Chronic Hepatitis B Infection: New Approaches towards Cure.

Chronic hepatitis B virus (HBV) infection leads to the development of cirrhosis and hepatocellular carcinoma. Lifelong treatment with nucleotides/nucleoside antiviral agents is effective at suppressing HBV replication, however, adherence to daily therapy can be challenging. This review discusses recent advances in the development of long-acting formulations for HBV treatment and prevention, which could potentially improve adherence. Promising new compounds that target distinct steps of the virus life cycle are summarized. In addition to treatments that suppress viral replication, curative strategies are focused on the elimination of covalently closed circular DNA and the inactivation of the integrated viral DNA from infected hepatocytes. We highlight promising long-acting antivirals and genome editing strategies for the elimination or deactivation of persistent viral DNA products in development.

Alcohol and HIV-Derived Hepatocyte Apoptotic Bodies Induce Hepatic Stellate Cell Activation.

Recently, we found that both HIV and acetaldehyde, an alcohol metabolite, induce hepatocyte apoptosis, resulting in the release of large extracellular vesicles called apoptotic bodies (ABs). The engulfment of these hepatocyte ABs by hepatic stellate cells (HSC) leads to their profibrotic activation. This study aims to establish the mechanisms of HSC activation after engulfment of ABs from acetaldehyde and HIV-exposed hepatocytes (ABAGS+HIV). In vitro experiments were performed on Huh7.5-CYP (RLW) cells to generate hepatocyte ABs and LX2 cells were used as HSC. To generate ABs, RLW cells were pretreated for 24 h with acetaldehyde, then exposed overnight to HIV1ADA and to acetaldehyde for 96 h. Thereafter, ABs were isolated from cell suspension by a differential centrifugation method and incubated with LX2 cells (3:1 ratio) for profibrotic genes and protein analyses. We found that HSC internalized ABs via the tyrosine kinase receptor, Axl. While the HIV gag RNA/HIV proteins accumulated in ABs elicited no productive infection in LX2 and immune cells, they triggered ROS and IL6 generation, which, in turn, activated profibrotic genes via the JNK-ERK1/2 and JAK-STAT3 pathways. Similarly, ongoing profibrotic activation was observed in immunodeficient NSG mice fed ethanol and injected with HIV-derived RLW ABs. We conclude that HSC activation by hepatocyte ABAGS+HIV engulfment is mediated by ROS-dependent JNK-ERK1/2 and IL6 triggering of JAK-STAT3 pathways. This can partially explain the mechanisms of liver fibrosis development frequently observed among alcohol abusing PLWH.

Bovine mammary alveolar MAC-T cells afford a tool for studies of bovine milk exosomes in drug delivery.

Bovine milk exosomes (BMEs) have attracted attention as vehicles for delivering RNA therapeutics. BMEs originate in mammary alveolar cells. Here, we determined whether bovine mammary alveolar MAC-T cells afford a tool to assess RNA delivery by BMEs. MAC-T cells exosomes (MAC-T BMEs) and BMEs were harvested by differential ultracentrifugation. Exosome size, morphology, microRNA content and marker proteins were assessed using nanoparticle tracking analysis, transmission electron microscopy, real-time PCR and immunoblot analysis, respectively. MAC-T cells were genetically engineered to secrete MAC-T BMEs endogenously labeled with a near-infrared fluorescent protein and tissue distribution was compared to fluorophore-labeled BMEs following intravenous injection in C57BL/6 mice. Morphology and size were similar in MAC-T BMEs and BMEs (94 ± 5.8 nm and 101 ± 4.2 nm, p > 0.05). Both preparations expressed miR-320a, miR-200c and let-7a-5p (positive controls) but not miR-1 (negative control). Exosome marker proteins, CD9, CD63, CD81 and Tsg101, were detected in both MAC-T BMEs and BMEs. Distribution in mouse tissues was similar for both preparations, with liver being the primary accumulation site. Collectively, MAC-T BMEs afford a tool for BMEs-based RNA delivery studies.