Guidelines for antiretroviral treatment (cART) are published regularly, but there is little information regarding the effect of adherence to guidelines on patient outcomes. We assessed the effect of following the "when-to-start" and "what-to-start" guidelines, on treatment modifications, and on immunological and virological outcome at 12 months in a cohort of HIV-1 infected patients initiating cART from 2000 to 2010.Consecutive HIV-1 infected patients, antiretroviral naive, initiating cART from 2000 to 2010 at a University Hospital were enrolled. HIV-2 infection, cART for prevention of mother-to-child transmission or during primary HIV-infection and unlicensed drugs were excluded. The respect or not of the "when-to-start" and "what-to-start" guidelines was based on French guidelines published from 2000 to 2010. Factors associated with cART modifications at 12 months and factors associated with an HIV viral load of <50 copies/mL at 12 months were assessed by univariate and multivariate logistic regression modeling. Variations in CD4 counts from baseline were assessed by univariate and multivariate linear regression.Of 1365 patients starting cART, 151 were treated outside "when-to-start" guidelines and 150 were treated outside "what-to-start" guidelines. Not using "when-to-start" guidelines was mainly related to early initiation in young men having sex with men, and was not associated with a significantly different outcome at 12 months. Treatments that did not follow "what-to-start" guidelines were not observed in any specific population and were associated with more treatment modifications and a poorer virological outcome at 12 months.Adherence to "what-to-start" guidelines is associated with a better outcome at 12 months in HIV-infected patients initiating antiretroviral therapy. Efforts should be made to promote adherence to these guidelines. The introduction of combined antiretroviral therapy (cART) in 1996 led to a dramatic improvement in HIV disease prognosis, from a deadly disease to a manageable chronic infection compatible with a normal or near-normal life expectancy . Since tFollowing the guidelines depends on the availability of the drugs and the organization of the health-care system, but also on physicians’ personal experience. For example, prescription of not recommended drugs may occur because of the guidelines lag behind clinical trial results but also in specific clinical situations such as HCV or HBV coinfections, renal insufficiency or treatment of opportunistic infections. The availability of numerous drugs facilitates the prescription of various combinations in industrialized countries, resulting in a significant proportion of prescriptions outside the recommendations .Few studies have reported adherence to guidelines in industrialized countries -15]. So. So15]. We conducted an analysis of first line cART in a large cohort of patients who initiated cART from 2000 to 2010. We hypothesized that good adherence to guidelines would improve patients' outcome, independent of the time period. One-year outcomes were studied according to whether or not the patient's treatment followed the French national guidelines applicable during the time of treatment.The Department of Infectious and Tropical Diseases at Croix-Rousse Hospital in Lyon, France, follows approximately 2,500 HIV-infected patients yearly. The Department provides care to patients from the diagnosis of HIV infection to complications from AIDS, leading to a high retention rate. This hospital cohort is included in the French Hospital Database on HIV and follows the protocols described elsewhere . patientDemographic and biological data at baseline and at 12 months were recorded. The time from HIV diagnosis to cART initiation was also recorded, as well as the duration of the first line of treatment and the reason for treatment modification or interruption if it occurred during the first year.The timing of cART initiation ("when-to-start" guidelines) was defined as "recommended", "optional", or "not recommended" on the basis of actual CD4 count, HIV viral load, and other specific conditions such as hepatitis B virus (HBV) or hepatitis C virus (HCV) coinfection, high cardio-vascular risk, or treatment as prevention -22]. Si. Si22]. The choice of antiretrovirals and the type of combination ("what-to-start" guidelines) was defined as "preferred", "alternative", or "not recommended". Since new drugs were often classified as "alternative" before being classified as "preferred", these 2 categories were grouped for analysis. Table cART modification was defined as any change of at least 1 antiretroviral during the first year of treatment. Dosage adjustments and switch to fixed-dose combinations using the same molecules were not considered modifications.The main reason for treatment modification or interruption was classified at the time of event as virological failure (defined by a confirmed HIV-RNA >200 copies/mL at 6 months or >50 copies/mL at 12 months), toxicity/tolerance issues, simplification of treatment, patient request (including observance issues), and other reasons, including pregnancy and pharmacological interactions.U Test. The Chi2 test for linear trends was used to assess the effect of the year of guideline publication on the outcomes.Qualitative variables were reported as numbers and percentages and were compared with the Chi2 test. Quantitative variables were reported as medians with interquartile range (IQR) and were compared with the Mann-Whitney Factors associated with cART modifications at 12 months and factors associated with an HIV viral load <50 copies/mL at 12 months were assessed by univariate and multivariate logistic regression modeling. Variations in CD4 counts from baseline were assessed by univariate and multivariate linear regression.The independent variables were whether the treatments followed the "when-to-start" and "what-to-start" guidelines. The following potential confounders were tested: year guidelines were published, age at cART initiation, risk factors for HIV infection, delay between HIV diagnosis and cART initiation, presence of HBV/HCV coinfection, Centers for Disease Control and Prevention (CDC) stage, HIV viral load, and CD4 count at cART initiation.P value was≤ .15; a backward stepwise process was then implemented and models were compared with the Wald test. Baseline CD4 count was forced in the predictive model of CD4 count variation at 12 months. Baseline HIV viral load was forced in the predictive model of undetectable HIV viral load at 12 month. All statistical tests were 2-tailed, and P < .05 was considered statistically significant. The viral suppression rate at 12 months was analyzed in an intention to treat (ITT) way . The data were analyzed by Stata software 11.0 . Missing data were avoided by complete case analysis.After univariate analysis, independent variables and potential confounders were entered in multivariate models if their From January 2000 to December 2010, 1365 patients initiated cART accordingly to the inclusion criteria. The demographics and clinical characteristics of the patients at baseline are summarized in Table P < .001). Factors associated with not following the "when-to-start" guidelines at cART initiation included younger age, male gender, men having sex with men, CDC stage A-B, higher CD4 count, and lower HIV viral load at baseline.cART not following the current "when-to-start" guidelines was initiated in 151 patients (11%). Treatment not following the guidelines declined from 22% in the 2000-2001 period to 6% in 2010 (P < .001). Factors associated with initiating cART that did not follow the "what-to-start" guidelines included older age, male gender, a shorter delay between HIV diagnosis and cART initiation, CDC stage C, lower CD4 count, and higher HIV viral load at baseline. Factors associated with following or not following the guidelines are summarized in Table One hundred and thirty-two different cART combinations were prescribed; 65 that followed the "what-to-start" guidelines and 67 that were outside the guidelines. These 67 regimens outside the guidelines represented 150 patients and included suboptimal combinations such as 2 or 3 nucleoside/nucleotide reverse transcriptase inhibitors (NRTI) (25 patients), more than 3 drugs (n = 69), 2 NRTI with a fusion inhibitor (n = 10) and other combinations of 3 drugs from 3 different classes (n = 46). cART that followed the current "what-to-start" guidelines was initiated in 1215 patients (89%) and treatments that were outside guidelines were initiated for 150 (11%). Prescription of drugs outside the current guidelines declined from 18% in the 2000-2001 period to 9% in 2010 . Similarly, the proportion of patients with CD4 counts >500/mm3 at 12 months increased from 28.6% to 43.2% (P < .001). Conversely, the proportion of patients reporting cART modification during the first year decreased from 52.1% to 22.6% (P < .001). Reasons for cART modification were virological failure in 6.6% of patients (n = 39), toxicity/tolerance issue in 41.0% (n = 244), simplification in 21.3% (n = 127), patient request in 7.9% (n = 47), and other reasons in 23.2% (n = 138).Eleven patients died during the first year of follow up. At 12 months, 64.3% of patients had an undetectable HIV-RNA <50 copies/mL, 31.9% had a CD4 count greater than 500/mmP < .001), whereas the CD4 increase from baseline was lower in these patients (P = .006).The proportion of patients with an HIV viral load <50 copies/mL at 12 months was similar for patients initiating cART with treatments that followed and did not follow the "when-to-start" guidelines, as well as the proportion of patients with a treatment modification during the first year. The median CD4 count was higher at 12 months for patients initiating cART that did not follow the "when-to-start" guidelines (P < .001), experienced undetectable HIV-RNA less frequently, and had lower CD4 counts and lower CD4 count increases at 12 months than patients initiating cART according to the guidelines (P < .001). The 12-month outcome data are summarized in Table Conversely, patients initiating cART with treatments that did not follow the "what-to-start" guidelines reported more treatment modifications (P = .004), but was not associated with treatment modification  or HIV-RNA <50 copies/mL at 12 months . Not following the "what-to-start" guidelines was associated with a higher treatment modification rate , decreased rate of HIV-RNA <50 copies/mL , and decreased CD4 count at 12 months .After univariate regression analysis, treatments not following the "when-to-start" guidelines were associated with decreased CD4 counts , and a treatment that did not follow the "what-to-start" guidelines. A sensitivity analysis was performed by excluding simplification of treatment from other causes of cART modification. This analysis gave similar results (data not shown).Factors independently associated with a greater increase in CD4 counts at 12 months were CDC stage A-B, baseline HIV-RNA <105 copies/mL, absence of HBV/HCV coinfection, pursuit of the same cART during the first year, and initiation of cART after 2004. Treatments that followed the "what-to-start" guidelines were not independently associated with CD4 count variation at 12 months.Factors independently associated with an undetectable HIV-RNA at 12 months were CDC stage A-B, pursuit of the same cART during the first year, initiation of cART after 2006, and treatments following the "what-to-start" guidelines.Treatments that followed the "when-to-start" guidelines were not independently associated with cART modification, CD4 increase, or undetectable HIV-RNA at 12 months. The results of these different models are summarized in Table The objective of this study was to identify the relationship in daily practice between following treatment guidelines and the outcome within 1 year of initiating cART. Following the guidelines appeared overall satisfactory in this well-defined population of cART-naïve HIV-infected adults. However, 11% of patients were treated outside of the "when-to-start" guidelines and 11% outside of the "what-to-start" guidelines. Similar results have been observed in other European and Australian studies ,12]-[14-[1412]-,[11], buThe reasons for initiating treatment outside the guidelines appeared remarkably different when considering whether or not the "when-to-start" or "what-to-start" guidelines were followed. Patients initiating cART outside the "when-to-start" guidelines were typically young men having sex with men, less immunocompromised than the other patients, who initiate cART at a higher CD4 count level than those recommended at that time. Since the recommended CD4 count threshold has increased over time, patients starting cART above this threshold generally anticipated the following guidelines. Initiating treatment above the given CD4 count threshold was also observed in Danish and Australian studies ,15], wi, wi15], The 1-year outcomes were quite similar whether or not the "when-to-start" guidelines were followed. Not surprisingly, patients initiating treatment outside these guidelines had higher 12 month CD4 counts but lower CD4 increases than other patients. The virological efficacy was, however, identical between groups, and there was a similar rate of treatment modification during the first year of treatment. Therefore, neither a beneficial nor a detrimental evolution was observed after 1 year of follow-up in patients initiating treatment outside the "when-to-start" guidelines, whereas long-term differences could not be ruled-out in this study. Additionally, initiating cART earlier in these patients could have been associated with other benefits not measured in this study, such as a reduction in infectiosity and contagiousness.Following the "when-to-start" guidelines was better during the studied period than during the pre-cART (<1996) and early-cART eras (1996-1999), when the rapid evolution of knowledge led physicians to largely anticipate the guidelines . Similar3 in this study, whereas more patients initiated cART with CD4 counts between 200 and 350/mm3 in our study. However, the distribution of HIV viral load at initiation of treatment was similar between the 2 cohorts.Twelve-month outcomes were remarkably different regarding whether or not "what-to-start" guidelines were followed. Patients initiating cART outside of these guidelines had significantly worse immunological and virological outcomes and more treatment modifications during the first year than patients treated within these guidelines. A similar result was observed in the Swiss Cohort  in whichTreatments outside of the "when-to-start" guidelines were not associated with virological or immunological outcome at 12 months. Conversely, complex interactions were observed between treatment modifications during the first year, immunological and virological response at 1 year, and following the "what-to-start" guidelines. CDC stage C and the year of guideline publication were common predictors of all 12-month outcomes. Similarly, cART modification was negatively associated with an undetectable HIV-RNA at 12 months and with CD4 count increases from baseline. Female gender was associated with more treatment modifications during the first year, but was not associated with immunological and virological response. As previously reported ,26], HB, HB26], Limitations of this study include the follow-up that was voluntarily limited to 1 year in order to assess the effect of the first line of cART. Data on adherence and compliance were not collected, as well as associated comorbidities, which could have influenced the timing of cART initiation or the choice of drugs. A center effect could not be excluded, but the monocentric nature of the study allowed us to better characterize the predictors of outcome, since the study was less susceptible to heterogeneity in patient care than multicentric ones. Since the French guidelines for treating HIV infection are widely recognized in France, we referred to these guidelines rather than those of the IAS-USA or other international organizations, but we speculate that given the convergence of these guidelines over time, our conclusions would have been the same using a different set of guidelines. Excluding acute infection from the study could be another possible limitation since many physicians have been treating acute infection far before guidelines indicated to do so. However, since recommendations on the treatment of primary infection evolved with time, excluding acute infections provided homogeneity in our study population.In conclusion, initiating treatments that did not follow the "when-to-start" guidelines was related to cART initiation at higher CD4 counts than the recommended level at the time, mainly in young men who have sex with men, and was not associated with a significantly different 12-month outcome than observed in the same population during that period of time. Conversely, not following the "what-to-start" guidelines was not observed in any specific population and led to significantly worse virological outcomes at 12 months and more treatment modifications during the first year of cART. Efforts should be made to promote adherence to the "what-to-start" guidelines and to assess the adequacy of prescriptions within these guidelines.LC designed the study, collected the data, participated to data analysis and interpretation, drafted and revised the article. TB analyzed the data, participated to data interpretation and revised the article. PV designed the study, analyzed the data participated to data interpretation and revised the article. CB collected the data, participated to data interpretation and revised the article. TP collected the data, participated to data interpretation and revised the article. TF collected the data, participated to data interpretation and revised the article. CC designed the study, collected the data, participated to data analysis and interpretation and revised the article. All authors read and approved the final manuscript.


Muscle invasive bladder cancer (MIBC) is often lethal and non-MIBC (NMIBC) can recur and progress, yet prognostic markers are currently inadequate. SAM68, a member of RNA-binding proteins, has been reported to contribute to progression of other cancers. The aim of this study is to investigate the potential utility of SAM68 in the progression and prognosis of bladder cancer.Quantitative PCR and immunohistochemistry were utilized to examine the expression of SAM68 in ten pairs of MIBC and adjacent normal bladder urothelium, and eight pairs of MIBC and non-MIBC (NMIBC) tissues from the same patient. Moreover, SAM68 protein expression level and localization were examined by immunohistochemistry in 129 clinicopathologically characterized MIBC samples. Prognostic associations were determined by multivariable analysis incorporating standard prognostic factors.SAM68 expression was elevated in MIBC tissues compared with adjacent normal bladder urothelium, and was increased at both transcriptional and translational levels in MIBC tissues compared with NMIBC tissues of the same patient. For MIBC, high expression and nucleus-cytoplasm co-expression of SAM68 were associated with higher T-stage, higher N-stage and worse recurrence-free survival. Five-year recurrence-free survival was 80% and 52.9% for MIBC patients with low and high SAM68 expression, respectively (p = 0.001). SAM68 nucleus-cytoplasm co-expression associated with worse 5-year recurrence-free survival rate (49.2%) than SAM68 expression confined to the nucleus (82.5%) or cytoplasm (75.5%) alone. On multivariable analysis SAM68 expression level, SAM68 nucleus-cytoplasm co-expression, T-stage, and N-stage were all independent prognostic factors for recurrence-free survival of MIBC patients.SAM68 expression is increased in MIBC when compared to normal urothelium and NMIBC, and appears to be a potentially useful prognostic marker for MIBC. Bladder cancer is the fourth most-common malignancy in men, accounting for 7% of all cancer cases. In 2014 there will be 74,690 new cases of bladder cancer in the USA alone, leading to 15,580 cancer-related deaths , and sucin vitro study revealed that down-regulation of SAM68 in breast cancer cells inhibited cell proliferation by blocking the transition from G1 to S phase, and the Akt/GSK-3β signaling and FOXO/p21/p27 pathway appeared to be involved [SAM68  is a member of the STAR  family of RNA-binding proteins , and hasinvolved . In earlinvolved . In the Patient consent and approval from the Sun Yat-sen University Cancer Center Institutional Review Board were obtained for the use of these clinical materials for research purposes. Ten pairs of MIBC tissue specimens and corresponding non-tumorous specimens were obtained from patients with bladder cancer who underwent radical cystectomy at the Cancer Center of the Sun Yat-sen University . Eight paired of non-muscle invasive (NMIBC) and MIBC tissues from the same patient were obtained from TURBT and radical cystectomy, respectively. All excised tissues were obtained within 1 h after surgery and were immediately placed in liquid nitrogen until further analysis. Immunohistochemistry analyses were performed on 129 paraffin-embedded radical cystectomy samples, which were histologically diagnosed as MIBC at the Cancer Center, Sun Yat-sen University, between 2000 and 2008. Tumor-node-metastasis (TNM) staging was determined according to the 2010 American Joint Committee on Cancer TNM classification of bladder cancer . The detTotal RNA from tumor and adjacent non-tumorous tissues was extracted using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Quantitative polymerase chain reaction (PCR) was performed according to standard methods as described previously . PCR priImmunohistochemistry (IHC) was performed to study altered SAM68 protein expression levels in 129 human MIBC tissues, as well as the ten pairs of MIBC tissue specimens and corresponding non-tumorous specimens, and eight paired of NMIBC and MIBC tissues. In brief, 4 μm-thick tissue sections were incubated with polyclonal rabbit antibody against SAM68  at 4°C overnight. Before incubation with the primary antibody, the sections were treated for antigen retrieval with ethylene diamine tetraacetic acid buffer followed by heating in a microwave oven. For negative controls, the rabbit anti-SAM68 antibody was restored with normal nonimmune serum. After washing, tissue pieces were treated with biotinylated anti-rabbit secondary antibody (Zymed), followed by further incubation with streptavidin -horseradish peroxidase complex (Zymed). Tissue sections were then immersed in 3,3′-diaminobenzidine and counterstained with 10% Mayer's hematoxylin, dehydrated, and mounted.The degree of immunostaining of paraffin-embedded sections was reviewed and scored independently by two observers based on the proportion of positively-stained tumor cells and the intensity of staining. The method has been introduced in detail previously . The staThe relationship between SAM68 expression and clinicopathological characteristics was analyzed by the chi-square and Fisher’s exact tests. Survival curves between subgroups that were divided according to SAM68 expression levels and localizations were drawn with the use of the Kaplan-Meier method, and significant differences among subgroups were compared by the log-rank test. Survival data were evaluated using univariate and multivariable Cox regression analyses. In all cases, P < 0.05 was considered to be statistically significant. All statistical analyses were conducted using the SPSS v.13.0 statistical software package .To determine the expression of SAM68 in MIBC tissues, quantitative PCR analyses were conducted on ten matched pairs of bladder cancer tissues and adjacent, nontumorous bladder urothelium from the same patient. As shown in Figure To explore the role of SAM68 in bladder cancer progression, we analyzed eight MIBC patients with a history of NMIBC, who underwent radical cystectomy for recurrent tumor infiltrated into or beyond muscularis propria. Paired of NMIBC and MIBC were obtained from TURBT and radical cystectomy, respectively, of the same patient. The results indicated that SAM68 was markedly up-regulated at both transcriptional and translational levels in MIBC tissues compared with NMIBC tissues by quantitative PCR and IHC Figure A and B. To study the clinicopathological significance of SAM68 in MIBC, we examined the SAM68 protein expression levels in 129 paraffin-embedded, archived MIBC, from radical cystectomy, with long term follow-up data. The IHC results are summarized in Table χ2 test and survival analysis with long-rank test were performed. As shown in Table To determine the association of SAM68 expression and clinical features and recurrence-free survival of MIBC, MIBC is typically managed with perioperative chemotherapy and radical cystectomy with extended pelvic lymph node dissection , yet almIn the current study, we evaluate SAM68 as a potential prognostic marker for MIBC, and report for the first time clinical and pathologic correlates for this malignancy. Using immunohistochemistry, we found that MIBC patients with high expression or nucleus-cytoplasm co-expression of SAM68 had lower recurrence-free survival at 5 years. Multivariable analysis revealed that SAM68 expression level and nucleus-cytoplasm co-expression were independent prognostic factors for recurrence-free survival, adding additional prognostic power above that provided by T-stage and N-stage, which are the well-established prognostic factors for this malignancy. Our data suggests that SAM68 may allow for refined risk stratification after radical cystectomy that could prove to be of clinical utility. In particular, patients with high expression or nucleus-cytoplasm co-expression of SAM68 may need adjuvant therapy and more intensive surveillance. If identified on biopsy, SAM68 could potentially influence decisions about neoadjuvant chemotherapy or even less intensive alternative strategies, such as chemo-radiation, although further studies will be required. In addition, we found that bladder cancer developing from NMIBC to MIBC may be associated with increased SAM68 expression, which if validated in independent and larger cohorts, might suggest a potential target for therapeutics.Our findings are consistent with recent data about SAM68 as a tumor-promoter in other cancers ,11,20, aAnother interesting finding was that almost 50% of MIBC cases demonstrated co-expression of SAM68 in both the nucleus and cytoplasm, which associated with advanced T and N stage and compromised recurrence-free survival, and co-expression proved to be an independent prognostic factor on multivariable analysis. Although SAM68 was initially reported to be predominantly localized to the nucleus ,23, cytoAlthough our study enrolled a large cohort of MIBC patients, there are still some limitations. Firstly, this was a retrospective study, and some bias is therefore inevitable. Secondly, the mechanism and function of the accumulation of SAM68 in the cytoplasm were not investigated in this study. Thirdly, the role of high expression of SAM68 in bladder cancer cell progression has not been illuminated. Finally, independent validation with larger cohorts of patients will be required to more definitively explore the potential value of SAM68 as a biomarker for bladder cancer.In summary, our study suggests that SAM68 expression is elevated in MIBC compared with normal urothelium and NMIBC and may have potential as a prognostic marker for MIBC. However, large scale prospective study will be required to validate our findings, and further studies regarding mechanisms of action are also indicated.


To investigate the serum levels of Interleukin (IL)-17 and eotaxin levels and the relationship between serum IL-17, eotaxin and pulmonary function in asthmatic patients with allergic rhinitis.Serum IL-17 and eotaxin levels in asthmatic patients with allergic rhinitis during attacking and remission and in healthy control subjects were measured using enzyme-linked immunosorbent assay (ELISA) kits. Then we studied the correlation between the serum IL-17, eotaxin levels and pulmonary function in patients.Serum IL-17 and eotaxin levels were significantly elevated in patients during asthma attack and remission compared with healthy control subjects. These levels in patients during asthma attack were much higher than those during remission. Furthermore, serum IL-17 and eotaxin levels were negatively correlated with pulmonary function in asthmatic patients with allergic rhinitis, respectively.Our findings suggest that IL-17 and eotaxin are important factors in asthma with allergic rhinitis, and the correlation between serum IL-17, eotaxin and lung function possibly lead to improvements in the diagnosis and treatment of asthma with allergic rhinitis and related diseases. Asthma is a chronic inflammatory disease of airways characterized by chronic inflammation, airway hyper-reactivity, and by symptoms of coughing, wheezing and chest tightness. Executive summary of the Global Initiative of Asthma (GINA) Dissemination Committee report in 2004 estimated that 300 million people of all ages, and all ethnic backgrounds, suffered from asthma, and there would be 400 million persons with asthma by 2025.2+ human progenitors into neutrophils.Interleukin (IL)-17, a newly described cytokine secreted by activated subtype of T helper lymphocytes Th17,However, the pathophysiological role of IL-17 and eotaxin in asthmatic patients with allergic rhinitis are still unclear. To determine whether IL-17 and eotaxin play a role in asthma with allergic rhinitis, we explored the serum of IL-17 and eotaxin levels and the relationship between serum IL-17, eotaxin and disease severity as measured by FEV1%pred.The study protocol was approved by the Ethics Committee of Taicang Hospital of Traditional Chinese Medicine, and informed written consents were obtained from all patients. Total twenty asthmatic patients with allergic rhinitis were enrolled in the study from the department of respiration of Taicang Hospital of Traditional Chinese Medicine, Suzhou, P. R. China. Diagnosis of asthma was performed according to the Chinese Guidelines on the Prevention and Management of Asthma. Diagnosis of allergic rhinitis was based on the allergic rhinitis diagnostic criteria established by the 2004 Otorhinolaryngology Society of the Chinese Medical Association . All patients had no oral steroid intake or change of asthma or allergic rhinitis medications four weeks prior to recruitment of study. Twenty-three sex- and age-matched healthy non-allergic healthy volunteers were enrolled as controls.We collected blood samples from healthy subjects, patients during asthma attack and remission. Blood samples were drawn in serum tubes, clotted at room temperature for 10-20 minutes and centrifuged for 20 minutes at 3000 rpm. Serum samples were immediately frozen at -80 °C for further assay.Immediately after the blood sampling, FEV1%pred was evaluated using a pneumotach meter system with Lily head .Serum IL-17 and eotaxin were measured by commercial enzyme-linked immunosorbent assay (ELISA) using reagent kits of Shanghai yifeng Biotechnology Co., Ltd.  according to the manufacturer’s instructions.t-test was used to compare normally distributed data between two groups, and the rank sum test was applied for nonnormally distributed data between two groups. The χ2 test was used to compare frequencies. All reported p values were two-sided, and p < 0.05 was considered statistically significant.Data were analyzed by the SPSS 18.0 software . Normally distributed data were expressed as the mean±SD and non-normally distributed data were expressed as the median (25-75th percentiles). The Student paired p< 0.05) compared to healthy control subjects (p< 0.05). Eosinophils are the primary effector cells in the mechanism of asthma. As shown in p< 0.05), the eosinophils (%) levels in patients with remission were greatly decreased than those during asthma attack (p< 0.05).The demographics and clinical characteristics of the healthy control subjects and asthmatic patients with allergic rhinitis are shown in subjects . Howeverp< 0.05) compared with healthy control subjects. In addition, serum IL-17 and eotaxin levels were much higher during asthma attack than those during remission (p< 0.05).Serum IL-17  and eotap< 0.05). Furthermore, in patients the serum eotaxin levels significantly correlated with FEV1%pred  of asthmatic patients.Similarly to IL-17, the meanserum levels of eotaxin increased in patients, increased transcription from eotaxin gene had been observed in the bronchial mucosa of asthmatic patients.In conclusion, this study showed that serum IL-17 and eotaxin levels were significantly elevated in asthmatic patients with allergic rhinitis during attacking, which were greatly decreased during remission, the serum IL-17 and eotaxin levels were negatively correlated with lung function in asthmatic patients with allergic rhinitis, respectively. Furthermore, serum levels of IL-17 and eotaxin are much easier measurable compared to these in sputum, BALFs or bronchial biopsies. Thus, the correlation between serum IL-17, eotaxin levels and lung function possibly leading to improvements in the diagnosis and treatment of asthma with allergic rhinitis and related diseases.We evaluated a limited number of patients, and further work is also needed to clarify the precise mechanism of action of IL-17 and eotaxin in asthmatic patients with allergic rhinitis.


Regular physical activity (PA) is recommended for persons with chronic obstructive pulmonary disease (COPD). Interventions that promote PA and sustain long-term adherence to PA are needed.We examined the effects of an Internet-mediated, pedometer-based walking intervention, called Taking Healthy Steps, at 12 months.Veterans with COPD (N=239) were randomized in a 2:1 ratio to the intervention or wait-list control. During the first 4 months, participants in the intervention group were instructed to wear the pedometer every day, upload daily step counts at least once a week, and were provided access to a website with four key components: individualized goal setting, iterative feedback, educational and motivational content, and an online community forum. The subsequent 8-month maintenance phase was the same except that participants no longer received new educational content. Participants randomized to the wait-list control group were instructed to wear the pedometer, but they did not receive step-count goals or instructions to increase PA. The primary outcome was health-related quality of life (HRQL) assessed by the St George’s Respiratory Questionnaire Total Score (SGRQ-TS); the secondary outcome was daily step count. Linear mixed-effect models assessed the effect of intervention over time. One participant was excluded from the analysis because he was an outlier. Within the intervention group, we assessed pedometer adherence and website engagement by examining percent of days with valid step-count data, number of log-ins to the website each month, use of the online community forum, and responses to a structured survey.P<.001), but approached zero in months 8-12. Within the intervention group, mean 76.7% (SD 29.5) of 366 days had valid step-count data, which decreased over the months of study (P<.001). Mean number of log-ins to the website each month also significantly decreased over the months of study (P<.001). The online community forum was used at least once during the study by 83.8% (129/154) of participants. Responses to questions assessing participants’ goal commitment and intervention engagement were not significantly different at 12 months compared to 4 months.Participants were 93.7% male (223/238) with a mean age of 67 (SD 9) years. At 12 months, there were no significant between-group differences in SGRQ-TS or daily step count. Between-group difference in daily step count was maximal and statistically significant at month 4  Physical activity (PA) is significantly reduced in persons with chronic obstructive pulmonary disease (COPD), even at the earliest stages of disease -3. Its c1) [Physical activity is a modifiable health behavior that affects COPD-specific outcomes -14. It h1) .Despite the evidence and recommendations, effective long-term PA interventions are lacking in the clinical care of patients with COPD. Most studies of long-term exercise interventions have examined methods to maintain exercise in the subset of persons with COPD who have completed a conventional pulmonary rehabilitation program -21. ThesWe developed an automated, Internet-mediated, pedometer-based walking program called Taking Healthy Steps to promote PA in persons with COPD. Taking Healthy Steps combines the Omron HJ-720 ITC pedometer  with a disease-specific website accessed via a URL. Taking Healthy Steps provides iterative step-count feedback, individualized step-count goals, education on disease self-management, motivational support, and an online community of social support -27. We sThe study design and methods have been reported previously ,27. PartA random sample of 28,957 Veterans  with a COPD diagnosis was sent a recruitment letter. Inclusion criteria included having access to a computer with an Internet connection, a USB port, and Windows XP, Vista, Windows 7, or Windows 8. Our a priori exclusion criteria excluded those who did not upload baseline step-count data or who did not complete the baseline survey to assess HRQL. Per study protocol, participants had to have baseline values for the primary (HRQL) and secondary outcome (daily step count) to be enrolled and randomized. Ultimately, 239 participants were enrolled and randomized in a 2:1 ratio to either Taking Healthy Steps  or wait-list control , stratified by Modified Medical Research Council (MMRC) dyspnea score and urban versus rural status . All parThe St. George’s Respiratory Questionnaire (SGRQ), a disease-specific instrument with 50 items that has been well validated in COPD ,30 was uDaily step count was assessed by the Omron HJ-720 ITC pedometer. Once participants completed the baseline survey, study staff mailed them a pedometer that had an embedded USB port, an upload cable, and detailed written instructions on how to install the Java software and upload pedometer data. For users who did not have Java already installed on their computers, the software installation was a one-time event. Thereafter, participants uploaded step-count data using the cable that connected the pedometer to their home computer. Research staff were available by telephone to assist with software installation and upload of step counts.A wear day with valid step-count data was defined as one having at least 100 steps and 8 hours of step counts recorded . At baseParticipants randomized to Taking Healthy Steps completed an intensive 4-month intervention period, followed by a distinct 8-month maintenance phase . During Participants randomized to the wait-list control group were instructed to wear the pedometer every day, reminded monthly to log in to the website to upload step-count data, and asked to report all adverse events. Veterans in the wait-list control group received neither instruction to increase PA nor step-count goals. They had access to a webpage that showed only a checklist of surveys completed and a count of what week they were in the study. After 12 months, they were given the option to use the Internet-mediated intervention.At baseline, participants answered questions online that assessed comorbidities, oxygen use, smoking status, and demographics. At study entry, 4 months, and 12 months, dyspnea was assessed using the MMRC scale (range 0-4 with 4 indicating the most severe level of dyspnea) . Events We examined device adherence, overall and by group, by calculating the percentage of days (of 366 days) that were wear days with valid step-count data. For the participants who uploaded valid step-count data at 12 months and completed the study, we also examined percentage of days (of 42 days) that were wear days during the last 6 weeks of the study.In the intervention group, we objectively examined website engagement by recording the number of log-ins to the website by month of study and assessing the frequency of use of the online community forum. In addition, at 4 and 12 months, participants in the Taking Healthy Steps group answered a structured survey eliciting feedback about their commitment to their step-count goals and various aspects of engagement with the intervention, including participants’ ease of finding time to log in to the website, knowledge of step-count goals, and use of the different components of the website.t tests and chi-square tests compared baseline characteristics between groups. The occurrence of COPD-related events (acute exacerbations or pneumonia), hospitalizations, emergency room visits, deaths, and adverse events during the study were each compared between groups using a logistic regression model. For the count of hospitalizations, a zero-inflated Poisson regression model was also used to assess the difference in the rate of hospitalizations between groups. These models adjusted for age, gender, treatment group, and oxygen use.Proportions, means, and standard deviations described baseline participant characteristics. Two-sample The primary analysis used the intention-to-treat approach, and used a linear mixed-effects model with baseline, 4-month, and 12-month outcome values  as dependent variables. No baseline variable was predictive of missingness in models adjusting for stratification variables and treatment group. Thus, the longitudinal data model included participants who had the dependent variable for at least one time point and was expected to give unbiased estimates of the intervention effect assuming missingness at random. The model included participants as random intercepts to adjust for within-participant correlations of repeated measures, fixed predictors of treatment group, 4- and 12-month time indicators, and treatment group by time indicator interactions, MMRC dyspnea score (dichotomized to 0-1 vs 2-4), and urban versus rural residence. We also analyzed the data excluding those who died. The proportion of participants who had at least a 4-unit improvement in SGRQ-TS at 12 months was compared between groups using a chi-square test . For theWe assessed website engagement in the intervention group by characterizing the number of log-ins to the website using the mean, median, and interquartile range, and assessed trends over months of study using a linear mixed-effects regression analysis with monthly number of log-ins for each participant as the outcome and time (month since randomization) as the predictor. Trends for device adherence over month of study were examined with percent of days with valid step-count data using a linear mixed-effects model and for use of the online community forum using a generalized mixed-effects model with logit link. The effect of time on participant responses to the online survey about goal commitment and intervention engagement at 4 and 12 months was estimated for each response variable using a mixed-effects model with 4- and 12-month survey data as the dependent variable and predictors including 12-month indicator, baseline dichotomized MMRC dyspnea score, and urban versus rural status. All models, including the model for the number of log-ins, were checked for model assumptions using residuals.One participant in the Taking Healthy Steps group was considered an outlier given that his change in SGRQ-TS was 4.0 standard deviations greater than the mean for change in SGRQ-TS and his change in daily step count was 8.1 standard deviations greater than the mean for change in daily step count. The extremely high step counts more likely reflected his occupational PA rather than any effects of our intervention. Our main analyses excluded the outlying individual, but we also repeated primary and secondary outcome analyses with this participant included. All analyses were performed with Stata 14.0 .No information is available on the persons to whom we mailed recruitment materials but who were not screened because they did not go to our website and did not call us . The topParticipants’ (N=238) characteristics include: mean age 67 (SD 9) years, male , rural residence , MMRC dyspnea score ≥2 , current smokers , and supplemental oxygen use  . There wP=.18) or baseline daily step count  between those for whom SGRQ-TS could not be calculated (n=29) versus those for whom SGRQ-TS was calculated at 12 months (n=209).At 12 months, 29 of 238 (12.2%) participants did not have sufficient data to calculate the SGRQ-TS: 19 Taking Healthy Steps participants and 10 controls. There was no significant difference in baseline SGRQ-TS . No between-group difference was found in the percent of participants with hospitalizations  or emergency room visits  during the 12-month study. For the count of hospitalizations, a zero-inflated Poisson regression model also found no between-group difference. The percent of participants who died during the study did not differ between groups . Finding no between-group differences in the percentage of participants who were hospitalized or died provided assurance that the censoring of the outcome variables (SGRQ-TS or daily step counts) due to these events was not likely to confound the assessment of the between-group outcome differences. However, we repeated the analyses with deaths excluded as well.The percent of participants with COPD-related events (acute exacerbations or pneumonia) during the study did not differ between groups  at 12 months (P=.08). There was no significant between-group difference in the SGRQ domain scores of symptoms , activities , and impact  at 12 months.There was no significant between-group difference in the primary outcome of SGRQ-TS  (P=.02), and impact improved by a mean 3.4 units  at 12 months. The control group showed no significant changes in the SGRQ-TS and domain scores at 12 months compared to baseline (P=.05). When the analysis excluded the eight deaths, results remained nearly identical.Intervention participants showed an improvement in SGRQ-TS of a mean 2.5 units (95% CI –4.5 to –0.6) at 12 months, compared to baseline (P=.01) . For dombaseline . When thP=.73) (P=.14) or in the control group (P=.52) at 12 months, compared to baseline . Analysis excluding the eight deaths did not change results.There was no significant difference between groups with respect to the secondary outcome of daily step count at 12 months (P=.73) . There wbaseline . Examinabaseline . Betweenhe study . AnalysiP=.002). For the 177 participants in both groups who uploaded valid step-count data at 12 months and completed the study, mean 83.1% (SD 21.9) of days in the last 6 weeks of the study had valid step-count data. In these last 6 weeks, mean 87.5% (SD 16.5) of days had valid step-count data in the intervention group, which was significantly higher than the mean 74.1% (SD 28.1) of days observed in the control group (P<.001). In the intervention group, device adherence decreased significantly over time (P<.001), with mean 92.1% (95% CI 86.6-97.6) of days having valid step-count data at month 1 versus 70.3% (95% CI 64.9-75.8) of days at month 12  of the 366 days having valid step-count data in the intervention group versus mean 63.7% (SD 32.9) of the 366 days having valid step-count data in the control group .In the intervention group, mean number of log-ins to the website decreased significantly over the months of study P<.001; . The numP=.06). Responses to questions about engagement with the use of Taking Healthy Steps were not significantly different at 12 months compared to 4 months . There were no differences between groups with respect to pulmonary, cardiac, or other adverse events during the 12 months.Adverse events were categorized as pulmonary, cardiac, musculoskeletal, or other. A significantly greater percent of participants in the intervention group  had minor musculoskeletal adverse events than in the control group  over time mirrors the declines in device adherence and website engagement over time.The reasons for the observed decline in daily step counts over time require further exploration. Participants may not have continued to wear the pedometer, log in to the website, and walk over the 12 months for a variety of possible reasons that we did not assess, such as waning interest with the intervention, progression of underlying COPD, flare-up of comorbidities, or occurrence of intercurrent life events . The effect of the intervention on daily step counts could potentially have been greater if the control group had not received a pedometer and monthly reminders to upload step counts. We are confident that battery life did not affect the results because we mailed a new battery with replacement instructions to each participant every 4 months. We replaced lost or broken devices reported to us.These results are similar to published data examining maintenance exercise programs after conventional pulmonary rehabilitation -21. TypiThe failure to obtain long-term benefits with our PA interventions parallels the literature studying other behavior changes, such as smoking cessation  and weigThe exact role of digital walking programs in starting and maintaining exercise in persons with COPD remains to be determined. Both acute and chronic models of digital walking programs are potentially useful. Acute intervention models are needed to initiate and promote PA in the vast majority of patients with COPD who cannot access a conventional pulmonary rehabilitation program . In addiThe potential full impact of our intervention can only be appreciated by performing a future cost-effectiveness analysis. Results from cross-sectional data from our group and others have shown that every step counts. We have not found a “threshold” or “optimal” daily step count to obtain clinical benefits. The benefits appear to be linear such that those with higher step counts have lower risks for acute exacerbations, hospitalizations, hospital admissions and readmissions, and death compared to those with lower step counts -14. FutuMajor strengths of our study include the randomized controlled trial design with balanced groups at baseline, objective data on device adherence and website engagement, and the long-term follow-up of 12 months. Our intervention is based on a theoretical model, and informed by previous work eliciting patient feedback to optimize user acceptability and develop the motivational and educational content ,45. Our Our study has several limitations. We studied primarily white male Veterans limiting the generalizability of our results. Spirometric confirmation of the COPD diagnosis was not made at study entry. However, any potential misclassification of asthma as COPD was most likely balanced between groups and would not bias the primary results. The vast majority of the patients had MMRC <2. It is justified to include patients with MMRC <2 because patients with newly diagnosed COPD have reduced PA even at the earliest stages of the disease . It is iAn Internet-mediated, pedometer-based PA intervention for persons with COPD does not maintain improvements in HRQL or daily step count at 12 months, despite demonstrated improvements at 4 months. In addition, waning engagement with the PA intervention support that future efforts should focus on improving features of PA interventions to enhance long-term behavior change and sustain engagement with PA. These findings need to be considered when designing future Internet-mediated PA interventions.


Food choices influence health status, but also have a great impact on the environment. The production of animal-derived foods has a high environmental burden, whereas the burden of refined carbohydrates, vegetables and fruit is low. The aim of this study was to investigate the associations of greenhouse gas emission (GHGE) and land use of usual diet with mortality risk, and to estimate the effect of a modelled meat substitution scenario on health and the environment.The usual diet of 40011 subjects in the EPIC-NL cohort was assessed using a food frequency questionnaire. GHGE and land use of food products were based on life cycle analysis. Cox proportional hazard ratios (HR) were calculated to determine relative mortality risk. In the modelled meat-substitution scenario, one-third (35 gram) of the usual daily meat intake (105 gram) was substituted by other foods.During a follow-up of 15.9 years, 2563 deaths were registered. GHGE and land use of the usual diet were not associated with all-cause or with cause-specific mortality. Highest vs. lowest quartile of GHGE and land use adjusted hazard ratios for all-cause mortality were respectively 1.00 (95% CI: 0.86-1.17) and 1.05 (95% CI: 0.89-1.23). Modelled substitution of 35 g/d of meat with vegetables, fruit-nuts-seeds, pasta-rice-couscous, or fish significantly increased survival rates (6-19%), reduced GHGE (4-11%), and land use (10-12%).There were no significant associations observed between dietary-derived GHGE and land use and mortality in this Dutch cohort. However, the scenario-study showed that substitution of meat with other major food groups was associated with a lower mortality risk and a reduced environmental burden. Especially when vegetables, fruit-nuts-seeds, fish, or pasta-rice-couscous replaced meat. Current climate changes and the increased need for food underlines the importance of a sustainable food system . Food chThe largest environmental impact from food comes from the production and consumption of meat and dairy products; the estimated GHGE is 50% and use of total farmlands is 80% -5. Meat A diet according to the Dutch national dietary guidelines would result in a 8% reduction in GHGE  when compared to the actual dietary pattern as observed in the Dutch national food consumption survey . A studyAlthough it is expected that a diet which is environmental friendlier also reduces mortality of chronic diseases, this has never been investigated in a real-life setting. Therefore, the aim of this study was to investigate to what extent an environmental friendlier diet is also a healthier diet by increasing survival rates in the EPIC-NL cohort study. The effects of food weight-based modelled substitution scenarios, in which part of the total meat intake was replaced by other food groups, on mortality and environmental impact were also investigated.EPIC-NL is the Dutch contribution to European Prospective Investigation into Cancer and Nutrition . EPIC-NLExclusion criteria for the present analyses were no informed consent for follow-up of vital status (n = 956) and missing follow-up data (n = 16). Participants with a history of cancer (n = 1640), diabetes (n = 781), myocardial infarction (n = 536), stroke (n = 458), or a combination of these diseases (n = 249) were excluded because the usual diet reported by these persons may not reflect their diet before diagnosis. In addition, these participants have a higher risk of death. Subjects without dietary information (n = 154) were excluded. To exclude implausible values, participants in the highest and lowest 0.5% of the ratio of reported energy intake (based on the food frequency questionnaire (FFQ)) on energy requirement ) were also excluded (n = 662). After these exclusions, 35 057 participants remained for the all-cause mortality survival analysis.Usual daily dietary intake was estimated by a 178-item FFQ that has been validated against twelve 24-h recalls, and biomarkers in 24-h urine and blood ,23. Spea2) emissions through the use of fossil fuels, methane (CH4) released during the rearing of cattle and the cultivation of certain crops, and nitrous oxide (N20) released from fertilizers, manure and ploughing of grassland [2-equivalents per day. Land use covers the surface needed for the production of food [2*year per day. These LCA data were combined with the EPIC-NL FFQ data to calculate individual daily greenhouse gas emission and land use for each of our participants. The LCA scores were based on current production practices and assumed equal in the nineties when the FFQ was assessed.Blonk Consultants assessed the environmental impact of the Dutch dietary habits . To estirassland ,25. GHGE of food ,25 and iAt baseline, study participants completed a questionnaire on the presence of chronic diseases and related potential risk factors, and medical and lifestyle factors . Body maVital status of all EPIC-NL participants was obtained through linkage with the municipal population registries. The information on vital status for the EPIC-NL cohort is complete until 11 April 2011 for MORGEN and until 4 July 2011 for PROSPECT. These data were retrieved from the GBA .Participants were followed for the occurrence of cancer, cardiovascular disease, respiratory disease and other causes by linkage to several disease registries . Primary cause of death was coded according to the International Classification of Diseases (ICD). Incidence of cancer deaths was coded as 140–239 (ICD-9) or C00-D48 (ICD-10), incidence of cardiovascular disease (CVD) deaths as 390–459 (ICD-9) or I00-I99 (ICD-10), incidence of respiratory system disease mortality as 460–519 (ICD-9) or J00-J99 (ICD-10). The remaining causes of death were merged into the category ‘other causes’. Cause-specific mortality data were available until 31 December 2010. This is the most recent linkage to the database of Statistics Netherlands.Participants were followed over time until death from any cause, loss to follow-up, or were censored on 11 April 2011 for MORGEN and 4 July 2011 for PROSPECT. In the cause-specific mortality analysis, the censor date was 31 December 2010 for both cohorts.2/kJ, showed very similar results (results not shown).Cox proportional hazard models were used to estimate crude and adjusted hazard ratios (HRs) with 95% confidence intervals (CI) for GHGE and land use in association with mortality. Using manual backward selection, covariates were excluded from the final model when the HR did not change ≥10% . This maTo study the effect of a modelled substitution of meat by other food components, both meat and the replacement component were added as continuous variables in the same multivariate model. Similar to previous studies, the difference in the parameter estimates and covariance was used to estimate HR and 95% CI ,17. The p-value of <0.05 was considered statistically significant.All statistical analyses were performed using SAS software . A two-sided 2-equivalents/d and for land use 3.61 m2*year/d. While contributing 3.6% of daily intake weight (and 11% of daily energy intake), total meat intake accounts for approximately 30% of total dietary-derived GHGE and land use  differed only slightly between the highest and lowest quartiles of GHGE and land use Table .In the crude Cox proportional hazards analyses, we observed an inverse association of total greenhouse gas emission of usual diet with all-cause mortality. The HR  of highest versus lowest quartile of GHGE was 0.76 (0.68-0.85) Table . After mIn crude analysis, total land use of usual diet was inversely associated with all-cause mortality (HR of highest versus lowest quartile: 0.74 (0.66-0.82)) , reduce GHGE (4-12%), and land use (10-12%).In this study, the environmental burden of the usual diet was divided into quartiles of total GHGE and land use to analyse the influence of diets with a higher impact on the relative risk for mortality. For this division no impact on mortality risk was observed in the Cox survival models. Other studies have suggested that a healthier diet may also be more sustainable ,15. A diet al. showed that a complete replacement of meat and dairy by a variety of plant-derived foods would not affect total iron intake, reduce saturated fatty acid intake, and reduce land use by around 50% in Dutch female young adults [The Dutch diet is relatively high in animal-derived products and refined carbohydrates and low in fruit and vegetables. Within the dietary range of this cohort, there was no significant association between the overall daily GHGE and land use and mortality. Although total GHGE and land use were not associated with mortality, modelling a one-third reduction of total meat, a major contributor to dietary GHGE and land use, resulted in both reduced mortality risk as well as reduced environmental impact. The 35-gram reduction of meat was well within the intake variation (standard deviation) of 55 gram and is thus a realistic scenario. Meat intake has been linked to an increased risk of mortality before . In addig adults .Substituting high-GHGE with low-GHGE meats could also contribute to increased survival rates and reduced environmental impact. Replacing red meat with poultry would reduce the environmental impact (data Blonk Consultants ) and is associated with reduced mortality risk . In addiA New Zealand study presented findings of scenario development with linear programming that determined several dietary patterns to cover nutrient intake at low cost and low GHGE profiles . The stuOur study has some strengths and limitations. The combination of sustainability of the usual diet and health was not previously studied in a large prospective cohort with a follow-up time of 16 years. The participants of this cohort were sampled from four different geographic areas in the Netherlands and therefore the results may be extrapolated to the Dutch population. In addition, mean GHGE and land use in our cohort were similar to the Dutch Consumption Survey of 1998 . The dieThe scope of this study is limited to substitutions of an equivalent quantity in grams. Future research may include iso-caloric substitutions or nutritional component equivalency of meat substitutions. In addition, within food groups the environmental impact can vary per product due to farming methods, animal feed, use of side products, transport, and growing conditions . Taking The Dutch diet is relatively high in animal-derived food products and refined carbohydrates and low in fruit and vegetables. Within the dietary range of this population-based cohort, there were no significant associations between overall daily dietary-derived GHGE and land use and mortality. However, a modelled reduction of 35 gram meat which was replaced with vegetables, fruits, fish, or cereal-rice-couscous resulted in lower GHGE and land use as well as decreased all-cause mortality risk. The results of our study emphasise that a healthier diet is not necessarily a more sustainable diet, and the other way around. Nevertheless, a reduction of meat consumption can influence both health and environmental aspects.BMI: Body mass index; CPAI: Cambridge physical activity index; EPIC: European Prospective Investigation into Cancer and Nutrition; FFQ: Food frequency questionnaire; GHGE: Greenhouse gas emission; HR: Hazard ratio; 95% CI: 95% confidence interval.The authors declare that they have no competing interests.SB carried out the statistical analysis, prepared the tables and figures, and wrote the paper, taking into account comments from all the co-authors. EHMT and HBB-d-M initiated and designed this study. EHMT was the overall project coordinator. HBB-d-M and EHMT were members of the writing group and gave input on the statistical analysis and interpretation of the results. HBB-d-M, PHMP, WMMV, and YTS are members of the EPIC-NL steering committee. All authors provided comments and suggestions on the manuscript and approved the final version.


High retention rates have been documented among patients receiving antiretroviral therapy (ART) in Myanmar. However, there is no information on human immunodeficiency virus (HIV)-infected individuals in care before initiation of ART (pre-ART care). We assessed attrition (loss-to-follow-up [LTFU] and death) rates among HIV-infected individuals in pre-ART care and their associated factors over a 4-year period.–June 2014) as part of an Integrated HIV Care (IHC) programme, Myanmar. Attrition rates per 100 person-years and cumulative incidence of attrition were calculated. Factors associated with attrition were examined by calculating hazard ratios (HRs).In this retrospective cohort study, we extracted routinely collected data of HIV-infected adults (>15 years old) entering pre-ART care (June 2011–80). The cumulative incidence of attrition was 70% at the end of a 4-year follow-up, of which nearly 90% occurred in the first 6 months. Male sex , WHO clinical Stage 3 and 4, CD4 count <200 cells/µL, abnormal BMI, and anaemia were statistically significant predictors of attrition.Of 18,037 HIV-infected adults enrolled in the IHC programme, 11,464 (63%) entered pre-ART care  cell count 160 cells/µL). Of the 11,464 eligible participants, 3,712 (32%) underwent attrition of which 43% were due to deaths and 57% were due to LTFU. The attrition rate was 78 per 100 person-years (95% CI, 75Pre-ART care attrition among persons living with HIV in Myanmar was alarmingly high – with most attrition occurring within the first 6 months. Strategies aimed at improving early HIV diagnosis and initiation of ART are needed. Suggestions include comprehensive nutrition support and intensified monitoring to prevent pre-ART care attrition by tracking patients who do not return for pre-ART care appointments. It is high time that Myanmar moves towards a ‘test and treat’ approach and ultimately eliminates the need for pre-ART care. Globally, human immunodeficiency virus (HIV) infection continues to be an important infectious disease causing mortality and morbidity, particularly in developing countries. The burden of HIV in the Asia-Pacific region is high, with an estimated 4.9 million people living with HIV (PLHIV) . The num3  (based on cluster of differentiation 4 CD4 cell counts less than 200 cells/µL), and 40,128 (44%) of them were receiving ART. Access to ART care in the country increased in 2012 with the National AIDS Programme (NAP) adopting the revised World Health Organization-2010 (WHO) recommendations of increasing the CD4 threshold for ART initiation to 350 cell/mmAlthough retention is high among patients who have started ART, there is no information about the retention of those in pre-ART care, that is, care before initiation of ART. Studies from India and Africa show that pre-ART care attrition could be as high as 40% at 1 year after registration –8. AnecdMyanmar is currently developing National Strategic Plans with the aim of achieving 90-90-90 goals related to HIV  and finally ending the HIV/AIDS pandemic in line with the United Nations’ Sustainable Development Goals by 2030 , Paris, France, approved the study and waived the need for individual informed consent because the study involved a review of existing programme records. The NAP of Myanmar permitted us to use the data for publication. To ensure confidentiality, we excluded patient identifiers from the final database prior to analysis.This is a retrospective cohort study using routinely collected data.The IHC programme is being implemented within the public sector by The Union, in collaboration with the Department of Health, National Tuberculosis (TB) Programme (NTP) and NAP since 2005 in Myanmar. Initial implementation was at a single ART clinic in Mandalay. Since then, there has been a steady expansion of the programme, and currently, ART is delivered through 18 ART centres and 14 ART decentralized sites. By the end of 2014, more than 20,000 patients were receiving ART in the IHC programme running across the country. Every month, around 400 patients are enrolled in the programme. Patients with a confirmed HIV diagnosis are referred from hospitals and health centres, STD /HIV-AIDS clinics, TB clinics, antenatal clinics and ART programmes run by other organizations apart from the IHC.Patient flow and assessment procedures after enrolment in IHC programme as well as the potential points of attrition in pre-ART care are shown in The PLHIV who are assessed and are not yet eligible for ART are usually clinically healthy and receive multivitamins, drugs for treatment of opportunistic infections, and counselling for psychosocial support. These patients are followed up after 3 months when ART eligibility is reassessed using the same clinical and immunological criteria described above. Subsequent follow-up visits of patients in pre-ART care usually occur at 6 monthly intervals but may be more frequent as per the discretion of the treating physician and as per patient needs.The PLHIV who are eligible for ART but have not yet started on treatment are usually ill and hence prioritized for receiving the next appointment in 1 or 2 weeks’ time. Patients who are not assessed for eligibility at enrolment are also scheduled for next appointment depending on their clinical condition. All of these services are provided free of cost to the patients.The PLHIV taking ART in IHC programme are also involved as volunteers, actively participating in clinic activities such as triage, registration, adherence counselling, and psychosocial support, and they play an important role in tracing patients who are lost to follow-up. Most of these efforts are targeted towards those already on ART and incorrect addresses pose major problems. Reasons include a migratory population, fear of discrimination, and long travel distances to receive care.Every patient has a dedicated file in which clinical details are entered by the treating doctors in IHC clinics. Data in these records are electronically captured in ‘IUATLD epi-concept’ database on a daily basis by trained data entry operators and medical doctors. Data quality control is performed at regular intervals to minimize errors in data entry and ensure data validity and consistency.st June 2011 and 30th June 2014. We excluded patients aged ≤15 years and women under a prevention of mother-to-child transmission (PMTCT) programme because of the different follow-up and clinical management schedule. Patients transferred out to other ART facilities were also excluded.We included all HIV-infected adults (>15 years of age) enrolled into pre-ART care of IHC programme, Myanmar, between 12]), haemoglobin level, and dates of enrolment, ART initiation, LTFU, last appointment, and hepatitis B and C infection status at enrolment. Based on haemoglobin (grams per decilitre) level, we divided the patients into four categories: normal , mild , moderate , and severe anaemia   was the date of censoring for patients who were retained in follow-up. Patients who were initiated on ART were censored on the date of ART initiation. Overall and stratum-specific cumulative attrition rates (death rates and LTFU rates) were estimated and expressed as attrition per 100 person-years (PYs). The cumulative incidence of attrition at monthly intervals for the first 6 months and then yearly from year 1 onwards was calculated as the proportion of patients in the cohort who had undergone attrition by the end of each period of follow-up. Associations between demographic, clinical, and immunological characteristics and attrition were studied, after excluding patients with missing values. Log-rank tests and bivariate unadjusted hazard ratios (HRs) were calculated. Characteristics found statistically significant during bivariate analysis (p<0.2) were entered into a Cox proportional hazard regression model, and adjusted HRs were calculated. Model selection was based on comparisons of change in model-2 log-likelihood in backward stepwise manner. Interaction between WHO Stage and CD4 counts was examined. Proportionality assumptions were tested using Schoenfeld residuals, log–log plots, and observed versus predicted survival plots. Kaplan–Meier survival curves (showing the cumulative incidence of attrition) across strata of key variables associated with attrition were plotted. Statistical significance was assessed at 5% probability of type-1 error and 95% confidence intervals (95% CI) were estimated for attrition rates and HRs.STATA  was used for data analysis. The baseline demographic, clinical, and immunological characteristics of patients were analysed. Survival analysis was undertaken to analyse the hazards of attrition after enrolment. The risk time of patients in pre-ART care started on their date of enrolment. For patients who developed attrition events (i.e. death or LTFU), the last scheduled appointment date (i.e. patient was scheduled to come but did not turn up) was the date of failure event. The last scheduled appointment date  were assessed for ART eligibility and 15,919 (90%) were found ART eligible. Among the ART eligible, only 6,573 (41%) were started on ART at enrolment. Thus, a total of 11,464 patients entered into pre-ART care group . Of thes2 and two-thirds were anaemic. The proportion of missing records was high for CD4 count (15%), BMI (21%), and haemoglobin level (16%)  .The pre-ART group had a total observation period of 4,788 PYs, during which 3,712 attrition events occurred: 1,585 (43%) deaths and 2,127 (57%) LTFU. The overall attrition rate was 78 per 100 PYs . Based on the nature of entry in the pre-ART cohort, the attrition rate among 343 patients not assessed for ART eligibility was 92 , 1,776 patients assessed for ART but not found eligible was 42 , and the 9,346 patients found ART eligible but not started was 98  per 100 PYs. Of the patients entering pre-ART care, 7,261 (64.4%) were subsequently initiated on ART at a median follow-up time interval of 77 days.The cumulative incidence of attrition and the nature of attrition are shown in 2, and had anaemia (2, and having moderate or severe anaemia, were factors independently associated with a higher risk of attrition. However, because the covariates, apart from the WHO staging, were found to violate the proportionality assumptions, the adjusted analysis results are not presented here. The unadjusted cumulative incidence of attrition by age, sex, WHO Stage, CD4 counts, BMI, and haemoglobin levels over the period of follow-up is presented in Stratum-specific attrition rates by patient characteristics and the estimated HRs for attrition are shown in  anaemia . In a muThis is the first study from Myanmar on pre-ART attrition among HIV-infected patients. Pre-ART patients are one of the most neglected subgroups of PLHIV. Attrition rates among pre-ART groups worldwide are higher as compared to those receiving ART , 13, 14.The median CD4 count was low at 160 cells/µL indicating advanced disease at presentation, delays in care seeking by PLHIV, and delayed diagnosis. This is possibly a reason for high dead cases during the first month. To address this, HIV testing should be made more widely available in the country. Currently, national guidelines in Myanmar recommend HIV testing of high-risk groups, pregnant women, new military recruits, and TB patients . EffortsEven among those found to be eligible for ART, more than half started on ART only 6 weeks after enrolment, suggesting delayed ART initiation. This might have contributed to the high mortality found in our study which is also in line with previous studies –27. The It appears that starting a patient on ART is the best way of retaining patients in care , 29. We Until the country moves into ‘test and treat’ strategy, we need to ensure that patients placed on pre-ART care are monitored well. We recommend introducing an indicator in the monthly report for monitoring retention and/or attrition among pre-ART cohort  because This study has several strengths. Firstly, the information is drawn from the second largest cohort of PLHIV in Myanmar with long follow-up data providing reliable estimates of attrition. Secondly, the data come from programmatic settings, thus reflecting the realities on the ground. Thirdly, this fills a knowledge gap in Myanmar about attrition among pre-ART care. Although there are many studies of ART cohorts, no previous study has examined pre-ART cohort in Myanmar . FourthlAs always, there are some limitations and they are mainly related to the operational nature of this study. Firstly, we used a definition of 90 days after the last appointed date to define LTFU. Given that the visits in pre-ART care were scheduled to be between 3 and 6 months apart for clinically healthy patients, we had to wait for 6 to 9 months before declaring LTFU. Hence, it is possible that some of the patients categorized as ‘retention in pre-ART’ at the time of assessment could actually be LTFU or dead. Thus, we could be underestimating our attrition rates. On the other hand, it is possible that many of those identified as having dropped out could be receiving HIV care in other facilities, without our knowledge , one couAttrition in a pre-ART HIV care cohort in Myanmar was alarmingly high – with most occurring within the first 6 months. Strategies such as improving early HIV diagnosis and the early initiation of ART, providing comprehensive nutrition support, implementing intensive monitoring using newly developed indicators, and tracking patients who do not return for their appointments are needed. It is most important that Myanmar moves towards universal ART and eliminate the need for pre-ART care.


The aims of this study were to 1) compare resting state functional connectivity (rs-fc) of the periaqueductal gray (PAG), a key region in the descending pain modulatory system (DPMS) between migraine without aura (MwoA) patients and healthy controls (HC), and 2) investigate how an effective treatment can influence the PAG rs-fc in MwoA patients. One hundred MwoA patients and forty-six matched HC were recruited. Patients were randomized to verum acupuncture, sham acupuncture, and waiting list groups. Resting state fMRI data were collected and seed based functional connectivity analysis was applied. Compared with HC, MwoA patients showed reduced rs-fc between the PAG and rostral anterior cingulate cortex/medial prefrontal cortex (rACC/mPFC), key regions in the DPMS and other pain related brain regions. The reduced rs-fc between the PAG and rACC/mPFC was associated with increased migraine headache intensity at the baseline. After treatments, rs-fc between the PAG and the rACC in MwoA patients significantly increased. The changes of rs-fc among the PAG, rACC and ventral striatum were significantly associated with headache intensity improvement. Impairment of the DPMS is involved in the neural pathophysiology of migraines. Impaired DPMS in migraine patients can be normalized after effective treatment. Migraine is a common chronic brain disorder with prevalence of 18.2% among females and 6.5% among males in the United StatesCurrent concepts of migraine suggest that the disturbed homeostasis of the trigeminovascular nociceptive pathway is a key factor for susceptibility to migraine headache. The pathophysiology of an imbalance in activity between the brainstem nuclei regulating antinociception and vascular control, especially in the ventrolateral periaqueductal gray (vlPAG) has been proposed679Despite the potential importance of the descending pain modulation system (DPMS) in the development of migraine13151513Thus, in this study, we first compared DPMS rs-fc between migraine patients  and matched healthy controls. We then explored if an effective non-pharmacological therapeutic modality, longitudinal acupuncture treatments, could modulate the rs-fc of DPMS. We hypothesize that 1) migraine is associated with dysfunction of DPMS as indicated by rs-fc changes between the PAG and key regions of DPMS; 2) an effective treatment can normalize the impaired rs-fc of DPMS.We choose acupuncture as treatment in this study because previous studies have found that longitudinal acupuncture can significantly reduce the symptoms of migraineurs17www.clinicaltrial.gov . Informed consent was obtained from all subjects. The recruitment started in June 2011 and ended in November 2013.The study protocol was approved by the Ethics Committee of the 1st Teaching Hospital of Chengdu University of Traditional Chinese Medicine. The experiment was performed in accordance with approved guidelines. Participants were enrolled from the outpatient department of the 3rd Teaching Hospital and the campus of Chengdu University of Traditional Chinese Medicine. This study was registered on Migraine without aura patients were included in this study. Diagnosis of MwoA was based on the International Classification of Headache Disorders, 2nd Edition ICHD-II MwoA criteriaRight-handed healthy controls (HC), aged between 17–45 years, free from any chronic pain conditions were recruited for this study as healthy controls. Each subject underwent a review of medical history evaluation, physical examination, hepatic function, renal function, and routine analysis of blood, urine, and stool to exclude organic disease carriers. Individuals with abnormal test results or a history of head trauma with loss of consciousness, pregnancy or lactation were excluded.The total observation period for MwoA patients of this study was eight weeks. After screening, all MwoA patients were randomized into five groups, including verum acupuncture groups 1, 2, 3 , sham acupuncture group (SA) and waiting-list (WT) group. Weeks one to four served as baseline phase were used to record baseline headache diaries. Weeks five to eight were served as intervention phase, during which patients in treatment groups received verum or sham acupuncture treatment. All patients continued recording headache diaries during this period. In addition, MRI scans were applied at the end of the forth and eighth weekends for migraine patients. All MwoA patients were migraine-free for at least 72 hours at the time of MRI scan. HC received only the baseline MRI scan .Acupoint and non-acupoint selection were similar to those in our previous RCT studies18Two licensed acupuncturists administrated all acupuncture treatment. All acupoints and non-acupoints were punctured bilaterally using disposable stainless steel fine needles. The needles were inserted perpendicularly at a penetration of 5 to 15 mm and were gently twisted, lifted, and thrust at an even amplitude, force, and speed to acquire deqi sensation  in all treatment groupsPatients were instructed and also agreed not to take any regular medications for the treatment of migraines. In cases of severe pain, ibuprofen (300 mg each capsule with sustained release) was allowed as rescue medication.The primary outcome of this study was the PAG rs-fc change. The clinical outcome were the headache intensity , 0 indicates no pain, 10 indicates worst pain ever) and frequency of migraine attacks  based on patients’ headache diary according to the guidelines of the IHS for Clinical Trials in Migraine233 employing an axial fast spoiled gradient recalled sequence . The BOLD resting-state functional images were obtained with echo-planar imaging .MRI data was acquired with a 3.0T magnetic resonance scanner  with an 8-channel phase-array head coil at the West China Hospital MRI center. Subjects were asked to stay awake and to keep their heads still during the scan, with their eyes closed and ears plugged. Prior to the functional run, a high-resolution structural image for each subject was acquired using a three-dimensional MRI sequence with a voxel size of 1 mmt-tests or χ2, as appropriate. The significant level used for the statistical analysis with 2-tailed testing was 5%. Continuous variables were presented as the mean with 95% confidence intervals (CI). Categorical variables were described as n (percentage).The clinical outcomes were analyzed using SPSS16.0 . Within and between-group comparisons were performed using paired or unpaired http://www.fil.ion.ucl.ac.uk/spm). The first ten volumes were not analyzed to allow for signal equilibration effects. The fMRI images were slice timing corrected, head motion corrected, coregistered to respective structural images for each subject, segmented, regressed out 6 head motion parameters, white matter signal and cerebrospinal fluid (CSF) signal, normalized by using structural image unified segmentation, and then re-sample to 3-mm cubic voxels. Subjects with head movements exceeding 2 mm on any axis or head rotation greater than 2° were excluded. Similar to our previous studyAll pre-processing steps and the rs-fc analysis were performed using DPARSFAWe chose the right vlPAG  as the seed. The rationales for choosing this seed are: 1) we found that increased levels of heat pain can evoke a significant fMRI signal increase in this regionGiven that the PAG seed region sits adjacent to a ventricle with significant pulsatile effect, we also chose a seed from ventricular aqueduct nearby the PAG seed  and seeds from the forth ventricle  as controls.Next, the averaged time course was obtained from the seed and the correlation analysis was performed in a voxel-wise way. Contrast images were generated for each subject by estimating the regression coefficient between all brain voxels and each seed’s time series, respectively. The correlation coefficient map was then converted into a Fisher-Z map by Fisher’s r-to-z transform to improve the normality. Group analysis was later applied using the random effect model with SPM8. We first compared the rs-fc difference between MwoA patients and healthy controls using two sample t-tests. Then, we compared the changes of rs-fc difference (post-treatment minus pre-treatment) between acupuncture (verum + sham) groups (AG) and waiting-list group in factorial design module in SPM8. To explore the association between the clinical outcomes and the PAG rs-fc, we also applied regression analyses between: 1) baseline PAG rs-fc and the corresponding migraine symptom (VAS), and 2) post- and pre-treatment rs-fc changes and corresponding VAS changes in acupuncture groups. For all group analysis, disease duration, SAS, and SDS are included as non-interest covariates. A threshold of a voxel-wise P < 0.005 uncorrected and P < 0.05 family wise error (FWE) correction at cluster level was applied for all analysis.150 patients were screened and 100 patients were recruited for this study. 46 age and sex-matched healthy controls were recruited in this study, 4 HCs were excluded due to excessive head movements. 88 patients participated in first fMRI scan, 81 patients participated in the second fMRI scan. 7 patients did not participate in the second fMRI scan, due to scheduling conflicts . Of the 81 patients who participated in the two fMRI scans, 9 patients were excluded from data analysis due to incomplete scans  and 10 patients were excluded due to excessive head movements  .We found no statistical difference among VA1, VA2, VA3, SA and waiting-list groups in age, sex, weight, height, duration of disease, headache intensity (VAS score), headache frequency, SAS and SDS (P > 0.05). We also found no statistical difference in age, gender, weight and height between all MwoA patients and healthy controls (P > 0.05) .Compared with the baseline group, VA1, VA2, and VA3 groups showed improvement in VAS score (P < 0.05). SA part28To explore the neural pathophysiology of migraine, we first applied a comparison between all MwoA patients and healthy controls using a two-sample t-test. Results indicated that compared with healthy controls, MwoA patients showed decreased rs-fc between the v1PAG and the bilateral medial prefrontal cortex (mPFC), left orbitofrontal cortex (OFC) and rostral anterior cingulate cortex (rACC), and increased rs-fc between the vlPAG and the bilateral adjacent PAG. Regression analysis indicated that the rs-fc between the vlPAG and left rACC/mPFC were negatively associated with VAS scores at baseline in MwoA patients &5. FigurWe then compared post- and pre-treatment differences in all treatment groups. Results revealed that after longitudinal acupuncture treatment, MwoA patients showed increased rs-fc between vlPAG and the bilateral middle cingulate cortex (MCC) and rACC, and left mPFC . InteresTo further explore the rs-fc changes at different conditions, we extracted the rs-fc Fisher-z value in the overlapping rACC/mPFC region and found that effective treatment can normalize the decreased rs-fc between the vlPAG and rACC . RegressIn the waiting-list group, comparison between the first and second fMRI scan showed reduced rs-fc between the vlPAG and the left inferior parietal lobe, precuneus, angular gyrus. Compared with the waiting-list group, the AG treatment groups showed greater vlPAG rs-fc increases in the bilateral rACC, and left mPFC and MCC after treatments . In addiAs an extra control, we also ran the above analysis using a seed from ventricular aqueduct nearby the PAG seed and seeds from the forth ventricle. Using the seed ventricular aqueduct nearby the PAG, we only found that from when compared with HCs, vlPAG in MwoA patients showed increased rs-fc with the adjacent PAG  at the threshold we set in rs-fc analysis; no result was found using the seeds from the forth ventricle, which further validates the findings of our experiment.In this study, we investigated the PAG rs-fc changes between migraine patients and healthy controls as well as the modulation effect of the effective treatment. We found that migraine patients are associated with reduced PAG-DPMS regions rs-fc as compared with healthy controls, and the longitudinal acupuncture treatment can normalize the decreased PAG-rACC rs-fc in migraine patients. Most importantly, these changes in the rs-fc were associated with alleviation  of migraine headache intensity.The PAG plays a central role in DPMS and is closely associated with opioid analgesia303118F-FDG), investigators found that affective, sensorimotor, and cognitive brain regions are involved in acupuncture treatment of migraine patients35363745Using resting state BOLD-fMRI and PET-CT There are several potential limitations in this study. 1) Sample size in each group was relatively small, which prevented us from testing the clinical outcome differences between different treatment groups. 2) Dropout rate in each group was relatively high, however, we would like to emphasize that the reasons for dropout do not seem to be associated with treatment response. Also, the aim of this study was to explore if an effective treatment could modulate the PAG rs-fc in migraine patients rather than to test the efficacy of acupuncture treatment. 3) We did not quantitatively record intensity of ‘deqi’ sensation, which is thought to be an important contributor to acupuncture effect131415We found that impairment of the DPMS is associated with neural pathophysiology of migraine during interictal period. Effective treatment can normalize the reduced rs-fc between the PAG and rACC/mPFC in migraine patients, which is associated with reduction of migraine intensity. Our result demonstrated the potential role of DPMS in monitoring the development of migraine.How to cite this article: Li, Z. et al. Altered periaqueductal gray resting state functional connectivity in migraine and the modulation effect of treatment. Sci. Rep. 6, 20298; doi: 10.1038/srep20298 (2016).