Hepatitis C virus infects and perturbs liver stem cells.

Hepatitis C virus (HCV) is the leading cause of death from liver disease. How HCV infection causes lasting liver damage and increases cancer risk remains unclear. Here, we identify bipotent liver stem cells as novel targets for HCV infection, and their erroneous differentiation as the potential cause of impaired liver regeneration and cancer development. We show 3D organoids generated from liver stem cells from actively HCV-infected individuals carry replicating virus and maintain low-grade infection over months. Organoids can be infected with a primary HCV isolate. Virus-inclusive single-cell RNA sequencing uncovered transcriptional reprogramming in HCV+ cells supporting hepatocytic differentiation, cancer stem cell development, and viral replication while stem cell proliferation and interferon signaling are disrupted. Our data add a new pathogenesis mechanism-infection of liver stem cells-to the biology of HCV infection that may explain progressive liver damage and enhanced cancer risk through an altered stem cell state.ImportanceThe hepatitis C virus (HCV) causes liver disease, affecting millions. Even though we have effective antivirals that cure HCV, they cannot stop terminal liver disease. We used an adult stem cell-derived liver organoid system to understand how HCV infection leads to the progression of terminal liver disease. Here, we show that HCV maintains low-grade infections in liver organoids for the first time. HCV infection in liver organoids leads to transcriptional reprogramming causing cancer cell development and altered immune response. Our finding shows how HCV infection in liver organoids mimics HCV infection and patient pathogenesis. These results reveal that HCV infection in liver organoids contributes to liver disease progression.

Cyclone: an accessible pipeline to analyze, evaluate, and optimize multiparametric cytometry data.

In the past decade, high-dimensional single-cell technologies have revolutionized basic and translational immunology research and are now a key element of the toolbox used by scientists to study the immune system. However, analysis of the data generated by these approaches often requires clustering algorithms and dimensionality reduction representation, which are computationally intense and difficult to evaluate and optimize. Here, we present Cytometry Clustering Optimization and Evaluation (Cyclone), an analysis pipeline integrating dimensionality reduction, clustering, evaluation, and optimization of clustering resolution, and downstream visualization tools facilitating the analysis of a wide range of cytometry data. We benchmarked and validated Cyclone on mass cytometry (CyTOF), full-spectrum fluorescence-based cytometry, and multiplexed immunofluorescence (IF) in a variety of biological contexts, including infectious diseases and cancer. In each instance, Cyclone not only recapitulates gold standard immune cell identification but also enables the unsupervised identification of lymphocytes and mononuclear phagocyte subsets that are associated with distinct biological features. Altogether, the Cyclone pipeline is a versatile and accessible pipeline for performing, optimizing, and evaluating clustering on a variety of cytometry datasets, which will further power immunology research and provide a scaffold for biological discovery.

Cyclone: an accessible pipeline to analyze, evaluate and optimize multiparametric cytometry data.

In the past decade, high-dimensional single cell technologies have revolutionized basic and translational immunology research and are now a key element of the toolbox used by scientists to study the immune system. However, analysis of the data generated by these approaches often requires clustering algorithms and dimensionality reduction representation which are computationally intense and difficult to evaluate and optimize. Here we present Cyclone, an analysis pipeline integrating dimensionality reduction, clustering, evaluation and optimization of clustering resolution, and downstream visualization tools facilitating the analysis of a wide range of cytometry data. We benchmarked and validated Cyclone on mass cytometry (CyTOF), full spectrum fluorescence-based cytometry, and multiplexed immunofluorescence (IF) in a variety of biological contexts, including infectious diseases and cancer. In each instance, Cyclone not only recapitulates gold standard immune cell identification, but also enables the unsupervised identification of lymphocytes and mononuclear phagocytes subsets that are associated with distinct biological features. Altogether, the Cyclone pipeline is a versatile and accessible pipeline for performing, optimizing, and evaluating clustering on variety of cytometry datasets which will further power immunology research and provide a scaffold for biological discovery.

Randomized Trial of Tenofovir With or Without Peginterferon Alfa Followed by Protocolized Treatment Withdrawal in Adults With Chronic Hepatitis B.

INTRODUCTION: Hepatitis B surface antigen (HBsAg) loss is associated with improved long-term outcomes of patients with chronic hepatitis B but is infrequently achieved with current monotherapies. We assessed whether combination strategies that included treatment withdrawal enhanced HBsAg loss. METHODS: A randomized (1:1) trial of tenofovir disoproxil fumarate (TDF) for 192 weeks with or without peginterferon (PegIFN) alfa-2a for the first 24 weeks, followed by withdrawal of TDF at week 192 with 48 weeks of off-treatment follow-up to week 240. The primary end point was HBsAg loss at week 240. RESULTS: Of 201 participants (52% HBeAg positive, 12%/6% genotype A/A2, 7% cirrhosis) randomized to TDF + PegIFN (n = 102) or TDF alone (n = 99), 6 participants had lost HBsAg at the end of the treatment phase (week 192), 5 (5.3%) in the combination group, and 1 (1.0%) in the TDF alone group ( P = 0.09). By week 240, 9 participants had cleared HBsAg, 5.3% in combination, and 4.1% in monotherapy arms ( P = 0.73). HBsAg decline and loss occurred earlier with TDF + PegIFN than TDF, with a ≥1-logIU/mL qHBsAg decline by week 24 in 28% in TDF + PegIFN compared with 6% in TDF ( P = 0.04). HBsAg loss occurred in 7 of 12 (58%) with hepatitis B virus subgenotype A2 (all HBeAg positive) compared with only 2 of 189 (1%) with other hepatitis B virus genotypes and in 8 of 93 (8.6%) HBeAg positive vs 1 of 87 (1.1%) HBeAg negative. DISCUSSION: PegIFN combined TDF followed by protocolized TDF withdrawal led to earlier but not higher percentages of HBsAg clearance. Pretreatment HBeAg positivity and subgenotype A2 were strongly associated with HBsAg clearance.

The Tenuous Role of Distress in the Diagnosis of Premature Ejaculation: A Narrative Review.

BACKGROUND: Unlike the other 2 criteria for diagnosing premature ejaculation (PE), namely lack of ejaculatory control and short ejaculation latency (EL), the role of bother/distress has received only minimal consideration and investigation. AIM: The specific aim was to determine both why distress is included in the PE diagnosis and whether such inclusion is advantageous to achieving better diagnostic outcomes. To this end, the review explored the historical and theoretical underpinnings of the inclusion of "bother/ distress" in the diagnosis of PE, with reference to the larger role that distress has played in the diagnosis of mental disorders, in an attempt to understand the utility (or lack thereof) of this construct in making a PE diagnosis. METHODS: We reviewed the role of bother/distress across current professional definitions for PE and then expanded this discussion to include the role of distress in other sexual dysfunctions. We then included a brief historical perspective regarding the role that distress has played in the diagnosis of PE. This discussion is followed by a deeper look at 2 nosological systems, namely DSM and ICD, to allow perspective on the inclusion of the bother/distress construct in the diagnosis of mental and behavioral disorders, including the assumptions/arguments put forward to include or exclude bother/distress as an important criterion underlying various professional assumptions. OUTCOME: Determination of the value and/or need of including bother/distress as a necessary criterion for the diagnosis of PE. RESULTS: Based on the research literature, bother/distress does not appear to be as critical for a PE diagnosis as either the lack of ejaculatory control or short EL. It is the weakest of the differences among men with and without PE, and recent evidence suggests that its inclusion is generally redundant with the severity of the 2 other criteria for PE, ejaculatory control and EL. CLINICAL TRANSLATION: Bother/distress appears to serve little purpose in the diagnosis of PE yet its assessment may be important for the treatment strategy and for assessing treatment effectiveness. STRENGTHS AND LIMITATIONS: This review did not provide a critical analysis of the literature regarding the role of bother/distress in PE, but rather focused on its potential value in understanding and diagnosing PE. CONCLUSION: Although bother/distress appears to add little to the improvement of accuracy for a PE diagnosis, understanding and assessing the man's or couple's experience of distress has important implications for the treatment strategy and focus, as well as for assessing treatment success. Rowland DL, Cooper SE. The Tenuous Role of Distress in the Diagnosis of Premature Ejaculation: A Narrative Review. Sex Med 2022;10:100546.

Modelling T-cell immunity against hepatitis C virus with liver organoids in a microfluidic coculture system.

Hepatitis C virus (HCV) remains a global public health challenge with an estimated 71 million people chronically infected, with surges in new cases and no effective vaccine. New methods are needed to study the human immune response to HCV since in vivo animal models are limited and in vitro cancer cell models often show dysregulated immune and proliferative responses. Here, we developed a CD8+ T cell and adult stem cell liver organoid system using a microfluidic chip to coculture 3D human liver organoids embedded in extracellular matrix with HLA-matched primary human T cells in suspension. We then employed automated phase contrast and immunofluorescence imaging to monitor T cell invasion and morphological changes in the liver organoids. This microfluidic coculture system supports targeted killing of liver organoids when pulsed with a peptide specific for HCV non-structural protein 3 (NS3) (KLVALGINAV) in the presence of patient-derived CD8+ T cells specific for KLVALGINAV. This demonstrates the novel potential of the coculture system to molecularly study adaptive immune responses to HCV in an in vitro setting using primary human cells.

Incidence and prediction of HBsAg seroclearance in a prospective multi-ethnic HBeAg-negative chronic hepatitis B cohort.

BACKGROUND AND AIMS: Achieving HBsAg loss is an important landmark in the natural history of chronic hepatitis B (CHB). A more personalized approach to prediction of HBsAg loss is relevant in counseling patients. This study sought to develop and validate a prediction model for HBsAg loss based on quantitative HBsAg levels (qHBsAg) and other baseline characteristics. METHODS: The Hepatitis B Research Network (HBRN) is a prospective cohort including 1240 untreated HBeAg-negative patients (1150 adults, 90 children) with median follow-up of 5.5 years. Incidence rates of HBsAg loss and hepatitis B surface antibody (anti-HBs) acquisition were determined, and a predictor score of HBsAg loss using readily available variables was developed and externally validated. RESULTS: Crude incidence rates of HBsAg loss and anti-HBs acquisition were 1.6 and 1.1 per 100 person-years (PY); 67 achieved sustained HBsAg loss for an incidence rate of 1.2 per 100 PY. Increased HBsAg loss was significantly associated with older age, non-Asian race, HBV phenotype (inactive CHB vs. others), HBV genotype A, lower HBV-DNA levels, and lower and greater change in qHBsAg. The HBRN-SQuARe (sex,∆quantHBsAg, age, race) score predicted HBsAg loss over time with area under the receiver operating characteristic curve (AUROC) (95% CIs) at 1 and 3 years of 0.99 (95% CI: 0.987-1.00) and 0.95 (95% CI 0.91-1.00), respectively. In validation in another cohort of 1253 HBeAg-negative patients with median follow-up of 3.1 years, HBRN SQuARe predicted HBsAg loss at 1 and 3 years with AUROC values of 0.99 (0.98-1.00) and 0.88 (0.77-0.99), respectively. CONCLUSION: HBsAg loss in predominantly untreated patients with HBeAg-negative CHB can be accurately predicted over a 3-year horizon using a simple validated score (HBRN SQuARe). This prognostication tool can be used to support patient care and counseling.

Remediation Strategies for Performance Anxiety across Sex, Sport and Stage: Identifying Common Approaches and a Unified Cognitive Model.

Strategies for addressing anxiety-related decrements in performance have been implemented across a variety of domains, including Sex, Sport, and Stage. In this review, we (1) iterate the dominant anxiety-related remediation strategies within each of these domains; (2) identify over-lapping and domain-specific strategies; and (3) attempt to unify the conceptualization of performance-related anxiety across these three areas under the information-processing framework of the Reflective/deliberative-Impulsive/automatic Model (RIM). Despite both diversity and similarity in remediation approaches across domains, we found that many strategies appear to share the common goal of maintaining a dominant automatic style of information processing in high performance demand situations. We then describe how various remediation strategies might hypothetically fit within the RIM framework and its subcomponents, identifying each intervention as falling into one or more broad categories related to achieving and/or maintaining dominance in automatic information processing. We conclude by affirming the benefit of adopting a unifying information-processing framework for the conceptualization of performance-related anxiety, as a way of both guiding future cross- and inter- disciplinary research and elucidating effective remediation models that share common pathways/mechanisms to improved performance.

Hepatitis B e antigen loss in adults and children with chronic hepatitis B living in North America: A prospective cohort study.

Hepatitis B e antigen (HBeAg) is a soluble viral protein in plasma of patients with hepatitis B virus infection. HBeAg loss is an important first stage of viral antigen clearance. We determined the rate and predictors of HBeAg loss in a North American cohort with chronic hepatitis B viral infection (CHB). Among children and adults with CHB and without HIV, HCV or HDV co-infection enrolled in the Hepatitis B Research Network prospective cohort studies, 819 were HBeAg positive at their first assessment (treatment naïve or >24 weeks since treatment). Of these, 577 (200 children, 377 adults) were followed every 24-48 weeks. HBeAg loss was defined as first HBeAg-negative value; sustained HBeAg loss was defined as ≥2 consecutive HBeAg-negative values ≥24 weeks apart. During a median follow-up of 1.8 years, 164 participants experienced HBeAg loss, a rate of 11.4 (95% CI, 9.8-13.3) per 100 person-years. After adjustment for confounders, HBeAg loss rate was significantly higher in males than females, in older than younger individuals, in Whites or Blacks than Asians, in those with genotype A2 or B versus C, and in those with basal core promoter/pre-core mutations versus wild type. Additionally, during follow-up, an ALT flare and a lower quantitative HBsAg, quantitative HBeAg or HBV DNA level predicted higher rates of HBeAg loss. The majority (88%) with HBeAg loss had sustained HBeAg loss. In conclusion, a number of specific demographic, clinical and viral characteristics impacted rate of HBeAg loss and may prove useful in design and interpretation of future therapeutic studies.

Clinical significance of quantitative e antigen in a cohort of hepatitis B virus-infected children and adults in North America.

BACKGROUND: In chronic hepatitis B (CHB) viral infection, e antigen positivity (HBeAg+) is associated with high levels of viral replication and infectivity. Furthermore, HBeAg-positive CHB is associated with a liver disease spectrum ranging from none to severe. AIM: To assess whether the level of circulating HBeAg is associated with different clinical presentations of HBeAg-positive CHB. METHODS: A cross-sectional analysis was conducted among HBV mono-infected participants enrolled in Hepatitis B Research Network (HBRN) cohorts to explore clinical and virological associations with quantitative HBeAg (qHBeAg). RESULTS: Among 763 HBeAg+ participants (56% female; 85% Asian; median age 26 years), multivariable median regression modelling significantly associated qHBeAg with liver injury (inverse qHBeAg association with ALT p<.001 and APRI p<.001), and with both race and age (p=0.01). Among Asians, qHBeAg was inversely related to age; a relationship less clear among Blacks and Whites. Among Asians also, median qHBeAg levels were higher among those infected with HBV genotype C versus B (p<0.001), suggesting causal virologic differences. Across all races, median qHBeAg was higher in women (p=.01). Independent of sex, age, race and HBV genotype, qHBeAg was higher in participants with predominant wild-type versus basal core promoter and/or precore 'stop' viral variants (p<0.001). CONCLUSION: Lower qHBeAg was observed among HBRN participants with the greatest degree of liver injury independent of demographics and HBV genotype. These data support longitudinal studies to examine the role of qHBeAg in modulating the host immune response and predicting the outcomes of chronic HBV infection.

Phase Transition Is Infrequent Among North American Adults With e-Antigen-Negative Chronic Hepatitis B and Low-Level Viremia.

INTRODUCTION: Patients with hepatitis B early antigen (HBeAg)-negative chronic hepatitis B (CHB) and low-level viremia are a heterogeneous group. Identifying those at risk of developing active CHB requiring antiviral therapy is important. In this study, we prospectively characterize incidence rates and predictors of transitioning from inactive to active CHB in a North American adult cohort. METHODS: Participants in the multicenter National Institute of Diabetes and Digestive and Kidney Diseases Hepatitis B Research Network cohort who were HBeAg negative with baseline hepatitis B virus (HBV) DNA ≤ 10,000 IU/mL were included in the study. Cox regression models were used to estimate the proportion of individuals in 3 baseline HBV DNA categories (≤100, 101 to ≤2,000, and 2,001 to ≤10,000 IU/mL) who developed phase transition defined by HBV DNA > 10,000 IU/mL and alanine aminotransferase (ALT) > 2× upper limit of normal or initiated treatment during follow-up. RESULTS: Of 970 participants meeting inclusion criteria, 15% experienced phase transition or initiated treatment over a median follow-up of 4 years: 9% of those with baseline HBV DNA ≤ 100 IU/mL, 14% with HBV DNA 101 to ≤2,000 IU/mL, and 24% with HBV DNA 2,001 to ≤10,000 IU/mL (P < 0.001). The overall rate of phase transition or treatment initiation was 7.6 per 100 person-years: 4.6 in those with HBV DNA ≤ 100 IU/mL, 6.8 in those with HBV DNA 101 to ≤2,000 IU/mL, and 12.2 in those with HBV DNA 2,001 to ≤10,000 IU/mL (P < 0.001). Factors independently associated with higher rate of phase transition or treatment initiation included HBV genotype B or C, higher baseline ALT and HBV DNA levels, lower platelet count, quantitative hepatitis B surface antigen > 1,000 IU/mL, and hyperlipidemia. Only higher ALT, higher HBV DNA, and lower platelets were associated with phase transition when patients starting treatment were censored. DISCUSSION: Most adults in this North American cohort with HBeAg-negative CHB and low-level viremia remained inactive and off treatment over 4 years. Transition from inactive to active CHB is infrequent and predominantly associated with viral rather than host factors.

Age, race and viral genotype are associated with the prevalence of hepatitis B e antigen in children and adults with chronic hepatitis B.

Hepatitis B e antigen (HBeAg) is an important serological marker of hepatitis B virus (HBV) infection and is associated with higher levels of viraemia, increased risk of infectivity to others and increased risk of hepatocellular carcinoma. We analysed HBeAg status in a large cohort of adults and children enrolled in Cohort Studies of the Hepatitis B Research Network, long-term natural history studies of chronic HBV infection. A cross-sectional analysis examined factors associated with HBeAg positivity, including demographic and virologic data, across the age spectrum. Among 2241 enrolled participants who met criteria for this analysis, 825 (37%) were seropositive for HBeAg. The prevalence of HBeAg was lower in those with older age, ranging from 85% among those up to 10 years of age to only 12% among those older than 50 years. In addition to age, both race and HBV genotype were independently associated with HBeAg positivity. There was a significant interaction between age and race; the prevalence of HBeAg was significantly higher among Asians > 10-30 years old vs Whites or Blacks who were >10 to 30 years old and those infected with HBV genotype C. Conversely, the presence of the basal core promoter and precore variants was associated with significantly lower prevalence of HBeAg, even when adjusted for age, race and genotype. These data will provide a better understanding of factors associated with seropositivity for HBeAg and may lead to better strategies for preventing HBV infection and broader indications for antiviral therapy.

Epigenomic signatures in liver and blood of Wilson disease patients include hypermethylation of liver-specific enhancers.

BACKGROUND: Wilson disease (WD) is an autosomal recessive disease caused by mutations in ATP7B encoding a copper transporter. Consequent copper accumulation results in a variable WD clinical phenotype involving hepatic, neurologic, and psychiatric symptoms, without clear genotype-phenotype correlations. The goal of this study was to analyze alterations in DNA methylation at the whole-genome level in liver and blood from patients with WD to investigate epigenomic alterations associated with WD diagnosis and phenotype. We used whole-genome bisulfite sequencing (WGBS) to examine distinct cohorts of WD subjects to determine whether DNA methylation could differentiate patients from healthy subjects and subjects with other liver diseases and distinguish between different WD phenotypes. RESULTS: WGBS analyses in liver identified 969 hypermethylated and 871 hypomethylated differentially methylated regions (DMRs) specifically identifying patients with WD, including 18 regions with genome-wide significance. WD-specific liver DMRs were associated with genes enriched for functions in folate and lipid metabolism and acute inflammatory response and could differentiate early from advanced fibrosis in WD patients. Functional annotation revealed that WD-hypermethylated liver DMRs were enriched in liver-specific enhancers, flanking active liver promoters, and binding sites of liver developmental transcription factors, including Hepatocyte Nuclear Factor 4 alpha (HNF4A), Retinoid X Receptor alpha (RXRA), Forkhead Box A1 (FOXA1), and FOXA2. DMRs associated with WD progression were also identified, including 15 with genome-wide significance. However, WD DMRs in liver were not related to large-scale changes in proportions of liver cell types. DMRs detected in blood differentiated WD patients from healthy and disease control subjects, and distinguished between patients with hepatic and neurologic WD manifestations. WD phenotype DMRs corresponded to genes enriched for functions in mental deterioration, abnormal B cell physiology, and as members of the polycomb repressive complex 1 (PRC1). 44 DMRs associated with WD phenotype tested in a small validation cohort had a predictive value of 0.9. CONCLUSIONS: We identified a disease-mechanism relevant epigenomic signature of WD that reveals new insights into potential biomarkers and treatments for this complex monogenic disease.

Entecavir and Peginterferon Alfa-2a in Adults With Hepatitis B e Antigen-Positive Immune-Tolerant Chronic Hepatitis B Virus Infection.

Monotherapy with interferon or nucleoside analog is generally not recommended during the immune-tolerant (IT) phase of chronic hepatitis B virus (HBV) infection. Recognition that high HBV DNA levels are associated with hepatocellular carcinoma has increased interest in treating HBV in the IT phase. Small pediatric studies reported efficacy with combination nucleoside analog and interferon therapy. The aim of this study was to evaluate the safety and efficacy of the combination of entecavir and peginterferon in adults in the IT phase of chronic HBV infection. Hepatitis B e antigen (HBeAg)-positive adults with HBV DNA > 107 IU/mL and alanine aminotransferase (ALT) ≤ 1.5 times the upper limit of normal (ULN) (male: ≤ 45, female: ≤ 30 U/L) received entecavir 0.5 mg daily for 8 weeks followed by the addition of peginterferon alfa-2a 180 µg/week to entecavir for an additional 40 weeks. The primary endpoint was HBeAg loss and HBV DNA ≤ 1,000 IU/mL 48 weeks after end of treatment (EOT). Among 28 participants from 11 sites, the median age was 37.2 (range: 22-61) years, 54% were male, and 96% were Asian. Nearly all were infected with genotype C (64%) or B (32%). Median baseline HBV DNA was 8.2 log10 IU/mL, and ALT was 0.9 times the ULN. Although one (4%) participant cleared HBeAg, none met the primary endpoint of both HBeAg loss AND HBV DNA ≤ 1,000 IU/mL 48 weeks post-EOT. ALT elevations > 5 times the ULN occurred in eight (29%) participants, and none were associated with icterus. Forty-eight weeks posttreatment, HBV DNA rebounded to baseline levels in all participants, including the participant who lost HBeAg, and ALT values returned to near baseline levels in all but four participants. Conclusion: A lead-in strategy of 8 weeks of entecavir followed by combination peginterferon and entecavir therapy for 40 weeks had limited efficacy in adults in the IT phase of chronic HBV infection and cannot be recommended.

Epigenetic changes of the thioredoxin system in the tx-j mouse model and in patients with Wilson disease.

Wilson disease (WD) is caused by mutations in the copper transporter ATP7B, leading to copper accumulation in the liver and brain. Excess copper inhibits S-adenosyl-L-homocysteine hydrolase, leading to variable WD phenotypes from widespread alterations in DNA methylation and gene expression. Previously, we demonstrated that maternal choline supplementation in the Jackson toxic milk (tx-j) mouse model of WD corrected higher thioredoxin 1 (TNX1) transcript levels in fetal liver. Here, we investigated the effect of maternal choline supplementation on genome-wide DNA methylation patterns in tx-j fetal liver by whole-genome bisulfite sequencing (WGBS). Tx-j Atp7b genotype-dependent differences in DNA methylation were corrected by choline for genes including, but not exclusive to, oxidative stress pathways. To examine phenotypic effects of postnatal choline supplementation, tx-j mice were randomized to one of six treatment groups: with or without maternal and/or continued choline supplementation, and with or without copper chelation with penicillamine (PCA) treatment. Hepatic transcript levels of TXN1 and peroxiredoxin 1 (Prdx1) were significantly higher in mice receiving maternal and continued choline with or without PCA treatment compared to untreated mice. A WGBS comparison of human WD liver and tx-j mouse liver demonstrated a significant overlap of differentially methylated genes associated with ATP7B deficiency. Further, eight genes in the thioredoxin (TXN) pathway were differentially methylated in human WD liver samples. In summary, Atp7b deficiency and choline supplementation have a genome-wide impact, including on TXN system-related genes, in tx-j mice. These findings could explain the variability of WD phenotype and suggest new complementary treatment options for WD.

An OX40/OX40L interaction directs successful immunity to hepatitis B virus.

Depending on age of acquisition, hepatitis B virus (HBV) can induce a cell-mediated immune response that results in either cure or progressive liver injury. In adult-acquired infection, HBV antigens are usually cleared, whereas in infancy-acquired infection, they persist. Individuals infected during infancy therefore represent the majority of patients chronically infected with HBV (CHB). A therapy that can promote viral antigen clearance in most CHB patients has not been developed and would represent a major health care advance and cost mitigator. Using an age-dependent mouse model of HBV clearance and persistence in conjunction with human blood and liver tissue, we studied mechanisms of viral clearance to identify new therapeutic targets. We demonstrate that age-dependent expression of the costimulatory molecule OX40 ligand (OX40L) by hepatic innate immune cells is pivotal in determining HBV immunity, and that treatment with OX40 agonists leads to improved HBV antigen clearance in young mice, as well as increased strength of T cell responses in young mice and adult mice that were exposed to HBV when they were young and developed a CHB serological profile. Similarly, in humans, we show that hepatic OX40L transcript expression is age-dependent and that increased OX40 expression on peripheral CD4+ T cells in adults is associated with HBV clearance. These findings provide new mechanistic understanding of the immune pathways and cells necessary for HBV immunity and identify potential therapeutic targets for resolving CHB.

Durability of Hepatitis B e Antigen Seroconversion in Chronic Hepatitis B Patients Treated with Entecavir or Tenofovir.

INTRODUCTION: Loss of HBeAg and development of anti-HBe (seroconversion) is seen as a milestone and endpoint in the treatment of HBeAg-positive patients with chronic hepatitis B (CHB). Among patients treated with nucleos(t)ide analogs (NA), recurrent viremia is common after discontinuation of therapy. Entecavir (ETV) and tenofovir (TDF) are highly potent NA. The durability of virological response and HBeAg seroconversion in patients treated with these agents is not well studied. METHODS: We retrospectively studied the outcomes of 54 HBeAg-positive CHB patients who were treated with either ETV (n = 30) or TDF (23) or both (n = 1) that achieved virological response and underwent seroconversion and consolidation therapy before cessation of treatment. RESULTS: Only 4 (7%) patients had sustained virological, serological, and biochemical remission. Thirteen patients (24%) continued to have HBV DNA levels below 2000 IU/mL and normal alanine aminotransferase activity (ALT). Thirty-seven patients (69%) developed HBV DNA >2000 IU/mL, with 20 having elevated ALT. Among these 37 patients, 23 (62%) remained HBeAg negative/anti-HBe positive, 12 (32%) became HBeAg positive, and 2 (5%) were HBeAg and anti-HBe negative. Duration of consolidation therapy did not correlate with low versus high level of virological relapse. CONCLUSIONS: Durability of HBeAg seroconversion associated with ETV or TDF was not superior to that reported in patients treated with less potent NA. Our results, aggregated with others, suggest HBeAg seroconversion should not be considered as a treatment endpoint for most HBeAg-positive patients treated with NA. Future updates of treatment guidelines should reconsider HBeAg seroconversion as an endpoint to therapy.

Oral prenylation inhibition with lonafarnib in chronic hepatitis D infection: a proof-of-concept randomised, double-blind, placebo-controlled phase 2A trial.

BACKGROUND: Therapies for chronic hepatitis delta virus (HDV) infection are unsatisfactory. Prenylation is essential for HDV and inhibition abrogates HDV production in experimental models. In a proof-of-concept study, we aimed to assess the effect on HDV RNA levels, safety, and tolerability of the prenylation inhibitor lonafarnib in patients with chronic delta hepatitis. METHODS: In this phase 2A double-blind, randomised, placebo-controlled study, patients aged 18 years or older with chronic HDV infection were randomly assigned (3:1 in group 1 and 2:1 in group 2) to receive lonafarnib 100 mg (group 1) or lonafarnib 200 mg (group 2) twice daily for 28 days with 6 months' follow-up. Participants were randomised by random-number tables blocked in groups of four without stratification. Both groups enrolled six treatment participants and two placebo participants. Group 1 placebo patients received open-label lonafarnib as group 2 participants. The primary therapeutic endpoint was a decrease in HDV RNA viral titre in serum and the primary safety endpoint was the ability to tolerate the drug at the prescribed dose for the full 4-week duration, defined as drug discontinuation due to intolerance or grade 3/4 adverse events. This trial is registered with ClinicalTrials.gov, number NCT01495585. FINDINGS: Between Jan 19, 2012, and April 28, 2014, 14 patients were enrolled, of whom eight were assigned to group 1 and six were assigned to group 2. At day 28, compared with placebo, mean log HDV RNA declines from baseline were -0·73 log IU/mL in group 1 (95% CI 0·17-1·31; p=0·03) and -1·54 log IU/mL in group 2 (1·21-1·93; p<0·0001). Lonafarnib serum concentrations correlated with HDV RNA change (r(2)=0·78, p<0·0001). Model fits show that hepatitis B surface antigen (HBsAg) remained stable after a short pharmacological delay (0·75 days [SE 0·24]), lonafarnib effectiveness in blocking HDV production was greater in group 2 than in group 1 (0·952 [SE 0·06] vs 0·739 [0·05], p<0·001), and the HDV half-life was 1·62 days (0·07). There was no evidence of virological resistance. Adverse events were mainly mild to moderate with group 1 patients experiencing diarrhoea in three patients (50%) and nausea in two patients (33%) and in group 2 with all patients (100%) experiencing nausea, diarrhoea, abdominal bloating, and weight loss greater than 2 kg (mean of 4 kg). No treatment discontinuations occurred in any treatment groups. INTERPRETATION: Treatment of chronic HDV with lonafarnib significantly reduces virus levels. The decline in virus levels significantly correlated with serum drug levels, providing further evidence for the efficacy of prenylation inhibition in chronic HDV. FUNDING: National Institute of Diabetes and Digestive and Kidney Diseases and National Cancer Institute, National Institutes of Health, and Eiger Biopharmaceuticals Inc.

Immune evasion mediated by tumor-derived lactate dehydrogenase induction of NKG2D ligands on myeloid cells in glioblastoma patients.

Myeloid cells are key regulators of the tumor microenvironment, governing local immune responses. Here we report that tumor-infiltrating myeloid cells and circulating monocytes in patients with glioblastoma multiforme (GBM) express ligands for activating the Natural killer group 2, member D (NKG2D) receptor, which cause down-regulation of NKG2D on natural killer (NK) cells. Tumor-infiltrating NK cells isolated from GBM patients fail to lyse NKG2D ligand-expressing tumor cells. We demonstrate that lactate dehydrogenase (LDH) isoform 5 secreted by glioblastoma cells induces NKG2D ligands on monocytes isolated from healthy individuals. Furthermore, sera from GBM patients contain elevated amounts of LDH, which correlate with expression of NKG2D ligands on their autologous circulating monocytes. NKG2D ligands also are present on circulating monocytes isolated from patients with breast, prostate, and hepatitis C virus-induced hepatocellular carcinomas. Together, these findings reveal a previously unidentified immune evasion strategy whereby tumors produce soluble factors that induce NKG2D ligands on myeloid cells, subverting antitumor immune responses.