text
string | predicted_class
string | confidence
float16 |
|---|---|---|
Hospitals have a special importance as the greatest and very expensive operational unit of healthcare and treatment systems. Hospitals use 50-80 percent of all the expenses in the whole healthcare section and have a great contribution of educated and highest level of personnel .
|
other
| 99.94 |
The authority of government places a lot of pressure on the policymaking, execution, and observation of this section and leads the hospital system to turn over some of its administrative activities to the non-governmental section, in order to improve its efficiency . Although, the procedure of variable the expenses and decreasing the resources is always increasing and the gap between the achievable and required resources is developing on a daily basis. What is more, is that the private hospitals, especially in expanding countries, which are directly governed by the state authorities, have weak performances, and efforts to improve their performance were not very efficient by applying internal management modifications . During recent years, Iran has turned over a piece of the healthcare services to the private section aiming to better the modality of healthcare and treatment services, increasing the patients’ satisfaction and decreasing expenses . Outsourcing includes the act of transferring some of the internal activities of an organization to its supplier outside of the organization and transferring the decision making right to the outside of the organization based on a contract. In fact, the outsourcing does not only imply activities but also manufacturing agents (human resources, equipments, facilities, technologies and other assets) and the authority of decision making in most cases is transferred .
|
other
| 99.9 |
Organizations try to turn over the internal affairs of the organization and make their body as small as possible due to various reasons . Alvani believes that benefits resulted by outsourcing are factors such as decreasing expenses, organization’s concentration on its main activities, saving the moment for making the internal affairs of the structure, decreasing the risk by entering in partnership with another module in an unsafe business environment, improving consumer service, decreasing the company’s employees, creating the sense of competition in various sections of the organization . Outsourcing was used to lead to an efficient management of the resources and increasing the modality and consent of various parties. Considering the existence of the facility of outsourcing in so many sections of the hospital, we could benefit from it in governing the hospital and we could evaluate the success rate by defining specific indexes . The patients’ satisfaction is an important scale to evaluate services or received product because satisfied patients are more eager to continue using medical and healthcare services, keeping their contact with the service supplier and following the medical and control regimes . Con Vikticle et al. defined the care quality as the satisfying of physical needs with providing professional care, social-mental support, satisfaction with care and ensuring the presence of general and multi-dimensional cares to the patient . Many expertises consider the patients’ rate of consent from the hospital aims as important indexes of efficiency and aim modality in various sections . It shows that studying the patients’ consent is important outputs of the healthcare systems and evaluating the care by the patient is also one of the major methods to measure and scale the quality of medical and healthcare services . Moreover, Peiravi also mentioned in his research that in Iran, the Ministry of Health has obligated all hospitals to do periodic evaluations regarding the patients’ satisfaction and also required interventions to increase the patients’ satisfaction, since 2011, so that it can comply with its main mission . Considering the mentioned issues about the patients’ satisfaction, we should also point out the important issue that although nowadays companies move toward outsourcing, it represents a part of their liability in the world, so that they can achieve benefits such as decreasing expenses to get hold of advanced technology. Nevertheless, the difference in organizational cultures and complexity of managing created relations means that outsourcing could guide to failure or lack of satisfaction . Therefore, all the above-mentioned issues confirmed that we should select a method that the present services to the patients should work with, having the lowest expense and highest quality, so that the hospital’s expenses could be minimized on one hand and the hospital should be enabled to continue working and present the highest quality services to the patients on the other hand. This way, not only their expenses decrease but they also achieve their satisfaction. Also, this study was executed while aiming to research the influence of outsourcing on the efficiency and affectivity of Radiology Unit services in Hospitals of Tehran City.
|
other
| 99.7 |
This study was an analytical and witness based study, its results being applicative, and its time duration being periodic, during the time period of June until January 2013. The current study was done in the Educational-Medical Hospitals of Tehran. The statistical society included all the patients during a month in the Radiology Unit in both hospitals (N=1200). Considering the fact that a load of people coming to the Radiology Unit was around 1200 individuals in any hospital, the case bulk to measure the patients’ consent rate was calculated to be equal to 291 individuals by means of Morgan table. 300 individuals (n+10) were questioned in each hospital to lower the error percentage and the simple accidental sampling method was used for sampling. The method to gather data for the study of the radiology patients’ consent was the field method and its tool was a questionnaire. This questionnaire consisted twelve asks concerning the condition of the unit, 2 related to the waiting duration. The mentioned questionnaire was scaled via Likert scale from 1 to 5 in such a manner that number 1 was relevant to the lowest level of satisfaction and number 5 was related to the great amount of satisfaction, the maximum point being 60 points. Questions relevant to the waiting time duration were separated to 2 sections. The first question was relevant to the time between the entrance and admission of the sick mans and another question was related to the time between the admission and receiving radiology services. The questionnaire was designed by Medical University lecturers and its admissibility–stability was also confirmed by lecturers and also hospitals clinical governance committee. To confirm the credibility and admissibility, the questionnaire was analyzed and confirmed by 10 experts. What is more, is the reality that to specify the consistency of the questionnaire, Cronbach alpha was applied. After gathering the research data, finally, the gathered data were excavated by SPSS copy 21 and, to excavate data, descriptive statistics was used in redundancy, median, standard deviation tables, and deductive statistics including Kelmogroph–Smirnoph test, Uman–Whitney test and K2.
|
study
| 100.0 |
Findings of the present research indicated that the median of the patients’ consent in the turned over module was equivalent to 41/ 46 (7/ 3±) from the maximum 60 points, equal to (69%) and the median of the patients’ satisfaction in the non-turned over unit was equal to 45 (6/ 94±) from the highest mark of 60 points (75%). This fact indicated a lower satisfaction of pointers in the turned over section in comparison with the general part, and, the minimum medians among all aspects, was relevant to the patients’ consent in turned over Radiology Units, also being related to giving turns to the system with the lowest median of 3.11, satisfaction of the existing comforting equipments in the unit with a median of 3.36, and personnel behavior while getting accepted, 3.41.
|
study
| 99.94 |
In order to determine the norm of distributing information relevant to the patients’ consent the climagraph-Smirnoph exam was applied. By means of this test, it was determined that the generalization mark of the patients’ consent was not normal (p-value=0.000). Also, to compare the median scores in the two units, nonparametric Uman-Vitney test for the patients’ consent score was used. A significant difference was observed between the patients in these two hospitals (p-value=0.000) and since the p-value was so small, it clearly showed the severity of differences among medians. Therefore, it was obvious that the outsourcing had a great impact on the patients’ consent.
|
study
| 100.0 |
Also, the results indicated that most patients were accepted in a time duration of 16 to 20 minutes after entering the unit in turned over units, while in non-turned over units, most of the patients (42%) were accepted in 5 to 10 minutes. Findings also showed that most patients (42%) of turned over units, were X-rayed in a 16-20 minutes time period, after they were settled, while in non-turned over units, most patients (33.3%) were X-rayed 5-10 minutes after they were received. Time duration patients waited for to be received in two units, showed a significant difference based on K2-test (p-value-0.000) and the waiting time in-between, and the X-ray in both units was less significant based on K2-test (p-value -0.000). Since the p-value was so small in both cases, it indicated that the outsourcing had a great influence on the patients’ waiting time duration since they entered, were received, and were X-rayed. Patients’ waiting time duration is presented in Table 1.
|
study
| 100.0 |
Results also indicated that 225 individuals among patients of turned over unit (75%) and 277 individuals between the sick mans of the non-turned over unit (92.3%) category suggested these modules to anthers and this disagreement was significant based on the J2-test (p-value-0.000). Therefore, the outsourcing had a great impact on suggesting the Radiology Unit to other patients.
|
study
| 96.4 |
Findings resulted in this current research, displayed that the median of other sectors authorities’ satisfaction with the turned over unit of 29.26 (5.65±) had the maximum score of 70 points and the median of other sectors authorities’ satisfaction of 28.97 (5.1±) had a maximum score of 70 points. The authorities’ satisfaction with the turned over section and non-turned over section was almost equal. The lowest median observed among items related to the authorities’ satisfaction with a turn over radiology unit was related to question number 3 in relation with on time access to portable radiology devices with the minimum median of (2.72) and accepting suggestions and also applying them by the median of (3.05).
|
study
| 99.25 |
The present research was done aiming to determine the influence source of out on the output and affective of hospitals Radiology Unit. Finding obtained to that the weight media of patients’ satisfaction score in turn over radiology units was (41.46) and in non-turned over was (45), being significant from the difference statistical point of view. Results indicated that the patients’ consent was higher with non-turned over radiology units and therefore the outsourcing had no positive affect on the patients’ satisfaction which was similar to studies of Baderaldin, Amerion and Mohaghegh and in contrast with primarily expectancy of executing outsourcing procedure. The most extended rate of discontent between patients in analyzing the major factors of discontent was the long time duration of waiting . Amerion considered the lack of sufficient explanation in order to get ready for radiology the reason for the minimum rate of satisfaction (7.8%) , while Mohaghegh et al. presented the insufficient time investment of pharmacy employees for consulting as being a reason. Meanwhile, the present research daresay that loss consent in turned over unit was the conclusion of giving the turn system the minimum median of (3.11) and, to confirm this, we could mention the lack of devoting enough work force for recipient part in turned over unit, existence of 5 secretaries in 3 working shifts for the whole unit, which included radiology, MRI, bone accumulation and another aims, led to the increment in work load, and, as a finding, caused problems in the system of giving turns.
|
study
| 99.94 |
The second factor, which had the lowest rate of satisfaction, was relevant to the welfare equipments, which was observed in both turned over and non-turned over units. The mentioned median was equivalent to (3.36) in non-turned over unit and this was more because of loss work force to move the wheelchair, while this median was equivalent to (3.54) in the turned over unit due to the lack of equipments inside the unit.
|
other
| 99.7 |
The third factor that showed the lowest median in analogy with the other factors in turned over units was the individual’s behavior while receiving the patient, with a median equal to (3.41) and such a claim could be justified by the reception of the personnel’s high work load and their exhaustion. Therefore, to fix this problem, not only should we decrease workload but it is also necessary to educate the critical communication skills to turn over units’ personnel.
|
other
| 99.9 |
The results of the present research also indicated that most of the patients (43%) were received in a time duration of 16-20 minutes while the receiving time duration in non-turned over for most of the patients (42%) was equal to 5-10 minutes after entering the unit, which showed a significant statistical difference. Badroldin concluded in a research entitled “Patients’ satisfaction with medical services in educational Saudi Arabia hospital” that the most important factor expressed by patients about their dissatisfaction with medical services was the long time duration of waiting to receive their medications . Moreover, Amerion found out a study entitled “Outpatients and inpatients’ satisfaction of army hospitals” that the largest dissatisfaction (19.2%) in the pharmaceutical sector, was the long waiting time to receive medication, and the minimum rate of dissatisfaction (19.2) in clinic, was relevant to the reality that doctors made patients wait and generally, most of significant reason of dissatisfaction in current study, was related to the waiting time to receive medication (19.2%) .
|
study
| 99.94 |
By the present research, we could consider that increasing the time duration to receive a patient is because of a loss enough workforce in the reception and, increasing the waiting time to be scanned is because of loss of enough workforce in the scanning section. Therefore, obtained results indicated a negative influence of outsourcing on the patients’ waiting time duration.
|
study
| 99.9 |
What is more important is the fact that based on obtained results, 225 individuals among the patients who went to turned over radiology (75%) and 277 individuals among the patients who went to non-turned over radiology (92.3%) recommended these hospitals to others and the upper percent of non-turned over radiology indicated the higher rate of patients’ satisfaction with these types of units.
|
other
| 99.9 |
The present research explained that the rate of patient’s satisfaction with radiology services was decreased and this showed that managers usually turn over services without any consideration related to human and organizational characteristics and dimensions, justifying it by decreasing costs. So, it is necessary and critical for authorities to consider not only the financial aspects but also the individual and human aspects of the procedure while settling an outsourcing contract.
|
other
| 99.9 |
There is a large heterogeneity in the manifestation and response to treatment in preschool wheeze . Early studies of montelukast [11, 17] a leukotriene receptor antagonist, showed it to be effective and is widely prescribed for preschool wheeze across the globe . In addition to the cost of montelukast, some children may suffer from side effects without clinical benefit.
|
review
| 99.9 |
A recent Cochrane review showed montelukast to be ineffective at reducing courses of oral corticosteroid (OCS) in viral-induced wheeze . The European Respiratory Society (ERS) has recently highlighted the overlap of viral-induced and multiple-trigger wheeze . This meta-analysis aims to investigate the effectiveness of montelukast in all wheezing preschool patients, which is a more clinically relevant and ‘real-life’ group.
|
review
| 99.9 |
Children must have been randomised to receive montelukast (compared to placebo), as an intermittent (during episodes of viral upper respiratory tract infections (URTI)) or continuous therapy for 12 months. Studies had to be conducted over a 12-month period to eliminate any seasonal variation.
|
other
| 99.9 |
Trials were identified from Cochrane Library, PubMed, Ovid MEDLINE and Ovid EMBASE databases by two independent reviewers (HH, CB). Keywords were a combination of free texts and MeSH subject headings (Online supplement). The search strategy included filters to limit the results by the study type (RCTs only) and subject age range: infant (0–23 months) and preschool (2–5 years). No date limits were applied. No language restriction was applied. The bibliography of eligible trials was searched for relevant papers. The most recent search was conducted in April 2017. The process of study selection is shown in Fig. 1.Fig. 1Flow chart for selection of studies
|
review
| 99.7 |
The risk of bias and the methodological quality of each study were assessed according to the Cochrane Collaboration’s tool. The trials have been evaluated for the presence of risk of bias in terms of allocation of randomisation sequence, concealment of allocation, blinding, handling of incomplete outcome data (online supplement), selective reporting bias and other sources of bias. The trials were of high methodological quality; therefore, the risk of bias among the studies was low. Summary assessment of the six key domains of risk of bias is presented in Fig. 2. The study by Bacharier et al. was supported by the National Heart, Lung and Blood Institute (NHLBI). The WAIT study was supported by the Medical Research Council and the National Institute for Health Research. Commercial sponsors provided the drugs and placebo, but all the final decisions were made by the NHLBI. Studies performed by Valovirta and Bisgaard et al. were sponsored by Merck & Co. Inc. The study by Robertson et al. was sponsored by Merck Sharp and Dohme (Australia) Pty. Ltd.Fig. 2Methodological quality summary: review authors’ judgments about each methodological quality item for each included study
|
review
| 99.9 |
Data for the selected outcomes were extracted and entered into Review Manager Software (version 5.3). Data were expressed as a weighted mean difference (MD) and 95% CI. Fixed-effect (FE) model was used to pool the data. Whenever there was a heterogeneity, random-effect (RE) model was applied. Standard deviations (SD), if they were not reported, were calculated from means and 95% CI. The study results were combined depending on the method of prescribing montelukast (intermittent or continuous). In one trial, preschool and school-age children were included ; we used the preschool data only.
|
study
| 99.94 |
One hundred sixteen records were identified from all databases. After completion of the study selection process, five studies (n = 3960) met the inclusion criteria (Table 1) [2, 4, 13, 15, 18]. All studies were conducted in high-income countries except one study with centres from Africa, South America and the Middle East. Montelukast was given intermittently in four studies [2, 13, 15, 18]. Intermittent montelukast therapy was started by parents/caregivers, as a chewable tablet or oral granules (4 or 5 mg) over 12 months. In one study, in addition to episode-driven montelukast, daily montelukast therapy was also investigated .Table 1Characteristics of included studiesAuthorNOutcomesSide effectsNotes/biasNwokoro et al. Mk, 669PBO, 677P: USMAS: Number and duration of WE, duration of hospital stay, number of OCS courses, time to first USMA, symptomatic daysNone● Intermittent use ● ALOX5 (5/5 and 5/x + x/x) strata● 71.5% EVW● 21 primary and 41 secondary care sites in the UKBacharier et al. MK (4 mg), 95 BIS (1 mg), 96PBO, 47P: Proportion of EFDsS: Symptom score, caregiver QOL, numbers and time to first OCS course, number of WEs, number of USMA, linear growth, days missed from day care and parental workN/A●Intermittent use● 2:1 randomisation● More dropouts in Mk group● 5 clinical centres in the USABisgaard et al. Mk, 278PBO, 271P: Number of AEES: Number of oral and ICS courses, duration of episodes, percentage of days without asthma, severity of AEE, blood eosinophil count, proportion of patients with an AEE, time to first AEE, asthma-related resource utilisation1 case of vomiting due to Mk overdose● Continuous use● Subgroup analysis based on atopic profile and blood eosinophil count● 68 sites in 23 countriesRobertson et al. Mk, 107 PBO, 113P: USMAS: Individual components of USMA, duration of episode, symptom score, O CS and β-agonist use, days missed from parental work and school or childcare, number of nights with disturbed sleepNone● Intermittent use● More children with history of atopy in Mk group● Country: AustraliaValovirta et al. Daily Mk, 58912-day Mk, 591PBO, 591P: Number of asthma episodes culminating in asthma attacksS: Symptom score, number of asthma attacks and episodes, percentage of EFDs, difference in efficacy between 2 regimens1 case of somnolence due to Mk overdose● Intermittent and continuous use● Double dummy● 111 multinational sitesAll double-blind randomised placebo-controlled trial Mk montelukast, PBO placebo, P primary, S secondary, USMA unscheduled medical attendance, WE wheezing episode, CS corticosteroid, EVS episodic viral wheeze, MTW multiple-trigger wheeze, ALOX5 arachidonate 5-lipoxygenase, BID budesonide inhalation suspension, EFD episode-free day, QOL quality of life, AEE asthma exacerbation episodes, ICS inhaled corticosteroid
|
review
| 99.75 |
Mk montelukast, PBO placebo, P primary, S secondary, USMA unscheduled medical attendance, WE wheezing episode, CS corticosteroid, EVS episodic viral wheeze, MTW multiple-trigger wheeze, ALOX5 arachidonate 5-lipoxygenase, BID budesonide inhalation suspension, EFD episode-free day, QOL quality of life, AEE asthma exacerbation episodes, ICS inhaled corticosteroid
|
other
| 99.94 |
The number of wheezing episodes was described in three studies [2, 13, 18]. The final study reported the number of wheezing episodes whereby a child was seen by a healthcare professional. Trials showed no effects of montelukast in preventing episodes of wheeze. The pooled estimate showed no statistically significant difference (MD = 0.07, 95% CI −0.14 to 0.29, mean for montelukast 2.68 vs placebo 2.54 (p = 0.5)). The figure shows no heterogeneity between the studies (Fig. 3a, p = 0.79, I 2 statistic = 0%).Fig. 3Intermittent montelukast vs placebo. a Numbers of wheezing episodes. b Unscheduled medical attendances. c Number of oral corticosteroid courses
|
study
| 99.56 |
All studies which used montelukast intermittently reported USMA [2, 4, 15, 18]. The overall effect was not statistically significant (MD = −0.13, 95% CI −0.33 to 0.07, mean for montelukast 1.62 vs placebo 1.78 (p = 0.21)). There was a low level of heterogeneity (Fig. 3b, p = 0.30, I 2 statistic 18%).
|
study
| 99.94 |
The effect of montelukast on the use of OCS was described in three trials [2, 13, 15]. In one trial , only the percentage of episodes that required OCS was presented (21% montelukast vs 24% placebo). Thus, the results could not be entered into the meta-analysis. This trial reported no significant difference between montelukast and placebo in reducing the number of OCS courses (OR, 0.80; 95% CI, 0.56 to 1.15; mean for montelukast 0.35 vs placebo 0.36 (p = 0.25)) .
|
study
| 95.8 |
The pooled estimate of the other two studies [2, 13] showed that montelukast did not significantly reduce the number of OCS courses (MD = −0.06, 95% CI −0.15 to 0.02, p = 0.14). There was no heterogeneity between the trials (Fig. 3c, p = 0.51, I 2 statistic 0.0%).
|
study
| 99.94 |
Only 2 of the 5 included studies investigated regular continuous montelukast (n = 1691) [4, 18]. The pooled estimate comparing the number of wheezing episodes was not statistically significant (MD = −0.40, 95% CI −1.00 to 0.19, mean for montelukast 2.05 vs placebo 2.37 (p = 0.18)); analysis showed that there was a substantial heterogeneity between the included studies (Fig. 4a, p = 0.04, I 2 statistic = 77%).Fig. 4Continuous montelukast vs placebo. a Number of wheezing episodes. b Unscheduled medical attendances . c Number of oral corticosteroid courses
|
study
| 99.94 |
One study described no statistically significant difference in the number of USMA between montelukast and placebo (Fig. 4b, MD = −0.04, 95% CI −0.26 to 0.18). The other showed no statistical significance in the number of patients presenting with at least one USMA (MD = −0.11, 95% CI −0.36 to 0.14). As outcome measures differed, the data could not be pooled.
|
study
| 99.56 |
There was no significant difference in the incidence of adverse events between the placebo and montelukast. Two participants were suffered from somnolence and vomiting due to the montelukast overdose [4, 18]. The trials indicated that adverse events related to the intervention rare and montelukast was safe in preschool children.
|
review
| 96.56 |
This systematic review of pooled data shows no evidence of benefit of the use of intermittent or continuous montelukast on the number of wheezing episodes, USMA or OCS use for recurrent wheeze in preschool children. However, there may be subgroups of children with preschool wheeze who do respond to montelukast, but there were insufficient data to determine specific phenotypes of responders, apart from one study which did suggest a genotype which was linked with greater response.
|
review
| 99.9 |
The clinical question is an important one. The use of montelukast in preschool wheeze is recommended in the British Thoracic Society (BTS) guidelines ; it is widely used across the world and should only be recommended if it is actually helpful. The added value of this study compared to the recent Cochrane review is the inclusion of all types of preschool wheeze; therefore, more akin to real life, it therefore makes the clinical question more relevant and clinically applicable. Our primary outcome measure was also very clinically relevant, being number of wheezy episodes.
|
review
| 99.7 |
The recent Cochrane review used courses of OCS as a primary outcome measure; however, evidence points to the ineffectiveness of OCS in preschool wheeze. Therefore, by using this as an outcome measure, subtle changes in outcome may not be picked up, due to inconsistencies of prescribing across healthcare professionals. The use of corticosteroids in this group of children may vary from clinician to clinician, whereas the frequency of wheezing episodes is more accurate and clinically applicable.
|
review
| 99.9 |
One of the disadvantages of our analysis was that there was some heterogeneity in the outcome set in the studies chosen for review; thus, some outcomes which we did not select were unable to be included within the pooled analysis. For example, in the study by Bisgaard et al. , we could not compare USMAs, because in this study, they compared the proportion of patients who had at least one USMA rather than the number of unscheduled visits (37% for montelukast; 42% for placebo).
|
review
| 99.9 |
A further disadvantage was that although the entry criteria of the primary studies were generally similar, there were some differences in the baseline characteristics of the populations of the studies. Analysis showed a significant heterogeneity between the two studies used in the meta-analysis of those given continuous montelukast (p = 0.04, I 2 statistic = 77%). The study performed by Bisgaard et al. included patients with quite mild symptoms of asthma, compared to those included in the study by Valovirta et al. . In the latter study, the patients had moderate-severe asthma (intermittent symptoms as well as one course of OCS or hospitalisation in the previous year) . There was another study which analysed relatively mild asthma .
|
review
| 99.7 |
The fact that our meta-analysis looked at preschool wheeze altogether, without separation for viral-induced or multiple-trigger wheeze, is a great strength of the current analysis compared to other meta-analyses . A recent international consensus report disputes the use of such terms in preschool wheeze , as it seems that there is huge overlap between these phenotypes as well as change over time; thus, separating patients in this way may not be clinically relevant. This makes our study more clinically applicable to the general population.
|
study
| 99.94 |
A further advantage was the strict criteria for selection meant that only those studies with the highest quality were included, and there was subsequently little bias or doubt of the validity of the data. However, with such stringent criteria set, some studies were excluded which may have been useful. This includes a number of studies which did not span 12 months. The reason for excluding such studies was to eliminate any seasonal variation. One excluded large multicentre multinational RCT which enrolled over 600 children, given montelukast or placebo for 12 weeks, showed improvement in episode-free days, symptoms, use of OCS, β-agonist use and serum eosinophil counts. The population studied seemed to be >50% atopic and many suffered from daily symptoms, maybe more akin to the previous term of multiple-trigger wheeze. It is important, however, to note that this particular study was conducted by the pharmaceutical company marketing montelukast.
|
review
| 99.8 |
In all primary studies, recording of the symptoms and initiating of the intervention in the intermittent montelukast studies were carried out by parents/caregivers. Although in all the trials, the parents were contacted either through telephone or visits, only one study provided an educational program to the parents on recognising symptoms which were more likely to represent respiratory tract infection and followed by wheezing . It is possible that the initiation of treatment was too long after the onset of symptoms, causing stimulation of the immune response by the virus and thus failure of the montelukast therapy .
|
review
| 99.44 |
Some of the trials included children aged 6–24 months, which could incorporate some patients with post bronchiolitis wheeze [2, 13, 18]. This may have led to negative findings, because a Cochrane review reported that montelukast was not effective in reducing the incidence of recurrent wheezing, symptom-free days or relevant usage of corticosteroid in patients with post-bronchiolitis wheezing . Interestingly, two included trials that excluded children younger than 2 years showed significant improvements in montelukast group compared with placebo group [4, 15]. Subgroup analysis showed better outcomes for children 2 years of age and older (p = 0.017) . It is thus possible that there may have been some exaggeration of negative results due to the inclusion of children less than 2 years of age.
|
review
| 99.9 |
We wanted to perform subgroup analysis to check which patients were ‘montelukast responders’, especially checking if serum eosinophil or atopy predicted response. Unfortunately, there were insufficient data available to analyse this. Nwokoro et al. did perform subgroup analysis and found that those patients with ALOX5 5/5 genotype had less USMA on montelukast compared with placebo, but there was some overlap between the groups. Repeatability in further large RCTs of such results would need to be performed for this to be convincing.
|
study
| 99.56 |
Since this meta-analysis has been performed, a further meta-analysis has been performed comparing the effectiveness of continuous and intermittent high-dose inhaled corticosteroid (ICS), with placebo and montelukast at preventing a severe exacerbation in preschool wheeze . Continuous ICS and intermittent ICS were both found to be effective but were also found to be significantly more effective than montelukast at preventing a severe attack . This meta-analysis illustrated the strong and consistent evidence that ICS is effective in preschool wheeze. This paper, as well as our findings, may influence a change in protocol for the treatment of preschool wheeze. There have also been some early data to suggest that azithromycin therapy in severe preschool wheeze may prevent a severe exacerbation [3, 19], but more work in this area is required, due to the increase in bacterial resistance following such medication.
|
review
| 99.9 |
This meta-analysis shows that, compared with placebo, 12 months of intermittent or continuous montelukast was not associated with significant reduction in the frequency of wheezing episodes, USMA or need for OCS use. This may call into question the BTS recommendations for the use montelukast in preschool wheeze.
|
review
| 98.94 |
No benefit was seen with montelukast for preschool wheeze from the limited well-conducted RCTs over at least 12 months in preschool children with recurrent wheeze. Future trials should be adequately powered for the predefined subgroup analysis to identify the subgroup of children most likely to exhibit a beneficial treatment response to montelukast.
|
review
| 57.44 |
Among all brain cancers arising from transformed glial cells, grade IV glioblastoma (GBM), as defined by the World Health Organization, is the most prevalent and aggressive . The diffusely invasive nature of these tumors precludes their complete surgical resection, which inevitably leads to tumor recurrence and patient death . Glioblastoma cells migrate onto normal brain microvessels for invasion and tumor growth . They communicate directly with surrounding normal cells such as astrocytes, glia and endothelial cells, through the formation of gap junctions. These cell-to-cell interactions modify astrocyte phenotype [4–6] and promote tumor angiogenesis and tumor growth . Therefore, gap junctions and their signaling are proposed as potential therapeutic targets in these patients.
|
review
| 99.8 |
Gap junctions are specific cell-to-cell channels formed by membrane proteins called connexins (Cx). Connexin43 (Cx43) is the major connexin expressed in human microvascular endothelial cells (HMEC), astrocytes and glioblastoma cells. Cx43 is specifically upregulated in the reactive astrocytes surrounding glioblastoma , suggesting that gap junctions at the tumor margins are involved in tumor cell invasion [4, 6, 8, 9] and tumor growth . The precise role of gap junctions remains poorly understood. Nevertheless, microRNAs (miRs) were observed to be exchanged between glioblastoma cells and normal astrocytes through gap junctions. MiRs are small non-coding RNAs that modulate gene expression by affecting the translation of messenger RNAs (mRNAs) into proteins and inducing target mRNA decay [12–14]. A miR is single-stranded and ~21nucleotides long, forming a linear molecule with a diameter of ~1.0 nm, which is in the same order of the gap junction channel pore size [15, 16]. We have recently reported that miR-145-5p, which reduces glioma growth , could be exchanged between HMEC and colon cancer cells through gap junctions formed by Cx43 .
|
study
| 100.0 |
Here, we examined whether the formation of gap junctions could permit the exchange of specific miRs between glioblastoma and microvascular cells, and how this transfer could influence the endothelial cell function in vitro (i.e., leading to angiogenesis). We used the U87 human glioblastoma cell line and HMEC, and focused on two human mature miRs, namely miR-145-5p which is expressed in HMEC but not in U87 , and miR-5096 which is expressed in U87 but not reported in HMEC. We demonstrate an exchange of these two miRs between the two cell types through the same gap junction pathway. The effects of miR-5096, whose transfer is transiently mediated by gap junctions, starts at earliest stages of cell-cell contact and may be amplified by signal spread among HMEC. Our results reveal that glioblastoma cells modify the behavior of microvascular cells through miR transfer.
|
study
| 100.0 |
We first explored the basal expression level of miR-145-5p and miR-5096 in HMEC and U87 cells, in homotypic cultures. We observed that miR-145-5p was almost exclusively expressed in HMEC and could not be detected at a significant level in U87 (Figure 1A). In contrast, miR-5096 was mainly expressed in U87 and poorly detected in HMEC (Figure 1B). We subsequently labelled U87 with the cell tracker DiL-C18 , and co-cultured these cells with HMEC before flow cytometry sorting. As an additional control, the expression levels of these miRs were measured in U87 and HMEC, sorted immediately after being mixed, and were similar to that measured in cells cultured separately, i.e. miR-145-5p and miR-5096 remained poorly detected in U87 and HMEC, respectively (not shown). After 12 hours of co-culture, mir145-5p expression level was increased in both U87 (by 40%) and HMEC (by 20%) (Figure 1A). At the same time, miR-5096 expression level was significantly increased in HMEC while decreased by 20% in U87 (Figure 1B). These observations led us to explore miR exchanges between these two cell types.
|
study
| 100.0 |
A, B. Expression level of miR-145-5p and miR-5096 was measured by qPCR in HMEC (black) and U87 (dashed), cultured separately (left) or co-cultured (right) for 12 h (means ± SD; *P<0.05; n = 3). Levels of miR-145 or −5096 were measured relative to levels of U6 snRNA, as an internal control. For co-cultures, U87 were labelled with the fluorescent dye DiL-C18 (red cells), plated with unlabeled HMEC at a 1:1 ratio, and sorted by flow cytometry before analysis.
|
study
| 100.0 |
To determine whether miR-145-5p was transferred from endothelial to cancer cells, we transfected HMEC with a miR-145-5p mimic (30 nM) before culturing them with DiL-C18-labelled U87 (ratio 1:1) for 12 hours. The two cell types were subsequently sorted by flow cytometry and miR145-5p expression was measured in each population. Both HMEC and U87 expressed high levels of miR-145-5p (Figure 2A). To evaluate the contribution of cell-to-cell contact in this transfer, we cultured U87 with miR-145-5p-transfected HMEC in transwell plates to prevent any cell-cell contact. In these non-contact conditions, we failed to detect any increase in miR-145-5p expression level in U87 (Figure 2B). These results indicate that U87 do not ingest extracellular miR-145-5p, either free or incorporated into soluble exosomes .
|
study
| 100.0 |
Donor cells were loaded or not (E) with miR-mimic (M; 30 nM, hatched) or miR-inhibitor (I; 30 nM, white). The miR levels were determined by qPCR in donors (left) and acceptors (right), after 12 h of co-culture, and measured relative to U6 snRNA. Values are means ± SD of triplicate measurements from three experiments; *P<0.05 vs empty (Mann-Whitney U test and Kruskal-Wallis test; n = 3). A. Transfer of miR-145 from HMEC to U87. B. Abolition of miR-145 transfer to U87 when cells are co-cultured in transwell plates (non-contact). C. Transfer of miR-5096 from U87 to HMEC. D. Inhibition of miR-5096 transfer to HMEC by carbenoxolone (100 μM), a gap junction blocker. Note that carbenoxolone does not affect miR-5096 level in transfected U87.
|
study
| 100.0 |
We performed the same experiment by transfecting U87 with miR-5096 mimic (30 nM) before culturing them with DiL-C18-labelled HMEC (ratio 1:1) for 12 hours. High levels of miR-5096 were also detected both in U87 and HMEC (Figure 2C, 2D). No increase in miR-5096 expression level in HMEC was observed in non-contact co-cultures with U87 (not shown). To evaluate the contribution of gap junctions to miR-5096 transfer from glioblastoma cells to HMEC, co-culture was made in the presence of carbenoxolone, in order to block the gap junction intercellular communication (GJIC) [11, 20]. Clearly, inhibition of GJIC prevented the miR-5096 increase in HMEC (Figure 2D), i.e.- the miR-5096 expression in HMEC remained very low in the presence of carbenoxolone (0.761±0.4 × 10-4) as compared to cells cultured alone (0.801±0.4 × 10-4, n=3; P>0.5). Because Cx43 is mostly involved in GJIC between HMEC and U87 , we knocked down Cx43 expression in U87 by using specific siRNA (see supplementary Figure S3A). The down regulation of Cx43 in U87 prevented the transfer of miR-5096 to HMEC (see supplementary Figure S3C). It is to note that carbenoxolone also prevented the transfer of miR-145-5p from HMEC to U87; the miR-145 expression levels in U87 were 2.2742±0.43 and 2.21±0.1 × 10-4, respectively in the absence and in the presence of carbenoxolone (means ± SD; P<0.05; n = 3), i.e. a miR-145 level similar to homotypic U87 culture (2.601±0.1 × 10-4, n=3; P>0.5). Thus, the two miRs were exchanged through the same pathway.
|
study
| 100.0 |
Transfected U87 were double loaded with calcein, a dye that passes through gap junctions, and DiL, a membrane-bound dye (Figure 3A; ). Labelled U87 were plated onto HMEC monolayers to which they rapidly adhered. The calcein transfer to HMEC was then measured, attesting the formation of heterocellular GJIC. When U87 were transfected with a miR-5096 mimic, calcein transfer was significantly increased within 5 hours. The gap junction blocker, carbenoxolone, did not affect cancer cell adhesion to endothelial cells (Figure 3A), but abolished calcein transfer from U87 to HMEC (Figure 3B). A similar result was obtained by inhibiting miR-5096 expression in glioblastoma cells. These results suggest that miR-5096 itself favors the communication between cancer and endothelial cells.
|
study
| 100.0 |
A. Functional GJIC between U87 and HMEC. Donor U87, transfected or not (Empty) with miR-5096-mimic (30 nM) or miR-5096-inhibitor (30 nM), were loaded with calcein and labelled with DiL-C18. Calcein diffuses through gap junctions, while DiL-C18 does not. These cells were then plated on unlabeled HMEC monolayer (acceptor). After 5 h of co-culture, HMEC establishing GJIC with U87 become fluorescent by calcein diffusion. Carbenoxolone (100 μM) prevented it without affecting the cell-to-cell adhesion. Dotted areas are enlarged in the right inserts (representative of 3 experiments; Bar 80 μm). B. Histogram shows the cell number of HMEC receiving dye (calcein) per U87 (mean ±SD; **P<0.01 vs control, n = 3). Carb.: 100 μM carbenoxolone. C. Immunoblot analysis of Cx43 protein in HMEC (left) and U87 (right) whole cell lysates after 12 h of co-culture. P0, P1 and P2 denote the three major Cx43 migration bands. One representative of 3 independent experiments is shown (β-actin as loading control). D. Histogram shows changes in all band intensity related to the total Cx43 expression level in the whole cell lysates (mean ± SD; *P<0.05 vs empty; Mann-Whitney U test and Kruskal-Wallis test; n = 3).
|
study
| 100.0 |
We analyzed the effect of miR-5096 on Cx43 expression in the two cell types (Figure 3C). Transfection of U87 with a miR-5096 mimic induced a 2-fold increase in Cx43 expression in U87, without modifying Cx43 expression in co-cultured HMEC for 12 h (Figure 3D). This is in agreement with the large transfer of miR-5096 from U87 to HMEC within 12 hours of co-culture (Figure 2C, 2D) and its inhibition by decreasing Cx43 expression using siRNA in U87 (see supplementary Figure S3C).
|
study
| 100.0 |
We subsequently used an in vitro matrigel tube formation assay to explore if miR-5096 could modulate the ability of HMEC to form capillary-like structures . Co-culture of U87 and HMEC initiated the formation of typical capillary-like structures within 5 hours (Figure 4A). When miR-5096 mimic-loaded U87 were co-cultured with HMEC, the formation of a capillary-like network at 5 hours was increased (Figure 4A, 4B). Such an effect was not observed when U87 were loaded with a miR-5096 specific inhibitor. Thus, miR-5096 expressed by glioblastoma cells could act as a proangiogenic factor, i.e., through increasing tubulogenesis (Figure 4B). The opposite effect was observed when miR-145-5p mimic-transfected HMEC were co-cultured with U87, as this transfection inhibited the formation of capillary-like structures (Figure 4B, see supplementary Figure S1). Altogether, these results suggest that, by modulating the tumor associated capillary-like network, the transfer of miR-145-5p from endothelial to cancer cells may decrease tumor growth whereas the transfer of miR-5096 from cancer to endothelial cells may have opposite effects by promoting angiogenesis.
|
study
| 100.0 |
A. In vitro tubulogenesis assay of HMEC plated on Matrigel with donor U87, loaded or not (empty) with miR-5096 mimic (30 nM) or inhibitor (30 nM), just after plating (upper panel) or after 5 h of co-culture. Representative micro-photographs of endothelial tube formation (Bar 80 μm; n=3 experiments triplicate). B. Comparative effects of miR-145 and miR-5096. Histogram shows the number of branch points per field of view (at least 80 single cells were scored). Donor HMEC were loaded or not (empty; E) with miR-145 mimic (M; 30 nM) or inhibitor (I; 30 nM), and co-cultured with U87 for 5 h (left). Acceptor HMEC were co-cultured with donor U87 as described in panel A (mean ± SD; **P<0.01 vs empty; Mann-Whitney U test and Kruskal-Wallis test; n = 3). C. miR-5096 did not affect the proliferation of HMEC (black) or U87 (dashed), when loaded or not (empty) with miR-5096 mimic (30 nM) or inhibitor (30 nM), and plated separately for 24 h in homotypic culture conditions (T0 initial time point of experiment; mean ± SD; P>0.05 vs empty; Mann-Whitney U test and Kruskal-Wallis test; n = 3). D. miR-5096 did not affect the VEGF release by loaded cells. Same experimental conditions in panel C (mean ± SD; P>0.05 vs C non-transfected cells; Mann-Whitney U test and Kruskal-Wallis test; n = 3).
|
study
| 100.0 |
As previously reported , miR-145-5p mimic also decreased U87 cell proliferation (not shown). Conversely, neither cell loading with a miR-5096 mimic nor a miR-5096 inhibitor did affect the proliferation of U87 and HMEC (Figure 4C). The VEGF soluble protein release was not influenced by miR-5096 (Figure 4D).
|
study
| 100.0 |
To determine whether miR-5096 effects are stable with time, its cell expression level was determined two days after cell loading. More precisely, donor U87 were plated 2 days after transfection with acceptor HMEC for 12 h (ratio 1:1). As shown in Figure 5A, miR-5096 expression level in U87 was lower after 2 days in culture than within the first day (by 30 %; see Figure 2C). Its expression level in HMEC was also decreased (by 70% of its value measured within the first day; see Figure 2D). Thus, after 2 days, U87 were still capable to transfer miR-5096 to co-cultured HMEC but to a lesser extent.
|
study
| 100.0 |
A. miR-5096 transfer from U87 to HMEC, two days after loading. Donor U87, either non-transfected (E) or transfected with mimic (M; 30 nM) or inhibitor (I; 30 nM), were cultured alone for 48 h, then co-cultured with acceptor HMEC (right panel) for 12 h. The miR-5096 levels, relative to U6 snRNA, are means ± SD (*P<0.05 vs empty; Mann-Whitney U test and Kruskal-Wallis test; n = 3). B. Blockage of functional GJIC between U87 and HMEC by miR-5096. Transfected U87 were cultured alone for 48 h, then loaded with calcein and DiL-C18. Labelled U87 (donor) were plated onto HMEC monolayer (acceptor) as described in Fig. 3A. Phase-contrast microphotographs after 5 h of co-culture (representative of 3 experiments; Bar 80 μm). Histogram shows the cell number of HMEC receiving dye (calcein) per U87 (mean ±SD, n=3; **P<0.01 vs control). C. Down-regulation of Cx43 expression in U87 by miR-5096. Immunoblot analysis of Cx43 in whole-cell lysates from transfected U87 (right), cultured alone for 48 h then co-cultured with HMEC for 12h (representative of 3 independent experiments; β-actin as loading control). Histogram shows the miR-5096-mediated down-regulation of Cx43 in U87 and the up-regulation of Cx43 in co-cultured HMEC (mean ± SD; *P<0.05 vs empty; Mann-Whitney U test and Kruskal-Wallis test; n = 3). D. miR-5096 mediated tubulogenesis. Representative micro-photographs of HMEC plated on Matrigel for 5 h with U87 previously loaded with mimic or inhibitor (Bar 80 μm). Histogram showing the number of branch points per field (mean ± SD; **P<0.01 vs empty; Kruskal-Wallis test; n = 3).
|
study
| 100.0 |
However, U87 loaded with miR-5096 mimic did not transfer calcein to HMEC monolayers (Figure 5B). Conversely, a large calcein transfer to HMEC, attesting the formation of heterocellular GJIC, was observed by inhibiting miR-5096 in U87. The GJIC capacity was increased by 4 fold with the miR-5096 inhibitor and was completely abolished with miR-5096 mimic (Figure 5B, right panel). We performed the same experiment by transfecting HMEC with miR-5096 mimic and inhibitor. Two days after transfection, loaded HMEC were plated onto HMEC monolayers and the calcein transfer was observed (see Supplementary Figure 2). Neither miR-5096 mimic nor inhibitor affected the GJIC established between transfected and non-transfected HMEC.
|
study
| 100.0 |
Since functional GJIC required Cx43 expression, we performed immunoblot analyses of whole-cell extracts in co-cultures, 2 days after loading (Figure 5C). Strikingly, miR-5096 mimic down-regulated Cx43 expression in transfected U87, while it up-regulated Cx43 expression in HMEC. The immunofluorescent labelling of Cx43 in U87 further demonstrated the time-dependent decrease in heterocellular GJIC (see supplementary Figure S4). These results suggest that miR-5096 modulates Cx43 expression and GJIC, in a time- and cell type-dependent manner. In spite of the decrease in Cx43 expression, U87 loaded with miR-5096 mimic were still able to increase the formation of a capillary-like network at 5 hours (Figure 5D).
|
study
| 100.0 |
Accumulated evidence indicate that non-coding microRNAs (miRs) have multiple effects on gene regulation during tumor progression [14, 21]. Here, we demonstrate the ability of gap junctions to drive miRs exchange between HMEC and the U87 human glioblastoma cell line and to modulate the behavior of target cells. First, miR-145, which is downregulated in early stages of glioma progression and behaves as a tumor suppressor , can migrate from endothelial to tumor cells and function as an “antiangiogenic” signal. Second, miR-5096, which is upregulated in glioma (as compared to normal brain tissues) and promotes glioma invasion , can migrate from tumor cells to endothelial cells in which it functions as a “proangiogenic” signal. The cell-to-cell transfer of these miRs is inhibited by the loss of contact between HMEC and U87 cells and by the presence of the GJIC blocker, carbenoxolone, indicating that miR-145 and miR-5096 use the same intercellular transfer pathway (GJIC) to mediate their opposite effects on angiogenesis.
|
study
| 99.94 |
The observation that miR-145 can be transferred from HMECs to U87 enforces our previous demonstration that functional gap junctions between colon carcinoma cells and HMEC could permit the transfer of miR-145-5p from cell to cell . Re-expression of miR-145 in U87 can inhibit glioma cells proliferation, invasion and angiogenesis in vitro and reduce glioma growth in vivo . This effect of miR-145 was initially related to its ability to decrease vascular endothelial growth factor (VEGF) expression levels . Actually, VEGF expression level also decreases when miR-145 expression is downregulated . Moreover, perivascular invasion by glioma cells increases when VEGF synthesis is deficient and when brain tumor xenografts are treated with anti-VEGF blocking antibodies [24, 25]. In patients GBMs that are resistant to bevacizumab therapy, demonstrated a tendency toward perivascular invasion . Blocking angiogenic signaling using antibodies targeting the VEGF-A axis failed to curb progressive tumor growth and meaningfully extend patient survival, suggesting that both perivascular brain tumor growth and invasion use a VEGF-independent mechanism of tumor vascularization . Conversely, we here demonstrate an angiogenic effect of miR-5096, which is independent of any change in VEGF release from U87 cells. Also, overexpression and downregulation of miR-5096 did not affect the proliferation of either U87 or HMEC in vitro. Altogether, the transfer of miR via functional gap junctions between tumor cells and endothelial cells may modulate the behavior of tumor cells and surrounding normal tissues and subsequently change tumor growth characteristics [4, 6, 14].
|
study
| 99.94 |
We also demonstrate that miR-5096 transfer via gap junctions is time-dependent. Both the GJIC and underlying Cx43 expression were transiently increased in transfected U87. After a few days in culture, gap junctions between U87 and HMEC have lost their functionality but miR-5096 is still transferred from U87 to HMEC, although less efficiently, suggesting that another still unidentified pathway is activated [27, 28]. Conversely, transfection of miR-5096 into HMEC does not affect the homocellular GJIC in HMEC monolayers (see Supplementary Figure S2), indicating that overexpression of miR differentially regulates Cx43 expression in HMEC and U87 [14, 29, 30].
|
study
| 100.0 |
Altogether, our results highlight a complex dialog between brain blood vessels and perivascular glioma cells that ends in invasion in a VEGF-independent manner. Future investigation will indicate if the manipulation of miR exchanges between these cells deserves to be developed as an alternative anti-GBM therapy.
|
study
| 99.94 |
Mouse monoclonal anti-Cx43 (610062) was purchased from BD Transduction Laboratories (Lexington, KY). Mouse anti-Hsc70 and anti-β-actin were from Santa Cruz Biotech. Vybrant cell labeling solution DiL-C18 was from Molecular Probes (Invitrogen; Life Technologies, Saint-Aubin, Fr). Rabbit polyclonal anti-HIF-1α (ab2185) was from Abcam (Paris, Fr). GW4869 was purchased from Calbiochem (Merck Chimie SAS, Fontenay-sous-Bois, Fr). Other chemicals were from Sigma-Aldrich.
|
other
| 99.94 |
Human hsa-miR-145-5p mimics (mirVana TM miRNA mimic, 4464066-MC11480), hsa-miR-145-5p inhibitors (mirVana TM miRNA mimic, 4464084-MH11480), hsa-miR-5096 mimics (mirVana TM miRNA mimic, 4464066-MC22429) and hsa-miR-5096 inhibitors (mirVana TM miRNA mimic, 4464084-MH22429) were purchased from Ambion (Invitrogen). Silencing RNA (siRNA) targeting the human Cx43 gene was purchased from Santa Cruz Biotech (GJA1_human mapping 6q22.31; Clinisciences; Nanterre, Fr) and control siRNA was from Dharmacon (ThermoFischer, Saint-Remy-les-Chevreuses, Fr). Cells were transfected by lipofectamine RNAiMAX according to the manufacturer's protocol (Invitrogen; Life Technologies).
|
study
| 99.9 |
Acceptor cells were labelled with DiL-C18, then washed and mixed with unlabeled cells (donors) in a ratio of 1:1. After co-culture, donors and acceptors were separated by flow cytometry based on the fluorescence dye. Cell sorts were carried out twice to guarantee 100% purity.
|
study
| 99.9 |
Total RNA was isolated using TRIzol reagent (Invitrogen). Expression of miR-145 was determined using TaqMan miRNA assay (Invitrogen) according the manufacturer's protocols. Level of miR-145 was expressed relative to the level of U6 snRNA (Ambion, 4427975-001973), used as internal control for each measurement. Relative values thus obtained were averaged.
|
study
| 99.94 |
Cells were trypsinized and resuspended in ECM gel with DMEM according to the manufacturer's instructions (Cell Biolabs, Inc) . Each well is duplicated for each experiment, and each experiment was repeated three times. For short term assays (after 4 hours of incubation at 37°C), 80 single HMEC cells were scored for the number of processes per cell. Cells were photographed at a magnification of x10 using Zeiss microscope, equipped with a video camera.
|
study
| 99.94 |
For collection of conditioned media (CM), cells were grown 6 h in FCS-free DMEM then fresh medium (3 ml/T-25 flask) was added for 12 h before to be collected. The quantitative determinations of human VEGF concentrations in CM were made by enzyme-linked immunosorbent assays (ELISAs; Quantikine; R&D Systems), according to the manufacturer's instructions.
|
study
| 99.94 |
Results are expressed as mean ± SD. Groups were compared using one-way analysis of variance (ANOVA; Statview Software). A Mann-Whitney U test was also used to compare data groups. In some cases, statistics were made with Tanagra software using a Kruskal-Wallis 1-way ANOVA. In all cases, *P values < 0.05 were significant.
|
study
| 99.9 |
Glioblastoma multiforme (GBM) is a highly invasive cancer that is characterized by changes in cerebral vessels and the gradual invasion of surrounding tissues along the perivascular space [1, 2]. In the last years, several immune regulators have been shown to contribute to GBM progression as immunossupressive mechanisms, including recruitment of tumor-associated macrophages (TAMs) and changes of the expression patterns of cytokines that modulate the anti-tumor T cell responses [3–6]. However, the origin of these initial mechanisms underlying the suppression of anti-tumor immune responses around the blood vessel, where GBM initiates and progresses, are poorly understood. Previous works suggest that glioblastoma cells can differentiate into cells of tumor vessels, providing a means of tumor vascularization ; and that the tumorogenic phenotype can be transferred from the tumor cell to host cells around the blood vessels during GBM progression [8, 9]. Thus, to better understand these processes, it is essential to determine the consequences of possible interactions between cancer cells and cells in the perivascular compartments.
|
study
| 62.66 |
Small blood vessels are composed of two different but interdependent cellular compartments, endothelial cells (ECs) and pericytes [10, 11]. Pericytes are perivascular stromal cells that are located on the abluminal vessel wall of brain capillaries and regulate vascular tone and morphology, in a similar way as vascular smooth muscle cells do in big blood vessels [10, 12]. Pericytes may have stem cell properties and represent an immunological defense in the brain [13, 14]. Indeed, they have phagocytic activity and express numerous macrophage markers, supporting an ability to acquire functional properties of macrophages [9, 15].
|
review
| 77.06 |
Immune activation of brain pericytes, which act as mediators of neuroinflammation, specifically leads to the expression of several pro-inflammatory cytokines, co-stimulatory molecules and Major Histocompatibility Complex (MHC) molecules, which may modulate T cell responses [16–19]. The role of pericytes in the regulation of T cell function is poorly understood. Some authors described the ability of pericytes pre-activated by inflammatory challenge to present antigens on MHC to T cells, modulating the responses of different T cell populations, including suppressive regulatory T cells [16, 20, 21]. However, it is not well known if brain pericytes are immunologically protective in response to tumor formation or if, by contrast, they might fail to activate antigenic T cell responses, preventing tumor clearance.
|
review
| 99.75 |
The lack of pericytes promotes endothelial hyperplasia and abnormal vascular morphogenesis, which contributes to increased transendothelial permeability [9, 22], Pericytes can also regulate the expression of cytokines, chemokines and proteases in the tumor cell niche, which may promote immunosuppression, tumor angiogenesis, growth and metastasis [23, 24]. Therefore, pericytes might provide a critical element for the local control of both vascular-tumor cell interaction and the modulation of the anti-tumor immune response. In this work, we show that glioblastoma cells induce immunotolerant properties in brain pericytes. This may explain, at least in part, why GBM cells are not detected by the immune system during perivascular inflitration and tumor growth. Thus interference with the immunossuppressive function of pericytes may represent a novel target for the development of new therapies against GBM.
|
study
| 99.94 |
With our work, we try to address the mechanisms that might explain how the interaction of glioblastoma cells with pericytes allows tumor to grow and infiltrate brain parenchyma; and to specifically determine the relevance of this intercellular interaction in the modulation of the anti-glioblastoma immune response. A non-immunosuppressed mouse Glioblastoma model has been defined as a useful tool to study human Glioblastoma . The use of xenografts of human glioblastoma cells makes our experimental model more relevant to demonstrate an immunoregulatory mechanism activated by human glioblastoma. We show that, even under those conditions, immunocompetent C57BL/6 wild type mice cannot reject human glioblastoma tumor, likely due to the potent ability of glioblastoma cells to induce immune tolerance. Our purpose was to use this system to identify those mechanisms that allow glioblastoma cells to induce such a profound tolerant state, and may also represent a new approach to develop new experimental designs to study GBM-immune system interactions.
|
study
| 99.94 |
The immune function of pericytes in response to tumor cells remains unknown. To characterize if brain pericytes may acquire an anti-inflammatory function in response to their interaction with GBM cells (glioblastoma-conditioned-pericytes: GBC-PC) during tumor progression, we determined first if changes in the expression levels of cytokines would occur in response to the interaction of pericytes with GBM cells in vitro. Purification of brain pericytes from transgenic mice that express actin fusioned to GFP (C57Bl/6-Tg(ACTB-EGFP)1Osb/J), was confirmed by immunofluorescence after five passages [9, 26, 27], using well characterized pericyte markers [28, 29], such as NG2, PDGFR-β, and RGS-5 (Figure 1A). Staining for control markers of astrocytes, microglia and endothelial cells was negative (not shown), which ensures that none other cell type without stem cells properties survived in a culture without specific growth factors [26, 27]. Analyses of cytokines secreted from pericytes co-cultured with a GBM human cell line (GBC-PC), revealed a significant increase in the production of IL-10 and TGF-β after 72 hours compared to basal levels of control pericytes (Figure 1B). The expression of pro-inflammatory cytokines, such as IL-1, IL-23 and IL-12 was, however, hardly detectable (not shown). GBC-PC also produced the pro-inflammatory cytokine TNF-α, no significant differences were detected compared to the control pericytes, until late time points of co-culture with GBM cells (Figure 1B). Importantly, the increase in the production of TNF-α, which showed just a doubling of its expression at 72 hours of co-culture, was much lower than the one detected when assessing the anti-inflammatory cytokines TGF-β and IL-10 (Figure 1B). These data correlated with similar changes in the levels of mRNA expression in GBC-PC compared to controls (Figure 1C). Analysis of cytokine gene expression in GBC-PC reflected upregulation of the mRNA of the anti-inflammatory cytokine genes Tgfb and Il10. Il1b and Il12 gene expression was not detected even in control pericytes (not shown), and the mRNA level of Tnfa, Il4 and Il23 in pericytes was not significantly affected by GBM cells. Surprisingly, mRNA and protein expression of the angiogenic cytokine IL-6 was clearly increased in GBC-PC compared to control pericytes after 72 hours of coculture (Figure 1C, Supplementary Figure 1) [30, 31]. No cytokine mRNA or protein were detected in control GBM cells (not shown). To determine if the marked rise of expression of TGF- β and IL-10 in GBC-PC requires direct cell-cell interaction or is mediated by soluble molecules expressed by GBM cells [32, 33], we incubated pericytes with sequential dilutions of supernatants from different lines of GBM cells. Our results showed the same levels of cytokine expression in supernatant-treated pericytes as in control pericytes, supporting that the acquired immunomodulatory phenotype in pericytes in response to GBM likely requires cell-to-cell interaction (Figure 1D).
|
study
| 100.0 |
(A) Expression of pericyte markers in pericytes expressing GFP. NG2 (scale bar, 50 μm), PDGFRβ and RGS5 (scale bar, 100 μm). The images are representative of at least, three independent experiments. (B) ELISA measuring IL-10, TGF-β and TNF-α levels in pericytes co-cultured with Glioblastoma cells (GBC-PC) at different time points, and at basal levels in control pericytes (PC), ** p<0.01 or ***p<0.001. All data represent mean ± Standard Deviation obtained from at least three independent experiments. (C) Quantitative analysis of cytokine mRNA expression in GBC-PC at different time points. Results are presented relative to those of basal levels in control pericytes at each time point, and normalized to the housekeeping reference gene expression, * p<0.01. All data represents mean ± Standard Deviation obtained from at least four independent experiments. (D) Quantitative analysis of IL-10 and TGF-β mRNA expression in pericytes (PC), after 72 hours in different conditions of culture (pericytes in presence of GBM cells: GBC-PC; pericytes in presence of several dilutions of GBM conditioned media: PC + ½, ½ GB media. Results are presented relative to those of basal levels in control pericytes at 72 hours of culture, and normalized to the housekeeping reference gene expression, * p<0.01. All data represents mean ± Standard Deviation obtained from at least, five independent experiments using U373 and U87 GBM lines independently.
|
study
| 100.0 |
Activated pericytes have been reported to present properties of myeloid cells, such as macrophages, expressing macrophage markers and acquiring phagocytic activity and the ability to present antigens to T cells [17, 18, 34]. To identify if pericytes might gain some of the immunosuppressive properties of TAMs in response to their interaction with GBM cells, we first analyzed the expression of several membrane molecules implicated in the inhibition of anti-tumor responses [24, 35]. Interestingly, we found high levels of Il4ra and Il1rn mRNA expression in pericytes, after 24 hours following interaction with GBM cells (Figure 2A). Then we determined if the immunosuppressive ligand of PD-1, PDL-1, which has been associated with glioblastoma progression [3, 36, 37], was expressed in pericytes, and if its levels changed in response to GBM interaction. We observed that PDL-1 was expressed in pericytes in resting conditions, but its level of expression was maintained upon GBM cell interaction (Figure 2B). Interestingly, we found that expression of the co-stimulatory molecules CD80 and CD86 was significantly reduced in GBC-PC compared to control pericytes (Figure 2C). To analyze if the ability of brain pericytes to present antigen to T cells might be affected by GBM cell interaction, we determined the expression levels of major histocompatibility complex class II molecules (MHC-II) in GBC-PC. We found a drastic and significant reduction of MHC-II expression in pericytes when cocultured with GBM cells compared to control pericytes (Figure 2D). None of these markers was detected in control GBM cells. These data support an immunomodulatory phenotype in pericytes in response to GBM cell interaction.
|
study
| 100.0 |
(A) Quantitative analysis of mRNA expression of Ilrn (IL-1RA) and Il4ra (IL-4RA) cytokine receptors in GBC-PC. Data are presented relative to basal levels in control pericytes at each time point of culture, and normalized to the housekeeping reference gene expression. Results are mean ± Standard Deviation obtained from at least, four independent experiments, * p<0.01. (B) Flow cytometry analysis in control PC or GBC-PC for PDL-1 expression after 72 h. Gating strategy for GFP+ pericyte population for further flow cytometry analyses is shown (upper panel). Solid and dotted histograms represent staining with PDL1 specific antibody and isotype control, respectively. Insert numbers inside histograms represent mean fluorescence intensity values. The bar graph represents percentages of expression of PDL-1 in PC compared to percentages in GBC-PC. (C) Flow cytometry analysis of expression of the co-stimulatory molecules CD80 and CD86. (D) Flow cytometry analysis of expression of MHC-II (I-A). Non-specific fluorescence was measured using specific isotype monoclonal antibodies and GBM cells were used as negative controls. All data of flow citometry represents mean ± Standard Deviation obtained from at least, three independent experiments, * p<0.05, or ** p<0.01.
|
study
| 100.0 |
It has been reported that pericytes activated by inflammatory challenge have the ability to present antigen on MHC molecules to T cells, regulating the activity of different T cells populations [16, 20, 21]. We observed that pericytes showed reduced expression of MHC-II molecules and an anti-inflammatory phenotype upon interaction with GBM cells. Therefore, we determined if, in response to GBM interaction, pericytes could instead impair T cell activation. For that purpose, we analyzed the ability of pericytes to present OVA323-339 peptide to T cells, and compared CD4+ T cell responses to peptide presented by GBC-PC with responses to antigen presentation by control pericytes (Figure 3A–3B). We found that pericytes were able to present OVA peptide and activate CD4+ T cells, which led to IL-2 production and cell proliferation. However, pericytes that were interacting with GBM cells showed a significantly impaired ability to activate T cells (Figure 3A–3B). As we had observed that GBC-PC produced high levels of anti-inflammatory cytokines, cytokines, expressed immunosuppresive molecules such as PDL-1 and showed reduced expression of co-stimulatory molecules, we analyzed whether GBM-PC would also affect T cell activation in response to antigen presented by professional antigen presenting cells (APCs). This would support that, when in contact with GBM, pericytes might not only show a reduced ability to cross-present tumor antigens but could also hinder the function of APCs. Corroborating our hypothesis, whereas CD4+ T cells produced high levels of IL-2 and proliferated in response to antigen presented by APCs, those T cells showed defective IL-2 production and proliferation when activated by antigen-loaded APCs in the presence of GBC-PC. T cell activation was not affected by control GBM cells or control pericytes (Figure 3C–3D). To further support that GBC-PC could release anti-inflammatory cytokines to the media that could explain their effect on APC-mediated activation of T cells, we determined whether GBC-PC-conditioned media would also be able to affect T cell activation. We then measured T cell responses to T Cell Receptor (TCR) engagement and co-stimulation, following activation with anti-CD3 and anti-CD28 antibodies, in the presence or absence of culture media from GBC-PC for 72 hours. Importantly, CD4+ T cells showed reduced IL-2 production and proliferation upon TCR and co-stimulation engagement in the presence of GBC-PC-conditioned media when compared to CD4+ T cells activated in presence of control pericyte media or control vehicle. IL-2 secretion and cell proliferation were not affected by control GBM cells (Figure 3E–3F). Importantly, impaired T cell responses caused by GBC-PC were not due to increased apoptosis, as there were no significant differences in cell death percentages between resting or activated T cells cultured with control pericytes or GBC-PC (Supplementary Figure 2). Therefore, our data support that GBC-PC reduces T cell responses through the induction of an anti-inflammatory response and the development of an immunosuppressive phenotype in response to interaction with GBM cells.
|
study
| 100.0 |
(A) IL-2 production was measured by ELISA in resting and 72-hours stimulated naïve CD4+ T cells in response to OVA323-339 peptide presented by control pericytes (PC) and GBC-PC. Results are mean + Standard Deviation from five to six different experiments, ***p<0.005. (B) T cell proliferation was measured by BrdU incorporation in response to antigen presentation by PC and GBC-PC. Results are mean + Standard Deviation from four different experiments, ***p<0.005. (C-D) Cell proliferation and IL-2 production were measured in resting and 72-hours stimulated naïve CD4+ T cells in response to antigen presentation by antigen presenting cells (APC) in presence or not of GBC-PC (APC:GBC-PC; APC). T cells ractivaton in response to antigen presentation was also measured in the presence of control Pericytes (APC:PC) and GBM cells (APC:GB). Results are mean + Standard Deviation from four different experiments. **p<0.01. (E-F) Cell proliferation and IL-2 production were measured in naïve CD4+ T cells stimulated for 72 hours with plate-bound anti-CD3 (αCD3) and anti-CD28 (αCD28) antibodies, in the presence or absence of media from GBC-PC diluted 1:2 in fresh pericyte media (vehicle). Control media from pericytes (PC) and GBM (GB) cell cultures was also diluted in fresh pericyte media and cytokine production and cell proliferation measured in these control conditions. Results are mean + Standard Deviation from three different experiments. **p<0.01. *p<0.05.
|
study
| 100.0 |
To determine if proliferation of GBM cells might be directly facilitated by pericytes, therefore enhancing tumor growth, we measured cell proliferation as cumulative population doublings in GBM cells interacting with pericytes in vitro. Interestingly, we found that GBC-PC did not proliferate at all, as opposed to control pericytes that proliferated, as expected, exponentially with the time of culture (Figure 4A). However, proliferation of GBM cells was not inhibited by the presence of pericytes (PCC-GB). PCC-GB seemed to reach even higher proliferation levels than control GBM cells, although this difference was not significant. In addition, we analyzed the cell survival of pericytes to confirm that the obtained data were due to inhibition of proliferation and not increased cell death. We found that pericytes survived after interaction with GBM cells during long time cultures (72 hours) as well as control pericytes (Figure 4B).
|
study
| 100.0 |
(A) Proliferation of GBM cells interacting with pericytes (PCC-GB) and pericytes interacting with GBM cells (GBC-PC), represented as cumulative population doubling (CPD), during different times of co-culture was measured and compared to proliferation of control GBM cells and pericytes. Results are mean + Standard Deviation of at least three different experiments, *p<0.05; **p<0.01. (B) Detection of death cell by blue-fluorescent reactive dye, of two representative populations of pericytes (PC1, PC2) compared to those same populations conditioned by GBM cells (GBC-PC1, GBC-PC2) (left panel). Quantification of dead cells percentage (right panel) from at least, four independent experiments. (C) GBM tumor growth in mice that were xenografted with GBM cells (GBM) compared with (D) xenografts of co-cultured PC and GBM cells (GBM+Pc) (scale bars, 100 μm). (E) Infiltration stream of GBM cells (GBM) together with a GFP+ Pericyte (Pc) in the perivascular space (scale bar, 25 μm). (F) Morphometric measurement reveals the average of tumor size in GBM grafted mice (GB) and GBM+Pc grafted mice (6 tumors / 5 grafted mice and 7 tumors / 5 grafted mice, respectively). (G) Representative detail of xenografts GBM+Pc shows proliferating GBM cells (GBM/Ki67+), which appear as intense RFP+ fluorescent spherical dividing cells, in which one of the sister cells is more intensely immunopositive (arrow) than the other (arrowhead) for both RFP (red) and Ki67 (green) (scale bar, 25 μm). (H) Proliferating cells (strong RFP+) in GBM xenografts and (I) GBM+Pc xenografts (scale bars, 50 μm). All results are shown using the U373 GBM line, 4 weeks after-graft, and are representative of at least four different experiments using U87 or U373 GBM lines independently. Control pericytes (0 tumors / 5 grafted mice (4weeks) and 0 tumors/ 5 grafted mice (11weeks)) not shown. (J) Relative quantification of number of dividing GBM cells in GBM+Pc and GB grafted mice. Results are mean+SD from at least four different experiments * p<0.05; *** p<0.0005. (K-P) IL-10 and TGF-β expression in control GBM xenografts (scale bars, 50 μm). (L, Q, S) Colocalization of the heterogeneous distribution of pericytes (GFP+, arrowheads) and (M, R, T) IL-10 and TGF-β expression (arrowheads) in GBM+Pc xenografts. (N, U) Merged pictures showing higher IL-10/ TGF-β expression in areas where grafted GBC-PC accumulate (arrows) (scale bars, 50 μm). (O, V) High-power confocal image (one micron-thick section) shows the colocalization of GFP and IL-10/ TGF-β in GBC-PC (arrows) (scale bars, 50 μm). Results are shown 4 weeks after-graft and are representative of at least, four independent experiments. (W) Quantification of IL-10 expression relative to total area of IL-10 immune-positive particules in GBC+Pc xenografts compared to GB control. (X) Relative quantification of TGF-β expression related to luminosity of TGFb immuno-processed sections in GBC+Pc xenografts compared to GB control. Results are mean+SD from at least four different experiments * p<0.05; *** p<0.0005.
|
study
| 100.0 |
To confirm in vivo these findings, and corroborate that tumor growth might be assisted by GBC-PC with immunosuppressive properties, GBM cell proliferation was studied in grafts of co-cultured human RFP-GBM cells and GFP-mouse pericytes (GBM+Pc) grafted into brain cortex of an immunocompetent C57Bl/6 mouse model . GBM tumors from both U373 and U87 cell lines (clonally labelled by RFP expression), were observed in most of the grafts with GBM cells alone (control grafts) or GBM+Pc at 4 and 11 weeks after graft (Figure 4C, 4D). Interestingly, pericytes were not observed when pericytes were grafted alone or together with GBM cells but without prior co-cultured (control grafts). As expected, these control mice showed the same tumor progression than mice grafted with glioblastoma alone (not shown). Increased perivascular infiltration of GBM cells was observed in GBM+Pc grafts (Figure 4E). The GBM tumor mass was larger in the brain of mice xenografted with GBM+Pc than in mice xenografted with GBM cells alone (Figure 4C, 4F). Due to the ovoid-like shape of the tumor mass we measured the tumor diameters in the central section and calculated the volume of the tumoral ovoid: 5.8+/−0.5×106 μm3 in GBM+Pc grafts [n=5] and 2.5+/−0.3×106 μm3 in GBM grafts [n=4] (Figure 4F). Supporting this data, we observed that dividing GBM cells, which appear as spherical strong RFP+ Ki67+ cells (Figure 4G), were more abundant in GBM+Pc grafts than in GBM grafts (Figure 4H, 4I). Relative quantification (counting strong fluorescent RFP cells in the same area of two consecutive sections of GB+PC grafts [90+/−10; n=5] and GBM grafts [30+/−7; n=5]; ImageJ-NHI software) showed that dividing cells were three times more numerous in GBM+Pc than in GBM grafts (Figure 4J). To determine if xenografts of GBM+Pc were able to acquire an anti-inflammatory phenotype in vivo, we analyzed the expression of IL-10 and TGF-β on the tumor/brain edge. Compared to GBM cell grafts (Figure 4K, 4P), we detected an increase of IL-10 and TGF-β expression in GBM+Pc grafts (Figure 4L–4M and 4Q–4TQ, respectively). This expression was particularly high in areas where grafted GBC-PC were identified surrounding blood vessel. The most intense fluorescent areas of Cy5-IL10 and Cy5-TGF-β colocalized with GFP expression corresponding to PC cellular mass (Figure 4N–4O and 4U–4VU, respectively). Quantitative analysis revealed that the expression of IL-10 (by measuring the area of immunopositive particles in equivalent areas of the central section of each xenograft [n=5] using ImageJ-NIH software) and TGF-β (by quatifying luminosity in the same area of the central section of each xenograft [n=5] using Adobe Photoshop software) were significantly higher in GBM+PC grafts than in GBM control grafts (Figure 4W and 4X, respectively). Supporting these data, we detected the appearance of inhibitory PD-1/PDL-1 interactions, which corresponded to perivascular lymphocytic infiltration and pericytes respectively in GBM+Pc grafts (Supplementary Figure 3), and compared it to host pericytes, where we did not find those interactions though perivascular PDL- 1 expression was present (not shown). Interaction between PD-1 receptor in lymphocytes and PDL-1 in GBM progression has been described, but it is not clear if, apart from GBM cells, other cell types may also present PDL-1 in perivascular areas . Our data showed pericytes expressing PDL-1 at the plasma membrane and interacting with PD-1 expressed in perivascular infiltrating T cells. Unspecific detection of high membrane-associated expression of PDL-1 was also detected in perivascular areas that did not correspond to pericytes but to GBM cells [36, 37] (Supplementary Figure 3).
|
study
| 100.0 |
Cellular interactions in the tumor microenvironment have been associated to immunosuppression and the induction of tolerance in the immune system against the tumor. In the immunosuppressive niche, in addition to cancer cells, tumor-associated macrophages (TAMs), glia cells, endothelial cells and T cells also infiltrate the tumor and secrete cytokines, chemokines, and proteases, thus promoting tumor angiogenesis, growth, metastasis, and immunosuppression . The inability to clear the tumor is known, in part, to be a consequence of an immunosuppressive response, with high levels of anti-inflammatory cytokines and reduced expression of T cell activation molecules [35, 38]. However, the mechanisms underlying that immunosuppressive response are not well understood yet, and even less in perivascular areas where tumor cells can infiltrate and metastasize. In this work, we describe a previously unknown immunosuppressive role of brain perivascular pericytes and demonstrate the importance of pericytes’ interaction with tumor cells during GBM progression. Our study reveal that brain pericytes show a pattern of immunosuppressive membrane surface molecule expression in response to GBM cell interaction. Analysis of cytokine expression from GBC-PC also shows high levels of expression of the anti-inflammatory cytokines IL-10 and TGF-β, low level of the pro-inflammatory cytokine TNFα and hardly any production of other important pro-inflammatory cytokines compared to basal levels detected in control pericytes. Interestingly, the increased production of anti-inflammatory cytokines correlates with an upregulation of those cytokines’ gene expression in pericytes, suggesting that pericytes are conditioned by GBM cells (GBC-PC) to produce those cytokines. Our findings indicate that Il1b and Il12 gene expression was not even detected in control pericytes (not shown), and that the mRNA level of Il4, Tnfa and Il23 in pericytes was barely affected by GBM cells. Although protein levels of TNFα are increased in GBC-PC compared to control pericytes, the doubling in amount of protein expression of this pro-inflammatory cytokine (reaching up to 45 pg/ml approximately) is much lower than the protein expression of the anti-inflammatory cytokines TGF-β and IL-10, which reaches higher levels of up to 100 and 400 pg/ml, respectivately in GBC-PC. Interestingly, mRNA and protein expression of the angiogenic cytokine IL-6 released by pericytes in response to neurovascular damage [30, 31] was surprisingly raised in GBC-PC. In a recent study in ovarian cancer, IL-6 was shown to induce defective angiogenesis through altered pericyte coverage in aortic ring vessels . Interestingly, treatment of pericytes with TGF-β has been recently associated with regulation of the neurovascular function, affecting the phagocitic ability of these cells, reducing pericytes proliferation and increasing IL-6 expression . The fact that IL-6 has been associated to an angiogenic gene program in pericytes and even affects pericytes proliferation during angiogenesis in cancer, supports our data and might also explain why we observed a reduced proliferation of GBC-PC in vitro. It is possible that IL-6 expression might be regulated by TGF-β signaling in GBC-PC, attenuating cell proliferation and other neurovascular functions that also affect the tumor progression.
|
study
| 99.94 |
It has been recently shown that GBM cells might interact with host cells, such as pericytes, to transfer malignant properties and affect their function [8, 9], supporting that GBM-pericytes cell-cell direct interaction are required to lead the changes in the pericytes immune phenotype.
|
study
| 100.0 |
Our hypothesis that GBC-PC might present similar immunosuppressive properties as TAMs, which are implicated in inhibitory anti-tumor responses, is supported by the clear upregulation of Il4ra and Il1rn gene expression in GBC-PC [24, 35]. Indeed, our results show that PDL-1, which is associated with glioblastoma progression and contribute to suppress anti-tumor T cell responses [3, 36, 37, 39], is expressed in pericytes and maintained upon GBM interaction. Furthermore, we show reduced expression of T cell co-stimulatory molecules, such as CD80 and CD86, in GBC-PC, supporting an immunosuppressive phenotype that might prevent tumor clearance. Our data confirm that brain pericytes express MHC molecules [16, 20, 21], but interestingly expression levels are drastically reduced in GBC-PC compared to PC, suggesting that the anti-tumor T cell response might be affected through inefficient antigen presentation. GBC-PC also shows impaired ability to present antigen to T cells and cell culture media from GBC-PC is enough to reduce T cell responses. In addition, T cell function in response to antigen presentation by APCs is also suppressed in the presence of GBC-PC. Therefore, these results suggest that GBC-PC could acquire suppressing properties and hinder T cell activation, contributing to tumor growth by preventing the activity of other APCs.
|
study
| 100.0 |
Brain pericytes preferentially cover ECs junctions in response to inflammation, having an important role in blood brain barrier disruption in inflammatory processes [28, 40]. Interestingly our studies reveal that GBC-PC remain in a dormant-like state and do not proliferate compared to control pericytes, which proliferate normally. In contrast, proliferation of GBM cells interacting with pericytes (PCC-GB) is not affected.
|
study
| 100.0 |
Supporting our in vitro studies, we demonstrate in vivo that GBM cell proliferation and, therefore, tumor progression, is assisted by the interaction with pericytes. Our results strongly suggest that GBM cell interaction with pericytes is required to maintain immunotolerance in the immunocompetent mouse brain . Indeed, our results analysing GBM+Pc grafts compared to control grafts, show high levels of anti-inflammatory cytokines in perivascular areas where grafted GBC-PC were found. Perivascular detection of less intense anti-inflammatory cytokine levels in GBM grafts might correspond to expression by endogenous pericytes.
|
study
| 100.0 |
Supporting our in vitro results, we have also seen the establishment of inhibitory interactions PD-1/PDL-1 between lymphocytes and pericytes, respectively, in perivascular areas. Interestingly, we don’t detect either the presence of grafted pericytes in grafts of control mice, or when pericytes are grafted with glioblastoma cells without being previously co-cultured. As it was expected, these last control mice showed the same tumor progression that control mice grafted with glioblastoma alone. However, we observe pericytes in grafted GBM+Pc mice, indicating they need to be conditioned by interaction with Glioblastoma previously to anchor in perivascular areas. This effect may be due to a decreased competence of grafted versus host pericytes for trophic factors to survive; and suggests that GBM cells confer increasing competence to grafted pericytes, as well as inducing immunotolerance, which in addition could facilitate the cell anchorage to perivascular areas.
|
study
| 100.0 |
In conclusion, our results indicate that brain pericytes show an immunosuppressive function as a consequence of their interaction with GBM cells, possibly assisting the tumor immune evasion and, consequently promoting GBM tumor. This finding identify pericytes as key cellular components of the GBM niche, which should lead to further studies to search for possible ways to regulate their interaction with GBM cells to modulate their immunosuppressive function and control tumor growth.
|
study
| 100.0 |
Six to eight-week-old wild type C57Black/6, C57Bl/6-Tg(ACTB-EGFP)1Osb/J (Charles River laboratory) and OT II TCR transgenic (Jackson laboratory) mice were maintained in pathogen-free conditions in the animal facility of University of Murcia. All animal procedures were approved and performed according to the guidelines set by University of Murcia Institutional Animal Care and Use Committee.
|
other
| 99.9 |
Primary brain pericytes from GFP-actin mice were isolated as described previously and according to the method of Oishi et al. [9, 26, 27]. Pericytes were used from 5th-9th passage and checked by pericytes markers. For the detailed cell culture, please see Supplementary Text.
|
study
| 99.94 |
Co-cultures of pericytes and GBM cells, at a ratio of 1:1, were plated in pericytes media for 24-72 hours. Cells were trypsinized, replated and identified by GFP- or RFP-protein labeling. Pericytes were sorted by GFP-protein with a Cell Sorter (Sony SH800).
|
study
| 99.94 |
Primary CD4+ T cells were isolated from lymph nodes and spleens of mice using anti-CD4-coupled magnetic beads (Life Technologies). In some cases, isolated T cells were stimulated with 0.5 μg/ml plate bound anti-CD3 and 0.5 μg/ml anti-CD28 (BD Biosciences). CD4+ T cells were growth in T cell media.
|
study
| 99.94 |
cDNA was synthesized from total mRNA, and gene expression was analyzed by real time PCR using SYBR Green in a Step One Plus Thermocycler (Applied Biosystems). Gene expression was normalized to mouse β-actin expression. Glioblastoma cells were used as negative control for cytokines expression. For primer sequence information, please see Supplementary Text.
|
study
| 99.8 |
Co-cultures of pericytes (1 × 105) and GBM cells in 1:1 proportion, were plated in 96 well plate. Pericytes and GBM cells cultures were used as controls. After 72 hours, T cells (5×104) were plated on attached pericytes or attached GBC-PC previously, in 96 well plate, in 5:1 proportion respectively and in presence or not of OVA323-339 peptide antigen (Sigma-Aldrich). Pericytes were detached with trypsin 0.05% after 72 hours and plated with T cells at the same time to confirm the same results. For suppression assay of T cell function, splenocytes and T cells were plated in 96 well plates, in 5:1 proportion, in presence or not of OVA323-339 peptide on GBC-PC or pericytes plated 72 hours before. In some cases, media from cultures of GBC-PC, control pericytes and control GBM cells for 72 hours, was recollected to add to stimulated T cells with plate-bound antibodies.
|
study
| 100.0 |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.