Jan M. Vrolijk

A replicon-based bioassay for the measurement of interferons in patients with chronic hepatitis C

Overall treatment results of chronic hepatitis C have improved markedly with the introduction of pegylated interferon-alpha (PEG-IFN-alpha) and ribavirin combination therapy. However, cure rates in the most common genotype 1 infection are still unsatisfactory. IFN-alpha dose-response studies on viral kinetics suggest that inadequate dosing might be a key factor but drug levels have hardly been tested, which is in part due to difficulties in measuring this cytokine in patient samples. We have shown recently that hepatitis C virus (HCV) replicons are highly sensitive to IFN-alpha. In this report we tested whether the replicon system could be used as a sensitive bioassay to determine the amount of biologically active IFN-alpha in serum or heparinized plasma of patients under therapy. To facilitate the measurements, a stably replicating subgenomic HCV RNA was developed that carries the gene encoding the firefly luciferase. Dose response studies with IFN-alpha demonstrate that the amount of expressed luciferase directly correlates with the level of HCV replication. By using this cell-based assay, serum samples of HCV patients treated with different types and doses of IFN-alpha were analyzed in parallel to IFN-alpha standards made by serial dilutions of the same type of IFN-alpha the patient was treated with. Based on nonlinear logistic models serum concentrations corresponding to 1.3-19 U/ml were determined in patients under standard or high dose IFN-alpha therapy, and from 3.8 to 4.1 ng/ml in patients treated with PEG IFN-alpha. In conclusion, the HCV-replicon based bioassay allows determining the levels of biologically active IFN-alpha in serum and heparinized plasma of patients under treatment.

Genome-Wide Association Study Identifies Variants Associated With Autoimmune Hepatitis Type 1

BACKGROUND & AIMS Autoimmune hepatitis (AIH) is an uncommon autoimmune liver disease of unknown etiology. We used a genome-wide approach to identify genetic variants that predispose individuals to AIH. METHODS We performed a genome-wide association study of 649 adults in The Netherlands with AIH type 1 and 13,436 controls. Initial associations were further analyzed in an independent replication panel comprising 451 patients with AIH type 1 in Germany and 4103 controls. We also performed an association analysis in the discovery cohort using imputed genotypes of the major histocompatibility complex region. RESULTS We associated AIH with a variant in the major histocompatibility complex region at rs2187668 (P = 1.5 × 10(-78)). Analysis of this variant in the discovery cohort identified HLA-DRB1*0301 (P = 5.3 × 10(-49)) as a primary susceptibility genotype and HLA-DRB1*0401 (P = 2.8 × 10(-18)) as a secondary susceptibility genotype. We also associated AIH with variants of SH2B3 (rs3184504, 12q24; P = 7.7 × 10(-8)) and CARD10 (rs6000782, 22q13.1; P = 3.0 × 10(-6)). In addition, strong inflation of association signal was found with single-nucleotide polymorphisms associated with other immune-mediated diseases, including primary sclerosing cholangitis and primary biliary cirrhosis, but not with single-nucleotide polymorphisms associated with other genetic traits. CONCLUSIONS In a genome-wide association study, we associated AIH type 1 with variants in the major histocompatibility complex region, and identified variants of SH2B3and CARD10 as likely risk factors. These findings support a complex genetic basis for AIH pathogenesis and indicate that part of the genetic susceptibility overlaps with that for other immune-mediated liver diseases.

γ‐Glutamyltransferase and rapid virological response as predictors of successful treatment with experimental or standard peginterferon‐α‐2b in chronic hepatitis C non‐responders

Background: High‐dose peginterferon‐α (PegIFN‐α) induction and prolongation of therapy may be an option to improve sustained virological response (SVR) rates among hepatitis C virus (HCV) non‐responders, although a higher and a longer dosing of PegIFN‐α may intensify side effects.

Pretreatment Intrahepatic CD8+ Cell Count Correlates with Virological Response to Antiviral Therapy in Chronic Hepatitis C Virus Infection

We analyzed the relationship between virological response and baseline immune factors in 17 patients chronically infected with hepatitis C virus (HCV) who received interferon-alpha-ribavirin therapy for 26 weeks. The number of intrahepatic CD8(+) cells present in the portal tract before the start of treatment was found to be significantly higher in patients who responded to treatment than in nonresponders. The relationship between portal CD8(+) cell counts and the response to therapy could be described by a logistic curve. Neither peripheral cytokine levels nor HCV-specific T cell reactivity in peripheral blood mononuclear cells showed a relationship to response to therapy.

High sustained virological response in chronic hepatitis C by combining induction and prolonged maintenance therapy

Summary.  Chronic hepatitis C patients with genotype 1 infection, liver cirrhosis, high viral load, or those who have not responded to anti‐viral treatment in the past have limited chances of clearing the virus, even with pegylated interferon–ribavirin therapy. In this study we treated such patients with a treatment schedule that combines high dose induction Interferon (IFN), prolonged daily IFN and ribavirin treatment. Twenty‐four consecutive patients were included in this study with either genotype 1 infection, cirrhosis, previous non‐response to IFN or a combination of these poor‐response characteristics. Patients were treated with 10 million units (MU) of IFN daily for 4 weeks followed by 5 MU/day until week 24, 3 MU/day until week 52 and 3 MU thrice weekly until week 76 in combination with 1–1.2 g ribavirin daily. HCV RNA levels were assessed weekly until week 4 and at least once every 3 months thereafter, by a validated assay with a detection limit below 500 copies/mL. Both intention to treat (ITT) and per protocol (PP) analysis showed a high sustained virological response (ITT 67%, PP 80%). A virological response occurred rapidly (before 8 weeks of treatment) in all patients with a sustained response. Relapse after stopping therapy was observed in only 5%. Side‐effects were observed frequently, and six patients had to be hospitalized. With this new treatment regimen that combines induction‐ and prolonged daily interferon treatment with ribavirin it seems possible to eliminate hepatitis C virus in the majority of patients that have an a priori limited chance of sustained response. Further clinical evaluation of intensive interferon and ribavirin combination therapy (now also including PEG‐interferon) is recommended in centres that can provide close patient monitoring and experienced hepatological support.

Cytotoxic T Lymphocyte Antigen–4 +49A/G polymorphism does not affect susceptibility to autoimmune hepatitis

Single nucleotide polymorphisms (SNP) in the Cytotoxic T lymphocyte antigen‐4 gene (CTLA‐4) have been associated with several autoimmune diseases including autoimmune Hepatitis (AIH). In this chronic idiopathic inflammatory liver disease, conflicting results have been reported on the association with a SNP at position +49 in the CTLA‐4 gene in small patient cohorts. Here, we established the role of this SNP in a sufficiently large cohort of AIH patients.

Psychiatric Side Effects and Fluctuations in Serotonergic Parameters in the Treatment of Chronic Hepatitis C Infection

Introduction: Treatment of hepatitis C with peginterferon induces psychiatric side effects. These might include changes in serotonergic function. Methods: Twenty-two hepatitis C patients were treated with peginterferon. At different time points, psychometric assessment was performed using the Profile of Mood States. Plasma samples were taken to study serotonergic parameters. Results: Anger and depression increased compared to baseline, starting with anger (from week 3 onwards), followed by depression (from week 7 onwards). Other scores did not show consistent changes. No consistent changes were observed in tryptophan, tryptophan/large neutral amino acids ratio, biopterin and 5-hydroxyindoleacetic acid. The tyrosine/large neutral amino acids ratio, neopterin, phenylalanine/tyrosine ratio, and prolactin concentrations increased compared to baseline. Prolactin levels were associated with the occurrence of depression and anger. Discussion: Particularly anger and depression increased during treatment. Neither a decrease in tryptophan and tryptophan availability was seen, nor a relationship between these parameters and the development of psychopathology. Therefore, other mechanisms in the induction of psychopathology should be considered. The observed increases in neopterin and phenylalanine/tyrosine ratio are indicative of changes in tetrahydrobiopterin, which is involved in the metabolism of serotonin, noradrenaline and dopamine, and possibly mediating the increase in prolactin. The increase in prolactin levels and its relationship with depression and anger needs further exploration.

Monitoring intrahepatic CD8+ T cells by fine-needle aspiration cytology in chronic hepatitis C infection

Summary.  Infection of the liver with hepatitis C virus (HCV) causes compartmentalization of CD8+ cytotoxic T cells to the site of disease. These cells are thought to be involved in viral clearance during interferon therapy. The repetitive analysis of the intrahepatic immune response is hampered by the difficulty to obtain the intrahepatic T cells. The fine‐needle aspiration biopsy (FNAB) technique was evaluated for its use to obtain liver‐derived CD8+ T cells in a minimally invasive way. In 26 chronic HCV patients who were evaluated for Peg‐interferon and ribavirin combination therapy, pre‐treatment FNABs and peripheral blood specimens were obtained simultaneously with liver tissue biopsies, and CD3+ and CD8+ T cells were quantified by immunocytochemistry. The CD8+/CD3+ ratio was significantly higher in the FNABs than in peripheral blood (P < 0.01), and similar to those in portal areas in the tissue biopsies. A significant correlation was observed between numbers of CD3+CD8+ T lymphocytes in the FNABs and the numbers of CD8+ cells in the lobular fields or in the portal tracts of the liver tissue biopsies, but not with CD3+CD8+ T lymphocytes in peripheral blood. Finally, the ratio of CD8+/CD3+ T lymphocytes in FNABs was significantly higher in those patients who responded rapidly to therapy when compared with slow responders at 4 weeks of treatment (P = 0.02). These findings demonstrate that the intrahepatic T‐cell composition is reflected in FNABs, and that the FNAB technique can be used for predicting early virological response to therapy of patients chronically infected with HCV.

Fluid hydration to prevent post-ERCP pancreatitis in average- to high-risk patients receiving prophylactic rectal NSAIDs (FLUYT trial) : study protocol for a randomized controlled trial

BackgroundPost-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (PEP) is the most common complication of ERCP and may run a severe course. Evidence suggests that vigorous periprocedural hydration can prevent PEP, but studies to date have significant methodological drawbacks. Importantly, evidence for its added value in patients already receiving prophylactic rectal non-steroidal anti-inflammatory drugs (NSAIDs) is lacking and the cost-effectiveness of the approach has not been investigated. We hypothesize that combination therapy of rectal NSAIDs and periprocedural hydration would significantly lower the incidence of post-ERCP pancreatitis compared to rectal NSAIDs alone in moderate- to high-risk patients undergoing ERCP.MethodsThe FLUYT trial is a multicenter, parallel group, open label, superiority randomized controlled trial. A total of 826 moderate- to high-risk patients undergoing ERCP that receive prophylactic rectal NSAIDs will be randomized to a control group (no fluids or normal saline with a maximum of 1.5 mL/kg/h and 3 L/24 h) or intervention group (lactated Ringer’s solution with 20 mL/kg over 60 min at start of ERCP, followed by 3 mL/kg/h for 8 h thereafter). The primary endpoint is the incidence of post-ERCP pancreatitis. Secondary endpoints include PEP severity, hydration-related complications, and cost-effectiveness.DiscussionThe FLUYT trial design, including hydration schedule, fluid type, and sample size, maximize its power of identifying a potential difference in post-ERCP pancreatitis incidence in patients receiving prophylactic rectal NSAIDs.Trial registrationEudraCT: 2015-000829-37. Registered on 18 February 2015.ISRCTN: 13659155. Registered on 18 May 2015.

Clinical impact of five large-scale screening projects for chronic hepatitis B in Chinese migrants in the Netherlands.

In low‐endemic countries it is debated whether first‐generation migrants should be screened for chronic hepatitis B infection. We describe the clinical impact of five large‐scale Dutch screening projects for hepatitis B in first‐generation Chinese migrants.