IL-17A increases TNF-α-induced COX-2 protein stability and augments PGE<inf>2</inf> secretion from airway smooth muscle cells: Impact on β<inf>2</inf>-adrenergic receptor desensitization

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. Background IL-17A plays an important role in respiratory disease and is a known regulator of pulmonary inflammation and immunity. Recent studies have linked IL-17A with exacerbation in asthma and COPD. We have shown that the enzyme cyclooxygenase-2 (COX-2) and its prostanoid products, prostaglandin E2 (PGE2) in particular, are key contributors in in vitro models of infectious exacerbation; however, the impact of IL-17A was not known. Methods and Results We address this herein and show that IL-17A induces a robust and sustained upregulation of COX-2 protein and PGE2 secretion from airway smooth muscle (ASM) cells. COX-2 can be regulated at transcriptional, post-transcriptional and/or post-translational levels. We have elucidated the underlying molecular mechanisms responsible for the sustained upregulation of TNF-α-induced COX-2 by IL-17A in ASM cells and show that is not via increased COX-2 gene expression. Instead, TNF-α-induced COX-2 upregulation is subject to regulation by the proteasome, and IL-17A acts to increase TNF-α-induced COX-2 protein stability as confirmed by cycloheximide chase experiments. In this way, IL-17A acts to amplify the COX-2-mediated effects of TNF-α and greatly enhances PGE2 secretion from ASM cells. Conclusion As PGE2 is a multifunctional prostanoid with diverse roles in respiratory disease, our studies demonstrate a novel function for IL-17A in airway inflammation by showing for the first time that IL-17A impacts on the COX-2/PGE2 pathway, molecules known to contribute to disease exacerbation

The human liver microenvironment shapes the homing and function of CD4+ T-cell populations.

OBJECTIVE: Tissue-resident memory T cells (TRM) are vital immune sentinels that provide protective immunity. While hepatic CD8+ TRM have been well described, little is known about the location, phenotype and function of CD4+ TRM. DESIGN: We used multiparametric flow cytometry, histological assessment and novel human tissue coculture systems to interrogate the ex vivo phenotype, function and generation of the intrahepatic CD4+ T-cell compartment. We also used leukocytes isolated from human leukocyte antigen (HLA)-disparate liver allografts to assess long-term retention. RESULTS: Hepatic CD4+ T cells were delineated into three distinct populations based on CD69 expression: CD69-, CD69INT and CD69HI. CD69HICD4+ cells were identified as tissue-resident CD4+ T cells on the basis of their exclusion from the circulation, phenotypical profile (CXCR6+CD49a+S1PR1-PD-1+) and long-term persistence within the pool of donor-derived leukcoocytes in HLA-disparate liver allografts. CD69HICD4+ T cells produced robust type 1 polyfunctional cytokine responses on stimulation. Conversely, CD69INTCD4+ T cells represented a more heterogenous population containing cells with a more activated phenotype, a distinct chemokine receptor profile (CX3CR1+CXCR3+CXCR1+) and a bias towards interleukin-4 production. While CD69INTCD4+ T cells could be found in the circulation and lymph nodes, these cells also formed part of the long-term resident pool, persisting in HLA-mismatched allografts. Notably, frequencies of CD69INTCD4+ T cells correlated with necroinflammatory scores in chronic hepatitis B infection. Finally, we demonstrated that interaction with hepatic epithelia was sufficient to generate CD69INTCD4+ T cells, while additional signals from the liver microenvironment were required to generate liver-resident CD69HICD4+ T cells. CONCLUSIONS: High and intermediate CD69 expressions mark human hepatic CD4+ TRM and a novel functionally distinct recirculating population, respectively, both shaped by the liver microenvironment to achieve diverse immunosurveillance

The human liver microenvironment shapes the homing and function of CD4+ T-cell populations

OBJECTIVE: Tissue-resident memory T cells (TRM) are vital immune sentinels that provide protective immunity. While hepatic CD8+ TRM have been well described, little is known about the location, phenotype and function of CD4+ TRM. DESIGN: We used multiparametric flow cytometry, histological assessment and novel human tissue coculture systems to interrogate the ex vivo phenotype, function and generation of the intrahepatic CD4+ T-cell compartment. We also used leukocytes isolated from human leukocyte antigen (HLA)-disparate liver allografts to assess long-term retention. RESULTS: Hepatic CD4+ T cells were delineated into three distinct populations based on CD69 expression: CD69−, CD69INT and CD69HI. CD69HICD4+ cells were identified as tissue-resident CD4+ T cells on the basis of their exclusion from the circulation, phenotypical profile (CXCR6+CD49a+S1PR1−PD-1+) and long-term persistence within the pool of donor-derived leukcoocytes in HLA-disparate liver allografts. CD69HICD4+ T cells produced robust type 1 polyfunctional cytokine responses on stimulation. Conversely, CD69INTCD4+ T cells represented a more heterogenous population containing cells with a more activated phenotype, a distinct chemokine receptor profile (CX3CR1+CXCR3+CXCR1+) and a bias towards interleukin-4 production. While CD69INTCD4+ T cells could be found in the circulation and lymph nodes, these cells also formed part of the long-term resident pool, persisting in HLA-mismatched allografts. Notably, frequencies of CD69INTCD4+ T cells correlated with necroinflammatory scores in chronic hepatitis B infection. Finally, we demonstrated that interaction with hepatic epithelia was sufficient to generate CD69INTCD4+ T cells, while additional signals from the liver microenvironment were required to generate liver-resident CD69HICD4+ T cells. CONCLUSIONS: High and intermediate CD69 expressions mark human hepatic CD4+ TRM and a novel functionally distinct recirculating population, respectively, both shaped by the liver microenvironment to achieve diverse immunosurveillance

Adjuvant Radio-chemotherapy for extrahepatic biliary tract cancers

<p>Abstract</p> <p>Background</p> <p>Extrahepatic biliary duct cancers (EBDC) are uncommon malignancies characterized by a poor prognosis with high rate of loco-regional recurrence. The purpose of the present study is to assess the feasibility and the potential impact of adjuvant radiotherapy (RT) in a series of patients treated in one institution.</p> <p>Methods</p> <p>Twenty three patients with non-metastatic bile duct cancer treated surgically with curative intent (4 gallbladder, 7 ampullary and 12 cholangiocarcinoma) received 3D conformal external beam RT to a median total dose of 50.4Gy. Concurrent chemotherapy based on 5-FU was delivered to 21 patients (91%). Surgical margins were negative in 11 patients (48%), narrow in 2 (9%), and microscopically involved in 8 (35%). Eleven patients (55%) had metastatic nodal involvement. The average follow-up time for all patients was 30 months (ranging from 3-98).</p> <p>Results</p> <p>Acute gastrointestinal grade 2 toxicity (RTOG scale) was recorded in 2 patients (9%). Nausea or vomiting grade 1 and 2 was observed in 8 (35%) and 2 patients (9%) respectively. Only one patient developed a major late radiation-induced toxicity. The main pattern of recurrence was both loco-regional and distant (liver, peritoneum and/or lung). No difference was observed in loco-regional control according to the tumor location. The 5-year actuarial loco-regional control rate was 48.3% (67% and 30% for patients operated on with negative and positive/narrow/unknown margins respectively, p = 0.04). The 5-year actuarial overall survival was of 35.9% for the entire group (61.4% in case of negative margins and 16.7% in case of positive/narrow/unknown margins, p = 0.07).</p> <p>Conclusions</p> <p>Postoperative RT with 50-60 Gy is feasible with acceptable acute and late toxicities. The potential benefit observed in our series may support the use of adjuvant RT in patients with locally advanced disease. Prospective randomized trials are warranted to confirm definitively the role of RT in this tumor location.</p

Cell-based analysis of CAD variants identifies individuals likely to benefit from uridine therapy.

PURPOSE: Pathogenic autosomal recessive variants in CAD, encoding the multienzymatic protein initiating pyrimidine de novo biosynthesis, cause a severe inborn metabolic disorder treatable with a dietary supplement of uridine. This condition is difficult to diagnose given the large size of CAD with over 1000 missense variants and the nonspecific clinical presentation. We aimed to develop a reliable and discerning assay to assess the pathogenicity of CAD variants and to select affected individuals that might benefit from uridine therapy. METHODS: Using CRISPR/Cas9, we generated a human CAD-knockout cell line that requires uridine supplements for survival. Transient transfection of the knockout cells with recombinant CAD restores growth in absence of uridine. This system determines missense variants that inactivate CAD and do not rescue the growth phenotype. RESULTS: We identified 25 individuals with biallelic variants in CAD and a phenotype consistent with a CAD deficit. We used the CAD-knockout complementation assay to test a total of 34 variants, identifying 16 as deleterious for CAD activity. Combination of these pathogenic variants confirmed 11 subjects with a CAD deficit, for whom we describe the clinical phenotype. CONCLUSIONS: We designed a cell-based assay to test the pathogenicity of CAD variants, identifying 11 CAD-deficient individuals who could benefit from uridine therapy

Phase III study of 5FU, etoposide and leucovorin (FELV) compared to epirubicin, cisplatin and 5FU (ECF) in previously untreated patients with advanced biliary cancer

The purpose of this study was to determine whether epirubicin, cisplatin and infused 5FU (ECF) improves overall survival (OS) compared to 5FU, etoposide and leucovorin (FELV) in patients with previously untreated advanced biliary cancer in a prospective randomised study. Patients were randomly assigned to receive epirubicin, cisplatin and infused 5FU ECF or bolus 5FU etoposide and leucovorin (FELV). The primary end point was OS with secondary end points of objective response rate (ORR), failure-free survival (FFS), quality of life (QOL) and toxicity. In all, 54 patients were recruited with 27 randomly assigned to each arm. The median OS for ECF was 9.02 months (95% confidence interval (CI): 6.46–11.51) and FELV 12.03 months (95% CI: 9.3–14.7), P=0.2059. Objective response rates were similar for both arms: ECF 19.2% (95% CI: 6.55–39.3); FELV 15% (95% CI: 3.2–37.9), P=0.72. There was significantly increased grade 3/4 neutropenia with FELV vs ECF (53.8 vs 29.5%, respectively, P=0.020). Symptom resolution was impressive for both regimens. This is the largest reported randomised study to date in this setting. ECF did not improve OS compared to FELV, but was associated with less acute toxicity. These data suggest that chemotherapy can prolong OS and achieve good symptomatic relief in advanced biliary cancer

Understanding Study Drug Discontinuation Through EUCLID

Introduction: Disparities in the care and outcomes of peripheral artery disease (PAD) have been well-established. In part this is due to disparities in enrollment of PAD trial cohorts. However, less attention has been paid to non-random protocol non-adherence after enrollment, which may lead to inaccurate estimates of treatment effects and reduce generalizability of study results. We aimed to ascertain characteristics associated with premature study drug discontinuation in a PAD cohort.Methods: Using data from EUCLID (Examining Use of Ticagrelor in Peripheral Artery Disease), factors associated with study drug discontinuation were assessed using univariable and multivariable Cox proportional hazards models with time to study drug discontinuation as the outcome of interest. Relationships between study drug discontinuation and major adverse cardiovascular events (MACE; cardiovascular death, myocardial infarction, ischemic stroke), major adverse limb events (MALE; acute limb ischemia, major amputation, and lower extremity revascularization), and all-cause hospitalization were assessed.Results: Of 13,842 eligible EUCLID participants, 3,886 (28.1%) prematurely and permanently discontinued study drug over a maximum follow-up of 42 months (annualized rate of 13.2 discontinuations per 100 patient-years). In a multivariable model, premature study drug discontinuation was associated with older age (aHR 1.16, 95%CI 1.14-1.19), eligibility based on prior lower extremity revascularization rather than ABI/TBI criteria (aHR 1.14, 95%CI 1.06-1.23), CLI status (aHR 1.23, 95%CI 1.06-1.42), COPD (aHR 1.36, 95%CI 1.24-1.49), and geographic region. In a multivariable analysis, study drug discontinuation was significantly associated with MACE (aHR 3.27, 95%CI 2.90-3.67, p p Conclusions: This analysis of EUCLID demonstrates that premature, permanent discontinuation of study drug is relatively common in more than a quarter of PAD patients, is unevenly distributed based on geography and other baseline characteristics, and is associated with worse outcomes in a clinical trial context. Study teams leading future PAD trials may want to address the possibility of study drug discontinuation prospectively, as a proactive approach may help investigators to maintain study cohort diversity and representativeness without sacrificing power and precision.</p