal landing zone ( plz ) for stent graft overrides the origin of the arch vessels , hybrid procedure ( i.e. surgical debranching of the arch vessels plus tevar ) has been recently developed . in this study
, we sought to determine the intermediate - term results of tevar for thoracic aortic aneurysm .
the study cohort comprises 16 patients who underwent tevar between october 2003 and april 2010 .
median age at tevar was 59 years ( 20~78 years ) , and 11 were males .
types of aortic lesion were descending thoracic aortic aneurysm in 4 , chronic aortic dissection in 4 , traumatic aortic rupture in 3 , aortic arch aneurysm in 2 , acute type b aortic dissection in 2 , and descending thoracic aorta pseudoaneurysm in 1 ( table 1 ) .
associated morbidities were systemic hypertension in 7 , multiple trauma in 3 , a history of ascending aortic replacement for previous type a aortic dissection in 2 , diabetes mellitus in 2 , and aortic rupture in 1 .
indications for tavar were dilated aortic diameter equal to or greater than 5.5 cm ( n=6 ) , increase in aortic size during the follow - up ( n=4 ) , traumatic aortic rupture ( n=3 ) , persistent chest pain ( n=2 ) , and ruptured aortic aneurysm ( n=1 ) ( table 2 ) .
mean diameter of the stent used for tevar was 33 mm ( 26~40 mm ) , and mean number of the stent per a patient was 1.25 ( 1~2 ) .
all patients underwent multi - detector computed tomography ( mdct ) for the assessment of thoraco - abdominal aorta , and sufficient plz was defined by aortic diameter ( 40 mm ) and length ( 2 cm ) on mdct .
if the length of normal aorta is insufficient for plz , tevar was preceded by debranching surgery of the arch vessels ( fig .
cerebrospinal fluid drainage was done in a patient whose distal landing zone was to be below the level of 8 thoracic vertebra , and intraoperative electroencephalography ( eeg ) monitoring was done for patients with debranching surgery . under general or epidural anesthesia ,
right femoral artery was exposed for the catheter sheath insertion , and left femoral artery or left radial artery was punctured to introduce a reference catheter , 5 french in size , which was used to determine the exact location of dlz . after 5,000 iu of heparin
was administered , a stent graft ( seal flex , s & g co , korea ) was introduced through the sheath in the right femoral artery up to the level of pre - determined plz and dlz . during the procedure , calcium channel blockers and remifentanil were used to lower the systolic systemic blood pressure down to 80 mmhg .
rapid right ventricular pacing to prevent windsock phenomenon , which is recommended by several programs , was not performed . after the stent graft was deployed , aortography was performed to ascertain the absence of endo - leak .
if endo - leak was suspected , balloon dilatation of the stent graft or additional stent graft implantation was performed .
follow - up mdct was done at one week after the procedure , and patients were follow - up in the out - patient department at postoperative 1 , 6 and 12 months .
one patient showed minimal type 1 endoleak , and has been closely followed up ever since the procedure without further deterioration .
cerebral embolism occurred in four patients : three recovered completely , and one developed mild right side motor weakness .
two patients developed left - sided pleural effusion , which was drained using a catheter in one .
there was no surgical mortality . during a mean follow - up duration of 18 months ( 1~73 months ) ,
the diameter of the thoracic aorta covered by the stent graft changed within 10% range in 12 patients , decreased by more than 10% in 3 , and increased by more than 10% in one ( fig .
stent graft tevar for various thoracic aortic diseases reportedly shows low morbidity and mortality compared to conventional surgical approach [ 1 - 4 ] .
technical success rate of tevar ( i.e. complete isolation of the anurtysmal cavity from the aortic circulation ) has reached up to 98% , while early mortality ( 1.9~2.9% ) is extremely lower than that ( 5.7~11.7% ) of surgical series [ 2 - 4 ] . as for the morbidity ,
a recent report claimed that tevar resulted in lower incidence ( 9.4% ) of morbidities , including myocardial infarction , respiratory complications and cerebrospinal injury compared to the outcome of surgical intervention ( 33% ) .
indications for tavar are almost the same as those for surgical intervention , that are dilated aortic diameter equal to or greater than 5.5 cm , increase in aortic size by greater than 1 cm per year during the follow - up , traumatic aortic rupture , persistent chest pain , peripheral perfusion failure from type b aortic dissection , and penetrating arotic ulcer larger than 2 cm in diameter .
traumatic aortic rupture is caused by an acute deceleration injury on the insertion site of ligamentum arteriosum , and frequently associated with multiple trauma , which may render the surgical outcome even worse . in this
setting , tevar is a formidable alternative to a surgical intervention by minimizing the invasiveness of the repair .
small sized stent grafts tend to be used for tevar for patients with traumatic aortic rupture because this condition frequently occurs in younger population whose pre - trauma aortic size could well be normal . with respect to the outcome after tevar for uncomplicated type b dissection , instead ( investigation of stent graft in patients with type b aortic dissection ) study conducted in europe showed that there was no difference in 1 year survival between tevar group and medical treatment group .
however , given that the size of the aorta increases as time passes , the longer term outcome may turn out to be in favor of tevar strategy . to the contrary ,
iris et al observed that there was no difference in the size of the false aortic lumen between tevar and medical treatment groups , and they asserted that tevar procedure never abolishes , albeit may delay , the manifestation of the adverse outcome from the natural progression of the aortic disease . in our series , there was one patient whose aortic diameter increased as time passed . as for chronic aortic dissection , partial thrombus formation in the false lumen is believed to increase the risk of rupture and death .
thus , the benefits from tevar in comparison to surgical approach is still controversial , because stent graft only obliterates the opening of the entry site while leaving reentry site uncovered , which may end up with partial thrombus formation , progressive aortic dilation and rupture of the thoracic aorta .
surgical correction of acute type b dissection involving the origin of arch vessels is associated with significant morbidity and high mortality rate .
arch vessels obstruction is caused by intimal flap occluding the origins of the vessels , and obstruction can be either static ( i.e. fixed obstruction ) or dynamic ( i.e. intermittent obstruction according to the cardiac cycle ) .
arch dissection with static obstruction of the arch vessels necessitate self - expandable stent insertion into the arch vessels , and communication between the true lumen and arch vessel origins is established either by fenestration of the stent graft or by the use of separate bare stent at the arch level . on the contrary ,
arch dissection with dynamic obstruction of the arch vessels only requires obliteration of the entry site by stent graft so that intermittent expansion of the initmal flap during the systolic phase is prevented .
early and late mortality after surgery for patients who had undergone tevar for arch dissection with arch vessel obstruction are reported to be 17% and 36% , respectively , which appears to be much better than the mortality after conventional surgical repair for acute arch dissection ( 40% ) .
this finding signifies that the application of tevar to arch dissection is designed to convert a complicated type b dissection involving arch vessels into an uncomplicated chronic type b dissection , which is much more amenable to a surgical intervention . in this
regards , szeto et al asserted that tevar should be considered as the fist - line treatment for complicated type b acute dissection ( i.e. rupture or arch obstruction ) , evidenced by the low procedure - related mortality ( 3% ) in 35 patients with complicated arch dissection .
however , some argue that the risk of false lumen rupture in type - b dissection with arch vessel obstruction remains significant even after tevar is performed . as for uncomplicated type b dissection ,
adsorb ( acute uncomplicated aortic dissection type b : evaluating stent - graft placement or best medical treatment alone ) study has investigated on the benefits of stent graft over medical treatment , which has not reached a conclusion yet .
timing of tevar for type b acute dissection is also controversial . some prefer to delay the procedure to 2~4 weeks after the onset , based on the findings that intimal flap is fragile and easily breakable during early period , while others believe that early intervention may promote the complete restoration of the aortic integrity , and
thus could improve the long - term outcome . in a study which compared the outcomes after tevar and medical treatment for acute type b dissection
, the authors insisted that there was no difference in the development of complication or mortality , and therefore tevar should be reserved only for complicated type b dissection .
akira et al stratified the patients according to the diameter of the aorta , and they concluded that tevar should be indicated for patients whose maximal aortic diameter is greater than 40 mm , based on their observations that 60% of the patients with large aorta ( 40 mm in diameter ) showed enlargement of the false lumen and underwent a surgical intervention while 94% of the patients with small aorta ( 40 mm in diameter ) showed spontaneous obliteration of the false lumen .
preoperative computed tomographic angiography can be utilized for the evaluation of the arch vessels , and , if basilar artery mal - perfusion after tevar is anticipated due to poor collateral circulation to the left subclavian artery or left dominance of vertebral artery circulation , graft interposition between the left subcalvian artery and left common carotid artery should be performed beforehand . if the stent graft is to cover the left common carotid artery too , graft interposition between both common carotid arteries should also be performed before tevar procedure .
if the innominate artery is to be covered , all three arch vessels should be debranched and reimplanted into the proximal ascending aorta through median sternotomy .
cerebral embolism of atheromatous plaques may take place during the catheter work or graft stenting , and left vertebral artery malperfusion may lead to posterior cerebral infarction when plz violates the origin of the left subclavian artery .
furthermore , spinal cord injury is likely to occur when more than 15 cm of the descending thoracic aorta is covered by the stent graft or dlz is less than 5 cm away from the origin of the celiac trunk . as to the complications after tevar
, post - implantation syndrome refers to inflammatory responses after tevar characterized by leukocytosis , mild fever and the elevation of the inflammatory markers , and is attributed to the activation of the intimal cells of the aorta .
distal migration of the stent graft more than 10 mm off the original site is observed in 1~2.8% of the patients , and too large stent graft or variability of the luminal curvature in the proximity of plz are thought to be the risk factors for stent graft migration .
postoperative evaluation is based on the findings of mdct , which is to be performed at postoperative 1,6 and 12 months , and annually from that on .
if type 1 endo - leak is detected on mdct , immediate intervention is recommended , and , if type 2 endo - leak is suspected , close follow - up focusing on the size change of the aneurysm is suggested .
traumatic aortic rupture and descending thoracic aneurysm seem to be best benefitted from this new therapeutic modality , and tevar is expected to rapidly replace the conventional surgical approach . as for aortic arch dissection involving arch vessels , employment of hybrid procedure ( i.e. tevar with surgical interventions for arch vessels ) will increase .
the efficacy of tevar for acute type b dissection is still under debate : some are insisting that tevar does not change the natural course of the false lumen while others claim that tevar may contribute to the regression of the false lumen by thrombus formation and absorption .
thus , further study with longer - term follow - up is necessary to delineate the benefits from tevar for this subset .
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backgroundthe number of cases employing thoracic endovascular aortic repair ( tevar ) has been increasing due to lower morbidity and mortality compared to open repair technique .
the aim of this study is to evaluate the outcome of tevar for thoracic aortic diseases.materials and methodssixteen patients underwent tevar from october 2003 to april 2010 .
mean age at operation was 59 years ( 20~78 years ) , and 11 were male .
indications for tevar were large aortic diameter ( > 5.5 cm ) upon presentation in 6 patients , increasing aortic diameter during the follow - up period in 4 , traumatic aortic rupture in 3 , persistent chest pain in 2 , and ruptured aortic aneurysm in one .
the mean diameter , length and the number of the stents were 33 mm ( 26~40 mm ) , 12 cm ( 9.5~16.0 cm ) , and 1.25 ( 1~2 ) , respectively .
aortography employing multi - detector computerized tomography ( mdct ) technique was performed at one week , and patients were followed up in the out - patient department at one month , 6 months , and one year postoperatively.resultsprimary technical success showing complete exclusion of the aneurysm was achieved in 15 patients .
one patient showed a small endo - leak ( type 1 ) .
four patients developed perioperative stroke : three recovered without sequelae , and one showed mild right - side weakness .
there was no operative mortality .
diameter of the thoracic aorta covered by stent graft changed within 10% range in 12 patients , decreased by more than 10% in 3 , and increased by more than 10% in one during mean follow - up duration of 18 months ( 1~73 months ) .
there was no recurrence - related death during this period.conclusionintermediate-term outcome after tevar was encouraging .
indications for tevar could be extended for other thoracic aortic diseases .
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modafinil ( 2-[(diphenylmethyl ) sulfinyl ] acetamide ) is a unique non - amphetamine wakefulness - promoting agent that was first marketed in france in the early 1990s for the treatment of excessive somnolence in patients with narcolepsy .
it was subsequently approved in the usa in 1998 under the brand name provigil and is used for the treatment of various sleep disorders characterized by excessive sleepiness , including narcolepsy , obstructive sleep apnea , and shift work sleep disorder .
it has steadily gained popularity among prescribers due to its wakefulness - promoting efficacy , presumed lower potential for abuse , and lack of peripheral sympathomimetic effects commonly observed with amphetamine stimulants . as a result , it has been studied and used off - label to treat sedation and fatigue in neurological and medical conditions including multiple sclerosis , parkinson s disease , hiv infection , cancer , and fibromyalgia . it has also shown promise in the treatment of cocaine dependence and withdrawal [ 68 ] , alcoholic organic brain disorder [ 9 , 10 ] , and in augmentation of antidepressant regimens .
it is known to cause an increase in extracellular concentrations of dopamine , norepinephrine , serotonin , glutamate , and histamine and to cause a decrease in extracellular gamma - aminobutyric acid in the neocortex .
it has been shown to bind to and inhibit the dopamine transporter and norepinephrine transporter at clinically relevant doses , which may be responsible in part for its therapeutic effects .
unlike amphetamines , modafinil has no effect on spontaneous dopamine release or turnover and lacks peripheral sympathomimetic effects [ 12 , 14 , 15 ] .
two small case series have been published in abstract form [ 16 , 17 ] , and a recent review of data from 11 states was published by spiller et al . .
during clinical trials , intentional acute overdoses as high as 4,500 mg were reported , with insomnia , tachycardia , agitation , and anxiety being the only side effects reported ; no life - threatening toxicity occurred , and to date , no fatal overdoses have occurred involving modafinil alone .
we performed a retrospective chart review of modafinil exposures reported to the california poison control system over an 11-year period .
our aim in this study was to characterize the clinical effects and toxicity associated with overdose of modafinil in the hope of improving treatment guidelines and recommendations for patients with modafinil ingestion .
the university of california san diego institutional review board approved this retrospective cohort study .
a retrospective chart review of the california poison control system ( cpcs ) electronic database ( visual dotlab ) for cases between the years 1998 and 2008 was performed .
cases were assessed by the principal and assistant investigators only after removal of all patient identifiers .
inclusion criteria included patients of any age with single substance exposure to modafinil and follow - up to a known outcome ( this cohort included patients exposed to modafinil who were described in abstract form in [ 16 , 17 ] ) .
exclusion criteria were history of any co - ingestants and inability to follow the patient to a known outcome .
data collected included date of occurrence , age , gender , weight ( where available ) , dose ingested by history ( or , in the case of a child , pill count or events witnessed by a parent ) , whether exposure was intentional or unintentional , symptoms , duration of clinical effects , site of exposure , management site , treatment , length of hospital stay , and clinical outcome . among intentional exposures ,
abuse was defined as use of the medication for purported psychotropic effects , while misuse was defined as use of the medication for its known stimulating effects for non - fda - approved indications ( e.g. , to facilitate studying ) .
tachycardia was defined as a heart rate > 100 bpm , and hypertension was defined as a systolic blood pressure > 140 mmhg or diastolic blood pressure > 90 mmhg .
outcomes were coded as no effect , minor effect , moderate effect , major effect , or death according to the criteria set forth by the american association of poison control centers .
all data abstracted were transcribed into a standardized microsoft excel 2008 for mac ( microsoft , redmond , wa ) spreadsheet .
a total of 257 cases of modafinil ingestion were reported to the cpcs between 1998 and 2008 .
eighteen cases were excluded because of confirmed non - exposure ( e.g. , all pills were later accounted for ) , 43 were excluded due to lack of follow - up to a known outcome , 108 were excluded due to reported co - ingestants , and one was a non - human ( canine ) exposure .
this left 87 cases that met inclusion criteria ( see fig . 1 ) .
1flow diagram of identified modafinil exposures flow diagram of identified modafinil exposures fifty - three patients ( 60.9% ) were female .
patient ages ranged from 1.25 to 72 years with a mean of 29.9 years .
of these , mean and median doses ingested were 939 and 400 mg , respectively , with a range of 2516,100 mg .
exposure management sites are listed in table 1 ; the majority of patients ( 56.3% ) were managed in a non - healthcare facility setting .
table 1exposure management sitesexposure management sitenumber of patients ( % ) on - site ( e.g. , home)49 ( 56.3)emergency department33 ( 37.9)critical care unit4 ( 4.6)physician s office1 ( 1.1 ) exposure management sites effects were classified as no effect ( n = 22 ) , minor ( n = 54 ) , and moderate ( n = 11 ) .
n = 7 ) , antihistamines ( n = 2 ) , intravenous fluids ( n = 2 ) , haloperidol ( n = 2 ) , and beta - blockers ( n = 1 ) .
table 2summary of medical outcome by dose of modafinil ingested no effectminor effectmoderate effectall patients ( n = 87)22 ( 25.3%)54 ( 62.1%)11 ( 12.6%)patients with known dose ( n = 81)22 ( 27.2%)50 ( 61.7%)9 ( 11.1%)dose range ( mg)50120025161004008,000mean dose ( mg)4119951,922median dose ( mg)2504001,200all patients 6 years old ( n = 17)11 ( 64.7%)6 ( 35.3%)patients 6 years old with known dose ( n = 16)11 ( 68.8%)5 ( 31.3%)dose range ( mg)50500251,000mean dose ( mg)186275median dose ( mg)100100 summary of medical outcome by dose of modafinil ingested clinical effects are summarized in table 3 .
the most frequently reported effect was tachycardia , which occurred in 23 patients ( 26.4% ) and ranged from 101 to 168 bpm .
there was one report of chest pain in a 65-year - old male who inadvertently took 800 mg of modafinil after mistaking it for acetaminophen ; vital signs revealed a heart rate of 64 bpm and blood pressure of 137/78 mmhg , and troponin measurements were negative .
table 3clinical effectsclinical effectnumber of patients ( % ) tachycardia23 ( 26.4)agitation14 ( 16.1)anxiety11 ( 12.6)headache8 ( 9.2)hypertension6 ( 6.9)dystonia / tremor6 ( 6.9)dizziness5 ( 5.7)insomnia4 ( 4.6)stomach upset / nausea4 ( 4.6)erythema / flushing2 ( 2.3)dysarthria2 ( 2.3)hallucinations2 ( 2.3)palpitations2 ( 2.3)chest pain1 ( 1.1)edema1 ( 1.1 ) neurologic effects following excessive modafinil ingestion also occurred frequently and manifested primarily as agitation , anxiety , and headache .
one 37-year - old female accidentally ingested 1,600 mg of modafinil and presented to an ed with agitation , visual hallucinations , and marked orofacial dyskinesia .
she was noted to have a heart rate of 115 bpm and blood pressure of 138/73 mmhg .
she remained tachycardic for 13 h and was ultimately discharged from the hospital 20 h post - ingestion without sequelae .
of these , five ( 5.7% ) were due to misuse , while six ( 6.9% ) were due to abuse .
of these , 19 were instances where a patient had inadvertently taken a double dose of their medication .
eighteen of these patients had no significant effects ; the one symptomatic patient was a 24-year - old female who accidentally ingested 400 mg instead of her usual single 200 mg tablet and who presented to an ed with a heart rate of 168 and blood pressure of 168/131 mmhg .
single - agent exposures to modafinil appear to occur infrequently . in this case series ,
our results are similar to those of the recent study by spiller et al . which examined data from poison control centers of 11 states .
as would be expected , increasing mean and median dosages were associated with more significant clinical effects .
modafinil has been classified as a schedule iv drug by the drug enforcement administration due to its potential to cause euphoria and alterations in mood , thought , and perception .
although a recent review concluded that modafinil has a low abuse potential , a recent volunteer study demonstrated that modafinil increases dopamine concentration in the nucleus accumbens area of the brain , an effect that has been associated with the potential for abuse .
additionally , reports abound in internet drug use forums of modafinil s misuse among students in order to facilitate studying and improve grades . in our case series , one third ( 11/33 ) of intentional exposures were due to abuse or misuse .
given modafinil s increasingly widespread use , it is likely that cases of toxicity will continue to be reported to poison control centers .
this was a retrospective study which limits the amount of data that we were able to retrieve from each case .
some symptoms may have been present but were not reported to cpcs , and our frequency of clinical effects may not represent actual frequency of effects .
it is very likely that our study did not capture all cases of modafinil toxicity or exposure , given that reporting of such cases to cpcs is voluntary .
additionally , it is not possible to confirm exposure in all cases since assays for measurement of plasma concentrations of modafinil are not widely available .
effects seen in our series of modafinil exposures were usually minor , with cns symptoms and tachycardia most commonly seen .
no significant toxicity occurred with ingestions < 400 mg ; this suggests that patients who ingest less than this amount may be managed safely at home . additionally , accidental
double - dosing of modafinil was not associated with clinical effects in the majority of cases , but did result in clinically significant effects requiring treatment in one patient .
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modafinil is a non - amphetamine wakefulness - promoting agent used for the treatment of various sleep disorders characterized by excessive daytime sleepiness .
there is little information in the medical literature with respect to supratherapeutic doses of this medication .
we performed a retrospective review of the california poison control system database for all cases of single - substance ingestion of modafinil with follow - up to a known outcome for the time period 19982008 .
data collected included age , gender , dose ingested , clinical effects , and medical outcome .
there were a total of 87 patients , 53 ( 61% ) of which were female .
patient ages ranged from 1.25 to 72 years with a mean of 30 years ; 17 ( 20% ) patients were aged 6 years or less . thirty - three ( 38% ) were intentional overdoses .
most commonly reported effects were tachycardia ( n = 23 ) , agitation ( n = 14 ) , anxiety ( n = 11 ) , headache ( n = 8) , hypertension ( n = 6 ) , dystonia / tremor ( n = 6 ) , and dizziness ( n = 5 ) .
forty - nine patients ( 56% ) were managed at home , and 38 ( 44% ) were managed in a healthcare setting .
therapies administered included activated charcoal ( n = 8) , benzodiazepines ( n = 7 ) , antihistamines ( n = 2 ) , intravenous fluids ( n = 2 ) , haloperidol ( n = 2 ) , and beta - blockers ( n = 1 ) .
effects were classified as none ( n = 22 ) , minor ( n = 54 ) , and moderate ( n = 11 ) . no major effects and no deaths occurred .
effects of modafinil overdose appear to be mild in most cases , with tachycardia and cns symptoms predominating .
clinically significant effects requiring treatment occurred in a small number of patients .
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the radioactivity level from the natural radionuclides is termed as background radiation which will depend on the amount of the radioactive materials in the environment .
the background radiation can be high if the environment is polluted either from man - made or natural activities .
it can also be high in regions with deposit of mineral resources such as uranium ores and phosphate .
materials from the deposit may be brought to the surface soil through processes such as weathering of rocks and soil formation .
they can also leach into the groundwater system , contaminate it , and lead to pollution far away from the source . in tanzania , high concentrations of uranium deposits of up to 464 ppm
this has brought concern about the level of natural radioactivity in the soil and drinking water at villages in the neighborhood of the deposit .
this is because there are reports in the literature which indicate high radioactivity levels in regions near uranium deposits [ 2 , 3 ] .
( 2009 ) in india reported enhanced level of u and th in the soil around uranium deposits as compared to the world average [ 2 , 4 ] .
( 2008 ) reported high activity concentration of ra , th , and k in soil around gurvanbulag uranium deposit in the eastern part of mongolia .
the present study has determined concentration levels of the u decay series and the th decay series as well as k in soil and water from likuyu village which is 54 km east of the mkuju uranium deposit in tanzania .
the paper will use u and th to indicate the concentrations of these decay series .
the data will offer useful and necessary information in the monitoring of environmental contamination which will provide appropriate and better protection guidelines to the public .
the likuyu - seka - maganga village is at namtumbo district in ruvuma region southern tanzania .
the village is situated at longitude 3616 east and latitude 1019 south of the equator .
it is near to the undendeule forest reserve ( ufr ) at the south and the selous game reserve ( sgr ) at the north .
the likuyu village is situated at about 54 km east of the mkuju uranium deposit .
the ambient temperature at the likuyu village varies from minimum of 18.8c to a maximum of 28.2c .
the wind blows from the mkuju uranium deposit through the likuyu village in the month of december to february with speed of up to 11.6 m / s .
two rivers , which are mkuju river and kilowelo river , flow from the mkuju uranium deposit to the northern and southern parts of the likuyu village , respectively .
the water from these rivers is domestically used by the people of the likuyu village .
the village has a total population of about 10,000 people settled in eight hamlets , seven of which lie along the road to the mkuju uranium deposit ( mantra , 2010 ) .
the village was divided into three sampling zones : the northern , the central , and the southern zones . ten samples of soil were collected randomly from each sampling area to make a total of 30 samples of soil . the samples were collected at 05 cm depth level as reported elsewhere .
the collected samples were then placed in labeled polythene bags and transferred to the taec laboratory for preparation and analysis .
the samples were filled in a one liter acid precleaned polyethylene container to avoid wall absorption ( iaea , 1989 ) and were wrapped using insulation tape to avoid spill during transport of the samples from sampling point to the taec laboratory for preparation and analysis . in the laboratory
, the soil samples were sieved by a 2 mm mesh to remove larger objects and then ground using mortar and pestle to fine powder in order to have the same matrix as the reference sample .
after that , the samples were dried in an oven at a temperature around 4550c for several hours until constant weights were attained and then placed in desissators to avoid moisture absorption as described elsewhere .
samples of about 500 g each were packed in marinelli beaker of about 500 cm volume and sealed using silicon and plastic tapes for air tight .
the samples were left for a minimum of 28 days for radioactive secular equilibrium between rn - radon gas and its decay products ( pb , bi , and ra ) , from the u decay series to be acquired .
the activity of u was determined using the gamma - lines of ra ( 186 kev ) , pb ( 295 kev and 351 kev ) , bi ( 1238 kev and 1378 kev ) , ac ( 338 kev , 911 kev , 965 kev , 969 kev ) , and tl ( 860 kev ) .
the gamma - lines of 609.3 kev , 1120 kev ( from u series ) , 583.2 kev , 727.3 kev , and 795 kev ( th series ) were omitted from the analyses as sensitivity analysis has shown elsewhere that they are affected by coincidence summing .
a lead - shielded coaxial high - purity germanium detector ( hpge ) of relative efficiency 51% and resolution of 2.1 kev for 1332 kev co gamma - ray source was used for low level counting of the samples .
the detector chamber was shielded with three layers of copper , cadmium , and lead of 30 mm , 3 mm , and 100 mm thick , respectively .
the -ray energy calibration was performed daily using standard radiation sources of ba , cd , mn , co , co zn , na , and cs .
efficiency calibration was determined using in situ object counting - system ( isocs ) of genie 2000 software . the activity concentration ( in bq kg ) , a
ei of a radionuclide i and for a peak of energy e , in a samples was calculated using the following formula :
( 1)aei = neidemst ,
where n
ei is the net peak area at energy e ,
e is the detection efficiency at the energy e , t is the counting live time in seconds ,
d is the gamma ray yield per disintegration of the specific radionuclide for a transition at energy e , and m
s is the mass of the dry weight in kg of the measured sample . the iaea soil 375 was used as a standard reference material to evaluate the precision and accuracy of the gamma results .
the standard reference material was counted at the same time as used for counting the samples ( 8 hours ) , and then its activity at various energies was calculated and compared with the certified value after correction for the decay by considering the date ( december 31st , 1991 ) provided in the data sheet . as shown in table 1 , the experimental concentration values agreed well with the recommended values within 10% accuracy .
the average natural radionuclide activity concentrations in the soil samples collected from the three zones at the likuyu village are reported in table 2 .
specific activities concentrations of u , th , and k are reported in bq kg dry weight .
the lowest mean values of all three radionuclides were found in samples from the central part of the village . the northern zone had the highest concentrations of u and k , which were each 1.2 times higher than their values determined in samples from the central zone .
this value was 1.3 times higher than the mean value obtained in samples collected from the central zone .
rivers mkuju and kilowero flow from the mkuju uranium deposit to the northern and southern zones , respectively .
higher values of radionuclides in samples from these zones compared to the values determined in samples from central zone might indicate the rivers transportation of radionuclides from mkuju uranium deposit to the village .
the mean activity concentrations of natural radioactivity found in the soil in the present study were compared with the range and average of natural radioactivity concentration levels in soils of eight countries reported by unscear ( 2000 ) ( table 3 ) .
the mean concentration of u found in this study was found to be higher than its mean concentrations in soil of 75% of the countries reported by unscear ( 2000 ) .
the value is also higher than the world average value ; however , it lies within the ranges of u revealed in soils of all countries in the unscear report ( unscear , 2000 ) .
the mean concentration of th obtained in the study is comparable to the world average value but lower than the mean concentrations in 63% of the countries reported by unscear ( 2000 ) .
the average natural radionuclide activity concentrations in the water samples collected from the two rivers in likuyu village are reported in table 4 .
specific activities concentrations of u , th , and k are reported in bq l. as table 4 shows , samples from w1 and w2 had similar concentration of each radionuclide u , th , and k. the concentration of u and th was similar to the concentration in the control sample collected from nduluma river in arusha , whilst the mean activity level of k was 5 times higher than that of the control sample .
figure 1 compares the mean concentration of radionuclides from the two rivers in this study with those obtained in rivers of 5 countries reported in the literature .
the mean concentration of u from the two rivers in likuyu was similar and approximately 4 times higher than the activity value reported by faanu et al .
this value was only 1.5 times higher than the value reported by el arabi et al .
the mean value reported in this study was also 3 times lower than the value reported in water samples collected from a river in jordan . the mean concentration level of th found in this study was lower than the mean activity reported in pakistan , jordan , and nigeria , but slightly higher than the value reported in ghana and egypt .
this study determined the activity concentrations of natural occurring radionuclides in soil and water samples collected at likuyu village which is 54 km east of the mkuju uranium deposit in southern tanzania .
the measurements were carried out using low level gamma ray spectrometry technique at the taec laboratory in arusha .
the mean concentration of u ( 51.7 bq / kg ) soil analyzed in this study was found to be higher than its mean concentrations in soil of 75% of the countries reported by unscear ( 2000 ) .
the value is also higher than the world average value ; however , it lies within the ranges of u shown in soils of all countries in the unscear report ( unscear , 2000 ) .
the mean concentration of th obtained in the study is comparable to the world average value but lower than the mean concentrations in 63% of the countries reported by unscear ( 2000 ) . in this study ,
the highest mean values of all three radionuclides were found in samples from northern and southern parts of the village , which might be influenced by the flow of rivers mkuju and kilowero from the mkuju uranium deposit to the village .
the results of radioactivity concentrations in the water sample obtained in this study were comparable with the concentration values of natural radioactivity reported in the literature .
moreover , their concentrations were similar to those obtained in the control sample collected from nduluma river in arusha .
this is because during rainy season rivers might have elevated concentration of radionuclides due to the soil erosion at mkuju uranium deposit .
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the discovery of high concentration uranium deposit at mkuju , southern part of tanzania , has brought concern about the levels of natural radioactivity at villages in the neighborhood of the deposit .
this study determined the radioactivity levels of 30 soil samples and 20 water samples from likuyu village which is 54 km east of the uranium deposit .
the concentrations of the natural radionuclides 238u , 232th , and 40k were determined using low level gamma spectrometry of the tanzania atomic energy commission ( taec ) laboratory in arusha .
the average radioactivity concentrations obtained in soil samples for 238u ( 51.7 bq / kg ) , 232th ( 36.4 bq / kg ) , and 40k ( 564.3
bq / kg ) were higher than the worldwide average concentrations value of these radionuclides reported by unscear , 2000 .
the average activity concentration value of 238u ( 2.35
bq / l ) and 232th ( 1.85
bq / l ) in water samples was similar and comparable to their mean concentrations in the control sample collected from nduluma river in arusha .
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the world health organization ( who ) estimated in a systemic review that glaucoma is the second commonest cause of blindness worldwide . in spite of this data
, glaucoma was not included in the top priority five diseases for the first phase of the vision 2020 program of africa .
this could be partly explained by the uncertainties that have existed concerning the evidence for case detection . with the scarcity of population - based surveys ,
many studies have assessed the clinical impact of glaucoma as regards visual fields and intraocular pressure as primary outcomes .
this is optimum for developed countries but for developing countries , visual acuity as a primary outcome of assessment for glaucoma needs to be also studied .
the aim of this clinic - based study is to assess patterns of visual acuity and blindness in glaucoma patients and to find out the bulk they represent in comparison to nonglaucoma patients in the same practice setting .
patients attending cairo university hospital 's ophthalmic clinics were enrolled in this cross - sectional clinic - based study .
inclusion criteria included all patients attending the ophthalmic outpatient clinic for one day of work , only one clinic for each unit , to avoid detection bias ( ophthalmology departments consist of subunits with each unit running separate clinics ) .
tenets of helsinki declaration and the world medical association declaration on ethical considerations regarding health databases were respected .
clinical data collected for these patients included their demographic data , best obtained visual acuity , and whether or not they have had an established diagnosis of glaucoma .
ophthalmologists who collected the data did not participate in data analysis nor their interpretation to avoid performance bias . in this study ,
legal blindness was defined as visual acuity worse than 3/60 in the better seeing eye irrespective of the status of the visual field .
visual impairment was defined as visual acuity 3/60 or better but worse than 6/18 in the better seeing eye irrespective of the status of the visual field .
a totally blind eye was defined as an eye with vision of hand motions or less . legal blindness and visual impairment
the definitions of legal blindness and visual impairment were guided by the egyptian law for the measurement of visual disability , which is exclusively dependent on the visual acuity to identify the degree of disability .
the primary outcome from this study was to identify the levels of visual acuity affection in glaucoma patients in comparison to visual acuity of those without glaucoma in the same practice setting .
the secondary outcome was to know the prevalence of glaucoma patients in this practice setting .
nine hundred and eighty - eight eyes of 494 patients who had the mean age of 44.02 ( sd 18.8 ) years were enrolled in this survey .
female to male ratio was 1.2 : 1 and their mean visual acuity was similar in either eye , 6/24 ( 3 snellen lines ) .
the prevalence of patients with glaucoma was 8.1% ( confidence ; level 95% , interval 2.14.1 ) .
the differences between glaucoma and nonglaucoma groups as regards age and sex were not statistically significant .
patterns of visual acuities for glaucoma patients and nonglaucoma ones are shown in figure 1 .
the weights of visual impairment and legal blindness when assessed per patient between either groups were not statistically significant ( p = 0.074 ) ( figure 2 ) . when analyzing eyes with total blindness , glaucoma ones had shown high odds of getting total blind eyes and that was statistically significant ( table 1 ) .
one percent of africans are blind in whom glaucoma is the leading cause of irreversible blindness [ 5 , 6 ] .
the earliest population - based survey for the patterns of visual acuity and blindness in egypt was done between 1965 and 1968 .
they reported that the prevalence of patients with vision 1/60 or less were 16.7% higher in rural areas when compared to the urban areas , but they did not report the rates of legal blindness nor total blind eyes from glaucoma . however , we can find in their published tables that the prevalence of glaucoma was 2% in urban population and 9% in the rural one .
few regional clinic - based reports have evaluated blindness of glaucoma in egypt . in these surveys from different egyptian cities ,
the prevalence of blindness from glaucoma was 12.1% at mansoura in 2002 , 19.7% at alexandria in 1987 , and 7.6% at al - azhar university study ( cairo ) in 1989 [ 810 ] .
similarly , the proportion of blindness from glaucoma in clinic - based surveys from different african countries ranged from 10% to 33% . in a hospital - based study in nigeria , researchers reported that 23.1% of blind patients were due to glaucoma .
we found highly significant odds for recording eyes with total blindness in eyes with glaucoma ( 30% ) . in a hospital - based study in addis ababa ,
this could be either due to a delay in presentation , diagnosis , or many other factors .
the definitions of blindness at which visual acuities were assessed were not uniform among the previous studies . in the current report which was done in cairo ,
the better figures of legal blindness rates compared to earlier studies , either reflects improvements in glaucoma health care or was underestimated because we did not include visual fields in our legal blindness assessment , which would have increased those who were legally blind . amending the national laws and considering the status of visual fields when assessing the visual disability
is recommended to fulfill the requirements advised by the who regarding legal blindness and visual impairment .
the high rates of the totally blind eyes found for glaucoma in this study mandates the importance to investigate the factors behind these figures on a wider scale .
this necessitates the need for interventional strategies to manage the problem of glaucoma among egyptian patients .
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purpose .
glaucoma is the second commonest cause of blindness worldwide .
visual fields and intraocular pressures are optimum outcomes to be assessed in developed countries . visual acuity as an outcome is the key player when assessing blindness in developing countries .
the aim of this study is to assess visual impairment and blindness in glaucoma patients and to identify the bulk they represent in comparison to nonglaucoma patients in the same practice setting
. methods .
patients attending outpatient clinics of cairo university hospitals were enrolled in this cross - sectional study .
clinical data collected for these patients included their demographic data , best obtained visual acuity , and whether or not they have had an established diagnosis of glaucoma .
results were compared at 95% confidence intervals .
results .
988 eyes of 494 patients were included for this study .
their mean visual acuity was 6/24 ( 3 snellen lines ) .
legal blindness was found in 5% and 4% of glaucoma and nonglaucoma groups , respectively . there were high odds for finding eyes with total blindness due to glaucoma .
that was statistically significant ( p < 0.05 ) .
conclusions . a large proportion of glaucoma patients suffered from blindness in at least one eye .
interventional strategies are recommended regarding visual disability among egyptian glaucoma patients .
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c - reactive protein ( crp ) is a classical plasma protein marker that is markedly elevated in the acute phase of inflammation , infection , and tissue damage and thus has been broadly used for monitoring and differential diagnosis [ 1 , 2 ] . the major functions of crp include its ability to bind to various ligands exposed to damaged tissue or bacteria ( opsonization ) for the enhancement of phagocytosis and activation of the complement pathway , thereby enabling it to exert both anti- and proinflammatory functions [ 2 , 3 ] .
crp is mainly expressed by hepatocytes , and its synthesis is regulated at the posttranscriptional level by cytokines .
ample data from both clinical and experimental studies have shown that a high level of plasma crp is a risk factor as well as marker for cardiovascular diseases [ 59 ] , although some studies failed to prove the risk of crp compared to other risk factors .
the jupiter trial ( justification for the use of statins in primary prevention : an intervention trial evaluating rosuvastatin ) showed that a lipid - lowering drug , rosuvastatin ( crestor ) , can significantly reduce the incidence of major cardiovascular events , even in apparently healthy subjects not exhibiting established risk factors such as hyperlipidemia , but with elevated high - sensitive crp levels . regardless of this controversy , emerging evidence indicates that high levels of crp may be potentially atherogenic [ 11 , 12 ] .
studies of transgenic mice ( expressing either human or rabbit crp ) along with human crp transgenic rabbits and crp - deficient mice failed to provide a clear conclusion regarding whether crp is atherogenic [ 1323 ] .
the major concerns about these animal studies are as follows : ( 1 ) mouse endogenous crp is not physiologically active in vivo and ( 2 ) transgenic proteins are exogenous to animals , which may complicate the evaluation of crp pathophysiological functions in these models . in our previous study , we found that whhl rabbits are an excellent model for the study of crp and its relationship with atherosclerosis because they have higher levels of plasma crp and immunoreactive crp proteins are present in lesions of atherosclerosis .
in addition , rabbit crp has 74% homology with human crp and rabbit crp levels are highly inducible and responsive during the inflammatory reaction . to examine whether crp is involved in the development of atherosclerosis and whether therapeutic strategies to lower crp levels are useful for treating atherosclerosis , we intravenously injected the rabbit crp antisense oligonucleotides ( asos ) into whhl rabbits
using two different - aged whhl models , we examined ( 1 ) whether crp asos could reduce the plasma levels of crp and ( 2 ) whether crp lowering would affect the initiation and progression of aortic atherosclerosis and coronary atherosclerosis .
however , we did not identity antiatherogenic effects of crp antisense , suggesting that crp is not an atherogenic factor or a therapeutic target for the treatment of atherosclerosis .
watanabe heritable hyperlipidemic ( whhl ) rabbits were bred in a closed colony at kobe university and housed in the animal facility of university of yamanashi with a 12 h light / dark cycle at 23c and 55% humidity .
they were fed with a standard chow diet ( cr-3 ) , containing 17.6% protein , 4.1% fat derived from soybean oil , and 10.1% fiber ( clea japan , inc . , tokyo , japan ) and had free access to water .
all animal experiments were performed with the approval of the animal care committee of the university of yamanashi and conformed to the guide for the care and use of laboratory animals published by the us national institutes of health . rabbit crp antisense oligonucleotides ( aso , 5ataagcaagcaaacaccc3 , no .
aso 280290 was selected among 100 candidate oligonucleotides and doses aimed at obtaining maximally inhibitory efficacy were screened using cultured rabbit hepatocytes . for in vivo studies ,
crp asos were dissolved in saline solution and intravenously injected into whhl rabbits through ear veins ( 60 mg / kg bw / week ) twice a week for 16 weeks .
to examine whether rabbit crp aso administration could affect the development of atherosclerosis , we designed and performed two experiments .
for the first experiment , we used young whhl rabbits aged 3~4 months ( n = 9 for each group , containing 5 males and 4 females ) .
these whhl rabbits started to develop aortic atherosclerosis , which represents the so - called early - stage lesions , such as fatty streaks in humans .
therefore , we could examine whether crp asos had any effects on the initiation and prevention of atherosclerosis .
for the second experiment , relatively old whhl rabbits ( 811 months old , n = 10 for each group ) were used .
these old whhl rabbits developed advanced lesions , which are similar to human atherosclerotic plaques ( including lipid cores , fibrous caps , and shoulders ) . in this way , we could clarify whether crp aso had any influence on the progression of the advanced lesions of atherosclerosis .
plasma total cholesterol ( tc ) , triglycerides ( tg ) , and hdl - cholesterol ( hdl - c ) were determined using wako assay kits ( wako pure chemical industries , osaka , japan ) . for the analysis of lipoprotein profiles ,
plasma was resolved by electrophoresis in 1% agarose universal gels ( helena laboratories , saitama , japan ) , and then the gels were stained with fat red 7b .
plasma lipoproteins were also analyzed using high - performance liquid chromatography ( hplc ) by skylight biotech , inc .
plasma crp levels were quantified using crp enzyme - linked immunosorbent assay ( elisa ) kits ( shibayagi co. , ltd . ,
liver mrna expression levels of crp were analyzed by real - time reverse transcriptase - polymerase chain reaction ( rt - pcr ) , as described previously .
all rabbits were sacrificed after 16 weeks and their aortas and hearts were dissected for examination of atherosclerotic lesions using the standard method established in our laboratory [ 23 , 31 ] .
all values are expressed as the mean sd and statistical significance was determined using student 's t - test . in all cases ,
as reported in our previous study , whhl rabbits at 4 months exhibited higher plasma levels of crp than jw wild - type rabbits .
as shown in figure 1 ( left panels ) , whhl rabbits had about 10-fold ( in males ) and 20-fold higher crp ( in females ) levels at 4 months than jw rabbits ( 3.15 0.8 mg
16 weeks later , plasma levels of crp were further increased by 143% ( on average in males and females ) , suggesting that these increased crp levels of whhl rabbits were accompanied by ( or correlated with ) the development of aortic lesions induced by hypercholesterolemia .
treatment with crp asos for 16 weeks led to the reduction of plasma crp by 61% in males and 56% in females compared with that of the controls , although the differences were not significant ( p = 0.06 ) . reduced plasma levels of crp were consistent with lower hepatic expression of crp mrna , as shown by rt - pcr analysis ( figure 1 , right panels ) . in spite of this , we found that crp aso - treated whhl rabbits had somewhat higher plasma lipids including tc ( starting from 3 weeks , p < 0.05 ) and tg ( starting from 10 weeks , p < 0.05 ) than controls , ( figure 2 ) while hdl - c levels were unchanged ( data not shown ) .
analysis of lipoprotein profiles revealed that increased plasma lipids in crp aso - treated whhl rabbits were mainly caused by significantly increased very low density lipoproteins ( vldls ) ( p < 0.01 ) and chylomicron remnant contents ( p < 0.05 ) ( figure 3 ) . to elucidate the possible mechanisms , we measured the rates of vldl secretion in fasting animals in vivo using triton wr-1339 to block hydrolysis of tg - rich lipoproteins by lipoprotein lipase .
the base line levels of vldl - tg of aso - treated whhl rabbits were higher than those of controls ; however , vldl synthesis rate afterwards was similar to that in controls ( figure 3 ) .
after rabbits were sacrificed , we compared the aortic lesions and examined the histological features under a light microscope .
as shown in figure 4 , aso treatment did not change the aortic en face lesion areas in all parts compared to those of controls .
quantitative analysis of the microscopic lesions and macrophage and smooth muscle cell contents along with crp immunoreactive protein deposition did not reveal any significant differences between crp aso - treated whhl rabbits and controls ( figure 5 ) . in the second experiment
these old rabbits showed extensive atherosclerotic lesions in both aortas and coronary arteries accompanied by high levels of crp .
after treatment with crp asos for 16 weeks , plasma crp levels were consistently lower than in controls ( figure 6 ) .
similar to the first experiment , plasma levels of tc were higher in the crp aso - treated group than that in controls ( figure 7 ) , while tg and hdl - c levels were unchanged ( data not shown ) .
regardless of prominently lower crp levels , we did not find any differences between aso - treated and control groups in both aortic gross and microscopic lesion areas ( figures 8 and 9 ) .
histological examination revealed that crp immunoreactive protein contents were slightly reduced in the lesions of crp aso - treated whhl rabbits , but without statistical significance ( figure 9 ) .
because old whhl rabbits developed coronary atherosclerotic lesions , we further compared the coronary lesions ( expressed as stenosis percentage ) and found that left coronary stenosis was slightly less in crp aso - treated whhl rabbits , although the difference was not statistically significant ( figure 10 ) .
c - reactive protein ( crp ) is not only a predictor but also a potential risk factor of cardiovascular events .
several lines of evidence showed that crp may modulate the vascular functions and thereby influence the initiation and progression of atherosclerosis [ 11 , 34 ] . on the other hand , many controversial and contradictory results from both human and experimental animals have been published on the effects of crp on atherosclerosis [ 18 , 23 , 35 ] . in the current study , we first developed rabbit crp antisense oligonucleotides and then evaluated their effects on whhl rabbits , a well - established model for the investigation of atherosclerosis .
although crp asos could reduce the plasma levels of crp through inhibiting hepatic crp synthesis , we failed to demonstrate any beneficial ( antiatherogenic ) effects caused by crp lowering : crp aso treatment did not change the aortic and coronary atherosclerosis in two groups of whhl rabbits compared with that of controls . in spite of this , crp aso did not affect the lesion cellular components as well .
therefore , these results are consistent with our previous study using cholesterol - fed human crp transgenic rabbits and further strengthen the notion that crp is not an atherogenic factor but rather an inflammatory marker .
it is also unlikely that crp can be a therapeutic target for the treatment of atherosclerosis .
these observations are consistent with the current clinical trial results showing that crp inhibitors can reduce plasma crp levels by ~80% in normal subjects , as well as , endotoxin challenged and atrial fibrillation patients while other key cytokines , signs , and symptoms remained entirely unchanged in the endotoxin challenged subjects ( graham , m. personal communications ) .
it should be pointed out , however , that the current results can not rule out the possibility that crp may be involved in other inflammatory diseases .
unexpectedly , we found that crp aso treatment elevated plasma lipids in whhl rabbits due to enhancement of apob - containing particle production .
crp aso - induced lipid raising effect was not found in human clinical trials using crp asos .
it is currently unknown whether elevated plasma lipids are caused by crp inhibition or crp asos themselves ( such as off - targeting effects ) . in conclusion
, we found that crp lowering does not have significant influence on the initiation and progression of atherosclerosis in whhl rabbits ; thus , crp may not be a therapeutic target for the treatment of atherosclerosis .
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increased plasma levels of c - reactive protein ( crp ) are closely associated with cardiovascular diseases , but whether crp is directly involved in the pathogenesis of atherosclerosis is still under debate .
many controversial and contradictory results using transgenic mice and rabbits have been published but it is also unclear whether crp lowering can be used for the treatment of atherosclerosis . in the current study , we examined the effects of the rabbit crp antisense oligonucleotides ( aso ) on the development of atherosclerosis in whhl rabbits .
crp aso treatment led to a significant reduction of plasma crp levels ; however , both aortic and coronary atherosclerotic lesions were not significantly changed compared to those of control whhl rabbits .
these results suggest that inhibition of plasma crp does not affect the development of atherosclerosis in whhl rabbits .
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in a study reported in this issue of critical care , van de leur and colleagues investigated the experience of critical illness and intensive care unit ( icu ) support from the patient 's perspective .
although many workers have documented memories of a period spent in the icu , van de leur and coworkers attempted to relate memories of discomfort in icu to patients ' factual recall of the icu .
good factual recall was associated with increased risk for having memories of physical and/or psychological discomfort .
the relative frequency of hallucinations as a source of icu discomfort ( 32% ) appears greater than that found by others , although it must be highlighted that this is the percentage in the subset of patients with recollection of the icu a stratification not considered by other studies .
recollections of pain , discomfort and other stressors suggest an undesirable experience in icu , and may be the origin of sleep disturbances and other ( post - icu ) problems that affect quality of life .
a significant strength of the study is the early assessment of patients ( within 3 days of icu discharge ) using a structured questionnaire administered at a face - to - face interview .
the methodology is further enhanced by the use of an assessment of factual recall in a matched control group of general ward patients .
the fact that they were interviewed 3 days after hospital discharge by telephone rather than face - to - face is probably of little significance .
the tool used to assess factual recall was simple and would be easily transferable between units .
the authors acknowledge that a lack of objective sedation scoring was a weakness in the study . more detail on the relative balance between the sedation and analgesia received by study patients
the finding that young patients have better factual recall and more memory of discomfort may be due in part to a decreased sensitivity to sedation / analgesia relative to older patients .
it may also be , in part , a reflection that younger patients may be less intellectually impaired by a given physiological insult than more elderly patients .
a measure of the intensity of the remembered discomfort would also be useful in a study of this type ( e.g. using a visual analogue scale ) .
we might judge the recollection of occasional mild discomfort as a less worrying problem and might even view it as ( potentially ) unavoidable .
illness severity in this study was moderate , as evidenced by the modest acute physiology and chronic health evaluation ii scores on day 1 , relatively short length of stay ( los ) in the icu , and the ability of almost all patients to complete a structured interview within 3 days of icu discharge .
although this might be viewed as a weakness , it might in fact be a strength because such patients , when interviewed during recovery , may be expected to remember discomfort from a previous phase of illness more accurately than patients with high sickness severity scores with a long los in the icu . a cynical view after reading this study ( specifically the finding that lack of factual recall
was associated with less recollection of discomfort ) might be that it is better to use deeper sedation in icu patients .
however , such sedative practice has been shown to increase the duration of mechanical ventilation and los in the icu , which are known risk factors for nosocomial pneumonia [ 9 - 11 ] .
absence of memory for a period may produce a condition similar to post - traumatic stress disorder .
therefore , the use of high - dose sedation might be counterproductive in terms of post - icu psychological health .
this appears to be a significant ' quality of life ' problem for many patients after discharge from the icu . the use of diaries completed by relatives and/or staff to reduce the memory deficit may be useful in this context . from this paper we may conclude that the ideal icu sedative regimen should produce good analgesia in a patient who is ' tranquil ' but who retains mental clarity .
van de leur and colleagues previously suggested that discomfort due to the presence of a tube in the trachea may in fact be due to memories of tracheal suction .
it is somewhat surprising that chest physiotherapy was not identified as a source of discomfort in postoperative surgical patients .
we need to explore the use of suction regimens that are limited in terms of frequency or invasiveness in appropriate patients .
the finding that more than 50% of patients who remembered the icu had memories of discomfort is disappointing . to evaluate this problem fully , we must study different patient populations , using similar methodology to that used in the study by van de leur and colleagues , but including an objective measure of sedation / analgesia and a means to quantify the degree of discomfort remembered .
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many studies have documented patients ' distressing recollections of the intensive care unit ( icu ) .
the study by van de leur and colleagues , conducted in a group of surgical icu patients with moderate severity of sickness , found that the frequency of such unpleasant memories was increased in those able to recall factual information about their stay in the icu .
the study did not include sedation scoring but it did use a simple tool to assess factual recall .
this tool appeared reliable and could be easily applied in any icu .
previous work strongly suggests that abolishing memory of icu by using deep sedation would not be an appropriate response to these findings .
rather , we need to work on strategies that reduce distress by improving analgesia , reducing noxious stimuli ( if possible ) and , potentially , using pharmacology to produce a calm patient with minimal sedation . achieving the latter is rarely possible today but it might become possible with future drug development .
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serial cross - sectional surveys of wild bird populations throughout northern england were undertaken from july 2004 through january 2007 .
samples were collected from 441 individual wild birds of 42 species , including both migratory and resident species ( table 1 ) .
fecal samples and oropharyngeal swabs were collected from live birds that had been caught primarily for ringing purposes ( 8) and from dead wildfowl and corvids provided by licensed shooters .
biometric data and information concerning the location where birds had been trapped or shot were recorded ( 8) .
samples were placed in virus transport media ( eagle minimum essential medium supplemented with 10% fetal calf serum , penicillin , and streptomycin [ 10,000 units penicillin , 10 g streptomycin / ml ] and amphotericin b [ 250 g / ml ] ) ; samples collected from the same wild bird species or genus were pooled in groups of 5 and frozen at 80c until required .
viral rna was extracted from pooled fecal samples and pooled oropharyngeal swabs by using a qiaamp viral rna mini kit ( qiagen , crawley , uk ) following the manufacturer s instructions .
the primers utr41 + ( 5-atgtctatcgccagggaaatgtc-3 ) and utr11- ( 5-gctctaactctatactagccta-3 ) targeted the 3 untranslated region ( utr ) of the coronavirus genome , which is highly conserved among all known types of ibv ( 9 ) .
this procedure was followed by use of a heminested pcr with the same forward primer but the reverse primer utr hemi- ( 5-cttaaactaaaatttagctcttcc-3 ) under the same reaction conditions as the initial pcr , which had an expected product size of 214 bp .
pcr products were purified by using a commercial purification kit ( qiaquick pcr purification kit ; qiagen ) according to the manufacturer s instructions and were sequenced commercially ( cogenics , essex , uk ) as recommended by the manufacturer ( abi 3730xl dna analyser ; applied biosystems , warrington , uk ) .
nucleotide sequences derived from this study have been deposited in the genbank sequence database under accession nos .
sequences were aligned with previously published coronavirus sequences obtained from genbank by using the clustal program within the mega 4.0 package ( 10 ) ( appendix figure ) .
phylogenies were estimated by using a minimum - evolution method ( 11 ) , and evolutionary distances were computed by using the tamura - nei method ( 12 ) .
phylogenetic trees were drawn to scale ; branch lengths in the same units as those of the evolutionary distances were used to infer the phylogenetic tree .
animal - level prevalences and confidence limits , based on pooled samples , were estimated by using a pooled prevalence calculator ( www.ausvet.com.au/pprev ) .
generalized linear modeling was used to calculate maximum - likelihood estimates of prevalence and confidence limits ( 14 ) .
coronavirus rna was detected in 7 fecal sample pools ( table 2 ) , giving an individual animal
level prevalence estimate of 1.6% ( 95% confidence interval 0.73.1 ) . of the pools with positive results for coronavirus ,
4 were collected from ducks ( designated anas / uk / p20/2005 , anas / uk / p33/2005 , anas / uk / p42/2005 and anas / uk / p71/2005 ; table 2 ) .
another pool contained samples from whooper swans ( cygnus cygnus ) ( whooper swan / uk / p3/2005 ) , 1 consisted of samples from red knots ( calidris canutus ) ( red knot / uk / p60/2006 ) , and 1 contained samples from eurasian oystercatchers ( haematopus ostralegus ) ( oystercatcher / uk / p17/2006 ) .
pcr - positive pools were from birds sampled in estuarine , salt marsh , or standing water habitats ( table 2 ) .
phylogenetic analyses were based on a final usable sequence of 146 nt after removal of primer sites ( appendix figure ) .
sequences detected in 3 pooled duck samples and a sequence derived from a pool of whooper swan samples clustered with sequence from an ibv h120 ( massachusetts ) vaccine strain .
sequences within this cluster were relatively homogenous with low within - group distance values ( 0.0%2.8% ) . within this cluster , bootstrap support for the individual nodes was relatively low ( figure ) .
minimum - evolution tree ( 11 ) of coronaviruses based on a 146-bp fragment of the 3 untranslated region of infectious bronchitis virus ( ibv ) .
evolutionary distances were computed by using the tamura - nei method ( 12 ) and are in the units of the number of base substitutions per site .
the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test ( 1,000 replicates ) is shown next to the branches ( 13 ) .
the tree is drawn to scale ; branch lengths are in the same units as those of the evolutionary distances used to infer the phylogenetic tree .
coronavirus sequences detected in red knots clustered with a previously described goose coronavirus ; divergence at the nucleotide level was 2.0% .
sequences from viruses detected in samples from eurasian oystercatchers and ducks clustered with sequence from a published duck coronavirus ( 7 ) .
the sequence from eurasian oystercatchers was identical to that of the published duck coronavirus ; distance values within this cluster were 0.0%1.0% .
although samples were collected from wild bird populations comprising 46 species of wild birds from numerous and diverse habitats , coronavirus rna was detected only in wildfowl ( anseriforms ) and waders ( charadriiformes ) .
coronaviruses have been detected previously in wildfowl species , rock doves , wild peafowl , and some passerine species ( 47 ) .
all wild birds from which coronaviruses were detected in this study appeared to be healthy . although ibv is recognized primarily as a respiratory agent , it has been demonstrated that certain strains are able to replicate in the chicken intestine without obvious clinical disease ( 15 ) .
virus sequences from 3 pools of fecal samples from ducks and whooper swans shared high nucleotide sequence identity with sequence from the ibv h120 vaccine strain , which is commonly used for the vaccination of commercial chickens worldwide . coronaviruses sharing a high degree of identity with the ibv h120 vaccine strain have been detected previously in healthy , unvaccinated , domestic peafowl and as well as wild peafowl in china ( 4,5 ) .
these viruses may be revertant attenuated vaccine strains that have arisen as a result of the widespread use of ibv vaccines in the local poultry population in china . to understand their potential role as reservoirs of ibv strains , further surveillance for coronaviruses in wild bird populations is needed
. it would be useful to determine the number and genome position of accessory genes of the coronaviruses detected in wild birds and to compare them with those of ibv .
more detailed genetic characterization of the viruses detected including , for example , the s1 spike gene , is also needed . the detection of coronaviruses that appear to be related to ibv in wild migratory birds raises interesting questions as to their role in the transmission , dissemination , and evolution of ibv strains .
untranslated region of coronaviruses detected in wild birds in this study and other previously published group 3 coronavirus sequences from wild birds and a beluga whale .
sequences were aligned using the clustal program within the mega 4.0 software package ( 10 ) .
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infectious bronchitis virus ( ibv ) causes a costly respiratory viral disease of chickens .
the role of wild birds in the epidemiology of ibv is poorly understood .
we detected diverse coronaviruses by pcr in wildfowl and wading birds in england .
sequence analysis showed some viruses to be related to ibv .
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annually more than 12 million neonates are born prematurely that include 10 - 12 percent of births worldwide . some of these neonates undergo mechanical ventilation including endotracheal tube suctioning ( ets ) that is one of the basic steps in caring and maintaining air way .
even though ets is performed to maintain upper air way permeability , it is not a safe procedure and may lead to short and long term detrimental effects .
ets is one of the most frequent interventions in nursing and in fact it is the most important responsibility of nurses in intensive care units .
however , studies have shown that performance of nurses in this procedure is weak and in most wards there is no manual based on evidence about ets to guide nurses performance .
studies on adults have revealed that ets caused moderate to severe pain . what is painful for adults is painful for neonates as well , even if the neonate does n't show the physiologic and behavioral symptoms .
today , there is sufficient evidence that pain , especially due to repeated routine painful procedures like ets is traumatic in neonates , increasing the likelihood of short and long term complications .
pain increases demands on cardio - respiratory system and elevates intracranial pressure , theoretically increasing the risk of intraventricular hemorrhage .
other studies have shown that there is a relationship between long term repeated pain during neonatal period and changes in pain sensitivity and pain processing later in life .
neonates , especially premature ones , are subjected to diagnostic and therapeutic procedures which are essential for their survival .
one of the most routine procedures done in nicu is nasal , pharyngeal and endotracheal tube suctioning . simons and his colleagues in 2003 showed that 63.6% of the procedures done on neonates are suctioning but often no releasing intervention was done for this repeated painful procedure .
lago et al showed that only 25% of nicus in italy had policies for acute pain control and 50% for chronic pain and health care providers took measures for pain control only in 14% of ets procedures .
studies showed that administering analgesic drug before ets and non - pharmacological intervention after that could not decrease the pain of ets .
therefore , given that the underlying factors , such as nurses skills in performing procedures , affect the pain , we made an attempt to improve the performance of the nurses by evidence based education and assessed the pain in neonates .
a quasi - experimental study carried out in august 2009 in nicu ( level 3 ) in zeinab ( pbuh ) hospital of shiraz .
this ward was equipped with 8 beds with mechanical ventilation , pulse oximetry and central monitoring .
the equipment were controlled before the study and all of them were calibrated and scot free .
there was no protocol for pain control in this ward and nothing was done to control the pain of neonates during ets .
inclusion criteria for nurses were consent form and working in nicu . first , one of the researchers observed the performance of 25 nurses during ets with a checklist including 43 items with maximum score of 43 which were developed by literature review and validated by expert panel .
its content validity was confirmed by a team of 7 nursing specialists and its inter - observer reliability was 93% .
the neonates had a gestational age of less than 37 weeks , were intubated ( at least for 8 hours and up to 24 hours ) and did not get any analgesics .
the exclusion criteria were suffering from ivh grade 3 and 4 , having chest tube and being complicated due to ets such as hemorrhage and pneuomothorax .
pain was assessed using premature infant pain profile ( pipp ) one minute before , during and 5 minutes after ets .
it has two physiologic indicators ( heart rate and oxygen saturation ) , three facial indicators ( brow bulge , eye squeeze and nasolabial furrow ) , and two contextual factors ( gestational age and behavioral state ) .
pain scores 6 indicate minimum pain or no pain , 7 - 12 indicate mild to moderate pain and scores > 2 indicate moderate to severe pain .
this tool was translated into persian and back translated to english by a professional translator ( content validity ) .
intra - reliability coefficients analysis was equally high at 0.94 - 0.98 . in a 6 hour workshop , the standard principle of ets in neonates
one day following the education , the performance of the nurses in ets was observed and pain score of the neonates was assessed using pipp .
data were gathered only in the morning for controlling confounding variables such as light , noise , crowding , etc , before and after education .
this study was approved by the research and ethics committee of shiraz university of medical sciences and consent forms were gathered from nursing staff .
ets is a routine procedure in nicu and the researchers did not interfere in it , so obtaining consent form from the parents was not necessary .
chi square and t - test were used for controlling confounding variables before and after education .
wilcoxon test was used for comparison of performance scores of nurses before and after education .
friedman tests were used for comparing pain before , during and after ets and mann - whitney tests were used for comparing pain of neonates before and after education .
all the nurses were female with a mean age of 30.16.8 years and mean experience of 6.95.6 years .
wilcoxon test showed statistically significant differences between the mean score of nurses performance in ets before ( 20.63.12 ) and after ( 39.143.06 ) education ( p=0.001 ) . before education , 4% of the nurses and after that 72% of them assessed the neonatal need for ets , showing significant differences with mcnemar test ( p=0.001 ) .
demographic variables such as sex , gestational age , and weight , and intubation period of neonates were not significantly different ( table 1 ) .
demographic characteristics of premature neonates in different groups result of friedman nonparametric test showed that pipp1 , pipp2 and pipp3 were significantly different before and after education and wilcoxon test affirmed that the differences were significant between all pairs except pipp1-pipp3 after education ( p=0.61 ) ( table 2 ) .
mann - whitney test showed no significant differences between pipp1 before and after education ( p=0.25 ) but pipp2 and pipp3 were significantly different before and after education ( p0.01 ) ( fig .
1 ) .
comparison of pain score before and after education comparisons of pain scores in different times of endotracheal tube suctioning pipp : premature infant pain profile
ets procedure was performed in each neonatal ward differently , even every nurse uses her own methods and the standard instructions were not used . in this study before education , nurses performance in ets was poor and theoretical and practical education improved the performance .
they showed that the performance of nurses were poor in ets and in addition to theoretical education , practical education , administrative and prevocational support are also required to improve the performance and decrease ets complications .
painful procedures in nicu may be inevitable , so it is necessary that nurses balance the painful , but medically necessary care and employ painful procedures based on neonatal needs
. an outstanding result of this study is that before education , a small number of nurses ( 4% ) assessed neonatal need for ets while after the education the majority of nurses ( 72% ) did this assessment .
it can be inferred that education can improve nursing performance and prevent pain due to unnecessary painful procedures .
wood in her study showed that assessing patients need for ets reduced the number of procedures and decreased its complications .
this study showed that pain scores were different before , during and after ets before education .
neonates had no pain before ets or they had minimum pain , but they experienced moderate to severe pain during and mild pain after ets .
arroyo - novoa and colleagues ( 2007 ) showed that in 169 hospitals of canada , australia , england and usa , the severity of pain just before , after and ten minutes after ets were different and pain severity just after ets was more .
in addition 64% of patients reported moderate to severe pain related to ets . in our study after education , neonates experienced pain during ets but this pain was mild , in addition pain severity after ets was similar to that before ets .
these results revealed that education had effect on reducing pain severity during and after ets .
saarenmaa et al ( 2001 ) in a clinical trial administered ketamine for relieving pain of ets in the neonatal intensive care unit but the result showed that ketamine could not relieve pain and it had even no effect on the heart rate and blood pressure changes due to ets procedure .
also , cignacco et al ( 2008 ) used morphine before and multisensorial stimulation after ets procedure but none of them had an effect on the pain of ets , concluding that due to concerns about potential complications of narcotics especially in premature neonates , non - pharmacological interventions should be considered and performed . according to the results of this research and other studies , various interventions and preferably a combination of different strategies are recommended to stimulate various senses and relieve pain .
our data support the assumption that education on principals of ets could relieve pain in neonates during and after this procedure .
the finding that education could decrease complications is consistent with the results of mohamadi et al ( 1391 ) indicating that education could control the changes in the heart rate and oxygen saturation due to ets and decrease its complications .
therefore , it can be concluded that education on ets principles could decrease pain in premature neonates .
it is recommended that such education become a part of in - service education programs in nicu , and manuals and policies must be prepared in these wards .
the main limitations of the present study were lack of a suitable control group and a sample size that did not permit the meaningful evaluation of the large number of variables that can influence pain score
ets caused moderate to severe pain in premature neonates but performing a standard procedure based on neonatal need and improving nursing performance could decrease this pain . despite these interventions , neonates will still experience mild pain .
there is a need for more research to illuminate optimal pain management and strategies to relieve pain in neonates .
further studies can compare two groups of neonates with administration of analgesics and other groups with skillful nurses in suctioning practices .
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objectiveendotracheal tube suctioning ( ets ) is a painful and invasive procedure .
studies have shown that the performance of nurses in this procedure is weak , so we conducted a study to evaluate the effect of ets education for nurses on neonates pain in neonatal intensive care units ( nicus).methodsin a quasi - experimental study , performance of 25 nurses working in nicu was assessed before and after ets education by a checklist . in addition pain score of 50 neonates
was measured using pain assessment tool ( pipp ) one minute before , during and 5 minutes after ets .
the neonates had a gestational age of less than 37 weeks and were intubated ( at least for 8 hours and up to 24 hours ) .
a p. value of less than 0.05 was considered as statistically significant.findingsmean scores of nurses performance were significantly different before and after education ( p0.001 ) by wilcoxon test .
friedman test revealed that pipps before , during and after ets were significantly different before and after education ( p0.05 ) .
mann - whitney test showed no significant differences between pipps before ets , before and after education ( p=0.2 ) , but pipps during and after ets were significantly different ( p0.01).conclusionets causes moderate to severe pain in neonates .
education improved performance of nurses and decreased pain in neonates during and after ets . despite education
, neonates will experience mild pain during ets , so other interventions need to be considerate .
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approximately 20 to 50 % of maintenance hemodialysis ( hd ) patients suffer from protein - energy wasting ( pew ) , a condition of decreased body protein and fat mass that potently predicts morbidity and mortality in this population [ 13 ] .
muscle wasting is a key component of pew , and it adversely affects multiple patient - centered outcomes including muscle function , exercise performance , physical function , and quality of life ( qol ) [ 4 , 5 ] .
numerous epidemiologic studies have also consistently shown that reduced muscle mass is associated with decreased survival in end - stage renal disease patients , including those receiving hd , peritoneal dialysis , or kidney transplantation [ 614 ] . while various body anthropometry surrogates ( e.g. , mid - arm muscle circumference ) and sophisticated equations have been utilized to estimate muscle mass in these studies , serum creatinine has been found to be a reliable marker of muscle mass in dialysis patients with little to no residual kidney function .
indeed , a number of studies have shown that lower serum creatinine levels in hd patients are associated with increased death risk [ 6 , 7 , 11 ] .
recent data also suggest that decreased muscle strength may be an even more potent mortality predictor than muscle mass .
given its high prevalence and dire sequelae , considerable study has been made to understand the underlying etiologies of muscle wasting in hd patients , in order to identify strategies that correct and/or prevent this complication ( fig . 1 ) .
the pathophysiology of muscle wasting and weakness appears to be complex and multifactorial and may be attributed to one or more of ( 1 ) insufficient nutritional intake ; ( 2 ) catabolic effects of dialysis therapy ; ( 3 ) hormonal aberrations including low levels or increased resistance to anabolic hormones ( e.g. , testosterone , growth hormone , insulin - like growth factor-1 [ igf-1 ] ) , increased levels of catabolic hormones ( e.g. , cortisol ) , and possible thyroid hormone deficiency ; ( 4 ) chronic inflammation ; ( 5 ) metabolic acidosis ; and ( 6 ) concurrent comorbidities ( e.g. , diabetic gastroparesis ) [ 1 , 4 , 16 , 17].fig .
1risk factors and sequelae of muscle wasting in hemodialysis patients risk factors and sequelae of muscle wasting in hemodialysis patients muscle disuse due to reduced physical activity ( defined as any bodily movement produced by contraction of skeletal muscle that increases energy expenditure above a basal level ) is another important yet relatively under - appreciated risk factor for muscle wasting in hd patients .
epidemiologic data show that physical activity levels in hd patients are exceedingly low . among 1,547 ambulatory dialysis patients in the comprehensive dialysis study ,
physical activity scores ascertained by the human activity profile were below the fifth percentile of healthy individuals based on age and sex norms .
similarly , among 134 patients in the dialysis outcomes and practice patterns study , 64 % of patients had sedentary or low physical activity levels . while the 2005 kidney disease outcomes quality initiative clinical practice guidelines on cardiovascular disease in dialysis patients
recommend that nephrology and dialysis staff routinely counsel dialysis patients on increasing their physical activity levels , survey data has shown that less than one - third of nephrologists routinely recommend exercise ( a form of physical activity that is planned , structured , repetitive , and purposive in the sense that the improvement or maintenance of one or more components of physical fitness is the objective [ 23 , 24 ] ) .
this may in part relate to providers uncertainties regarding the benefits of exercise upon short- and long - term outcomes in hd patients as well as its optimal modality , prescription , and associated risks in this population .
a number of studies have examined various modalities of exercise training in dialysis patients over the past three decades .
while most studies have focused on aerobic exercise , which primarily improves cardio - respiratory endurance and fitness , there have been fewer studies of resistance exercise , which promotes muscle growth , mass , and strength ; has been shown to be an effective anabolic intervention in elderly patients and other chronic disease populations ; and is theoretically a more optimal exercise modality in enhancing physical function [ 4 , 26 , 28 ] .
furthermore , there has been particular interest in studying resistance exercise during or around the time of dialysis treatment , which is thought to enhance compliance and to counter - act muscle wasting at a time when catabolism is at its peak . in a sentinel study by johansen et al . , among 79 hd patients randomized to a 2 2 factorial trial of moderate - intensity intra - dialytic resistance exercise training and anabolic steroid administration ( nandrolone decanoate ) for 12 weeks , exercise resulted in increased quadriceps muscle cross - sectional area measured by mri , increased strength , and improved self - reported physical function .
however , resistance exercise did not increase lean body mass detected by dual - energy x - ray absorptiometry nor improve physical performance ( e.g. , walk test , stair climb , chair rise ) . shortly thereafter ,
an elegant study conducted by kopple et al . showed that , among 80 patients randomized to intra - dialytic strength / resistance training , endurance training , strength / resistance + endurance training , vs. no exercise training , those assigned to resistance or endurance training experienced increases in muscle mrna , muscle igf-1 protein , and lean body mass ascertained by anthropometric measurements .
however , other randomized controlled trials of moderate- to high - intensity resistance training alone , or in combination with intra - dialytic nutritional supplementation , have not shown improvements in muscle cross - sectional area nor lean body mass and have been inconsistent with respect to effects upon muscle strength [ 3133 ] .
hence , the utility of intra - dialytic resistance exercise as an anabolic intervention in hd patients has remained unclear . a recent study by kirkman et al . published in the journal of cachexia , sarcopenia , and muscle entitled anabolic exercise in haemodialysis patients : a randomized controlled pilot study has sought to address this knowledge gap by examining the impact of a novel intra - dialytic progressive resistance exercise training ( pret ) program on muscle volume , strength , and physical function in hd patients as well as in non - hd healthy patients during university visits . in this single - blind controlled study , 23
hd and 9 non - hd patients were randomized to pret , which consisted of thrice - weekly high - intensity leg press exercises ( three sets of eight to ten repetitions at 80 % of their predicted one - repetition maximum , which is the maximum weight that can be lifted one time with proper technique ) , vs. control ( sham ) therapy , which consisted of low - intensity lower body stretching activities using ultra - light resistance bands .
an important innovation of this study was to incorporate an incremental increase in the weekly training load / volume in the pret arm of the trial .
after a 12-week interventional period , pret resulted in a significant increase in ( 1 ) the primary outcome of thigh muscle volume ascertained by mri and ( 2 ) the secondary outcome of knee extensor strength measured by isometric dynamometer in both hd and non - hd patients .
furthermore , patients randomized to sham therapy , particularly those in the hd group , experienced clinically significant amounts of muscle volume loss
. however , pret did not enhance hd patients ( 3 ) performance in physical function tests ( e.g. , sit - to - stand , get - up - and - go , walk test ) or ( 4 ) self - reported qol ascertained by the short form-36 , although improvements in non - hd patients were observed .
this stands in contrast to prior studies in the elderly , those with other catabolic states , and dialysis patients in whom similar interventions have resulted in improved physical function [ 27 , 34 ] .
this study s discrepant findings may have been due to ( 1 ) selection of particular physical function assessment tests that have a high degree of variability , are subject to a ceiling effect , or have limited ability to capture improvements in activities of daily living as compared to other physical function instruments and metrics ( i.e. , self - reported physical function ) ; ( 2 ) type 2 error due to the small sample size of the pilot study ; or ( 3 ) true absence of effect on physical function and qol . several important contributions made by kirkman et al.s study should be noted .
first , their incorporation of a graded increase in weekly training load / volume in the study arm may be necessary to promote an adequate anabolic response in hd patients , and the absence of this intervention in prior studies of resistance training may in part explain the inconsistencies across these collective data [ 19 , 2933 ] .
second , while earlier studies of intra - dialytic resistance have employed combination anabolic interventions ( i.e. , anabolic hormone administration , intra - dialytic nutritional supplementation ) given concerns that exercise both stimulates muscle protein synthesis and breakdown , the findings of the kirkman et al .
third , while there may be theoretical concerns related to the risk of exercise in hd patients ( e.g. , particularly musculoskeletal injury , cardiac ischemia , and sudden cardiac death as well as exercise - related hypo- and hypertension , electrolyte abnormalities , and hypoglycemia [ 26 , 28 ] ) , the low frequency and rather mild nature of adverse events observed in hd patients assigned to the pret arm provides further reassurance that resistance exercise is safe in this population .
however , it is of our opinion that hd patients with underlying history or risk factors for cardiopulmonary disease and related comorbidities should undergo exercise testing prior to the initiation of a resistance exercise training program . taken together , these data corroborate that resistance exercise training administered at incrementally higher levels is an effective and safe anabolic intervention in hd patients . at this time , further study is needed to determine if these short - term augmentations in muscle volume and strength translate into improved physical function , qol , hospitalization and mortality risk , and cost - effectiveness .
|
muscle wasting is a common complication afflicting maintenance hemodialysis ( hd ) patients , and it is associated with decreased muscle function , exercise performance , physical function , and quality of life .
meanwhile , numerous epidemiologic studies have consistently shown that greater muscle mass ( ascertained by body anthropometry surrogates , body composition tests such as dual x - ray absorptiometry , and/or serum creatinine in patients with little to no residual kidney function ) is associated with increased survival in this population .
the pathophysiology of muscle wasting in hd patients is complex and may be caused by poor dietary intake , catabolic effects of dialysis therapy , hormonal alterations ( e.g. , decreased levels or resistance to anabolic hormones , increased levels of catabolic hormones ) , inflammation , metabolic acidosis , and concurrent comorbidities .
muscle disuse resulting from low physical activity is an important yet under - appreciated risk factor for muscle wasting .
intra - dialytic resistance exercise training has been suggested as a potential strategy to correct and/or prevent this complication in hd patients , but prior studies examining this exercise modality as an anabolic intervention have shown mixed results . in a recently published 12-week randomized controlled trial of a novel intra - dialytic progressive resistance exercise training ( pret ) program vs. control therapy conducted in hd and non - hd patients , pret resulted in increased muscle volume and strength in both groups . at this time
, further study is needed to determine if anabolic improvements imparted by resistance exercise translates into improved physical function and quality of life , decreased hospitalization and mortality risk , and greater cost - effectiveness in hd patients .
|
n - methylation
of lysine residues
on histone tails is a pivotal epigenetic mark that
is involved in defining both transcriptionally active and inactive
chromatin .
for instance , methylation at
lysine 4 of histone 3 ( h3k4 ) is associated with actively transcribed
gene loci , whereas methylation at h3k9 and h3k27 leads to reduction
in transcription .
methylation of histone
lysine residues is reversible and is mediated by histone lysine methyltransferases
and histone lysine demethylases ( kdms ) .
one class comprises the kdm1s ,
which are homologues
of the flavin - containing amine oxidases .
the other class comprises jumonji c ( jmjc)-domain containing demethylases ,
which belong to the family of fe(ii)/-ketoglutarate - dependent
oxygenases .
human jmjc - domain containing
demethylases can be divided into five subfamilies , i.e. , the kdm2/7 ,
kdm3 , kdm4 , kdm5 , and kdm6 subfamilies , according to sequence and
structural similarities . several
jmjc - domain containing demethylases such as kdm2 , kdm4 ,
kdm5 , and kdm7
for example , it has been reported that kdm7b ( also known
as jhdm1f , phf8 , kiaa1111 ) is associated with proliferation of prostate
cancer cells and osteosarcoma cells .
therefore ,
kdm2/7-subfamily inhibitors are of interest , both as tools for probing
the biological functions of kdm2/7 subfamily and also as candidate
anticancer agents .
several types of kdm inhibitors have been found
so far by us and others , including n - oxalylglycine ( nog , 1 ) , 2,4-pyridinedicarboxylic acid
( pca , 2 ) , ncdm-32 ( 3 ) , daminozide ( 4 ) , and gsk - j1 ( 5 ) ( chart 1 ) . among these , daminozide ( 4 ) has been reported as
a kdm2/7-subfamily selective inhibitor . daminozide ( 4 )
was once widely used as a plant growth retardant but later withdrawn
because of genotoxic concerns .
the genotoxicity
of daminozide ( 4 ) is proposed to be derived from its
1,1-dimethylhydrazine structure .
therefore ,
it is desirable to identify novel kdm2/7-inhibitors without the 1,1-dimethylhydrazine
structure for use as functional probes and for evaluation in cancer
therapies .
here we describe the identification of a novel kdm2/7 subfamily
inhibitor that shows antiproliferative activity .
the routes used for the synthesis of compounds 713 ( chart 2 ) ,
which were prepared for
this study , are shown in schemes 1 and 2 .
michael addition
of o - benzylhydroxylamine to tert - butyl acrylate 14 afforded amine 15 .
9-bromononanoic
acid sodium salt 16 was converted to alkyl carboxylic
acids 1922 by treatment with an
alkyl magnesium bromide 17 and dilithium tetrachlorocuprate .
9-phenylnonanoic acid 23 was obtained by the treatment
of 16 with phenyl magnesium bromide and ferric acetylacetonate
in the presence of tetramethylethylenediamine and hexamethylenetetramine
in thf .
carboxylic acids 1823 were
treated with amine 15 in the presence of edci and hobt
to give amides 2429 .
the benzyl
group of compounds 2429 was removed
by hydrogenation to give hydroxamates 3035 .
removal of the tert - butyl group of 3035 using hydrochloric acid afforded
the desired compounds 813 .
reagents and conditions : ( a ) o - benzylhydroxylamine ,
et3n , 1,4-dioxane , reflux ,
44% ; ( b ) rmgbr ( 17 ) , li2cucl4 ,
thf , 10 c , 7491% ; ( c ) phmgbr , fe(acac)3 , tmeda , hmta , thf , 0 c , 67% ; ( d ) edci , hobt , dmf , room temp ,
2472% ; ( e ) h2 , pd / c , acoet , room temp , 3495% ;
( f ) hcl , ch2cl2 , room temp , 2359% . reagents and conditions : ( a ) o - benzylhydroxylamine ,
et3n , 1,4-dioxane , reflux ,
59% ; ( b ) prmgbr , li2cucl4 , thf , 10 c , 29% ; ( c ) edci , hobt , dmf , room
temp , 65% ; ( d ) h2 , pd / c , acoet , room temp , 61% .
michael addition
of o - benzylhydroxylamine
to methyl acrylate 36 afforded amine 37 .
10-methylundecanoic acid 38 was prepared using the procedure
described for the synthesis of 1922 .
amine 37 was treated with 38 in the presence
of edci and hobt to yield compound 39 .
with the aim of identifying potent
kdm2/7 inhibitors , hydroxamate
derivatives , which have been previously prepared in work on kdm4 inhibitors , were screened for kdm7b- , kdm4a ( also known
as jmjd2a , jhdm3a)- , kdm4c ( also known as jmjd2c , jhdm3c , gasc1)- ,
and kdm5a ( also known as jarid1a , rbp2)-inhibitory activity .
we selected
kdm4a , kdm4c , and kdm5a for the primary inhibition selectivity studies
because they are relatively similar to kdm7b in that they have a plant
homeodomain .
compounds 14 were evaluated as positive controls for inhibition . in our
enzyme assays ,
compound 4 selectively inhibited kdm7b
in preference to kdm4 and kdm5 as reported before ( table 1 ) .
we also found that
compounds 1(7a ) and 2(7b ) did not inhibit kdm7b ( at 250 m ) ,
and compound 2 showed selectivity for the kdm4-subfamily
over kdm7b as for the reported kdm4 inhibitor 3(7d ) under the conditions tested . among the tested
compounds ,
compound 6 was found to inhibit kdm7b relatively
selectively although its potency was moderate ( ic50 = 24
m ) .
values are means of two experiments
that in all cases varied by 19% or less . the ic50 of 4 for kdm7b was reported
to be 0.55 m in alphascreen assays . to improve the potency
and selectivity of 6 for kdm7b ,
we investigated the binding mode of 6 with kdm7b by modeling
( figure 1 ) .
an
inspection of the simulated kdm7b / compound 6 complex
indicates that the hydroxamate group of compound 6 coordinates
to the fe(ii ) in a bidentate manner , via its carbonyl and hydroxyl
groups , and that its carboxylate group will bind to form hydrogen
bonds / electrostatic interactions with asn 189 , thr 244 , and lys 264
at the kdm7b active site , in a similar manner to the c-5 carboxylate
of 2-oxoglutarate .
most importantly , it is also suggested that the
alkyl chain of compound 6 binds to a hydrophobic pocket
formed by the side chains of ile 191 , tyr 234 , leu 236 , phe 250 , val
255 , tyr 257 , and phe 359 , which we propose is unique to kdm2 and
kdm7 ( supporting information figures s1s6 ) .
view of the conformation of compound 6 ( ball - and - stick )
docked into the kdm7b active site . on the basis of the structure of the modeled kdm7b / compound 6 complex , we designed and synthesized compounds 813 ( table 2 ) in which
the isopropyl group of compound 6 was replaced with various
hydrophobic groups that might interact with hydrophobic amino acid
residues more efficiently than compound 6 .
compound 7 was prepared to investigate the importance of the carboxylic
acid of the inhibitor .
values are means of two experiments
that in all cases varied by 30% or less . the ic50 of 4 for kdm7b was reported
to be 0.55 m in alphascreen assays . as shown in table 2 ,
the conversion of carboxylic
acid ( 6 ) to a methyl ester ( 7 ) significantly
reduced the kdm7b - inhibitory activity , supporting the proposed binding
mode of compound 6 .
compounds 813 were found to be kdm7b inhibitors . in particular , a pronounced
inhibitory effect ( ic50 = 1.2 m ) was observed with
the cyclopropyl - containing compound 9 , which was 20-fold
more active than 6 in kdm7b in maldi assays .
docking
of compound 9 in the active site of kdm7b suggested that
the cyclopropyl group of compound 9 interacts with the
phenyl - group of phe 250 through ch or cyclopropyl
interactions ( figure 2 ) .
furthermore , compound 9 inhibited kdm2a ( also known as jhdm1a , fbxl11 ) , kdm7a ( also
known as jhdm1d , kiaa1718 ) , and kdm7b , demonstrating inhibition of
all kdm2/7 subfamily members tested ( tables 2 and 3 ) .
compound 9 displayed
selectivity for kdm2/7 over kdm4a ( ic50 > 120 m ) ,
kdm4c ( ic50 = 83 m ) , kdm5a ( ic50 = 55
m ) , and kdm6a ( also known as utx ) ( ic50 > 100
m ) ;
note however different assays conditions were used ( see experimental section ) .
thus , compound 9 was the
most potent kdm7b inhibitor identified in the enzyme assays .
in addition ,
compound 13 showed comparatively high selectivity for
kdm2a over kdm7b and the other isozymes tested .
because the molecular
modeling suggest that the space around the cyclopropane ring of 9 is not so large in the hydrophobic pocket of kdm7b ( figure 2 ) , it may be difficult for compound 13 bearing a phenyl ring to have a conformation which can efficiently
interact with phe of the pocket .
on the other hand , kdm2a has a hydrophobic
pocket more spacious than kdm7b ( supporting information
figure s2 ) , in which the phenyl ring can efficiently interact
with hydrophobic amino acid residues of kdm2a
. these may be the reason
that compound 13 shows selectivity for kdm2a over kdm7b .
view of
the conformation of compound 9 ( ball - and - stick )
docked into the kdm7b active site .
values are means of two experiments
that in all cases varied by 30% or less .
to investigate whether compound 9 is
active as an
inhibitor of kdm7a and kdm7b in cells , we performed a cellular assay
with western blot analysis . because kdm7 is known to catalyze the
demethylation of h3k27me2 , the methylation level
of h3k27 in cells was analyzed . in this study , we used n2a cells because
it has been reported that kdm7 is expressed in the cells .
as figure 3 shows , the
level of h3k27me2 was dose - dependently elevated in the presence of 9 .
although the interpretation of changes in global histone n - methylation status can be complex , the elevation in the
h3k27me2 levels is consistent with kdm7 inhibition .
these results
suggest that h3k27me2 demethylation is inhibited by compound 9 in cells , and it looks to be useful as a tool for probing
the biological role of kdm7 .
western blot detection of h3k27me2 levels in
n2a cells after 24
h incubation with dimethyl ester prodrug of nog ( dmog ) and compound 9 .
although it has been reported
that rnai - mediated knockdown of kdm7b
suppresses the growth of some cancer cells , it remains unclear whether the demethylase function of kdm7b is
responsible for the suppression because rnai - mediated knockdown of
kdm7b should cause loss of not only the demethylase function but also
other functions of kdm7b , including those
relating to noncatalytic binding domains .
initially , we investigated
the n2a cell growth inhibition activity of compound 9 . n2a cell growth suppression by compound 9 was observed
( gi50 = 86 m ) ( figure 4 )
at the concentration range in which distinct h3k27 methylation was
detected on western blot analysis ( figures 3 ) .
thus , this may suggest the demethylase function of kdm7 is involved
in this cell growth , however , more potent and selective compounds
will be needed to fully elucidate this fact .
next , we carried out
cell growth inhibition assays of compound 9 as well as
prodrugs of 1 , 2 , and 3 using
hela cells and kyse150 cells ( table 3 ) .
it
has also been reported that knockdown of kdm4c decreases cell proliferation , however , the kdm4c inhibitors 2 , 3 ( table 3 ) , and their methyl
ester prodrugs ( gi50 > 500 m ) did not show any
effects
on the growth inhibition of tested cancer cells although they are
cell membrane permeable .
these results suggest
that the demethylase activity of kdm4c is not directly associated
with cancer cell growth , at least in some cell types . on the other
hand , cell growth suppression by the kdm2/7-subfamily inhibitor 9
furthermore , compound 9 caused h3k27
methylation both in hela cells and in kyse150 cells at the concentration
range in which the cell growth inhibition was observed ( figure 6 ) .
the data shown in table 3 indicate that kdm2/7 inhibitors are worthy of evaluation as candidate
anticancer agents .
growth - inhibitory activity
of compound 9 toward kyse150
and hela cell lines .
western blot detection of h3k27me2 levels in hela cells and kyse150
cells after 24 h incubation with compound 9 .
a recent study reported that kdm7b activates the
transcription
of the e2f1 transcription factor in hela cells , which promotes cell
cycle progression .
because compound 9 decreases the growth of hela cells with h3k27me2 accumulation
( table 3 ; figures 5 and 6 ) , we examined whether compound 9 down - regulates
the expression of e2f1 in hela cells by quantitative rt - pcr .
as shown
in figure 7 , compound 9 significantly
decreases the mrna level of e2f1 at 80 m in which the growth
of hela cells was affected .
these data suggest that the kdm7b - mediated
regulation of e2f1 gene expression may be one of the mechanisms of
growth regulation in some cancer cells .
we also investigated the effect of compound 9 on cell
cycle progression by facs analysis .
hela cells and kyse150 cells incubated
with 10 or 100 m of compound 9 for 24 h showed
a dose - dependent reduction in g2m phase , whereas
there was a dose - dependent increase in g0g1 phase ( figure 8) .
these results revealed
that hela cells and kyse150 cells cultured with compound 9 arrested in the g0/g1 phase of the cell cycle ,
which is consistent with the down - regulation of e2f1 by compound 9 ( figure 7 ) .
induction
of cell cycle arrest in hela cells and kyse150 cells
by compound 9 .
we have identified a novel kdm2/7 subfamily
inhibitor 9 , which should be useful as a lead structure
in the development of
more potent and selective kdm2/7 inhibitors .
such inhibitors are candidates
for anticancer agents as well as tools for studying the biological
roles of kdm2/7 subfamily in cells .
melting points were determined using a yanagimoto
micro melting point apparatus or a bchi 545 melting point
apparatus .
proton nuclear magnetic resonance spectra ( h nmr ) and carbon nuclear magnetic resonance spectra ( c nmr ) were recorded using a jeol jnm - la500 , jeol jnm - a500 , or bruker
avance600 spectrometer in solvent as indicated .
chemical shifts ( )
are reported in parts per million relative to the internal standard
tetramethylsilane .
elemental analysis was performed with a yanaco
chn corder nt-5 analyzer , and all values were within 0.4% of
the calculated values , which indicates > 95% purity of the tested
compounds .
high - resolution mass spectra ( hrms ) and fast atom bombardment ( fab )
mass spectra were recorded on a jeol jms - sx102a mass spectrometer .
purity tests by analytical hplc used a shimazu instrument equipped
with a ods-3 ( 4.6 mm 150 mm , gl science ) and eluted at 1 ml / min
with milli - q water and ch3cn , and all values 95%
purity .
preparative hplc was performed with a jasco instrument equipped
with a inertsil ods-3 ( 20 mm 250 mm , gl science ) and eluted
at 10 ml / min with milli - q water and ch3cn .
gradient conditions
of hplc were as follows : ( a is ch3cn containing 0.1% tfa
( cf3co2h ) , b is milli - q water containing 0.1%
tfa , c is ch3cn containing 0.1% fa , d is milli - q water
containing 0.1% fa ( hcooh ) ) ; gradient ( i ) , a 50% ( 02 min ) ,
a 50% to a 80% ( 220 min ) ,
a 80% ( 20 30 min ) , a 80%
to a 50% ( 3 40 min ) water b ; gradient ( ii ) , c 60% ( 0
2 min ) , c 60% to c 80% ( 220 min ) , c 80% ( 2030 min ) ,
c 80% to c 50% ( 3040 min ) water d ; gradient ( iii ) , c 50% ( 02
min ) , c 50% to c 80% ( 220 min ) , c 80% ( 2030 min ) ,
c 80% to c 50% ( 3040 min ) water d. reagents and solvents were
purchased from aldrich , tokyo kasei kogyo , wako pure chemical industries ,
and kanto kagaku and used without purification .
flash column chromatography
was performed using silica gel 60 ( particle size 0.0460.063
mm ) supplied by merck .
a solution of tert - butyl
acrylate ( 11.5 g , 90 mmol ) , benzylhydroxylamine hydrochloride ( 3.6
g , 26 mmol ) , and triethylamine ( tea ) ( 6.0 ml ) in dioxane ( 60 ml ) was
stirred with reflux for 19 h. the reaction mixture was poured into
water and extracted with acoet . the organic layer was separated and
washed with brine and dried over na2so4 .
filtration ,
concentration in vacuo , and purification by flash column chromatography
( acoet / n hexane = 1/4 ) gave 3.3 g ( 44% ) of 15 as yellow oil .
h nmr ( cdcl3 , 500
mhz , ; ppm ) 7.35 ( 5h , m ) , 5.83 ( 1h , s ) , 4.70 ( 2h , s ) , 3.17
( 2h , m ) , 2.50 ( 2h , m ) , 1.44 ( 9h , s ) . to the suspension
of 9-bromononanoic acid sodium salt
( 16 )
( 400 mg , 1.5 mmol ) in anhydrous thf ( 3 ml ) was added
the solution of li2cucl4 in thf ( 200 l ,
0.02 mmol ) at 10 c .
then , cyclopropyl magnesium bromide
( 4.6 ml , 2.3 mmol ) was added dropwise with vigorous stirring . the
color of the mixture changed from orange through green and blue to
white .
after addition of the grignard reagent , the temperature was
kept at 10 c for another 2 h. the reaction mixture was
then diluted with water and extracted three times with et2o , and the organic extracts were washed by saturated aqueous solution
of na2co3 .
the water layer was then acidified
by hcl and washed three times by et2o , and the combined
organic layers were washed with water and dried over na2so4 .
filtration and concentration in vacuo gave 19 ( 226 mg , 74% ) as a white solid .
h nmr ( cdcl3 , 500 mhz , ; ppm ) 2.35 ( 2h , t , j =
7.5 hz ) , 1.661.59 ( 2h , m ) , 1.321.21 ( 12h , m ) , 1.181.14
( 2h , m ) , 0.670.60 ( 1h , m ) , 0.410.35 ( 2h , m ) . a mixture of 19 ( 221 mg , 1.10 mmol ) , 15 ( 232 mg , 1.10 mmol ) , 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
( edci ) ( 320 mg , 1.70 mmol ) , and hobth2o ( 226 mg ,
1.70 mmol ) in dmf ( 5 ml ) was stirred at 80 c for 15 h. the mixture
was poured into water and extracted with acoet .
filtration , concentration in vacuo , and purification by silica gel
flash column chromatography ( acoet / chcl3 = 1/10 ) gave 115
mg ( 24% ) of 25 as a colorless oil .
h nmr
( cdcl3 , 500 mhz , ; ppm ) 7.38 ( 5h , m ) , 4.82 ( 2h ,
s ) , 3.943.88 ( 2h , m ) , 2.53 ( 2h , t , j = 7.0
hz ) , 2.35 ( 2h , t , j = 7.0 hz ) , 1.601.54 ( 4h ,
m ) , 1.41 ( 9h , s ) , 1.301.24 ( 10h , m ) , 1.191.15 ( 2h ,
m ) , 0.660.63 ( 1h , m ) , 0.400.36 ( 2h , m ) .
to a solution of 25 ( 116 mg , 0.27 mmol ) in 3 ml of acoet
was added 50 mg of 5% pd / c .
the mixture was stirred under h2 at room temperature for 1 h and then filtered through celite .
the
filtrate was concentrated in vacuo and purified by flash column chromatography
( acoet / chcl3 = 3/1 ) to give 72 mg ( 78% ) of 31 as a white solid .
h nmr ( cdcl3 , 500 mhz ,
; ppm ) 8.36 ( 1h , broad s ) , 8.13 ( 1h , broad s ) , 3.893.83
( 2h , m ) , 2.65 ( 2h , t , j = 6.0 hz ) , 2.462.40
( 2h , m ) , 1.601.54 ( 4h , m ) , 1.46 ( 9h , s ) , 1.401.25
( 10h , m ) , 1.191.15 ( 2h , m ) , 0.650.62 ( 1h , m ) , 0.400.36
( 2h , m ) . to a solution of 31 ( 69
mg , 0.20 mmol ) in dcm ( 1 ml ) was added 4n hcl in dioxane ( 1 ml ) in
a dropwise fashion with ice - bath cooling ; the mixture was stirred
at room temperature for 16 h. concentration in vacuo gave a colorless
solid .
the solid was recrystallized from acoet to give 13 mg ( 23% )
of 9 as colorless crystals : mp 8687 c .
h nmr ( cdcl3 , 500 mhz , ; ppm ) 3.94 ( 2h ,
m ) , 2.79 ( 2h , m ) , 2.42 ( 2h , m ) , 1.64 ( 4h , m ) , 1.371.29 ( 10h ,
m ) , 1.191.15 ( 2h , m ) , 0.64 ( 1h , m ) , 0.400.36 ( 2h ,
m ) .
c nmr ( cdcl3 , 125 mhz , ; ppm ) 176.88 ,
173.92 , 44.62 , 44.26 , 38.06 , 34.75 , 29.63 , 29.47 , 29.36 , 26.50 , 25.27 ,
24.63 , 10.96 , 4.36 . ms ( fab ) m / z 286 ( mh ) .
compound 8 was prepared from 18 using the procedure described for 9 ( steps
35 ) : yield 14% from 18 ( 800 mg , 4.6 mmol ) ; a
pale - pink solid ; mp 8485 c .
h nmr ( cdcl3 , 500 mhz , ; ppm ) 3.963.90 ( 2h , m ) ,
2.802.76
( 2h , m ) , 2.472.42 ( 2h , m ) , 1.701.50 ( 2h , m ) , 1.301.27
( 12h , m ) , 0.88 ( 3h , t , j = 7.0 hz ) .
c nmr ( cdcl3 , 125 mhz , ; ppm ) 177.20 , 174.61 , 44.50 ,
32.42 , 31.88 , 31.60 , 29.46 , 29.37 , 29.28 , 25.31 , 24.66 , 22.67 , 14.11 .
ms ( fab ) m / z 260 ( mh ) .
hplc tr = 10.37 min ( gradient
( i ) , purity 96.8% ) .
compound 10 was prepared
from 16 ( 400 mg , 1.5 mmol ) using the procedure described
for 9 ( steps 25 ) : yield 14% ; a white solid ; mp
101102 c .
h nmr ( cdcl3 , 500 mhz ,
; ppm ) 3.93 ( 2h , m ) , 2.79 ( 2h , m ) , 2.42 ( 2h , m ) , 1.72 ( 4h ,
m ) , 1.601.55 ( 3h , m ) , 1.531.48 ( 2h , m ) , 1.301.26
( 12h , m ) , 1.061.04 ( 2h , m ) .
c nmr ( cdcl3 , 125 mhz , ; ppm ) 176.76 , 174.28 , 44.62 , 44.23 , 40.18 , 36.26 ,
32.74 , 32.44 , 32.17 , 31.32 , 31.05 , 29.90 , 29.53 , 29.38 , 28.79 , 25.21 ,
24.62 . ms ( fab ) m / z 314 ( mh ) .
calcd for c17h31no4 : c , 65.14 ; h , 9.97 ; n , 4.47 . found : c , 64.75 ;
h , 9.95 ; n , 4.53 .
compound 11 was prepared
from 16 ( 2.0 g 7.7 mmol ) using the procedure described
for 9 ( steps 25 ) : yield 21% ; a white solid ; mp
9496 c .
h nmr ( cdcl3 , 500 mhz ,
; ppm ) 3.94 ( 2h , m ) , 2.81 ( 2h , m ) , 2.43 ( 2h , m ) , 1.691.67
( 7h , m ) , 1.261.17 ( 16h , m ) , 0.86 ( 2h , m ) .
c nmr
( cdcl3 , 125 mhz , ; ppm ) 174.35 , 168.35 , 44.57 , 44.33 ,
37.68 , 37.55 , 33.47 , 32.20 , 31.36 , 31.04 , 29.94 , 29.53 , 29.37 , 26.86 ,
26.78 , 26.47 , 25.31 , 24.63 . ms ( fab )
compound 12 was prepared
from 16 ( 400 mg , 1.5 mmol ) using the procedure described
for 9 ( steps 25 ) : yield 13% ; a white solid ; mp
8687 c .
h nmr ( cdcl3 , 500 mhz ,
; ppm ) 3.94 ( 2h , m ) , 2.79 ( 2h , m ) , 2.41 ( 2h , m ) , 1.681.55
( 8h , m ) , 1.491.37 ( 5h , m ) , 1.301.25 ( 10h , m ) , 1.181.11
( 4h , m ) .
c nmr ( cdcl3 , 125 mhz , ; ppm )
174.01 , 168.27 , 60.43 , 44.57 , 44.25 , 39.28 , 38.24 , 34.68 , 32.17 , 31.32 ,
31.04 , 29.96 , 29.55 , 29.37 , 28.58 , 27.42 , 26.59 , 25.29 , 24.63 . ms
( fab ) m / z 342 ( mh ) .
hplc tr = 21.64 min ( gradient
( iii ) , purity 98.9% ) . to a solution of 9-bromononanoic acid sodium
salt ( 16 )
( 1.0 g , 3.9 mmol ) , fe(acac)3 ( 67
mg , 0.19 mmol ) ,
tetramethylethylenediamine ( 57 l , 0.38 mmol ) , and hexamethylenetetramine
( 27 mg , 0.19 mmol ) in dry thf ( 6.0 ml ) was added phenylmagnesium bromide
( 4.87 ml , 5.0 mmol ) at 0 c .
the reaction mixture was then stirred
for 2.5 h , after which it was diluted with water and extracted three
times with et2o .
the aqueous
layer was then acidified by hcl and extracted three times with et2o , and the combined organic layers were washed with water
and dried over na2so4 . filtration and concentration
in vacuo gave 23 ( 610 mg , 67% ) as a yellow oil .
h nmr ( cdcl3 , 500 mhz , ; ppm ) 7.297.25
( 2h , m ) , 7.187.16 ( 3h , m ) , 2.60 ( 2h , t , j = 7.3 hz ) , 2.34 ( 2h , m ) , 1.641.61 ( 4h , m ) , 1.391.28
( 8h , m ) .
compound 13 was prepared
from 23 ( 604 mg , 2.6 mmol ) using the procedure described
for 9 ( steps 35 ) : yield 19% ; a white solid ; mp
8788 c .
h nmr ( cdcl3 , 500 mhz ,
; ppm ) 7.27 ( 3h , m ) , 7.187.17 ( 2h , m ) , 3.93 ( 2h , m ) ,
2.80 ( 2h , m ) , 2.59 ( 2h , t , j = 7.6 hz ) , 2.462.41
( 2h , m ) , 1.60 ( 4h , m ) , 1.31 ( 8h , m ) .
c nmr ( cdcl3 , 125 mhz , ; ppm ) 177.15 , 174.53 , 142.88 , 128.40 , 128.23 ,
125.57 , 44.40 , 35.96 , 31.83 , 31.47 , 29.34 , 29.27 , 29.16 , 25.28 , 24.68 ,
22.65 . ms ( fab ) m / z 322 ( mh ) .
compound 37 was prepared from
methyl
acrylate ( 36 ) ( 4.4 g , 51 mmol ) using the procedure described
for 9 ( step 1 ) : yield 59% ; a yellow oil .
h nmr ( cdcl3 , 500 mhz , ; ppm ) 7.377.28
( 5h , m ) , 5.82 ( 1h , broad s ) , 4.68 ( 2h , s ) , 3.67 ( 3h , s ) , 3.20 ( 2h ,
t , j = 6.4 hz ) , 2.60 ( 2h , t , j =
6.4 hz ) . compound 7 was prepared from 37 ( 3.1 g , 15 mmol ) using the procedure described for 9 ( steps 24 ) : yield 11% ; a colorless oil .
h nmr ( cdcl3 , 500 mhz , ; ppm ) 3.94 ( 2h , m ) , 3.73
( 3h , s ) , 2.75 ( 2h , m ) , 2.502.44 ( 2h , m ) , 1.621.56
( 4h , m ) , 1.51 ( 1h , sep , j = 6.7 hz ) , 1.301.25
( 8h , m ) , 1.201.10 ( 2h , m ) , 0.86 ( 2h , d , j = 6.7 hz ) .
c nmr ( cdcl3 , 125 mhz , ;
ppm ) 52.39 , 44.59 , 39.04 , 32.57 , 29.88 , 29.54 , 29.39 , 27.98 , 27.39 ,
25.30 , 24.68 , 22.66 . ms ( ei ) m / z 301 ( m ) .
the x - ray structure of kdm7b ( pdb
code 3kv4 ) was
used as a model for docking .
protein preparation , receptor grid generation ,
and ligand docking were performed using the software glide 3.5 .
the standard precision mode of glide was used to determine
favorable binding poses , which allowed the ligand conformation to
be flexibly explored while holding the protein as a rigid structure
during docking .
kdm7b ( 0.5 mg / ml ) was incubated
with 150 mm kcl , 2.5% glycerol , 0.5 mm dithiothreitol , 0.05 mm pmsf ,
2.5 mm glutathione reduced form , 20 m ( + ) -fe(ii)-l - ascorbic acid , 20 m zncl2 , 0.5 mm ascorbic acid ,
0.5 mm 2-oxoglutarate , and 5 m h3k4me3k9me2 ( art(kme3)qtar(kme2)stggkaprkql - cys )
for 1 h at 37 c in 8 l of 10 mm tris - hcl buffer ( ph 8.0 ) .
the reaction was stopped by adding 75 l of matrix - solution
( 5 mg / ml -cyano-4-hydroxycinnamic acid , 37% acetonitrile , and
0.12% trifluoroacetic acid ) and then sonicated for 30 s. then 1 l
of the reaction mixture was spotted on the sample plate , dried , and
analyzed by matrix - assisted laser desorption ionization time - of - flight
mass spectrometry ( maldi - tof ms ) using voyager - de pro ( applied biosystems ) .
the kdm7b inhibition activity of the test compounds was calculated
from the remaining amount of h3k9me2 .
the 50% inhibitory concentration
( ic50 ) of the test compounds was calculated as the concentration
at which the half amount of h3k9me2 was removed compared to that removed
when the enzyme was added ( supporting information
figure s7 ) .
the kdm4c - inhibitory
activity
was measured using 0.6 mg / ml enzyme .
the final concentrations of dmso in the reaction mixtures
were less than 3.3% , and it was confirmed that 3.3% dmso did not affect
the kdm4c activity .
reaction mixtures ( 94.6 l ) , containing all of the materials
except h3k9me3 peptide and 2-oxoglutarate , were preincubated for 5
min .
then the reactions were started by the addition of 5.4 l
of a solution of 0.93 mm h3k9me3 peptide and 3.7 mm 2-oxoglutarate
.
the enzyme activity was determined as described above . the ratio
of the enzyme activity measured in the presence of inhibitor to that
of the control was plotted against log [ inhibitor ] . to confirm that
the reduction of the kdm4c activity by test compounds was not due
to inhibition of the coupled enzyme fdh , we examined the effects of
the test compounds on fdh activity .
the reaction mixture ( 0.1 ml )
contained 20 mm hepes - koh , ph 7.5 , 50 m formaldehyde , 1 mm
3-acetylpyridine adenine dinucleotide , 1 mm reduced glutathione , 0.1
mg / ml bsa , 0.1 mg / ml fdh , and a fixed concentration of 123 m
test compound .
the fdh activity in the presence of test compounds
was similar to that in the absence of the compounds .
the kdm4a activity was measured
by the fdh - coupled assay as described for kdm4c except that reactions
were performed in a final volume of 30 l in 384-well plate
( nunc ) and a final concentration of the kdm4a was 0.37 mg / ml . the kdm5a activity was measured
by the fdh - coupled assay as described for kdm4c except that reactions
were performed with h3k4me3 peptide in a final volume of 30 l
in 384-well plate ( nunc ) and a final concentration of the kdm5a was
0.64 mg / ml . the inhibitory
activities of
test compounds against kdm2a was assayed according to the method reported
in ref ( 4 ) .
the epigenase jmjd3/utx demethylase
activity / inhibition assay kit ( epigentek group inc . ) was used for
kdm6a enzyme assay .
the materials supplied with the kit , 100 m
of test compounds and kdm6a ( human , recombinant , bps bioscience , inc . )
( 300 ng / well ) were added to wells coating trimethylated histone substrate ,
according to the supplier s protocol .
the resulting mixtures
were incubated at 37 c for 120 min . after enzyme reaction ,
each
well was reacted with capture antibody for 60 min and with detection
antibody for 30 min .
finally , developer solution and stop solution
were added to wells in sequence and the absorbance ( 450 nm ) in each
well was measured with an arvo x3 microplate reader .
the kdm7a assay was run using
maldi and k27me2 peptide as a substrate under the same conditions
as used in ref ( 4 ) .
n2a cells , hela cells , or kyse150
cells ( 5 10 ) were treated for 24 h with inhibitors
at the indicated concentrations in opti - mem medium , then collected
and extracted with sds buffer .
protein concentrations of the lysates
were determined using a bradford protein assay kit ( bio - rad laboratories ) ;
equivalent amounts of proteins from each lysate were resolved in 420%
sds - polyacrylamide gel and then transferred onto nitrocellulose membranes
( bio - rad laboratories ) .
after having been blocked for 30 min with
tris - buffered saline ( tbs ) containing 3% skim milk , the transblotted
membrane was incubated overnight at 4 c with dimethylated h3k27
antibody ( cst ) ( 1:1000 dilution ) or h3 antibody ( abcam ) ( 1:100000
dilution ) in tbs containing 3% skim milk or tbs - tween 20 ( tbs - t ) .
the membrane was probed with the primary antibody , then washed twice
with tbs or tbs - t , incubated with goat antirabbit igg - horseradish
peroxidase conjugates ( diluted 1:2500 ) for 1.5 h at room temperature ,
and again washed twice with tbs and once with tbs - t .
the cells were plated at initial densities
of 5000 cells / well ( 100 l / well ) in 96-well plates in rpmi 1640
with 10% fetal bovine serum and allowed to attach overnight .
the cells
were exposed to inhibitors for 48 h at 37 c in 5% co2 incubator . a solution ( 5 mg / ml ) of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide ( mtt ) ( sigma ) was added ( 10 l / well ) and incubated with
the cells for 3 h before solubilization buffer ( 0.04 mol / l hcl2-propanol )
was added ( 100 l / well ) onto the cultured cells .
the solubilized
dye was quantified by colorimetric reading at 560 nm using a reference
wavelength of 750 nm .
absorbance values of control wells ( c ) and test wells ( t ) were measured . moreover ,
absorbance of the test wells ( t0 ) was
also measured at time 0 ( addition of compounds ) .
using these measurements ,
cell growth inhibition ( percentage of growth ) by a test inhibitor
at each concentration used was calculated as : % growth = 100[(t t0)/(c
t0 ) ] , when t > t0 and % growth = 100[(t t0)/t ] , when t < t0 .
computer
analysis
of the % growth values afforded the 50% growth inhibition parameter
( gi50 ) .
hela cells were treated
for 48 h with 0.238% dmso or compound 9 at the concentration
of 30 and 80 m , respectively .
total rna was isolated from hela
cells using rnazol ( molecular research center , inc . ) following the
manufacturer s protocol .
first - strand cdna synthesis from total
rna was carried out using revertra ace ( toyobo ) .
resulting cdna was
then analyzed by semiquantitative pcr ( semi - qpcr ) using 2720 thermal
cycler ( applied biosystems ) .
primers are specific for genes tested ,
and their sequences are as follows : gapdh 450bp primer(f ) : 5-tccaccaccctgttgctgta-3
( 20mer ) primer(r ) : 5-accacagtccatgccatcac-3 ( 20mer ) e2f1 435bp primer(f ) : 5-actcctcgcagatcgtcatcatct-3(24mer )
primer(r ) : 5-ggacgttggtgatgtcatagatgcg-3(25mer ) cycle parameters were 94 c for 2 min , followed by 28 ( e2f1 ) ,
20 ( gapdh ) cycles of 98 c for 10 s , 60 c for 30 s , and
68 c for 30 s , with a final extension at 68 c for 1 min . cells ( 5 10 ) were
treated for 24 h with compound 9 at the indicated concentrations
in rpmi 1640 with 10% fetal bovine serum , then harvested by trypsinization .
the cells were collected by centrifugation , fixed with ice - cold 70%
ethanol , washed with phosphate - buffered saline , and resuspended in
0.5 ml of phosphate - buffered saline containing propidium iodide ( 10
g / ml ) and rnase a ( 0.2 mg / ml ) . after a final incubation at
25 c for 30 min , the cells were analyzed using a jsan flow cytometer
( bay bioscience ) .
|
histone n-methyl lysine demethylases
kdm2/7 have been identified as potential targets for cancer therapies .
on the basis of the crystal structure of kdm7b , we designed and prepared
a series of hydroxamate analogues bearing an alkyl chain .
enzyme assays
revealed that compound 9 potently inhibits kdm2a , kdm7a ,
and kdm7b , with ic50s of 6.8 , 0.2 , and 1.2 m , respectively .
while inhibitors of kdm4s did not show any effect on cancer cells
tested , the kdm2/7-subfamily inhibitor 9 exerted antiproliferative
activity , indicating the potential for kdm2/7 inhibitors as anticancer
agents .
|
neck pain and its disability result in a significant burden on individuals and family
members , communities , health - care systems , and businesses1,2,3 .
recently , studies have been conducted in an attempt to identify the
disability of deep cervical muscles contributing to stabilization of the cervical spine .
a
comparison study reported that people with neck pain have reduced activity of deep neck
flexor ( dcf ) muscles with excessive activity of long - lever superficial muscles ( e.g. ,
sternocleidomastoid and anterior scalene muscles ) , resulting in unstable segmental motion in
the intervertebral joint during repetitive upper limbs tasks4 .
disability of the dcf muscles in people with neck pain is regarded as a general
consideration that a health professional might consider in planning a treatment strategy . in
a control trial ,
jull et al.5 reported
that targeted training of the dcf muscles in people with neck pain demonstrated a greater
reduction in score for the neck disability index ( ndi ) and intensity of neck pain .
however ,
for health professionals , the only feasible assessment method known to detect the activity
of the dcf muscles in a clinical setting , due to their location , is electromyographic
testing by applying needle or tube insertions , which is not a simplistic approach clinically
and legally .
therefore , in a clinical setting , an easy to apply approach may benefit rehabilitation
practices for the imbalance of muscle recruitment in neck flexor muscles due to lack of dcf
muscle activity .
ultrasonography is currently receiving attention as a tool for examination
of deep layer muscles , but few studies have successfully reported muscle recruitment and
neuromuscular responses of dcf muscles using ultrasonography6 .
the purpose of this study was to identify the imbalance of muscle
recruitment in cervical flexor muscles during the craniocervical flexion test ( ccft ) using
ultrasonography in people with neck pain and to use the findings to further guide explicit
exercise management for neck flexor disabilities .
the study subject were 18 students ( 9 males ) from d university gyeongbuk province with a
mean age , weight , and height of 21.03 2.67 years , 57.62 12.9 kg , and 168.01 7.61 cm ,
respectively .
participants were invited to participate in the study if they reported a
current neck pain score of 5 points or greater for the ndi ( total score is 50 points)7 .
participants were excluded if they reported
signs of neurological disorders , visited a health professional , or had experienced whiplash .
in accordance with the ethical standards of the declaration of helsinki , information on the
study
was provided to all the subjects , and informed consent was received from them prior to
participation in the study .
real - time ultrasonography images were obtained from the longitudinal cross section of the
right deep cervical flexor unit ( longus colli and capitis ) and right sternocleidomastoid
during the ccft in accordance with the previously established protocol6 .
a 7.5 mhz transducer of a z.one ultra convertible
ultrasound system ( zonare medical systems , inc .
, mountain view , ca , usa ) was positioned
diagonally against the front of the neck area running along the trachea s line and 5 cm away
from the midline of the cervical spine .
subjects were positioned in
the supine position with the head and neck in a mid position , and the task required subjects
to perform five gradually increasing movements of craniocervical flexion motion adjusted by
visual feedback from an air - filled pressure sensor right underneath their neck ; the applied
pressures were 22 , 24 , 26 , 28 , and 30 mmhg the images obtained from the trial were transmitted to the nih imagej software ( version
1.44 for windows ) and subjected to image analysis process to measure the muscle thicknesses .
a four - line grid with a 0.5 cm interval between lines was drawn on each image , and the first
line was centered in the middle of each image .
the average value for distance estimated from
the second , third , and fourth line was designated as the thickness of each muscle .
the
amount of change in muscle thickness at each pressure level from the reference pressure ( 20
mmhg ) was calculated by the following formula : ( muscle thickness at the target pressure
muscle thickness at the reference pressure ) / muscle thickness at the reference
pressure8 .
the imbalance of muscle
recruitment at each pressure level was represented as the ratio of the change in muscle
thickness of the sternocleidomastoid muscle ( scm ) to the change in muscle thickness in the
dcf muscles ( imbalance ratio : thickness of scm / thickness of dcf ) .
this study used ibm spss statistics for windows ( version 20.0 ) to analyze the data .
repeated measures analysis of variance ( anova ) was used to examine the ratio of the amount
of muscle thickness changes in two comparison muscles according to incremental cervical
flexion movements .
the muscle thickness ratio of the scm / dcf according to the incremental pressure showed
significant differences between 22 mmhg and 24 mmhg , between 24 mmhg and 28 mmhg , between 24
mmhg and 30 mmhg , and between 26 mmhg and 28 mmhg ( p<0.05 ) .
the muscle thickness ratio of
the scm / dcf was smallest at 24 mmhg ( table
1table 1.comparison of the ratio of dcf thickness to scm thickness according to pressure
increasepressure22 mmhg24 mmhg26 mmhg28 mmhg30 mmhg22 mmhg2.281.271.330.42 * 1.470.471.940.602.040.8124 mmhg1.470.471.940.60 * 2.040.81 * 26 mmhg1.940.60 * 2.040.8128 mmhg2.040.81*p<0.05 ( meansd ) .
in this study , ultrasonographic assessment of the muscle recruitment in cervical flexor
muscles was conducted in people with neck pain .
the secondary aim of this study was to
determine the clinical applicability of using ultrasonography in evaluating the disability
of cervical flexor muscles .
multiple studies have successfully visualized the recruitment
pattern of deep cervical muscles in noninvasive and low - cost manners6 , 9,10,11 , and the present study
highly suggested that ultrasonography could be used for this purpose .
the real values for the thicknesses of the dcf and scm gradually increased as the pressure
increased .
heightened recruitment of the scm during the ccft , was observed in the present
study , which is consistent with the results of previous studies5 , 12 , 13 .
nonetheless , the aim of this study , unlike the previous
studies , was to compare the muscle recruitment ratios of the superficial and deep cervical
muscles and to suggest the optimal level of pressure for balanced muscle recruitment during
the ccft .
as depicted in table 1 , the greatest imbalance
in muscle recruitment between the deep and superficial muscles during the ccft was revealed
at a pressure of 22 mmhg due to considerable thickness changes in the scm .
the second and
third greatest imbalances , also due to increased changes in recruitment of the scm , were 30
mmhg and 28 mmhg , respectively , while the dcf muscles maintained steady amounts of
recruitment changes at both of these pressure levels .
this finding suggests that health
professionals need to take into consideration differences in applied pressures during
interventions for patients with a disability of the dcf . as the activation of the scm during
the ccft is smallest at the pressures of 24 mmhg and 26 mmhg , a recommendation for an
adequate exercise intervention for dcf training might be established at a pressure level
between 24 mmhg and 26 mmhg .
although this study found great imbalances at pressure levels of 22 mmhg , 28 mmhg , and 30
mmhg , the standard muscle recruitment ratio between deep and superficial cervical muscles
was not evaluated in a healthy population . for a comprehensive assessment of the muscle
recruitment between symptomatic and non - symptomatic populations ,
|
[ purpose ] the purpose of this study was to identify the imbalance of muscle recruitment
in cervical flexor muscles during the craniocervical flexion test by using ultrasonography
and to propose the optimal level of pressure in clinical craniocervical flexion exercise
for people with neck pain .
[ subjects and methods ] a total of 18 students ( 9 males and 9
females ) with neck pain at d university in gyeongsangbuk - do , south korea , participated in
this study .
the change in muscle thickness in superficial and deep cervical flexor muscles
during the craniocervical flexion test was measured using ultrasonography .
the ratio of
muscle thickness changes between superficial and deep muscles during the test were
obtained to interpret the imbalance of muscle recruitment in cervical flexor muscles .
[ results ] the muscle thickness ratio of the sternocleidomastoid muscle / deep cervical
flexor muscles according to the incremental pressure showed significant differences
between 22 mmhg and 24 mmhg , between 24 mmhg and 28 mmhg , between 24 mmhg and 30 mmhg , and
between 26 mmhg and 28 mmhg .
[ conclusion ] ultrasonography can be applied for examination
of cervical flexor muscles in clinical environment , and practical suggestion for
intervention exercise of craniocervical flexors can be expected on the pressure level
between 24 mmhg and 26 mmhg enabling the smallest activation of the sternocleidomastoid
muscle .
|
a slow - growing member of the normal microbiota of sebaceous glands which may be beyond the reach of topical antiseptics , propionibacterium acnes , has long been recognized as a contaminant in clinical cultures . in specific clinical scenarios , however , p. acnes
recognition of the pathogenicity of p. acnes in such cases is dependent on its recovery from multiple separately collected specimens from a site expected to be sterile , in association with clinical symptoms . because p. acnes has traditionally been considered a contaminant , its recovery has not been historically emphasized in clinical microbiology laboratories . and
despite its recognition as a pathogen , it is also frequently isolated as a contaminant .
recent studies have informed strategies for ideal recovery of pathogenic p. acnes from clinical specimens [ 1 , 2 ] , but this issue remains a topic of controversy .
other findings classically associated with infection , including fever and elevated white blood count ( wbc ) , sedimentation rate ( esr ) , and c - reactive protein ( crp ) , are uncommon .
the absence of abnormal inflammatory markers and other typical signs of bacterial infection may lead to p. acnes infection being disregarded because , despite repeated isolation of this organism , other findings pointing to a diagnosis of implant - associated infection may be lacking .
current guidelines for prosthetic joint infection ( pji ) diagnosis rely on features that may be absent in p. acnes pji .
the reason for the paucity of acute inflammatory markers associated with p. acnes infection remains incompletely defined .
p. acnes secretes a number of proteins ; the p. acnes secretome includes 20 proteins .
the identity and function of only a few of these proteins have been described and characterized . among the proteins in the secretome
are the enzymes hyaluronidase , chondroitin sulfatase , and gelatinase [ 5 , 6 ] .
hyaluronic acid is present in tissues throughout the body , including bones and joints , contributing to the viscosity of synovial fluid and the lubrication of joints .
it is also a part of the extracellular structure of chondrocytes involved in tissue healing .
the ability of bacteria to degrade hyaluronic acid may be a virulence factor , enabling penetration of hyaluronidase - producing organisms into tissues rich in hyaluronic acid , including joints , creating an advantage for establishing growth in articular spaces .
in addition to p. acnes , other gram - positive pathogens , such as streptococcus pneumoniae , streptococcus intermedius , streptococcus constellatus , streptococcus dysgalactiae , staphylococcus aureus , peptostreptococcus species , clostridium perfringens , and clostridium septicum , as well as other species of propionibacterium , including p. granulosum , produce hyaluronidase . herein , we defined the prevalence of a hyaluronidase gene and of hyaluronidase production in a collection of clinical isolates of p. acnes .
we also defined the time course of in vitro hyaluronidase production by p. acnes using a subset of three clinical isolates .
197 clinical isolates of p. acnes were studied , including isolates associated with clinically significant monomicrobial infections , such as pji , spine infections , and endocarditis , as well as isolates from clinical specimens with polymicrobial growth and isolates representing contamination ( table 1 ) .
of the 197 isolates , 71 were associated with clinically significant growth , defined as growth of 20 colony forming units/10 ml sonicate fluid or 3 positive cultures .
all 197 isolates underwent molecular testing for a p. acnes hyaluronidase gene and phenotypic testing for hyaluronidase production ; three isolates underwent time - course studies for assessment of kinetics of hyaluronidase production .
p. acnes isolates were grown under anaerobic conditions in 2 ml brain heart infusion broth supplemented with 1% glucose , with agitation .
dna was extracted using the qiaamp ucp pathogen mini kit ( qiagen , valencia , ca ) with a final elution into 50 l elution buffer .
a 451-base - pair region of the hyaluronidase gene sequence was targeted by polymerase chain reaction ( pcr ) primers , forward primer 5-tttcgggatccttgtggta-3 , and reverse primer 5-tctggacaacaaacctgt-3. primer sequences were selected from the published p. acnes hyaluronidase gene sequence .
basic local assignment search tool ( blast ) analysis showed that the primer sequences had complete matches only with the two published p. acnes hyaluronidase gene sequences
. 5 l of isolated dna was amplified using 42 cycles as follows : melting at 95c for 30 seconds , annealing at 54c for 60 seconds , and amplifying at 72c for 120 seconds .
the final product was run on a 1% agarose gel and the product identified based on amplified product size . amplified dna from a representative isolate
hyaluronidase production was evaluated as described by smith and willett and used as a binary determinant of the ability of each isolate to produce hyaluronic acid .
briefly , 100 ml brain heart infusion media was prepared with 1 g of agar , autoclaved at 121c for 15 min , and cooled to 46c ; then , a 2 mg / ml solution of sodium hyaluronidase , boiled for 10 minutes to achieve solubility , was added to the cooled medium to a final concentration of 400 g / ml .
5% bovine albumin fraction v was added under constant stirring to yield a final concentration of 1% .
plates were poured to a depth of 3 - 4 mm and allowed to solidify at 4c .
p. acnes isolates were grown on sheep blood agar under anaerobic conditions until growth was observed .
a single colony was streaked onto the hyaluronic acid medium , incubated under anaerobic conditions , and observed daily until growth was observed ( 24 to 72 hours ) . on the day growth
was first observed , the plate was flooded with 2 n glacial acetic acid , which binds hyaluronic acid and albumin forming a white precipitate . hyaluronidase production was considered to have been present if a zone of clearing was observed . the method was modified from that described by tolksdorf and mccready .
the assay measures turbidity resulting from hyaluronic acid mixed with albumin in an acidic solution ; hyaluronidase activity is measured as lack of turbidity .
louis , mo ) was used for constructing the standard curve and for experimental readings , and hyaluronidase from bovine testes ( sigma ) was used to define a standard curve of enzyme concentration / activity ( figures 1(a ) and 1(b ) ) .
based on these standardized curves , the limit of detection was 0.0125 g/l hyaluronidase , and the assay was fully saturated at 0.05 g/l hyaluronidase ( figure 1(b ) ) . to quantify production of hyaluronidase over time , three clinical isolates of p. acnes
one was selected based on its lack of production of hyaluronidase using the plate method .
the other two produced hyaluronidase , as determined using the plate method , one of which was observed to grow quickly ( idrl-7844 ) and the other of which took longer than 48 hours to exhibit visible growth ( idrl-7751 ) .
the isolates were suspended in brain heart infusion broth supplemented with 1% glucose to a turbidity of 1.0 mcfarland and diluted 1 : 50 with glucose - supplemented brain heart infusion broth .
200 l was inoculated in triplicate in a 96-well tissue culture plate , with 7 identical plates prepared in duplicate .
one half of the plates was incubated anaerobically using anaeropack system ( mitsubishi gas chemical america , inc .
, new york , ny ) , and one half was incubated aerobically . at 6 , 12 , 24 , 48 , 72 , 96 , 120 , and 144 hours
, a single plate was removed from each incubator , the od600 documented to assess planktonic growth , and the supernatant broth collected and filtered using a 0.2 m membrane filter to abort further enzyme production by removing viable bacteria and prevent turbidity of bacterial growth from influencing the od540 reading done to detect enzyme activity based on precipitation .
85 l of a 0.4 mg / ml solution of hyaluronic acid was combined with 85 l of sample broth supernatant and allowed to react at 37c for 10 minutes .
the samples were then treated with 135 l of an acidic albumin reagent described by tolksdorf and mccready made of 2.5 g bovine serum albumin , fraction v in 1000 ml of 0.5 m sodium acetate buffer at ph 4.2 , for 10 minutes , following which the od540 was measured ( figure 2 ) .
of the 197 clinical isolates evaluated , all ( 100% ) were positive for hyaluronidase gene ( figure 3 ) .
of the 197 isolates tested , 191 ( 97% ) produced hyaluronidase , characterized by a zone of clearing around colonies ( figure 4 ) .
the clinical isolates of p. acnes showed some variation in time to achieve robust bacterial growth .
most achieved detectable plate growth by 24 hours ; however , some took as long as 72 hours of incubation before detectable plate growth was observed
. when the two hyaluronidase - producing p. acnes isolates were grown anaerobically , hyaluronidase was detectable in solution by 96 hours and continued to accumulate over time ( figure 2 ) , suggesting that hyaluronidase production begins when the organism reaches logarithmic phase growth .
hyaluronic acid is a component of skin and extracellular matrix found throughout the body , primarily in soft tissue .
there are several distinct hyaluronidases including enzymes found in mammalian spermatozoa , in the saliva of parasites , such as leeches , and in bacteria .
bacterial hyaluronidases are similar to one another in that the end product of bacterial hyaluronidases is unsaturated disaccharides .
this is distinct from mammalian and parasitic hyaluronidases . a variety of gram - positive human pathogens excrete hyaluronidase , an enzyme that may enable bacterial spreading and tissue penetration .
these include species of streptococcus , staphylococcus , peptostreptococcus , and clostridium listed previously , in addition to p. acnes .
gram - negative bacteria may also produce hyaluronidase ; however , rather than excreting the enzyme extracellularly , the hyaluronidase they produce is a periplasmic enzyme and as such is less likely to aid in tissue penetration than is the enzyme produced by gram - positive bacteria .
approximately 50% of the hyaluronic acid in the human body is found in the skin , though it is rich in articular spaces as well . as such , organisms able to cleave hyaluronic acid
may be favored as agents of infection in these locations , aided by their enhanced ability to penetrate these spaces .
results of our investigation show that all tested clinical isolates of p. acnes have the described hyaluronidase gene , and most have the ability to cleave hyaluronic acid .
organisms that tested positive by pcr , but which were unable to cleave hyaluronic acid , may have mutations in the hyaluronidase gene or its regulators .
the ubiquity of the amplified genetic sequence is noteworthy as it suggests that the hyaluronidase is important to the core genome of p. acnes .
the widespread presence of this gene in p. acnes makes it appealing to consider it as a potential genetic target for detection of p. acnes in clinical specimens [ 11 , 12 ] . the nearly universal phenotypic ability to cleave
hyaluronic acid is notable not only as a potential functional virulence mechanism but also as a potential tool for determining whether p. acnes isolated from a clinical specimen is a contaminant or a pathogen ; given the results of the time - course studies in liquid medium , the enzyme may only be present in clinical tissue specimens in true infections . in our experiments using liquid media ,
this contrasted with the observed 24 to 72 hours it took for p. acnes to produce detectable quantities of hyaluronidase using the plate method .
the reason for the difference in time to detectable hyaluronidase production may be attributable to the lower inoculum used in the studies in liquid compared to solid media .
alternatively , it may be a consequence of the density of growth , which would be higher sooner on plates than in liquid medium .
in clinical specimens collected from discrete physiologic spaces from which p. acnes is isolated , the presence of hyaluronidase may , hypothetically , support the sustained presence and growth of p. acnes in that site , which would not be expected to be the case when the p. acnes isolated is a contaminant , since a newly introduced contaminant would not have had sufficient time to accumulate detectable quantities of hyaluronidase .
this may provide a tool for differentiating between contamination and sustained growth , even if no living organism is isolated from a specimen . of note
, other organisms are known to produce hyaluronidase , though their requisite growth conditions for hyaluronidase production and kinetics thereof are outside the scope of this paper . demonstrating that an enzyme such as hyaluronidase can be detected as a byproduct of p. acnes growth is a novel potential approach to detecting and characterizing infections and could set precedent for using byproducts of growth as a means of differentiating between contamination and sustained growth of this organism and others in clinical specimens . describing the secreted and excreted products associated with bacterial growth
the p. acnes secretome has been described and 20 proteins have been identified , though not all have been characterized .
exactly how unique each organism 's secretome is , is unknown ; however , it is likely that the secretome varies from organism to organism and could potentially be used as a fingerprint of growth to signify specific organism - types in clinical specimens , even in the case of nonculturable organisms . given a lack of information on the secretome of common pathogens , a discussion of the broader application of using secreted or excreted proteins for detecting and characterizing bacterial growth from clinical specimens remains academic , aside from the established use of urease production for diagnosing helicobacter pylori infection .
this may represent an approach to more broadly detect and characterize infection , particularly in cases where there is a question of potential contamination .
the environment of the articular space has been a subject of debate and the question has been raised over time as to what oxygen tension is present in the joint space .
it has been suggested that articular spaces may have lower oxygen content than other locations in the body .
our observation that p. acnes hyaluronidase is produced under anaerobic but not aerobic conditions ( figure 4 ) may have clinical relevance in the context of the environment in joint spaces and possibly in biofilms .
there are several limitations to our study . in cases where the gene segment of interest was present , but there was phenotypic negativity , we did not sequence the gene . also , quantification of hyaluronidase production was performed on just three isolates . further characterization of more isolates under a variety of conditions might be informative .
most clinical isolates of p. acnes are capable of cleaving hyaluronic acid , and all tested clinical isolates of p. acnes had a hyaluronidase gene .
the ubiquity of this characteristic in p. acnes isolates and the finding that the studied subset of isolates of p. acnes produced detectable quantities of hyaluronidase by 96 hours of sustained growth could hypothetically have diagnostic utility in differentiating between contamination with p. acnes and its presence as a pathogen in clinical specimens . in vivo and clinical studies would be useful to characterize the translation of our findings and to explore the use of excreted proteins in characterizing the significance of bacterial growth .
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objectives . we sought to describe the prevalence of a hyaluronidase gene and hyaluronidase production in 197 clinical isolates of p. acnes
; we assessed kinetics of hyaluronidase production in a subset of three isolates .
methods .
the hyaluronidase gene was detected using polymerase chain reaction .
hyaluronidase production was detected by growing isolates on bhi agar containing 400 g / ml hyaluronic acid and 1% albumin and flooding plates with 2 n glacial acetic acid to precipitate unbound hyaluronic acid , with a zone of clearing representing a positive phenotype .
hyaluronidase production kinetics were measured as a function of hyaluronic acid digestion over time in a liquid medium . results .
a hyaluronidase gene and hyaluronidase production were detected in 100 and 97% of p. acnes isolates , respectively .
hyaluronidase production in liquid medium was detectable after 96 hours of growth .
conclusions .
hyaluronidase production is nearly universal among p. acnes isolates .
three days appear to be required for significant hyaluronidase production in a liquid medium .
detection of hyaluronidase in tissue specimens may be a strategy to differentiate p. acnes infection from colonization when p. acnes is isolated from a clinical specimen .
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the small round cell tumors of kidney include wide range of entities with overlapping histomorphology and they have different prognostic values .
these are monophasic wilm s tumor , neuroblastoma , non - hodgkin s lymphoma , embryonal rhabdomyosarcoma , small cell neuroendocrine carcinoma , synovial sarcoma and ewing s sarcoma / peripheral primitive neuro ectodermal tumor ( pnet ) . among these entities ewing s sarcoma / pnet is an extraordinally rare tumor .
this aggressive tumor is more prevalent in adolescent and young adults with slight male preponderance [ 1 , 3 ] .
herein we report the clinical , histopathological and immunohistochemical finding in a new case of primary ewing s sarcoma / primitive neuroectodermal tumor of kidney .
a 24 year old young female has presented with pain in right lower abdomen for 2 months and a vaguely palpable lump in right lumber region .
ct scan of whole abdomen has shown a heterogenous enhancing mass of 7 cm 4.1 cm , involving right mid pole and indenting the calyceal system ( figure 1 ) .
no radiological evidence of hepatic or pulmonary metastasis , and perirenal and hilar lymphadenopathy has seen .
the patient has undergone radical nephrectomy . on gross examination after removal of renal capsule a 6 cm 5 cm mass has seen in the medial part of the kidney .
the cut section of the mass was grayish white with areas of focal hemorrhage , extending into renal pelvis .
both the upper and lower poles of the kidney have uninvolved ( figure 2 ) .
microscopic examination has shown proliferation of monomorphic small round cells arranged in vague lobulated pattern separated by thin firbrovascular septae .
the individual cells were small having scanty cytoplasm , round nucleus and small nucleoli . in areas these neoplastic cells
degenerated malignant round cells have seen which have associated by distinct vascular network , with thick wall and dilated lumina giving filigree pattern ( figure 3 , 4 ) .
immunohistochemically the tumor cells were diffusely and strongly positive for cd 99 ( figure 5 ) and negative for cytokeratin ae1/ae3 , cd 45 and wt-1 .
a diagnosis of extraskeletal ewing s sarcoma / pnet of kidney has made . the patient has undergone 6 cycles of chemotherapy with vac -ie protocol using cyclophosphamide ( 1200 mg / m2 i.v ) , doxorubicin ( 250 mg/ m2 i.v bolus ) , vincristine ( 1.4 mg / m2 i.v ) alternating with ifosfamide ( 1800mg / m2 ) and etoposide ( 100 mg / m2 i.v ) within two weeks interval .
the patient could not afford treatment after that and remained apparently disease free for 5 months .
the disease has continued to progress and later on , she has developed bilateral pulmonary metastasis and died on the 15th months after initial diagnosis due to several terminal complications .
apart from bony structures of pelvis , it might arise from non - osseous structures like gastrointestinal tract , kidney or adrenal glands .
25% of the patients have presented with metastasis at the time of diagnosis and the frequent sites of metastasis would be the lung , bone , and bone marrow .
the most important prognostic factors were the presence of metastasis at the time of presentation , location of the tumour , tumour size and the serum ldh level .
pelvic primary tumours have been more aggressive and have shown a poor prognosis in comparison with tumors of extremity .
primary renal ewing s sarcoma / pnet was an aggressive malignant tumor , and has male predominance with mean age of 28 - 34 years .
the previous reports have revealed the aggressive behaviour of this neoplasm with variability of its presentations .
this highly aggressive tumour has been often diagnosed in advanced stage when it has already involved adjacent structures of kidney or liver , spleen , peritoneum , and lungs .
the 5-years disease free survival rate would be around 45 - 55% in well - confined cases .
cases with advanced stage at presentation have shown a median relapse - free survival of only 2 years .
so the distinction from the other malignant neoplasms of kidney has been too much essential from the prognostic point of view .
small blue round cell tumor would be a diagnostic challenge in the field of renal histopathology because great number of other tumors have presented in almost a similar histomorphology under microscope .
this group includes ewing s sarcoma / pnet , non - hodgkin lymphoma , alveolar rhabdomyosarcoma , renal neuroblastoma , wilm s tumor , monophasic synovial sarcoma , desmoplastic small roumd cell tumor , carcinoid tumors , clear cell sarcoma of kidney .
the blastemal cells of wilm s tumor might mimic renal pnet , but presence of epithelial component especially tubular differentiation has ruled out renal pnet .
the homer wright type of rosettes commonly has scared of number or less defined in extra skeletal ewing s sarcoma were a typical histological feature for pnet and could address the diagnosis but they could also be found in neuroblastoma . apart from rosettes neuroblastomas might show neurofibrillary background .
most of the non - hodgkin s lymphomas have shown polymorphic histology with small lymphocytes in the background .
but filigree pattern in ewing s sarcoma/ pnet has not seen in the other small round cell tumors of kidney . for a better diagnostic approach
for this purpose the commonly used immunohistochemical markers were cytokeratin ( for nephroblastoma , small cell carcinoma , synovial sarcoma ) , cd45 or leucocyte common antigen ( for lymphoma ) , chromogranin or neuron specific enolase ( for neuroblastoma ) , cd 99 ( for ewing s sarcoma / pnet ) and wt-1 ( for wilm s tumor ) .
the bases of diagnosis of the present case as ewing s sarcoma / pnet were histomorphology of small blue round call tumor and cd 99 positivity immunohistochemically .
pnet of soft tissue and ewing s sarcoma of bone were pathologically equivalent entities as they share common genetic alteration .
bony counterparts have been more undifferentiated than the pnet of soft tissue which shown more neuroectodermal differentiation .
histogenesis of the renal ewing s sarcoma / pnet has been from the neural ramification investing the kidney .
the innervations of kidney have come from the adrenergic fibres originating be from the celiac plexus and accompanying efferent arterioles and descending vasa recta .
another possibility of histogenesis was the migration of the embryonic neural crest cells into kidney and subsequent malignant transformation .
apart from histopathology and immunohistochemistry , molecular analytic methods would be also important requirements for better confirmation of the diagnosis of es / pnet group of tumors .
more than 85% of ewing s sarcoma / pnet have characterized by presence of t(11;22 ) ( q24;q12 ) translocation that results fusion of ews gene located on chromosome 22 to fli1 gene on chromosome 11 resulting formation of chimeric product of ews - fli1 .
but here we could not perform molecular genetic methods because of poor socioeconomic condition of the patient and patient party .
management of renal ewings sarcoma / pnet has included surgery , chemotherapy , and radiation .
surgery would be an important method for local control of the disease . according to some school local radiotherapy to the surgical bed
the survival rate of the patients with local disease without metastasis at presentation , and who has received multimodality treatment was almost 70% where it has come down to 9 - 41% among the patients with metastasis at presentation . in the study of mukkunda et al .
7 patients with renal ewing s sarcoma have undergone median follow - up of 36 months ( range from 5 to 149 ) and had a median disease - free survival ( in patients with nonmetastatic disease ) of 30.35 months ( range from 5.1 to 149 ) with a 5-years overall survival rate of 42% .
99 study has recommended a multidrug chemotherapy , delivered before and continued after local control .
no randomized studies have been published in the setting of the rarity of renal ewings ; sarcoma / pnet .
most of the regimens have contained vincristine , doxorubicin , cyclophosphamide , ifosfamide and etoposide similar to gpoh - ewing sarcoma study protocols .
but the combination of used multiple chemotherapeutic agents have differed extensively inter - individually . despite receiving vac - ie chemotherapy protocol ,
our patient had extensive pulmonary metastasis with an apparent disease free period of 5 month proving the high fatality of the disease entity .
it has been much important to distinguish primary renal ewing s sarcoma / pnet from the other small blue round cell tumours by histopathological , immunohistochemical methods for its aggressive behaviour and therapeutic response . in absence of molecular genetic analysis in poor socioeconomic conditions
, careful histopathological and immunohistochemical studies might be valuable in the differential diagnosis of small blue round cell tumours of kidney and to overcome the diagnostic dilemma in cases of ewing s sarcoma / pnet .
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among the group of small round cell tumors of kidney ewing s sarcoma / pnet is a very rare entity which has aggressive clinical course .
we report a case of renal mass in 24 years old male which was histologically diagnosed as small round cell tumor of kidney .
arrangement of the malignant cell along with vascular network in a filigree pattern was suspicious for a diagnosis of ewing s sarcoma / pnet which was confirmed by positivity of cd 99 immunohistochemically .
thus careful histological observation and immunohistochemical stains can give the proper diagnosis of primary ewing s sarcoma / pnet thus removing the diagnostic dilemma in the group of small round cell tumors .
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flybase is a comprehensive resource of information on the insect family drosophilidae , and one of the major model organism database projects that support biomedical research ( see http://www.nih.gov/science/models/ ) .
although its principal focus is drosophila melanogaster , which has been used for genetic research since the beginning of the 20th century ( 1 ) , flybase also incorporates information on all drosophilidae , including the 11 additional drosophila species that were recently sequenced ( 2,3 ) . the steady increase in publications relating to drosophila in conjunction with the whole genome sequence and other high - throughput data
an important priority for flybase over the last few years has been an extensive revision of data management methods , which resulted in the storage of practically all flybase data sets in a single relational database using the chado schema ( 4 ) .
the new database was used to produce the fb2006_01 release of flybase in december 2006 , which also included a major redesign of the user interface ( 5 ) .
one of the key challenges facing any database is to enable the efficient retrieval of information , and also allow the discovery of relationships between different information in the database .
the search tools that flybase has developed serve a number of distinct functions that enable the scientific community to take full advantage of the way the information is stored in the database .
the simple search tools quicksearch and jump to gene are designed to help users navigate to a report page where information related to the object is presented , such as the size of an mrna transcript or the orthologs of a gene .
other tools , such as gbrowse and the new interactions browser , highlight relationships between objects through a graphical interface , while querybuilder provides users with the ability to perform complex multi - step queries across all fields and different data sets . in this article
, we present an overview of the new and improved flybase search tools , which take advantage of the integration of molecular and genetic data curated from the literature with annotated gene models and reagents aligned to the sequence in the database , and discuss the most important new features , including how results can be passed between tools for further analysis .
a google flybase site search is provided for searching documentation and other non - data - driven content of the flybase site .
although report pages are also searchable via google , this is typically an inefficient approach and does not allow the creation and manipulation of coherent sets of biologically related information .
google is blind to the underlying relationships of the data in flybase and without the ability to target a search to defined data types , a search , such as for the gene name cortex , or the symbol cnn , can produce many spurious hits .
flybase provides two simple search tools , quicksearch on the homepage and jump to gene in the menu bar of every page , that use knowledge of data classes and common data entry points to either take you directly to a gene or other record , or to produce a short list of relevant options .
jump to gene , which has no user - selected settings , attempts to identify a single best match based on unique identifiers , gene symbols or synonyms or gene name , in that order .
quicksearch is a little more sophisticated providing access to different data classes , and enabling expansion of the search to all species and all report text .
one notable improvement to quicksearch is that the symbol of a gene can now be entered under many data classes to list all objects of the data class that are associated with the gene .
clones as the data class and entering dpp will list all of the genomic and cdna clones of the decapentaplegic gene . quicksearch and jump to gene are highly effective tools and are the principal entry points to the flybase data for the majority of our users .
more complex queries that target other data fields or integrate several search criteria can be accomplished with querybuilder .
querybuilder supports sophisticated searches that take full advantage of how the data are stored in flybase .
a search can be focused to a particular piece of data within a report page , such as the mapped features and mutations associated with a gene , and boolean operators can be used to combine two or more searches .
for example , you can use querybuilder to find gal4 insertions that are expressed in the wing ( figure 1 ) .
the results of a two step querybuilder search to identify gal4 insertions expressed in the wing or which are associated with wing phenotypes are shown .
each column can be sorted by clicking the small arrows on either side of the column header .
records are selected for further manipulation via the checkboxes at the left ; all hits are selected by default .
the results of a two step querybuilder search to identify gal4 insertions expressed in the wing or which are associated with wing phenotypes are shown .
each column can be sorted by clicking the small arrows on either side of the column header .
records are selected for further manipulation via the checkboxes at the left ; all hits are selected by default . a complex query comprised of several individual parts
can be run at any stage of its construction to ensure that the chosen constraints are operating as expected at each step .
the search results are shown in the form of a standard hit list , described below .
if the number of leads found during the search is not excessive , querybuilder also displays buttons above the hit list that provide access to data related to the results of your initial search .
for example , data sets related to a list of genes could include the alleles of those genes , related clones , and available stocks .
another useful option of querybuilder is that a list of flybase identifiers or valid symbols can be imported from an external file to use as a query segment .
this provides an easy way to explore bulk data such as genes identified in a microarray experiment . for example
the resulting data set can then be examined for the frequency of genes annotated with a given molecular function or biological process controlled vocabulary term by using the results analysis and refinement tool of the hit list . with the release of flybase fb2007_01 we revised the querybuilder interface to make it more intuitive to use .
the search is now specified within a single window , which contains a series of tabs that lead you through the specification of a query term .
all fields in a report are available for targeted searches and they are listed as they appear on the report pages to make them easier to understand .
the help documentation describes many features of querybuilder , such as its ability to perform calculations on the data .
a notable feature we would like to emphasize is that querybuilder can be used to refine a list of results generated by another search tool .
a set of results can be exported to querybuilder as described under the hit list section , and then modified to refine the search by adding additional query segments .
thus , querybuilder is a very powerful tool that can be used in many different ways to explore the data in flybase and is now tightly integrated with other flybase query tools .
the search results page , the hit list , is presented when you perform any search that returns multiple hits ( figure 1 ) . by default ,
all records are selected for inclusion in subsequent manipulations , but the checkboxes allow user - defined subsets to be created .
the first data column links directly to the report for each record that matched your search .
other columns link to gbrowse or to searches that return hits directly related to that record . in addition to these links
, the hit list provides a set of powerful tools for query refinement or batch processing .
the show related drop - down menu enables you to see all objects of a particular class that are related to the hits selected in your list .
show related menu of a genes search will return a list of clones that are related to the selected genes .
results analysis / refinement button provides the frequency of values within your selected hits for a predefined list of fields . selecting biological process , for example , from the results analysis / refinement tool for a list of genes involved in the notch signaling pathway will result in a page listing the distribution of the different biological process controlled vocabulary terms associated with the list ( figure 2 ) .
lastly , the hitlist conversion tools button allows you to send the selected hits to the batch download tool or to a new querybuilder session for further advanced queries , to download ids to a local file on your computer , or to view html tables of various third - party data sources linked to the hits in your result list .
the results analysis / refinement tool has been used to display the distribution of biological process controlled vocabulary terms applied to a list of the components of the notch signaling pathway .
the next three columns show a graphical display , the raw size , and the relative size of the term in the distribution . clicking on the number in the related records column will return a hit list containing the individual records that make up that bin .
results analysis / refinement tool . the results analysis / refinement tool has been used to display the distribution of biological process controlled vocabulary terms applied to a list of the components of the notch signaling pathway . the second column displays the term for which the distribution was created .
the next three columns show a graphical display , the raw size , and the relative size of the term in the distribution . clicking on the number in the
related records column will return a hit list containing the individual records that make up that bin .
the batch download tool provides bulk access to a variety of data and data formats , such as fasta sequence data and xml files , for a specified list of unique ids ( secondary ids , synonyms or full names are not allowed because they are not unique ) .
ids can be sent from a flybase hit list , uploaded from a local file , or entered manually .
notably , the field data output format provides access to two types of data : data from our set of precomputed flat files and data from the html reports . any line from a precomputed file that matches the list of ids you supplied
the html table option allows you to create a custom report with only the fields you want while preserving hyperlinks for direct navigation to other flybase data .
recently , the html table option has been improved by listing all fields as they appear on the report pages , and making them easier to identify by categorizing them as cv ( controlled vocabulary ) , symbol , date or text .
tools menu that provides a graphical way of exploring the genetic interactions reported in the allele reports .
the browser works in two modes : you can either search for the interactions of an allele ( figure 3 ) , or the interactions of a gene .
each node of an interaction diagram is a hyperlink , which enables you to navigate and browse the complex web of known genetic interactions .
placing your cursor over the center of a node activates a pop - up window that in the case of a network of gene interactions contains a summary of the function of that particular gene , while in the case of interactions between alleles shows the context in which the interactions of that allele have been reported .
the options at the top of the page of the interactions browser permit the selection of different types of interaction data and adjustment of the scan depth , which refers to the number of interaction steps shown from the query .
the auto value set for the scan depth by default chooses a number based on the complexity of the data . in the diagram ,
the alleles or genes that interact directly with the query allele or gene are shown in dark blue , and alleles or genes that interact with them are shown in light blue if the scan depth is set to 2 .
the program uses different colored lines to indicate the type of interaction and arrows to denote the direction in which the interaction was recorded . in some interaction diagrams that have many alleles or genes
, the symbols can overlap making it hard to identify them . to view this data the picture
the options at the top of the page of the interactions browser permit the selection of different types of interaction data and adjustment of the scan depth , which refers to the number of interaction steps shown from the query .
the auto value set for the scan depth by default chooses a number based on the complexity of the data . in the diagram ,
the alleles or genes that interact directly with the query allele or gene are shown in dark blue , and alleles or genes that interact with them are shown in light blue if the scan depth is set to 2 .
the program uses different colored lines to indicate the type of interaction and arrows to denote the direction in which the interaction was recorded . in some interaction diagrams that have many alleles or genes
, the symbols can overlap making it hard to identify them . to view this data the picture
the interactions browser can also be invoked from the results analysis / refinement button above a hit list produced by another query tool to analyze the relationships between alleles or genes in the results list . in this case , every item in the list is treated as a primary query term and several diagrams will be drawn if the list of results contains distinct groups of alleles or genes that do not interact or share interaction partners . the interactions browser offers an intuitive way to learn about the genetic interactions known between alleles , and
gbrowse is a gmod tool ( 6 ) that displays features of the genome aligned to the genomic sequence . by default
, flybase presents a view of d. melanogaster that displays gene models , transcript and polypeptide data , natural transposon insertion sites , and cdnas .
these , and many additional tracks ( 67 options at present ) , are easily configured to create a customized view of the data .
you can navigate to a specific location by entering a precise sequence range , or any valid flybase identifier for a gene , gene product or insertion in the landmark or region box .
additionally , flybase blast output includes gbrowse links that display each blast alignment as a highlighted feature in the context of neighboring gene models and other features of the region .
this is an extremely useful entry path into the sequence data of species other than d. melanogaster , which in some cases is comprised of a large number of relatively short unlinked scaffolds .
development of flybase gbrowse over the last year has focused on facilitating exploration of the newly sequenced drosophila species genomes . by adding
similarity tracks to the d. melanogaster genome view you can use the resulting ortholog links to navigate to orthologs in the other species . you can also find an ortholog by selecting the species from the data source menu and entering the d. melanogaster gene symbol or fbgn i d in the landmark or region box . for genomes other than d. melanogaster gbrowse is configured by default to show two windows that indicate how the region displayed is related to d. melanogaster .
orthoview displays a representation of the genome of the selected species showing the predicted gene .
if this window contains a putative ortholog , a second window will appear with the d. melanogaster genome aligned to the ortholog closest to the center of the upper window .
the relationship between the genomes is shown by sets of green lines that connect the orthologs in the region displayed ( figure 4 ) .
furthermore , we have added pop - ups to gbrowse to provide a gene summary when you mouse - over a gene span , and information on an insertion when you hover over the transgene insertion site .
this is helpful if you are unfamiliar with a gene , or when viewing sequence ranges over 100 kbp , where the symbols of genes and insertions can become hard to read or may not be displayed at all .
these enhancements to gbrowse not only make it easy to obtain information about genes and their orthologs , but also enable the identification of regions of the genomes of the newly sequenced species that appear to have undergone rearrangements .
the orthoview of gbrowse shows an alignment of one of the newly sequenced drosophila genomes with the genome of d. melanogaster .
the alignment of putative orthologs within the region displayed is indicated by green lines connecting the two genome views .
information about the d. melanogaster genes displayed is provided in a pop - up window when you place your mouse over a gene span .
each gene and transcript within the d. melanogaster genome view is linked to a detailed report page and this function will be extended to the other genomes when report pages are created .
the orthoview of gbrowse shows an alignment of one of the newly sequenced drosophila genomes with the genome of d. melanogaster .
the alignment of putative orthologs within the region displayed is indicated by green lines connecting the two genome views .
information about the d. melanogaster genes displayed is provided in a pop - up window when you place your mouse over a gene span .
each gene and transcript within the d. melanogaster genome view is linked to a detailed report page and this function will be extended to the other genomes when report pages are created .
the tighter integration , additional features and improvement in usability of flybase search tools offer biologists many more options to analyze their results and discover relationships between genes and the different types of data available in the database .
this is particularly important , as recently discussed by zhong and sternberg ( 7 ) , given the increase in sequence data and the number of system - wide data sets available .
this enables the results to be refined by adding additional criteria in querybuilder , or the creation of a customized view of specific data fields associated with results by using the field data option of the batch download tool .
the latter option is particularly useful for rapidly comparing information across a set of results , such as the predicted molecular weight of proteins found in a search . in addition , the development of the interactions browser and enhancements to gbrowse , which both display relationships between objects in the database graphically , make it much easier for users to explore the data .
all search tools are obviously limited by the quality of the data stored in the database , and we encourage the members of our user community to report any errors or omissions they find .
in addition , when interpreting the results of a search , it is important to consider the nature of the underlying data .
for example , the interactions shown for genes by the interactions browser represent the data of all alleles , which may include antimorphic or neomorphic alleles whose functions do not reflect the wild - type role of the gene .
the improvements to the set of flybase query tools should enable users to fully exploit the information contained in the database and we hope that they will prove effective at generating new hypothesis and stimulating further experiments .
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flybase ( http://flybase.org ) is the primary resource for molecular and genetic information on the drosophilidae .
the database serves researchers of diverse backgrounds and interests , and offers several different query tools to provide efficient access to the data available and facilitate the discovery of significant relationships within the database .
recently , flybase has developed interactions browser and enhanced gbrowse , which are graphical query tools , and made improvements to the search tools quicksearch and querybuilder .
furthermore , these search tools have been integrated with batch download and new analysis tools through a more flexible search results list , providing powerful ways of exploring the data in flybase .
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rotator cuff tendon tears occur frequently , and surgical repair of the torn tendons is often necessary to relieve symptoms and restore function . approximately , 15 - 20% of the repairs may re - tear , and regular radiological monitoring may be necessary in post - operative period for early detection and treatment of these failed repairs .
we describe a simple technique of radio - opaque marking of the rotator cuff margins using metal markers during surgical repair of the tears ; the markers can be monitored on simple radiographs , and on sonography , and failure in cuff repair integrity may be diagnosed at an early stage .
the patient is positioned in the beach - chair position , and a standard posterior portal is used to evaluate the tear pattern and associated lesions . if a mini - open technique is used , a 2-inch incision is used along langers lines , and the deltoid muscle is split between its anterior and middle segments .
. the footprint of the tendons along the greater tuberosity is prepared , and medial and lateral row anchors are placed .
medial sutures are passed in a mattress configuration through the tendons , and lateral sutures are passed as simple sutures , or in a mason - allen configuration .
prior to suture knot tying , the healthy tendon edge is marked with a metal marker .
the marker may be prepared using a metal wire that may be passed through the tendon edge and twisted around to form a closed loop ; this is possible when a mini - open deltoid - splitting approach is utilized .
alternately , titanium hemostatic clips may be used ; these may be clamped over the tendon edge using special forceps , and this may be performed as an arthroscopic procedure .
secure placement of the marker is crucial to prevent post - operative dislodgement , and it is useful to test the security with a probe .
thereafter , the medial and lateral sutures are tied ; the double - row repair configuration adds to the fixation of the marker , and this is now securely placed between the tendon and the rotator cuff footprint [ figure 1a - c ] .
intraoperative images show the technique of radio - opaque marking of the torn cuff tendon .
( a ) a special forceps are used to grasp the titanium clip during the procedure , ( b ) the position of the clip is shown prior to tying the medial and lateral row sutures , ( c ) the final position of the clip is shown after tying the sutures from the medial and lateral row anchors .
( c : clip , s : supraspinatus , h : grasping forceps , f : rotator cuff footprint , a : sutures from anchors ) the post - operative rehabilitation protocol is similar to that of a double - row rotator cuff repair . in the immediate post - operative period , an anteroposterior shoulder radiograph is obtained with the shoulder in neutral rotation and the arm by the side , and the position of the metal marker is documented for future reference [ figure 2 ] .
thereafter , a simple anteroposterior radiograph may be obtained at any time in the post - operative and follow - up visits to evaluate the position of the marker .
the usual protocol is to obtain a true anteroposterior radiograph at 3 weeks and 6 weeks after surgery to detect early failures of repair that may be diagnosed in this period .
thereafter , 3 - 6 monthly radiographs or sonography may be performed to monitor cuff integrity .
the follow - up radiographs are compared to the immediate post - operative radiograph , and displacement of the marker from the medial edge of the greater tuberosity footprint is measured .
significant displacement of the marker on early radiographs or progressive displacement on successive radiographs is indication for further evaluation and treatment [ figure 3a and b ] .
revision surgery for failed repairs has shown that the marker remains attached to the torn tendon , and is easily retrieved without additional damage to the cuff [ figure 4a and b ] .
immediate post - operative radiograph demonstrates the position of the metal marker ( c ) relative to the anchors ( a ) and humeral head ( h ) anteroposterior ( a ) and axillary - lateral radiographs ( b ) show the medialized position of the metal marker ( c ) , and are suggestive of failure of repair .
( h : humeral head , g : glenoid ) ( a ) arthroscopic evaluation of a failed cuff repair shows the torn tendon ( t ) of the supraspinatus ( s ) , and the metal marker ( c ) is seen attached to the torn edge of the cuff .
( f : cuff footprint , a : sutures from anchors ) , ( b ) arthroscopic retrieval of the metal marker ( c ) is shown prior to final repair .
( f : cuff footprint , a : sutures from anchors , s : supraspinatus , t : cuff tear )
surgically repaired rotator cuff repairs may fail to heal or re - tear in the post - operative and rehabilitation periods , and regular periodic imaging is useful to detect these failures .
magnetic resonance imaging is the ideal modality ; however , this may not be available readily and costs involved are often prohibitive .
ultrasonography is non - invasive , and may be the preferred mode of regular imaging of rotator cuff repairs ; however , ultrasonography is operator dependent , and useful in experienced hands .
the radiographic technique described here is simple , and re - tears can be detected on simple radiographs that may be obtained readily .
further studies are necessary to analyze the utility and cost - effectiveness of this technique in comparison to other modalities .
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surgically repaired rotator cuff repairs may re - tear in the post - operative follow - up phase , and periodic imaging is useful for early detection .
the authors describe a simple surgical technique that provides a visible clue to the tendon edge on an anteroposterior radiograph of the shoulder .
the technique involves arthroscopic or mini - open radio - opaque tagging of the tendon edge using a metal marker , and followed by a double - row rotator cuff repair using suture anchors .
serial post - operative radiographs may then be used to monitor the position of the marker .
progressive or marked displacement of the marker suggests a failure of cuff repair integrity and should be evaluated further .
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thromboembolic events are major causes of morbidity and mortality in the united states ( us ) .
atrial fibrillation , which is associated with a 5-fold increase in the risk of cardioembolic stroke , is a leading cause of death and permanent disability.13 venous thromboembolism ( vte ) , including deep vein thrombosis ( dvt ) and pulmonary embolism ( pe ) , are likewise a significant cause of mortality , with an estimated prevalence of over 100,000 per year.4 the health care costs associated with vte are considerable , exceeding $ 1.5 billion annually in the us.5,6 historically , anticoagulant therapy with warfarin has been considered the standard of care for prevention of thromboembolic events associated with vte and atrial fibrillation.3,7 physician familiarity , clinical experience , and efficacy continue to influence prescribing patterns of warfarin .
in addition , the ability to monitor the degree of anticoagulation , reversibility of effects , and low - cost generic availability appeal to both patients and providers . however , several disadvantages exist for use of warfarin including significant drug - drug and drug - food interactions , delayed onset and offset of action , complex dosing with genetic variances , narrow therapeutic index , and frequent monitoring with patient - specific dose adjustments.3,4,811 despite demonstrated efficacy in preventing thrombotic disease , the practical challenges associated with the utilization of warfarin have led to the search for new treatment options.3 direct oral anticoagulants ( doacs ) , such as dabigatran , rivaroxaban , and apixaban , are newer agents approved for prevention of thrombosis in both nonvalvular atrial fibrillation ( nvaf ) and vte .
randomized controlled trials have demonstrated non - inferiority to warfarin in preventing thromboembolic events.1214 advantages of these newer agents compared to warfarin include no routine monitoring of anticoagulant effect due to more predictable pharmacological profiles , fewer drug - drug and drug - food interactions , and no required bridge therapy during brief interruptions due to rapid onset and offset of action.810 challenges associated with doacs include lack of clinical experience , concerns related to safety considering no antidote is available for reversal of anticoagulant effects for most agents , and cost .
furthermore , strict adherence is crucial as missed doses greatly increase the risk of thromboembolism due to a much shorter half - life compared to warfarin.3,4,810,15 current guidelines support the use of doacs for patients with dvt , pe , and nvaf and a cha2ds2-vasc score of 2 or greater ( level b recommendation).811,15 they are also recommended for patients unable to maintain therapeutic international normalization ratio ( inr ) levels with warfarin therapy . before initiation of any doac
, it is recommended that kidney function be evaluated to determine if dose reductions are indicated.15 although doacs have provided possible solutions to several challenges associated with warfarin therapy , some prescribers remain unaware of potential drug - drug interactions , dose adjustments based on kidney function , and cost implications .
current literature evaluating the utilization , prescribing patterns , and appropriateness of dabigatran and rivaroxaban in patients greater than 65 years of age with atrial fibrillation have indicated a high prevalence of inappropriate prescribing , which may lead to adverse events ( aes).1618 however , literature evaluating prescribing of all doacs for patients with either atrial fibrillation or dvt / pe is lacking . therefore , determining the utilization and prescribing patterns of doacs within a broader patient population could have a positive impact on patient outcomes and overall health costs .
the primary aim of this study was to assess utilization and prescribing patterns of doacs in patients with nvaf and vte ( ie , pe and dvt ) in an outpatient practice setting .
secondary aims of this study were to determine if differences exist in utilization and prescribing patterns of doacs between internal medicine ( i m ) and family medicine ( fm ) teaching clinics , characterize potentially inappropriate prescribing based on drug interactions , contraindications , dosing , cost / access to treatment , and identify factors ( eg , inappropriate prescribing , kidney function , age , agent selection ) associated with undesirable treatment outcomes .
this was a retrospective , cohort analysis of adults receiving a doac with a diagnosis of nvaf or vte .
patients were identified through a query of outpatient fm and i m clinic medical records and confirmed by manual chart review .
patients were eligible for inclusion if the following criteria were met : 18 years of age or greater , diagnosis of nvaf , pe , or dvt ( based on icd-9 codes ) , documented clinic visit with fm or i m from 10/19/2010 to 10/23/2014 , and documented prescription for dabigatran , rivaroxaban , or apixaban as anticoagulant therapy for prevention or treatment of thrombosis .
patients were excluded if they were less than 18 years of age , pregnant , or had inadequate documentation in the medical record to meet inclusion criteria .
all patient data were maintained in a microsoft excel ( redmond , wa , usa ) spreadsheet . to provide quality assurance , individuals responsible for collecting data were trained in the use of the database and random audits
baseline demographics collected included age , race , sex , height , weight , body mass index , relevant past medical history , insurance status , indication(s ) for anticoagulant therapy , prescriber specialty ( fm vs i m ) , prescriber status ( resident , faculty , or midlevel [ ie , nurse practitioner or physician assistant ] ) , and atrial fibrillation thromboembolic risk scores chads2 and cha2ds2-vasc . additional data obtained from subject medical records included laboratory markers to evaluate kidney function ( ie , calculated creatinine clearance [ crcl ] using the cockcroft - gault equation ) , concomitant medications , duration of anticoagulant therapy , drug - drug or drug - disease interactions , and history of anticoagulant and antiplatelet therapy . if subjects were transitioned between anticoagulants within the study period , the most recent doac data were utilized .
inpatient and outpatient records were evaluated to identify aes ( eg , hemorrhagic and/or thrombotic events ) , hospital admissions , or emergency room visits related to anticoagulant use during the study period .
finally , an adapted medication appropriateness index ( mai ) tool was utilized to evaluate inappropriate prescribing .
the mai is a comprehensive tool that has been previously validated in the elderly population to assess inappropriate prescribing of medications.19,20 for this study , a similar methodology to larock et al was utilized.18 it included an adapted mai for doac prescribing with 9 categories of appropriateness : indication , choice , dosage , modalities and practicability of administration , drug - drug interactions , drug - disease interactions , duplication , and duration .
each mai criterion was evaluated using detailed instructions that were developed based on us food and drug administration ( fda)-approved doac prescribing information and guideline recommendations ( table s1 ) . for each criterion , the evaluator rated a ( appropriate ) , b ( inappropriate with limited clinical importance ) , or c ( inappropriate ) . only inappropriate ratings ( c ) were considered for the primary outcome .
the study was approved by the texas tech university health sciences center institutional review board and patient consent was not required .
patient data were converted from microsoft excel to ibm spss 23 ( ibm corp , armonk , ny , usa ) for analysis .
descriptive statistics , including means , standard deviations , and percentages were generated for the sample .
binary logistic regression was implemented to determine the relationship of continuous and dichotomous independent variables with dichotomous dependent variables .
categorical independent variables used in the logistic regression models were dummy coded prior to analysis . for the logistic regression models , goodness of fit was assessed and residuals were reviewed . for all analyses ,
this was a retrospective , cohort analysis of adults receiving a doac with a diagnosis of nvaf or vte .
patients were identified through a query of outpatient fm and i m clinic medical records and confirmed by manual chart review .
patients were eligible for inclusion if the following criteria were met : 18 years of age or greater , diagnosis of nvaf , pe , or dvt ( based on icd-9 codes ) , documented clinic visit with fm or i m from 10/19/2010 to 10/23/2014 , and documented prescription for dabigatran , rivaroxaban , or apixaban as anticoagulant therapy for prevention or treatment of thrombosis .
patients were excluded if they were less than 18 years of age , pregnant , or had inadequate documentation in the medical record to meet inclusion criteria .
all patient data were maintained in a microsoft excel ( redmond , wa , usa ) spreadsheet . to provide quality assurance , individuals responsible for collecting data were trained in the use of the database and random audits
baseline demographics collected included age , race , sex , height , weight , body mass index , relevant past medical history , insurance status , indication(s ) for anticoagulant therapy , prescriber specialty ( fm vs i m ) , prescriber status ( resident , faculty , or midlevel [ ie , nurse practitioner or physician assistant ] ) , and atrial fibrillation thromboembolic risk scores chads2 and cha2ds2-vasc .
additional data obtained from subject medical records included laboratory markers to evaluate kidney function ( ie , calculated creatinine clearance [ crcl ] using the cockcroft - gault equation ) , concomitant medications , duration of anticoagulant therapy , drug - drug or drug - disease interactions , and history of anticoagulant and antiplatelet therapy . if subjects were transitioned between anticoagulants within the study period , the most recent doac data were utilized .
inpatient and outpatient records were evaluated to identify aes ( eg , hemorrhagic and/or thrombotic events ) , hospital admissions , or emergency room visits related to anticoagulant use during the study period .
finally , an adapted medication appropriateness index ( mai ) tool was utilized to evaluate inappropriate prescribing .
the mai is a comprehensive tool that has been previously validated in the elderly population to assess inappropriate prescribing of medications.19,20 for this study , a similar methodology to larock et al was utilized.18 it included an adapted mai for doac prescribing with 9 categories of appropriateness : indication , choice , dosage , modalities and practicability of administration , drug - drug interactions , drug - disease interactions , duplication , and duration .
each mai criterion was evaluated using detailed instructions that were developed based on us food and drug administration ( fda)-approved doac prescribing information and guideline recommendations ( table s1 ) . for each criterion , the evaluator rated a ( appropriate ) , b ( inappropriate with limited clinical importance ) , or c ( inappropriate ) . only inappropriate ratings ( c ) were considered for the primary outcome .
the study was approved by the texas tech university health sciences center institutional review board and patient consent was not required .
patient data were converted from microsoft excel to ibm spss 23 ( ibm corp , armonk , ny , usa ) for analysis .
descriptive statistics , including means , standard deviations , and percentages were generated for the sample .
binary logistic regression was implemented to determine the relationship of continuous and dichotomous independent variables with dichotomous dependent variables .
categorical independent variables used in the logistic regression models were dummy coded prior to analysis . for the logistic regression models , goodness of fit was assessed and residuals were reviewed . for all analyses ,
a total of 26 patients were excluded due to lack of indication ( nvaf or vte ) for a doac , leaving a study population of 120 patients .
of the 120 patients included in the analysis , 40 patients ( 62.5% ) with nvaf met at least 1 inappropriate criterion compared to 35 ( 58.3% ) with vte .
thirty - six patients ( 30.0% ) met 1 inappropriate criterion , 23 ( 19% ) had 2 inappropriate criteria , 9 ( 7.5% ) had 3 inappropriate criteria , and 4 ( 3.3% ) met 4 out of 9 inappropriate criteria .
the 3 most common inappropriate categories were dosage ( 33% ) , duration of therapy ( 18.4% ) , and correct administration ( 18% ) . for patients in whom inappropriate dosing
was noted ( n=39 ; 33.3% ) , most were associated with apixaban ( n=5 ; 41.7% ) , followed by dabigatran ( n=11 ; 28.2% ) and rivaroxaban ( n=23 ; 34.8% ) .
no difference was found between fm and i m clinics within the most prevalent category , inappropriate dosage ( n=16 ; 35.6% vs n=23 ; 31.9% , p=0.840 ) .
additionally , differences in kidney function did not confer a difference in appropriateness of dose prescribed ( 37.5% when crcl < 60 ml / min vs 31.1% when crcl 60 ml / min ; p=0.544 ) .
two statistically significant predictors of appropriate choice of doac utilization over vitamin k antagonist were identified .
patients with prescription insurance had approximately 6 times the odds of appropriately receiving a doac compared to those without prescription insurance ( adjusted odds ratio [ or]=6.298 ; 95% confidence interval [ ci ] 1.11235.658 ; p=0.038 ) .
additionally , the model suggested that patients prescribed apixaban had a much lower odds of meeting appropriate choice criterion compared to those receiving rivaroxaban ( adjusted or=0.088 ; 95% ci 0.0080.964 ; p=0.047 ) .
further analysis indicated patients with a history of bleeding were more likely to meet 1 or more categories of inappropriateness compared to those without a previous bleeding event ( 86% vs 57% , respectively ; p=0.037 ) .
an ae was noted in 27 patients ( 22.5% ) , among whom 20 patients ( 16.7% ) experienced a bleeding event , 4 ( 3.4% ) suffered from a thrombotic event ( stroke or vte ) , and 3 ( 2.5% ) had a non - thrombotic or non - bleeding event while taking a doac .
according to the logistic regression model ( table 3 ) , significant predictors of a bleeding ae include number of inappropriate criteria met , age > 60 years , and calculated crcl .
the analysis suggests that after controlling for covariates , the odds of a bleeding event roughly doubled with each additional inappropriate criterion met ( or=1.949 ; 95% ci 1.1903.190 ; p=0.008 ) .
additional analysis suggested that patients with a history of bleeding were more likely to experience a bleeding ae compared to those without a prior bleeding event ( 36% vs 14% , respectively ; p=0.042 ) .
the current study found a high degree of potentially inappropriate prescribing of doacs in primary care teaching clinics .
the most common issues noted in prescribing were dosage , correct administration , and duration of therapy .
findings of our study are consistent with previously published literature,18 although there are some notable differences .
first , we did not consider the choice of a doac compared to warfarin inappropriate based on extreme weight ( ie , < 50 and > 110120 kg ) due to lack of a specific contraindication in the approved package labeling .
while extremes in weight may present challenges regarding the predictability of anticoagulation activity , and many prescribers prefer not to use doacs in these populations , there are currently no differences in dosing in the package insert based solely on weight.2123 in our study , approximately one - fifth of the subjects who received a doac weighed > 110 kg , and of these , 2 had an ae ( 1 on dabigatran suffered a major bleed and 1 on rivaroxaban had a stroke / transient ischemic attack ) . in a study that assessed the safety , tolerability , pharmacokinetics , and pharmacodynamics of rivaroxaban in subjects of normal weight ( 7080 kg ) compared to subjects with extreme body weight ( < 50 and > 120 kg ) ,
no differences were found.24 scant literature is available assessing safety and efficacy in patients with low weight .
further studies are needed to evaluate the use of doacs in patient populations with extremes in weight . regarding dosing of doacs ,
these findings are similar to a recent analysis of apixaban and rivaroxaban prescriptions written by us cardiologists , where a high percentage ( ~20% ) was written for a reduced dose.25 our findings of rivaroxaban being more frequently dosed lower than current prescribing recommendations are likewise consistent with their findings .
apixaban was more frequently dosed inappropriately compared to other doacs , with most events comprising doses lower than the fda - approved prescribing recommendations .
this result may be due to the complexity of recommended kidney function dose adjustments associated with apixaban , which requires an evaluation of serum creatinine , weight , and age compared to simply calculating crcl with other doacs .
caution should be exercised when prescribing doacs , and particular care should be taken regarding the dose used in patients with kidney dysfunction . in our study
, the risk of bleeding was increased in patients with higher crcl , contrary to what one would expect .
however , given the adjusted odds ratio ( 1.028 ) , this may be of limited clinical relevance .
issues related to administration could be explained by differing dosing regimens based on the indication , agent , or patient demographics .
duration of therapy was commonly potentially inappropriate ( 18.4% ) , with most prescribers extending anticoagulant therapy beyond recommendations for vte .
recommendations included in the mai used to evaluate the appropriateness of duration were obtained from manufacturers labeled indications and evidence - based guidelines .
our results suggest that some prescribers are uncomfortable following chest guidelines for length of doac therapy after a vte event and prolonged therapy.810 while this practice may be suitable in various patient scenarios , a thorough risk - benefit analysis should be performed , along with a joint prescriber - patient discussion and detailed documentation .
alternatively , inappropriate duration of therapy could have been unintentional with poor documentation of doac start date , targeted duration of therapy not clearly stated in initial or follow - up care plan , or due to loss of follow - up . according to our logistic regression , significant predictors of a bleeding ae
were the number of inappropriate criteria met , age greater than or equal to 60 years , and calculated crcl . while increasing age and impaired kidney function are primarily fixed risk factors associated with increased bleeding with anticoagulants , appropriate patient selection and dosing are modifiable factors that could be improved through specific educational and practice initiatives .
in addition , patients with a history of prior bleed were significantly associated with more categories of inappropriateness and greater incidence of a bleeding ae .
these patients should be identified early and closely monitored for the duration of doac therapy .
while the current study provides additional data related to factors associated with potentially inappropriate doac prescribing and variables that may increase this risk , there are limitations .
thus , it was difficult for co - investigators to assess patient compliance with the doacs , which may have impacted the observed results
. however , it did evaluate use in 2 different clinics and is the first study evaluating multiple categories of appropriateness for the 3 doacs currently commercially available . secondly ,
although explicit instructions were provided for each category within the mai , the evaluation of appropriateness could be viewed as subjective , especially in situations where clear evidence - based guideline recommendations were lacking . however , when questions arose related to appropriateness in a particular case , co
third , due to the retrospective nature of this study , our results may be confounded by inaccurate or incomplete documentation .
this limitation was mitigated by verifying data with multiple sources , such as reviewing both outpatient and inpatient records for documentation of an ae .
finally , investigators noted that a large volume of doac prescribing ( 21.7% ) was performed by outside providers or consultants ( eg , cardiologists ) . in these cases , patients were seen at fm and i m clinics for their primary care , but anticoagulation therapy may have either been started or managed by specialists .
therefore , the primary care provider may have been reluctant to change therapy . nevertheless , the investigators chose to preserve these data when analyzing utilization , as appropriate use of pharmacotherapy is a shared responsibility .
first , strict adherence is critical , even compared to warfarin , due to differences in the pharmacokinetic and pharmacodynamic profiles .
therefore , the fact that 2 doacs are dosed twice daily may be a likely source of confusion among prescribers leading to inappropriate dosing .
additionally , marketing strategies that emphasize doacs as a simple alternative to warfarin do not take into account the real - world challenge for providers to remember precise prescribing details , contraindications and precautions , and required dose adjustments .
improvements in clinical decision support and expansion of the pharmacist s role in prescribing and utilization of doacs may help address many of the challenges associated with their use .
clinical decision support tools provided in electronic medical records could be developed to detect potential medication inappropriateness .
likewise , the involvement of a clinical pharmacist in the care of patients on doacs could also improve their use .
a pharmacist may be more likely to have expertise in the medication and guidelines for their use , the time to focus specifically on drug therapy management and educating the patient rather than multiple competing issues that a patient may have , and can assist in transitions of care and communication with the patient s other health care providers .
an evaluation of the utilization of doacs in patients with nvaf and vte within outpatient clinic settings suggests frequent potentially inappropriate prescribing , which may lead to increased aes , such as bleeding .
these results underscore the importance of prescriber education regarding appropriateness , dosing , and monitoring of these agents .
additional research is needed to identify effective interventions to reduce the likelihood of potentially inappropriate prescribing and resultant adverse sequelae .
adapted medication appropriateness indexa notes : a = appropriate , b = inappropriate with limited clinical importance , c = inappropriate .
ann pharmacother , 2014;48(10),12581268 , copyright 2014 by sage publications , adapted by permission of sage publications.inc.1 abbreviations : bid , twice daily ; ddi , drug - drug interaction ; inr , international normalization ratio ; doac , direct oral anticoagulant ; vka , vitamin k antagonist ; vte , venous thromboembolism .
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backgroundscant literature exists evaluating utilization patterns for direct oral anticoagulants ( doacs).objectivesthe primary objective was to assess doac prescribing in patients with venous thromboembolism ( vte ) and nonvalvular atrial fibrillation ( nvaf ) in outpatient clinics .
secondary objectives were to compare utilization between family medicine ( fm ) and internal medicine ( i m ) clinics , characterize potentially inappropriate use , and identify factors associated with adverse events ( aes).methodsthis was a retrospective cohort study of adults with nvaf or vte who received a doac at fm or i m clinics between 10/19/2010 and 10/23/2014 .
descriptive statistics were utilized for the primary aim .
fisher s exact test was used to evaluate differences in prescribing using an adapted medication appropriateness index .
logistic regression evaluated factors associated with inappropriate use and aes.resultsone-hundred twenty patients were evaluated .
at least 1 inappropriate criterion was met in 72 patients ( 60.0% ) .
the most frequent inappropriate criteria were dosage ( 33.0% ) , duration of therapy ( 18.4% ) , and correct administration ( 18.0% ) .
apixaban was dosed inappropriately most frequently .
there was no difference in dosing appropriateness between fm and i m clinics .
the odds of inappropriate choice were lower with apixaban compared to other doacs ( odds ratio [ or]=0.088 ; 95% confidence interval [ ci ] 0.0080.964 ; p=0.047 ) .
twenty - seven patients ( 22.5% ) experienced an ae while on a doac , and the odds of bleeding doubled with each inappropriate criterion met ( or=1.949 ; 95% ci 1.1903.190 ; p=0.008).conclusionpotentially inappropriate prescribing of doacs is frequent with the most common errors being dosing , administration , and duration of therapy .
these results underscore the importance of prescriber education regarding the appropriate use and management of doacs .
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over the last decades , there has been a worldwide growing prevalence of overweight and obesity among people of all ages [ 1 , 2 ] .
the real risk factor of obesity is an excess in adiposity which is strongly associated with adverse health outcomes , including diabetes mellitus , dyslipidemia , blood pressure coronary disease , kidney disease , cancer , musculosketal consequences , asthma , and decreased fertility . given the major short- and long - term consequences of childhood obesity on health , well - being , and costs to health care and social security systems , as well as the better chances for intervention at young ages , public and private funding agencies should give a high priority to research on obesity in children and adolescents .
early identification of adolescents at risk for excess adiposity and its related metabolic complications requires reliable , simple , and specific measures of excess body fat for this age group . to this end , a set of techniques are used to assess obesity such as isotope dilution and dual energy x - ray absorptiometry ( dexa ) .
these techniques offer accurate measurement of adiposity but they are expensive and can not be used everywhere .
thus , measuring body fat in most clinical and epidemiological settings is relatively difficult , and surrogate anthropometric measures , such as body mass index ( bmi ; kg / m ) and waist circumference ( wc ) are used for assessment of obesity in children and adolescents .
a number of studies have explored the relationship between bmi and adiposity measures and showed high degree of correlation between them in children and adolescents [ 912 ] .
however , bmi provides misleading information about body fat [ 13 , 14 ] and its clinical interpretation remains controversial .
other studies have reported strong positive correlation between wc and body fat [ 16 , 17 ] , and wc has been advocated as an indicator of central obesity [ 18 , 19 ] . however , it was indicated that wc may overestimate total and trunk fat and it is not clear which of the central adiposity measures best predict the overall adiposity .
the goals of this study were to assess , for the first time in morocco and north africa : ( a ) the relationship between body fat , assessed by isotope dilution technique , and each of bmi and wc in adolescents , and ( b ) the effectiveness of fat mass ( fm ) , percent body fat ( pbf ) , bmi , and wc to predict adolescents with high - blood glucose level as health risk related to excess body fat .
the study was carried out in rabat region ( morocco ) after receiving the institutional approval from the ministry of national education .
a total of 167 adolescents ( 123 girls and 44 boys ) aged 1117 years were recruited from seven randomly selected secondary schools .
the adolescents who participated in the study were selected by their teacher based on their weight status ( overweight / obese and normal weight ) .
a written consent was obtained from the parents or tutors , and verbal consent was provided by each subject .
fat mass , percent body fat and fasting blood glucose level were determined at the laboratory of unit mixte de recherche en nutrition et alimentation , urac39 ( universit ibn tofal - centre national de l'energie , des sciences et techniques nuclaires - cnesten - rabat ) .
body weight was measured to the nearest 0.1 kg using portable scale ( seca , germany ) with minimal clothing and no shoes .
height was measured to the nearest 0.1 cm using a height bar ( 2 meters , dismantling ) without shoes .
bmi was calculated as weight in kilogram divided by the square of height in meter ( kg / m ) .
wc was measured to the nearest 0.1 cm in standing position at the midpoint between the lowest rib and the iliac crest and at the end of normal expiration , using a measuring tape .
using these measurements and the new who growth reference 519 years , the weight status of each subject was categorized : obese ( z - score > + 2sd , equivalent to bmi > 30 kg / m at 19 years ) , overweight ( z - scores > + 1sd , equivalent to bmi > 25
kg / m at 19 years ) , and normal weight ( 2sd z - scores
+ 1sd , equivalent to 18 25 kg / m < bmi 25 kg / m at 19 years ) . in our study ,
naturally , the body water pool contains a small amount of deuterium ( h ) .
the amount of deuterium in body water above that naturally present is known as the enrichment of body water that reaches a plateau
each adolescent received orally a dose of h2o ( 0.5 g / kg body weight ) .
the saliva samples were taken at baseline , after an overnight fast , and at 3 h after ingesting the h ( endpoint ) .
the level of h in saliva samples was measured by fourier transform infrared spectroscopy ( ftir ) .
tbw was calculated from the saliva sample by the plateau method , assuming that this plateau was reached at 3 hours .
the following equations were used :
( 1)deuterium space(l)=dose amount ( mg)(enrichment h2 in saliva ( mg / kg ) ) .
in order to correct the in vivo isotope exchange in calculating tbw , it is necessary to divide by 1.041
( 2)tbw ( l)=deuterium space ( l)1.041 .
fm and pbf were calculated from tbw using the following equations :
( 3)fat free mass ( ffm ) ( kg)=tbw ( l)hydration factor , fm ( kg)=weight ( kg)ffm ( kg),pbf ( % ) = fm ( kg)weight ( kg)100 .
in this study , we used the hydration factors ( see table 1 ) for children and adolescents .
blood samples were stored in ice till the delivery to the laboratory ( within 4 hours ) , and subsequently stored at 80c until analysis .
two - way anova was used to examine the effect of gender , weight status , and their interaction .
pearson 's correlation was used to assess the relationship between body fat ( fm and pbf ) and each of bmi and wc , and their association with fbg .
all statistical analyses were performed using spss ( statistical package for social sciences , version 17.0 ) .
the kolmogorov - smirnov normality test was used to determine whether data set was well modeled by a normal distribution or not .
42% were overweight or obese and 58% had normal weight . since the number of obese and overweight subjects was low , boys and girls of the study sample
the mean and standard deviations ( sd ) of age , weight , height , bmi , wc , fm , pbf , and fbg are presented in table 2 .
statistical analyses showed that pbf was significantly higher in girls than boys ( p = 0.001 ) , while there was no gender effect on the other variables .
weight , bmi , wc , fm , and pbf were significantly higher in overweight - obese groups compared to normal weight groups ( p < 0.0001 ) ; however weight status had no effect on fbg .
regarding the interaction between gender and weight status , it had a significant effect on bmi ( p = 0.025 ) , while it had no effect on wc , fm , pbf , and fbg .
bmi was positively correlated to fm , with pearson 's correlation coefficients ( r ) above 0.57 , in both genders of the study sample ( boys , r = 0.850 ; girls , r = 0.896 : all p < 0.0001 ) and in different weight status groups ( boys : normal weight , r = 0.770 ; overweight - obese , r = 0.739 ; girls : normal weight , r = 0.690 ; overweight - obese , r = 0.799 : all p < 0.0001 ) .
bmi was also significantly correlated with pbf in both genders of the study sample ( boys , r = 0.711 ; girls , r = 0.724 : all p < 0.0001 ) and in normal weight boys ( r = 0.648 , p = 0.005 ) , overweight - obese boys ( r = 0.413 , p = 0.032 ) , normal weight girls ( r = 0.505 , p < 0.0001 ) , and overweight - obese girls ( r = 0.488 , p = 0.001 ) .
on the other hand , wc showed significant positive correlation with fm in both genders of the study sample ( boys , r = 0.717 ; girls , r = 0.824 : all p < 0.0001 ) and in different weight status groups ( boys : normal weight , r = 0.513 , p = 0.035 ; overweight - obese , r = 0.571 , p = 0.002 ; girls : normal weight , r = 0.626 , p < 0.0001 ; overweight - obese , r = 0.628 , p < 0.0001 ) .
similarly , significant correlation between wc and pbf was seen in both genders of the study sample ( boys , r = 0.575 ; girls , r = 0.677 : all p < 0.0001 ) , in normal weight and overweight - obese girls ( r = 0.434 , p < 0.0001 ; r = 0.404 , p = 0.008 , resp . ) .
however , there was no significant correlation between pbf and wc in normal weight and overweight - obese boys .
overall , the relationships of bmi and wc with each of fm and pbf were found to be dependent on gender and weight status .
the relationship between bmi and pbf was stronger in overweight - obese girls than overweight - obese boys .
similarly , the relationship between wc and pbf was stronger in both normal weight and overweight - obese girls than normal weight and overweight - obese boys . on the other hand , the association of bmi and wc with fm was observed to be more significant than with pbf mainly in overweight - obese boys .
table 4 shows the pearson 's correlation coefficients of fbg with fm , pbf , bmi , and wc .
fbg was found to be strongly correlated with fm in girls of the study sample ( r = 0.241 , p = 0.007 ) and in overweight - obese girls ( r = 0.583 , p < 0.0001 ) .
similar positive correlation was observed between fbg and pbf in girls of the study sample ( r = 0.246 , p = 0.006 ) and in overweight - obese girls ( r = 0.561 , p < 0.0001 ) .
in addition , a significant correlation was found between fbg and both bmi and wc in overweight - obese girls ( r = 0.330 , p = 0.033 and r = 0.528 , p = 0.004 , resp . ) . however , the relationship of fbg to fm , pbf , bmi and wc was not significant in boys .
on the other hand , there was a trend toward a negative correlation between fbg and fm , in overweight - obese group of boys , and both bmi and wc in normal weight girls , but these correlations were not significant .
bmi is commonly used as an indicator of overall obesity in adults due to its simplicity and correlation with percent body fat , but its use in children and adolescents is still a controversial issue because it seems to give a limited insight of excess body fat degree [ 8 , 30 ] .
children with the same bmi may show a noticeable variation in total body fat . unlike adults ,
annual increase in bmi during childhood is generally attributed to the lean rather than to the fat component of bmi [ 3133 ] .
the association between bmi and pbf in young subjects differs among ethnic groups , and bmi does not fully explain differences in pbf .
our results showed a high significant relationship between bmi and each of fm and pbf in both boys and girls .
these results are in agreement with previous studies , suggesting that bmi is highly related to adiposity and may be useful in identifying excess body fat in children and adolescents [ 3538 ] and that correlation of bmi with fm is greater than with pbf [ 9 , 38 ] , we have now confirmed this in a different ethnic sample of adolescents from north africa . in addition , and most of all , our findings confirm the results of previous studies , indicating the high positive relationship between bmi and pbf among adolescents , the role of bmi as a predictor of pbf , and the gender differences in the relationship between pbf and bmi . a number of studies have reported the strong positive correlation between wc and body fat [ 16 , 17 ] .
wc rather than bmi agrees with perception of body size , possibly due to its relation with abdominal fat at different ages , and could serve better than bmi and skin fold thickness for identifying central adiposity .
wc has been shown to have a significant role in identifying overweight and obese individuals .
however , it was indicated that wc may overestimate total and trunk fat [ 5 , 20 ] and that the relationship between wc and body fat could be influenced by weight status and gender in youth .
wc was found to be closely associated to fm and pbf in both boys and girls of the study sample , and in normal weight and overweight - obese girls .
our results are consistent with those of previous studies which suggest wc as a good diagnostic test for fatness in adolescents [ 17 , 20 ] . regarding the effect of weight status on the relationship between wc and adiposity measures , our results differ in some respects from earlier studies , probably relating to the low number of overweight and obese adolescents who participated in the study . however , these results confirm that the relationship between wc and direct measures of overall adiposity may be influenced by gender .
this gender difference was apparent in our study as the correlations were stronger in girls compared to those observed in boys .
these finding agree with a previous study which showed that girls have a higher fm than boys and wc may not reflect total fat . the current study aimed to test the effectiveness of fm , pbf , bmi , and
wc in predicting high levels of fbg as health risk related to excess body fat in adolescents .
many studies support the hypothesis that the relationship between adiposity and risk of disease begins early in life [ 43 , 44 ] .
the increased intra - abdominal adipose tissue is the most clinically relevant type of body fat that is associated with metabolic complications and adverse health effects including hyperinsulinemia and type 2 diabetes in childhood [ 45 , 46 ] .
however , it is not yet clear whether this association can be found in youth of all ethnic groups .
our results showed high positive association of fbg with fm and pbf in girls of the study sample and in overweight - obese girls as well .
similar positive association of fbg with bmi and wc was observed in overweight - obese girls .
our findings are in line with available data from previous studies on the relationship between adiposity and blood glucose level .
it has been reported that the level of fbg was found to be higher in overweight and obese children compared to the normal children and that adolescents with high levels of overall and abdominal adiposities had the least favorable glucose levels .
independently of the amount of fat mass , intra - abdominal fat accumulation was found to be strongly related to insulin resistance and hyperglycemia in obese adolescents .
moreover , it was indicated that overweight and obesity were associated with increased risk for developing type 2 diabetes [ 5052 ]
. the high significant relationship of fbg to bmi , wc , fm and pbf seen in the current study , especially in overweight and obese girls , may be due to the decreased insulin sensitivity which was found to be strongly associated with excess body fat in previous studies [ 5356 ] , while weight loss was found to be associated with a decrease in insulin concentration and an increase in insulin sensitivity in adolescents [ 57 , 58 ] .
also such positive relationship in overweight - obese group of girls may be explained by the clustering of metabolic syndrome factors which place individuals at risk for type 2 diabetes as it has been reported in another study .
on the other hand , there was no significant correlation between fbg and bmi , wc , fm , and pbf in boys , may be due to the small sample size .
our study had some limitations such as the small size of the whole sample and weight status groups particularly for boys .
our ability to recruit more subjects was hampered by the surge of influenza a / h1n1 during the course of the study . also the authorization to access to schools
has not been renewed by the concerned authorities for 2010 - 2011 academic year .
another limitation is that the relationship between adiposity measures and each of bmi and wc , in one hand , and between fbg and other variables , on the other hand , may depend on pubertal stages ( ps ) that were not addressed in our study .
for instance , it was observed in a previous study that the relationship between wc and pbf changes with sexual maturity , and that the normal pattern from ps1 to ps5 is for pbf to decrease and wc to increase .
bmi and wc were closely associated with fm and pbf , derived from isotope dilution technique , in a sample of moroccan adolescents from rabat .
it should be noted , however , that these associations depend on gender and weight status , and that bmi may provide a better proxy estimate of overall obesity than wc .
nevertheless , both of them appear to be reasonable surrogate for fm and pbf , particularly in epidemiological studies , as screening tools to identify adolescents at increased risk of developing excess body fat and high levels of fasting blood glucose .
further research is needed for this group of population and should include ( 1 ) studies to confirm our results taking into account the puberty stage , ( 2 ) investigation of the association of overall and central obesity with fasting blood glucose level among girls in different age or ethnic groups and the mechanism that produces the gender difference observed in the current study , and ( 3 ) relationship of bmi , wc , fm , and pbf with other metabolic abnormalities for early prevention of health risks related to overweight and obesity .
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objectives . the study aimed to assess the relationship between body fat and each of body mass index ( bmi ) and waist circumference ( wc ) , and to test the effectiveness of fat mass ( fm ) , percent of body fat ( pbf ) , bmi , and wc in predicting high levels of fasting blood glucose ( fbg ) . methods .
a total of 167 adolescents aged 1117 years were recruited from rabat region .
bmi and wc were determined using standard equipments .
fm and pbf were derived from isotope dilution technique .
fbg was determined by the hexokinase method .
results .
regardless of the weight status , bmi showed a strong positive correlation with fm and pbf in both genders .
wc was significantly correlated with fm in boys and girls , and with pbf in different groups of girls and boys of the study sample .
however , there was no significant relationship between wc and pbf in normal weight and overweight - obese groups of boys .
fbg was highly correlated with fm and pbf in girls of the study sample and in overweight - obese girls .
similar significant relationship between fbg and both bmi and wc was observed in overweight - obese girls , while there was no significant association between fbg and other variables in boys and normal - weight girls .
conclusion .
bmi and wc were closely associated with fm and pbf , respectively .
however , the degree of these associations depends on gender and weight status .
bmi may provide a better proxy estimate of overall adiposity than wc ; nevertheless , both of them would appear to be a reasonable surrogate for fm and pbf as screening tools to identify adolescents at risk of developing excess body fat and high level of fbg .
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toxic neuropathies are often misdiagnosed as there are no easily available specific or biological tests for the diagnosis .
toxic neuropathies are suspected on the basis of clinical examination and electrodiagnostic features . in india
ayurveda , homeopathy , siddha and unani are practiced and serve as a source of poisoning and drug toxicity .
we report a 30 year - old male who presented with numbness of palms and soles followed by weakness of upper limbs and lower limbs .
the present case study emphasizes the need to consider heavy metal intoxication in patients presenting with acute demyelinating neuropathies and history of use of indigenous medications .
numbness of palms and soles 5 daysweakness of upper limbs and lower limbs 4 daysdysphagia 1 day .
numbness of palms and soles 5 days weakness of upper limbs and lower limbs 4 days this gentleman initially developed bilateral upper limb and lower limb numbness followed by progressive weakness of the lower limbs ascending to the upper limbs .
there was history of swaying while walking , buckling of knees and loosening of slippers from foot .
he developed nasal regurgitation , nasal tone on talking and dysphagia to liquids after 1 day .
he had history of oral and genital ulcers three months ago which was evaluated and diagnosed as ?
he was given ayurvedic medicines in the form of powders for 10 days following which he developed his present symptoms .
on examination , he had bilateral lower motor neuron facial palsy along with bilateral vagal palsy .
his blood samples and the ayurvedic drug samples were sent for toxicological analysis [ tables 24 ] .
csf study done in the second week toxicological analysis of ayurvedic samples permissible limits of heavy metals in ayurveda / siddha / unani / homeo medicines with only herbal ingredients as per who / fda34 toxicological analysis of blood sample he was treated initially with intravenous immunoglobulin , but did not show much improvement .
this gentleman initially developed bilateral upper limb and lower limb numbness followed by progressive weakness of the lower limbs ascending to the upper limbs .
there was history of swaying while walking , buckling of knees and loosening of slippers from foot .
he developed nasal regurgitation , nasal tone on talking and dysphagia to liquids after 1 day .
he had history of oral and genital ulcers three months ago which was evaluated and diagnosed as ?
he was given ayurvedic medicines in the form of powders for 10 days following which he developed his present symptoms .
on examination , he had bilateral lower motor neuron facial palsy along with bilateral vagal palsy .
his blood samples and the ayurvedic drug samples were sent for toxicological analysis [ tables 24 ] .
csf study done in the second week toxicological analysis of ayurvedic samples permissible limits of heavy metals in ayurveda / siddha / unani / homeo medicines with only herbal ingredients as per who / fda34 toxicological analysis of blood sample
toxic neuropathies are often misdiagnosedin a survey , 14 of the 70 herbal medical products available commercially were found to contain lead in 13 ( median concentration , 40 mg / g ; range , 537,000 ) , mercury in 6 ( median concentration , 20,225 mg / g ; range , 28104,000 ) , and/or arsenic in 6 ( median concentration , 430 mg / g ; range , 378130 ) .
if taken as recommended by the manufacturers , each of these 14 drugs could result in heavy metal intakes above published regulatory standardsusers of herbal medical products may be at risk for heavy metal toxicity ; therefore , testing of ayurvedic drugs for toxic heavy metals should be mandatory .
toxic neuropathies are often misdiagnosed in a survey , 14 of the 70 herbal medical products available commercially were found to contain lead in 13 ( median concentration , 40 mg / g ; range , 537,000 ) , mercury in 6 ( median concentration , 20,225 mg / g ; range , 28104,000 ) , and/or arsenic in 6 ( median concentration , 430 mg / g ; range , 378130 ) . if taken as recommended by the manufacturers , each of these 14 drugs could result in heavy metal intakes above published regulatory standards users of herbal medical products may be at risk for heavy metal toxicity ; therefore , testing of ayurvedic drugs for toxic heavy metals should be mandatory .
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case : a 30 year old male presented with numbness of palms and soles followed by weakness of upper limbs and lower limbs of 5 days duration , which was ascending and progressive .
three months back he was treated for oral and genital ulcers with oral steroids .
his ulcers improved and shifted to indigenous medication .
his clinical examination showed polyneuropathy .
csf study did not show albuminocytological dissociation .
nerve conduction study showed demyelinating polyneuropathy .
his blood samples and the ayurvedic drug samples were sent for toxicological analysis .
inference : acute toxic neuropathy - arsenic
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an air space in the lung measuring more than 1 centimeter in diameter is defined as a bulla , which consists of nonventilated air pockets formed by compressed and attenuated lung tissue.1,2 this entity is highly associated with emphysema , which is most commonly caused by smoking .
air fluid levels inside bullae are an unusual complication of chronic obstructive pulmonary disease and the therapeutic antibiotics or broncoscopy have not been evaluated.3 giant bullous emphysema , occupying large portions of the hemithorax , can be treated with bullectomy when lung - function is affected and improvement has been seen in the intermediate and long - term follow up.2,47 localized bullae have shown better results with bullectomy as oppose to medical management.8 few reports have shown spontaneous collapse of bullae in long term follow - up.3,9 in our report , we present a case that highlights a unique aspect of conservative antibiotic therapy for a community - acquired pneumonia - related infectious process that voided the need for surgical intervention .
in september 2009 , a 49-year - old african - american man presented with shortness of breath , non - productive cough , and a fever of 1-week duration . he had seen his primary care doctor 1 week prior for similar symptoms and was prescribed antibiotics for a suspected infectious process .
his medical history was significant for coronary artery disease , peripheral vascular disease , hypertension , hypercholesterolemia , sleep apnea , and history of bilateral apical bullae .
he had a surgical history of disk herniation surgery and percutaneous coronary intervention for right femoral stent .
he had a 22 pack - year smoking history , but had quit using tobacco products 20 years before and denied any alcohol or narcotic use .
the patient has been following up with his pulmonologist for the bilateral apical bullae . a chest x - ray ( cxr ) taken in november 2006 ( fig .
1a ) showed hyperinflated lungs with bilateral upper lobe bullae , which was again seen by computed tomography on admission ( fig .
at admission in 2010 , the cxr and the ct - scan showed upper lobe bullae with air fluid levels in the left upper lobe with a small left plural effusion .
the cxr showed bilateral bullae , with an air fluid level on the left side ( fig .
2a ) , and the ct - scan of the chest confirmed multiple bilateral upper lobe bullae with air fluid levels in the left upper lobe bullae .
the infectious disease department was consulted on the case and ceftriaxone , azithromycin , and clindamycin were started for the presumed diagnosis of pneumonia . upon feeling better ,
he was discharged on 2 g ceftriaxone iv piggyback once a day for 3 more weeks ( he was already on it for 9 days previously ) and clindamycin , 450 mg , every 8 hours . a pulmonology consult
was also called , and it was suggested that the patient had infected bullae , with possible community acquired pneumonia . for teaching purposes ,
alpha-1 antitrypsin levels were 232 ( normal range : 83199 mg / dl ) and alpha-1-antitrypsin deficiency phenotype test confirmed no mutations ; these tests ruled out any suspicion of emphysema .
his physicians had a presumptive diagnosis of community - acquired pneumonia for which he would be treated with a prolonged course of antibiotics .
the patient was discharged on hospital day 9 with clindamycin and ceftriaxone for 3 more weeks .
he was also encouraged to consult with cardiothoracic surgery for evaluation of the possibility for bullectomy , in order to limit future complications of infection or pneumothorax . a follow - up chest x - ray 2 weeks after discharge revealed bilateral bullae with air fluid levels on the left and a new air fluid level on the right ( fig .
at home , the patient was still complaining of shortness of breath and cough . given this information , he was still not a candidate for any type of surgical intervention , but the antibiotics course was continued .
6 weeks after the patient was discharged , the chest x - ray showed a resolution of the left apical air - fluid level and an expansion of the right sided bullae , with a larger air fluid level ( fig .
the patient continued to complain of sporadic symptoms ; therefore , his antibiotic course was continued . at 4-month follow - up ,
a chest x - ray revealed a complete resolution of all air fluid levels and bullae ( fig .
a long - term follow - up ct - scan at 16 months ( july 2012 ) revealed no change in the radiographic findings from the ct - scan done at the 4-month follow up appointment ( fig .
the patient was asymptomatic with no identifiable radiographic pathology and did not qualify for surgical intervention .
the patient had complete clearance of symptoms and radiographic pathology , managed solely with antibiotics .
antibiotic treatment was initiated after the patient clinically developed pneumonia with evidence of air fluid levels on chest x - ray . a thorough work - up
was performed showing no evidence of lung abscess or empyema as evidenced by ct - scan ( fig .
. peters et al discusses a series of patients that underwent serial follow - up with chest x - rays and bronchoscopy , while being managed with antibiotic therapy.10 all of these patients had a complete resolution of symptoms and air fluid levels in a time period of 2 to 32 weeks .
this patient was not initially ordained for surgery due to his pneumonia , and at the very least , we anticipated bullectomy would be indicated after its resolution .
much to our surprise , conservative management yielded results that precluded him from having any indication for surgical intervention .
a surveillance chest x - ray performed at 4 months time from admission demonstrated absolute clearance of his bilateral upper lobe bullae ( fig .
long - term follow - up with a ct - scan was completed , as it is unclear whether the bullae would recur with time and continued air trapping . a follow up ct - scan performed on june 2012 revealed no regression of the lung markings and no expansion of the bullae in the upper lobes ( fig .
4 ) . this case presents a paralleled progression of clinical symptoms with chest x - ray findings .
the conservative management with antibiotic treatment resolved the symptoms of shortness of breath and fever .
the cardiothoracic surgery department was consulted , as bullectomy is indicated for patients who have complications related to bullous disease such as infection or pneumothorax.11 surprisingly , as the patient was followed by the multi - disciplinary team , there was an eventual spontaneous regression of all lung pathology and clinical symptoms .
we all understand that even those patients who undergo bullectomy may not have outright improvement in clinical symptoms . with this knowledge ,
rubin and buchberg showed that fluid resorption may be associated with significant resolution of the bulla ( autobullectomy).12 this case portrays a similar scenario .
it is not clear if the antibiotic therapy had a factor in the overall improvement of his condition , but based on other similar reports , this possibility can not be ruled out.9,13,14 the regression of the bullae is shown in a clear manner through ct and x - ray images .
the 1-year follow - up to monitor continued air trapping , which was completed because the resorption could reverse with time , supports the therapeutic management taken .
this case supports the implementation of conservative management in patients with significant chronic bullous disease .
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we report a case of bilateral apical lung bullae that collapsed following an episode of community - acquired pneumonia with bilateral air fluid levels . with standard treatment for community - acquired pneumonia , management of a patient that may have qualified for bullectomy , ( as in our case )
showed complete resolution of all pathology without surgical intervention .
conservative management took precedence in alleviating pathology over surgical intervention .
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low birth weight and other indicators of reduced fetal growth are associated with adult cardio - metabolic and psychiatric diseases .
this association is the result of developmental programming , whereby a stimulus during a sensitive period of early development exerts permanent effects on structure , physiology or metabolism
. the environmental mechanisms of developmental programming identified so far can be simplified into two major groups : fetal stress exposure and maternal nutrition , although changes in glucocorticoids appear to underpin the programming effects of both . in many mammals , including mice and humans , there is an increased exposure of the developing fetus to glucocorticoids late in pregnancy , as they have a crucial role in the structural development and functional maturation of fetal organs .
however , glucocorticoid overexposure of the fetus can be detrimental as glucocorticoids cause a shift from cell proliferation to differentiation .
therefore , exposure to excess glucocorticoids in utero alters fetal organ growth and maturation patterns , which can result in adverse consequences in later life . in humans ,
the actions of glucocorticoids are exploited for preterm births to advance fetal lung maturation , although this may set the stage for adverse effects in later life .
this review addresses one of the potential adverse effects of glucocorticoid overexposure in utero , i.e. increased incidence of affective behaviours , and the mechanisms underlying these behaviours including alteration of the hypothalamic - pituitary - adrenal ( hpa ) axis and serotonergic and catecholamine pathways .
as glucocorticoids are highly lipophilic , they readily diffuse across biological membranes and , therefore , control of intracellular levels of bioactive glucocorticoids is critical .
this control arises from the enzyme 11-hydroxysteroid dehydrogenase ( 11-hsd ) which interconverts the active glucocorticoids cortisol and corticosterone with their biologically inactive forms , cortisone and 11-dehydrocorticosterone , respectively .
there are two distinct forms of 11-hsd : 11-hsd1 is a low - affinity , nadp(h)-dependent bidirectional enzyme , although in vivo it appears to act predominantly as an 11-oxoreductase to enhance glucocorticoid activity .
11-hsd2 is a high - affinity nad - dependent enzyme which exhibits exclusive 11-dehydrogenase activity ( conversion of corticosterone to 11-dehydrocorticosterone ) to reduce glucocorticoid potency .
11-hsd2 is highly expressed in aldosterone - selective target tissues such as the distal nephron , colon , salivary glands and skin , thus serving to confer aldosterone specificity on the mineralocorticoid receptor ( mr ) to which both corticosterone and aldosterone can bind .
importantly , 11-hsd2 does not always colocalise with mr , such as within placental and fetal tissues , and so its function has expanded beyond its involvement in the electrolyte transport to include regulation of corticosteroid action . during much of normal pregnancy , circulating levels of glucocorticoids in the fetus are substantially lower than in the mother .
this difference arises in part from the high expression of 11-hsd2 in both the placenta and fetus , and this 11-hsd2 expression serves as a glucocorticoid barrier , thus enabling a tight regulation of the materno - fetal glucocorticoid transfer . within the placenta , 11-hsd2
is highly expressed at the interface between maternal and fetal circulations , in the syncytiotrophoblast in humans and the labyrinthine zone in rodents . in the rodent ,
11-hsd2 expression within the labyrinth zone of the placenta falls during late gestation , which may facilitate glucocorticoid passage to the fetus and thus lung maturation . within fetal tissues , 11-hsd2
11-hsd2 is abundant in the neuroepithelium throughout mid - gestation and then strikingly and rapidly declines , coinciding with the terminal stage of neurogenesis .
similar patterns of expression occur in the human fetal brain , with 11-hsd2 silenced between gestational weeks 19 and 26 .
thus , this abundance and expression pattern of 11-hsd2 suggests that 11-hsd2 acts to protect immature mitotically active brain cells from premature exposure to the maturational effects of glucocorticoids . after birth , high levels of 11-hsd2
are localised in mice to only the proliferating external granular layer of the cerebellum and in several nuclei of the thalamus .
therefore , in the early postnatal period the cerebellum is sensitive to glucocorticoid - induced remodelling caused either by exogenous administration or in response to the stress induced by maternal separation .
the high expression of 11-hsd2 in placenta and fetal tissues and the growth retarding and maturational effects of glucocorticoids on the fetus have led to the proposal that variations in feto - placental 11-hsd2 may underlie developmental programming .
thus , placental 11-hsd2 activity correlates with birth parameters in rodents and , less consistently , in humans , suggesting that normal variation in fetal exposure to maternal glucocorticoids may impact on fetal growth .
numerous studies have shown that inhibition , deficiency or bypass ( poor substrate steroids such as dexamethasone or betamethasone ) of 11-hsd2 in gestation in rodents and humans associates with alterations in pregnancy duration , birth weight and programmed outcomes in the offspring .
furthermore , maternal stress of rodents during pregnancy has been associated with a decreased expression of placental 11-hsd2 .
interestingly , in programming models involving maternal low - protein diet , there is an increase in maternal and fetal glucocorticoid levels in addition to a decrease in placental 11-hsd2 activity and/or expression .
consequently , there seems to be considerable overlap in mechanisms by which maternal undernutrition and fetal glucocorticoid overexposure elicit developmental programming .
the developing brain , as other fetal tissues , is extremely sensitive to glucocorticoids , which are crucial for normal cellular and biochemical maturation .
thus , glucocorticoids initiate terminal maturation , remodel axons and dendrites and determine programmed cell death . in sheep
, prenatal glucocorticoid administration retards brain weight at birth , delaying maturation of neurons , myelination , glia and vasculature .
the perinatal hippocampus is especially sensitive to glucocorticoids , with consequences for subsequent memory and behaviour .
thus , antenatal treatment of rhesus monkeys with dexamethasone causes a dose - associated degeneration of hippocampal neurones and reduced hippocampal volume which persists at 20 months of age .
prenatal stress ( induced by repeated restraint of the pregnant female in the last week of pregnancy ) reduces actively proliferating hippocampal cells and feminises sexually dimorphic parameters of the adult hippocampus .
a critical outcome of excess glucocorticoid exposure in early life is the programming of affective function . in the rat , central programming by glucocorticoids ,
be it from maternal administration of dexamethasone or prenatal stress , produces offspring that appear more anxious as adults .
thus , late - gestational dexamethasone exposure in rats impairs the offspring 's coping behaviours in aversive situations later in life , as exemplified by reduced exploration in the open field test and elevated plus maze . such increase in anxiety - like
behaviour is evident as early as postnatal week 10 in rats prenatally exposed to dexamethasone . in rodents , prenatal stress increases depressive - like symptoms with an increased immobility time in the forced swim test and tail suspension test and anhedonia , although not always .
furthermore , offspring of prenatally stressed rats are anxious , with less time spent in the anxiogenic open arms of the elevated plus maze and altered behaviour in the open field test . in animal models of antenatal glucocorticoid administration , prenatal stress and maternal dietary restriction , these programmed changes in behaviour are frequently accompanied by alterations in the hpa axis .
thus , maternal dexamethasone treatment increases corticosterone and adrenocorticotrophic hormone ( acth ) levels in the adult offspring , although , interestingly , mostly in males .
these effects seem to reflect a change in the feedback of the hpa axis at the level of the hypothalamus , as corticotrophin - releasing hormone ( crh ) mrna increases in the paraventricular nucleus , whereas the hippocampal mr and glucocorticoid receptor ( gr ) both decrease .
furthermore , the hpa axis period of hyporesponsiveness in early postnatal life is abolished in adult rats exposed to prenatal stress , whilst normal age - related hpa axis dysfunction is accelerated by prenatal stress . in sheep ,
a single injection of betamethasone on gestational day 104 alters the hpa axis function in offspring at 1 year of age , with elevated basal and stimulated plasma cortisol concentrations .
in contrast , repeated maternal betamethasone injections elevated the offspring 's acth responses to a crh / arginine vasopressin challenge in addition to increased basal acth levels but decreased basal and stimulated cortisol levels
. in primates , offspring of mothers treated with dexamethasone during late pregnancy have elevated basal and stress - stimulated cortisol levels . moreover ,
prenatal stress and alterations in the offspring 's hpa axis function have also been associated in humans .
thus , children of mothers present at or near to the world trade center atrocity on 9/11 , who themselves developed symptoms of post - traumatic stress disorder ( ptsd ) , had lower cortisol levels .
importantly , these changes were most apparent in babies born to mothers who were in the last 3 months of their pregnancies when the trauma occurred , suggesting these observations can be attributed to developmental programming phenomena rather than to a genetic susceptibility or the presence of ptsd per se .
such effects may transmit into subsequent generations , since healthy adult children of holocaust survivors with ptsd ( and therefore lower plasma cortisol levels ) themselves have lower cortisol levels though no ptsd .
this appears to be confined to the children of holocaust - exposed mothers with ptsd .
in contrast to ptsd , maternal anxiety and depression seem to elevate cortisol in the child .
therefore , the mechanisms of prenatal stress programming hpa axis function in humans seem complex , with possibly different pathways involved .
intriguingly , in finland , women who voluntarily ingest liquorice - containing foodstuffs ( that potently inhibit placental 11-hsd2 ) in pregnancy have somewhat shorter gestations and their 8-year - old offspring show altered cognitive function , affective disturbances ( notably markedly increased rates of attention - deficit / hyperactivity disorder ) , hpa axis hyperactivity and sleep disturbances .
however , while it is tempting to conclude that an altered hpa axis response is the underlying mechanism to anxiety - related behaviour , it is important to note that these behavioural changes can occur in the absence of hpa axis alteration .
as 11-hsd2 appears to be a hub for eliciting programming effects , genetically modified mouse models have provided useful insight into underlying mechanisms .
an initial mouse model of targeted 11-hsd2 disruption on an outbred mf1 background revealed mice with an apparently normal phenotype at birth ; however , within 48 h , 50% exhibit motor deficiencies , perhaps due to hypokalaemia , and die . survivors
are fertile , but exhibit severe hypertension , hypokalaemia and polyuria , all typical characteristics of apparent mineralocorticoid excess and , thus , apparent mineralocorticoid actions of corticosterone were revealed by 11-hsd2 deficiency .
interestingly , these mice did not exhibit reduced fetal weight , although this was clearly apparent in later studies on a 11-hsd2 knockout model congenic on a c57bl/6j background . in assessing the relevance of 11-hsd2 in developmental programming ,
male and female mice null or wild - type for 11-hsd2 are mated , although this experimental model is complicated by the potential effects that life - long loss of 11-hsd2 has on maternal care of offspring . to eliminate these effects ,
a heterozygous mating approach has also been taken and has been the main method for assessing the importance of 11-hsd2 in developmental programming . with regard to neurodevelopment , cerebellar size
is reduced in homozygously bred 11-hsd2 mice in early postnatal life due to a decrease in the molecular and internal granular layers .
this associates with a delay in the attainment of neurodevelopmental landmarks such as negative geotaxis and eye opening .
thus , the timing of exposure of the developing brain to glucocorticoids seems to be tightly regulated by the presence of local 11-hsd2 and the cell - specific patterns of its downregulation during maturation . as adults , 11-hsd2 offspring generated from either a homozygous or heterozygous mating approach exhibit increased anxiety .
thus , exploration of the anxiogenic open arm of the elevated plus maze is reduced in 11-hsd2 offspring in comparison to wild types .
additionally , open field exploration is altered in the homozygously bred 11-hsd2 offspring , with them being more reluctant to explore the anxiogenic central field .
interestingly , open field exploration is unaltered in heterozygously bred 11-hsd2 offspring , which implies that aspects of adult behaviour are influenced by maternal factors in this model .
the behavioural phenotype of heterozygously bred 11-hsd2 offspring has been subsequently extended with the observation that 11-hsd2 offspring exhibit depressive - like behaviour .
indeed , 11-hsd2 offspring spent a greater percentage of total time immobile during the tail suspension test ( 11-hsd2 : 52.73 3.5% vs. 11-hsd2 : 70.92 3.7% ; p < 0.05 , unpaired t test ; unpubl .
data ) and the forced swim test ( 11-hsd2 : 54.98 2.4% vs. 11-hsd2 : 68.88 4.8% ; p < 0.05 , unpaired t test ; unpubl . data ) , both indicating increased depressive - like behaviour .
however , it is unknown if similar behaviour is exhibited by homozygously bred 11-hsd2 offspring .
support of the notion that altered 11-hsd2 activity contributes to affective behaviour has also been found clinically .
thus , measurements of urinary glucocorticoids and their metabolites in depressed patients reveal changes in the intracellular regulation of glucocorticoid activity , in particular 11-hsd2 , in comparison to healthy controls .
it is unclear , however , if this alteration in steroid metabolism is a consequence or cause of depression .
interestingly , despite increased anxiety , the hpa axis activity of 11-hsd2 offspring appears unaffected , perhaps a reflection of the additional effects of attenuated hpa axis reactivity due to the reduced glucocorticoid clearance in the absence of renal 11-hsd2 .
however , as predicted , adrenal size is reduced and hence resetting of the hpa axis may have occurred during development .
this , together with a decreased degradation of corticosterone , means that less corticosterone needs to be produced .
consistent with this , 11-hsd2 mice exhibit no differences in the limbic expression of gr , mr or crh during adulthood , but there are some transient changes within the postnatal period . in homozygous matings of 11-hsd2 mice , transient elevations in the gr transcript were observed in situ in all hippocampal subfields of 11-hsd2 offspring at postnatal day 14 .
it should be noted though that while no overt changes in adult hpa axis function are apparent , as 11-hsd2 is widely expressed in the cns during development , it still remains to be determined if the observed behavioural effects are mediated by local fine - tuning of glucocorticoids .
as described above , placental 11-hsd2 may underpin aspects of developmental programming by allowing excess glucocorticoid passage from the
glucocorticoid maternal circulation to the low glucocorticoid fetal environment and , thus , impair fetal growth by direct effects of glucocorticoids on the fetus .
fetal growth is , however , dependent on an array of maternal , placental and fetal endocrine signals , and glucocorticoid - mediated fetal growth retardation must also be related , at least in part , to disturbances in placental growth and function . indeed ,
maternal treatment with dexamethasone impairs normal vascular growth in the rat placenta and has marked effects on the amino acid and glucose transport .
furthermore , an elegant recent study has revealed that the placenta is a source of serotonin for the fetal forebrain , which , while yet to be investigated in the 11-hsd2 model , could also potentially impact on the development of adult affective behaviours .
nonetheless , the current data provide a convincing argument that while maternal glucocorticoids could play a direct role in programming the fetus , notably its brain , placental development and function additionally play a key role
. it must be noted , however , that until tissue - specific knockouts of 11-hsd2 in placenta and fetal tissues are developed , the differential significance of feto - placental 11-hsd2 for development can not be elucidated .
in addition to altered hpa axis function , early - life glucocorticoid exposure can also affect serotonergic and catecholamine pathways .
indeed , in a mouse model of prenatal stress , a depression - like phenotype was accompanied by increased serotonin ( 5-ht ) output and decreased reuptake , as indicated by reduced 5-ht transporter levels in the hippocampus and a trend for decreased tryptophan hydroxylase-2 expression in the dorsal raphe .
in contrast , another model of strong prenatal restraint stress increased both serotonin and tryptophan hydroxylase expression within the dorsal raphe nuclei , whilst others have shown prenatal stress to decrease hippocampal 5-ht1a receptor binding in young male offspring . neonatal handling , a model of early - life stress , increases hippocampal 5-ht levels and turnover .
maternal separation also has dramatic effects on the adult offspring , with an increase in the inhibitory effect of the 5-ht reuptake inhibitor citalopram on serotonergic neuron firing frequency in the dorsal raphe , a reduced sensitivity of 5-ht1a receptors in the dorsal raphe and layer ii / iii cortical pyramidal neurons .
furthermore , perinatal glucocorticoid exposure increases the size and alters the distribution of adult dopaminergic populations within the substantia nigra pars compacta and the ventral tegmental area , while maternal dexamethasone treatment reduces serotonin turnover in the offspring .
maternal protein restriction also alters serotonergic and dopaminergic systems , with the normal rise in dopamine following restraint stress of adult offspring being dampened and serotonin release enhanced .
thus , serotonin , via 5-ht7 receptors , is involved in mediating the permanent upregulation of gr following neonatal handling .
furthermore , in the rat , allelic variations in the serotonin transporter alter the changes in hippocampal gr mrna and corticosteroid stress response that occur during postnatal stress .
preliminary data suggest that altered 5-ht and catecholamine pathways in 11-hsd2 adult brains may be responsible , at least in part , for anxiety - related behaviour [ c.s.w .
thus , levels of 5-ht and its metabolite 5-hydroxyindole acetic acid were measured using high - performance liquid chromatography , as was dopamine and its metabolites
dihydroxyphenylacetic acid , homovanillic acid and noradrenaline in homogenates of the cortex , hippocampus , hindbrain and diencephalon of 8-month - old male mice from 11-hsd2 heterozygous matings .
significant changes were found within the diencephalon region only , with levels of 5-ht increased by 1.6-fold in 11-hsd2 offspring in comparison to 11-hsd2 littermates ( p < 0.05 ) , with no change in metabolites , suggesting increased 5-ht synthesis and/or impairment of 5-ht breakdown ( fig .
1 ) . furthermore , in the same region increases in the dopamine metabolites dihydroxyphenylacetic acid ( 1.5 fold ; p < 0.05 ) , homovanillic acid ( 1.6 fold ; p < 0.05 ) and noradrenaline ( 1.4 fold ; p < 0.05 ) , yet no alteration in dopamine , were found in 11-hsd2 offspring , indicative of increased dopamine release and/or breakdown ( fig .
the significance of these results , particularly given that the changes were observed in the diencephalon , is currently uncertain but is suggestive of increased 5-ht synthesis and/or impaired 5-ht breakdown in addition to increased dopamine release and/or dopamine breakdown .
however , these results need to be followed up with further studies to place these current findings in context , in particular , characterising dopamine and serotonin transporters , receptors and enzymes . furthermore , while the observed monoaminergic changes in 11-hsd2 offspring are unaccompanied by alteration in the hpa axis function , this does not discount a direct role for glucocorticoids in altering monoamines , as local , intracellular glucocorticoid regulation has yet to be investigated in this model .
in summary , prenatal exposure to glucocorticoids exerts long - term effects on the offspring , altering affective behaviours .
while changes in the hpa axis are often attributed to underlying these altered behaviours , changes also occur within the serotonergic and catecholamine pathways .
furthermore , the development of mice in the absence of 11-hsd2 has proven instrumental in ascertaining the significance of 11-hsd2 for the development of affective disorders .
it is interesting that a common pathway altered in the developmental programming of affective disorders , the hpa axis , is in fact marginally affected in the 11-hsd2 mouse .
however , preliminary data suggest that changes in serotonergic and catecholamine pathways may , at least in part , underlie the altered behaviour of 11-hsd2 mice .
it should be noted though that these findings are potentially not just a consequence of feto - placental 11-hsd2 loss but also of life - long renal 11-hsd2 loss .
therefore , the development of tissue - specific knockouts of 11-hsd2 will aid in eliminating this confounder .
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fetal glucocorticoid exposure is a key mechanism proposed to underlie prenatal programming of adult affective behaviours such as depression and anxiety .
indeed , the glucocorticoid metabolising enzyme 11-hydroxysteroid dehydrogenase type 2 ( 11-hsd2 ) , which is highly expressed in the placenta and the developing fetus , acts as a protective barrier from the high maternal glucocorticoids which may alter developmental trajectories .
the programmed changes resulting from maternal stress or bypass or from the inhibition of 11-hsd2 are frequently associated with alterations in the hypothalamic - pituitary - adrenal ( hpa ) axis .
hence , circulating glucocorticoid levels are increased either basally or in response to stress accompanied by cns region - specific modulations in the expression of both corticosteroid receptors ( mineralocorticoid and glucocorticoid receptors ) . furthermore , early - life glucocorticoid exposure also affects serotonergic and catecholamine pathways within the brain , with changes in both associated neurotransmitters and receptors .
indeed , global removal of 11-hsd2 , an enzyme that inactivates glucocorticoids , increases anxiety and depressive - like behaviour in mice ; however , in this case the phenotype is not accompanied by overt perturbation in the hpa axis but , intriguingly , alterations in serotonergic and catecholamine pathways are maintained in this programming model .
this review addresses one of the potential adverse effects of glucocorticoid overexposure in utero , i.e. increased incidence of affective behaviours , and the mechanisms underlying these behaviours including alteration of the hpa axis and serotonergic and catecholamine pathways .
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the pathway interaction database ( pid , http://pid.nci.nih.gov ) is a growing collection of human signaling and regulatory pathways curated from peer - reviewed literature and stored in a computable format .
pid was designed to deal with two issues affecting the representation of biological processes : the arbitrariness of pathway boundaries and the need to capture knowledge at different levels of detail .
pathway boundaries are often arbitrary and overlapping : different biologists might include different biochemical interactions in , for example , the p53 signaling pathway ; and it is not unusual for two pathways representing distinct processes to have one or more interactions in common .
this fuzziness simply reflects the fact that terms like the p53 signaling pathway and the bcr signaling pathway are high - level concepts of convenience , designating slices through the very complex mix of concurrent processes in the cell .
an important goal of pid is to provide an operational definition of high - level concepts like the bcr signaling pathway ( figure 1 ) in the form of predefined pathways , while at the same time allowing a user to explore novel networks composed computationally from the universe of interactions underlying the predefined pathways .
for example , for some protein interactants the precise posttranslational modifications might be known , while for other interactants perhaps the only sure knowledge is that the protein is
the ability to represent information at different levels of detail is also useful in communicating generalizations .
for example , it is sometimes useful to encapsulate a complex process , such as cytoskeleton reorganization as a single event or to treat as a single entity a set of proteins , such as class 1a pi3ks that are functionally equivalent in catalyzing a given event .
pid has mechanisms for dealing with incomplete knowledge , for encapsulating complex events and for expressing generalizations .
the pathway header information includes the date of the latest revision ; the data curation or import source ; the curator ; the reviewers ; the stable pathway identifier ; links to a pathway - specific molecule list and a pathway - specific references list ; and links to pathway graphic and text data exchange format options .
the pathway header information includes the date of the latest revision ; the data curation or import source ; the curator ; the reviewers ; the stable pathway identifier ; links to a pathway - specific molecule list and a pathway - specific references list ; and links to pathway graphic and text data exchange format options .
pid has adopted a network - level representation , similar to reactome ( 1 ) , humancyc ( 2 ) and kegg ( 3 ) . like reactome and humancyc , pid annotates interactions with citations to the literature .
pid differs from reactome , humancyc and kegg in its focus on signaling and regulatory pathways ; it does not attempt to cover metabolic processes or generic mechanisms like transcription and translation ( see table 1 for a comparison of pid with other publicly accessible pathway databases ) .
pid contains only structured data and it links to but does not reproduce molecular information readily available from other sources , such as nucleotide or amino acid sequence , molecular weight and chemical formula .
the principal source of data in pid is the highly curated nci - nature curated collection of pathways , but pid also includes two other sources of data : data imported into the pid data model from reactome 's biological pathways exchange ( biopax ) level 2 ( 4 ) export , and an import of information from the biocarta collection of pathways ( table 2 ) .
bulk downloads are available in biopax level 2 , a standard format for exchanging pathway information , and a pid - specific xml format at http://pid.nci.nih.gov/pid/download.shtml .
table 1.open access human pathway databasesbiocartahumancyckeggpantherpidreactomescope of contentmetabolic and signaling pathwaysmetabolic pathwaysmetabolic , regulatory , signaling , disease and drug pathwaysmetabolic and regulatory pathwayssignaling and regulatory pathwaysmetabolic , signaling and regulatory pathwayshuman pathways3532262081657763human events>300014935083150043732806curation typemanualpart manual / part computationalmanualmanualmanualmanual expert reviewnonononoyesyes literature reference annotationfor pathwaysfor pathwaysfor pathwaysoptionalfor each eventfor each event capture of evidence typesnoyesnonoyesno data visualizationnon interactive predefined pathwaysinteractive predefined pathwaysinteractive predefined pathwaysnoninteractive predefined pathwaysinteractive
predefined pathways and dynamically generated interactive network mapsinteractive predefined pathwaysdata from http://www.biocarta.com/ ( as of september , 2008).data from http://humancyc.org/ ( as of september , 2008).data from http://www.genome.jp/kegg/xml/hsa/index.html ( as of september , 2008).data from nucleic acids research 2007 , 35(database issue):d247d252.data from http://www.reactome.org/ ( as of september , 2008 ) .
table 2.summary of all data sourcesnci - nature curatedreactome importedbiocarta importedpathways7763254subpathways427530interactions437334663003proteins260726924218small molecules135617205complexes19491897880 open access human pathway databases data from http://www.biocarta.com/ ( as of september , 2008 ) .
pid recognizes four kinds of molecules : small molecules ( called compounds ) , rna , proteins and complexes .
pid recognizes five kinds of events : gene regulation ( called transcription , but encompassing both transcription and translation ) , molecule transport ( called translocation ) , small - molecule conversion ( called reaction ) , protein protein interactions ( called modification ) and black - box processes whose internal composition is not provided ( called macroprocesses ) .
in addition , an entire pathway can be abstracted and used as a single event in another pathway . as a participant in an event
, a molecule may have one of four roles : input , output , positive regulator and negative regulator .
these roles define simple relations : an interaction consumes its inputs ( but not its regulators ) and produces its outputs ; and the inputs , positive regulators and the absence of negative regulators are jointly the necessary and sufficient causes of the interaction . each molecule in pid
basic molecules are distinguished by their nucleotide or amino acid sequence ( for macromolecules ) or by their chemical formula ( for small molecules ) .
while pid does not record the sequence of a macromolecule or the chemical formula for a small molecule , each protein or rna is associated with a uniprot or entrez gene accession and most small molecules are associated with chemical abstracts service ( cas ) registry numbers .
each molecule use , as an interactant in an interaction or as a component of a complex , references its corresponding basic molecule .
each molecule use may have additional information , including posttranslational modifications ( for proteins ) and cellular location and activity state ( for all molecule types ) .
a basic protein molecule has a single identifying uniprot accession associated with a particular amino acid sequence .
if the particular isoform of a protein used in an interaction is not known , then the basic protein molecule may be associated with an entrez gene identifier instead of a uniprot accession ; in pid , this method of identifying proteins is restricted almost entirely to the uncurated section of the database imported from biocarta . a use of a protein as a participant in an interaction or component of a complex may have additional attributes : posttranslational modifications , an abstract activity - state attribute and a cellular location attribute .
currently , pid uses 13 types of posttranslational modifications , with phosphorylation being by far the most frequently used modification ( table 3 ) .
the abstract activity - state attribute , with values such as active and inactive , allows curators to distinguish functionally different forms of a protein even if the precise covalent modifications are not known .
values for the cellular location attribute are drawn from the gene ontology ( go ) cellular component vocabulary ( 5 ) .
cleaved subunits of a precursor protein are not distinguished by the posttranslational modification mechanism ; rather they are treated as basic protein molecules separate from each other and from the precursor .
however , pid explicitly relates the cleaved subunit to its precursor and records the cleavage coordinates when these are known .
a pid protein corresponds roughly to a biopax level 3 protein reference , while a biopax level 3 protein corresponds to a pid protein use ( with posttranslational modifications and cellular location ) .
table 3.posttranslational modifications in nic - nature curated data sourcemodification typeall usesunique modificationsacetylation11146farnesylation74geranylgeranylation22glycosaminoglycan92glycosylation13515hydroxylation133methylation132myristoylation154oxidation84palmitoylation5214phosphorylation74031070sumoylation5010ubiquitination14952 posttranslational modifications in nic - nature curated data source pid allows the definition of generic proteins , complexes , small molecules and rna molecules .
a generic molecule is called a family , but is not restricted to the traditional protein families defined by sequence similarity : any set of proteins ( or other type of molecule ) that are in some respect functionally equivalent may be grouped in a family .
the family itself can be used as a participant in an interaction , or as a component of a complex .
because data are entered by multiple curators and because the database contains data from multiple sources , pid needs rules for determining equivalence of molecules .
two basic molecules that are neither families nor complexes are equivalent if they have the same external database accession ( e.g. uniprot or entrez gene ) , or if , in cases where neither has an external database accession , they have the same name . two molecule uses ( as participant in an interaction or component of a complex or member of a family )
are equivalent if they refer to the same basic molecule , and have the same ( or no ) posttranslational modifications , and have the same ( or no ) activity - state attribute , and have the same ( or no ) cellular location attribute .
two basic families ( or complexes ) are equivalent , if they have the same number of members ( or components ) and if for each member ( component ) of one , there is an equivalent member ( component ) in the other .
these rules are applied recursively to define , for example , equivalent uses of complexes with components that are families .
equivalence of molecule uses is the basis on which novel networks are constructed : any two interactions in the database may be joined in a network if one interaction has a participant that is equivalent to a participant in the other interaction .
analogous rules of equivalence are implemented for interactions and entire networks , allowing equivalent ( redundant ) interactions to be pruned from the novel networks .
currently , interactions in the nci - nature curated data source are annotated with 3105 distinct pubmed references .
in addition , an interaction may be annotated with one or more evidence codes that specify the kind of evidence adduced in the citations in support of the interaction ( table 4 ) .
table 4.evidence in nci - nature curated data sourcecodeevidence kindusesiaeinferred from array experiments3idainferred from direct assay1780iepinferred from expression pattern37ifcinferred from functional complementation13igiinferred from genetic interaction12impinferred from mutant phenotype1689iosinferred from other species1139ipiinferred from physical interaction1203rgeinferred from reporter gene expression311 evidence in nci - nature curated data source a predefined pathway is a curated pathway representing a known biological process .
at present , every pathway stored in the pid database is a predefined pathway and every interaction in the database is a member of at least one predefined pathway .
however , the search and retrieval tools allow the user to compose novel pathways from interactions defined in the predefined pathways .
this ability to recombine interactions and to thus create novel pathways is a distinguishing feature of pid .
since the original biocarta diagrams were not associated with an explicit data model , the import of the biocarta pathway data did not challenge the pid data model .
the original biocarta diagrams show protein protein interactions , but the semantics of the connecting arrows are implicit .
the import of these pathways into pid required the interpretation of each interaction and the manual encoding of the semantics in the pid data model .
this was tedious , but since the original biocarta pathways were underspecified , the process did not entail loss of information .
in contrast , the import of the reactome data is automated but does entail some loss of information .
however , since biopax level 2 lacks the means to specify an entity set , this information was lost in the import process . along with other important enhancements , this is being corrected in biopax level 3 .
on the other hand , reactome has some features that are expressible in biopax level 2 but have no correspondence in pid .
for example , in reactome it is possible to explicitly specify that one interaction is a predecessor ( preceding event ) of another , and this is also directly expressible in biopax level 2 .
however , in pid the predecessor relation is implicit , inferred from the identity of interactants and the directionality of inputs and outputs .
consequently , the predecessor relation between two reactome interactions that do not share an interactant is lost in the pid import .
pathways selected for curation are based on potential drug targets , suggestions made by users and reviewers , and other molecules known to be of interest to the cell signaling community . a list of nci - nature curated pathways , along with a list of the pathways imported from reactome and biocarta ,
can be found on the browse pathways page of the pid website : http://pid.nci.nih.gov/browse_pathways.shtml in curating , editors synthesize meaningful networks of events into defined pathways and adhere to the pid data model for consistency in data representation : molecules and biological processes are annotated with standardized names and unambiguous identifiers ; and signaling and regulatory events are annotated with evidence codes and references . to ensure accurate data representation , editors assemble pathways from data that is principally derived from primary research publications .
the majority of data in pid is human ; however , if a finding discovered in another mammal is also deemed to occur in humans , editors may decide to include this finding , but will also record that the evidence was inferred from another species . prior to publication ,
all pathways are reviewed by one or more experts in a field for accuracy and completeness .
any pathway curation effort must decide to what extent to annotate pathways and interactions with information about the larger biological context , including the cell types in which a set of interactions is known to occur and the role of individual pathways in specific pathologies . using the
go biological process vocabulary , pid curators are able to connect interactants and interactions to macroprocesses that characterize particular cell types ( e.g. endothelial cell migration ) .
while , it would be desirable to annotate each interaction with the list of cell types in which the interaction has been observed , pid does not at present attempt to supply this level of detail . given the sparseness of available data , it would be necessary to specify not only positive findings but also negative findings in order to prevent a lack of data from being misinterpreted as the absence of the interaction in a particular cell type .
pid has a few pathways that explicitly describe a particular pathological response ( e.g. cellular roles of anthrax toxin ) ; however , the focus of pid is normal human biology .
the assumption is that a pathological pathway relies on the signaling topology of normal biology but deviates from normal biology in quantity ( e.g. overabundance of a protein ) , in co - occurrence ( abnormal presence of two reactants at the same time in the same cellular compartment ) , or in the introduction of specific abnormal reactants ( e.g. the anthrax toxin or the constitutively active bcr / abl fusion protein ) .
curators are able to specify abnormal reactants ; however , this feature has not been used extensively to date .
as described in the next section , an important use of the batch query is for a researcher to overlay experimental information about such deviations onto the normal signaling topology and thus visualize possible perturbations .
pid provides several query options : a simple query , an advanced query , a connected molecules query and a batch query . in the simple query , the user provides the name , alias or accession of a molecule or biological process ; wildcarding is permitted .
the query will return a list of all uses of the molecule , as simplex or as participant in a complex , and all uses of the biological process , in the database , with hyperlinks to visualizations of the relevant predefined pathways containing the queried entities .
the user also has the option to visualize the novel network(s ) that include all interactions using the queried entities .
the advanced query allows the user to construct the set of novel networks from interactions that : ( i ) involve any of a set of user - specified molecules , or ( ii ) are part of any predefined pathway whose name includes a user - specified key word , or ( iii ) have a user - specified go biological process term or national cancer institute ( nci ) thesaurus ( 6 ) term as their event type or condition .
an important feature of the advanced query is the provision for including interactions that are immediately upstream or downstream of the set of interactions retrieved by molecule , pathway name or go / nci thesaurus term .
the connected molecules query allows a user to find a novel network that connects two or more molecules specified by name , alias or accession .
the query will find only one of the possibly many networks satisfying the constraint , but the one found will have the minimum number of interactions .
finally , the batch query allows a user to upload one or two lists of molecule identifiers ( name , alias or accession ) .
the user has two options : to analyze the number of molecules in the lists that
hit each predefined pathway or to construct the novel network(s ) that include all interactions using any of the listed molecules .
for the first option , the query uses a hypergeometric distribution to compute the probability that each pathway in the database is hit by molecules in either of the lists .
the query returns a list of pathways ordered by p - value . in the visualization of a predefined pathway ( first option ) or novel pathway ( second option ) , molecules from the first list are colored blue , molecules from the second list are colored red and any molecules appearing in both lists are colored purple .
supplementary figure 1 presents an example of invoking the batch query with a single molecule list , the 120 protein kinases found by greenman et al .
( 7 ) to have at least one cancer - predisposing mutation . selecting the predefined pathways option
, one can see that this list samples a small number of pathways , biased toward immune cell signaling , at a p < 0.0001 . while pid associates a single external database accession ( typically a uniprot accession ) with a protein , the query interface searches pid not only by uniprot accession , but also by related gene identifiers ( hugo symbol , alias , entrez gene identifier ) . any predefined pathway or novel network
can be visualized in either gif ( graphics interchange format ) or svg ( scalable vector graphics ) graphic mode .
network graphics are all automatically constructed from the underlying data using the graphviz package ( 8) .
events and molecule uses in the graphics are hyperlinked to html pages of information about the interaction or molecule .
in addition , any predefined pathway or novel network can be exported in native pid xml or biopax level 2 formats . using the biopax export
, a user can also visualize pid pathways in cytoscape ( http://cytoscape.org ) a popular third - party network visualization tool ( 9 ) .
for any predefined pathway , the user can obtain ( and export to tab - separated format ) a list of literature citations and participating molecules .
pid is a highly structured , curated database of molecular interactions and events that compose human cell signaling and regulatory pathways .
a particular strength of pid is the ability to create novel networks that can reveal parallel alternative paths to events of interest , like activation of a protein or disassembly of a complex in the dna repair process . in cancer biology ,
such a view can elucidate the variety of strategies that a given type of cancer may adopt , explain why a single - agent therapy is not effective and suggest potential multi - agent therapies .
for example , by using pathways as the integrating framework , the cancer genome atlas project has mapped genomic abnormalities of different types copy number , mutation and methylation to a set of oncogenic processes ( 10 ) . at present ,
most attempts to profile tumor subtypes have relied on dna and rna assays . however , as high - throughput proteomic methods improve the kind of detailed information on posttranslational modifications of proteins available in pid will be essential in mapping more accurately the state of a cell .
consistent with its focus on interactions and events derived from curated signaling cascades and regulatory processes , pid does not at present include interaction data deriving from high - throughput protein
this reflects not a judgment on the quality of high - throughput data , but a recognition that there are databases specifically designed to provide access to this data ( 11 , 12 , 13 ) .
however , while it does not lead directly to the construction of signaling cascades , information from high - throughput protein protein interaction experiments can be useful in interpreting the curated pathways and assessing their completeness .
for example , a high - throughput protein protein interaction experiment can identify an unexpected binding partner for a catalyst , suggesting the possibility that the in vivo presence of the partner can sequester the catalyst and thus turn off downstream interactions . in the future , pid will allow users to take advantage of high - throughput protein
protein interaction data , either by allowing users to upload interaction sets to be added to the novel networks created by pid queries or by querying other data sources ( such as pathway commons , http://pathwaycommons.org ) as needed to support a user query .
the pid data model is currently being integrated with nci 's cancer bioinformatics infrastructure objects model ( cabio ) ( 14 ) , thereby making pid data accessible on nci 's cagrid ( 15 ) .
the national cancer institute . funding for open access charge : national cancer institute . conflict of interest statement .
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the pathway interaction database ( pid , http://pid.nci.nih.gov ) is a freely available collection of curated and peer - reviewed pathways composed of human molecular signaling and regulatory events and key cellular processes .
created in a collaboration between the us national cancer institute and nature publishing group , the database serves as a research tool for the cancer research community and others interested in cellular pathways , such as neuroscientists , developmental biologists and immunologists .
pid offers a range of search features to facilitate pathway exploration .
users can browse the predefined set of pathways or create interaction network maps centered on a single molecule or cellular process of interest .
in addition , the batch query tool allows users to upload long list(s ) of molecules , such as those derived from microarray experiments , and either overlay these molecules onto predefined pathways or visualize the complete molecular connectivity map .
users can also download molecule lists , citation lists and complete database content in extensible markup language ( xml ) and biological pathways exchange ( biopax ) level 2 format .
the database is updated with new pathway content every month and supplemented by specially commissioned articles on the practical uses of other relevant online tools .
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we obtained a tracheal aspirate specimen from the lower respiratory tract of the patient with the first reported a(h10n8 ) virus infection on day 7 after illness onset ( 4 ) .
the tracheal aspirate specimen was inoculated on monolayers of mdck cells and into the allantoic cavities of 10-day - old embryonated chicken eggs .
the isolated novel a(h10n8 ) virus strain ipb13 was serially passaged in mdck cells every 72 hours for a total of 3 passages .
after 1 blind passage , cytopathic effects were clearly visible in 80% of the cells .
the supernatants of the second and third passages in mdck cells and the allantoic fluids of the inoculated chicken eggs were individually harvested and processed for full genome sequencing .
we extracted total rna from each sample using a qiaamp viral rna mini kit ( qiagen , hilden , germany ) and amplified it by sequence - independent pcr with a onestep rt - pcr kit ( qiagen ) .
we determined the viral genome sequence of each isolate using an abi 3730xl automatic dna analyzer ( life technologies , grand island , ny , usa ) .
potential heterogeneous sites in each genome were initially identified by manual inspection of the sequence trace data and confirmed by clone sequencing as previously described ( 7 ) .
in addition , the tracheal aspirate sample was processed for direct deep sequencing as previously described ( 8) but with a slight modification .
we used an illumina / hiseq2500 sequencer ( illumina , san diego , ca , usa ) to generate 100-bp single - end reads according to the manufacturer s instructions .
the deep sequencing reads were screened for quality control and removal of human contamination as previously described ( 9 ) .
the valid reads were aligned to the previously determined genome sequence of the novel a(h10n8 ) virus strain jx346 ( gisaid [ global initiative on sharing all influenza data ] accession no .
we obtained a total of 2,629,199 reads that were successfully aligned to the jx346 genome .
then , we used the samtools package to detect possible polymorphic or heterogeneous sites from the alignment ( 11 ) .
we deposited the complete genome sequences of all isolates in genbank under accession nos . kj406531kj406562 .
the change of host environment from the in vivo human respiratory tract to the in vitro cell lines might introduce mutations in the virus genome during culture .
indeed , in our previous study , we observed dozens of point mutations in the genome of novel a(h7n9 ) virus obtained from embryonated chicken eggs compared with that derived directly from clinical sample ( 8) .
however , in this study , we obtained exactly the same genome sequences of the novel a(h10n8 ) virus from the clinical sample and that cultured in chicken eggs ( table ) .
moreover , the 2 heterogeneous sites in the clinical sample were retained during culture in embryonated chicken eggs . in contrast , 10 heterogeneous sites , 8 of which resulted in point mutations , were observed in the virus genomes derived from cultures in mdck cells ( table ) .
additionally , no genomic difference was visible between viruses in the second and third passages in mdck cells . * ha , hemagglutinin ; na , neuraminidase ; np , nucleocapsid protein ; ns , nonstructural protein ; pa , polymerase acidic ; pb , polymerase basic ; , synonymous substitution without amino acid change .
site positions are numbered from the start codon ( m ) ; h3 and n2 numbering for ha and na , respectively .
the heterogeneous site in polymerase basic ( pb ) 2 resulted in a mixture of glutamic acid and lysine at residue 627 of the pb2 protein in the clinical sample ( table ) ; this mixture might have partially contributed to the severity of the patient s illness ( 4 ) .
indeed , the heterogeneity in this site vanished during culture in mdck cells , leading to complete lysine substitution at residue 627 of the pb2 protein as previously observed ( 12 ) .
this finding is consistent with the established hypothesis that the e627k substitution in pb2 was associated with the increased transmissibility and pathogenicity of avian influenza viruses in mammals ( 13,14 ) .
notably , a heterogeneous site in the neuraminidase ( na ) segment led to a mixture of arginine and lysine at residue 292 ( n2 numbering ) of the na protein in the viruses cultured in mdck cells ( table ) .
the r292k substitution in the na protein of avian influenza a(h7n9 ) virus was able to reduce the antiviral efficacy of na inhibitors , especially oseltamivir ( 15 ) .
therefore , although the novel a(h10n8 ) virus was sensitive to oseltamivir in vitro ( 4 ) , its potential to develop antiviral drug resistance in mammalian cells requires further attention .
we investigated the genomic mutations and heterogeneities of the novel influenza a(h10n8 ) virus during culture in embryonated chicken eggs and mdck cells compared with the genome sequence obtained directly from the clinical specimen .
the viral genome was highly conserved during culture in embryonated chicken eggs , and no mutations were identified .
this result suggests that the novel a(h10n8 ) virus might have been highly adapted to an avian - like host before it was transmitted to the human host ( i.e. , the first patient ) .
in contrast , substantial genetic mutations were observed in the viral genome during culture in mdck cells ; this finding implies an ongoing adaptive microevolution of the virus in a mammalian environment . taken together
, our results favor the proposal that the novel influenza a(h10n8 ) virus has an avian origin ; however , more research is required to establish the definite origin of the emerging influenza virus .
furthermore , the substitutions e627k ( in the pb2 protein ) and r292k ( in the na protein ) observed in the cultures of the mdck cells indicate that the virus might be undergoing rapid adaptation to mammals and developing antiviral drug resistance .
although only 3 human cases of infection with the novel a(h10n8 ) virus have been reported , the potential for this virus to threaten public health should not be underestimated .
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the recent emergence of human infection with influenza a(h10n8 ) virus is an urgent public health concern .
genomic analysis showed that the virus was conserved in chicken eggs but presented substantial adaptive mutations in mdck cells .
our results provide additional evidence for the avian origin of this influenza virus .
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participants were a non treatment - seeking sample of children taking part in a longitudinal study designed to investigate risk factors for excessive weight gain and adverse metabolic outcomes ( clinical trial reg .
nos . nct00001522 and nct00001195 , clinicaltrials.gov ) between july 1996 and november 2010 . by design ,
the sample was oversampled to include children at increased risk for the development of adult obesity by virtue of either the child s overweight status ( bmi 85th percentile ) or a parental history of being overweight ( bmi 25 kg / m ) .
participants were recruited through mailings to pediatricians , family physicians , and two waves of notices to families with elementary school aged youth in maryland and the washington , dc , metropolitan area .
advertisements requested the participation of children willing to undergo phlebotomy and x rays for studies investigating hormones and metabolic functioning in children .
approximately 7% of families responded to each of the school mailings , and subjects recruited directly from these mailings constituted 88% of all subjects studied .
youth were in good general health and were not taking medications known to affect body weight or metabolism for at least 2 weeks prior to study entry .
exclusion criteria included a significant medical health problem , such as renal , hepatic , most endocrinologic ( e.g. , hyperthyroidism , cushing syndrome , or type 2 diabetes ) , or pulmonary disorders ( other than mild asthma not requiring chronic medication ) or major psychiatric illnesses requiring treatment .
children were financially compensated for their participation at baseline ( $ 120 ) and follow - up ( $ 120 ) .
all study procedures were approved by the eunice kennedy shriver national institute of child health and human development institutional review board . at a baseline assessment visit
, participants underwent a physical examination that included pubertal staging by a pediatric endocrinologist or trained pediatric or family nurse practitioner .
parents reported the child s and the family medical history , including parental height and weight and family history of type 2 diabetes .
each child s height was measured three times to the nearest millimeter by a stadiometer ( holtain , crymmych , wales , u.k . )
weight was measured to the nearest 0.1 kg with a calibrated digital scale ( scale - tronix , wheaton , il ) .
bmi z ( sd ) scores for sex and age were calculated according to the centers for disease control and prevention 2000 standards .
participants completed the 27-item children s depression inventory ( cdi ) to assess the extent and severity of depressive symptoms ( 14 ) .
younger children ( aged 8 years ) had the questions read to them so that any difficult concepts could be explained .
a total raw score , ranging from 0 to 54 , is derived from the sum of its items . a total score that exceeded 12
was proposed as the cutoff for screening for at risk for clinical depression ( 15 ) .
we examined the cdi total score both as a continuous variable and as a dichotomous variable ( lower depressive symptoms [ cdi < 13 ] vs. elevated depressive symptoms [ cdi 13 ] ) .
the cdi is reliable and well validated ( 14 ) , and the total score has demonstrated adequate internal reliability in studies oversampled to include youth aged 14 years who were at risk for adult obesity in our laboratory ( = 0.79 ) .
any child who endorsed active suicidal ideation was referred for an immediate psychiatry consultation at the national institutes of health clinical center and was excluded from participation in the current study .
each participant provided fasting blood samples for serum insulin and glucose after an overnight fast .
a fasted state was encouraged to be reported truthfully by providing compensation for the visit even if subjects reported not having fasted .
youth consumed their habitual diet ( invariably reported on a food - frequency questionnaire as 35% energy from carbohydrates ) during the week before they were studied .
glucose was measured using a hitachi 917 analyzer using reagents from roche diagnostics ( indianapolis , in ) .
insulin concentrations were determined using a commercially available immunochemiluminometric assay purchased from diagnostic product corporation ( los angeles , ca ) and calibrated against insulin reference preparation 66/304 .
the insulin assay uses a monoclonal anti - insulin antibody and was run on an immulite2000 machine ( diagnostic product corporation ) .
the cross - reactivity of the insulin assay with proinsulin was < 8% and with c - peptide was < 1% , sensitivity was 2 u / ml , and the mean inter- and intra - assay coefficients of variation were 5.8 and 3.6% , respectively .
insulin resistance was estimated with the homeostasis model assessment of insulin resistance ( homa - ir ) index , calculated as follows : ( fasting insulin [ u / ml ] fasting glucose [ mmol / l])/22.5 .
although homa - ir as a continuous variable was considered the primary outcome measure in the current study , secondary outcomes included insulin resistance defined dichotomously ( absence [ homa - ir < 3.16 ] vs. presence [ homa - ir 3.16 ] ) , impaired fasting glucose ( absence [ fasting glucose < 100 mg / dl ] vs. presence [ fasting glucose 100 mg / dl ] ) , and hyperinsulinemia ( fasting insulin 15 u
/ l ) . at a follow - up appointment intended to take place 5 years later ,
participants again provided fasting blood samples for serum insulin and glucose , which were used to reassess homa - ir . for ~2% of participants , plasma glucose ( collected in tubes containing powdered sodium fluoride )
was used in place of serum glucose when the latter was not available . for participants who did not complete a 5-year follow - up , available anthropometric and
phlebotomy data were used either from a somewhat shorter or longer follow - up period , whenever such data were available .
study variables were examined to determine whether the assumptions of univariate and multivariate analyses were met .
the skew and kurtosis were satisfactory on all variables , and outliers ( < 3% of all data points ) were adjusted to fall 1.5 times the interquartile range below or above the 25th or 75th percentile .
this strategy was used because it minimizes outliers influence on the characteristics of the distribution , minimally changes the distribution overall , and avoids potential bias associated with eliminating outliers altogether .
missing data patterns were characterized to test baseline differences in study variables between children who did and those who did not complete a follow - up assessment .
correlations or independent - sample t tests were used to examine the bivariate associations among baseline demographic and anthropometric characteristics , depressive symptoms , and insulin resistance .
hierarchical multiple regressions were conducted regressing follow - up insulin resistance on baseline depressive symptoms , including in the model baseline insulin resistance , sex , race ( non - hispanic white vs. other ) , first- or second - degree family history of type 2 diabetes ( presence vs. absence ) , baseline age ( years ) , baseline bmi ( kg / m ) , change in bmi from baseline to follow - up , and time in study ( years between baseline and follow - up ) .
baseline pubertal staging was considered to be a covariate but was removed because it was nonsignificant in all models ( p > 0.65 ) .
parallel analyses also were conducted for the secondary outcomes of fasting insulin and fasting glucose .
ancova was used to test whether similar relationships between depressive symptoms and insulin resistance , fasting insulin , or glucose were observed if depressive symptoms were considered categorically as children with lower depressive symptoms ( cdi total score < 13 ) versus elevated symptoms ( cdi total score 13 ) .
logistic regressions were conducted to test whether depressive symptoms predicted greater odds of categorical outcomes of insulin resistance ( absence [ homa - ir < 3.16 ] vs. presence [ homa - ir 3.16 ] ) , hyperinsulinemia ( fasting insulin 15 u / l ) , and impaired fasting glucose ( absence [ fasting glucose < 100 mg / dl ] vs. presence [ fasting glucose 100 mg / dl ] ) .
, we also reran the primary analyses using multiple imputation with sas 18.0 to handle missing data .
following standard multiple imputation procedures , each dataset was analyzed separately , and then the effects were combined using the sas mianalyze procedure . because these results did not significantly differ from the nonimputed findings ,
at a baseline assessment visit , participants underwent a physical examination that included pubertal staging by a pediatric endocrinologist or trained pediatric or family nurse practitioner .
parents reported the child s and the family medical history , including parental height and weight and family history of type 2 diabetes .
each child s height was measured three times to the nearest millimeter by a stadiometer ( holtain , crymmych , wales , u.k . )
weight was measured to the nearest 0.1 kg with a calibrated digital scale ( scale - tronix , wheaton , il ) .
bmi z ( sd ) scores for sex and age were calculated according to the centers for disease control and prevention 2000 standards .
participants completed the 27-item children s depression inventory ( cdi ) to assess the extent and severity of depressive symptoms ( 14 ) .
younger children ( aged 8 years ) had the questions read to them so that any difficult concepts could be explained .
a total raw score , ranging from 0 to 54 , is derived from the sum of its items . a total score that exceeded 12
was proposed as the cutoff for screening for at risk for clinical depression ( 15 ) .
we examined the cdi total score both as a continuous variable and as a dichotomous variable ( lower depressive symptoms [ cdi < 13 ] vs. elevated depressive symptoms [ cdi 13 ] ) .
the cdi is reliable and well validated ( 14 ) , and the total score has demonstrated adequate internal reliability in studies oversampled to include youth aged 14 years who were at risk for adult obesity in our laboratory ( = 0.79 ) .
any child who endorsed active suicidal ideation was referred for an immediate psychiatry consultation at the national institutes of health clinical center and was excluded from participation in the current study .
each participant provided fasting blood samples for serum insulin and glucose after an overnight fast .
a fasted state was encouraged to be reported truthfully by providing compensation for the visit even if subjects reported not having fasted .
youth consumed their habitual diet ( invariably reported on a food - frequency questionnaire as 35% energy from carbohydrates ) during the week before they were studied .
glucose was measured using a hitachi 917 analyzer using reagents from roche diagnostics ( indianapolis , in ) .
insulin concentrations were determined using a commercially available immunochemiluminometric assay purchased from diagnostic product corporation ( los angeles , ca ) and calibrated against insulin reference preparation 66/304 .
the insulin assay uses a monoclonal anti - insulin antibody and was run on an immulite2000 machine ( diagnostic product corporation ) .
the cross - reactivity of the insulin assay with proinsulin was < 8% and with c - peptide was < 1% , sensitivity was 2 u / ml , and the mean inter- and intra - assay coefficients of variation were 5.8 and 3.6% , respectively .
insulin resistance was estimated with the homeostasis model assessment of insulin resistance ( homa - ir ) index , calculated as follows : ( fasting insulin [ u / ml ] fasting glucose [ mmol / l])/22.5 .
although homa - ir as a continuous variable was considered the primary outcome measure in the current study , secondary outcomes included insulin resistance defined dichotomously ( absence [ homa - ir < 3.16 ] vs. presence [ homa - ir 3.16 ] ) , impaired fasting glucose ( absence [ fasting glucose < 100 mg / dl ] vs. presence [ fasting glucose 100 mg / dl ] ) , and hyperinsulinemia ( fasting insulin 15 u / l ) . at a follow - up appointment intended to take place 5 years later , participants height and weight were reassessed to calculate bmi , as completed at baseline .
participants again provided fasting blood samples for serum insulin and glucose , which were used to reassess homa - ir . for ~2% of participants ,
plasma glucose ( collected in tubes containing powdered sodium fluoride ) was used in place of serum glucose when the latter was not available . for participants who did not complete a 5-year follow - up , available anthropometric and
phlebotomy data were used either from a somewhat shorter or longer follow - up period , whenever such data were available .
study variables were examined to determine whether the assumptions of univariate and multivariate analyses were met .
the skew and kurtosis were satisfactory on all variables , and outliers ( < 3% of all data points ) were adjusted to fall 1.5 times the interquartile range below or above the 25th or 75th percentile .
this strategy was used because it minimizes outliers influence on the characteristics of the distribution , minimally changes the distribution overall , and avoids potential bias associated with eliminating outliers altogether .
missing data patterns were characterized to test baseline differences in study variables between children who did and those who did not complete a follow - up assessment .
correlations or independent - sample t tests were used to examine the bivariate associations among baseline demographic and anthropometric characteristics , depressive symptoms , and insulin resistance .
hierarchical multiple regressions were conducted regressing follow - up insulin resistance on baseline depressive symptoms , including in the model baseline insulin resistance , sex , race ( non - hispanic white vs. other ) , first- or second - degree family history of type 2 diabetes ( presence vs. absence ) , baseline age ( years ) , baseline bmi ( kg / m ) , change in bmi from baseline to follow - up , and time in study ( years between baseline and follow - up ) .
baseline pubertal staging was considered to be a covariate but was removed because it was nonsignificant in all models ( p > 0.65 ) .
parallel analyses also were conducted for the secondary outcomes of fasting insulin and fasting glucose .
ancova was used to test whether similar relationships between depressive symptoms and insulin resistance , fasting insulin , or glucose were observed if depressive symptoms were considered categorically as children with lower depressive symptoms ( cdi total score < 13 ) versus elevated symptoms ( cdi total score 13 ) .
logistic regressions were conducted to test whether depressive symptoms predicted greater odds of categorical outcomes of insulin resistance ( absence [ homa - ir < 3.16 ] vs. presence [ homa - ir 3.16 ] ) , hyperinsulinemia ( fasting insulin 15 u / l ) , and impaired fasting glucose ( absence [ fasting glucose < 100 mg / dl ] vs. presence [ fasting glucose 100 mg / dl ] ) . parallel sets of covariates were used in these analyses .
, we also reran the primary analyses using multiple imputation with sas 18.0 to handle missing data .
following standard multiple imputation procedures , each dataset was analyzed separately , and then the effects were combined using the sas mianalyze procedure . because these results did not significantly differ from the nonimputed findings ,
a total of 198 children ( 51.9% female ) aged 8.6 1.7 years ( range 613 ) completed a baseline assessment visit .
fifty percent of participants were obese ( bmi 95th percentile ) , 16% were overweight ( bmi 85th and < 95th percentile ) , and 34% were nonoverweight ( bmi < 85th percentile ) but had at least one overweight parent .
demographic , anthropometric , and metabolic characteristics of the study participants by depressive symptom status are described in table 1 . compared with children with lower depressive symptoms ( n = 160 ) , children with elevated depressive symptoms ( n = 38 ) were slightly , but significantly , younger ( aged 8.1 1.5 years vs. 8.7 1.6 years , p = 0.03 ) , were more likely to have hyperinsulinemia ( 31.6 vs. 14.4% , p = 0.01 ) , and were more likely to have elevated insulin resistance ( homa - ir 3.16 ; 34.2 vs. 16.9% , p = 0.02 ) at baseline .
fifty - eight percent of children ( n = 115 ) returned for a follow - up phlebotomy appointment an average of 5.8 1.3 years ( range 3.18.3 ) later .
mean age at follow - up was 14.6 2.3 years ( range 8.920.3 ) .
youth who did not complete a follow - up appointment had greater baseline depressive symptoms ( 8.4 6.5 vs. 6.8 5.2 , p = 0.05 ) , higher baseline fasting insulin ( 11.0 7.7 vs. 8.5 5.6 , p = 0.01 ) , and higher baseline insulin resistance ( 2.5 1.8 vs. 1.9 1.3 , p = 0.01 ) than youth who did return for a follow - up .
participant characteristics at baseline assessment data are means se ( range ) , unless otherwise indicated .
when race / ethnicity was defined dichotomously as non - hispanic white vs. other , its association with depressive symptoms status remained nonsignificant ( p = 0.10 ) .
table 2 summarizes the analyses examining baseline depressive symptoms as a predictor of follow - up insulin resistance , fasting insulin , and fasting glucose .
controlling for all other variables in the models , children s baseline depressive symptoms predicted greater insulin resistance and higher fasting insulin and glucose at follow - up ( p < 0.01 ) .
baseline depressive symptoms explained 8% of the variance in follow - up insulin resistance ( p < 0.001 ) , 7% of the variance in follow - up fasting insulin ( p < 0.001 ) , and 4% of the variance in follow - up fasting glucose ( p = 0.019 ) , after accounting for the other variables in the model . an identical pattern of results was observed when depressive symptoms were considered categorically .
youth with elevated depressive symptoms at baseline ( n = 17 ) had higher insulin resistance , fasting insulin , and fasting glucose at follow - up than youth with lower depressive symptoms at baseline ( p 0.001 ) ( fig .
, we also tested whether baseline insulin resistance predicted follow - up depressive symptoms , accounting for baseline depressive symptoms and a parallel set of covariates .
insulin resistance was not a significant predictor of follow - up depressive symptoms ( p = 0.99 ) .
multiple hierarchical regressions examining children s baseline depressive symptoms as a predictor of follow - up insulin resistance , fasting insulin , and fasting glucose compared with children with lower depressive symptoms at baseline ( n = 97 ; cdi total score < 13 ) , youth with elevated depressive symptoms at baseline ( n = 18 ; cdi total score 13 ) had greater follow - up : insulin resistance ( homa - ir : [ means se ] 2.8 0.2 vs. 4.3 0.4 , p < 0.001 ) ( a ) , fasting insulin ( 13.2 0.8 u / l vs. 19.4 1.7 u / l , p = 0.001 ) ( b ) , and fasting glucose ( 85.3 0.8 mg / dl vs. 92.9 1.7 mg / dl , p < 0.001 ) ( c ) , adjusting for the respective baseline values , sex , race , family history of type 2 diabetes , baseline age , baseline bmi , bmi change , and time in study . at follow - up , 43 ( 37.4% ) children met the criteria for elevated insulin resistance , 43 ( 37.4% ) for hyperinsulinemia , and 4 ( 3.8% ) for impaired fasting glucose .
table 3 presents a summary of analyses examining baseline depressive symptoms as a predictor of elevated insulin resistance , hyperinsulinemia , and impaired fasting glucose .
accounting for all other variables in the model , each one - unit increase in cdi total score at baseline was associated with a 1.14 greater odds of elevated insulin resistance at follow - up ( 95% ci 1.011.28 , p < 0.05 ) .
when depressive symptoms were considered categorically , those with and without elevated depressive symptoms at baseline did not significantly differ in their odds of elevated insulin resistance ( p = 0.10 ) , hyperinsulinemia ( p = 0.52 ) , or impaired fasting glucose ( p = 0.98 ) , adjusting for all of the same covariates .
children s baseline depressive symptoms as a predictor of follow - up elevated insulin resistance , hyperinsulinemia , and impaired fasting glucose data are odds ratios ( 95% cis ) .
the current study provides evidence that children s depressive symptoms are a prospective risk factor for worsening insulin resistance .
even when accounting for known additional risk factors , including family history of type 2 diabetes , children s baseline bmi , and changes in children s bmi over time , depressive symptoms were associated with greater insulin resistance ~6 years later .
depressive symptoms impact on insulin resistance was clinically meaningful such that depressive symptoms were associated with a significantly greater likelihood of developing clinically elevated homa - ir ( defined as 3.16 ) .
of note , children s degree of insulin resistance is a significant predictor of type 2 diabetes onset in young adulthood , even after accounting for bmi ( 17 ) .
the current findings are consistent with previous cross - sectional studies demonstrating a link between depressive symptoms or negative affect and insulin resistance in youth independent of body composition ( 12,13 )
. moreover , the present results are consistent with adult data demonstrating that depressive symptoms are related to greater odds of developing type 2 diabetes ( 9 ) .
the mechanisms explaining the relationship between depressive symptoms and insulin resistance are not well understood .
symptoms of depression , including fatigue , lack of energy , or anhedonia ( referring to loss of pleasure over activities that one previously found enjoyable ) , may prompt behavioral decreases in voluntary energy expenditures , such as exercise , which , in turn , may heighten insulin resistance .
consistent with this notion , adolescent depressive symptoms are associated with poorer cardiorespiratory fitness ( 18 ) .
depressive symptoms also have been concurrently related to emotional eating patterns ( 19 ) , which possibly may promote insulin resistance independent of weight gain . from a neurohumoral framework ,
depressive symptoms are hypothesized to promote insulin resistance by upregulating cortisol and enhancing its downstream effects , including increasing the production of the neurotransmitter neuropeptide y ( 10,20,21 ) .
strengths of the current investigation include the longitudinal nature of data , the examination of depressive symptoms and insulin resistance in a sizeable sample of children , and the adjustment for important covariates , including measured bmi and bmi change .
the measure of insulin resistance was derived from fasting values , which , although highly related to clamp - derived measures , is not considered as precise an assessment . likewise , although the cdi is a widely used , reliable , and valid measure of depressive symptoms , it does not provide a diagnostic assessment of clinical depression .
future longitudinal studies examining the impact of depressive symptoms on insulin resistance using criterion measures are warranted .
another significant study shortcoming was the very high degree of attrition that diminished the sample size at follow - up .
although the greater likelihood of dropout among youth with greater baseline depressive symptoms and higher insulin resistance could be expected to attenuate the significance of the results , this pattern , as well as the nature of the sample being oversampled to include youth at risk for adult obesity , may limit the generalizability of the findings .
in addition , the effect of depressive symptoms on insulin resistance was small relative to the effect of anthropometric variables .
examination of the depression - insulin resistance relationship in samples of adolescents with clinically elevated symptomatology may shed more light on the magnitude of the depression - insulin relationship .
adolescence marks a developmental period notable for a normative increase in insulin resistance that typically resolves by the end of puberty ( 2225 ) . yet
, youth vulnerable for type 2 diabetes may display the largest increases in insulin resistance and continued progression of worsening insulin resistance throughout late adolescence and possibly into young adulthood .
therefore , investigation of the impact of child or adolescent depressive symptoms on the progression of insulin resistance during an even longer follow - up interval would be important to clarify the role of pediatric depressive symptoms in the development of type 2 diabetes .
an equally important task for future research is to elucidate the mechanisms by which depressive symptoms may impact insulin resistance .
an understanding of the putative behavioral and/or physiological factors that explain how depression relates to insulin resistance is crucial to the design of effective interventions . in the current study
, we observed that depressive symptoms begin to exert an impact on insulin resistance early in life .
among adults , interventions targeting depressive symptoms among adults with type 2 diabetes and/or major depression have been shown to improve indices of glucose impairment or insulin resistance even without altering body weight or adiposity ( 1 ) .
research is needed to determine whether early interventions to decrease youths depressive symptoms will ameliorate worsening insulin resistance and consequently lessen the risk of developing type 2 diabetes .
if treating or preventing the onset of major depression in adolescents improves insulin resistance , routine depression screening in primary care settings , especially among youth at risk for type 2 diabetes , might have the potential to delay or possibly prevent type 2 diabetes onset in a considerable subset of individuals .
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objectivethe purpose of this study was to determine whether having childhood depressive symptoms is a risk factor that prospectively predicts impairment in glucose homeostasis.research design and methodsa non treatment - seeking sample of 115 children ( aged 513 years ) , oversampled for being at risk for adult obesity , was assessed at baseline and again ~6 years later .
children self - reported depressive symptoms using the children s depression inventory at baseline .
insulin resistance was assessed at baseline and follow - up with the homeostasis model assessment of insulin resistance index ( homa - ir).resultschildren s depressive symptoms were a significant predictor of follow - up homa - ir , fasting insulin , and fasting glucose in models accounting for baseline homa - ir , insulin , or glucose values ; sex ; race ; baseline age ; baseline bmi ; change in bmi at follow - up ; family history of type 2 diabetes ; and time in the study ( p < 0.01).conclusionsin this study , depressive symptomatology at baseline predicted the progression of insulin resistance during child and adolescent development independent of changes in bmi .
research is needed to determine whether early intervention to decrease elevated depressive symptoms in youth ameliorates later development of insulin resistance and lessens the risk of type 2 diabetes .
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these samples came from hunter - harvested red deer in 21 wild or semifree ranging populations ( 892 deer ) and from 2 farms ( 76 deer ) .
sampling sites were representative of a variety of habitats and climates , which can be simplified into 5 different bioregions ( figure ) ( 12 ) .
sampling sites were grouped into 7 areas and 2 red deer farms ( table ; figure ) .
management conditions of red deer were classified as open ( no fencing and no management , 9 sites ) , fenced ( fencing and artificial feeding , 12 sites ) , and farmed ( livestock - like management , 2 farms ) . to analyze time trends , we classified samples collected during 20002005 as time 1 and those collected during 20062009 as time 2
. only sites where sampling occurred in both periods and with comparable sampling sizes were included in the time - trend analysis .
broken line borders indicate open sites ; solid lines indicate fenced estates ; asterisks indicate the 2 red deer farms . *
ig , immunoglobulin ; hev , hepatitis e virus ; ci , confidence interval ; rt - pcr , reverse transcription pcr . red deer farms .
serum samples were tested for hev immunoglobulin ( ig ) g by using elisa as described ( 4,13 ) , except for including protein g horseradish peroxidase ( sigma chemical , st .
louis , mo , usa ) as a conjugate , as in previous studies of red deer ( 12 ) .
anti - hev positive serum was obtained from domestic swine that were positive for hev by elisa and reverse transcription
anti - hev negative serum was obtained from previous studies ( 14 ) and negative controls from hev - negative cattle ( 13 ) .
results were expressed as the percentage of optical density ( % od ) by using the formula [ % od = 100 sample od / sum of negative controls od ] .
serum samples with % od values > 100% were considered positive . for the rt - pcr ,
viral rna was extracted from 150 ml of serum with nucleospin rna virus kit ( macherey - nagel , dren , germany ) by following the manufacturer s instructions .
hev was detected by using a seminested rt - pcr as described ( 14 ) . in each run ,
hev sequences were identified by using the blast algorithm ( www.ncbi.org ) against hev sequences available in genbank ( on january 25 , 2010 ) .
tests were used to analyze the association of age , sex , sampling site , and management conditions with serologic and rt - pcr results .
association between seropositivity and hev rna in the serum was also analyzed by using pearson test .
overall , 101 ( 10.4% , 95% ci 8.612.5 ) serum samples were positive for igg ( table ) .
hev seroprevalence did not differ significantly between sex ( = 0.894 , 1 df , p>0.05 ) and age classes ( = 12.436 , 3 df , p>0.05 ) .
seroprevalence in time 2 ( 12.2% , 95% ci 9.815.0 ) was significantly higher than seroprevalence in time 1 ( 7.5% , 95% ci 5.110.8 ) ( = 5.181 , 1 df , p<0.05 ) .
local igg seroprevalences ranged from 0 ( 95% ci 020.8 ) to 31.4% ( 95% ci 21.343.5 ; figure ) .
igg seroprevalence differed significantly by management types ( 6.876 , 2 df , p<0.05 ) , with higher values in open ( 14.9% , 95% ci 11.319.4 ) than in fenced ( 9.1% , 95% ci 7.011.7 ) and farmed ( 2.6% , 95% ci 0.59.0 ) areas .
eleven ( 13.6% , 95% ci 7.422.7 ) of 81 samples were rt - pcr positive .
local viral rna prevalence ranged from 4.5% ( 95% ci 2.422.21 to 38.5% ( 95% ci 16.665.8 ; table ) .
seven samples belonging to sequence hm113374 shared 99% nucleotide identity with domestic swine strains from spain .
one sample , sequence hm113373 , showed similarity ( 91% ) with a strain from acute hepatitis e in a person in marseille , france , according to genbank .
our findings of hev infection confirm that hev circulates actively among red deer in the iberian peninsula , as described for wild boar ( 9 ) .
red deer can be infected with hev ( 7,8 ) , and the results of our large serosurvey in this species in europe show an increasing prevalence trend in the last decade .
de deus et al . found higher igg seroprevalences in estates with higher wild boar densities ( 9 ) .
however , in the present study , mean seroprevalence rates were lowest in red deer farms , where densities were the highest and red deer had no contact with wild boar or domestic swine .
in contrast , the highest seroprevalence rates were reported in open areas where contact with suids may have occurred . however
, wild boar densities also are high in fenced hunting estates ( 15 ) , and hev antibody prevalence rate was intermediate in deer from these sites
. these differences could indicate that red deer may need a source of infection and thus act as spillover hosts more frequently than as true reservoirs .
presence of hev rna in 13% of deer serum implies that deer represent a risk for zoonotic transmission , and consequently , handling of live animals and carcasses is a risk activity .
red deer are infected with hev at lower rates than are wild boar and domestic pigs but may act as a potential source of hev infection in humans .
further studies are needed to fully elucidate the epidemiology of hev in wildlife and the foodborne zoonotic transmission risks .
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to describe the epidemiology of hepatitis e virus ( hev ) in red deer in mainland spain , we tested red deer for hev rna and antibodies .
overall , 10.4% and 13.6% of serum samples were positive by elisa and reverse transcription
pcr , respectively .
the increasing prevalence suggests a potential risk for humans .
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patients use a purgative the day before the procedure to improve the accuracy of the diagnosis .
oral sodium phosphate ( osp ) and polyethylene glycol ( peg ) solutions are commonly used for bowel preparation because of their ease of use and their safety profile .
osp has an advantage over peg in terms of a lower fluid volume to be ingested ( two bottles of 45 ml each ) .
initially , the adverse effects of this agent were unremarkable , especially in patients with normal kidney function . however , because of cases of osp - induced acute renal failure , the safety of osp has been questioned .
furthermore , it was noted that acute renal failure can progress to chronic kidney disease ( ckd ) .
here , we report a case of ckd caused by acute phosphate nephropathy ( aphn ) following use of osp as a bowel purgative .
a 51-year - old woman was admitted to inha university hospital for evaluation of renal failure that was recently found at a local clinic .
she had been diagnosed with essential hypertension 3 years previously , which was managed with hydrochlorothiazide 12.5 mg and candesartan 16 mg once a day .
she took ibuprofen 400 mg on the initial day of menstruation on a monthly basis for 30 years to relieve abdominal pain .
bowel preparation involved ingestion of osp ( phospho soda , fleet , usa ) . after 8 hours of npo , she visited the health promotion center at 09:00 hours .
then , esophagogastroduodenoscopy ( egd ) and colonoscopy were performed under anesthesia using intravenous midazolam 5 mg .
serum blood urea nitrogen ( bun ; 8.8 mg / dl ) , creatinine ( 0.86 mg / dl ) , calcium ( 9.0 mg / dl ) , phosphate ( 4.7 mg / dl ) , and albumin ( 4.3 g / dl ) ) were all normal at that time .
blood pressure was 130/70 mmhg , pulse rate was 72/minute , and body temperature was 37 c .
blood tests yielded the following results : hemoglobin 8.3 g / dl , platelets 350,000/l , white blood cells 8170/l , bun 42.5 mg / dl , creatinine 2.95 mg / dl , calcium 8.5 mg / dl , phosphate 3.7 mg / dl , total protein 7.9 g / dl , albumin 4.0 g / dl , glucose 102 mg / dl , sodium 140 meq / l , potassium 4.6 meq / l , chloride 103 meq / l , and total co2 21.4 meq / l .
c3 was 134 mg / dl , c4 was 34 mg / dl , and antinuclear antibody , anti - neutrophil cytoplasm antibody , cryoglobulin , ra factor , anti - ds dna antibody , and anti - gbm antibody tests were all negative .
serum iron was 40 g / dl , total iron - binding capacity was 303 g / dl , ferritin was 28.7 g / ml , and stool occult blood was negative according to egd .
the 24-hour urine volume was 3700 ml , with protein of 595 mg and creatinine of 1.0 g. a bone marrow biopsy showed no evidence of multiple myeloma or other diseases .
however , renal contour and size were normal ( right 11.9 cm , left 11.3 cm , long axis ; fig . 1 ) and there was no evidence of a renal stone or hydronephrosis .
l of normal saline for 10 day . however , her serum creatinine level remained elevated ( 2.74 mg / dl ) . to find the cause of renal failure , a kidney biopsy was performed on day 10 .
multifocal scattered calcifications were present within the tubules , mainly in the distal tubules ( fig .
the interstitium showed a moderate degree of fibrosis and inflammatory cell infiltration , mainly of lymphocytes and some neutrophils ( fig .
3 ) . the tubular calcifications did not polarize and were positive to von kossa stain ( fig .
3 ) , which confirmed their composition as calcium phosphate . the glomeruli appeared mildly enlarged and there were no capillary wall changes .
after kidney biopsy , a further examination was performed to rule out other causes of nephrocalcinosis .
serum intact parathyroid hormone ( ipth ) decreased from an initial level of 186 pg / ml to 54.6 pg / ml after 2 months .
results for 24-hour urinary analytes were as follows : sodium , 98 meq / day ( normal range 40220 meq / day ) ; uric acid , 425 mg / day ( 250750 mg / day ) ; citric acid , 16 mg / day ( 3201240 mg / day ) ; oxalate , 3.36 mg / day ( 16.2053.30 mg / day ) ; calcium , 35.0 mg / day ( 70.0180.0 mg / day ) ; and creatinine , 0.9 g / day ( 1.02.0 g / day ) .
supportive treatment was given and the patient 's kidney function remained unchanged 3 months later , with a creatinine level of 2.21 mg / dl .
this case shows that exposure to osp as a bowel purgative is associated with the development of aphn in patients with risk factors and can progress to ckd .
furthermore , in ckd patients with unknown cause , clinicians should consider that use of an osp bowel purgative may be one of the possible causes of the development of ckd .
osp preparations are osmotic purgatives that obligate water excretion into the intestinal lumen to maintain its isotonicity with plasma .
each 45-ml dose of phospho soda contains 5 g of sodium and 17.8 g of phosphate , yielding a solution with osmolarity of 16,622 mosm / l .
various complications , including hyperphosphatemia , hypocalcemia , hypernatremia , hypokalemia , metabolic acidosis , and acute kidney injury ( aki ) , have been reported in association with osp ingestion .
therefore , desmeules et al . used the term aphn for aki following osp use as a bowel purgative . the incidence has been reported as 1.295.0% , .
calcium phosphate deposition in renal tubules and interstitium appears to be a mechanism underlying aphn and has been demonstrated histopathologically using von kossa stain .
the phosphates in calcium phosphate deposits are detected in paraffin sections using von kossa stain , which involves a precipitation reaction in which silver ions react with phosphate to yield a black product .
, calcium salts are typically found along medullary tubular basement membranes , as concretions within tubules , and in the interstitium .
by contrast , the calcium phosphate deposits seen in aphn are found primarily in the tubular lumen and the cytoplasm of tubular epithelial cells , with rare interstitial deposits as well .
hypercalciuria and hyperoxaluria are also risk factors for nephrocalcinosis . in our case , the elevated serum ipth level seemed to be due to ckd , and biopsies in such cases show calcium phosphate deposits , primarily in the tubular lumen .
our case did not show hypercalcuria , hyperoxaluria or a history of herb ingestion . among patients with ckd of unknown cause
, it has been reported that chinese herb nephropathy may be an etiologic agent , in which progressive tubulointerstitial nephritis and development of end - stage renal disease within 12 years are characteristics , .
following intake of a large amount of phosphate , proximal tubular phosphate reabsorption is decreased , leading to a rapid increase in delivery to the distal tubule .
diarrhea - induced hypovolemia leads to avid salt and water reabsorption in both the proximal tubule and the descending limb of henle 's loop , which is relatively impermeable to calcium and phosphate .
the net effect is a decrease in phosphate reabsorption in the proximal tubule , leading to a marked increase in calcium
hypovolemia - associated tubular injury may also precondition the distal tubular epithelium , leading to surface expression of hyaluronan and osteopontin , which in turn creates a suitable environment for adherence of calcium phosphate crystals .
a direct toxic effect of hyperphosphatemia on renal tubules is proposed as one of the mechanisms of renal failure after osp use . in a rat model of aphn involving intravenous administration of phosphorus ,
zarger et al . observed vacuolization of tubular cells without calcium phosphate deposition in renal biopsies .
old age , female gender , liver disease , renal disease , decreased bowel motility , hypertension , diabetes , and medications contributing to renal hypoperfusion , such as diuretics , angiotensin - converting enzyme inhibitors and angiotensin ( at)ii receptor blockers , could predispose to this disorder . in our case ,
the patient had risk factors of female gender , hypertension , and at - ii receptor blocker use .
the pathophysiology has not been elucidated yet , but it appears to be associated with activation of the innate immune system in mediating the pro - inflammatory effects of tissue crystal deposition .
pattern recognition receptors , such as the toll - like receptor ( tlr ) , mediate monosodium urate monohydrate - dependent nitric oxide and il-1 release from chondrocytes and macrophages , respectively . given the ability of specific tlrs to recognize calcium crystals and the expression of a variety of tlrs in adult kidney , it is possible that intraluminal calcium phosphate crystal formation after osp is recognized by specific epithelial tlrs , with subsequent activation of the innate immune response .
persistence of these crystals could result in chronic inflammation with extracellular matrix deposition and interstitial fibrosis .
we speculate that iron deficiency anemia in our case was caused by a decreased iron intake due to persistent anorexia .
there are only a few reports in terms of ckd after aphn following osp ingestion as a bowel purgative so far in korea .
kim et al . reported a case of aphn following osp use as a bowel purgative in a 66-year - old female .
we report a case of aphn that progressed to ckd , as proven by renal biopsy , in a female patient of normal renal function prior to colonoscopy .
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acute phosphate nephropathy ( aphn ) following oral sodium phosphate solution ( osp ) ingestion as a bowel purgative has been frequently reported .
it was recently suggested that aphn could progress to chronic kidney disease ( ckd ) and a history of aphn might be considered as one of the causes of ckd .
however , there are few reports proving aphn as a cause of ckd . here
, we report a case of aphn that progressed to ckd , as proven by renal biopsy .
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syphilis , a sexually transmitted infection , is caused by the spirochete bacterium treponema pallidum ( tp ) subspecies palladium , which is strongly contagious and capable of infecting multiple tissues and organs ( 1 ) . since the year 2000
, the incidence of syphilis has been increasing in the usa , canada , the uk , australia , and europe , and it has a mortality of 8% to 58% , with a greater death rate in males ( 2 ) . in sub - saharan africa
, it caused about 113,000 deaths , with more than 90% of the cases in developing countries ( 4 ) .
syphilis has complex clinical manifestations and tp can not be cultured in vitro ; therefore , serological tests for syphilis are key elements of the current diagnostic and therapeutic monitoring ( 5 ) .
serological testing for syphilis can be divided into two categories : the tp antigen serological test and the non - tp antigen serological test ( 6 ) .
specific antibodies against tp are first detected during the incubation period of syphilis infection and the positive response remains high for an extended period after the cure , sometimes even for the lifetime of the patient ( 7 ) .
the live or dead tp or its ingredients are used as the antigen in laboratory tests , with the common tests being the tp enzyme - linked immunosorbent assay ( tp - elisa ) , the tp hemagglutination test ( tpha ) , the tp particle agglutination test ( tppa ) , the fluorescent treponemal antibody absorption test ( fta - abs ) , the 19s - igm tp hemagglutination test ( 19s - igm ) , and the tp hemagglutination test ( mha - tp ) ( 8) . the non - treponemal antigen serum tests , which include the venereal disease research laboratory test ( vdrl ) , the unheated serum reagin test ( usr ) , the syphilis rapid plasma reagin test ( rpr ) , and the toluidine red unheated serum test ( trust ) , use a cardiolipin antigen to detect serum anti - cardiolipin antibodies .
a quantitative non - treponemal test of antibody titers is also important because antibody titers usually correlate with disease activity and show responses to treatment ( 9 ) .
after proper treatment , the antibody level ( titer ) decreases in the serum , so the titer can be used as a marker of the treatment efficacy or reinfection .
in recent years , the articles on serologic testing for syphilis in hospitalized patients have focused on the tp antibody positive rate . however , syphilis serological titer distributions are rarely reported .
therefore , in our study , we analyzed the tp - elisa results obtained for inpatients in wenzhou central hospital from january 2010 to december 2012 and the distribution of trust titers among the tp - antibody - positive patients .
the study was approved by the ethics committee of wenzhou central hospital , wenzhou , china .
the syphilis test results were collected for inpatients ( 34,165 males and 47,781 females ) treated in our hospital from january 2010 to december 2012 .
the tp - elisa was performed with a tp - elisa kit ( livzon diagnostics , inc . , zhuhai , china ) according to the manufacturer s instructions
. diluted sample ( 50 l ) was added to each well of 96 well plates and incubated for 1 hour at 37c .
the tp antigen ( 50 l ) , conjugated with horseradish peroxidase ( hrp ) , was then added to each well and incubated for 1 hour at 37c .
the wells were then washed with pbs , and 50 l of reagent a and b were added to each well and incubated in the dark for 30 minutes .
the positivity of the tp - antibody - positive patients was reaffirmed with a tppa test ( fujirebio diagnostics inc . ,
tokyo , japan ) based on the agglutination of colored gelatin particle carriers sensitized with tp ( nichols strain ) antigen .
particles sensitized by tp antigen and nonsensitized particles were added ( 25 l ) to 50 l of the 1:80 and 1:40 dilutions , respectively .
the mixtures were oscillated for 30 seconds , incubated at room temperature for 2 hours , and then each well was examined for agglutination .
the particle agglutination that appeared polygonal , coarse , and macrocyclic in the 1:80 dilution and small , circular , and smooth in the 1:40 dilution were designated as positive , while the particle agglutination that appeared as small , circular , and smooth in the 1:80 dilution was designated as negative .
serum samples ( 50 l ) were dropped onto paper cards and 20 l trust reagent ( rongsheng biological pharmaceutical co. , ltd . ,
the samples were shaken at 100 rpm for 8 minutes and then examined for agglutination . if the result was positive , the test sera were serially diluted with saline and re - tested as described above .
the differences in tp - positive rate and trust titer < 1:8 rate between different years , genders , and age groups were analyzed by the chi - square test .
the study was approved by the ethics committee of wenzhou central hospital , wenzhou , china .
the syphilis test results were collected for inpatients ( 34,165 males and 47,781 females ) treated in our hospital from january 2010 to december 2012 .
the tp - elisa was performed with a tp - elisa kit ( livzon diagnostics , inc . , zhuhai , china ) according to the manufacturer s instructions .
diluted sample ( 50 l ) was added to each well of 96 well plates and incubated for 1 hour at 37c . the tp antigen ( 50 l ) , conjugated with horseradish peroxidase ( hrp ) , was then added to each well and incubated for 1 hour at 37c .
the wells were then washed with pbs , and 50 l of reagent a and b were added to each well and incubated in the dark for 30 minutes .
the positivity of the tp - antibody - positive patients was reaffirmed with a tppa test ( fujirebio diagnostics inc . ,
tokyo , japan ) based on the agglutination of colored gelatin particle carriers sensitized with tp ( nichols strain ) antigen .
particles sensitized by tp antigen and nonsensitized particles were added ( 25 l ) to 50 l of the 1:80 and 1:40 dilutions , respectively .
the mixtures were oscillated for 30 seconds , incubated at room temperature for 2 hours , and then each well was examined for agglutination .
the particle agglutination that appeared polygonal , coarse , and macrocyclic in the 1:80 dilution and small , circular , and smooth in the 1:40 dilution were designated as positive , while the particle agglutination that appeared as small , circular , and smooth in the 1:80 dilution was designated as negative .
serum samples ( 50 l ) were dropped onto paper cards and 20 l trust reagent ( rongsheng biological pharmaceutical co. , ltd . ,
the samples were shaken at 100 rpm for 8 minutes and then examined for agglutination . if the result was positive , the test sera were serially diluted with saline and re - tested as described above .
the differences in tp - positive rate and trust titer < 1:8 rate between different years , genders , and age groups were analyzed by the chi - square test .
the gender / age distributions of hospitalized patients from 2010 to 2012 are shown in table 1 . a total of 81,946 inpatients , with slightly more female than male patients , agreed to serological testing for syphilis .
patients aged 20 - 39 years accounted for the largest proportion ( 33.8% ) , while patients older than 80 made up the smallest proportion ( 8% ) . of the 81,946 inpatients , 1618 ( 1.97% ) patients were identified as tp antibody - positive ( table 2 ) . among these tp - positive patients , 549 had early primary syphilis , 130 had late syphilis , and 939 had latent syphilis .
about 2.27% of the cases ( 566/24920 ) in 2010 , 1.58% cases ( 460/29077 ) in 2011 , and 2.11% cases ( 592/27949 ) in 2012 were identified as positive .
a significant difference was observed in the positive rate between 2011 and 2010 ( = 34.20 , p < 0.01 ) , as well as between 2011 and 2012 ( = 22.62 , p < 0.01 ) . however , no significant difference was noted between 2010 and 2012 ( = 1.442 , p = 0.23 ) . across the three years , the tp antibody positive rate was lower in female than in male patients . among the total 34165 male inpatients , 812 cases were tp antibody positive , whereas 806 of the 47781 female inpatients were tp antibody positive .
the positive rate was higher among male inpatients ( 2.37% ) than among female inpatients ( 1.68% ) ( = 48.97 , p < 0.01 ) .
patients older than 80 years showed the highest positive rate in 2010 and 2012 , whereas the positive rate was the lowest among patients aged 20 - 39 years across all 3 years . in total , the positive rate for the group aged 80 years ( 3.63% ) was the highest , followed by the groups aged 19 years ( 2.83% ) , 60 - 79 years ( 2.38% ) , 40 - 59 years ( 2.14% ) , and 20 - 39 years ( 0.94% ) , in decreasing order .
the group aged 60 - 79 years had the greatest number of positive cases , whereas the group aged 19 years had the fewest cases . among the positive male patients , the group aged 60 - 79 years
had the highest positive rate ( 35.96% ) , while the group aged 20 - 39 years had the lowest rate ( 6.77% ) .
in contrast , the female group aged 40 - 59 years had the highest positive rate ( 26.30% ) and the female group aged 19 years had the lowest positive rate ( 10.30% ) ( table 3 ) .
the number of trust positive cases was lower in 2012 than in 2010 and 2012 ( table 4 and figure 1 ) .
the proportions of patients with trust titers of 1:8 or less in 2010 , 2011 , and 2012 were 81.45% , 79.78% , and 89.53% , respectively , and the differences were statistically significant between 2012 and 2010 , as well as between 2012 and 2011 ( = 15.30 , p < 0.01 ; = 19.56 , p < 0.01 , respectively ) . compared with 2010 , 2011 , p < 0.01 * ; compared with the 20 - 39 group , p < 0.01#. values are expressed as no .
no statistical significance was observed for the changes in or the distribution of the serum trust titers between male and female tp antibody positive patients ( = 11.23 , p = 0.19 ) ( table 4 and figure 2 ) .
the proportion of low serum titers ( < 1:8 ) in men was 83.25% and in women was 84.62% , and this difference was not statistically significant ( = 0.55 , p = 0.45 ) .
the proportion of trust serum titers of 1:8 or less for the group aged 80 years was the highest ( 87.45% ) , followed by the group aged 60 - 79 years ( 86.12% ) , the group aged 19 years ( 84.65% ) , the group aged 40 - 59 year ( 81.69% ) , and the group aged 20 - 39 years ( 79.69% , figure 3 ) . a statistically significant difference was determined between the group aged 80 years and the group aged 20 - 39 years ( = 5.418 , p = 0.02 ) .
the gender / age distributions of hospitalized patients from 2010 to 2012 are shown in table 1 . a total of 81,946 inpatients , with slightly more female than male patients , agreed to serological testing for syphilis .
patients aged 20 - 39 years accounted for the largest proportion ( 33.8% ) , while patients older than 80 made up the smallest proportion ( 8% ) . of the 81,946 inpatients , 1618 ( 1.97% ) patients were identified as tp antibody - positive ( table 2 ) . among these tp - positive patients , 549 had early primary syphilis , 130 had late syphilis , and 939 had latent syphilis .
about 2.27% of the cases ( 566/24920 ) in 2010 , 1.58% cases ( 460/29077 ) in 2011 , and 2.11% cases ( 592/27949 ) in 2012 were identified as positive .
a significant difference was observed in the positive rate between 2011 and 2010 ( = 34.20 , p < 0.01 ) , as well as between 2011 and 2012 ( = 22.62 , p < 0.01 ) . however , no significant difference was noted between 2010 and 2012 ( = 1.442 , p = 0.23 ) . across the three years , the tp antibody positive rate was lower in female than in male patients . among the total 34165 male inpatients , 812 cases were tp antibody positive , whereas 806 of the 47781 female inpatients were tp antibody positive .
the positive rate was higher among male inpatients ( 2.37% ) than among female inpatients ( 1.68% ) ( = 48.97 , p < 0.01 ) .
patients older than 80 years showed the highest positive rate in 2010 and 2012 , whereas the positive rate was the lowest among patients aged 20 - 39 years across all 3 years . in total , the positive rate for the group aged 80 years ( 3.63% ) was the highest , followed by the groups aged 19 years ( 2.83% ) , 60 - 79 years ( 2.38% ) , 40 - 59 years ( 2.14% ) , and 20 - 39 years ( 0.94% ) , in decreasing order .
the group aged 60 - 79 years had the greatest number of positive cases , whereas the group aged 19 years had the fewest cases . among the positive male patients , the group aged 60 - 79 years
had the highest positive rate ( 35.96% ) , while the group aged 20 - 39 years had the lowest rate ( 6.77% ) .
in contrast , the female group aged 40 - 59 years had the highest positive rate ( 26.30% ) and the female group aged 19 years had the lowest positive rate ( 10.30% ) ( table 3 ) .
in the 1618 tp antibody - positive patients , a total of 1395 were diagnosed as trust positive .
the number of trust positive cases was lower in 2012 than in 2010 and 2012 ( table 4 and figure 1 ) .
the proportions of patients with trust titers of 1:8 or less in 2010 , 2011 , and 2012 were 81.45% , 79.78% , and 89.53% , respectively , and the differences were statistically significant between 2012 and 2010 , as well as between 2012 and 2011 ( = 15.30 , p < 0.01 ; = 19.56 , p < 0.01 , respectively ) . compared with 2010 , 2011 , p < 0.01 * ; compared with the 20 - 39 group , p <
no statistical significance was observed for the changes in or the distribution of the serum trust titers between male and female tp antibody positive patients ( = 11.23 , p = 0.19 ) ( table 4 and figure 2 ) .
the proportion of low serum titers ( < 1:8 ) in men was 83.25% and in women was 84.62% , and this difference was not statistically significant ( = 0.55 , p = 0.45 ) .
the proportion of trust serum titers of 1:8 or less for the group aged 80 years was the highest ( 87.45% ) , followed by the group aged 60 - 79 years ( 86.12% ) , the group aged 19 years ( 84.65% ) , the group aged 40 - 59 year ( 81.69% ) , and the group aged 20 - 39 years ( 79.69% , figure 3 ) . a statistically significant difference was determined between the group aged 80 years and the group aged 20 - 39 years ( = 5.418 , p = 0.02 ) .
the tp antibody positive rate among the inpatients was statistically the highest ( 2.27% ) in 2010 , and it dropped to 1.58% in 2011 , followed by a tendency toward an increase in 2012 .
the total tp antibody positive rate was lower than that reported by ganesan et al .
the number of tp antibody positive patients with serum trust titers was significantly increased in 2012 compared to 2010 and 2011 .
the trust positive rates were 82.32% , 81.38% , and 83.33% in the studies conducted by yin et al .
( 12 ) , jian and mingxia ( 13 ) , and wang et al .
( 15 ) reported that the proportion of low serum titers of 1:8 or less was 84.28% in men and 83.45% in women , which was similar to the proportion and distribution of positive trust titers found in the tp - positive patients in our study .
the reason for the difference between tp antibody positive rate and serum trust titers was that the trust titer negative patients can not be excluded for the diagnosis of stage i and iii syphilis ( 16 , 17 ) . the trust titers are not sensitive for latent syphilis and neurosyphilis , and the patients with these conditions were negative after treatment .
the detected targets of trust were non - specific antibodies that emerged later than the specific antibodies .
the tp antibody positive and trust titer negative patients may be associated with aids and syphilis immunosuppression ; however , the b. burgdorferi antigen serum test should also be considered a false positive .
the tp antibody positive rate in men was 2.37% , which was significantly higher than that in women ( 1.68% ) . in china ,
the male population has relatively open sexual attitudes , and the relative mobility of the population is higher , so the probability of infection in men is relatively higher . no statistical significance was noted for the change and distribution of the serum trust titers between male and female positive patients , in agreement with the reports of yin et al .
the trust method is likely to miss the detection of positive cases , and the early diagnosis of syphilis is poor with the trust method .
the trust method detects stage ii syphilis , but is not sensitive to stage i and iii syphilis ( 16 , 17 ) .
some diseases , including autoimmune diseases , malaria , and sexually transmitted diseases , may cause false positives , as patients with underlying diseases may release anti - lipid or anti - tp antibodies that lead to cross - false positives ( 18 ) .
thus , the trust and tp - elisa , which are both convenient to perform and moderately priced , are suitable for screening of syphilis , but any trust - positive or tp - elisa - positive patients should then be confirmed by a specific method , such as tppa .
this conclusion is validated by the finding in the present study , where the tp antibody positive patients , who were all confirmed by tppa , did not all give positive results for the trust test .
the tp antibody positive rate in the group aged 80 year was the highest , which was consistent with the studies of bosshard et al .
( 20 ) , who reported that the population aged 70 years had the highest positive rate , but about eighteen percent were false positives . the incidence of false positives is less for the spiral antigen serum test than for the non - helical antigen serum test , as is commonly observed in autoimmune disease , lyme disease , leprosy , cancer , genital herpes , diabetes , lymphoma , meningioma , hypergammaglobulinemia , infectious mononuclear cell histiocytosis , heroin addiction , and pregnancy ( 21 ) .
the incidence of these diseases is also higher in elderly patients than in young patients , so the syphilis seropositivity is also higher . despite the high false positive rate in laboratory test results , the elderly infected patients , especially those with asymptomatic latent syphilis , can not be ignored . among the elderly people with positive syphilis serology , a portion also suffers from underlying diseases that induce the body to produce anti - lipid antibodies or anti - tp cross antigens and cause false positives ( 22 , 23 ) .
elderly patients generally have low levels of education and lack capabilities for disease prevention . for economic reasons and the fear of moral , family , and community condemnation ,
the elderly patients are often afraid to commit to active treatment ( 24 , 25 ) .
virtually , any acute sexually transmitted disease ( std ) may produce acute biological false positive results , but the serum responses of these cases are low , rarely exceeding 1:8 ( 26 , 27 ) .
when we applied specific tests such as the tpha , the serum response was negative .
chronic biological false positives can be sustained for several months or years , or even for a lifetime ; this is common in patients with autoimmune diseases , leprosy , cirrhosis , or narcotics addiction , and in pregnant women ( 28 , 29 ) .
the trust titer of 1:8 or less in the oldest group ( 80 years ) accounted for the highest proportion ( 87.45% ) , followed by the group aged 60 - 79 years ( 86.12% ) .
the tp antibody positive rate in the group aged 20 - 39 years was the lowest ( 0.94% ) , but the trust titer of 1:8 or more in this group accounted for the highest proportion of the population ( 20.31% ) .
the population in this group has relatively open sexual attitudes and the relative mobility of the population is higher , so the population of this group has important epidemiological significance as a source of infection .
in addition , the tp antibody positive rate of group aged 19 years was relative higher , and the newborns accounted for the vast majority ( 188/202 ) , so women of childbearing age should be conclusively diagnosed before marriage or pregnancy , or during the prenatal period , to reduce neonatal syphilis infection . in summary ,
the rate of tp antibody positivity was highest for the elderly group , followed by the group aged 19 years , although the highest serum trust titers were detected mainly in the group aged 20 - 39 years .
therefore , in addition to focusing on the prevention and treatment of syphilis in young patients and pregnant women , prevention work should further expand to the elderly .
the trust test is better used in syphilis screening and to judge the curative effects of treatment , but syphilis diagnosis needs specific methods such as tp - elisa and the tppa test . the diagnosis of syphilis should be made cautiously , and must be combined with clinical examination and medical history .
furthermore , we should strengthen the popularity of sex education as well as the knowledge of sexually transmitted diseases for the elderly .
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backgroundthe high prevalence of syphilis among inpatients is an important concern in clinical settings .
thus , a better understanding of the serological test would be valuable.objectiveswe analyzed the serological test results for syphilis among the inpatients in wenzhou central hospital , china , to estimate the distribution of syphilis this chinese population.patients and methodsthe blood samples of 81946 inpatients at the hospital from january 2010 to december 2012 were collected and retrospectively analyzed .
syphilis testing was conducted using a treponema pallidum enzyme - linked immunosorbent assay ( tp - elisa ) and a tp particle agglutination ( tppa ) assay . a toluidine red unheated serum test ( trust )
was then used to determine the titer of tp antibody in the tp- elisa - positive samples.resultsin total , 1618 of the 81946 inpatients showed positive syphilis serology ; the positive rates in 2010 , 2011 , and 2012 were 2.27% , 1.58% , and 2.11% , respectively .
males had a significantly higher positive rate when compared to females .
surprisingly , the highest positive rate was observed among patients older than 80 years , followed by patients younger than 19 years , while patients aged 20 - 39 years had the lowest positive rate .
the trust titer of most tp - positive cases was less than 1:8 .
patients aged 20 - 39 years showed the highest percentage of trust titer values 1:8 , while patients older than 80 years showed the lowest percentage ; the differences between these two groups were statistically significant.conclusionsthe serological characteristics of syphilis varied with gender and age .
syphilis screening and control should be conducted for young patients and pregnant women , but special attention should also be paid to elderly inpatients .
the trust assay is better used in syphilis screening and for judgment of curative effects , but the diagnosis needs specific methods , such as the tp - elisa and the tppa test .
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obesity is a significant public health problem in the us and worldwide ( 5 ) .
clinical guidelines define obesity in terms of excess body weight adjusted for height ( i.e. , body mass index [ bmi ] ) 25 ( 21 ) .
therefore , overweight in adults is defined as a bmi between 25.0 kg / m and 29.9 kg / m , and adults are considered obese if their bmi is at or above 30 kg / m , regardless of gender , age , or race - ethnicity ( 21 ) .
obesity is associated with increased risk of multiple chronic diseases , and elevated abdominal obesity , assessed by measuring waist circumference ( wc ) , is also associated with increased chronic disease risk .
clinical guidelines for excess wc are gender - specific and have been set at 102 cm for men and 88 cm for women ( 21 ) . despite a high correlation between both bmi and wc with total percent body fat ( % bf ) estimated from dual - energy x - ray absorptiometry ( dxa ) ( 7 ) ,
field - based body fat prediction equations can be particularly useful for health professionals given that bmi and wc are merely proxies for excess body fat , which varies considerably by age , gender , and race - ethnicity ( 2 , 3 , 4 , 35 9 , 10 , 12 ) .
therefore , the purpose of this study was to develop and compare two body fat prediction equations based on bmi or wc , gender , age , and race - ethnicity , using multiply imputed dxa data obtained from participants 20 + years of age that participated in the 19992004 national health and nutrition examination survey ( nhanes ) .
the nhanes is a continuous data collection initiative conducted by the national center for health statistics ( nchs ) of the centers for disease control and prevention ( cdc ) ( 17 ) .
the nhanes design is a stratified , multistage probability sample based on counties , blocks , 11 households , and the number of people within households .
data are collected via house hold interviews and health examinations at a mobile examination center ( mec ) .
detailed information regarding the nhanes survey design , procedures , and protocols can be found online ( 17 ) . due to the nature of the analysis ( secondary data analysis ) , and the lack of personal identifiers ,
this study was deemed exempt by the louisiana state university agricultural center institutional review board . beginning in 1999 , nhanes began collecting whole - body dxa scans on survey subjects 8 years of age and older to provide nationally representative data on body composition .
these data were collected across three survey cycles ( 19992000 , 20012002 , and 20032004 ) and were subsequently released as public - use data files in 2008 ( 16 ) .
the 19992004 nhanes dxa data sets contain estimates of absolute and relative total and regional body composition including fat mass , bone - free lean - tissue mass , and bone mineral .
trained technicians obtained whole - body dxa scans in a mec using a hologic qdr 4500a fan - beam densitometer ( hologic , inc . ,
briefly , participants were positioned supine on the tabletop with their feet in a neutral position and hands flat by their side .
all scans were reviewed and analyzed ( using hologic software version 8.26:a3 ) by the nhanes quality control center at the university of california , san francisco , department of radiology .
further details of the dxa examination protocol are documented in the nhanes body composition procedures manual ( 14 , 15 ) . of the 21,230 examined participants age 8 years of age and older who were eligible for the dxa scan
, 19,040 completed dxa scans , and valid total body measurements ( all regions 12 were able to be analyzed accurately ) were obtained from 16,973 or 80% of examined participants ( 1 ) . beyond planned exclusions ( i.e. , pregnant females ) ,
the percentage of participants with valid data decreased with increasing age , primarily because of implants ( i.e. , pacemakers , stents , hip replacements ) and other artifacts such as metal which can affect the accuracy of the dxa results .
additionally , the dxa scanner can not penetrate much thicker than 15 cm , after which the accuracy of segmenting bone is limited .
similarly , height ( 65 ) and weight ( 300 lbs . ) limits of the dxa machine precluded certain subjects from being eligible to participate . because data were not missing completely at random , and therefore could not be treated as a random subset of the population , missing dxa values were imputed in the public - use data sets .
multiple imputation was carried out by the nchs to allow analyses to reflect additional variability due to the use of imputed values for the missing data ( 16 , 20 ) . with multiple imputation , m ( in this case , m = 5 ) versions of the imputed values
each data set is analyzed separately and the m sets of results are combined , with the variability across the m analyses measuring the additional variability due to missing data .
briefly , a sequential regression multivariate imputation procedure ( 19 ) was implemented separately within 10 age - by - gender groups .
all analyses were conducted using sas version 9.2 ( sas institute , cary , nc ) and sas89 callable sudaan version 10.0.1 ( rti , research triangle park , nc ) to adjust the variance for the complex survey design of nhanes .
age - adjusted , sample - weighted means and standard 13 errors of the variables were calculated using proc descript .
t - tests with bonferroni adjustments for multiple comparisons were used to assess the significance of differences between point estimates . the bonferroni adjustment is a statistical adjustment for significance across the entire analysis , performed by dividing the nominal p value of 0.05 for a single test by the number of comparisons performed .
global differences for categorical variables were tested using tests , and wald f tests were used to test for differences among means of continuous variables .
mean ( se ) unadjusted body composition , and t test statistics for differences between means were calculated for continuous variables using proc descript of sudaan .
the standard errors of the means were estimated by taylor series linearization ( 11 ) , a method that incorporates the sample weights and accounts for the complex sample design of nhanes .
for the 6-years 19992004 , a 6-year mec weight variable was created by assigning 2/3 of the 4-year weight for 19992002 if the person was sampled in 19992002 or assigning 1/3 of the 2-year weight for 20032004 if the person was sampled in 20032004 .
six - year mec weights were applied to the data to account for differential probabilities of selection , nonresponse , and non - converage , as recommended .
multiple linear regression analyses were used to describe the strength of association of dxa - measured percent fat as the dependent variable with bmi or wc , age , gender , and race ethnicity as predictor variables .
gender was included as a dichotomous variable ( i.e. , 0 for women , 1 for men ) and race - ethnicity was coded from 1 to 3 with non - hispanic whites as the reference to account for differences in % bf by gender and race - ethnicity .
the subpopn statement of sudaan was used to restrict the analysis to adults 20 + years of age , and to three race - ethnicity groups .
age in 14 years was calculated at the time of the interview and race - ethnicity was self - reported . for the purposes of this study , race - ethnicity was restricted to the following three major us ethnic groups : non - hispanic whites ( nhw ) , non - hispanic blacks ( nhb ) , and mexican americans ( ma ) .
height and weight were measured using a seca electronic stadiometer and a toledo electronic weight scale , respectively , using standardized techniques and equipment ( 14 , 15 ) .
the total number of non - pregnant subjects 20 + years of age with non - missing percentbody fat data was 13,091 .
of these participants , 1,019 were excluded based on self - reported race ethnicity of hispanic or other ; and among the remaining 12,072 participants , 165 were missing bmi data .
the nhanes is a continuous data collection initiative conducted by the national center for health statistics ( nchs ) of the centers for disease control and prevention ( cdc ) ( 17 ) .
the nhanes design is a stratified , multistage probability sample based on counties , blocks , 11 households , and the number of people within households .
data are collected via house hold interviews and health examinations at a mobile examination center ( mec ) .
detailed information regarding the nhanes survey design , procedures , and protocols can be found online ( 17 ) . due to the nature of the analysis ( secondary data analysis ) , and the lack of personal identifiers ,
this study was deemed exempt by the louisiana state university agricultural center institutional review board .
beginning in 1999 , nhanes began collecting whole - body dxa scans on survey subjects 8 years of age and older to provide nationally representative data on body composition .
these data were collected across three survey cycles ( 19992000 , 20012002 , and 20032004 ) and were subsequently released as public - use data files in 2008 ( 16 ) .
the 19992004 nhanes dxa data sets contain estimates of absolute and relative total and regional body composition including fat mass , bone - free lean - tissue mass , and bone mineral .
trained technicians obtained whole - body dxa scans in a mec using a hologic qdr 4500a fan - beam densitometer ( hologic , inc . ,
briefly , participants were positioned supine on the tabletop with their feet in a neutral position and hands flat by their side .
all scans were reviewed and analyzed ( using hologic software version 8.26:a3 ) by the nhanes quality control center at the university of california , san francisco , department of radiology .
further details of the dxa examination protocol are documented in the nhanes body composition procedures manual ( 14 , 15 ) . of the 21,230 examined participants age
8 years of age and older who were eligible for the dxa scan , 19,040 completed dxa scans , and valid total body measurements ( all regions 12 were able to be analyzed accurately ) were obtained from 16,973 or 80% of examined participants ( 1 ) . beyond planned exclusions ( i.e. , pregnant females )
the percentage of participants with valid data decreased with increasing age , primarily because of implants ( i.e. , pacemakers , stents , hip replacements ) and other artifacts such as metal which can affect the accuracy of the dxa results .
additionally , the dxa scanner can not penetrate much thicker than 15 cm , after which the accuracy of segmenting bone is limited .
limits of the dxa machine precluded certain subjects from being eligible to participate . because data were not missing completely at random , and therefore could not be treated as a random subset of the population , missing dxa values were imputed in the public - use data sets .
multiple imputation was carried out by the nchs to allow analyses to reflect additional variability due to the use of imputed values for the missing data ( 16 , 20 ) . with multiple imputation ,
m ( in this case , m = 5 ) versions of the imputed values are created randomly and independently , resulting in m completed data sets .
each data set is analyzed separately and the m sets of results are combined , with the variability across the m analyses measuring the additional variability due to missing data .
briefly , a sequential regression multivariate imputation procedure ( 19 ) was implemented separately within 10 age - by - gender groups .
all analyses were conducted using sas version 9.2 ( sas institute , cary , nc ) and sas89 callable sudaan version 10.0.1 ( rti , research triangle park , nc ) to adjust the variance for the complex survey design of nhanes .
age - adjusted , sample - weighted means and standard 13 errors of the variables were calculated using proc descript .
t - tests with bonferroni adjustments for multiple comparisons were used to assess the significance of differences between point estimates .
the bonferroni adjustment is a statistical adjustment for significance across the entire analysis , performed by dividing the nominal p value of 0.05 for a single test by the number of comparisons performed .
global differences for categorical variables were tested using tests , and wald f tests were used to test for differences among means of continuous variables .
mean ( se ) unadjusted body composition , and t test statistics for differences between means were calculated for continuous variables using proc descript of sudaan .
the standard errors of the means were estimated by taylor series linearization ( 11 ) , a method that incorporates the sample weights and accounts for the complex sample design of nhanes . for the 6-years 19992004 ,
a 6-year mec weight variable was created by assigning 2/3 of the 4-year weight for 19992002 if the person was sampled in 19992002 or assigning 1/3 of the 2-year weight for 20032004 if the person was sampled in 20032004 .
six - year mec weights were applied to the data to account for differential probabilities of selection , nonresponse , and non - converage , as recommended .
multiple linear regression analyses were used to describe the strength of association of dxa - measured percent fat as the dependent variable with bmi or wc , age , gender , and race ethnicity as predictor variables .
gender was included as a dichotomous variable ( i.e. , 0 for women , 1 for men ) and race - ethnicity was coded from 1 to 3 with non - hispanic whites as the reference to account for differences in % bf by gender and race - ethnicity .
the subpopn statement of sudaan was used to restrict the analysis to adults 20 + years of age , and to three race - ethnicity groups .
age in 14 years was calculated at the time of the interview and race - ethnicity was self - reported . for the purposes of this study , race - ethnicity was restricted to the following three major us ethnic groups : non - hispanic whites ( nhw ) , non - hispanic blacks ( nhb ) , and mexican americans ( ma ) .
height and weight were measured using a seca electronic stadiometer and a toledo electronic weight scale , respectively , using standardized techniques and equipment ( 14 , 15 ) .
the total number of non - pregnant subjects 20 + years of age with non - missing percentbody fat data was 13,091 .
of these participants , 1,019 were excluded based on self - reported race ethnicity of hispanic or other ; and among the remaining 12,072 participants , 165 were missing bmi data .
the sample consisted of 5,981 men and 5,926 women . of these , 80% were nh - whites ( nhw ) , 12% nh - blacks ( nhb ) , and 8% mexican americans ( ma ) .
mean values and standard errors ( se ) for age , bmi , and percent body fat among men and women were : 46.3 0.4 and 46.4 0.5 years ; 28.0 0.10 and 28.2 0.16 kg / m2 ; 28.2 0.09 and 39.8 0.17 % , respectively .
table 2 shows comparisons in age and body composition by race - ethnicity and stratified by gender .
compared to nhw and nhb , ma men and women were significantly younger and shorter .
weight was similar between nhw and nhb men , and between nhw and ma women .
in contrast , nhb women had significantly higher mean bmi values than ma and nhw women , and ma women also had significantly higher mean bmi values than nhw women .
finally , percent body fat was lowest among nhb men and nhw women , when compared to the other two groups ( table 2 ) .
the combination of gender , bmi or wc , age , and race - ethnicity explained 82% of the variance in percent body fat .
body mass index explained 35% of the variance in % bf , compared to 20% by waist circumference .
however , when gender was included in the models , the r values increased to 0.81 in the wc equation and 0.78 in the bmi equation .
interestingly , age explained an additional 3% of the variance in % bf when added to the bmi equation , compared to only 1% in the wc equation .
finally , race contributed minimally to both equations , with negative beta coefficients for non - hispanic blacks which equates to less % bf compared to the other groups ( table 3 ) .
the formulas for calculating % bf based on bmi and wc are shown below : % bf = ( 0.8 bmi ) ( 11.5 gender ) + ( 0.1 age ) ( 2.0(nhb ) [ or + 0.7(ma ) ] ethnicity ) + 13.45 and % bf = ( 0.3 wc ) ( 14.0 gender ) + ( 0.04 age ) ( 1.0(nhb ) [ or + 1.2(ma ) ] ethnicity ) + 6.6 where gender = 0 for women , 1 for men ; race - ethnicity = 0 for nhw , 1 for nhb , and 2 for ma . a plot of % bf versus bmi ( fig
as expected , men had lower bmi and waist circumference values across the entire range of body fat percentages .
additionally , the bmi - % bf relationship appeared to be somewhat curvilinear , whereas the wc - % bf relationship appeared to be slightly more linear for both men and women .
the aim of this study was to develop and compare two body fat prediction equations appropriate for use with a nationally representative sample of the us adult population and based on bmi and wc .
the primary finding was that both equations predicted percent body fat with approximately the same group accuracy .
the nhanes data indicate that the combination of bmi or wc , along with age , gender , and race - ethnicity significantly improve the prediction of dxa - measured percent fat . given the widespread use of bmi and wc as proxies for obesity , future studies should consider including underrepresented minority groups with bmi or wc when developing body fat equations in other populations .
body mass index and wc are simple , easy - to - use methods of estimating obesity in population - based studies .
several other studies have developed generalized body fat prediction equations for adults based on bmi , and a meta - analysis has been done ( 4 ) .
( 8) reported an r2 of 0.67 and a see of 5.68% when combining bmi with age and gender . in that study which included 504
white and 202 black males and females aged 2094 , race - ethnicity did not significantly influence the % bf bmi relationship .
furthermore , significant differences were noted in weight , bmi , fat mass , percent fat , and fat - free mass between white and black females .
these findings are similar to those obtained from this study , with several differences noted in females but not males .
while the use of bmi has shown to be a reasonable measure of adiposity in adults , some research suggests that bmi may be a poor indicator of body fatness in certain population subgroups , such as ethnic minorities ( 2 ) , and individuals with a large body build ( 3 ) .
men generally have less body fat than women , and nh - blacks typically have higher bone mineral density than other ethnic groups ( 14 , 8 , 9 , 12 , 13 ) . using the same data set reported on in this study , flegal and colleagues observed correlations between bmi and % bf ranging from 0.72 among males and females 80 + years of age , to 0.84 in young adult females 2039 years ( 7 ) .
similar patterns were noted for wc and % bf , with correlations ranging from 0.65 in females 80 + years of age , to 0.86 in young adult males 2039 years ( 7 ) .
the nhanes dxa data have been reported by several others to compare dxa measured % bf to bmi and other anthropometric measures in children ( 6 ) and adults ( 7 ) ; to describe differences in whole body and regional bone mineral density ( 13 ) ; and to develop a body composition reference database including an obesity classification scheme based on fat mass divided by height squared ( 10 ) .
finally , these data have been examined by the nhanes study group and released on the cdc website to fully describe population estimates of fat , lean , and bone among the us population by age , gender , and race - ethnic group ( 1 ) .
unique to this study was the use of the nhanes dxa data set to provide field - based prediction equations to estimate percent body fat . equipped with measurements of height , weight , and
wc which are easily obtained in the field , combined with age , gender , and race 195 ethnicity , health professionals in a variety of settings can utilize these results to predict percent body fat .
although other studies have developed prediction equations using more complex models ( i.e. , four - component [ 4-c ] models ) based on anthropometric measures ( 18 , 22 ) , the results observed from this study in terms of model prediction are comparable to previous findings , in the sense that the prediction equations explained 82% of the variance in % bf from dxa .
the utility of these results are further strengthened by the use of a nationally representative sample of us adults . from a practical standpoint ,
the prediction models developed are parsimonious , while maintaining a high level of accuracy and precision .
the limitations of this study include its cross - sectional design to derive models to predict percent fat .
such a design limits conclusions about causal inference and generalizability . furthermore , because the nhanes 19992004 datasets were designed to over sample mexican americans and non - hispanic blacks , the results can not be generalized to individuals of other race - ethnic groups such as asians or hispanics .
additional studies are needed to cross - validate the equations developed to assess intra - individual accuracy within the group . in conclusion
, this study found that bmi and wc predicted % bf equally well among a representative sample of the us population .
this study provides an additional tool for health professionals to use to assess individual health status based on excess body fat rather than excess weight adjusted for height or waist circumference alone .
both equations developed are easy to use , and could be developed as an application on a smartphone or other handheld device , or simply integrated into a spreadsheet for use as an additional tool for health professionals to assess the current health status of individuals based on predicted body fat from bmi , wc , and demographics .
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clinical guidelines define obesity in terms of excess body weight adjusted for height ( i.e. , bodymass index [ bmi ] categories ) and/or gender - specific waist circumference ( wc ) cut - point values . since body composition , particularly fat mass , is the most variable among individuals due to differences by gender , age , and race , and total percent body fat ( % bf ) can be estimated accurately using dual - energy x - ray absorptiometry ( dxa ) , the purpose of this study was to develop and compare two field - based body fat prediction equations suitable for a nationally representative sample of the us adult population .
data were analyzed from subjects 20 + years of age ( n = 11,907 ) with bmi and wc values , and that participated in dxa scans as part of the 19992004 national health and nutrition examination survey ( nhanes ) . multiple linear regression was used to develop and compare dxa - estimated % bf as the dependent variable versus bmi or wc , gender , age , and race as predictor variables .
mean values for age , bmi , wc , and % bf among the sample were 46.84 0.30 years , 28.17 0.11 kg / m2 , 96.69 0.27 cm , and 34.19 0.14 % , respectively .
both equations were similar in terms of explained variance , with r2 values of 0.82 for the bmi and wc equations , respectively .
both equations are easy to use , and could easily be developed as an application on a smartphone or other handheld device , or simply integrated into a spreadsheet for use as an additional tool for health professionals to assess the current health status of individuals based on predicted body fat from bmi , wc , and demographics .
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animals : male ddy mice ( 1619 g body weight ) were obtained from japan slc
inc .
the animals were group - housed under a light - dark cycle ( light on :
06:0018:00 ) with free access to food and water .
all animal experimental procedures were
carried out according to a protocol approved by the animal care and use committee of teijin
institute for bio - medical research .
partial sciatic nerve ligation : the partial sciatic nerve ligation ( psl )
was made according to the method of seltzer et al . and
briefly , the mice were anesthetized with isoflurane in oxygen during surgery . the right
sciatic nerve was exposed and ligated with a 9 - 0 silk suture around approximately one - half
of the nerve diameter under a light microscope .
a sham operation was performed in the same
manner , except for the sciatic nerve ligation .
mechanical allodynia - like response : the paw withdrawal threshold ( pwt ) for
mechanical stimulation was determined using calibrated von frey hair ( vfh ) filaments ( 0.008 ,
0.02 , 0.04 , 0.07 , 0.16 , 0.4 , 0.6 , 1.0 and 1.4 g ; semmes - weinstein monofilaments , north coast
medical inc .
the mice were individually placed in suspended cages with wire mesh bottoms
for at least 60 min .
the plantar surface of the hind paw was stimulated with a vfh filament
in ascending order .
once the withdrawal response was observed , the paw was re - tested with the same vfh
filament to confirm the response .
the lowest amount of force required to elicit a response
was recorded as the pwt ( g ) .
a mechanical allodynia - like response was defined when the
animals responded to a vfh of 0.04 g or below , which was innocuous for normal or
sham - operated mice .
materials : igg ( sulphonated human igg ; venilon ) was kindly supplied by the
chemo - sero - therapeutic research institute ( kaketsuken ) ( kumamoto , japan ) . in previous
studies
, we showed that sulphonated igg is converted back into the non - sulphonated igg both
in vitro and in vivo and that sulphonated igg exhibits almost the same antibody activities against
various antigens as those of the non - sulphonated igg .
the sulphonated igg was administered intraperitoneally daily at doses of 400
or 1,000 mg / kg / day ( 8 ml / kg or 20 ml / kg , respectively )
according to previous studies [ 11 , 14 ] .
physiological saline was used as a vehicle control
( 20 ml / kg ) .
although the human igg used in this study was xenogenic to the
host , no abnormal symptom , such as decreased body weight or body weight gain , was noted
during the experiment ( data not shown ) , suggesting that igg antigenicity between different
species was not likely to be a problem under this experimental condition .
this finding is
consistent with those of previous reports [ 3 , 31 ] .
test protocol : to examine the effects of prophylactic treatment with igg
on psl - induced mechanical allodynia - like response , 45 mice were used .
the pwts of these mice
were tested 2 days before surgery to obtain baseline values , and the mice were randomized to
3 groups ( n=15/group ) in which they received vehicle or sulphonated igg ( 400 or 1,000
mg / kg / day ) .
1 mouse in the
sulphonated igg 1,000 mg / kg / day group died from anesthesia overdose .
sulphonated igg was
administered for 5 days , from 1 day before surgery to post - operative day ( pod ) 3 .
the pwts
of these mice were examined on pods 5 , 10 , 14 , 21 and 28 . to examine the effect of therapeutic treatment with igg on the psl - induced mechanical
allodynia - like response ,
the pwts of these mice were tested on 1 day
before surgery to obtain baseline values .
47 psl mice showed
a reduction in the pwt to 0.04 g or a lower force , and 36 psl mice were then selected .
these
36 psl mice were randomized to 3 groups ( n=12/group ) in which they received vehicle or
sulphonated igg ( 400 or 1,000 mg / kg / day ) .
sulphonated igg was administered for 5 days , from
pod 11 to pod 15 .
the pwts of these mice were measured on pods 18 , 24 , 30 and 36 . to explore the mechanism of the effect of igg on the psl - induced mechanical allodynia - like
response , the mrna expression of inflammatory cytokines in the injured sciatic nerve on pod
1 and pod 5
the mice were divided into
3 groups ( n=20/group ) ; sham / vehicle , psl / vehicle and psl / sulphonated igg ( 1,000 mg / kg / day ) .
the mice were treated with vehicle or sulphonated igg for 3 days , from 1 day before surgery
to pod 1 .
the sciatic nerve segments ( about 1-cm long ) proximal to the ligatures were
harvested on pod 1 .
the sciatic nerves from 2 mice were pooled and analyzed using
quantitative real - time reverse transcription polymerase chain reaction ( qrt - pcr ) . for pod 5
sampling , 60 mice were divided into 3 groups ( sham / vehicle , psl / vehicle and psl / sulphonated
igg 1,000 mg / kg / day , n=20/group ) that were treated with vehicle or sulphonated igg for 5
days , from 1 day before surgery to pod 3 .
the sciatic nerve was harvested on pod 5 and
analyzed as for the pod 1 samples .
quantitative real - time reverse transcription polymerase chain reaction :
the qrt - pcr analyses were performed using an abi prism 7500 sequence detection system
( applied biosystems , foster city , ca , u.s.a . ) and sybr green mix ( applied biosystems )
according to the manufacturer s instructions .
briefly , total rna was extracted from the
sciatic nerve within 1 cm of the ligation site using the isogen reagent ( nippon gene ,
toyama , japan ) .
first strand cdna was synthesized from 1,000 ng of total
rna in a final volume of 12 l using the omniscript rt kit ( qiagen gmbh ,
hilden , germany ) , rnaseout recombinant ribonuclease inhibitor ( invitrogen , carlsbad , ca ,
u.s.a . ) and random primer ( takara bio inc . ,
the reaction conditions were as follows : 50c for 2 min and then 95c for
10 min , followed by 40 cycles at 95c for 15 sec ( denaturation ) and 60c for 1 min
( annealing and elongation ) .
messenger rna ( mrna ) expression for each sample was calculated
by using the ct procedure ( ct = ct of the target mrna ct of the control mrna ) .
glyceraldehyde 3-phosphate dehydrogenase ( gapdh , taqman rodent gapdh control
reagents , thermo fisher scientific inc . ,
waltham , ma , u.s.a . ) was used as a reference
control gene .
the primer sets used
were as follows : tumor necrosis factor- ( tnf- ) ,
( 5-aagcctgtagcccacgtcgta-3 and 3-ggcaccactagttggttgtctttg5 ) , interleukin-1 ( il-1 )
( 5-tgtgaaatgccaccttttga-3 and
3-agcttctccacagccacaat-5 ) , monocyte chemotactic protein-1 ( mcp-1 ) ( 5-aactgcatctgccctaaggtct-3 and 3-ggaaaaggtagtggatgcattagc-5 ) and
macrophage inflammatory protein-1 ( mip-1 ) ( 5-
tgcccttgctgttcttctct-3 and 3-caggcattcagttccaggtc-5 ) .
statistical analysis : the data relating to pwt are expressed as the
medians and the 1st and 3rd quartiles that indicate the range of median values .
the qrt - pcr data are expressed as
means sd and were subjected to a parametric - type or a nonparametric - type tukey test .
all
data were statistically analyzed using sas software for windows , release 9.2 ( sas institute
inc .
effects of prophylactic treatment with sulphonated igg on psl - induced mechanical
allodynia - like responses : in the paw ipsilateral to the psl , the pwts in mice
with psl were significantly decreased from pod 5 to pod 28 compared with the pwts before
surgery ( p<0.01 ) .
this result indicated that a mechanical allodynia - like
response had developed on pod 5 and was sustained until the end of the study ( pod 28 ) . in
psl
/ sulphonated igg groups , these decreases in pwts were attenuated , and significant
differences were noted between the pwts of psl / vehicle and psl / sulphonated igg ( 400 or 1,000
mg / kg / day ) on pods 5 , 10 and 14 ( p<0.05 for all comparisons ) ( fig
1.effects of sulphonated igg on the development of a mechanical allodynia - like
response .
vehicle or sulphonated igg ( 400 or 1,000 mg / kg / day ) was intraperitoneally
administered to mice with psl from 1 day before surgery to pod 3 .
the pwt was measured
using a von frey hair filament on the ipsilateral ( a ) and contralateral ( b ) paw of a
mouse with psl . in the ipsilateral paw ,
pwts in the psl / vehicle group were
significantly decreased from pod 5 to pod 28 ( # # p<0.01 ) compared
with the pwts before surgery , suggesting that a mechanical allodynia - like response had
developed .
data are expressed as the median force ( g ) required to induce paw
withdrawal in 1415 animals per group ( parenthesis indicates the number of animals
used ) , and vertical bars represent 1st and 3rd quartiles . *
p<0.05 ,
* * p<0.01 vs. vehicle , and # # p<0.01 vs.
before surgery ( dunnett s test ) . ) . in the contralateral paw
although a significant difference was
noted between the pwts before surgery and the pwts in the psl / sulphonated igg 400 mg / kg / day
group on pod 21 , this difference was not considered to be related to sulphonated igg
treatment , because there was no significant difference between the pwts of the psl / vehicle
group and the pwts of the psl / sulphonated igg 400 mg / kg / day group on pod 21 .
effects of sulphonated igg on the development of a mechanical allodynia - like
response .
vehicle or sulphonated igg ( 400 or 1,000 mg / kg / day ) was intraperitoneally
administered to mice with psl from 1 day before surgery to pod 3 .
the pwt was measured
using a von frey hair filament on the ipsilateral ( a ) and contralateral ( b ) paw of a
mouse with psl . in the ipsilateral paw ,
pwts in the psl / vehicle group were
significantly decreased from pod 5 to pod 28 ( # # p<0.01 ) compared
with the pwts before surgery , suggesting that a mechanical allodynia - like response had
developed .
data are expressed as the median force ( g ) required to induce paw
withdrawal in 1415 animals per group ( parenthesis indicates the number of animals
used ) , and vertical bars represent 1st and 3rd quartiles . *
p<0.05 ,
* * p<0.01 vs. vehicle , and # # p<0.01 vs.
before surgery ( dunnett s test ) .
effects of therapeutic treatment with sulphonated igg on psl - induced mechanical
allodynia - like responses : in the paw ipsilateral to the psl , the pwts in mice
with psl were significantly decreased from pod 11 to pod 36 compared with the pwts before
surgery ( p<0.05 ) , suggesting that a mechanical allodynia - like response
had developed by pod 11 ( before sulphonated igg treatment ) .
when sulphonated igg was
administered from pod 11 to pod 15 , it did not affect the pwts at either dose ( 400 or 1,000
mg / kg / day ) ( fig .
2.effects of sulphonated igg on the maintenance of a mechanical allodynia - like
response . vehicle or sulphonated
igg ( 400 or 1,000 mg / kg / day ) was intraperitoneally
administered to mice with psl from pod 11 to pod 15 .
the pwt was measured using a von
frey hair filament on the ipsilateral ( a ) and contralateral ( b ) paw of a mouse with
psl . in the ipsilateral paw ,
pwts in the psl / vehicle group were significantly
decreased from pod 11 to pod 36 ( # # p<0.01 ) compared with the pwts
before surgery , suggesting that a mechanical allodynia - like response had developed by
pod 11 .
data are expressed as the median force ( g ) required to induce paw withdrawal
in 12 animals per group ( parenthesis indicates the number of animals used ) , and
vertical bars represent 1st and 3rd quartiles .
# p<0.05 and
# # p<0.01 vs. before surgery ( dunnett s test ) . ) . in the contralateral paw , sulphonated igg did not affect the pwts at either dose
( fig .
although significant differences were
noted between the pwts before surgery and the pwts in the psl / sulphonated igg group on pod
11 ( 400 mg / kg / day ) and on pod 30 ( 400 or 1,000 mg / kg / day ) , these differences were not
considered to be related to sulphonated igg treatment .
this is because significant
differences between the pwts of the psl / vehicle and the psl / sulphonated igg groups were not
observed at those time points .
effects of sulphonated igg on the maintenance of a mechanical allodynia - like
response .
vehicle or sulphonated igg ( 400 or 1,000 mg / kg / day ) was intraperitoneally
administered to mice with psl from pod 11 to pod 15 .
the pwt was measured using a von
frey hair filament on the ipsilateral ( a ) and contralateral ( b ) paw of a mouse with
psl . in the ipsilateral paw ,
pwts in the psl / vehicle group were significantly
decreased from pod 11 to pod 36 ( # # p<0.01 ) compared with the pwts
before surgery , suggesting that a mechanical allodynia - like response had developed by
pod 11 .
data are expressed as the median force ( g ) required to induce paw withdrawal
in 12 animals per group ( parenthesis indicates the number of animals used ) , and
vertical bars represent 1st and 3rd quartiles .
# p<0.05 and
# # p<0.01 vs. before surgery ( dunnett s test ) .
effects of sulphonated igg on the expression of inflammatory cytokines in mice with
psl : to explore the mechanism of the above sulphonated igg effect , the mrna
expression of inflammatory cytokines ( tnf- , il-1 , mip-1 and mcp-1 ) in the injured sciatic
nerve was examined using qrt - pcr . on pod 1 , inflammatory cytokine mrnas
were significantly
up - regulated in psl / vehicle when compared with those of sham / vehicle . in the psl /
sulphonated
igg group , the up - regulation of tnf- and mcp-1 mrnas was significantly attenuated
( p<0.05 ) ( fig . 3fig .
3.effects of sulphonated igg on the expression of inflammatory cytokine mrnas on pod 1 .
vehicle or sulphonated igg ( 1,000 mg / kg / day ) was intraperitoneally administered to
mice with psl from 1 day before surgery to pod 1 .
the mice were sacrificed on pod 1 , and total rna was isolated from the sciatic
nerves .
the mrna expression levels of tnf- , il-1 , mip-1 and mcp-1 were measured
using qrt - pcr .
the amount of the target mrna relative to the expression of gapdh mrna
is expressed as the meansem ( n=10 ) .
* p<0.05 ,
* * p<0.01 and * * * p<0.001 ( tukey s test ) . ) . on pod 5 , tnf- and mcp-1
mrnas were still up - regulated , and pre - treatment with
sulphonated igg up to pod 3 showed a tendency to suppress the up - regulation of the mcp-1
mrna ( p=0.0542 ) ( fig .
4.effects of sulphonated igg on the expression of inflammatory cytokine mrnas on pod 5 .
vehicle or sulphonated igg ( 1,000 mg / kg / day ) was intraperitoneally administered to
mice with psl from 1 day before surgery to pod 3 .
the mice were sacrificed on pod 5 ,
and total rna was isolated from sciatic nerves .
the mrna expression levels of tnf- ,
il-1 , mip-1 and mcp-1 were measured using qrt - pcr .
the amount of the target mrna
relative to the expression of gapdh mrna is expressed as the meansem ( n=10 ) .
effects of sulphonated igg on the expression of inflammatory cytokine mrnas on pod 1 .
vehicle or sulphonated igg ( 1,000 mg / kg / day ) was intraperitoneally administered to
mice with psl from 1 day before surgery to pod 1 .
the mice were sacrificed on pod 1 , and total rna was isolated from the sciatic
nerves .
the mrna expression levels of tnf- , il-1 , mip-1 and mcp-1 were measured
using qrt - pcr .
the amount of the target mrna relative to the expression of gapdh mrna
is expressed as the meansem ( n=10 ) .
* p<0.05 ,
* * p<0.01 and * * * p<0.001 ( tukey s test ) .
effects of sulphonated igg on the expression of inflammatory cytokine mrnas on pod 5 .
vehicle or sulphonated igg ( 1,000 mg / kg / day ) was intraperitoneally administered to
mice with psl from 1 day before surgery to pod 3 .
the mice were sacrificed on pod 5 ,
and total rna was isolated from sciatic nerves .
the mrna expression levels of tnf- ,
il-1 , mip-1 and mcp-1 were measured using qrt - pcr .
the amount of the target mrna
relative to the expression of gapdh mrna is expressed as the meansem ( n=10 ) .
the present study showed that prophylactic treatment with igg may attenuate the development
of a mechanical allodynia - like response in mice with neuropathic pain .
the reasons why we
used a psl model and detected a mechanical allodynia - like response were as follows ; psl
model was widely used as a mouse model of neuropathic pain like chronic constriction injury
( cci ) model , mechanical allodynia , an exaggerated sensory response to non - noxious stimuli ,
is one of the representative symptom in patients with neuropathic pain , detection of the
mechanical allodynia - like response is a very popular assay performed in an animal model of
neuropathic pain , and previous experiment conducted in our laboratory demonstrated that
gabapentin , positive control drug , significantly suppressed mechanical allodynia - like
response in this model .
in contrast to our findings , sommer et al . reported that igg did not show
any effects on the development of a mechanical allodynia - like response in mice with cci
.
although the exact reason for the discrepancy
between our findings and theirs is unclear , it might be due to , at least in part , the
different dose of igg used in each study and/or the different igg preparations .
the
sulphonated igg used in this study was prepared from pooled human plasma from thousands of
donors .
it is known that igg prepared from a large donor pool contains variable amounts of
igg dimer , whereas igg isolated from the plasma of a single individual , which was used in
the previous report , is essentially monomeric [ 28 ,
29 ] .
. demonstrated
that therapeutic effects of igg preparations on the platelet counts of rats with immune
thrombocytopenia depended on the amount of igg dimers in the preparations . in this study , sulphonated igg significantly attenuated the up - regulation of tnf- and
mcp-1 mrna in mice with psl .
wagner and zelenka
showed that a single injection of tnf- into a
rat sciatic nerve decreased the mechanical withdrawal threshold for a week .
czeschik
et al . reported that tnf- increased voltage - gated sodium channels
currents and potassium ion conductance in a non - voltage gated fashion in isolated rat drg
neurons and leading to neuronal
hyper - excitability .
abbadie et al . also demonstrated that an intraplantar
injection of mcp-1 induced a mechanical allodynia - like response for more than 180 min in
mice .
it is therefore postulated that the
reduction in inflammatory cytokine expression induced by sulphonated igg on pod 1
contributes to its attenuation of the development of a mechanical allodynia - like response on
pod 5 in mice with psl .
however , we can not rule out any other factors to contribute the
prophylactic effects of sulphonated igg .
it is not yet known how igg may attenuate cytokine expression in the injured sciatic nerve .
it has been reported that igg reduces tnf- and il-1 production by human peripheral blood
mononuclear cells in the presence of lipopolysaccharide .
kishimoto et al . reported that treatment with sulphonated igg
decreased t - lymphocyte infiltration and serum cytokine levels ( tnf- , inf- and mip-2 ) , and
attenuated the development of myocarditis induced by encephalomyocarditis virus in mice
.
therefore , an inhibitory effect of igg on
inflammatory cell infiltration into the injured sciatic nerve and/or on cytokine expression
in macrophages might be considered as a mechanism by which igg attenuates neuropathic
pain .
in contrast to clinical studies , therapeutic treatment with sulphonated igg has little
effects on the maintenance of a mechanical allodynia - like response in mice with psl .
the
differences of the relative contributions of inflammatory cytokines on the maintenance of
neuropathic pain might cause the discrepant results . although we have not examined the
temporal profile of inflammatory cytokines , several studies have reported that cytokine
mrnas are immediately elevated in the injured sciatic nerve ( from 12 hr to 7 days after
nerve injury ) and then gradually return to the control level by 714 days in mice with psl
[ 9 , 10 , 20 ] . the relative contribution of inflammatory cytokines
on maintenance of mechanical allodynia - like response might be small in our experimental
conditions . in the clinical studies ,
igg treatment attenuates some types of neuropathic
pain , whose pathophysiology may involve immune changes in the peripheral tissues and/or cns ,
and the analgesic action of igg in these conditions was thought to be the suppression of
cytokine expression . in summary ,
our results have provided the first preclinical evidence that prophylactic
treatment with sulphonated igg attenuates the development of a mechanical allodynia - like
response via an inhibition of inflammatory cytokine expression in mice with psl .
|
human immunoglobulin g ( igg ) concentrates are immune - modulating ,
anti - inflammatory plasma - derived products .
clinical studies in recent years have suggested
that igg attenuates neuropathic pain . in this study ,
effects of sulphonated igg on the
development and maintenance of a mechanical allodynia - like response were examined in mice
with neuropathic pain induced by a partial sciatic nerve ligation ( psl ) .
when sulphonated
igg ( 400 or 1,000 mg / kg / day , i.p . )
was administered for 5 days , from 1 day before surgery
to post - operative day ( pod ) 3 , the development of a mechanical allodynia - like response was
attenuated
. on the other hand , sulphonated igg had little effect on the maintenance of a
mechanical allodynia - like response when administered for 5 days , from pod 11 to pod 15 , at
which time a mechanical allodynia - like response had already been developed . to explore the
mechanism of sulphonated igg , the mrna expression of inflammatory cytokines
was evaluated
in the injured sciatic nerve .
sulphonated igg ( 1,000 mg / kg / day , i.p . ) that was
administered for 3 days , from 1 day before surgery to pod 1 , significantly attenuated the
up - regulation of tumor necrosis factor- and monocyte chemotactic protein-1 mrnas on pod
1 .
these results suggest that prophylactic treatment with sulphonated igg attenuates the
development of mechanical allodynia - like response by inhibition of inflammatory cytokine
expression in mice with psl .
|
all synthetic and enzymatic reaction products were characterized by nmr spectroscopy and/or high - resolution mass spectrometry .
a typical nmr assay contained 0.25 mm hppe , 0.25 mm fe(nh4)2(so4)26h2o , 7.5 mm fmn , 25 mm substrate ( ( r)-7 , ( s)-7 , 11 , 12 , ( rr)-23 , ( rs)-23 , or 27 ) , and 25 mm nadh in 700 l of 20 mm tris buffer ( ph 7.5 ) . the conversion of ( rr)-23/(rs)-23 to 26 by hppe was confirmed by spiking the reaction mixture with an authentic standard of 26 .
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( s)-2-hydroxypropylphosphonate ( ( s)-2-hpp ) epoxidase ( hppe ) is a mononuclear non - heme iron - dependent enzyme1,2,3 responsible for the last step in the biosynthesis of the clinically useful antibiotic fosfomycin4
. enzymes of this class typically catalyze oxygenation reactions that proceed via the formation of substrate radical intermediates .
in contrast , hppe catalyzes an unusual dehydrogenation reaction while converting the secondary alcohol of ( s)-2-hpp to the epoxide ring of fosfomycin1,5 .
hppe is shown here to also catalyze a biologically unprecedented 1,2-phosphono migration with the alternative substrate ( r)-1-hpp . this transformation likely involves an intermediary carbocation based on observations with additional substrate analogues , such as ( 1r)-1-hydroxy-2-aminopropylphosphonate , and model reactions for both radical- and carbocation - mediated migration .
the ability of hppe to catalyze distinct reactions depending on the regio- and stereochemical properties of the substrate is given a structural basis using x - ray crystallography .
these results provide compelling evidence for the formation of a substrate - derived cation intermediate in the catalytic cycle of a mononuclear non - heme iron - dependent enzyme .
the underlying chemistry of this unusual phosphono migration may represent a new paradigm for the in vivo construction of phosphonate - containing natural products that can be exploited for the preparation of novel phosphonate derivatives .
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avian influenza ( ai ) is a highly contagious disease primarily of birds , and caused by influenza a viruses .
the exceptionally virulent viruses cause highly pathogenic avian influenza ( hpai ) , with mortality in affected flock as high as 100% .
this group belongs to subtypes h5 and h7 , but it is worth noting that not all h5 and h7 viruses ' infection lead to hpai .
all other subtypes cause a milder , primarily respiratory , disease , that is , low pathogenic avian influenza ( lpai ) unless exacerbated by secondary infections .
avian influenza is one of the greatest concerns for public health that has emerged from the animal reservoir .
the spread of hpai ( h5n1 ) to countries in which hygienic standards are deficient increases the virus 's pandemic potential and raises concerns about food security particularly in rural villages .
aquatic birds like ducks , geese , swans ( anseriformes ) , and gulls , terns ( charadriiforms ) are thought to be natural reservoirs for avian influenza viruses and are capable of shading the viruses asymptomatically [ 5 , 6 ] .
hpai viruses have also been isolated from free - flying wild birds ; for example , 2 genotypes of hpai ( h5n1 ) viruses were isolated from apparently healthy tree sparrows ( p. montanus ) in china in 2003 and 2004 . also , large - scale outbreak of hpai occurred in a zoological institution in cambodia that affected a variety of captive wild - bird species including eagles and owls .
these findings suggested that land - based wild birds might also be a major reservoir of influenza viruses .
additionally , they intermingle freely with wild and domestic populations of waterfowl and poultry making them potentially important bridge in the transmission of avian influenza viruses from aquatic birds to poultry and vice versa . therefore , these species may be relevant in virus transmission and should form an integral part of avian influenza surveillance and monitoring programs [ 8 , 9 ] .
but , there are limited data describing their vulnerability to influenza virus ( h5n1 ) infection or potential to transmit the viruses . in nigeria ,
the highly pathogenic avian influenza ( hpai ) virus of the h5n1 subtype was first detected in february 2006 , in chickens at a commercial poultry farm in kaduna state , northern nigeria .
the infection spread and persisted for 21 months , and a fatal human case was reported .
the ai viruses of the h5n2 subtypes had been detected by means of molecular tests in free - living and apparently healthy white - faced whistling - duck ( dendrocygna viduata ) and spur - winged goose ( plectropterus gambensis ) in nigeria ; supporting the view that migratory birds may serve an important role in the introduction of hpai ( h5n1 ) viruses into nigeria .
introduction and spread of the virus through illegal trade in poultry and poultry products have not been ruled out .
illegal poaching of wild birds and other wild - life species for food , traditional medicine , and certain belief are not uncommon in nigeria .
captive wild birds are usually kept in rural community setting for a while , awaiting market days and may in the process come in contact with domestic poultry .
mention that trade in wildlife commonly provides disease - transmission avenues at scales that not only cause human disease outbreaks but also threaten livestock , international trade , rural livelihoods , native wildlife populations , and the health of ecosystems . from october 2007 to june 2008 , despite intensive surveillance for ai , there had been no reported cases in nigeria .
this development helped to ease out fear among poultry farmers and had a positive effect on restoration of consumers ' confidence in poultry products , but the relief was for a short period . in july 2008 , new hpai - virus isolates were obtained .
two of the isolates from kano and katsina states belonged to clade 2.2 , previously isolated in nigeria , but isolates from gombe and kebbi states were of a new sublineage of clade 2.2.1 , which was new to the african continent [ 13 , 15 ] .
the factors responsible for the spread and sustenance of the hpai viruses in infected states of nigeria are still not clear .
the issue is , do avian influenza viruses persist and circulate at reasonably detectable levels in apparently healthy potential bridge
ultimately , the objective of this study was to determine whether avian influenza viruses were present at reasonably detectable levels ( prevalence of 0.5% ) in apparently healthy possible bridge species of wild and domestic birds around poultry houses and in captive wild birds markets .
it was envisaged that the study would bring about better understanding of the possible roles played by bridge species in the re - occurrence and spread of avian influenza viruses among poultry . as well as early detection of the possible risks posed by their activities to the poultry sector .
findings would also be useful in setting priorities for surveillance and evaluation of control programmes .
the target was wild birds , scavenging around active poultry houses , moving inbetween poultry farms , and that might come intact with aquatic birds at water bodies and rice fields .
majority of wild birds in captivity were captured at the wetlands or acquired from other sources such as neighbouring countries .
these categories were considered as potential bridge between aquatic birds and domestic poultry . using available and relevant information such as lists of states , infected poultry farms , and wild bird markets ; a multistage simple random - sampling technique with cluster sampling at primary sampling units was adopted .
briefly , 8 states ( borno , gombe , kaduna , kano , plateau , ogun , oyo , and yobe ) were selected at random from the list of 25 avian influenza infected states . in each selected state ,
1 poultry farm was selected from list of previously infected but restocked farms , then , all wild birds trapped by mist net around the farm were sampled .
also , in selected sates that had captive live wild birds markets , 1 captive wild bird trader was selected randomly from the list of traders in each market , and all wild birds in his position were sampled . in total , 8 poultry farms and 8 wild birds ' markets were surveyed . as data on population sizes are lacking in wild animals , including wild birds species , due to their remoteness , varying populations , and the migratory behaviour these animals constitute a major challenge for designing disease monitoring systems .
furthermore , in this study , prevalence of avian influenza viruses in the targeted species in nigeria was unavailable and it was considered unreasonable to extrapolate based on prevalence obtained in other places . therefore , as in majority of similar studies conducted elsewhere [ 6 , 9 , 12 , 1719 ] , it was considered convincible to constrain the sample size to available resources .
the team for the study was composed of veterinarians , disease surveillance agents , laboratory diagnosticians , and an ornithologist . in collaboration with fao nigeria and rome and the ornithological research institute , jos ,
the team was given a refreshing training on biosecurity measures , sensitization of poultry farmers , captive wild - bird traders , leaders of local communities , and on capturing and handling wild birds , sampling , and sample preservation .
all activities were simulated by participants on the last day of the training and appropriate corrections made on observed lapses .
prior to the commencement of field activities , owners of all selected poultry farms , leaders of traders , and the host communities were contacted , visited , and sensitized on the objectives of the study , its benefits , and their expected roles such as winning the cooperation of their workers and members .
also , the schedules for the survey exercise was discussed with them and agreed upon .
visits to poultry farms were made twice every day , early in the morning and late in the evening from about 5:30 to 8:30 am and from 5:10 to 7:10 pm ( local time ) , respectively , being periods of maximum activities for majority of birds and less stress to birds during handling .
birds were captured using mist nets , harvested and sampled according to standard practices [ 18 , 20 ] . both pharyngeal and cloacal swabs collected from each bird were stored separately in a cryovial tube containing viral transport media as described by gaidet et al . .
briefly , the viral transport media consisted of isotonic phosphate - buffered saline , ph 7.07.4 , containing antimicrobial agents ( penicillin 10,000 u / ml , streptomycin 10 mg / ml , amphotericinb 25 g / ml , and gentamycin 250 g / ml ) supplemented with 10% glycerol .
samples were labelled ( date , species of birds , sample type ( tracheal or cloacal ) , and specific identity number ) using indelible marker .
the coordinates of the sample points were recorded using garmin etrex legend by garmin international inc .
samples were parked , stored on ice park in a colman box , sealed , and transported to the laboratory with completed data forms at most within 36 hours of collection .
all samples were tested at the national veterinary research institute ( nvri ) , vom , using viral isolation in embryonated chicken eggs in line with standard procedures and described by joannis et al . .
virus isolation was done in 911-day - old embryonated chicken eggs ( incubated at 37c for 5 days ) .
the eggs were candled daily to determine viability and eggs with dead embryos were removed and kept at + 4c .
all eggs were opened aseptically and the allantoic fluids ( alfs ) were harvested and tested by haemagglutination test .
the chorioallantoic membranes ( cams ) of positive eggs were tested by agar gel immunodiffusion ( agid ) to detect influenza a virus group antigen and haemagglutination inhibition ( hi ) test to determine the virus subtype .
all negative alf ( from all the samples in this case ) were further passaged in a second set of embryonated chicken eggs .
any negative samples after the second passage were declared as such . the calculations for the confidence interval were done in r 2.10.1 program for exact binomial ci ( ) function .
6 samples ( afterthought ) were positive for newcastle disease virus ( ndv ) but all tested samples were negative for avian influenza viruses .
the overall ci was 00.007406 ( upper and lower limits , resp . ) . among the states , ogun had the lowest samples 6.9% , ci ( 00.101466 ) ; plateau the highest 21.6% , ci ( 00.033848 ) . in the overall species , sampled , tawny eagle ( aquila rapax ) was the lowest sampled 0.3% and
red - billed firefinch ( lagonosticta senegala ) was the highest 11.7% . among the 281 wild birds category sampled , tawny eagle ( a. rapax ) was the lowest sampled 0.4% , ci ( 00.95 ) ; while the highest was red - billed firefinch ( l. senegala ) 16.7% , ci ( 00.06175 ) . in domestic birds category , 122 were sampled , out of this , black - crowned crane ( blearica pavonina ) ( in captivity ) was the lowest sampled 1.6% , ci ( 00.776393 ) ; domestic duck ( anas platyrhynchos ) was the highest sampled 35.3% , ci ( 00.067297 ) . in both cases ,
the lowest sampled had wider ci , an indication of higher uncertainty , while the highest sampled had relatively narrower ci indicative of relatively lesser uncertainty .
this may be attributable to differences in the sample sizes , as bigger sample size tends to reduce the uncertainty in a survey .
. it can be applied in the verification or rejection of a hypothesis about a given system and in describing the proportion of the disease impact that remains undetected and which may lead or contribute to unobserved spread of a disease , in this study , the latter may be considered applicable .
the manifestation , host invasiveness , ecological spread , and associated risks of highly pathogenic avian influenza ( hpai ) h5n1 subtype have resulted in increased concerns globally .
similarly , the devastating agricultural and economic problems encountered in domestic poultry subsector , as well as fatal human cases resulting from the infection , have been substantially overwhelming . the situation is worse in majority of developing countries , where poultry rearing particularly at rural areas is a major source of livelihood and in majority of cases the most viable and sustainable source . over the years ,
the complex role played by wild birds in the introduction , spread , sustenance , and incidences of avian influenza viruses particularly h5n1 has been a subject for in - depth exploration .
surveillance for avian influenza viruses in wild and domestic birds provides an exclusive opening to enhance our knowledge not only of ecology and epidemiology of the hpai virus subtypes but also that of lpai viruses in their natural hosts , at the same time and for the same cost .
this approach enables timely detection of palpable signals for the introduction of the disease into new regions and may provide access to updated data and strains of the viruses for categorization .
however , for proper evaluation of control programmes , risk - based decision making , and prioritization of utilization of scarce resources , we also need a better understanding of the roles of interfaces between wild and domestic birds .
for example , we need clearer insight of the possible role played by potential bridge
species of wild and domestic birds , aimed at the identification of virus permissiveness of these species and their relative likelihood to develop disease and patterns of virus secretion .
the occurrence and distribution of subtypes of influenza a viruses broadly , may vary between different surveillance studies depending on target species , time , and place [ 6 , 23 ] .
previous studies have attributed this phenomenon to differences in taxonomy and behaviour such as colony breeding , feeding patterns , and aggregation at stopover or wintering sites which encourages mixing of different population of birds . also the deferential attention by researchers to aquatic birds ' species believed to harbour the viruses .
for example , previous studies have demonstrated that virus isolations from other wild birds have been completely surpassed by the number , variety , and widespread distribution of influenza viruses in waterfowl , order anseriformes [ 24 , 25 ] .
this could be a possible explanation for limited data on avian influenza viruses in other wild birds species particularly land - based species .
it is acknowledged that this study had short - comings of relatively small sample size , attributable to limited resources particularly funds available for the study , number of days allotted for the study , and the timing . while the limitation of resources on the study was obvious , that of timing was also prominent .
a period when possible bridge species targeted had alternative sources of water and feeds .
as such , were dispersed and made less frequent visits in search for feeds to poultry houses where majority of captures were made .
more so , the timing coincided with period of increased demand for poultry eggs in nigeria .
therefore , farmers would not allow extended surveillance activities around their farms for fear that the birds would be disturbed which might lead to drop in production .
also , the limitations of designing effects including clustering had not been accounted for due to the all negative outcome ( lack of variance ) of the data .
this result could as well have been influenced by factors such as high virulence of hpai virus in these species , such that infected birds could not survive long enough , extremely low prevalence of the viruses in these species that lead to failed detection , or the outcome was by chance .
for instance , unlike aquatic wild birds that congregate during breeding seasons and at roosting sites leading to the build up of a local population density that facilitates spread of diseases through contact , land - based wild birds rarely congregate at particular spots but are dispersed majority of the time , allowing less time for spread of diseases through contact .
it could as well be that these species lacked the ability to harbour the viruses particularly lpai subtypes .
this was with the assumption that if the study had been repeated several times , the true values of the data would have been captured within the confidence intervals .
for example out of more than 10,000 samples collected from wild birds in 15 orders other than the anseriformes , charadriiformes , and gruiformes , no influenza a viruses were detected .
in contrast , in another study out of 939 samples from land - based wild birds , order passeriformes , 24 were positive for influenza a viruses giving a prevalence of 2.6% .
the latter , establishes the possible occurrence of these viruses in wider wild birds species other than aquatic birds and suggests the need for widening up of surveillance activities to incorporate more species .
it seems most likely ( at least from previous studies ) that the situation of avian influenza viruses in land - based wild birds which form majority of possible bridge species remain poorly understood or largely unknown and need to be explored .
the need for this is more urgent in developing countries where contacts between wild birds and domestic poultry are more rampant than would be expected due to the production system . to obtain a clearer picture of avian influenza viruses in potential
bridge species of birds , a multidisciplinary approach with effective collaborations is required .
this has the advantage of maximising the utilization of scarce human expertise and financial resources particularly in developing countries .
for example a team could be composed of a veterinarian , statistician , ornithologist , laboratory diagnostician , and rural sociologist .
this approach , combined with a community - oriented approach to disease reporting , would most probably yield the desired result of better understanding .
while the human communal life style in african villages encourages the comingling of household flocks , it , most importantly , provides the social setting convenient for collective action to prevent diseases , improve poultry production systems , and disease reporting .
the major contributions of this study are as follows : firstly , targeted surveillance of avian influenza viruses in this group of birds should be given special consideration of supplementation with extensive passive surveillance normally targeting sick or recently dead birds .
this view is consistent with the findings by hesterberg et al . and feare that active surveillance for avian influenza viruses is essential in providing important information on lpai viruses ' circulation , including h5 and h7 subtypes that have the potential to become highly pathogenic in poultry . while vigilance for dead birds through passive surveillance may remain the best means of detecting hpai viruses such as h5n1 subtypes in wild birds . the possible explanation to this theory which also concurs with that highlighted by breed et al .
is that ; on one hand , passive surveillance is more responsive for the detection of hpai viruses , as birds may be either sick or dead .
however , the limitation is that the method seems to exclude the detection of the hpai viruses in healthy birds that may harbour them . on the other hand
, active surveillance appears more responsive for the detection of lpai viruses , as the viruses are confined , compared with the overwhelming hpai viruses resulting in a lower chance of finding lpai viruses in dead birds .
however , knight - jones et al . believe that sentinel surveillance is effective in detection of both lpai and hpai viruses . integrating the two surveillance methods ( passive and active ) , therefore , seems reasonable and may be more result oriented .
bridge species of birds for the spread of avian influenza viruses comprising those that frequented active poultry houses , and species that were sold in live wild birds markets .
this may serve as base - line information for more elaborate study in nigeria and elsewhere in future targeting these species .
majority of species trapped were seed predators ( granivores ) , since poultry feed constitutes mainly of grains .
however , because of the location of certain farms plus their integrated nature , some insectivorous birds like cattle egret and brown babblers were also trapped .
thirdly , as mentioned in the result , the afterthought ( not initially considered in the study ) findings of 6 positive samples for newcastle disease virus ( ndv ) , apart from being an indication that samples were in good conditions , it suggested that two or more closely related diseases could be studied concurrently in wild birds .
this approach particularly in developing countries has the advantage of being less time consuming , more cost effective , and informative .
more so , isolation of ndv further implicated wild bird 's species in the spread of other diseases like newcastle disease .
this scenario has contributed to complications in behavioural changes due to wrong perception of avian influenza , particularly amongst rural populations .
they find it extremely difficult to believe that avian influenza is different from newcastle disease .
the study motivated by existing concerns on avian influenza has demonstrated surveillance for avian influenza viruses in wild and domestic birds ' species that might serve as bridge at the interfaces between migratory birds and domestic poultry .
thus , it calls for caution in making strong conclusions and corroborates the need for broader coverage and blending of active and passive surveillances for avian influenza viruses in these species . considering the dynamic nature of the avian influenza viruses ,
it would seem most probable that future study in this area will require concerted efforts , holistic and multidisciplinary approach focusing on appropriate ornithological and epidemiological variables such as type , census , and movement of birds , and distribution of avian influenza incidences in time and space in high - risk areas .
these approaches , over a longer time frame , are likely to convey a better understanding of the possible roles played by potential bridge species of birds in the epidemiology of avian influenza viruses in nigeria .
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the highly pathogenic avian influenza ( h5n1 subtype ) recurred in nigeria after 9 months period of no reported case .
a critical look at possible sources of the re - occurrence was desirable .
the objective of this study was to determine whether avian influenza viruses were present at reasonably detectable levels ( 0.5% ) in possible bridge species of wild and domestic birds .
the study was conducted in 8 nigerian states .
a total of 403 birds from 40 species were sampled .
virus isolation was done in embryonated chicken eggs according to standard protocols .
the test results were all negative for avian influenza viruses .
the overall confidence interval ( ci ) calculated in r using the exact binomial confidence interval function was 00.007406 .
tawny eagle ( aquila rapax ) was the lowest sampled 0.3% ( 1/403 ) and red - billed firefinch ( lagonosticta senegala ) the highest 11.7% ( 47/403 ) .
the limitations of the sample size and possibly designing effects on the study , as to make concrete conclusions were acknowledged .
species of wild birds , so identified in the study could be useful in future surveys . furthermore , multidisciplinary and community oriented approach , blending targeted and passive surveillances was suggested .
this approach was envisaged to bring about wider coverage of bridge species and clearer insight of their possible roles in avian influenza re - occurrences and spread in nigeria .
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dissecting ventricular septal hematoma ( dvsh ) after repair of ventricular septal defect ( vsd ) is a rare , potentially life threatening [ 2 , 3 ] complication initiated by surgical disruption of the coronary microcirculation .
the resultant bleeding dissects along a plane beneath the endocardium resulting in a hematoma that bulges out into ventricular cavity .
the following is a case report of the diagnosis and management of acute , severe , left ventricular outflow tract ( lvot ) obstruction caused by the development of a dvsh after vsd repair .
a 7-week - old male infant was noted to be hypoxemic prior to repair of an inguinal hernia .
a postoperative echocardiogram demonstrated a large membranous vsd and a long segment coarctation of the aorta .
three months after repair of the coarctation the infant was in congestive heart failure and was brought to the operating room for vsd repair .
preoperative transesophageal echocardiography ( tee ) demonstrated a large membranous vsd that extended into the inlet septum and a widely patent left ventricular outflow tract ( figure 1 ) .
the echocardiographic findings were confirmed at surgery , and the vsd was closed in the standard manner using a dacron patch and 50 prolene pledgeted suture .
the initial suture line was carried clockwise avoiding the crest of the ventricular septum . near the septal leaflet of the tricuspid valve
the vsd was then closed in a counterclockwise fashion staying away from the crest of the ventricular septum , across the ventricular infundibular fold avoiding the aortic annulus , and eventually transitioning to the septal leaflet of the tricuspid valve .
the remainder of the vsd underneath the septal leaflet of the tricuspid valve was closed by weaving in and out of the valve leaflet and the vsd patch .
postoperative tee ( figure 2 ) , performed immediately after coming off of cardiopulmonary bypass , demonstrated an echo lucent region beneath the vsd patch that was surrounded by a thin membrane protruding into the lvot , findings consistent with a dvsh . during systole the anterior mitral leaflet / chordal apparatus came in contact with the dvsh resulting in severe lvot obstruction with a peak instantaneous doppler gradient between 7080 mmhg which was confirmed by direct pressure measurement .
the patient was placed back on cardiopulmonary bypass and an oblique aortotomy was made allowing retraction of the aortic valve leaflets to inspect the ventricular septum .
the hematoma was identified and the thin membrane overlying the hematoma was incised and evacuated .
the remainder of the hematoma was unroofed and lvot patency was confirmed with a 9 mm dilator .
postevacuation tee confirmed a widely patent lvot with no residual gradient ( figure 3 ) .
although rare , dvsh has the potential to cause significant hemodynamic perturbations and may be life threatening .
this process , initiated by surgical disruption of the coronary microcirculation , creates a form of myocardial rupture as blood dissects along the spiral planes of the cardiac muscle beneath the endocardium .
the resulting hematoma bulges out into the right , left , or both ventricular cavities .
this may lead to septal rupture which may create a vsd , extension of the hematoma onto the lv free wall with the potential for myocardial rupture , development of a communication between the ventricles across their inferior walls without septal rupture , cardiogenic shock , heart block , outflow obstruction , cardiac tamponade , abscess transformation , and death .
dvsh after vsd repair can be readily diagnosed during intraoperative tee , and we agree with previous recommendations that intraoperative tee should be performed in all patients undergoing vsd repair .
both the right and left sides of the ventricular septum must be thoroughly evaluated and the finding of an echo lucent region surrounded by a membrane ( the thickness of the membrane varies depending on the level of the hematoma , those deep within the septum have a very thick membrane while those close to the endocardium have a very thin membrane ) that protrudes into the cavity of the ventricle ( figure 2 ) should raise suspicion that there is a septal hematoma present .
it must be remembered that since the hematoma is caused by disruption of the coronary microcirculation in a patient that is anticoagulated , small hematomas have the potential to enlarge and cause arrhythmogenic / hemodynamic abnormalities , even after the patient leaves the operating room .
for this reason , any suspicious lesion should be thoroughly evaluated and a management strategy determined prior to the patient leaving the operating room .
management strategies of dvsh include evacuation [ 13 ] or observation [ 5 , 8 ] of the hematoma .
since conservative treatment of dvsh is associated with a mortality rate reported to be as high as 90% , we recommend immediate evacuation of the hematoma if the diagnosis is made in the operating room during intraoperative tee .
because of the potential for reaccumulation of the dvsh if it is evacuated by needle aspiration , we recommend that complete unroofing of the hematoma be performed by placing the patient back on cardiopulmonary bypass .
if the dvsh is diagnosed after the patient has left the operating room , serial echocardiograms should be performed until the size of the dvsh has stabilized and periodically thereafter until the hematoma has either resolved or is noted to be enlarging .
if the hematoma enlarges , or causes any other complications , consideration should be given to surgically evacuating the hematoma .
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dissecting ventricular septal hematoma ( dvsh ) rarely occurs after repair of a ventricular septal defect ( vsd ) but can lead to serious complications such as septal rupture , myocardial rupture , cardiogenic shock , heart block , outflow obstruction , cardiac tamponade , abscess transformation , and death .
this paper describes the diagnosis and management of acute , severe , left ventricular outflow tract obstruction caused by the development of a dvsh after vsd repair .
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before 1980 , the names of genes and classification of their encoded proteins were highly variable and non - systematic -- especially to anyone slightly outside a particular field or to a new graduate student entering the field .
professor margaret oakley dayhoff was a pioneer in attempting to create order out of chaos in the naming of genes and gene families by means of computerised protein alignments .
she was widely recognised as the founder in this new field of gene / protein classification , before her untimely death in 1983 .
cytochrome p450 ( cyp ) genes are conveniently arranged into families and subfamilies based on the percentage amino acid sequence identity [ 2 - 7 ] .
enzymes that share approximately 40 per cent identity are assigned to a particular family designated by an arabic numeral , whereas those sharing approximately 55 per cent identity are grouped into a particular subfamily designated by a letter .
for example , the sterol 27-hydroxylase enzyme and the 25-hydroxy - vitamin d3 1-hydroxylase enzyme are both assigned to the cyp27 family because they share > 40 per cent sequence identity .
furthermore , the sterol 27-hydroxylase is assigned to the cyp27 ' a ' subfamily and the 25-hydroxy - vitamin d3 1 -hydroxylase to the cyp27 ' b ' subfamily because their protein sequences are < 55 per cent identical .
if an additional enzyme were to be discovered that shared > 55 per cent identity with the sterol 27-hydroxylase , then it would be named cyp27a2 .
if an additional enzyme were to be discovered that shared < 55 per cent but > 40 per cent identity with the sterol 27-hydroxylase as well as the 25-hydroxy - vitamin d3 1-hydroxylase , then it would be named cyp27c1 .
the development and application of this delightfully logical system of nomenclature to the genes of many animals , plants and bacteria has eliminated the confusion that often had plagued the naming of gene families and superfamilies .
subsequently , this ' divergent evolution ' nomenclature system was adopted for several hundred other gene families and superfamilies -- including the olfactory receptor superfamily .
vertebrate olfactory receptor ( or ) genes represent a category of g - protein - coupled receptors ( gpcrs ) that contain seven transmembrane -helical domains and function in the reception of innumerable odour molecules in the environment .
the or gene superfamily is the largest in vertebrate genomes [ 10 - 13 ] .
the genomic architecture of mammalian or gene clusters shows an ancient evolutionary origin , preceding the marsupial - eutherian split ; species - specific evolution has further shaped the different or gene families , by means of both gains and losses of complete clusters , as well as expansion and contraction of existing clusters .
this dynamic flexibility is also reflected among individual humans ; examining 51 candidate or genes on dna chips in 189 ethnically diverse subjects , a striking amount of population diversity was found . segregating pseudogenes ( spgs )
are genes that segregate in populations between intact genes and pseudogenes -- due to a disruptive single nucleotide polymorphism ( snp ) .
a range of 16 - 24 functional or genes was found , just in this study alone , indicating that the or gene superfamily is among the most pronounced examples of functional population diversity in the human genome .
copy number variations ( cnvs ) , another type of polymorphism , are also highly prevalent among human or genes .
all these genomic events are evidence of a relatively recent process , whereby the extreme diminution of a functional repertoire in humans has occurred -- a process which is presumably still ongoing . for most mammalian species ,
based on recent or gene mining data in the platypus , opossum , cow and dog genomes -- compared with that in the rat , mouse , macaque and human genomes -- we are now certain that there has been a substantial expansion of the or gene superfamily since the mammalian radiation ~ 100 million years ago .
the evolutionary change in the number of or genes in insects is not nearly as extensive as that in mammals .
a comparison of 12 drosophila species , encompassing ~ 60 million years of divergence , shows that the number of functional or genes has remained fairly stable .
caenorhabditis elegans has a highly developed chemosensory system , which enables it to detect a wide variety of volatile ( olfactory ) and water - soluble ( gustatory ) cues associated with food , danger or other animals ; between 500 and 1,000 different gpcrs are expressed in chemosensory neurones , and these may be supplemented by alternative sensory pathways as well .
the vertebrate or gene repertoire has thus evolved from a subset of ancestral genes in the fly and worm .
there appear to be three important periods in the evolution of the vertebrate olfactory system , as evidenced by comparative genomics : ( 1 ) the adaptation to land in amphibian ancestors ; ( 2 ) the decline of olfaction in primates ; and ( 3 ) the delineation of putative pheromone receptors concurrent with rodent speciation .
the gene : pseudogene ratio is lowest in human , higher in chimpanzee and highest in rat and mouse .
this most likely reflects the necessity of olfaction for survival -- more so in the rodent than in the human .
whereas the chicken , platypus and primate genomes carry < 400 functional or genes , the opossum and rodent genomes , not surprisingly , contain between 1,000 and 1,210 functional or genes .
curiously , however , it is difficult to explain why the cow genome , with 970 functional or genes , shows more than the dog genome , with ~811 functional or genes , when dogs are considered to have such a keen sense of olfaction .
thus , the number of or genes in a species does not appear to be directly related to the environmental ' requirement ' or to lifestyle .
the or gene superfamily comprises 18 gene families and 300 subfamilies ( table 1 ) .
presently , there are 390 putatively functional ( protein - coding ) or genes and 465 or pseudogenes located in multiple clusters of varying sizes scattered throughout all autosomes except chromosome ( chr ) 20 , and on the x but not the y chromosome [ 21 - 23 ] . the members of each subfamily have been placed therein because of divergent evolution , as described above .
these subfamilies differ from cyp subfamilies , in that individual members within one subfamily are often located on two or more different chromosomes .
the or2 t ( table 2 ) subfamily contains 16 functional genes -- more than in any other subfamily .
a phylogenetic analysis of one representative from each family of the human or gene repertoire . in this tree
, one can see the following : ( 1 ) a general guideline for how the different families relate to one another ( although this is very general , and the branching is not always this well defined ) ; ( 2 ) the numbers near each branch denote the or family number ; ( 3 ) each pie chart size is scaled to represent the number of the or genes inside that family ( black = functional genes , grey = pseudogenes , yellow = segregating pseudogenes [ spgs ] ) .
spgs are genes that segregate in populations between intact genes and pseudogenes -- due to a disruptive snp .
this disruptive mutation can introduce a stop codon , or alter a highly conserved amino acid that is important for proper function of the protein . in tables 1 - 5 ,
the spgs are counted as ' functional genes ' or ' pseudogenes ' , according to the human genome project public version .
summary of the olfactory receptor ( or ) gene superfamily ( 18 families ) summary of or genes in families 1 to 4 family ori genes are located on chromosomes 9 , 17 , 19 , 11 , 16 , 5 , 1 , 6 and x. family or2 genes are located on chromosomes 1 , 6 , 7 , 11 , 5 , 9 , 12 , 16 , 19 and x. family or3 genes are located on chromosomes 17 , 1 and x. family or4 genes are located on chromosomes 11 , 14 , 15 , 1 , 19 , 17 , 18 , 21 , 6 , 4 , 5 , 8 and x. note that , in many instances , some subfamilies contain only a single gene or only a single pseudo - gene ( tables 2 - 5 ) . in fact , the or7e subfamily has only one functional gene , and all the other 85 members are pseudogenes ( table 3 ) .
the or7e subfamily is the largest subfamily in the human or gene repertoire , and probably has expanded in the human genome through a series of segmental gene duplication events .
the newly described human or14 gene family ( table 4 ) was realised after analysis of the platypus and opossum or gene repertoires .
this analysis revealed that six human or functional genes and one or pseudogene ( which previously had been classified within the or5 family ) are actually derived from a distinct platypus or gene family .
summary of or genes in families 5 to 8 family or5 genes are located on chromosomes 11 , 3 , 12 , 6 , x , 14 , 19 , 2 , 4 and 9 .
family or6 genes are located on chromosomes 12 , 1 , 11 , 14 , 10 , 7 , 2 and 8 .
family or7 genes are located on chromosomes 19 , 11 , 3 , 8 , 4 , 13 , 2 , 12 , 7 , 10 , 14 , 9 , 21 , 5 and x. family or8 genes are located only on chromosomes 11 and 12 .
summary of or genes in families 9 to 13 family or9 genes are located on chromosomes 11,7 , 12 , 1 and 2 .
family or10 genes are located on chromosomes 11 , 1 , 19 , 12 , 14 , 7 and 6 .
family or11 genes are located on chromosomes 14 , 15 , 1 , x , 6 , 12 and 22 .
the ' shotgun ' splattering of or genes throughout the human genome must have happened before speciation of homo sapiens and the development of its 22 autosomes plus the x and y chromosomes ; this can be inferred from the high conservation of the or genes ' genomic organisation among marsupial and eutherian mammals , and the phylogenetic analysis of the platypus or gene repertoire -- by comparison with that in mammals .
in contrast to this or gene arrangement would be the establishment of the cyp gene subfamilies , which arose as syntenic clusters of members within a single chromosomal segment .
this finding suggests that gene duplication events within cyp subfamilies occurred after mammalian speciation and development of the autosomes and sex chromosomes .
the two largest or gene clusters are located on chr 11 , with 38 functional genes ( 51 per cent of total ) on 11q ( cluster 11@5.0 ) and 44 functional genes ( 45 per cent ) on 11p ( cluster 11@55.6 ) .
these genes are predominantly in or families 51 , 52 , 55 and 56 ( table 5 ) . immersed within these two clusters are dozens of other non - or - related genes .
this intrusion of other non - or - related genes can also be seen in all other or gene clusters throughout the genome .
summary of or genes in families 51 , 52 , 55 and 56 the or51 , or52 , or55 and or56 genes are located only on chromosome 11 .
a recently appreciated discovery in olfaction is the unique specialisation of sensory neurones , such that each individual sensory neurone is stochastically chosen to express usually only one odourant receptor allele .
this mechanism of ' allelic exclusion ' , by which mutually exclusive expression of odourant receptor genes is regulated , remains unclear at present .
the vomeronasal-1 receptor genes ( vn1r ) also encode gpcrs and , while they encode odourant receptors , they are evolutionarily distinct from the very large or gene superfamily
the vn1r1 , vn1r2 and vn1r4 genes and vn1r6p pseudogene are located at chr 19q13.42 ; the vn1r10p , vn1r11p , vn1r12p , vn1r13p and vn1r14p pseudogenes are located on chr 6p21 ; vn1r7p and vn1r8p are on chr 21p11.2;vn1r3 is alone on chr 16p11.2 ; vn1r5 is alone on chr 1q44 ; vn1r9p is alone on chr 22 . a phylogenetic analysis of platypus , opossum and human or genes for the new family 14 only .
ori4 is an expansion of three ancestral or gene subfamilies ( a , b and c ) ; the expansion , in both platypus and opossum , took place after speciation , whereas only one branch shows an orthologous relationship between platypus and human ( marked with * ) .
the tree was generated with mega4 , using the nearest - neighbour - joining algorithm , and distances with the poisson correction model .
this tree is grounded with the or5iei gene . only genes with no more than two frame disruptions were considered in the analysis . at the present time , information about the ligands for mammalian or genes is very limited .
the smell of lemons ( limonene ) , the perception of a floral or woody smell ( acetophenone ) and the ability to smell isovaleric acid have been mapped in the mouse to two specific genomic loci on chr 4 ( ivat1 ) and chr 6 ( iva2 ) . in humans ,
isovaleric acid was found to be highly associated with the or11h7p segregating pseudogene , which is not syntenic with either ivat1 or iva2 .
another recent study found that human or7d4 is selectively activated in vitro by androstenone ; interestingly , this study found that two non - synonymous snps account for a significant proportion of the variance in smell perception of androstenone .
members of the gustatory receptor ( gr ) gene family in drosophila are expressed in chemosensory neurones and are known to mediate the perception of sugars , bitter substrates , carbon dioxide and pheromones .
intriguingly , some of these gr genes have now been shown to be expressed in abdominal multi - dendritic neurones , hygroreceptive neurones of the arista , peripheral proprioceptive neurones in the legs , neurones in the larval and adult brain , and oenocytes .
along these same lines , we and others have observed several or genes being significantly up- or downregulated in the liver or kidney of knockout mouse lines -- that is , in tissues not normally known to be involved in olfaction . it is therefore tempting to speculate that the receptors encoded by or genes , as well as by gr genes , might participate in the roles of detecting endogenous ligands .
the writing of this article was funded , in part , by nih grant p30 es06096 ( d.w.n . ) .
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the olfactory receptor gene ( or ) superfamily is the largest in the human genome .
the superfamily contains 390 putatively functional genes and 465 pseudogenes arranged into 18 gene families and 300 subfamilies .
even members within the same subfamily are often located on different chromosomes . or genes are located on all autosomes except chromosome 20 , plus the x chromosome but not the y chromosome . the gene : pseudogene ratio is lowest in human , higher in chimpanzee and highest in rat and mouse -- most likely reflecting the greater need of olfaction for survival in the rodent than in the human .
the or genes undergo allelic exclusion , each sensory neurone expressing usually only one odourant receptor allele ; the mechanism by which this phenomenon is regulated is not yet understood . the nomenclature system ( based on evolutionary divergence of genes into families and subfamilies of the or gene superfamily ) has been designed similarly to that originally used for the cyp gene superfamily .
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the primary cause of cancer death is lung cancer . the estimated new cases and deaths because of lung cancer in the united states in 2015 were 221,200 and 158,040 , respectively , whereas the 5-year survival rate for patients with lung cancer is 17.4% .
including small - cell lung carcinoma , only 37% of lung cancer is diagnosed while it is still confined to the primary site or spread to regional lymph nodes or directly surrounding tissues ; 5-year survival rates for patients with localized and regional tumors are 54% and 26.5% , respectively .
it is essential to manage the disease locoregionally to increase the survival rates of patients who have locally advanced disease ( la - nsclc ) with no distant metastasis .
although the management of localized or metastatic disease is well established , there are major ongoing controversies for locally advanced disease .
although randomized trials failed to show any survival benefit provided by surgery in stage iiia disease , surgery may be still an option for patients with single n2 node ( <3 cm ) .
for the remaining patients , neoadjuvant chemoradiotherapy ( crt ) or chemotherapy ( ct ) is recommended , whereas in cases with multiple > 3-cm pathologically proven malignant lymph nodes , definitive crt is more convenient .
furthermore , a recently published meta - analysis that included 7 trials with 1049 patients revealed that neoadjuvant ct / crt followed by surgery was not superior to neoadjuvant ct / crt followed by radical rt in patients with stage iiia disease .
the combined crt was shown to be superior to rt alone in unresectable stage iiia - iiib nsclc in several studies .
in addition , concurrent crt is more effective than sequential crt at the expense of an increase in grade 34 esophagitis .
however , one of the main debates centers on which ct regimen is more efficient with concomitant crt . to date
, the suggested ct regimens for all histologies include cisplatin / etoposide ( ep ) , cisplatin / vinblastine ( pv ) , and carboplatin / paclitaxel ( pc ) , and a platin combination with pemetrexed may be used for patients with nonsquamous nsclc .
nevertheless , there is no current consensus on which ct regimen is the best to administer concomitantly with rt in patients with la - nsclc .
several recent studies compared pe with pc in terms of efficiency and produced conflicting results .
however , docetaxel is a third - generation ct agent and its effectiveness has been demonstrated in many studies indicating that the docetaxel - cisplatin ( dp ) regimen was equal or superior to a pv combination .
in addition , docetaxel proved its superiority to vinca - alkaloids in combinations in both metastatic and la - nsclc .
furthermore , a meta - analysis revealed that docetaxel - based ct provided longer survival than vinca alkaloid - containing regimens .
the phase ii southwest oncology group s9504 trial , which added docetaxel consolidation to concomitant ep , notified a median survival of 26 months .
however , no studies have compared ep regimen with dp in la - nsclc . the only trial that compared ep with a cisplatin - taxane combination including paclitaxel but not docetaxel was conducted by the eastern cooperative oncology group .
their study comprised 599 patients with stage iiib - iv nsclc who received either ep or a cisplatin - paclitaxel regimen ; the authors reported a survival benefit in favor of paclitaxel .
thus , we conducted the present study to address the question as to whether dp is superior to ep in patients with la - nsclc who are concurrently treated with crt in terms of survival and safety .
the medical records of approximately 10,000 patients with lung cancer who were referred to istanbul university institute of oncology from 2004 to 2012 were evaluated . among them , patients with stage iib , iiia , or iiib histopathologically confirmed nsclc whose medical records were available in detail were included in the study .
the 10 edition of american joint committee on cancer ( ajcc ) tumor - node - metastasis ( tnm ) systems was used while staging according to radiologic and pathologic findings .
adverse effects were evaluated based on the common terminology criteria for adverse events ( ctcae ) version 4 .
the treatment decision was taken by a multidisciplinary team that included a medical oncologist , radiation oncologist , pathologist , chest disease specialist , thoracic surgeon , radiologist , and a nuclear medicine specialist .
patients who were inoperable were treated with radical crt , crt followed by consolidation ct , or induction ct followed by crt .
the pretreatment evaluation included a detailed clinical history and physical examination with a series of biochemistry tests and complete blood cell counts .
selection for treatment required an eastern cooperative oncology group ( ecog ) performance score of 0 to 2 , and appropriate bone marrow ( absolute neutrophil count > 1500 cells/l , and platelet count > 100,000 cells/l ) , and cardiac , renal , and hepatic function .
patients were treated with either etoposide ( 50 mg / m on days 15 and 2933 ) and cisplatin ( 50 mg / m on days 1,8,29 , and 36 ) ( ep ) or 6 cycles of weekly dp ( each 20 mg / m ) concurrently with radiotherapy up to a 60 gy .
linear accelerators ( simens / oncor impression , varian dbx 600c and varian trilogy - rapid arc ) with a 6 to 15 mv photon beam were used for radiotherapy .
all patients underwent 3-dimensional treatment planning using either cms - xio ( cms inc , st louis , mo ) or varian eclipse tps station version 8.9 ( varian medical systems , palo alto , ca ) treatment planning systems .
treatment planning was based on ct scans with 5-mm section thickness and 5-mm intervals obtained in a treatment position .
radiotherapy was administered with an angled field technique to include the entire planning tumor volume ( ptv ) in the isodose 95% area . gross tumor volume ( gtv )
ptv consisted of ctv plus 1 cm to the superior - inferior direction and 0.5 cm to the anterior - posterior and left - right directions .
all patients had pretreatment imaging of primary tumors with magnetic resonance imaging ( mri ) or computed tomography . for patients with evaluable imaging studies before and after treatment
, radiologic response was recorded according to the response evaluation criteria in solid tumors ( recist ) or pet response criteria in solid tumors ( percist ) , and classified as follows : complete response ( cr ) , partial response ( pr ) , stable disease ( sd ) , or progressive disease .
disease follow - up programs included physical examination , laboratory tests , and computed tomography scan or mri depending on which imaging methods were indicated , and performed every 3 months for the first 2 years , every 6 months for the following 3 years , and annually thereafter with no anticancer treatment .
baseline characteristics of patients and diseases including age , sex , weight loss , ecog performance status , histologic type , clinical t and n stages , as well as treatment type , ct regimen , operability , progression during follow - up , metastasis , hematologic and nonhematologic adverse effects of treatment were obtained retrospectively in detail for all patients from their medical records .
all procedures performed in studies involving human participants were in accordance with the ethics standards of the institutional research committee and with the 1964 helsinki declaration and its later amendments or comparable ethics standards .
formal consent is not required for this type of study . the statistical package for the social sciences ( spss ) for windows version 16.0 ( spss inc . , chicago , il ) was used for data analysis .
student t test was used to compare continuous variables between the groups , and and/or kruskal wallis tests were performed for the comparison of categorical variables . overall survival ( os ) was calculated from the date of first admission to disease - related death or date of last contact with the patient or any family member .
progression - free survival ( pfs ) was calculated from the date of admission to the date of first radiologic progression .
meier analysis was performed for the estimation of survival distribution and differences in pfs and os .
the log - rank test was used to assess statistical significance in univariate comparisons . to adjust for potential covariate effects ,
the medical records of approximately 10,000 patients with lung cancer who were referred to istanbul university institute of oncology from 2004 to 2012 were evaluated . among them , patients with stage iib , iiia , or iiib histopathologically confirmed nsclc whose medical records were available in detail were included in the study .
the 10 edition of american joint committee on cancer ( ajcc ) tumor - node - metastasis ( tnm ) systems was used while staging according to radiologic and pathologic findings .
adverse effects were evaluated based on the common terminology criteria for adverse events ( ctcae ) version 4 .
the treatment decision was taken by a multidisciplinary team that included a medical oncologist , radiation oncologist , pathologist , chest disease specialist , thoracic surgeon , radiologist , and a nuclear medicine specialist .
patients who were inoperable were treated with radical crt , crt followed by consolidation ct , or induction ct followed by crt .
the pretreatment evaluation included a detailed clinical history and physical examination with a series of biochemistry tests and complete blood cell counts .
selection for treatment required an eastern cooperative oncology group ( ecog ) performance score of 0 to 2 , and appropriate bone marrow ( absolute neutrophil count > 1500 cells/l , and platelet count > 100,000 cells/l ) , and cardiac , renal , and hepatic function .
patients were treated with either etoposide ( 50 mg / m on days 15 and 2933 ) and cisplatin ( 50 mg / m on days 1,8,29 , and 36 ) ( ep ) or 6 cycles of weekly dp ( each 20 mg / m ) concurrently with radiotherapy up to a 60 gy .
linear accelerators ( simens / oncor impression , varian dbx 600c and varian trilogy - rapid arc ) with a 6 to 15 mv photon beam were used for radiotherapy .
all patients underwent 3-dimensional treatment planning using either cms - xio ( cms inc , st louis , mo ) or varian eclipse tps station version 8.9 ( varian medical systems , palo alto , ca ) treatment planning systems .
treatment planning was based on ct scans with 5-mm section thickness and 5-mm intervals obtained in a treatment position .
radiotherapy was administered with an angled field technique to include the entire planning tumor volume ( ptv ) in the isodose 95% area . gross tumor volume ( gtv )
ptv consisted of ctv plus 1 cm to the superior - inferior direction and 0.5 cm to the anterior - posterior and left - right directions .
all patients had pretreatment imaging of primary tumors with magnetic resonance imaging ( mri ) or computed tomography . for patients with evaluable imaging studies before and after treatment
, radiologic response was recorded according to the response evaluation criteria in solid tumors ( recist ) or pet response criteria in solid tumors ( percist ) , and classified as follows : complete response ( cr ) , partial response ( pr ) , stable disease ( sd ) , or progressive disease .
disease follow - up programs included physical examination , laboratory tests , and computed tomography scan or mri depending on which imaging methods were indicated , and performed every 3 months for the first 2 years , every 6 months for the following 3 years , and annually thereafter with no anticancer treatment .
baseline characteristics of patients and diseases including age , sex , weight loss , ecog performance status , histologic type , clinical t and n stages , as well as treatment type , ct regimen , operability , progression during follow - up , metastasis , hematologic and nonhematologic adverse effects of treatment were obtained retrospectively in detail for all patients from their medical records .
all procedures performed in studies involving human participants were in accordance with the ethics standards of the institutional research committee and with the 1964 helsinki declaration and its later amendments or comparable ethics standards .
the statistical package for the social sciences ( spss ) for windows version 16.0 ( spss inc . , chicago , il ) was used for data analysis .
student t test was used to compare continuous variables between the groups , and and/or kruskal wallis tests were performed for the comparison of categorical variables . overall survival ( os )
was calculated from the date of first admission to disease - related death or date of last contact with the patient or any family member .
progression - free survival ( pfs ) was calculated from the date of admission to the date of first radiologic progression .
meier analysis was performed for the estimation of survival distribution and differences in pfs and os .
the log - rank test was used to assess statistical significance in univariate comparisons . to adjust for potential covariate effects ,
in total , 50 patients ( median age 54 years ; range , 3270 years ) who were given concurrent ep , and 55 patients ( median age 55 years ; range , 3773 years ) who were given concurrent dp were enrolled in the analyses .
there was no statistically significant difference in baseline clinicopathologic features including age ( p = 0.41 ) , sex ( p = 0.33 ) , weight loss ( p = 0.20 ) , ecog performance status ( p = 0.19 ) , histologic type ( p = 0.38 ) , lung site ( p = 0.35 ) , clinical t and n stage ( p = 0.59 and p = 0.12 , respectively ) , disease stage ( p = 0.96 ) , surgery ( p = 0.30 ) , radiation dose ( < 60 vs. 60 gy ) ( p = 0.95 ) , and treatment response ( p = 0.99 ) between the groups .
however , the mean radiation dose was higher in the ep group compared with the dp group ( 59 vs. 54 gy ; p = 0.02 ) . in addition ,
induction or consolidation ct administration was more common in the dp group than in the ep group ( 84% vs. 34% ; p
the comparison of baseline characteristics of patients and treatment details of the 2 groups is shown in table 1 .
comparison of clinical and pathological variables of non - small cell lung cancer patients treated with concomitant chemoradiotherapy either with etoposide and cisplatin or docetaxel and cisplatin .
the median ( min max ) follow - up time of patients was 27 months ( range , 1132 months ) in the ep group and 19 months ( range , 196 months ) in the dp group . in the univariate analysis , median pfs of patients treated with ep
was found higher than that of patients treated with dp ( pfs = 19 months , 95% confidence interval [ ci ] 434 vs. 10 months 95% ci 512 ;
p = 0.02 ) ( table 2 ) , as well as in patients with treatment response ( pfs = 22 months , 95% ci 1133 vs. 6 months 95% ci 48 ; p < 0.001 ) .
univariate analysis of progression free survival of patients . in the univariate analysis , the median os of patients treated with ep
was found higher than that of patients treated with dp ( os = 41 months ; 95% ci 2656 vs. 20 months , 95% ci 1426 ; p = 0.003 ) ( fig .
1 ) . furthermore , right - sided tumor ( os = 30 months ; 95% ci 1644 vs. 19 months , 95% ci 1226 ; p = 0.029 ) , surgery performance ( os = 52 months ; 95% ci 1392 ] vs. 23 months , 95% ci 2026 ] ; p = 0.023 ) , and treatment response ( os = 40 months ; 95% ci 2983 vs. 17 months , 95% ci 172 ; p <
there was no statistical significance in terms of age ( p = 0.07 ) , sex ( p = 0.98 ) , weight loss ( p = 0.68 ) , ecog performance status ( p = 0.51 ) , histologic type ( p = 0.63 ) , clinical t and n stage ( p = 0.33 and p = 0.32 , respectively ) , disease stage ( p = 0.45 ) , induction or consolidation therapy ( p = 0.06 ) , and radiation dose ( p = 0.12 ) significance ( table 3 ) . the comparison of overall survival between etoposide and cisplatin and docetaxel and cisplatin groups .
multivariate analysis further revealed survival advantage with ep compared with dp ( hazard ratio [ hr ] = 0.46 ; 95% ci 0.250.83 ; p = 0.009 ) .
significant variables were stratified with respect to potential confounding factors such as age , weight loss , t stage , histology , and stage of nodal involvement .
in addition , both right - sided tumors ( hr = 0.58 ; 95% ci 0.350.98 ; p = 0.041 ) and treatment response ( hr = 0.26 ; 95% ci 0.150.48 ; p <
neither surgery ( hr = 0.65 ; 95% ci 0.321.28 ; p = 0.22 ) nor induction or consolidation therapy was associated with survival in the multivariate analysis ( hr = 0.84 ; 95% ci 0.541.65 ; p = 0.94 ) . the results of multivariate analysis are shown in table 4 .
the toxicity profile of 2 treatment groups including anemia ( p = 0.91 ) , thrombocytopenia ( p = 0.47 ) , nausea / vomiting ( p = 0.69 ) , esophagitis ( p = 0.69 ) , neurotoxicity ( p = 0.20 ) , nephrotoxicity ( p = 0.38 ) , hepatotoxicity ( p = 0.37 ) , and diarrhea ( p = 0.76 ) was found similar except that pulmonary ( grade 34 : 0% vs. 6% , p = 0.02 ) and skin toxicities ( grade 12 : 4% vs. 24% ,
p = 0.01 ) were higher in the dp group compared with ep .
although neutropenia incidence was slightly higher in the ep group than with dp ( grade 3 , 8% vs. 0% , p = 0.055 ) , the infection rate was similar between the 2 groups ( grade 34 , 0% vs. 6% , p = 0.08 ) .
there are several ct combinations choices to consider for concomitant treatment , among which the most accepted and commonly used is ep .
the other frequent options are combinations of platin with taxanes and vinorelbine ; however , the best remains to be elucidated because of a lack of large , randomized trials . nonetheless , there is some evidence for which combination might be more effective .
for instance , in a meta - analysis by shen et al that included 9 randomized controlled trials with 1741 patients , the dp combination was revealed to be superior to cisplatin and vinorelbine in advanced nsclc concerning response to treatment and survival rates , and with a better safety profile .
the current controversies continue as to whether standard ep regimen is superior to a platin plus taxane combination .
santana - davila et al reported that cp was as effective as ep regimen based on the data of the large database of the veterans health administration .
however , the results of the only randomized trial that compared ep with cp showed that ep provided higher survival time and less pulmonary but higher hematologic toxicity than ep , which is fully compatible with our results . the conflicting results of these 2 studies gave rise to the following question in our minds : was the superiority of the ep regimen over cp combination caused by the superiority of cisplatin over carboplatin in the study of wang et al ? because the lung adjuvant cisplatin evaluation meta - analysis demonstrated that carboplatin was not as efficient as cisplatin at eliminating micrometastasis in the adjuvant setting of stage ii and iii nsclc .
accordingly , we decided to compare 2 cisplatin - containing regimens with standard etoposide or docetaxel , and our results indicated that ep was still superior to dp , which verified the findings of wang et al .
concurrent chemoradiotherapy with ep may provide more favorable outcomes than that of dp with a better safety profile .
the median os and pfs achieved with ep in our group of patients were 40 months and 19 months , respectively , which were considerably higher compared without comes reported in the int 139-patient trial ( 23.6 months and 12.8 months , respectively ) that used the same ct protocol .
furthermore , both ct regimens resulted in median os rates that were better than expected on the basis of data from previously reported studies in patients with stage iii nsclc .
the reason for this inconsistency may be related to the fact that patients enrolled in our study with mediastinal nodal ( n2 ) or n3 involvement were judged to be fit for rigorous therapy .
additionally , pathologic confirmation of mediastinal staging could not be performed to all patients ; this may clinically result in upstaging of patients whose disease stages may actually be earlier .
it is estimated that the frequency of this complication is between 10% and 30% depending on factors such as volume of the irradiated area , irradiation technique , the type of ct agents given , as well as the previous pulmonary comorbidities of the patient .
taxanes are well known antineoplastics that have the potential to induce pulmonary injury through a variety of mechanisms . among the third - generation agents used with radiation in current practice , docetaxel might be the most toxic .
this drug has resulted in a relatively high rate of serious ( nci - ctc grade 3 ) pneumonitis , reported to occur in as many as 47% of patients treated concurrently with radiotherapy . in our study , the frequency ( 6% ) of severe pulmonary toxicity seen with weekly docetaxel and cisplatin was lower than the previously reported data , which may be because of careful patient selection , meticulous rt volume planning , as well as prompt supportive treatment initiated at the first sign of pneumonitis . although most adverse effects were comparable between the 2 groups , grade 3 neutropenia was more common among patients who received ep concomitantly , which is similar to previous reports .
the incidence of severe neutropenia and thrombocytopenia was considerably lower in our study ( 12% and 0% , respectively ) compared with other studies including pe regimen ( 39% vs. 10%).variations in terms of efficiency and toxicity have been reported between different nations receiving the same ct regimens .
these differences mainly emerge from ethnic instabilities in single - nucleotide polymorphism distributions that affect ct transport and metabolism .
it is well - established that the pharmocogenomic variability in genes may result in inter - racial and individual disparity concerning toxicity and outcomes .
our population comprised only turkish patients ; however , no pharmocogenomic study has investigated the distribution of gene polymorphisms in the turkish population .
additionally , severe skin toxicity was not observed in any of the treatment arms ; however , grade 2 skin toxicity was more frequent in patients who received dp ( 12% vs. 0% , p = 0.017 ) .
increased cutaneous toxicity with docetaxel has been reported , especially in patients with breast cancer .
first , the population of our study mainly consisted of men ( > 90% ) .
the incidence of nsclc has increased recently , and the rate of women diagnosed with nsclc is about 35% to 40% .
second , > 45% of patients had squamous cell lung cancer in the present study , whereas adenocarcinoma histology is the most common subtype among patients with la - nsclc referred to oncology centers .
in conclusion , we reported the toxicities and clinical outcomes of patients with unresectable stage iii nsclc treated with concurrent thoracic rt with either pe or dp . given that pe provided better outcomes with less pulmonary but greater hematologic toxicity , we believe that pe might be more appropriate to administer concomitantly with close monitoring for neutropenia .
still , the results of our study warrant further investigation in randomized studies to make a final decision .
david f. chapman who is a native - english speaker with scientific expertise for editing the language of the manuscript .
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abstractpresently , there is no consensus regarding which chemotherapy regimen is best to administer with radiotherapy in patients with locally advanced non - small - cell lung cancer ( la - nsclc ) .
herein , our aim was to compare the outcome of patients treated with either etoposide cisplatin ( ep ) or docetaxel cisplatin ( dp ) in this curative setting.patients treated with either ep or dp and concurrent radiotherapy from 2004 to2012 were identified and their detailed medical records and follow - up information were obtained for analysis in this retrospective study .
survival rates were compared using cox proportional hazards regression models with adjustments for confounding parameters provided by propensity score methods.a total of 105 patients were treated with concurrent chemoradiotherapy for la - nsclc ( stage iib - iiia - iiib ) .
the median ages were 54 years ( range , 3270 years ) and 55 years ( range , 3773 years ) in the ep ( n = 50 ) and dp ( n = 55 ) groups , respectively .
the median follow - up time was 27 months ( range , 1132 months ) in the ep group and 19 months ( range , 196 months ) in dp group .
there was no significant difference in baseline clinicopathologic features including age , sex , performance status , histologic subtype , and clinical tnm stages between groups . in the univariate analysis ,
the median overall survival of patients treated with ep was higher than that of patients treated with dp ( 41 vs. 20 months , p = 0.003 ) .
multivariate analysis further revealed a survival advantage with ep compared with dp ( hazard ratio [ hr ] , 0.46 ; 95% confidence interval : 0.250.83 ; p = 0.009 ) .
the toxicity profile of the 2treatment groups was similar except that pulmonary toxicity was higher in the dp group ( grade 34 : 0% vs. 6% , p = 0.024).concurrent chemoradiotherapy with ep may provide more favorable outcomes than dp and with an acceptable safety profile .
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overweight women have a higher incidence of menstrual dysfunction and anovulation , possibly because of altered secretion of gonadotropin releasing hormone , sex hormone binding globulin , ovarian and adrenal androgen , and luteinising hormone and also because of altered insulin resistance . a body mass index that was either high or low
mechanisms through which body mass affects reproduction that have been cited include menstrual disturbance and anovulation but these problems can be overcome through assisted reproduction treatment .
there is no evidence that body mass affects the quality of the embryo and therefore the pregnancy rate .
other mechanisms may be proposed such as altered receptivity of the uterus after transfer of embryos , possibly because of disturbed endometrial function .
the prevalence of obesity in infertile women is high , and there is growing evidence that bmi is associated with pregnancy outcome after art .
several recent and previous studies have linked overweight and obesity to low pregnancy rate [ 48 ] and spontaneous abortion in art programs .
obesity is also potentially modifiable , possibly amenable to low cost , noninvasive self - management by patients .
these studies , however , did not examine the interaction ( effect modification ) between bmi and other related factors on the probability of pregnancy outcome in these women . according to published studies [ 10 , 11 ]
, patient 's age , infertility type , and number of transferred embryos appeared to be the most important factors affecting pregnancy outcome after icsi .
the current study aimed to examine the association of pregnancy outcome with bmi and to assess the possible effect modification of women 's age , infertility type , and number of transferred embryos on the probability of positive pregnancy outcome associated with body mass index in infertile women receiving assisted reproduction treatment .
a retrospective study was designed to examine the association of pregnancy outcome with bmi and to examine the possible effect modification of women 's age , infertility type , and number of transferred embryos on the observed association .
the study extracted data from the files of 349 infertile women who underwent assisted fertilization at the art unit , the international islamic center for population researches and studies ( iicprs ) , al - azhar university , during 2011 .
three hundred forty - nine files were selected randomly from the art medical data archives according to the estimated sample size . because of missing outcome data , only one file was excluded from study analyses .
the sample size is calculated according to the estimated average pregnancy rate in the studied center during the previous years ( 25% ) and to an assumed precision of 0.04 with confidence interval of 95% and probability value of 0.05 .
according to pregnancy outcome , a nested case - control approach is designed where women with positive pregnancy outcome were considered as cases and those with negative pregnancy outcome as controls .
urine and/or serum human chorionic gonadotrophin ( hcg ) measurement was routinely obtained on days 1417 after embryo transfer , and positive pregnancy is confirmed following hcg rise .
the main studied variables were categorized for the clarity of data analysis and presentation of results as follows : age is classified into three categories according to tertile distribution of the studied women : < 27 years , 2733 years , and > 33 years ; and three bmi subgroups were formed : < 25 kg / m ( normal ) , 25>30 kg / m ( overweight ) , and 30 kg / m(obese ) .
the number of transferred embryos was classified into three categories ( one embryo transfer , two embryos transfer , and three and more embryos transfer ) .
the collected data were analysed by using the statistical analysis system ( sas ) software package .
chi - square and t - tests were used as appropriate to compare the studied women by bmi in relation to their characteristics .
logistic regression models were used to estimate odds ratios and their 95% confidence intervals for the association of positive pregnancy outcome and bmi . to explore the possible effect modification of patient 's age , type of infertility , and number of embryo transfer on the probability of positive pregnancy outcome associated with bmi
, we incorporated the main effect variables and their cross - product terms into logistic regression models for testing of interaction based on a multiplicative model .
the positive pregnancy rate among the whole studied women was 21% ( 75 of 349 ) .
the positive pregnancy rate is varied by bmi where it was 33% , 22% , and 18% in normal , overweight , and obese women , respectively .
there were significant differences among bmi strata regarding age , infertility duration , cycle length , and luteinising hormone ( lh ) level , where the highest mean age , infertility duration , cycle length , and the lowest mean lh level were among obese women .
on the other hand , no significant differences among the three bmi strata were observed regarding other studied factors .
table 2 shows odds ratios ( ors ) and their 95% confidence intervals for the association of positive pregnancy outcome with bmi in the unadjusted and adjusted logistic model . the results of both models were nearly the same with no confounding effects of the controlled confounders being observed .
the odds ratio of positive pregnancy is found to be negatively and significantly associated with overweight and obesity . among overweight and obese women , the probability of positive pregnancy is reduced by 50% and 55% , respectively , in overweight and obese women ( or = 0.50 ; 95% ci = 0.250.99 among overweight women ; or = 0.45 ; 95% ci = 0.200.99 among obese women ) .
table 3 shows the adjusted ors of positive pregnancy outcome and their 95% cis for the two - way interactions between bmi and patient 's age , infertility type , and et .
although no relation was detected between bmi and pregnancy outcome among women aged 2733 years , a varying degree of reduction was observed for other age groups .
the highest reduction was among overweight women aged > 33 years ( 80% ) and obese women aged < 27 years ( 77% ) .
also , there has been a reduction in pregnancy outcome among overweight and obese women with primary and secondary infertility with the highest and significant reductions being for primary infertile women ( or = 0.27 ; 95% ci = 0.150.98 for overweight , and or = 0.35 ; 95% ci = 0.120.99 for obese women ) .
the reduction in pregnancy rate among overweight and obese women who had one or two et was not seen among women who had more than two embryos transferred . in this group ,
the probability of positive pregnancy outcome was increased by 1.6-fold ( or = 1.60 ; 95% ci = 0.744.68 ) .
this study contributes to the previous reports of decreased probability of positive pregnancy outcome in overweight and obese women after infertility treatment [ 4 , 68 ] .
the endocrinological and/or biochemical milieu associated with obesity , operating through a functional state such as insulin resistance , can create a hostile intraovarian or intrauterine environment for the oocytes or embryos . also , plausibility for a causal link between obesity and reproductive hormones can be found in studies that link modest weight loss and a reduction of central fat to improved insulin sensitivity , resulting in ovulation and pregnancy in overweight infertile women .
moreover , obesity is associated with alterations in carbohydrate and fat metabolism central to the development of insulin resistance .
a diet with a high - glycemic index has been associated with infertility , fetal loss , congenital anomalies , and prematurity , as well as macrosomia .
greater carbohydrate intake and dietary glycemic load have been associated with an increased risk of infertility due to anovulation .
the striking negative association of positive pregnancy outcome with overweight and obesity , observed in our study as in previous ones [ 3 , 5 , 16 , 17 ] , directed us to examine if the probability of positive pregnancy varies according to other known risk factors .
the results suggest the reducing effect of overweight and obesity on the probability of positive pregnancy outcome to increase more in primary infertile women and among those aged < 27 years and more than 33 years .
however , this reducing effect disappeared among women aged 2733 years and those have had two et . the practice of multiple embryos transfer in art has also been suggested to decrease early pregnancy loss among normal and overweight women [ 18 , 19 ] .
the mechanism by which age , infertility type , and number of et can modify the probability of positive pregnancy associated with overweight and obesity is unclear .
but these results may open a number of avenues for subsequent research to shed more light on such mechanisms . however , there is a still controversy about the dangerous effect of transferring multiple embryos .
multiple birth is related to the number of transferred embryos and is associated with adverse fetal and infant outcomes , and they also pose increased risks of maternal morbidity and mortality [ 20 , 21 ] .
accordingly , weight reduction among obese women undergoing art may be of value in reducing the number of transferred embryos and hence reducing the associated complications .
however , a recent study reported that short - term weight loss was unrelated to positive -hcg , clinical pregnancy , or live birth rates , particularly among obese and overweight women . to our knowledge , this study is the first to search for the possible effect modification of some women 's characteristics as well as the number of embryo transfer on the reported negative association between bmi and positive pregnancy outcome . a potential concern in this study is the lack of data , due to the retrospective nature of the study .
accordingly , other factors were not available about women lifestyle factors ( such as smoking , physical exercise , and dietary habit ) to investigate their modification effect on the probability of pregnancy outcome associated with bmi . in summary
, this study confirms the reported reducing effect of overweight and obesity on positive pregnancy outcome in infertile women after assisted reproduction treatment .
our study also suggests a possible effect modification of patient 's age , infertility type , and number of et on such association .
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the present study aimed to evaluate the impact of body mass index ( bmi ) on pregnancy outcome after intracytoplasmic sperm injection ( icsi ) .
the study analyzed pregnancy outcome of 349 women who underwent icsi by their bmi : < 25 , 25<30 , and 30 kg / m2 .
the associations were generated by applying logistic regression models . a significant reduction in positive pregnancy outcome
was observed among overweight and obese women ( odds ratio ( or ) = 0.50 ; 95% confidence interval ( ci ) = 0.250.99 for overweight women and or = 0.45 ; 95% ci = 0.200.89 for obese women ) .
these estimates show that the pregnancy rates are reduced with increasing bmi .
the effect of obesity on pregnancy outcome was absent when three and more embryos were transferred .
our study contributes to the reports linking overweight and obesity with decreased positive pregnancy outcome after icsi and suggests women 's age , infertility type , and number of embryos transferred to modify this reducing effect .
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we
have identified short n , c - capped peptides that selectively inhibit
the proteasome of the malaria - causing pathogen plasmodium
falciparum .
these compounds are highly potent in culture
with no toxicity in host cells .
one cyclic biphenyl ether compound
inhibited intraerythrocytic growth of p. falciparum with an ic50 of 35 nm , and we show that even a pulse
treatment with this cyclic peptide induced parasite death due to proteasome
inhibition .
these compounds represent promising new antimalarial agents
that target the essential proteasomal machinery of the parasite without
toxicity toward the host .
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the gwtg - stroke registry is an initiative of the aha and the american stroke association , with the goal of improving the care and outcomes of patients with stroke and transient ischemic attacks .
details of the design and conduct of the gwtg - stroke program have been published previously.6 gwtg - stroke uses a web - based patient management tool ( outcome sciences , inc ) to collect clinical data on consecutively admitted patients , to provide decision support , and to enable real - time online reporting features.6,13 hospital personnel collect data on patients admitted with the principal clinical diagnosis of acute stroke or transient ischemic attack by prospective clinical identification , retrospective identification through the use of discharge codes , or a combination thereof .
outcome sciences , inc , serves as the data collection and coordination center for gwtg - stroke .
the duke clinical research institute serves as the gwtg data analysis center and has an agreement to analyze the aggregate deidentified data for research purposes .
the duke university health system institutional review board determined that this project met the criteria for a declaration of exemption under the code of federal regulations 45 cfr 46.101(b)(2 ) .
patient data , such as demographics , medical history , onset time of stroke symptoms ( recorded as last known well time ) , arrival time , treatments and procedures , tpa treatment initiation time , and tpa complications , were abstracted from the gwtg - stroke registry .
data on hospital - level characteristics ( eg , bed size , geographical region , teaching status , and annual stroke volume ) were gathered from the aha hospital database.6 a structured survey tool consisting of 70 items regarding components of stroke care management , including communication and teamwork , current process of care , organizational culture , performance monitoring and feedback , and overcoming of barriers was used for this study .
the survey was a standardized telephone questionnaire ; each item had 5 likert - type answer options , and respondents chose the option that most accurately fit the site s status .
the survey questions were developed based on a previous qualitative study conducted by our research group and content experts.14 the questions were then reviewed by 10 stroke neurologists , stroke advanced practice nurses , and coordinators all considered experts in the field . based on this feedback ,
the survey was refined and then given to 5 stroke nurse coordinators for additional input .
the survey was field tested through telephone interviews with medical and nursing staff at community hospitals , further refined through factor analysis , and then finalized.15 three questions were created to elicit each site s perception of tpa administration and dtn performance , which was defined as the percentage of patients who received tpa within 60 minutes of arrival among all patients who received tpa at the institution .
respondents were asked to quantify both their tpa administration rate among eligible patients and their dtn performance as 0% to 20% , 20% to 40% , 40% to 60% , 60% to 80% , or 80% to 100% .
they were further asked to rank themselves as a low , below - average , average , above - average , or top performer in terms of their dtn performance on a national level .
accuracy of perception was defined as the respondent s ability to correctly identify their performance status relative to all gwtg - stroke hospitals ( below average , average , above average ) .
responses overestimating or underestimating performance were scored as inaccurate ( eg , perceiving hospital performance as average when it is actually below average ) .
a factor analysis of the other survey items has been published separately.15 survey targets were key personnel in stroke care at eligible gwtg - stroke sites .
contact was established through information from the gwtg - stroke database ( as available ) or from aha quality improvement directors or by direct calling of the hospital .
we interviewed a representative from each institution who identified himself or herself as a person familiar with the hospital s tpa administration process .
these interviewees included stroke neurologists , stroke coordinators , and hospital - based quality - improvement staff who were considered experts in the field .
interviews were 15 minutes long and were conducted by telephone between january 2011 and july 2011 .
patient sociodemographic and clinical variables , hospital characteristics , and quality - of - care measures were compared among hospitals classified according to performance ranking .
performance rank , categorized as top , middle , and low performers , was considered to be ordinal .
categorical variables were reported as percentages , and continuous variables were reported as medians and 25th and 75th percentiles .
further analysis of sites focused on the dichotomized ranking of their relative performance into overestimation versus no overestimation .
top - performing hospitals were excluded for this analysis because their ranking could not be overestimated .
top performer were categorized as overestimation , whereas those that answered average , below average , or
top performer were labeled as overestimation , whereas those that answered below average or
sociodemographic factors , clinical variables , hospital characteristics , and quality - of - care measures were compared between these 2 groups .
pearson tests and wilcoxon rank - sum tests were used to compare categorical and continuous variables , respectively .
a multivariable logistic regression model was used to determine which factors were independently associated with dtn performance overestimation .
variables that were likely potential confounders were identified a priori to be included in this model based on literature review and clinical judgment .
these variables were joint commission primary stroke center status , geographic region , annual volume of tpa , annual volume of ischemic stroke , percentage of dtn time within 60 minutes , and years that the survey respondent had been with the site s stroke team .
a structured survey tool consisting of 70 items regarding components of stroke care management , including communication and teamwork , current process of care , organizational culture , performance monitoring and feedback , and overcoming of barriers was used for this study .
the survey was a standardized telephone questionnaire ; each item had 5 likert - type answer options , and respondents chose the option that most accurately fit the site s status .
the survey questions were developed based on a previous qualitative study conducted by our research group and content experts.14 the questions were then reviewed by 10 stroke neurologists , stroke advanced practice nurses , and coordinators all considered experts in the field . based on this feedback ,
the survey was refined and then given to 5 stroke nurse coordinators for additional input .
the survey was field tested through telephone interviews with medical and nursing staff at community hospitals , further refined through factor analysis , and then finalized.15 three questions were created to elicit each site s perception of tpa administration and dtn performance , which was defined as the percentage of patients who received tpa within 60 minutes of arrival among all patients who received tpa at the institution .
respondents were asked to quantify both their tpa administration rate among eligible patients and their dtn performance as 0% to 20% , 20% to 40% , 40% to 60% , 60% to 80% , or 80% to 100% .
they were further asked to rank themselves as a low , below - average , average , above - average , or top performer in terms of their dtn performance on a national level .
accuracy of perception was defined as the respondent s ability to correctly identify their performance status relative to all gwtg - stroke hospitals ( below average , average , above average ) .
responses overestimating or underestimating performance were scored as inaccurate ( eg , perceiving hospital performance as average when it is actually below average ) .
a factor analysis of the other survey items has been published separately.15 survey targets were key personnel in stroke care at eligible gwtg - stroke sites .
contact was established through information from the gwtg - stroke database ( as available ) or from aha quality improvement directors or by direct calling of the hospital .
we interviewed a representative from each institution who identified himself or herself as a person familiar with the hospital s tpa administration process .
these interviewees included stroke neurologists , stroke coordinators , and hospital - based quality - improvement staff who were considered experts in the field .
interviews were 15 minutes long and were conducted by telephone between january 2011 and july 2011 .
patient sociodemographic and clinical variables , hospital characteristics , and quality - of - care measures were compared among hospitals classified according to performance ranking .
performance rank , categorized as top , middle , and low performers , was considered to be ordinal .
categorical variables were reported as percentages , and continuous variables were reported as medians and 25th and 75th percentiles .
further analysis of sites focused on the dichotomized ranking of their relative performance into overestimation versus no overestimation .
top - performing hospitals were excluded for this analysis because their ranking could not be overestimated .
top performer were categorized as overestimation , whereas those that answered average , below average , or
low - performing hospitals that answered average , above average , or top performer were labeled as overestimation , whereas those that answered below average or
sociodemographic factors , clinical variables , hospital characteristics , and quality - of - care measures were compared between these 2 groups .
pearson tests and wilcoxon rank - sum tests were used to compare categorical and continuous variables , respectively .
a multivariable logistic regression model was used to determine which factors were independently associated with dtn performance overestimation .
variables that were likely potential confounders were identified a priori to be included in this model based on literature review and clinical judgment .
these variables were joint commission primary stroke center status , geographic region , annual volume of tpa , annual volume of ischemic stroke , percentage of dtn time within 60 minutes , and years that the survey respondent had been with the site s stroke team .
all 1395 hospitals participating in gwtg - stroke between october 1 , 2009 , and september 30 , 2010 , were included as potential survey sites .
low - volume sites , defined as those treating < 6 acute ischemic stroke patients with tpa during the study period , were excluded from the sampling frame .
the 100 highest and 100 lowest performing hospitals from this ranked list were selected , with dtn performance ranging from 45.2% to 92.3% and from 0.0% to 0.0% , respectively .
fifty additional hospitals above and below the median dtn performance of the ranked list ( 20.0% ) were selected to form 100 middle - performing hospitals ( dtn performance range 16.7% to 25.0% ) . of these 300 sites , 157 responded to initial site contact . among these ,
16 sites were excluded : 2 sites refused to complete a telephone interview , and an additional 14 sites did not respond to key questions or had incomplete data ( > 10% of responses missing ) .
the final sample represented 141 hospitals that treated 48 201 stroke patients during the study period .
surveyed hospitals tended to have a higher number of eligible patients receiving tpa ( p=0.001 ) and a higher annual tpa volume ( p=0.006 ) but did not have significant differences in other hospital characteristics , including annual stroke volume or rate of dtn 60 minutes ( table1 ) .
most patient demographics queried showed statistically significant differences , including race / ethnicity , insurance status , and components of medical history , although many of these differences were modest ( table2 ) .
hospital characteristics of surveyed versus nonsurveyed sites 25th to 75th p indicates 25th to 75th percentile ; dtn , door - to - needle ; tjc , the joint commission ; tpa , tissue plasminogen activator .
patient characteristics of surveyed versus nonsurveyed sites 25th to 75th p indicates 25th to 75th percentile ; cad , coronary artery disease ; mi , myocardial infarction ; tia , transient ischemic attack ; va , veterans affairs .
the study population consisted of 49 top performers , 52 middle performers , and 40 low performers .
most personnel interviewed from each hospital were nursing staff ( 85.0% ) . on average , respondents had been working with their current stroke teams for 4 years ( 25th to 75th percentile 2 to 6 years ) .
comparisons of hospital and patient characteristics among these performance groups are shown in tables3 and 4 .
significant hospital - level factors that varied among the 3 performance groups were volume of tpa administration during the performance evaluation period ( october 1 , 2009 , to september 30 , 2010 ; p<0.001 ) , annual volume of tpa administration ( p<0.001 ) , and annual ischemic stroke admissions ( p=0.045 ) .
top - performing hospitals tended to have more ischemic stroke admissions per year and administered tpa to a greater number of patients .
in addition , most patient - level variables showed statistically significant differences among the 3 performance groups .
these variables included median age , gender , race / ethnicity , insurance status , and comorbidities such as previous stroke or transient ischemic attack ( table4 ) .
hospital characteristics by performance on dtn 60 minutes 25th to 75th p indicates 25th to 75th percentile ; dtn , door - to - needle ; tjc , the joint commission ; tpa , tissue plasminogen activator .
baseline patient characteristics by performance on dtn 60 minutes 25th to 75th p indicates 25th to 75th percentile ; cad , coronary artery disease ; dtn , door - to - needle ; mi , myocardial infarction ; tia , transient ischemic attack ; va , veterans affairs .
almost two - thirds of respondents ( 65.3% ) correctly estimated the percentage of eligible patients at their institution who received tpa .
figure1 shows a significant trend toward lower accuracy on this measure as dtn performance declined ( p=0.002 ) . regardless of performance category
, respondents reported less accurately on their performance on the dtn 60 minutes metric ( figure1 ) .
overall , 29.1% of hospitals were able to accurately quantify their dtn 60 minutes ability , and this rate of accuracy was not statistically different among performance groups ( p=0.853 ) .
percentage of accurate responses regarding tpa administration and rates of dtn 60 minutes at respondent s home institution .
although the median dtn 60 minutes rate among top - performing hospitals was 57.1% of treated patients , the majority ( 67.4% ) of top - performing hospitals estimated their performance on dtn 60 minutes to be > 60% ( figure2 ) . similarly , 67.5% of low - performing hospitals perceived their performance to be > 20% ( actual median dtn 60 minutes rate 0.0% ) . the majority ( 81.6% ) of top performers
recognized themselves as above average or top in their ability to deliver timely tpa relative to other sites ( figure3 ) .
in contrast , low performers tended to overestimate their relative ability to provide timely tpa compared with others : 85.0% reported their dtn 60 minutes performance to be average , above average , or top in comparison to other us hospitals .
perception of performance on door - to - needle ( dtn ) 60 minutes by quartile for hospital ranking .
distribution of responses regarding perceived achievement rate of dtn 60 minutes at respondent s home institution divided by hospital ranking , based on actual performance data , and comparison of response distribution with actual median within performance subgroups .
* indicates category for median rate of actual dtn 60 minutes for each classification of performers .
perceived relative performance on door - to - needle ( dtn ) 60 minutes by hospital ranking .
distribution of responses regarding perceived success rate of dtn 60 minutes at respondent s home institution relative to other us institutions divided by hospital ranking based on actual performance data . among all middle- and
low - performing hospitals surveyed ( n=92 ) , respondent overestimation occurred at 56 sites ( 60.9% ) .
patient and hospital characteristics differed slightly for sites that overestimated their relative performance versus those that did not .
hospitals with staff that overestimated performance had higher percentages of minority patients ; slightly younger median age populations ; and higher combined percentages of patients on medicaid , medicare , or with no insurance .
hospitals with staff that reported accurately or that underestimated their performance relative to others had a slightly higher percentage of patients with almost every medical condition collected .
statistically significant percentages between the 2 groups were found for patients with a history of atrial fibrillation or flutter , coronary artery disease or previous myocardial infarction , peripheral vascular disease , smoking , dyslipidemia , and heart failure . at hospitals with staff that were accurate or that underestimated their performance ,
higher percentages of patients were on antiplatelet medications , cholesterol - lowering medications , and anticoagulation medications prior to admission .
sites with overestimated dtn performance tended to have lower volumes of tpa administration during the performance evaluation period ( median 10.0 [ 25th to 75th percentile 8.0 to 15.5 ] versus median 14.5 [ 25th to 75th percentile 9.0 to 22.5 ] , p=0.022 ) , smaller percentages of eligible patients receiving tpa ( 84.7% versus 89.8% , p=0.008 ) , and smaller percentages of dtn 60 minutes ( 10.6% versus 16.6% , p=0.002 ) ( table5 ) .
other hospital characteristics , such as hospital size , joint commission primary stroke center status , academic affiliation , geographic region , and hospital location ( rural versus urban ) were not found to be associated with accuracy of performance perception .
hospital characteristics of overestimation versus no overestimation 25th to 75th p indicates 25th to 75th percentile ; dtn , door - to - needle ; tjc , the joint commission ; tpa , tissue plasminogen activator . in a multivariable analysis of factors associated with relative dtn performance overestimation ,
sites were less likely to overestimate their performance for every 10-case increase in annual tpa volume ( odds ratio 0.31 , 95% ci 0.10 to 0.94 ) and for every 10-percentage - point increase in the percentage of dtn times within 60 minutes of arrival ( odds ratio 0.59 , 95% ci 0.36 to 0.96 ) .
multivariable model of overestimation dtn indicates door - to - needle ; or , odds ratio ; tjc , the joint commission ; tpa , tissue plasminogen activator .
our study demonstrates that staff perception of their own hospital s performance in terms of timeliness of tpa administration in acute stroke care is generally more optimistic .
less than one - third of identified hospital - based stroke - care quality leaders could accurately quantify their institution s rate of dtn 60 minutes .
furthermore , hospital personnel tended to overestimate their dtn performance relative to peers . among low - performing hospitals ,
85% overestimated their institution s performance of dtn time , with almost 5% of low performers believing their dtn performance was superior on a national level .
sites with higher annual volumes of tpa administration and higher percentages of dtn times within 60 minutes were less likely to overestimate their relative dtn performance .
we found that institutions often did not recognize their underperformance relative to other sites , and that is consistent with prior studies .
a survey of us hospital board chairs found that two - thirds of respondents rated their institution s joint commission or hospital quality alliance performance as better or much better than that of other hospitals . among low - performing hospitals ,
58% reported their performance to be better or much better than the national average ; none of these hospitals reported their performance to be worse or much worse than their us peers.10 a swedish survey by pukk et al found similar results ; respondents tended to rate their institution as equal to or better than others.12 nonetheless , previous studies have noted that the impetus to change is 1 of the top 5 elements critical to the successful and sustainable transformation of patient care.16 consequently , addressing misperceptions that one s performance is average or above par when it actually is not is an important step in addressing this motivation for change .
in addition , previous studies have demonstrated that having leadership and a culture of continuous quality improvement , as well as a financial commitment to quality , are highly associated with hospital improvement in the care of patients with acute coronary syndromes.17 providing service - line directors , nursing analytic tools , and comparative data further assist in perpetuating interest in continuous quality improvement.18 a study of hospital board chairs showed that chairs of top - performing hospitals were more likely to report being familiar with quality measures and to regularly review performance reports.10 prior systematic reviews have also demonstrated the impact of audit and feedback on health care professionals compliance with desired practice and shown that feedback is more effective when baseline performance is low.19 consequently , it appears that with access to comparative data , respondents are more realistic about their performance , whereas in instances in which no or little data were available , respondents tended to overestimate their own performance.12,20 in our study , overall stroke volume and tpa volume were related to performance of dtn time , and top performers were more accurate in their perceptions of performance , suggesting that they may track this information and that these metrics are important .
in contrast , lower performers overestimated their institution s performance of tpa use relative to peers .
this gap between performance and perception could be the result of better communication of internal and relative performance at high - performing hospitals ; this communication may increase awareness and engage all stroke - care staff in quality improvement .
our study provides further insights into the challenges of feedback and provider self - reflection .
critical to the success and interest in gwtg - stroke as a voluntary quality - improvement initiative is the ability for hospitals to submit their institutional stroke care data and to have this information benchmarked against similar peers .
the basis for the program is to provide hospital physicians and leadership with the ability to assess their quality - improvement efforts relative to secular and policy trends that influence all hospitals care of stroke patients .
all institutions in our study were able to download routine site - specific performance data , yet it is unclear how individual hospitals used this information and shared metrics among personnel in stroke care for assessment of quality gaps and case review .
the striking discordance among all hospitals and the care provided suggests that opportunities exist to focus communication of individual performance and relative performance across personnel involved in stroke care . even though our survey efforts selected a single member of a larger team or a person with limited experience , training or exposure to these data and the fragmentation in the understanding of relative performance
further studies are needed to evaluate practices used by stroke teams to stay abreast of their performance on a national scale and will help implementation of targeted interventions to address the misperceptions identified through our research .
participation in the gwtg - stroke registry is voluntary ; therefore , despite participation from > 1000 hospitals across the united states , this study could select centers with greater interest in stroke - care quality improvement . consequently
, the accuracy of perception of quantitative performance may not be applicable to non gwtg - stroke hospitals .
second , as with all surveys , the data presented are dependent on the subjective interpretation and biases of interview participants .
furthermore , only 1 representative was interviewed from each institution ; obtaining perceptions from a larger number of team members from each hospital may have produced different findings .
third , although our response rate was quite high among those we were able to successfully contact ( 141 of 157 ; 89% ) , we failed to find correct contact information for many sites that were originally selected .
finally , although hospitals were identified based on their performance in 20092010 , interviews were conducted between january 2011 and july 2011 , and that may have created some historical bias .
quality - improvement initiatives implemented during this time lag may have affected both measured and perceived performance .
participation in the gwtg - stroke registry is voluntary ; therefore , despite participation from > 1000 hospitals across the united states , this study could select centers with greater interest in stroke - care quality improvement .
consequently , the accuracy of perception of quantitative performance may not be applicable to non gwtg - stroke hospitals .
second , as with all surveys , the data presented are dependent on the subjective interpretation and biases of interview participants .
furthermore , only 1 representative was interviewed from each institution ; obtaining perceptions from a larger number of team members from each hospital may have produced different findings .
third , although our response rate was quite high among those we were able to successfully contact ( 141 of 157 ; 89% ) , we failed to find correct contact information for many sites that were originally selected .
finally , although hospitals were identified based on their performance in 20092010 , interviews were conducted between january 2011 and july 2011 , and that may have created some historical bias .
quality - improvement initiatives implemented during this time lag may have affected both measured and perceived performance .
personnel at low - performing hospitals had a tendency to overestimate their performance in timely tpa administration in stroke care relative to other sites participating in gwtg - stroke .
such overestimation may have important implications for the low motivation to investigate improvement barriers and to implement site - specific quality - improvement initiatives .
potential changes in benchmarked performance feedback reports should be considered to ensure broader distribution of improvement initiatives .
this project was supported by cooperative agreement number u18hs016964 from the agency for healthcare research and quality .
the content is solely the responsibility of the authors and does not necessarily represent the official views of the agency for healthcare research and quality .
dr lin , dr olson , dr britz , and mr constable have no relevant disclosures to report . ms cox is an employee of the duke clinical research institute , which serves as the aha gwtg data coordinating center .
dr fonarow receives research support from the national institutes of health ( significant ) , and is an employee of the university of california , which holds a patent on retriever devices for stroke ( significant ) .
dr schwamm serves as chair of the aha gwtg stroke clinical workgroup ; serves as a consultant to the massachusetts department of public health and serves on the steering committee for lundbeck s dias4 clinical trial .
he is pi of an ninds - funded trial of extended window thrombolysis for which genentech provides alteplase free of charge and some supplemental site payments .
dr peterson has received research grants from lilly , johnson & johnson , and bristol - myers squibb , sanofi - aventis , and merck - schering plough partnership .
dr peterson serves as principal investigator ( pi ) of the data analytic center for american heart association s get with the guidelines and the pi for the certs program , which funded this study . dr shah reports being a consultant for castlight health , llc .
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backgroundtimely thrombolytic therapy can improve stroke outcomes .
nevertheless , the ability of us hospitals to meet guidelines for intravenous tissue plasminogen activator ( tpa ) remains suboptimal .
what is unclear is whether hospitals accurately perceive their rate of tpa door - to - needle ( dtn ) time within 60 minutes and how dtn rates compare across different hospitals.methods and resultsdtn performance was defined by the percentage of treated patients who received tpa within 60 minutes of arrival .
telephone surveys were obtained from staff at 141 get with the guidelines hospitals , representing top , middle , and lowdtn performance . less than one - third ( 29.1% ) of staff accurately identified their dtn performance . among middle- and low - performing hospitals
( n=92 ) , 56 sites ( 60.9% ) overestimated their performance ; 42% of middle performers and 85% of low performers overestimated their performance .
sites that overestimated tended to have lower annual volumes of tpa administration ( median 8.4 patients [ 25th to 75th percentile 5.9 to 11.8 ] versus 10.2 patients [ 25th to 75th percentile 8.2 to 17.3 ] , p=0.047 ) , smaller percentages of eligible patients receiving tpa ( 84.7% versus 89.8% , p=0.008 ) , and smaller percentages of dtn 60 minutes among treated patients ( 10.6% versus 16.6% , p=0.002).conclusionshospitals often overestimate their ability to deliver timely tpa to treated patients .
our findings indicate the need to routinely provide comparative provider performance rates as a key step to improving the quality of acute stroke care .
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breast cancer is the major form of female cancer that is responsible for 548,000 deaths or 7% of all cancer deaths in women [ 1 , 2 ] . in malaysia ,
breast cancer is the most common form of cancer in all age groups of women and infiltrating ductal carcinoma ( idc ) is the most common form of breast cancer that made up ~85% of all breast cancer cases . over these years
, progress has been made on the study of molecular pathways , including the roles of hormones and receptors in cancer development and progression . however , the exact roles these molecules play in breast cancer growth remain inconclusive and poorly understood [ 5 , 6 ] . therefore , identifying the differentially expressed proteins in breast cancer would be useful in understanding how the disease forms and advances .
identification of these proteins would also enable treatments that are specific to breast cancer to be formulated .
proteomics is a large - scale study of the proteome , which is the entire compliment of expression of proteins by a living organism .
proteomics uses tools such as two - dimensional polyacrylamide gel electrophoresis ( 2d - page ) and liquid chromatography / mass spectrometry ( lc / ms ) for protein separation and analysis , respectively .
this approach has been used to identify protein biomarkers in breast cancers [ 913 ] and other types of cancer [ 1417 ] .
identification of protein biomarkers may have important prognostic values in the diagnosis and treatment of the disease [ 18 , 19 ] .
the classification and prognosis of breast cancer are based on stage and grade of the tumour [ 20 , 21 ] .
both the information for grade and stage of the cancer are used to plan treatment for the patient . in this study
, we aim to identify the differentially expressed aqueous soluble proteins between cancerous and normal idc breast tissues and to relate the expression of these proteins to the grade and stage of idc .
breast cancer patients were diagnosed with idc and had undergone surgical treatment at penang general hospital , penang , malaysia .
the patients were divided by stage and grade into 4 cohorts ; stages ii , iii and grades ii , iii , respectively .
stage ii , iii cohorts contained 7 patients and 10 patients , respectively while grade ii and iii cohorts contained 7 patients and 9 patients , respectively .
the tissues were confirmed as cancerous and normal , respectively , by the hospital 's pathologist . the information on patients ' age , stage , tnm , grade , estrogen receptor , progesterone receptor , and c - erb - b2 oncoprotein status is listed in table 1 .
human ethical clearance from the human ethical clearance committee of universiti sains malaysia and the ministry of health , malaysia was received prior to conducting the study .
normal and cancerous breast tissues samples from idc patients were obtained from the penang general hospital , penang , malaysia .
frozen section of tissue morphology was taken from the cancerous tissues from the anterior and deep region to ensure tumour adequacy and only the part of the cancerous tissue that had greater than 90% malignant cells was used in this study .
frozen tissues were thawed at room temperature ( 25c ) , rinsed with distilled water and homogenized on ice for 5 min .
proteins were extracted by addition of tris buffer ( tris ) ( ( 40 mm tris , 1 mm 4-(2-aminoethyl ) benzenesulfonyl fluoride ( aebsf ) ) to the homogenized tissue .
after centrifugation ( 13000 rpm , 20 min , 20c ) , the supernatant was collected and the protein concentration was determined in duplicates by rc - dc protein assay ( bio - rad , usa ) . prior to isoelectric focusing , tris protein extracts were first precipitated by using trichloroacetic acid ( tca)/acetone method .
ten percent tca in ice - cold acetone containing 20 mm dithiothreitol ( dtt ) was added to the tris extracts and incubated at 20c for 1.5 h. after centrifugation ( 13000 rpm , 15 min , 4c ) , the pellet was washed with ice - cold acetone containing 20 mm dtt before being centrifuged ( 13000 rpm , 15 min , 4c ) .
the supernatant was discarded and the pellet was resolubilized in thiourea lysis buffer ( tlb ) ( 8 m urea , 2 m thiourea , 4% ( w / v ) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate ( chaps ) , 0.4% ( w / v ) carrier ampholyte , 50 mm dtt , 1 mm aebsf ) and incubated for 1 h at 25c .
a protein extract containing 250 mg protein was passively rehydrated unto an 11 cm ph 4 - 7 ipg strips ( bio - rad , usa ) for 15 h at 20c .
isoelectric focusing ( ief ) was performed using the protean ief cell ( bio - rad , usa ) at 20c for 15 min at 250 v , 2.5 h at 8000 v and held at 8000 v for 30 kvh .
the proteins were equilibrated by adding in equilibration buffer i ( 6 m urea , 0.375 m tris - hcl , ph 8.8 , 2% sds , 20% glycerol , 1% ( w / v ) dtt ) to the focused ipg strip for 10 min at 25c , followed by the addition of equilibration buffer ii ( 6 m urea , 0.375 m tris - hcl , ph 8.8 , 2% sds , 20% glycerol , 2.5% ( w / v ) iodoacetamide ) for 10 min at 25c .
the ipg strip was positioned on top of a 10% sodium dodecyl sulfate - polyacrylamide gel ( sds - page ) ( 135 160 1 mm ) and electrophorised in a protean ii xi cell ( bio - rad , usa ) at a constant voltage of 200 v for 3 hours according to the method of laemmli .
after electrophoresis , the gels were stained using coomassie brilliant blue 250 ( cbr-250 ) solution ( 0.1% ( w / v ) cbr-250 , 40% ( v / v ) methanol , 10% ( v / v ) acetic acid ) for 4 h. the staining background was removed by incubating the gel in a destaining solution ( 40% ( v / v ) methanol , 2% ( v / v ) acetic acid ) twice for 2 h each .
the images of 2d - page gels were captured by using versadoc system ( bio - rad , usa ) .
the gel images were processed and analyzed by using pdquest software 7.3 ( bio - rad , usa ) .
the software created a match set to compare the images of cancerous and normal breast tissues , which was then used to analyze quantitative and qualitative differences in protein spots between the images .
each protein spot was normalized by dividing the spot intensity with the total intensity value of all the pixels in the gel image in order to quantitate the spot and to correct slight variations in protein loading .
a protein was considered as up - regulated if its expression level in cancerous tissues was increased 1.5-fold or more as compared to normal breast tissue : it was considered as down - regulated if its expression level in cancerous tissues was decreased 1.5-fold or more as compared to the normal breast tissue .
the statistical significance of the protein 's differential expression was determined by using the wilcoxon signed - rank test available in the pdquest software . in gel digestion
they were washed with deionized water , cut in fine pieces , hydrated and dehydrated with 100 mm ammonium bicarbonate ( nh4hco3 ) and acetonitrile ( acn ) , respectively , in order to remove the stain material .
the protein was reduced in situ with dtt and alkylated with iodoacetamide and finally digested into peptides by treating it with trypsin .
the tryptic peptides were eluted from the gel and dried under the continuous flow of nitrogen gas .
peptides were reconstituted in 30 l of 0.1% ( v / v ) formic acid in a solution of deionized water : acetonitrile ( 85 : 15 ) and were fractionated by rp - hplc ( c18 , 150 0.3 mm , 5 m 300 ) using an agilent 1100 series .
the mobile phases a ( 0.1% formic acid in deionized water ) and b ( 0.1% formic acid in acn ) are pumped at a constant flow rate of 4 l / min .
the peptides were eluted by a linear gradient of 5% b to 95% b in 70 min and held constant at 95% b for 5 min .
the hplc was interfaced to an esi - ion trap mass analyzer ( agilent ) .
two types of scan were performed : full scan ms and full scan ms / ms , where the two most intense ions in an ms scan that exceeded the set threshold of 5000 counts will be isolated for ms / ms scan to produce a series of product ions spectrum for protein identification .
the instrumental parameters used were nebulizer pressure at 20.0 psi , auxiliary dry gas flow of 6.0 l / min , auxiliary dry gas temperature at 300c , capillary voltage at 3.5 kv , exit capillary voltage 84.5 v , skimmer 1 voltage at 17.2 v , skimmer 2 voltage at 6.0 v. ms scan region from 2001800 m / z with a scan time 1 s and a interscan time of 0.1 s. the ms / ms scan parameters were default collision energy ( voltage ) of 1.15 v , charge state of 2 , minimum threshold of 5000 counts , and isolation width of 2 m / z .
protein identification was done by submitting the ms / ms data to a mascot database search engine at http://www.matrixscience.com/. the search parameters used were homo sapiens for taxonomy , carboxymethyl for fixed modifications , peptide tolerance of 2 da , ms / ms tolerance of 0.8 da , and average experimental mass value .
the gel was incubated in a cold transfer buffer ( 25 mm tris , 192 mm glycine and 1.3 mm sds , ph 8.3 ) for 30 min .
the proteins were transferred from the gel to a nitrocellulose membrane ( bio - rad , usa ) in a te 70 semiphor semidry transfer unit ( hoefer scientific , germany ) at 134 ma for 1.5 h. the membrane was incubated with blocking buffer ( 3% ( w / v ) bovine serum albumin ( bsa ) in phosphate buffered saline ( pbs ) ) for 2 h at 25c . after washing with pbs ,
the membrane was incubated in 20 ml of mouse antiannexin v antibody ( abnova , taiwan ) with a 1 : 2000 dilution in antibody diluent buffer ( 0.1% ( w / v ) bsa , 0.1% tween 20 , 0.02% sodium azide in pbs ) overnight at 25c .
after pbs washing , the membrane was incubated in 50 ml of horseradish peroxidase ( hrp ) conjugated anti - mouse secondary antibody ( bio - rad , usa ) at 1 : 3000 dilution for 2 h at 25c . after being washed with pbs , 20 ml of 4-chloro naphthol ( 4cn ) , substrate solution ( bio - rad , usa )
in this study , similar amount of protein and identical separation and staining conditions were applied to all the samples .
although the pattern of the 2d images for cancerous and normal breast tissues was relatively similar , the intensities of the spots varied between the tissues types .
figure 1 shows examples of 2d images of cancerous and normal tissues obtained from the same patient .
the difference in spots intensities indicates the differentially expressed proteins between the cancerous and normal tissues , these spots were excised and subjected to mass spectrometry analysis for protein identification .
a total of 24 differentially expressed protein spots were subjected to further analysis and the protein identities were listed in table 2 .
the grand average of hydropathy ( gravy ) scores indicate the polarity of the proteins , the more negative the score represents the higher hydrophilicity of the protein .
figure 3 shows the ms and ms / ms spectra of one of the proteins . after being subjected to mascot protein database search
, the peptide was found to belong to annexin v ; the identity of the protein was further confirmed by western blotting experiment shown in figure 4 . out of the 24 proteins , four proteins were up - regulated while one protein was down - regulated in > 75% of patients tested .
the up - regulated proteins was sec13-like 1 ( isoform b ) , calreticulin , 14 - 3 - 3 protein zeta and 14 - 3 - 3 protein eta .
the wilcoxon signed - rank test was used to determine the statistical significance of the changes in protein expression levels between the two tissues types .
the differential expression of these proteins were statistically significant with a 95% confidence level ( p < .05 ) .
the expression levels of the rest of the proteins identified were inconsistent between patients , where their expression levels range from 6% to 67% variation .
figure 5 shows the expression levels of the 24 proteins in all the patients ; the expression levels were categorized as upregulation , equal expression , downregulation or nonexpression in the patients tested .
nine up - regulated proteins and one down - regulated proteins were detected at a significant level ( p < .05 ) in > 70% of the stage ii patients ( n = 7 ) . the proteins that up - regulated were superoxide dismutase , peroxiredoxin-2 , serum albumin precursor , class iv beta tubulin , sec13-like 1 ( isoform b ) , calreticulin , unidentified protein , 14 - 3 - 3 protein zeta and 14 - 3 - 3 protein eta while fibrinogen beta chain precursor was significantly down - regulated .
haptoglobin precursor which was down - regulated in > 70% stage ii patients was not statistically significant .
in contrary , hypothetical protein ( protein spot 16 ) was down - regulated in < 70% of stage ii patients , but the change in its expression level was statistically significant .
figure 6 shows the percentage of upregulation , equal expression , downregulation and nonexpression of the proteins in stage ii patients .
four proteins were up - regulated and one protein was down - regulated significantly ( p > .05 ) in > 70% of the stage iii patients ( n = 10 ) tested .
these up - regulated proteins were sec13-like 1 ( isoform b ) , calreticulin , 14 - 3 - 3 protein zeta and 14 - 3 - 3 protein eta , while the down - regulated protein was fibrinogen beta chain precursor .
glutathione transferase was up - regulated in > 70% of stage iii patients but the change in its expression level was not statistically significant .
on the other hand , superoxide dismutase , serum albumin precursor , class iv beta tubulin , and f - actin capping protein were up - regulated in < 70% of stage iii patients , however , their upregulations were statistically significantly in stage iii patients .
haptoglobin precursor was significantly down - regulated in < 70% of stage iii patients .
figure 7 shows the percentage of upregulation , equal expression , downregulation and nonexpression of the proteins for stage iii patients .
five proteins were significantly ( p < .05 ) up - regulated in > 70% of the grade ii patients ( n = 7 ) .
these proteins were superoxide dismutase , class iv beta tubulin , sec - like 1 ( isoform b ) , calreticulin and f - actin capping protein .
glutathione transferase , 14 - 3 - 3 protein zeta , and 14 - 3 - 3 protein eta were up - regulated in > 70% of grade ii patients but their upregulation was not statistically significant . on contrary , peroxiredoxin-2 was significantly up - regulated in < 70% of grade ii patients .
figure 8 shows the percentage of upregulation , equal expression , downregulation and nonexpression of the proteins in grade ii patients .
seven up - regulated proteins and one down - regulated proteins were significantly expressed in > 70% of grade iii patients ( n = 9 ) .
the up - regulated proteins were peroxiredoxin-2 , sec - like 1 ( isoform b ) , calreticulin , unidentified protein , hypothetical protein , 14 - 3 - 3 protein zeta and 14 - 3 - 3 protein eta while the down - regulated protein was fibrinogen beta chain precursor . additionally , superoxide dismutase , serum albumin precursor and class iv beta tubulin were significantly up - regulated while haptoglobin precursor was significantly down - regulated in < 70% of grade iii patients .
figure 9 shows the percentage of upregulation , equal expression , downregulation and nonexpression of the proteins in grade iii patients .
there were 12 proteins that were up - regulated significantly in at least one of the stage and grade .
figure 10 shows the upregulation of these proteins in stage ii , stage iii , grade ii , and grade iii patients .
superoxide dismutase was up - regulated at 71% in stage ii and grade ii patients and at 60% in stage iii and grade iii patients .
peroxiredoxin-2 was up - regulated at 86% in stage ii patients and at 50% , 57% and 50% in stage iii , grade ii and grade iii patients , respectively .
serum albumin precursor was up - regulated at 86% in stage ii patients and at 50% , 57% and 67% in stage iii , grade ii and grade iii patients , respectively .
glutathione transferase was up - regulated at 70% and 71% in stage iii and grade ii patients , respectively .
its upregulation in stage ii and grade iii patients were 57% and 67% , respectively .
class iv beta - tubulin was up - regulated at 71% in both stage ii and grade ii patients and at 50% and 67% in stage iii and grade iii patients , respectively .
sec13-like 1 was up - regulated at 71% , 90% , 86% and 78% in stage ii , iii , grade ii and iii patients , respectively .
calreticulin was up - regulated at 86% , 100% , 100% and 89% in stage ii , iii , grade ii and iii patients , respectively .
the unidentified protein ( protein spot 16 ) was up - regulated at 86% and 78% of stage ii and grade iii patients , respectively and at 50% and 57% in stage iii and grade ii patients , respectively .
hypothetical protein was up - regulated at 78% in grade iii patients and at 57% , 60% and 43% in stage ii , iii and grade ii patients , respectively .
14 - 3 - 3 protein zeta was up - regulated in all stage and grade groups at 100% , 80% , 71% and 100% in stage ii , iii , grade ii and iii patients , respectively .
14 - 3 - 3 protein eta was up - regulated in 85% , 80% and 100% of stage ii , iii and grade iii patients , respectively .
f - actin capping protein was up - regulated at 85% in grade ii patients and at 57% , 60% and 56% in stage ii , iii and grade iii patients , respectively .
tissues heterogeneity is the main problem encountered when conducting proteomic analysis on tissue specimens . in this study , only the part of cancerous tissues with > 90% malignancy was used to ensure tumour adequacy .
we used the normal tissue adjacent to the tumour tissue for comparison purpose , we found that this is crucial due to tissue heterogeneity between patients , although there was a consistent protein profiles obtained from the patients , the profiles were not identical .
this can be explained as proteins are expression components of cells , and therefore they will be expressed differently under different environment which caused as variations between tissues from different patients ; therefore , the best comparison for protein expression would be between the cancerous and normal tissues from the same patients . despite this , the drawback of using such normal tissues as the control is that these tissues may possess some molecular and epigenetic abnormalities .
we overcome this problem by only collecting the normal tissues that were confirmed normal by pathologist ; a minute contamination that derived from tissue abnormalities would be excluded as the minute proteins derived from the abnormal tissues would not be detected under commassie blue staining .
in addition , we focused only on the proteins that showed consistent expression in the patients in order to minimize false identification of protein that resulted from tissue heterogeneity between patients or sample handling ( seng et al . ) .
furthermore , normalization of the gel intensity was carried out prior to the determination of the target protein spots intensities , where the intensity of each protein spot was calculated as the percentage to the total intensity of all the spots on the same image .
this approach was taken to reduce the variation in protein spot intensity that may be caused by variability in sample loading .
the main limitation of this study is the number of tissues used , nevertheless , potential biomarkers with consistent and significant upregulation in patients were identified that worth further investigation with a greater number of tissues . since proteins are the functional components of cells that regulate cell 's activity , it would be interesting to relate the expression of the differentially expressed proteins with the various stages and grades of tumour .
we have identified a few proteins that can be strongly and significantly related with certain stages and grades of idc .
14 - 3 - 3 protein zeta was found up - regulated significantly ( p < .05 ) in all ( 100% ) the patients with grade iii cancer and also in all the patients with stage ii cancer while 14 - 3 - 3 protein eta was detected up - regulated significantly in all ( 100% ) the grade iii patients tested .
calreticulin was found to be a good biomarker for all the grades and stages tested in this study , where its expression levels were 100% in grade ii patients and also stage iii patients , respectively , and as for grade iii patients and stage ii patients , its expression levels were greater than 80% in both the two cohorts .
other proteins with > 80% expression levels in grade ii patients were f - actin capping protein and sec13-like 1 protein . in stage iii patients , 14 - 3 - 3 protein eta , 14 - 3 - 3 protein zeta and sec13-like 1 were expressed at 80% .
14 - 3 - 3 protein eta , an unidentified protein ( 60 kda ) , calreticulin , serum albumin precursor and peroxiredoxin-2 were expressed at > 80% in stage ii patients . in general , we have identified four proteins that can be strongly and significantly related to at least one of the stages or grades tested in this study , these proteins were sec13-like 1 , calreticulin , 14 - 3 - 3 protein zeta and 14 - 3 - 3 protein eta .
there was an interesting trend of expressions shown by these four proteins , whereby both the 14 - 3 - 3 protein eta and zeta were shown to be strongly related with grade iii patients and stage ii patients indicating that the proteins may be associated with the aggressiveness of the tumour before the tumour spread .
on the other hand , calreticulin and sec13-like 1 were related more strongly with grade ii patients and stage iii patients indicating that as the tumour has spread to lymph nodes , the expression of these proteins increases . besides stage and grade , the expression levels of a few receptors such as progesterone
, estrogen and c - erb - b2 and the combinations of the three receptors as triple positive and triple negative grading were also used in the prediction of the prognosis of the disease . in this study
, we found that within the stages and grades tested , the expressions of the four proteins were not correlated with the expressions of these receptors ( see supplementary material available online at doi : 10.1155/2010/516469 ) .
calreticulin is a 48 kda multifunctional calcium ion - binding protein that regulates cellular activities in the endoplasmic reticulum ( er ) .
it binds to misfolded proteins to prevent them from being transported to the golgi apparatus .
calreticulin has been reported to be highly expressed in human breast ductal carcinoma cells compared to normal breast cells [ 2830 ] .
14 - 3 - 3 eta protein ( ywah ) and 14 - 3 - 3 zeta protein ( ywaz ) are subunits of the 14 - 3 - 3 isoform proteins ( 14 - 3 - 3 ) .
these proteins bind to proteins which are involved in the signaling pathway , including kinases and transmembrane receptors .
14 - 3 - 3 interact with signaling proteins to regulate a wide variety of cellular events [ 3135 ] .
14 - 3 - 3 regulate cancer - related processes , where they inhibit apoptosis , act as cell cycle checkpoints [ 36 , 37 ] and control oncogene products that are overexpressed in cancer cells .
14 - 3 - 3 were up - regulated in breast ductal carcinoma tumour cells .
protein sec13 homolog ( sec13 ) belongs to the sec13 family of wd - repeat proteins .
sec13 is required in the production of vesicles from the er to the golgi apparatus for the transport of proteins .
serum albumin precursor is used for the synthesis of human serum albumin ( hsa ) , an endogenous plasma protein and it is the main component of the blood transport system .
hsa transports and binds with water , hormones , fatty acids , metal ions and drugs .
it also maintains blood osmotic pressure and ph besides acting as antioxidant in removing reactive oxygen species in the blood plasma .
nevertheless , hsa levels and characteristics in cancer cells might have been modified from normal hsa .
the albumin portion of hsa exerts a growth inhibition in breast cancer cells that may affect cell proliferation by modulating the activities of growth factors .
hsa was a potent stimulator of estrone sulphatase activity and therefore contributes directly to the high estrogen concentrations found in breast cancer .
in addition to breast cancer , hsa was found accumulated in tumours in ovarian cancer .
the major limitation of this study is its sample size , nevertheless , the proteins identified were found consistently up - regulated in the patients tested and their upregulations were significant statistically ( p < .05 ) indicating their possible roles in the development of the disease .
the expressions levels of sec13-like 1 ( isoform b ) , calreticulin , 14 - 3 - 3 protein zeta , and 14 - 3 - 3 protein eta were found significantly related to certain stages or grades of the idc breast tumour tissues . in view of their consistent ( 100% )
upregulated expression in specific stage or grade of idc breast tumour , their potential use as biomarkers for diagnosis , prognosis , or treatment of idc breast cancer is undeniable .
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breast cancer is a leading cause of female deaths worldwide . in malaysia
, it is the most common form of female cancer while infiltrating ductal carcinoma ( idc ) is the most common form of breast cancer .
a proteomic approach was used to identify changes in the protein profile of breast cancerous and normal tissues .
the patients were divided into different cohorts according to tumour stage and grade .
we identified twenty - four differentially expressed hydrophilic proteins .
a few proteins were found significantly related to various stages and grades of idc , amongst which were sec13-like 1 ( isoform b ) , calreticulin , 14 - 3 - 3 protein zeta , and 14 - 3 - 3 protein eta . in this study , we found that by defining the expression of the proteins according to stages and grades of idc , a significant relationship between the expression of the proteins with the stage or grade of idc can be established , which increases the usefulness of these proteins as biomarkers for idc .
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it is a real ileal diverticulum usually located on the anti - mesenteric edge in the last meter of the small bowel .
the location on the ileal mesenteric edge has been rarely reported and remains controversial [ 1 , 2 ] .
indeed , it raises the question about the differential diagnosis with bowel duplication which is still not elucidated .
we report a case of perforated meckel 's diverticulum that was located on the mesenteric edge of the ileum .
a 45-year - old patient admitted to the emergency unit was complaining of abdominal pain lasting for 3 days .
initially , the pain was located in the epigastric region , then secondarily generalized to the whole abdomen .
physical examination revealed a good general condition , a temperature of 38.8 c , a pulse rate of 120 heart beats / min , a blood pressure of 100/90 mmhg and a respiratory frequency of 28 cycles / min .
plain abdominal radiography showed air - fluid levels and was suggestive of abdominal effusion ( fig . 1 ) .
on biology , blood cell count was 12 400 cells / mm ( normal range : 400010 000 ) and creatinine was 16.8 mg / l ( normal range : 612 ) .
a generalized peritonitis was then suspected , and a laparotomy was performed . on exploration , we found 400 ml of seropurulent peritoneal fluid and a perforated meckel 's diverticulum of 3.5 cm in size , which was located 60 cm from the ileocecal junction on the mesenteric edge of the ileum ( fig . 2 ) .
the patient underwent a removal of the diverticulum by a segmental resection of the ileum with an end - to - end anastomosis .
the postoperative course was marked by persistent peritonitis , which motivated a revision surgery that involved a peritoneal lavage and drainage .
it results from the incomplete regression of the omphalomesenteric duct that connects the primitive intestine to the yolk sac during the embryonic period [ 1 , 3 ] .
the location of this diverticulum on the anti - mesenteric ileal edge has been for a long recognized as diagnostic criterion [ 1 , 2 , 4 ] . however ,
some cases of location on the mesenteric ileal edge have been reported since 1941 and so were suggestive of possible ileal duplication [ 1 , 2 , 4 ] .
meckel 's diverticulum with mesenteric location can be subserosal or intra - mesenteric . in our patient
, it was a subserous diverticulum . while in intra - mesenteric variety , transillumination of the mesentery
the persistence of a short vitelline artery is thought to be implicated in the pathophysiology of mesenteric diverticula [ 2 , 5 ] .
indeed , this artery yolk could in part explain the diverticular meso , which , in case of rapid growth , causes a pull on the diverticulum and then a migration of its base to the mesenteric edge [ 2 , 5 ] . the main differential diagnosis in these cases is the ileal duplication where vascularization is dependent on the ileal vascularization , while the mesenteric diverticulum 's vascularization is most often provided by a vitelline artery [ 1 , 2 ] .
however , most authors do not consider vascularization as a differential diagnosis tool because the vitelline artery presents in only 10% of cases [ 1 , 2 ] .
this hypothesis is supported by kurzbart et al . , who reported a case of omphalomesenteric duct persistence detected in the neonatal period that fell to the age of 3 months and became a meckel 's diverticulum without mesenteric diverticular meso .
in our patient , as well as in buke 's one , pathological examination of the surgical specimen did not show meso or diverticular sac artery .
, meanwhile , found a vitelline artery on ct scan . according to buke et al .
, a complete regression of the vitelline artery or a clot in utero between the mesentery and the omphalomesenteric duct might also explain this anatomical variant .
perforation of meckel 's diverticulum is a main cause of right lower quadrant pain as well as appendicitis .
if the systematic resection of incidental meckel diverticula is not unanimous , resection of complicated diverticula is widely agreed . according to park et al .
, a diverticulectomy can be achieved if there is no mass suggestive of heterotopic tissue . however , given that the risk of degeneration of these tissues that may go unnoticed , segmental ileal resection seems to be the best option to adopt .
it may be followed by an ileal anastomosis when local conditions allow it . in our patient , it was realized a segmental ileal resection followed by an end - to - end anastomosis .
the postoperative course was marked by a persistent peritonitis probably due to a peritoneal toilet failure . in case of massive contamination of the peritoneal cavity ,
segmental ileal resection seems to be the ideal therapeutic attitude given the possible presence of heterotopic tissues . in case of perforation , resection and ileostomy
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a meckel 's diverticulum is the most common congenital anomaly of the gastrointestinal tract .
it is a real diverticulum that is usually located on the anti - mesenteric edge in the last meter of the ileum .
its location on the mesenteric edge has been rarely reported .
it may lead to several complications including perforation that may be life - threatening for the patient .
we report herein a case of perforated mesenteric meckel 's diverticulum in an adult patient . upon surgical exploration by laparotomy
, we found a perforated meckel 's diverticulum located on the mesenteric edge of the ileum 60 cm from the ileocoecal junction and 400 ml of seropurulent peritoneal fluid .
the patient underwent a segmental ileal resection and an end - to - end anastomosis .
the postoperative outcomes were marked by a persistent peritonitis that required successful revision surgery .
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laparoscopic inguinal herniorrhaphy has become widely accepted as an effective alternative to the treatment of inguinal hernias compared with the anterior approach owing to its minimal invasiveness and its ability to achieve identical success and quicken the recovery by decreasing time to return to work or physical activities . in australia ,
the rate of laparoscopic inguinal herniorrhaphy , in 2012 , was 48% of the total numbers of inguinal hernias being repaired . in australia , from 2010 to 2011 , there were 46,651 hospital separations for inguinal hernia and 3,711 ( 7.9% ) were for those specified as recurrent , although these figures do not differentiate between the rates of recurrence for each type of repair .
it is generally accepted that the best repair for a recurrent inguinal hernia after the laparoscopic repair is the anterior repair , whereas that after an anterior repair is the laparoscopic repair . however , there is currently no consensus as to the best repair for multiply recurrent hernias after a failed anterior and laparoscopic repair .
part of the reason for this is that not all surgeons performing laparoscopic inguinal hernia repair perform laparoscopic ventral hernia repair and vice versa .
consequently , there are experts in laparoscopic inguinal hernia repair who have successfully attempted relaparoscopic repair , but this practice is confined to very few surgeons in specialized hernia centers . on the other hand , surgeons who are confident with laparoscopic ventral hernia repair and total extraperitoneal ( tep ) or transabdominal preperitoneal ( tapp ) repair might consider the intraperitoneal onlay mesh ( ipom ) repair as merely an extension of laparoscopic ventral hernia repair , although detailed knowledge of the laparoscopic extraperitoneal inguinal anatomy would be essential . in an attempt to further reduce parietal trauma , single - incision laparoscopic surgery ( sils )
has been touted as the most important innovation in laparoscopic surgery since the latter 's popularization with the first laparoscopic cholecystectomy in 1988 . indeed , since the first commercial availability of the sils port in 2007 , multiple different single ports have been made available .
multiple prospective randomized controlled trials , mainly for cholecystectomy and appendectomy , mostly with small numbers of patients , but more significantly during the learning curve , comparing single - port and multiport surgery , have shown consistent safety and effectiveness of the single - port approach .
similarly , single - port laparoscopic inguinal herniorrhaphy has been shown to be safe and effective .
however , data regarding the superiority the single - port ( over conventional multiport ) surgery , other than cosmesis , are still lacking , although it is hoped that with increasing experience with sils , more high - powered randomized controlled trials will provide us with a clearer picture of the place of sils .
our unit has been offering routine laparoscopic herniorrhaphy for inguinal hernias since 1991 and ventral hernias since 2003 . since december 2009
, we have routinely treated virtually all ventral ( including parastomal ) and inguinal hernias with the single - port approach .
the treatment of multiply recurrent inguinal hernias after conventional anterior and laparoscopic approaches with laparoscopic ipom repair represents an obvious choice .
in addition , parietal trauma could now be reduced with single - port compared with multiport surgery . to our knowledge , this is the first case series of sil - ipom repair for the treatment of recurrent inguinal hernias after both failed conventional anterior and failed laparoscopic repairs with mesh .
between november 2009 and october 2013 , all patients referred with inguinal / femoral hernias underwent sil inguinal herniorrhaphy . for this study ,
inclusion criteria include patients presenting with recurrent inguinal hernias after both failed anterior and failed laparoscopic repairs with mesh .
patients were informed of our practice of performing laparoscopic ipom repair and that this could now be achieved with sils .
after induction with general anesthetic , patients were prepped and draped with iodine from epigastrium to mid thighs , and then draped with iodine - impregnated adhesive cover ( ioband ; 3 m , st paul , minnesota ) to expose the entire abdomen and both groins ( figure 1 ) .
a preoperative intravenous dose of cephalosporin was given , and the patient was routinely catheterized .
after infiltration with 20 ml of bupivacaine 0.5% with 1:200,000 ephedrine in the umbilical area , a 2- to 2.5-cm ( depending on the laxity of the skin ) crescentic infraumbilical incision was made and the anterior rectus sheath was incised transversely and the rectus sheath was retracted laterally .
this site of entry was made on the contralateral side from the previous laparoscopic entry ( if total extraperitoneal approach was used ) to avoid scar tissues .
the posterior rectus sheath and the peritoneum were then entered for placement of a sils port ( covidien , norwalk , connecticut ) .
the patient was then placed in a trendelenburg position at 10 to 15 ( figure 1 ) .
laparoscopy was performed with 52 cm/30 angled laparoscope to assess the amount of adhesions ( figure 2 ) and these were meticulously divided by sharp dissection with avoidance of electrocautery ( figure 3 ) .
chopsticks and inline , were employed to overcome the relative loss of triangulation . the pubic symphysis was identified and the peritoneum was incised 2 cm superior to this and extended laterally , or superior to a direct sac , if present ( figure 4 ) .
no attempt was made to incise through ( or remove any part of ) the previously placed ( extraperitoneal ) mesh , and the dissection was performed from the inferior aspect of this mesh and continued proximally .
care was taken to stay below the inferior epigastric vessels as the dissection continued laterally .
the peritoneum was then reflected inferiorly over the pubic symphysis and continued laterally over the spermatic cord and its structures , reducing any direct , femoral , and indirect hernia and lipoma of the cord akin to the dissection during a tapp inguinal hernia repair .
extreme care was taken to preserve retroperitoneal nerves in the vicinity ( figure 4 ) .
no attempt was made to dissect the superior flap of peritoneum overlying the previous laparoscopically placed mesh .
often the previously placed extraperitoneal mesh had folded up during placement or deflation , causing the recurrence , and consequently the inferior peritoneal flap was usually surprisingly easy to raise ( figures 3 and 4 ) .
after deflation to 8 mm hg , measurements were taken externally for the size of the mesh ( gore - tex dualmesh ; w. l. gore & associates , flagstaff , arizona ) , which was at least 5 cm longer craniocaudally to extend inferior to the pubic symphysis .
a pds 0 suture ( ethicon , somerville , new jersey ) was placed in the medial and superior corner of the mesh , and the mesh was marked 5 cm up from its inferior and medial corner to correspond to the superior edge of the symphysis pubis ( figure 5 ) .
the mesh was rolled inward along its horizontal axis , like a scroll , and placed intraperitoneally via a 12-mm trocar , which had temporarily replaced the 5-mm camera trocar .
one of the 5-mm trocars was temporarily withdrawn until it was outside of the fascial defect to facilitate insertion of the 12-mm trocar .
a stab incision was then made in the midline and inferior to the umbilicus to retrieve the pds suture in the superior and medial corner of the mesh with a suture passer .
this allowed the mesh to be more easily maneuvered into the correct position before nonabsorbable tacks ( protack ; covidien ) were placed on the pubic bone and along the pubic ramus , taking care to stay clear of the external iliac vein ( figures 2 and 5 ) .
the mesh was then tacked medially , superiorly , and cautiously laterally to avoid the nerves in the vicinity .
this was aided by the fact the mesh used was sufficiently sized in the craniocaudal dimension such that its superior edge was well above the iliohypogastric nerve ; hence , the tacks would be unlikely to be accidentally placed into it , the ilioinguinal nerve , the genital branch of the genitofemoral nerve , or the lateral cutaneous nerve of the thigh .
fibrin sealant ( 2 ml ) ( tisseel duo ; baxter ag , vienna , austria ) was sprayed along the inferior edge of the mesh ( figure 5 ) .
the inferior peritoneal flap was then reflected up and tacked lightly onto the mesh with care being taken not to leave any significant gaps that would allow herniation of the bowel loops .
fibrin sealant ( 2 ml ) was also then sprayed along the mesh - peritoneum interface , periphery of the mesh , and over the tacks to minimize the risk of adhesions ( figure 5 ) .
the fascial defect in the umbilical wound was closed in layers with interrupted pds 0 sutures and absorbable sutures subcutaneously and subcuticularly .
the urinary catheter was left in place overnight and removed before the patient was discharged home .
all patients were seen at 1 week and 4 weeks with plans to see them annually for 5 years .
setup for single - incision laparoscopic intraperitoneal onlay mesh repair for a left multiply recurrent inguinal hernia ; the extra long laparoscope was used to prevent clashing of the handles of the conventional straight dissecting instruments with the side arm of the scope .
laparoscopic findings in patients with multiply recurrent inguinal hernias after tep / tapp and a mean of 2 anterior repairs : rolled up mesh after tep repair exposing a direct inguinal defect ( a ) ; a pantaloon hernia after a tapp repair ( b ) ; a cupped intraperitoneally placed mesh exposing a suprapubic defect ( c ) ; and a direct defect after tep repair with the inferolateral aspect of mesh rolled up ( d ) .
intraoperative photos of dissection of a pantaloon hernia after previous tep and 3 anterior repairs : incarcerated loop of sigmoid colon ( a ) ; previous extraperitoneal mesh , which has been displaced upward allowing indirect and direct recurrence ( b ) ; incision of peritoneum along inferior edge of mesh , which is then extended medially and laterally ( c ) ; and complete reduction of direct and indirect defects with preservation of cord structures ( d ) .
intraoperative findings on multiply recurrent inguinal hernias : extensive bowel adhesions to the rolled up mesh , which must be meticulously divided by sharp dissection ( a , b ) and exposure of retroperitoneal nerves , which can be at risk of damage during dissection and hence no mesh fixation with tacks in these areas ( c , d ) .
single - incision laparoscopic intraperitoneal onlay mesh repair for multiply recurrent inguinal hernias : arrow on the mesh corresponding to top of pubic symphysis with mesh stapled into pubic ramus ( a ) ; stay suture placed in the midline and inferior to the umbilicus to aid orientation of the mesh ( a inset ) ; inferior edge of mesh glued with fibrin sealant ( b ) ; the inferior peritoneal fold tacked back onto the mesh ( c ) ; and fibrin sealant sprayed along mesh - peritoneum interface ( d ) .
between november 2009 and october 2013 , there were 9 patients with recurrent inguinal hernias after previous failed anterior and laparoscopic repairs with mesh with no patients excluded from the study .
this was part of a cohort of 415 patients , over the same period , who had undergone sil inguinal herniorrhaphy .
each patient had had 1 laparoscopic repair ( 5 teps and 4 tapps ) and a mean of 2 anterior repairs ( range 14 ) .
the mean age was 55 years ( range 4274 ) ; all patients were men .
the mean body mass index was 26.5 kg / m ( range 24.528.4 kg / m ) ( table 1 ) .
all patients were found to have direct hernias with 4 patients having incarcerated hernias containing small bowel / colon that needed to be reduced .
furthermore , there were always omental adhesions in the inguinal region ( even in the 5 patients who had had laparoscopic extraperitoneal approach ) that had to be divided . in all patients ,
the mesh was found to be deficient medially with the mesh either within the direct defect or that it had folded up exposing a direct defect . in 4 patients ,
two patients also had a contralateral primary hernia , which was also treated by ipom repair at the same time .
there were no mortalities , morbidities , port - site hernias , or recurrences with a mean follow - up of 20 months ( range 148 months ) .
bmi , body mass index ; ipom , intraperitoneal onlay mesh repair ; tapp , transabdominal preperitoneal ; tep , totally extraperitoneal .
since the first laparoscopic extraperitoneal inguinal hernia repair by ger et al in 1989 , there has been an exponential increase in the uptake of the laparoscopic repair for inguinal hernias worldwide .
data from medicare australia ( www.medicareaustralia.gov ) showed that the rates of laparoscopic versus conventional anterior repair were 9.4% in 1994 , 20.5% in 2000 , and 48% in 2012 . the latter figure is reflected in the same percentage of surgeons performing laparoscopic repair ( as defined by any surgeon who lodged a claim to medicare australia with the item number 30609 , which corresponded to laparoscopic inguinal hernia repair ) .
this exponential rise in the uptake of laparoscopic inguinal herniorrhaphy is remarkable given that the vast majority of the laparoscopic repairs are only performed in private hospitals where surgical trainees are not normally trained and the surgery is normally only performed by the consultants .
it is possible that many surgeons go overseas to attend hands - on animal workshops in tep / tapp repair , possibly due to the lack of such courses offered in australia due to the stringent animal ethics requirements to run such courses in australia .
it has been estimated that the recurrence rates for inguinal hernias range from 7% to 10% in australia and this means that there are increasingly more patients with multiply recurrent inguinal hernias after failed anterior and laparoscopic repairs .
the international endohernia society guidelines suggest that the best repair for the treatment of an inguinal hernia recurrence after an anterior approach is the laparoscopic approach and vice versa . yet , there are no specific recommendations for the treatment of multiply recurrent hernias after both anterior and laparoscopic approaches
. anecdotal reports of a relaparoscopic approach , usually tapp , have come from highly specialized centers but the incidence of complications are much higher even though the success rates are higher than another anterior approach .
van den heuvel and dwars reported a series of 51 patients who underwent a tapp repair for a recurrent inguinal hernia after previous posterior hernia repair . in two - thirds of the patients ,
however , there were 9 postoperative adverse events , 4 port - site hernias , and 4 with postoperative pain , which restricted daily activities .
the concept of ipom in the management of inguinal hernia is not new . in 1998 , kingsley et al . demonstrated the feasibility of inguinal hernia repair using ipom with polytetrafluoroethylene mesh ( 1015 cm ) but the recurrence rate was 43% at 41-month follow - up .
a total of 76 patients underwent tapp and 72 underwent ipom ; 10 7 cm polytetrafluoroethylene mesh was used for ipom and 15 12 cm polypropylene mesh was used for tapp .
there were no recurrences in tapp at 32 months compared with an 11.1% recurrence rate for ipom .
neuralgia was noted with 3 tapp and 11 ipom patients ( p < .05 ) . as a results of these and other studies ,
clearly , multiple factors have contributed to these poor results , including no reduction of the hernia sacs , inadequate mesh size , lack of permanent bony fixation as well as lack of tissue glue fixation of the inferior edge of the mesh .
central to the conventional laparoscopic approach is the attempt to place the mesh in the extraperitoneal position , and this means having to raise the peritoneal flaps sufficiently to cover the new mesh .
this is almost impossible , as often the peritoneum is so densely adherent to the previous mesh that one ends up with multiple defects in the peritoneal flaps .
this would then expose the normal mesh to bowel - causing adhesions with possible deleterious sequelae .
indeed , lo menzo et al showed , in a series of 6 patients with 7 recurrent inguinal hernias after laparoscopic repairs , there were 2 cases where the peritoneal flap was not able to cover the mesh and a tissue - separating mesh with fibrin sealant had to be used to cover the myopectineal orifice .
additionally , the tapp repair involves placement of a 10-mm trocar through the linea alba in the umbilical region and 2 5 mm trocars more inferiorly ( although some surgeons prefer to place these latter trocars laterally on each side of the abdomen ) , all of these trocar sites are at risk of port - site hernia formation .
an extraperitoneal approach , with the inferiorly placed 5-mm trocars , would be difficult , if not impossible , because the extraperitoneal space is likely to be obliterated from the previous laparoscopic repair , and this is the reason why most relaparoscopic repairs have been performed as a tapp procedure where the 5-mm trocars can be placed laterally on either side of the umbilical camera port . in our study , the umbilical ( and only ) port was placed via transverse incisions in the anterior and posterior rectus sheaths with retraction of the rectus muscle laterally , and this was closed with slowly absorbable monofilament sutures .
these entry and closure techniques prevented port - site hernia formation . with the introduction of the first commercial single - port device , sils port , in 2007 , there has been an exponential rise in the number and variety of sil procedures performed .
it has been estimated the learning curve for sils for an experienced laparoscopic surgeon is between 25 and 50 cases , and this means that it should not take more than a year for a general surgeon in australia , who would , on average , perform 26 inguinal hernias per annum ( www.medicareaustralia.gov ) , to be competent with sils .
while the conventional laparoscopic ventral hernia repair involves placement of a 10-mm camera port in the upper outer quadrant and 2 5 mm ports more inferiorly in the anterior axillary line , such a configuration of ports would be quite a distance from the inguinal region , causing poor ergonomics for the dissecting instruments as well as the fact that longer dissecting instruments would have to be used , which would further compromise the ergonomics of dissecting instruments .
while sils suffers from the relative lack of triangulation , this can be overcome by modifying dissection techniques , the use of a smaller and longer laparoscope , and with increased experience .
our recently completed prospective randomized controlled study comparing single - port and multiport laparoscopic totally extraperitoneal inguinal herniorrhaphy demonstrated similar safety , efficacy , and additional cosmetic and noncosmetic benefits of the single - port technique beyond the learning curve . to date
, the principal author has performed > 700 sil - tep repairs and 100 sil
ventral hernia repairs , with the latter including some of the most difficult abdominal wall hernias , namely , parastomal hernias .
therefore , it became a natural progression to treat the multiply recurrent inguinal hernias after open and laparoscopic repairs with sils , using the umbilicus as the only point of access for placement of the single port .
the sils port allows for placement of a 12-mm trocar for ease of placing an antiadhesive mesh intraperitoneally .
the sil - ipom repair follows closely the dissection of the inferior flap during the tapp repair as meticulous dissection of the inferior flap , below the inferior border of the previous extraperitoneally placed mesh , is important to reduce any direct , indirect , and femoral hernias , as well as any lipoma of the cord with preservation of the latter and its structures . because virtually all of these hernias have a direct component , fixation of the mesh into the pubic ramus with nonabsorbable tacks
is an important aspect of the repair to ensure permanent fixation to prevent any further mesh displacement or eventration into the direct defect .
however , unlike a tapp repair of a recurrent inguinal hernia , the sil - ipom does not interfere with the previously placed extraperitoneal mesh , but it aims to cover the defective inferior / medial border of the previous mesh with an antiadhesive mesh that is then extended well above the previous extraperitoneally placed mesh in an attempt to prevent accidental stapling of the relevant nerves in the groin with consequent severe postherniorrhaphy chronic pain .
the fact that multiply recurrent hernias seem to have a direct component suggests an intrinsic weakness of the myopectineal orifice .
indeed , henriksen et al found a consistent , significant increase in immature type iii collagen relative to the stronger type i collagen in patients with a hernia , and these changes were most pronounced in patients with a direct inguinal hernia than in those with an indirect inguinal hernia .
furthermore , although the inferior edge of the mesh can not be tacked to avoid damage to vital neurovascular structures , it can be fixed with fibrin sealant . additionally ,
reflection of the inferior peritoneal fold back onto the mesh and its fixation with tacks prevents any further folding of the mesh , which should minimize the risks of recurrence .
lau , in a prospective randomized study of mesh fixation with either fibrin sealant or tacks in laparoscopic inguinal hernia repair , showed that fibrin sealant reduced the incidence of postherniorrhaphic chronic pain . in our study , we used fibrin sealant to fix the inferior edge of the mesh . but additionally , we also used fibrin sealant for its antiadhesive property based on our experimental and clinical research .
indeed , since 2007 , for all our patients undergoing laparoscopic ventral hernia repair , we have sprayed fibrin sealant along the periphery of the mesh , where adhesions are likely to take place , as well as on the tacks , which are known to cause adhesions .
furthermore , the application of fibrin sealant along the mesh - peritoneum interface also reduces the risk of bowel loops herniating through undetected gaps .
one important technical aspect in our study relates to the use of the mesh , which must be big enough in the craniocaudal dimension such that the superior edge of the mesh was tacked well above the relevant nerves in the inguinal region to avoid nerve entrapment even though , theoretically , only a 5-cm overlap of the defect is normally required for a sound repair .
the single - port approach not only allowed the instruments to be inserted sufficiently far from the inguinal region , hence facilitating ease of dissection , but also permitted the mesh to be tacked more superiorly than would have been possible with the multiport approach . in addition , the inferior peritoneal flap had to be meticulously raised , with avoidance of electrocautery , to prevent accidental damage to the retroperitoneal nerves that otherwise could have caused chronic pain postoperatively . in our case series of 9 patients over 4 years , all were successfully treated with sil - ipom repair without any complication or recurrence with a mean follow - up of 20 months .
this compares favorably with the alternative tapp repair , which would not be suitable for surgeons only trained in the tep repair . on the other hand ,
successful sil - ipom repair demands the highest level of competence in laparoscopic surgery to achieve safe adhesiolysis , avoidance of inadvertent nerve damage or entrapment through detailed knowledge of laparoscopic inguinal anatomy , and the use of sufficiently large antiadhesive mesh , which must be judiciously fixed to achieve successful repair .
multiply recurrent inguinal hernias following failed conventional anterior and laparoscopic repairs can be safely and effectively treated with laparoscopic ipom repair . when combined with sils , the umbilicus can be used as the only incision site , which apart from having the potential to reduce port - site complications also allowed improved ergonomics of the dissecting instruments , albeit with modified dissection techniques , by being at optimal proximity to the inguinal regions , where bilateral repairs can be performed safely and effectively .
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introduction : despite an exponential rise in laparoscopic surgery for inguinal herniorrhaphy , overall recurrence rates have remained unchanged . therefore , an increasing number of patients present with recurrent hernias after having failed anterior and laparoscopic repairs .
this study reports our experience with single - incision laparoscopic ( sil ) intraperitoneal onlay mesh ( ipom ) repair for these hernias.materials and methods : all patients referred with multiply recurrent inguinal hernias underwent sil - ipom from november 1 2009 to october 30 2013 .
a 2.5-cm infraumbilical incision was made and a sil surgical port was placed intraperitoneally .
modified dissection techniques , namely , chopsticks and inline dissection , 5.5 mm/52 cm/30 angled laparoscope and conventional straight dissecting instruments were used .
the peritoneum was incised above the symphysis pubis and dissection continued laterally and proximally raising an inferior flap , below a previous extraperitoneal mesh , while reducing any direct / indirect / femoral / cord lipoma before placement of antiadhesive mesh that was fixed into the pubic ramus as well as superiorly with nonabsorbable tacks before fixing its inferior border with fibrin sealant . the inferior peritoneal flap was then tacked back onto the mesh.results:there were 9 male patients who underwent sil - ipom .
mean age was 55 years old and mean body mass index was 26.8 kg / m2 .
mean mesh size was 275 cm2 .
mean operation time was 125 minutes with hospital stay of 1 day and umbilical scar length of 21 mm at 4 weeks ' follow - up .
there were no intraoperative / postoperative complications , port - site hernias , chronic groin pain , or recurrence with mean follow - up of 20 months.conclusions:multiply recurrent inguinal hernias after failed conventional anterior and laparoscopic repairs can be treated safely and efficiently with sil - ipom .
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electroosmosis in a circular cylindrical pore of finite length h differs from that in an infinitely long pore due to end
effects . if the cylinder length h = 0 , then the pore
consists of a hole in a charged membrane of zero thickness , and electroosmosis
can be considered to be entirely due to end effects .
when the
cylindrical pore is infinitely long , end effects are negligible , and
the computation of the electroosmotic volumetric flow rate q , for arbitrary debye lengths and surface charge densities ,
is standard ( with similar results available for infinitely
long planar channels ) . here , we are interested in intermediate values of h. full numerical computation of the poisson nernst planck
( pnp ) equations for ionic motion is , of course , possible , and some
typical results were reported by mao et al .
such numerical computations , however , do not identify the mechanisms
underlying the qualitative features of the physical system . here ,
we discuss how simple models , based on continuity of electric current
and volumetric flow rate , can be combined in order to estimate end
effects for pore lengths h > 0 .
we assume that
the
zeta potential on the surface of the membrane is small , so that the
poisson boltzmann equation governing the equilibrium charge
cloud can be linearized , and the electroosmotic velocity can be determined
by an analysis equivalent to that of henry for electrophoresis , i.e. , fluid motion is generated by the effect
of the applied electric field acting on the equilibrium charge cloud
( which is not deformed either by the applied electric field or by
fluid motion ) . in this limit , the electroosmotic volumetric flow rate q through the hole in the membrane can be determined by
means of the reciprocal theorem .
the cylindrical pore cd has radius a and length h. the cylindrical surface
cd of the pore has surface charge density c ,
and the membrane surfaces bc and de have surface charge density m .
an electrical potential difference is applied between the
fluid reservoirs at either side of the membrane , and electroosmotic
flow is generated by the resulting electric field acting on the charge
cloud adjacent to the charged surfaces .
the analysis of mao et al . assumed that the external reservoirs on either
side of the pore were unbounded , with radius b =
. for the numerical computations presented in section 3 , the external reservoirs were bounded by uncharged
cylinders of radius b a , sufficiently large that numerical results when h = 0 differed little from the analytic results for h = 0 and b infinite .
there have been many studies
in which flow is generated in cylinders of different dimensions , connected
either in series or in networks intended
to represent porous media . here
, however ,
we are interested in the effect of the surfaces bc and de of the membrane
on electroosmotic flow within the cylindrical pore , and any boundaries ,
ab and ef , of the external reservoirs are so far away that they can
be neglected .
cylindrical pore cd , of length h and
radius a with surface charge density c ,
passing through the membrane with surface charge density m on the two surfaces bc and de .
the reservoirs on either side
of the membrane are large ( b a ) .
we shall allow the surface charge
density m on
the membrane to differ from the charge density c on the wall of the cylindrical pore .
there have been previous detailed
studies of the effect of a discontinuity in surface charge density
on electroosmosis .
the fine details of the charge
cloud and fluid motion around such a discontinuity will be lost by
the simple models presented here .
they are , of course , fully taken
into account in the numerical computations discussed in section 3 . in section 2.2 , we
set up the approximate
analysis of end effects and compare results to those obtained from
full numerical computations .
the analysis is presented from first
principles , but it can alternatively be set within the framework of
the reciprocal theorem , as explained in section 2.6 .
the agreement between the approximate analysis and full
computation is , in general , good , except for large debye lengths
a. in section 4 , we consider this case in more detail in order
to evaluate how much of the charge cloud due to the charged walls
of the cylindrical pore lies within the pore and how much spills out
beyond the ends of the pore .
when this overspill is taken into account ,
the agreement between the computations and the approximate model is
improved .
we use cylindrical polar coordinates ( r , z ) , with the z axis along the axis of symmetry
and z = 0 at the midpoint
of the cylindrical pore , the ends of which are at z = h/2 .
when h = 0 , we shall
also use oblate spherical coordinates ( , ) , with1where <
<
and 0 < /2 . the cylindrical pore
and the reservoirs at either end
are filled with liquid with electrical
conductivity and viscosity . the wall cd of the cylindrical
pore is charged , with uniform surface charge density c , and the surface charge density over the membrane surfaces ,
bc and de , is m
. the electrical permittivity s of the membrane will be typically much smaller
than the permittivity of the liquid , and we assume s = 0 . we assume that the reservoir boundaries
ab and ef are uncharged and at infinity
. we shall occasionally refer
to the surface potential , which will not , in general , be uniform ,
but which is required to be small , with kt / e , where e is the elementary charge
and kt the boltzmann temperature .
the electrical
potential 0 within the equilibrium charge cloud
therefore satisfies the linearized poisson
boltzmann equation
so that2where is the debye
length , and the charge density in the equilibrium charge cloud is3 the applied
electric field is e = , where
the potential satisfies the laplace equation4with gradient5normal to the walls of the membrane and of
the cylindrical pore . in z
> 0 , the electric potential
far from the membrane is = 1/2 , and the
potential far from the membrane in z < 0 is
= 1/2 .
when the membrane thickness h = 0 , the potential can be expressed explicitly as6on the plane of the membrane , within the circular
opening,7the liquid within the pore has
electrical
conductivity ; we have assumed that surface charge density
( and hence the density of charge in the cloud of counterions ) is small ,
so that surface conductivity may be neglected . indeed ,
if the mobilities
of the various ionic species are identical , then the surface conductivity
due to the mobile charge cloud given by the linearized model ( 2 ) at o(e/kt ) is zero .
the total electric current i m flowing through the hole in the membrane is therefore8 if h > 0 , then we assume
that the potential within
the cylindrical pore varies linearly and approximate the potential
within the pore as9as would be expected in the absence of any
end effects .
the potential in z > h/2 is approximated by that outside a membrane ( with a hole ) of zero
thickness10with (r , z ) = (r , z ) .
this approximation ( 9 ) and ( 10 ) is continuous at z = h/2 where the potential is assumed
to be 2/2 across the entire width of the opening
( by eq 7 ) .
the as yet unspecified potential
2 is
determined by requiring continuity of the electrical current at z = h/2 .
the current ic through the cylindrical pore is11and the electrical current through the reservoir
in z > h/2 is , by eq 8,12equating ic ( 11 ) and i m ( 12 ) , we find13this computation suggests that the system
can be treated as two resistors in series , with composite resistance14however , this estimate
assumes a uniform potential
over the ends of the pore at z = h/2 , and we have effectively inserted thin , perfectly conducting sheets
over the pore ends .
removal of these sheets can only increase the
resistance and hence rcomp is an underestimate
for the true total resistance rtot .
figure 2a shows rtot/(a ) computed numerically by means of the freefem++
finite element package , together with rcomp/(a ) .
( a ) nondimensional
ohmic resisistance r/(a )
of a hole of radius a in a membrane
of thickness h , as a function of h / a . solid line , rtot/(a ) computed numerically ; dashed line , the
approximation rcomp/(a ) ( 14 ) .
we assume throughout this article that the perturbation
of the equilibrium charge cloud by the applied electric field and
by fluid motion is negligibly small .
the force acting on the ions
in the charge cloud due to the applied electric field
is therefore 0. the
equilibrium potential within an infinite cylindrical pore is15 in the absence of any end effects , if the
electric field e0 = 2/h is applied along the length of the cylindrical
pore , then the fluid velocity is16and the
total electroosmotic volumetric flow
rate is17where the electroosmotic
coefficient18a18bthe total
current through the cylindrical
pore is ic ( 11 ) ,
so the ratio between volume flux and current is19a19b it was shown by mao et al . that if
the equilibrium charge density is 0 , the imposed
electric field is e =
and the fluid velocity generated by a pressure difference p1 across a pore ( of arbitrary geometry ) is20then the reciprocal
theorem for stokes flows can be used
to show that electroosmotically
generated volumetric flow rate through the pore is21where the integral is over all the fluid .
the fluid velocity generated by the pressure difference p1 across a circular hole in a membrane of zero thickness
is22an explicit expression
for g(r , z ) is available , and the potential is
given by eq 6 .
the charge density in the equilibrium
charge cloud around a membrane
of zero thickness is23which
consists of the charge density adjacent
to a uniform charged surface , from which has been subtracted the charge
density around a uniformly charged disk .
the integral ( 21 ) can be evaluated numerically , and the electroosmotic flow rate through a hole in a membrane of
zero thickness can be expressed in the form24where25with26the ratio of the electroosmotic volume flux qme to the electrical current i m is27where28with29 figure 3 shows a log log plot of
results for hm / h0 obtained by mao et al .
the continuous
line shows the analytic result ( 21 ) obtained
via the reciprocal theorem , and the asymptote ( 25 ) for a 1 is indicated .
electroosmotic
coefficient hm , scaled
by h0 ( 26 ) , for
a membrane of thickness h = 0 , as a function of a. solid line , analytic result ( 21 ) ; dashed line , asymptote ( 25 ) for a 1 ; triangles , full pnp numerical computation
( h = 0 ) .
the dot dashed line shows hm / h0 = 6/(a ) with the expected slope for large a. squares and circles show electroosmotic coefficients h / h0 for nonzero membrane thickness h > 0 , computed by numerical integration of the full
pnp
equations : solid circles , h / a =
0.06 ; open squares , h / a = 0.1 . the membrane has zero thickness ,
so there is always a region near
the edge of the pore where the debye length can not be considered small compared with h ; smoluchowski s
analysis for thin charge clouds , which would predict h = 6h0/(a ) if
m took the uniform value m , therefore , can not automatically be invoked when a 1 . however ,
if we set up a local coordinate s indicating distance from the edge of the pore , then both
the electric potential ( 6 ) and the fluid
velocity g ( 22 ) vary
as s when s a ( i.e. , near the pore edge ) . the charge cloud density
0 decays over a length scale , and
only counterions of membrane surface charge within a distance from the edge contribute to 0 within
the hole .
the contribution of the edge to the integral ( 21 ) is therefore o((a ) ) , as was similarly found for the electrophoretic
velocity of a charged disk .
the data in figure 3 do not extend
to sufficiently high values of a to allow
us to estimate the asymptote with any accuracy , and for the figure ,
we simply indicate the line hm / h0 = 6/(a ) suggested
by the smoluchowski analysis .
a similar reduction in the broadside
electrophoretic velocity of a disk below the value predicted by smoluchowski
was noted by sherwood and stone .
individual
points in figure 3 indicate results obtained
from full numerical solutions of the poisson nernst
planck
equations in a symmetric electrolyte at low applied potential and
low surface charge . in the computations , the length of the reservoirs
in the z direction was equal to their radius b , with b = max(10a,10 ) .
when h > 0 , it
is natural to suppose that the electric field outside the membrane
pumps fluid toward the cylindrical pore at a rate30and the electric field within the cylindrical
pore pumps fluid through the pore at a rate31however , in general , qme ( 30 ) and qce ( 31 ) differ , and a pressure p2/2 builds up at z = h/2 ( i.e. , at the entrance and exit to the cylindrical pore )
in order to ensure that the volumetric flow rate is continuous .
consider a membrane of zero thickness ( h = 0 ) , with
pressure p = p1/2 ( above
the reference ambient pressure ) at infinity on the side z > 0 and with p = p1/2 at infinity on the other side .
the pressure within the
hole in the membrane is32the fluid velocity generated by the pressure
difference p1 across the membrane is u = p1 g ( 22 ) , and the corresponding volumetric flow
rate is33 if h > 0 , then we approximate
the pressure field
in the fluid in much the same way as we approximated the electrical
potential within the fluid : we patch a linearly varying pressure p(z ) within the cylindrical pore to the
pressure field outside a membrane of zero thickness , and we take the
pressure over the two ends z = h/2 of the cylindrical pore to be p2/2 .
thus , the pressure within the pore is approximated as34the fluid velocity within the pore
is35and the volumetric
flow rate within the pore
is36outside
the cylindrical pore , the fluid velocity
is now assumed to be37with ur(r , z ) = ur(r , z ) and uz(r , z ) = uz(r , z ) . the volumetric
flow rate outside the membrane is now38we have ensured that the
pressure ( but not
the fluid velocity or the volumetric flow rate ) is continuous across
the ends z = h/2 of the
cylindrical pore . when an electric field generates an electroosmotic
velocity , the
volumetric flow rates within the cylindrical pore and outside the
membrane are identical if p2 is such that qmh + qme = qch + qce ,
i.e. , if39but the pressure at infinity is zero in the
electroosmotic problem , so p1 = 0 , and
2 is given by eq 13 .
hence40and the total electroosmotic flow
is41an alternative derivation of this
approximate
composite hcomp ( 41 ) is given in the next section .
inserting into eq 41 the various estimates
for gm ( 38 ) , gc ( 36 ) , rm ( 8) , and rc ( 11 ) , we obtain42for small h / a , the approximate composite hcomp is
larger than hm if43experimental arrangements sometimes involve
measurements at fixed current , and a coefficient kcomp that gives the electroosmotic flux per unit current
is therefore useful .
this quantity may be obtained readily from eqs 11 , 13 , and 4144which changes from km when h = 0 to kc when h a. we now show
that approximations to the electric potential
and pressure - driven velocity g within a pore of nonzero
length h > 0 , when inserted into the integral
expression
( 21 ) for the electroosmotic volume flux , lead
to an approximate electroosmotic coefficient identical to hcomp ( 42 ) obtained in
the previous section .
we have already shown that we may approximate
the electric potential by a composite potential ( 9 ) and ( 10 ) of the form45a45b45cwe now create a similar approximation
for
the fluid velocity for flow through a membrane of thickness h subjected only to a pressure drop p1 but no applied potential drop .
we suppose that in z > h/2 the fluid velocity is given
by
eq 37 , corresponding to flow outside a membrane
of zero thickness , and that within the cylindrical pore the fluid
velocity is given by eq 35 .
continuity of the
volumetric flow rates ( 36 ) and ( 38 ) at the entrance to the cylindrical pore requires that the
pressure p2/2 at the two ends of the pore satisfies46so that47hence , our approximation to the fluid velocity
is u = gp1 ,
with48a48bthe ( small ) errors
involved in this approximation
are discussed by dagan et al .
we
now use approximations ( 45 ) and ( 48 ) in integral ( 21 ) in order
to compute the electroosmotic volumetric flow rate .
but the integration
splits naturally into an integral over the cylindrical pore and an
integral over the regions outside the membrane .
the integral over
the cylindrical pore is exactly the integral required to determine
the electroosmotic flow rate hc ( 17 ) in a cylinder , and the integral outside the membrane
is exactly that required to determine hm ( 24 ) .
hence , the integral yields the composite
electroosmotic flow rate49identical to ( 41 ) , obtained
in section 2.5 by elementary methods .
figure 4 shows hcomp ( 42 ) as a function
of h / a for four different values
of a , with m = c .
also shown are the results of full numerical computations
based on the poisson nernst
the coefficient hc ( 17 ) is proportional to h and is very large when the pore length h is small ,
leading to a large electroosmotic coefficient hcomp .
the action of the electric field acting on charge confined
within the cylindrical pore is much more efficient at creating fluid
motion than is the weaker electric field acting on charge outside
the pore .
we see that for a 1 the
approximate analysis captures the main features of the full numerical
results , and it is clear from ( 42 ) that it also
has the correct limits as h / a
0 and h / a . however ,
it is also evident from figure 4d that the
theory is unsatisfactory when a 1 .
electroosmotic
coefficient h scaled by h0 ( 26 ) for m = c , as a function of h / a , for ( a ) a = 10 , ( b ) a
= 2 , ( c ) a = 1 , and ( d ) a = 0.1 .
solid line , hcomp ( 42 ) ; solid circles , full pnp numerical computation .
the results of figure 4 are presented in
terms of the coefficient kcomp ( 44 ) in figure 5 .
both kcomp and the full numerical results now increase
monotonically with h , with a final end point kcomp = kc that is
independent of h when h a. figure 5 , like figure 4 , shows that the theory leading to kcomp is inadequate when a
1 .
we discuss this limit in section 4 , where
we shall show that when a 1 some
of the charge cloud of ions that neutralizes the surface charge on
the cylindrical wall of the pore spills out of the ends of the pore ,
where it is less effective at generating electroosmotic flow .
results of figure 4 , presented in terms
of the electroosmotic coefficient k = hrtot scaled by k0 ( 29 ) for m = c ,
as a function of h / a , for ( a ) a = 10 , ( b ) a = 2 , ( c ) a = 1 , and ( d ) a = 0.1 .
solid
line , kcomp ( 44 ) ; solid circles , full
pnp numerical computation . when the debye length is large compared
with the pore radius a , the cloud of counterions
associated with the charged cylindrical wall of the pore spills out
of the ends of the pore .
the applied
electric field is e = , where
the potential satisfies the laplace equation4with gradient5normal to the walls of the membrane and of
the cylindrical pore . in z
> 0 , the electric potential
far from the membrane is = 1/2 , and the
potential far from the membrane in z < 0 is
= 1/2 .
when the membrane thickness h = 0 , the potential can be expressed explicitly as6on the plane of the membrane , within the circular
opening,7the liquid within the pore has
electrical
conductivity ; we have assumed that surface charge density
( and hence the density of charge in the cloud of counterions ) is small ,
so that surface conductivity may be neglected . indeed , if the mobilities
of the various ionic species are identical , then the surface conductivity
due to the mobile charge cloud given by the linearized model ( 2 ) at o(e/kt ) is zero .
the total electric current i m flowing through the hole in the membrane is therefore8 if h > 0 , then we assume
that the potential within
the cylindrical pore varies linearly and approximate the potential
within the pore as9as would be expected in the absence of any
end effects .
the potential in z > h/2 is approximated by that outside a membrane ( with a hole ) of zero
thickness10with (r , z ) = (r , z ) .
this approximation ( 9 ) and ( 10 ) is continuous at z = h/2 where the potential is assumed
to be 2/2 across the entire width of the opening
( by eq 7 ) .
the as yet unspecified potential
2 is
determined by requiring continuity of the electrical current at z = h/2 .
the current ic through the cylindrical pore is11and the electrical current through the reservoir
in z > h/2 is , by eq 8,12equating ic ( 11 ) and i m ( 12 ) , we find13this computation suggests that the system
can be treated as two resistors in series , with composite resistance14however , this estimate
assumes a uniform potential
over the ends of the pore at z = h/2 , and we have effectively inserted thin , perfectly conducting sheets
over the pore ends .
removal of these sheets can only increase the
resistance and hence rcomp is an underestimate
for the true total resistance rtot .
figure 2a shows rtot/(a ) computed numerically by means of the freefem++
finite element package , together with rcomp/(a ) . the difference
is small and
( a ) nondimensional
ohmic resisistance r/(a )
of a hole of radius a in a membrane
of thickness h , as a function of h / a . solid line , rtot/(a ) computed numerically ; dashed line , the
approximation rcomp/(a ) ( 14 ) .
the time - independent
pnp stokes equations governing the
electrical potential , the ionic number density of the ith ionic species n ( i = 1 , ... , n ) , the fluid
velocity u and fluid pressure p are50515253where zi is the valence of the ith ionic species and
is its mobility . here ,
we restrict
our attention to the case n = 2 , with z1 = z2 = 1 .
we used a finite volume numerical scheme to solve the system of
coupled eqs 5053 in the axisymmetric geometry depicted in figure 1 .
thus , we considered a cylindrical pore of radius a and length h connecting two large cylindrical
reservoirs of radius b. the lengths of ab and ef
in our simulation were also taken to be b , which
was kept much larger than either a or the debye length
so that the reservoirs were effectively
infinite . at a and f , the
two ends of the reservoirs , ion concentrations were set equal to the
concentration in the bulk electrolyte ( i.e. ,
n = ni ) ; a potential
difference v was applied across the system
by setting to v/2 , respectively ,
at a and f , where the pressures were set equal to the bulk pressure , p = p. at ab and ef ,
the side walls of the cylindrical reservoirs , the radial component
of the electric field , ionic flux , and velocity were all set to zero ,
as was the tangential shear stress , in order to minimize the effect
of these boundaries .
the last condition was imposed as the cylindrical
reservoirs merely represent a convenient computational domain ; the
walls of the real physical reservoir are far enough away from the
pore to be essentially irrelevant . at the membrane and pore surfaces ,
bc , cd , and de
, a no - flux condition was used for ( 51 ) , and a no - slip condition was used for the flow . at solid fluid
interfaces ( with unit normal n ) , the electric
potential is continuous , but the normal component of the electric
field undergoes a jump , with [ en ] = m at bc and de and [ en ] = c at cd .
an electrohydrodynamic
solver was implemented to solve the system described above using the
openfoam cfd library , a c++ library designed
for computational mechanics .
a structured mesh was constructed by
means of the polymesh meshing tool within openfoam .
the grid was refined
near the membrane and pore surfaces to resolve the debye layer .
grid
independence was checked in all cases by refining the grid and verifying
that the solution did not change within specified tolerances . for the finite volume discretization of the governing equations ,
central differences were used for all diffusive terms in ( 51 ) and viscous terms in ( 52 ) .
a second - order upwind scheme was used for the convective terms
in ( 51 ) .
the discretized linear system was solved
using a preconditioned conjugate gradient solver if the matrix was
symmetric or a preconditioned biconjugate gradient solver if the matrix
was asymmetric .
an iterative scheme
was used to solve the pnp stokes equations .
initially , the flow velocity was set to zero .
equations 50 and 51 were then solved sequentially
in a loop with under - relaxation ( to ensure stability of the nonlinear
pnp system ) until the absolute residual was smaller than a specified
tolerance , in our case , 10 .
the electric force
density izien was then obtained from this solution
and used as an explicit external forcing in the solution of the incompressible
stokes flow problem , ( 52 ) and ( 53 ) , solved by means of the simple algorithm .
the flow field
so computed was then substituted into ( 51 ) ,
and the pnp equations were solved again using the updated flow field .
an outer loop was constructed to iterate over the pnp loop and stokes
flow module until the solution changed negligibly between two outer
iterations .
our main object of interest is the volumetric flux , q. this was obtained by numerically integrating the axial
velocity
over the plane z = 0 . at the low voltages employed
,
the linear relation found between q and v leads to the electroosmotic coefficient h = q/v , shown as discrete
points in figures 35 and 7 .
the amount of charge within
the pore was determined by numerical integration and used to obtain
the quantities hlost and hgained reported in table 1 .
we consider a cylindrical pore of radius a , with surface charge density c . when the debye
length a , the
equilibrium potential 0 ( 15 ) in an infinitely long cylinder can be expanded as54where55thus ,
the equilibrium potential 0 and charge density 0 = 0 within
the charge cloud vary little over
the cross - section of the pore . on the other hand , if the cylinder
is not infinitely long and uniform , 0 and 0 vary in the axial ( z ) direction with a length
scale . we can therefore consider the equilibrium
potential 0 within the cylindrical pore to be a
function only of z. we first consider a semi - infinite , charged cylindrical pore going
from z = 0 to z = . the
equilibrium potential 0 satisfies a one - dimensional
poisson
boltzmann equation56the solution that tends to the uniform potential
a within the pore as z far from the pore end at z =
0 , is57for some unknown constant a. the charge density within the charge cloud inside the pore is 0 , and when the cylindrical pore is infinite
( and hence uniform ) the charge per unit length in the charge cloud
is aa = 2ca , equal and opposite to the charge
per unit length on the pore walls .
when the pore is semi - infinite ,
with a nonuniform charge cloud ( 57 ) , the total
charge that is lost from within the pore is58at the end of the pore ( z = 0 ) , the potential
is = a a. in z < 0 , the charge cloud
is no longer confined
by the walls of the cylindrical pore and spreads out radially : it
is no longer possible to assume that 0 is a function
of z alone . we therefore need to solve the linearized
poisson boltzmann equation in the half - space z < 0 , with 0 = a a over the region z = 0 , r < a and 0/z = 0 on z = 0 , r > a. at large distances from the end
of the pore , the potential decays as exp(r)/r , where r = ( z + r ) is a
spherical polar coordinate , but in the important region r = o(a ) , the potential can be approximated
by the electrostatic potential corresponding to a solution of the
laplace equation ( i.e. , = 0 ) .
hence , from ( 6),59to relate
the potential ( 59 ) to the amount of charge in
the overspilling charge cloud
( in z < 0 ) , we note that the charge on one side
of a charged disk at uniform potential ( a a ) in unbounded space is q = 4a(a a ) .
alternatively , one can argue
that far from the plane z = 0 , the spherical distance r a cosh , so
that the potential ( 59 ) is approximately60 in a spherically symmetric geometry this field
corresponds to the far field around a point charge of magnitude 8a(a a ) , and the total surface charge on one side of the disk
is q = 4a(a a ) , in agreement with
the charge obtained by considering the capacitance of the disk .
the
charge in the overspill charge cloud in z < 0
is equal and opposite to q and is therefore61but the
total charge ( 61 ) in the overspill outside the
end of the pore must be equal to the
charge ( 58 ) that has been lost from within the
pore .
that63and the potential at the end of the pore is64the charge that
has been lost from the end
of the pore is equivalent to the charge usually found in a pore of
length65the loss of charge implies that the combined
charge cloud and wall surface charge over a cross - section of constant z are no longer electrically neutral , as pointed out by
baldessari and santiago . we can now perform the
same analysis for a pore that occupies the
region h/2 < z < h/2 .
the equilibrium potential within the pore has the form66where we have chosen the solution
that is
symmetric about the center of the pore at z = 0 .
the charge that has been lost from within the pore is67the total
flux of electric
field through the two ends of the pore is68comparing
( 67 ) and ( 68 ) , we conclude
that c = a .
the
potential over the ends of the pore
is69the total charge in the two overspill
charge
clouds is therefore , by ( 61),70and
this must be equal to
the charge ( 67 ) lost from within the pore .
hence71and72the total charge that has been lost ( from
the two ends ) is equivalent to a total lost length737475we see from eqs 65 and 74 that when h 1
the lost charge is twice that lost from a single end of a pore .
hlost > 0 , and that when the
pore is short ( h 1 ) the amount of
charge remaining within the cloud within the pore is proportional
to76 if the cylindrical pore itself is uncharged , but the
membrane surfaces are charged , ions from the charge cloud adjacent
to the membrane surface are able to move into the ends of the pore .
if the membrane has zero thickness , then the charge density 0 in the equilibrium charge cloud is given by ( 23 ) , and both 0 and the potential 0 = 0/( ) vary over the area of the pore .
nevertheless , we may work out the
mean potential over the circular pore77where , when a 178thus , when the membrane has zero thickness
( and there is no cylindrical pore into which ions can escape ) the
absence of surface charge over the area of the pore changes the average
potential over the opening from the value 0 = m/( ) due to a uniformly charged surface to m/( ) , where79 we now consider the charge that leaks
into a pore of length h > 0 from the charge clouds
on either side of the membrane .
we suppose that the potential on the planes z = h/2 is perturbed by an amount d and becomes80within the pore ,
the potential obeys the one - dimensional
poisson boltzmann equation ( 52 ) , with
solution81and the additional charge
within the pore
is82outside the pore , the perturbed
potential
( 80 ) is associated with a total additional charge
( 61)83on the two sides of the membrane .
but the
total change in charge caused by this redistribution must be zero ,
i.e. , qin + qout = 0 .
hence84i.e.85the total
charge qin = qout ( 83 ) that leaks into the pore
at the two ends corresponds to the charge
inside a uniformly charged cylinder with surface charge density m , of length868788thus , hgained ( 86 ) is smaller than hlost ( 73 ) by a factor a/2 .
we can compare predictions ( 73 ) and ( 86 ) against results obtained from full numerical solution
of the nonlinear poisson boltzmann equation with either m = 0 and aec/(kt ) = aec/(kt ) = 0.00273 or c =
0 and aem/(kt ) = 0.00273 : results for a = 0.1 are given
in table 1 .
we see
that there is excellent agreement between the numerical computations
and the analysis presented above . in terms of
an equivalent pore
length hlost ( 73 ) . in terms of an equivalent pore
we first consider how the electroosmotic coefficients hc and hm are modified by the
overspill of the charge cloud from inside the cylindrical pore to
outside the membrane . if a uniform electric field of strength e = 1/h is applied
between the ends of the pore , then the navier
stokes equations
for steady flow yield the axial velocity profile89so that the volumetric flow rate is90but q is independent of z ( by incompressibility ) , and the difference in pressure p between the two ends of the capillary is zero . hence ,
integrating ( 90 ) along the length h of the cylindrical
pore and noting that the total amount of charge
in the charge cloud remaining within the pore is 2ac(h hlost ) , we find91which may be compared to the result
( 18b ) which ignores overspill .
the charge cloud
outside
the pore is enhanced by the overspill and becomes ( in z > 0)92with
the final term [ a b cosh ( h/2 ) ] ,
corresponding to the overspill charge cloud ( 72 ) , being approximately valid in a volume o(a ) around the pore , but invalid at large distance o( ) from the pore , where the
exponential decay of the charge density is not captured by the solution
( 59 ) of the laplace equation .
the volumetric
flow rate through a pore of zero thickness created by a potential
difference 1 is given by the integral ( 21 ) and was shown by mao et al .
to be93where94hence , the electroosmotic
flow rate q = hm1 due
to the charge cloud outside the membrane is modified , and hm becomes95 if m is comparable to c , then we saw
in section 4.3 that the change in the charge
within the pore
due to the charge cloud outside the membrane entering the pore is o(a ) smaller than the loss of charge
from the charge cloud within the pore to the regions outside the membrane .
however , this contribution can be included with very little effort
and becomes important in the limit h 0 ,
when the gain ( 87 ) in charge within the pore
from the outside surface charge density m is proportional
to hgained h , whereas the charge cloud ( due to c within the
pore ) remaining within the pore is proportional to h
hlost h , by ( 76 ) . the electroosmotic coefficient hc for the cylindrical pore ( 91 ) becomes96and the electroosmotic coefficient hm for the charge cloud outside the membrane
( 95 ) becomes97
now
that hc ( 96 ) and hm ( 97 ) have
been corrected for the effects of overspill in the two directions ,
they can be inserted into expression ( 42 ) for
the composite electroosmotic coefficient hcomp .
results are shown in figure 7a , together with full numerical solutions of the
poisson nernst planck equations .
we see that the agreement
between theory and computation is much better than when overspill
is ignored ( figure 4d ) .
charge overspill or
underspill causes the total charge of mobile ions within the pore
to differ from what might be expected on the basis of net electroneutrality
of the pore .
thus , the driving force is modified , leading to deviations
from the calculated result that ignores such effects .
figure 7b shows
the results of figure 7a expressed in terms
of k , rather than h , and there is
again good agreement between the theoretical kcomp and full numerical results .
( a ) electroosmotic coefficient h scaled by h0 ( 26 ) for m = c , as a function
of h / a , for a
= 0.1 , including
the effect of overspilling charge clouds . solid line , hcomp ( 42 ) , using hc given by ( 96 ) and hm given by ( 97 ) ; solid circles ,
full pnp numerical computation ( cf . figure 4d , in which overspill was neglected ) .
( b ) the same results , presented
in terms of k = rtoth scaled by k0 ( 29 ) .
solid line , kcomp ( 44 ) , using kc = rchc and km = rmhm ; solid circles , full computation ( cf .
note that when h the effective length of the cylindrical pore h hlost ah/2 , by ( 76 ) .
the approximation
( 96 ) for hc is therefore
dominated by the term hgained and gives hc am/(8h ) , with hc / hm 3a/(8h ) .
we conclude from ( 43 ) that hcomp is a decreasing function of h near h = 0 , as seen in figure 7a .
the analysis presented here shows
that it is possible to use simple
analyses based on continuity of volumetric flow rate and electric
current to estimate electroosmotic end effects in a charged cylindrical
pore traversing a membrane of thickness h > 0 .
note
that we have made repeated use of the assumption that surface charge
densities , and corresponding zeta potentials , are small .
boltzmann equation
( 2 ) , but have also used superposition to combine
various contributions to the charge clouds due to overspill of the
clouds from one region ( inside / outside the pore ) to the other . at
high potentials , it would also be necessary to keep track of the fluxes
of individual ion species , rather than simply ensuring that the total
electrical current is continuous .
the assumption of small potentials also justifies our neglect of
other nonlinear electrokinetic effects such as induced charge electroosmosis
( iceo ) , which can produce vortices in the vicinity
of sharp corners or near rapid constrictions
in channels when the permittivity of
the solid s > 0 . however , numerical
solutions confirm the expectation that the flow rate is only weakly
affected by such vortices , particularly under conditions of small
potentials . in recent experiments on nanopores ,
potential differences 0200
mv were applied across the pore . here , we have assumed that
, where itself is assumed small in comparison
with the thermal voltage kt / e
25 mv .
thus , our results can only be expected to describe the initial
linear part of the current voltage and flow voltage
characteristics , even though numerical simulations seem to show that this linear regime extends to applied voltages
100 mv . finally , we point out that the correction factor
( 79 ) reminds us that the hole in the
charged membrane
removes a circular region of surface charge and reduces the equilibrium
potential at the entrance to the pore .
the introduction of
< 1 improved the agreement between theoretical and numerical results
for hgained in table 1 .
however , the analysis is not rigorous , since the equilibrium
potential across the hole is not uniform .
the o(1
) correction to the equilibrium potential corresponds
to an o(1 ) correction to the charge
density 0 .
if we use this in the integral expression
( 93 ) in order to determine a correction to the
electroosmotic flow rate through a membrane of zero thickness , then
the analysis suggests that the correction to the leading order result
( 25 ) for a
1 should be o((a ) ) , whereas investigation of the difference ( seen in figure 3 ) between numerical results and the asymptote ( 25 ) indicates additional corrections o((a ) ln a ) .
|
a theoretical model of electroosmosis
through a circular pore of
radius a that traverses a membrane of thickness h is investigated .
both the cylindrical surface of the pore
and the outer surfaces of the membrane are charged .
when h a , end effects are negligible , and the
results of full numerical computations of electroosmosis in an infinite
pore agree with theory . when h = 0 , end effects dominate ,
and computations again agree with analysis . for intermediate values
of h /
a , an approximate analysis
that combines these two limiting cases captures the main features
of computational results when the debye length 1 is small compared with the pore radius a. however ,
the approximate analysis fails when 1 a , when the charge cloud due to the charged cylindrical
walls of the pore spills out of the ends of the pore , and the electroosmotic
flow is reduced . when this spilling out is included in the analysis ,
agreement with computation is restored .
|
soybean- and cruciferous vegetable - enriched
diets seem to be particularly protective , but the mechanisms for this
anticarcinogenicity are not fully understood .
genistein , a dietary
phytoestrogen belonging to the isoflavone class of flavonoids , is thought to
have anticarcinogenic activities , particularly for breast and prostate cancer
[ 1 , 2 ] . dietary soy has been shown in
mice to inhibit prostate tumor growth through inhibition of cell proliferation ,
increased apoptosis , and reduced microvessel density .
epidemiology studies of asian women indicate
that consumption of a traditional diet high in soy confers significant
protection against breast cancer .
3,3-diindolylmethane
( dim ) , a breakdown product of glucobrassicins , which are found in cruciferous
vegetables , has been shown in vivo to have protective effects for breast
cancer .
we have shown previously
that dim lowers the levels of cxcr4 and cxcl12 , a chemokine receptor and its
unique ligand required for the metastasis of breast cancer [ 6 , 7 ] . in
addition to mediating the directional homing of primary breast cancer cells to
secondary organ sites , cxcr4 and cxcl12 are important in other aspects of
cancer progression , such as adhesion , proliferation , and angiogenesis .
these effects are not limited to breast
cancer , as the interaction between cxcr4 and cxcl12 is implicated in the
progression of many different types of cancer .
the
biological effects of genistein are extensive and include antioxidant activity ,
weak estrogenic / antiestrogenic activity , upregulation of apoptosis , inhibition
of angiogenesis , inhibition of dna topoisomerase ii , and inhibition of protein
tyrosine kinases ( ptks ) .
genistein has
been shown to regulate specific sex steroid receptors , inhibit nfkb ,
downregulate tgf- , and inhibit efg - stimulated growth .
furthermore , genistein inhibits
2,3,7,8-tetrachlorodibenzo - p - dioxin
( dioxin)-induced cyp1a1 activity , and isoflavones can prevent the
cyp1a1-mediated binding of benzo[a]pyrene ( b[a]p ) metabolites to dna .
our
previous observations that dim downregulates cxcr4 and cxcl12 in breast and
ovarian cancer cells represent a novel mechanism for the chemoprotective
effects of this phytochemical . here , we
demonstrate that these effects are not unique to dim , but can also be seen with
genistein .
interestingly , we see that
the combined effect of dim and genistein elicits a greater downregulation of
cxcr4 and cxcl12 than either compound alone , indicating that the phytochemicals
used in combination may be even more potent in their chemoprotective
properties .
we also demonstrate that like dim , genistein specifically inhibits
chemotaxis and chemoinvasion of breast and ovarian cancer cells toward cxcl12 in vitro .
genistein was purchased from sigma ( st . louis , mo , usa ) , dim was
purchased from lkt laboratories ( st .
paul , minn , usa ) , and the remaining
phytochemicals were a kind gift from dr .
david heber ( university of california ,
los angeles , calif , usa ) .
kenneth korach ( national institute of environmental health
sciences , nc , usa ) .
mcf-7 and mda - mb-231
cells were purchased from the american type culture collection ( atcc , manassas ,
va , usa ) .
mda - mb-231
cells were maintained in dulbecco 's
modified eagle 's medium containing 4 mm l - glutamine .
all media were purchased from invitrogen
( carlsbad , calif , usa ) and supplemented with 10% fetal bovine serum ( fbs ; omega ,
tarzana , calif , usa ) , 100 u / ml penicillin/100 g / ml
streptomycin solution
( gemini bio - products , west sacramento , calif ,
usa ) , and 0.25 u / ml amphotericin b ( omega )
for all experiments , chemicals were dissolved in dmso and administered to cells with a
final concentration of dmso at 0.1% in the medium .
rna isolation , cdna
synthesis , and taqman multiplex real - time
pcr were performed as previously described .
cxcr4 and cxcl12 cdnas were amplified using assays on demand ( affinity bioreagents , golden , colo , usa ; product
numbers hs00607978_s1 and hs00171022_m1 , resp . ; sequences proprietary ) .
the forward and reverse primers used for 36b4
quantification were 5-ccacggtgctgaacatgct-3 and 5-tcgaacacctgctggatgac-3 ,
respectively .
the 36b4 probe sequence
was 5-texas red - accatctcccccttctcctttgggct - iowa black-3. all primers and
probes were purchased from integrated dna technologies ( coralville , iowa , usa ) .
real - time pcr was carried out using the
icycler iq ( biorad , hercules , calif , usa ) or 7500 fast ( applied biosystems ,
foster city , calif , usa ) under standard protocols .
data were analyzed using the icycler or abi
software and microsoft excel , and significance was evaluated using student 's t - test . for surface staining of cxcr4 and
intracellular staining of cxcl12 , cells were grown to 70% confluence and
treated with genistein for 24 or 48 hours .
in the case of cxcl12 quantification , the cells were also cotreated with
1 g / ml brefeldin a ( glogiplug ; bd
pharmingen , franklin lakes , nj , usa ) for the final six hours of incubation to
inhibit protein secretion .
cells were
harvested and stained as previously described [ 6 , 9 ] with either primary cxcl12
antibody ( r&d cat .
mab350 ; minneapolis , minn , usa ) , primary cxcr4
antibody ( affinity bioreagents cat .
opa1 - 01101 ; ill , usa ) or the
appropriate primary igg isotype control antibody , followed by staining with
either goat antimouse or antirabbit igg - fitc secondary antibodies
( bd - pharmingen and caltag , carlsbad , calif , usa , resp . ) .
fluorescence was quantified using a facscan analytic
flow cytometer ( becton dickinson , ucla flow cytometry core facility ) .
cells
were pretreated with genistein , dim , or the combination , followed by chemotaxis
and invasion assays , performed as we have described previously . after migration / invasion , mts assays
( promega , madison , wis ) were performed in each individual transwell to control
for small variations in cell number .
after detection of the formazan product at 490 nm , cells on the inserts
were washed with pbs and those on the upper layer were gently removed with a
prewet q - tip .
cells on the lower layer
were fixed in 100% methanol and stained with crystal violet .
membranes were manually excised from the
inserts , mounted on microscope slides , and divided into 8 equal sections .
cells in one random viewing field from each
section were counted at 40x magnification , and the average was calculated .
data are
expressed as chemotaxis or chemoinvasion indices , which were defined as the
normalized number of cells in the experimental group relative to the control
group .
statistical analyses were
performed using a two - tailed student 's t - test .
mcf-7 and mda - mb-231 breast cancer cells and
bg-1 ovarian cancer cells were treated with concentrations of genistein ranging
from 1100 m ( at a
constant final dmso concentration ) for 24 hours , after which time cxcr4 and
cxcl12 mrnas were quantified by real - time pcr .
cxcr4 mrna levels were significantly decreased at 1 m genistein in all
three cell lines ( figures 1(a)1(c ) ) .
maximal downregulation was seen at 100 m , 50 m , and 30 m genistein in each cell line , respectively .
significant
downregulation of cxcl12 mrna occurred at a concentration of 50 m genistein in
mcf-7 cells and 10 m genistein in bg-1 cells ( figures 1(a)1(c ) ) .
interestingly ,
low doses of genistein ( 110 m )
significantly increased cxcl12 mrna
levels in mcf-7 cells , but had the opposite effect in bg-1 cells at these
doses .
flow
cytometric analysis was used to quantify surface cxcr4 and intracellular cxcl12
expression .
cells were treated with 30 ,
70 , or 100 m genistein for 24 hours , harvested , stained , and analyzed for
surface expression levels of cxcr4 .
relative to dmso - treated controls , cxcr4 was found to be downregulated
by genistein in mcf-7 , mda - mb-231 , and bg-1 cells at all three doses ( figures 2(a)2(c ) ) .
analysis of cxcl12 intracellular expression
showed that relative to dmso - treated control cells , cxcl12 levels were reduced
by all three doses of genistein in both mcf-7 and bg-1 cells ( figures 2(a ) , 2(c ) ) .
we previously found that dim downregulates cxcr4 and cxcl12 mrnas and proteins in invasive breast and ovarian
cancer cells .
we therefore tested
whether the downregulation elicited by cotreatment with both dim and genistein
was additive or synergistic in these cell lines . using real - time pcr , we quantified gene
expression after treatment of mda - mb-231 ( cxcr4 only ) or bg-1 ( both cxcr4 and
cxcl12 ) with 20 m dim , 100 m genistein , or the combination .
we found that in
both cell lines , the degree of downregulation of cxcr4 was greater after
treatment with both phytochemicals in combination ( figures 3(a)-3(b ) ) . in mda - mb-231 cells ,
dim reduced levels of
cxcr4 by 57% , genistein by 51% , and the combination by 85% ( figure 3(a ) ) . in bg-1 cells , dim downregulated cxcr4 by
49% ,
similarly , dim lowered the levels of cxcl12
by 50% , genistein lowered levels by 84% , and genistein by 91% ( figure 3(b ) ) .
these effects can not be attributed to
cytotoxicity , since neither dim , genistein , nor the combination was cytotoxic
at these doses for the 24 hour time period ( data not shown ) . since
cxcr4 and cxcl12 mediate directional migration , and since we saw previously
that dim inhibited chemotaxis and chemoinvasion of these cells , we performed
assays to determine whether genistein could inhibit migration through
fibronectin and/or invasion through matrigel .
we found that pretreatment with
genistein inhibited the directional migration of mcf-7 cells , with a resulting
migration rate similar to the background rate ( dmso - treated cells exposed to no
cxcl12 gradient , figure 4(a ) ) .
however ,
we did not see a further reduction in migration after cotreatment with
genistein and dim , presumably because both chemicals used independently at
these doses fully reduced migration to background levels .
this result indicates
that the degree to which genistein and dim downregulate surface cxcr4 levels at
these doses may be sufficient to significantly impact the homing of breast
cancer cells to areas of high cxcl12 expression .
as we have seen previously with dim , the
inhibition of chemotaxis by genistein specifically affects migration toward
cxcl12 , since an inhibition of mcf-7 cell chemotaxis toward fbs was not
observed .
mcf-7
cells do not invade through matrigel ( a synthetic extracellular matrix ) , and we were therefore unable to
evaluate invasive potential in these cells .
however , mda - mb-231 breast cancer cells are invasive and we were able to
quantify both chemotactic and chemoinvasive potential in this cell line .
we found that genistein , dim , and the
combination significantly inhibited chemotaxis and chemoinvasion of these cells
toward cxcl12 , but not toward il-6 , a known in vitro chemoattractant for mda - mb-231 cells ( figures 4(b)-4(c ) ) .
this result indicates that the inhibitory
effects of dim and genistein are specific for cxcl12-induced
chemoattraction / chemoinvasion and is not merely a general effect on migration
or invasion .
a similar result was seen with bg-1 ovarian cancer cells , which
are also invasive in vitro
( figure 4(d ) ) .
these cells do not migrate through matrigel toward il-6 ( data not shown ) , but did exhibit moderate chemoinvasion toward fbs .
the
antiestrogenic activity of genistein may mediate , in
part , the protective effects of soy for breast and other cancers .
although other nonestrogenic mechanisms of
action such as ptk and topoisomerase ii inhibition likely play a role in these
protective effects , we describe here an additional novel mechanistic pathway in
which genistein and possibly other phytochemicals may be protective .
we have
found that genistein downregulates cxcr4 in the er - positive ( er+ ) breast cancer
cell line , mcf-7 , the er - negative ( er ) breast cancer cell line , mda - mb-231 ,
and the er+ ovarian cancer cell line , bg-1 .
furthermore , cxcl12 , the unique
ligand for cxcr4 , is downregulated by genistein in both mcf-7 and bg-1
cells .
we show that this downregulation
results in a subsequent inhibition of migration and invasion of these cells
toward cxcl12 in vitro .
we
found previously that dim downregulates cxcr4 and cxcl12 in mcf-7 , mda - mb-231 ,
and bg-1 cells .
we show here that when used in combination , the effects of
dim and genistein on cxcr4 and cxcl12 mrna levels are greater than with either
compound alone , suggesting that phytochemicals used in combination may increase
the efficacy of their protective effects .
at 20
m dim and 70 m genistein , we did not find the combination to
further inhibit chemotaxis or chemoinvasion of breast or ovarian cancer cells
since these concentrations of the phytochemicals fully inhibit these processes .
however , at the slightly lower doses of 10 m dim and 50 m genistein , the
combination does appear to be more effective at inhibiting chemotaxis of
mda - mb-231 cells . at these latter doses
, the individual phytochemicals did not
fully reduce chemotaxis of these cells to background levels .
these results
therefore suggest that lower doses of the phytochemicals used in combination
may be equally or more effective in chemoprotection than a higher dose of a
single phytochemical . the
mechanisms of action of dim and genistein in the downregulation of cxcr4 and
cxcl12 remain to be determined .
dim and genistein both act as weak
agonists / antagonists of the estrogen receptor , and it is therefore possible
that the compounds can act through the same mechanism .
importantly however , we
see downregulation of cxcr4 and a subsequent inhibition of chemotaxis and
chemoinvasion in mda - mb-231 cells , which do not express the estrogen receptor .
on the other hand , dim is a ligand for the ahr , whereas genistein has not been
shown to bind the ahr in the cell lines we used .
genistein is a specific ptk
inhibitor as well as an inhibitor of topoisomerase ii , and may modulate tgf-
signal transduction [ 11 , 12 ] .
interestingly , both genistein and dim are known to inhibit nfkb in
breast , prostate , and pancreatic cancer cells [ 1315 ] . since cxcr4 is regulated at the
transcriptional level by nfkb
, it is plausible that the mechanism of cxcr4
and/or cxcl12 downregulation by dim and/or genistein could be through nfkb
inactivation or downregulation [ 16 , 17 ] .
condoning
the general use of genistein as a supplement is at this point
controversial .
soy products are
extensively consumed in asian populations without apparent adverse effects , but
experimental data have led to concerns about the safety of genistein and other
constituents of soy . although it was
well tolerated , high doses of genistein in chronic studies caused an increase in
the weights of the kidney , spleen , adrenal , and testes in male rats and an
increase in liver , kidney , spleen , ovary , and uterus weights in female rats
.
in the same study , histological
changes were seen in the reproductive organs of both male and female rats .
genistein can behave as both an estrogen and
an antiestrogen , and the net estrogenic effect of the chemical has proven
difficult to quantify .
a chronic
exposure study very recently carried out by the national toxicology program
showed a significant increase in the incidence of mammary gland adenoma and
adenocarcinoma ( combined ) .
importantly ,
the time of administration appears to significantly impact whether genistein
elicits a protective , adverse , or no effect .
for example , dimethylbenzanthracene
( dmba)-induced mammary cancer was reduced after prepubertal and combined
prepubertal and adult administration of genistein , but not after prenatal - only
or adult - only treatments .
dose also plays a critical role in the biological effects of
genistein ; although doses greater than 10 m over an extended period of time
inhibit the growth of both er+ and er breast cancer cells , low doses of
genistein ( < 1 m ) appear to stimulate the growth of er+ breast cancer cells [ 21 , 22 ] .
interestingly , cxcl12 has been shown to mediate the proliferative
effects of estradiol in breast cancer cells .
we noted a significant increase in cxcl12
mrna after treatment with low doses ( 110 m ) of genistein
in mcf-7 cells , although this effect was not seen in mda - mb-231 or bg-1 cells .
an upregulation of cxcl12 by low doses of genistein remains to be confirmed in
tissues that serve as common sites of metastasis such as the lung and bone .
however , in light of what is known about the
toxic and potentially carcinogenic effects of genistein , this observation underscores the importance of thorough
safety analyses prior to condoning the use of phytochemicals as dietary
supplements .
in particular , it highlights the importance of
dose , especially at the tissue level , when assessing the impact of genistein on
the development and progression of breast and other cancers .
the doses of
genistein and dim described here are likely achievable in humans upon
supplementation , especially at the tissue level .
total genistein plasma concentrations of up
to 20 m were obtained after feeding human volunteers a genistein supplement
[ 2426 ] .
furthermore , genistein was
found to accumulate in certain organs to considerably higher concentrations
, and this is likely to be the case for fatty tissues , such as the breast ,
in particular . when mice were
administered a single oral dose of dim , a serum concentration of approximately
5 m was achieved .
thus , the concentrations of genistein and dim
that we have used in our studies are likely to reflect attainable doses in the
human . since cxcr4 and cxcl12 are implicated in the
progression of many different cancers , it is of interest to determine whether
dim and/or genistein downregulate these proteins in cancer cell lines of other
origins .
it will also be important to investigate whether cxcl12 is
downregulated by phytochemicals in tissues that serve as preferred sites of metastasis
for these cancers , such as the lung and bone . furthermore , since many other phytochemicals
have been implicated in cancer protection , it will be prudent to determine
whether phytochemicals other than dim and genistein exert similar effects on cxcr4
and cxcl12 levels .
finally , phytochemicals in combination should be tested in vivo to determine whether a
potentiated effect can be achieved .
dim
has been suggested as a potential chemotherapeutic for er+ breast cancers . surprisingly however , we have found that dim
and genistein downregulate cxcr4 and cxcl12 in both er+ and er cell lines ,
indicating that these phytochemicals may be effective in the treatment of both
early- and late - stage breast cancers .
effective therapies for advanced disease
are lacking , and the potential use of compounds as innocuous as phytochemicals
for treatment of either early- or late - stage cancers is an attractive
alternative . furthermore , the increased
response of cxcr4 and cxcl12 downregulation by dim and genistein in combination
may prove useful in eliciting a potentiated effect in vivo and perhaps
allow for optimization of the associated biological responses .
|
cxcr4 is a chemokine receptor frequently overexpressed on primary tumor cells
. organs to which these cancers metastasize secrete cxcl12 , the unique ligand for cxcr4 , which stimulates invasion and metastasis to these sites .
similar to our previous work with the chemoprotective phytochemical , 3,3-diindolylmethane ( dim ) , we show here that genistein also downregulates cxcr4 and cxcl12 and subsequently lowers the migratory and invasive potentials of breast and ovarian cancer cells .
moreover , genistein and dim elicit a significantly greater cumulative effect in lowering cxcr4 and cxcl12 levels than either compound alone .
our data suggest a novel mechanism for the protective effects of phytochemicals against cancer progression and indicate that in combination , these compounds may prove even more efficacious .
|
pulpal diseases leading to inflammation of periapical tissues show the presence of bacteria in the root canal system .
teeth with pulpal or periapical pathology have a complex microbial flora consisting of cocci , rods , spirochetes , and fungi .
studies have shown that there is a difference between the microbial flora of root canal in cases of primary endodontic infection and in cases of reinfection .
the microorganisms in retreatment cases possess greater resistance to intracanal medicaments . also , it is found that microbes not only grow in planktonic cells or in aggregates but also forms biofilms .
since then studies have shown that the endodontic environment is selective and it supports the specific microorganisms to grow .
in 1982 showed that strict anaerobes succeed over facultative anaerobes in root canal due to changes in root canal ecology .
studies have also shown that in post treatment diseased teeth , gram positive microorganisms predominate .
some of these microorganisms have the capability to survive in harsh and nutrient limited condition in root canal filled teeth .
teeth with persistent disease showed a high prevalence of enterococci ranging from 29 to 77% .
enterococcus faecalis can withstand high ph of intracanal dressings like calcium hydroxide , and is hence found in higher concentration in reinfection cases . the other common organism which persists in post treatment
both these organisms can survive as monoinfection and invade dentinal tubules . hence , amongst the 24 million endodontic treatment performed on an annual basis , 5.5% procedures involve endodontic surgery and perforation repair .
when endodontic surgeries are performed at the apex , it involves placement of the material at apical end of the root to seal the root canal from the periapex .
historically , a plethora of materials have been used from amalgam , zinc oxide eugenol , composite resins , and glass ionomer cements .
unfortunately these materials failed to satisfy the ideal requirements of root - end filling material . later in 1990s
, mineral trioxide aggregate ( mta ) was introduced as a root - end filling material . till date it is being used as a material of choice for root - end filling .
recently , two new materials have been introduced into market , namely , mta like material mta plus ( compounded by prevest denpro , jammu , india for avalon biomed , inc usa ) and a calcium silicate based material biodentine ( septodont , saint - maur - des - fosses , france ) .
manufacturers claim that these materials can be used as a root - end filling material .
thus , the aim of the study is to evaluate and compare the antimicrobial efficacy of proroot mta , mta plus , and biodentine against the e. faecalis and c. albicans .
the materials evaluated for antimicrobial efficacy were proroot mta ( dentsply ) , mta plus ( compounded by prevest denpro , jammu , india for avalon biomed , inc usa ) , and biodentine ( septodont , saint - maur - des - fosses , france ) .
two hundred milligrams of the material was dissolved in 20 ml of sterile brain heart infusion ( bhi ) broth .
this solution now contained 10 mg of material in 1 ml of broth and is called master dilution .
2 to 6 such that subsequent test tubes had half the concentration of material than the previous . hence , tube no .
ten microliter of standardized e. faecalis was added to tube no . 1 - 6 and tube no . 8
tube no . 8 and 9 were used as positive and negative control , respectively .
subcultures from each of the tubes were made on mac conkey 's agar and read at the end of 24 , 48 , and 72 h of incubation .
ten different strains of e. faecalis were used for evaluation of antibacterial efficacy of the materials in a similar manner .
sabouraud 's dextrose broth ( sdb ) and sabouraud 's dextrose agar ( sda ) was used for dilutions and culture , respectively .
ten different strains of c. albicans were used for evaluation of antifungal efficacy of the materials .
the results were tabulated and statistically analyzed using one - way analysis of variance ( anova ) and scheffe 's post - hoc tests using statistical package for social sciences ( spss ) 20#.
results of antimicrobial efficacy were evaluated between different time intervals andbetween different materials using one - way anova followed by scheffe 's post - hoc test . there was a statistically significant difference between the materials at all time intervals ( 24 , 48 , and 72 h ) with p - value 0.019 , 0.000 , and 0.043 , respectively [ table 1 ] .
scheffe 's post - hoc test revealed that there was statistically significant difference between biodentine and mta plus at 24 and 48 h with p - value of 0.022 and 0.002 , respectively .
biodentine showed statistically significant difference with prorootmta at 48 h with p - value 0.000 .
one - way anova test between three materials at different time intervals prorootmta showed statistically significant difference at different time intervals with p - value 0.001 [ table 2 ] .
scheffe 's post - hoc tests revealed there was statistically significant difference in antibacterial efficacy of proroot mta between 24 and 48 h ( p - value 0.04 ) and between 24 and 72 h ( p - value 0.004 ) .
there was statistically no significant difference for biodentine and mta plus at different time intervals .
one - way anova test for materials between different time intervals there was no statistically significant difference between mta plus , biodentine , and mta at all time intervals ( 24 , 48 , and 72 h ) .
there was a statistically significant difference between the materials at all time intervals ( 24 , 48 , and 72 h ) with p - value 0.019 , 0.000 , and 0.043 , respectively [ table 1 ] .
scheffe 's post - hoc test revealed that there was statistically significant difference between biodentine and mta plus at 24 and 48 h with p - value of 0.022 and 0.002 , respectively .
biodentine showed statistically significant difference with prorootmta at 48 h with p - value 0.000 .
one - way anova test between three materials at different time intervals prorootmta showed statistically significant difference at different time intervals with p - value 0.001 [ table 2 ] .
scheffe 's post - hoc tests revealed there was statistically significant difference in antibacterial efficacy of proroot mta between 24 and 48 h ( p - value 0.04 ) and between 24 and 72 h ( p - value 0.004 ) .
there was statistically no significant difference for biodentine and mta plus at different time intervals .
there was no statistically significant difference between mta plus , biodentine , and mta at all time intervals ( 24 , 48 , and 72 h ) .
the most common method for assessing the antimicrobial activity of root - end filling materials is agar diffusion test . however , this method has disadvantages like lack of standardization of inoculums density , adequate culture medium , agar viscosity , etc . in this study ,
it is an efficient method for evaluating as there is a direct contact between the microorganisms and experimental materials which allows a more realistic interaction .
various dilutions of the material are prepared and the concentration at which the material was able to inhibit the growth of microorganism was recorded as the minimal inhibitory concentration ( mic ) of the material .
the results of antibacterial efficacy against e. faecalis showed that proroot mta and biodentine inhibited the growth of majority of strains of e. faecalis ; whereas , mta plus was not that effective .
the mic at which proroot mta and biodentine were effective in inhibiting e. faecalis is 5 mg / ml . whereas , mta plus inhibited a few strains at 10 mg / ml . for antifungal efficacy , there was no statistically significant difference between proroot mta , mta plus , and biodentine .
the mic at which proroot mta , mta plus , and biodentine were effective in inhibiting c. albicans is 2.5 mg / ml .
the antimicrobial efficacy of proroot mta is due to release of calcium ions and hydroxyl ions which results in increase in ph .
this release of calcium is when calcium silicate gets hydrated and calcium hydroxide is released as a byproduct .
. the antimicrobial effect of proroot mta against e. faecalis and c. albicans obtained in this study is similar to other studies .
mta plus is available in the powder form which is sealed in a desiccant container .
it consists of tricalcium silicate , dicalcium silicate , bismuth oxide calcium sulfate , and silica .
it can be mixed with either water or an antiwashout gel . on hydration of mta plus , strong peak of calcium hydroxide
the release of calcium ions and subsequent increase in ph leading to alkaline nature of the material is significant only after 7 and 14 days .
this could be the reason why mta plus did not prove to have a good antibacterial property .
a calcium silicate - based material biodentine was introduced in 2010 by gilles and olivier .
biodentine showed higher release of free calcium ions when compared with proroot mta , and hence higher alkalinizing capacity .
the high release of calcium in biodentine is attributed to presence of calcium silicate and calcium chloride .
within the limitations of the study , proroot mta and biodentine proved to have antifungal and antibacterial property .
this study shows that mta plus was a good antifungal agent , but not an efficient antibacterial .
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aim : the aim of the study is to evaluate and compare the antimicrobial efficacy of two new materials mta plus and biodentine with proroot mta using tube dilution method.materials and methods : the materials used were proroot mta ( dentsply ) , mta plus ( compounded by prevest denpro , jammu , india for avalon biomed inc , usa ) and a calcium silicate based material biodentine ( septodont , saint - maur - des - fosses , france ) .
doubling dilutions of the material were prepared in sabouraud 's dextrose broth ( sdb ) and brain heart infusion ( bhi ) broth for candida albicans and enterococcus faecalis , respectively .
the minimal concentration at which inhibition of microorganism occurred was measured and noted as minimal inhibitory concentration ( mic ) of the material.results:there was no statistically significant difference between the materials against c. albicans .
biodentine was statistically significant than mta plus against e. faecalis ( p - value-0.022 ) .
proroot mta was statistically significant at different time intervals against e. faecalis ( p - value-0.001).conclusion : proroot mta and biodentine proved to have antimicrobial property .
mta plusproved as a good antifungal agent .
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there is a paucity of information on management of forearm fractures through pre - existing ischemic contractures .
the prevention of a volkmann s contracture in forearm compartment syndrome requires vigilant clinical evaluation and emergent fasciotomy , but many of the patients who develop these contractures often do so as a result of delayed presentation due to substance abuse and intoxication .
this case describes the first report and management of a severely displaced both bone ( bb ) forearm fracture through a chronic forearm volkmann s contracture .
a 39 year old female presented with an acute both bone forearm fracture in the setting of a volkmann s contracture .
although very limited in use , the arm was functional for holding objects and was determined to be important in her activities of daily living .
the surgical management involved open reduction internal fixation with radial and ulnar shortening osteotomies to restore cortical alignment secondary to the extensive overlying soft tissue contractures .
restoration of the radial bow and other standard principles of open reduction and internal fixation of bb fractures may not be as important as obtaining fracture union in patients with these complicated injuries due to the pre - existing functional limitations of the limb .
the soft tissue envelope in a contracted forearm is commonly fibrotic and provides an abnormal fracture healing environment .
this report details the first case , technical difficulties , and subsequent management of a bb fracture in the setting of subsequent management of a both bone fracture in the setting of a chronic volkmann s contracture .
in 1881 , volkmann first described an ischemic forearm contracture that resulted as a consequence of an untreated forearm compartment syndrome [ 1 , 2 ] . increased pressure within the osseofascial compartments leads to decreased capillary perfusion , resulting in progressive ischemia and eventual muscle necrosis [ 3 , 4 ] .
the moderate type affects both the wrist and finger flexors , while the severe type involves extensor muscles in addition to both the wrist and finger flexors [ 5 , 6 ] .
contracture prevention requires astute clinical evaluation and emergent operative intervention in the setting of compartment syndrome .
adult patients that develop this contracture often do so as a result of delayed presentation , such as in cases of intoxication or refusal of surgical intervention [ 7 , 8 ] .
prolonged external pressure on the forearm during a drug - induced haze was the most common cause for the development of forearm compartment syndrome and subsequent volkmann s contracture .
owen et al . studied changes in forearm compartment pressures in volunteers placed in positions typical of patients found after drug overdose .
this study demonstrated that pressures in limbs compressed between a hard surface and the body easily reached pressures that resulted in muscle ischemia . in 2012 , statistics showed a nationwide increase in illegal drug use in the united states , with approximately 9.2% of the population using illicit drugs within the last month . as drug use continues to be prevalent in society
, orthopedic surgeons must be aware of the management of potential sequelae of drug abuse , including volkmann s contracture .
although both bone ( bb ) forearm fractures are common injuries in the pediatric population , adult bb fractures are less common . while the mainstay of treatment for pediatric patients is nonoperative or with less invasive reduction techniques , the adult population frequently requires open reduction internal fixation ( orif ) .
adherence to operative principles such as restoration of radial bow and compression plating has been shown to be critical in the adult functional outcome .
a bb fracture in a patient with a volkmann s contracture presents a formidable surgical undertaking .
restoration of length , sagittal , coronal , and rotational alignment of the radius and ulna can be technically challenging in the setting of a severely contracted forearm .
the patients with a patients with a volkmann s contracture have a variable range of motion , and depending on preoperative function of the contracted forearm , re - establishment of the radial bow or alignment may not be as crucial as obtaining a solid union .
in addition to fracture care , soft tissue management is essential as the surrounding soft tissues are often compromised and may demonstrate scars from prior surgeries , skin contractures or skin grafts .
the surgeon must take all of these variables into consideration , when formulating a plan of care for these complicated patients .
close attention to post - operative wounds and osseous union is important as the decreased muscle viability overlying the fracture site can delay healing and tenuous soft tissues can be compromised .
the management of an adult bb forearm fracture in the setting of a pre - existing volkmann s contracture has not been previously discussed .
this report describes the management of a 39-year - old female with a chronic volkmann s contracture who presented with an acute bb forearm fracture after a motor vehicle accident .
a 39-year - old female presented as an unrestrained driver involved in a high - velocity motor vehicle accident . after initial stabilization , secondary survey demonstrated an acute , osseous deformity of the right forearm with a concomitant volkmann s contracture .
1 ) . past medical history revealed longstanding substance abuse with a previous right forearm compartment syndrome .
6 years prior , she was found unconscious for an unknown duration on her right arm while in a drug - induced haze . despite emergent dorsal and volar fasciotomy ,
extensive myonecrosis was observed intraoperatively and she , subsequently , developed a volkmann loped a volkman .
clinical examination of the contracture demonstrated a static wrist flexion contracture of 90 with clawing deformities of the small and ring fingers lacking any functional pronation or supination .
the thumb was held in a fixed flexion deformity , while the index and long fingers exhibited a 35 extension contracture at the metacarpal phalangeal joints with limited motion at the distal interphalangeal and proximal interphalangeal joints .
her elbow range of motion was limited to a 35 arc of motion ( 75 - 110 ) . despite these deformities ,
the patient reported her right upper extremity was integral in her activities of daily living .
she has been able to drive an automatic transmission vehicle using her left upper extremity without difficulty despite her contracture on the right .
anteroposterior and lateral radiographs of the right forearm demonstrating a severely displaced , rotated , and shortened both bone forearm fracture .
a dorsal approach between extensor carpi radialis brevis and extensor digitorum comminus was utilized as previous skin grafting and fasciotomy scarring precluded the use of a volar incision ( fig .
2 ) . despite a general anesthesia , paralytics and a supraclavicular block , intraoperative restoration of forearm length was impossible due to the forearm contracture .
ulnar and radial osteotomies of 2.1 cm were required to achieve reduction of the forearm with two 7-hole limited contact dynamic compression plates ( fig .
the osteotomy of 2.1 cm was the minimal amount of bone resection possible that would allow us to realign the fracture .
no soft tissue , release was performed given her longstanding contractures and to minimize compromise of soft tissues for fracture site healing .
post - operative evaluation demonstrated no complications , well - healed incisions , intact hardware , and a maintained forearm reduction ( fig .
her range of motion of the elbow and wrist were unchanged from her preoperative examination ( 75 - 110 elbow motion and a fixed 90 wrist flexion contracture ) , and she fully regained her preoperative functional ability .
posteroanterior and lateral postoperative radiograph of the right forearm demonstrating restoration of cortical alignment of radius and ulna with intact hardware .
treating patients with fractures through a contracted extremity is a complicated process requiring careful preoperative planning . to obtain a successful outcome
, the surgeon requires an understanding of not only the acute injury but also the details surrounding the chronic contracture . in the described case ,
previous medical documentation detailed the prior fasciotomy incisions and the status of the volar compartment . applying this historical knowledge to the current injury
, it was determined that a volar approach to the radius would be compounded by abnormal anatomy , fibrotic muscle , and potential wound coverage issues .
a surgical approach minimizing exposure through contracted tissues is important for surgical anatomy and post - operative wound healing .
for these reasons , a dorsal approach to the radius afforded a safer and less complicated approach for fracture fixation in this patient . while restoration of forearm length , radial bow , and rotation are the principles of bb fracture care in the noncontracted adult patient , a bb fracture in a volkmann s contracture may require less emphasis on these principles given each patient s preoperative function . in a contracted forearm , fibrotic musculature maintains a set length due to the radius and ulna being intact . in the setting of a bb fracture ,
this fibrotic , inelastic muscle mass no longer maintains its length , and shortening of the fracture segments occurs .
when traditional methods of restoring length and rotation fail due to the pre - existing contracture , achieving union must become the most important goal .
this may require radial and ulnar shortening osteotomies at the cost of restoring an anatomic radial bow . while the postoperative function of noncontracted patients with a bb fracture has been correlated with the restoration of the radial bow
an understanding of preoperative function in the fractured , contracted extremity can help dictate intraoperative decision making when preinjury length can not be obtained .
the patient in this report had limited function of the contracted extremity but stated she routinely used her contracted forearm for tasks such as carrying groceries .
thus , it was determined that achieving a bony union with excellent cortical contact would continue to afford the same postoperative function once healed , despite the loss of anatomic radial bow . while post - operative vigilance is required for any patient with a surgically treated bb fracture , more frequent clinical and radiographic follow - up is required in the setting of a bb fracture through a contracted forearm
. decreased vascularity at the fracture site and the soft tissues as a result of previous compartment syndrome and fasciotomy scarring can compromise fracture and wound healing .
the dermis in volkmann s contracture may exhibit areas of skin grafts , contractures , and inelastic scar tissue . in normal fracture environments ,
this blood supply provides progenitor cells that differentiate into osteoclasts and osteoblasts to contribute to fracture healing [ 13 , 14 , 15 ] . in the setting of a volkmann contracture ,
these abnormal soft tissue conditions result in decreased cutaneous vascularity , which can compromise osseous and wound healing .
depending on the extent of muscle necrosis and vascular damage , the treating surgeon must remain vigilant for the development of a delayed or atrophic nonunion .
alteration of normal bb postoperative protocols may require prolonged immobilization , and weight bearing restrictions to reflect this delayed osseous healing .
although the overall incidence of untreated adult forearm compartment syndrome is rare , drug - induced unconsciousness and prolonged external pressure on the forearm has become a more common cause of volkmann s contracture in the adult population . as illicit substance abuse continues to rise , so does the risk of untreated forearm compartment syndrome . a general knowledge of operative care and potential pitfalls in the treatment of bb fractures in a contracted upper extremity must be understood .
this is the first report in the literature describing , the management of a bb fracture through a pre - existing volkmann s ischemic contracture .
this patient demonstrated significant pain , fracture displacement , and acceptable pre - operative function , which precluded conservative management .
orif was performed with an ulnar and dorsal forearm approach , which minimized exposure through previously operated volar contracted tissues . when managing a bb fracture in a volkmann s contracture , extensive radial and ulnar shortening osteotomies may be required to regain cortical contact , fracture compression , and an acceptable alignment .
frequent radiographic and clinical evaluation in the postoperative period is imperative for managing these complex upper extremity injuries .
it is important to determine preoperative function in developing a surgical plan to treat the patients with fractures through a contracted extremity .
restoration of length , radial bow , and rotation often becomes secondary to achieving union in these patients , and shortening osteotomies may be necessary to restore bony apposition .
careful pre - operative planning and consideration of the soft tissue envelope is critical in a successful approach .
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introduction : there is a paucity of information on management of forearm fractures through pre - existing ischemic contractures .
the prevention of a volkmann s contracture in forearm compartment syndrome requires vigilant clinical evaluation and emergent fasciotomy , but many of the patients who develop these contractures often do so as a result of delayed presentation due to substance abuse and intoxication .
this case describes the first report and management of a severely displaced both bone ( bb ) forearm fracture through a chronic forearm volkmann s contracture.case report : a 39 year old female presented with an acute both bone forearm fracture in the setting of a volkmann s contracture .
although very limited in use , the arm was functional for holding objects and was determined to be important in her activities of daily living .
the surgical management involved open reduction internal fixation with radial and ulnar shortening osteotomies to restore cortical alignment secondary to the extensive overlying soft tissue contractures.conclusion:restoration of the radial bow and other standard principles of open reduction and internal fixation of bb fractures may not be as important as obtaining fracture union in patients with these complicated injuries due to the pre - existing functional limitations of the limb .
the soft tissue envelope in a contracted forearm is commonly fibrotic and provides an abnormal fracture healing environment .
careful attention to osseous and soft tissue healing in the postoperative period is recommended .
this report details the first case , technical difficulties , and subsequent management of a bb fracture in the setting of subsequent management of a both bone fracture in the setting of a chronic volkmann s contracture .
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body weight change in patients with rheumatoid arthritis ( ra ) is a complex issue .
on one hand , active disease could lead to weight loss and frank wasting , termed rheumatoid cachexia , which correlates with the intensity of systemic inflammation . on the other hand
, some studies showed that a higher body mass index ( bmi ) is associated with less severe radiographic joint damage in the early phases of the disease .
even , some evidence showed that all - cause mortality and cardiovascular - related mortality rates decrease with increasing bmi in ra patients .
the subject becomes more complex when considering the adipose tissue as an active site that , by releasing adipocytokines , has some immunological effects .
therefore , it seems to be a bi - directional relationship between body weight change and disease activity in patients with ra , which is not only affected by biological factors but also by patients behaviors and life style .
the association of bmi and the amount of body fat with clinical course of ra is still a matter of debate , and clinical applications of current data are not clear . some studies with long follow - up periods have showed that obesity is associated with worse outcomes , while others have found lower bmi to be associated with decreased survival , and higher bmi as being protective . also , some data are available on the association between bmi and treatment response in ra patients . in this
regard , recent studies reported that a higher bmi is associated with less well response to antitumor necrosis factor alpha ( anti - tnf ) agents .
considering the controversial and complex results of the previous studies and lack of data , the primary aim of this prospective study was to determine whether body fat composition affects response to combination therapy with disease - modifying antirheumatic drugs ( dmards ) including methotrexate plus hydroxychloroquine in ra patients .
moreover , ra patients may encounter a decrease in muscle mass that can mask the increase in fat mass and result in having a normal bmi .
therefore , bmi can not distinguish between the tissues that comprise it and may not accurately reflect the body fat content .
hence , the secondary aim of this study was to evaluate , in addition to bmi , the association of other anthropometric measurements including waist and hip circumferences and waist to hip ratio with treatment response , because they can provide more accurate information about the body fat distribution .
this observational cohort study was conducted on patients with ra ( 16 to 80 year old ) referred to the rheumatology clinic of a university hospital in isfahan ( central iran ) in 2012 .
consecutive patients who met the american college of rheumatology ( acr ) criteria for the classification of ra and had active disease based on the disease activity score using 28 joint counts ( das28 ) > 2.6 were included .
those with previous toxicity to combination therapy with methotrexate plus hydroxychloroquine , and those with significant change of bmi ( 10% ) in the preceding three months were not included . also , we excluded patients whose treatment contained dmards or drugs other than methotrexate plus hydroxychloroquine ( e.g. sulfasalazine ) . considering type - i error = 0.05 ,
study power = 0.8 , and expecting correlation between bmi and das28 as at least 0.3 , the sample size was calculated as 106 cases .
the ethics committee of the isfahan university of medical sciences approved the study and informed consent was obtained from all patients .
an internist interviewed with all patients and gathered demographic data ( age , gender ) and clinical data ( disease duration , previous therapies ) from patients history and documents .
height , weight , and waist and hip circumferences were measured by an educated nurse .
body mass index ( calculated as weight in kilograms divided by the square of height in meters ) was categorized into underweight ( < 18.5 kg / m ) , normal ( 18.5 - 24.9 kg / m ) , overweight ( 25 - 29.9 kg / m ) , and obese ( 30 kg / m ) .
patients were examined by a single rheumatologist who evaluated the number of swollen / tender joints , patient 's global activity ( general health ) on a visual analog scale ( vas , 0 to 100 ) , and disease functional class based on the american college of rheumatology 1991 revised criteria .
all patients were referred to a reference laboratory for measurement of erythrocyte sedimentation rate ( esr ) and c - reactive protein ( crp ) . finally , das28 was calculated based on the formula of das28-esr ; 0.56 (tjc28 ) + 0.28 (sjc28 ) + 0.70 lognat ( esr ) + 0.014 vas , and classified as in remission ( < 2.6 ) , low ( 2.6 to < 3.2 ) , moderate ( 3.2 to 5.1 ) , and high ( > 5.1 ) disease activity .
most of the patients have been under treatment with methotrexate monotherapy or combination therapy with methotrexate plus hydroxychloroquine when they were evaluated for entering to our study .
considering response to previous therapies , the clinician started combination therapy ( or increased drugs dose ) with methotrexate ( 7.5 to 10 mg / week ) plus hydroxychloroquine ( 200 to 400 mg / day ) and prednisolone ( 2.5 to 10 mg / day ) based on the clinical course .
dosage of treatment and change of the treatment for patients was registered in data gathering form .
patients were followed for 24 weeks and das28 was calculated with the same rheumatologist again every 12 weeks .
correlation between variables was evaluated using the pearson test if data were parametric and spearman test if not normally distributed .
comparisons between those with and without response to treatment were done using chi - square test for qualitative variables and independent sample t - test and mann - whitney test ( if not normally distributed ) for quantitative variables . because the clinical response was defined based on the change in the das28 values , ancova and regression analyses were applied to correct for the baseline das28 while analyzing the effect of bmi on treatment response ( das28 )
this observational cohort study was conducted on patients with ra ( 16 to 80 year old ) referred to the rheumatology clinic of a university hospital in isfahan ( central iran ) in 2012 .
consecutive patients who met the american college of rheumatology ( acr ) criteria for the classification of ra and had active disease based on the disease activity score using 28 joint counts ( das28 ) > 2.6 were included .
those with previous toxicity to combination therapy with methotrexate plus hydroxychloroquine , and those with significant change of bmi ( 10% ) in the preceding three months were not included . also , we excluded patients whose treatment contained dmards or drugs other than methotrexate plus hydroxychloroquine ( e.g. sulfasalazine ) . considering type - i error = 0.05 ,
study power = 0.8 , and expecting correlation between bmi and das28 as at least 0.3 , the sample size was calculated as 106 cases .
the ethics committee of the isfahan university of medical sciences approved the study and informed consent was obtained from all patients .
an internist interviewed with all patients and gathered demographic data ( age , gender ) and clinical data ( disease duration , previous therapies ) from patients history and documents .
height , weight , and waist and hip circumferences were measured by an educated nurse .
body mass index ( calculated as weight in kilograms divided by the square of height in meters ) was categorized into underweight ( < 18.5 kg / m ) , normal ( 18.5 - 24.9 kg / m ) , overweight ( 25 - 29.9 kg / m ) , and obese ( 30 kg / m ) .
patients were examined by a single rheumatologist who evaluated the number of swollen / tender joints , patient 's global activity ( general health ) on a visual analog scale ( vas , 0 to 100 ) , and disease functional class based on the american college of rheumatology 1991 revised criteria .
all patients were referred to a reference laboratory for measurement of erythrocyte sedimentation rate ( esr ) and c - reactive protein ( crp ) . finally , das28 was calculated based on the formula of das28-esr ; 0.56 (tjc28 ) + 0.28 (sjc28 ) + 0.70 lognat ( esr ) + 0.014 vas , and classified as in remission ( < 2.6 ) , low ( 2.6 to < 3.2 ) , moderate ( 3.2 to 5.1 ) , and high ( > 5.1 ) disease activity .
most of the patients have been under treatment with methotrexate monotherapy or combination therapy with methotrexate plus hydroxychloroquine when they were evaluated for entering to our study .
considering response to previous therapies , the clinician started combination therapy ( or increased drugs dose ) with methotrexate ( 7.5 to 10 mg / week ) plus hydroxychloroquine ( 200 to 400 mg / day ) and prednisolone ( 2.5 to 10 mg / day ) based on the clinical course .
dosage of treatment and change of the treatment for patients was registered in data gathering form .
patients were followed for 24 weeks and das28 was calculated with the same rheumatologist again every 12 weeks . a decrease of > 1.2 score in das28 was defined as response .
correlation between variables was evaluated using the pearson test if data were parametric and spearman test if not normally distributed .
comparisons between those with and without response to treatment were done using chi - square test for qualitative variables and independent sample t - test and mann - whitney test ( if not normally distributed ) for quantitative variables . because the clinical response was defined based on the change in the das28 values , ancova and regression analyses were applied to correct for the baseline das28 while analyzing the effect of bmi on treatment response ( das28 ) .
from a total of 140 patients investigated during the study period , 19 patients were under treatment with dmards or drugs other than methotrexate plus hydroxychloroquine including sulfasalazine , tacrolimus , and cyclosporine .
finally , 106 patients were included to the analyses and completely studied ; ( 87.7% female ) with mean age of 48.5 13.8 years and mean disease duration of 4.4 years ( se = 0.48 ) .
demographic data and clinical characteristics of all participants are presented in [ table 1 ] .
demographic data and disease characteristics of the patients including all patients , there was a negative correlation between bmi and baseline das28 ( r = 0.217 , p = 0.026 ) .
das28 score was decreased from 4.5 1.6 to 3.3 1.5 after 12 weeks of treatment ( p = 0.005 ) , and then was decreased to 2.9 1.4 after 24 weeks from starting the treatment ( p < 0.001 ) .
the association between bmi and das28 after therapy was weak and not significant ( r = 0.132 , p = 0.177 ) .
regarding other anthropometric measures , there was no association of waist circumference or waist to hip ratio with baseline disease activity or with das28 after therapy [ table 2 ] .
correlation of demographic data with baseline and change in disease activity with regards to das28 components , bmi was associated with baseline vas score ( r = 0.198 , p = 0.040 ) .
also , bmi ( r = 0.301 , p = 0.001 ) and waist circumference ( r = 0.185 , p = 0.020 ) were associated with the baseline number of swollen joint [ table 2 ] . regarding changes in das28 components after therapy
, there was only a week association between bmi and changes in esr ( r = 0.196 , p = 0.043 ) . defining response as a decrease of >
1.2 score in das28 , comparison of responders and non - responders is presented in [ table 3 ] .
responders were not different from non - responders in terms of age , gender , disease duration , and anthropometric data .
although there was a trend for non - responders to be more obese ( 41.3% vs. 25% , p = 0.184 ) and having higher bmi ( 28.9 5.5 vs. 27.6 4.0 , p = 0.179 ) than responders , this differences were not statistically significant .
comparison of responders with nonresponders we categorized the patients to those with disease duration of 2 years indicating early disease and those with disease duration of > 2 years . in those with early disease stage , analysis showed a significant and strong association of bmi ( r = 0.415 , p = 0.005 ) and waist circumference ( r = 0.296 , p = 0.05 ) with baseline das28 .
bmi was also associated with the number of swollen joints ( r = 0.415 , p = 0.005 ) and tender joints ( r = 0.338 , p = 0.025 ) , and with vas score ( patient 's global activity ) ( r = 0.447 , p = 0.002 ) . in this group of patients ,
bmi ( r = 0.337 , p = 0.025 ) and waist circumference ( r = 0.315 , p = 0.038 ) were also correlated with das28 after therapy [ table 4 ] . also , responders had higher bmi ( 31.0 5.5 vs. 27.1 4.2 ) and greater waist circumference ( 95.8 11.5 vs. 87.7 11.6 ) , [ table 5 ] .
none of the aforementioned associations or differences was present in patients with disease duration of > 2 years .
correlation of demographic data with baseline and change in disease activity in patients of early disease stage comparison of responders with nonresponders in those with early disease stage considering the difference between responders and non - responders in baseline das28 and also considering correlation between bmi and baseline das28 , ancova was applied to correct for the baseline das28 .
this analysis showed no influence of bmi on das28 ( f = 1.523 , p = 0.223 ) . also , a linear regression analysis controlling for age , gender , disease duration , baseline das28 , and methotrexate , hydroxychloroquine , and prednisolone total received dose showed no association between bmi and the amount of change in das28 ( = 0.019 , 95% ci : 0.058 to 0.073 ) , but there was a significant association between baseline das28 with das28 ( = 0.819 , 95% ci : 0.714 to 1.145 ) , [ table 6 ] .
by separate insertion of waist circumference and waist to hip ratio instead of bmi into the linear regression model , there was no association between these anthropometric measures and das28 after therapy .
also , separate linear regression analysis in those with disease duration of 2 years and > 2 years did not change these results .
from a total of 140 patients investigated during the study period , 19 patients were under treatment with dmards or drugs other than methotrexate plus hydroxychloroquine including sulfasalazine , tacrolimus , and cyclosporine .
finally , 106 patients were included to the analyses and completely studied ; ( 87.7% female ) with mean age of 48.5 13.8 years and mean disease duration of 4.4 years ( se = 0.48 ) .
demographic data and clinical characteristics of all participants are presented in [ table 1 ] .
including all patients , there was a negative correlation between bmi and baseline das28 ( r = 0.217 , p = 0.026 ) .
das28 score was decreased from 4.5 1.6 to 3.3 1.5 after 12 weeks of treatment ( p = 0.005 ) , and then was decreased to 2.9 1.4 after 24 weeks from starting the treatment ( p < 0.001 ) .
the association between bmi and das28 after therapy was weak and not significant ( r = 0.132 , p = 0.177 ) .
regarding other anthropometric measures , there was no association of waist circumference or waist to hip ratio with baseline disease activity or with das28 after therapy [ table 2 ] .
correlation of demographic data with baseline and change in disease activity with regards to das28 components , bmi was associated with baseline vas score ( r = 0.198 , p = 0.040 ) .
also , bmi ( r = 0.301 , p = 0.001 ) and waist circumference ( r = 0.185 , p = 0.020 ) were associated with the baseline number of swollen joint [ table 2 ] . regarding changes in das28 components after therapy
, there was only a week association between bmi and changes in esr ( r = 0.196 , p = 0.043 ) . defining response as a decrease of > 1.2 score in das28 , comparison of responders and non - responders
responders were not different from non - responders in terms of age , gender , disease duration , and anthropometric data .
although there was a trend for non - responders to be more obese ( 41.3% vs. 25% , p = 0.184 ) and having higher bmi ( 28.9 5.5 vs. 27.6 4.0 , p = 0.179 ) than responders , this differences were not statistically significant .
we categorized the patients to those with disease duration of 2 years indicating early disease and those with disease duration of > 2 years . in those with early disease stage , analysis showed a significant and strong association of bmi ( r = 0.415 , p = 0.005 ) and waist circumference ( r = 0.296 , p = 0.05 ) with baseline das28 .
bmi was also associated with the number of swollen joints ( r = 0.415 , p = 0.005 ) and tender joints ( r = 0.338 , p = 0.025 ) , and with vas score ( patient 's global activity ) ( r = 0.447 , p = 0.002 ) . in this group of patients ,
bmi ( r = 0.337 , p = 0.025 ) and waist circumference ( r = 0.315 , p = 0.038 ) were also correlated with das28 after therapy [ table 4 ] .
also , responders had higher bmi ( 31.0 5.5 vs. 27.1 4.2 ) and greater waist circumference ( 95.8 11.5 vs. 87.7 11.6 ) , [ table 5 ] .
none of the aforementioned associations or differences was present in patients with disease duration of > 2 years .
correlation of demographic data with baseline and change in disease activity in patients of early disease stage comparison of responders with nonresponders in those with early disease stage considering the difference between responders and non - responders in baseline das28 and also considering correlation between bmi and baseline das28 , ancova was applied to correct for the baseline das28 .
this analysis showed no influence of bmi on das28 ( f = 1.523 , p = 0.223 ) . also , a linear regression analysis controlling for age , gender , disease duration , baseline das28 , and methotrexate , hydroxychloroquine , and prednisolone total received dose showed no association between bmi and the amount of change in das28 ( = 0.019 , 95% ci : 0.058 to 0.073 ) , but there was a significant association between baseline das28 with das28 ( = 0.819 , 95% ci : 0.714 to 1.145 ) , [ table 6 ] . by separate insertion of waist circumference and waist to hip ratio instead of bmi into the linear regression model
, there was no association between these anthropometric measures and das28 after therapy . also , separate linear regression analysis in those with disease duration of 2 years and > 2 years did not change these results .
the purpose of this study was to determine if bmi or other anthropometric measures including waist circumference and waist to hip ratio are associated with disease activity and clinical response to combination therapy with methotrexate and hydroxychloroquine in ra patients .
with regard to the association between obesity and disease activity at the study entry , we found an inverse correlation of bmi with baseline disease activity . after separating patients based on disease duration , this association was only present in patients with disease duration of 2 years .
the association between waist circumference and baseline disease activity was also only present in these patients .
these results were similar to some previous studies that have found a possible protective role for higher bmi in ra patients at early disease stages . also , we found an association of bmi with clinically swollen and tender joint counts and also with patient 's global activity in early disease stage .
this finding was similar to radiologic studies in patients with early ra , of up to 3-year duration , indicating the protective role of higher bmi against joint damage .
however , it must be noted that bmi was no longer associated with disease activity or separately with its components in patients with disease duration of more than 2 years .
this finding indicates that the possible protective effects of obesity in early ra may be diminished later in the course of the disease .
even , some studies have showed a direct association between obesity and disease activity in patients with long - time ra . in advanced ra ,
both underweight and obese states are associated with worse disease activity ; an active disease lead to loss of lean body tissue and better control of the disease is associated with weight gain .
these findings highlight the complex relationship between adiposity and obesity and clinical course in ra patients .
adipose tissue produces adipocytokines with various pro - inflammatory and anti - inflammatory effects , but the exact mechanisms behind the immune - modulatory effects of adipose tissue in ra patients are yet unexplored . with regard to the effects of obesity on treatment response , we found an inverse correlation of bmi and waist circumference with the amount of change in das28 after therapy in patients at early disease stage .
however , after controlling baseline disease activity , these correlations were no longer existed , indicating a large confounding effect of the baseline disease activity .
in contrast to these results , previous studies showed a high bmi associated with less well response to anti - tnf agents even after adjustment for the baseline disease activity .
found that ra patients with a higher bmi have a more active disease at study entry and respond less well to infliximab .
the study by gremese and colleagues in a larger sample of patients ( n = 641 ) and longer treatment period ( 12 months ) with anti - tnf blockers ( adalimumab , etanercept , and infliximab ) found no association between bmi and disease activity at baseline , but better response to infliximab in those with lower bmi and lower disease activity at baseline . in another recent study by heimans and colleagues ,
508 patients were allocated to initial monotherapy with methotrexate or combination therapy of methotrexate with prednisone or infliximab for 1 year .
authors found that higher bmi was associated with failure to achieve response ( a das 2.4 ) on initial therapy with methotrexate . in this study , high bmi ( 25 kg / m )
was also associated with failure on delayed combination therapy with infliximab , and in the first year after starting the study , patients with a high bmi had higher das , worse functional ability , more tender joints and a higher vas global health .
but , high bmi in this study was not associated with more swollen joints or systemic inflammation .
differences between these findings and ours may be related to different patients characteristics and different treatments .
patients in the mentioned studies had longer disease duration . also , our patients had significantly higher bmi ( 28.2 kg / m ) compared with the mentioned studies ( 24.9 and 26 ) , while disease activity score was lower in our study ( 4.5 vs. 5.6 and 5.9 ) .
anyway , the exact role of bmi in response to various treatments in patients with various disease stages is yet to be clarified .
it has been suggested that the association of obesity with disease activity and response to treatment in ra patients may be due to high levels of proinflammatory cytokines produced by adipocytes .
waist circumferences and waist to hip ratio provide more accurate information about the body fat distribution .
hence , we evaluated these parameters to better investigate if adipose tissue has a role in creating an inflammatory and therapy - resistant state in ra patients .
however , compared with bmi , we found smaller correlation coefficient of waist circumference and no association of waist to hip ratio with disease activity . in our study as well as in previous studies , bmi and waist circumference
these findings are not in favor of a mechanism involving adipose tissue - derived mediators of inflammation .
it should be considered that clinical synovitis might be less easy to assess in ra patients with obesity and it is possible that our study as well as others underestimated joint swelling , associated with a local inflammation , in patients with a high bmi .
more investigations including advanced imaging and biomarker studies are needed to further elucidate the relation between bmi and disease activity as well as treatment response .
the association between obesity and treatment response in our study has been confounded by baseline disease activity .
indeed , a larger sample of patients was required for more precise evaluation of the role of obesity on various components of ra activity as well as in different disease stages , and consideration of possible confounders .
moreover , our study period was six months which , compared to other studies , was not long enough . although , in addition to bmi , we investigated waist circumference and waist to hip ratio which provide more accurate information about the body fat distribution , these are not the most accurate measures , and also we did not evaluate adipocytokines , the hypothesized underlying mediators .
our study results showed that obesity is associated with less severe disease activity in early stage of ra , but is not associated with response to combination therapy with methotrexate plus hydroxychloroquine in ra patients .
however , due to our study limitations , these results should be considered with cautious .
further cohorts with larger sample size and longer follow - ups , and with more accurate investigation of body adipose composition and possible immunological mechanisms , are warranted in this regards .
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background : the role of obesity in clinical curse of rheumatoid arthritis ( ra ) is not clear . we investigated the association of obesity and adiposity with disease activity and clinical response to combination therapy in ra patients.materials and methods : active ra patients with the disease activity score using 28 joint counts ( das28 ) > 2.6 were studied .
height , weight , and waist and hip circumferences were measured and body mass index ( bmi ) and waist to hip ratio were calculated .
patients were treated with methotrexate ( 7.5 to 10 mg / week ) plus hydroxychloroquine ( 200 to 400 mg / day ) and prednisolone ( 2.5 to 10 mg / day ) and were followed by das28 for up to 24 weeks.results:one hundred and six patients were studied ; age = 48.5 13.8 years , 87.7% female , disease duration = 4.4 years [ se = 0.48 ] .
das28 was decreased from 4.5 1.6 to 2.9 1.4 ( p <
0.001 ) after 24 weeks of treatment . only in patients with disease duration of 2 years , bmi ( r = 0.415 , p = 0.005 ) and waist circumference ( r = 0.296 , p = 0.05 ) were correlated with baseline das28 .
although bmi ( r = 0.337 , p = 0.025 ) and waist circumference ( r = 0.315 , p = 0.038 ) were correlated with change in das28 after therapy , these correlations were disappeared after controlling for baseline das28.conclusion:obesity and adiposity are associated with less severe disease activity in early stage of ra , but are not associated with response to combination therapy with methotrexate plus hydroxychloroquine in ra patients .
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fg was first described by baurienne in 1764 and is named after a french venereologist , jean alfred fournier , who in 1883 , described a rapidly progressive gangrene of the penis and scrotum without apparent cause .
fournier 's gangrene ( fg ) is a potentially life threatening progressive infection of the perineum and genitalia . despite aggressive modern management , the mortality of fg ranges from 16% to 40% .
anatomically , the disease affects male external genitalia at the level of the superficial and deep fascial plains , often sparing the deep muscular structures and to variable degrees internal organs .
branches from the inferior epigastric and deep circumflex iliac arteries supply the lower aspect of the anterior abdominal wall .
moreover , branches of the external and internal pudendal arteries supply the scrotal wall . with the exception of the internal pudendal artery ,
each of these vessels travels within camper`s fascia and can therefore become thrombosed in the progression of fournier 's gangrene .
pathologically , the condition is a polymicrobial necrotizing fasciitis of the perineal , perianal , and external genitalia that might extend up to the anterior abdominal wall skin , superficial and deep structures .
the testes are seldom involved as they are primarily supplied by direct branches of the abdominal aorta .
diagnosis is clinically based as patients present with intense pain and tenderness in the genitalia .
the affected area is swollen , dusky and covered by macerated skin and presents with a feculent odor .
the medical and operative records from january 2010 to january 2013 were reviewed for those pertaining to fg at our hospital .
data was collected on patients demographics , comorbidity , survivals , fsi at presentation , the source of infection , length of hospital stay ( los ) , the number of icu and operation room ( or ) entries exclusively for urologic service , the total phalli and testes removed , colostomies created , and cystostomies performed . as the infection is polymicrobial , we standardized the antibiotic protocol to cover gram positive , gram negative , and anaerobic bacteria ; providing tazocin ( piperacillin sodium and tazobactam sodium ) 4.5 g intravenously , every eight hours , plus dalacin c ( clindamycin ) 600 mg intravenously , every six hours .
aziz hospital at holy makkah , is a general hospital based on the high traffic junction between jeddah and al madenah highways .
the human geography of the hospital put a special emphasis on our study ; as the number of hajji`s , and the admission dates in relation to hajj season were considered .
all 20 cases ( 100% ) collected for the study survived , despite the known fatality of the condition .
the median age of the patients was 55.95 years ( 3978 years ) on the day of admission .
only one patient ( 5% ) was admitted to intensive care unit ( icu ) post operatively because the operation aimed to track and cleanse the offensive discharge up to the left renal fossa .
penile amputation was carried out in three out of the 20 cases , accounting ( 15% ) of the total , in where the pathology was limited to the phallus .
the total number of or entries for wound debridement by a urologist was 34 entries making an average of 1.7 entry per patient .
remarkably , half of the candidates involved in the study , ( ten victims ) were hajji`s .
hajj months ( the month 11 and 12 of hijri islamic calendar ) was 14 ( 70% ) , constituting a noticeable seasonal peak .
a majority of 12 patients , forming ( 60% ) of the total , were diabetic and admitted with poorly controlled type ii dm and sepsis .
a sum of six testes extirpated from the whole study group were due to unusual involvement of testes in the gangrene process , as testes blood supply are derived separately from the abdominal aorta via paired gonadal arteries ( also called the internal spermatic arteries in older texts ) .
none of the patients required colostomy creation . only one patient constituting ( 5% ) required a cystostomy tube insertion to divert the urine suprapubically in an attempt to deal with a cleaner wound .
the sources were anorectal in five cases , dermal in three cases and urologic in three cases . in remaining nine cases ,
the average length of hospital stay was 22.3 days ranging from 1 to 76 days ( table 1 ) .
although fg is a rare life threatening disease , it is not uncommon for a practitioner in our hospital to share fg management on the surgical floor , critical care unit , operating theater , or emergency room . as fg is a urologic emergency ,
the sufferer will be booked for urgent surgical debridement at day one of the admission .
these patients will often require a second look operation after 24 to 48 hours to exclude further disease progression .
we believe that the excellent survival rates together with the low number of debridement attempts are attributed to aggressive initial debridement shortly after admission and to the daily or twice daily bedside dressing coupled with the comprehensive intravenous antibiotic coverage .
the standard of antibiotics we set was piperacillin 4 g ( an extended spectrum beta lactam antibiotic of the ureidopenicillin class ) coupled with tazobactam 0.5 g ( beta lactamase inhibitor ) in a single formula ; in combination with clindamycin ( a lincosamide antibiotic used to treat infections with anaerobic bacteria ) .
our antibiotics standard therefore , prevent gram negative and positive bacteria including pseudomonas aeruginosa and anaerobes .
we do not use hyperbaric oxygen , nor vacuum assisted closure ; we would rather use povidone
iodine 10% , anti septic solution after excising visibly dead tissue tags as a daily bedside wound refreshment procedure under narcotic injection .
we save both testes in the inguinal canal marked by a long silk tie in order to bring them down later in the plastic reconstruction operation .
cases where the infection started anorectally were more difficult , as general surgeons had to share in the initial debridement procedure to secure the anal sphincter and to create a colostomy if needed .
it is worth mentioning that the fsi calculation did not add a significant value to our study , as fsi parameters are focused on the acuity of the case upon admission regarding vitals and electrolytes imbalance ; in other words , no tangible reflection or clue to surgical morbidity .
no case was sent for plastic reconstruction prior to havinging clean wound with three negative wound
swab cultures and normal albumin value . although in 1924 meleney attributed necrotizing infections to mainly streptococcal species , in our study acinetobacter species recovered .
despite the fact that fg is not exclusive for males , there were no female patients in our study .
the multinationality of the candidates and the seasonal peak of the disease occurrence were striking features .
nowadays , al hajj season corresponds to october and november in gregorian months when the weather is modest .
we expect to face more fg cases if the study was to be conducted in summer months ; as in al
hajj dynamic , heat and strenuous physical activities demand more cleanliness and better hygiene ( figures 16 ) .
in comparison to what is published in literature , fg is not considered rare for a practitioner in king abdul
the excellent survival rate recorded is believed to be due to the moderate severity of the cases treated , the comprehensive antibiotic coverage and mostly , to the surgical intervention timing and the extensive initial surgical debridement of dead and marginally viable tissues .
the figures presented do not reflect saudi nationals , as the study candidates were multinational .
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introductionfourniers gangrene ( fg ) , is a rare life threatening urologic emergency that requires immediate admission of metabolic stabilization and surgical debridement .
the mortality rate ranges from 16% to 40% .
this study was conducted to investigate the reasons behind the excellent survival rate in our center.materials and methoda retrospective analysis of the medical records of 20 fg cases from january 2010 to january 2013 was conducted .
data was collected on patients comorbidity , survivals , fourniers severity index ( fsi ) at presentation , length of hospital stay ( los ) , the number of intensive care unit ( icu ) and operating room ( or ) entries , the total phalli and testes removed , colostomies created , and cystostomies performed.resultsall 20 cases ( 100% ) collected for the study survived .
all patients were male .
the median age of the patients was 55.95 years ( 3978 year ) .
only one patient ( 5% ) was admitted to icu post operatively .
penile amputation was carried out in three of the 20 cases ( 15% ) .
the total number of or entries was 34 ( average of 1.7 ) .
a majority of 12 patients ( 60% ) were diabetic .
a total of six testes were extirpated from the study group .
none of the patients required colostomy creation .
only one patient ( 5% ) required a cystostomy tube insertion .
fsi was 5.65 ranging from 0 to 14 .
the average length of hospital stay was 22.3 days.conclusionin contrast to what is published in the literature , fg is not rare in our center .
perfect survival rate owes to the moderate severity of the cases treated , but mostly to the urgent surgical intervention with extensive debridement .
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fibromyalgia ( fm ) is predominantly found in women1 ,
2 and is characterized by widespread
chronic pain3 .
other symptoms including
allodynia , sleep disturbances , fatigue , depression , and cognitive impairment4 are also observed .
patients with fm are treated with pharmacotherapy , functional therapy , cognitive behavioral
therapy and alternative therapies5,6,7,8 . however , because the cause of fm is
unclear , there is no clinically effective treatment method at present . for most patients
,
pharmacotherapy can not reduce the pain as markedly as they expect , and reduction of pain is
difficult because of the indefinite nature of the symptoms .
bengtsson et al . performed stellate ganglion block ( sgb ) in eight fm patients and reported
its effects on reducing both the number of tender points and pain at rest9 . however , noninvasive methods for sgb have
been suggested for several reasons .
first , fm patients tend to feel excessive pain from the
needle injection during sgb .
second , complications can occur during sgb such as intravenous
or intra - arterial injection .
third , serious adverse effects following sgb , such as death
caused by retropharyngeal hematoma , have been reported10,11,12 .
yoshida et al . reported that xenon light irradiation on the skin surface at the needle
insertion site in sgb produced sympathetic block effects similar to those of conventional
sgb13 .
many studies have shown the
effects of stellate ganglion irradiation ( sgi ) with various types of light in patients with
diverse diseases who responded to sgb14 , 16 .
sgi , with its lack of serious side
effects , may be a promising treatment method that could replace sgb13 , 15 .
we performed sgi in patients with primary fm by using xenon light generated by
high - intensity electrical stimulation of xenon gas and evaluated its effects on pain .
this study was initiated after obtaining approval from the ethics committee of daiichi
rehabilitation hospital on may 17 , 2013 ( fms-0003 ) .
the study group consisted of 5 men and
22 women ( age , 56.4 16.3 years [ range , 2584 years ] ; fm duration , 3.6 2.4 years [ range ,
110 years ] ) ( table 1table 1.patients characteristics , changes in the visual analogue scale ( vas ) score , and
duration of the immediate effects of stellate ganglion irradiation ( sgi)patient no.agesexage of onsetduration of fm ( year)j - fiq%vasduration of immediate effects155f51460.1 100225f21442.1 100365f63232.5 100445fncnc27.6
100545f37870.9 9024 hours658f56283.9 100784f83184.1 81no answer844f43159.2 6324 hours984m83158.8 8023 hours1049m47275.6 9424 hours1171f66570.0 9012 hours1240m37376.9 92no answer1338m35348.2 1001474f641079.2
1002142f37545.6 1002271f70168.3 87no answer2371fncnc46.9 72no answer2474f73128.4 1062536m28862.0 1002680f77339.9 1002751f48395.6 100%vas = ( vas score after sgi / that before sgi ) 100 nc : not clear ) who visited the fm clinic of daiichi rehabilitation hospital ( kochi , japan )
between july 2013 and august 2013 .
patients were diagnosed with fm according to the modified
2010 american college of rheumatology ( acr ) diagnostic criteria for fibromyalgia and gave
written consent to participate in this study after proper explanation of the procedure . at
the time of the first consultation , patients were asked about their age and duration since
onset .
bilateral xenon light sgi was performed by physical therapists ( pt ) in the supine
position on a bed ( fig .
1.a : use of emission probes of the xenon phototherapy device for irradiation around the
stellate ganglion region .
b : the emission spectrum of xenon consists of continuous
complex wavelengths ( 3801,100 nm ) .
c : characteristics of the xenon phototherapy
device ( nihon iko co. , ltd . ) ) using a xenon phototherapy device ( auve , nihon iko co. , ltd . ,
tokyo , japan ) for
15 min ( once per second during the first 1-minute period and once every 4 s for the
subsequent 14-minute period ) . before and after irradiation
, the effects on pain were
evaluated by the physical therapists using a visual analogue scale ( vas ) consisting of 101
levels ( 0100 ) .
in addition , the japanese version of the fibromyalgia impact questionnaire
( j - fiq)17 was completed before
irradiation . in responders to xenon light sgi , the duration of the immediate effects ( time
until pain returned to the pre - sgi level ) was evaluated using a selection - type
questionnaire . because all patients were also receiving drug therapy , evaluation of the
long - term effects of xenon - light sgi alone was impossible .
% vas = ( vas score after sgi / that before sgi ) 100 nc : not clear a : use of emission probes of the xenon phototherapy device for irradiation around the
stellate ganglion region .
b : the emission spectrum of xenon consists of continuous
complex wavelengths ( 3801,100 nm ) .
c : characteristics of the xenon phototherapy
device ( nihon iko co. , ltd . ) xenon light , which is generated by high - intensity electrical stimulation of xenon gas , is
used for wound - healing and analgesia , including analgesia against joint pain , as a low - level
light therapy ( lllt)13 .
the emission
spectrum of xenon consists of continuous complex wavelengths ( 3801,100 nm ) ( fig.1b ) .
ultraviolet rays of 400 nm or less are cut
using filters in this device ( auve , nihon iko co. , ltd .
in addition , because
the wavelength range is in the near - infrared region , the risk of burns is low , and
absorption by water , melanin , and hemoglobin is minimal . therefore , xenon reaches deep areas
of the body , with excellent permeability . in general , the light emission pattern is
continuous or pulsed . for treatment of deep areas of the body ,
high energy is necessary .
when light energy is increased using continuous waves , the local temperature rises .
light
energy can be increased while thermal energy is inhibited by using pulse waves employing a
pulse width shorter than the time required for thermal diffusion .
reviewed
studies on lllt performed between 1970 and 2010 and reported stronger effects for pulse
waves than for continuous waves18 .
this
xenon phototherapy device provides high - energy light ( 18 j / pulse ) by reducing the pulse
width to 5/1,000 s. fig.1c shows the device
characteristics ( nihon iko co. , ltd . ) . in the present study , this effect was examined using
the cochran - armitage test for trend ( ca - trend test ) and jmp10 ( sas institute inc . , cary , nc ,
usa ) .
table 1 shows the patients characteristics ,
the changes in the vas score ( % vas = [ vas score after sgi / vas score before sgi ] 100 ) , and
the duration of the immediate effects of sgi . there were 4 patients who did not complete the
questions on the duration of the immediate effects in the questionnaire because they had
only a vague memory of the effect and
could not accurately remember the duration of the
immediate effect when answering the questionnaire .
one patient showed an increase in the vas
score after the therapy , which was considered to result from neck dorsiflexion during
irradiation compared with the posture in daily life .
this adverse effect can likely be
improved by adjusting the pillow height during irradiation .
patients were classified according to the duration after onset into 10 groups ( 110 years )
and according to the severity as established by the j - fiq into 3 groups : ( j - fiq 70.0
( severe ) , 70.0 > j - fiq > 50.0 ( moderate ) , and j - fiq 50.0 ( mild)19 . no clear relationship was detected between the change in
vas score and the duration of fm .
( tables 1 and
2table 2.changes in the visual analogue scale ( vas ) score according to the duration of
fibromyalgia ( fm)duration of fm ( year)12345678910%vas<100 ( people)4121100102%vas=100 ( people)3332100100 ) .
however , a clear relationship was detected between the change in vas score
and the severity of fm .
table 3table 3.changes in the visual analogue scale ( vas ) score according to the severity of
fibromyalgia ( fm)severity%vas=100%vas<100mild82moderate43severe28 shows the number of patients with a decrease or no change in the vas score
after sgi in each group . the vas score decreased in 81% of the patients with a j - fiq score
of 70.0 , in 43% of those with a score of < 70.0 and > 50.0 , and in 11% of those with a
score of 50.0 .
the trend was statistically examined using the cochran - amitage test for
trend , and with a higher pre - sgi j - fiq score , the vas score tended to significantly decrease
after sgi ( p = 0.0036 ) .
next , we used the paired t - test to compare the vas scores before and
after sgi in patients with severe fm .
the main finding in this study is that the vas score decreased significantly after sgi in
patients with a higher j - fiq score .
because the vas for pain , used as the outcome measure in this study , is subjective , a true
double - blind trial with a placebo would have been desirable20 , 21 .
however , placebo
irradiation could not be performed because irradiation produces a specific , warm sensation
in the upper half of the body , so it would be obvious to subjects when the irradiation was a
placebo . in addition , although subjects can be blindfolded so that they can not see light
emission during irradiation , the operator can readily determine whether or not the
irradiation is a placebo based on light emission . in this study ,
only immediate effects were evaluated based on vas scores immediately before
and immediately after sgi . although the vas score decreased compared with the pre - sgi score ,
the effects were transient .
however ,
continuation of this treatment at regular intervals is expected to gradually reduce the
level of pain returning after sgi compared with the pre - sgi level22 .
therefore , if the vas score is measured before each sgi
when sgi is performed regularly , the pre - sgi vas score may decrease in the long term .
however , because all patients were also receiving drug therapy , evaluation of the long - term
effects of xenon sgi alone was impossible .
moreover , the number of patients was not
sufficient for statistical analysis ; therefore , the results of the cochran - amitage test for
trend may be inaccurate .
although our results may have been influenced by the placebo effect
and by an inaccurate statistical analysis , we strongly suggest that sgi may be a beneficial
treatment for fm .
because the cause of fm is unclear at present , there is no definitive
treatment method for fm , and drug therapy also only serves as a palliative method to relieve
pain5 .
some patients endure severe pain
for a long period because drug therapy can not reduce the pain to the degree they
expected23 . in such patients ,
methods
such as sgi that reduce pain ( even slightly ) are beneficial because they facilitate pain
control to improve the quality of life ( qol)24 .
our study suggested stronger effects of sgi in patients with more
severe symptoms , i.e. , with a higher j - fiq score .
treatment methods that reduce pain without side effects
and do not require special training help to improve the qol of fm patients with severe
symptoms24 .
however , studies in various fields have suggested it is caused
by neuroendocrine abnormalities25 ,
neurotransmitter abnormalities26 , or
sympathetic hyperactivity27 . it is
possible that sgi reduces sympathetic overactivity13 ,
which may be more closely associated with pain in fm patients with
a higher j - fiq score , as suggested by our results . in this study , sgi did not induce horner syndrome . it is speculated that sgi has a much
weaker effect than sgb , which may be why the effect observed in the present study was weaker
than that obtained by bengtsson et al9 . in conclusion , in this study , we demonstrated that irradiation of the stellate ganglion
region with xenon ( 0.381.1 m ) decreases the vas score in patients with fm having a higher
j - fiq score , which suggests that stronger effects could be obtained in patients with more
severe fm .
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[ purpose ] the aim of the study was to determine the effect of xenon irradiation of the
stellate ganglion region on fibromyalgia . [ subjects ] the study included 5 men and 22 women
( age , 56.4 16.3 years [ range , 2584 years ] ) who were diagnosed with fibromyalgia
according to the modified 2010 criteria of the american college of rheumatology between
july and august 2013 .
[ methods ] bilateral xenon light irradiation ( 0.381.1 m ) around the
stellate ganglion was performed in the supine position by physical therapists using a
xenon phototherapy device .
we evaluated pain before and after irradiation using the visual
analogue scale .
[ results ] we did not observe a relationship between the change in the
visual analogue scale score and duration of fibromyalgia . however , we observed a
relationship between the change in the visual analogue scale score and the score for the
japanese version of the fibromyalgia impact questionnaire using the cochran - armitage test
for trend .
[ conclusion ] xenon light irradiation of the stellate ganglion significantly
decreased the visual analogue scale score in patients with fibromyalgia having a higher
score in the fibromyalgia impact questionnaire , suggesting that a stronger effect could be
obtained in patients with more severe fibromyalgia .
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peripheral nerves which connect the brain and spinal cord to the body , if injured can lead to neuropathic pain , a chronic debilitating condition manifested as allodynia and hyperalgesia ( brookoff , 2000 ) .
it is a common clinical problem associated with 87% of traumatic conditions and 12% owing to surgeries .
peripheral nerve lesions occur approximately in 2.8% of multiple trauma patients , or 5% root and plexus injured patients ( hulsebosch et al . , 2009 ) .
clinical symptoms are characterized by abnormalities in the pain sensation which may be sensations like shooting pain , burning , tingling , numbness , allodynia and hyperalgesia ( baron et al . , 2010 ) .
peripheral nerve injury results in orchestrated changes similar to the wallerian degeneration leading to structural and functional alterations which affect the whole peripheral nervous system including peripheral nerve endings , afferent fibers , dorsal root ganglion ( drg ) and also central afferent terminals in the spinal cord ( austin et al . ,
the changes include cell body swelling , loss of nissl bodies , and displacement of the nucleus from the center of the neuron to a position near the cell membrane .
after peripheral nerve injury , the nerve derangement , axon degeneration , endoneurial edema and massive demyelination were observed in peripheral nerves whereas there was axon degeneration , swelling and immune cell infiltration in drg and dorsal horns of lumbar spinal cord ( zochodne , 2012 ) .
aftermath of nerve injury includes functional and behavioural deficits which pose challenges for the identification of novel therapeutic strategies for the treatment of neuropathic pain . unfortunately , despite several years of research experience in repair of peripheral nerve , functional recovery after the injury is disappointing .
the available drugs provide symptomatic relief from neuropathic pain and suffer from several limitations like resistance ( opioids ) , dose - limiting side effects ( antidepressants and anticonvulsants ) and no uniform success ( nickel et al . , 2012 ) .
therefore , understanding the exact pathomechanism is necessary for finding better treatment options as well as the development of novel pharmacological interventions .
the next question was to select a suitable experimental model which can be as close as possible to human pathophysiology of trauma / injury induced neuropathy and neuropathic pain .
the animal model of chronic constriction injury ( cci ) of the sciatic nerve was preferred , as it is one of the most commonly used peripheral neuropathic pain models which is a reliable and easily reproducible model ( bennett and xie , 1988 ; austin et al . , 2012 ) .
the constriction of the sciatic nerve by placing 4 loose ligatures around the sciatic nerve proximal to the trifurcation is associated with an epineural inflammatory lesion , intraneural edema , focal ischemia and wallerian degeneration .
the behavioral signs of spontaneous pain , excessive guarding and licking , limping off the ipsilateral hind paw and avoidance of placing weight on the injured paw have been reported ( wang and wang , 2003 ) .
various other behavioral changes like mechanical and thermal hyperalgesia , chemical cold allodynia have been noted to develop within a week with maximal pain - related behaviors and postural asymmetries during the second week of post - surgery .
the pathological alterations in both myelinated and non - myelinated neurons may be responsible for this decrease in nerve conduction velocities ( gabay and tal , 2004 ) .
partial damage to the nerve leads to sensitization of both a and c fibers and thus plays a major role in initiating and maintenance of pain behavior .
hence , it produces unilateral peripheral mononeuropathy and it has been observed that symptoms in this rat model match to causalgia or complex regional pain syndrome is seen in patients suffering from trauma - induced neuropathic pain ( campbell and meyer , 2006 ) .
therefore , evaluating the therapeutic potential of pharmacological interventions in the animal model of cci can be correlated to the clinical trauma - induced neuropathy and neuropathic pain .
then we took an overview of all the known mechanisms reported for contributing to the genesis of neuropathy associated changes .
pathogenesis of nerve injury involves peripheral and central sensitization in which oxidative / nitrosative stress play a major role .
prominently the role of various transcription pathways like nuclear factor-b ( nf-b ) , p38 mitogen - activated protein ( map ) kinase ( mapk ) , jun amino - terminal kinases ( jnk ) , wnt/-catenin has been well studied and reported ( hulsebosch et al . , 2009 ) .
under such extreme stress conditions , hydroxyl radicals , superoxide and nitric oxide are produced in the cytoplasm by various enzymatic reactions , which are activated by increased intracellular calcium ( areti et al . , 2014 ) .
oxidative modification of neuronal biomolecules leads to mitochondrial dysfunction and drives the cell towards apoptosis ( ott et al . , 2007 ) .
superoxide and nitric oxide can readily convert to a highly reactive and toxic peroxynitrite that attacks nucleic acids .
peroxynitrite , a strong oxidizing and nitrating agent attacks various biomolecules like proteins , enzymes and nucleic acids etc . and results in their malfunctioning ( sandireddy et al . , 2014 ) .
nitrite species generally attack the proteins and render them nonfunctional due to various modifications including nitrosylation .
especially , peroxynitrite radicals attack mitochondrial superoxide dismutase and cause its inactivation ( macmillan - crow et al . , 1998 ) .
it also alters the glutamatergic transmission and inactivates glutamine transferase which helps in the synthesis of endogenous antioxidant , glutathione ( little et al . , 2012 ) .
this results in loss of antioxidant defenses and alters the neurotransmission which aggravates the vicious cycle of oxidative / nitrosative stress and loss of nerve functionality .
in addition to protein nitrosylation , peroxynitrite also attacks dna and can possibly amplify poly(adp - ribose ) polymerase ( parp ) overactivation ( moylan et al . , 2014 ) .
parp is a ubiquitous enzyme linked to dna repair and associated with cellular functions such as preservation of genomic stability and cell death ( jagtap and szabo , 2005 ) . but oxidative dna damage overactivated parp which rapidly depletes the cellular nad and atp leading to the bioenergetic crisis .
parp acts on mitochondria and initiates cell death process through mitochondrial depolarization / membrane permeability transition ( mpt ) and release of cytochrome c ( cyt c ) , apoptosis - inducing factor ( aif)/endonuclease g into the cytosol .
parp activation also leads to transport and binding of poly ( adp ) ribosyl ( par ) units to mitochondrial membranes thereby catalyzes mitochondrial mpt and initiates apoptotic cell death ( galluzzi et al . , 2011 ) .
the cellular dysfunction is further enhanced by the activation of pro - inflammatory gene expression by parp , through the promotion of map kinase , nf-b and activator protein-1 ( ap-1 ) activation .
the parp overactivation especially in neuronal tissues results in an increased expression of ap-1 and nf-b dependent genes such as inducible nitric oxide synthase ( inos ) , intracellular adhesion molecule-1 ( icam-1 ) , monocyte chemotactic protein-1 ( mcp-1 ) , complement proteins ( c3 ) , cyclooxygenase -2 ( cox-2 ) and proinflammatory cytokines ( such as il-1 , tnf- ) ( de la lastra et al . , 2007 ) .
activation of transcription factor nf-b induces inflammatory process through pro - inflammatory cytokines plays a critical role in the maintenance of persistent pain .
cox-2 enhances the synthesis of prostaglandins and sensitize the nociceptors whereas tnf- and il-6 modulate excitatory and inhibitory synaptic transmission , respectively in spinal dorsal horn causing hyperalgesia and allodynia ( komirishetty et al . , 2016b ) .
hence , parp overactivation causes energy depletion , mitochondrial dysfunction through excess ribosylation of proteins and neuroinflammation , thus aggravates the cycle of nerve damage ( tentori et al . , 2002 ) .
this consequently forces the cell to undergo death because of bioenergetic crisis & neuronal dysfunction .
parp overactivation is associated with several nervous system disorders like neurodegenerative diseases , ischemia - reperfusion and traumatic injury ( jagtap and szabo , 2005 )
. there has been substantial research on the role of peroxynitrite , overactivation of parp and inflammatory mediators in the progression of different types of neuropathies and neuropathic pain ( ilnytska et al . , 2006 ) .
mounting evidence indicates that targeting parp overactivation could counteract the pathogenic effects against diabetic and chemotherapy induced neuropathies and interestingly parp inhibitors also provide neuroprotection from the unanticipated pathomechanisms in neuropathy conditions ( obrosova et al . , 2005 ) .
but the role of parp overactivation and related neuroinflammation has not been clearly explored for their involvement in the peripheral nerve injury - induced neuropathy .
hence , we have explored the role of oxidative / nitrosative stress and parp overactivation in the animal model of cci - induced neuropathic pain .
cci produced significant rise in the levels of oxidative stress markers like nitrites and malondialdehyde ( mda ) , an indicative of nitro - oxidative stress in the tissues of the sciatic nerve and spinal cord ( komirishetty et al .
the extreme nitrosative stress can be explained by the expression of nitrotyrosine positive cells in both the tissues .
parp overactivation showed the mitochondrial dysfunction through the loss of membrane potential and electron transport chain inactivation in the sciatic nerve ( komirishetty et al . , 2016c ) .
oxidant - induced dna damage and parp - induced neuronal apoptosis were prominently observed in three types of neuronal tissues i.e. , ipsilateral sciatic nerve , l46 drg and lumbar spinal cord through tunel positive cells ( komirishetty et al .
increased par immunoreactivity in sciatic nerve and lumbar spinal cord sections indicated the oxidative / nitrosative stress induced parp overactivation .
it also led to nad and atp depletion driving the cell towards bioenergetic crisis induced cell death .
hence , parp overactivation after peripheral nerve injury has been demonstrated to switch cell death pathways from the well - controlled and highly regulated apoptosis to the more inflammatory necrosis in the event of high levels of reactive oxygen species .
it also serves as a co - factor for nf-b thus its activation elevates the expression levels of proinflammatory cytokines tnf- , il-6 , inos , cox-2 in the peripheral nerve tissues ( kumar et al . , 2011 , 2012 ) .
therefore , parp - induced neuroinflammation aggravates the neurodegeneration process and challenges for the cell survival after the peripheral injury .
cci - induced alterations in the expression levels of oxidative / nitrosative stress markers and inflammatory markers can modulate excitatory and inhibitory synaptic transmission in spinal dorsal horn which can be correlated to the observed hyperalgesia and allodynia ( komirishetty et al . , 2016a , b ) . functional and behavioural deficits after the nerve injury can be explained through the parp - induced bioenergetic failure and neuronal dysfunction .
parp inhibition ameliorates par accumulation in neuronal vasculature and axons , thus restores normal neuronal functions like conduction velocities and nerve blood flow ( obrosova et al . , 2004 ) .
sciatic functional evaluation is important to know the extent of myelin degradation and nerve injury ( jessen and mirsky , 2008 ) . a significant sciatic functional loss and
cci of sciatic nerve induced morphological & structural changes like axon degeneration , ganglia nucleolar , nuclear and somatic size reduction with nucleolar segregation induced by damage to cell bodies in dorsal root ganglia , peripheral sensory and motor neurons makes them more susceptible to oxidant - induced nervous damage ( komirishetty et al . , 2016b ) .
the recent findings also suggest the nitro - oxidative stress and parp overactivation is bidirectional rather than unidirectional ( obrosova et al . , 2005 ) .
hence , parp overactivation after the nerve injury feed forwards the neurodegenerative mechanisms leading to the functional , behavioural and biochemical deficits ( figure 1 ) . identifying the pathological role of parp overactivation in trauma - induced neuropathy ,
our research group evaluated the neuroprotective potential of parp inhibitors like morin hydrate , 3-aminobenzamide ( 3-ab ) , 1,5-isoquinolinediol ( iso ) and 4-amino 1,8-napthalimide ( 4-ani ) in the cci model of neuropathic pain .
interestingly , administration of parp inhibitors for 14 days in rats recovered the sciatic functional index , foot posture .
they also attenuated the cci - induced behavioral changes including thermal hyperalgesia , cold allodynia , dynamic mechanical allodynia , mechanical hyperalgesia , mechanical allodynia and spontaneous pain .
improved functional and behavioural deficits with the treatment indicate the therapeutic potential of parp inhibitors against sciatic nerve constriction induced axon degeneration , demyelination and disturbed sensory motor inputs .
our studies also demonstrated that treatment with parp inhibitors attenuated the cci - induced bioenergetic crisis , neuronal apoptosis , cellular dysfunction and neuroinflammation through improved nad & atp levels , reduced tunel positive cells , par immunoreactivity and nf-b activation respectively in the sciatic nerve and lumbar spinal cord after the nerve injury ( komirishetty et al .
schematic representation of the role of oxidative / nitrosative stress & parp overactivation and therapeutic potential of parp inhibitors in cci - induced peripheral neuropathy .
atp : adenosine triphosphate ; cci : chronic constriction injury ; gsh : glutathione ; il-6 : interleukin 6 ; inos : inducible nitric oxide synthase ; mda : malondialdehyde ; nad : nicotinamide adenine dinucleotide ; nf-b : nuclear factor-b ; no : nitric oxide ; parp : poly ( adp - ribose ) polymerase ; sod : superoxidase dismutase ; tnf- : tumor necrosis factor . in summary , our studies demonstrated the role of nitro - oxidative stress and parp overactivation in an experimental model of trauma - induced neuropathic pain .
parp inhibitors mitigated the neuroinflammation and nitro - oxidative stress induced functional , behavioural and biochemical deficits , thus showed protection against parp - overactivation induced neuronal damage in an experimental model of trauma - induced neuropathy .
hence , it can be speculated that parp inhibitors may facilitate promising therapeutic benefits in the treatment of peripheral nerve injury - induced neuropathic pain .
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neuropathic pain is triggered by the lesions to peripheral nerves which alter their structure and function .
neuroprotective approaches that limit the pathological changes and improve the behavioral outcome have been well explained in different experimental models of neuropathy but translation of such strategies to clinics has been disappointing .
experimental evidences revealed the role of free radicals , especially peroxynitrite after the nerve injury .
they provoke oxidative dna damage and consequent over - activation of the poly(adp - ribose ) polymerase ( parp ) upregulates pro - inflammatory pathways , causing bioenergetic crisis and neuronal death . along with these changes
, it causes mitochondrial dysfunction leading to neuronal apoptosis . in related preclinical studies agents that neutralize
the free radicals and pharmacological inhibitors of parp have shown benefits in treating experimental neuropathy .
this article reviews the involvement of parp over - activation in trauma induced neuropathy and therapeutic significance of parp inhibitors in the experimental neuropathy and neuropathic pain .
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currently , menopause affects the lives of millions of women globally and will be an issue of increasing concern as the population ages over the next few decades .
the overall health and well - being of mid - aged women has become a major public health concern around the world .
more than 80% of women experience physical or psychological symptoms in the year approaching menopause , leading to decrease in quality of life ( qol ) .
various studies existing from foreign countries have indicated that menopause is negatively related to qol by menopausal symptoms based on their severity .
the study of qol in the post - menopause has become an essential component in clinical practice .
thus , present study was undertaken to determine the menopause related symptoms and its impact on qol in urban and rural post - menopausal women .
a cross - sectional 1-year study was conducted in the department of obstetrics and gynecology , smgs hospital , government medical college , jammu and in the rural field practice area , ranbir singh pura .
any women who has achieved menopause naturally , irrespective of her marital status and parity , was a potential participant of the study .
women undergoing treatment for cancer , or in remission , women with history of using oral contraceptives pills , hormone replacement therapy , and phytoestrogens were excluded from the current analysis .
the participant women of urban area attending the outpatient department , where as rural women were interviewed with a house - to - house survey , with the help of a pretested semi - structured standard questionnaire .
information regarding socio - demographic profile and reproductive parameters ( such as parity , age of menarche , regularity of menses , age of menopause , and years since last menstruation ) were recorded . for assessment of the menopausal symptoms menopause rating scale ( mrs ) was used .
each of the 11 symptoms in mrs contained in the scale can get 0 ( no complaints ) or up to 4 scoring points ( severe symptoms ) depending on the severity of the complaints perceived by the women completing the scale .
the composite scores for each of the dimensions ( subscales ) are based on adding up the scores of each item of the respective dimensions .
the composite score ( total score ) is the sum of the dimension scores , and is proportional to the severity of subjectively perceived symptoms . for the assessment of health - related quality of life ( hrqol ) .
the world health organization quality of life scale ( who qol - bref ) questionnaire in english version translated to local language was used for it .
the scores were calculated according to the standard methods where , the raw scores were converted to transformation scores .
the first transformation converts scores to range of 4 - 20 and the second transformation converts domain scores to 0 - 100 scale .
higher scores reflect better qol . the who qol - bref contained 26 items , categorized under 4 main domains : physical , psychological , social , and environmental .
a separate 5-point scale ranging from never ( 4 ) to always ( 0 point ) was used for the measurement of each item .
total score of each domain was 108 ; the higher score indicating a good qol , a lower score indicating a poor qol and high effect of menopausal symptoms on qol . those who obtained scores from 0 to 33.3% were considered poor qol , from 33.3 to 66.7% were considered average qol , and more than 66.7% were considered to have good qol .
the data was analyzed by computer software ms excel and statistical package for the social sciences ( spss ) version 21.0 for windows .
the data was analyzed by computer software ms excel and statistical package for the social sciences ( spss ) version 21.0 for windows .
there was a significant difference between the mrs total scores of the urban ( 14.67 6.64 ) and rural ( 16.08 7.65 ) group .
the somatic , psychological , and urogenital symptoms were high in rural women than in urban women .
comparison of urban and rural menopausal women according to menopause rating scale comparison of urban and rural women according to mean menopause rating scale scores per subscale and symptoms comparison of urban and rural women according to menopause rating scale scores and percentages per subscale and symptoms comparison of menopausal women from urban and rural areas according to world health organization quality of life scale ( who qol - bref ) raw score mean scores of hot flushes and sweating was more in rural ( 1.66 1.25 ) as compared to urban ( 1.45 1.03 ) women .
the mean score of heart discomfort was 1.20 1.04 in urban women which is more as compared to 1.16 1.06 of the rural women .
the score of sleeping problems was more , i. e. , 1.44 1.29 in the rural women compared to 1.30 1.08 in urban women .
muscle and joint problems had a score of 1.61 1.16 in urban women which was less as compared to that of rural women ( 1.77 1.22 ) [ tables 13 ] .
depressive mood score was 1.33 1.16 in urban women which was less compared to 1.57 1.25 of the rural women .
irritability was in 1.34 1.10 urban women as compared to 1.44 1.29 rural women .
anxiety score was 1.30 1.06 in urban women as compared to 1.42 1.13 in the rural women [ table 3 ] .
physical and mental exhaustion score was 1.59 1.14 in urban women as compared to 1.93 1.18 in the rural women .
sexual problems score was 1.96 1.51 in urban women as compared to 1.91 1.69 in the rural women [ tables 4 and 5 ] .
bladder problems score was in 0.87 1.01 in urban women as compared to 1.01 1.10 in the rural women .
scores for dryness of vagina was 0.68 0.92 in urban women as compared to 0.71 1.02 in the rural women .
comparison of menopausal women from urban and rural areas according to world health organization quality of life scale ( who qol - bref ) transformed score ( 4 - 20 ) comparison of menopausal women from urban and rural areas according to world health organization quality of life scale ( who qol - bref ) transformed score ( 0 - 100 ) the mean raw scores of physical health , psychological , social relationships , and environmental domains is more in urban than in rural women .
the mean transformed scores ( 4 - 20 ) of physical health , psychological , social relationships , and environmental domains is more in urban than in rural women .
the mean transformed scores ( 0 - 100 ) including the physical health , psychological , social relationships , and environmental domains is more in urban than in rural women .
the present study revealed that proportion of menopausal symptoms was significantly high in both rural and urban women .
the findings of the present study showed that the , women in the rural area suffered from severe menopausal symptoms such as : musculoskeletal , hot flushes , vasomotor , sexual , genito - urinary , and sweating as compared to urban women .
this was in concordance to the finding made by the yohanis et al . , but contradictory to martinez as in their study in rural women menopausal symptoms were less severe .
however , with regards to frequency of symptoms , a higher proportion of urban women have been reported to suffer as put by various workers compared to the rural women .
this result was in concordance to sagdeo and arora , the percentage of all menopausal symptoms were more in urban women as compared to rural .
martinez too stated that there was a greater frequency of menopausal symptoms in urban women .
the joint and muscle problems , physical and mental exhaustion , sleep disorders , hot flushes were the most common symptoms reported in our study which was similar to various studies conducted in india and abroad by sagdeo and arora , jahanfar et al . , nisar and sohoo , poomalar and bupathy , and ashrafi et al .
but the results are not consistent with the finding of other studies of madhukumar et al .
, sharma et al . , puri et al . , vijayalakshmi et al . , and borker et al .
another aspect worth mentioning is that the urogenital symptoms including sexual problems , bladder problems , and dryness of vagina were less frequent both in the rural and in urban women ; the individual and overall scores of mrs were also low for urogenital domain especially more so in urban women but the difference was not found to be statistically significant .
vasomotor instability or menopausal hot flushes was believed to be due to sympathetic discharge but its hormonal basis can not be ruled out .
our study is line with adhi et al . , chuni and sreeramareddy , jacob et al . , and nisar and sohoo on hot flushes but not in line with the studies of mahajan et al . , and sharma et al .
these differences in frequencies of symptoms may be because of differences of race , life style , culture , genetics , diet , and the co - morbid diseases . in the present study
, we have found the who qol - bref scores for all domain lower for rural women ; it indicates that not only the menopausal symptoms but aging , increasing frequency of chronic illness , and social deprivation may have negative impact on qol of menopausal women .
we did not find significant difference in scores for physical domain of who qol - bref .
the physical domain access the influences on the qol of factors like activities of daily living , dependence on medicinal substances and medical aids , energy and fatigue , mobility , pain and discomfort , sleep and rest , and work capacity can be explained by the fact that the urban sample was hospital - based which could be the reason for lower score on the physical health . in our study , the rural women showed a considerably worse qol in the psychological , social , and the environmental domains of the who qol questionnaire when compared to their urban counterparts and the results were statistically significant .
rural women were more of low socio - economic status so were more likely to encounter financial problems , issues of unemployment or underemployment , discrimination ; there was the lack of education , poorer access to health services , higher levels of personal health risk factors , and environmental issues linked to road travel and occupation , whereas the women living in the urban area were having have more opportunities to be involved in social , cultural , or economic activities .
in contrast , women in rural areas are more labile to a diminished self - esteem at the end of their child - bearing age . in professionally non - active psychosomatic syndromes , such as anxiety , depression ,
enhance a greater discomfort , which makes them limit their contacts with acquaintances or friends .
sleep disorders particularly , insomnia as well as sleep continuity disorders , sexual disorders influence women 's everyday functional life and their lifestyle . in this respect , the investigated women also reported problems of different intensity . therefore , it was not unexpected that rural females possess higher risk of anxiety and depression social and environmental problems .
, estimated that scores for depression and anxiety were higher in rural women . but were contradictory to yohanis et al . , who concluded the qol showed no significant difference between rural and urban women .
the reason for the discrepancy because of the character and culture of rural communities in their study that they were more accepting to the differences they have experienced since menopause , the idea that menopause is a natural thing that must happen in every menopausal woman and not just a peruses aging or loss of beauty , but also a process of maturation .
but there was lack of awareness of its cause , effect , and management pertaining to it .
a wide gap in the knowledge has been documented in women from rural and urban area in developing countries .
the current study had some limitations as secondary causes of amenorrhea were not ruled out and co - morbidities and medication play an important role on qol issues which was not addressed in the current analysis .
the high proportions and the scores of mrs were observed in both rural and the urban women .
the qol in urban society where the symptoms experienced were less severe was average and better than the qol in rural women having severe menopause symptoms .
thus , the current study warrants an urgent need to create awareness about menopausal health among both urban and rural post - menopausal women .
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aim and objective : to analyze the menopause - related symptoms and its impact on quality of life in post - menopausal women from urban and rural area.materials and methods : a cross - sectional 1-year study was carried among women of urban ( n = 490 ) and rural ( n = 380 ) areas , attending the outpatient department in the urban area and a house - to - house survey in rural areas , by interviews with the help of a pretested semi - structured standard questionnaire .
for assessment of the menopausal symptoms menopause rating scale ( mrs ) and for quality of life , world health organization quality of life scale ( who qol - bref ) questionnaire was used.results:there was a significant difference between the mrs total scores of the urban ( 14.67 6.64 ) and rural ( 16.08 7.65 ) group .
the somatic , psychological , and urogenital symptoms were high in rural women than in urban women .
the results were not significant for urogenital subscale .
the mean raw scores of physical health , psychological , social relationships , and environmental domains was more in urban than in rural women .
the mean transformed scores ( 4 - 20 ) of physical health , psychological , social relationships , and environmental domains was more in urban than in rural women .
the mean transformed scores ( 0 - 100 ) including the physical health , psychological , social relationships , and environmental domains was more in urban than in rural women .
the result was not significant for physical health.conclusion:the high proportions and the scores of mrs were observed in both rural and the urban women .
the severity of symptoms was found more distressing for rural women .
the quality of life in urban society was average and better than in rural women .
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hepatocellular adenoma ( hca ) is a rare benign tumour ( incidence of 1/1,000,000 ) that is mainly found in women of child - bearing age ( second most frequent hepatocellular tumor in young women after focal nodular hyperplasia ) .
there is evidence that hca is strongly related to current and recent ( first generation , high dose ) oral contraceptives ( oc ) use .
recent , low - dose ocs appear less strongly , at all , related to hca .
non - oc - related causes of hca include familial insulin - dependent diabetes , fanconi anaemia , glycogen storage diseases , and hormonal stimulation from other sources , for instance , anabolic steroid use by body builders , gynaecological tumours , or pregnancy [ 26 ] .
right upper abdominal quadrant fullness or discomfort is present in 40% of cases due to mass effect .
typical clinical manifestation is spontaneous rupture or haemorrhage leading to acute abdominal pain with possible progression to hypotension and even death .
laboratory tests for liver function are usually normal . because of the different therapeutic options hca must be distinguished from other hypervascular lesions ( hcc , fibrolamellar hcc , focal nodular hyperplasia ( fnh ) , and metastases ) that may occur in young adults without cirrhosis . steroid related adenoma is typically detected when it reaches about 5 cm in diameter but the size could be 630 cm ( large and multiple lesions are more prone to spontaneous haemorrhage ) . in 75% of cases
the lesion involves the subcapsular region of right lobe of liver , in 10% it is intraparenchymal or pedunculated .
a so - called adenomatosis is present in subjects observed to have more than 10 lesions : this entity is independent of gender or hormone therapy [ 7 , 8 ] .
treatment criteria include number and size of lesions , presence of symptoms , and surgical risk .
the possible role for elective surgery mainly depends upon the risk of complications , but data concerning its actual incidence are presently lacking .
in fact , both selection and spectrum bias negatively affect the retrospective surgical case series that represent most of the available literature on this topic .
moreover , concerning the two other common indications for surgery , represented by the uncertainty of diagnosis and the presence of symptoms related to tumour size , the role of surgery can be challenged .
in fact , the continuous improvement in diagnostic techniques , particularly magnetic resonance , has the consequence that one can restrict surgical resection to very few participants for which the procedure has not only a curative target , but also the need for a precise pathologic definition .
also the role of surgery for symptomatic tumors has been claimed only by small uncontrolled case series .
however , as a general rule , if hca is < 5 cm in size , discontinuation of oc and radiological follow - up are acceptable ; if the lesion is > 5 cm or near hepatic surface , due to the recognized risk of rupture and haemorrhage , surgical resection is the treatment of choice .
pregnancy should be avoided due to increased risk of rupture . according to recent studies hcas
are currently categorized in four distinct genetic and pathologic subtypes as follows : inflammatory hepatocellular adenomas , hepatocyte - nuclear - factor-1-alpha- ( hnf-1-mutated ) hepatocellular adenomas , and -catenin - mutated hepatocellular adenomas .
finally hcas without any genetic abnormalities are categorized in the unclassified subtype [ 1013 ] .
although this classification is new and not yet widely accepted , it has definitive management implications .
image - guided biopsy or surgical resection with histopathologic and immunohistochemical analysis is necessary for complete characterization of hcas but mr imaging plays an important role in diagnosis and subtype characterization as well as identification of complications and surveillance [ 14 , 15 ] . the purpose of this paper is to present mr imaging characteristics of specific subtypes of hcas by using dual phase contrast agents and to discuss differential diagnosis with the most common benign and malignant lesions mimicking hcas .
gadobenate dimeglumine ( gd - bopta , multihance , bracco ) and gadoxetic acid ( gd - gd - eob - dtpa , primovist , bayer ) differ from purely extracellular gadolinium agents as they combine the properties of conventional nonspecific gadolinium agents with those of an agent targeted specifically to hepatocytes : for this reason they are also called hepatospecific contrast agents . with these contrast media
it is possible to perform dynamic phase imaging as performed with conventional gadolinium agents and delayed - phase imaging as performed with mangafodipir trisodium ( teslascan , ge healthcare ) . using gd - bopta arterial , portal venous , and equilibrium phase images are readily obtainable using identical sequences , the same contrast speed injection to those employed with nonspecific gadolinium agents . in a different way ,
considering the very fast hepatocyte intake of gd - eob - dtpa , the equilibrium phase is spurious because of the overlap between interstitial and hepatocyte times . indeed , hepatocyte phase after gd - eob - dtpa starts after 3 minutes of contrast medium injection .
there is an overlap between equilibrium and early hepatocyte phases ; therefore , equilibrium appearance after gd - eob - dtpa is spurious . unlike the conventional agents , approximately 35% and 50% , respectively , of the injected doses of gd - bopta and gd - eob - dtpa
the result of the hepatocytic uptake is that the normal liver parenchyma shows strong enhancement on delayed t1w images that is maximal approximately 2 - 3 hours after gd - bopta injection and 20 minutes after gd - eob - dtpa administration .
a second feature unique to gd - bopta and gd - eob - dtpa is that the contrast - effective moiety of these agents interacts weakly and transiently with serum albumin .
this interaction shows the tumbling rate of the gd - bopta and gd - eob - dtpa chelates and results in longer rotation correlation times in shell water protons for gd - bopta and gd - eob - dtpa compared to generic gadolinium agents that do not interact with serum albumin .
this in turn results in a t1 relaxivity in human plasma at 37c at 1.5 t that is approximately that of conventional gadolinium agents ( r1 7 , 3 and 6 , 7 for gd - eob - dtpa and gd - bopta , resp .
, in comparison to 4 , 24 , 6 with conventional gd - chelates ) .
not only does this increased relaxivity permit lower overall doses to be used to acquire the same information of dynamic as available with conventional agents at a standard dose , but it also facilitates the improved performance of the gd - bopta and gd - eob - dtpa for both intra- and extrahepatic vascular imaging .
a principal advantage of the selective uptake by functioning hepatocytes is that the normal tissue enhances while tumors of nonhepatocytic origin such as metastases and cholangiocarcinoma , as well as nonfunctioning tumor that are unable to uptake gd - bopta and gd - eob - dtpa , remain unenhanced , thereby increasing the liver contrast - noise ratio ( cnr ) and hence the ability to detect lesion .
mr imaging is typically performed , being gd - bopta and gd - eob - dtpa t1 relaxation time contrast agents , with 2d or 3d gradient - echo sequences while the use of fat saturation has been shown to raise cnr on dynamic and delayed hepatobiliary phase imaging .
clinical studies and routine clinical practice have shown that the dynamic phase imaging is particularly important for lesion characterization while delayed phase imaging in the hepatobiliary phase increases the sensitivity of mri for liver detection
. however , delayed phase imaging also contributes to improving the characterization of lesions , particularly when results of unenhanced and dynamic sequences are equivocal or when atypical enhancement patterns are noted on dynamic imaging [ 1721 ] .
inflammatory hca is the most common subtype and accounts for about 30%50% of all hepatocellular adenomas .
these tumors are mainly seen in women , in association with obesity , hepatic steatosis , diabetes mellitus , glycogenesis ( in particular , type i glycogen storage diseases ) , and alcohol abuse .
patients with inflammatory hca may present with signs of chronic anemia and/or systemic inflammatory syndrome , characterized by fever , leukocytosis , and elevated serum levels of c - reactive protein .
inflammatory hcas are associated with a definitive increased risk of bleeding ( > 30% ) and a risk of malignant transformation ( 510% ) [ 10 , 12 ] .
they comprise a prototype example of tumours induced by hepato - biliary inflammation : more than 40% of genes overexpressed in inflammatory hcas are associated with inflammation and immune response . around 10% of inflammatory hcas may also show mutation involving catenin gene .
histologically , inflammatory hcas are characterized by significant sinusoidal dilatation , polymorphous inflammatory infiltrates , areas of peliosis , and thickened tortuous arteries .
steatosis within nodule is variable and less extensive compared with hnf-1-mutated hcas [ 15 , 24 ] . on plain mr imaging
inflammatory hca is often hyperintense on t2w images and hypointense on t1w sequence , frequently with heterogeneous signal intensity .
hyper- and hypointensity on t2w and t1w images , respectively , correspond mainly to areas of sinusoidal dilatation and inflammatory infiltrates .
focal areas of fat may be seen as hypointense areas on t1 out - phased images due to signal drop .
inflammatory hca may appear as a hypervascular mass with persistent enhancement during dynamic evaluation and may show a variable uptake in the hepatobiliary phase specially at the periphery ( figure 1 ) .
sometimes because of sinusoidal dilatation , inflammatory component and ductal reaction , in the hepato - biliary phase image areas of hypointensity in adenomas , mainly in the periphery , may be seen [ 25 , 26 ] . marked t2 hyperintensity associated with delayed persistent enhancement has a sensitivity of as much as 85% and a specificity of 87% for the diagnosis of inflammatory hca .
peripheral hyper - intensity ( atoll sign ) reflects the abnormal ductal reaction with alterated biliary excretion ( figure 2 ) [ 13 , 14 ] . in a small percent of cases
, inflammatory hca may appear isointense on t2 and t1w images with discrete enhancement in arterial phase and quite rapid wash out ( figure 3 ) .
hnf-1-mutated hca is the second most common type ; it constitutes about 3035% of all hcas and arises because of biallelic inactivation of transcription factor 1 gene located in chromosome twelve .
this kind of adenoma is nearly exclusively seen in women , except for rare hca with germline hfn-1 mutations which can be also observed in men .
the final outcome of this mutation is the production of nonfunctioning hnf-1 protein which promotes lipogenesis by suppression of gluconeogenesis , activation of glycolysis , and promotion of fatty acid biosynthesis .
the reduction of fatty acid binding protein 1 leads to faulty transport of fatty acids and to intracellular deposition of fat .
hnf-1 mutation may be the primary inciting event that results in the accumulation of estrogen metabolites that unconditionally stimulate hepatocyte proliferation [ 27 , 28 ] . on mr examination ,
hnf-1-mutated hca often shows heterogeneous hypointensity areas on t1 out - phased sequences with significant signal drops on out - phased in comparison with in - phased sequences , corresponding to fatty deposition .
hyperintensity on t1 in - phased and out - phased images signal drop may correspond to glycogen component or less commonly haemorrhage .
on t2w images the lesion tends to appear as iso- or hypointense nodule without significant restriction on dwi .
this is true in uncomplicated adenomas ; conversely , complicated adenomas or adenomas containing different tissues may show restriction .
adc maps may appear aspecific , with positive or negative values between 0.9 and 1.3 [ 29 , 30 ] .
on dynamic evaluation after gd - bopta and gd - eob - dtpa , hnf-1-mutated hca appears hypervascular with variable degrees , but usually less evident than inflammatory adenoma .
on portal venous and equilibrium phases the lesion tends to be hypointense ; on hepatobiliary phase the mass appears hypointense in almost all cases with homogeneous appearance ( figure 4 ) . significant signal drop in t1 out - phased imaging for predictive hnf-1-mutated hca
is reported to be 85% , 100% , 100% , 94% of sensitivity , specificity , predictive positive , and negative predictive value , respectively .
-catenin - mutated hcas constitute about 1015% of all hcas ; they originate from sustained activation of -catenin because of mutations involving the ctnnb1 gene ( catenin 1 ) .
these tumors primarily involve patients with glycogen storage disease and on androgen treatment and have a greater propensity to undergo malignant transformation to hccs .
-catenin plays a major role in hepatocyte development , differentiation , proliferation , and regeneration .
activation of -catenin in normal hepatocyte is usually transient and is regulated by its rapid degradation .
excessive nuclear accumulation and sustained activation of -catenin may result from mutation in -catenin itself or from mutation involving cytoplasmic degradation complex .
excessive -catenin activity results in autonomous growth of hepatocyte and accelerates hca formation [ 22 , 27 , 31 ] .
although -catenin mutation is implicated in malignant transformation of hca , only 2030% of malignant hcas show -catenin mutation .
up to 75% of patients , glycogen storage disease may develop hca . on mr imaging -catenin - mutated hca appears as homogeneous or heterogeneous hypervascular mass with persistent or nonpersistent enhancement during the delayed - phase images .
signal intensity on t2 and on t1 precontrast sequences is variable but mainly heterogeneously hyper - and hypointense , respectively .
malignant transformation simulates hcc on imaging and does not show peculiar findings ( figure 5 ) .
frequently , the presence of haemorrhage may be one of the reasons that justify the unclassified categorization of the lesion ( figure 6 ) .
no specific mr imaging patterns have yet been proposed to identity unclassified hcas also because imaging experience is very limited [ 10 , 22 ] .
no immunohistochemical analyses were performed to compare with mri features , allowing to assert the diagnosis of hca subtype .
focal nodular hyperplasia is the second most common benign hepatic tumor ( 8% of primary hepatic tumors at autopsy ) .
fnh represents an hyperplastic response to a localized vascular abnormality ; consequently it is not a true benign tumor but a benign congenital hamartomatous malformation .
fnh is predominantly found in the same group of patients as well as hca : female patients ( m : f = 1 : 8) , usually in 3rd-4th decades of life , with history of oc consumption .
multiple fnhs are found in 1020% of cases and association with hemangioma occurs in 510% of cases .
pathologically fnh is usually a solitary ( 80% ) , subcapsular , and nodular homogeneous mass .
fnhs could be subdivided into fnh with sinusoidal dilatation ( most of the so - called telangiectatic fnh are now recognized as inflammatory hca ) and fnh with cytological atypia .
on cut section in the majority of large fnhs , pathological features are fibrous septa and cellular areas of hepatic proliferation .
lesions more than 5 cm frequently show a central fibrous scar which consists of fibrous connective tissue , cholangiocellular proliferation , and malformed vessels ( arteries , capillaries , and veins ) .
on mr imaging , classic fnh appears as homogeneously isointense or slightly hyperintense on t2w images and isointense or slightly hypointense on t1w images before contrast medium administration .
typical behaviours during dynamic study are marked and homogeneous enhancement during the arterial phase , rapid wash out during the portal phase , and isointensity ( with the exception of the scar ) during the equilibrium phase . in hepatobiliary phase fnh
in contrast , on delayed liver specific phase images after gd - bopta and gd - eob - dtpa administration , the common appearance of hcas is hypointensity of the solid , nonhemorrhagic components of the lesions , with the exception of the inflammatory subtype .
when present , in fnh a typical scar appears as hyperintense or hypointense stellate area , respectively , on t2 and t1-weighted images ; it is hypointense during the arterial and portal - venous phases and slightly hyperintense during the equilibrium phase ( figure 7 ) [ 3537 ] .
large regenerative hyperplasia ( lrh ) is another generally asymptomatic rare condition ( 0.52.5% on autopsy series ) characterized by diffuse micronodular transformation of the hepatic parenchyma , without fibrous septa between nodules .
disorders in the hepatic microcirculation ( chronic ischemia ) with hyperplastic parenchyma response seem to be the primary cause of lrh .
myeloproliferative and lymphoproliferative disorders , chronic vascular and rheumatologic syndromes , and some drugs used are related to lrh .
adults ( mean age 50 ) , without gender predilection known , are more affected by lrh , rarely reported in childhood ( e.g. , in congenital absence of portal vein ) .
acute abdominal pain occurs in case of rupture of a large subcapsular nodule with hemoperitoneum . if central scar is present nodules can be indistinguishable from fnh .
portal vein obstruction and secondary hepatic arterial dilatation can be observed . in case of splenomegaly and
increased flow in portal vein , several nodules can be observed with patent portal branches . when thrombosis of a large portal branch occurs , remnant portal flow is redirected in the perihilum parenchyma with large regenerative nodules ( of several cm ) near the large portal tracts and small nodules with atrophy in the peripheral parenchyma .
t1w mr images lrh is generally almost isointense or slightly hyperintense compared to the surrounding liver parenchyma while on unenhanced t2w images the nodule appears iso- or slightly hypointense .
a pheripheral hypointense rim is often visible in large lesions on t1- and t2-weighted images . on dynamic mr imaging ,
lrh is usually hyperintense in the arterial phase and iso- or slightly hyperintense in the portal - venous and equilibrium phases . in the delayed
, liver specific phase after gd - bopta or gd - eob - dtpa the lesion appears isointense or hyperintense because it consists of benign hepatocytes with abnormal biliary system drainage ( figure 8) [ 40 , 41 ] .
the main diagnostic dilemma is between lrh and inflammatory hca ; the key elements to achieve the differential diagnosis are the different signal on t2 images ( isointense or slightly hypointense in lrh , hyperintense in hca ) ; the dissimilar contrast behaviour in dynamic study .
hcc in noncirrhotic patients and fibrolamellar hepatocellular carcinoma ( fl - hcc ) are rare malignant primary liver tumors which arise in young healthy patients of both sexes ( m : f = 1 : 1 ) .
hepatomegaly , malaise , pain in right upper quadrant , fever , and/or weight loss may be present .
the lesion becomes symptomatic only when the mass reaches very big size and compresses adjacent structures or invades vascular or biliary vessels ( rarely jaundice can reveal the presence of tumour ) .
both tumors can present with metastatic disease ( lymph nodes and lungs are the most frequent sites ) .
alpha - fetoprotein is usually elevated in conventional hcc but often normal in fl - hcc .
generally there is a large ( average mean size > 10 cm ) , single ( 8090% ) , well - demarcated , lobulated , noncapsulated mass ( incomplete capsule in 1/3 of cases ) ; in 20% of cases it can be pedunculated . in fl - hcc coarse calcifications had been depicted in more than 50% of lesions and , at cut section , lobular arrangement fibrous septa with radial disposition and central scar are seen in 6070% of cases ; abdominal lymphadenopathy had been detected in > 60% of patients .
central scar , fibrosis , and calcification are rare in conventional hcc whereas necrosis hemorrhage and intratumoral fat are much more common than in fl - hcc .
the surgical resectability is high ( 50% ) ; multiple or too large tumors can be treated with liver transplantation .
fl - hcc is frequently recurrent , but 5-year survival is about 5060% . on mr imaging , hcc in non - cirrhotic patients depends largely on tumor size that is generally large because the lesion is not detected in early stage .
generally , most hccs , because of their hypervascular nature , are hyperintense compared to the liver in the arterial phase and hypointense in the portal - venous and equilibrium phases .
irregular mosaic - like or pheripheral enhancement is usually seen in large neoplasms , depending on the internal architecture . in the delayed liver - specific phases after
gd - bopta or gd - eob - dtpa well - differentiated and moderately differentiated hccs may show in a small percentage of cases superior signal enhancement ratios to poorly differentiated hccs ( figure 9 ) .
differential diagnosis between hcc and hca , subtypes inflammatory and -catenin , is very difficult : both adenomas may be heterogeneously hyperintense on t2-weighted images and hypointense on t1-weighted sequences ; they may show intense and heterogeneous enhancement in the arterial phase of dynamic study , subsequent wash out , and/or hypointense signal in hepatobiliary phase .
fl - hcc is usually either hypointense or , rarely , isointense compared to the liver on t1-weighted images . on t2-weighted images ,
the purely fibrous nature of the scar means that it is hypointense on both t1- and t2-weighted images .
fl - hcc becomes heterogeneously hyperintense during the arterial phase after the administration of gadolinium and appears as isointense or slightly hypointense during the portal - venous and equilibrium phase .
the central scar shows minimal or no enhancement in arterial and portal - venous phase images but may sometimes show persistent enhancement in equilibrium phase . in hepatobiliary phases
, fl - hcc usually appears as heterogeneously hypointense with areas of low signal intensity due to necrosis or , less frequently , haemorrhage ( figure 10 ) [ 4244 ] . besides the key elements already described for hcc in non cirrhotic patients , fl - hcc may be differentiated from hca for areas of fibrotic tissue which are hypointense on t1 and t2w images and hyperintense in the delayed phases of the dynamic studies .
signs of malignancy ( vascular and biliary invasion ) present in hcc and fl - hcc are other helpful elements in differential diagnosis .
cholangiocarcinoma ( cc ) is the primary malignancy arising from the bile duct epithelium and it is the second most common liver malignancy after hcc .
incidence of cc among primary liver tumor ranges from 5 to 30% , with an average age of 5060 years , seen slightly more often in men ( m : f = 3 : 2 ) .
risk factors are primary sclerosing cholangitis , bile stasis , recurrent cholangitis , infections with clonorchis sinensis and opisthorchis viverrini , hepatolithiasis , congenital bile ducts anomalies , familiar polyposis , thorotrast deposition , and caroli 's syndrome .
recent findings indicate that hcv - hbv infection conferred a more than twofold elevated risk of icc . according to the site of origin cc
can be classified into two types as follows : intrahepatic or peripheral ( pcc ) and extra - hepatic .
pcc presents as large mass because tumor does not cause clinical symptoms in early stages .
the initial symptoms are abdominal pain , malaise , anorexia , weight loss , fever , palpable mass , and jaundice ( rare ) .
the peripheral mass appears as a large white - grey lesion characterized by fibrosis ( fibrotic core ) and associated with capsular retraction ; calcifications are rare .
sometimes there is concomitant dilatation of adjacent bile ducts and atrophy of corresponding liver segments .
metastatic spread is common : lung , bone , pancreas , adrenals , kidney , spleen , and peritoneum .
according to the liver cancer study group of japan ( lcsgj ) and based on the gross appearance icc can be categorized in three patterns of growth that can be present alone or in to combination : mass - forming type ( mf ) , intraductal growth ( ig ) type , and periductal infiltrating type ( pi ) .
the prognostic significance of such classification has been confirmed in several clinical studies [ 45 , 46 ] .
curative resection is the most effective treatment and the only therapy associated with prolonged disease - free survival ; the rate of radical resection is extremely variable in the literature , ranging between 30% and 80% .
imaging pcc is either isointense or hypointense relative to the normal liver on t1w mr images but may range from mildly to markedly hyperintense on t2w images .
the signal intensity of the tumour is variable and depends on the amount of mucinous material , fibrous tissue , haemorrhage , and necrosis within the lesion . on dynamic study after injection of gd - bopta or gd - eob - dtpa , minimal or moderate incomplete enhancement
is seen at the periphery on early images , whereas progressive central contrast enhancement is seen on later images .
generally , on delayed phase images lesions show peripheral hypointensity and central iso- or hyperintensity due to central pooling of contrast medium within central desmoplastic reaction .
satellite nodules are seen in about 1020% of pcc cases and are chiefly responsible for the poor prognosis of this tumour ( figure 11 ) [ 47 , 48 ] .
primary lymphoma ( pl ) of the liver ( confined to the liver without involvement of lymph nodes or spleen or bone marrow ) is very rare ( 0.4% of extranodal non - hodgkin 's lymphomas and 0.2% of all non - hodgkin 's lymphomas ) .
pl can arise at every age but it is more frequent in childhood - adolescence or in middle - old age ( m : f = 4 : 1 ) . it can be associated with epstein - barr infection ( ebv ) , and more frequently in patients with chronic hepatitis or cirrhosis ( by hbv and/or hcv infections ) , with autoimmune disorders or with aids manifestations ; it can also be associated with immunosuppression in transplanted patients .
liver involvement ( 50% of cases ) in haematological diseases ( secondary liver lymphoma ) is more common .
hepatomegaly , presence of hepatic mass or masses , and pain in upper right quadrant are the most frequent signs and symptoms in primary lymphoma .
pl can present as multiple , small nodular lesions ( well - differentiated b - cell lymphoma ) or as diffuse infiltration of hepatic parenchyma ( undifferentiated subtypes ) ; in some patients with hodgkin 's lymphoma peliosis hepatis can be associated .
surgical resection followed by chemotherapy and/or radiation gives the best prognosis in primary lymphoma [ 49 , 50 ] . on mr imaging , pl
is generally seen as homogenously / heterogeneously hypointense compared to the normal parenchyma on unenhanced t1-weighted images and hyperintense on t2-weigthed images .
dynamic imaging after gd - bopta or gd - eob - dtpa typically reveals a hypointense appearance on arterial phase images , followed by homogeneous , delayed enhancement on portal - venous phase images , and isointensity on equilibrium phase images .
. the equivocal behavior of pl does not offer important elements for differential diagnosis with hca .
hnf-1-mutated hca may be differentiated for the fatty intralesional component which determines signal patterns on plain mr and dynamic study .
metastases are the most common cause of malignant focal liver lesions ( 1820 times more frequent than primary malignant tumors ) . at autopsy
liver metastases originate predominantly from primary tumors localized in gastrointestinal tract ( colon , stomach , and pancreas in decreasing order of frequency ) , by hematogenous spread , via portal vein .
other frequent secondary lesions are from breast and lung cancers ( but also endocrine / neuro - endocrine tumors and melanoma ) : primary neoplasms probably give metastases by hematogenous spread , via the arterial blood supply to the liver .
lymphogenous spread occurs along bile ducts from near organs ( pancreas ) or from haematological diseases ( lymphoma , leukaemia ) .
direct invasion from nearest organs ( gallbladder or bile duct cancer , pancreatic neoplasms ) can also be the cause of liver metastases .
liver involvement by intra - abdominal dissemination can also be observed ( ovarian cancer ) .
most patients with metastases to the liver present with symptoms related only to the primary tumor ; the asymptomatic hepatic involvement is discovered in the course of clinical evaluation .
sometimes there are no specific symptoms : weakness , weight loss , fever , and loss of appetite .
rarely , hepatomegaly , hepatic mass , or pain in upper right quadrant can be the first symptoms of liver involvement mostly when lesion / s are huge and multiple .
metastases can vary in size , number , consistency , uniformity of growth , vascularity , and stromal response .
the outcome depends not only on primitive tumor , but even on number , size , and tissue component of metastases .
if resectable , hepatic metastases have 2040% survival rate at 5 years ( colon cancer metastases ) .
the differential diagnostic dilemma between hca and metastases is mainly in the case of hypervascular , solitary lesion in patients with unknown primary cancer .
generally , on unenhanced t1w images metastases have low signal intensity compared to the surrounding parenchyma . on t2w sequences
the lesions demonstrate high signal intensity , although the signal is generally lower than that typically observed in hemangiomas .
hypervascular metastases tend to reveal strong transient enhancement in the arterial phase followed by hypointensity in the portal and equilibrium phases ; they appear hypointense in hepatobiliary phase ( figure 13 ) [ 41 , 53 , 54 ] . the most important elements to achieve the differential diagnosis between hca and each type of hypervascular lesions in non
according to recent studies hcas are currently categorized in to four distinct genetic and pathologic subtypes : inflammatory hepatocellular adenomas , hepatocyte - nuclear - factor-1-alpha ( hnf-1-mutated ) hepatocellular adenomas , -catenin - mutated hepatocellular adenomas , and unclassified adenomas .
mr imaging plays an important role in diagnosis and characterization , particularly in inflammatory and steatotic subtypes , as well as in identification of complications and surveillance .
image - guided biopsy or surgical resection with histopathologic and immunohistochemical analysis is necessary for complete characterization of hcas and in some differential diagnostic dilemmas .
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hepatocellular adenomas ( hcas ) are currently categorized into distinct genetic and pathologic subtypes as follows : inflammatory hepatocellular adenoma , hepatocyte - nuclear - factor-1-alpha ( hnf-1-mutated ) hepatocellular adenoma , and -catenin - mutated hepatocellular adenomas ; the fourth , defined as unclassified subtype , encompasses hcas without any genetic abnormalities .
this classification has accepted management implications due to different risks of haemorrhage and malignant transformation of the four subtypes .
imaging guided biopsy and/or surgical resection very important in obtaining definitive characterization ; nevertheless , mri with intra - extravascular and hepatobiliary ( dual phase ) agents , is an important tool not only in differential subtypes definition but even in surveillance with early identification of complications and discovery of some signs of hca malignant degeneration .
inflammation , abnormal rich vascularisation , peliotic areas , and abundant fatty infiltration are pathologic findings differently present in the hca subtypes and they may be detected by multiparametric mri approach .
lesion enlargement and heterogeneity of signal intensity and of contrast enhancement are signs to be considered in malignant transformation .
the purpose of this paper is to present the state of the art of mri in the diagnosis of hca and subtype characterization , with particular regard to morphologic and functional information available with dual phase contrast agents , and to discuss differential diagnosis with the most common benign and malignant lesions mimicking hcas .
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recent studies indicate lower hepcidin gene expression in the heart , kidney , pancreas , adipose tissue , as well as pathogen - activated neutrophiles and macrophages [ 2 , 3 , 4 ] .
hepcidin is synthesized as a 84-amino acid pre - pro - peptide , that undergoes two enzymatic cleavages .
however , only mature 25-amino acid hepcidin has assigned biological function [ 5 , 6 ] . the small size of hepcidin-25 ( approximately 2 kda ) and its highly cross - linked structure is the main problem in developing a reliable method for quantification of serum , plasma and urine hepcidin . for this reason the majority of clinical research , measure a high molecular weight precursor- prohepcidin by elisa kits [ 7 , 8 , 9 , 10 ] .
hepcidin acts by binding to ferroportin , a cellular iron exporter , inducing its internalization and subsequent proteolytic degradation in the cytoplasm .
thus , overall , hepcidin inhibits iron absorption in the small intestine , iron release from the reticuloendothelial macrophages and iron transport across the placenta .
there are many different factors which can regulate hepcidin expression , including iron excess , tissue hypoxia , erythropoietin and cytokines [ 12 , 13 ] .
this seems to occur at the level of gene transcription via different , closely coordinated signaling pathways [ 14 , 15 ] . since the discovery of hepcidin
, experimental and clinical studies have been carried out to determine the relationships between hepcidin ( or prohepcidin ) and iron - related disorders , i.e. haemochromatosis , thalassaemia intermedia , renal anaemia and anaemia of chronic disease [ 10 , 16 , 17 , 18 ]
. however , clinical research pertaining to healthy individuals are unique . as a consequence , a scientific knowledge of hepcidin s physiology and pharmacokinetics hasnt increased significantly over the past few years .
little information exists regarding hepcidin and prohepcidin levels in infants , men during transient hypoxia and women of reproductive age , including those who are pregnant [ 19 , 20 , 21 ] .
moreover , the data available are based on small study groups . within the last 8 years , it has become evident that hepcidin has a diagnostic and therapeutic potential .
therefore , it is a great need to establish the reference interval for this peptide .
the aim of this study was to assess the impact of sex and age on serum prohepcidin concentration in healthy adults .
a total of 100 healthy adult volunteers were recruited from blood donor candidates from the regional blood transfusion center in bydgoszcz ( poland ) . during the 18 months of the study 56 males ( aged 19 - 60 yrs ) and 44 females ( aged 19 - 50 yrs ) were enrolled .
those who had taken any iron supplements or been vegetarian were excluded from this study .
university in bydgoszcz approved a study protocol and a written informed consent was obtained from the subjects .
blood samples were drawn from an antecubital vein between 8 and 10 a.m. with minimal venous stasis and centrifuged .
serum prohepcidin concentration was measured by elisa using commercially available kit ( drg international , marburg , germany ) .
serum iron- , unsaturated iron binding capacity- ( uibc ) and total iron binding capacity ( tibc ) concentrations were measured on architect ci8200 system ( abbott laboratories , il , usa ) according to the manufacturer s instructions .
the percentage of transferrin saturation ( tfs ) levels was determined by the ratio of serum iron levels to tibc .
serum ferritin was measured using drg ferritin kit ( eia-1872 , drg intl , inc . ,
usa ) and soluble transferrin receptor ( stfr ) concentrations were examined by an stfr elisa ( biovendor laboratory medicine , inc . , czech republic ) .
plasma erythropoietin ( epo ) was determined by the epo elisa kit ( roche diagnostics gmbh , mannheim , germany ) .
data are reported as mean 1sd for parametric data and median ( q1-lower quartile ; q3-upper quartile ) for nonparametric data .
differences were evaluated by the independent - sample t test or the mann - whitney u test as appropriate .
the correlation of serum prohepcidin with iron indices was examined using the spearman rank correlation coefficient .
a total of 100 healthy adult volunteers were recruited from blood donor candidates from the regional blood transfusion center in bydgoszcz ( poland ) . during the 18 months of the study 56 males ( aged 19 - 60 yrs ) and 44 females ( aged 19 - 50 yrs ) were enrolled .
those who had taken any iron supplements or been vegetarian were excluded from this study .
university in bydgoszcz approved a study protocol and a written informed consent was obtained from the subjects .
blood samples were drawn from an antecubital vein between 8 and 10 a.m. with minimal venous stasis and centrifuged . serum and plasma were stored at -80c until analysis .
serum prohepcidin concentration was measured by elisa using commercially available kit ( drg international , marburg , germany ) .
serum iron- , unsaturated iron binding capacity- ( uibc ) and total iron binding capacity ( tibc ) concentrations were measured on architect ci8200 system ( abbott laboratories , il , usa ) according to the manufacturer s instructions .
the percentage of transferrin saturation ( tfs ) levels was determined by the ratio of serum iron levels to tibc .
serum ferritin was measured using drg ferritin kit ( eia-1872 , drg intl , inc . ,
usa ) and soluble transferrin receptor ( stfr ) concentrations were examined by an stfr elisa ( biovendor laboratory medicine , inc . , czech republic ) .
plasma erythropoietin ( epo ) was determined by the epo elisa kit ( roche diagnostics gmbh , mannheim , germany ) .
data are reported as mean 1sd for parametric data and median ( q1-lower quartile ; q3-upper quartile ) for nonparametric data .
differences were evaluated by the independent - sample t test or the mann - whitney u test as appropriate .
the correlation of serum prohepcidin with iron indices was examined using the spearman rank correlation coefficient .
serum ferritin levels were significantly higher in males ( me=15.3 ng / ml ) than in females ( me=8.1 ng / ml ) ( p=0.0006 ) .
to our surprise , depletion of body iron stores , defined as serum ferritin below 10 ng / ml , was observed in 20% of male and 44% of female healthy volunteers .
no significant differences in serum iron , stfr , epo and transferrin saturation between males and females were detected .
prohepcidin levels were significantly higher in males ( median 145.7ng / ml ; ( 123,3 - 213,7 ) than in females ( median 127.3 ng / ml ; 107,4 - 158,6 ) ( fig .
serum prohepcidin levels were not associated with age in both sexes ( r=-0.2064 , p=0,1270 ; r=-0.0196 , p=0,8996 , respectively for males and females ) .
linear regression analyses on serum prohepcidin concentrations with iron metabolism parameters revealed statistically significant negative correlations between prohepcidin and ferritin in males ( r=-0.52 ; p<0.01 ) ( fig .
hepcidin measurement might turn to be a useful tool in the differential diagnosis of iron deficiency or iron overload disorders in the near future [ 22 , 23 , 24 , 25 ] .
it is required to establish a reference interval for this hormone and to investigate important aspects of hepcidin biology i.e. diurnal variations , the half - life , age- , sex- , and race- related differences .
we undertook the present study to examine the changes in serum prohepcidin concentration as a function of gender and age in healthy adults .
our studies show that prohepcidin levels range from 74.9 ng / ml to 300.4 ng / ml in healthy adults of both sexes .
2004 ) observed lower prohepcidin levels in the group of 26 healthy volunteers ( 51.6 153.4 ng / ml ) . on the contrary ,
significantly higher results were reported in studies on newborns , non - anemic infants and premenopausal women indicating that prohepcidin shows an extensive physiological variation in serum concentration in healthy subjects [ 6 , 19 , 27 ] .
differences in serum prohepcidin concentrations between healthy males and females were observed by luukkonen et al . in 2006 .
they reported mean serum prohepcidin concentrations of 254 ng / ml and 227 ng / ml , respectively , for 16 males and 37 females .
however , the difference was not statistically significant because of the large between - subject variation .
serum prohepcidin levels were significantly higher in males than in females healthy individuals ( 145.7 ng / ml vs 127.3 ng / ml ) .
lower prohepcidin concentration in women compared with men may be attributable in part to the high incidence of iron depletion observed in women ( 44% ) .
it is known , that menstruating women are especially prone to develop iron deficiency because of ongoing blood loss , recent pregnancies , and inadequate dietary iron [ 29 , 30 ] .
we suggest therefore , that downregulation of serum prohepcidin is perhaps a physiological reaction of the organism to a low - grade iron deficiency observed in young females .
our study group consisted of 100 healthy individuals between 18 to 60 years of age .
nevertheless , analysis revealed no significant correlations between prohepcidin and age in both male and female subjects . instead
, prohepcidin levels in serum of healthy adults of both sexes were negatively correlated with ferritin . the well - established negative relation between prohepcidin and ferritin , observed in the present study ,
could be explained by the physiological feedback loop where increased hepcidin levels through degradation of ferroportin leads to systemic iron deficiency ( low ferritin concentration ) .
in contrast to our results , some clinical studies indicated positive correlations between prohepcidin and ferritin [ 31 , 32 , 33 , 34 ] .
its synthesis is induced by proinflammatory cytokines such as il-1 , il-6 or tnf-. therefore , during inflammation both hepcidin and ferritin increase significantly and are thus positively correlated .
the results of our study should be taken into consideration while establishing reference intervals for serum prohepcidin .
further studies are required to examine serum hepcidin levels and their mutual relations with prohepcidin .
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introductionwithin the last 8 years , it has become evident that hepcidin has a diagnostic and therapeutic potential .
therefore , it is a great need to establish the reference interval for hepcidin and its precursor .
the aim of this study was to assess the impact of age and sex on serum prohepcidin concentration in healthy adults .
material and methods : 100 healthy volunteers were enrolled during the 18 months of the study - 56 males and 44 females , mean age 34.810.1 yrs .
serum prohepcidin , ferritin , soluble transferring receptor ( stfr ) and plasma erythropoietin were examined by enzyme - linked immunosorbent assay ( elisa ) kits .
serum iron and unsaturated iron binding capacity were determined on architect ci8200 ( abbott diagnostics ) according to the manufacturer s instructions.resultsserum prohepcidin concentrations ranged from 74.9 ng / ml to 300.4 ng / ml in healthy adults of both sexes . however , prohepcidin levels were significantly higher in males ( median value 145.7 ng / ml ) than in females ( median 127.3 ng / ml ) ( p=0.0016 ) .
serum prohepcidin was not associated with age in the group of healthy adults.conclusionsserum prohepcidin concentrations were found to be related to sex .
significantly lower prohepcidin levels were observed in females compared with males .
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with the development of gas and petrochemical industries in the persian gulf region , many workers are required to do jobs of different intensities in the hot and humid weather conditions . among the different workers , those who work in the construction of buildings , installation of technical equipments , welding , and driving
although the amount of physical demand has been decreased due to the technological progression in construction industry and equipment installation , the task performed is severe or moderate physical activities in many cases .
doing tasks in hot conditions results in increased blood supply required by large muscles , also the exposure to sultry weather and heat excretion through sweating and evaporation of sweat increases the potential of cardiac strain incidence .
the prevalence of overweight and obesity is increasing at an alarming rate in developing and industrial countries .
have reported that obesity and overweight increased in iran from 13.6% and 32.2% in 1999 to 19.6% and 35.8% in 2005 and 22.3% and 36.3% in 2007 , respectively .
janghorbani et al . have reported the mean overweight and obesity among men as 42.8% and 11.1% , respectively .
the epidemiological studies indicate that overweight and obesity are important risk factors for some diseases such as diabetes , cardiovascular disease , cancer , and premature death .
furthermore , considering the fact that fat tissue is a good thermal insulator and has less density and blood vessels compared to other tissues , heat transfer coefficients for muscle and skin tissue are 95% and 85% , respectively . also , for fat tissue , it is 36% .
although fat tissue plays a positive role in cold strain , it has a negative effect on heat strain .
moreover , in many people , low level of physical fitness causes overweight , which increases the heart rate at the time of physical activity .
overweight can be delineated by higher body mass index ( bmi ) as well as fat tissue percentage .
the reduction of performance in hot weather in obese people is due to higher metabolism rate and slower loss of heat load caused by low area / weight ratio , which has a considerable influence on the capacity and appropriateness of the job . with regard to the high prevalence of obesity and overweight in adults population , as well as the fact that a high percentage of this population is exposed to hot weather and high humidity simultaneously in the south of iran , particularly in the hot seasons of the year , the question that comes to mind
is whether this coincidence , hot weather and high humidity , augments the occurrence of cardiac strain among workers .
thus , considering the limitation of relevant studies in such climatic conditions during the warm months in the persian gulf region , this study was aimed to determine the relationship between the severity of cardiac strain and overweight in the real field work carried out in very hot and humid weather in the south of iran .
this cross - sectional study was conducted on 71 ( a total of 350 ) workers during 2 months , from june to september 2010 in the south of iran .
subjects were engaged in outdoor jobs at two sites of petrochemical industrial complex that were exposed to heat stress .
subjects were selected by simple random sampling method . out of the 76 workers in the study ,
the mean ( standard deviation ) of age , height , weight , and bmi were 31.6 ( 8.6 ) years , 171 ( 5.9 ) cm , 73.5 ( 13.3 ) kg , and 25.0 ( 4.0 ) kg / m , respectively .
inclusion criteria were voluntary participation from workers , heat acclimatization , absence of diseases such as cardiovascular disease , respiratory disease , infectious disease , diabetes , and hyperthyroidism , and they were not under any medication .
therefore , participants were medically screened by physician ; at the time of study , all subjects were healthy .
this study was approved by the medical ethics committee of the faculty of medical science at the tarbiat modares university and all subjects signed a consent form in accordance with the helsinki declaration . after informing the individuals about the aim of the study ,
all subjects were instructed not to drink coffee or alcohol at the night before the day of testing .
after resting for 30 min in a cool room ( wet bulb globe temperature index = 22.6 1.9 ) , body height was measured in the erect position without shoes to the nearest 0.1 cm using wall - mounted stadiometer .
body weight was measured in minimum clothing to the nearest 0.1 kg using a electronic personal scale ( hamilton sh 223 china ) , heart rate at 20 , 25 , and 30 min ( as baseline ) was measured by a heart rate monitor ( polar electro rs100 , finland ) .
then , without removing the measuring devices , the subjects were asked to begin their work .
if the work location was farther than 50 m from the cool room , the subjects were transported by car . after they started the work , the researcher measured and recorded the heart rate continuously .
simultaneous measurement of heart rate , dry bulb temperatures , wet bulb temperature , and globe temperature was carried out .
wbgt index at rest and work was also measured by microtherm wbgt ( casella cel u.k ) .
after 60 min of heat exposure , the subjects were asked to stop their work and sit on a footstool in the same work station for 5 min .
heart rate was measured at the last 30 sec of each minute of the recovery period , that is , from 30 sec to 1 min ( p1 ) after stopping work , and from 2.5 to 3 min ( p3 ) and from 4.5 to 5 min ( p5 ) .
assessment of heart rate recovery and reaching the normal rate was done in comparison with p1 and p3 : if p1
p3 < 10 and p3 < 90 beats per minute ( bpm ) , the heart rate reduction pattern was normal ; if p1
p3 > 10 and p3 < 90 bpm , the duration of the heart rate returning to the normal pattern was long and conditions required further analysis ; and if p1
p3 < 10 and p3 > 90 bpm , it shows that heart rate returning to normal did not happen and this no recovery pattern indicates too much strain . to estimate the effect of physical activities , the persian version of rating perceived exertion of the eston
, all measurements were performed outdoor from 9.00 am to 12.00 pm and from 3.00 to 6.00 pm . at the end of measurements , bmi and body surface area ( bsa )
were calculated , according to the equations bmi = height ( m)/weight ( kg ) and bsa = 0.20247 weight height , respectively .
the maximum heart rate ( mhr ) was estimated from the formula 220 age .
heart rate reserve ( hrr ) was calculated as the difference between mhr and resting heart rate ( rhr ) .
net cardiac cost ( ncc ) was calculated as the difference between working heart rate ( whr ) and rhr .
relative cardiac cost ( rcc ) was obtained by expressing the ncc as the percentage of the hrr of the participants by using the following equation : rcc = ncc / hrr 100 . the relative cardiovascular load ( % cvl )
was evaluated on the basis of hr as follows : % cvl = 100 ( ( whr rhr)/hrmax ( 8 hours ) ) where , hrmax ( 8 hours ) is the maximum acceptable hr for a work shift of 8 hours , that is , 1/3 ( 220 age ) + rhr .
the cvl evaluates the cardiovascular load or aerobic strain and can be classified as follows : < 30% cvl , acceptable level , no actions required ; 30 - 60% cvl , moderate level , peak loads should be reduced within a period of weeks ; 61 - 100% cvl , high level , peak loads should be reduced within a period of months ; > 100% cvl , intolerable high level , peak loads should be reduced immediately or work must be stopped . with regard to the fact that heart rate is affected by the activity intensity , to neutralize the effect of intense activity ,
heart rate was compared in two groups with different bmi in sitting , low mobility standing , and high mobility standing postures .
the obtained data were analyzed with descriptive statistics , pearson correlation , and t - tests using spss-16 . the significance level equal to 0.05 was considered .
participants were workers in jobs such as welding ( n = 17 ) , construction ( n = 19 ) , assembly of steel structures and components ( n = 10 ) , driving and operating ( n = 15 ) , or supervising ( n = 10 ) .
bmi was greater than 25 kg / m in 42% of workers , and based on world health organization criteria , were classified as overweight or obesity groups .
individual characteristics of the workers in three postures , sitting , standing with low mobility , and standing with high mobility , in two groups based on bmi are shown in table 1 .
the mean ( sd ) , maximum , and minimum bmi was 25 ( 4 ) , 17.5 , and 37 kg / m , respectively . all the three levels of working , the means of bmi , weight , and bsa in the two bmi groups were significantly different ( p > 0.001 ) . in the average of age and height in the two bmi groups , there was no significant difference ( except age in low mobility group ) .
in the two groups with different bmi , the mean of rhr in sitting posture ( p = 0.021 ) , and whr ( p = 0.002 ) and hrr ( p = 0.008 ) in low mobility posture were significantly different .
physical and physiological characteristics of subjects in different bmi and posture groups the average ( sd ) of rhr in the normal weight and overweight groups were 70 ( 11.9 ) and 75 ( 9.6 ) , respectively .
their means were not significantly different ( p = 0.06 ) . the average ( sd ) of mhr in the normal weight and
overweight groups were 190 ( 8.3 ) and 186 ( 8.4 ) , respectively ; the difference between their means was significant ( p = 0.023 ) .
the average ( sd ) of heart rate in the normal weight and overweight groups were 120 ( 14.5 ) and 110 ( 10.0 ) , respectively , and the difference was significant ( p = 0.002 ) .
the physiological strain indices in the normal weight and overweight groups at different levels of activity are presented in table 2 .
physical workload in two different bmi groups in relation to work posture the mean standard deviation of dry bulb temperature , wet bulb natural temperature , relative humidity , globe temperature , and wet bulb globe temperature index were 37.4 3.0 , 31.0 2.0 , 62 12.8 , 30.0 3.9 , and 33.3 2.0 , respectively .
the means at the three levels of postures in the normal weight and overweight groups are shown in table 3 .
the average of all temperatures and wbgt index at the three levels of postures in the two bmi groups was not significantly different , and therefore the heat stress was almost the same in all cases .
heat stress in two different bmi groups in relation to work posture the average ( sd ) of whr in the normal weight and overweight groups were 101 ( 20.3 ) and 112 ( 18.9 ) , respectively , and the difference in means was significant ( p = 0.026 ) .
the means of ncc in the normal weight and overweight groups were 30.5 ( 17.6 ) and 36.3 ( 19.0 ) , respectively , and the difference in means was not significant .
the average ( sd ) of rcc in the normal weight and overweight groups were 26.6 ( 15.1 ) and 32.4 ( 16.2 ) , respectively , and the difference was not significant ( p = 0.073 ) .
the average ( sd ) of heart rate recovery indicator ( p1 p3 ) in the normal weight and overweight groups were 6.5 ( 6.9 ) and 6.6 ( 6.3 ) , respectively , and the difference was not significant . according to the data in table 4 , all the three levels of activity and the average of whr , the ncc , and the rcc in the overweight group compared with those in the normal weight group were higher . but the mean of whr was statistically different ( p = 0.042 ) .
the data illustrated in table 3 show that the average of heart rate recovery after 3 and 5 min of resting in the overweight group compared with that in the normal weight group tended to increase , but it was statistically significant only in the low mobility standing posture ( p = 0.015 ) .
percentage of subjects exceeding the recommended limits of cardiac strain the pearson correlation between bmi and rhr , ncc , rcc , and whr , adjusted for age , wbgt index , and activity intensity was 0.25 , 0.27 , 0.31 ( p < 0.01 ) , and 0.37 ( p = 0.002 ) , respectively .
based on the data in table 4 , it can be seen that despite the adjusted wbgt index and activity intensity variables , the percentage of cardiac strain indices was higher than the recommended limits in the overweight group .
participants were workers in jobs such as welding ( n = 17 ) , construction ( n = 19 ) , assembly of steel structures and components ( n = 10 ) , driving and operating ( n = 15 ) , or supervising ( n = 10 ) .
bmi was greater than 25 kg / m in 42% of workers , and based on world health organization criteria , were classified as overweight or obesity groups .
individual characteristics of the workers in three postures , sitting , standing with low mobility , and standing with high mobility , in two groups based on bmi are shown in table 1 .
the mean ( sd ) , maximum , and minimum bmi was 25 ( 4 ) , 17.5 , and 37 kg / m , respectively . all the three levels of working , the means of bmi , weight , and bsa in the two bmi groups were significantly different ( p > 0.001 ) . in the average of age and height in the two bmi groups , there was no significant difference ( except age in low mobility group ) .
in the two groups with different bmi , the mean of rhr in sitting posture ( p = 0.021 ) , and whr ( p = 0.002 ) and hrr ( p = 0.008 ) in low mobility posture were significantly different .
physical and physiological characteristics of subjects in different bmi and posture groups the average ( sd ) of rhr in the normal weight and overweight groups were 70 ( 11.9 ) and 75 ( 9.6 ) , respectively .
their means were not significantly different ( p = 0.06 ) . the average ( sd ) of mhr in the normal weight and
overweight groups were 190 ( 8.3 ) and 186 ( 8.4 ) , respectively ; the difference between their means was significant ( p = 0.023 ) .
the average ( sd ) of heart rate in the normal weight and overweight groups were 120 ( 14.5 ) and 110 ( 10.0 ) , respectively , and the difference was significant ( p = 0.002 ) .
the physiological strain indices in the normal weight and overweight groups at different levels of activity are presented in table 2 .
the mean standard deviation of dry bulb temperature , wet bulb natural temperature , relative humidity , globe temperature , and wet bulb globe temperature index were 37.4 3.0 , 31.0 2.0 , 62 12.8 , 30.0 3.9 , and 33.3 2.0 , respectively .
the means at the three levels of postures in the normal weight and overweight groups are shown in table 3 .
the average of all temperatures and wbgt index at the three levels of postures in the two bmi groups was not significantly different , and therefore the heat stress was almost the same in all cases .
the average ( sd ) of whr in the normal weight and overweight groups were 101 ( 20.3 ) and 112 ( 18.9 ) , respectively , and the difference in means was significant ( p = 0.026 ) .
the means of ncc in the normal weight and overweight groups were 30.5 ( 17.6 ) and 36.3 ( 19.0 ) , respectively , and the difference in means was not significant .
the average ( sd ) of rcc in the normal weight and overweight groups were 26.6 ( 15.1 ) and 32.4 ( 16.2 ) , respectively , and the difference was not significant ( p = 0.073 ) .
the average ( sd ) of heart rate recovery indicator ( p1 p3 ) in the normal weight and overweight groups were 6.5 ( 6.9 ) and 6.6 ( 6.3 ) , respectively , and the difference was not significant . according to the data in table 4 , all the three levels of activity and the average of whr , the ncc , and the rcc in the overweight group compared with those in the normal weight group were higher .
the data illustrated in table 3 show that the average of heart rate recovery after 3 and 5 min of resting in the overweight group compared with that in the normal weight group tended to increase , but it was statistically significant only in the low mobility standing posture ( p = 0.015 ) .
percentage of subjects exceeding the recommended limits of cardiac strain the pearson correlation between bmi and rhr , ncc , rcc , and whr , adjusted for age , wbgt index , and activity intensity was 0.25 , 0.27 , 0.31 ( p < 0.01 ) , and 0.37 ( p = 0.002 ) , respectively . based on the data in table 4 , it can be seen that despite the adjusted wbgt index and activity intensity variables , the percentage of cardiac strain indices was higher than the recommended limits in the overweight group .
in this cross - sectional study , the level of cardiac strain among workers exposed to very humid climatic conditions was found to be higher among overweight or obese people than in those with normal weight , particularly in standing posture with low / high activity . on the other hand , out of all parameters showing cardiac strain , higher activity exceeded the acceptable limits in overweight and obese people than in those with normal weight [ table 4 ] . nevertheless , all the climatic parameters of the workplace were not significantly different for all activities in both the groups .
therefore , based on the comparison between the two groups with the same activity , it can be concluded that the differences between the parameters representing cardiac strain were caused by neither the climatic conditions nor the severity of activity , but were more influenced by bmi .
first of all , for doing a certain job , compared to the normal weight , overweight and obesity increases the metabolism of work as well as makes the rate of heat conduction of central parts of the body lower due to the decrease in the surface area to mass ratio since the fat tissue heat capacity ( 0.2 w / m c ) is lesser than that of the muscular tissue ( 0.5 - 0.6 w / m c ) .
furthermore , considering that fat tissue possesses lesser heat capacity ( 0.4 kcal / kg c ) than the whole body ( 0.82 kcal / kg c ) , saving certain amount of heat in the body enhances the temperature of fat tissue .
also , peripheral blood flow ( skin ) in thin people is lesser than in obese people at the time of working .
hence , the potential of heat accumulation enhances in the central parts of the body in obese or overweight people , which itself leads to heart rate increase in order to speed up the peripheral blood flow and this mechanism might have led to increased bmi as a risk factor in job disease incidence caused by hot weather of the workplace .
a significant correlation between heart rate at rest and relative obesity in young and middle - aged men has been reported in sitting posture , which aligns with the result of this study .
besides , a significant correlation has been reported at the time of activity between the body fat content and heart rate increase in experimental conditions .
pin and chung studied 218 soldiers who suffered from thermal disorders and 537 soldiers as the control group with regard to age and gender . in this study , they reported that the odd ratio in obese people ( bmi > 27 kg / m ) for the occurrence of thermal disorders was 3.53 . under the experimental conditions ,
it was also reported that body weight , bmi , body fat percent , and a decline in the surface area to mass ratio were significantly related with cardiac strain increase ( deep body temperature and heart rate ) . in the evaluation of the relation between bmi and thermal fatigue , bate and donoghue
reported that , with increasing bmi , the risk of thermal fatigue occurrence was obviously increased , as the odd ratios for bmi < 27 , 27 < bmi < 32 , and bmi > 32 were 1.0 , 2.94 , and 3.63 , respectively .
according to the study results , the intensity of cardiac strain among overweight or obese workers was significantly higher than it was in those with normal weight . therefore , in order to control workers cardiac strain , employing people with bmi more than 25
kg / m in hot humid conditions should be avoided while monitoring people before employment .
furthermore , it is proposed that to decline the intensity of cardiac strain among overweight employees , implementation of some essential interventions such as nutrition education and regular physical activity encouragement seems to be essential .
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background : in hot weather , overweight and obesity are considered as significant risk factors for the incidence of cardiac strain in workers . this study was aimed to compare cardiac strain among overweight and normal - weight workers in hot , humid conditions in the south of iran.methods:this cross - sectional study was conducted on 71 workers in the south of iran in summer 2010 .
the heart rate was measured at rest and at actual work .
cardiac strain based on working heart rate ( whr ) , the relative cardiac cost ( rcc ) , the net cardiac cost ( ncc ) , load relative cardiovascular ( cvl ) , and heart rate reduction was analyzed in 35 normal - weight people ( body mass index ( bmi ) < 25 kg / m2 ) and 36 overweight people ( bmi > 25 kg / m2 ) using descriptive statistics.results:in 42% of the total workers , bmi was > 25 kg / m2 .
the average of wet bulb globe temperature index ( wbgt index ) in the two groups was not significantly different .
the mean whr in the two groups was 101 20.3 and 112 18.9 , respectively ( p = 0.026 ) .
percentages exceeded the acceptable limits in the parameters ncc , rcc , whr , cvl , and brouha index , which were significantly higher in overweight people than in those with normal weight.conclusions:based on the study results , it is concluded that the severity of cardiac strain was higher in overweight workers compared with that in normal weight workers .
hence , in order to decrease the cardiac strain , selecting overweight individuals for these jobs should be avoided and also some vital intervention for losing weight should be implemented such as nutrition education and encouraging them regarding physical activity .
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cerebral cysticercosis is caused by infection of the central nervous system by the larvae of the pork tapeworm , taenia solium .
the parasites can be located in the brain parenchyma , the subarachnoid space and the ventricular system in highly variable combinations .
the frequency of the diagnosis has been increasing , presumably as a result of the widespead availability of computed tomography .
empty - sella syndrome is the name given by busch to an enlargement of the sella due to bulging of the arachnoid through a defect of the dural diaphragm .
pressure and pulsation of the cerebrospinal fluid supposedly act on the walls of the sella turcica so as to flatten the pituitary gland .
the primary empty sella syndrome has been a subject of much interest and research in recent years on account of its clinical correlations with headache , visual defect , papilledema and endocrine disorders . in this paper
we report a very rare case of total empty sella syndrome associated with cerebral cysticercosis .
a 55-year - old korea woman , previously in good health , presented with severe recurrent headache and dizziness .
she had no previous parasitic infections and had not ingested any raw or undercooked pork .
the headache , which was localized on the frontal and the occipital areas , had a throbbing characteristic .
it was not associated with any other specific signs or symptoms and there was also no evidence of migraine .
six moths prior to admission , she had once visited a private clinic because of headache .
she refused the recommended brain ct scan , but took some medicine prescribed by the clinic .
examination of csf showed an opening pressure of 130mmcsf , 9 leukocytes / mm3 , a protein level of 30mg / dl and a glucose level of 49mg / dl .
a ct scan showed multiple low - density cystic lesions in the suprasellar and left sylvian cisternae compatible with empty - sella syndrome ( fig .
1 ) . in order to evaluate the communication between the cisternae and the suprasellar cyst ,
2 ) . enzyme linked immunosorbent assay ( elisa ) for anti - cysticerci igg in the patient s csf and serum gave titers of 1.14 and 0.95 , respectively , consistent with active infection ( table 2 ) .
the reserve capacities were all prompt except for the tsh response which was blunted ( table 1 ) .
t4 5ug / dl , ft4 0.86ng / ml , estradiol 25 pg / dl and prolactin 4.31ng / ml . after the patient gave an informed consent , praziquantel therapy was started at 50mg / kg body weight / day in three divided doses for 15 days .
on the first day of praziquantel treatment , the patient complained of diffuse headache , and a low - grade fever and signs of mild meningismus developed .
the therapy was continued with dexamethasone added to the original regimen from the second day on .
she was discharged on an anticonvulsant medication with a tapering course of steroid . a follow - up ct scan , done one month after praziquantel therapy , showed a slight reduction in the size of all intracranial cysts ( fig .
the term empty sella was applied by busch to an autopsy material in which the diaphragma sella was incomplete regardless of whether there had been prior surgical or radiotherapeutic interventions .
later , weiss and raskind emphasized the need to distinguish primary ( without any prior surgical or - radiotherapeutic procedures ) from secondary ( following such procedures ) cases .
because this patient had no past history of operation or radiotherapy , this case would be classified as primary empty - sella syndrome associated with multilobulated cystic lesions in the suprasellar cistern and the left sylvian cistern .
the total empty sella syndrome associated with cns cysticercosis has not been reported so far .
headache is by far the most frequent clinical manifestation of empty - sella syndrome ; on review of previous case studies , it appears to occur in approximately 80% of cases , while papilledema , visual defects and endocrine disorders occur in no more than 8% , 16% and 25% , respectively . in the case of neurocysticercosis ,
headache together with seizure is one of the most frequently manifested symptoms . in this patient
, however , the origin of the headache might be the combination of the above two conditions , namely the empty sella and the neurocysticercosis , and it was very difficult to confirm whether the one was mainly caused by the other or the two were just incidentally associated with each other .
several mechanisms of the pathogenesis of the empty - sella have been postulated : 1 ) rupture of intrasellar or parasellar cyst 2 ) infarction of the sellar contents , 3 ) pituitary hypertrophy and subsequent atrophy , and 4 ) transmission of cerebrospinal pressure through a congenitally defective sellar diaphragm of these etiologic hypotheses , the most commonly accepted one might be the transmission of either normal or elevated cerebrospinal pressure .
an incomplete sellar diaphragm is an essential prerequisite to the development of the empty sella according to this hypothesis although it was not confirmed in this particular patient .
kaufman called attention to the documented or suspected elevation of csf pressure , but in this patient the elevated csf pressure was not documented .
it is speculated however , that a slightly longstanding elevation of csf pressure by the cyst of the pork tapeworm might have resulted in the enlargement and remodeling of the sellar turcica and in the flattening of the pituitary contents against the floor in the setting of the incomplete sellar diaphragm .
we do not exclude , however , the possibility of a coincidental association of the total empty - sella syndrome with cysticercosis in this particular case . for the evaluation of pituitary functional reserve , a combined pituitary stimulation test was done .
the reserve capacities were all within normal limits , but tsh response by trh was blunted . in the empty sella syndrome , abnormalities of pituitary function have been confined to failure of the growth hormone to increase following insulin induced hypoglycemia , arginine and glucose loads , diminished corticoid response , imparied tsh response , hypogonadotropism and virilization .
surgery has been generally disappointing and largely limited to managing the secondary effect of the infection , and it is not helphul for the most common presentation of the disease , namely , diffuse parenchymal infection as observed in our patient . the medical treatment of neurocysticercosis is greatly improved by praziquantel , the first effective drug aganist cysticerocosis .
the treatment with praziquantel clearly elicited an immunogenic effect , as evidenced by the devolpment of the cerebrospinal fluid syndrome accompanied by a significant rise in the levels of specific igg that followed treatment .
most authors have stressed the importance of simultaneous use of steroid given with parziquantel to prevent the dampening of the inflammatory reaction that often follows the action of the antiparasitic agent on the larvae .
early in the course of the praziquantel treatment without dexamethasone , this patient showed symptoms of cerebrospinal reaction syndrome such as headache , meningismus and fever . in this patient
there would be no way of determining , retrospectively , if the cerebrospinal fluid reaction was really diminished in its intensity by the steroid therapy , but we were able to complete the praziquantel course in this patient without further complication . in patients with cns cysticercosis , the pituitary gland should be evaluated with a sella ct scan which can diagnose empty sella syndrome . inevitably , the reserve capacity of the pituitary gland should be investigated for the patient with empty sella syndrome .
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a 55-year - old woman presented with severe recurrent headache accompanied by dizziness .
the brain ct scan showed multiple low - density cystic lesions in the suprasellar and left sylvian cisternae with total empty sell syndrome .
the communication between the cisternae and the suprasellar cyst was not verified on the metrizamide ct scan .
treatment with praziquantel resulted in headache inilially and a rise in specific igg .
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freshwater aquaculture plays a very significant role in global aquaculture production . in 2011 , 56.8% of the global aquaculture production was freshwater fishes , and output amounted to 35.6 million tons .
grass carp ( ctenopharyngodon idellus ) is one of the most important freshwater fish that is native to china , and it plays an important role in aquaculture with 4.57 million tons produced in 2011 , the highest in fish production worldwide . in china ,
the grass carp industry aims to increase the production of value - added products in order to improve profitability , and crisp grass carp is a kind of high value - added fishes which have firmer muscle and higher contents of crude protein , fat , and amino acids than grass carp [ 35 ] .
currently in guangdong province of china , the crisp grass carp has become an economically important freshwater fish because of its increased muscle firmness ( hardness ) .
fillet firmness of fish is an important quality trait for consumer acceptance in many studies with chinook and atlantic salmon [ 6 , 7 ] , channel catfish , and gilthead sea bream .
muscle firmness is associated with the intrinsic structure and properties of components of the flesh .
it has been found in many studies that firmness is influenced by muscle fiber density , muscle fiber diameter , and intermyofibrillary spaces and gaps [ 1013 ] .
these factors are determined by changes in the cellularity of skeletal muscle [ 14 , 15 ] .
the changes in cellularity will contribute to changes in the quality of the skeletal muscle , and since this tissue is the part of the fish destined for human consumption , it may have important economic value . sole faba bean ( vicia faba )
feeding differentially enhances muscle firmness of grass carp and the grass carp with higher muscle firmness is called crisp grass carp .
although the composition of faba bean is complex , common characteristic of muscle firmness increase is also demonstrated in other fishes feeding on faba bean including european seabass ( dicentrarchus labrax ) [ 18 , 19 ] and channel catfish ( ictalurus punctatus ) . in the previous studies of crisp grass carp muscle , it has been found that the diameter of muscle fibers was decreased and the content of the ecm was increased [ 2 , 4 , 17 ] , and our team also found that the increase in the expression of type i collagen in crisp grass carp is higher than those of grass carp .
however , the regulatory mechanism of muscle firmness increase in crisp grass carp is still unclear . given that the expression of muscle firmness increase was regulated by multiple genes network , it is expedient to analyze systematically muscle firmness increase of crisp grass carp in the gene levels using microarray technology , which may help to explore signal transduction pathways of nutritional regulation of fish muscle firmness .
microarray technology presents a powerful tool for revealing expression patterns and genes associated with phenotypic characteristics . by determination of expression levels of thousands of genes simultaneously in muscle tissue
, it could be effective to reveal global gene expression patterns and to identify genes or groups of genes associated with texture variations of atlantic salmon . in this study , crisp grass carp ,
having higher muscle firmness , is used to characterize the global gene expression profile in the muscle in comparison with that of grass carp and analyze the biological functions and possible signal transduction pathways that address muscle firmness of crisp grass carp .
the grass carp and crisp grass carp are raised in six enclosures in the dongsheng aquatic breeding base ( zhongshan , guangdong , china ) , and the diet of grass carp is artificial feed and the diet of crisp grass carp is sole faba bean ( vicia faba ) .
the average weights of the specimens were 3.98 0.36 kg ( n = 60 ) for crisp grass carp and 3.45 0.52 kg ( n = 60 ) for grass carp . in this paper , living fishes
were directly dissected and the white muscle tissues of three fishes were obtained for crisp grass carp and grass carp , respectively .
the obtained samples were snap - frozen in liquid nitrogen and stored at 80c for rna extraction .
total rna was isolated from white muscle using the trizol reagent ( invitrogen , carlsbad , ca ) according to the manufacturer 's instruction .
the concentration of the isolated rna was determined by measuring absorbance at 260 nm .
the integrity of the rna was determined by agarose gel electrophoresis and agilent bioanalyzer 2100 .
affymetrix zebrafish chip containing oligonucleotides representing 14,900 transcripts was used to profile the differences in genes expression of the muscles between crisp grass carp and grass carp .
gene expression levels were determined by comparing the amount of mrna transcript present in the experimental sample to the control .
hybridizations were performed in the fluidics station 450 and chips were scanned using the affymetrix scanner 3000 .
fluorescent signal intensities for all spots on the arrays were analyzed using the gene chip operating system ( gcos ; affymetrix ) .
microarray data were deposited ( according to microarray gene expression data society standards ) in the ncbi gene expression omnibus ( geo , http://www.ncbi.nlm.nih.gov/geo/ ) with the series accession number ( gse4787 ) .
the categorization of biological process go ( gene ontology ) was analyzed using david bioinformatics resources 6.7 ( http://david.abcc.ncifcrf.gov/ ) . within the significant category , the enrichment re was given by re = ( nf / n)/(nf / n ) , where nf was the number of differential genes within the particular category , n was the total number of genes in the same category , nf was the number of differential genes in the entire microarray , and n was the total number of genes in the microarray .
the pathway analysis was conducted using kegg ( kyoto encyclopedia of genes and genomes ) database . the false discovery rate ( fdr )
p value < 0.05 and fdr < 0.05 were used as the threshold to select significant go categories and kegg pathways . to validate microarray data , the expression levels of six genes of interest were quantified using real - time pcr with -actin as the internal control .
these genes included myostatin ( mstn ) , collagen type i alpha-1 ( col1a1 ) , collagen type i alpha-2 ( col1a2 ) , and calreticulin ( calr ) , heat shock cognate 70-kd protein ( hsp70 ) , and heat shock protein 90 kda alpha ( hsp90 ) ( table 1 ) .
the cdna synthesis was performed using 0.5 g of dnase - treated total rna ( turbo dna - free ; ambion , austin , tx , usa ) using taqman gold reverse transcription kit ( applied biosystems , foster city , ca , usa ) and oligo - dt primers .
pcr primers ( table 1 ) were designed using vector nti and synthesized by invitrogen . the amplicon lengths were checked on 1% agarose gel .
pcr efficiency was calculated from tenfold serial dilutions of cdna for each primer pair in triplicate .
real - time pcr assays were conducted using faststart sybr green master ( roche diagnostics , mannheim , germany ) in an optimized 12 l reaction volume , using 1 : 10 diluted cdna , with primer concentrations of 0.40.6 m .
pcr was performed in duplicate in 96-well optical plates on light cycler 480 ( roche diagnostics , mannheim , germany ) under the following conditions : 95c for 5 min ( preincubation ) , 95c for 5 s , 60c for 15 s , 72c for 15 s ( amplification ) , 95c for 5 s , and 65c for 1 min ( melting curve ) .
statistical differences were determined by one - way anova followed by duncan 's multiple range test ( p < 0.05 ) .
the microarray analysis demonstrated that expressions of 127 genes were upregulated and 114 genes were downregulated in the muscle of crisp grass carp compared with the control group . according to the genes of differential expression , the biological processes go terms mainly consisted of protein metabolism , muscle development and growth , carbohydrate metabolism , and so on ( figure 1 ) .
differentially expressed genes involved in protein metabolism in the muscle of crisp grass carp and grass carp were shown in table 2 .
expressions of collagen type i alpha-1 and alpha-2 , type ii alpha-1a were upregulated in the muscle of crisp grass carp . differentially expressed genes involved in the protein metabolism were clustered into biological categories including protein transport ( 9 genes ) , proteolysis ( 9 genes ) , and regulation of cellular protein metabolic process ( 4 genes ) .
the 11 genes that regulate the glycoproteins were found with nine notably upregulated and two downregulated .
the genes involved in muscle development and growth were classified into developmental growth ( 4 genes ) , muscle cell differentiation ( 4 genes ) , skeletal system development ( 4 genes ) , and cytoskeleton organization ( 14 genes ) in the crisp grass carp .
above all , transcription of mstn , which was tightly related to muscle development , was upregulated in the muscle of crisp grass carp ( table 3 ) .
downregulated expressions of glycolytic enzymes were detected in the muscle of crisp grass carp ( table 4 ) .
these enzymes include enolase-3 , hexokinase-1 , hexokinase-2 , phosphofructokinase , pyruvate dehydrogenase , glycerophosphodiester phosphodiesterase and phosphatase , and tensin homolog - b . in the muscle of crisp grass carp ,
the gos of these genes included zinc ion binding , calcium and iron ion binding ( table 5 ) .
as genes involved in vitamin metabolism , cysteine conjugate - beta lyase and katiii were upregulated .
the differential expression of genes involved in protein biosynthesis occurred at multiple levels , including regulation of transcription ( 31 genes ) , rna processing ( 6 genes ) , and tetratricopeptide - like helical domain ( 8 genes ) ( table 6 ) .
fifty - two differentially expressed genes were included in the go terms of immune system development and immunoglobulin - like domain ( 13 genes ) , embryonic morphogenesis ( 9 genes ) , golgi apparatus ( 6 genes ) , neuron differentiation ( 8 genes ) , organelle membrane ( 13 genes ) , and fin morphogenesis ( 3 genes ) ( table 7 ) . to further analyze the interactional relation of all differentially expressed genes ,
the results of pathway analysis found that downregulated signals in glycolysis / gluconeogenesis pathway happened in the crisp grass carp ( p value < 0.01 ) .
the detailed information of glycolysis / gluconeogenesis pathway was shown in figure 2 , which was formed from all differentially expressed genes . to verify the data obtained by microarray analysis , quantitative real - time pcr was performed for six genes , including five upregulated genes and one downregulated gene , with a -actin gene used as an internal control .
the relative hybridization intensities of the six selected genes are basically consistent with those analyzed by real - time pcr , thus confirming that use of the zebrafish genome array was suitable for this study ( figure 3 ) .
in this study , we show that muscle firmness increase of crisp grass carp is tightly related to the genes of differential expression in the functional groups including differentiation of muscle fibers , deposition of extracellular matrix ( ecm ) , glycolysis / gluconeogenesis pathway , and calcium metabolism .
the decrease in the diameter of muscle fibers in crisp grass carp may be related to the downregulated expressions of mstn and axin and differentially expressed genes involved in diminution of actin filaments .
mstn , known as growth differentiation factor ( gdf)-8 , was reported to inhibit the proliferation and differentiation of resident muscle cell . in this study ,
evidence that the growth of muscle cell is inhibited in the muscle of crisp grass carp is that , in the muscle of crisp grass carp , the transcription level of mstn is elevated from microarray expression , and the mrna expression of mstn is 3.4 times that of grass carp by gene quantitative analysis .
it was found that the muscle fibres of crisp grass carp were less than those of grass carp . as it is an important protein constituting muscle , diminution of actin filaments in crisp grass carp muscle
the downregulation of actin related protein 2/3 complex and nexilin and upregulation of capping protein in this paper suggested the diminution of the actin filaments in crisp grass carp muscle [ 24 , 25 ] . in our experiments ,
muscle firmness increase of the crisp grass carp has been demonstrated in the increasing deposition of ecm , which includes upregulated expressions of collagen and differential expressions of transforming growth factor-1 ( tgf-1 ) , and the genes related to fibroblasts . as an important protein of ecm
, collagen has been proven to be closely related to the firmness of muscle in fish [ 26 , 27 ] . in zebrafish ,
it was documented that downregulated expression of collagen and collagenase-3 enzyme elicited reduction in the firmness of atrophying muscle of rainbow trout .
our results that the expressions of type i alpha-1 and alpha-2 and type ii alpha-1a collagen are upregulated in crisp grass carp are consistent with the fact that collagen content in the muscle of crisp grass carp was 1.36 times greater than that of grass carp .
and the increase in the collagen content plays an important role in firmness increase of crisp grass carp muscle and resultant texture characteristics [ 2 , 4 ] . for the upregulated expressions of type i and type ii collagen , we speculate that the genes of inf-7 and procollagen - proline 4-hydroxylase ( an enzyme hydrolyzing the collagen ) probably play important roles in crisp grass carp .
the basal expression of type i collagen was inhibited by inf-7 acting in not definitively located promoter region . in the muscle of crisp grass carp , the mrna level of inf-7 was 0.18 times that of grass carp and the transcription levels of type i and type ii collagen increased .
the results suggest that the inhibition of expression of inf-7 in the muscle of crisp grass carp promotes the synthesis of type i and type ii collagen .
the expression of procollagen - proline 4-hydroxylase was downregulated in the muscle of crisp grass carp , and this may help understand the increase in the expression of type i and type ii collagen .
the increasing deposition of ecm in crisp grass carp muscle can also be suggested by enhanced tgf-1 signaling .
enhanced tgf-1 signaling in crisp grass carp could be demonstrated in the differential expressions including upregulated expression of tgf-induced factor homeobox-1 and downregulated expressions of both interferon regulatory factor-7 and interferon - inducible protein - kinase which are closely related to interferon inhibition of tgf-1 signaling pathway .
tgf-1 is a pleiotropic cytokine known to play an important role in cell growth , embryonic development , and tissue repair and could induce the synthesis and accumulation of components of the extracellular matrix ( ecm ) in the muscle .
it was found that upregulated expression of tgf-1 correlated with increase of ecm in the muscle .
in addition , the significantly upregulated expression of activin a receptor in crisp grass carp in this study , a downstream gene of tgf-1 signaling pathway , further confirms enhanced tgf-1 signaling . besides tgf-1 , the genes related to fibroblasts may play important roles in the increasing deposition of ecm .
fibroblasts were proven to produce an accumulation of fibrotic interstitial ecm components such as collagen and fibronectin , growth factors , and cytokines . in the muscle of crisp grass carp ,
the mrna level of fibroblast growth factor receptor 4 was more than that of grass carp , suggesting that fibroblast was activated .
some evidences had shown that mstn could directly stimulate muscle fibroblast proliferation , and the enhancement in the transcripts levels of mstn further demonstrated fibroblast proliferation in the muscle of crisp grass carp .
stromal cell - derived factor ( sdf ) was also involved in the activation , proliferation , and migration of fibroblast and secretion of ecm and played important roles in fibrosis .
both sdf-1 and sdf-4 had been demonstrated to be highly expressed in the muscle of crisp grass carp than those in grass carp , suggesting that these two genes may be responsible for the increased firmness in the muscle of crisp grass carp .
downregulated expressions of genes involved in glycolysis , as a major source of energy in the muscle , could result in the lower expression of myofiber proteins which were closely related to muscle firmness increase . in this study ,
downregulated glycolysis / gluconeogenesis pathway in the crisp grass carp may contribute to the muscle firmness increase of crisp grass carp .
the evidence that glycolysis pathway in the muscle of crisp grass carp is downregulated is the decrease in the expressions of five glycolytic enzymes in addition to aldehyde dehydrogenase which acts on products of glycolysis .
the evidence that crisp grass carp have lower levels of anaerobic metabolism is that they have lower velocity of both glycolysis and tca cycle . on the contrary ,
higher rates of aerobic metabolism in crisp grass carp are demonstrated by the upregulated expressions of mitochondrial genes .
larsson et al . also found that the firmness of atlantic salmon muscle was associated with high rates of aerobic metabolism .
in addition , differential expression of genes involved in glucose utilization has also been found in the change of muscle texture under nutrition restriction [ 26 , 37 , 38 ] .
however , the function and mechanism of glucose utilization acting in the muscle firmness increase still need further exploration .
calcium could activate the increase in the density of filamentous myosins , and the increasing density of filamentous myosins contributes to the muscle firmness increase .
the previous results that the calcium content in crisp grass carp and the density of filamentous myosins were increased could further help to understand the muscle firmness increase in crisp grass carp . in this paper , the expressions of seven genes related to calcium including calreticulin ( crt ) , calmodulin ( cam ) , and cadherin protein ( cad ) were found with upregulated mrna expression .
crt , as one of the major calcium - binding proteins of the endoplasmic reticulum , was involved in the regulation of intracellular ca homeostasis and endoplasmic reticulum ca storage capacity , and its overexpression increased calcium fluxes across endoplasmic reticulum .
cam , a protein that binds calcium with high affinity and specificity , serves as an intracellular ca - receptor and mediates the ca regulation of cyclic nucleotide and glycogen metabolism , secretion , motility , and ca transport .
thus , an increase in the ca content of crisp grass carp muscle further contributed to increased expression levels of calcium - dependent proteins including cam and cad .
in addition , downregulated expressions of three genes including desmocollin , guanylate cyclase activator , and zgc:136759 would help the study of calcium regulation in crisp grass carp .
affymetrix zebrafish chip has been proven to be a valid way to examine the gene expression profiling of grass carp muscle .
because dna microarrays are unavailable for grass carp , the affymetrix zebrafish chip was used in this study .
grass carp is near to the zebrafish in an evolutionary sense , and these two species were a family of cyprinidae .
the affymetrix zebrafish array had been used to screen gene transcript profiles of grass carp recently , and the 416 genes of differential expressions were found to be related to the use of ls as an alternative dietary antibiotic in fish .
use of cross - hybridization with microarrays for analysis of closely related species also had been reported by other researchers .
a cdna microarray from african cichlid fish , astatotilapia burtoni , had been proven to be a powerful tool for analyzing the transcription profile of other cichlid species including enantiopus melanogenys and neolamprologus brichardi and oreochromis niloticus .
the microarray composed of channel catfish ( ictalurus punctatus ) transcripts was effectively used to analyze gene expression profiling of blue catfish ( ictalurus furcatus ) .
the affymetrix zebrafish array was also used to screen gene expression profiles of distantly related species , and it was found that 375 genes were significantly expressed in the muscle tissues of chinese mandarin fish ( siniperca chuatsi ) .
such applications indicated that use of the zebrafish genome array could be a valid way to examine grass carp , and a conclusion was strongly supported in the current study by real - time rt - pcr validation . in conclusion , during the muscle firmness increase from grass carp to crisp grass carp , a total of 127 transcripts were found to be upregulated and a total of 114 transcripts were downregulated . strong correlation with muscle firmness increase of crisp grass carp
was found for these genes from differentiation of muscle fibers and deposition of ecm , and also glycolysis pathway and calcium metabolism may contribute to muscle firmness increase .
however , a number of genes with unknown functions may be related to muscle firmness , and these genes can be regarded as candidate markers of nutritional regulation of grass carp muscle firmness .
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grass carp ( ctenopharyngodon idellus ) is one of the most important freshwater fish that is native to china , and crisp grass carp is a kind of high value - added fishes which have higher muscle firmness . to investigate biological functions and possible signal transduction pathways that address muscle firmness increase of crisp grass carp , microarray analysis of 14,900 transcripts
was performed .
compared with grass carp , 127 genes were upregulated and 114 genes were downregulated in crisp grass carp .
gene ontology ( go ) analysis revealed 30 gos of differentially expressed genes in crisp grass carp . and
strong correlation with muscle firmness increase of crisp grass carp was found for these genes from differentiation of muscle fibers and deposition of ecm , and also glycolysis / gluconeogenesis pathway and calcium metabolism may contribute to muscle firmness increase .
in addition , a number of genes with unknown functions may be related to muscle firmness , and these genes are still further explored .
overall , these results had been demonstrated to play important roles in clarifying the molecular mechanism of muscle firmness increase in crisp grass carp .
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pharmacopuncture therapy is a new acupuncture therapy based on herbal medicine , acupuncture & moxibustion medicine , and meridian theory . through a single procedure
as the pharmacopuncture does not pass through the digestive system , it works fast and it has effects that oral administration does not have .
pharmacopuncture fluid is extracted from these selected herbs and injected into the meridian points or sore spots .
the constituents of the chukyu ( spine - healing ) pharmacopuncture are cervi parvum cornu ( deer antlers ) , moschus ( musk ) , angelicae gigantis radix , cnidii rhizoma , ostericii radix , angelicae pubescentis radix , astragali radix , and scolopendra .
these were extracted at low temperature and low pressure in an aseptic room at the korean pharmacopuncture institute . in the name
yu means healing , chukyu means spinal healing . actually , chukyu ( spine - healing ) pharmacopuncture is known clinically to have effects on lumbago and skelalgia .
several studies on the relation between the toxicity and the composition of chukyu pharmacopuncture have been conducted .
koo et al . conducted a philological study on the toxicity of cnidii rhizoma , roh et al . conducted a philological study on the toxicity and side effects of angelicae sinensis radix and park et al .
choi et al . reported that in toxicological tests , astragali radix showed no toxicity in rats and byun et al .
reported that in an analysis by using microculture tetrazolium ( mtt ) method , the cervi parvum cornu pharmacopuncture was not able to cause cell cytotoxicity in the hepatocytes of rats .
conducted a safety study of scolopendrid pharmacopuncture and showed no abnormal findings after scolopendrid pharmacopuncture treatment .
nevertheless , objective single - dose toxicity testing of chukyu pharmacopuncture , which is complex combination of herbs , has not been conducted yet .
the current research trend for single - dose toxicity testing of extracts is to study acute and subacute toxicity through good laboratory practice ( glp ) regulations .
all the experiments for this research were conducted at biotoxtech , a non - clinical studies authorized institution , under the glp .
this study was performed to analyze the single - dose toxicity and the lethal dose of chukyu pharmacopuncture in rats .
the chukyu pharmacopuncture was prepared in a sterile room at the korean pharmacopuncture institute ( k - gmp ) . after the mixing process with pure water
the reason sprague - dawley rats were chosen is that they have been widely used in safety test in the field of medicine , so the results can be easily compared with many other data bases .
the mean weights of the rats were 179.8 - 198.5 g and 143.8 - 173.1 g , respectively , for the male and the female rats at the time of injection .
for all animals , a visual inspection was conducted ; all animals were weighed using a cp3202s system ( sartorius , germany ) . during 7 days of acclimatization
the temperature of the lab was 20.0 - 22.8 , and the humidity was 48.5 - 65.9% . enough food ( teklad certified irradiated global 18% protein rodent diet 2918c ) and uv - filtered water were provided .
animals were selected if their weights were close to the mean weight . in total , 20 male rats and 20 female rats were selected .
the animals were randomly distributed into 4 groups ( 5 mice per group ) as shown in , table 1 . in clinical applications ,
no death occurred in the pilot test in which 1.0 ml of chukyu pharmacopuncture was injected into each male and female rat . in this study 1.0
ml / animal was set as a high - dose , and 0.5 ml/ animal and 0.1 ml / animal were set as mid and low doses , respectively . in the control group , 1.0 ml of normal saline solution was administered .
a single dose , 0.1 and 0.5 ml/ animal , was injected into the left thigh muscle of the rats in the low and mid - dose groups , respectively , and 0.5 ml of chukyu pharmacopuncture was injected into each thigh muscles of the rats in the high - dose groups , for a total of 1.0 ml / animal , by disposable syringes .
this study was conducted under the approval of the institutional animal ethic committee of biotoxtech co. , ltd .. from the 1st day to 14th day after treatment , the general symptoms were examined once a day . on the day of dosing ( day 0 ) , the general symptoms ( side effects , revealing time , recovery time , etc . ) , as well as mortality , were examined at 30 minutes and at 1 , 2 , 3 , and 4 hours after injection .
the weights were measured immediately before treatment and at 3 , 7 and 14 days after treatment . after fasting for more than 18 hours
blood samples were taken from the abdominal aorta on the day of necropsy ( 15 days after injection ) .
a 1-ml blood sample was analyzed by using an automatic hematology analyzer ( advia 120 , siemems , germany ) .
a 2.0-ml blood sample was centrifuged for the blood coagulation test ( 3,000 rpm , 10 minutes ) .
the results were measured by using an automated coagulation analyzer ( coapresta 2000 , sekisui , japan ) .
the results were measured by using an automatic analyzer ( 7180 , hitachi , japan ) and electrolyte analyzer ( avl9181 , roche , germany ) .
after observations had been terminated , the organs and tissues of all surviving animals were visually inspected and microscopically examined .
the weight , hematologic examination and blood chemical test results from the experiments were analyzed by using sas ( statistical analysis system , version 9.2,9.3 , sas institute inc .
a bartlett test was conducted to evaluate the homogeneity of the variance and the significance . the one - way anova test
was conducted when the homogeneity of the variance was recognized , and the kruskal - wallis test was conducted post - hoc .
in this study , no deaths or abnormalities occurred in any of the groups ( tables 2 , 3 ) in addition , no changes in weight were observed in any of the groups ( table 4 ) finally , no meaningful changes in the hematological examination , blood chemical test or necropsy were noted ( tables 5 , 6 , 7 ) on histopathological examination , interstitial infiltrating macrophages were found in one female rat in the 0.5- ml / animal group , but no significant changes in the brain , lungs , liver , kidney and spinal cord related to the injections were found in the other groups ( table 8)
, there has been only one clinical review on the effects of chukyu pharmacopuncture . on the other hand ,
cervi parvum cornu which is a component of chukyu pharmacopuncture has anti - arthritis effects , as well as analgesic effects [ 11 , 12 ] .
angelicae gigantis radix , cnidii rhizoma , angelicae pubescentis radix , and astragali radix are known to have effects on arthritis in rats .
in addition , scolopendra , through its anti - inflammatory activity , has been reported to have therapeutic effects on patients with herniated intervertebral discs of the lumbar spine or cauda equine syndrome [ 18 - 20 ] .
thus , chukyu pharmacopuncture and its component herbs have been reported to have many effects on several disorders . although it is used in clinics , safety studies on chukyu pharmacopuncture are insufficient , so more safety studies are needed .
the toxicity test is an important data base and is essential for evaluating the safety of test substances in medications . *
doses of 0.1 , 0.5 , 1.0 ml of chukyu pharmacopuncture were administered to the experimental groups , and a 1.0-ml dose of normal saline solution were administered to the control group . in all four groups ,
no significant changes in the clinical signs , weights , hematologic examination results and blood chemical test results were noted between the control group and the experimental groups . in necropsy for checking for abnormalities in organs and tissue , no significant histopathological findings
were noted except for one case where interstitial infiltrating macrophages were found in one female rat in the 0.5-ml / animal group . to assess the toxicity of chukyu pharmacopuncture , we need to study its acute and chronic side effects and its relations with capacity reaction more .
the korea food & drug administration has testing protocol guidelines for the study of toxicity , and all experiments should be conducted following good laboratory practice ( glp ) regulations . in this study ,
a 1.0-ml dose of chukyu pharmacopuncture caused no considerable side effects in either male or female rats , which indicates that this dose is safe to use and does not cause severe histological abnormalities .
further studies on the subject should be conducted to yield more concrete evidence to support this finding .
the results showed that administration of 1.0-ml / animal chukyu pharmacopuncture did not cause any changes in weight or in the results of hematological , blood chemical , and necropsy examinations .
it also did not result in any mortality , which indicates that chukyu pharmacopuncture administration can be used as a safe treatment .
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objectives : this study was performed to analyze the single dose toxicity of chukyu ( spine - healing ) pharmacopuncture.methods:all experiments were conducted at the biotoxtech , an institution authorized to perform non - clinical studies under the regulations of good laboratory practice ( glp ) regulations .
sprague - dawley rats were chosen for the pilot study .
doses of chukyu ( spine - healing ) pharmacopuncture , 0.1 , 0.5 and 1.0 ml , were administered to the experimental groups , and a dose of normal saline solution , 1.0 ml , was administered to the control group .
this study was conducted under the approval of the institutional animal ethic committee.results:no deaths or abnormalities occurred in any of the four groups .
no significant changes in weight , hematological parameters or clinical chemistry between the control group and the experimental groups were observed . to check for abnormalities in organs and tissues ,
we used microscopy to examine representative histological sections of each specified organ ; the results showed no significant differences in any of the organs or tissues except in one case , where interstitial infiltrating macrophages were found in one female rat in the 0.5-ml / animal experimental group.conclusion:the above findings suggest that treatment with chukyu ( spine - healing ) pharmacopuncture is relatively safe .
further studies on this subject are needed to yield more concrete evidence .
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progressive supranuclear palsy ( psp ) is a rapidly progressing neurodegenerative disease with a median survival after onset of symptoms of around 6 years.1,2 the classic clinical picture of psp includes progressive gait disturbance , early falls , vertical ophthalmoparesis , akinetic - rigidity , prominent bulbar dysfunction and fronto - subcortical dementia.25 the loss of independent gait and the inability to stand unassisted occurs less than 3 years after disease onset6 with patients often wheelchair - bound by this time . with an estimated annual incidence of 5.3 per 100,000 in europe and an age - adjusted prevalence of approximately 6.4 per 100,000 , psp is as common as motor - neuron disease or multiple system atrophy.7 however , this rate is almost certainly underestimated as many patients may be incorrectly diagnosed with parkinson s disease ( pd ) , corticobasal degeneration ( cbd ) or alzheimer s disease ( ad ) .
psp is characterized by the pathological accumulation of hypherphosphorylated tau protein in the basal ganglia , brainstem and cerebral cortex .
severe neuronal loss in the substantia nigra , globus pallidus , subthalamic nucleus , midbrain , and pontine reticular formation accompanies neurofibrillary tangles that are composed of straight tau filaments.8 these neuronal tau deposits , and the coiled bodies and tufted astrocytes that are pathognomonic on psp , are predominantly composed of four - repeat ( 4r ) tau , in distinction to pick s disease which is mostly three - repeat ( 3r ) tau isoforms , and ad which is a mixture of 3r and 4r tau isoforms.9 in addition to the extensive and multifocal neuropathological changes there are multiple neurotransmitter abnormalities , including dopamine , acetylcholine , gamma - aminobutyric acid and the noradrenaline systems.10 the onset of symptoms is usually between the ages of 5070 years with both genders being nearly equally affected .
many patients report an insidious onset of disequilibrium and balance problems leading to postural instability , which is one of the early and most important clinical features . swallowing liquids and solids
often becomes difficult as the disease progresses , leading to aspiration pneumonias which are the main cause of death in advanced psp .
there are no approved treatments for psp and the negative outcome of the majority of therapeutic studies precludes recommending an evidence - based standard therapy.11 drug treatment usually starts with levodopa ( l - dopa ) at doses as high as 1500 mg which may alleviate some of the bradykinesia or rigidity , but rarely in a clinically meaningful way .
levodopa treatment may be accompanied by other therapies that are used for symptomatic improvement in pd.12 however , none of these therapies provide effective control of symptoms , particularly in the early phase of the illness .
psp rapidly progresses over time and worsens dramatically within 13 years after symptom onset.6 dopaminergic drug responses deteriorate and the need for alternative approaches increases . in the absence of an identified biological marker for psp pathology , early diagnosis and intervention before symptoms develop is not possible at this time .
secondary prevention to slow down , stop , or even reverse neuronal death after diagnosis would be a significant advance for the scientific and medical community.3 until the recent publication of the neuroprotection and natural history in parkinson plus syndromes ( nnipps ) trial , large scale controlled studies in patients with psp had not been conducted . although the nnipps trial2 has provided useful insights into atypical parkinsonian disorders , it failed to detect an effect of riluzole on disease progression or mortality .
similarly , a small study examining the effect of coenzyme q10 produced interesting preliminary data for efficacy , but additional studies are needed to conclude a therapeutic benefit.13 other pilot therapeutic interventional studies in the treatment of psp include lithium,14 rasagiline,15 and valproic acid.16 this situation puts a high priority on clinical trials in psp using novel drug candidates with a special focus on postural instability including the potential for disease modifying drugs
. clearly , neuroprotective drugs that can impact the underlying disease pathology would be of particular importance .
tideglusib ( noscira , madrid , spain ) is a gsk-3 inhibitor in phase ii clinical development for ad and psp based on the hypothesis that preventing tau phosphorylation should benefit psp patients .
another advanced product in psp clinical trials , davunetide , takes an alternative approach to slow disease progression by stabilizing microtubules and presumably reducing tau pathology ( allon therapeutics inc , vancouver , canada ) . in the following sections , we review the nonclinical and clinical pharmacology of davunetide with particular attention paid to the rationale for testing davunetide in psp in addition to providing data that support its use in other neurodegenerative disorders characterized by dysfunctional microtubules .
davunetide ( also referred to as napvsipq , nap or al-108 ) was originally described as a neuroprotective fragment of activity - dependent neuroprotective protein ( adnp).17 the parent protein was discovered as a glial - derived neuroprotectant and it has since been shown to be essential for fetal development18 due , at least in part , to its interaction with chromatin19 and its regulation of the expression of multiple neurogenesis and organogenesis - related genes.20 davunetide , an eight amino acid fragment of adnp , was found to be neuroprotective and is being developed as a therapeutic .
davunetide has been extensively investigated and has demonstrated neuroprotection and improved cognitive performance in multiple in vitro and in vivo preclinical studies.2124 in these experiments , davunetide has been shown to protect neurons and glial cells25 against a broad range of toxins including those relevant to ad and other neurodegenerative disorders .
the activity of davunetide also extends to promoting neuritic outgrowth2630 and axon growth or branching.31 such an effect of davunetide was also confirmed in vivo in a model of diabetes associated cognitive - dysfunction.25 with respect to in vivo testing of davunetide , activity has been seen in a wide range of disease models23,32,33 including , most recently , two models of chronic neurodegeneration that are relevant to tauopathies , such as psp , and to pd , respectively ( see below ) .
davunetide crosses the blood brain barrier whether administered intranasally or parenterally and demonstrates distribution throughout all areas of the brain34 suggesting that it will act upon compromised neurons regardless of their location .
the potential impact of davunetide on the progressive degeneration mediated by tau was explored in transgenic mice expressing a mutated form of the human tau protein [ p301s;k257t].33 following daily davunetide treatment for approximately 5 months , drug treated animals showed statistically significant improvement , compared to controls , in the morris water maze . following an additional 5 months of treatment ,
the analysis showed that davunetide treatment reduced the levels of hyperphosphorylated tau compared to vehicle treated animals .
these data , coupled with a decrease in staining of neurofibrillary tangles in these mice , provide support for davunetide s ability to reduce tau pathology and improve cognitive performance .
the potential of davunetide to treat sporadic pd was investigated in mice overexpressing human wild - type -synuclein under the thy1 promoter ( thy1-asyn ) and littermate wild type controls .
this mouse model accumulates -synuclein aggregates which are also seen in several neurodegenerative disorders ( synucleinopathies ) , including pd.32 two - month - old mice were treated daily with intranasal davunetide for 2 months and subjected to a variety of motor function tests at 4.5 months of age , and their brains were examined for proteinase k - resistant -synuclein inclusions in the substantia nigra and olfactory bulb .
davunetide treated thy1-asyn mice showed improved motor performance ( errors per step in the challenging beam traversal test ) and a statistically significant reduction in -synuclein inclusions in the substantia nigra .
since overexpression of -synuclein negatively impacts microtubule - dependent transport,35,36 these data suggest that compounds like davunetide that act on the microtubule cytoskeleton have potential for the treatment of synucleinopathies .
cumulative evidence suggests that some neurodegenerative diseases and psychiatric illnesses are associated with cytoskeletal alterations which result in a loss of synaptic structures and impaired synaptic transmission . as such
, the neuronal cytoskeleton can be a target for drug therapy for these diseases and illnesses .
disruption of the cytoskeletal architecture and microtubule function has been associated with poor cognitive performance in animal models.37 furthermore , microtubule dependent transport is impaired in ad38 and axonopathies have been reported in different ad animal models,39 suggesting that perturbed microtubule transport may underlie cognitive deficits observed in ad .
the overlap between microtubule biochemistry and cell death / survival signaling in neurons is exemplified by work demonstrating the traditional role ascribed to tau as a microtubule stabilizing protein is incomplete , and that this protein in fact also acts as a scaffold or adaptor protein that is critical to signal transduction in dendritic spines.40 in this context , the cumulative data summarized here for davunetide shows that this molecule is acting on cellular pathways at the intersection of mechanisms critical to synaptic plasticity and neurotransmission .
davunetide s activity in cellular and animal models appears to be linked to maintenance of microtubule function and on inhibition of apoptosis.41,42 this effect on the microtubular network is based , in particular , on observations that davunetide has protective effects against zinc intoxication in glial and neuronal cells that is accompanied by restoration of an intact microtubular cytoskeleton and apparent inhibition of toxic tubulin - zinc aggregate formation.29,43 similarly , davunetide is able to promote repolymerization of microtubules after nocodazole treatment in primary neurons.44 the hypothesis that davunetide stabilizes microtubules is supported by the observation that davunetide increases the ratio of total tau to phosphorylated tau in vitro,45 an effect that is associated with stabilization of microtubules .
incubation with davunetide protected microtubules against the overexpression of katanin , a protein that severs microtubules playing a major role in sculpting microtubular structures.46 in this respect , davunetide was able to compensate for katanin - induced defective axonal branching in the presence of reduced tau expression
. in vivo confirmation comes from several animal models in which davunetide reduces the level of phosphorylated tau in models of ad,47,48 tauopathy33,49 and adnp deficiency.50 a link between davunetide s effects on microtubules and cell survival pathways is suggested by studies involving neuroprotection against toxic insults .
for instance , the protective effect of davunetide in support of neuronal survival in the presence of compromised glia ( as a consequence of the glial prenatal ethanol exposure , pee ) has been described.31 co - cultures of astrocytes from pee animals with neurons from nave animals resulted in a decreased number of viable neurons and of their synaptic connections compared to glial controls .
davunetide was able to rescue neurons from the effects of pee - damaged astrocytes , with a concomitant increase in the phosphorylation of akt , erk mapk and creb . blocking the activity of pi-3-kinase or erk with small molecule inhibitors ( wortmannin and pd 98059 , respectively ) prevented the neuroprotective effects of davunetide on cell viability and synaptic connection numbers .
31 similarly , in a mouse model of excitotoxic brain lesion mimicking brain damage associated with cerebral palsy , it was recently demonstrated that davunetide reduced the number of apoptotic neurons through activation of pi-3k / akt pathway in the cortical plate or both pi-3k / akt and mapk / mek1 kinases in the white matter.51 further , davunetide potentiated axon outgrowth in cerebellar granule neurons ( cgns ) in serum - free conditions,52 increasing the length of axons by about 70% relative to controls .
davunetide effects were blocked by an inhibitor of the src family of kinases ( sfks ) , and inhibition of mitogen - activated protein kinase kinase ( mek ) but was unaffected by inhibition of protein kinase a ( pka ) and cytochalasin d ( an inhibitor of f - actin polymerization ) .
knockdown of fyn kinase using sirna abrogated the effect of davunetide on axons suggesting fynk and downstream mediators are required for davunetide s activity .
the ultimate downstream impact of such effects on intracellular signaling may be the modulation of gene expression as suggested by activation of creb28 and polyadp - ribosylation.27 in conclusion , the data described in the preceding section indicate that davunetide affects multiple pathways that have direct translation to neurodegenerative disease at three levels ( see figure 1 for a schematic ) : davunetide is able to modulate cytoskeletal structures within neurons and glia , at least in part due to a reduction in tau phosphorylation .
davunetide treatment reduces the phosphorylation of tau , in association with the modulation of intracellular signaling cascades critical to cell survival , synaptic plasticity and microtubule stabilization .
these cellular events ultimately result in maintenance of neurotransmission and cell viability which in turn provides a drug - dependent impact on disease progression .
furthermore , preclinical pharmacology studies have shown that davunetide improves outcomes in models of diseases where neurofibrillary tangles are not a major hallmark . in such cases , we posit that the ability of davunetide to modulate microtubule - associated cell survival pathways accounts for the beneficial effects .
davunetide s effect on human pharmacology was explored in a phase ii clinical study of amnestic mild cognitive impairment ( amci ) patients.53,54 amci is a prodromal state of ad characterized by significant memory impairment but in which other cognitive functions remain relatively intact.55 the neuropathology of amci which contributes to the memory impairment appears to involve microtubule dysfunction resulting in neurofibrillary tangles composed of tau.5557 davunetide consistently improves microtubule structure and function leading to a reduction in tau pathology in a variety of cell - based and animal models .
therefore , the hypothesis is davunetide can improve cognitive performance in amci patients . a double blind , placebo - controlled study randomized 144 subjects into three groups , placebo , 5 mg daily and 15 mg twice daily for 12 weeks of treatment .
davunetide was generally safe and well - tolerated with an equal percent ( approximately 50% ) of adverse events reported by placebo subjects and davunetide - treated subjects . a slightly higher incidence of headache and nasopharyngeal adverse events was reported by davunetide - treated subjects compared to placebo ( 13% versus 6% for headache and 8%9% versus 2% for nasopharyngitis ) ; however the total number was small and the severity was mild - to - moderate .
five cognitive tests were administered throughout the study ( digit span , delayedmatch- to - sample , spatial working memory , paired associates learning , and one - touch stockings of cambridge ; cambridge cognition ltd , uk ) .
davunetide 15 mg twice daily had a statistically significant impact on two measures , namely digit span and delayed - match - to - sample , tests of verbal recall and visual working memory , respectively .
these exploratory data from the amci study serve as proof - of - principle and support the preclinical mechanism of action postulated for davunetide .
while davunetide has also shown activity in an exploratory trial in cognitive impairment in schizophrenia,58 the data in amci provide the most relevant translation to psp .
the opportunity to treat psp , given its neuropathology , with davunetide follows from the clinical confirmation of activity obtained in the amci study .
since the distribution of tau pathology in the brain of psp patients correlates well with the clinical presentation of symptoms , a reduction of the tau burden should translate into a reduced rate of progression in the clinical outcome measures .
a pivotal phase ii / iii study of psp patients opened for enrollment in the fourth quarter of 2010 under a special protocol assessment granted by the united states food and drug administration .
the placebo - controlled study enrolled approximately 300 patients from the usa , canada , australia , germany , france and the united kingdom .
subjects in the study will be treated with placebo or davunetide 30 mg twice daily for 1 year .
davunetide ( also referred to as napvsipq , nap or al-108 ) was originally described as a neuroprotective fragment of activity - dependent neuroprotective protein ( adnp).17 the parent protein was discovered as a glial - derived neuroprotectant and it has since been shown to be essential for fetal development18 due , at least in part , to its interaction with chromatin19 and its regulation of the expression of multiple neurogenesis and organogenesis - related genes.20 davunetide , an eight amino acid fragment of adnp , was found to be neuroprotective and is being developed as a therapeutic .
davunetide has been extensively investigated and has demonstrated neuroprotection and improved cognitive performance in multiple in vitro and in vivo preclinical studies.2124 in these experiments , davunetide has been shown to protect neurons and glial cells25 against a broad range of toxins including those relevant to ad and other neurodegenerative disorders .
the activity of davunetide also extends to promoting neuritic outgrowth2630 and axon growth or branching.31 such an effect of davunetide was also confirmed in vivo in a model of diabetes associated cognitive - dysfunction.25 with respect to in vivo testing of davunetide , activity has been seen in a wide range of disease models23,32,33 including , most recently , two models of chronic neurodegeneration that are relevant to tauopathies , such as psp , and to pd , respectively ( see below ) .
davunetide crosses the blood brain barrier whether administered intranasally or parenterally and demonstrates distribution throughout all areas of the brain34 suggesting that it will act upon compromised neurons regardless of their location .
the potential impact of davunetide on the progressive degeneration mediated by tau was explored in transgenic mice expressing a mutated form of the human tau protein [ p301s;k257t].33 following daily davunetide treatment for approximately 5 months , drug treated animals showed statistically significant improvement , compared to controls , in the morris water maze . following an additional 5 months of treatment ,
the analysis showed that davunetide treatment reduced the levels of hyperphosphorylated tau compared to vehicle treated animals .
these data , coupled with a decrease in staining of neurofibrillary tangles in these mice , provide support for davunetide s ability to reduce tau pathology and improve cognitive performance .
the potential of davunetide to treat sporadic pd was investigated in mice overexpressing human wild - type -synuclein under the thy1 promoter ( thy1-asyn ) and littermate wild type controls .
this mouse model accumulates -synuclein aggregates which are also seen in several neurodegenerative disorders ( synucleinopathies ) , including pd.32 two - month - old mice were treated daily with intranasal davunetide for 2 months and subjected to a variety of motor function tests at 4.5 months of age , and their brains were examined for proteinase k - resistant -synuclein inclusions in the substantia nigra and olfactory bulb .
davunetide treated thy1-asyn mice showed improved motor performance ( errors per step in the challenging beam traversal test ) and a statistically significant reduction in -synuclein inclusions in the substantia nigra .
since overexpression of -synuclein negatively impacts microtubule - dependent transport,35,36 these data suggest that compounds like davunetide that act on the microtubule cytoskeleton have potential for the treatment of synucleinopathies .
cumulative evidence suggests that some neurodegenerative diseases and psychiatric illnesses are associated with cytoskeletal alterations which result in a loss of synaptic structures and impaired synaptic transmission . as such
, the neuronal cytoskeleton can be a target for drug therapy for these diseases and illnesses .
disruption of the cytoskeletal architecture and microtubule function has been associated with poor cognitive performance in animal models.37 furthermore , microtubule dependent transport is impaired in ad38 and axonopathies have been reported in different ad animal models,39 suggesting that perturbed microtubule transport may underlie cognitive deficits observed in ad .
the overlap between microtubule biochemistry and cell death / survival signaling in neurons is exemplified by work demonstrating the traditional role ascribed to tau as a microtubule stabilizing protein is incomplete , and that this protein in fact also acts as a scaffold or adaptor protein that is critical to signal transduction in dendritic spines.40 in this context , the cumulative data summarized here for davunetide shows that this molecule is acting on cellular pathways at the intersection of mechanisms critical to synaptic plasticity and neurotransmission .
davunetide s activity in cellular and animal models appears to be linked to maintenance of microtubule function and on inhibition of apoptosis.41,42 this effect on the microtubular network is based , in particular , on observations that davunetide has protective effects against zinc intoxication in glial and neuronal cells that is accompanied by restoration of an intact microtubular cytoskeleton and apparent inhibition of toxic tubulin - zinc aggregate formation.29,43 similarly , davunetide is able to promote repolymerization of microtubules after nocodazole treatment in primary neurons.44 the hypothesis that davunetide stabilizes microtubules is supported by the observation that davunetide increases the ratio of total tau to phosphorylated tau in vitro,45 an effect that is associated with stabilization of microtubules .
incubation with davunetide protected microtubules against the overexpression of katanin , a protein that severs microtubules playing a major role in sculpting microtubular structures.46 in this respect , davunetide was able to compensate for katanin - induced defective axonal branching in the presence of reduced tau expression . in vivo confirmation comes from several animal models in which davunetide reduces the level of phosphorylated tau in models of ad,47,48 tauopathy33,49 and adnp deficiency.50 a link between davunetide s effects on microtubules and cell survival pathways is suggested by studies involving neuroprotection against toxic insults .
for instance , the protective effect of davunetide in support of neuronal survival in the presence of compromised glia ( as a consequence of the glial prenatal ethanol exposure , pee ) has been described.31 co - cultures of astrocytes from pee animals with neurons from nave animals resulted in a decreased number of viable neurons and of their synaptic connections compared to glial controls .
davunetide was able to rescue neurons from the effects of pee - damaged astrocytes , with a concomitant increase in the phosphorylation of akt , erk mapk and creb . blocking the activity of pi-3-kinase or erk with small molecule inhibitors ( wortmannin and pd 98059 , respectively ) prevented the neuroprotective effects of davunetide on cell viability and synaptic connection numbers .
31 similarly , in a mouse model of excitotoxic brain lesion mimicking brain damage associated with cerebral palsy , it was recently demonstrated that davunetide reduced the number of apoptotic neurons through activation of pi-3k / akt pathway in the cortical plate or both pi-3k / akt and mapk / mek1 kinases in the white matter.51 further , davunetide potentiated axon outgrowth in cerebellar granule neurons ( cgns ) in serum - free conditions,52 increasing the length of axons by about 70% relative to controls .
davunetide effects were blocked by an inhibitor of the src family of kinases ( sfks ) , and inhibition of mitogen - activated protein kinase kinase ( mek ) but was unaffected by inhibition of protein kinase a ( pka ) and cytochalasin d ( an inhibitor of f - actin polymerization ) .
knockdown of fyn kinase using sirna abrogated the effect of davunetide on axons suggesting fynk and downstream mediators are required for davunetide s activity .
the ultimate downstream impact of such effects on intracellular signaling may be the modulation of gene expression as suggested by activation of creb28 and polyadp - ribosylation.27 in conclusion , the data described in the preceding section indicate that davunetide affects multiple pathways that have direct translation to neurodegenerative disease at three levels ( see figure 1 for a schematic ) : davunetide is able to modulate cytoskeletal structures within neurons and glia , at least in part due to a reduction in tau phosphorylation .
davunetide treatment reduces the phosphorylation of tau , in association with the modulation of intracellular signaling cascades critical to cell survival , synaptic plasticity and microtubule stabilization .
these cellular events ultimately result in maintenance of neurotransmission and cell viability which in turn provides a drug - dependent impact on disease progression . furthermore
, preclinical pharmacology studies have shown that davunetide improves outcomes in models of diseases where neurofibrillary tangles are not a major hallmark . in such cases , we posit that the ability of davunetide to modulate microtubule - associated cell survival pathways accounts for the beneficial effects .
davunetide s effect on human pharmacology was explored in a phase ii clinical study of amnestic mild cognitive impairment ( amci ) patients.53,54 amci is a prodromal state of ad characterized by significant memory impairment but in which other cognitive functions remain relatively intact.55 the neuropathology of amci which contributes to the memory impairment appears to involve microtubule dysfunction resulting in neurofibrillary tangles composed of tau.5557 davunetide consistently improves microtubule structure and function leading to a reduction in tau pathology in a variety of cell - based and animal models .
therefore , the hypothesis is davunetide can improve cognitive performance in amci patients . a double blind , placebo - controlled study randomized 144 subjects into three groups , placebo , 5 mg daily and 15 mg twice daily for 12 weeks of treatment .
davunetide was generally safe and well - tolerated with an equal percent ( approximately 50% ) of adverse events reported by placebo subjects and davunetide - treated subjects . a slightly higher incidence of headache and nasopharyngeal adverse events was reported by davunetide - treated subjects compared to placebo ( 13% versus 6% for headache and 8%9% versus 2% for nasopharyngitis ) ; however the total number was small and the severity was mild - to - moderate .
five cognitive tests were administered throughout the study ( digit span , delayedmatch- to - sample , spatial working memory , paired associates learning , and one - touch stockings of cambridge ; cambridge cognition ltd , uk ) .
davunetide 15 mg twice daily had a statistically significant impact on two measures , namely digit span and delayed - match - to - sample , tests of verbal recall and visual working memory , respectively . these exploratory data from the amci study
serve as proof - of - principle and support the preclinical mechanism of action postulated for davunetide . while davunetide has also shown activity in an exploratory trial in cognitive impairment in schizophrenia,58 the data in amci provide the most relevant translation to psp .
the opportunity to treat psp , given its neuropathology , with davunetide follows from the clinical confirmation of activity obtained in the amci study .
since the distribution of tau pathology in the brain of psp patients correlates well with the clinical presentation of symptoms , a reduction of the tau burden should translate into a reduced rate of progression in the clinical outcome measures . a pivotal phase ii / iii study of psp patients opened for enrollment in the fourth quarter of 2010 under a special protocol assessment granted by the united states food and drug administration .
the placebo - controlled study enrolled approximately 300 patients from the usa , canada , australia , germany , france and the united kingdom .
subjects in the study will be treated with placebo or davunetide 30 mg twice daily for 1 year .
the relationship between tau , psp and ad59 provides drug developers with strong translational rationale to explore tau - directed approaches , with some of these programs either in clinical development or close to entering that stage .
psp has emerged as an attractive clinical trial population for three main reasons : ( 1 ) it is a debilitating and life - threatening disease for which there is a desire to develop effective treatment options that currently do not exist ; ( 2 ) from a scientific point of view , it serves to prove whether a tau - directed therapy can affect a primary neurodegenerative disease , thereby reducing the risk of failure in a related condition , namely ad ; and ( 3 ) it is a rare disease that qualifies as an orphan indication thereby providing certain commercial incentives and allows sponsors to pursue development with more limited resources . with this background ,
it is necessary to consider some key factors that impact the quality of clinical trials in psp .
issues that are often particularly challenging when working in rare diseases in general are : ( 1 ) a well - defined patient population ; ( 2 ) validated and appropriate outcome instruments ; and ( 3 ) an intervention with a clear pharmacological rationale and data to support further testing . in the context of psp , these three factors are interrelated and present substantial challenges in the design , execution and interpretation of results from a targeted clinical study . the following discussion deals with these issues in turn , and describes how they can be overcome . for clinical trials , and especially for a disease modifying compound ,
the optimal study population is both homogeneous and at very early stages of the disease .
currently used diagnostic criteria2 have high levels of sensitivity and specificity for psp compared to pathological diagnosis . however , because psp may present with features of pd or other tauopathies , many patients are initially diagnosed with pd and only after they have failed treatment trials with dopaminergic compounds does their psp diagnosis becomes apparent.60 while there are putative cerebrospinal fluid ( csf)61 and imaging biomarkers,6264 a consequence of atypical or overlapping presentations is that many psp patients are misdiagnosed at the earliest stages of the disease .
other potential pitfalls have included : ( a ) including subjects with putative psp even if not all inclusion / exclusion criteria are met ; ( b ) starting or adjusting dopaminergic medications in order to ensure that a patient is indeed non - responsive to dopaminergic medications and requiring subjects to undergo a prolonged stabilization period ; ( c ) subjects with presentations that overlap cbd / ftd ( fronto - temporal dementia ) requiring further evaluations ; and ( d ) the lack of a validated biomarker precluding a population enrichment strategy or a pharmacodynamic outcome instead of a clinical outcome . in the allon - sponsored study evaluating davunetide in psp ,
several study design features were incorporated to help enroll as homogeneous a psp population as possible .
firstly , the inclusion / exclusion criteria used were derived from several long - term clinical trials in psp where the sensitivity and specificity of diagnostic criteria were established .
secondly , the investigators selected for this study were all psp experts and intimately familiar with the diagnostic criteria , natural history and management of psp as well as outcome instruments commonly used with these patients . in cases where the diagnosis was ambiguous ,
the study medical monitor ( specialized in movement disorders ) , the coordinating investigator and members of the study steering committee were available to discuss a given case with an investigator and reach a consensus about diagnosis and lastly the sponsor did not grant protocol exemptions based on diagnostic uncertainty .
with respect to the selection of outcome instruments , clinical trials often adopt scales or instruments developed for the diagnosis or staging of a disease as outcome instruments for interventional studies .
this practice , while apparently driven by common sense , does not deal with the fact that the psychometric properties of an instrument designed for staging a disease may not be appropriate for use in detecting a response to intervention.66 issues such as floor or ceiling effects are problematic in interventional studies .
accurate and precise data on rate of progression are critical when designing studies aimed at disease modification ; however , scales initially designed for staging are not ideal for determining rates of progression .
a clear example of this issue is the rate of progression seen on the alzheimer s disease sssessment scale - cognitive subscale ( adas - cog ) in ad when compared to baseline severity .
the instrument is insensitive to progression in either very mildly affected or severely affected patients . in the first case , the test may be too simple or fails to test affected domains whereas in the latter , the subject is so impaired that they can not reliably be tested .
the net effect is that the scale is sensitive to change in a relatively narrow band of severity .
this is clearly a problem when one wants to test the effect of a disease modifying compound in subjects at the earliest possible stages of the clinical syndrome .
the situation in psp has evolved significantly from the use of pd - derived instruments67 to the use of time - to - event outcomes2 ( ie , survival , inability to walk or inability to stand ) to validated composite scales such as the psp rating scale ( psprs ) derived from longitudinal observational4 or interventional cohorts.68 most of the currently used outcome instruments appear to have acceptable psychometric properties ( ie , inter- , intrarater [ by the same rater or in between two raters assessing clinical phenotypes ] reliability , stability etc ) ; however , these traditional metrics are insufficient to characterize an outcome instrument for interventional clinical trials and calls for the use of more appropriate analyses such as item response or rasch analyses .
these potential issues have been addressed in the assessment of davunetide in psp through the exclusion of patients with severe psp that might have introduced overt floor effects , exclusion of subjects with dementia to reduce the impact of cognitive impairment on subject - reported outcomes and the implementation of a strict standardized training program for psprs raters .
in addition , demographic and psprs data at baseline were compared to data from previous interventional studies or longitudinal cohorts using the psprs to determine whether substantial differences were apparent .
lastly , the choice of compound to be taken into clinical trials is also a critical factor in the rationale for , and design of , a clinical trial .
the most obvious gap in this area is the lack of validated models of psp or other sporadic tauopathies and the resultant dependence on transgenic models . drawing from the lessons learned in the ad arena
, it appears that transgenic models , while useful as a way to validate the pharmacology of a given compound , should be used with a clear understanding of their limitations . in the absence of validated models , and an incomplete understanding of the pathophysiological cascade underlying sporadic psp
, a pragmatic approach is to target the salient pathological features in psp using a variety of different models in order to determine whether the compound has consistently neuroprotective effects at concentrations that are achievable in the human brain .
the clinical development of davunetide has encompassed elements of both strategies22 using transgenic models directed at tau as well as a broad range of other models ; all of which have consistently yielded evidence of a neuroprotective effect . early clinical experience with davunetide in amci subjects
while both patients and relatives frequently seek medical treatment , their willingness to participate in clinical trials has been surprisingly low historically.69 in studies requiring a complex therapeutic regimen , ensuring patient compliance is critically important ; therefore , ensuring the cooperation of a reliable caregiver , such as a relative or a nursing - home employee , to accompany patients to all study visits is mandatory .
individuals who want to participate in clinical trials must be fully aware of the purpose , procedures , and possible benefits of the study.70 study staff must provide sufficient time to allow for regular breaks and the transitions between different study investigations very carefully given the hypokinetic and bradyphrenic nature of psp patients . if a study has included a large number of procedures it might be useful to perform them on two days , especially since cognitive tests may be flawed due to fatigue .
this review lays out some of the challenges encountered in interventional trials in psp and the experience of recruiting the trial for the assessment of davunetide s efficacy in psp .
in general , patients with psp and their relatives or caregivers strongly adhere to the tasks associated with clinical trial protocols .
they are reliable partners in clinical trials since their ultimate hope is to assist in finding a drug that will ameliorate the symptoms of the disease and possibly even modify disease progression . as more experience has been gathered and novel treatment options have appeared , the understanding of operational issues in psp trials has led to better trial methodology and provides a basis on which to test novel pharmacologic treatments such as davunetide .
these advances , coupled with the support of key opinion leaders and advocacy groups , have been important in enrollment in the davunetide study being completed ahead of schedule suggesting an eagerness for robust studies that have the potential to bring therapies to patients as quickly as possible .
since no therapy exists to slow disease progression in psp , there is an urgent need to identify compounds and to evaluate their potential in clinical studies .
the pharmacological profile of davunetide suggests that this compound may be effective in slowing down the neurodegenerative process observed in psp and it is therefore important to study this drug in psp patients .
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progressive supranuclear palsy ( psp ) is a rare neurodegenerative disease characterized by the accumulation of tau protein aggregates in the basal ganglia , brainstem and cerebral cortex leading to rapid disease progression and death .
the neurofibrillary tangles that define the neuropathology of psp are comprised of aggregated 4r tau and show a well - defined distribution .
classically , psp is diagnosed by symptoms that include progressive gait disturbance , early falls , vertical ophthalmoparesis , akinetic - rigid features , prominent bulbar dysfunction and fronto - subcortical dementia .
there are currently no effective therapies for the treatment of this rapidly degenerating and debilitating disease .
davunetide is a novel neuroprotective peptide that is thought to impact neuronal integrity and cell survival through the stabilization of microtubules .
preclinical activity in models of tauopathy has been translated to clinical studies , demonstrating pharmacologic activity that has supported further development .
davunetide s efficacy and tolerability are being tested in a placebo - controlled study in psp patients , making it the most advanced drug candidate in this indication .
this review examines the disease characteristics of psp , the rationale for treating psp with davunetide and assesses some of the challenges of clinical trials in this patient population .
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cefepime is a broad - spectrum fourth - generation cephalosporin with enhanced coverage against gram - positive and gram - negative bacteria.1 cefepime is predominantly cleared from the body by renal secretion , therefore the elimination half - life is increased in patients with renal impairment.2 cefepime - induced neurotoxicity , including nonconvulsive status epilepticus ( ncse ) , has been reported especially in patients with renal impairment .
we present a rare case of aphasic status epilepticus ( se ) mimicking acute stroke in a patient with nephrotic syndrome and urinary tract infection treated by cefepime .
a 36-year - old right - handed man was admitted to internal medicine department with 3 months history of generalized edema and decreased urine output .
he was diagnosed as nephrotic syndrome and urinary tract infection . because his urine culture demonstrated pseudomonas aeruginosa , intravenous cefepime 2 g
his creatinine clearance calculated by modification of diet in renal disease ( mdrd ) formula was 49 ml / min/1.73 m , suggesting mild renal impairment .
he was consulted to neurology department for acute onset global aphasia and right hemiplegia during admission .
although he had a past medical history of intracerebral hemorrhage ( ich ) due to left middle cerebral artery ( mca ) aneurysmal rupture about 7 years ago , he did not have any language difficulties , hemiplegia , and seizures . at first , under the suspicion of acute stroke , an emergent brain magnetic resonance imaging ( mri ) with angiography was done .
however , there were no acute lesions in brain imaging , except old encephalomalacia in left fronto - temporal area and clipping of left mca aneurysm ( fig .
1 ) . there was no significant alteration of perfusion status on perfusion - weighted mri .
although right hemiplegia recovered completely within a few hours , motor aphasia was persistent . after ruling out acute stroke , we performed electroencephalography ( eeg ) due to the persistent aphasia .
eeg revealed continuous 23 hz rhythmic spike - and - waves in left hemisphere , especially in fronto - temporal area ( fig .
2a ) , with clinical ictal phenomena ( aphasia ) during the eeg patterns , compatible with the working clinical criteria for ncse with focal onset.3 because he has been treated with high dose of cefepime since 2 days ago from the onset of neurological symptoms , we suspected cefepime - induced ncse , particularly aphasic se .
2b ) and clinical symptoms made a gradual recovery within 3 days after the discontinuation of cefepime .
follow - up brain diffusion - weighted mri after 5 days from symptom onset also did not show the acute lesions .
various antibiotics have been reported to be associated with se , and these drugs representatively include third and fourth generation cephalosporins and quinolones.4 patients with prior central nervous system ( cns ) disease , advanced age , and particularly renal impairment may be vulnerable.2,47 although there have been various case reports and series of cefepime - induced neurotoxicity , including ncse , aphasic se itself is a rare phenomenon , and cefepime - induced aphasic se proven by eeg has never been reported to our knowledge . although our patient had a past medical history of ich , there were no acute lesions in brain imaging and he has never experienced a seizure before the administration of cefepime .
therefore the diagnosis of cefepime - induced ncse was more relevant than acute stroke or simple post - stroke seizures .
our case was a high - risk patient having renal impairment and prior cns disease simultaneously .
renal impairment may increase the concentration of cefepime in the serum , cerebrospinal fluid , and brain tissue.2,6 in addition , the increased permeability of blood - brain barrier ( bbb ) and decreased albumin binding of antibiotics in the presence of uremia , as well as the presence of endogenous uremic toxins might increase a patient s vulnerability to neurotoxicity.6 our patient s brain , especially the left hemisphere , might be particularly vulnerable because bbb was locally disrupted by the prior ich .
the most widely accepted theory on the pathogenesis of seizures induced by beta - lactam antibiotics including cefepime involves the interference or inhibition of gamma - aminobutyric acid ( gaba ) binding to gabaa receptors.6 gabaa receptor inhibition can lead to increased neuronal excitability which can cause se .
the focal - aphasic - se was thought to be the result of this hyperexcitability in the vulnerable brain area in our case .
therefore focal ncse can be induced by cefepime in the presence of prior cns disease , although the most of previous reports of cefepime - induced ncse delineated generalized epileptiform discharges on eeg with decreased consciousness . in conclusion , as the use of cefepime is increasing , clinicians should be aware of various cefepime - induced neurotoxicity .
when encountered with unexplained neurologic deficits , eeg should be considered to diagnose ncse in in patients receiving cefepime especially with renal impairment and prior cns disease .
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cefepime - induced neurotoxicity , including nonconvulsive status epilepticus , has been reported especially in patients with renal impairment .
however , focal nonconvulsive status epilepticus is very rare and cefepime - induced aphasic status epilepticus proven by electroencephalography have never been reported to our knowledge .
we present an interesting case of aphasic status epilepticus mimicking acute stroke in a patient treated by cefepime .
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a beautiful smile is the best ornament for the face and is the most primitive forms of human communication .
the harmony of the smile is determined especially by the shape , the position and the color of the teeth .
people of all ages are increasingly concerned about their smile and overall appearance . an adequate mucogingival complex , in which the mucogingival tissues can sustain their biomorphologic integrity and maintain an enduring attachment to the teeth as well as
. when a mucogingival problem occurs , there are basically two ways in which it presents itself : ( a ) as a close disruption of the mucogingival complex resulting in pocket formation .
( b ) as an open disruption of the mucogingival complex resulting in gingival clefts and gingival recession .
gingival recession is the term used to characterize the apical shift of the marginal gingiva from its normal position on the crown of the tooth to the levels on the root surface beyond the cemento - enamel junction .
although many dental conditions go unnoticed by patients , gingival recession can often be visible to patients and for which they may seek advice of a dentist .
gingival recession usually creates an esthetic problem , especially when such problem affects the anterior teeth and anxiety about tooth loss due to progression of the destruction .
it may also be associated with dentine hypersensitivity , root caries , abrasion and/or cervical wear , erosion because of exposure of the root surface to the oral environment and an increase in accumulation of dental plaque . despite the frequent observation in adult subjects ,
the occurrence and severity of the gingival recession presents considerable differences between study populations . a limited amount of prevalence and etiology related studies on gingival recession
therefore , it is important to collect detailed information , to assess the tendency and epidemiology of this condition , identify the etiological factors and establish preventive measures .
hence , the aim of the present study was to determine the occurrence of gingival recession and to identify the most common factor associated with the cause of gingival recession in the patients .
subjects of this study were selected from the outpatients attending the department of periodontology , yenepoya dental college , mangalore .
assuming that the values obtained are z = a point on normal distribution with 95% confidence , p = prevalence from pilot study , q = 100-p , d = admissible error that is 10% of prevalence .
the study was reviewed and approved by the institutional review board of yenepoya university , mangalore , india .
a written informed consent was taken from every participant prior to the study and at the end of the study all the patients were treated .
patients aged between 15 and 60 years who had a minimum of 20 permanent teeth and no histories of periodontal therapy undertaken during the past 6 months were included in the study .
an observational study was performed to find out the most common cause and occurrence of gingival recession during may 2011 to january 2012 .
a random sampling technique was used to select 710 subjects of both genders aged between 15 years and 60 years from the outpatient department of periodontology , yenepoya dental college , mangalore .
data were collected by an interview with the help of a proforma prepared for this study , and dental examination was carried out .
the proforma of the study contained a detailed history of personal habits and brushing habits .
each subject was examined in a dental chair by a single , trained , and calibrated examiner using dental chair light , mouth mirror , explorer , william 's periodontal probe and cpi probe .
presence of gingival recession was recorded using miller 's classification ( 1985 ) of gingival recession . following clinical parameters were recorded - silness and loe plaque index ( 1967 ) , loe and silness gingival index ( 1963 ) , community periodontal index ( cpi ) ( 1997 ) .
the tooth malalignment was observed by viewing the teeth from occlusal plane and position of each tooth was classified according to its relation to the regular curve of the arch either correctly positioned or outstanding ( labially placed ) and instanding ( lingually placed ) in all subjects .
diagnosis of faulty toothbrushing in this study was done by examining the facial surface of the tooth for the presence of cervical abrasion .
the upper and lower frenum was examined for the site of attachment and presence of frenal pull according to placek et al .
tension test was carried out to confirm the adequacy of the width of the attached gingiva .
history of smoking and brushing habits ( the type of brushing method , duration of brushing , frequency of brushing ) was included in the questionnaire .
the data obtained were subjected to statistical analysis using chi - square test and students unpaired t - test .
of 710 subjects , 291 ( 40.98% ) had gingival recession . in total , 1152 teeth had gingival recession [ table 1 ] .
occurrence of gingival recession in age group 1525 years , the gingival recession was 26.9% ( n = 78 ) ; age group 2535 years , it was 41.5% ( n = 113 ) ; age group 3545 years , it was 66.1% ( n = 74 ) ; and in age group 4560 years , it was 70.3% ( n = 26 ) .
it shows that as age increases there is increase in gingival recession which was of statistical significance ( p < 0.001 ) [ figure 1 ] .
occurrence of gingival recession according to age males were mostly affected by gingival recession 60.5% compared to females 39.5% which was of statistical significance [ figure 2 ] .
gingival recession was commonly seen in mandibular incisors ( 43.0% ) followed by maxillary molar ( 13.2% ) , mandibular premolar ( 12.2% ) , maxillary incisor and premolar ( 8.9% ) , mandibular molar ( 4.9% ) , maxillary canine ( 4.6% ) , mandibular canine ( 4.3% ) [ figure 3 ] .
recession was commonly observed in the mandibular arch ( 66% ) than maxillary arch ( 34% ) [ figure 4 ] .
occurrence of gingival recession according to gender occurrence of gingival recession according to the type of teeth occurrence of gingival recession according to the type of arch the most commonly seen was miller 's class i ( 59.5% ) followed by class ii ( 35.3% ) , class iii ( 2.7% ) and class iv ( 2.5% ) [ figure 5 ] . according to cpi , in recession group ,
occurrence of gingival recession according to millers class of gingival recession when the etiologic factors causing gingival recession were examined in 1152 teeth of 710 subjects , the most common factor was found to be dental plaque accumulation ( 44.1% ) followed by faulty toothbrushing ( 42.7% ) , habits such as smoking and use of smokeless tobacco ( 7.1% ) , malocclusion ( 4.6% ) , high frenal attachment ( 0.4% ) and others like inadequate attached gingiva , occlusal trauma ( 1% ) [ figure 6 ] .
etiology of gingival recession in percentage the mean plaque index was 1.35 in subjects without gingival recession compared to 1.96 in subjects with gingival recession .
it was statistically significant in recession group compared to without recession group ( p < 0.001 )
. horizontal type of brushing was followed by 64.9% of subjects with recession ( p < 0.001 ) . about 46.4% of subjects with recession brushed for > 3 min ( p < 0.001 ) .
about 62.2% of subjects with recession brushed twice daily ( p < 0.001 ) the type of brushing method , duration of brushing , frequency of brushing are statistically significant in subjects with recession compared to subjects without recession .
of 710 subjects , 291 ( 40.98% ) had gingival recession . in total , 1152 teeth had gingival recession [ table 1 ] .
occurrence of gingival recession in age group 1525 years , the gingival recession was 26.9% ( n = 78 ) ; age group 2535 years , it was 41.5% ( n = 113 ) ; age group 3545 years , it was 66.1% ( n = 74 ) ; and in age group 4560 years , it was 70.3% ( n = 26 ) .
it shows that as age increases there is increase in gingival recession which was of statistical significance ( p < 0.001 ) [ figure 1 ] .
occurrence of gingival recession according to age males were mostly affected by gingival recession 60.5% compared to females 39.5% which was of statistical significance [ figure 2 ] .
gingival recession was commonly seen in mandibular incisors ( 43.0% ) followed by maxillary molar ( 13.2% ) , mandibular premolar ( 12.2% ) , maxillary incisor and premolar ( 8.9% ) , mandibular molar ( 4.9% ) , maxillary canine ( 4.6% ) , mandibular canine ( 4.3% ) [ figure 3 ] .
recession was commonly observed in the mandibular arch ( 66% ) than maxillary arch ( 34% ) [ figure 4 ] .
occurrence of gingival recession according to gender occurrence of gingival recession according to the type of teeth occurrence of gingival recession according to the type of arch the most commonly seen was miller 's class i ( 59.5% ) followed by class ii ( 35.3% ) , class iii ( 2.7% ) and class iv ( 2.5% ) [ figure 5 ] . according to cpi , in recession group ,
when the etiologic factors causing gingival recession were examined in 1152 teeth of 710 subjects , the most common factor was found to be dental plaque accumulation ( 44.1% ) followed by faulty toothbrushing ( 42.7% ) , habits such as smoking and use of smokeless tobacco ( 7.1% ) , malocclusion ( 4.6% ) , high frenal attachment ( 0.4% ) and others like inadequate attached gingiva , occlusal trauma ( 1% ) [ figure 6 ] .
etiology of gingival recession in percentage the mean plaque index was 1.35 in subjects without gingival recession compared to 1.96 in subjects with gingival recession .
it was statistically significant in recession group compared to without recession group ( p < 0.001 ) . horizontal type of brushing was followed by 64.9% of subjects with recession ( p < 0.001 ) .
about 46.4% of subjects with recession brushed for > 3 min ( p < 0.001 ) .
about 62.2% of subjects with recession brushed twice daily ( p < 0.001 ) the type of brushing method , duration of brushing , frequency of brushing are statistically significant in subjects with recession compared to subjects without recession .
epidemiology is a useful means of establishing a need for treatment or for preventive intervention .
however , correlations are not a means of showing cause and effect but only a means of showing a relationship .
the present study was designed to determine the occurrence and probable etiology of gingival recession among 710 individuals in the age group between 15 and 60 years . of the 710 subjects examined for the study , 291 ( 40.98% ) subjects showed gingival recession .
similar results were seen in previous studies . whereas a higher prevalence of ( > 50% ) gingival recession was seen in other studies . a recent study conducted in greece showed an overall prevalence of 63.9% . on the contrary , a study by mathur et al
. this might be due to the very young age group ( 1015 years ) of subjects in the study . in the present study , frequency of gingival recession
was found to increase with age . in younger age group ( 1525 years ) ,
the gingival recession was 26.9% ; and in older age group ( 4560 years ) , it was 70.27% .
investigators have found that in most of the studies , the frequency of recession was 100% for older age group .
this relationship between the occurrence of gingival recession and age may be because of the longer period of exposure to the agents that cause gingival recession ; associated with intrinsic changes in the organism , both local and systemic , besides the cumulative effects of the lesion itself . according to loe et al .
the occurrence of gingival recession in young patients is usually localized and seems to comprise isolated etiologic factors . on the other hand , a more generalized distribution , as observed among older subjects , might indicate the associated and cumulative effect of several factors such as previous periodontal disease associated with toothbrushing trauma .
a high frequency of gingival recession was seen in males ( 60.5% ) compared to females ( 39.5% ) .
ainamo et al . on the other hand found that gingival recession was equally common in both the genders in 17 years age group . in our study ,
mandibular incisors ( n = 495 ) had the highest prevalence of gingival recession when compared to other teeth .
recessions may be found in teeth that are prominently positioned that is , the alveolar bone is thin or absent , and the gingival tissue is thin in these areas .
areas with deficient keratinized mucosa have been demonstrated to be more susceptible to gingival recession , especially due to the smaller amount of connective tissue available in the area .
this leads to localized inflammatory reactions , which are triggered by different processes that affect the entire extension of the tissue , ultimately leading to gingival recession .
this might be one of the most common reasons for the occurrence of gingival recession in the mandibular anterior teeth .
but the findings in the few other studies showed that the gingival recession is more in maxillary first molar .
this may be due to the angulations of the root in the bone , which has got an influence on recession and is often observed in maxillary molars area . in this study ,
, it was also seen that millers class i recession was more common due to the presence of plaque and millers class ii recession was due to faulty toothbrushing .
it should be stressed that the distribution pattern of gingival recessions has been related to different etiologic factors .
gingival recessions on the mandibular incisors have been primarily associated with poor oral hygiene , whereas those on the premolars would be originated by traumatic toothbrushing . concerning the maxillary
first molars , some authors believe the cause would be traumatic toothbrushing , while others state that it would be the outcome of poor oral hygiene , demonstrated by the presence of dental plaque and calculus .
the cpi of the subjects of two groups was also recorded . in recession group ,
63.6% of subjects had score 3 and 99.7% had loss of attachment more than 3 mm .
the severity of periodontal disease can be quantified with measurements of the pocket depth and by loss of supporting structures around tooth , which is indexed clinically as attachment loss .
hence , to know the severity of the loss of attachment in recession group , this index was recorded .
studies by dodwad , bindu and cheru chrysanthakopoulos have found the etiology of gingival recession to be multifactorial like faulty toothbrushing , tooth malposition , lack of function , frenal pull , habits , poor oral hygiene , etc .
, with one type being associated with the other . in our study , the most common factor associated with gingival recession was seen to be plaque followed by faulty toothbrushing and this similar result has been found by several other authors as well
. localized inflammatory process due to plaque causes the breakdown of connective tissue and proliferation of the epithelium into the site of connective tissue destruction .
proliferation of epithelial cells into the connective tissue brings about a subsidence of the epithelial surface , which is manifested clinically as gingival recession . in the present study ,
subjects using horizontal method of toothbrushing showed the second common associated factor of gingival recession than those following either vertical or circular methods ( 11.78% ) .
vigorous and forceful use of hard and medium stiff - bristled brushes in a horizontal direction can cause minor lacerations , contusions or abrasions of the gingiva with the resultant cleavage detachment or atrophy of the same as well as resorption of the underlying alveolar plate which lead to gingival recession .
have reported a significantly increased odds ratio of 2.1 for the likelihood of developing gingival recession in those subjects who brush more than once a day over less frequent brushers .
the duration of toothbrushing was implicated in a study by tezel et al . in which both males and females who brushed for > 3 min had approximately twice the mean severity of gingival recession than those subjects who brushed for < 1 min . in a systematic review ,
it was concluded that toothbrushing factors that had been associated with the development and progression of gingival recession were duration and frequency of brushing , technique , brushing force , frequency of changing toothbrushes and hardness of the bristles . on the contrary ,
bindu and cheru found malalignment of teeth to be the most common etiologic factor associated with recession .
banihashemrad et al . found gingival recession more common in smokers . in this study , it was also found that severe gingivitis was seen in recession group compared to moderate gingivitis in the other group .
the information gathered would probably help in evolving long - term strategies to prevent the occurrence of mucogingival defects and enable us to predict the rate of success of therapeutic measures .
in addition to the ones mentioned in this study , there are few more implicating factors such as gingival biotype , chemical trauma , lack of function in the initiation of gingival recession that have not been considered here .
the etiology of gingival recession is multifactorial , and its appearance is always the result of more than one factor acting together . at the community level , adequate awareness and education in oral hygiene maintenance should prove to be fruitful in long run .
|
objectives : gingival recession is the term used to characterize the apical shift of the marginal gingiva from its normal position on the crown of the tooth . it is frequently observed in adult subjects . the occurrence and severity of the gingival recession present considerable differences between populations . to prevent gingival recession from occurring ,
it is essential to detect the underlying etiology .
the aim of the present study was to determine the occurrence of gingival recession and to identify the most common factor associated with the cause of gingival recession.methods:a total of 710 subjects aged between 15 years to 60 years were selected .
data were collected by an interview with the help of a proforma and then the dental examination was carried out .
the presence of gingival recession was recorded using miller 's classification of gingival recession .
the silness and loe plaque index , loe and silness gingival index , community periodontal index were recorded .
the data thus obtained were subjected to statistical analysis using chi - square test and student 's unpaired t-test.results:of 710 subjects examined , 291 ( 40.98% ) subjects exhibited gingival recession .
the frequency of gingival recession was found to increase with age .
high frequency of gingival recession was seen in males ( 60.5% ) compared to females ( 39.5% ) .
gingival recession was commonly seen in mandibular incisors ( 43.0% ) .
miller 's class i gingival recession was more commonly seen .
the most common cause for gingival recession was dental plaque accumulation ( 44.1% ) followed by faulty toothbrushing ( 42.7%).conclusion : approximately half of the subjects examined exhibited gingival recession .
the etiology of gingival recession is multifactorial , and its appearance is always the result of more than one factor acting together .
|
the genus leishmania spp . describes a cluster
of protozoan parasites that cause a variety of related disease patterns
with main relevance in low income countries of the tropics and subtropics
and around the mediterranean basin .
more recent figures indicate 50000
deaths and 2 million new infections each year mainly due to suboptimal disease management including diagnosis ,
prognosis , and treatment .
infective leishmania promastigotes are injected
by the sandfly vector ( lutzomya spp . and phlebotomus spp . ) into the skin of the mammalian host and
is
the macrophage phagolysosome where the
physicochemical conditions , in particular the high temperature and
low ph initiate transformation from flagellated promastigote to immobile
amastigote stage which proliferates via binary fission .
completion
of the life cycle is granted when parasite - containing macrophages
are taken up by the vector during a blood meal and the amastigotes
are transformed back into promastigotes in the midgut .
the promastigotes
differentiate into infective metacyclics and migrate to the proboscis
to be injected into the mammalian host .
disease development is strongly linked to parasite species
and
immune status of the host .
while l. aethiopica , l. major and l. tropica are dermotropic species for which
proliferation is usually restricted to the primary site of inoculation ,
the dermo - mucotropic ( l. braziliensis , l. panamanensis ) and viscerotropic species ( l. donovani , l. infantum ) can spread to mucosa and internal organs , respectively . although the tropism of the various leishmania strains is a major contributor to disease outcome , the state of
the host s immune system is also influential and can , especially
in the immune - suppressed individual , induce a spread of parasites
to unusual locations such as the gastro - intestinal or respiratory
tract during the visceral form of disease .
early observations on murine l. major infection
models showed a varying disease outcome in different mouse strains ,
whereby the self - healing course in c57bl/6 and the progressive infection
in balb / c mice have probably been most characterized and scientifically
exploited . due to the clear distinctive course of pathology depending on the
rodent model chosen
, l. major has been an important
tool in defining the mechanisms of an effective immune response to
intracellular pathogens .
the system has substantially contributed
to basic immunological understanding in infection by confirming the
general mechanistic trends of t helper cell subpopulations and their specific role in micro- and macro - parasite
defense . the original classification into two dichotomous t helper
( th ) cell populations had to be reshaped in the past decade due to
other emerging th subsets including th17 and t regulatory cells and the substantiation of the emerging concept
of t cell plasticity .
however , the original simplification
can still offer a gross mechanistic guidance on the cellular as well
as on the metabolic level .
the l - arginine pathway is
a crucial immune - metabolic cascade
responsible for either control or explosion of intracellular parasite
populations , which is tightly controlled by the th cell response and
the corresponding cytokine environment .
classically activated macrophages
( cam ) , stimulated with tnf- , il-12 , and ifn- ,
typically referred to as th1 cytokines , will generate reactive nitrogen
species including nitric oxide ( no ) and its derivatives no2 , hno2 and onoo , via the action of inducible nitric oxide synthase ( inos ) .
by contrast ,
macrophage exposure to il-4 , il-13 and il-21 , classically representing
th2 cytokines , stimulates transcription of arginase and the subsequent
generation of polyamines via l - ornithine , which support parasite
growth and replication .
the
major underlying immune - metabolic processes of macrophage mediated
defense to l. major have been described in depth ,
particularly with regard to the l - arginine pathway and its
major metabolic regulators including picolinic acid , an intermediate
of the tryptophan degradation ( l - kynurenine pathway ) , phenylalanine and taurine .
substantial progress has been made indeed to further dissect the
role of selected pathway components , particularly of the role of l - arginine , arginase and inos .
however , an approach for simultaneous global metabolic assessment
of all participating metabolic entities , that is , parasite , macrophage
and cell environment has not been described in depth so far .
metabolic profiling based on h nuclear magnetic resonance
( nmr ) spectroscopy combined with multivariate modeling strategies
and correlation analysis has shown the capacity to shed light on parasite - host
interaction in vivo in an integrative and nonassumptive
way . despite being applied only in recent years within parasitology
,
this approach has opened a new avenue to assess parasite - host interplay
from a global perspective and allowed unexpected insight into remote
parasite effects and systemic metabolic events in the host . by coanalyzing and correlating relative cytokine levels with
the h nmr derived metabolic information , new comprehension on
immune - metabolic processes in the host can potentially be obtained .
although in vivo research has been the main
focus
of interest in the field of metabolic profiling , the importance and
simplicity of in vitro systems has been acknowledged
and has offered a simple tool to address more targeted questions .
such research has been introduced more recently as a complementary
component in a variety of disease scenarios , including infectious
disease and cancer . here
we assess a well - defined cytokine - regulated
macrophage in vitro system and combine targeted enzymatic
and biochemical
assays with a h nmr - based screening of the single compartments
involved , including parasite , external and internal macrophage metabolome ,
and cell growth supernatant , in order to comprehend the metabolic
communication between the metabolically active entities .
three different
macrophage activation states were characterized and compared , specifically
cam , aam and nonactivated m ( nam ) in an
uninfected state and during l. major exposure .
our
studies define the wider metabolic impact of the cytokine induced
activation on parasite , macrophage and cell environment .
bone marrow
derived macrophages were obtained from nave 68
week old female balb / c mice ( charles river , u.k . ) .
bone marrow was
harvested by flushing femurs and tibias and 5 10 bone marrow precursor cells were differentiated in hydrophobic teflon
bags in 50 ml of macrophage differentiation medium into mature macrophages .
the macrophage differentiation medium used was dulbecco s modified
eagle medium ( dmem , gibco ) supplemented with 10% heat - inactivated
fetal calf serum ( fcs ) , 5% horse serum , 50 iu / ml penicillin , 50 g / ml
streptomycin , 2 mm glutamine , ( gibco ) , 0.05 mm -mercaptoethanol ( sigma ) and
( 10% v / v ) l929 fibroblast culture supernatant , the latter
contains macrophage colony stimulating factor ( m - csf ) necessary for
the differentiation of the precursor cells into macrophages .
the single
cell suspensions were differentiated for 8 days in teflon bags , harvested ,
washed in dmem , counted and adjusted to the required cell concentration .
mature bone marrow
derived macrophages ( m ) were activated in the presence or absence
of l. major lv39 ( mrho / su/59/p - strain ) promastigotes
at a multiplicity of infection of 10 parasites per m. macrophage
cultures were prepared in 6 replicates in 6-well tissue culture plates
( costar ) and seeded at 2.5 10 cells / well in 5 ml
dmem , supplemented with heat - inactivated 10% fcs , 50 iu / ml penicillin ,
50 g / ml streptomycin , 2 mm glutamine , ( gibco ) , and 0.05 mm
-mercaptoethanol ( sigma ) .
six different groups were assessed
and compared , namely alternatively activated macrophages ( aam ) ,
classically activated macrophages ( cam ) and nonactivated macrophages
( nam ) .
each activation state was set up in absence and presence
of l. major and all
cultures were incubated for 48 h at 37 c , 5% co2 .
the 48 h culture period was selected to ensure optimal viability of
macrophages used in the six different groups .
activation stimuli included
ifn- and tnf- for cam ( 5 10/ml mature m with 100 u / ml recombinant murine ifn- and 500 u / ml recombinant
mouse tnf- ;
peprotech ) whereas no stimuli were used for nam. il-4 was supplemented
for generation of aam ( 5 10/ml mature m
with 20 u / ml recombinant mouse il-4 peprotech ) .
l. major parasites lv39 ( mrho / su/59/p - strain ) were maintained in a virulent
state by monthly passage in balb / c mice .
parasites were isolated monthly
from lesions of l. major infected balb / c mice , and
used for an average of 68 in vitro passages .
the freshly isolated parasites were cultured and maintained at 26
c , 5% co2 in solid phase blood agar , overlaid with
5 ml dmem containing 50 iu / ml penicillin and 50 g / ml streptomycin
( p / s ) .
late log phase / stationary phase l. major promastigotes
were collected from culture , washed three times in dmem and an aliquot
of the parasite suspension was fixed in 2% paraformaldehyde and used
for counting parasites using a hemacytometer and adjusted to the required
density .
after 48 h of incubation at
37 c , 10% co2 infected macrophages were washed and
incubated with freshly prepared sterile lysis medium ( dmem supplemented
with 0.008% w / v sds ) for 720 min at 37 c , 10% co2 . during this time , macrophage disintegration was monitored
in regular intervals and when the host cells were lysed , the reaction
was stopped by adding dmem supplemented with 20% heat - inactivated
fcs , 20 mm hepes , 4 mm sodium bicarbonate , 50iu / ml penicillin and
50 g / ml streptomycin . the lysates containing the liberated l. major amastigotes were transferred into blood agar cultures
and incubated at 26 c , 5% co2 to allow the amastigotes
to transform back into promastigotes and to proliferate .
leishmania promastigotes were counted in a separate experiment based on four
replicates per activation group , using a hemacytometer after 35
days of culture . for metabolic profile assessment ,
l.
major parasites were counted , adjusted to 4.4 10 promastigotes and washed three times in ice - cold sterile
pbs .
supernatant was removed and the residual parasite pellet was
snap frozen on dry ice and subsequently stored at 40 c
prior to nmr analysis .
a volume of 5 ml of the cell culture medium (= supernatant ) was
transferred from tissue culture plates into sterile 15 ml falcon tubes
and centrifuged at 2500 rpm at room temperature ( rt ) to pellet any
cell debris .
supernatants were transferred again into a new set of
tubes and stored at 40 c prior to h nmr acquisition .
after cell culture
medium was removed , macrophage monolayers were quickly washed once
with 1 ml / well pbs ( kept at rt ) which was discarded .
this was followed
by a cell quenching step using 960 l of ice cold hplc grade
methanol ( sigma - aldrich ) per well and subsequently placing the plates
at 4 c for 25 min to permit cell lysis .
lysed cells
and debris were harvested from the bottom of the wells using a cell
scraper and the suspension was transferred into 2 ml eppendorf tubes ,
whereby the recovered amount was 600650 l from
each well due to the rapid evaporation of the methanol .
a further
460 l methanol were used to rinse each well and subsequently
transfer to the cell lysate suspension kept on ice to obtain maximum
metabolic yield .
lysates were dried overnight in the speedvac at 45
c , followed by 80% methanol extraction , performed twice , as
follows : dried lysates were resuspended in 500 l cold hplc
grade methanol and vortexed for 30 s ; 130 l of distilled
water was added into each tube and samples were vortexed again for 30
s. tubes were kept on ice for 1020 min , spun down at 13000
rpm for 10 min and lysate supernatants transferred into new tubes
on ice .
extraction with 80% methanol was performed a second time on
the remaining pellets , as described .
supernatants were pooled with cell lysate samples
from the first methanol extraction and dried overnight in the speedvac
at 45 c . dried cell extracts were stored at 40 c
prior to preparation for h nmr analysis .
equal amounts
of cell supernatant and phosphate buffer ( 0.2 m na2hpo4 , 0.043 m nah2po4 , d2o : h2o = 7:3 , v / v , 0.01% of sodium 3-(trimethylsilyl ) propionic
acid 2,2,3,3-d4 ( [ tsp ) ] , ph = 7.4 ) , were mixed to a total amount of
600 l in eppendorf tubes by vortexing .
samples were spun down
at 13000 rpm for 5 min and 550 l was transferred into 5 mm
nmr tubes .
samples were spun down at 13000 rpm for 5 min
and 550 l of the supernatant was transferred into 5 mm nmr
tubes .
a frozen pellet of 4.4 10 parasites
was transferred
immediately into a zro2 rotor and 25 l d2o were added . after inserting a spacer to adjust for the small
amount of biomass , the rotor was sealed with a kel - f rotor cap .
all cell culture supernatants were assessed on a bruker avance
600 nmr spectrometer with txi probe head ( bruker ; rheinstetten , germany )
operating at 600.13 mhz for proton frequency , whereby 128 scans per
sample were sufficient for obtaining optimal signal output .
the cell extracts were acquired using a cryo probe head ( co tci )
in order to minimize electric noise and augment the signal - to - noise
ratio , whereby the number of scans was adjusted to 384 for each sample .
a standard 1-dimensional ( 1d ) pulse program was applied ( recycle delay
( rd)-90-t1 - 90-tm-90-acquisition time ( aq ) ) and the
water impact on the baseline was minimized by irradiating the water
frequency during the rd ( 2 s ) .
the 90 pulse length was set to
14.75 s for all cell supernatants and 14.62 s for the cell extracts .
the spectra were recorded in 32768 data points within a spectral width
of 20 ppm and a 0.3 hz line - broadening factor was applied to the free
induction decays ( fids ) prior to fourier transformation . in
contrast to supernatants and cell extracts ,
the l. major pellet was acquired via high resolution magic angle spinning ( mas)-nmr
to maximize metabolic information from such a small amount of biomass .
the rotor was therefore placed in a mas probe
at an angle of 54.7 and spun at 5000 khz in order to compensate
for the line broadening effects introduced by the restricted motion
of the analyte and the sample heterogeneity .
additionally to 1d spectral
acquisition , a carr purcell meibom gill ( cpmg ) pulse sequence was applied to the parasite pellet
in order to account for the smaller metabolic weight components .
mas
spectra were acquired in 256 scans and recorded in 65536 data points ,
whereby a 90 pulse length of 10.43 s was applied .
spectral assignments
were made by consulting the literature and
in - house databases confirmed via statistical total correlation spectroscopy
( stocsy ) and using nmr suite profiler
software ( chenomx , usa ) .
spectral
data were processed using nmrproc , an in - house developed matlab algorithm
( dr . tim ebbels and dr .
hector keun , mathworks ) which performs optimized
phasing , baseline alignment and automatic calibrating to the tsp reference
peak at 0.00 .
to exclude the reference peak and residual resonances
of the water peak from the data modeling process , only the spectral
region from 0.59 was imported into matlab and the
region affected by the water was cut out , that is , 4.544.98
for extracts and 4.75 for the supernatants .
an in
house adapted script for probabilistic quotient normalization was
applied prior to statistical analysis ( dr . kirill veselkov ) .
multivariate analysis including principal component analysis
( pca ) and partial least - squares ( pls)-related methods were applied
to the data using a simca ( umetrics , sweden ) and matlab interface .
as unsupervised method
, pca offers an unbiased overview on the general
data distribution of a data matrix ( x ) by compressing
the total amount of information into one single data point onto a
plane .
the resulting scores plot allows visualization
of groupings , outliers and time trajectories hence pca is the ideal
tool for defining further analytical strategy .
pls on the other hand
relates the data in x to a second matrix ( y ) containing a separate set of information which can be
quantitative , such as cytokine levels , parasitaemia , inflammatory
markers , etc . , or qualitative ( e.g. , affiliation to class ) in which
case a pls discriminant analysis ( pls - da ) would be applied .
o - pls - da is a further optimization and is currently being applied
for biomarker identification , comparing two classes of samples . whereas the pls algorithm maximizes the class differences and facilitates
identification of the loadings ( metabolites ) responsible , the inbuilt
orthogonal filter removes systemic variation unrelated to class - affiliation .
integrals from selected nonoverlapped regions
from each potential biomarker were therefore calculated in matlab
and imported into graphpad prism ( graphpad , usa ) and spss ( ibm corporation ,
usa ) for graphical representations and univariate statistical tests
which included two tailed mann
in addition to the 6 well cultures
for metabolic profiling , macrophage cultures ( 1 ml / well ) were set
up in four replicates in 24 well tissue culture plates and stimulated
and infected in parallel .
bone - marrow derived macrophages ( 5
10/ml ) were assessed in unstimulated , classically , and
alternatively activated state ( as above ) in the absence or presence
of l. major promastigotes at a multiplicity of infection
( moi ) of 10:1 .
after 48 h of culture at 37 c , 10% co2 , macrophages were washed and lysed .
arginase activity was measured
in macrophage lysates by measuring the conversion of l - arginine
to urea , as described previously .
briefly , cells
were lysed with 0.1% triton x-100 ( sigma ) and 50 mm tris - hcl ( ph 7.5 ,
sigma ) , which contains the arginase cofactor manganese chloride ( sigma )
and the enzyme activated at 56 c for 7 min .
the hydrolysis was
conducted by the addition of 0.5 m l - arginine ( ph 9.7 ) and
incubated at 37 c for 15120 min .
the reaction was stopped
using an acid mix consisting of phosphoric acid ( 85% , fluka ) , sulphuric
acid ( 98% , fluka ) and distilled water at a v / v / v ratio of 1/3/7 .
the
urea production via the hydrolysis of l - arginine by arginase
was measured by the addition of 6% -isonitrosopropiophenone
and incubated at 100 c for 45 min .
urea production by l - arginine hydrolysis in samples was measured against a urea standard
and the od of the reactions was measured at 550 nm .
arginase activity
was calculated in mu per sample and expressed per 1 10 macrophages , whereby the lowest detectable level of arginase
was 2.1 mu/1 10 macrophages .
one unit of enzyme
activity defined as the amount of enzyme that catalyzes the formation
of 1 m urea per minute .
after two days at 37 c , 10%
co2 , culture supernatants were harvested from the different
macrophage cultures in 24 well plates .
nitrite accumulation was used
as an indicator of nitric oxide ( no ) production in culture supernatants
and measured by using the griess reagent .
equal volumes of macrophage
culture supernatants and griess reagent ( 1% sulphanilamide/0.1% n-(1-naphtyl ) ethlyenediamine dihydrochloride/2.5% h3po4 ) were incubated at room temperature for 10
min .
spectral data from macrophage extracts and cell supernatants ( figure 1 ) were assessed separately for differences between
infection and activation state .
pca of cell extract spectra revealed
clear groupings between the different macrophage activation states
as well as between l. major infected
and uninfected cell cultures ( figure 2a ) .
the
most striking separation was observed between cam
and all other in vitro conditions .
analysis of the
cell culture supernatants also indicated a metabolic separation between
the activation states along the first principal component ( pc1 ) , again
showing a distinction between cam from all other data points
( figure 2b ) .
infection induced a shift of the
aam data points along pc1 but no clear infection - related group
separation was found within cam and nam spectra .
h nmr spectrum of cell extract acquired by using a
standard pulse sequence on cell extracts ( red ) and cell culture supernatants
( blue ) .
the metabolite concentration differes substantially between
the two matrixes assessed which leads to a generally lower signal - to - noise
ration in the spectra acquired from the cell extracts .
key : aa s ,
amino acids ; bcaa , branched chain amino acids ; gpc , glycerophosphocholine .
the
groups in the scores plots represent different infection and activation
states of the bone marrow derived macrophages .
key : blue , aam infected ; green ,
aam uninfected ; yellow , nam infected ; pink , nam
uninfected ; red , cam infected ; black , cam uninfected . to identify the metabolites responsible for group
separation o - pls - da
was applied in a first step ( figure 3 ) . to
further validate the statistical significance of the identified metabolic
markers , the integrals were calculated for each , and subjected to
univariate nonparametric tests ( see methods ) . in table 1 , those metabolites are represented that were found to be
statistically different between infected and noninfected samples ,
whereas tables 2 and 3 show the metabolic differences between activation states , that is ,
aam , cam and nam. only metabolites that reached
statistical significance as validated via uni- and multivariate methods
are documented .
o - pls - da analysis comparing spectra of cell supernatants
of infected
and uninfected aam. metabolites which are different between
the two states are color coded according to significance , whereby
red denotes highest and blue no discriminatory power .
upward pointing
peaks are positively correlated with the infection , whereas downward
pointing peaks are anti correlated with l. major infection .
, arbitrary units ; bcaa , branched chain amino acids ; 3hb ,
3-hydroxybutyrate ; uk , unknown metabolite ; * , tentatively assigned .
the infection state showed particular discrimination
in cell culture
supernatants of aam and the cell extracts of nam ( table 1 ) , whereas the nam supernatants did not appear
to differ significantly between the infected and uninfected state
with the exception of increased succinate after l. major exposure .
the cam cell extracts did not seem to undergo major
metabolic changes either upon infection and showed solely an increase
of creatine .
the cell supernatants on the other hand revealed elevated
levels of acetate , alanine , fumarate , pyruvate and succinate in those
samples which have been incubated with l. major .
metabolites were selected based
on the o - pls - da output and the difference in between the median values
of metabolite concentrations ( in mm , estimated from integral calculations ,
relative to tsp ) between infected group and non - infected group was
calculated for each .
changes in median concentration and fold changes
were calculated relative to non - infected states , whereby increases
are shown as positive values and decreases are shown as negative values .
statistical significance ( p )
measured via two - tailed
mann - whitney test ( with bonferroni correction for individual m
subsets ) whereby * , p < 0.05 ; * * , p < 0.01 ; * * * , p < 0.001 ; * * * * , p < 0.0001 ; and ns , not statistically significant .
metabolite levels are displayed
as median concentration and range per condition ( in mm ) , estimated
from integral calculations , relative to tsp .
statistical significance
( p ) measured via kruskal - wallis test across three
activation classes , whereby * , p < 0.05 ; * * , p < 0.01 ; * * * , p < 0.001 ; and ns ,
not statistically significant . tentatively assigned .
metabolite levels are displayed
as median concentration and range per condition ( in mm ) , estimated
from integral calculations , relative to tsp .
statistical significance
( p ) measured via kruskal - wallis test across three
activation classes , whereby * , p < 0.05 ; * * , p < 0.01 ; * * * , p < 0.001 ; and ns ,
not statistically significant .
the activation state was reflected strongly in the
metabolic background ,
that is , cell extracts and culture supernatant in both l.
major infected and uninfected macrophage cultures ( tables 2 and 3 ) .
a depletion of glucose ,
alanine , and glycine ( cell culture supernatant ) and a subsequent increase
of lactate ( cell culture supernatant or extract ) was the most general
change comparing cam with either nam or aam in
both states , infected and uninfected ( tables 2 and 3 , figures 3 and 4 ) .
more specific differences included relative lower
acetate levels in aam cell supernatants compared to nam
supernatants during infection , or a general higher intracellular level
of taurine in both aam and nam compared to cam
during infection .
biomarkers represented in a schematic in vitro context arranged in pairwise comparisons : those metabolites within
the macrophage were identified as biomarkers derived from the analysis
on the cell extracts , whereas the metabolites outside the cell denote
changes characterized in the corresponding cell supernatants .
a ) effect
of infection within each activation state , whereby metabolites in
red indicate a relative increase of the metabolite during infection
whereas blue metabolites were found to be relatively lower in macrophages
infected with l. major .
b ) effect of activation state
within uninfected cells ( top row ) and infected macrophages ( bottom
row ) .
the color code ( red , increased ; blue , decreased ) referes to
the first mentioned activation state in each pairwise comparison ,
for example , in cam vs nam , the red metabolites would
refer to cam , indicating that in cam red is relatively
increased compared to nam , etc .
a few biomarkers that were identified in the cell
culture supernatants
were also found in the cell extracts , namely acetate , citrate and
lactate , whereas others were specific to extracts ( e.g. , creatine ,
creatine phosphate and choline derivatives ) and supernatants ( e.g. ,
succinate , alanine ) ( tables 13 ) , which is in line with the general metabolite
recovery ( table 4 ) . the color code indicates presence
( red ) and no confirmed detection of a metabolite ( blue ) .
the metabolites detected by h nmr spectroscopy of macrophage
extracts , macrophage culture supernatants , fresh cell culture growth
media and l. major showed a high overlap .
lactate and the branched chain amino acids ( bcaa )
leucine , isoleucine and valine , were identified in all four compartments ,
and a large amount of metabolites was observed in three compartments ,
including acetate , glucose , glycine , phenylalanine and tyrosine .
uracil
on the other hand was only present in the l. major metabolome and formate and fumarate were specific to the external
macrophage metabolome ( figures 1 and 5 and table 4 ) .
some residual
lipid resonances were observed only in mas nmr spectra of l. major ( figure 5 ) as a result of
analyzing intact parasites and not further discussed in comparisons
to solution - state analyses of cell extracts which focused only on
polar metabolites .
alanine , acetate , citrate , succinate , 3-hydroxybutyrate ,
formate and fumarate were all found in the supernatant after incubation
with cellular material .
high resolution h mas nmr acquisition
of l.
major pellet using a cpmg pulse sequence to obtain overview
on the small metabolic weight components of the parasite .
the top
figure indicates the alphatic region of a h nmr spectrum
and the bottom represents ch groups in aromatic systems .
the standards
for the bcaa assignements were used and overlapped in amix , version
3.9.3 .
key : aa , amino acid ; bcaa , branched chain amino acid ; gpc ,
glycerophosphocholine ; pc , phosphocholine .
arginase activity , no production and parasite counts were assessed
in two separate experiments including four replicates in order to
confirm that the different stimulation assays resulted in the expected
macrophage activation state and the expected effect to infection intensity .
the nitric oxide assay did not show detectable amounts of no in nam
and aam ( limit of detection 5 m ) but revealed higher
levels in l. major infected cells ( 76.1 4.38
m ) compared to the uninfected control samples ( 58.1
1.99 m ) as validated by the nonparametric mann
. pronounced arginase activity was detected
in aam ( l. major ( ) : 580.8
16.40 ; l. major ( + ) : 666.0 2.06 mu/10 cells ) , whereas nam ( l. major ( ) :
17.6 0.29 ; l. major ( + ) : 21.1 0.17
mu/10 cells ) and cam ( l. major ( ) : 6.7 1.59 ; l. major ( + ) : 47.8
0.78 mu/10 cells ) only expressed background levels
of arginase activity .
additional mann whitney tests between infection and
control groups within each activation class revealed that no and arginase
activity were significantly different between infected and uninfected
m throughout all activation groups in which detectable levels
were obtained . whereas no was found to be relatively higher in l. major ( + ) cam than in l. major ( ) cam ,
arginase levels were significantly higher
in l. major ( + ) aam and nam compared
to each corresponding uninfected set of cells but were found to be
relatively lower in l. major ( + ) cam compared
to l. major ( ) cam. parasite
transformation and determination of promastigote survival
after 48 h intracellular exposure to the different activation states
confirmed the well established association of no with parasite killing
and arginase with promotion of parasite growth .
about 70% fewer promastigotes
were recovered from cam ( 5 10 0.66
10 ) whereas higher promastigote numbers were recovered
from aam ( 28.3 10 3.12 10 ) as compared to baseline levels in nam ( 22.8
10 1.32 10 ) .
macrophage activation plays a critical role
in determining disease
progress during the course of l. major infection .
whereas classical activation leads to clearance of the parasite via
generation of reactive nitrogen species , in particular no , alternative
activation will induce increased arginase - mediated conversion of l - arginine to ornithine and polyamines , which are used as substrates
for parasite growth and replication .
the measured no production and arginase activity
in the here presented study confirmed that the different stimulation
conditions used , resulted in alternatively or classically activated
macrophages . the parasite counts furthermore confirmed the leishmanicidal
effect of the classical activation pathway products .
the metabolic
screening of cell extracts via h nmr has revealed clear
metabolic differences between aam s , cam s
and nam s in l. major - infected and
uninfected state , whereby the classical activation state separated
most clearly from all other conditions .
the corresponding cell culture
supernatant confirmed the striking separation of the cam s
from all other states in the global pca ( figure 2 ) , indicating that classical activation of the macrophages has a
more profound effect on the global cellular metabolism than the coexistence
of an intracellular pathogen .
the more obvious group separation of l. major - exposed cells in the extracts , compared to the
cell supernatants , might be due to the exposure of any secreted components
to the relatively large volume of external growth medium .
a potential
dilution of cell derived metabolites might render the detection of
more subtle changes to intracellular metabolic pathways more difficult
in the extracellular milieu .
however , subsequent in depth analysis
using o - pls discriminant analysis was able to detect those subtle
differences and reveal the biomarkers responsible for discrimination
of infection and activation state .
moreover the biomarker yield in
each compartment was dependent on activation state but overall quantitatively
comparable ( figure 4 ) .
o - pls - da assessment
of infection markers within each of the three
activation states separately , showed that l. major infection induced a similar metabolic fingerprint in all macrophage
populations , which included different combinations of relatively elevated
levels of actetate , alanine , pyruvate , succinate and lactate in internal
and external macrophage metabolome and may , as main end products of leishmania , reflect a direct contribution of the parasite
( figures 3 and 4 , table 1 ) . the strongest biomarkers
that were recovered from the analysis
of the macrophage extracts included glycerophosphocholine , phosphocholine ,
creatine phosphate and creatine which were measured at relative higher
concentrations in infected samples from aa and nam , compared
to the noninfected corresponding samples ( figure 4 ; table 1 ) .
the increase of choline
species might indicate a certain degree of membrane decomposition
and subsequent lipid degradation during invasion of l. major , which did not seem to have been the
case after classical activation .
the higher cellular uptake of creatine
( aa , ca . and nam s ) and creatine phosphate ( aa and nam s ) ,
on the other hand might reflect the higher energy demand of the macrophages
in order to cope with the infection , since creatine phosphate represents
a major cellular short - term energy reserve for atp generation .
the cam showed , however , overall the
least metabolic differences between l. major - infected and uninfected state , which may be explained
by the confirmed effective intracellular killing of the parasite and
hence a relatively minor accumulation of metabolic end products and
intracellular pathway interruptions in contrast to the uncontrolled
growth and replication of l. major in nam
and aam. parallel evaluations of arginase and no indeed confirmed
relatively higher leishmanicidal activity and significantly lower
parasite counts in cam. the most striking effect related
to macrophage activation state
was the clear separation of the cam s from both aa and
nam s ( figure 2 , tables 2 and 3 ) .
biomarker assessment
within each infection state ( l. major present and
absent ) revealed that a depletion of glucose in the cell culture supernatants
and a subsequent increase of lactate in both macrophage extract and
culture supernatants were the main metabolic drivers for the separation
and indicate that cam has a significantly higher energy demand
compared to the other two groups , to successfully defend infection .
an increased amount of atp is necessary , for instance , for enzyme
activity such as the nadph oxidase which is crucial for the production
of reactive oxygen and nitrogen species .
it has been shown that the excessive no production upon classical
activation can lead to complete arrest of atp production via the respiratory
chain .
. the
increased glycolytic rate and the subsequent accumulation of lactate ,
in turn , might be directly responsible for the observed ph decrease
in the cell supernatants of infected cam ( cam 67 ;
aa and nam s 89 ) .
the potential contribution
of parasitic d - lactate seems
herby unlikely , since no lactate increase was observed in infected
cam compared to uninfected samples ( figure 4 , table 1 ) . the majority of metabolites
which were found prevalent in all or
most compartments assessed were amino acids and glucose to serve as
source for energy and anabolic pathways .
choline and its derivatives
phosphocholine and glycerophosphocholine ( gpc ) , on the other hand ,
were found in the intracellular metabolome of parasite and macrophage
but not in the external fluids , which might reflect accumulation of
the intermediates for the intracellular recycling process of remodelling
the cell membrane , since choline is substrate for the phospatidylcholine
anabolism .
the tricarboxylic acid cycle intermediates
citrate , fumarate , pyruvate
and succinate were unequally distributed throughout the compartments
whereby presence in the external macrophage metabolome was the only
commonality .
only succinate was detected in the protozoa via h nmr , which might reflect back to the fact that the compound
is one of the main metabolic end products of l. major with relatively higher accumulation in the parasite when compared
with fumarate and citrate for instance .
the
culture growth medium and the external macrophage metabolome
showed the highest degree of intercompartment overlap ( i.e. , 79.4% ) .
qualitative differences were detected in only 7 out of 34 metabolites
and include acetate , alanine , citrate , succinate , 3-hydroxybutyrate ,
formate and fumarate which were all present in the macrophage footprint
but absent in pure dmem .
whereas acetate , alanine and succinate are
major end products of the leishmania metabolism and
might be directly derived from the parasite , the remaining components
are likely to reflect metabolic waste products of both , macrophage
and parasite metabolism .
work in previous parasite - rodent models
has shown the capacity
of metabolic profiling in discovery of infection - specific candidate
diagnostic biomarkers and in characterizing immune - metabolic codevelopment
during infection .
whereas in vivo , immune measures
such as cytokines are being coanalyzed with the metabolic background
in a systemic , untargeted manner , the present in vitro approach is exploring metabolic change in macrophages upon specific
cytokine stimulation , which represents a more targeted and complementary
approach .
we are describing a highly efficient and reproducible read
out system for characterizing the gross metabolic background of all
metabolically relevant compartments involved ( i.e. , macrophage , parasite
and growth media ) via h nmr spectroscopy .
the well - defined
compartmentalization and the known baseline metabolic compositions
allow much clearer conclusion toward biomarker origin and metabolic
translocation across the compartments , compared to complex mammalian
systems .
the approach therefore holds promise to offer substantial
support in identifying parasite - derived compounds as candidate diagnostic
biomarkers .
moreover , the efficiency of the presented testing system
may be further exploited for evaluating potential parasiticidal metabolic
supplements , novel drug candidates , and varying cytokine stimuli and
can be easily adaptable to other metabolic profiling platforms if
need be .
we believe that integration of this more focused in vitro strategy into the landscape of untargeted metabolic
profiling will help us to access the wealth of information waiting
to be explored within the immune - metabolic interface .
|
infection with leishmania spp . can lead
to a range
of symptoms in the affected individual , depending on underlying immune - metabolic
processes .
the macrophage activation state hereby plays a key role .
whereas the l - arginine pathway has been described in detail
as the main biochemical process responsible for either nitric oxide
mediated parasite killing ( classical activation ) or amplification
of parasite replication ( alternative activation ) , we were interested
in a wider characterization of metabolic events in vitro .
we therefore assessed cell growth medium , parasite extract , and
intra- and extracellular metabolome of activated and nonactivated
macrophages , in presence and absence of leishmania major .
a metabolic profiling approach was applied combining 1h nmr spectroscopy with multi- and univariate data treatment .
metabolic
changes were observed along both conditional axes , that is , infection
state and macrophage activation , whereby significantly higher levels
of potential parasite end products were found in parasite exposed
samples including succinate , acetate , and alanine , compared to uninfected
macrophages .
the different macrophage activation states were mainly
discriminated by varying glucose consumption . the presented profiling
approach allowed us to obtain a metabolic snapshot of the individual
biological compartments in the assessed macrophage culture experiments
and represents a valuable read out system for further multiple compartment in vitro studies .
|
thyroid - related ophthalmopathy ( tro ) usually follows rundle 's curve , with early inflammatory phase , followed by stability and often improvement with some residual , inactive disease .
signs and symptoms range from dry eye to dysthryoid optic neuropathy ( don ) . both disease activity and severity
werner proposed the first systematic classification under the acronym nospecs , using characteristic ocular signs and symptoms .
the classification of tro has evolved over time to include more objective criteria and guidelines for therapeutic management , most notably the clinical activity score ( cas ) used by the european graves orbitopathy group .
more recently , dolman and rootman amalgamated these concepts under their visa classification , which divides tro into four disease end points : vision , inflammation , strabismus , and appearance [ fig
measurements obtained in each section allow monitoring of disease activity , grading of severity , and appropriate treatment selection .
visa classification : this classification system divides thyroid - related ophthalmopathy into four disease end points : vision , inflammation , strabismus , and appearance all patients with tro should be referred to an internist and/or an endocrinologist for management of the systemic dysthyroid state .
systemic symptomatic control may be achieved with a beta - blocker , whereas hyperthyroidism may be treated by radioactive iodine ablation ( rai ) , surgical thyroidectomy , or with a thionamide such as propylthiouracil .
patients with vision - threatening or severe tro activity should avoid rai if possible because of the risk worsening of tro after therapy .
thereafter , thyroid levels should be monitored every 4 - 8 weeks until normalized , then every 2 - 3 months .
smoking cessation should be recommended to all patients ; it is among the most important risk factors for tro progression , with the effect being proportional to the number of cigarettes smoked daily .
orbital imaging should be obtained with ultrasound , computed tomography ( ct ) , or magnetic resonance imaging ( mri ) .
ultrasound provides fast in - office evaluation of the extraocular muscles but requires an experienced echographer .
ct and mri have the added benefit of visualizing the entire orbit , although ct has greater sensitivity in detecting extraocular muscle enlargement .
t2-weighted mri sequences may show hyperintensity within the extraocular muscles as evidence of disease activity .
active disease is defined as progression of any of the visa parameters or cas of 4 or more ( max score = 8) , with the converse true of inactive disease .
the level of activity can be further divided into mild ( cas 4 or less without deterioration ) , moderate to severe ( cas 5 or more , or progressive disease ) , or vision threatening .
topical antibiotics and/or hourly preservative free lubrication are helpful but often inadequate alone , and the clinician should not hesitate to perform tarsorrhaphy or periorbital botulinum toxin for mechanical protection of the eye . severe ulceration or perforation may require glue , amniotic membrane , and/or corneal grafting with the aid of an anterior segment specialist .
( iv ) steroid therapy given weekly at 0.25 - 0.5 g / week , as in moderate to severe disease below or urgent posterior orbital decompression . in a prospective trial comparing surgical decompression with pulsed iv steroid therapy , there was no apparent advantage for either method , and some surgical patients still required subsequent iv steroids or radiotherapy .
the individual patient 's medical co - morbidities and severity of optic neuropathy must be considered in deciding on surgical versus medical treatment .
consecutive day dosing of iv steroid may be considered for more severe don or as a temporizing measure prior to decompression , administered for daily 3 days followed by another dose a week later .
if the response to iv steroid is poor after one to 2 weeks , surgical decompression should be offered . additional iv steroid therapy with or without radiotherapy may be needed after surgery if the disease is still active .
standard therapy for mild tro includes observation and supportive measures to alleviate dry eye symptoms ( ocular lubrication , moisture goggles , sunglass wear , wind avoidance ) and periocular swelling ( elevation of the head of bed , humidification ) .
a prospective study of oral selenium supplementation ( sodium selenite 100 ug twice a day ) in newly diagnosed patients with mild tro showed a reduction in cas activity and improved quality of life ( go - qol ) , lid retraction , and soft tissue edema when compared to placebo after 6 or 12 months of treatment , this study was conducted in a population that is often deficient in selenium , so generalization to a broader population should be made with caution . as the treatment carries little to no risk ,
it has become popular with physicians . unless contraindicated , glucocorticoid ( gc ) therapy ,
either oral or iv , is usually the first choice for active and moderate to severe disease .
both modalities have side effects , with prolonged oral gc usage causing an increased risk of osteoporosis versus iv gcs , which carry a risk of worsening hepatic dysfunction or failure .
the advantages iv therapy has over oral are an overall reduction in total steroid burden and decreased chronic oral steroid intake , more rapid improvement in cas , better tolerability , and lower risk of osteoporosis .
the optimal dosing schedule is not established ; in our practice we prescribe 500 mg of iv methylprednisolone given weekly for 6 weeks followed by 250 mg of iv methylprednisolone weekly for 6 more weeks .
hepatic function should be monitored , and the total dose of methylprednisolone should not exceed eight grams because of significantly increased risk of hepatic failure . alternatively , oral gc therapy , in the form of prednisone , can be given at 1 mg / kg / day , followed by a prolonged taper of 10 mg / week until a dose of 20 mg / day is reached , then decreased 5 mg every week . during this time blood sugar , especially with concurrent diabetes mellitus , blood pressure , and monthly hepatic function should be followed .
bisphosphonates are recommended especially if duration of gc therapy exceeds 3 months or an average daily dose greater than 5 mg of prednisone is consumed . due to data showing superior efficacy of iv gc
glucocorticoid treatment is not effective in all patients with active disease , and side - effects may limit their use .
thus , the addition or substitution of immunosuppressive drugs has been studied with varying results .
cyclosporine monotherapy was found to be inferior to oral prednisone , but in combination appeared to be more effective than prednisone alone and potentially useful in those not responding to steroid therapy .
this treatment regimen has not had widespread use , and disappointing results have been seen with azathioprine and methotrexate .
rituximab has shown promise in the treatment of severe steroid - refractory tro , with several case reports and recent case series showing a substantial reduction in disease activity and severity . in the united states , rituximab carries a black box warning , as patients have contracted progressive multifocal leukoencephalopathy with chronic therapy for other diseases . as a biologic agent
, the drug also remains extremely expensive , and its role in the overall treatment algorithm remains very limited .
determining the efficacy of orbital radiotherapy ( or ) has been hampered by the lack of controlled studies and the frequent co - administration of gc during radiotherapy , which confounds retrospective data analysis .
the available data do not support using or in isolation or in the initial management of don , and it appears beneficial only for moderate or worse active disease or with confirmed disease progression .
the standard dosing regimen is 20 gy / orbit in ten fractions administered over two weeks , although 10 gy may be as effective .
a temporary worsening of inflammation often occurs after or and can be prevented with concurrent gc use .
in fact , combination therapy with gc may be superior to either treatment alone , especially in improving diplopia . or carries an extremely low risk of causing either optic neuropathy or retinopathy , although the latter has been reported and may be related to concurrent diabetes and/or treatment error . in general , or is relatively contraindicated in diabetic patients and those with concurrent vascular disease due to the risk of worsening retinal microvascular function , and should be used with caution in patients under 35 years of age due to the theoretic risk of secondary malignancy . aside from urgent treatment of don ( see above ) ,
surgical options usually are pursued when tro is inactive , defined as no activity or progression over 6 - 12 months , and strabismus and/or disfiguring changes persist .
the sequence of surgery for inactive tro , if necessary , is typically decompression , followed by strabismus surgery , and finally eyelid repair .
principles of orbital decompression include reducing the volume of the orbital contents by removing intraconal and extraconal fat ; space is increased as well via bone removal while sparing the orbital rim as much as possible to preserve eyelid and canthal tendon positions .
a number of approaches to orbital decompression have been described , with medial wall removal ( especially posteriorly ) most effective in relieving orbital apex crowding , and balanced decompression with medial and lateral wall removal and/or preservation of the inferomedial orbital strut most effective in minimizing postoperative diplopia .
decompression alone rarely corrects strabismus when it already exists , and new strabismus may be seen in up to 30% of decompressions .
fat removal alone has the lowest incidence of postoperative diplopia but requires greater surgical skill and experience to obtain an adequate result .
we typically perform either a balanced decompression ( with endoscopic medial wall removal ) or traditional 2- or 3-wall decompression through a lateral canthal and inferior transconjunctival approach .
strabismus surgery is aimed at correcting diplopia in the primary position and downgaze by recessing the restricted muscles , frequently with adjustable sutures to improve postoperative alignment .
eyelid surgery may involve recession of the upper eyelid retractors , recession of the lower eyelid retractors with or without a spacer graft , and conservative blepharoplasty .
standard therapy for mild tro includes observation and supportive measures to alleviate dry eye symptoms ( ocular lubrication , moisture goggles , sunglass wear , wind avoidance ) and periocular swelling ( elevation of the head of bed , humidification ) .
a prospective study of oral selenium supplementation ( sodium selenite 100 ug twice a day ) in newly diagnosed patients with mild tro showed a reduction in cas activity and improved quality of life ( go - qol ) , lid retraction , and soft tissue edema when compared to placebo after 6 or 12 months of treatment , this study was conducted in a population that is often deficient in selenium , so generalization to a broader population should be made with caution . as the treatment carries little to no risk ,
unless contraindicated , glucocorticoid ( gc ) therapy , either oral or iv , is usually the first choice for active and moderate to severe disease .
both modalities have side effects , with prolonged oral gc usage causing an increased risk of osteoporosis versus iv gcs , which carry a risk of worsening hepatic dysfunction or failure .
the advantages iv therapy has over oral are an overall reduction in total steroid burden and decreased chronic oral steroid intake , more rapid improvement in cas , better tolerability , and lower risk of osteoporosis .
the optimal dosing schedule is not established ; in our practice we prescribe 500 mg of iv methylprednisolone given weekly for 6 weeks followed by 250 mg of iv methylprednisolone weekly for 6 more weeks .
hepatic function should be monitored , and the total dose of methylprednisolone should not exceed eight grams because of significantly increased risk of hepatic failure .
alternatively , oral gc therapy , in the form of prednisone , can be given at 1 mg / kg / day , followed by a prolonged taper of 10 mg / week until a dose of 20 mg / day is reached , then decreased 5 mg every week . during this time blood sugar , especially with concurrent diabetes mellitus , blood pressure , and monthly hepatic function should be followed .
bisphosphonates are recommended especially if duration of gc therapy exceeds 3 months or an average daily dose greater than 5 mg of prednisone is consumed . due to data showing superior efficacy of iv gc
glucocorticoid treatment is not effective in all patients with active disease , and side - effects may limit their use .
thus , the addition or substitution of immunosuppressive drugs has been studied with varying results .
cyclosporine monotherapy was found to be inferior to oral prednisone , but in combination appeared to be more effective than prednisone alone and potentially useful in those not responding to steroid therapy .
this treatment regimen has not had widespread use , and disappointing results have been seen with azathioprine and methotrexate .
rituximab has shown promise in the treatment of severe steroid - refractory tro , with several case reports and recent case series showing a substantial reduction in disease activity and severity . in the united states , rituximab carries a black box warning , as patients have contracted progressive multifocal leukoencephalopathy with chronic therapy for other diseases . as a biologic agent
, the drug also remains extremely expensive , and its role in the overall treatment algorithm remains very limited .
determining the efficacy of orbital radiotherapy ( or ) has been hampered by the lack of controlled studies and the frequent co - administration of gc during radiotherapy , which confounds retrospective data analysis .
the available data do not support using or in isolation or in the initial management of don , and it appears beneficial only for moderate or worse active disease or with confirmed disease progression .
the standard dosing regimen is 20 gy / orbit in ten fractions administered over two weeks , although 10 gy may be as effective .
a temporary worsening of inflammation often occurs after or and can be prevented with concurrent gc use .
in fact , combination therapy with gc may be superior to either treatment alone , especially in improving diplopia . or carries an extremely low risk of causing either optic neuropathy or retinopathy , although the latter has been reported and may be related to concurrent diabetes and/or treatment error . in general , or is relatively contraindicated in diabetic patients and those with concurrent vascular disease due to the risk of worsening retinal microvascular function , and should be used with caution in patients under 35 years of age due to the theoretic risk of secondary malignancy .
aside from urgent treatment of don ( see above ) , surgical options usually are pursued when tro is inactive , defined as no activity or progression over 6 - 12 months , and strabismus and/or disfiguring changes persist .
the sequence of surgery for inactive tro , if necessary , is typically decompression , followed by strabismus surgery , and finally eyelid repair .
principles of orbital decompression include reducing the volume of the orbital contents by removing intraconal and extraconal fat ; space is increased as well via bone removal while sparing the orbital rim as much as possible to preserve eyelid and canthal tendon positions .
a number of approaches to orbital decompression have been described , with medial wall removal ( especially posteriorly ) most effective in relieving orbital apex crowding , and balanced decompression with medial and lateral wall removal and/or preservation of the inferomedial orbital strut most effective in minimizing postoperative diplopia .
decompression alone rarely corrects strabismus when it already exists , and new strabismus may be seen in up to 30% of decompressions .
fat removal alone has the lowest incidence of postoperative diplopia but requires greater surgical skill and experience to obtain an adequate result .
we typically perform either a balanced decompression ( with endoscopic medial wall removal ) or traditional 2- or 3-wall decompression through a lateral canthal and inferior transconjunctival approach .
strabismus surgery is aimed at correcting diplopia in the primary position and downgaze by recessing the restricted muscles , frequently with adjustable sutures to improve postoperative alignment .
eyelid surgery may involve recession of the upper eyelid retractors , recession of the lower eyelid retractors with or without a spacer graft , and conservative blepharoplasty .
a standardized treatment algorithm for thyroid ophthalmopathy does not exist , in part because the natural history of the disease is often favorable and thus allows observation of many patients . when treatment is indicated because of vision - threatening disease or documented disease progression , corticosteroids are first - line therapy for most cases and are effective . in refractory cases , other drugs
may be needed , and surgery in the quiescent phase is directed at correcting the sequelae of the active disease phase .
|
aims : the aim was to highlight recent advances in the treatment of thyroid eye disease.settings and design : review article.materials and methods : existing literature and the authors experience was reviewed.results:thyroid ophthalmopathy is a disfiguring and vision - threatening complication of autoimmune thyroid disease that may develop or persist even in the setting of well - controlled systemic thyroid status .
treatment response can be difficult to predict , and optimized algorithms for disease management do not exist .
thyroid ophthalmopathy should be graded for both severity and disease activity before choosing a treatment modality for each patient .
the severity of the disease may not correlate directly with the activity ; medical treatment is most effective in active disease , and surgery is usually reserved for quiescent disease with persistent proptosis and/or eyelid changes.conclusions:intravenous pulsed corticosteroids , orbital radiotherapy , and orbital surgical techniques form the mainstay of current management of thyroid ophthalmopathy .
immunosuppressive and biologic agents may have a role in treating active disease although additional safety and efficacy studies are needed .
|
weaning from invasive mechanical ventilation ( mv ) may be defined as the process of abrupt or gradual withdrawal of ventilator support , thereby shifting the work of breathing from machine to man .
more than 40% of the time that a patient spends on mv is constituted by the weaning period , and around 20% of mechanically ventilated patients will fail their first attempt at weaning .
the corresponding figures for patients of chronic obstructive pulmonary disease ( copd ) are 59% and 50% , respectively .
thus , achieving a careful balance between early versus delayed weaning is a must to minimize the risk of complications associated with either of the two .
moreover , overloading and fatigue of respiratory muscles together with an inability to protect the airway leads to reintubation .
the latter by itself is associated with an increase in morbidity , mortality , duration of mv , and length of intensive care unit ( icu ) and hospital stay . on the other hand ,
if initiated late , weaning may be unsuccessful because of respiratory muscle weakness caused by deconditioning and disrupted breathing regulation .
further , prolonged mv , as often seen in copd patients , is itself associated with complications like nosocomial pneumonia , cardiac morbidity , gastrointestinal bleeding , deep vein thrombosis and death .
thus , choosing the right time and right weaning strategy forms a crucial part of the management of such critically - ill patients and certainly affects their outcome .
several studies have been conducted previously to compare different weaning methods for patients on mv , with variable results .
two large randomized controlled trials ( rcts ) conducted separately by brochard et al . , and esteban et al .
, have found the superiority of pressure support ventilation ( psv ) and t - piece , respectively , over other methods of weaning .
however , these studies do not help in arriving at a consensus regarding the best mode of weaning .
the present study was conducted with the objective to compare the usefulness of nippv with conventional psv as two different weaning modalities in mechanically ventilated copd patients .
this prospective , two - group , parallel rct was conducted in the icu of a tertiary care referral medical institution of north india over a period of 18 months .
the study protocol was approved by the ethics committee of the hospital and informed consent was taken from the patients kin . known cases of copd ( confirmed clinico - radiologically and with available pulmonary function test ( pft ) reports ) who were admitted to our icu in an acute exacerbation requiring endotracheal intubation ( eti ) , were eligible for the study .
type ii respiratory failure was defined as sudden , severely worsened dyspnoea without any other obvious cause and with room - air arterial blood gas ( abg ) analysis showing ph < 7.35 , arterial partial pressure of carbon dioxide ( paco2 ) > 45 mm hg and arterial partial pressure of oxygen ( pao2 ) < 60 mm hg .
major criteria for eti were respiratory arrest , loss of consciousness / gasping for breath , psychomotor agitation requiring sedation , heart rate ( hr ) < 50 beats / min with loss of alertness , and hemodynamic instability with arterial systolic blood pressure ( sbp ) < 70 mm hg or > 180 mm hg .
minor criteria were a respiratory rate ( rr ) > 35 breaths / min , arterial ph < 7.30 , ( pao2 ) < 45 mm hg despite supplemental oxygen , worsened neurological status ( encephalopathy score ) and inability to expectorate due to weakened cough reflex .
the presence of one major criterion before initiating treatment or two minor criteria an hour after initiation of treatment was considered as an indication for eti .
non - intubated copd patients were excluded from the study , as were those having neurological alteration unrelated to hypercapnoeic encephalopathy , cranio - facial deformity , upper airway obstruction , cardiogenic pulmonary edema , cardiac arrest , cardiogenic shock , acute myocardial infarction , pneumonia , pneumothorax , pulmonary neoplasm , pulmonary thromboembolism , gastrointestinal bleeding , and post - operative respiratory failure .
all subjects were evaluated by thorough history taking , clinical examination , chest radiography , abg analysis , and other routine hematological investigations , as needed .
abg was done at 0 , 1 , 4 , 8 , and 12 hours after initiation of mv and also during weaning when required .
similar medications were used in all patients depending upon their requirement , like intravenous antibiotics , bronchodilators , corticosteroids , aerosolized bronchodilators and corticosteroids , correction of electrolytes and intravascular volume abnormalities .
orotracheal route was utilized for eti and control mode of ventilation / assist control mode of ventilation ( cmv / acv ) was used initially for mechanical ventilation , depending upon the patients mentation and abg parameters .
12 - 16/min , tidal volume 8 - 10 ml / kg , inspired oxygen fraction ( fio2 ) to achieve an oxygen saturation ( sao2 ) of 90% with a positive end - expiratory pressure ( peep ) of 5 cm h2o , and an inspiratory : expiratory ratio of 1:2 - 2.5 . use of muscle relaxants and sedatives was limited to an as and when required basis . weaning criteria used when giving an initial t - piece trial to patients were : an adequate mentation and cough reflex , clinical stability , and adequate oxygenation as reflected by sao2 of > 90% at fio2 0.4 , after at least 48 hours of invasive mv .
criteria for failure of the t - piece trial were ph < 7.35 , pao2 < 50 mm hg at fio2 of 0.4 , rr > 35/min , hr > 145/min , sbp > 180 mm hg or < 70 mm hg , severe cardiac arrhythmias , agitation / anxiety / diaphoresis .
complete rest was given to patients who failed the initial t - piece trial by putting them back on cmv / acv mode of ventilation till previous ph and paco2 values were attained and rr was 30/min . thereafter , the patients were considered for weaning and randomized into two groups
group i , weaned by nippv ; and group ii , weaned by invasive psv .
the randomization was done by using two closed , non - transparent identically looking envelopes , each containing information on one of the weaning methods being investigated . after a patient was included in the study , a third party not involved in the study was asked to choose one of the envelopes .
depending on the information in the chosen envelope , the patient was allocated to undergo weaning by either of the two methods .
following randomization , group i patients were extubated and applied nippv with a full - face mask using bilevel positive airway pressure ( bipap ) system ( resmed , vpap iii sta ) .
initial levels of inspiratory and expiratory positive airway pressure ( ipap and epap respectively ) were decided on the basis of achieving acceptable abg parameters , rr < 25/min , and patient 's tolerance and comfort .
further adjustment in bipap setting was done by decreasing the pressure support by 2 cm h2o every 4 hours , with close monitoring for worsening of sao2 and rr .
when ipap and epap were reduced to 8 and 4 cm h2o respectively with satisfactory blood gases , ph > 7.35 , stable vitals , sao2 90% on fio2 0.4 , rr <
30/min , and absence of severe dyspnea / depressed sensorium , the nippv support was removed and patients were allowed to breathe spontaneously on a venturimask .
patients of group ii received invasive psv ( by servo - s macquet ventilator system ) with an initial level of pressure support that was decided on the basis of achieving acceptable abg parameters , rr < 25/min , and patient 's tolerance and comfort .
subsequently , the pressure support was decreased by 2 cm h2o every 4 hours with close monitoring for worsening of sao2 and rr .
when the pressure support and peep reached 10 and 5 cm of h2o respectively , with satisfactory blood gases , ph > 7.35 , stable vitals , sao2 90% on fio2 0.4 , rr < 30/min , and absence of severe dyspnea / depressed sensorium , patients were extubated and allowed to breathe spontaneously on a venturimask .
successful when , after two hours of spontaneous breathing on a venturimask , the patient maintained a sao2 90% on fio2 0.4 , ph > 7.35 , rr < 30/min , no dyspnea , intact sensorium and stable hemodynamic status .
failure when one or more of these criteria were absent , or if the patient could not be extubated even after 30 days of invasive mv , or had to be re - intubated within 72 hours of extubation , or if death occurred due to mv per se .
causes of the latter were nosocomial infection , pneumothorax , cardiac ischemia or fatal arrhythmias .
nosocomial / ventilator - associated pneumonia ( vap ) was defined as the presence , during mv , of new and persistent opacities on chest x - ray along with fever and total leukocyte count ( tlc ) >
the outcome variables used to compare the two groups were duration of mv ( in group i , the duration before randomization ; in group ii , the duration before randomization in addition to the weaning duration after randomization ) , duration of weaning ( in both groups , the duration after randomization ) , duration of icu stay ( in both groups , from day of admission to day of discharge ) , occurrence of nosocomial pneumonia , and patient outcome in terms of survival or death at discharge .
data are expressed as mean standard deviation , numbers ( n ) or percentage ( % )
. the standard normal variate test ( z score , applicable for quantitative data ) for testing the significant difference between double sample mean was applied to find the significant difference for different variables between the two groups .
all the values of zcalculated were compared with ztabulated value of 1.96 at 5% level of significance .
chi - square ( ) test was used for categorical data , wherever applicable .
statistical package for social sciences software ( spss inc , chicago il , usa ) was used for all statistical calculations .
one hundred and fifty patients of acute exacerbation of copd with type ii respiratory failure were admitted in the icu of our hospital during the 18 month study period .
fifteen were excluded from the study due to concomitant pneumonia ( n = 6 ) , lung neoplasm ( n = 5 ) , acute myocardial infarction ( n = 2 ) and cardiogenic pulmonary edema ( n = 2 ) . of the remaining 135 patients ,
ninety five patients were initially given a trial of nippv of which 25 required invasive mv within an hour of the trial .
thus , the total number of patients who needed mv and were eligible for the study were 65 .
of the remaining 57 patients , seven were successfully extubated after the initial t - piece trial and were excluded .
remaining 50 patients were randomized into two groups of 25 each and compared for the outcome variables [ figure 1 ] .
methodology of patients inclusion no difference was observed between the two groups in terms of demographic , clinical and biochemical parameters at the time of admission and randomization [ table 1 ] .
medical therapy was optimized for patients of both groups and comprised of 2 agonists , theophylline and anti - cholinergic agents .
demographic , clinical and biochemical parameters of the two groups the initial mean ipap and epap used in patients of group i were 14.79 1.38 and 5.99 0.73 cm of h2o respectively .
initial mean pressure support used in patients of group ii was 18.51 1.23 cm of h2o above a peep of 5 cm of h2o .
statistically significant difference was found between the two groups in terms of the durations of ventilation , weaning and icu stay [ table 2 ] .
occurrence of nosocomial pneumonia was also significantly lower in patients weaned by nippv than those by psv ( 2 patients-8% vs 8 patients-32% , respectively ) [ table 2 ] .
fewer deaths occurred in the icu at discharge among group i patients than among group ii patients ( 2 patients-8% vs 8 patients-32% , respectively ) [ table 2 ] .
more patients were successfully discharged from the icu in group i than in group ii ( 23 patients-92% vs 17 patients-68% , respectively ) [ table 2 ] .
our study shows that nippv appears to be a promising weaning modality for mechanically ventilated hypercapneic copd patients who fail an initial t - piece breathing trial .
occurrence of bronchospasm , increased airway mucus and airway inflammation in copd produces air trapping , diaphragmatic flattening and increased airway resistance .
these in turn increase the elastic recoil , intrinsic peep , work of breathing , produce muscle weakness , recruitment of accessory muscles , changes in shape of the rib - cage , and ultimately respiratory muscle failure occurs .
these factors are responsible for prolongation of mv and weaning failure in copd patients . during acute decompensation of copd ,
the goal is to reduce carbon dioxide levels by unloading the respiratory muscles and augmenting alveolar ventilation , thereby stabilizing arterial ph until the underlying problem can be reversed .
nippv achieves this by resting the muscles of respiration thus reducing their fatigue , improving the pattern of respiration and facilitating efficient gas exchange .
thus it may be beneficial in hypercapneic respiratory failure which is frequently encountered in copd and in weaning failure .
role of nippv in acute exacerbation of copd has been proven by previously conducted rcts and meta - analyses .
however , its use as an early weaning / extubation technique from invasive mv remains controversial .
there are a few studies highlighting the role of nippv in weaning patients with respiratory failure due to copd .
nava et al . , found that nippv reduces weaning time , shortens the time in icu , decreases the incidence of nosocomial pneumonia , and improves 60-day survival rates , thus concluding that nippv is more effective than conventional psv in weaning copd patients from invasive mv .
gamal made similar observations and concluded that noninvasive ventilation ( niv ) should be considered as an early extubation and weaning technique in patients with copd with acute - on - chronic respiratory failure who are difficult to wean .
hilbert demonstrated that nippv was effective in correcting gas - exchange abnormalities in copd patients with post - extubation hypercapneic respiratory insufficiency and also significantly reduced the need for eti .
prospective rct by girault and associates has shown that nippv is a useful early extubation and weaning technique in intubated acute - on - chronic respiratory failure patients who are difficult to wean .
trevisan concluded that the combination of early extubation and nippv is a useful and safe alternative for ventilation in patients who fail initial weaning attempts .
, demonstrated a consistent positive effect of non - invasive weaning on mortality in mechanically ventilated patients with predominant copd .
these studies suggest that use of nippv is associated with decreased duration of ventilatory support , length of icu stay , incidence of vap and survival benefit , when compared to conventional methods of weaning . in our study , the above results were confirmed and we found that nippv significantly reduced the duration of weaning , duration of ventilation , length of icu stay , incidence of nosocomial pneumonia , and improved survival .
however , few studies partially or wholly contradict the above benefits of nippv in weaning .
girault has shown that nippv used as an early weaning / extubation technique in difficult - to - wean patients with chronic respiratory failure did not reduce the reintubation rate within seven days as compared with conventional weaning .
nevertheless , they found that nippv may improve the weaning results in these patients by shortening the intubation duration and reducing the risk of post - extubation acute respiratory failure .
esteban concluded that nippv is not effective in averting the need for reintubation in un - selected patients with post - extubation respiratory failure .
contrarily , nippv does not improve survival and may in fact be harmful , although selected patients in specialized centers may benefit from this therapy .
another study by keenan also showed no benefit from the addition of nippv to standard medical therapy in patients who develop post - extubation respiratory distress .
thus , in light of the conflicting evidence from this literature review , further large scale studies are a need of the hour to conclusively derive a consensus regarding the usefulness of nippv in weaning copd patients .
the relatively higher mortality in the present study as compared to previously published studies may be explained by the occurrence of vap , as both the patients of group i and all eight patients of group ii who developed vap , subsequently expired .
greater severity of disease at presentation ( as evident from low fev1 % and gcs scores , and severe respiratory acidosis ) may also be responsible for greater mortality among our patients .
vap occurred four times lesser in patients weaned by nippv than those weaned by psv in our study ( 2/25 vs 8/25 , p < 0.05 ) .
in contrast , nava , girault and chen have respectively reported the incidence of vap as 0/25 vs 7/25 , 0/17 vs 2/16 and 0/12 vs 7/12 in the study vs control groups .
lesser occurrence of vap in the nippv arm may be because nippv reduces the duration of exposure to artificial airway , facilitates expectoration and deglutition , thus reducing the chances of aspiration and consequent development of vap
. limitations of our study are that the sample size was relatively small , patients were monitored only during their icu stay , and no data were collected after they were discharged from the icu .
further , the study was conducted in a selected group of severely hypercapneic copd patients , and its success in sicker patients with serious co - morbidities or severe hypoxemia merits further investigation . to conclude , based on the shorter time needed for weaning from mv , duration of ventilation , icu length of stay , incidence of nosocomial pneumonia and reduced mortality
, nippv has been shown in the present study as a better weaning modality in a selected group of mechanically ventilated , hypercapneic copd patients than conventional weaning by psv .
thus , nippv should be tried in resource - limited settings especially in developing countries . however , keeping in mind the conflicting evidences observed from previously published studies , the need for well - designed , multicenter , prospective rcts to arrive at more reliable conclusions regarding the benefits or otherwise of nippv in weaning can not but be overemphasized .
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background : chronic obstructive pulmonary disease ( copd ) patients frequently pose difficulty in weaning from invasive mechanical ventilation ( mv ) .
prolonged invasive ventilation brings along various complications .
non - invasive positive pressure ventilation ( nippv ) is proposed to be a useful weaning modality in such cases.objective:to evaluate the usefulness of nippv in weaning copd patients from invasive mv , and compare it with weaning by conventional pressure support ventilation ( psv).materials and methods : for this prospective randomized controlled study , we included 50 copd patients with type ii respiratory failure requiring initial invasive mv . upon satisfying weaning criteria and failing a t - piece weaning trial
, they were randomized into two groups : group i ( 25 patients ) weaned by nippv , and group ii ( 25 patients ) weaned by conventional psv .
the groups were similar in terms of disease severity , demographic , clinical and biochemical parameters .
they were compared in terms of duration of mv , weaning duration , length of intensive care unit ( icu ) stay , occurrence of nosocomial pneumonia and outcome.results:statistically significant difference was found between the two groups in terms of duration of mv , weaning duration , length of icu stay , occurrence of nosocomial pneumonia and outcome.conclusion:nippv appears to be a promising weaning modality for mechanically ventilated copd patients and should be tried in resource - limited settings especially in developing countries .
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