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The hepatitis B virus (HBV) and hepatitis D virus (HDV) infections cause liver disease, including hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). HBV infection remains a major global health problem. In 2019, 296 million people were living with chronic hepatitis B and about 5% of them were co-infected with HDV. In vitro cell culture systems are instrumental in the development of therapeutic targets. Cell culture systems contribute to identifying molecular mechanisms for HBV and HDV propagation, finding drug targets for antiviral therapies, and testing antiviral agents. Current HBV therapeutics, such as nucleoside analogs, effectively suppress viral replication but are not curative. Additionally, no effective treatment for HDV infection is currently available. Therefore, there is an urgent need to develop therapies to treat both viral infections. A robust in vitro cell culture system supporting HBV and HDV infections (HBV/HDV) is a critical prerequisite to studying HBV/HDV pathogenesis, the complete life cycle of HBV/HDV infections, and consequently identifying new therapeutics. However, the lack of an efficient cell culture system hampers the development of novel antiviral strategies for HBV/HDV infections. In vitro cell culture models have evolved with significant improvements over several decades. Recently, the development of the HepG2-NTCP sec+ cell line, expressing the sodium taurocholate co-transporting polypeptide receptor (NTCP) and self-assembling co-cultured primary human hepatocytes (SACC-PHHs) has opened new perspectives for a better understanding of HBV and HDV lifecycles and the development of specific antiviral drug targets against HBV/HDV infections. We address various cell culture systems along with different cell lines and how these cell culture systems can be used to provide better tools for HBV and HDV studies.
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Vaccination against hepatitis B using a dissolving microneedle patch (dMNP) could increase access to the birth dose by reducing expertise needed for vaccine administration, refrigerated storage, and safe disposal of biohazardous sharps waste. In this study, we developed a dMNP to administer hepatitis B surface antigen (HBsAg) adjuvant-free monovalent vaccine (AFV) at doses of 5 µg, 10 µg, and 20 µg, and compared its immunogenicity to vaccination with 10 µg of standard monovalent HBsAg delivered by intramuscular (IM) injection either in an AFV format or as aluminum-adjuvanted vaccine (AAV). Vaccination was performed on a three dose schedule of 0, 3, and 9 weeks in mice and 0, 4, and 24 weeks in rhesus macaques. Vaccination by dMNP induced protective levels of anti-HBs antibody responses (≥10 mIU/ml) in mice and rhesus macaques at all three HBsAg doses studied. HBsAg delivered by dMNP induced higher anti-HBsAg antibody (anti-HBs) responses than the 10 µg IM AFV, but lower responses than 10 µg IM AAV, in mice and rhesus macaques. HBsAg-specific CD4+ and CD8+ T cell responses were detected in all vaccine groups. Furthermore, we analyzed differential gene expression profiles related to each vaccine delivery group and found that tissue stress, T cell receptor signaling, and NFκB signaling pathways were activated in all groups. These results suggest that HBsAg delivered by dMNP, IM AFV, and IM AAV have similar signaling pathways to induce innate and adaptive immune responses. We further demonstrated that dMNP was stable at room temperature (20 °C-25 °C) for 6 months, maintaining 67 ± 6 % HBsAg potency. This study provides evidence that delivery of 10 µg (birth dose) AFV by dMNP induced protective levels of antibody responses in mice and rhesus macaques. The dMNPs developed in this study could be used to improve hepatitis B birth dose vaccination coverage levels in resource limited regions to achieve and maintain hepatitis B elimination.
Asunto(s)
Vacunas contra Hepatitis B , Hepatitis B , Animales , Ratones , Macaca mulatta , Antígenos de Superficie de la Hepatitis B , Vacunación/métodos , Anticuerpos contra la Hepatitis B , Hepatitis B/prevención & control , Adyuvantes InmunológicosRESUMEN
DNA mismatch repair (MMR) relies on MutS and MutL ATPases for mismatch recognition and strand-specific nuclease recruitment to remove mispaired bases in daughter strands. However, whether the MutS-MutL complex coordinates MMR by ATP-dependent sliding on DNA or protein-protein interactions between the mismatch and strand discrimination signal is ambiguous. Using functional MMR assays and systems preventing proteins from sliding, we show that sliding of human MutSα is required not for MMR initiation, but for final mismatch removal. MutSα recruits MutLα to form a mismatch-bound complex, which initiates MMR by nicking the daughter strand 5' to the mismatch. Exonuclease 1 (Exo1) is then recruited to the nick and conducts 5' â 3' excision. ATP-dependent MutSα dissociation from the mismatch is necessary for Exo1 to remove the mispaired base when the excision reaches the mismatch. Therefore, our study has resolved a long-standing puzzle, and provided new insights into the mechanism of MMR initiation and mispair removal.
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Disparidad de Par Base , Reparación de la Incompatibilidad de ADN , Adenosina Trifosfatasas , ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Humanos , Proteína MutS de Unión a los Apareamientos Incorrectos del ADN/genéticaRESUMEN
[This corrects the article DOI: 10.1038/s41421-019-0126-6.].
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Oral squamous cell carcinoma (OSCC) is a common subtype of head and neck squamous cell carcinoma (HNSCC), but the pathogenesis underlying familial OSCCs is unknown. Here, we analyzed whole-genome sequences of a family with autosomal dominant expression of oral tongue cancer and identified proto-oncogenes VAV2 and IQGAP1 as the primary factors responsible for oral cancer in the family. These two genes are also frequently mutated in sporadic OSCCs and HNSCCs. Functional analysis revealed that the detrimental variants target tumorigenesis-associated pathways, thus confirming that these novel genetic variants help to establish a predisposition to familial OSCC.
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Osteopontin (OPN) is critical for ischemia-induced neovascularization. Unlike rodents, humans express three OPN isoforms (a, b, and c); however, the roles of these isoforms in post-ischemic neovascularization and cell migration remain undefined. Our objective was to determine if OPN isoforms differentially affect post-ischemic neovascularization and to elucidate the mechanisms underlying these differences. To investigate if human OPN isoforms exert divergent effects on post-ischemic neovascularization, we utilized OPN-/- mice and a loss-of-function/gain-of-function approach in vivo and in vitro. In this study OPN-/- mice underwent hindlimb ischemia surgery and 1.5 × 106 lentivirus particles were administered intramuscularly to overexpress OPNa, OPNb, or OPNc. OPNa and OPNc significantly improved limb perfusion 30.4% ± 0.8 and 70.9% ± 6.3, respectively, and this translated to improved functional limb use, as measured by voluntary running wheel utilization. OPNa- and OPNc-treated animals exhibited significant increases in arteriogenesis, defined here as the remodeling of existing arterioles into larger conductance arteries. Macrophages play a prominent role in the arteriogenesis process and OPNa- and OPNc-treated animals showed significant increases in macrophage accumulation in vivo. In vitro, OPN isoforms did not affect macrophage polarization, whereas all three isoforms increased macrophage survival and decreased macrophage apoptosis. However, OPN isoforms exert differential effects on macrophage migration, where OPNa and OPNc significantly increased macrophage migration, with OPNc serving as the most potent isoform. In conclusion, human OPN isoforms exert divergent effects on neovascularization through differential effects on arteriogenesis and macrophage accumulation in vivo and on macrophage migration and survival, but not polarization, in vitro. Altogether, these data support that human OPN isoforms may represent novel therapeutic targets to improve neovascualrization and preserve tissue function in patients with obstructive artery diseases.