Faculty Bibliography

BACKGROUND:Sexual transmission chains of Ebola virus (EBOV) have been verified and linked to EBOV RNA persistence in semen, post-recovery. The rate of semen persistence over time, including the average duration of persistence among Ebola virus disease (EVD) survivors, is not well known. This cohort study aimed to analyze population estimates of EBOV RNA persistence rates in semen over time, and associated risk factors in a population of survivors from Sierra Leone. METHODS AND FINDINGS/RESULTS:In this cohort study from May 2015 to April 2017 in Sierra Leone, recruitment was conducted in 2 phases; the first enrolled 100 male participants from the Western Area District in the capital of Freetown, and the second enrolled 120 men from the Western Area District and from Lungi, Port Loko District. Mean age of participants was 31 years. The men provided semen for testing, analyzed by quantitative reverse transcription PCR (qRT-PCR) for the presence of EBOV RNA. Follow-up occurred every 2 weeks until the endpoint, defined as 2 consecutive negative qRT-PCR results of semen specimen testing for EBOV RNA. Participants were matched with the Sierra Leone EVD case database to retrieve cycle threshold (Ct) values from the qRT-PCR analysis done in blood during acute disease. A purposive sampling strategy was used, and the included sample composition was compared to the national EVD survivor database to understand deviations from the general male survivor population. At 180 days (6 months) after Ebola treatment unit (ETU) discharge, the EBOV RNA semen positive rate was 75.4% (95% CI 66.9%-82.0%). The median persistence duration was 204 days, with 50% of men having cleared their semen of EBOV RNA after this time. At 270 days, persistence was 26.8% (95% CI 20.0%-34.2%), and at 360 days, 6.0% (95% CI 3.1%-10.2%). Longer persistence was significantly associated with severe acute disease, with probability of persistence in this population at 1 year at 10.1% (95% CI 4.6%-19.8%) compared to the probability approaching 0% for those with mild acute disease. Age showed a dose-response pattern, where the youngest men (≤25 years) were 3.17 (95% CI 1.60, 6.29) times more likely to be EBOV RNA negative in semen, and men aged 26-35 years were 1.85 (95% CI 1.04, 3.28) times more likely to be negative, than men aged >35 years. Among participants with both severe acute EVD and a higher age (>35 years), persistence remained above 20% (95% CI 6.0%-50.6%) at 1 year. Uptake of safe sex recommendations 3 months after ETU discharge was low among a third of survivors. The sample was largely representative of male survivors in Sierra Leone. A limitation of this study is the lack of knowledge about infectiousness. CONCLUSIONS:In this study we observed that EBOV RNA persistence in semen was a frequent phenomenon, with high population rates over time. This finding will inform forthcoming updated recommendations on risk reduction strategies relating to sexual transmission of EBOV. Our findings support implementation of a semen testing program as part of epidemic preparedness and response. Further, the results will enable planning of the magnitude of testing and targeted counseling needs over time.

Adenosine A2a receptor (A2aR) stimulation promotes the synthesis of collagens I and III, and we have recently demonstrated that there is crosstalk between the A2aR and WNT/beta-catenin signaling pathway. In in vitro studies, A2aR signaling for collagen III expression was mediated by WNT/beta-catenin signaling in human dermal fibroblasts; we further verified whether the crosstalk between A2aR and Wnt/beta-catenin signaling was involved in diffuse dermal fibrosis in vivo. Wnt-signaling reporter mice (Tcf/Lef:H2B-GFP) were challenged with bleomycin and treated with the selective A2aR antagonist istradefylline (KW6002) or vehicle. Dermal fibrosis was quantitated and nuclear translocation of beta-catenin in fibroblasts was assessed by double-staining for Green fluorescent protein or dephosphorylated beta-catenin or beta-catenin phosphorylated at Ser552, and vimentin. KW6002 significantly reduced skin thickness, skinfold thickness, breaking tension, dermal hydroxyproline content, myofibroblast accumulation, and collagen alignment in bleomycin-induced dermal fibrosis. Also, there was increased expression of Tcf/Lef:H2B-GFP reporter in bleomycin-induced dermal fibrosis, an effect that was diminished by treatment with KW6002. Moreover, KW6002 significantly inhibited nuclear translocation of Tcf/Lef:H2B-GFP reporter, as well as dephosphorylated beta-catenin and beta-catenin phosphorylated at Ser552. Our work supports the hypothesis that pharmacologic blockade of A2aR inhibits the WNT/beta-catenin signaling pathway, contributing to its capacity to inhibit bleomycin-induced dermal fibrosis.

BACKGROUND: Fibrosing diseases are a leading cause of morbidity and mortality worldwide and, therefore, there is a need for safe and effective antifibrotic therapies. Adenosine, generated extracellularly by the dephosphorylation of adenine nucleotides, ligates specific receptors which play a critical role in development of hepatic and dermal fibrosis. Results of recent clinical trials indicate that tenofovir, a widely used antiviral agent, reverses hepatic fibrosis/cirrhosis in patients with chronic hepatitis B infection. Belonging to the class of acyclic nucleoside phosphonates, tenofovir is an analogue of AMP. We tested the hypothesis that tenofovir has direct antifibrotic effects in vivo by interfering with adenosine pathways of fibrosis using two distinct models of adenosine and A2AR-mediated fibrosis. METHODS: Thioacetamide (100mg/kg IP)-treated mice were treated with vehicle, or tenofovir (75mg/kg, SubQ) (n = 5-10). Bleomycin (0.25U, SubQ)-treated mice were treated with vehicle or tenofovir (75mg/kg, IP) (n = 5-10). Adenosine levels were determined by HPLC, and ATP release was quantitated as luciferase-dependent bioluminescence. Skin breaking strength was analysed and H&E and picrosirus red-stained slides were imaged. Pannexin-1expression was knocked down following retroviral-mediated expression of of Pannexin-1-specific or scrambled siRNA. RESULTS: Treatment of mice with tenofovir diminished adenosine release from the skin of bleomycin-treated mice and the liver of thioacetamide-treated mice, models of diffuse skin fibrosis and hepatic cirrhosis, respectively. More importantly, tenofovir treatment diminished skin and liver fibrosis in these models. Tenofovir diminished extracellular adenosine concentrations by inhibiting, in a dose-dependent fashion, cellular ATP release but not in cells lacking Pannexin-1. CONCLUSIONS: These studies suggest that tenofovir, a widely used antiviral agent, could be useful in the treatment of fibrosing diseases.

INTRODUCTION: Adenosine, acting through the A2A receptor, promotes tissue matrix production in the skin and the liver and induces the development of dermal fibrosis and cirrhosis in murine models. Since expression of A2A receptors is increased in scleroderma fibroblasts, we examined the mechanisms by which the A2A receptor produces its fibrogenic effects. METHODS: The effects of A2A receptor ligation on the expression of the transcription factor, Fli1, a constitutive repressor for the synthesis of matrix proteins, such as collagen, is studied in dermal fibroblasts. Fli1 is also known to repress the transcription of CTGF/CCN2, and the effects of A2A receptor stimulation on CTGF and TGF-beta1 expression are also examined. RESULTS: A2A receptor occupancy suppresses the expression of Fli1 by dermal fibroblasts. A2A receptor activation induces the secretion of CTGF by dermal fibroblasts, and neutralization of CTGF abrogates the A2A receptor-mediated enhancement of collagen type I production. A2AR activation, however, resulted in a decrease in TGF-beta1 protein release. CONCLUSIONS: Our results suggest that Fli1 and CTGF are important mediators of the fibrogenic actions of adenosine and the use of small molecules such as adenosine A2A receptor antagonists may be useful in the therapy of dermal fibrosis in diseases such as scleroderma.