Analysis of antibodies from HCV elite neutralizers identifies genetic determinants of broad neutralization.

The high genetic diversity of hepatitis C virus (HCV) complicates effective vaccine development. We screened a cohort of 435 HCV-infected individuals and found that 2%-5% demonstrated outstanding HCV-neutralizing activity. From four of these patients, we isolated 310 HCV antibodies, including neutralizing antibodies with exceptional breadth and potency. High neutralizing activity was enabled by the use of the VH1-69 heavy-chain gene segment, somatic mutations within CDRH1, and CDRH2 hydrophobicity. Structural and mutational analyses revealed an important role for mutations replacing the serines at positions 30 and 31, as well as the presence of neutral and hydrophobic residues at the tip of the CDRH3. Based on these characteristics, we computationally created a de novo antibody with a fully synthetic VH1-69 heavy chain that efficiently neutralized multiple HCV genotypes. Our findings provide a deep understanding of the generation of broadly HCV-neutralizing antibodies that can guide the design of effective vaccine candidates.

Only repeatedly elevated IgG4 levels in primary sclerosing cholangitis may distinguish a particular patient phenotype.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a chronic liver disease leading to inflammation with scaring and strictures of bile ducts, which can lead to liver cirrhosis. A subtype of PSC characterized by high serum IgG4 (sIgG4) levels has been reported to be associated with poor outcomes, but the exact role and the longitudinal development of sIgG4 levels in PSC progression remains to be clarified. The aim of this study was to investigate if subsequent analysis of sIgG4 levels allows the identification of the PSC phenotype with high sIgG4. METHODS: sIgG4 values were repeatedly analysed in a well-characterized European PSC cohort of 110 individuals. Biochemical parameters, clinical endpoints, death and liver transplantation were compared between PSC subgroups. RESULTS: 12.7% (n = 14) of PSC patients showed increased sIgG4 levels (PSC-IgG4). The values normalized in 57.1% (n = 8; PSC-IgG4norm) during follow-up measurements, whereas the values remained permanently elevated in 42.9% (n = 6; PSC-IgG4const). Serum values of AP and γGT were significantly higher in PSC-IgG4const compared to PSC-IgG4norm at final blood sampling. Furthermore, mean age at PSC diagnosis was markedly lower in PSC-IgG4const compared to PSC-IgG4norm. CONCLUSIONS: This is the first study analyzing longitudinal development of sIgG4 in PSC. Our data indicate that only sequential determination of sIgG4 levels allow to accurately distinguish between the PSC phenotype with high sIgG4 and PSC with low sIgG4.

Role of PAR1 -506 deletion/insertion polymorphism in primary sclerosing cholangitis.

AIM: Primary sclerosing cholangitis (PSC) is a rare cholestatic liver disease characterized by inflammation of the intra- and extrahepatic bile ducts. Pathogenesis of PSC is still enigmatic but is likely to be multifactorial. Recently, we identified an interleukin-6 (IL-6)-dependent signal transducer and activator of transcription 3 (STAT3) activation in CD4+ TH1 and TH17 cells in PSC. The IL-6/STAT3 pathway was shown to be regulated by protease-activated receptor 1 (PAR1) contributing to inflammation. The role of the PAR1 -506 deletion/insertion (Del/Ins) polymorphism in PSC has not yet been investigated. METHODS: Two hundred eighty four PSC patients (200 patients with inflammatory bowel diseases [IBD] and 84 without IBD) and 309 healthy controls were genotyped for PAR1 rs11267092 (-506 Del/Ins -13 bp). Results were correlated with clinical characteristics and transplant-free survival. RESULTS: The frequency of PAR1 -506 Ins allele carriers (Del/Ins and Ins/Ins) was significantly higher in PSC patients (57.0%) compared to healthy controls (39.8%). Furthermore, carriers of PAR1 -506 Ins allele were more likely to have PSC than noncarriers (odds ratio 2.01; 95% confidence interval, 1.45-2.79). Patients with PSC carrying the PAR1 -506 Ins allele showed significantly higher alanine aminotransferase serum levels (p = 0.0357) and a trend toward shorter transplant-free survival time compared to noncarriers (8.9 ± 6.6 years vs. 10.5 ± 7.1 years; p = 0.076). CONCLUSIONS: Our study shows that PAR1 -506 Ins is significantly more frequent in people with PSC. As PAR1 -506 Ins allele carriers tended to have a shorter transplant-free survival, PAR1 might play a role in the development and course of PSC.

Circulating levels of endotrophin and cross-linked type III collagen reflect liver fibrosis in people with HIV.

BACKGROUND AND AIMS: Liver-associated complications still frequently lead to mortality in people with HIV (PWH), even though combined antiretroviral treatment (cART) has significantly improved overall survival. The quantification of circulating collagen fragments released during collagen formation and degradation correlate with the turnover of extracellular matrix (ECM) in liver disease. Here, we analysed the levels of ECM turnover markers PC3X, PRO-C5, and PRO-C6 in PWH and correlated these with hepatic fibrosis and steatosis. METHODS: This monocentre, retrospective study included 141 PWH. Liver stiffness and liver fat content were determined using transient elastography (Fibroscan) with integrated CAP function. Serum levels of formation of cross-linked type III collagen (PC3X), formation of type V collagen (PRO-C5) and formation type VI collagen (PRO-C6), also known as the hormone endotrophin, were measured with ELISA. RESULTS: Twenty-five (17.7%) of 141 PWH had clinical significant fibrosis with liver stiffness ≥ 7.1 kPa, and 62 PWH (44.0%) had steatosis with a CAP value > 238 dB/m. Study participants with fibrosis were older (p = 0.004) and had higher levels of AST (p = 0.037) and lower number of thrombocytes compared to individuals without fibrosis (p = 0.0001). PC3X and PRO-C6 were markedly elevated in PWH with fibrosis. Multivariable cox regression analysis confirmed PC3X as independently associated with hepatic fibrosis. PRO-C5 was significantly elevated in participants with presence of hepatic steatosis. CONCLUSION: Serological levels of cross-linked type III collagen formation and endotrophin were significantly associated with liver fibrosis in PWH receiving cART and thus may be suitable as a non-invasive evaluation of liver fibrosis in HIV disease.

Hepatitis C reference viruses highlight potent antibody responses and diverse viral functional interactions with neutralising antibodies.

OBJECTIVE: Neutralising antibodies are key effectors of infection-induced and vaccine-induced immunity. Quantification of antibodies' breadth and potency is critical for understanding the mechanisms of protection and for prioritisation of vaccines. Here, we used a unique collection of human specimens and HCV strains to develop HCV reference viruses for quantification of neutralising antibodies, and to investigate viral functional diversity. DESIGN: We profiled neutralisation potency of polyclonal immunoglobulins from 104 patients infected with HCV genotype (GT) 1-6 across 13 HCV strains representing five viral GTs. Using metric multidimensional scaling, we plotted HCV neutralisation onto neutralisation maps. We employed K-means clustering to guide virus clustering and selecting representative strains. RESULTS: Viruses differed greatly in neutralisation sensitivity, with J6 (GT2a) being most resistant and SA13 (GT5a) being most sensitive. They mapped to six distinct neutralisation clusters, in part composed of viruses from different GTs. There was no correlation between viral neutralisation and genetic distance, indicating functional neutralisation clustering differs from sequence-based clustering. Calibrating reference viruses representing these clusters against purified antibodies from 496 patients infected by GT1 to GT6 viruses readily identified individuals with extraordinary potent and broadly neutralising antibodies. It revealed comparable antibody cross-neutralisation and diversity between specimens from diverse viral GTs, confirming well-balanced reporting of HCV cross-neutralisation across highly diverse human samples. CONCLUSION: Representative isolates from six neutralisation clusters broadly reconstruct the functional HCV neutralisation space. They enable high resolution profiling of HCV neutralisation and they may reflect viral functional and antigenic properties important to consider in HCV vaccine design.

Primary sclerosing cholangitis with moderately elevated serum-IgG4 - characterization and outcome of a distinct variant phenotype.

BACKGROUND AND AIMS: Immunoglobulin G4-associated cholangitis (IAC) is characterized by distinctly elevated immunoglobulin G4 in serum (sIgG4) and responds well to corticosteroid therapy. Primary Sclerosing Cholangitis (PSC) is a progressive liver disease without causal treatment options usually not responding to immunosuppression. Increased serum levels of sIgG4 in patients with PSC, that do not meet criteria of IAC, have been reported in 10%-25%. Therefore, we aimed to characterize this subgroup of patients in a retrospective, multicenter study. METHODS: sIgG4 values of 289 patients with PSC from three German university hospitals were analysed. Patients with elevated sIgG4 levels were identified and further characterized by clinical and biochemical parameters and by cholangiographic presentation. Clinical endpoints, death and liver transplantation were compared between groups. Parameters associated with outcome were identified with Cox regression analysis. RESULTS: 14.5% of patients with PSC showed increased sIgG4 levels (PSC-IgG4), presented with significantly higher (P < .02) albumin, aspartate-aminotransferase, bilirubin and alkaline phosphatase and had a significant lower prevalence of a concomitant autoimmune hepatitis (P = .025). Cholangiogram obtained via ERC showed extrahepatic dominant strictures more often in the PSC-IgG4 subgroup (P = .047). The disease severity models Amsterdam-Oxford-Score (P = .018) and Mayo-Risk-Score (P = .025) predicted lower survival rates for the PSC-IgG4 subgroup. Transplant-free survival after first diagnosis of PSC was shorter in patients with elevated sIgG4 (11.6 vs 15.1 years, P = .001). CONCLUSION: Patients with PSC and elevated sIgG4 should be considered as a distinct subgroup, characterized by different clinical and cholangiographical features and are associated with an inferior outcome.

Diagnostic value of magnetic resonance parametric mapping for non-invasive assessment of liver fibrosis in patients with primary sclerosing cholangitis.

BACKGROUND: Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease, characterized by bile duct inflammation and destruction, leading to biliary fibrosis and cirrhosis. The purpose of this study was to investigate the utility of T1 and T2 mapping parameters, including extracellular volume fraction (ECV) for non-invasive assessment of fibrosis severity in patients with PSC. METHODS: In this prospective study, patients with PSC diagnosis were consecutively enrolled from January 2019 to July 2020 and underwent liver MRI. Besides morphological sequences, MR elastography (MRE), and T1 and T2 mapping were performed. ECV was calculated from T1 relaxation times. The presence of significant fibrosis (≥ F2) was defined as MRE-derived liver stiffness ≥ 3.66 kPa and used as the reference standard, against which the diagnostic performance of MRI mapping parameters was tested. Student t test, ROC analysis and Pearson correlation were used for statistical analysis. RESULTS: 32 patients with PSC (age range 19-77 years) were analyzed. Both, hepatic native T1 (r = 0.66; P < 0.001) and ECV (r = 0.69; P < 0.001) correlated with MRE-derived liver stiffness. To diagnose significant fibrosis (≥ F2), ECV revealed a sensitivity of 84.2% (95% confidence interval (CI) 62.4-94.5%) and a specificity of 84.6% (CI 57.8-95.7%); hepatic native T1 revealed a sensitivity of 52.6% (CI 31.7-72.7%) and a specificity of 100.0% (CI 77.2-100.0%). Hepatic ECV (area under the curve (AUC) 0.858) and native T1 (AUC 0.711) had an equal or higher diagnostic performance for the assessment of significant fibrosis compared to serologic fibrosis scores (APRI (AUC 0.787), FIB-4 (AUC 0.588), AAR (0.570)). CONCLUSIONS: Hepatic T1 and ECV can diagnose significant fibrosis in patients with PSC. Quantitative mapping has the potential to be a new non-invasive biomarker for liver fibrosis assessment and quantification in PSC patients.

Functional and immunogenic characterization of diverse HCV glycoprotein E2 variants.

BACKGROUND & AIMS: Induction of cross-reactive antibodies targeting conserved epitopes of the envelope proteins E1E2 is a key requirement for an hepatitis C virus vaccine. Conserved epitopes like the viral CD81-binding site are targeted by rare broadly neutralizing antibodies. However, these viral segments are occluded by variable regions and glycans. We aimed to identify antigens exposing conserved epitopes and to characterize their immunogenicity. METHODS: We created hepatitis C virus variants with mutated glycosylation sites and/or hypervariable region 1 (HVR1). Exposure of the CD81 binding site and conserved epitopes was quantified by soluble CD81 and antibody interaction and neutralization assays. E2 or E1-E2 heterodimers with mutations causing epitope exposure were used to immunize mice. Vaccine-induced antibodies were examined and compared with patient-derived antibodies. RESULTS: Mutant viruses bound soluble CD81 and antibodies targeting the CD81 binding site with enhanced efficacy. Mice immunized with E2 or E1E2 heterodimers incorporating these modifications mounted strong, cross-binding, and non-interfering antibodies. E2-induced antibodies neutralized the autologous virus but they were not cross-neutralizing. CONCLUSIONS: Viruses lacking the HVR1 and selected glycosylation sites expose the CD81 binding site and cross-neutralization antibody epitopes. Recombinant E2 proteins carrying these modifications induce strong cross-binding but not cross-neutralizing antibodies. LAY SUMMARY: Conserved viral epitopes can be made considerably more accessible for binding of potently neutralizing antibodies by deletion of hypervariable region 1 and selected glycosylation sites. Recombinant E2 proteins carrying these mutations are unable to elicit cross-neutralizing antibodies suggesting that exposure of conserved epitopes is not sufficient to focus antibody responses on production of cross-neutralizing antibodies.

Role of regulatory T cells and checkpoint inhibition in hepatocellular carcinoma.

Immune checkpoint inhibition suggests promising progress for the treatment of advanced hepatocellular carcinoma (HCC). However, the underlying cellular mechanisms remain unclear because liver cancer cells apparently do not upregulate inhibitory checkpoint molecules. Here, we analysed whether regulatory T cells (Tregs) can alternatively trigger checkpoint inhibition pathways in HCC. Using flow cytometry we analysed expression of checkpoint molecules (PD-1, PD-L1, CTLA-4, GITR, Tim-3) on peripheral CD4+CD25+Foxp3+ Tregs and their secretion of inhibitory mediators (IL-10, IL-35, TGF-beta, galectin-9) in 116 individuals (50 patients with HCC, 41 non-tumour bearing liver disease controls, 25 healthy controls). Functional activity of Tregs on T effector cells (IFN-gamma production, cytotoxicity) was characterized in vitro using a lectin-dependent cellular cytotoxicity (LDCC) assay against checkpoint inhibitor-negative P815 target cells. Unlike liver patients without malignancy and healthy controls, the frequency of checkpoint inhibitor-positive Tregs inversely correlated to age of patients with HCC (PD-L1, p = 0.0080; CTLA-4, p = 0.0029) and corresponded to enhanced numbers of Tregs producing IL-10 and IL-35 (p < 0.05 each). Tregs inhibited IFN-gamma secretion and cytotoxicity of CD8+ T cells when added to LDCC against P815 cells. Treg-induced inhibition of IFN-gamma secretion could be partially blocked by neutralizing PD-1 and PD-L1 antibodies specifically in HCC patients. In HCC peripheral Tregs upregulate checkpoint inhibitors and contribute to systemic immune dysfunction and antitumoural activity by several inhibitory pathways, presumably facilitating tumour development at young age. Blocking PD-L1/PD-1 interactions in vitro selectively interfered with inhibitory Treg -T effector cell interactions in the patients with HCC and resulted in improved antitumoural activity also against checkpoint inhibitor-negative tumour cells.

Fibroblast growth factor 21 is independently associated with severe hepatic steatosis in non-obese HIV-infected patients.

BACKGROUND: Severe hepatic steatosis shows a high prevalence and contributes to morbidity and mortality in human immunodeficiency virus (HIV) infected patients. Known risk factors include obesity, dyslipidaemia and features of metabolic syndrome. Fibroblast growth factor 21 (FGF-21) is involved with hepatic glucose and lipid metabolism. This study aimed to evaluate FGF-21 as a biomarker for severe hepatic steatosis in non-obese HIV-infected patients. METHODS: This is a prospective, cross-sectional, monocentric study including HIV-infected out-patients. Hepatic steatosis was measured via controlled attenuation parameter (CAP) using FibroScan 502 touch (ECHOSENS, France). Severe hepatic steatosis was defined at CAP ≥ 253 dB/m. Peripheral blood samples were collected and plasma was analysed for FGF-21. Demographic and clinical characteristics were collected from patient's health records. RESULTS: In total, 73 non-obese HIV-monoinfected patients were included in this study. Prevalence of severe hepatic steatosis was 41%. Patients with severe hepatic steatosis showed significantly higher levels of FGF-21. Univariate analysis revealed FGF-21, BMI, hyperlipidaemia, ALT levels and arterial hypertension as significant, while multivariate analysis showed only FGF-21, arterial hypertension and ALT levels as significant independent risk factors for severe hepatic steatosis. CONCLUSION: This study presents FGF-21 as an independent and stronger predictor of severe hepatic steatosis than blood lipids in HIV-infected patients. Moreover, arterial hypertension and ALT levels predict severe steatosis even in non-obese HIV-monoinfected patients. Furthermore, this study supports existing metabolic risk factors and expands them to non-obese HIV-infected patients.

Increased peripheral CD4+ regulatory T cells persist after successful direct-acting antiviral treatment of chronic hepatitis C.

BACKGROUND & AIMS: CD4+ regulatory T cells (Tregs) expand during chronic hepatitis C virus (HCV) infection, inhibit antiviral immunity and promote fibrosis. Direct-acting antiviral agents (DAA) have revolutionized HCV therapy. However, it is unclear if Tregs are normalized after DAA-induced HCV elimination. METHODS: We analyzed Tregs before (baseline), at end of therapy (EOT), 12 and 24weeks (SVR12, SVR24) and long-term (51±14weeks) after EOT in 26 genotype-1-infected patients who were successfully treated with sofosbuvir (SOF) plus interferon (IFN)/ribavirin (n=12) and IFN-free DAA regimens (SOF plus daclatasvir or simeprevir; n=14). Frequency, phenotype and suppressor function of peripheral Foxp3+ CD25+ CD4+ T cells were studied by multi-color flow cytometry and co-culture inhibition assays. RESULTS: Frequencies and activation status of Foxp3+ CD25+ CD4+ T cells remained elevated above those of normal controls in both treatment groups even long-term after HCV elimination. Co-culture assays indicated a dose-response relationship for functional inhibition of autologous CD4+ effector T cells and confirmed that activation of Tregs remained largely unchanged over the observation period. Unlike IFN-free regimens, SOF plus IFN/ribavirin induced a transiently increased frequency of Foxp3+ CD25+ CD4+ T cells at EOT (5.0% at baseline to 6.1% at EOT; p=0.001). These Foxp3+ CD25+ CD4+ T cells co-expressed the activation markers glycoprotein A repetitions predominant (GARP; p=0.012) and tumor necrosis factor receptor superfamily, member 4 (OX-40; p=0.001) but showed unchanged in vitro inhibitory activity. CONCLUSION: Although IFN-based DAA therapy induced transient expansion of activated Foxp3+ CD25+ CD4+ T cells, neither IFN-based nor IFN-free DAA regimens normalized frequencies and activation status of Tregs one year after viral elimination. Persistence of immunosuppressive Tregs may thus contribute to complications of liver disease even long-term after HCV cure. LAY SUMMARY: In chronic hepatitis C virus (HCV) infection, CD4+ regulatory T cells (Tregs) can reduce antiviral immune responses, promote liver fibrosis and may increase the risk for liver cancer, because they gradually expand during disease. Modern direct-acting antiviral agents (DAA) can "cure" hepatitis C in almost all treated patients. However, our study shows that DAA do not normalize the increased frequency and activation status of Tregs even long-term after HCV elimination. Tregs may persistently modulate functions of the immune system even after "cure" of hepatitis C.

Circulating microRNAs as a marker for liver injury in human immunodeficiency virus patients.

UNLABELLED: Human immunodeficiency virus (HIV) and hepatitis virus coinfection amplify and accelerate hepatic injury. MicroRNAs (miRNAs) are small regulatory RNAs suggested as biomarkers for liver injury. We analyzed the circulating levels of miRNAs in HIV patients with regard to the extent and etiology of liver injury. Total RNA was extracted from 335 serum samples of HIV patients and 22 healthy control participants using Qiazol. Comprehensive polymerase chain reaction (PCR) array analyses (768 miRNA) were performed in serum samples of eight HIV, eight HIV/HCV (hepatitis C virus), six HCV patients, and three healthy controls. Reverse transcription (RT)-PCR measured levels of miRNA-122, miRNA-22, and miRNA-34a in serum samples of 335 patients and 19 healthy control participants. Liver injury and fibrosis in these patients were defined using aspartate aminotransferase (AST) levels, fibrosis-4 (FIB-4) index and AST-to-platelet ratio index (APRI) score. The miRNA pattern of HIV/HCV samples showed altered expression of 57 and 33 miRNA compared to HCV and HIV infection, respectively. miRNA-122, miRNA-22, and miRNA-34a were highly up-regulated in HIV/HCV patients. Analyzing the entire cohort, these miRNAs were correlated with liver function tests and were independent predictors of liver injury (AST >2 × ULN). miRNA-122 and miRNA-22 were associated with relevant fibrosis (FIB-4 >1.45; APRI >1). Circulating levels of miRNA-122 were independent predictors for relevant fibrosis in HIV patients. Interestingly, miRNA-122 and miRNA-34a levels were higher in HIV/HCV patients, miRNA-22 levels were highest in HIV/HBV patients, and circulating levels of miRNA-34a correlated positively with illicit drug use and ethanol consumption. CONCLUSION: Circulating miRNA-122, miRNA-22, and miRNA-34a correlates with the etiology of liver injury in HIV patients. These biomarkers not only mirror different mechanisms of hepatic injury, but also are independent predictors of liver injury in HIV patients.

Primary sclerosing cholangitis with IgG4-positive plasma cells in bile duct biopsies - Frequency and characterization.

OBJECTIVES: Patients diagnosed with primary sclerosing cholangitis (PSC) but with characteristics of immunoglobulin G4 (IgG4)-associated cholangitis (IAC) have been described. IAC often presents with biliary IgG4-positive plasma cell (IgG4+ PC) infiltration and responds to corticosteroids. In PSC, the frequencies or implications of biliary IgG4+ PC are unknown. We aimed to characterize the phenomenon of biliary IgG4+ PC in patients with an established PSC diagnosis. METHODS: Bile duct biopsies from 191 surveillance or therapeutic endoscopic retrograde cholangiography of 58 PSC patients were retrospectively analyzed for IgG4+ PC infiltration. Patients with ≥10 IgG4+ PC per high-power field (HPF) were identified and characterized by clinical parameters, including serum IgG4 and cholangiographic presentations. RESULTS: Altogether 39.7% of the PSC patients showed ≥10 IgG4+ PC/HPF in bile duct biopsies. Patients with biliary IgG4+ PC infiltration were significantly younger at diagnosis of PSC (P = 0.023). There was no association between biliary IgG4+ PC infiltration and transplant-free survival (P = 0.618). Patients with IgG4+ PC infiltration in bile duct biopsies showed significantly higher baseline (P = 0.002) and maximum (P = 0.001) serum IgG4 compared to those without. Biliary IgG4+ PC infiltration was associated with high-grade bile duct strictures (P = 0.05). IgG4-positive plasma cell infiltrations were found multifocally in 72.7% of this subgroup of PSC patients. CONCLUSIONS: IgG4+ PC ≥10/HPF can be found abundantly in bile duct biopsies in PSC. Histological findings correlated with serum IgG4, age, and high-grade bile duct strictures. IgG4+ PC was located multifocally, hinting at a systemic biliary phenotype.

STAT activation in regulatory CD4+ T cells of patients with primary sclerosing cholangitis.

INTRODUCTION: Regulatory CD4+ T cells (Tregs) are pivotal for inhibition of autoimmunity. Primary sclerosing cholangitis (PSC) is an autoimmune cholestatic liver disease of unknown etiology where contribution of Tregs is still unclear. Activation of the JAK-STAT pathway critically modifies functions of Tregs. In PSC, we studied activation of STAT proteins and Treg functions in response to cytokines. METHODS: In 51 patients with PSC, 10 disease controls (chronic replicative hepatitis C), and 36 healthy controls we analyzed frequencies of Foxp3+CD25+CD127lowCD4+ Tregs, their expression of ectonucleotidase CD39, and cytokine-induced phosphorylation of STAT1, 3, 5, and 6 using phospho-flow cytometry. In parallel, we measured cytokines IFN-gamma, interleukin (IL)-6, IL-2, and IL-4 in serum via bead-based immunoassays. RESULTS: In patients with PSC, ex vivo frequencies of peripheral Tregs and their expression of CD39 were significantly reduced (p < .05 each). Furthermore, serum levels of IFN-gamma, IL-6, IL-2, and IL-4 were markedly higher in PSC (p < .05 each). Unlike activation of STAT1, STAT5, and STAT6, IL-6 induced increased phosphorylation of STAT3 in Tregs of PSC-patients (p = .0434). Finally, STAT3 activation in Tregs correlated with leukocyte counts. CONCLUSIONS: In PSC, we observed enhanced STAT3 responsiveness of CD4+ Tregs together with reduced CD39 expression probably reflecting inflammatory activity of the disease.

Increased type-I interferon level is associated with liver damage and fibrosis in primary sclerosing cholangitis.

BACKGROUND: The level of type-I interferons (IFNs) in primary sclerosing cholangitis (PSC) was investigated to evaluate its association with disease activity and progression. METHODS: Bioactive type-I IFNs were evaluated in a murine model of PSC and human patients' sera using a cell-based reporter assay and ELISA techniques. In total, 57 healthy participants, 71 PSC, and 38 patients with primary biliary cholangitis were enrolled in this study. RESULTS: Bioactive type-I IFNs were elevated in the liver and serum of multidrug resistance protein 2-deficient animals and showed a correlation with the presence of CD45+ immune cells and serum alanine transaminase levels. Concordantly, bioactive type-I IFNs were elevated in the sera of patients with PSC as compared to healthy controls (sensitivity of 84.51%, specificity of 63.16%, and AUROC value of 0.8267). Bioactive IFNs highly correlated with alkaline phosphatase (r=0.4179, p<0.001), alanine transaminase (r=0.4704, p<0.0001), and gamma-glutamyl transpeptidase activities (r=0.6629, p<0.0001) but not with serum bilirubin. In addition, patients with PSC with advanced fibrosis demonstrated significantly higher type-I IFN values. Among the type-I IFN subtypes IFNα, ß and IFNω could be detected in patients with PSC with IFNω showing the highest concentration among the subtypes and being the most abundant among patients with PSC. CONCLUSIONS: The selectively elevated bioactive type-I IFNs specifically the dominating IFNω could suggest a novel inflammatory pathway that might also have a hitherto unrecognized role in the pathomechanism of PSC.

Probabilities of developing HIV-1 bNAb sequence features in uninfected and chronically infected individuals.

HIV-1 broadly neutralizing antibodies (bNAbs) are able to suppress viremia and prevent infection. Their induction by vaccination is therefore a major goal. However, in contrast to antibodies that neutralize other pathogens, HIV-1-specific bNAbs frequently carry uncommon molecular characteristics that might prevent their induction. Here, we perform unbiased sequence analyses of B cell receptor repertoires from 57 uninfected and 46 chronically HIV-1- or HCV-infected individuals and learn probabilistic models to predict the likelihood of bNAb development. We formally show that lower probabilities for bNAbs are predictive of higher HIV-1 neutralization activity. Moreover, ranking bNAbs by their probabilities allows to identify highly potent antibodies with superior generation probabilities as preferential targets for vaccination approaches. Importantly, we find equal bNAb probabilities across infected and uninfected individuals. This implies that chronic infection is not a prerequisite for the generation of bNAbs, fostering the hope that HIV-1 vaccines can induce bNAb development in uninfected people.

Myosteatosis independently predicts transplant-free survival in patients with primary sclerosing cholangitis.

BACKGROUND: Primary Sclerosing Cholangitis (PSC) is a progressive cholestatic liver disease with liver transplantation (LT) as the only curative therapy. Some regions use body-weight-loss as standard-exception criteria for organ allocation but data on the impact of body composition on survival of patients with PSC is scarce. METHODS: Abdominal MRI of PSC patients were quantitatively analyzed for intramuscular fat fraction (IMFF) as surrogate of myosteatosis. Clinical and laboratory data were retrieved from patient records. Primary outcome was transplant-free survival (TFS). RESULTS: 116 PSC patients were included. Median age was 38 (18-71) years with 74 (64%) male patients. 15 (13%) patients had significant weigh loss. IMFF was significantly associated with survival. Multivariate regression analysis showed IMFF ≥ 15% as independent predictor for TFS (p = 0.032, HR 3.215 CI 1.104-9.366), but not significant weight loss (p = 0.618). CONCLUSION: IMFF is independently associated with TFS in patients with PSC and may identify patients with more urgent need for LT. NCT03584204.

IL-6-Dependent STAT3 Activation and Induction of Proinflammatory Cytokines in Primary Sclerosing Cholangitis.

INTRODUCTION: Primary sclerosing cholangitis (PSC) is a rare cholestatic liver disease with periductal inflammation and fibrosis. Genetic studies suggest inflammatory cytokines and IL-6-dependent activation of transcription factor STAT3 as pivotal steps in PSC pathogenesis. However, details of inflammatory regulation remain unclear. METHODS: We recruited 50 patients with PSC (36 with inflammatory bowel disease, 14 without inflammatory bowel disease), 12 patients with autoimmune hepatitis, and 36 healthy controls to measure cytokines in the serum, bile, and immune cell supernatant using bead-based immunoassays and flow cytometry and immunohistochemistry to analyze phosphorylation of STATs in immune cells. Finally, we analyzed cytokines and STAT3 phosphorylation of T cells in the presence of JAK1/2 inhibitors. RESULTS: In PSC, IL-6 specifically triggered phosphorylation of STAT3 in CD4 + T cells and lead to enhanced production of interferon (IFN) gamma and interleukin (IL)-17A. Phospho-STAT3-positive CD4 + T cells correlated with systemic inflammation (C-reactive protein serum levels). Combination of immunohistology and flow cytometry indicated that phospho-STAT3-positive cells were enriched in the peribiliary liver stroma and represented CD4 + T cells with prominent production of IFN gamma and IL-17A. JAK1/2 inhibitors blocked STAT3 phosphorylation and production of IFN gamma and IL-6, whereas IL-17A was apparently resistant to this inhibition. DISCUSSION: Our results demonstrate systemic and local activation of the IL-6/STAT3 pathway in PSC. Resistance of IL-17A to STAT3-targeted inhibition points to a more complex immune dysregulation beyond STAT3 activation.