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Abstract The incidence of hepatocellular carcinoma (HCC) in non-cirrhotic livers is rising significantly, but clear risk factors for screening remain elusive. This study sought to characterize non-cirrhotic HCC etiologies. HCC cases from 2009 to 2020 in a Dutch referral center were examined, revealing 371 out of 1654 cases (22%) as non-cirrhotic. Notably, the incidence of non-cirrhotic HCC increased by 61% in the time frame between 2009 and 2020. Interestingly 39% of non-cirrhotic HCC cases had cryptogenic origins. Cryptogenic non-cirrhotic HCC exhibited similarities with non-cirrhotic NAFLD HCC, but displayed advanced tumor stages, lower surgical rates, and a more frequent presence of symptoms, which substantiated in poor survival rates. Advanced cryptogenic non-cirrhotic HCC stages exhibited elevated serum interleukin-6 levels compared to non-cirrhotic HCC with defined etiologies. Comparative analysis encompassing cryptogenic and NAFLD non-cirrhotic HCC cohorts and controls unveiled comparable circulating immune biomarker profiles and PNPLA3 polymorphisms. To conclude, the primary etiology of non-cirrhotic HCC in our cohort has not defined risk factors. This cryptogenic variant exhibits distinct traits, such as advanced tumors and increased symptoms, and most resemble burned-out NAFLD. Understanding this HCC variant is crucial for improving screening and management strategies.

Introduction and Objectives: Tolloid like protein 1 (TLL1) rs17047200 has been reported to be associated with HCC development and liver fibrosis. However, to our knowledge, no studies have been performed on Latin Americans and comparative differences between TLL1 rs17047200 in HCC patients from Latin America and Europe are undefined. Materials and Methods: Cross-sectional analysis was performed on Latin American and European individuals. We analyzed TLL1 rs17047200 on DNA from 1194 individuals, including 420 patients with HCC (86.0 % cirrhotics) and 774 without HCC (65.9 % cirrhotics). Results: TLL1 rs17047200 genotype AT/TT was not associated with HCC development in Latin Americans (OR: 0.699, 95 %CI 0.456-1.072, p = 0.101) or Europeans (OR: 0.736, 95 %CI 0.447-1.211, p = 0.228). TLL1 AT/TT was not correlated with fibrosis stages among metabolic dysfunction-associated steatotic liver disease (MASLD) patients from Latin America (OR: 0.975, 95 %CI 0.496-1.918, p = 0.941). Among Europeans, alcohol-related HCC had lower TLL1 AT/TT frequencies than cirrhosis (18.3 % versus 42.3 %, OR: 0.273, 95 %CI 0.096-0.773, p = 0.015). Conclusions: We found no evidence that the TLL1 rs17047200 AT/TT genotype is a risk factor for HCC development in Latin Americans or Europeans. A larger study integrating ethnic and etiology backgrounds is needed to determine the importance of the TLL1 SNP in HCC development.

Background & Aims: HBsAg secretion may impact immune responses to chronic HBV infection. Thus, therapeutic approaches to suppress HBsAg production are being investigated. Our study aims to examine the immunomodulatory effects of high and low levels of circulating HBsAg and thereby improve our understanding of anti-HBV immunity. Methods: An optimized 10x Genomics single-cell RNA sequencing workflow was applied to blood samples and liver fine-needle aspirates from 18 patients undergoing tenofovir/entecavir (NUC) treatment for chronic HBV infection. They were categorized based on their HBsAg levels: high (920-12,447 IU/ml) or low (1-100 IU/ml). Cluster frequencies, differential gene expression, and phenotypes were analyzed. Results: In the blood of HBV-infected patients on NUC, the proportion of KLRC2+ “adaptive” natural killer (NK) cells was significantly lower in the HBsAg-high group and, remarkably, both KLRC2+ NK and KLRG1+ CD8 T cells display enrichment of lymphocyte activation-associated gene sets in the HBsAg-low group. High levels of HBsAg were associated with mild immune activation in the liver. However, no suppression of liver-resident CXCR6+ NCAM1+ NK or CXCR6+ CD69+ CD8 T cells was detected, while memory B cells showed signs of activation in both the blood and liver. Conclusions: Among NUC-treated patients, we observed a minimal impact of HBsAg on leukocyte populations in the blood and liver. However, for the first time, we found that HBsAg has distinct effects, restricted to NK-, CD8 T-, and memory B-cell subsets, in the blood and liver. Our findings are highly relevant for current clinical studies evaluating treatment strategies aimed at suppressing HBsAg production and reinvigorating immunity to HBV. Impact and implications: This study provides unique insight into the impact of HBsAg on gene expression levels of immune cell subsets in the blood and liver, particularly in the context of NUC-treated chronic HBV infection. It holds significant relevance for current and future clinical studies evaluating treatment strategies aimed at suppressing HBsAg production and reinvigorating immunity to HBV. Our findings raise questions about the effectiveness of such treatment strategies and challenge the previously hypothesized immunomodulatory effects of HBsAg on immune responses against HBV.

Background & Aims: Pretreatment predictors of finite nucleo(s)tide analogue (NUC) therapy remain elusive. We studied the association between pretreatment HBV DNA levels and outcomes after therapy cessation. Methods: Patients with chronic hepatitis B who were HBeAg negative at the start of NUC treatment were enrolled from sites in Asia and Europe. We studied the association between pretreatment HBV DNA levels and (1) clinical relapse (defined as HBV DNA >2,000 IU/ml + alanine aminotransferase >2 × the upper limit of normal or retreatment) and (2) HBsAg loss after NUC withdrawal. Results: We enrolled 757 patients, 88% Asian, 57% treated with entecavir, with a median duration of treatment of 159 (IQR 156–262) weeks. Mean pretreatment HBV DNA levels were 5.70 (SD 1.5) log IU/ml and were low (20,000 IU/ml) in 607 (80%). The cumulative risk of clinical relapse at 144 weeks after therapy cessation was 22% among patients with pretreatment HBV DNA levels 20,000 IU/ml, whereas the cumulative probabilities of HBsAg loss were 17.5% vs. 5% (p

Introduction and Objectives: New peripheral markers are needed for the early detection of hepatocellular carcinoma (HCC). Currently, the only accepted biomarker is alpha-fetoprotein (AFP) which by itself is suboptimal for early HCC detection. We investigated peripheral immune markers to detect HCC in a large cohort of South American patients and a sub-group of viral hepatitis-related HCC. Materials and Methods: Through the ESCALON network, we prospectively evaluated 127 individuals with HCC and 113 cirrhotic controls from 3 countries in South America (Argentina, Brazil and Ecuador). 42% of HCC cases were related to viral hepatitis B or C. Blood samples were analyzed for 37 unique interleukins, chemokines and growth factors using a multiplex Bio-Rad platform. We used leave-one-out cross-validation (LOOCV) to compute a ROC curve. Results: Median age for HCC patients was 68 y/o and for controls 62 y/o. 70% of cases and 55% of controls were males. 55% of HCCs were under 5cm in diameter. The most common causes of HCC were viral hepatitis (42%) and NAFLD (23%). Twenty-two markers showed a significant difference between cases and controls. Three markers (IL-12p40, Beta-NGF and Gro-alpha) were exclusively dysregulated in viral hepatitis related HCC compared to other HCCs. From all causes of HCCs, we identified five cytokines (MIP-3a, MIG, CCL-25, MDC, and HGF) that were differentially regulated in HCCs compared to cirrhosis controls. ROC analysis of the top-5 markers in HCC cases exclusively related to viral hepatitis showed an AUROC of 0.816 (CI 0.783-0.886). The same panel applied to HCC

Introduction and Objectives: Hepatocellular carcinoma (HCC) is the third cause of cancer-related death worldwide. Assessment of genetic components has been used to better stratify those at risk. However, most studies have been performed in Asian or Caucasian populations. Tolloid-like protein 1 (TLL1) is one such SNP that has been shown to increase risk in hepatitis C virus (HCV)-associated HCC. We evaluated the risk association of TLL1 in a South American cohort. Materials and Methods: This is a cross-sectional analysis performed in South Americans with HCC as well as cirrhotic controls through the ESCALON network. We analyzed 120 HCC blood samples and 293 cirrhotic controls from Argentina, Chile, Brazil, Colombia, Ecuador and Peru. The pathogenic variant of TLL1 (rs1704200) was evaluated using TaqMan-genotyping assay. Multiple logistic regression was used to establish the association between TLL1 and HCC. Results: The median age of HCC patients was 68 years (IQR 62-72) and of cirrhotics 64 years IQR 68-70). The most common underlying liver disease in both groups was Non-alcoholic fatty liver disease (NAFLD) at 58% and 59%, respectively. The proportion of individuals who developed HCC with a TLL1 pathogenic variant (AT/TT) was 18.2% in the South American cohort. The calculated Odds-Ratio (OR) for HCC among South Americans with the TLL1 variant was 0.69 (CI 0.37-1.29), suggesting a non-significant decrease odds for HCC. Interestingly, different results were found when examining HCV-associated HCC (11% of the cases and 6% of controls). The OR for HCV-associated HCC in Latin Americans was 2.07 (CI 0.93-4.58), suggesting a non-significant increased odd of being diagnosed with HCC in South Americans with the variant. Conclusions: TLL1 mutations do not seem to associate with HCC development in South American patients with liver disease. However, preliminary results show that the presence of TLL1 SNP could confer an increased risk for HCC in South Americans with HCV infection.

Introduction and Objectives: Hepatocellular carcinoma (HCC) has a strong genetic component and single nucleotide polymorphisms (SNPs) have been consistently associated with HCC risk. Genetic variants in PNPLA3 have been shown to be frequent in South American populations related to non-alcoholic fatty liver disease (NAFLD). In Caucasian populations, the variant has been shown to increase the risk for HCC when included in Genetic Risk Scores (GRS). Whether this risk applies to other Latino or other populations is unclear. Materials and Methods: We analyzed blood samples of 217 HCC cases, 120 from South American patients (Argentina, Ecuador, Colombia, Chile and Peru) and 97 from Europeans (Netherlands), as well as 326 cirrhotic controls through the ESCALON network. Genotyping for PNPLA3 was performed using TaqMan-genotyping assay. Associations between HCC and each SNP were evaluated using logistic regression models. Results: The median age for HCC in South Americans was 68 y/o (IQR 62-72) and in Europeans, 69 y/o (IQR 60-74), with 59% and 69% of males, respectively. The etiology of liver disease was similar in both groups except for NAFLD/NASH, which accounted for 59% of Hispanics with HCC vs. 25% of Europeans. Proportions of the risk allele of PNPLA3 were more prevalent among Hispanics (90%) than Europeans (57%). PNPLA3 G/G was present in 22% of Europeans with HCC compared to 57% of Hispanics. The presence of 2 risk alleles for PNPLA3 was not associated with a higher risk of HCC in South Americans, OR 1.19 (CI 0.58-2.46) or Europeans OR 1.1o (CI 0.34-3.58). When PNPLA3 was added in a GRS with TM6SF2 and HSD17B13, calculating different allele combinations did not associate either with HCC in South Americans, Conclusions: Our results show that the prevalence of risk alleles in PNPLA3 differs between South Americans and Europeans. An SNP in PNPLA3 does not seem to confer an increased risk for HCC in South Americans.

Introduction and Objectives: Worldwide, there is incomplete information about the epidemiology of hepatitis D virus (HDV), a hepatotropic satellite pathogen with an RNA genome, which requires the hepatitis B virus (HBV) as a collaborating agent for its transmission and spread. HDV genotypes have a defined geographical distribution. Very few studies have been carried out in South America. This study aimed to study the circulation of HDV in subjects with chronic HBV from South America. Materials and Methods: We studied 38 samples obtained between 2019 and 2021 from individuals chronically infected with HBV by assessing the ESCALON network (a cross-sectional and prospective study addressing hepatobiliary disease in South America). Samples were from Argentina (n=12), Peru (n=11), Colombia (n=4), Ecuador (n=4), Chile (n=4), and Brazil (n=3). Total anti-HDV antibody detection was performed using the Liaison XL Murex anti-HDV kit (DiaSorin). Positive samples were subjected to viral RNA detection by RT-PCR, and genotyped by Sanger sequencing. Results: Median age was 59 years old (IQR 48.5-67.3); 75% of the individuals were males and 25% were females. Three samples were positive for anti-HDV antibody detection (8%). Two of them, from Colombia and Chile, belonged to individuals with cirrhosis, while the third one, from Ecuador, originated from an individual with hepatocellular carcinoma (HCC). This sample could be amplified by RT-PCR, corresponding to a 44 years-old male. The sequencing showed HDV genotype 3. Conclusions: The results show circulation of HDV in South America, with a prevalence close to that estimated by the WHO (5%). The detections were performed in patients with severe liver disease, likely secondary to the presence of the two viral agents (HDV+HBV). Although our cohort is small, its strength lies in the geographical amplitude of the samples (6 countries). The study remains active and is expected to substantially increase the sample size over the coming year.

Chronic viral hepatitis infections, caused by the hepatitis B or C virus, are a major global health problem causing an estimated one million deaths each year. Immunological studies have classically focused on T cells, while B cells have largely been neglected. Emerging evidence, however, highlights a role for B cells in the immunopathogenesis of chronic hepatitis B and C infections. B cell responses appear to be altered across different clinical phases of chronic HBV infection and across stages of disease in chronic HCV infection. These B cell responses show signs of a more activated state with a simultaneous enrichment of phenotypically exhausted atypical memory B cells. Despite the fact that studies show an activating B cell signature in chronic viral hepatitis infection, antibody responses to HBsAg remain impaired in chronic HBV infection, and glycoprotein E2-specific neutralizing antibody responses remain delayed in the acute phase of HCV infection. At the same time, studies have reported that a subset of HBV- and HCV-specific B cells exhibit an exhausted phenotype. This may, at least in part, explain why antibody responses in chronic HBV and HCV patients are suboptimal. Here, we summarize recent findings and discuss upcoming research questions while looking forward to how new single-cell technologies could provide novel insights into the role of B cells in chronic viral hepatitis infections.

Introduction: Cholangiocarcinoma (CCA) includes a heterogeneous group of biliary cancers with dismal prognosis and increasing incidence. Information on epidemiology and risk factors are scarce, particularly in Latin America. Aim: Describe and analyze a multicentric cohort of CCA patients from Latin America. Methods: The Ibero-Latin American Research Network on Cholangiocarcinoma (ILARN-CCA) Registry and ESCALON consortium (www.escalon.eu) collected data from patients diagnosed from 2010 and onwards. Results: 183 patients with histologically/cytologically confirmed CCA were included from 5 tertiary hospitals (Brazil,Argentina,Chile,Ecuador and Peru). Median age at diagnosis was 62 years-old (IQR:25-87) and 55.7% were women. Most frequent risk factors were overweight/obesity (n=68;31.1%), diabetes (n=35;19.1%), NAFLD (n=14;7.7%), viral hepatitis (n=5;2.7%), cirrhosis (n=4;2.2%), gallstones (n=10;5.5%), primary sclerosing cholangitis (n=11;6%) and 21.3%(n=39) had no known-risk factor. Intrahepatic CCA was the predominant type (n=73;39.9%), followed by distal (n=49;26.8%) and perihilar (n=38;20.8%). Regional lymph-node invasion was found in 74 (40.4%) and metastasis in 79 (43.2%) patients. Upon diagnosis, 88 patients (48.1%) required upfront biliary stenting prior to main treatments, consisting in resection (n=39;21.3%) or palliative modalities (n=135;73.8%). Recurrence occurred in 64.1%(n=25), with median time-to-recurrence of 13.5 months (95%CI:6.5-18.8). Chemotherapy was delivered to 120 patients (Gemcitabine+Cisplatin:n=105;87.5%) with a median progression-free survival of 4.2 months (95%CI:3.4-4.9). Median overall survival of the entire cohort was 8.2 months (n=183;95%CI:6.3-10.2), 22.5 (n=39;95%CI:11.6-34.1) under surgery, 10.4 (n=87;95%CI:8.4-13.6) under chemotherapy and 2.5 (n=30;95%CI:1.5-3.9) without active treatments (log-rank p

Objective: We report the immune response during a case of acute HEV response in a patient with an acute hepatitis E virus (HEV) genotype 3 infection in the Netherlands. Methods: Cytokine evaluation was performed via multiplex cytokine array for 65 immune markers in plasma during the different phases of hepatitis. Results: The patient initially presented with features typical of acute viral hepatitis, with detectable HEV RNA in blood. This evolved into a cholestatic disease following peripheral clearance of the virus, leading to the demise of the patient. Real time PCR revealed the presence of HEV in liver tissue, suggestive of active intrahepatic infection despite clearance in blood. During the phase of detectable HEV RNA in serum, there was a surge in T-cell-related immune mediators, as well as interferon alpha and interferon gamma-induced protein 10 (IP-10), characteristic of a viral infection. After clearance of the virus in the blood and development of cholestatic hepatitis, several inflammatory markers subsided, followed by an increase in immune factors related to anti-inflammatory activity, as well as monocyte/macrophage-related markers, likely due to the intrahepatic presence of the virus. Conclusions: This report describes the dissociation of intra- and extra-hepatic immune responses during acute HEV infection. As shedding of the virus became solely intrahepatic, an immune profile reflective of the activity of hepatic resident cells was observed. Keywords: HEV, Immune-regulation, Cholestatic hepatitis

Humanized liver mouse models are crucial tools in liver research, specifically in the fields of liver cell biology, viral hepatitis and drug metabolism. The livers of these humanized mouse models are repopulated by 3-dimensional islands of fully functional primary human hepatocytes (PHH), which are notoriously difficult to maintain in vitro. As low efficiency and high cost hamper widespread use, optimization is of great importance. In the present study, we analyzed experimental factors associated with Hepatitis E virus (HEV) infection and PHH engraftment in 2 xenograft systems on a Nod-SCID-IL2Ry -/- background: the alb- urokinase plasminogen activator mouse model (uPA-NOG, n=399); and the alb- HSV thymidine kinase model (TK-NOG, n = 198). In a first analysis, HEV fecal shedding in liver humanized uPA-NOG and TK-NOG mice with comparable human albumin levels was found to be similar irrespective of the mouse genetic background. In a second analysis, sex, mouse age at transplantation and hepatocyte donor were the most determinant factors for xenograft success in both models. The sexual imbalance for xenograft success was related to higher baseline ALT levels and lower thresholds for ganciclovir induced liver morbidity and mortality in males. These data call for sexual standardization of human hepatocyte xenograft models, but also provide a platform for further studies on mechanisms behind sexual dimorphism in liver diseases.

Hepatocellular carcinoma (HCC) is estimated to be the fourth leading cause of cancer-related deaths worldwide. HCC patients face a dismal prognosis because symptoms usually appear in an advanced stage of disease. The detection of early stage HCC allows for curative surgical treatment and therefore saves lives. Specific non-invasive or diagnostic markers for HCC may represent a valuable tool for detecting these tumors at an early stage. The clinically most established serological biomarker alpha-fetoprotein shows only limited diagnostic performance, however novel candidate biomarkers and biomarker panels for detecting HCC at early stages of development are being studied. In this review we will discuss the findings of these studies.

We recently published a report validating point-of-care rapid diagnostic tests (RDT) for the diagnosis of Hepatitis B virus (HBV) infection in serum. In the current report, we validated a whole-blood RDT for HBsAg in the form of a test-strip. The test was validated in 55 HBV positive individuals across all genotypes other than F, and in 20 HBV negative individuals in the Netherlands. The RDT showed 100% sensitivity and specificity. The low cost and use in whole blood allows this RDT to be useful in resource-limited locations, further validation in such settings will be of importance.

Abstract Antiviral treatment options for chronic Hepatitis E Virus (HEV) infections are limited and immunological determinants of viral persistence remain largely unexplored. We studied the antiviral potency of pegylated interferon-α (pegIFNα) against HEV infections in humanized mice and modelled intrahepatic interferon stimulated gene (ISG) responses. Human gene expression levels in humanized mouse livers were analyzed by qPCR and Nanostring. Human CXCL10 was measured in mouse serum. HEV genotype 3 (gt3) infections were cleared from liver and feces within 8 pegIFNα doses in all mice and relapsed after a single pegIFNα injection in only half of treated animals. Rapid viral clearance by pegIFNα was confirmed in HEV gt1, but not in Hepatitis B Virus infected animals. No ISG induction was observed in untreated HEV gt3 and gt1 infected humanized livers compared to control chimeric mice, irrespective of the human hepatocyte donor, viral isolate or HEV infection duration. Human specific ISG transcript levels in mouse liver increased significantly after pegIFNα treatment and induced high circulating human CXCL10 in mouse serum. In conclusion, HEV gt1 and gt3 infections do not elicit innate intrahepatic immune responses and remain highly sensitive to pegIFNα in immunocompromised humanized mice.

Chronic Hepatitis B (HBV) is the most important cause of liver disease worldwide. There is a need for low-cost tests to aid in diagnosis and management of HBV infection in resource-limited settings. We evaluated the utility of several rapid diagnostic tests (RDT) in three different continents (Europe, South America, Africa). The HBsAg RDT showed optimal sensitivity and specificity. The anti-HBeAb RDT showed acceptable sensitivity and excellent specificity. Our results suggest that these RDTs could be used for screening and management of HBV.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Interestingly, the great majority of individuals affected by the tumor have underlying liver disease, therefore narrowing the population to be screened. Still, however, there is a clear lack of blood biomarkers, and surveillance in those at risk is performed by frequent imaging of the liver. A variety of multinational collaborations are currently invested in finding biomarkers for HCC based on liver-produced proteins. A new approach with assessment of peripheral proteins might be necessary for the successful early detection of this malignancy.

Chronic hepatitis B is a highly heterogeneous liver disease characterized by phases with fluctuations in viral replication and progressive liver damage in some, but not all infected individuals. Despite four decades of research, insight into host determinants underlying these distinct clinical phases-immunotolerant, immune active, inactive carrier, and HBeAg-negative hepatitis-remains elusive. We performed an in-depth transcriptome analysis of archived FFPE liver biopsies of each clinical phase to address host determinants associated with the natural history. Therefore, we determined, for the first time, intrahepatic global expression profiles of well-characterized chronic HBV patients at different clinical phases. Our data, obtained by microarray, demonstrate that B cells and NK/cytotoxic-related genes in the liver, including CD19, TNFRSF13C, GZMH, and KIR2DS3, were differentially expressed across the clinical HBV phases, which was confirmed by modular analysis and also Nanostring arrays in an independent cohort. Compared to the immunotolerant phase, 92 genes were differentially expressed in the liver during the immune active phase, 46 in the inactive carrier phase, and 71 in the HBeAg-negative phase. Furthermore, our study also revealed distinctive transcription of genes associated with cell cycle activity, NF-κB signaling, cytotoxic function and mitochondrial respiration between clinical phases. Our data define for the first time using microarray unique transcriptomes in the HBV-infected liver during consecutive clinical phases. We demonstrate that fluctuations of viral loads and liver damage coincide with fluctuations in the liver transcriptome and point to functional- immune and non-immune- components contributing to the clinical phenotype in patients.

Hepatitis E Virus (HEV) mutations following ribavirin treatment have been associated with treatment non-response and viral persistence, but spontaneous occurring genomic variations have been less well characterized. We here set out to study the HEV genome composition in 2 patient sample types and 2 infection models. Near full HEV genome Sanger sequences of serum- and feces-derived HEV from two chronic HEV genotype 3 (gt3) patients were obtained. In addition, viruses were sequenced after in vitro or in vivo expansion on A549 cells or a humanized mouse model, respectively. We show that HEV acquired 19 nucleotide mutations, of which 7 nonsynonymous amino acids changes located in Open Reading Frame 1 (ORF1), ORF2, and ORF3 coding regions, after prolonged in vitro culture. In vivo passage resulted in selection of 8 nucleotide mutations with 2 altered amino acids in the X domain and Poly-proline region of ORF1. Intra-patient comparison of feces- and serum-derived HEV gt3 of two patients showed 7 and 2 nucleotide mutations with 2 and 0 amino acid changes, respectively. Overall, the number of genomic alterations was up to 1.25× per 1000 nucleotides or amino acids in in vivo samples, and up to 2.84× after in vitro expansion of the same clinical HEV strain. In vitro replication of a clinical HEV strain is therefore associated with more mutations, compared to the minor HEV genomic alterations seen after passage of the same strain in an immune deficient humanized mouse; as well as in feces and blood of 2 immunosuppressed chronically infected HEV patients. These data suggest that HEV infected humanized mice more closely reflect the HEV biology seen in solid organ transplant recipients.

Mucosal-associated invariant T (MAIT) cells comprise a subpopulation of T cells that can be activated by bacterial products and cytokines to produce IFN-γ. Since little is known on MAIT cells during HCV infection, we compared their phenotype and function in comparison to HIV and HCV/HIV co-infected patients, and determined the effect of IFN-α-based and direct-acting antiviral therapy on MAIT cells of HCV patients.Blood samples from patients with chronic HCV (CHCV), virologically suppressed HIV, acute HCV/HIV co-infection (AHCV/HIV) and healthy individuals were examined by flowcytometry for phenotype and function of MAIT and NK cells.Compared to healthy individuals, the frequency of CD161+Vα7.2+ MAIT cells was significantly decreased in patients with CHCV, HIV and AHCV/HIV co-infection. CD38 expression on MAIT cells was increased in AHCV/HIV patients. MAIT cells were responsive to IFN-α in vitro as evidenced by enhanced frequencies of IFN-γ producing cells. IFN-α-based therapy for CHCV decreased the frequency of IFN-γ+ MAIT cells, which was still observed 24 weeks after successful therapy. Importantly, even after successful IFN-α-based as well as IFN-α-free therapy for CHCV, decreased frequencies of MAIT cells persisted. We show that the frequencies of MAIT cells are reduced in blood of patients with CHCV, HIV and in AHCV/HIV co-infection compared to healthy individuals. Successful therapy for CHCV did not normalize MAIT cell frequencies at 24 weeks follow up. The impact of HIV and HCV infection on the numbers and function of MAIT cells warrant further studies on the impact of viral infections and the antimicrobial function of MAIT cells.

Due to the scarcity of immunocompetent animal models for chronic viral hepatitis, little is known about the role of the innate intrahepatic immune system during viral replication in the liver. These insights are however fundamental for the understanding of the inappropriate adaptive immune responses during the chronic phase of the infection. We apply the Lymphocytic Choriomenigitis Virus (LCMV) clone 13 mouse model to examine chronic virus-host interactions of Kupffer cells (KC) and infiltrating monocytes (IM) in an infected liver. LCMV infection induced overt clinical hepatitis, with rise in ALT and serum cytokines, and increased intrahepatic F4/80 expression. Despite ongoing viral replication, whole liver transcriptome showed baseline expression levels of inflammatory cytokines, interferons, and interferon induced genes during the chronic infection phase. Transcriptome analyses of sorted KC and IMs using NanoString technology revealed two unique phenotypes with only minimal overlap. At the chronic viral infection phase, KC showed no increased transcription of activation markers Cd80 and Cd86, but an increased expression of genes related to antigen presentation, whereas monocytes were more activated and expressed higher levels of Tnf transcripts. Although both KCs and intrahepatic IM share the surface markers F4/80 and CD11b, their transcriptomes point towards distinctive roles during virus-induced chronic hepatitis.

BACKGROUND/OBJECTIVE:Genetic polymorphisms in the inosine triphosphatase (ITPA) gene have been associated with the protection from early ribavirin(RBV)-induced hemolytic anemia among patients with chronic hepatitis C virus (HCV) infection. The aim of the present study was to investigate the association between the functional ITPA variants and hematological side effects during antiviral therapy with pegylated interferon (PegIFN) and RBV. PATIENTS AND METHODS:This cohort study included all consecutive Caucasian patients treated for chronic HCV infection with PegIFN and RBV between 2000 and 2009 for whom a serum sample was available for genetic testing. The predicted inosine triphosphate pyrophosphatase (ITPase) activity was based on the genotypes of the SNPs rs1127354 and rs7270101. Decline in hemoglobin (Hb) during antiviral therapy, as well as dose reductions, blood transfusions and use of erythropoietin were assessed. RESULTS:In total, 213 patients were included. The predicted ITPase activity was normal among 152 (71%) patients; 61 (29%) patients had ITPase deficiency. By multivariable linear regression, RBV dose in mg per kilogram (Beta 0.09, 95%CI 0.04-0.13, p

In humans, clearance of hepatitis C virus (HCV) infection is associated with genetic variation near the IL-28B gene and the induction of interferon-stimulated genes, like IP-10. Also in chimpanzees spontaneous clearance of HCV is observed. To study whether similar correlations exist in these animals, a direct comparison of IP-10 and IL-28B polymorphism between chimpanzees and patients was performed. All chimpanzees studied were monomorphic for the human IL-28B SNPs which are associated with spontaneous and treatment induced HCV clearance in humans. As a result, these particular SNPs cannot be used for clinical association studies in chimpanzees. Although these human SNPs were absent in chimpanzees, gene variation in this region was present however, no correlation was observed between different SNP-genotypes and HCV outcome. Strikingly, IP-10 levels in chimpanzees correlated with HCV-RNA load and γGT, while such correlations were not observed in humans. The correlation between IP-10, γGT and virus load in chimpanzees was not found in patients and may be due to the lack of lifestyle-related confounding factors in chimpanzees. Direct comparison of IP-10 and IL-28B polymorphism between chimpanzees and patients in relation to HCV infection, illustrates that the IFN-pathways are important during HCV infection in both species. The Genbank EMBL accession numbers assigned to chimpanzees specific sequences near the IL-28B gene are HE599784 and HE599785.

Abstract Accurate non-invasive biomarkers to diagnose metabolic dysfunction-associated steatotic liver disease (MASLD)-related fibrosis are urgently needed. This study applies a translational approach to develop a blood-based biomarker panel for fibrosis detection in MASLD. A molecular gene expression signature identified from a diet-induced MASLD mouse model (LDLr−/−.Leiden) is translated into human blood-based biomarkers based on liver biopsy transcriptomic profiles and protein levels in MASLD patient serum samples. The resulting biomarker panel consists of IGFBP7, SSc5D and Sema4D. LightGBM modeling using this panel demonstrates high accuracy in predicting MASLD fibrosis stage (F0/F1: AUC = 0.82; F2: AUC = 0.89; F3/F4: AUC = 0.87), which is replicated in an independent validation cohort. The overall accuracy of the model outperforms predictions by the existing markers Fib-4, APRI and FibroScan. In conclusion, here we show a disease mechanism-related blood-based biomarker panel with three biomarkers which is able to identify MASLD patients with mild or advanced hepatic fibrosis with high accuracy.