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ObjectiveA comprehensive immune landscape for HBV infection is pivotal to achieve HBV cure.DesignWe performed single-cell RNA sequencing of 2 43 000 cells from 46 paired liver and blood samples of 23 individuals, including six immune tolerant, 5 immune active (IA), 3 acute recovery (AR), 3 chronic resolved and 6 HBV-free healthy controls (HCs). Flow cytometry and histological assays were applied in a second HBV cohort for validation.ResultsBoth IA and AR were characterised by high levels of intrahepatic exhausted CD8+ T (Tex) cells. In IA, Tex cells were mainly derived from liver-resident GZMK+ effector memory T cells and self-expansion. By contrast, peripheral CX3CR1+ effector T cells and GZMK+ effector memory T cells were the main source of Tex cells in AR. In IA but not AR, significant cell–cell interactions were observed between Tex cells and regulatory CD4+ T cells, as well as between Tex and FCGR3A+ macrophages. Such interactions were potentially mediated through human leukocyte antigen class I molecules together with their receptors CANX and LILRBs, respectively, contributing to the dysfunction of antiviral immune responses. By contrast, CX3CR1+GNLY+ central memory CD8+ T cells were concurrently expanded in both liver and blood of AR, providing a potential surrogate marker for viral resolution. In clinic, intrahepatic Tex cells were positively correlated with serum alanine aminotransferase levels and histological grading scores.ConclusionOur study dissects the coordinated immune responses for different HBV infection phases and provides a rich resource for fully understanding immunopathogenesis and developing effective therapeutic strategies.

Priority of antiviral treatment for patients with chronic hepatitis B (CHB) is to increase the probability of functional cure. We aimed to synthesize evidence regarding the efficacy of different combination strategies of antiviral treatment based on interferon (IFN) and nucleos(t)ide analogues (NAs) in adults with CHB. PubMed, Web of Science and Embase databases were searched from inception to May 26, 2019. Three types of combination strategies were studied: initial combination (IFN or NAs monotherapy as control), add-on (I: IFN add-on NAs vs. NAs; II: NAs add-on IFN vs. IFN), switch-to (I: IFN switch-to NAs vs. IFN; II: NAs switch-to IFN vs. NAs). Compared to NAs monotherapy, initial combination strategy improved the probability of HBeAg loss (RR: 1.62, 95% CI 1.33–1.97) and HBsAg loss (RR: 15.59, 95% CI 3.22–75.49), while compared to IFN monotherapy, no higher rates in the loss of HBsAg or HBeAg for initial combination. Compared to NAs monotherapy, IFN add-on NAs strategy had a higher rate of HBsAg loss (RR: 4.52, 95% CI 1.95–10.47), while compared to IFN monotherapy, NAs add-on IFN had a similar outcome. Compared to NAs monotherapy, NAs switch-to IFN strategy improved HBsAg loss (RR: 12.15, 95% CI 3.99–37.01); while compared to IFN monotherapy, IFN switch-to NAs had no improved rate of HBsAg clearance but higher rates in undetectable HBV DNA, and HBeAg loss. IFN add-on NAs, or NAs switched to IFN could significantly improve the probability of HBsAg loss compared to NAs monotherapy.

Toxic elements can be transported to polar regions by long-range atmospheric transport from mid and low latitudes, leading to enrichment of elements in the polar environment, especially in the Arctic. The plants can be ideal bioindicators of element contamination in environments, but information on the element enrichment and sources of plants remains limited in polar regions. Here, concentrations of 15 metals and metalloids (Pb, Ni, Cr, Cu, Co, As, Cd, Sb, Hg, Se, Fe, Zn, Mn, Al, and Ti) in six species of plants, Deschampsia caespitosa (Tufted Hair Grass), Puccinellia phryganodes (Creeping Alkaligrass), Saxifraga aizoides (Yellow Mountain Saxifrage), Dicranum angustum (Dicranum Moss), Salix Polaris (Polar Willow), and Cerastium arcticum (Arctic Mouse-Ear Chickweed), collected from Ny-&Aring, lesund, the Arctic, were determined, and enrichment and sources of elements were assessed. Results show that element concentrations vary in different plant species, and element levels in D. angustum and C. arcticum are generally higher. In spatial terms, elevated element concentrations were found near residential areas, while low element levels were present at the sites far from settlement points. Enrichment assessment shows that Cd, Hg, and Zn are the most enriched elements, with enrichment factors above 30, suggesting sources other than soil dust control their concentrations. Principal component analysis (PCA) showed that the extracted three components can explain 82% of the total variance in element concentrations. The elements Ni, Cr, As, Sb, Fe, Al, Ti, and to a lesser extent Co are highly loaded in PC1, possibly associated with continental crust particles. PC2 is closely correlated with Cd, Se, Mn, Cu, and Zn, while Hg and Pb have high loadings on PC3. The elements highly loaded on PC2 and PC3 are likely associated with pollutants from atmospheric transportation. Together with enrichment assessment, the investigated plants have a great potential for monitoring atmospheric Cd, Hg, and Zn pollution in Ny-&Aring, lesund, and D. angustum and D. caespitosa are the more sensitive species. The results would be of significance for monitoring element contamination in the pristine Arctic environments using the bioindicator plants.

Coronavirus disease 2019 (COVID-19) not only causes pulmonary inflammation but also causes multiple organ damages, including the kidney. ACE2, as one of the receptors for SARS-CoV-2 intrusion, is widely distributed in kidney tissues. Currently, the diagnosis and treatment of SARS-CoV-2 infection in patients with chronic kidney disease (CKD) are still unclear. Here, we review the recent findings of characteristics of COVID-19 in CKD patients and highlight the possible mechanisms of kidney injury caused by SARS-CoV-2 infection. We then discuss the emerging therapeutic approaches aimed at reducing kidney damage and protecting kidney function including virus removal, immunotherapy, supporting treatment, special blood purification therapy, etc. Problems unresolved and challenges ahead are also discussed.

Liver cirrhosis is associated with defective vaccine responses and increased infections. Dysregulated B cell compartments in cirrhotic patients have been noticed but not well characterized, especially in the spleen. Here, we comprehensively investigated B cell perturbations from the spleens and peripheral blood of cirrhotic patients. We found that liver cirrhosis significantly depleted both switched and nonswitched splenic memory B cells, which was further confirmed histologically. Bulk RNA-seq revealed significant metabolic defects as the potential mechanism for the impaired splenic B cell functions. Functionally, the splenic memory B cells from cirrhotic patients showed strong metabolic defects and reduced proliferation compared with those from healthy controls. Thus, liver cirrhosis extensively disturbs the splenic and peripheral B cell compartments, which may contribute to defective humoral immunity during liver cirrhosis.

The study aimed to characterize rtA181T/sW172stop (*) and rtA181T/sW172non-stop mutations of hepatitis B virus (HBV). Total of 22,009 patients who visited Beijing 302 Hospital from 2007 to 2016 were enrolled. These patients all received nucleos(t)ide analogues (NAs) treatment and their serum samples were collected for sequence analysis of HBV reverse-transcriptase (RT) and S regions. The rtA181T mutation was detected in 5.37% (1182/22,009) of the patients' samples. The rtA181T-causative sW172*, sW172non-stop (sW172 L/S), and mixed sW172*/non-stop mutations occupied 82.91%, 7.70%, and 9.39%, respectively. The patients with rtA181T/sW172non-stop mutants had a higher HBV DNA level compared to those with rtA181T/sW172* mutants. 44.33% (524/1182) rtA181T-positive samples were detected with signature drug-resistant mutations, including 325 with adefovir-resistant mutation rtA181V/N236T, 57 with lamivudine-resistant mutation rtM204V/I, 99 with entecavir-resistant mutation rtM204V/I plus rt184/202/250 substitution(s), and 43 with multidrug-resistant mutation rtA181V/N236T + rtM204V/I ± rt184/202/250 substitution(s). The rtA181T/sW172non-stop mutation had a higher ratio of coexistence with adefovir-resistant mutation compared to rtA181T/sW172* mutation (42.86% vs. 24.59%, P 0.05). rtA181T/sW172S + rtN236T and rtA181T/sW172L + rtN236T mutants exhibited higher HBV DNA production and adefovir resistance fold than that of rtA181T/sW172* + rtN236T mutant (98.02% and 85.5% vs. 42.1% in HBV DNA production, and 7.38-fold and 5.49-fold vs. 3.69-fold in half maximal effective concentration of wild-type strain); rtA181T/sW172L + rtS202G + rtM204V strain exhibited higher HBV DNA production and entecavir resistance fold than that of rtA181T/sW172* + rtS202G + rtM204V strain (50.98% vs. 34.49%, 524.00-fold vs. 69.33-fold). In conclusion, rtA181T/sW172non-stop mutation may increase resistance fold of adefovir- and entecavir-resistant mutants compared to rtA181T/sW172* mutation and might influence clinical presentation of NAs-treated patients.

Limited data are available regarding the association of hepatitis B virus (HBV) mutations with liver fibrosis in HBV infection. The study aimed to clarify whether HBV preS deletion mutation is associated with liver fibrosis progression. A total of 469 patients were enrolled, including 324 with chronic hepatitis B (CHB), 28 with HBV-related compensated liver cirrhosis (LC), and 117 with HBV-related decompensated LC. All CHB and compensated LC patients received liver biopsy. Fibrosis grade was assessed using METAVIR score. HBV preS deletion was determined by direct sequencing and verified by clonal sequencing. Overall preS deletion was detected in 12.6% (59/469) patients, specifically, in 7.51% (13/173), 10.60% (16/151), and 20.69% (30/145) of patients with no-to-mild liver fibrosis (F0–1), moderate-to-severe liver fibrosis (F2–3), and cirrhosis (F4), respectively (p

The aim of this study was to investigate the differentiation potential of induced pluripotent stem cells (iPSCs) derived from human foreskin fibroblasts (HFFs) into hepatocyte-like cells (HLCs). The iPSCs were firstly induced by transduction of OCT4, SOX2, KLF4, and c-MYC into HFFs using retrovirus. Afterwards, expressions of pluripotency factors were identified by semiquantitative reverse transcription–polymerase chain reaction and immunofluorescence staining, and karyotype, embryoid, and teratoma were observed by microscope. Then, iPSCs were gradually differentiated into endoderm cells, hepatic progenitor cells, and mature HLCs by special culture medium. During this process, differentiation efficiency into each kind of cells was evaluated by detecting SOX17, HNF4a, and ALB using flow cytometry, respectively. Besides, enzyme-linked immunosorbent assay was conducted to detect the secretion of ALB in iPSC-induced HLCs and quantitative reverse transcription–polymerase chain reaction was performed to detect the expression levels of hepatocyte-specific genes. The iPSCs were successfully induced by HFFs, which exhibited typical embryonic stem cells morphology, positive alkaline phosphatase staining, normal diploid karyotype, and positive expression of various pluripotency factors. Meanwhile, spherical embryoid and teratoma with 3 germ layers were formed by iPSCs. The iPSCs were consecutively induced into endoderm cells, hepatic progenitor cells and mature HLCs, and the differentiation efficiency was 55.7 ± 2.9%, 45.7 ± 4.8%, and 35.0 ± 3.9%, respectively. Besides, the secretion of ALB and expression of various hepatocyte-specific genes was highly detected in iPSC-induced HLCs. The iPSCs were successfully derived from HFFs and then differentiated into HLCs, which proved a new source for hepatocyte transplantation. Highlights HFFs were successfully induced into iPSCs by transduction of OCT4, SOX2, KLF4, and c-MYC. Positive expressions of various pluripotency factors were exhibited in HFFs-induced iPSCs. The iPSCs were consecutively induced into endoderm cells, hepatic progenitor cells, and mature HLCs. Various hepatocyte-specific genes were highly expressed in iPSC-induced HLCs.

Mutations in hepatitis B virus (HBV) S gene are one of factors contributing to occult HBV infection (OBI). The study aimed to uncover the impact of OBI-related S-gene mutations on the detectability of hepatitis B surface antigen (HBsAg). Nine representative mutations within the major hydrophilic region of the S region were investigated. These included six (M1 - M6) from an OBI patient with HBV-related hepatocellular carcinoma, and three (M7 - M9) from 3 OBI blood donors. Recombinant plasmids on the basis of pTriEx-mod-1.1 HBV and pcDNA3.1(-)/myc-His A vectors were constructed for each and transfected into HepG2 or Huh7 cells, respectively. Electrochemical luminescence, ELISA, Western blotting, and confocal immunofluorescence were used to examine HBsAg expression and antigenicity. In comparison to wild-type strain, supernatant and intracellular HBsAg levels of the nine mutants were reduced by 56.39% - 99.09% and 42.76% - 99.77% upon Roche quantitative Elecsys assay, respectively. Confocal immunofluorescence showed that relative intensity ratios of HBsAg-myc-His fusion protein detected by anti-HBs and anti-His-tag were lower by 11.87% - 76.27% for the nine mutants compared to the wild-type strain. Specifically, M1 - M5 mutants that we firstly found recently were 33.14%, 76.27%, 57.93%, 53.37%, and 40.88% lower, respectively. Consistent results were obtained using double-antibody sandwich ELISA assays (anti-myc + anti-HBs vs. anti-myc + anti-His). Antigenicity reduction played a major role for the poor detectability of HBsAg caused by the OBI-related mutations, although decreased HBsAg expression of some mutants and anti-HBs in samples might play coordinated roles. Taken together, antigenicity reduction contributes mostly to poor detectability of HBsAg caused by these OBI-related mutations. This article is protected by copyright. All rights reserved.

Natural killer (NK) cells can induce liver fibrosis remission by killing hepatic stellate cells (HSCs) and producing interferon (IFN)-γ in a mouse model; however, their anti-fibrotic immune-characteristics and regulatory mechanisms by HSCs remain to be determined, especially in livers from HBV-infected liver cirrhosis (LC) patients. We analyzed frequency, phenotype and anti-fibrotic function of hepatic and peripheral NK subsets in 43 HBV-LC patients. We found that hepatic NK subsets from LC patients displayed a decreased frequency, activation status and anti-fibrotic activity compared with those from chronic hepatitis B patients, which were mainly mediated by increased intrahepatic tumour-growth factor (TGF)-β because blockade of TGF-β significantly reversed NK anti-fibrotic function in vitro. In vivo, hepatic NK cells were enriched in proximity to the α-smooth muscle actin (α-SMA+) area within mild fibrosis regions; while in severe fibrotic areas, they were either directly attached to or separated from the α-SMA+ region. NK cells from LC patients could enter HSCs to form emperipolesis (a cell-in-cell structure) and become apoptotic; anti-TGF-β treatment ameliorated this emperipolesis. This finding suggested a novel mechanism by which activated HSCs impair NK cells’ anti-fibrosis capacity through a TGF-β-dependent emperipolesis in LC patients, providing an anti-fibrotic rational by enhancing NK cell activity.

The aim of this study was to investigate the differentiation potential of induced pluripotent stem cells (iPSCs) derived from human foreskin fibroblasts (HFFs) into hepatocyte-like cells (HLCs). The iPSCs were firstly induced by transduction of OCT4, SOX2, KLF4, and c-MYC into HFFs using retrovirus. Afterwards, expressions of pluripotency factors were identified by semiquantitative reverse transcription-polymerase chain reaction and immunofluorescence staining, and karyotype, embryoid, and teratoma were observed by microscope. Then, iPSCs were gradually differentiated into endoderm cells, hepatic progenitor cells, and mature HLCs by special culture medium. During this process, differentiation efficiency into each kind of cells was evaluated by detecting SOX17, HNF4a, and ALB using flow cytometry, respectively. Besides, enzyme-linked immunosorbent assay was conducted to detect the secretion of ALB in iPSC-induced HLCs and quantitative reverse transcription-polymerase chain reaction was performed to detect the expression levels of hepatocyte-specific genes. The iPSCs were successfully induced by HFFs, which exhibited typical embryonic stem cells morphology, positive alkaline phosphatase staining, normal diploid karyotype, and positive expression of various pluripotency factors. Meanwhile, spherical embryoid and teratoma with 3 germ layers were formed by iPSCs. The iPSCs were consecutively induced into endoderm cells, hepatic progenitor cells and mature HLCs, and the differentiation efficiency was 55.7 ± 2.9%, 45.7 ± 4.8%, and 35.0 ± 3.9%, respectively. Besides, the secretion of ALB and expression of various hepatocyte-specific genes was highly detected in iPSC-induced HLCs. The iPSCs were successfully derived from HFFs and then differentiated into HLCs, which proved a new source for hepatocyte transplantation.HFFs were successfully induced into iPSCs by transduction of OCT4, SOX2, KLF4, and c-MYC. Positive expressions of various pluripotency factors were exhibited in HFFs-induced iPSCs. The iPSCs were consecutively induced into endoderm cells, hepatic progenitor cells, and mature HLCs. Various hepatocyte-specific genes were highly expressed in iPSC-induced HLCs.