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Secondary bacterial infections are a potentially fatal complication of influenza infection. We aimed to define the impact of secondary bacterial infections on the clinical course and mortality in coronavirus disease 2019 (COVID-19) patients by comparison with influenza patients. COVID-19 (n = 642) and influenza (n = 742) patients, admitted to a large tertiary center in Israel and for whom blood or sputum culture had been taken were selected for this study. Bacterial culture results, clinical parameters, and death rates were compared. COVID-19 patients had higher rates of bacterial infections than influenza patients (12.6% vs. 8.7%). Notably, the time from admission to bacterial growth was longer in COVID-19 compared to influenza patients (4 (1–8) vs. 1 (1–3) days). Late infections (> 48 h after admission) with gram-positive bacteria were more common in COVID-19 patients (28% vs. 9.5%). Secondary infection was associated with a higher risk of death in both patient groups 2.7-fold (1.22–5.83) for COVID-19, and 3.09-fold (1.11–7.38) for Influenza). The association with death remained significant upon adjustment to age and clinical parameters in COVID-19 but not in influenza infection. Secondary bacterial infection is a notable complication associated with worse outcomes in COVID-19 than influenza patients. Careful surveillance and prompt antibiotic treatment may benefit selected patients.

Acute liver failure (ALF) is a fulminant complication of multiple etiologies, characterized by rapid hepatic destruction, multi-organ failure and mortality. ALF treatment is mainly limited to supportive care and liver transplantation. Here we utilize the acetaminophen (APAP) and thioacetamide (TAA) ALF models in characterizing 56,527 single-cell transcriptomes to define the mouse ALF cellular atlas. We demonstrate that unique, previously uncharacterized stellate cell, endothelial cell, Kupffer cell, monocyte and neutrophil subsets, and their intricate intercellular crosstalk, drive ALF. We unravel a common MYC-dependent transcriptional program orchestrating stellate, endothelial and Kupffer cell activation during ALF, which is regulated by the gut microbiome through Toll-like receptor (TLR) signaling. Pharmacological inhibition of MYC, upstream TLR signaling checkpoints or microbiome depletion suppress this cell-specific, MYC-dependent program, thereby attenuating ALF. In humans, we demonstrate upregulated hepatic MYC expression in ALF transplant recipients compared to healthy donors. Collectively we demonstrate that detailed cellular/genetic decoding may enable pathway-specific ALF therapeutic intervention. A single-cell map of transcriptomic changes during acute liver failure unveils new insights into pathogenesis and potential therapeutic targets.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer related deaths. Patients with advanced HCC are treated with sorafenib. A recent randomized controlled trial demonstrated a survival benefit for regorafenib treatment in patients with advanced HCC who had progressed on sorafenib. We aimed to evaluate the cost-effectiveness of this approach. To evaluate the cost effectiveness of regorafenib, we used a Markov model that incorporates health outcomes, measured by life-years and quality-adjusted life-years (QALYs). Drug costs were based on 2017 discounted prices. Model robustness was validated by probabilistic sensitivity analyses using Monte Carlo simulations. The use of regorafenib results in a gain of 19.76 weeks of life (0.38 Life Years) as compared to placebo. When adjusted for quality of life, using regorafenib produced a gain of 0.25 quality adjusted life years (QALYs). The incremental cost-effectiveness ratio for regorafenib compared with best supportive care was between $201,797 and $268,506 per QALY. The modest incremental benefit at a relatively high incremental cost of regorafenib treatment suggests that it is not cost-effective at commonly accepted willingness to pay thresholds.

Patients with chronic hepatitis B (CHB) are regularly monitored for HBV DNA and liver enzymes in order to assess disease progression and the need for antiviral therapy. Identifying patients with a stable course of disease can potentially prolong the intervals between visits, withhold unnecessary tests and save money. Accordingly, we aimed to find predictors for a stable disease course in patients with CHB. 579 patients with CHB, who were followed in a tertiary referral center between January 2004–December 2018, were retrospectively analyzed. Patients with low and steady viral load titer (< 2000 IU/ml) and normal ALT levels (< 40 IU/ml) in 6 consecutive clinic encounters were considered to have a stable course of CHB. A stepwise multivariate logistic regression analysis and a decision tree model were used to identify predictors of a stable disease course. Following exclusion of ineligible patients, a total of 220 patients were included in the final analysis. 64/220 patients had a stable disease course. Patients with a stable disease were older (62.99 ± 12.36 Vs. 54.07 ± 13.64, p  < 0.001) with a higher percentage of women (53% vs. 38%) and had lower baseline levels of AST, ALT and viral load (VL). In a multivariate analysis, age (OR 0.94, 95% CI 0.91–0.98), baseline ALT (OR 1.06, 95% CI 1.01–1.1) and VL (OR 1.05 95% CI 1.02–1.08), were significantly associated with a stable disease. In a decision tree model, patients 46–67 years old, with baseline VL < 149 IU/mL and ALT < 40 IU/mL had the best probability (91%) for a stable disease course over 4.4 ± 2.2 years. We conclude that integrating patients’ age with baseline VL and ALT can predict a stable disease course in patients with CHB off treatment.

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.

Chronic hepatitis B virus (HBV) infection is prevalent, deadly and seldom cured due to the persistence of viral episomal DNA (cccDNA) in infected cells. Newly developed genome engineering tools may offer the ability to directly cleave viral DNA, thereby promoting viral clearance. Here, we show that the CRISPR/Cas9 system can specifically target and cleave conserved regions in the HBV genome, resulting in robust suppression of viral gene expression and replication. Upon sustained expression of Cas9 and appropriately chosen guide RNAs, we demonstrate cleavage of cccDNA by Cas9 and a dramatic reduction in both cccDNA and other parameters of viral gene expression and replication. Thus, we show that directly targeting viral episomal DNA is a novel therapeutic approach to control the virus and possibly cure patients.

Introduction The SARS-CoV-2 pandemic imposes an unprecedented burden on hospitals treating coronavirus disease 2019 (COVID-19) patients. [...]clinical parameters accurately predicting disease outcome are needed. Among the parameters tested (lowest values of albumin, lymphocyte count, blood oxygen saturation (BOS) and systolic blood pressure, peak levels of lactate dehydrogenase (LDH), C-reactive protein (CRP), ferritin, white blood cell count, and fever), only BOSmin (R = 0.07, p = 0.0004) showed significant correlation (Fig. 1). [...]IL-6 is not routinely tested at admission and might reflect other inflammatory conditions. [...]in spite of differences in test kits and procedures between different laboratories and institutions, viral load might provide a rapid screening tool for COVD-19 severity among admitted patients. Rights and permissions Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.

Liver fibrosis predicts liver-related morbidity and mortality in patients with non-alcoholic fatty liver disease (NAFLD). Non-invasive scores correlate with the degree of liver fibrosis in these patients. To investigate the accuracy of noninvasive scoring systems in predicting long-term outcomes and cancer incidence of patients with NAFLD, we performed a single-center retrospective study of patients with biopsy proven NAFLD. Mean follow up period was 100 months. Outcomes included liver-related complications, hospitalizations, overall mortality and the development of any malignancies. 32 patients had advanced fibrosis (F3-F4) per biopsy at baseline and 121 patients had mild to moderate fibrosis (F0-F2). Both advanced histologic fibrosis stage as well as higher non-invasive scores predicted repeated hospitalizations and longer hospitalization stays. In a multivariate analysis, liver fibrosis (p = 0.002), FIB-4 score (p<0.001), NFS (p<0.001) but not APRI score (p = 0.07) were predictors of overall mortality, and the occurrence of malignancies was associated with higher APRI (p<0.001), FIB-4 (p<0.001) and NFS (p = 0.008) scores, but not with advanced fibrosis, as determined by liver biopsy (p = 0.105). In NAFLD patients, noninvasive scoring systems are good predictors of morbidity and mortality and may have an additive value in predicting the development of hepatic and extra-hepatic cancers.

This protocol uses micropatterned co-cultures comprising 2D islands of primary human hepatocytes surrounded by supportive fibroblast cells to model liver infection by the hepatitis B and C viruses or Plasmodium pathogens in vitro . The development of therapies and vaccines for human hepatropic pathogens requires robust model systems that enable the study of host-pathogen interactions. However, in vitro liver models of infection typically use either hepatoma cell lines that exhibit aberrant physiology or primary human hepatocytes in culture conditions in which they rapidly lose their hepatic phenotype. To achieve stable and robust in vitro primary human hepatocyte models, we developed micropatterned cocultures (MPCCs), which consist of primary human hepatocytes organized into 2D islands that are surrounded by supportive fibroblast cells. By using this system, which can be established over a period of days, and maintained over multiple weeks, we demonstrate how to recapitulate in vitro hepatic life cycles for the hepatitis B and C viruses and the Plasmodium pathogens P. falciparum and P. vivax . The MPCC platform can be used to uncover aspects of host-pathogen interactions, and it has the potential to be used for drug and vaccine development.

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 hepatocytetargeted gene therapy approaches.