Ludger Leifeld

The HLA-A2 Restricted T Cell Epitope HCV Core35–44 Stabilizes HLA-E Expression and Inhibits Cytolysis Mediated by Natural Killer Cells

Impaired activity of natural killer cells has been proposed as a mechanism contributing to viral persistence in hepatitis C virus (HCV) infection. Natural cytotoxicity is regulated by interactions of HLA-E with inhibitory CD94/NKG2A receptors on natural killer (NK) cells. Here, we studied whether HCV core encodes peptides that bind to HLA-E and inhibit natural cytotoxicity. We analyzed 30 HCV core-derived peptides. Peptide-induced stabilization of HLA-E expression was measured flow cytometrically after incubating HLA-E-transfected cells with peptides. NK cell function was studied with a (51)chromium-release-assay. Intrahepatic HLA-E expression was analyzed by an indirect immunoperoxidase technique and flow cytometry of isolated cells using a HLA-E-specific antibody. We identified peptide aa35-44, a well-characterized HLA-A2 restricted T cell epitope, as a peptide stabilizing HLA-E expression and thereby inhibiting NK cell-mediated lysis. Blocking experiments confirmed that this inhibitory effect of peptide aa35-44 on natural cytotoxicity was mediated via interactions between CD94/NKG2A receptors and enhanced HLA-E expression. In line with these in vitro data we found enhanced intrahepatic HLA-E expression on antigen-presenting cells in HCV-infected patients. Our data indicate the existence of T cell epitopes that can be recognized by HLA-A2 and HLA-E. This dual recognition may contribute to viral persistence in hepatitis C.

Autoimmunity induced by interferon-α therapy for chronic viral hepatitis

Type I interferons, which are mostly alpha-interferons (either as single agents or in combination with antiviral drugs), are currently the standard therapy for chronic viral hepatitis B, B/D, and C. Side-effects are not uncommon and include exacerbation of pre-existing autoimmune disorders or the de novo induction of autoimmunity. These adverse effects are attributed to the immunomodulatory properties of type I interferons, and should be distinguished from autoimmunity associated with chronic viral hepatitis in which interferon treatment may indeed be beneficial. The major autoimmune side-effects of interferon therapy for chronic viral hepatitis are thyroid or liver disease. Therefore, screening for thyroid antibodies and auto-antibodies indicative of autoimmune hepatitis both before, during, and after interferon therapy is strongly recommended. The presence of high concentrations of thyroid auto-antibodies or antibodies associated with autoimmune hepatitis can be contraindicative to interferon therapy. However, treatment is not contraindicated in viral hepatitis (in particular chronic hepatitis C) associated with autoimmune phenomena--including low-titer thyroid antibodies or other non-organ specific auto-antibodies. If interferon-induced autoimmunity occurs, the necessity of therapy has to be balanced carefully against the risks of autoimmune disease. Further research is needed to identify the factors which determine susceptibility to interferon-associated autoimmunity in individual patients.

Early up-regulation of chemokine expression in fulminant hepatic failure

CC‐chemokines recruit and activate macrophages and T lymphocytes, the major components of inflammatory infiltrates in fulminant hepatic failure (FHF). To analyse the role of CC‐chemokines in the pathogenesis of FHF, this study examined serum levels and intrahepatic expression of MCP‐1, MIP‐1α, MIP‐1β, and RANTES in the livers and sera of patients with FHF and controls by ELISA, immunohistochemistry, and competitive RT‐PCR. Serum levels and intrahepatic expression of all chemokines studied in FHF exceeded the levels in chronic liver diseases and normal controls. Distinct patterns of expression of each chemokine were noted on Kupffer cells, sinusoidal endothelial cells, hepatocytes, lymphocytes, and bile ducts. Intrahepatic chemokine expression correlated closely with the extent of infiltration by macrophages and T lymphocytes (r = 0.65–0.95, p < 0.001). The functional relationship between intrahepatic chemokine release and infiltration was confirmed in chemotaxis assays by inhibiting chemotaxis induced by homogenates of liver tissue obtained from FHF patients with neutralizing MCP‐1, MIP‐1α, MIP‐1β, and RANTES antibodies. The time course of CC‐chemokine release was studied in the concanavalin A and the galactosamine/LPS mouse models of FHF. In both models, intrahepatic chemokine up‐regulation occurred as an early event prior to hepatic infiltration and liver damage. The data indicate that an abundant intrahepatic release of CC‐chemokines is an early and pivotal step in the pathogenesis of FHF. Copyright

Hepatitis C virus E2 and CD81 interaction may be associated with altered trafficking of dendritic cells in chronic hepatitis C

Dendritic cells (DC) are crucially involved in the induction of immune responses; however, reports on DC functions in chronic hepatitis C are controversial. Function of DC includes proper cell trafficking between sites of infection and lympho‐cellular compartments. Thus, we analyzed DC compartmentalization and changes in DC migration in hepatitis C virus (HCV)‐infected patients. We found significantly lower numbers of circulating BDCA1+ and BDCA2+ DC in HCV(+) patients (n = 20) than in healthy controls (n = 12) (P < .05). Analyzing liver samples from HCV(+) patients (n = 15), HCV(−) controls (n = 15), and disease controls (n = 10), we demonstrated chronic hepatitis C to be associated with intrahepatic DC enrichment (P < .05). In vitro studies indicated that HCV E2‐induced secretion of RANTES efficiently attracts CCR5(+) immature DC. Incubation of DC with sera derived from HCV(+) patients made DC unresponsive to CCL21, the chemokine recruiting DC to lymphoid tissues for T cell priming. Unlike attraction of CCR5+ DCs via RANTES, direct inhibition of DC migration in response to CCL21 was specific for patients with chronic hepatitis C and could be attributed to interaction of HCV E2 with CD81 on DC. In conclusion, migration of DC is markedly affected by interaction of HCV E2 with CD81. Failure of DC to recirculate to lymphoid tissue may be critically involved in impaired T cell priming during HCV infection. (HEPATOLOGY 2006;44:945–954.)

Intrahepatic upregulation of RhoA and Rho-kinase signalling contributes to increased hepatic vascular resistance in rats with secondary biliary cirrhosis

Background and aims: Portal hypertension in cirrhosis is mediated in part by increased intrahepatic resistance, reflecting an increased sensitivity of the hepatic microvasculature to vasoconstrictors. Activation of the RhoA/Rho-kinase pathway is essential for contraction of vascular smooth muscle. The aim of this study was to investigate RhoA/Rho-kinase mediated regulation of the intrahepatic vascular tone in cirrhotic rats. Methods: Cirrhosis was induced by bile duct ligation (BDL). Hepatic RhoA and Rho-kinase expressions were studied by real time reverse transcription polymerase chain reaction and western blot analysis. Hepatic Rho-kinase activity in rat and human livers was assessed as phosphorylation of the Rho-kinase substrate moesin. The effect of the Rho-kinase inhibitor Y-27632 on hepatic perfusion pressure was measured in livers perfused at constant flow. The in vivo effect of intravenous application of Y-27632 was studied by haemodynamic measurements. Results: Hepatic expressions of RhoA and Rho-kinase were increased at mRNA and protein level in BDL rats. Intrahepatic moesin phosphorylation was increased in livers from cirrhotic rats and patients with alcohol induced cirrhosis. Y-27632 reduced the basal perfusion pressure of in situ perfused livers in BDL rats but not in sham operated rats. Y-27632 reduced the sensitivity to methoxamine in isolated perfused livers in sham operated rats more than in BDL rats. In vivo, Y-27632 reduced portal pressure to a greater extent in BDL rats than in sham operated rats. Intrahepatic vascular resistance was decreased in response to bolus injection of Y-27632 in BDL rats but not in sham operated rats. Conclusions: Upregulation of RhoA and Rho-kinase contributes to increased intrahepatic resistance in cirrhotic rats and to an increased sensitivity of cirrhotic livers to vasoconstrictors.

Hemodynamic effects of urotensin II and its specific receptor antagonist palosuran in cirrhotic rats

In cirrhosis, splanchnic vasodilation contributes to portal hypertension, subsequent renal sodium retention, and formation of ascites. Urotensin II(U‐II) is a constrictor of large conductive vessels. Conversely, it relaxes mesenteric vessels, decreases glomerular filtration, and increases renal sodium retention. In patients with cirrhosis, U‐II plasma levels are increased. Thus, we investigated hemodynamic and renal effects of U‐II and its receptor antagonist, palosuran, in cirrhotic bile duct–ligated rats (BDL). In BDL and sham‐operated rats, we studied acute effects of U‐II (3 nmol/kg; intravenously) and palosuran (10 mg/kg; intravenously) and effects of oral administration of palosuran (30 mg/kg/day; 3 days) on hemodynamics and renal function. We localized U‐II and U‐II‐receptor (UTR) in livers and portal veins by immunostaining. We determined U‐II‐plasma levels by enzyme‐linked immunosorbent assay (ELISA), and mesenteric nitrite/nitrate‐levels by Griess‐reaction. RhoA/Rho‐kinase and endothelial nitric oxide synthase (eNOS) pathways were determined by western blot analysis and reverse transcription polymerase chain reaction (RT‐PCR) in mesenteric arteries. U‐II plasma levels, as well as U‐II and UTR‐receptor expression in livers and portal veins of cirrhotic rats were significantly increased. U‐II administration further augmented the increased portal pressure (PP) and decreased mean arterial pressure (MAP), whereas palosuran decreased PP without affecting MAP. The decrease in PP was associated with an increase in splanchnic vascular resistance. In mesenteric vessels, palosuran treatment up‐regulated expression of RhoA and Rho‐kinase, increased Rho‐kinase‐activity, and diminished nitric oxide (NO)/cyclic guanosine 3′,5′‐monophosphate (cGMP) signaling. Moreover, palosuran increased renal blood flow, sodium, and water excretion in BDL rats. Conclusion: In BDL rats, U‐II is a mediator of splanchnic vasodilation, portal hypertension and renal sodium retention. The U‐II‐receptor antagonist palosuran might represent a new therapeutic option in liver cirrhosis with portal hypertension. (HEPATOLOGY 2008.)

Inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) expression in fulminant hepatic failure

BACKGROUND/AIMS Inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) have important functions in inflammation and vasoregulation but their role in fulminant hepatic failure (FHF) is not well understood. METHODS Intrahepatic in situ staining and semi-quantification of iNOS and eNOS by immunohistochemistry in 25 patients with FHF, in 40 patients with chronic liver diseases (CLD) and in ten normal controls (NC). RESULTS Expression patterns of iNOS and eNOS differed. While in NC only faint iNOS expression was found in some Kupffer cells/macrophages and hepatocytes, eNOS was expressed constitutively in sinusoidal and vascular endothelial cells. In CLD, iNOS expression was induced in Kupffer cells/macrophages and hepatocytes, representing the main iNOS expressing cell types. Additionally, bile ducts, vascular endothelial cells and lymphocytes also expressed iNOS (P = 0.001). In contrast, no differences were found between eNOS expression in CLD and NC (P = 0.64). The same cell types expressed eNOS and iNOS in FHF but numbers of both were significantly enhanced, exceeding the levels seen in CLD (P < 0.001, P = 0.017). CONCLUSIONS Our data demonstrate that iNOS and eNOS are differently regulated in physiologic conditions and in liver disease. While eNOS seems to be involved in the physiological regulation of hepatic perfusion, strong upregulation of iNOS might contribute to inflammatory processes in FHF.

Diverticular disease: guidelines of the german society for gastroenterology, digestive and metabolic diseases and the german society for general and visceral surgery.

Background: Diverticular disease is one of the most common disorders of the gastrointestinal tract. 28-45% of the population develop colonic diverticula, while about 25% suffer symptoms and about 5% complications. Aim: To create formal guidelines for diagnosis and management. Methods: Six working groups with 44 participants analyzed key questions in subject areas assigned to them. Following a systematic literature search, 451 publications were included. Consensus was obtained by agreement within the working groups, two Delphi processes and a guideline conference. Results: Targeted management of diverticular disease requires a classificatory diagnosis. A new classification was created. In addition to the clinical examination, intestinal ultrasound or computed tomography is the determining factor. Interval colonoscopy is recommended to exclude comorbidities. A low-fiber diet, obesity, lack of exercise, smoking and immunosuppression have an adverse impact on diverticulosis. This can lead to diverticulitis. Antibiotics are no longer recommended in uncomplicated diverticulitis if no risk factors such as immunosuppression are present. If close monitoring is ensured, uncomplicated diverticulitis can be treated on an outpatient basis. Complicated diverticulitis should be treated in hospital, involving broad-spectrum antibiotic therapy, where necessary abscess drainage, and surgery, if possible laparoscopically. In the case of chronic relapsing diverticulitis, the risk of perforation decreases with each episode, so that surgery is no longer recommended after the second episode but only following individual assessment. Conclusions: New findings on diverticular disease call into question the overuse of antibiotics and excessive indications for surgery. Targeted treatment requires a precise diagnosis and intensive interdisciplinary cooperation. i 2014 S. Karger AG, Basel

Reduced CC chemokine receptor (CCR) 1 and CCR5 surface expression on peripheral blood T lymphocytes from patients with chronic hepatitis C infection

T cell recruitment to the infected liver is an essential step for the efficient elimination of hepatitis viruses. The surface expression of CC chemokine receptor (CCR) 1, CCR4, and CCR5 on peripheral blood T lymphocytes and their responsiveness to the chemokines macrophage inflammatory proteins (MIP)-1alpha, MIP-1beta, and RANTES (regulated on activation, normally T cell-expressed and secreted) was analyzed in patients with chronic hepatitis C and hepatitis B infection and compared with healthy subjects. Although CCR4 surface expression was not altered, hepatitis C virus (HCV)-infected patients had lower proportions of CD8 T cells with CCR1 and CCR5 surface expression (P<.05). Migration of CD8 T cells in response to MIP-1alpha, MIP-1beta, and RANTES was significantly reduced in HCV-infected patients (P<.05). Intracellular CCR1 and CCR5 protein and messenger RNA levels in peripheral blood T cells did not indicate reduced chemokine receptor biosynthesis in hepatitis C infection. Thus, chronic hepatitis C, but not hepatitis B, infection alters surface expression of distinct CCRs, resulting in lower CC chemokine responsiveness.

TH1 to TH2 shift of cytokines in peripheral blood of HIV-infected patients is detectable by reverse transcriptase polymerase chain reaction but not by enzyme-linked immunosorbent assay under nonstimulated conditions.

Background: Dysregulation of cytokines has been implicated in the pathogenesis of HIV infection. Therefore, we determined tumor necrosis factor‐a (TNF‐&agr;), interleukin‐l&bgr; (IL‐1&bgr;), IL‐4, IL‐10, and interferon‐&ggr; (IFN‐&ggr;) mRNA and serum levels in HIV‐infected patients under nonstimulated conditions. Material and Methods: Blood samples of 32 HIV‐infected patients and 10 healthy HIV‐negative controls were analyzed. Cytokine serum levels were quantified by enzyme‐linked immunosorbent assay (ELISA). Cytokine mRNA levels were determined semiquantitatively by competitive reverse transcriptase polymerase chain reaction (RT‐PCR) and expressed as ratios relative to those of &bgr;‐actin. Results: Competitive RT‐PCR was shown to be more sensitive than protein ELISA in analyzing cytokine production. We found a significant correlation between steady‐state mRNA ratios and serum protein levels for TNF‐&agr;. Significantly higher cytokine mRNA ratios were found in those patients with IL‐10 and IFN‐&ggr; levels detectable by ELISA. Steady‐state mRNA ratios of TNF‐&agr;, IL‐4, and IL‐10 were significantly increased in patients with highly replicative HIV‐infection. Furthermore, elevated IL‐4:IFN‐&ggr; ratios were related to both high viral load and loss of CD4 cells. Discussion: Determination of steady‐state mRNA ratios by semiquantitative RT‐PCR represents a sensitive method to analyze cytokines in peripheral blood of HIVinfected patients under nonstimulated conditions. The data obtained with this technique provide further evidence for a TH1 to TH2 cytokine shift with progressive HIV disease.