Co-delivery of Cas9 mRNA and guide RNAs edits hepatitis B virus episomal and integration DNA in mouse and tree shrew models.

With 296 million chronically infected individuals worldwide, hepatitis B virus (HBV) causes a major health burden. The major challenge to cure HBV infection lies in the fact that the source of persistence infection, viral episomal covalently closed circular DNA (cccDNA), could not be targeted. In addition, HBV DNA integration, although normally results in replication-incompetent transcripts, considered as oncogenic. Though several studies evaluated the potential of gene-editing approaches to target HBV, previous in vivo studies have been of limited relevance to authentic HBV infection, as the models do not contain HBV cccDNA or feature a complete HBV replication cycle under competent host immune system. In this study, we evaluated the effect of in vivo codelivery of Cas9 mRNA and guide RNAs (gRNAs) by SM-102-based lipid nanoparticles (LNPs) on HBV cccDNA and integrated DNA in mouse and a higher species. CRISPR nanoparticle treatment decreased the levels of HBcAg, HBsAg and cccDNA in AAV-HBV1.04 transduced mouse liver by 53%, 73% and 64% respectively. In HBV infected tree shrews, the treatment achieved 70% reduction of viral RNA and 35% reduction of cccDNA. In HBV transgenic mouse, 90% inhibition of HBV RNA and 95% inhibition of DNA were observed. CRISPR nanoparticle treatment was well tolerated in both mouse and tree shrew, as no elevation of liver enzymes and minimal off-target was observed. Our study demonstrated that SM-102-based CRISPR is safe and effective in targeting HBV episomal and integration DNA in vivo. The system delivered by SM-102-based LNPs may be used as a potential therapeutic strategy against HBV infection.

Limited disassembly of cytoplasmic hepatitis B virus nucleocapsids restricts viral infection in murine hepatic cells.

BACKGROUND AND AIMS: Murine hepatic cells cannot support hepatitis B virus (HBV) infection even with supplemental expression of viral receptor, human sodium taurocholate cotransporting polypeptide (hNTCP). However, the specific restricted step remains elusive. In this study, we aimed to dissect HBV infection process in murine hepatic cells. APPROACH AND RESULTS: Cells expressing hNTCP were inoculated with HBV or hepatitis delta virus (HDV). HBV pregenomic RNA (pgRNA), covalently closed circular DNA (cccDNA), and different relaxed circular DNA (rcDNA) intermediates were produced in vitro . The repair process from rcDNA to cccDNA was assayed by in vitro repair experiments and in mouse with hydrodynamic injection. Southern blotting and in situ hybridization were used to detect HBV DNA. HBV, but not its satellite virus HDV, was restricted from productive infection in murine hepatic cells expressing hNTCP. Transfection of HBV pgRNA could establish HBV replication in human, but not in murine, hepatic cells. HBV replication-competent plasmid, cccDNA, and recombinant cccDNA could support HBV transcription in murine hepatic cells. Different rcDNA intermediates could be repaired to form cccDNA both in vitro and in vivo . In addition, rcDNA could be detected in the nucleus of murine hepatic cells, but cccDNA could not be formed. Interestingly, nuclease sensitivity assay showed that the protein-linked rcDNA isolated from cytoplasm was completely nuclease resistant in murine, but not in human, hepatic cells. CONCLUSIONS: Our results imply that the disassembly of cytoplasmic HBV nucleocapsids is restricted in murine hepatic cells. Overcoming this limitation may help to establish an HBV infection mouse model.

SARS-CoV-2 Antibody Seroprevalence in Wuhan, China, from 23 April to 24 May 2020.

The outbreak of coronavirus disease 2019 (COVID-19) was first reported in Wuhan, China, in December 2019. To investigate the prevalence of COVID-19 in Wuhan, we conducted serologic tests on 35,326 individuals from four different communities to estimate cumulative incidence of infection. Our results showed that 1,332 individuals (3.77%) showed positive COVID-19 antibody (either IgM or IgG). Males had a lower positivity rate than females (3.02% versus 4.52%). The antibody positivity rates showed a clear trend of increase according to patients' ages and varied among different communities. The results indicate that public health interventions may play important roles in the control of COVID-19.IMPORTANCE Coronavirus disease 2019 (COVID-19) was first detected in December 2019 in Wuhan, China. Afterwards, a number of public health interventions were implemented, including lock-down, face mask ordinances, and social distancing. Studies that rely on viral RNA testing of symptomatic patients have shown that these multifaceted interventions contributed to the control of the COVID-19 outbreak in Wuhan and delayed the epidemic's progression. However, these estimates of confirmed cases may miss large numbers of asymptomatic patients and recovered symptomatic patients who were not tested. To investigate the prevalence of COVID-19 in Wuhan, we conducted serologic tests on 35,326 individuals to estimate the cumulative incidence of infection. The results suggest that public health interventions may play important roles in the control of COVID-19.

IL-1ß enhances human placenta-derived mesenchymal stromal cells ability to mediate Th1/Th2 and Th1/CD4+IL-10+ T cell balance and regulates its adhesion, proliferation and migration via PD-L1.

Human placenta-derived mesenchymal stromal cells (hPMSCs) are promising candidates for the treatment of graft-versus-host disease (GVHD), which is associated with high IL-1ß levels. In this study, the effects of IL-1ß and hPMSCs on each other were investigated by analyzing the proportion of Th1, Th2 and CD4+IL-10+ T cells and PD-L1 expression, as well as the adhesion, migration, and proliferation of hPMSCs. The results showed that hPMSCs decreased IL-1ß levels and downregulated Th1/Th2 and Th1/CD4+IL-10+ T cells ratios in the GVHD model. The in vitro results revealed that IL-1ß strengthened the hPMSCs capacity to reduce the Th1/Th2 and Th1/CD4+IL-10+ T cell ratios, inhibited the adhesion and proliferation of hPMSCs and increased PD-L1 expression on hPMSCs via the JAK and NF-κB pathways. Overall, these findings suggested that hPMSCs alleviate GVHD by decreasing IL-1ß level and maintaining the balance among different T cell subsets. IL-1ß enhanced the ability of hPMSCs to balance different T cell subsets and inhibited hPMSCs adhesion and proliferation by regulating PD-L1 expression via the JAK and NF-κB pathways.

IL-27 Promotes Human Placenta-Derived Mesenchymal Stromal Cell Ability To Induce the Generation of CD4+IL-10+IFN-γ+ T Cells via the JAK/STAT Pathway in the Treatment of Experimental Graft-versus-Host Disease.

Human mesenchymal stromal cells (MSCs) harbor immunomodulatory properties to induce the generation of suppressive T cells. MSCs have been successfully used in treating graft-versus-host disease (GVHD) accompanied by abundant inflammatory cytokines such as IL-27. This study investigated the effects of IL-27 on the human placenta-derived MSCs (hPMSCs) to induce generation of CD4+IL-10+IFN-γ+ T cells in vitro and in the humanized xenogenic GVHD NOD/SCID model. The results showed that the percentages of CD4+IL-10+IFN-γ+ T cells were significantly increased in activated human PBMC from both healthy donors and GVHD patients with hPMSCs and in the liver and spleen of hPMSC-treated GVHD mice, and the level of CD4+IL-10+IFN-γ+ T cells in the liver was greater than that in the spleen in hPMSC-treated GVHD mice. The serum level of IL-27 decreased and the symptoms abated in hPMSC-treated GVHD. Further, in vitro results showed that IL-27 promoted the regulatory effects of hPMSCs by enhancing the generation of CD4+IL-10+IFN-γ+ T cells from activated PBMC. Activation occurred through increases in the expression of programmed death ligand 2 (PDL2) in hPMSCs via the JAK/STAT signaling pathway. These findings indicated that hPMSCs could alleviate GVHD mice symptoms by upregulating the production of CD4+IL-10+IFN-γ+ T cells in the spleen and liver and downregulating serum levels of IL-27. In turn, the ability of hPMSCs to induce the generation of CD4+IL-10+IFN-γ+ T cells could be promoted by IL-27 through increases in PDL2 expression in hPMSCs. The results of this study will be of benefit for the application of hPMSCs in clinical trials.

Inhibition of Glutathione Synthesis via Decreased Glucose Metabolism in Stored RBCs.

BACKGROUND/AIMS: Although red blood cells (RBCs) transfusions can be lifesaving, they are not without risk. RBCs storage is associated with the abnormal metabolism of glutathione (GSH), which may increase the risk of the oxidative damage of RBCs after transfusion. The responsible mechanisms remain unknown. METHODS: We determined the L-cysteine efflux and influx by evaluating the changes of free -SH concentrations in stored RBCs. The glutamate cysteine ligase (GCL) activities and protein content in stored RBCs was determined by fluorescence assay and western blotting. In addition, the glucose metabolism enzyme activity of RBCs was measured by spectrophotometric assay under in vitro incubation conditions. RESULTS: We found that both L-cysteine transport and GCL activity significantly declined, thereby inducing the dysfunction of GSH synthesis during blood storage, which could be attenuated by ATP supplement and DTT treatment. In addition, the glycometabolic enzyme (G6PDH, HK, PK and LDH) activity significantly decreased after 6 weeks storage. Oxidant stress-induced dysfunction in glucose metabolism was the driving force for decreased GSH synthesis during storage. CONCLUSION: These experimental findings reflect an underlying molecular mechanism that oxidant stress induced glucose metabolism dysfunction contribute to decreased GSH synthesis in stored RBCs.

Interferon-γ suppresses the proliferation and migration of human placenta-derived mesenchmal stromal cells and enhances their ability to induce the generation of CD4+CXCR5+Foxp3+Treg subset.

We investigate the effects of interferon (IFN)-γ on human placenta-derived mesenchymal stromal cells (hPMSCs), in particular, their adhesion, proliferation and migration and modulatory effects on the CD4+CXCR5+Foxp3+Treg subset. And we compared hPMSCs ability to induce the generation of different Treg subsets in response to treatment with IFN-γ. We found that IFN-γ suppressed the proliferation and migration for hPMSCs. The ability of hPMSCs to induce the generation of CD4+CXCR5+Foxp3+Treg subset was enhanced by IFN-γ. And maximal effectiveness of IFN-γ treated hPMSCs upon inducing the generation of Treg subsets was for CD4+CXCR5+Foxp3+Treg subset as compared with that of CD4+CD25+Foxp3+, CD8+CD25+Foxp3+, CD4+IL-10+ and CD8+IL-10+Treg subsets. These results have important implications for the development and application of hPMSCs in clinical use.

IL-27 regulates the adherence, proliferation, and migration of MSCs and enhances their regulatory effects on Th1 and Th2 subset generations.

Interleukin 27 (IL-27) regulates T cell function and is involved in inflammation. It has been reported that human placenta-derived mesenchymal stromal cells (hPMSCs) can inhibit T cell responses and attenuate inflammation reactions. However, it is unclear whether IL-27 can regulate hPMSC function. Here, we examined the effects of IL-27 upon adherence, migration, and proliferation as well as the immunomodulatory effects of hPMSCs. The results show that IL-27 receptor α chain (IL-27Rα) is expressed in hPMSCs. IL-27 at 30 ng/ml inhibited hPMSC adherence and proliferation, while the migration of hPMSCs was promoted with IL-27 at doses of 20 or 30 ng/ml, as determined with use of real-time cell analysis (RTCA). Moreover, IL-27 promoted regulatory effects of hPMSCs through enhancing Th2 and suppressing Th1 subset generation from activated T cells in human peripheral blood. IL-27 also enhanced the ability of hPMSCs to secrete IL-10 from CD4+T cells through increased expression levels of the programmed death ligand 1 (PDL1) in hPMSCs via the Janus kinase (JAK)/signal transducer and activator of transcription 1 (STAT1) signaling pathway. In conclusion, IL-27 has significant modulatory effects on adherence, proliferation, and migration of hPMSCs. IL-27 increased PDL1 expression levels in hPMSCs via the JAK/STAT1 pathway, which then enhanced the regulatory effects of hPMSCs upon Th1 and Th2 cell generations and IL-10 secretion from CD4+T cells.

Involvement of soluble B7-H3 in combination with the serum inflammatory cytokines interleukin-17, -8 and -6 in the diagnosis of hepatocellular carcinoma.

Previous studies have demonstrated that B7-H3, and the inflammatory cytokines interleukin (IL)-17, IL-8 and IL-6, are involved in the development of a variety of tumors. The objectives of the present study were: i) To investigate the association between soluble B7-H3 (sB7-H3) and cytokine levels of IL-17, IL-8 and IL-6 in the serum of patients with hepatocellular carcinoma (HCC); and ii) to determine their potential value for use in HCC diagnosis. Serum sB7-H3, IL-17, IL-8 and IL-6 levels in the HCC patients and healthy control subjects were measured using ELISA. The accuracy of each of these biomarkers in HCC diagnosis was compared using a receiver operating characteristic curve and the area under the curve (AUC). A logistic regression model was used to investigate the accuracy of diagnosing HCC when evaluated using combined determinations of sB7-H3, IL-17, IL-8 and IL-6 levels. The data demonstrated that serum levels of sB7-H3, IL-17, IL-8 and IL-6 were significantly increased in HCC patients compared with those in the healthy control group. Serum sB7-H3 levels were positively associated with serum IL-17, whereas serum IL-8 levels were negatively correlated with serum IL-17 levels. The AUC values for sB7-H3, IL-17, IL-8 and IL-6 were 83.2, 65.7, 95.3 and 97.0%, respectively, and indicated that all four biomarkers exhibited a statistically significant capacity for diagnosing HCC. Using the logistic regression model, the AUC value, sensitivity and specificity, as determined for the combination of the four biomarkers, were 99.2, 96.67 and 97.14%, respectively. This was significantly greater than that achieved when any single biomarker was used alone in the logistic regression model to assess their accuracy in HCC diagnosis. The optimum cutoff value of the predicted probability obtained by the combination of sB7-H3, IL-17, IL-8 and IL-6 in the regression model was 0.5745. To conclude, the present study revealed that there exists a positive association between serum sB7-H3 and IL-17 levels in HCC patients. Determinations involving the combination of serum sB7-H3, IL-17, IL-8 and IL-6 levels demonstrate great potential for use in HCC diagnosis.

[IFN-γ up-regulates PD-L1 expression in human placenta mesenchymal stem cells and enhances cell ability to induce the differentiation of IL-10+ T cells from cord blood- and peripheral blood-derived T cells].

OBJECTIVE: To compare the differentiation, inducing effects of human placenta mesenchymal stem cells (hPMSCs) on IL-10(+) T cells derived from cord blood and peripheral blood, and investigate the effect of IFN-γ on the induction. METHODS: The hPMSCs were isolated from human placenta and cultured. The expression of programmed death ligand 1 (PD-L1) in hPMSCs was detected by reverse transcriptase PCR and flow cytometry (FCM), respectively. Mononuclear cells were isolated from cord blood and peripheral blood of healthy donors by Ficoll density gradient centrifugation, and T cells were purified by sheep red blood cells. Then hPMSCs, pretreated with PD-L1 mAb or IFN-γ, were co-cultured with phytohaemagglutinin (PHA)-activated T cells. Percentages of CD4(+)IL-10(+) and CD8(+)IL-10(+) T cells in cord blood and peripheral blood T cells were analyzed by FCM. RESULTS: hPMSCs could induce the differentiation of CD4(+)IL-10(+) and CD8(+)IL-10(+) T cells from cord blood or peripheral blood T cells, and the number of IL-10(+) T cells in the peripheral blood T cells was significantly higher than that in the cord blood T cells. Pretreatment with IFN-γ markedly enhanced the differentiation, inducing ability of hPMSCs. PD-L1 was highly expressed in hPMSCs, and the expression was also significantly promoted by IFN-γ. After the expression of PD-L1 was blocked in hPMSCs, the percentages of CD4(+)IL-10(+) and CD8(+)IL-10(+) T cells obviously decreased in cord blood and peripheral blood T cells. CONCLUSION: The ability of hPMSCs to induce the differentiation of IL-10(+) T cells from peripheral blood T cells was apparently stronger than that in cord blood T cells. IFN-γ could up-regulate the number of IL-10(+)T cells differentiated from cord blood and peripheral blood T cells in the present of hPMSCs by enhancing the expression of PD-L1 in hPMSCs.