Hd Nischalke

Natural killer p46High expression defines a natural killer cell subset that is potentially involved in control of hepatitis C virus replication and modulation of liver fibrosis

Natural killer (NK) cells play a role in the early control and natural course of hepatitis C virus (HCV) infection. NK cell function is regulated by a multitude of receptors, including activating NKp46 receptor. However, reports on NKp46 in hepatitis C are controversial. Therefore, we investigated the hepatic recruitment and function of NKp46(+) NK cells, considering differential surface expression of NKp46 resulting in NKp46High and NKp46Dim subsets. Intra‐ and extrahepatic NK‐cell subsets from HCV‐infected patients were characterized by flow cytometry. Cytotoxic activity and interferon‐gamma (IFN‐γ) secretion were studied using K‐562, P815, and primary hepatic stellate cells as targets. Anti‐HCV activity of NK‐cell subsets was studied using the replicon system. Density of NKp46 surface expression clearly segregated NKp46Dim and NKp46High subsets, which differed significantly with respect to the coexpression of maturation markers and NK‐cell receptors. More important, NKp46High NK cells showed a higher cytolytic activity and stronger IFN‐γ secretion than NKp46Dim NK cells. Accordingly, NKp46High NK cells efficiently blocked HCV replication in vitro. Blocking experiments confirmed an important role for the NKp46 receptor. Furthermore, we found an intrahepatic accumulation of NKp46High NK cells. Of note, high cytolytic activity of NKp46High NK cells was also confirmed in the intrahepatic NK‐cell population, and the frequency of intrahepatic NKp46High NK cells was inversely correlated with HCV‐RNA levels and fibrosis stage. Conclusions: NKp46High expression defines a specific NK‐cell subset that may be involved in both the suppression of HCV replication and HCV‐associated liver damage underpinning the role of NK cells in the immunopathogenesis of HCV. (HEPATOLOGY 2012)

The toll‐like receptor 2 (TLR2) ‐196 to ‐174 del/ins polymorphism affects viral loads and susceptibility to hepatocellular carcinoma in chronic hepatitis C

Chronic hepatitis C virus (HCV) infection is a major risk factor for hepatocellular carcinoma (HCC). HCV proteins core and NS3 can bind to toll‐like receptor 2 (TLR2) and trigger inflammatory responses. Polymorphisms in the TLR2 gene predispose to various forms of malignancy but have not been studied in HCV‐associated HCC. Here, we investigated whether single nucleotide polymorphisms (SNPs), rs4696480, rs5743708, rs5743704 and the ‐196 to ‐174 del/ins polymorphism of the TLR2 gene affect the risk for HCC in chronic hepatitis C. The study involved 189 and 192 HCV genotype 1 infected patients with and without HCC, respectively, as well as 347 healthy controls. TLR2 alleles were determined by hybridization probe assays and allele‐specific short fragment polymerase chain reaction on a LightCycler system. All TLR2 polymorphisms matched the Hardy–Weinberg equilibrium in each study group. Although TLR2 SNPs showed no effect, the frequency of the TLR2 ‐196 to ‐174 del allele was significantly higher in patients with HCV‐associated HCC (22.5%) than in HCV‐infected patients without HCC (15.6%, p = 0.016) and healthy controls (15.3%, p = 0.003). HCV‐infected carriers of a TLR2 ‐196 to ‐174 del allele had significantly higher HCV viral loads than TLR2 ‐196 to ‐174 ins/ins homozygous patients (p = 0.031). Finally, in carriers of the TLR2 ‐196 to ‐174 del allele, stimulation of monocytes resulted in significantly lower TLR2 expression levels and interleukin‐8 (IL‐8) induction than in individuals with the TLR2 ‐196 to ‐174 ins/ins genotype (p < 0.05). Our data suggest the TLR2 ‐196 to ‐174 del allele to affect HCV viral loads and to increase the risk for HCC in HCV genotype1‐infected patients.

Genetic Analyses Reveal a Role for Vitamin D Insufficiency in HCV-Associated Hepatocellular Carcinoma Development

Background Vitamin D insufficiency has been associated with the occurrence of various types of cancer, but causal relationships remain elusive. We therefore aimed to determine the relationship between genetic determinants of vitamin D serum levels and the risk of developing hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). Methodology/Principal Findings Associations between CYP2R1, GC, and DHCR7 genotypes that are determinants of reduced 25-hydroxyvitamin D (25[OH]D3) serum levels and the risk of HCV-related HCC development were investigated for 1279 chronic hepatitis C patients with HCC and 4325 without HCC, respectively. The well-known associations between CYP2R1 (rs1993116, rs10741657), GC (rs2282679), and DHCR7 (rs7944926, rs12785878) genotypes and 25(OH)D3 serum levels were also apparent in patients with chronic hepatitis C. The same genotypes of these single nucleotide polymorphisms (SNPs) that are associated with reduced 25(OH)D3 serum levels were found to be associated with HCV-related HCC (P = 0.07 [OR = 1.13, 95% CI = 0.99–1.28] for CYP2R1, P = 0.007 [OR = 1.56, 95% CI = 1.12–2.15] for GC, P = 0.003 [OR = 1.42, 95% CI = 1.13–1.78] for DHCR7; ORs for risk genotypes). In contrast, no association between these genetic variations and liver fibrosis progression rate (P>0.2 for each SNP) or outcome of standard therapy with pegylated interferon-α and ribavirin (P>0.2 for each SNP) was observed, suggesting a specific influence of the genetic determinants of 25(OH)D3 serum levels on hepatocarcinogenesis. Conclusions/Significance Our data suggest a relatively weak but functionally relevant role for vitamin D in the prevention of HCV-related hepatocarcinogenesis.

Hepatic and HSC-specific sorafenib effects in rats with established secondary biliary cirrhosis

Portal hypertension in cirrhosis depends on increased intrahepatic vascular resistance, which is explained by fibrosis and intrahepatic hyperresponsiveness to vasoconstrictors. Both are caused by activation and proliferation of hepatic stellate cells (HSCs). Portal hypertension of cirrhotic rats can be reduced by the multikinase inhibitor sorafenib, due to a reduction of intrahepatic vascular resistance. Therefore, the hepatic effects of sorafenib require further understanding. Here, we investigated hepatic and HSC-specific sorafenib effects in cirrhotic rats. Animal models of bile duct ligation-induced secondary biliary cirrhosis in rats were studied. The rats were treated with sorafenib (60 mg/kg/day) for 1 week, starting after established cirrhosis. Histological evaluation was carried out using hemalaun and eosin (HE) staining. Apoptosis was studied by PARP cleavage, colorimetric caspase-3 assay, and electrophoretic DNA detection. HSC activation was studied by hepatic Sirius red and immunohistochemical αSMA (α-smooth muscle actin) staining, and by in vitro experiments with culture-activated primary HSCs. Biochemical serum parameters suggested the occurrence of sorafenib-induced liver damage. HE staining revealed histological changes in livers of sham-operated and bile duct-ligated (BDL) rats in response to sorafenib, which were different in both groups. In BDL rats and isolated HSCs, the treatment with sorafenib reduced hepatic αSMA and procollagen-1α mRNA expression. As shown by immunohistochemical staining, perisinusoidal αSMA expression was reduced by sorafenib in BDL rats. This was associated with reduced perisinusoidal deposition of extracellular matrix, as revealed by Sirius red staining. Although no change in PARP cleavage and only a minor increase in hepatic caspase-3 activity were detected in BDL rats in response to sorafenib, livers of sorafenib-treated BDL rats contained small DNA fragments, which were not observed in untreated BDL rats. In conclusion, sorafenib treatment reduces the number of activated HSCs in cirrhotic livers. This leads to the decrease in intrahepatic vascular resistance, but also to liver damage in the dosage we used. Therefore, any translation to portal hypertensive patients who may profit from sorafenib should be done with particular care.

Binding of HCV E2 to CD81 induces RANTES secretion and internalization of CC chemokine receptor 5

Summary.  Hepatitis C virus (HCV) infection has been shown to be associated with reduced expression of the CC chemokine receptor (CCR) 5, and reduced responsiveness of lymphocytes to chemokines. However, the mechanism by which HCV alters CCR5 expression remains unclear. Here, we investigated whether altered CCR5 expression in hepatitis C results from interactions of CD81 with the HCV E2 protein. Peripheral blood mononuclear cells (PBMC) from HCV‐negative individuals were prepared by Ficoll density gradient separation. PBMC subpopulations (CD4+, CD8+ lymphocytes, CD19+ B cells, natural killer (NK) cells and monocyte‐derived dendritic cells) were isolated and stimulated with immobilized HCV E2, and changes in CCR5 expression and CC‐chemokine secretion were determined. Migration assays were performed using a 5‐μm nitrocellulose filter microchamber system according to the manufacturers recommendations. Exposure of PBMC to HCV E2 induced a dose‐dependent release of regulated on activation normal T‐cell‐expressed and secreted (RANTES), down‐regulation of CCR5 expression and intracellular accumulation of CCR5. This effect was blocked by preincubation of PBMC with anti‐CD81. RANTES release following exposure to HCV E2 was mainly attributable to CD8+ cells. After exposure to HCV E2 markedly fewer CD8‐positive lymphocytes were attracted by RANTES when compared with CD8+ cells that were studied in the absence of HCV E2. Our results suggest that interaction of HCV E2 with CD81 leads to increased RANTES secretion by CD8+ lymphocytes which induces down‐regulation of CCR5 surface via receptor internalization resulting in altered lymphocyte migration.

Selective pressures of HLA genotypes and antiviral therapy on human immunodeficiency virus type 1 sequence mutation at a population level

ABSTRACT The objective of this study was a comprehensive analysis of the immune-driven evolution of viruses of human immunodeficiency virus type 1 (HIV-1) clade B in a large patient cohort treated at a single hospital in Germany and its implications for antiretroviral therapy. We examined the association of the HLA-A, HLA-B, and HLA-DRB1 alleles with the emergence of mutations in the complete protease gene and the first 330 codons of the reverse transcriptase (RT) gene of HIV-1, studying their distribution and persistence and their impact on antiviral drug therapy. The clinical data for 179 HIV-infected patients, the results of HLA genotyping, and virus sequences were analyzed using a variety of statistical approaches. We describe new HLA-associated mutations in both viral protease and RT, several of which are associated with HLA-DRB1. The mutations reported are remarkably persistent within our cohort, developing more slowly in a minority of patients. Interestingly, several HLA-associated mutations occur at the same positions as drug resistance mutations in patient viruses, where the viral sequence was acquired before exposure to these drugs. The influence of HLA on thymidine analogue mutation pathways was not observed. We were able to confirm immune-driven selection pressure by major histocompatibility complex (MHC) class I and II alleles through the identification of HLA-associated mutations. HLA-B alleles were involved in more associations (68%) than either HLA-A (23%) or HLA-DRB1 (9%). As several of the HLA-associated mutations lie at positions associated with drug resistance, our results indicate possible negative effects of HLA genotypes on the development of HIV-1 drug resistance.

The tandem-repeat polymorphism of the DC-SIGNR gene in HCV infection

Summary.  The C‐type lectin DC‐SIGNR has been shown to bind hepatitis C virus (HCV). Here, we analysed the tandem‐repeat polymorphism of the DC‐SIGNR gene with respect to intraindividual HCV replication. In a cross‐sectional comparison HCV‐infected patients (n = 430) and healthy subjects (n = 100) were genotyped for the DC‐SIGNR polymorphism using PCR. The distribution of DC‐SIGNR alleles did not differ significantly between the two groups. However, HCV‐infected patients with 5‐, 6‐, and 7‐repeat alleles had higher HCV‐RNA levels when compared with carriers of 4‐ and 9‐repeat alleles (P < 0.05). Thus, the DC‐SIGNR polymorphism might affect HCV loads supporting the concept that DC‐SIGNR contributes to HCV replication efficacy.

Regulation of NK cell trafficking by CD81

NK cells, a heterogeneous sub‐population of lymphocytes, are critically involved in the regulation of both innate and adaptive immune responses in humans. Besides their participation in the control of tumors and viral infections, they also regulate inflammatory processes, mediating both beneficial and detrimental effects. To effectively fulfil their role in immune surveillance, proper trafficking of NK cells is essential. However, the mechanisms and factors governing NK cell recruitment are only poorly dissected. Here, we describe the functional role of tetraspanins, a family of evolutionary conserved cell‐surface proteins, in modulating migration and transmigration of human NK cells. We demonstrate expression of various tetraspanins on NK cells. Furthermore, we show that stimulation of the NK cell‐expressed tetraspanin CD81 induces phosphorylation of ezrin/radixin/moesin proteins and leads to NK cell polarization thereby facilitating NK cell migration toward various chemokines/cytokines. Finally, we provide evidence for a role of CD81 in promoting adhesion of NK cells to components of the extracellular matrix, a prerequisite for extravasation of lymphocytes in inflamed tissues. Thus, our data suggest that the tetraspanin CD81 is importantly involved in the regulation of NK cell recruitment.

CD27(+)CD56Bright natural killer cells may be involved in spontaneous clearance of acute hepatitis C in HIV-positive patients.

Objective:The objective of this study was to analyse the potential role of CD27 in natural killer (NK) cell-mediated control of hepatitis C virus (HCV) infection in HIV-positive patients. Design:Frequency of CD27-expressing CD56Bright NK cells was analysed in HIV mono-infected individuals and HIV-positive patients with acute or chronic hepatitis C. Anti-HCV activity of CD27(+) and CD27(−) NK cells was compared. Methods:NK cell mediated inhibition of HCV replication was analysed using the HUH7 HCV Replicon model. NK cell phenotype and interferon (IFN) secretion was studied by flowcytometry. Results:High frequency of CD27(+)CD56Bright NK cells is associated with spontaneous clearance of acute hepatitis C in HIV-positive patients. Accordingly, we found CD27(+)CD56Bright NK cells to display strong anti-HCV activity. Conclusion:Our results underline the important role of NK cells in modulating outcome of HCV infection.

Genetic polymorphisms associated with fatty liver disease and fibrosis in HIV positive patients receiving combined antiretroviral therapy (cART)

Hepatic steatosis can occur with any antiretroviral therapy (cART). Although single nucleotide polymorphisms (SNPs) have been identified to predispose to alcoholic and non-alcoholic fatty liver disease, their role for treatment-associated steatosis in HIV-positive patients remains unclear. We determined the frequency of PNPLA3 (rs738409), CSPG3/NCAN (rs2228603), GCKR (rs780094), PPP1R3B (rs4240624), TM6SF (rs8542926), LYPLAL1 (rs12137855) and MBOAT7 (rs626283) by RT-PCR in 117 HIV-positive patients on cART and stratified participants based on their “controlled attenuation parameter” (CAP) into probable (CAP: 215–300 dB/m) and definite (CAP >300 dB/m) hepatic steatosis. We analyzed CAP values and routine metabolic parameters according to the allele frequencies. Sixty-five (55.6%) and 13 (11.1%) patients were allocated to probable and definite steatosis. CAP values (p = 0.012) and serum triglycerides (p = 0.043) were increased in carriers of the GCKR (rs780094) A allele. Cox logistic regression identified triglycerides (p = 0.006), bilirubin (p = 0.021) and BMI (p = 0.068), but not the genetic parameters as risk factors for the occurrence of hepatic steatosis. Taken together, according to the limited sample size, this exploratory study generates the hypothesis that genetic polymorphisms seem to exert minor effects on the risk for fatty liver disease in HIV-positive patients on cART. Nevertheless, SNPs may modify metabolic complications once metabolic abnormalities have developed. Hence, subsequent analysis of a larger cohort is needed.