Einfluss einer Hepatitis C-Virus-Infektion auf die Interaktion von natürlichen Killerzellen mit aktivierten hepatischen Sternzellen

Im Mausmodell konnte gezeigt werden, dass natürliche Killerzellen (NK)-Zellen in der Lage sind, durch Apoptose-Induktion in aktivierten hepatischen Sternzellen anti-fibrotisch zu wirken. Allerdings blieb die Rolle von humanen NK-Zellen bei der Fibroseprogression weiterhin unklar. Darüber hinaus fehlen Untersuchungen, in wieweit sich eine Hepatitis C-Virus (HCV)-Infektion auf die anti-fibrotische Aktivität von NK-Zellen auswirkt, obwohl Infektionen mit HCV zu den häufigsten Ätiologien einer Leberfibrose gehören. Um dies zu klären, wurden NK-Zellen aus gesunden Spendern und HCVPatienten mit primären aktivierten hepatischen Sternzellen koinkubiert und anschließend der Anteil der apoptotischen Sternzellen durchflusszytometrisch mittels intrazellulärer aktiver Caspase-3 Färbung ermittelt. Anhand dieser Experimente konnte gezeigt werden, dass NK-Zellen aus HCV-Patienten im Vergleich zu NK-Zellen aus gesunden Probanden effektiv Apoptose in primären aktivierten hepatischen Sternzellen auslösen. Dabei korrelierte die anti-fibrotische NK-Zell-Aktivität invers mit dem Fibrosegrad des Patienten, da NK-Zellen aus HCV-Patienten mit einer geringen Leberfibrose deutlich effektiver Apoptose in hepatischen Sternzellen induzierten als NK-Zellen aus Patienten mit einer fortgeschrittenen Fibrose. Zusätzlich ergaben Blockierungsexperimente eine wichtige Rolle von NKG2D, TRAIL und FasL bei der NK-Zell-vermittelten Apoptose-Induktion in hepatischen Sternzellen, wobei weitere Faktoren involviert sein dürften. Zudem induzierten NK-Zellen aus Interferon-α therapierten Patienten am effizientesten Apoptose in hepatischen Sternzellen. Im Einklang mit dieser Beobachtung führte die in vitro Stimulation von NK-Zellen mit rekombinantem Interferon-α zu einer gesteigerten anti-fibrotischen NK-Zell-Aktivität, die durch eine Hochregulation von TRAIL vermittelt wurde. Somit konnte nachgewiesen werden, dass auch humane NK-Zellen eine anti-fibrotische Aktivität besitzen und eine wichtige Rolle bei der Modulation der HCV-induzierten Leberfibrogenese spielen. Im zweiten Teil dieser Arbeit wurden die Auswirkungen einer HIV/HCV-Koinfektion auf die Fibroseprogression untersucht, da infolge der zusätzlichen HIV-Infektion die HCV-induzierte Leberfibrose bei diesen Patienten deutlich schneller voranschreitet als bei HCV-monoinfizierten Patienten. In diesem Zusammenhang deckten eine ganze Reihe von epidemiologischen Studien auf, dass eine geringe CD4+ T-Zellzahl mit einem fortgeschrittenen Fibrosestadium assoziiert ist. Des Weiteren entwickelt sich die Fibrose bei Patienten mit niedrigen CD4+ T-Zellzahlen deutlich schneller, als bei Patienten mit einer physiologisch normalen CD4+ T-Zellzahl. Allerdings war bisher völlig unklar, welche Rolle die CD4+ T-Zellen bei der Modulation der Leberfibrose einnehmen. Um dieser Fragestellung nachzugehen, wurden NK-Zellen mit dem Überstand von CD3/CD28 aktivierten CD4+ T-Zellen stimuliert und anschließend mit primären aktivierten hepatischen Sternzellen koinkubiert. Die nachfolgende Analyse der apoptotischen Sternzellen ergab, dass CD4+ TZellen in der Lage sind, über eine IL-2 abhängige Hochregulation von NKG2D, die anti-fibrotische Aktivität von NK-Zellen zu stimulieren. Darüber hinaus wurde beim Vergleich von CD4+ T-Zellen aus gesunden und HCV-infizierten Probanden eine gestörte IL-2 Sekretion bei CD4+ T-Zellen infolge einer HIV/HCV-Koinfektion beobachtet, die in vitro mit einer verminderten Stimulation der antifibrotischen NK-Zell-Aktivität einherging. Daher könnte der HIV-assoziierte Verlust von CD4+ T-Zellen sowie die reduzierte IL-2 Sekretion zu einer beschleunigten Fibroseprogression bei einer HIV/HCV-Koinfektion beitragen. Außerdem konnte damit erstmalig eine mögliche Erklärung geliefert werden, warum die CD4+ T-Zellzahl mit der Fibroseprogression bei einer HIV/HCV-Koinfektion assoziiert ist

Future perspectives on in-vitro diagnosis of drug allergy by the lymphocyte transformation test

Acknowledgement We thank the European fund for regional development (EFRE), the German Federal State North Rhine-Westphalia (LeitmarktAgentur.NRW), Federal Institute for Drugs and Medical Devices (BfArM), and the Leibniz Institute for Analytical Sciences - ISAS-e.V. for the research project grant. The position of A.F. is financed by the research grant. Funding source The manuscript was written in context with a study related to the improvement of the lymphocyte transformation test which was funded by the European Fund for Regional Development (EFRE) and the German Federal State North Rhine-Westphalia (LeitmarktAgentur.NRW) (funding number: EFRE-0801755) and from own resources of the Federal Institute for Drugs and Medical Devices (BfArM) and the Leibniz Institute for Analytical Sciences - ISAS-e.V., Dortmund, Germany.Peer reviewedPublisher PD

TRAIL receptor I (DR4) polymorphisms C626G and A683C are associated with an increased risk for hepatocellular carcinoma (HCC) in HCV-infected patients

<p>Abstract</p> <p>Background</p> <p>Tumour surveillance via induction of TRAIL-mediated apoptosis is a key mechanism, how the immune system prevents malignancy. To determine if gene variants in the TRAIL receptor I (<it>DR4</it>) gene affect the risk of hepatitis C virus (HCV)-induced liver cancer (HCC), we analysed <it>DR4 </it>mutations C626G (rs20575) and A683C (rs20576) in HCV-infected patients with and without HCC.</p> <p>Methods</p> <p>Frequencies of <it>DR4 </it>gene polymorphisms were determined by LightSNiP assays in 159 and 234 HCV-infected patients with HCC and without HCC, respectively. 359 healthy controls served as reference population.</p> <p>Results</p> <p>Distribution of C626G and A683C genotypes were not significantly different between healthy controls and HCV-positive patients without HCC. <it>DR4 </it>variants 626C and 683A occurred at increased frequencies in patients with HCC. The risk of HCC was linked to carriage of the 626C allele and the homozygous 683AA genotype, and the simultaneous presence of the two risk variants was confirmed as independent HCC risk factor by Cox regression analysis (Odds ratio 1.975, 95% CI 1.205-3.236; p = 0.007). Furthermore HCV viral loads were significantly increased in patients who simultaneously carried both genetic risk factors (2.69 ± 0.36 × 10⁶ IU/ml vs. 1.81 ± 0.23 × 10⁶ IU/ml, p = 0.049).</p> <p>Conclusions</p> <p>The increased prevalence of patients with a 626C allele and the homozygous 683AA genotype in HCV-infected patients with HCC suggests that these genetic variants are a risk factor for HCC in chronic hepatitis C.</p

Lymphocyte transformation test for drug allergy detection: When does it work?

BACKGROUND: The lymphocyte transformation test (LTT) is an in vitro test system for the detection of a sensitization in the context of allergies to drugs. Its reported sensitivity varies largely and seems to be affected by different parameters. In review articles, the average LTT performance was often calculated by combining overall mean sensitivities of various published studies, but without considering different patient characteristics or varying patient numbers per publication. OBJECTIVE: To investigate the impact of different patient-specific and methodological parameters on the sensitivity of the LTT based on data on the level of the individual patient extracted from single studies. METHODS: We performed an advanced literature search in PubMed and screened the identified publications according to previously defined inclusion criteria. In total, individual patient data from 721 patients were extracted from 30 studies. Random-effects meta-regression analyses were performed. RESULTS: The analysis indicate that the enzyme-linked immunosorbent assay–based read-out is more sensitive compared with the classical radioactivity method (enzyme-linked immunosorbent assay: 80% vs radioactivity: 66%; P = .08). Interestingly, drug reaction with eosinophilia and systemic symptoms/drug-induced hypersensitivity syndrome is associated with a higher probability of a positive LTT test result compared with other investigated clinical phenotypes (“drug reaction with eosinophilia and systemic symptoms/drug-induced hypersensitivity syndrome” vs “bullous reaction”; odds ratio, 2.52; P value = .003). Our analysis also revealed an impact of the time to testing period after the occurrence of the allergic event (“&lt; 2 weeks” vs “2 weeks-2 months”; odds ratio, 2.12; P value = .03). CONCLUSION: The read-out method and relevant clinical parameters affect the sensitivity of the LTT. These findings are based on a meta-analysis providing a higher level of evidence than a single study or previous reviews not considering individual patient data

The CXCR3(+)CD56Bright phenotype characterizes a distinct NK cell subset with anti-fibrotic potential that shows dys-regulated activity in hepatitis C.

BACKGROUND: In mouse models, natural killer (NK) cells have been shown to exert anti-fibrotic activity via killing of activated hepatic stellate cells (HSC). Chemokines and chemokine receptors critically modulate hepatic recruitment of NK cells. In hepatitis C, the chemokine receptor CXCR3 and its ligands have been shown to be associated with stage of fibrosis suggesting a role of these chemokines in HCV associated liver damage by yet incompletely understood mechanisms. Here, we analyzed phenotype and function of CXCR3 expressing NK cells in chronic hepatitis C. METHODS: Circulating NK cells from HCV-infected patients (n = 57) and healthy controls (n = 27) were analyzed with respect to CXCR3 and co-expression of different maturation markers. Degranulation and interferon-γ secretion of CXCR3(+) and CXCR3(-) NK cell subsets were studied after co-incubation with primary human hepatic stellate cells (HSC). In addition, intra-hepatic frequency of CXCR3(+) NK cells was correlated with stage of liver fibrosis (n = 15). RESULTS: We show that distinct NK cell subsets can be distinguished based on CXCR3 surface expression. In healthy controls CXCR3(+)CD56Bright NK cells displayed strongest activity against HSC. Chronic hepatitis C was associated with a significantly increased frequency of CXCR3(+)CD56Bright NK cells which showed impaired degranulation and impaired IFN-γ secretion in response to HSC. Of note, we observed intra-hepatic accumulation of this NK cell subset in advanced stages of liver fibrosis. CONCLUSION: We show that distinct NK cell subsets can be distinguished based on CXCR3 surface expression. Intra-hepatic accumulation of the functionally impaired CXCR3(+)CD56Bright NK cell subset might be involved in HCV-induced liver fibrosis

The ratio of calprotectin to total protein as a diagnostic and prognostic marker for spontaneous bacterial peritonitis in patients with liver cirrhosis and ascites

AbstractDiagnosis of spontaneous bacterial peritonitis (SBP) is based on a differential ascites leukocyte count which does not provide prognostic information. We performed a pilot study to assess calprotectin in ascites as an alternative diagnostic and prognostic marker.We collected ascites from patients with liver cirrhosis from March 2012 to July 2013. Routine clinical and laboratory data of the patients were recorded. Ascites calprotectin levels were determined by ELISA.Overall, we collected 120 ascites samples from 100 patients with liver cirrhosis and from eight patients with malignant peritoneal effusion as disease control. Samples without infection had significantly lower calprotectin levels (median 34 ng/mL, range 5–795) than SBP samples (median 928 ng/mL, range 21–110,480; p&lt;0.001) and malignant effusions (median 401, range 47–2596 ng/mL; p&lt;0.001). In non-infected ascites, calprotectin levels were higher in Child-Pugh stage B versus C (median 57 ng/mL vs. 17 ng/mL; p&lt;0.001) and in alcoholic versus viral cirrhosis (median 37 ng/mL vs. 10 ng/mL; p=0.015). The ratio of ascites calprotectin to total protein was a better marker for SBP than calprotectin alone (AUROC=0.93; p&lt;0.001; sensitivity 93%, specificity 79%; positive predictive value 60%; negative predictive value 97%). In addition, high levels of the ratio to total protein were associated with poor 30-day survival (p=0.042).The ratio of ascites calprotectin to total protein may be a promising new diagnostic and prognostic marker in patients with liver cirrhosis and SBP and should be evaluated further.</jats:p