Hepatology (Baltimore, Md.)
Zhao, K;Guo, F;Wang, J;Zhong, Y;Yi, J;Teng, Y;Xu, Z;Zhao, L;Li, A;Wang, Z;Chen, X;Cheng, X;Xia, Y;
PMID: 35718932 | DOI: 10.1002/hep.32622
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.Cells expressing hNTCP were inoculated with HBV or hepatitis delta virus (HDV). HBV pre-genomic 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.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.This article is protected by
Clin Gastroenterol Hepatol.
Sun Y, Wu X, Zhou J, Meng T, Wang B, Chen S, Liu H, Wang T, Zhao X, Wu S, Kong Y, Ou X, Wee A, Theise ND, Qiu C, Zhang W, Lu F, Jia J, You H
PMID: 32147592 | DOI: 10.1016/j.cgh.2020.03.001
BACKGROUND & AIMS:
Progression of liver fibrosis still occurs in some patients with chronic hepatitis B virus (HBV) infection despite antiviral therapy. We aimed to identify risk factors for fibrosis progression in patients who received antiviral therapy.
METHODS:
We conducted a longitudinal study of patients with chronic HBV infection and liver biopsies collected before and after 78 weeks of anti-HBV therapy. Fibrosis progression was defined as Ishak stage increase ? 1 or as predominantly progressive classified by P-I-R system (Beijing Classification). Levels of HBV DNA and HBV RNA in blood samples were measured by real-time quantitative PCR. HBV RNA in liver tissue was detected by in situ hybridization.
RESULTS:
A total of 239 patients with chronic HBV infection with paired liver biopsies were included. Among the 163 patients with significant fibrosis at baseline (Ishak ? stage 3), fibrosis progressed in 22 patients (13%), was indeterminate in 24 patients (15%), and regressed in 117 patients (72%). Univariate and multivariate analyses revealed that independent risk factors for fibrosis progression were higher rate of detected HBV DNA at week 78 (odds ratio, 4.84; 95% CI, 1.30-17.98; P = .019) and alcohol intake (odds ratio, 23.84; 95% CI, 2.68-212.50; P = .004). HBV DNA was detected in blood samples from a significantly higher proportion of patients with fibrosis progression (50%) at week 78 than patients with fibrosis regression (19%) or indeterminate fibrosis (26%) (P = .015), despite low viremia (20-200 IU/mL) in all groups. The decrease of serum HBV RNA from baseline in the fibrosis regression group was larger than that in the fibrosis progression group.
CONCLUSIONS:
In a longitudinal study of patients with chronic HBV infection, we associated liver fibrosis progression at week 78 of treatment with higher rates of detected HBV DNA. We propose that a low level of residual HBV may still promote fibrosis progression, and that patients' levels of HBV DNA should be carefully monitored.
J Clin Oncol. 2018 Sep 28:JCO2018789602.
Mitchell TC, Hamid O, Smith DC, Bauer TM, Wasser JS, Olszanski AJ, Luke JJ, Balmanoukian AS, Schmidt EV, Zhao Y, Gong X, Maleski J, Leopold L, Gajewski TF.
PMID: 30265610 | DOI: 10.1200/JCO.2018.78.9602
Abstract PURPOSE: Tumors may evade immunosurveillance through upregulation of the indoleamine 2,3-dioxygenase 1 (IDO1) enzyme. Epacadostat is a potent and highly selective IDO1 enzyme inhibitor. The open-label phase I/II ECHO-202/KEYNOTE-037 trial evaluated epacadostat plus pembrolizumab, a programmed death protein 1 inhibitor, in patients with advanced solid tumors. Phase I results on maximum tolerated dose, safety, tolerability, preliminary antitumor activity, and pharmacokinetics are reported. PATIENTS AND METHODS: Patients received escalating doses of oral epacadostat (25, 50, 100, or 300 mg) twice per day plus intravenous pembrolizumab 2 mg/kg or 200 mg every 3 weeks. During the safety expansion, patients received epacadostat (50, 100, or 300 mg) twice per day plus pembrolizumab 200 mg every 3 weeks. RESULTS: Sixty-two patients were enrolled and received one or more doses of study treatment. The maximum tolerated dose of epacadostat in combination with pembrolizumab was not reached. Fifty-two patients (84%) experienced treatment-related adverse events (TRAEs), with fatigue (36%), rash (36%), arthralgia (24%), pruritus (23%), and nausea (21%) occurring in ≥ 20%. Grade 3/4 TRAEs were reported in 24% of patients. Seven patients (11%) discontinued study treatment because of TRAEs. No TRAEs led to death. Epacadostat 100 mg twice per day plus pembrolizumab 200 mg every 3 weeks was recommended for phase II evaluation. Objective responses (per Response Evaluation Criteria in Solid Tumors [RECIST] version 1.1) occurred in 12 (55%) of 22 patients with melanoma and in patients with non-small-cell lung cancer, renal cell carcinoma, endometrial adenocarcinoma, urothelial carcinoma, and squamous cell carcinoma of the head and neck. The pharmacokinetics of epacadostat and pembrolizumab and antidrug antibody rate were comparable to historical controls for monotherapies. CONCLUSION: Epacadostat in combination with pembrolizumab generally was well tolerated and had encouraging antitumor activity in multiple advanced solid tumors.
Allweiss L, Volz T, Giersch K, Kah J, Raffa G, Petersen J, Lohse AW, Beninati C, Pollicino T, Urban S, Lütgehetmann M, Dandri M.
PMID: 28428345 | DOI: 10.1136/gutjnl-2016-312162
Abstract
OBJECTIVE:
The stability of the covalently closed circular DNA (cccDNA) in nuclei of non-dividing hepatocytes represents a key determinant of HBV persistence. Contrarily, studies with animal hepadnaviruses indicated that hepatocyte turnover can reduce cccDNA loads but knowledge on the proliferative capacity of HBV-infected primary human hepatocytes (PHHs) in vivo and the fate of cccDNA in dividing PHHs is still lacking. This study aimed to determine the impact of human hepatocyte division on cccDNA stability in vivo.
METHODS:
PHH proliferation was triggered by serially transplanting hepatocytes from HBV-infected humanised mice into naïve recipients. Cell proliferation and virological changes were assessed by quantitative PCR, immunofluorescence and RNA in situ hybridisation. Viral integrations were analysed by gel separation and deep sequencing.
RESULTS:
PHH proliferation strongly reduced all infection markers, including cccDNA (median 2.4 log/PHH). Remarkably, cell division appeared to cause cccDNA dilution among daughter cells and intrahepatic cccDNA loss. Nevertheless, HBV survived in sporadic non-proliferating human hepatocytes, so that virological markers rebounded as hepatocyte expansion relented. This was due to reinfection of quiescent PHHs since treatment with the entry inhibitor myrcludex-B or nucleoside analogues blocked viral spread and intrahepatic cccDNA accumulation. Viral integrations were detected both in donors and recipient mice but did not appear to contribute to antigen production.
CONCLUSIONS:
We demonstrate that human hepatocyte division even without involvement of cytolytic mechanisms triggers substantial cccDNA loss. This process may be fundamental to resolve self-limiting acute infection and should be considered in future therapeutic interventions along with entry inhibition strategies.
Bustamante-Jaramillo, LF;Fingal, J;Blondot, ML;Rydell, GE;Kann, M;
PMID: 35336964 | DOI: 10.3390/v14030557
Hepatitis B virus infections are the main reason for hepatocellular carcinoma development. Current treatment reduces the viral load but rarely leads to virus elimination. Despite its medical importance, little is known about infection dynamics on the cellular level not at least due to technical obstacles. Regardless of infections leading to extreme viral loads, which may reach 1010 virions per mL serum, hepatitis B viruses are of low abundance and productivity in individual cells. Imaging of the infections in cells is thus a particular challenge especially for cccDNA that exists only in a few copies. The review describes the significance of microscopical approaches on genome and transcript detection for understanding hepatitis B virus infections, implications for understanding treatment outcomes, and recent microscopical approaches, which have not been applied in HBV research.
British journal of cancer
Girithar, HN;Staats Pires, A;Ahn, SB;Guillemin, GJ;Gluch, L;Heng, B;
PMID: 37041200 | DOI: 10.1038/s41416-023-02245-7
Breast cancer (BrCa) is the leading cause of cancer incidence and mortality in women worldwide. While BrCa treatment has been shown to be highly successful if detected at an early stage, there are few effective strategies to treat metastatic tumours. Hence, metastasis remains the main cause in most of BrCa deaths, highlighting the need for new approaches in this group of patients. Immunotherapy has been gaining attention as a new treatment for BrCa metastasis and the kynurenine pathway (KP) has been suggested as one of the potential targets. The KP is the major biochemical pathway in tryptophan (TRP) metabolism, catabolising TRP to nicotinamide adenine dinucleotide (NAD+). The KP has been reported to be elevated under inflammatory conditions such as cancers and that its activity suppresses immune surveillance. Dysregulation of the KP has previously been reported implicated in BrCa. This review aims to discuss and provide an update on the current mechanisms involved in KP-mediated immune suppression and cancer growth. Furthermore, we also provide a summary on 58 studies about the involvement of the KP and BrCa and five clinical trials targeting KP enzymes and their outcome.
Hepatology communications
Zhang, H;Zhang, M;Zhang, Q;Yu, Y;Zhang, F;Wang, J;Zhou, M;Yu, T;Shen, C;Yu, S;Huang, Y;Huang, Y;Zhang, J;Jin, J;Qiu, C;Guojun, L;Zhang, W;
PMID: 36995994 | DOI: 10.1097/HC9.0000000000000111
Chronic HBV infection evolves through different phases. Interactions between viral replication and the host immune response in the liver underlie the pathogenesis of this disease. The aim of this study was to directly visualize the HBV replication intermediates at a single-cell resolution inscribed on morphological changes corresponding to disease activity.A set of archived formalin-fixed paraffin-embedded liver needle biopsies from treatment-naïve patients were collected and categorized into phases according to the American Association for the Study of the Liver Diseases (AASLD) guidelines. HBV RNA and DNA were detected using in situ hybridization assays.The hepatocytes were ubiquitously infected in subjects with immune tolerance, and their percentage was gradually decreased in immune-active and inactive chronic hepatitis B phases. HBV-infected hepatocytes were prone to localize close to fibrous septa. The subcellular distribution of signals was able to distinguish hepatocytes with productive infection from those harboring HBV integrants and transcriptionally inactive covalently closed circular DNAs. A smaller number of hepatocytes with productive infection and more harboring transcriptionally inactive covalently closed circular DNA or HBV integrants became apparent in the inactive chronic hepatitis B phase.An atlas of in situ characteristics of viral-host interactions for each phase is described, which sheds light on the nature of viral replication and disease pathogenesis among the phases of chronic HBV infection.
Xia Y, Cheng X, Li Y, Valdez K, Chen W, Liang TJ.
PMID: 30021897 | DOI: 10.1128/JVI.00722-18
Hepatitis B virus (HBV) infection is a major health problem worldwide and chronically infected individuals are at high risk of developing cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms whereby HBV causes HCC are largely unknown. By using a biologically relevant system of HBV infection of primary human hepatocytes (PHHs), we studied how HBV perturbs gene expressions and signaling pathways of infected hepatocytes, and whether these effects are relevant to productive HBV infection and HBV-associated HCC. Using a human growth factor antibody array, we first showed that HBV infection induced a distinct profile of growth factor production by PHHs, marked particularly by significantly lower levels of transforming growth factor (TGF)-β family of proteins in the supernatant. Transcriptome profiling next revealed multiple changes in cell proliferation and cell cycle control pathways in response to HBV infection. A human cell cycle PCR array validated deregulation of more than 20 gene associated with cell cycle in HBV-infected PHHs. Cell cycle analysis demonstrated that HBV-infected PHHs are enriched in the G2/M phase as compared to the predominantly G0/G1 phase of cultured PHHs. HBV proviral host factors, such as PPARA, RXRA and CEBPB, were up-regulated upon HBV infection and particularly enriched in cells at the G2/M phase. Together, these results support that HBV deregulates cell cycle control to render a cellular environment that is favorable for productive HBV infection. By perturbing cell cycle regulation of infected cells, HBV may coincidently induce a premalignant phenotype that predispose infected hepatocytes to subsequent malignant transformation.IMPORTANCE Hepatitis B virus (HBV) infection is a major health problem with high risk of developing hepatocellular carcinoma (HCC). By using a biologically relevant system of HBV infection of primary human hepatocytes (PHHs), we studied how HBV perturbs gene expressions, and whether these effects are relevant to HBV-associated HCC. HBV induced a distinct profile of growth factor production, marked particularly by significantly lower levels of transforming growth factor (TGF)-β family of proteins. Transcriptome profiling revealed multiple changes in cell proliferation and cell cycle control pathways. Cell cycle analysis demonstrated that HBV-infected PHHs are enriched in the G2/M phase. HBV proviral host factors were up-regulated upon infection and particularly enriched in cells at the G2/M phase. Together, these results support that HBV deregulates cell cycle control to render a cellular environment that is favorable for productive infection. This may coincidently induce a premalignant phenotype that predispose infected hepatocytes to subsequent malignant transformation.
The Journal of infectious diseases
Yu, T;Zhang, M;Zhang, H;Zheng, J;Shen, C;Jiang, N;Zou, L;Wang, J;Yu, Y;Zhang, Q;Yu, S;Huang, Y;Huang, Y;Zhang, J;Qiu, C;Zhang, W;Meng, Z;
PMID: 36546708 | DOI: 10.1093/infdis/jiac493
Chronic hepatitis B is usually treated with nucleos(t)ide analogues (NAs). However, a cure is rarely achieved even with years of treatment. Here, we investigated whether viral replication is completely halted and how long covalently closed circular DNA (cccDNA) persists in NAs successfully treated patients.A series of longitudinal serum samples and a collection of cross-sectional liver biopsies were obtained from NAs successfully treated patients. Viral variants in serum HBV RNA were enumerated by deep sequencing. Viral replication intermediates in hepatocytes were directly visualized by in situ hybridization. The apparent half-life of each cccDNA was estimated.Three out of six successfully treated patients demonstrated clear evidence of a small proportion of virus evolution, although the overwhelming variants were identical or possessed a similar degree of divergence through time. The apparent half-life of variants was estimated from approximately 7.42 weeks to infinite. Moreover, hepatocytes remained positive for cytoplasmic nucleocapsids-associated relaxed circular DNA (rcDNA) in four out of seven liver needle biopsies.We therefore conclude that even after prolonged treatment, a small proportion of the cccDNA reservoir is constantly replenished by continued low-level HBV replication, whereas the greatest amount of cccDNA reservoir persists over time.
Biswas, S;Rust, LN;Wettengel, JM;Yusova, S;Fischer, M;Carson, JN;Johnson, J;Wei, L;Thode, T;Kaadige, MR;Sharma, S;Agbaria, M;Bimber, BN;Tu, T;Protzer, U;Ploss, A;Smedley, JV;Golomb, G;Sacha, JB;Burwitz, BJ;
PMID: 35637225 | DOI: 10.1038/s41467-022-30593-0
Hepatitis B virus has infected a third of the world's population, and 296 million people are living with chronic infection. Chronic infection leads to progressive liver disease, including hepatocellular carcinoma and liver failure, and there remains no reliable curative therapy. These gaps in our understanding are due, in large part, to a paucity of animal models of HBV infection. Here, we show that rhesus macaques regularly clear acute HBV infection, similar to adult humans, but can develop long-term infection if immunosuppressed. Similar to patients, we longitudinally detected HBV DNA, HBV surface antigen, and HBV e antigen in the serum of experimentally infected animals. In addition, we discovered hallmarks of HBV infection in the liver, including RNA transcription, HBV core and HBV surface antigen translation, and covalently closed circular DNA biogenesis. This pre-clinical animal model will serve to accelerate emerging HBV curative therapies into the clinic.
International journal of tryptophan research : IJTR
Abu Hejleh, AP;Huck, K;Jähne, K;Tan, CL;Lanz, TV;Epping, L;Sonner, JK;Meuth, SG;Henneberg, A;Opitz, CA;Herold-Mende, C;Sahm, F;Platten, M;Sahm, K;
PMID: 36798537 | DOI: 10.1177/11786469231153111
The vascular niche of malignant gliomas is a key compartment that shapes the immunosuppressive brain tumor microenvironment (TME). The blood-brain-barrier (BBB) consisting of specialized endothelial cells (ECs) and perivascular cells forms a tight anatomical and functional barrier critically controlling transmigration and effector function of immune cells. During neuroinflammation and tumor progression, the metabolism of the essential amino acid tryptophan (Trp) to metabolites such as kynurenine has long been identified as an important metabolic pathway suppressing immune responses. Previous studies have demonstrated that indoleamine-2,3-dioxygenase-1 (IDO1), a key rate-limiting enzyme in tryptophan catabolism, is expressed within the TME of high-grade gliomas. Here, we investigate the role of endothelial IDO1 (eIDO1) expression for brain tumor immunity. Single-cell RNA sequencing data revealed that in human glioma tissue, IDO1 is predominantly expressed by activated ECs showing a JAK/STAT signaling pathway-related CXCL11+ gene expression signature. In a syngeneic experimental glioma model, eIDO1 is induced by low-dose tumor irradiation. However, cell type-specific ablation of eIDO1 in experimental gliomas did not alter frequency and phenotype of tumor-infiltrating T cells nor tumor growth. Taken together these data argue against a dominant role of eIDO1 for brain tumor immunity.
Cortez, V;Livingston, B;Sharp, B;Hargest, V;Papizan, JB;Pedicino, N;Lanning, S;Jordan, SV;Gulman, J;Vogel, P;DuBois, RM;Crawford, JC;Boyd, DF;Pruett-Miller, SM;Thomas, PG;Schultz-Cherry, S;
PMID: 37290501 | DOI: 10.1016/j.mucimm.2023.05.011
Astroviruses cause a spectrum of diseases spanning asymptomatic infections to severe diarrhea, but little is understood about their pathogenesis. We previously determined that small intestinal goblet cells were the main cell type infected by murine astrovirus-1. Here, we focused on the host immune response to infection and inadvertently discovered a role for indoleamine 2,3-dioxygenase 1 (Ido1), a host tryptophan catabolizing enzyme, in the cellular tropism of murine and human astroviruses. We identified that Ido1 expression was highly enriched among infected goblet cells, and spatially corresponded to the zonation of infection. Because Ido1 can act as a negative regulator of inflammation, we hypothesized it could dampen host antiviral responses. Despite robust interferon signaling in goblet cells, as well as tuft cell and enterocyte bystanders, we observed delayed cytokine induction and suppressed levels of fecal lipocalin-2. Although we found Ido-/- animals were more resistant to infection, this was not associated with fewer goblet cells nor could it be rescued by knocking out interferon responses, suggesting that IDO1 instead regulates cell permissivity. We characterized IDO1-/- Caco-2 cells and observed significantly reduced human astrovirus-1 infection. Together this study highlights a role for Ido1 in astrovirus infection and epithelial cell maturation.
