Glycyrrhizin Exerts Antioxidative Effects in H5N1 Influenza A Virus-Infected Cells and Inhibits Virus Replication and Pro-Inflammatory Gene Expression

Glycyrrhizin is known to exert antiviral and anti-inflammatory effects. Here, the effects of an approved parenteral glycyrrhizin preparation (Stronger Neo-Minophafen C) were investigated on highly pathogenic influenza A H5N1 virus replication, H5N1-induced apoptosis, and H5N1-induced pro-inflammatory responses in lung epithelial (A549) cells. Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher). Glycyrrhizin also diminished monocyte migration towards supernatants of H5N1-infected A549 cells. The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen species and (in turn) reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication. Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease

Determination of hepatitis C virus-specific CD4/CD8 T-lymphocytes using flow cytometry

Das Hepatitis C-Virus ist eines der am weitesten verbreiteten persistierenden humanen Viren, mit dem weltweit über 170 Millionen Menschen chronisch infiziert sind. Bei über 80 Prozent der infizierten Personen kommt es zu einer chronischen Infektion, 20 bis 30 Prozent der Patienten erkranken an Leberzirrhose und bei 2,5 Prozent treten hepatozelluläre Karzinome auf. Gegenwärtig existiert keine Impfung gegen HCV, die derzeitige antivirale Therapie ist mit starken Nebenwirkungen verbunden, und überhaupt nur bei 50-60 Prozent der behandelten Patienten erfolgreich. Zudem stellen die Folgen einer Infektion mit HCV heute in den USA und Europa die vorherrschende Ursache für die Notwendigkeit einer Lebertransplantation dar. Es ist anzunehmen, dass im Verlauf einer Infektion mit dem Hepatitis C-Virus der zellulären Immunantwort eine Schlüsselrolle zukommt. Ziel dieser Arbeit war es, ein Testsystem zu etablieren, mit dessen Hilfe HCV-spezifische T-Lymphozyten mittels Durchflusszytometrie gemessen werden können, um so einen Beitrag zum besseren Verständnis für die Immunantwort gegen das Hepatitis C-Virus zu leisten. Das System des Lymphozytenstimulationstestes soll außerdem als Modell für die Messung der Immunantwort gegen weitere Viren dienen, wie das verwandte Klassische Schweinepest-Virus oder das Bovine Virusdiarrhoe/Mucosal Disease (BVD/MD)-Virus. Das bessere Verständnis der zellulären Immunantwort kann zur Entwicklung von Impfstoffen sowie zur Prävention und Therapie einer Erkrankung entscheidend beitragen. Die vorliegende Arbeit etabliert ein Testsystem zur Messung HCV-spezifischer T-Lymphozyten. Da das Hepatitis C-Virus nur sehr ineffizient in Zellkultur repliziert, gibt es verschiedene Surrogatmodelle, um das Virus und seine Pathogenese genauer zu untersuchen. Vielfach werden verschiedene rekombinante HCV-Proteine eingesetzt, um die Lymphozytenfunktion zu überprüfen. Es bleibt allerdings unklar, inwieweit solche Antigene in vivo tatsächlich den T-Zellen präsentiert werden. Als neues Stimulationsmodell setzt diese Arbeit HCV-Pseudotypen ein. Dabei bestehen die HCVpp aus lentiviralen Core-Partiken, die an ihrer Oberfläche unmodifizierte HCV E1- und E2-Hüllglykoproteine tragen, um so als Pseudotypen das native Hepatitis C-Virus zu imitieren. Die Verwendung von HCVpp im Lymphozytenstimulationstest führte zu vergleichbaren Ergebnissen wie die Protein-Stimulation, wobei die HCVpp zu einer stärkeren Stimulation der CD4-Lymphozyten führten. Falls sich diese Überlegenheit der HCVpp in weiteren Studien bestätigen würde, wäre dies ein großer Vorteil gegenüber der herkömmlichen Stimulation mit HCV-Proteinen, da die CD4-Lymphozyten eine entscheidende Rolle im Verlauf der HCV-Infektion spielen. Die Auswertung der Patientendaten ergab eine Th2-Verschiebung der Lymphozytenantwort bei denjenigen Patienten, die das Virus nicht beseitigen konnten. Zudem lassen die Ergebnisse vermuten, dass eine antivirale Therapie zu einer Rekonstitution oder Entwicklung einer Th1-Antwort und damit zur erfolgreichen Überwindung der HCV-Infektion führen könnte. Warum bei einigen Patienten die Therapie jedoch versagt, bleibt in weiteren Studien zu klären.The hepatitis C virus is one of the most prevalent persistent human viruses, more than 170 million humans worldwide are chronically infected. In more than 80 percent of infected persons the infection causes chronicity, which leads in 20 to 30 percent of patients to cirrhosis of the liver and in 2.5 percent to hepatocellular carcinoma. There is still no vaccine against HCV, the current antiviral therapy has strong side effects, and the treatment is successful in only 50 to 60 percent of patients. An infection with HCV is the leading cause for liver transplantation in the United States and Europe. The cellular immune response is supposed to play an important role in the course of infection with the HCV. The objective of this work was to establish a test system for the analysis of HCV-specific T lymphocytes by flow cytometry to achieve a better understanding of the immune response against the hepatitis C virus. In addition, the system could serve as a model for analysing the immune response against other viruses such as the related Classical Swine Fever Virus or Bovine Virus Diarrhea/Mucosal Disease (BVD/MD) Virus. The enhanced understanding of the cellular immune response can support the development of vaccines as well as be helpful for the prevention and treatment of illness. In this study, a test system for the analysis of HCV-specific T lymphocytes could be established. Given that the hepatitis C virus replicates very insufficiently in a cell culture, there are several surrogate markers for the virus and its pathogenesis. Different recombinant HCV proteins are used for the investigation of lymphocyte function. But it still remains unclear, how such antigens are presented to the T cells in vivo. In this study, HCV pseudoparticles (HCVpp) are used as a new model for the specific stimulation of T cells. The HCVpp consist of lentiviral core particles which are carrying unmodified HCV E1 and E2 glycoproteins on their surface. This pseudoparticles mimic the native hepatitis C virus. The use of HCVpp for the stimulation of antigen specific T lymphocytes led to similar results in comparison to the stimulation with HCV proteins. Notably, in CD4+ lymphocytes the stimulation with HCVpp resulted in stronger cytokine responses. As CD4+ lymphocytes play a crucial role in the course of HCV infections, this superiority of the HCVpp is a big advantage over conventional stimulation approaches with HCV proteins. However, these enhanced stimulation properties remain to be confirmed in further studies. The analysis of patient data showed a shift to Th2 response for those patients who could not eliminate the virus. Moreover, the results suggest that the antiviral therapy leads to a reconstitution or development of a Th1 response, and thus a successful resolution of HCV infection could result. The reason for therapy failure in some patients remains to be scrutinized in further studies

Measurement of cytotoxic T lymphocyte activity of human cytomegalovirus seropositive individuals by a highly sensitive coupled luminescent method.

A coupled luminescent method (CLM) based on glyceraldehyde-3-phosphate dehydrogenase released from injured target cells was used to evaluate the cytotoxicity of antigen-specific HLA class I-restricted CTLs. In contrast to established methods, CLM does not require the pretreatment of target cells with radioactive or toxic labeling substances. CTLs from healthy HLA-A2 positive donors were stimulated by autologous dendritic cells (DCs) pulsed with HLA-A2 restricted HCMV-pp65 nonamer peptides. HLA-A2 positive T2 cells or autologous monocytes pulsed with HCMV-pp65 nonamer peptide served as target cells. Lysis was detected only in HCMV-pp65-pulsed target cells incubated with CTLs from seropositive donors stimulated by HCMV-pp65-pulsed DCs. After 3 days, stimulation 38% of T2 cells and 17% of monocytes were lysed at an effector to target ratio of 8:1. In conclusion, CLM represents a highly sensitive, fast, material-saving and non-toxic/non-radioactive method for the measurement of antigen-specific CTL cytotoxic activity

Glycyrrhizin inhibits highly pathogenic H5N1 influenza A virus-induced pro-inflammatory cytokine and chemokine expression in human macrophages.

Hypercytokinaemia is thought to contribute to highly pathogenic H5N1 influenza A virus disease. Glycyrrhizin is known to exert immunomodulatory and anti-inflammatory effects and therefore a candidate drug for the control of H5N1-induced pro-inflammatory gene expression. Here, the effects of an approved parenteral glycyrrhizin preparation were investigated on H5N1 virus replication, H5N1-induced pro-inflammatory responses, and H5N1-induced apoptosis in human monocyte-derived macrophages. Glycyrrhizin 100 ?g/ml, a therapeutically achievable concentration, impaired H5N1-induced production of CXCL10, interleukin 6, and CCL5 and inhibited H5N1-induced apoptosis but did not interfere with H5N1 replication. Global inhibition of immune responses may result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8(+) T-lymphocytes. Notably, glycyrrhizin concentrations that inhibited H5N1-induced pro-inflammatory gene expression did not affect cytolytic activity of natural killer cells. Since H5N1-induced hypercytokinaemia is considered to play an important role within H5N1 pathogenesis, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease

N-acetyl-L-cysteine (NAC) inhibits virus replication and expression of pro-inflammatory molecules in A549 cells infected with highly pathogenic H5N1 influenza A virus.

The antioxidant N-acetyl-L-cysteine (NAC) had been shown to inhibit replication of seasonal human influenza A viruses. Here, the effects of NAC on virus replication, virus-induced pro-inflammatory responses and virus-induced apoptosis were investigated in H5N1-infected lung epithelial (A549) cells. NAC at concentrations ranging from 5 to 15 mM reduced H5N1-induced cytopathogenic effects (CPEs), virus-induced apoptosis and infectious viral yields 24 h post-infection. NAC also decreased the production of pro-inflammatory molecules (CXCL8, CXCL10, CCL5 and interleukin-6 (IL-6)) in H5N1-infected A549 cells and reduced monocyte migration towards supernatants of H5N1-infected A549 cells. The antiviral and anti-inflammatory mechanisms of NAC included inhibition of activation of oxidant sensitive pathways including transcription factor NF-kappaB and mitogen activated protein kinase p38. Pharmacological inhibitors of NF-kappaB (BAY 11-7085) or p38 (SB203580) exerted similar effects like those determined for NAC in H5N1-infected cells. The combination of BAY 11-7085 and SB203580 resulted in increased inhibitory effects on virus replication and production of pro-inflammatory molecules relative to either single treatment. NAC inhibits H5N1 replication and H5N1-induced production of pro-inflammatory molecules. Therefore, antioxidants like NAC represent a potential additional treatment option that could be considered in the case of an influenza A virus pandemic