Expression Of Human Tim-1 And Tim-3 In PBMC From Hepatitis B Patients

Tim family members are type I membrane glycoproteins expressed on T cells and containing common structural motifs, namely Ig V domain, highly glycosylated mucin domain and cytoplasmic domain. Tim family consists of 3 genes on syntenic human chromosome 5q33.2 with no other intervening genes. The human Tim family members are Tim-1, Tim-3 and Tim-4. All of them characterized thus far appear to have a role in the regulation of Th1 and Th2 differentiation and T cell mediated immune responses. Tim-1 is expressed on all activated T cells and at a higher level on Th2 than on Th1 cells.Tim-3 protein is specifically expressed on Th1 cells and negatively regulates Th1 responses. It has reported that the expression of Tim family members in some autoimmune disease patients is different from that of controls and some SNPs of Tims have relationship with autoimmune diseases, both of which indicate that they are related to autoimmune diseases.Hepatitis B is hepatocytic inflammation caused by hepatitis B vims (HBV) which is widespread in our country with serious influence. Chronic HBV infections can lead to liver failure with cirrhosis and hepatocellular carcinoma. At present, it is known that hepatitis B virus is not directly cytopathic for the infected hepatocytes. HBV-induced liver injury and viral clearance are mediated by the host's immune response to various components of this vims. Human CD4 T cells are known to be functionally heterogenous, containing two distinct Th subsets: Th1 producing interferon-gamma (IFN-γ) and IL-2 which are important for the activation of macrophages and for the clearance of intracellular pathogens, and Th2 releasing IL-4, IL-5 and EL-10 which are especially important for IgE production and for eosinophilic inflammation. The imbalance of Th1/Th2 cytokines plays an important role for viral clearance and disease pathogenesis in HBV infection. It is still unclear whether Tims play role in hepatitis B.In this paper, the expression of Tim-1,Tim-3 in PBMC (peripheral blood monoculear cells) from hepatitis B patients and healthy controls are assayed by fluorescent quantitative real time RT-PCR (FQ real time RT-PCR) and half quantitative RT-PCR .This work provides a new insight to study the mechanism of hepatitis B and reveal the relationship between Tim gene and hepatitis B.AIMTo analyze the expression of Tim-1 and Tim-3 in PBMC from hepatitis B patients , and to explore the mechanism of Tim-1, Tim-3 in hepatitis B development.MATERIAL AND METHODS1 COLLECTION OF THE SAMPLESThe studies were conducted with a group of 63 patients (57 men and 6 wemen), age 16-69 years (mean age of 38±14.8 years), with well documented diagnosis of chronic hepatitis B. In none of the cases infection with type C virus and other autoimmune diseases co-existed. The control group consisted of 34 healthy men and women with no history of HBV infection matched at ages with patients group. They were negative for HBV infection markers and showed normal serum ALT levels. Patients of the control group were studied as negative controls.2 MATHONDSAccording to the cDNA sequences of human Tim-1 ,Tim-3 a in PUBMED ,the primers were designed by Primier Primer 5.0 software. The mRNA expression of Tim-1 , Tim-3 in PBMCs from hepatitis B patients were assayed by FQ real time RT-PCR and relatively Quantitative RT-PCR.RESULTS1 Comparison of the concordance of two methods, fluorescent quantitative real time PCR and routine relatively Quantitative RT-PCR, which is used to detect Tim-1 and Tim-3In this study, we used two methods, fluorescent quantitative real time RT-PCR and routine relatively Quantitative RT-PCR, to detect the expression of Tim-1 and Tim-3, and to compare the concordance between these two methods. The results showed fluorescent quantitative real time RT-PCR had good specificity and compare to routine relatively Quantitative RT-PCR, fluorescent quantitative real time RT-PCR had very favorable concordance.2 Expression of Tim-1 and Tim-3 mRNA in PBMC from hepatitis B patientsThe expression of Tim-1 , Tim-3 in PBMCs from hepatitis B patients were assayed by FQ real time RT-PCR. 2^ΔCT is used to denote the expression value of Tim-1 and Tim-3. Results showed that the expression value of Tim-1 in PBMC from chronic hepatitis B patients is 1. 405 + 0. 6307, which is higher than health controls (0.8908±0.3881) (t test, P<0.01) , while there is no difference of the expression of Tim-1 between acute hepatitis B patients( 2.381±1.05)and health controls (t test, P= 0.20) . In parallel, the expression value of Tim-3 in PBMC from acute hepatitis B patients and chronic hepatitis B patients isl.48±0.6724 and 1.502±0.7619 respectively, which are all higher than health controls(0.7294±0.6297) (t test, P< 0.01 and P< 0.05) .3 Analysis of the correlation between the expression of Tim-1 ,Tim-3 from hepatitis B patients and ALTThe result showed that the expression of Tim-3 mRNA and ALT from chronic hepatitis B patients had positive relation, and the correlation coefficient (r) is 0.317, P<0.05, and there is no correlation between the expression of Tim-1 and ALT from chronic hepatitis B patients.4 Analysis of the correlation between the expression of Tim-1/Tim-3 and HBV DNA in peripheral blood from chronic hepatitis B patientsExpression of Tim-1 mRNA from chronic hepatitis B patients correlated well with HBV DNA at r=0.357 (P< 0.05). There is no correlation of the expression of Tim-3 from chronic hepatitis B patients with HBV DNA.5 The correlation between the expression of Tim-1 and Tim-3 mRNA in PBMC and HBeAg(+)/HBeAg(-)groupsThere is fairly difference in epidemiology, clinical character, prognosis and therapy. So chronic hepatitis B patients were divide to tow groups according to HBeAg and analyzed the expression of Tim-1 and Tim-3 in HBeAg(+)/HBeAg(-) groups respectively. Then the result show the expression value of Tim-3 in PBMC from HBeAg(-) chronic hepatitis B patients was conspicuous higher than HBeAg(+) group(P< 0.05), while there was no difference of the expression of Tim-1 between the two groups.6 Comparison and analysis the expression of Tim-1 and Tim-3 mRNA in peripheral blood from patients before and after clinic treatmentAfter analysis the expression of Tim-1 and Tim-3 mRNA before and after the clinic treatment in the same individual (4 groups) among the chronic HBV infected patients, we found that 3 of them who covered from liver damage had decreased Tim-3 expression while the other one who still had high ALT expressed higher Tim-3. This results together with result 3 strongly suggested Tim-3 correlated with ALT and could be used as a marker of liver tissue damage.CONCLUSIONThe results of this study showed, in the process of HBV onset and chronicity, Tim-1 and Tim-3 participate in development of the disease and play very important role.Tim-3 mRNA and ALT in HBV patients peripheral blood has positive correlation, so Tim-3 could be used to detect active extent of chronic HBV disease.