| Objective1. To investigate the functions of NK cells in hepatitis C virus infection and outcome after antiviral therapy.2. To compare functional differences of NK cells between patients with HCV infection and healthy controls both in vivo and in vitro.3. To analyse the functional changes of NK cells in HCV RNA+ patients before and during antiviral therapy, especially in patients with different viral load and genotype.Methods1. Collecting fresh heparinized venous blood of subjects including: 1) Patients in chronic HCV infection (n=49), those who were not on IFN-αtreatment currently or recently (n=30) and who were receiving antiviral therapy (n=19) constituted this group. 2) Patients who achieved spontaneous (untreated) viral clearance after HCV infection (n=5). 3) Patients in sustained virological response (SVR) after antiviral treatment (n=18). 4) Patients not in SVR after standard therapy (n=17). 5) Healthy controls (n=18).2. Hepatitis C markers were determined by commercial enzyme immunoassay kits. HCV genotype was performed by PCR-SSP. Patients who were receiving antiviral therapy were detected viral titer at baseline, 4 week, 12 week, 24 week of treatment, using Real time Quantitative Polymerase Chain Reaction, and other patients were detected when they were enrolled into this study.3. PBMCs were isolated from subjects by EZ-SepTM Human Lymphocyt Separation tube. Cytotoxicity of NK cells against the target cell lines K562 were assayed using CFSE/PI by flow cytometry.4. The analysis of frequency, phenotypes, receptors and cytokines secretion of NK cells was performed by flow cytometry via stained PBMCs using antibodies.5. PBMCs from some healthy and patients were cultured in the presence of rhIL-2 and rhIL-15 for 72 hours, then the receptors and cytokines of NK cells were determined by flow cytometry.6. Figures were processed by Flowjo softwere, datas were analysed using apporopriate descriptive statistics by Statistical Package for Social Sciences (SPSS) version 13.0, and p-values of<0.05 were considered significant.Results1. The frequency, phenotypes, cytotoxicity and cytokine profiles of NK cells in different groups(1).Untreated HCV patients showed a strikingly reduced proportion of cytotoxicity, NKG2D and IFN-γ, TNF-αin comparison with healthy controls (p<0.01); had a significant lower level of NKG2C compared to those who cleared virus spontaneously (p<0.05), presented significantly decreased percentage of NKp30, NKp44 and NKG2A compared to patients who were in SVR (p<0.05).(2).Compared to those who were in SVR, patients who were not in SVR presented a prominent decreased cytotoxicity; showed less frequency of NKG2D, IFN-γand TNF-αthan healthy controls, NKG2A was opposite simultaneously. (p<0.05).(3).Patients in SVR showed more percentage of NKp46, NKp30, NKp44, NKG2D and NKG2A than those who were not in SVR, also with a parallel cytokine production of NK cells, but there was no statistic difference.(4).We found those who achieved spontaneous viral clearance had a significant higher level of NK cells, NKp46, NKG2C expression,accompanied with vigonus TNF-αsecretion compared to patients not in SVR (p<0.05).2. After cultivation, PBMCs from untreated HCV infected patients showed less frequencies of NK cell than controls(p<0.01), on the contrary, higher proportion of NKG2A and NKG2C than controls (p<0.05). Patients not in SVR displayed a diminished ability to secrete cytokines compared to healthy controls (p<0.05).3. Viral titer and ALT showed no correlation with functional markers of NK cells in untreated patients and those who were not in SVR.4. Changes of phenotypes, receptors and functions of NK cells in patients being on antiviral treatment.(1). Patients with Genotype 2 or 3 and in lower viral levels showed expanded CD56bright subset and reduced CD56dim subset during therapy, however patients with Genotype 1 and higher viral titer not.(2). Expansion of proportion of NKG2A with diminished frequences of active receptors was found in patients with Genotype 1 and higher viral load during treatment.(3). NK cells presented reduced cytotoxicity and ability to produce cytokine, especially in patients with Genotype 1 and in higher viral levels.Conclusion1. Functions of NK cells were associated with HCV infection and outcome. Impaired activities of NK cells were observed in patients with chronic HCV infection. But those who eliminated HCV spontaneously or in SVR displayed more active functions of NK cells than chronically infected patients.2. Activities of NK cells both in vivo and in vitro were different between patients with HCV infection and healthy controls.3. Viral titer and ALT showed no correlation with functional markers of NK cells in untreated patients and those who were not in SVR.4. Functions of NK cells were not restored, even severely reduced by treatment in patients with Genotype 1 and higher viral load compared to patients with Genotype 2 or 3 and lower virus titer who showed slightly changes.
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