Jerome Schlue

Hepatitis B surface antigen (HBsAg) levels in the natural history of hepatitis B virus (HBV)-infection: a European perspective.

BACKGROUND & AIMS The quantifiable level of HBsAg has been suggested as a predictor of treatment response in chronic hepatitis B. However, there is limited information on HBsAg levels considering the dynamic natural course of HBV-infection. This study aimed to determine HBsAg levels in the different phases of HBV-infection in European HBsAg-positive patients. METHODS 226 HBV-monoinfected patients, not undergoing antiviral therapy, were analyzed in a cross-sectional study. Patients were categorized according to the phase of HBV-infection: HBeAg(+) immune tolerance phase (IT, n=30), immune clearance phase (IC, n=48), HBeAg(-) low-replicative phase (LR, n=68), HBeAg(-) hepatitis (ENH, n=68), and acute hepatitis B (n=12). HBsAg was quantified and correlated with HBV-DNA, HBV-genotypes and clinical parameters. In addition, 30 LR-patients were followed longitudinally. RESULTS HBsAg levels were higher in IT-patients and IC-patients compared to LR-patients and ENH-patients (4.96/4.37/3.09/3.87-log(10)IU/ml, p<0.001). HBsAg showed a strong correlation with HBV-DNA during acute hepatitis B (R=0.79, p<0.01). Correlation of HBsAg and HBV-DNA was weak or missing when analyzing different phases of persistent HBV-infection separately. However, associations between HBsAg and HBV-DNA were observed in patients infected with HBV-genotype D but not with HBV-genotype A. LR-patients with HBV-reactivation during follow-up (increase of HBV-DNA >2000IU/ml) showed >3-fold higher baseline HBsAg levels with a NPV of 95% for an HBsAg cut-off of 3500IU/ml. CONCLUSIONS HBsAg levels show significant differences during the natural course of HBV-infection and between HBV-genotypes. These findings may have important implications for understanding the natural history of HBV-infection and for using quantitative HBsAg as a diagnostic tool, i.e. as a marker for predicting HBV-reactivation.

Impact of patatin‐like phospholipase‐3 (rs738409 C>G) polymorphism on fibrosis progression and steatosis in chronic hepatitis C

Only 20% of patients with chronic hepatitis C (CHC) will develop cirrhosis, and fibrosis progression remains highly unpredictable. A recent genome‐wide association study identified a genetic variant in the patatin‐like phospholipase‐3 (PNPLA3) gene (rs738409 C>G) associated with steatosis that was further demonstrated to influence severity of fibrosis in nonalcoholic fatty liver disease. The aim of this study was to assess the impact of this polymorphism on histological liver damage and response to antiviral therapy in CHC. We recruited 537 Caucasian CHC patients from three European centers (Brussels, Belgium [n = 229]; Hannover, Germany [n = 171]; Lyon, France [n = 137]); these patients were centrally genotyped for the PNPLA3 (rs738409 C>G) polymorphism. We studied the influence of rs738409 and other variants in the PNPLA3 region on steatosis and fibrosis assessed both in a cross‐sectional and longitudinal manner. Seven other variants previously associated with fibrosis progression were included. Finally, we explored the impact of rs738409 on response to standard antiviral therapy using the interferon lambda 3 (IL28B) [rs12979860 C>T] variant both as a comparator and as a positive control. After adjustment for age, sex, body mass index, alcohol consumption, and diabetes, rs738409 mutant G allele homozygote carriers remained at higher risk for steatosis (odds ratio [OR] 2.55, 95% confidence interval [CI] 1.08‐6.03, P = 0.034), fibrosis (OR 3.13, 95% CI 1.50‐6.51, P = 0.002), and fibrosis progression (OR 2.64, 95% CI 1.22‐5.67, P = 0.013). Conversely, rs738409 was not independently associated with treatment failure (OR 1.07, 95% CI 0.46‐2.49, P = 0.875) and did not influence clinical or biological variables. Conclusion: The PNPLA3 (rs738409 C>G) polymorphism favors steatosis and fibrosis progression in CHC. This polymorphism may represent a valuable genetic predictor and a potential therapeutic target in CHC liver damage. (HEPATOLOGY 2011;)

Hepatitis E virus infection as a cause of graft hepatitis in liver transplant recipients

Hepatitis E virus (HEV) infection induces self‐limiting liver disease in immunocompetent individuals. Cases of chronic hepatitis E have recently been identified in organ transplant recipients. We questioned if chronic hepatitis E plays a role in graft hepatitis after liver transplantation in a low endemic area. Two hundred twenty‐six liver transplant recipients, 129 nontransplanted patients with chronic liver disease, and 108 healthy controls were tested for HEV antibodies. HEV RNA was investigated in all sera from transplanted patients. HEV antibodies were detected in 1 healthy control (1%), 4 patients with chronic liver disease (3%), and 10 liver transplant recipients (4%). Three liver transplant patients also tested positive for HEV RNA. Two of them developed persistent viremia with HEV genotype 3. The patients were anti‐HEV immunoglobulin G–negative and HEV RNA–negative before transplantation and had an episode of acute hepatitis 5 or 7 months after transplantation, which led to advanced liver fibrosis after 22 months in 1 patient. Seroconversion to anti‐HEV occurred not before 4 months after the first detection of HEV RNA. The possibility of reverse zoonotic transmission was experimentally confirmed by the infection of 5 pigs with a patients serum. The pigs showed histological inflammation in the liver, and HEV RNA was detectable in different organs, including muscle. In conclusion, the prevalence of HEV infection in Central European liver transplant recipients is low; however, chronic hepatitis E may occur and needs to be considered in the differential diagnosis of graft hepatitis. The diagnosis of HEV infection should be based on HEV RNA determination in immunosuppressed patients. We suggest that immunocompromised individuals should avoid eating uncooked meat and contact with possibly HEV‐infected animals. Liver Transpl 16:74–82, 2010.

Prospective biopsy-controlled evaluation of cell death biomarkers for prediction of liver fibrosis and nonalcoholic steatohepatitis

Fibrosis and steatosis are major histopathological alterations in chronic liver diseases. Despite various shortcomings, disease severity is generally determined by liver biopsy, emphasizing the need for simple noninvasive methods for assessing disease activity. Because hepatocyte cell death is considered a crucial pathogenic factor, we prospectively evaluated the utility of serum biomarkers of cell death to predict different stages of fibrosis and steatosis in 121 patients with chronic liver disease. We compared the M30 enzyme‐linked immunosorbent assay (ELISA), which detects a caspase‐cleaved cytokeratin‐18 (CK‐18) fragment and thereby apoptotic cell death, with the M65 ELISA, which detects both caspase‐cleaved and uncleaved CK‐18 and thereby overall cell death. Both biomarkers significantly discriminated patients with different fibrosis stages from healthy controls. However, whereas both markers differentiated low or moderate from advanced fibrosis, only the M65 antigen could discriminate even lower stages of fibrosis. The M65 assay also performed better in distinguishing low (≤10%) and higher (>10%) grades of steatosis. In a subgroup of patients, we evaluated the biomarkers for their power to predict nonalcoholic steatohepatitis (NASH). Importantly, both markers accurately differentiated healthy controls or simple steatosis from NASH. However, only serum levels of M65 antigen could differentiate simple steatosis from healthy controls. Conclusion: Cell death biomarkers are potentially useful to predict fibrosis, steatosis, or NASH. Compared with the widely used apoptosis marker M30, the M65 assay had a better diagnostic performance and even differentiated between lower fibrosis stages as well as between healthy individuals and patients with simple steatosis. (HEPATOLOGY 2012)

Chronic Hepatitis E in Heart Transplant Recipients

Chronic courses of hepatitis E virus (HEV) infections have been described in immunosuppressed patients. We aimed to study the role of HEV infections in heart transplant recipients (HTR). 274 HTR were prospectively screened for HEV infection using an anti‐HEV‐IgG ELISA and HEV‐PCR. In addition, 137 patients undergoing cardiac surgery (non‐HTR) and 537 healthy subjects were studied cross‐sectionally. The anti‐HEV‐IgG seroprevalence was 11% in HTR, 7% in non‐HTR and 2% in healthy controls (HTR vs. healthy controls p<0.0001; non‐HTR vs. healthy controls p<0.01). Anti‐HEV tested positive in 4.0% in control cohorts of other immunocompromised patients (n = 474). Four HTR (1.5%) were chronically infected with HEV as shown by HEV‐PCR and all four patients had liver transaminases of >200 IU/L and histological or clinical evidence of advanced liver disease. In three patients ribavirin treatment was successful with a sustained biochemical and virological response while treatment failed in one cirrhotic patient after ribavirin dose reduction. Heart transplant recipients and patients undergoing cardiac surgery have an increased risk for HEV infections. Chronic hepatitis E may explain elevated liver enzymes in heart transplant recipients. Treatment of HEV infection with ribavirin is effective but the optimal dose and duration of ribavirin therapy remains to be determined.

JAK2V617F allele burden discriminates essential thrombocythemia from a subset of prefibrotic-stage primary myelofibrosis

OBJECTIVE Among Philadelphia chromosome-negative myeloproliferative neoplasms (Ph(-) MPN), essential thrombocythemia (ET) and the prefibrotic phase of primary myelofibrosis (PMF) represent two subtypes with considerable overlap. MATERIALS AND METHODS In this study, histopathological classification of 490 MPN cases was correlated with the allelic burden of JAK2(V617F) and MPL(W515L). RESULTS Ph(-) MPN entities largely overlap with regard to JAK2(V617F) and MPL(W515L) allele burden, but ET displayed mutant allele burden <50%. PMF with different stages of myelofibrosis all yielded similar JAK2(V617F) allele burden. At initial presentation one-quarter of prefibrotic PMF cases exhibited an allele burden exceeding 50% (38% median JAK2(V617F) alleles, n=102). In ET, its main differential diagnosis, not a single case was found with >40% JAK2(V617F) alleles (median, 24% JAK2(V617F) alleles; n=90; p<0.001). Increase in JAK2(V617F) alleles during follow-up could not be linked to fibrosis or blastic progression but was related to polycythemic transformation in ET. MPL(W515L) was found in 3% of ET and 8% of PMF, with a significantly higher percentage of mutated alleles in fibrotic than prefibrotic PMF (median, 78% MPL(W515L) alleles; p<0.05). CONCLUSION Histopathological categories ET and prefibrotic PMF correlate with significant differences in mutant allelic burden of JAK2(V617F), but not of MPL(W515L) which, by contrast to JAK2(V617F), shows a higher percentage of mutated alleles in fibrotic than in prefibrotic cases. Thus, for Ph(-) MPN in which ET and prefibrotic PMF represent the most probable diagnoses, a JAK2(V617F) allele burden >50% favors a diagnosis of prefibrotic PMF.

Bone Morphogenetic Proteins Are Overexpressed in the Bone Marrow of Primary Myelofibrosis and Are Apparently Induced by Fibrogenic Cytokines

Primary myelofibrosis (PMF) is a myeloproliferative neoplasia characterized by progressive deposition of extracellular matrix components in the bone marrow. The involvement of members of the bone morphogenetic protein (BMP) family in aberrant bone marrow matrix homeostasis in PMF has not yet been investigated. Therefore, we analyzed expression of BMP1, an activator of latent transforming growth factor beta-1 (TGFbeta-1) and processor of collagen precursors, and other BMPs in bone marrow from PMF patients and controls (n = 95). Expression of BMP1, BMP6, BMP7, and BMP-receptor 2 was significantly increased in advanced stages of myelofibrosis compared with controls (P < or = 0.01), and enhanced levels of BMP6 expression were already evident in prefibrotic stages of PMF. Immunohistochemistry showed that bone marrow stromal cells and megakaryocytes were the major cellular sources of BMP1 protein. Because TGFbeta-1 and basic fibroblast growth factor have been shown to be important in the development of myelofibrosis, we studied the induction of BMPs by these cytokines in cultured fibroblasts. Fibroblasts treated with TGFbeta-1 showed a pronounced up-regulation of BMP6, suggesting that stromal cells may be susceptible to BMP activation by cytokines with a proven role in the pathogenesis of PMF. We conclude that BMP family members may play an important role in the pathogenesis of myelofibrosis in PMF and are apparently induced by cytokines such as TGFbeta-1.

Intrahepatic regulatory T cells in autoimmune hepatitis are associated with treatment response and depleted with current therapies

BACKGROUND & AIMS Autoimmune hepatitis (AIH) is a chronic autoimmune liver disease usually requiring life-long immunosuppression. The mechanisms for disease initiation and chronicity are largely unknown. A contribution of deficient regulatory T cells (Tregs) in the blood was controversially discussed recently. So far investigations in the target organ have been limited to single parameter analysis in untreated AIH. METHODS We retrospectively analysed the pattern of liver infiltrating T, B and regulatory T cells quantitatively with simultaneous multicolour immunofluorescence before (n=45) and under (n=31) therapy in adult AIH type 1 (AIH-1) patients. RESULTS Intrahepatic CD4(+) cells dominate over CD8(+) at diagnosis, but with increasing disease activity the CD4(+)/CD8(+) ratio approached one. While there is no change of Tregs in the blood, they are enriched with effector T cells (Teffs) within the liver of patients with untreated AIH-1 with a constant Treg/Teff ratio. Even more importantly, immunosuppression mostly with steroids and azathioprine caused a disproportional loss of intrahepatic Tregs. Patients reaching biochemical remission had higher intrahepatic Treg/Teff and Treg/B cell ratios compared to patients failing to reach remission. In vitro proliferation of Tregs seemed to be more suppressed by prednisolone than expansion of Teffs. Furthermore, intraportal B cells correlated with serum IgG suggesting an autochthonous intrahepatic IgG production. CONCLUSIONS Intrahepatic Tregs are rather enriched than numerically deficient in untreated AIH-1. The disproportional decrease of intrahepatic Tregs during therapy might explain high relapse rates after discontinuation of immunosuppression. Thus, future therapies increasing intrahepatic immunoregulation might be better suited for long-term control of AIH.

Genetic predisposition and environmental danger signals initiate chronic autoimmune hepatitis driven by CD4+ T cells

Autoimmune hepatitis (AIH) is defined as a chronic liver disease with loss of tolerance against liver tissue eventually leading to cirrhosis if left untreated. 80%‐90% of patients can be treated with a life‐long immunosuppression. Unfortunately, there are strong drug‐related side effects and steroid‐refractory patients. Therefore, there is a need for a model system to investigate the complex immunopathogenesis of this chronic disease and subsequently to develop new therapeutic interventions. We developed a new model of experimental murine AIH (emAIH) by a self‐limited adenoviral infection with the hepatic autoantigen formiminotransferase cyclodeaminase (FTCD). After an initial transient hepatitis there was a chronic evolving AIH, finally leading to portal and lobular fibrosis. We could show that the genetic predisposition provided by the NOD background was essential for creating a fertile field for the development of liver‐specific autoimmunity. However, a strong environmental trigger was additionally necessary to initiate the disease. Besides the break of humoral tolerance, T‐cell tolerance against hepatic self‐antigens was also broken and CD4+ T cells were identified as essential drivers of the disease. As the disease was successfully treated with prednisolone and budesonide, the model will be helpful to develop and test new therapeutic interventions. Conclusion: We developed a new murine AIH model closely resembling AIH in patients that explains the mechanisms of AIH pathophysiology. In addition, emAIH provides options to test therapeutic alternatives for patients not achieving remission, with reduced side effects of chronic nonspecific immunosuppression. (Hepatology 2013;58:718–728)

Increased HEV Seroprevalence in Patients with Autoimmune Hepatitis

Background Hepatitis E virus (HEV) infection takes a clinically silent, self-limited course in the far majority of cases. Chronic hepatitis E has been reported in some cohorts of immunocompromised individuals. The role of HEV infections in patients with autoimmune hepatitis (AIH) is unknown. Methods 969 individuals were tested for anti-HEV antibodies (MP-diagnostics) including 208 patients with AIH, 537 healthy controls, 114 patients with another autoimmune disease, rheumatoid arthritis (RA), and 109 patients with chronic HCV- or HBV-infection (HBV/HCV). Patients with AIH, RA and HBV/HCV were tested for HEV RNA. HEV-specific proliferative T cell responses were investigated using CFSE staining and in vitro stimulation of PBMC with overlapping HEV peptides. Results HEV-antibodies tested more frequently positive in patients with AIH (n = 16; 7.7%) than in healthy controls (n = 11; 2.0%; p = 0.0002), patients with RA (n = 4; 3.5%; p = 0.13) or patients with HBV/HCV infection (n = 2; 2.8%; p = 0.03). HEV-specific T cell responses could be detected in all anti-HEV-positive AIH patients. One AIH patient receiving immunosuppression with cyclosporin and prednisolone and elevated ALT levels had acute hepatitis E but HEV viremia resolved after reducing immunosuppressive medication. None of the RA or HBV/HCV patients tested HEV RNA positive. Conclusions Patients with autoimmune hepatitis but not RA or HBV/HCV patients are more likely to test anti-HEV positive. HEV infection should been ruled out before the diagnosis of AIH is made. Testing for HEV RNA is also recommended in AIH patients not responding to immunosuppressive therapy.