Deep mutational scanning of hepatitis B virus reveals a mechanism for cis-preferential reverse transcription.

Hepatitis B virus (HBV) is a small double-stranded DNA virus that chronically infects 296 million people. Over half of its compact genome encodes proteins in two overlapping reading frames, and during evolution, multiple selective pressures can act on shared nucleotides. This study combines an RNA-based HBV cell culture system with deep mutational scanning (DMS) to uncouple cis- and trans-acting sequence requirements in the HBV genome. The results support a leaky ribosome scanning model for polymerase translation, provide a fitness map of the HBV polymerase at single-nucleotide resolution, and identify conserved prolines adjacent to the HBV polymerase termination codon that stall ribosomes. Further experiments indicated that stalled ribosomes tether the nascent polymerase to its template RNA, ensuring cis-preferential RNA packaging and reverse transcription of the HBV genome.

Steatosis, HBV-related HCC, cirrhosis, and HBsAg seroclearance: A systematic review and meta-analysis.

BACKGROUND AND AIMS: NAFLD and chronic hepatitis B (CHB) infection are common etiologies of HCC. The impact of hepatic steatosis on HCC in CHB, as well as its relationship with the development of cirrhosis, fibrosis, and HBsAg seroclearance, remains controversial. APPROACH AND RESULTS: Data from observational studies were collected through PubMed, EMBASE, and the Cochrane Library from inception to February 1, 2022. Outcomes of interest included the association of hepatic steatosis with HCC, cirrhosis, advanced fibrosis, and HBsAg seroclearance, expressed in terms of pooled ORs. Additional subgroup and sensitivity analyses were performed to validate the robustness of findings. A total of 34 studies with 68,268 patients with CHB were included. Hepatic steatosis was associated with higher odds of HCC (OR, 1.59; 95% CI, 1.12-2.26; I2  = 72.5%), with the association remaining consistent in Asia (OR, 1.56; 95% CI, 1.08-2.25), studies with a median follow-up duration of ≥5 years (OR, 2.82; 95% CI, 1.57-5.08), exclusion of alcohol use (OR, 1.71; 95% CI, 1.01-2.91), and biopsy-proven steatosis (OR, 2.86; 95% CI, 1.61-5.06), although no significant association was noted among nucleos(t)ide analogue-treated patients (OR, 1.05; 95% CI, 0.62-1.77). Steatosis was associated with the development of cirrhosis (OR, 1.52; 95% CI, 1.07-2.16; I2  = 0%) and HBsAg seroclearance (OR, 2.22; 95% CI, 1.58-3.10; I2  = 49.0%). CONCLUSIONS: Hepatic steatosis was associated with an increased risk of HCC and cirrhosis among patients with CHB but with a higher chance of achieving a functional cure, highlighting the importance of identifying concomitant steatosis in CHB.

A genome-wide CRISPR activation screen reveals Hexokinase 1 as a critical factor in promoting resistance to multi-kinase inhibitors in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is often diagnosed at an advanced stage and is, therefore, treated with systemic drugs, such as tyrosine-kinase inhibitors (TKIs). These drugs, however, offer only modest survival benefits due to the rapid development of drug resistance. To identify genes implicated in TKI resistance, a cluster of regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 activation screen was performed in hepatoma cells treated with regorafenib, a TKI used as second-line therapy for advanced HCC. The screen results show that Hexokinase 1 (HK1), catalyzing the first step in glucose metabolism, is a top candidate for conferring TKI resistance. Compatible with this, HK1 was upregulated in regorafenib-resistant cells. Using several experimental approaches, both in vitro and in vivo, we show that TKI resistance correlates with HK1 expression. Furthermore, an HK inhibitor resensitized resistant cells to TKI treatment. Together, our data indicate that HK1 may function as a critical factor modulating TKI resistance in hepatoma cells and, therefore, may serve as a biomarker for treatment success.

Expansion, in vivo-ex vivo cycling, and genetic manipulation of primary human hepatocytes.

Primary human hepatocytes (PHHs) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture, and resistance to genetic manipulation have limited their use. Here, we show that the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These mouse-passaged (mp) PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum mpPHH can be efficiently genetically modified in culture, mobilized, and then recultured as spheroids or retransplanted to create highly humanized mice that carry a genetically altered hepatocyte graft. Together, these advances provide flexible tools for the study of human liver disease and evaluation of hepatocyte-targeted gene therapy approaches.

Elevated Serum Amyloid A Levels Contribute to Increased Platelet Adhesion in COVID-19 Patients.

Coronavirus disease-19 (COVID-19) patients are prone to thrombotic complications that may increase morbidity and mortality. These complications are thought to be driven by endothelial activation and tissue damage promoted by the systemic hyperinflammation associated with COVID-19. However, the exact mechanisms contributing to these complications are still unknown. To identify additional mechanisms contributing to the aberrant clotting observed in COVID-19 patients, we analyzed platelets from COVID-19 patients compared to those from controls using mass spectrometry. We identified increased serum amyloid A (SAA) levels, an acute-phase protein, on COVID-19 patients' platelets. In addition, using an in vitro adhesion assay, we showed that healthy platelets adhered more strongly to wells coated with COVID-19 patient serum than to wells coated with control serum. Furthermore, inhibitors of integrin aIIbß3 receptors, a mediator of platelet-SAA binding, reduced platelet adhesion to recombinant SAA and to wells coated with COVID-19 patient serum. Our results suggest that SAA may contribute to the increased platelet adhesion observed in serum from COVID-19 patients. Thus, reducing SAA levels by decreasing inflammation or inhibiting SAA platelet-binding activity might be a valid approach to abrogate COVID-19-associated thrombotic complications.

Association Between Fibrosis Stage and Outcomes of Patients With Nonalcoholic Fatty Liver Disease: A Systematic Review and Meta-Analysis.

BACKGROUND & AIMS: Biopsy-confirmed liver fibrosis is a prognostic factor for patients with nonalcoholic fatty liver disease (NAFLD). We performed a systematic review to quantify the prognostic value of fibrosis stage in patients with NAFLD and the subgroup of patients with nonalcoholic steatohepatitis (NASH) and to assess the evidence that change in fibrosis stage is a surrogate endpoint. METHODS: We searched the MEDLINE, Embase, Cochrane Library, and trial registry databases through August 2018 for prospective or retrospective cohort studies of liver-related clinical events and outcomes in adults with NAFLD or NASH. We collected data on mortality (all cause and liver related) and morbidity (cirrhosis, liver cancer, and all liver-related events) by stage of fibrosis, determined by biopsy, for patients with NAFLD or NASH. Using fibrosis stage 0 as a reference population, we calculated fibrosis stage-specific relative risk (RR) and 95% confidence interval (CI) values for mortality and morbidities. We performed fixed-effect and random-effect model meta-analyses. Metaregression was used to examine associations among study design (prospective vs retrospective cohort), overall risk of bias (medium or high), and mean duration of follow-up (in years). RESULTS: Our meta-analysis included 13 studies, comprising 4428 patients with NAFLD; 2875 of these were reported to have NASH. Compared with no fibrosis (stage 0), unadjusted risk increased with increasing stage of fibrosis (stage 0 vs 4): all-cause mortality RR, 3.42 (95% CI, 2.63-4.46); liver-related mortality RR, 11.13 (95% CI, 4.15-29.84); liver transplant RR, 5.42 (95% CI, 1.05-27.89); and liver-related events RR, 12.78 (95% CI, 6.85-23.85). The magnitude of RR did not differ significantly after adjustment for confounders, including age or sex in the subgroup of NAFLD patients with NASH. Three studies examined the effects of increasing fibrosis on quality of life had inconsistent findings. CONCLUSIONS: In a systematic review and meta-analysis, we found biopsy-confirmed fibrosis to be associated with risk of mortality and liver-related morbidity in patients with NAFLD, with and without adjustment for confounding factors and in patients with reported NASH. Further studies are needed to assess the association between fibrosis stage and patient quality of life and establish that change in liver fibrosis stage is a valid endpoint for use in clinical trials.

Modeling Social Distancing Strategies to Prevent SARS-CoV-2 Spread in Israel: A Cost-Effectiveness Analysis.

OBJECTIVES: While highly effective in preventing SARS-CoV-2 spread, national lockdowns come with an enormous economic price. Few countries have adopted an alternative "testing, tracing, and isolation" approach to selectively isolate people at high exposure risk, thereby minimizing the economic impact. To assist policy makers, we performed a cost-effectiveness analysis of these 2 strategies. METHODS: A modified Susceptible, Exposed, Infectious, Recovered, and Deceased (SEIRD) model was employed to assess the situation in Israel, a small country with ∼9 million people. The incremental cost-effectiveness ratio (ICER) of these strategies as well as the expected number of infected individuals and deaths were calculated. RESULTS: A nationwide lockdown is expected to save, on average, 274 (median 124, interquartile range: 71-221) lives compared to the "testing, tracing, and isolation" approach. However, the ICER will be, on average, $45 104 156 (median $49.6 million, interquartile range: 22.7-220.1) to prevent 1 case of death. CONCLUSION: A national lockdown has a moderate advantage in saving lives with tremendous costs and possible overwhelming economic effects. These findings should assist decision makers dealing with additional waves of this pandemic.

Early post-liver transplantation infections and their effect on long-term survival.

BACKGROUND: Infections post-liver transplantation are major drivers for morbidity and mortality. However, the impact of infections within 180 days post-liver transplantation on long-term survival is not clear. METHODS: We present a retrospective cohort of 317 liver transplant patients for whom all infectious episodes were prospectively collected during a mean follow-up of 4.4 years. RESULTS: A total of 143/317 (45%) of patients suffered from any infectious episode during the first 6 months following liver transplantation. Patients with surgical site infections have a reduced survival compared to those with no infection (HR 0.33, 95% CI 0.172-0.636, P = .001), whereas infections from other sources, including pneumonia, UTI, and line-related infections, were not associated with increased mortality. Furthermore, even though the presence of any infection within 30 days or 6 months post-transplantation did not affect survival, more than a single infectious episode per patient was significantly associated with increased mortality (HR 1.70, CI 1.12-2.60, P = .013). In a multivariate analysis, the number of infectious episodes remained statistically significant (HR 1.58, 95% CI 1.03-2.43, P = .035) upon adjustment for other major variables associated with comorbidities and infection risk. CONCLUSIONS: Surgical site infections and the number of infectious episodes within 180 days post-liver transplantation are major determinants of long-term survival among these patients.

Ombitasvir/paritaprevir/ritonavir & dasabuvir ± ribavirin following protease inhibitors failure - a prospective multi-centre trial.

BACKGROUND: Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease and hepatocellular carcinoma. Treatment with first generation protease inhibitors (PI) + peg-interferon (pegIFN) and ribavirin (RBV) achieved sustained virologic response (SVR) rates of 65-75% but was associated with multiple side effects. The aim of this study was to evaluate safety and efficacy of Ombitasvir/Paritaprevir/Ritonavir and Dasabuvir (3D) ± RBV in HCV genotype 1 patients that failed previous treatment with first generation PIs. METHODS: An investigator-initiated, open-label, multi-centre clinical trial. HCV Genotype 1 patients who were previously null/partial responders or relapsers to telaprevir, boceprevir or simepravir+pegIFN/RBV and met eligibility criteria were included. 3D ± RBV were administrated for 12 or 24 weeks according to label. The primary outcome was antiviral response (SVR12); Secondary outcomes were patient reported outcomes, adverse events and resistance associated variants. RESULTS: Thirty-nine patients initiated treatment according to study protocol (59% men, age 54.0 ± 8.7 years, BMI 28.7 ± 4.5 kg/m2). Thirty-seven (94.9%) completed the study. Thirty-five patients had genotype 1b (9 cirrhotics) and 4 had genotype 1a (2 cirrhotics). Intention-to-treat SVR12 was 92.3% and per-protocol SVR12 was 97.3%. The rate of advanced fibrosis (FibroScan® score F3-4) declined from 46.2 to 25.7% (P = 0.045). Abnormal ALT levels declined from 84.6 to 8.6% (P < 0.001). Seven patients (17.9%) experienced serious adverse events (3 Psychiatric admissions, 1 pneumonia, 1 ankle fracture, 2 palpitations), and 12 patients (30.8%) experienced self-reported adverse events, mostly weakness. CONCLUSION: 3D ± RBV is safe and effective in achieving SVR among patients with HCV genotype 1 who failed previous first-generation PI treatment. TRIAL REGISTRATION: NCT02646111 (submitted to ClinicalTrials.gov, December 28, 2015).

Post-Liver Transplantation Anemia and Its Correlation with Mortality and Graft Failure.

BACKGROUND: In adults, post-liver transplantation anemia (PLTA) is common, but its characteristics and long-term influence on major outcomes have yet to be elucidated. AIM: We aimed to assess prevalence, characteristics, predictors, and outcomes of PLTA at 6 months (early PLTA) and at 2 years (late PLTA). METHODS: A single-center retrospective cohort study using prospectively collected data from liver transplantations in adults during January 2007-December 2015. PLTA impact on various long-term outcomes was assessed, including mortality, composites of mortality or graft failure, cardiovascular outcomes, and malignancy occurrences. RESULTS: Hundred and fifty liver transplanted individuals were included. There was a 79% prevalence of anemia pre-transplantation, whereas early and late PLTA were evident in 58% and 40% of patients, respectively. Pre-transplantation anemia was associated with development of early PLTA which was associated with late PLTA. In a multivariate analysis, early PLTA was significantly associated with mortality or graft failure at a follow-up of 3 years (odds ratio 3.838, 95% CI 1.114-13.226). Late PLTA was not significantly associated with worse long-term outcomes. CONCLUSIONS: Early and late PLTA are prevalent among liver transplanted patients. Early PLTA is associated with long-term mortality or graft failure.

Liver steatosis is a major predictor of poor outcomes in chronic hepatitis C patients with sustained virological response.

Sustained virological response (SVR) results in reduced incidence of hepatocellular carcinoma (HCC) and mortality among chronic hepatitis C (CHC) patients with advanced fibrosis. Since both advanced fibrosis and liver steatosis (LS) may coexist in CHC patients, we evaluated their individual effects on a composite outcome of all-cause mortality and HCC in CHC patients with SVR following direct-acting antivirals (DAA) treatment. We retrospectively evaluated inception cohort of 515 CHC patients who achieved SVR following treatment with DAA, with a mean follow-up of 24 months. Baseline liver fibrosis was assessed by transient elastography, and LS was validated by at least three independent ultrasonographic examinations. 211 of 515 patients (41%) had baseline LS. Patients with LS had a higher cumulative rate of all-cause mortality and HCC at 2 years of follow-up compared to patients without LS (15.75% and 2.79%, respectively, P < 0.001), although they did not have increased incidence of advanced fibrosis or cirrhosis. Consistently, multivariate analysis showed that LS was associated with a significant 7.5-fold increased risk of all-cause mortality and HCC (HR 7.51, 95% C.I 3.61-13.36, P < 0.001) even upon adjustment to components of the metabolic syndrome, whereas advanced fibrosis showed only a trend towards statistical significance (HR 2.32, 95% C.I 0.97-6.59, P = 0.06). In conclusion, LS is a major predictor of all-cause mortality and HCC in patients who achieved SVR following DAA treatment regardless of fibrosis stage. These patients should be rigorously screened for HCC.

The Pattern of Elevated Liver Function Tests in Nonalcoholic Fatty Liver Disease Predicts Fibrosis Stage and Metabolic-Associated Comorbidities.

BACKGROUND: Patients with nonalcoholic fatty liver disease (NAFLD) and with abnormal liver function tests (LFTs) most commonly present with elevated hepatocellular enzymes (H pattern), but a subset of patients is found to have elevated cholestatic enzymes (C pattern) or a mixed (M) pattern. AIMS AND METHODS: To determine whether the epidemiologic background and comorbidities, as well as the degree of liver fibrosis, differ between NAFLD patients with different patterns of elevated LFTs by retrospectively analyzing data of 106 patients with a biopsy-proven diagnosis of NAFLD. The pattern of elevated LFTs was determined by adopting the "R-Ratio" formula commonly used for drug-induced liver injury. RESULTS: Advanced fibrosis (F > 2) was found in 15 out of 48 (31.3%) patients with a C pattern of elevated LFTs as compared to 2 out of 44 (4.5%) in M patients and 2 out of 11 (18.2%) in H patients (p = 0.004). Group C patients are older and also had a higher prevalence of diabetes, a higher mean hemoglobin A1c, and a higher prevalence of hypertension, as well as a trend for a higher prevalence of hypertriglyceridemia. CONCLUSIONS: Using a simple formula incorporating routine LFTs can help to categorize NAFLD patients as low or high risk for advanced fibrosis stage and metabolic-associated comorbidities.

The effect of cause, timing, kidney function recovery, and recurrent events on the prognosis of acute kidney injury in kidney transplant recipients.

AIM: To assess the incidence of acute kidney injury (AKI) and its common etiologies in kidney transplant recipients and the effect of AKI's characteristics on graft survival. METHODS: In a retrospective longitudinal cohort study, all serum creatinine (SCr) values of patients that had kidney transplantation between 01/2002-12/2010 were retrieved. AKI was defined as a 50% increase in SCr. Etiologies, recurrence, timing, and kidney function dynamics during the event were evaluated. The primary endpoint was defined as graft loss. Time-varying Cox model was used for the analysis. RESULTS: Of 659 patients, 208 (31.6%) patients had 321 documented AKI events. Of these, 138 (66.4%) patients had one event, and 70 (33.6%) patients had recurrent events. The leading etiologies of the first AKI event were as follows: infection (33.4%), hypovolemia (14.3%), and unknown etiology (16.8%). Both first and recurrent AKI events were associated with an increased risk of graft loss (HR: 2.76, 95% CI: 1.95-3.89) and (HR: 4.54, 95% CI: 2.59-7.93), respectively. This deleterious association was lower within three months after transplantation, compared to later events. Patients in whom kidney function returned to baseline were less prone to graft loss. CONCLUSIONS: Late-onset, incomplete recovery, and recurrent AKI events are associated with increased graft loss.

Characterization of hepatitis B and delta coinfection in Israel.

BACKGROUND: Characteristics of hepatitis B (HBV) and delta (HDV) coinfection in various geographical regions, including Israel, remain unclear. Here we studied HDV seroprevalence in Israel, assessed HDV/HBV viral loads, circulating genotypes and hepatitis delta antigen (HDAg) conservation. METHODS: Serological anti HDV IgG results from 8969 HBsAg positive individuals tested in 2010-2015 were retrospectively analyzed to determine HDV seroprevalence. In a cohort of HBV/HDV coinfected (n=58) and HBV monoinfected (n=27) patients, quantitative real-time PCR (qRT-PCR) and sequencing were performed to determine viral loads, genotypes and hepatitis delta antigen (HDAg) protein sequence. RESULTS: 6.5% (587/8969) of the HBsAg positive patients were positive for anti HDV antibodies. HDV viral load was >2 log copies/ml higher than HBV viral load in most of the coinfected patients with detectable HDV RNA (86%, 50/58). HDV genotype 1 was identified in all patients, most of whom did not express HBV. While 66.6% (4/6) of the HBV/HDV co-expressing patients carried HBV-D2 only 18.5% (5/27) of the HBV monoinfections had HBV-D2 (p=0.03). Higher genetic variability in the HDAg protein sequence was associated with higher HDV viral load. CONCLUSIONS: The overall significant prevalence of HDV (6.5%) mandates HDV RNA testing for all coinfected patients. Patients positive for HDV RNA (characterized by low HBV DNA blood levels) carried HDV genotype 1. Taken together, the significant HDV seroprevalence and the lack of effective anti-HDV therapy, necessitates strict clinical surveillance especially in patients with higher HDV viral loads and increased viral evolution.

Transplantation of Livers from Old Donors: Pushing the Envelope Beyond the Seventh Decade.

BACKGROUND: The lack of organs for liver transplantation has prompted transplant professionals to study potential solutions, such as the use of livers from donors older than 70 years. This strategy is not widely accepted because potential risks of vascular and biliary complications and recurrence of hepatitis C. OBJECTIVES: To examine the efficacy and safety of liver grafts from older donors for transplantation. METHODS: A retrospective analysis of data on 310 adults who underwent deceased donor liver transplantation between 2005 and 2015 was conducted. We compared graft and recipient survival, as well as major complications, of transplants performed with grafts from donors younger than 70 years (n=265, control group) and those older than 70 years (n=45, older-donor group), followed by multivariate analysis, to identify risk factors. RESULTS: There was no significant difference between the control and older-donor group at 1, 5, and 10 years of recipient survival (79.5% vs. 73.3%, 68.3% vs. 73.3%, 59.2% vs. 66.7%, respectively) or graft survival (74.0% vs. 71.0%, 62.7% vs. 71.0%, 54.8% vs. 64.5%, respectively). The rate of biliary and vascular complications was similar in both groups. Significant risk factors for graft failure were hepatitis C (hazard ratio [HR] = 1.92, 95% confidence interval [95%CI] 1.16-2.63), older donor age (HR = 1.02, 95%CI 1.007-1.031), and male gender of the recipient (HR = 1.65, 95%CI 1.06-2.55). CONCLUSIONS: Donor age affects liver graft survival. However, grafts from donors older than 70 years may be equally safe if cold ischemia is maintained for less than 8 hours.

Patients' Age Rather Than Model of End-Stage Liver Disease Score Predicts Survival After Liver Transplantation.

BACKGROUND: The model of end-stage liver disease (MELD) score is the standard tool for prioritizing patients awaiting liver transplantation. There is currently no definite high cutoff score reflecting disease severity that might exclude patients from transplantation. Furthermore, the age limit that used to disqualify patients from eligibility to transplantation was recently withdrawn in several countries. AIMS: The aims of this study were to assess the effects of MELD score and age at time of transplantation on patients' short- and long-term survival. METHODS: We conducted a retrospective single-center study on a cohort of patients transplanted for the first time due to non-fulminant liver failure. RESULTS: Four hundred and seventeen patients (mean age 50.2 years, 63% males) who underwent liver transplantation for the first time were included. Both higher patients' and donors' age were significantly associated with increased long-term mortality (p = 0.007, 95% CI 1.006-1.038 for patient age, p = 0.02, 95% CI 1.002-1.023 for donor age). Patients' age remained significantly associated with survival at 1 year post-transplantation, as well. We found no association between higher MELD score at transplantation and long-term mortality (p = 0.189, 95% CI 0.99-1.051) irrespective of patients' age. Specifically, when patients were divided according to their MELD score at transplantation (MELD < 15, MELD 15-25 and MELD > 25), no significant differences in long-term survival were detected between these three subgroups. Results did not differ significantly in a subgroup analysis of patients without hepatocellular carcinoma at the time of transplantation. CONCLUSIONS: Patients' and donors' age rather than patients' MELD score at transplantation determine survival following liver transplantation.

Modeling host interactions with hepatitis B virus using primary and induced pluripotent stem cell-derived hepatocellular systems.

Hepatitis B virus (HBV) chronically infects 400 million people worldwide and is a leading driver of end-stage liver disease and liver cancer. Research into the biology and treatment of HBV requires an in vitro cell-culture system that supports the infection of human hepatocytes, and accurately recapitulates virus-host interactions. Here, we report that micropatterned cocultures of primary human hepatocytes with stromal cells (MPCCs) reliably support productive HBV infection, and infection can be enhanced by blocking elements of the hepatocyte innate immune response associated with the induction of IFN-stimulated genes. MPCCs maintain prolonged, productive infection and represent a facile platform for studying virus-host interactions and for developing antiviral interventions. Hepatocytes obtained from different human donors vary dramatically in their permissiveness to HBV infection, suggesting that factors--such as divergence in genetic susceptibility to infection--may influence infection in vitro. To establish a complementary, renewable system on an isogenic background in which candidate genetics can be interrogated, we show that inducible pluripotent stem cells differentiated into hepatocyte-like cells (iHeps) support HBV infection that can also be enhanced by blocking interferon-stimulated gene induction. Notably, the emergence of the capacity to support HBV transcriptional activity and initial permissiveness for infection are marked by distinct stages of iHep differentiation, suggesting that infection of iHeps can be used both to study HBV, and conversely to assess the degree of iHep differentiation. Our work demonstrates the utility of these infectious systems for studying HBV biology and the virus' interactions with host hepatocyte genetics and physiology.

Humanized mice efficiently engrafted with fetal hepatoblasts and syngeneic immune cells develop human monocytes and NK cells.

BACKGROUND & AIMS: Human liver chimeric mice are useful models of human hepatitis virus infection, including hepatitis B and C virus infections. Independently, immunodeficient mice reconstituted with CD34(+) hematopoietic stem cells (HSC) derived from fetal liver reliably develop human T and B lymphocytes. Combining these systems has long been hampered by inefficient liver reconstitution of human fetal hepatoblasts. Our study aimed to enhance hepatoblast engraftment in order to create a mouse model with syngeneic human liver and immune cells. METHODS: The effects of human oncostatin-M administration on fetal hepatoblast engraftment into immunodeficient fah(-/-) mice was tested. Mice were then transplanted with syngeneic human hepatoblasts and HSC after which human leukocyte chimerism and functionality were analyzed by flow cytometry, and mice were challenged with HBV. RESULTS: Addition of human oncostatin-M enhanced human hepatoblast engraftment in immunodeficient fah(-/-) mice by 5-100 fold. In contrast to mice singly engrafted with HSC, which predominantly developed human T and B lymphocytes, mice co-transplanted with syngeneic hepatoblasts also contained physiological levels of human monocytes and natural killer cells. Upon infection with HBV, these mice displayed rapid and sustained viremia. CONCLUSIONS: Our study provides a new mouse model with improved human fetal hepatoblast engraftment and an expanded human immune cell repertoire. With further improvements, this model may become useful for studying human immunity against viral hepatitis. LAY SUMMARY: Important human pathogens such as hepatitis B virus, hepatitis C virus and human immunodeficiency virus only infect human cells which complicates the development of mouse models for the study of these pathogens. One way to make mice permissive for human pathogens is the transplantation of human cells into immune-compromised mice. For instance, the transplantation of human liver cells will allow the infection of these so-called "liver chimeric mice" with hepatitis B virus and hepatitis C virus. The co-transplantation of human immune cells into liver chimeric mice will further allow the study of human immune responses to hepatitis B virus or hepatitis C virus. However, for immunological studies it will be crucial that the transplanted human liver and immune cells are derived from the same human donor. In our study we describe the efficient engraftment of human fetal liver cells and immune cells derived from the same donor into mice. We show that liver co-engraftment resulted in an expanded human immune cell repertoire, including monocytes and natural killer cells in the liver. We further demonstrate that these mice could be infected with hepatitis B virus, which lead to an expansion of natural killer cells. In conclusion we have developed a new mouse model that could be useful to study human immune responses to human liver pathogens.