A simple methodology to assess endolysosomal protease activity involved in antigen processing in human primary cells

Background: Endolysosomes play a key role in maintaining the homeostasis of the cell. They are made of a complex set of proteins that degrade lipids, proteins and sugars. Studies involving endolysosome contribution to cellular functions such as MHC class I and II epitope production have used recombinant endolysosomal proteins, knockout mice that lack one of the enzymes or purified organelles from human tissue. Each of these approaches has some caveats in analyzing endolysosomal enzyme functions. Results: In this study, we have developed a simple methodology to assess endolysosomal protease activity. By varying the pH in crude lysate from human peripheral blood mononuclear cells (PBMCs), we documented increased endolysosomal cathepsin activity in acidic conditions. Using this new method, we showed that the degradation of HIV peptides in low pH extracts analyzed by mass spectrometry followed similar kinetics and degradation patterns as those performed with purified endolysosomes. Conclusion: By using crude lysate in the place of purified organelles this method will be a quick and useful tool to assess endolysosomal protease activities in primary cells of limited availability. This quick method will especially be useful to screen peptide susceptibility to degradation in endolysosomal compartments for antigen processing studies, following which detailed analysis using purified organelles may be used to study specific peptides

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Application of Earth Observation Data and Standardized Precipitation Index Based Approach for Meteorological Drought Monitoring, Assessment and Prediction Over Kutch, Gujarat, India

Drought is a natural phenomenon which differs from other natural hazards by its slow accumulating process and its indefinite commencement and termination. The present study addresses water deficiency and drought occurrence over Kutch district, Gujarat, because nearly 45% of the whole Kutch district is severely suffering by deficiency of water. Earth observation data (LANDSAT ETM+) and Standardized Precipitation Index were used to analyze drought severity. Daily rainfall data over the study area were obtained from Indian Meteorological Department (IMD) for the period of study (1990-2014) and geo-referenced for further analyses. Using Remote Sensing and GIS techniques, rainfall variability map over the period of study has been prepared to show rainfall distribution and land use and land cover map is prepared to show the area under different land use classes and impacts of drought over land uses. Standardized Precipitation Index (SPI) was generated for each block wise and scenario of drought development has been analyzed using decadal data set for the study period (1990-2014). The present study suggests method and techniques for continuous drought monitoring by linking temporal earth observation and rainfall data. The methodology will be very useful for the development of a regional drought monitoring syste

Barriers to hepatitis C direct‐acting antiviral therapy among HIV/hepatitis C virus‐coinfected persons

BACKGROUND AND AIM: Direct-acting antivirals (DAAs) have increased HCV treatment opportunities for vulnerable HIV/HCV coinfected persons. The aim of this study was to identify the frequency of and potential barriers to DAA prescription in HIV/HCV patients during the first few years of DAA availability in the United States. METHODS: The AIDS Healthcare Foundation (AHF) electronic medical record system was queried to identify all HCV viremic HIV-infected patients in care at AHF Healthcare Centers January 2015-August 2017 and compare characteristics by receipt of a DAA prescription. Multivariate logistic regression analyses were conducted to examine factors associated with DAA prescription. RESULTS: Of 826 eligible patients, 355 (43%) were prescribed a DAA; among those not prescribed a DAA, 301 (64%) had well-controlled HIV (HIV RNA ≤200 copies/mL). In multivariate logistic regression analysis, patients with a history of substance use (odds ratio (OR), 0.51 [95% confidence interval 0.35–0.73]) or on select HIV antiretroviral (ARV) regimens were less likely to be prescribed a DAA. Those who had well-controlled HIV (OR, 5.03 [3.06–8.27]), CD4+ T cell count >200 cells/mm(3) (OR, 1.85 [1.04–3.30]), estimated glomerular filtration rate >60 mL/min/1.73m(2) (OR, 3.32 [1.08–10.15]), or established care prior to January 2015 (OR, 1.57 [1.08–2.29] were more likely to be prescribed a DAA. CONCLUSIONS: In addition to lack of HIV suppression, select ARV regimens, substance use, and kidney disease appeared to limit DAA prescription in the early interferon-free DAA era. Many were not prescribed DAAs despite HIV suppression. Further research is needed to determine if the observed associations persist today

Multi-institutional analysis of aneuploidy and outcomes to chemoradiation and durvalumab in stage III non-small cell lung cancer

There is a need to identify predictive biomarkers to guide treatment strategies in stage III non-small cell lung cancer (NSCLCs). In this multi-institutional cohort of 197 patients with stage III NSCLC treated with concurrent chemoradiation (cCRT) and durvalumab consolidation, we identify that low tumor aneuploidy is independently associated with prolonged progression-free survival (HR 0.63; p=0.03) and overall survival (HR 0.50; p=0.03). Tumors with high aneuploidy had a significantly greater incidence of distant metastasis and shorter median distant-metastasis free survival (p=0.04 and p=0.048, respectively), but aneuploidy level did not associate with local-regional outcomes. Multiplexed immunofluorescence analysis in a cohort of NSCLC found increased intratumoral CD8-positive, PD-1-positive cells, double-positive PD-1 CD8 cells, and FOXP3-positive T-cell in low aneuploid tumors. Additionally, in a cohort of 101 patients treated with cCRT alone, tumor aneuploidy did not associate with disease outcomes. These data support the need for upfront treatment intensification strategies in stage III NSCLC patients with high aneuploid tumors and suggest that tumor aneuploidy is a promising predictive biomarker

Treatment outcomes and costs of a simplified antiviral treatment strategy for hepatitis C among monoinfected and HIV and/or hepatitis B virus-co-infected patients in Myanmar.

Access to hepatitis C virus&nbsp;(HCV) testing and treatment is limited in Myanmar. We assessed an integrated HIV and viral hepatitis testing and HCV treatment strategy. Sofosbuvir/velpatasvir (SOF/VEL)&nbsp;±&nbsp;weight-based ribavirin for 12&nbsp;weeks was provided at three treatment sites in Myanmar and sustained virologic response (SVR) assessed at 12&nbsp;weeks after treatment. Participants co-infected with HBV were treated concurrently with tenofovir. Cost estimates in 2018 USD were made at Yangon and Mandalay using standard micro-costing methods. 803 participants initiated SOF/VEL; 4.8% were lost to follow-up. SVR was achieved in 680/803 (84.6%) by intention-to-treat analysis. SVR amongst people who inject drugs (PWID) was 79.7% (381/497), but 92.5% among PWID on opioid substitution therapy (OST) (74/80), and 97.4% among non-PWID (298/306). Utilizing data from 492 participants, of whom 93% achieved SVR, the estimated average cost of treatment per patient initiated was $1030 (of which 54$1109 and real-world estimate of $1250. High SVR rates were achieved for non-PWID and PWID on OST. However, the estimated average cost of the intervention (under the assumption of no genotype testing and reduced real-world effectiveness) of$1250/patient is unaffordable for a national elimination strategy. Reductions in the cost of antivirals and linkage to social and behavioural health services including substance use disorder treatment to increase retention and adherence to treatment are critical to HCV elimination in this population

Development and Validation of a Clinical Prediction Rule for Angiotensin-converting Enzyme Inhibitor-induced Cough

BACKGROUND: Angiotensin-converting enzyme inhibitors are effective for many cardiovascular diseases and are widely prescribed, but cough sometimes necessitates their withdrawal. OBJECTIVE: To develop and validate a model that predicts, by using information available at first prescription, whether a patient will develop cough within 6 months. DESIGN: Retrospective cohort study with derivation and validation sets. SETTING: Outpatient clinics affiliated with an urban tertiary care hospital. PATIENTS: Clinical data were collected from electronic charts. The derivation set included 1,125 patients and the validation set included 567 patients. INTERVENTIONS: None. MEASUREMENTS: Angiotensin-converting enzyme inhibitorinduced cough assessed by predetermined criteria. RESULTS: In the total cohort, 12% of patients developed angiotensin-converting enzyme inhibitor-induced cough. Independent multivariate predictors of cough were older age, female gender, non-African American (with East Asian having highest risk), no history of previous angiotensin-converting enzyme inhibitor use, and history of cough due to another angiotensin-converting enzyme inhibitor. Patients with a history of angiotensin-converting enzyme inhibitor-induced cough were 29 times more likely to develop a cough than those without this history. These factors were used to develop a model stratifying patients into 4 risk groups. In the derivation set, low-risk, average-risk, intermediate-risk, and highrisk groups had a 6%, 9%, 22%, and 55% probability of cough, respectively. In the validation set, 4%, 14%, 20%, and 60% of patients in these 4 groups developed cough, respectively. CONCLUSIONS: This model may help clinicians predict the likelihood of a particular patient developing cough from an angiotensin-converting enzyme inhibitor at the time of prescribing, and may also assist with subsequent clinical decisions