Interferon-alpha treatment rapidly clears Hepatitis e virus infection in humanized mice

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

DNGR1-mediated deletion of A20/Tnfaip3 in dendritic cells alters T and B-cell homeostasis and promotes autoimmune liver pathology

Dendritic cells (DCs) are central regulators of tolerance versus immunity. The outcome depends amongst others on DC subset and activation status. Whereas CD11b+ type 2 conventional DCs (cDC2s) initiate proinflammatory helper T (Th)-cell responses, CD103+ cDC1s are crucial for regulatory T-cell (Treg) induction and CD8+ T-cell activation. DC activation is controlled by the transcription factor NF-κB. Ablation of A20/Tnfaip3, a critical regulator of NF-κB activation, in DCs leads to constitutive DC activation and development of systemic autoimmunity. We hypothesized that the activation status of cDCs controls the development of autoimmunity. To target cDCs, DNGR1(Clec9a)-cre-mediated excision of A20/Tnfaip3 was used through generation of Tnfaip3fl/flxClec9a+/cre (Tnfaip3DNGR1−KO) mice. Immune cell activation was evaluated at 31-weeks of age. We found that DNGR1-cre-mediated deletion of A20/Tnfaip3 resulted in liver pathology characterized by inflammatory infiltrates adjacent to the portal triads. Both cDC subsets as well as monocyte-derived DCs (moDCs) in Tnfaip3DNGR1−KO livers harbored an activated phenotype. Specifically, the costimulatory molecule CD40 in liver cDCs and moDCs was regulated by A20/Tnfaip3 expression. Livers from Tnfaip3DNGR1−KO mice had augmented prop

Unique intrahepatic transcriptomics profiles discriminate the clinical phases of a chronic HBV infection

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/cytotoxicrelated 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 functionalimmune and non-immune-components contributing to the clinical phenotype in patients

Liver monocytes and kupffer cells remain transcriptionally distinct during chronic viral infection

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 cli

Point-of-Care Tests for Hepatitis B Are Associated with A Higher Linkage to Care and Lower Cost Compared to Venepuncture Sampling During Outreach Screenings in an Asian Migrant Population

Background: This study compares venepuncture versus point-of-care (POC) HBsAg tests on screening cost and linkage to care in prospective outreach screenings in an Asian population in three major cities in Belgium between 10/2014 and 5/2018. Methods: Two community outreach screening programs were organised between 10/2014 and 5/2018. The first screening program used venepuncture and serologic testing for HBsAg. In the second program, HBsAg was tested in finger stick blood POC tests. Positive results were confirmed during outpatient visits with serologic testing. Linkage to care was defined as having received specialist care follow-up with at least one abdominal ultrasound within three months of screening. Results: For 575 participating individuals, 571 valid results were obtained, 456 with venepuncture, and 115 using POC testing. Overall HBsAg seroprevalence was 6.8%. Linkage to care was higher when using POC testing compared to venepuncture (86% or n = 6/7 versus 34% or n = 11/32; p = 0.020). The POC screening program was economically more attractive with a total cost of € 1,461.8 or € 12.7 per person screened compared to € 24,819 or € 54.0 per person screened when using venepuncture testing. Results and an appointment for specialist care follow-up were given onsite with POC testing, while with venepuncture testing; results were sent within 20-45 days. Conclusion: In an Asian migrant population in Belgium with an HBsAg seroprevalence of 6.8%, HBV screening based on POC tests resulted in lower costs per person screened (76.5% lower), and higher linkage to care (2.5 times)

Biobanking for Viral Hepatitis Research

Introduction: Viral hepatitis is a worldwide, important health issue. The optimal management of viral hepatitis infections faces numerous challenges. In this paper, we describe how biobanking of biological samples derived from viral hepatitis patients collected both in-hospital and during community outreach screenings provides a unique collection of samples. Materials and Methods: All samples and materials were provided with a study code within the SLIMS system Study protocols and an informed consent form were approved by the Antwerp University Hospital/University of Antwerp Ethical Committee. Systematic biobanking was initiated in October 2014. Collected sample types include: (1) serum and plasma of all newly diagnosed HBV, HCV, HDV, and HEV positive patients; (2) left-over serum and plasma samples from all PCR analyses for HBV and HCV performed in the context of routine clinical care; (3) left-over liver tissue not needed for routine histological diagnosis after liver biopsy; and (4) additional virus-specific, appropriate sample types using a scientific rationale-based approach. A community outreach screening program was performed in three major Belgian cities. Serum, EDTA, Tempus Blood RNA and BD Vacutainer CPT were collected. CPT tubes were centrifuged on-site and mononuclear cells collected within 24 h. Results: Concerning community screening: 298 individuals supplied all 4 sample types. Samples were stored at −150◦C and were logged in the biobank SLIMS database. Samples were used for HBV-related immunological and biomarker studies. DNA isolated from plasma samples derived from chronic HBV patients was used to investigate Single Nucleotide Polymorphism rs 1790008. Serum samples collected from chronic hepatitis C patients were used to assess the efficacy of HCV treatment. Peripheral Blood Mononuclear Cells (PBMC) isolated from chronic HBV patients and healthy controls were used for different immunological study purposes. Virus isolated from biobanked stool of a chronic hepatitis E patient was used to establish a mouse model for Hepatitis E infections, allowing further HEV virology studies. Conclusion: The establishment of a biobank with samples collected both in-hospital and during community-outreach screening resulted in a unique, continuously expanding collection of biological samples which provides an excellent platform for prompt answers to clinically and translational relevant research questions

Liver grafts procured and discarded by all Belgian centers and transplanted within Eurotransplant network: analysis of cause to decline, a Be-LIAC study

The annual balance between imports and exports of grafts is a matter of debate. We examined the Eurotransplant database of all liver grafts procured within Belgium and Luxemburg which were exported and transplanted abroad. The aim of our study was to analyse the reasons for graft refusal by all Belgian transplant centres and early postoperative evolution. Database between 2015 and 2019 included donor characteristics, reason of offer decline, graft and recipient survival. During the 4 year period 329 grafts were procured in Belgium and transplanted abroad. 163 were exported for HU recipients, 17 no national match recipients (8 AB group, 2 pediatric and 7 other reasons), 19 pay back, 15 splits, 11 not mentioned. Hundred and four grafts were declined by all Belgian centres. Forty seven were declined primary offers and fifty seven livers were distributed by extended allocation. Between them we find out four DCD donors, 83 for medical reasons (age, cytolysis, size mismatch and steatosis). Thirteen livers were accepted and declined at arrival for size mismatch (kept as rescue offer in the same centre). One donor was unstable and two were rejected for positive HCV virology. Only one liver who was primary accepted for a split was transplanted as a whole liver. Two patients presented primary graft nonfunction and three primary graft dysfunction. All of them were retransplanted. Thirteen patients died in the early 3 month postoperative period. Even though higher mortality is expected from marginal grafts, better acceptance rate could be achieved at a national level

Assessment of portal hypertension severity using machine learning models in patients with compensated cirrhosis

Background & Aims: In individuals with compensated advanced chronic liver disease (cACLD), the severity of portal hypertension (PH) determines the risk of decompensation. Invasive measurement of the hepatic venous pressure gradient (HVPG) is the diagnostic gold standard for PH. We evaluated the utility of machine learning models (MLMs) based on standard laboratory parameters to predict the severity of PH in individuals with cACLD. Methods: A detailed laboratory workup of individuals with cACLD recruited from the Vienna cohort (NCT03267615) was utilised to predict clinically significant portal hypertension (CSPH, i.e., HVPG ≥10 mmHg) and severe PH (i.e., HVPG ≥16 mmHg). The MLMs were then evaluated in individual external datasets and optimised in the merged cohort. Results: Among 1,232 participants with cACLD, the prevalence of CSPH/severe PH was similar in the Vienna (n = 163, 67.4%/35.0%) and validation (n = 1,069, 70.3%/34.7%) cohorts. The MLMs were based on 3 (3P: platelet count, bilirubin, international normalised ratio) or 5 (5P: +cholinesterase, +gamma-glutamyl transferase, +activated partial thromboplastin time replacing international normalised ratio) laboratory parameters. The MLMs performed robustly in the Vienna cohort. 5P-MLM had the best AUCs for CSPH (0.813) and severe PH (0.887) and compared favourably to liver stiffness measurement (AUC: 0.808). Their performance in external validation datasets was heterogeneous (AUCs: 0.589-0.887). Training on the merged cohort optimised model performance for CSPH (AUCs for 3P and 5P: 0.775 and 0.789, respectively) and severe PH (0.737 and 0.828, respectively). Conclusions: Internally trained MLMs reliably predicted PH severity in the Vienna cACLD cohort but exhibited heterogeneous results on external validation. The proposed 3P/5P online tool can reliably identify individuals with CSPH or severe PH, who are thus at risk of hepatic decompensation. Impact and implications: We used machine learning models based on widely available laboratory parameters to develop a non-invasive model to predict the severity of portal hypertension in individuals with compensated cirrhosis, who currently require invasive measurement of hepatic venous pressure gradient. We validated our findings in a large multicentre cohort of individuals with advanced chronic liver disease (cACLD) of any cause. Finally, we provide a readily available online calculator, based on 3 (platelet count, bilirubin, international normalised ratio) or 5 (platelet count, bilirubin, activated partial thromboplastin time, gamma-glutamyltransferase, choline-esterase) widely available laboratory parameters, that clinicians can use to predict the likelihood of their patients with cACLD having clinically significant or severe portal hypertension

Diagnostic criteria and long-term outcomes in AIH-PBC variant syndrome under combination therapy

Background &amp; Aims: Autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) can co-exist in AIH-PBC, requiring combined treatment with immunosuppression and ursodeoxycholic acid (UDCA). The Paris criteria are commonly used to identify these patients; however, the optimal diagnostic criteria are unknown. We aimed to evaluate the use and clinical relevance of both Paris and Zhang criteria. Methods: Eighty-three patients with a clinical suspicion of AIH-PBC who were treated with combination therapy were included. Histology was re-evaluated. Characteristics and long-term outcomes were retrospectively compared to patients with AIH and PBC. Results: Seventeen (24%) patients treated with combination therapy fulfilled the Paris criteria. Fifty-two patients (70%) fulfilled the Zhang criteria. Patients who met Paris and Zhang criteria more often had inflammation and fibrosis on histology compared to patients only meeting the Zhang criteria. Ten-year liver transplant (LT)-free survival was 87.3% (95% CI 78.9–95.7%) in patients with AIH-PBC. This did not differ in patients in or outside the Paris or Zhang criteria (p = 0.46 and p = 0.40, respectively) or from AIH (p = 0.086). LT-free survival was significantly lower in patients with PBC and severe hepatic inflammation – not receiving immunosuppression – compared to those with AIH-PBC (65%; 95% CI 52.2–77.8% vs. 87%; 95% CI 83.2–90.8%; hazard ratio 0.52; p = 0.043). Conclusions: In this study, patients with AIH-PBC outside Paris or Zhang criteria were frequently labeled as having AIH-PBC and were successfully treated with combination therapy with similar outcomes. LT-free survival was worse in patients with PBC and hepatic inflammation than in those treated as having AIH-PBC. More patients may benefit from combination therapy. Impact and implications: This study demonstrated that patients with AIH-PBC variant syndrome treated with combined therapy consisting of immunosuppressants and ursodeoxycholic acid often do not fulfill the Paris criteria. They do however have comparable response to therapy and long-term outcomes as patients who do fulfill the diagnostic criteria. Additionally, patients with PBC and additional signs of hepatic inflammation have poorer long-term outcomes compared to patients treated as having AIH-PBC. These results implicate that a larger group of patients with features of both AIH and PBC may benefit from combined treatment. With our results, we call for improved consensus among experts in the field on the diagnosis and management of AIH-PBC variant syndrome.</p

Diagnostic criteria and long-term outcomes in AIH-PBC variant syndrome under combination therapy

Background &amp; Aims: Autoimmune hepatitis (AIH) and primary biliary cholangitis (PBC) can co-exist in AIH-PBC, requiring combined treatment with immunosuppression and ursodeoxycholic acid (UDCA). The Paris criteria are commonly used to identify these patients; however, the optimal diagnostic criteria are unknown. We aimed to evaluate the use and clinical relevance of both Paris and Zhang criteria. Methods: Eighty-three patients with a clinical suspicion of AIH-PBC who were treated with combination therapy were included. Histology was re-evaluated. Characteristics and long-term outcomes were retrospectively compared to patients with AIH and PBC. Results: Seventeen (24%) patients treated with combination therapy fulfilled the Paris criteria. Fifty-two patients (70%) fulfilled the Zhang criteria. Patients who met Paris and Zhang criteria more often had inflammation and fibrosis on histology compared to patients only meeting the Zhang criteria. Ten-year liver transplant (LT)-free survival was 87.3% (95% CI 78.9–95.7%) in patients with AIH-PBC. This did not differ in patients in or outside the Paris or Zhang criteria (p = 0.46 and p = 0.40, respectively) or from AIH (p = 0.086). LT-free survival was significantly lower in patients with PBC and severe hepatic inflammation – not receiving immunosuppression – compared to those with AIH-PBC (65%; 95% CI 52.2–77.8% vs. 87%; 95% CI 83.2–90.8%; hazard ratio 0.52; p = 0.043). Conclusions: In this study, patients with AIH-PBC outside Paris or Zhang criteria were frequently labeled as having AIH-PBC and were successfully treated with combination therapy with similar outcomes. LT-free survival was worse in patients with PBC and hepatic inflammation than in those treated as having AIH-PBC. More patients may benefit from combination therapy. Impact and implications: This study demonstrated that patients with AIH-PBC variant syndrome treated with combined therapy consisting of immunosuppressants and ursodeoxycholic acid often do not fulfill the Paris criteria. They do however have comparable response to therapy and long-term outcomes as patients who do fulfill the diagnostic criteria. Additionally, patients with PBC and additional signs of hepatic inflammation have poorer long-term outcomes compared to patients treated as having AIH-PBC. These results implicate that a larger group of patients with features of both AIH and PBC may benefit from combined treatment. With our results, we call for improved consensus among experts in the field on the diagnosis and management of AIH-PBC variant syndrome.</p