Limited time and lack of knowledge are barriers to physical activity counseling in primary care. The objective of this study was to examine the effectiveness of a clinician-targeted intervention that used the 5As  approach to physical activity counseling in a medically underserved patient population. Family medicine clinicians at 2 community health centers were randomized to Group 1 or Group 2 intervention. Both clinician groups participated in 4 training sessions on the 5As for physical activity counseling; Group 2 training took place 8 months after Group 1 training. Both groups were trained to refer patients to a community exercise program. We used a pre–post analysis to evaluate the effectiveness of the intervention on clinician use of 5As. Eligible patients (n = 319) rated their clinicians’ counseling skills by using a modified Physical Activity Exit Interview (PAEI) survey. Clinicians (n = 10) self-assessed their use of the 5As through a survey and interviews.P = .01) and was 8.2 (P = .09) at 6-month follow-up; most of the improvement in PAEI score was due to increased use of 5As skills by Group 2 clinicians. Group 1 reported difficulty with problem solving, whereas Group 2 reported ease of referral to the community exercise program.Both patient and clinician groups had similar sociodemographic characteristics. The PAEI score for both groups combined increased from 6.9 to 8.6  from baseline to immediately postintervention (A clinician training intervention showed mixed results for 5As physical activity counseling. Americans made 560 million visits to primary care physicians in 2010 , Advise on, Agree upon, Assist with, and Arrange follow-up on patients’ behavior-change efforts –11. The Despite recommendations to use the 5As, evidence for the feasibility of implementing the 5As in “real-world” settings is limited. In 2010, the US Preventive Services Task Force updated its recommendations on physical activity counseling , concludThe objective of this study was to examine the effectiveness of a clinician-targeted intervention that used the 5As approach to physical activity counseling in a medically underserved patient population. The study design and protocol are described in detail elsewhere . BrieflyClinicians and patients were recruited through 2 federally qualified health centers in Rochester, New York, serving a predominantly low-income, racially/ethnically diverse population of 14,000 patients. Details on inclusion and exclusion criteria for participants are described elsewhere . CliniciPatients were eligible if they were currently enrolled at one of the health centers, were aged 18 years or older, were able to provide informed consent, and made a health maintenance visit or follow-up visit for a chronic condition for which physical activity counseling would be appropriate. Nurse assistants mentioned the study to the patients when they were in the examination room. If the patient expressed interest, a research assistant entered the room to provide additional details about the study; patients who agreed to participate signed an informed consent form. For ethical and regulatory reasons, patients were not blinded to study topic and objective; as part of the informed consent process, they were made aware of these. However, they were blinded to time point  and did not know whether or when their clinicians took part in the intervention; patient participants were paid $20 for participation.During the study period, 1,029 patient participants were assessed for eligibility. Of these, 481 (or 46.7% of those assessed) were ineligible. Among the remaining 548 patients, 325 consented to participate . Of the The intervention consisted of 4 one-hour training sessions for clinicians at the health center sites . CliniciFor each clinician, patients completed a survey at 3 points: baseline, immediately postintervention, and at 6-month follow-up. Each patient completed 1 survey, so different groups of patients provided surveys at each point. Patients completed the survey, a modified version of the Physical Activity Exit Interview (PAEI) , after tEach clinician completed baseline and 6-month follow-up surveys. The surveys asked about demographic characteristics, current practice of physical activity counseling, confidence in using 5As in counseling on physical activity, and knowledge of resources that could meet their patients’ needs. Their confidence in using counseling skills was self-rated on a 5-point Likert scale . At follow-up, each clinician also completed a 30-minute interview to provide feedback about the overall experience in the intervention, changes in skills, problems or difficulties, training techniques, satisfaction with the intervention, and suggestions for improvement.t tests for continuous variables and χ2 tests for categorical variables were used to evaluate the success of the randomization in balancing baseline covariates between groups. From the power and sample-size analysis, given our sample size, we could detect a difference of 1.5 or greater in PAEI scores between baseline and follow-up with 80% power. Data were analyzed with generalized estimating equations (GEE) models with patients nested within clinician using a negative-binomial link to account for the distribution of PAEI scores. We chose to use GEE models to account for nesting of patients within clinician and a possible unknown correlation between outcomes and to obtain robust standard errors. Also, GEE models provide a practical method for analyzing conditional responses that may depart from the normality assumption required for general linear models. Finally, we determined effect sizes and categorized them as small, medium, or large , and the average length of practice was 15 years . Group 1 and Group 2 patients did not differ significantly by any sociodemographic or health characteristic, so data for both groups were combined . PatientP = .01) (P = .09). The average PAEI score did not change significantly at either point for Group 1, whereas the average PAEI score increased at both points (from baseline) for Group 2  . The med Group 2 . The PAE Group 2 . Of the  Group 2 .From baseline to 6-month follow-up, clinician confidence increased significantly for assessing an exercise history, negotiating an exercise plan, turning setbacks into learning, helping patients cope with barriers, counseling in a cost-effective way, and having knowledge of community resources that could meet patients’ needs .From the clinician interviews, some differences emerged for Groups 1 and 2 in their perceptions of their 5As counseling skills. Group 1 clinicians (whose score did not change) commented frequently on the challenges of problem solving with patients (“getting lost in the barriers” as stated by 1 clinician). Group 1 clinicians felt that the training was too brief and that booster trainings would have been helpful. They requested more feedback on their counseling skills (either from the research team or their peers) and support beyond the office visit to encourage patients and help with problem solving. In contrast, Group 2 clinicians (whose PAEI score was lower than Group 1’s at baseline but increased at follow-up) mentioned the value and ease of referral to the community exercise program. The partnership for referral to the community exercise program was newly initiated when Group 1 took part in the study and was more established by the time Group 2 took part. Group 2 clinicians also commented that the 5As framework was useful for raising awareness and reminding them of the importance of counseling on physical activity.This study evaluated the effect of a clinician-targeted intervention on patient- and clinician-reported use of the 5As for physical activity counseling. The intervention used innovative, interactive clinician training techniques and focused exclusively on a medically underserved population not well represented in this type of research. The main finding was that the intervention increased patient-reported improvements in 5As counseling on physical activity immediately postintervention but not at 6-month follow-up, although this improvement was primarily due to higher scores for Group 2 clinicians. Group 1 clinicians had a higher baseline PAEI score, which may have limited the effectiveness of the intervention for them. Also, the community exercise program may have provided a greater incentive for Group 2 clinicians to use the 5As because the program was better established for them than for Group l clinicians.There are some practical lessons learned from this study. First, the development of the referral process for the community exercise program took time to function effectively, and this delay affected clinician behavior . Research on the effectiveness of community exercise programs for clinician referrals is lacking; others have commented on the potential demand and the difficulty of establishing effective partnerships for referral. A recent meta-analysis of primary care–based physical activity counseling  and receSecond, the intervention might have been more effective in increasing clinicians’ use of the 5As if it had included strategies such as booster trainings, reminders or prompts about the ongoing availability of the community exercise program, and additional staff support to help with problem solving . ConsistThis study has several limitations. Although clinicians were randomized for practical reasons, we had inadequate control group data and therefore used a pre–post analysis. Although the patient sample size was larger than is typically reported for this type of research, patients were nested within a small sample of clinicians from a single geographic location. Participants’ awareness of the study topic and objective could have increased the likelihood of patient bias to overreport physical activity counseling. However, the blinding of patients to time point and participation of clinicians in the intervention increased the likelihood that any bias was evenly distributed among time points and clinicians. Finally, by design, this study did not assess patients’ behavior change during the study; instead, it aggregated patient ratings nested by clinician. This trade-off was made for practical and logistical reasons because of the focus on an underserved population, but future research would be enhanced by longitudinal assessments of changes in physical activity among patients.Strengths of the study are that, to our knowledge, this is the first study to assess the effectiveness of a clinician intervention on use of the 5As for changing physical activity counseling in a medically underserved population. This study is relevant for several reasons. First, it addressed the high prevalence of lifestyle-related chronic conditions in a medically underserved population and the need to eliminate racial/ethnic disparities . Second,This study has 2 main clinical implications. First, clinicians can be taught to improve their physical activity counseling by using the 5As framework. Second, the intervention can improve confidence in counseling skills among physicians, especially by educating them about community resources for physical activity.This study also has policy relevance. Primary care is undergoing transformation in the United States. The patient-centered medical home (PCMH) initiative has created a resurgence of interest in helping primary care patients change health behaviors  to qualiThe results of a clinician-directed intervention designed to increase patient reports of physical activity counseling were mixed. Group 2 clinicians (who took part in the intervention after Group 1) increased their use of the 5As more than did Group 1; the difference in use between the 2 groups was due primarily to greater use of Assess and Assist skills among Group 2. Group 2 clinicians improved their awareness of the community exercise program for referral, whereas Group 1 clinicians reported difficulty with problem-solving skills. Future directions are to explore the association between 5As counseling and patient outcomes such as enrollment and participation in community exercise programs.


A protein profile based on the expression of a subset of 25 factors could accurately separate resistant from sensitive patients with a success rate of approximately 90%. The protein profile corresponding to the “sensitive” subset was associated with significantly longer PFS  and OS . This prognostic performance was superior to that of stage, histology and residual disease after cytoreductive surgery and the levels of vascular endothelial growth factor (VEGF) in ascites. In conclusion, we developed an “angiogenic signature” for patients with AOC, which can be used, after appropriate validation, as a prognostic marker and a tool for selection for anti-angiogenic therapies.Advanced ovarian cancer (AOC) is one of the leading lethal gynecological cancers in developed countries. Based on the important role of angiogenesis in ovarian cancer oncogenesis and expansion, we hypothesized that the development of an “angiogenic signature” might be helpful in prediction of prognosis and efficacy of anti-angiogenic therapies in this disease. Sixty-nine samples of ascitic fluid- 35 from platinum sensitive and 34 from platinum resistant patients managed with cytoreductive surgery and 1 Ovarian cancer is the most lethal gynecological type of cancer in developed countries. According to SEER data, approximately 23000 women will be diagnosed with ovarian cancer in the forthcoming years and about 15000 of them will die of the disease –8. NeverAngiogenesis, is a process of production of new blood vessels and is a hallmark of cancer related to tumor survival and induction of tumor metastasis . It consFor the above reasons, we hypothesized that an “angiogenic signature”, consisting of a panel of angiogenic factors that may be present in the patients’ ascitic fluid, might be an accurate prognostic tool as well as a means of selection of ovarian cancer patients likely to benefit from anti-angiogenic therapies. We hereby report the development of such “signature” based on the expression of 55 putative ovarian cancer markers of angiogenesis in ascites. In order to develop our model, we used resistance to chemotherapy as the discriminating factor to identify a favorable vs. an unfavorable angiogenic protein profile. The association of increased angiogenesis with chemoresistance is supported by a significant body of evidence from pre-clinical studies. Indicatively, pre-treatment of cancer cells with cisplatin induces a pro-angiogenic expression shift and leads to increased angiogenic activity when cells were treated with different chemotherapeutics including cisplatin . In addiWe found that a reduced marker set can form a promising tool, which is strongly associated with prognosis following cytoreductive surgery and standard cytotoxic chemotherapy. Following appropriate validation, this approach could be used for molecularly-directed anti-angiogenic therapy in ovarian cancer.st-line chemotherapy; patients had ascites at presentation; no anti-cancer treatment had been given before ascites collection. Ascites was prospectively collected but retrospectively analysed. All patients were treated at the 1st Dept of Obstetrics and Gynaecology  and the Dept of Clinical Therapeutics . The study protocol had appropriate Institutional Review Board approval  and written informed consent was given by all subjects for the collection and study of the ascitic fluid. The study was conducted according to the principles expressed in the declaration of Helsinki.Sixty-nine patients suffering from ovarian cancer and fulfilling the following criteria participated in this observational, single-institution study: Stage III to IV (according to FIGO guidelines); patients should be scheduled to receive carboplatin-based 1Ascitic fluid was collected prior to the administration of systemic therapy (either during primary cytoreductive surgery or paracentesis if laparotomy was not performed). It was mixed in sterile conditions with heparin (2u/ml) , centrifuged at 4°C at 1600 rpm, pellet was discarded and the supernatant was stored and cooled successively to -20°C for 1–2 hours and then transferred to of -80°C where it remained until used.After treatment, all patients received follow up checks with CT scan every 6 months and CA-125 measurement every 3 months according to institutional protocol. Patients who relapsed during or within six months after completion of first-line chemotherapy were classified as platinum resistant, while patients exceeding 6 months without relapse were classified as platinum sensitive according to GCIG classification . RelapseArray detection was performed in 69 samples of ascitic fluid. In order to develop a prognostic “signature”, platinum resistance was used as a surrogate of prognosis: 35 samples from platinum sensitive patients and 34 samples from platinum resistant patients were analysed.TM Human Angiogenesis Array Kit,  [ in the patients’ ascitic fluid according to the manufacturer’s instructions. Briefly, 1ml of the supernatant from ascitic fluid is incubated with 500 μl of a relevant buffer and 15 μl of a mixture of biotinylated antibodies (specific for each of the 55 factors) for 1 hour. The mixture of ascitic fluid supernatant-antibodies-buffer is incubated for 16 hours at 4°C with a cellulose membrane coated with the same antibodies. Non-specific binding of antibodies to proteins of interest is avoided by pre-incubation of the membrane with a blocking buffer for 1 hour. The membrane is then incubated with streptavidin-HRP for 30 minutes. Light development is achieved through incubation with an ECL chemical reagent (R&D Systems) for 5 min. The emission of light is detected by using a special photographic film which depicts the protein trace. Thus the angiogenic proteins present in ascitic fluid are detected . Quantitation of the detected spots was performed via standard densitometry using the Quantity One Software 4.4.1 .The Proteome ProfilerRRY 007)  was emplIn order to confirm our array findings, we studied 6 of the factors included in the array in 20 samples using enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions . We chose factors showing significant differences in their array results between the two groups. In order to maintain a balance between the two groups, 3 factors showing increased expression among sensitive and 3 with increased expression among resistant patients were used.The expression levels of the 55 angiogenic factors were used for feature selection and classification between platinum positive/resistant patients. The expression values were normalized according to the array specifications. The selection of the subset of factors that can most effectively separate the two classes was performed by recursive feature elimination with embedded cross-validation (RFECV) and using a Support Vector Machines (SVM) classifier. In order to estimate the best parameters for the SVM classifier, a grid scan approach was used, which consisted in automatically testing different combinations of parameters with the aim to maximize the Area Under Curve (AUC) criterion. Finally, the best performance was reported for the SVM classifier with the following parameters: linear kernel, C 0.001, gamma 0.001. Additional filtering was applied to the final subset using the entropy-based filtering methodology of Information Gain (IG), which measures the expected reduction in entropy caused by partitioning the samples according to a given attribute ,20. FactFor the selection of the best classifier to train with the reduced dataset of 25 factors see , a grid All aforementioned feature selection and classification procedures were implemented in the Python scikit-learn package.For the HeatMap representation the normalized expression levels of the 25 angiogenic factors were first log2 transformed and the relative expressions were estimated according to the median expression of each factor. One way  hierarchical clustering was performed in GeneARMADA .VEGF in ascites was measured by ELISA according to the manufacturer's instructions  as previously reported .st-line chemotherapy and date of death from any cause and relapse of the disease; patients not dead or without relapse were censored on the date of last contact. Survival distributions were estimated using the Kaplan-Meier method; log-rank tests for equality of survivor functions between treatment groups were used. Cox proportional hazards regression analysis (univariate and multivariate) was performed to assess the influence on OS and PFS of the protein profile (“sensitive” vs. “resistant”), VEGF (> median vs. ≤ median), histology (serous vs. non serous), age (>60 vs. ≤60) and residual disease after primary cytoreduction (≤ 1cm vs. > 1cm). Variables with p<0.1 in univariate analysis were entered into the multivariate models.The STATA/SE 11.2 SE software was used for statistical analysis. OS and PFS were defined as the time interval between the start of 1Table 2. In all cases, ascites was obtained at initial diagnosis. In four cases, histology was not determined because no tumor biopsy was obtained during laparoscopy and the diagnosis of adenocarcinoma was based on the cytology of peritoneal fluid. All patients underwent primary cytoreductive surgery. All patients received first-line chemotherapy following cytoreductive surgery. Sixty—five patients received combination chemotherapy with Carboplatin / Paclitaxel, while 4 received Carboplatin monotherapy.From March 2009 to January 2013 samples of ascites from 69 patients (35 platinum sensitive and 34 platinum resistant) with ovarian cancer were obtained. Their baseline characteristics are shown in Table 3).All protein array data for the 55 factors are included in , which together maximized the 4-fold cross validation score .Despite the low dimensionality of the initial dataset (69 samples), an optimal combination of factors was found, the discriminative potential of which was evaluated with the utilization of four different classification algorithms. Feature selection was performed to find the optimal factor subset for correct classification, and resulted to a subset of 25 factors  The SVM classifier had the maximum mean AUC (0.85). The other classifiers yielded lower scores but confirmed the discriminative potential of the factors subset, especially LDA with 0.84 mean AUC.The performances of the different classifiers, trained with the subset, are shown in . For each patient, the expression level of each factor was compared to the median expression among all 69 patients. Since the visualization of the separation of the two groups was not ideal, we further filtered and ranked the 25 factors by applying an additional, entropy-based filtering method (information gain), which measures the importance of the factors with respect to patients classes using an entropy-based criteria. For the new heatmap representation we retained the 5 factors , that are identified by both Feature Selection methodologies and were ranked higher than the others in their contribution in the final signature. Hence the new heatmap with 5 factors depicts a subspace of the final signature which shows a tendency of over-expression of those factors in the Sensitive class and was more prone to visualization .The differences in the expression levels of these 25 factors among the patients are depicted in the HeatMap of . By this approach the clinical value of the array kit as a diagnostic tool for sensitive/resistant patient classification is assessed more accurately, because LOOVC mitigates more effectively the phenomenon of “over-fitting” , in whicTable 5. With the SVM classifier, 62 out of the 69 patients were classified correctly using the subset of 25 factors. This success rate (~ 90%) confirms that the angiogenic factor array kit can be used for accurate platinum sensitivity prediction in combination with the SVM algorithm. On the contrary, the subset of 5 factors resulted in low prediction performances.The classifiers' performances are shown in For these analyses only the optimal “signature” resulting from the subset of the 25 factors was used. Median follow-up was 71 months (95% CI 48–99). During follow-up, 59 patients relapsed and 46 died from ovarian cancer. Median OS was 35 (95% CI: 24–41), while median PFS was 12 months (95% CI: 9.5–15). Histology, grade and residual disease after debulking surgery were not associated with PFS or OS. There was a trend towards shorter PFS and OS for women older than 60 years .. Multivariate analyses including age and protein profile showed that only protein profile retained its prognostic significance both for PFS  and OS .Median VEGF ascites level was 1777 pg/ml. There was no association of VEGF level (below or above the median) with PFS (p = 0.836) or OS (p = 0.977). This result did not change when other cut off levels between 1000 and 2500 pg/ml were studied. There was no significant difference of the median VEGF level between the “resistant” and the “sensitive” protein profile . On the contrary, the “resistant signature” was associated with significantly inferior median PFS  and OS  In the current study, we developed and tested a bedside-compatible “angiogenic signature”, which was strongly associated with PFS and OS in women suffering from advanced ovarian cancer. The methodology we used has produced promising prognostic tools in other types of cancer –23, althTable 4). Although VEGF plays a major role in the angiogenic process, it was not among them and VEGF levels in ascites did not predict for PFS or OS The latter could be attributed to the relatively small sample size but it should also be stressed that the prognostic and predictive value of this factor in ovarian cancer has been controversial. We and others have shown the prognostic significance of VEGF in serum or ascites in advanced ovarian cancer [Angiogenic profiling was based on the expression of 25 angiogenic factors , is a powerful mitogenic peptide produced by different tumors including ovarian cancer. ET-1 was found to be over expressed in patients who do not respond to chemotherapy. This is probably associated with the interference of ET-1 on cell functions such as proliferation, drug-induced apoptosis, invasiveness and epithelial-mesenchymal transition –38. SimiFinally, two factors of the epidermal growth factor family (EGF) were also included among the 25 significant factors. Tanaka et al  have shoTechnical issues, such as the addition of heparin in the collected ascites, are unlikely to have affected our results. Although theoretically the presence of heparin might interfere with HB-EGF or VEGF levels, the amount of heparin we used is much lower than that used by standard manufacturers for effective coagulation, while the isoforms present may not be binding the ligand with high affinity as shown in other studies . Our arrThere are certain limitations associated with this report. The number of patients included was relatively low. Furthermore, a commercially available kit was used because our intention was to leverage protein profiling for eventual and widespread use in everyday practice. It is possible that other factors might also be useful but this would complicate the practicability of this method. In spite of the limited number of patients, the results are robust, suggesting that this method may be used to develop a reliable tool for defining prognosis and make therapeutic decisions in advanced ovarian cancer. For this reason validation in independent datasets is underway. It is important to underline that no anti-angiogenic therapies were used in the current cohort. In order to assess the potential of this model as a means of selection for anti-angiogenic therapies, ideally evaluation in a randomized population should be sought . NeverthMolecular factors not included in our array, such as gp100, ERCC, CD44, CD147, c-met, IL-6, ALDH, CD117 and BMP2 have also been studied in the context of chemoresistance in ovarian cancer. –51. AlthIn conclusion, the panel of 25 angiogenesis-related factors provides a readily available tool, which can improve outcome prediction beyond that provided by age, grade, histotype and quality of primary surgery in patients with stage III and IV ovarian cancer. We believe that these findings may lead to the development of a tool to be used for patients’ selection for anti-angiogenic therapies. Future work is necessary to validate this model in prospective trials including patients treated with modern anti-angiogenic therapies.S1 File(RAR)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(DOC)Click here for additional data file.


The exacerbation of HIV-1 associated dyslipidemia seen in a subset of patients receiving anti-retroviral therapy suggests that genetic factors put these individuals at greater risk of cardiovascular disease. Single nucleotide polymorphisms (SNPs) within genes of and influencing the reverse cholesterol transport (RCT) pathway are associated with lipid levels but little is known regarding their copy number variation (CNV). This form of quantitative genetic variation has the potential to alter the amount of gene product made, thereby also influencing lipid metabolism.CETP gene, which showed a loss of copy in 1 of the 320 samples (0.3 %) in our study. The genes in our study showed little variation in expression between individuals, and the variation seen was not related to any detected CNV.To examine if CNV in RCT pathway genes was associated with altered serum lipid profiles in HIV-positive individuals receiving therapy, we designed a custom multiplex ligation-dependent probe amplification assay to screen 16 RCT genes within a subset of individuals from the Multicenter AIDS Cohort Study who show extreme lipid phenotypes. Verification of CNV was performed using a custom NanoString assay, and the Illumina HT-12 mRNA expression microarray was used to determine the influence of copy number on gene expression. Among the RCT genes, CNV was observed to be extremely rare. The only CNV seen was in the Whole gene CNV is uncommon in RCT pathway genes, and not a major factor in the development of highly active antiretroviral therapy (HAART) associated dyslipidemia.The online version of this article (doi:10.1186/s13104-015-1665-z) contains supplementary material, which is available to authorized users. Individuals infected with HIV-1 exhibit changes in serum lipid levels seen as hypercholesterolemia and hypertriglyceridemia . FollowiPrevious studies have shown that this dyslipidemic profile is associated with greater risk for cardiovascular disease (CVD), myocardial infarction and atherosclerosis in HIV-positive individuals , 5, 8–11APOA5 gene, was associated with increased triglycerides and decreased HDL-C, while a SNP in the LPL gene, rs328, was associated with increased levels of HDL-C [APOE were found to be associated with lipid levels where the E2 allele has a protective effect against dyslipidemia while the E4 is indicative of more risk [LDLR can be modified by mutations in the proprotein-convertase subtilisin-kexin type 9 gene (PSCK9), ultimately resulting in altered levels of LDL-C [PCSK9 show decreased amounts of LDL-C while those with gain of function mutations have increased amounts [Furthermore, several genome-wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) associated with CVD risk –25, manyof HDL-C . Additioore risk . Furtherof LDL-C , 36, 37. amounts , 33, 38.PCSK9, protein levels of RCT gene products could also be influenced by copy number variation (CNV). This type of genetic variation includes duplications, deletions, and inversions of DNA segments greater than 50 bp in size [CCL3L1 and DEFB4 genes illustrate that an increase in transcriptionally available copies of a gene not only results in increased expression levels but also increases in protein levels directly proportional to the number of copies [LDLR gene associated with Familial Hypercholesterolaemia [LPL, ABCA1, and LIPC [In addition to posttranslational protein regulation such as that seen with  in size –42. Prevf copies –46. Suchf copies –32, 47, rolaemia –51 and tand LIPC , 52. Furand LIPC .Here, we designed a study employing custom Multiplex Ligation-dependent Probe Amplification (MLPA) and NanoString probes to screen for CNV in 16 RCT associated genes in participants from the Multicenter AIDS Cohort Study (MACS), to identify if CNV is present, the degree to which it varies, and whether it has an association with the abnormal lipid metabolism observed in HIV-positive individuals undergoing antiretroviral therapy.Using the 2005 clinic measurements, and the NCEP/ATP III report criteria  HDL-C ≤. The demOf the individuals who were HIV-positive during 2005, over 70 % were receiving a type of antiretroviral therapy . Of thoAs reference samples with known copies of the RCT genes are not available, we identified experimental samples whose normalized peak height for each probe was similar to the sample set mean height. Using these samples as references, the coffalyser.net software calculated the probe ratios for each sample relative to the reference samples. Assuming that the most frequently observed ratio corresponded to two copies per diploid genome, ratios above 0.7 and below 1.3 are considered to be within the normal range of two copies . AnythinAPOA4, CETP, and ABCA1) showed any signs of CNV, and in each case the CNV was extremely rare. For each of these genes, a few individuals showed ratios that crossed or were at the lower threshold  while sample 157 had a loss of CETP copy number with a ratio of 0.65. Sample 209 had a normalized ratio for ABCA1 that fell on the 0.7 threshold indicating a possible loss. The standard deviations for each of these outlying probes were relatively small (<±0.05) indicating that the decrease in ratio observed was likely genuine. When MLPA was performed for a second time on these 4 samples, the observations were consistent with the first run. No clear association between any of these losses and lipid levels (Table Two samples 123 and 367) had a loss of 3 and 367ABCA1 and SRBI. While their IQRs were slightly broader than the other RCT probes, this spread of the ratio distribution was also seen in the properly functioning reference probes suggesting that this was within the normal range of our experiment (data not shown).The interquartile ranges (IQR) for most of the RCT gene probes were narrow (0.04–0.09), and similar to those of the two-copy reference probes. This tight clustering of ratios around the mean of each probe further suggests that CNV is not common in the RCT genes. The only probes with wider IQRs were DEFB103A and CCR5 genes, we were able to verify that our assay can identify a range of CNV even when the sample mean is used to define a referent  contained no SNPs within their ligation sites.To determine whether the rare loss for the three RCT genes identified during MLPA reflected true CNV or problems with probe binding, Sanger sequencing was performed to examine the probe binding site for those individuals who showed losses, plus several control individuals who showed no changes in copy number. We determined that individuals who showed a loss in signal for CETP in sample 157 (copy number ratio of 0.58) but did not observe the losses for ABCA1 in sample 209 (1.06) or APOA4 for both samples 123 and 367 (1.10 and 1.04). As the MLPA-derived ratio for ABCA1 in sample 209 fell on the threshold value of 0.7, it is likely that this sample does not in fact have a true loss in copies. It is also possible that MLPA probe used for ABCA1 is picking up a rare small CNV that is not detected by the NanoString probe, as the probes for these assays bind in different regions of the gene. The loss observed in APOA4 only by MLPA is attributed to the ligation-site SNP identified in the MLPA probe.We confirmed our findings by using a custom NanoString assay to measure CNV of the RCT genes for 267 of the samples analyzed by MLPA. The CNV ratios generated mirrored those seen with MLPA (data not shown). We replicated the loss in copy number of We also determined the expression levels of the RCT genes in our study, using data extracted from a whole-genome transcription dataset obtained using the Illumina HT-12 platform. Gene expression levels on 127 samples were compared to both MLPA and NanoString CNV ratios. As expected, comparisons of MLPA- and NanoString-generated CNV ratios to log transformed mRNA expression levels yielded no significant associations Fig. .Fig. 3ExThe contribution of host genetic variation to the development of the CVD-associated side effects seen in response to antiretroviral therapy is still not fully understood. We have previously studied the roles of Biogeographical Ancestry , and of CCL3L1 and DEFB4 genes, where increases in gene products correspond to copies present for each gene up to a plateau point [LDLR, LPL, ABCA1, and LIPC) [While previous studies have already found an association between sequence variation in genes within, and influencing, the RCT pathway and lipoprotein levels, we theorized that CNV in the RCT pathway could play a role in these extreme lipoprotein phenotypes. A region of duplication encompassing an entire gene and its regulatory regions has the capability to alter expression and protein levels in a manner directly proportional to the amount of copies present. Such a relationship is observed for the au point , 46. Evend LIPC) , 52.vs. strongly atherogenic). Our hypothesis was that CNV encompassing the full length of a RCT gene would result in an increased or decreased amount of transcribed protein product directly proportional to the amount of copies present, thus impacting serum cholesterol levels. Further, we wished to investigate whether individuals with CNV in the central range would have normal lipid levels while those whose CNV was in the outermost edges of the range would have a dysregulated lipid metabolism leading to the extreme lipid profiles.Data currently available within the Database of Genomic Variants  shows a CETP showed a loss in a single individual, and two genes (ABCA1 and APOA4) showed apparent copy number losses with MLPA. The apparent loss of copy number seen for APOA4 was determined to be due to a SNP in the ligation site of its MLPA probe while the apparent loss seen at ABCA1 is suspected to be not genuine. The small standard deviation seen, along with the reproducibility of the significant loss of signal during additional MLPA runs, indicates that the loss for the CETP probe was valid. Coupled with the tight clustering around the normalized ratio of 1.0 for the non-outlying points of all of the RCT probes, these results strongly suggest that whole gene CNV is not present in the RCT genes at anything above very low levels, and is therefore not likely to be a major influence on lipid levels in either the normal population or those infected with HIV and receiving antiretroviral therapy.Our results in this study identified rare loss variants in 3 of the RCT genes. Out of 267 individuals and 16 genes studied with two different CNV assay procedures, CETP. The ABCA1 gene was observed to have a wide variety of insertions and deletions within its bounds, including five losses in the region of our MLPA probe, although none of them encompass the entire gene. All of these reported variants were extremely rare, with only a few individuals having the variant in studies containing several thousand participants. For those with higher frequencies, the study sizes were too small to conclude that a common variant was observed.These findings are consistent with previous reports of limited structural variation in the RCT genes, as presented in the Database of Genomic Variants , 57. WitWe also compared the ratios obtained with both CNV assays to gene expression data available for a subset of our samples. None of the genes for which we had available CNV data showed variation in expression level. This further suggests that it is unlikely that significant CNV is present in these genes that might affect expression.LPA gene contains many kringle repeats that were not a focus of our study.Our study was designed to detect any gene-specific CNV in members of the RCT pathway that may impact levels of gene expression and subsequent enzymatic activity that therefore affect lipid levels and lead to dyslipidemia in members of our study cohort. Our study does have some limitations. As no prior survey of CNV in these genes had been attempted, the frequency of such variants was unknown and our study was exploratory. As such, it had limited statistical power to assess the impact of CNV between the atherogenic and atheroprotective groups. Also, the sizes of the atheroprotective (n = 23) and atheroprotective (n = 7) groups are not evenly matched, which would preclude more advanced statistical analysis were we to have found CNV in our samples, but the overall sample size of 319 individuals successfully typed is large enough to have detected any common variants and we can conclude that CNV of these genes is rare or absent. As we only designed one CNV probe set per gene it is also possible that variation within a gene could have been missed—for example, the Our study was also limited by the type of clinical data available. In each biannual visit, serum cholesterol and triglyceride measurements are obtained from our study participants, and were used to define our atherogenic and atheroprotective groups. Other measurements, such as lipoprotein particle size and number, and HDL efflux capacity, have been shown to be stronger predictors of cardiovascular disease risk , 59, andIn this study we were able to develop a MLPA assay capable of detecting CNV when it is present. Using this assay we have illustrated that whole gene CNV is present only at very low levels in the RCT genes, and is not a major factor in the development of HAART-associated dyslipidemia. Thus, other host genetic influences exist and need to be identified before we are able to understand fully the ways in which host, viral, and therapeutic environmental factors interact to determine the outcome of antiretroviral therapy in HIV-positive individuals.Experimental samples were obtained from the Multicenter AIDS Cohort Study (MACS). The MACS is a multicenter  ongoing prospective study, founded in 1984, of the natural and treated histories of HIV-1 infection in men who have sex with men. Participants attend clinics bi-annually for a physical exam and sample collection, and complete extensive questionnaires about their medical history, behavior changes, and overall quality of life. All samples were obtained from volunteer participants who had read and agreed to the consent policy implemented by the MACS for the protection of human subjects, and approved by the Institutional Review Board (IRB) at each MACS site .Samples (n = 366) were identified on the basis of serum lipid measures obtained in their 2005 visit. We used the Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III)  to identDEFB103A gene was already known [We also obtained 4 DNA control samples  from the Coriell Institute cell repository. Three of these individuals served as reference samples, as the CNV of their dy known . We alsohttp://bioinform.arcan.stonybrook.edu/mlpa2/cgi-bin/mlpa.cgi) [MLPA was performed using the MRC-Holland SALSA MLPA P300 Human DNA Reference-2 (Version: A1-0410) probemix kit, in conjunction with 16 custom probes for RCT pathway genes (Table lpa.cgi) , 65. BLAMLPA reactions were performed following the standard 1 tube protocol  with an 18–19 h hybridization and amplification step using the new universal primers (released in June 2011). Fragment separation was performed at the University of Pittsburgh Genomics and Proteomics Core Laboratories.http://wiki.coffalyser.net) was used to perform fragment and comparative analysis. Initially, no reference samples were indicated, and samples with average signal across all probes were selected as references for each of the four 96 sample runs. Fragment analysis was then performed for a second time with default settings. Samples with poor reference probe quality and reproducibility were removed before comparative analysis was performed for a second time. Final ratios and standard deviations were analyzed with the R statistical software package [The Coffalyser.net software , defined by MRC-Holland and based on a 2-copy reference sample  , were us.3, definA NanoString nCounter custom CNV CodeSet was designed containing the 16 RCT genes analyzed in our MLPA assay, and used to type 267 of the experimental samples previously typed by MLPA. DNA was processed and analyzed using a NanoString Technologies nCounter system at the University of Pittsburgh Genomics and Proteomics Core Laboratories. The NanoString nSolver Analysis Software (v1.1) was used to generate normalized ratios from raw counts, with 34 of the original MLPA samples set as RCT references.Gene expression data were available for 127 of the samples studied by MLPA and NanoString. These were obtained from whole blood collected into PAXgene tubes, from which RNA was extracted using the PAXgene Blood RNA Kit IVD. Samples were quantified, processed, and analyzed using the Illumina Human HT-12 v.4 whole-genome expression array at the University of Pittsburgh Genomics and Proteomics Core Laboratories. Raw data was exported from Illumina Genome Studio and further data analysis was performed using the Bioconductor R modules lumi  and Arrahttp://statepi.jhsph.edu/macs/forms.html.Data are held by the Center for Analysis and Management of the Multicenter AIDS Cohort Study (CAMACS). For access to the MACS data, please complete the collaboration concept sheet and identify the article for which the data were used. This form and instructions may be found at:


Aeromonas salmonicida and the virus VHSV, using microarray analysis at four early life history stages; eyed egg, post hatch, first feeding and three weeks post first feeding when adaptive immunity starts to be effective. All stages responded to both infections, but the complexity of the response increased with developmental stage. The response to virus showed a clear interferon response only from first feeding. In contrast, bacterial infection induced a marked response from early stages, with modulation of inflammatory, antimicrobial peptide and complement genes across all developmental stages. Whilst the viral and bacterial responses were distinct, there were modulated genes in common, mainly of general inflammatory molecules. This work provides a first platform to explore the development of fish immunity to infection, and to compare the age-dependent changes (from embryo to adults) across vertebrates.During early stages of development vertebrates rely on an immature immune system to fight pathogens, but in non mammalian species few studies have taken an in-depth analysis of the transition from reliance on innate immune mechanisms to the appearance of adaptive immunity. Using rainbow trout as a model we characterized responses to two natural pathogens of this species, the Gram negative bacterium  In most non mammalian vertebrates, pre- and post- hatch life occurs in an unpredictable, hostile external environment. This is in contrast to mammals where the embryo is in an almost sterile environment where pathogen exposure is controlled by the mother. Hence, fish eggs and embryos have to be able to fight efficiently against infections, and must possess active defence mechanisms.In teleost eggs, the first line of defence against invading pathogens is the physical barrier of the chorion and membranes1456A number of critical transitions occur during embryo and fry development. Hatching is a first drastic change. Mouth and gut opening and first feeding stages represent another critical transition: as the gut microbiota is establishedIn fish as in other vertebrates, innate immunity is particularly important in early phases of development, when adaptive immunity is still not (fully) established and functioning. Triggering of innate receptors specific for pathogen-specific molecular patterns activates a variety of signalling pathways. Generic defence mechanisms like proinflammatory responses lead to wide cell activation, but pathogen type-specific mechanisms like the type I interferon (IFN) response are also triggered. Essential players of this pattern recognition receptor (PRR) system are the Toll-like receptors (TLRs) and the retinoic acid-inducible gene I (RIGI)-like receptors (RLR)910111314Here we examine for the first time the divergence between responses to bacterial and viral infections during early development in a teleost fish. We exposed key development stages from eyed eggs to free feeding animals to two pathogenic models; a virus and a bacterial species. Transcriptomic profiling demonstrated bacterial-induced and common pathogen defences at early stages with a clearer specific response to the virus at the older stages. We found at the time of first feeding that components of the acquired immune system also become more responsive. The profiles substantially increase the understanding of complex molecular responses to pathogens, and also represent a valuable tool to aid improvement of disease control and vaccine development in aquaculture. There are common themes that appear conserved on an evolutionary scale with mammals, in addition to unique features in relation to early protection required for independent living.A. salmonicida, for which comprehensive descriptions of infection-induced modifications of the transcriptome have been reported in adult tissues16viperin/rsad2 and IL-1β, these markers indicating either an induced antiviral response (viperin) or proinflammatory response to bacteria (IL-1β). No clear response could be detected after bath infection, indicating that eyed egg to fry stages are well-protected from natural infection. In contrast, the injection route of infection led to up-regulation of the marker genes , VHSV and er genes  and to aer genes .A. salmonicida, we used a microarray platform enriched for immune related genes to identify genes differentially expressed between infected and uninfected control animals at each stage for each pathogen. In this way we were able to analyse the pathogen-specific response at each time and also examine how the different pathogens induced common or specific responses during ontological development (Table S1 and S2).To perform a comprehensive analysis of the transcriptional response induced by injection of VHSV and A. salmonicida, a completely different pattern was obtained, with a clear discrimination of infected and non-infected fish already at EE and H stages. Unlike the viral infection, the magnitude of the response at the FF and 3WFF did not appear to increase compared to earlier life stages.To gain an overview of the global responses, principal component analysis (PCA) was separately performed with hybridization datasets for viral and bacterial infections . The traPval < 1%, fold change >2 or <0.5) due to the infection at each stage  whereas the number of GO enriched biological processes in bacterial infected fish was much more consistent over age with 38 in EE and 59 in 3wFF.To further characterise the responses we used both gene ontology  enrichment analysis and KEGG pathway analysis to identify key functional groups that were enriched across each developmental stage. Results from this analysis showed a clear difference between the antiviral and antibacterial transcriptional response . The enrFor the viral responses, GO terms that were significantly enriched confirmed a major change in transcriptional activity. The magnitude of the enrichment  is shown in To test this hypothesis, we performed a specific analysis of gene expression in Toll-like receptor and RIG-I signalling pathways. We mapped up- and down-regulated genes on KEGG for TLR and RLR pathways at each developmental stage, using a combination of DavidA. salmonicida at any stage of development (set 1) and (2) genes that are among the “top20” for both pathogens for at least at one stage (set 2)  is shown in  (set 2) .When arranged from early to late genes, the expression heatmap reveals significant patterns. Within set 1 (“VHSV specific Top20 genes”) that comprises many well known interferon stimulated genes (ISGs) including viperin, CD9, ISG15 and others, fold changes greatly increase at the later stages. These genes are generally not induced by the bacterial infection, or only at low magnitude in later stages. These observations indicate that a strong, typical type I IFN response is triggered only at FF and 3wFF stages.A. salmonicida shared Top20 genes”) comprises genes up-regulated at most stages and do not generally show higher fold changes at later stages. These genes are also well-induced by A. salmonicida infection at most stages, and are mainly inflammatory and acute phase effectors such as antimicrobial peptides, serum amyloid proteins, and metalloproteinases. A few genes of set 2 (bottom of the panel) show more stage-restricted induction patterns, but these patterns are parallel after viral and bacterial infection which also suggests roles in general inflammation.In contrast, set 2 (“VHSV and ie. not by the bacteria at any stage), were induced only at 3wFF (46%) or only at FF and 3wFF (39%). Furthermore, among genes modulated by both VHSV and A. salmonicida, 70% of genes modulated at three or four stages after viral infection were also modulated at least at three stages after bacterial infection.Importantly, this was not restricted to the “top20” gene list. Indeed, 85% of genes modulated by the virus only . These GO identifiers include many key genes well characterised in inflammation such as IL-1β, transcription factors such as CEBP and cell surface lectins. We found that there is little consistent increase in the significance of the enrichment with ontological age and that many of the key genes up-regulated show a similar response at all stages. We did observe that the number and significance of enriched biological processes was reduced at the hatch stage; however, this stage had the highest number of genes altered in response to the infection which appears to impact the relative enrichment value. The major host response to To counteract the inflammatory response, transcripts encoding anti-inflammatory factors such as SOCS3 were increased in expression at all stages, however IL-10 was not increased in these fish. The GO identifiers related to cellular homeostasis are enriched at the later stages of development and include transferrin, ferritin and other serum proteins such as serum amyloid A , two patterns could be distinguished: (1) genes that are induced only at early stages (EE and H) by bacterial infection ; they are generally not induced by VHSV and appear as specific markers of the “early” response to bacterial infection, and (2) the other genes of set 3 which are induced at the later stages (FF and 3wFF) by A. salmonicida; these genes are often also up-regulated by VHSV at these stages although to a lower magnitude. This pattern is consistent with distinct transcriptional responses to the A. salmonicida infection at early and late stages.Among the list of the “top20” genes up-regulated by Aeromonas infection there are a number of genes that are consistently decreased in expression in all the early life stages. The collagens are the most pronounced genes with col1, 2, 4, 11 and 12 all being reduced in expression. These collagen genes are found in the GO analysis under “extracellular matrix organization” and “collagen metabolic process”. A second group of genes consistently down regulated encode structural proteins and are related to the GO term “muscle system process”. They include a variety of myosin heavy chain genes in several stages and myomesin, a protein key to myofibril development in striated muscle, which is decreased at all stages following bacterial infection. Together these results show that muscle growth and structural organization is being inhibited, most likely due to energy (incl. cellular energy) being directed towards immune function.Although the majority of genes are increased in expression following the A. salmonicida do not represent a large generic set of permanent inflammatory markers inducible from an early stage. On the contrary, this subset of dual responsive genes greatly expands at later stages.To investigate further the extent to which transcriptional responses to viral and bacterial challenge overlap during early trout development, we determined the numbers of genes co-modulated at each stage of development . The proportion of genes up- or down-regulated by both pathogens represented 10 to 30% of the total number of responsive genes at the studied stages . In fact, the large majority of the genes modulated by both infections was expressed at FF and/or 3wFF only (253 out of 326), and only 16 genes were common to the intersect at all stages . Overall, these observations indicate that genes common to the transcriptome response to VHSV and Thus, two main sets of co-modulated genes can be distinguished:(1) Forty-two genes that are co-induced by viral and bacterial infection in at least three stages . These gA. salmonicida. Several lectins also participate in both responses, reflecting their role in general inflammation.(2) A larger set of genes (247 genes) are co-modulated in viral and bacterial infection only at FF and/or 3wFF stages . This seThe remaining genes are induced with moderate fold changes (2 to 3), and although most are not directly associated with immune function, demonstrate that many other biological processes alter in the animals during the response to pathogens. They may be up-regulated as a side effect of the inflammatory response, or be involved in metabolic changes related to energy reallocation and lipid metabolism.A. salmonicida only at later stages, which comprises ISGs that are highly up-regulated by the virus, and somewhat induced by the bacterial infection.Overall, our data identify a gene subset modulated at all developmental stages by both pathogens in which antibacterial peptides appear as key protective factors, and a gene subset induced by VHSV and Teleost eggs and fry come into contact with many pathogens as their immune system matures and whilst their early defence mechanisms are mainly restricted to innate immunity. However such early stages of development are not particularly vulnerable to infections, and we could not infect trout eggs by bath challenge in concentrated viral or bacterial suspensions. In fact, the egg envelope is very effective at preventing pathogen entry, and hatching and first feeding therefore constitute key transitional stages with respect to the interactions of the developing fish with the pathogen world.ie. just around the time of mouth and gut opening/first feeding or before23Innate immune pathways triggered by viruses and extracellular bacteria are generally considered to be distinct but their role in the defence of the developing fish remains poorly characterized. As zebrafish larvae have been used to set up models for human infectious diseases2123242526Our data show that the development of virus- and bacteria- induced responses follow different dynamics, highlighting the importance of studying various life stages. The antiviral response shows a progressive development, with a few typical ISGs induced at EE and H stages, and a typical IFN based response at FF, which expands further at 3wFF. Importantly, the master antiviral cytokines, type I IFNs, are not induced at the early stages, raising the issue of the differentiation of IFN- and ISG- producing cells.The trout data presented here regarding the response to VHSV suggest that IFN is not induced in all cell types and significant transcriptional response is observed between H and FF stages. Later expansion of specialized cells involved in adaptive immunity between FF and 3wFF, which is reflected in expression of genes involved in antigen presentation, likely explains the increase of the ISG repertoire expressed at the most mature stage analyzed. Accordingly, previous studies on response to CHIKV infection, which induced a very strong IFN response in zebrafish larvae (around 5dpf) underlined the key role of neutrophils in IFN productionA. salmonicida is already considerable at the EE stage. However, the transcriptome response goes through large-scale and unexpected transitions across the four analyzed developmental stages with about half of the genes induced at EE and H being stage specific. Interestingly, very few of the “top20” induced genes at EE were specific to this stage; thus, genes induced by A. salmonicida only at the EE stage are up-regulated (moderately) either in EE stage-specific cells, or by cells that become unresponsive at later stages. In fact, some of the genes that were induced only at the EE stage may be members of gene families in which distinct members with redundant functions are induced at different stages. In this respect, our data provide a resource for understanding natural resistance at early stages and guiding selection.In sharp contrast, the response to While specialized responses to viral and bacterial infections trigger different pathways and cell types/tissues, some mechanisms were triggered by both types of pathogen. It is well known that proinflammatory cytokines such as IL-1β and TNFα, which are involved in fever in homeotherms, are induced by many different infectious pathogens. In fact, the actual impact of inflammation on the infecting pathogen and on the well-being/survival of the host is a delicate equilibrium. In our data, two main pathways might be of special importance for the effective defence against both types of pathogens: the complement cascade and the IFN pathway itself.Our data not only represent the first diachronic comparison between fish responses against a virus and an extracellular bacterium, they also provide an excellent platform to compare the maturation of the pathogen sensor-induced responses between fish and mammals. In humans, four main phases have been proposed recently in the development of the immune systemin utero, and with a need for mutual tolerance between mother and foetus. In teleosts, the EE stage is also well protected by its envelope, but does not need to develop any tolerance to mother. However, vertical transmission might occur for fish bacterial pathogens, when capacity to respond is required. Our data suggest that the antibacterial response is significant, and we did not detect any induction of IL-10 or other regulatory cytokine; the IL-10R is in fact down-regulated in eggs injected with A. salmonicida. The hatching of fish eggs is hardly equivalent to birth in humans and there are certainly no mature T cells at this stage. However, the type I IFN response is still low, while orthologues of several interleukins or interleukin receptors that may be involved in Th17 differentiation in mammals are induced at this stage by A. salmonicida (IL-11 and IL-21R)A. salmonicida did not clearly increase in trout from EE to 3wpFF. In contrast, the IL-2 receptor (CD132) was not modified during the EE stage but was increased at later stages, which might suggest a greater level of maturation of the Th1 cell lineage.This immunosuppressed pattern of the first phase is consistent with the quasi sterile environment of the foetus We find that during the ontological development of teleosts the responses take different trajectories depending on whether the immune stimulus is viral or bacterial. Our data allow us to draw a parallel between these changes to the mammalian “phases” of immune ontology.All animals were handled in strict accordance with good animal practice as defined by the European Union guidelines for the handling of laboratory animals5 pfu per ml), or with A. salmonicida (106 pfu per ml) for 24 h, and samples collected at 3 and 6 days post infection. For the injection challenge, 20 individuals of each stage were injected with 1 μl of PBS as uninfected treatment control, 1 μl of VHSV (108 pfu per ml), or 1 μl of A. salmonicida (106 pfu per ml), and collected 3 days post infection. At the EE stage there was 30% and 50% mortality within 24 h in the VHSV and A. salmonicida groups respectively, suggesting mechanical damage to these eggs. No significant mortality due to the injection was observed in other groups. The experiments were performed at 10°C.A rainbow trout double haploid clone, named B57http://www.ebi.ac.uk/arrayexpress/ : “Trout_imm_v1”_(Agilent array design 028918) with platform accession A-MEXP-2315. The hybridizations were performed as described in The microarray platform was custom designed and derived from rainbow trout EST tentative contigs (TC) from Rainbow Trout Gene Index Release 7.0 (July 2008). 40,125 oligos from this source were the basis of the array, and were supplemented with further immune related transcripts from more recent releases to NCBI designed using the Agilent e array oligo design software . A further 1,417 Agilent in house control features were included. Full details of the array platform are available at EBI array express .


Alcohol misuse in England costs around £7.3 billion (US$12.2 billion) annually from lost productivity and absenteeism. Delivering brief alcohol interventions to employees as part of a health check may be acceptable, particularly with online delivery which can provide privacy for this stigmatised behaviour. Research to support this approach is limited and methodologically weak. The aim was to determine the effectiveness of online screening and personalised feedback on alcohol consumption, delivered in a workplace as part of a health check.This two-group online individually randomised controlled trial recruited employees from a UK-based private sector organisation . 3,375 employees completed the online health check in the three week recruitment period. Of these, 1,330 (39%) scored five or more on the AUDIT-C  and were randomised to receive personalised feedback on their alcohol intake, alongside feedback on other health behaviours (n = 659), or to receive feedback on all health behaviours except alcohol intake (n = 671). Participants were mostly male (75%), with a median age of 48 years and half were in managerial positions (55%). Median Body Mass Index was 26, 12% were smokers, median time undertaking moderate/vigorous physical activity a week was 173 minutes and median fruit and vegetable consumption was three portions a day. Eighty percent  of participants completed follow-up questionnaires at three months. An intention to treat analysis found no difference between experimental groups for past week drinking (primary outcome) ), AUDIT  and health utility (EQ-5D).There was no evidence to support the use of personalised feedback within an online health check for reducing alcohol consumption among employees in this organisation. Further research is needed on how to engage a larger proportion of employees in screening.ISRCTN50658915International Standard Randomised Controlled Trial Number Register  Alcohol misuse is among the leading risk factors for disease burden across the globe, after high blood pressure and smoking There have been relatively few trials evaluating the effectiveness of SBI for alcohol misuse in the workplace setting. In 2009, a systematic review of workplace interventions for alcohol-problems One of the challenges with delivering SBI to employees in the workplace is the stigma associated with accessing services for alcohol misuse in this setting Another way of addressing the stigma surrounding SBI for alcohol in the workplace may be to deliver it in the context of a health check The aim of this study was to determine the effectiveness and cost of screening and personalised feedback on alcohol consumption, delivered as part of an online health check in a workplace setting. It was hypothesised that participants receiving the personalised feedback on alcohol consumption would reduce their alcohol intake more than those not receiving the feedback.www.controlled-trials.com/ISRCTN50658915). The protocol for this trial and supporting CONSORT checklist are available as supporting information; see This was a two-group, individually randomised controlled trial, conducted entirely online. Ethical approval was granted from University College London (UCL) Research Ethics Committee (4213/001) and the trial was registered with UCL's data protection officer. The trial protocol has been published This study was conducted online in a large UK-based organisation with an international workforce of around 100,000, with the majority of employees based in the UK. The company has an active occupational health team which runs frequent campaigns aimed to increase awareness and understanding of health behaviours, engage people to take personal responsibility for their health, and to change attitudes and behaviours. Campaigns often include online information, assess risk, facilitate monitoring activity, share information, present prizes to winners of competitions, and include: virtual gyms, road shows, health fairs and articles in newsletters. The organisation has worked with other academic institutions, which meant its employees were familiar with the process of taking part in research and answering questions about their health via the workplace. Employees aged 18 years and above were eligible to take part if their drinking put them at increased risk of alcohol-related harm, as indicated by a score of five or more on the Alcohol Use Disorders Identification Test – Consumption  Employees were invited to take part in a confidential online health check and receive personalised feedback on a range of behaviours known to impact on their health and wellbeing. The study advertisement was edited by the organisation's occupational health and communications teams into their “language” and posted on the company's web-portal in August 2012 for three weeks. All employees login through this portal to access any resource or service. We had intended to recruit participants to the study via individual email sent from the occupational health team to all employees The online health check asked employees about their height and weight , alcohol consumption, smoking status, fruit and vegetable consumption and level of physical activity. Respondents were then asked for some basic demographic information (see below for further details), before receiving immediate online tailored feedback which either 1) reinforced healthy behaviour and reminded people of recommended guidelines, or 2) encouraged a change in behaviour by highlighting the risk associated with not meeting the recommended guidelines. Feedback for BMI was categorised as underweight, healthy weight, overweight, obese or morbidly obese. Feedback was accompanied by links to relevant NHS Choices webpages and the organisation's own behaviour specific webpages. Employees scoring less than five on the AUDIT-C received feedback that their drinking was within recommended limits, a reminder of these limits, and feedback on the other health behaviours; they were not eligible for the trial and had no further contact with the study team. Employees scoring five or more on the AUDIT-C were automatically entered into the trial and randomised to the intervention or the control group.www.downyourdrink.org.uk) The intervention group received feedback on all health behaviours assessed in the health check, as detailed above, the only difference being their alcohol feedback, which provided criterion or risk-based feedback on the potential harm of drinking above recommended limits The control group received feedback on all health behaviours except alcohol consumption, in a wait-list design. Participants in both arms of the trial received feedback on their alcohol intake after completion of three month follow-up measures. It was not possible to assess long-term differences between groups as both experimental arms received the intervention  after three months.Baseline data were collected in the form of an online health check which asked employees about a range of behaviours, as described above. Alcohol consumption questions were the 3-item AUDIT-C questionnaire All baseline data were collected prior to randomisation. Respondents scoring five or more on the AUDIT-C were randomised by simple randomisation via computer-generated randomisation software to experimental groups in an automated process, therefore concealing allocation. It was not possible to blind participants to randomised groups as they either received the alcohol feedback or they did not. Participants received automated feedback online without facilitation by a researcher, health professional or member of occupational health; therefore obviating the need for blinding intervention providers. In addition, outcome data were self-completed by the participant eliminating bias introduced by an outcome assessor.Participants were contacted by email three months after completing the online health check and asked to complete follow-up questionnaires online via a hyperlink. The primary outcome measure was the TOT-AL, an online measure of self-reported past week alcohol intake that calculates UK units consumed per week (where 1 UK unit  = 8 g of ethanol) Participants received up to three emails at five day intervals, followed by a letter and a phone call. The letter provided the URL to the data collection page, but also contained a paper-based version of past week drinking and the health service utilisation questions. Postal questionnaires displayed the participant's unique identifier and were returned in pre-paid envelopes. Postal questionnaire data were entered into a separate database by an independent researcher and amalgamated into the main database by the trial statistician. Telephone calls served as prompts to complete either online or paper-based measures.736 participants in each group at 3 month follow up would have provided 90% power with 5% significance to determine a 20% reduction in alcohol consumption. Making a 25% allowance for loss to follow-up required randomising 920 participants to each group . Extensive steps were taken to minimise loss to follow-up through tailored reminders as described above.Analyses compared the primary and secondary outcomes between groups at 3 months, following the intention to treat principle. The principal analysis was conducted using a generalised linear model with identity link and Gaussian error. The baseline AUDIT-C score was included as a patient level explanatory variable, along with a classification variable for workplace and for randomised group. Analogous models were conducted for secondary outcomes.Self-reported use of health care resources and number of sick days were used to calculate the mean cost per participant to the English National Health Service (NHS) and employer. Unit costs for health care resource use were obtained from published sources A total of 3,375 employees completed the online health check in the three week recruitment period, which constituted around 3% of the organisation's total international workforce.Of these, 1,330 (39%) scored five or more on the AUDIT-C and therefore entered the trial. Of those participants who were randomised to the intervention group and received feedback on their drinking (n = 659)  19 (3%) Participants were mostly male (75%), in their middle years (median age 48 years), married (77%), with children (68%). Half of participants were in managerial positions (55%). Around 12% of participants were smokers, median fruit and vegetable consumption was three portions a day and median time spent undertaking moderate and/or vigorous activity was 173 minutes a week. Participants in this study were slightly overweight (average BMI 26). All participants scored 5 or more on the AUDIT-C  as this was necessary for inclusion in the trial. The median AUDIT-C score at baseline was six .At three month follow-up, 1,066 (80%) participants completed follow-up questionnaires: 906 (68%) after email prompts, 71 (5%) after letter, 83 (6%) after telephone call, 6 (0.5%) unclear due to missing date data. Alcohol consumption (UK units consumed in the past week) was 5.6% higher in the intervention group who received instantaneous feedback on their drinking alongside feedback on other health behaviours compared with the wait-list control group who received no alcohol feedback, although this was not statistically significant . The median number of units consumed a week in both intervention and control groups at follow-up was within recommended weekly limits  women: intervention median 14 (Interquartile range 9 to 20) units a week, control median 12 (Interquartile range 7 to 17) units a week; men: intervention median 15 (Interquartile range 10 to 24) units a week, control median 15 (Interquartile range 9 to 23) units a week .There were no significant differences between experimental groups for the secondary outcomes. There was a difference of 0.01%  in the AUDIT score. Health utility was −0.2% lower in the intervention group .A post-hoc analysis was conducted to explore a possible floor effect by determining the difference in past week drinking between experimental groups among those participants scoring eight or more on the AUDIT-C at baseline. The analysis compared the 117 participants in the intervention group and 133 participants in the control group and found 14% higher alcohol consumption associated with the intervention  however this was not statistically significant.There were no significant differences in costs between groups . The totThe offer of an online health check attracted 3,375 (3%) employees from a large UK-based organisation in the three week recruitment period. Nearly 40% of employees who completed the health check scored five or more on the AUDIT-C . At three month follow-up, there was no statistically significant difference in past week alcohol consumption between employees who did and did not receive online personalised feedback on their drinking in the context of a health check. There was also no difference between groups in the post-hoc analysis or in any of the secondary outcome measures, namely the AUDIT, EQ-5D index and costs. Nineteen (3%) participants in the intervention group accessed additional help to reduce their drinking via the Down Your Drink website.The findings of this trial do not support the existing literature which shows computer-based interventions may be effective in reducing alcohol consumption when compared with minimally active comparator groups, such as assessment-only or information-only websites A challenge that faces the interpretation of many trials of online interventions is that they evaluate access to the intervention, rather than engagement or use of it To increase the acceptability of the intervention and reduce selection bias, participants were invited to take part in a health check and receive personalised feedback as part of a study; they did not know they were taking part in a trial and that the focus of the study was on their alcohol consumption. The trial has low risk of bias from randomisation sequence generation, allocation to experimental groups and blinding of intervention facilitator and outcome assessor, as outlined in the Only 3% of the total workforce took part in the health check. Comparing the health behaviours of the participants in this study with the general adult population in England suggests that fewer of the participants were smokers Click here for additional data file.Protocol S1Trial Protocol.(PDF)Click here for additional data file.


Here we investigated whether main metabolites of metamizole (dipyrone) in human could be involved in the immune response using the basophil activation test (BAT). We studied subjects with confirmed selective immediate hypersensitivity to metamizole and performed BAT with metamizole and its metabolites: 4-methylamino-antipyrine (MAA), 4-aminoantipyrine (AA), 4-acetylamino-antipyrine (AAA) and 4-formylamino-antipyrine (FAA). BAT results showed an increase of positive results from 37.5% to 62.5% using metamizole plus metabolites as compared with the BAT carried out only with the parent drug, demonstrating that metamizole metabolites have a role in the reaction and can induce specific basophil activation in patients with immediate hypersensitivity to this drug. Our findings indicate that pyrazolone metabolites are useful for improving the in vitro diagnosis of allergic reactions to metamizole.Non-steroidal anti-inflammatory drugs (NSAIDs) are the most common cause of hypersensitivity reactions, with pyrazolones the most frequent drugs inducing selective reactions. Immediate selective hypersensitivity to pyrazolones is thought to be mediated by specific-IgE. Sensitivity of  Adverse drug reactions (ADRs) constitute an important public health issue, causing 3 to 6% of all hospital admissions and occur in 10 to 15% of hospitalized patientsin vitro diagnostic tests for evaluating these patients.The mechanism involved in non-immunological reactions is believed to be based on the inhibition of cyclooxygenase (COX) enzymes in NSAID-sensitive patientsThe mechanism involved in immunological reactions, named single NSAIDs-induced reactions or selective reactions713141516in vitro method which safely substitutes to the direct application of the drug and additionally provides “proof of mechanism” in these reactionsThe diagnosis of selective allergic reactions to pyrazolones is mainly based on the clinical history, skin testing and/or drug provocation test (DPT)142122The aim of this study was to analyze whether metamizole metabolites could be recognised by IgE bound to basophil surface and induce their activation in subjects with confirmed immediate hypersensitivity to these drugs.in vivo method that confirms an IgE mechanism when positive.In order to perform this pilot study, a group of patients with a confirmed selective immediate allergic reaction to metamizole, and a group of tolerant controls were selected. The diagnosis of allergy was based on an immediate skin test, since it is the only With the aim of evaluating which structures derived from metamizole were recognized by IgE from allergic patients to this drug, BAT was performed using metamizole and four metabolites purified from human urine samples  at different concentrations. The optimal cut-off point that corresponds to the best sensitivity and specificity for the native metamizole and its metabolites was calculated by receiver operating characteristic (ROC) curve analysis. With this method we needed to make a balanced decision between sensitivity and specificity. In the case of drug allergy, and especially in severe reactions, the decision must be chosen to increase the sensitivity in spite of a loss of specificity. Finally, in order to confirm an IgE-mediated basophil activation, the phosphatidylinositol 3-kinase (PI3-K) inhibitor, wortmannin (WTM), was used in BAT for those metabolites that showed to induce relevant specific basophil activation.The study included 16 patients, 13 women and 3 men, the median age was 57.5 years (interquartile range (IR): 46.5–63.5 years), 8 had developed anaphylaxis and 8 urticaria accompanied or not by angioedema, and the median interval between the reaction and the study was 109 days (IR: 59.25–365 days). All patients were skin test positive in immediate reading to metamizole  and demonstrated good tolerance to ASA, therefore they were classified as selective reactors to metamizole . The stuDose-response curves studies (0.125 mg/mL–2.5 mg/mL) for the metabolites analyzed showed that the lowest concentration tested (0.125 mg/mL) was not relevant for all the metabolites and the highest concentration (2.5 mg/mL) produced non-specific basophil activation only for the metabolite MAA . BAT resWe tried to confirm the involvement of specific IgE by using a PI3-K inhibitor, WTM. BAT inhibition with WTM was performed with samples from four positive patients to MAA and found a mean percentage of inhibition of basophil activation of 73.96% for MAA and of 85.16%, for the positive control anti-IgE. As expected, no significant inhibition was observed with the positive control N-formyl-Met-Leu-Phe (fMLP), which is a positive control of non-mediated IgE basophil activation .in vitro tests such as BAT are also used for diagnosis with variable results in different studies142027et al.et al.vs. 74.5%).NSAIDs are the drugs most frequently involved in hypersensitivity drug reactions14These tests used the parent drug only, limiting their power to detect the relevant specific IgE antibodies. In the present work we verified if, in addition to metamizole, metabolites of this drug could induce basophil activation. Experimental evidence indicates that several pyrazolone structures can be recognised by a given antibody, indicating cross-reactivity3031Considering the response to the native molecule, metamizole, the sensitivity achieved here was lower than in previous studies14in vivo metabolite of metamizole. Although none of the metabolites are bound to more than 60% of plasma proteinsIt is thought that the molecules that induce basophil activation require covalent binding to proteins. In fact it has been suggested that pyrazolones become antigenic after binding to biomacromolecules following metabolic activationThere is some controversy as to whether selective reactions to NSAIDs are IgE-mediated because specific IgE antibodies had not been identified directly. However, in the case of immediate reactions to metamizole, the results of skin testing and BAT indirectly support that these reactions are IgE-mediated. Further support is given by the tendency for BAT results to decrease over time in these patientsin vitro for propyphenazoneGiven these findings, the next step would be to put these metabolites with the appropriate carriers in different solid phases and test for the presence of specific IgE antibodies using immunoassays. To our knowledge, specific IgE antibodies have only been detected in vivo events that occur after drug administration, including drug biotransformation, in order to improve the sensitivity of the test and to make it clinically viable.In summary, we have shown that metamizole metabolites can be specifically recognised by IgE antibodies bound to the surface of basophils, inducing their activation in subjects with anaphylactic reactions to metamizole. These results deserve further study with an increased number of patients in order to further explore whether MAA is a relevant structure recognized by IgE antibodies. Moreover, more studies are needed to identify the full range of metamizole metabolites and to assess their utility in testing for metamizole allergy. This is the first study demonstrating the role of metamizole metabolites in immediate reactions and the findings obtained here support the hypothesis that drug metabolites may underlie allergic reactions. This may be a major cause of the lack of sensitivity that has been observed when BAT is performed using the parent drug only. It is highly recommended to design BAT to mimic as much as possible the We selected patients with confirmed immediate allergic reactions to pyrazolones. The diagnosis was established by the presence of clinical symptoms of anaphylaxis or immediate urticaria after a pyrazolone derivative intake  and a positive skin test . To ruleThe study was conducted according to the Declaration of Helsinki principles and was approved by the Malaga Research Ethics Committee. All subjects were informed orally about the study and signed an informed consent form.Skin testing was carried out as previously described14To exclude a cross-intolerant response we carried out a single-blind procedure in all patients, as previously describedg. The organic phase was transferred to fresh tubes and dried by evaporation under a nitrogen stream. Then, the tubes containing the evaporated metabolites were closed to maintain the nitrogen environment, and kept frozen at −80 °C until use. The concentration and purity of the metabolites was checked by HLPC analysis as described elsewhere30Metamizole metabolites were purified from human urine samples, obtained from healthy individuals who received an oral dose of 575 mg of metamizole , with the exception of 4-aminoantipyrine (AA) which was purchased from SIGMA chemical Co. . Because the vast majority of metabolites are recovered in urine in the first 24 h302 (w/v); 0.033% MgCl2 (w/v); 0.1% HSA (w/v); 1 M HEPES and 10 μL/mL IL-3) for 10 min at 37 °C. Then, metamizole  and their metabolites  obtained as described above, were added at different concentrations chosen from dose-response curves and cytotoxicity studies .