Synergistic effect of calcitriol and interferon-α on hepatitis C virus replication in vitro

Background and aims: V itamin D is an important modulator o fnumerous c ellular processes, including innate and adaptive immunepathways. A recent large-scale genetic validation study performed withinthe framework of the Swiss Hepatitis C Cohort S tudy has demonstratedan association between t he 1α-hydroxylase promoter single nucleotidepolymorphism CYP27B1-1260 rs10877012 and sustained virologicresponse (SVR) after pegylated interferon-α ( PEG-IFN-α) plus ribavirintreatment of c hronic hepatitis C in patients w ith a p oor-response IL28Bgenotype. This suggests an intrinsic role o f vitamin D signaling in theresponse t o treatment of chronic hepatitis C, especially in patients withlimited sensitivity to IFN-α. In the present study, we investigated theeffect of 1,25-(OH)2 v itamin D3 (calcitriol) alone or in combination withIFN-α on the hepatitis C virus (HCV) life cycle in vitro.Methods: H uh-7.5 cells harboring Con1- or JFH-1-derived HCVreplicons or cell culture-derived HCV were exposed to 0.1-100 nMcalcitriol ± 1 -100 IU/ml IFN-α. The effect on HCV RNA replication andviral particle production was investigated by quantitative r eal-time PCR,immunoblot analyses, and infectivity titration analyses. The expression ofinterferon-stimulated genes (ISGs) and of calcitriol target genes wasassessed by quantitative real-time PCR.Results: Calcitriol had no relevant effect on the viability of Huh-7.5 cells.Calcitriol strongly induced and repressed the expression of the calcitrioltarget genes CYP24A1 and CCNC, respectively, confirming that Huh-7.5cells c an respond to c alcitriol signaling. P hysiological doses of calcitrioldid not significantly a ffect HCV RNA replication or i nfectious particleproduction in vitro, and calcitriol alone h ad no significant effect on theexpression of several ISGs. However, calcitriol in combination with IFN-αsubstantially increased the expression of ISGs compared to IFN-α alone.In addition, calcitriol plus IFN-α s ynergistically inhibited HCV RNAreplication.Conclusions: C alcitriol at physiological concentrations and IFN-α a ctsynergistically on the expression of I SGs and HCV RNA replication i nvitro. Experiments exploring the underlying mechanisms are underway.

Importance of IL28B gene polymorphisms in hepatitis C virus genotype 2 and 3 infected patients.

BACKGROUND & AIMS: Genetic variation in the interleukin 28B (IL28B) gene has been associated with the response to interferon-alfa/ribavirin therapy in hepatitis C virus (HCV) genotype 1-infected patients. The importance of three IL28B single nucleotide polymorphisms (rs8099917, rs12980275 and rs12979860) for HCV genotype 2/3-infected patients is unknown. METHODS: In patients with chronic hepatitis C genotype 2/3 (n=267), IL28B host genotypes (rs8099917, rs12980275 and rs12979860) were analyzed for associations with sustained virologic response (SVR) to antiviral therapy with (pegylated) interferon-alfa and ribavirin and with respect to epidemiological, biochemical, and virological parameters. For comparison, hepatitis C genotype 1 patients (n=378) and healthy controls (n=200) were included. RESULTS: The rs12979860 CC genotype, lower age, and genotype 2 were significantly associated with SVR in HCV genotype 2/3-infected patients (p=0.01, p=0.03 and p=0.03, respectively). No association was observed for rs8099917 and rs12980275. In addition, an SVR in patients with rapid virologic response (RVR) was associated with the rs12979860 CC genotype (p=0.05), while for non-RVR no association was found. Furthermore, a significant association with a higher baseline viral load was observed for all three IL28B genotypes in genotype 1/2/3-infected patients. Finally, increasing frequencies of the rs12979860 CC genotypes were observed in genotype 1- (33.9%), genotype 3- (38.9%), and genotype 2-infected (51.9%) patients in comparison with healthy controls (49.0%) (p<0.01). CONCLUSIONS: In genotype 2/3-infected patients, rs12979860 was significantly associated with SVR. The frequency of the rs12979860 CC genotype is lower in HCV genotype 1 vs. genotype 2/3 patients. All major IL28B genotypes are associated with HCV-RNA concentration.

Reduced IFNλ4 activity is associated with improved HCV clearance and reduced expression of interferon-stimulated genes

Hepatitis C virus (HCV) infections are the major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma worldwide. Both spontaneous and treatment-induced clearance of HCV depend on genetic variation within the interferon-lambda locus, but until now no clear causal relationship has been established. Here we demonstrate that an amino-acid substitution in the IFNλ4 protein changing a proline at position 70 to a serine (P70S) substantially alters its antiviral activity. Patients harbouring the impaired IFNλ4-S70 variant display lower interferon-stimulated gene (ISG) expression levels, better treatment response rates and better spontaneous clearance rates, compared with patients coding for the fully active IFNλ4-P70 variant. Altogether, these data provide evidence supporting a role for the active IFNλ4 protein as the driver of high hepatic ISG expression as well as the cause of poor HCV clearance.

Ex vivo analysis of human antigen-specific CD8+ T-cell responses: quality assessment of fluorescent HLA-A2 multimer and interferon-gamma ELISPOT assays for patient immune monitoring.

The authors developed a standardized approach for immune monitoring of antigen-specific CD8+ T cells within peripheral blood lymphocytes (PBLs) that combines direct ex vivo analysis of Melan-A/MART-1 and influenza-specific CD8+ T cells with HLA-A2/peptide multimers and interferon-gamma ELISPOT assays. Here the authors assessed the quality of results obtained with 180 PBLs from healthy donors and melanoma patients. Reproducibility of the multimer assay was good (average of 15% variation). In the absence of in vivo antigen-specific T-cell responses, physiologic fluctuations of multimer-positive T cells was low, with variation coefficients of 20% for Melan-A and 28% for influenza-specific T cells. In contrast, patients with vaccination-induced T-cell responses had significantly increased T-cell frequencies clearly exceeding physiologic fluctuations. Comparable results were obtained with ELISPOT assays. In conclusion, this approach is well suited to assess T-cell responses as biologic endpoints in clinical vaccine studies.

Alfa-toksisen Staphylococcus aureuksen aiheuttama nekrotisoiva mastiitti : tapausselostus

Summary: Gangrenous mastitis caused by alpha-toxic Staphylococcus aureus - a case report

Cloning of an interferon-gamma regulated human melanoma-associated antigen: identity to the intercellular adhesion molecule ICAM-1.

The human melanoma-associated antigen identified by the monoclonal antibody (mAb) Me14-D12 is a cell surface protein whose expression is induced by interferon-gamma (IFN-gamma). We have recently reported the molecular cloning of a genomic probe specific for the gene and mRNA of this protein. By screening with the genomic probe, we have now isolated a full length 3.0 kb cDNA from a Raji cell line-derived lambda-gt10 library. Sequence analysis of this cDNA showed a 99.8% homology with the intercellular adhesion molecule-1 (ICAM-1). Mouse Ltk- cells stably transfected with the human cDNA clone were found to express the ICAM-1 antigenic determinants detected by mAb Me14-D12 and a reference anti-ICAM-1 mAb, as judged by surface immunofluorescence. Immunoprecipitation of surface-iodinated proteins with mAb Me14-D12 revealed the presence of a 90 kD molecule with identical mobility to ICAM-1. In addition, mAb Me14-D12 could inhibit the phorbolester-stimulated aggregation of U937 cells. The findings show that the human melanoma-associated Me14-D12 antigen is the adhesion molecule ICAM-1.

Heterogeneity of the induction of HLA-DR expression by human immune interferon on glioma cell lines and their clones.

The modulation of HLA-DR and HLA-A, -B, and -C by human recombinant immune interferon (IFN-gamma) was studied on 10 malignant glioma cell lines established in our laboratory, on 8 clones or subclones derived from these lines, and on a fetal astrocyte cell line. Comparative studies were performed with recombinant leukocyte interferon (IFN-alpha). The results not only confirmed the selective activity of IFN-gamma on the modulation of HLA-DR expression, as opposed to that of IFN-alpha, but also demonstrated a marked heterogeneity in the response of glioma cell lines and their clones to the two types of IFN tested. For example, all 3 clones of an inducible cell line could be modulated to express HLA-DR, whereas only 2 of 5 clones derived from a noninducible line were modulated. This heterogeneity did not seem to be due to the absence of the receptor for IFN-gamma on the surface of these cells, since almost all of the cell lines or clones tested (17 of 19) responded to IFN-gamma by the induction or enhancement of the expression for either HLA-DR or HLA-A, -B, and -C (or both). The heterogeneity of induction was also demonstrated between clones derived from a glioma line that did not express HLA-DR after IFN-gamma treatment. The production of HLA-DR by one of the clones was abundant enough to be confirmed by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis.

Induction of macrophage nitric oxide production by interferon-gamma and tumor necrosis factor-alpha is enhanced by interleukin-10.

Interleukin-10 (IL-10) has been reported to inhibit nitric oxide (NO) synthesis and microbicidal activity of interferon-gamma (IFN-gamma)-stimulated macrophages (M phi) by preventing the secretion of tumor necrosis factor-alpha (TNF-alpha) which serves as an autocrine activating signal. We have examined the effects of recombinant IL-10 on the capacity of IFN-gamma together with exogenous TNF-alpha to induce NO synthesis by bone marrow-derived M phi. Under these conditions and in contrast to its reported deactivating potential, IL-10 strongly enhanced NO synthesis measured as nitrite (NO2-) release (half maximal stimulation at approximately 10 U/ml). IL-10 further increased NO2- production by M phi stimulated in the presence of optimal concentrations of prostaglandin E2, a positive modulator of M phi activation by IFN-gamma/TNF-alpha. Increased steady state levels of NO synthase mRNA were observed in 4-h IFN-gamma/TNF-alpha cultures and enhanced NO2(-)-release was evident 24 h but not 48 h after stimulation. These results suggest that the effects of IL-10 on M phi function are more complex than previously recognized.

Elderly age is not a negative predictive factor for virological response to therapy with pegylated interferon-α and ribavirin in chronic hepatitis C virus patients.

BACKGROUND & AIMS: Age is frequently discussed as negative host factor to achieve a sustained virological response (SVR) to antiviral therapy of chronic hepatitis C. However, elderly patients often show advanced fibrosis/cirrhosis as known negative predictive factor. The aim of this study was to assess age as an independent predictive factor during antiviral therapy. METHODS: Overall, 516 hepatitis C patients were treated with pegylated interferon-α and ribavirin, thereof 66 patients ≥60 years. We analysed the impact of host factors (age, gender, fibrosis, haemoglobin, previous hepatitis C treatment) and viral factors (genotype, viral load) on SVR per therapy course by performing a generalized estimating equations (GEE) regression modelling, a matched pair analysis and a classification tree analysis. RESULTS: Overall, SVR per therapy course was 42.9 and 26.1%, respectively, in young and elderly patients with hepatitis C virus (HCV) genotypes 1/4/6. The corresponding figures for HCV genotypes 2/3 were 74.4 and 84%. In the GEE model, age had no significant influence on achieving SVR. In matched pair analysis, SVR was not different in young and elderly patients (54.2 and 55.9% respectively; P = 0.795 in binominal test). In classification tree analysis, age was not a relevant splitting variable. CONCLUSIONS: Age is not a significant predictive factor for achieving SVR, when relevant confounders are taken into account. As life expectancy in Western Europe at age 60 is more than 20 years, it is reasonable to treat chronic hepatitis C in selected elderly patients with relevant fibrosis or cirrhosis but without major concomitant diseases, as SVR improves survival and reduces carcinogenesis.

Treatment of Recurrent Hepatitis C after Liver Transplantation with PEG-Interferon and Ribavirin.

Background and aim: Recurrent hepati tis C is a major cause of morbidity and mortality after li ver transpl ant ati on (LT), and optimal treatm ent algorithms have yet to be defined. Here, we present our experience of 22 patients with recurrent hepatitis C treated in our institution .Patients and methods: Twenty-two patients with hi stology-proven recurrent hepati tis Cafter LT were treated since 2003. Treatment was ini ti ated with pegylated interferon-a2a 135 IIg per week and ribavirin 400 mg per day in the majority of patients, and subsequent doses were adapted individllally based on on-treatment virologieal responses and c1inical and/or biochemical si de effeets.Results: On an intention-to-treat basis, ustained virological re ponse(SVR) was achieved in 12/21 (54.5%) patie nts (5/12 [41 .6%], 2/3 [67%], 4/5 [80%] and 1/2 [50%] of patients infected with genotypes 1,2,3 and 4, respectively). Two patients experieneed relap e and 6 did not respond to treatm ent (NR). Treatment duration ranged from 24 to 90 weeks. It was stopped prematurely due to adverse events in 6/22 (27.2%) patients (with SVR achieved in 2 patients, NR in 2 patients, and death of 2 patients: one patient awaiting retransplantation and a second patient with HCV-HJV co-infection and fibrosing cholestat ic hepatiti s, nine months after transplantation). Of note, SVR was achi eved in a patient \Vi th combined liver and kidney transplantation. Importantly, SVR \Vas ach ieved in some patients despite the lack ofan early virological response or HCV RNA negativity at week 24. Darbepoetin a and fil ~,'rasti m were used in 36% and 18%, respectively.Conclusion: Individually adapted treatment of recurrent hepatitis C canachieve SVR in a substantial proponion ofLT patients. Conventional stopping rules do not apply in this setting so that prolonged therapy may be useful in selected patients.

Interferon-γ Induces Expression of MHC Class II on Intestinal Epithelial Cells and Protects Mice from Colitis

Immune responses against intestinal microbiota contribute to the pathogenesis of inflammatory bowel diseases (IBD) and involve CD4(+) T cells, which are activated by major histocompatibility complex class II (MHCII) molecules on antigen-presenting cells (APCs). However, it is largely unexplored how inflammation-induced MHCII expression by intestinal epithelial cells (IEC) affects CD4(+) T cell-mediated immunity or tolerance induction in vivo. Here, we investigated how epithelial MHCII expression is induced and how a deficiency in inducible epithelial MHCII expression alters susceptibility to colitis and the outcome of colon-specific immune responses. Colitis was induced in mice that lacked inducible expression of MHCII molecules on all nonhematopoietic cells, or specifically on IECs, by continuous infection with Helicobacter hepaticus and administration of interleukin (IL)-10 receptor-blocking antibodies (anti-IL10R mAb). To assess the role of interferon (IFN)-γ in inducing epithelial MHCII expression, the T cell adoptive transfer model of colitis was used. Abrogation of MHCII expression by nonhematopoietic cells or IECs induces colitis associated with increased colonic frequencies of innate immune cells and expression of proinflammatory cytokines. CD4(+) T-helper type (Th)1 cells - but not group 3 innate lymphoid cells (ILCs) or Th17 cells - are elevated, resulting in an unfavourably altered ratio between CD4(+) T cells and forkhead box P3 (FoxP3)(+) regulatory T (Treg) cells. IFN-γ produced mainly by CD4(+) T cells is required to upregulate MHCII expression by IECs. These results suggest that, in addition to its proinflammatory roles, IFN-γ exerts a critical anti-inflammatory function in the intestine which protects against colitis by inducing MHCII expression on IECs. This may explain the failure of anti-IFN-γ treatment to induce remission in IBD patients, despite the association of elevated IFN-γ and IBD.

Cleavage of mitochondrial antiviral signaling protein in the liver of patients with chronic hepatitis C correlates with a reduced activation of the endogenous interferon system.

Hepatitis C virus (HCV) infection induces the endogenous interferon (IFN) system in the liver in some but not all patients with chronic hepatitis C (CHC). Patients with a pre-activated IFN system are less likely to respond to the current standard therapy with pegylated IFN-alpha. Mitochondrial antiviral signaling protein (MAVS) is an important adaptor molecule in a signal transduction pathway that senses viral infections and transcriptionally activates IFN-beta. The HCV NS3-4A protease can cleave and thereby inactivate MAVS in vitro, and, therefore, might be crucial in determining the activation status of the IFN system in the liver of infected patients. We analyzed liver biopsies from 129 patients with CHC to investigate whether MAVS is cleaved in vivo and whether cleavage prevents the induction of the endogenous IFN system. Cleavage of MAVS was detected in 62 of the 129 samples (48%) and was more extensive in patients with a high HCV viral load. MAVS was cleaved by all HCV genotypes (GTs), but more efficiently by GTs 2 and 3 than by GTs 1 and 4. The IFN-induced Janus kinase (Jak)-signal transducer and activator of transcription protein (STAT) pathway was less frequently activated in patients with cleaved MAVS, and there was a significant inverse correlation between cleavage of MAVS and the expression level of the IFN-stimulated genes IFI44L, Viperin, IFI27, USP18, and STAT1. We conclude that the pre-activation status of the endogenous IFN system in the liver of patients with CHC is in part regulated by cleavage of MAVS.

Minocycline promotes remyelination in aggregating rat brain cell cultures after interferon-γ plus lipopolysaccharide-induced demyelination.

Minocycline has been shown to inhibit microglia reactivity, and to decrease the severity and progression of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. It remained to be examined whether minocycline was also able to promote remyelination. In the present study, myelinating aggregating brain cell cultures were used as a model to study the effects of minocycline on microglial reactivity, demyelination, and remyelination. Cultures were treated simultaneously with two inflammatory agents, interferon-γ (IFN-γ) and lipopolysaccharide (LPS), which caused an inflammatory response accompanied by demyelination. The inflammatory response was characterized by microglial reactivity, upregulation of inflammatory cytokines and iNOS, and increased phophorylation of P38 and P44/42 mitogen activated protein (MAP) kinases. Minocycline inhibited microglial reactivity, and attenuated the increased phophorylation of P38 and P44/42 MAP kinases. Demyelination, determined by a decrease in myelin basic protein (MBP) content and immunoreactivity 48 h after the treatment with the inflammatory agents, was not prevented by minocycline. However, 1 week after demyelination was assessed, the MBP content was restored in presence of minocycline, indicating that remyelination was promoted. Concomitantly, in cultures treated with minocycline, the markers of oligodendrocyte precursors cells (OPCs) and immature oligodendrocytes NG2 and O4, respectively, were decreased compared to cultures treated with the inflammatory agents only. These results suggest that minocycline attenuates microglial reactivity and favors remyelination by enhancing the differentiation of OPCs and immature oligodendrocytes.

Reticulocyte dynamic and hemoglobin variability in hemodialysis patients treated with Darbepoetin alfa and C.E.R.A.: a randomized controlled trial.

BACKGROUND: In a simulation based on a pharmacokinetic model we demonstrated that increasing the erythropoiesis stimulating agents (ESAs) half-life or shortening their administration interval decreases hemoglobin variability. The benefit of reducing the administration interval was however lessened by the variability induced by more frequent dosage adjustments. The purpose of this study was to analyze the reticulocyte and hemoglobin kinetics and variability under different ESAs and administration intervals in a collective of chronic hemodialysis patients. METHODS: The study was designed as an open-label, randomized, four-period cross-over investigation, including 30 patients under chronic hemodialysis at the regional hospital of Locarno (Switzerland) in February 2010 and lasting 2 years. Four subcutaneous treatment strategies (C.E.R.A. every 4 weeks Q4W and every 2 weeks Q2W, Darbepoetin alfa Q4W and Q2W) were compared with each other. The mean square successive difference of hemoglobin, reticulocyte count and ESAs dose was used to quantify variability. We distinguished a short- and a long-term variability based respectively on the weekly and monthly successive difference. RESULTS: No difference was found in the mean values of biological parameters (hemoglobin, reticulocytes, and ferritin) between the 4 strategies. ESAs type did not affect hemoglobin and reticulocyte variability, but C.E.R.A induced a more sustained reticulocytes response over time and increased the risk of hemoglobin overshooting (OR 2.7, p = 0.01). Shortening the administration interval lessened the amplitude of reticulocyte count fluctuations but resulted in more frequent ESAs dose adjustments and in amplified reticulocyte and hemoglobin variability. Q2W administration interval was however more favorable in terms of ESAs dose, allowing a 38% C.E.R.A. dose reduction, and no increase of Darbepoetin alfa. CONCLUSIONS: The reticulocyte dynamic was a more sensitive marker of time instability of the hemoglobin response under ESAs therapy. The ESAs administration interval had a greater impact on hemoglobin variability than the ESAs type. The more protracted reticulocyte response induced by C.E.R.A. could explain both, the observed higher risk of overshoot and the significant increase in efficacy when shortening its administration interval.Trial registrationClinicalTrials.gov NCT01666301.