Distinct cytokine patterns in Occult Hepatitis C and Chronic Hepatitis C Virus Infection

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Background&Aim: The immunopathogenesis of chronic hepatitis C virus (HCV)
infection is a matter of great controversy. The imbalance of T-helper lymphocyte cells cytokine production were believed to play an important pathogenic role in chronic viral hepatitis. Occult hepatitis C infection is regarded as a new entity that should be considered when diagnosing patients with a liver disease of unknown origin. The aim of this study was to determine serum T-helper 1 and T-helper 2 cytokine production in patients with occult HCV infection and its role in its pathogenesis versus chronic viral hepatitis C infection. Key words: Occult HCV, Chronic hepatitis C, Cytokines.

Introduction:
Hepatitis C virus (HCV) is a commonly encountered pathogen in medical practice. It is estimated that 2-3% of the world is affected by HCV, with a prevalence of 170 million people (3% of the world's population) and incidence of 3-4 million per year (1). Chronic HCV infection is a progressive disease that may lead to liver cirrhosis and hepatocellular carcinoma. The hallmark of chronic hepatitis C is the presence of anti-HCV and HCV RNA in serum for more than 6 months after acute infection (2,3). Patients with occult HCV infection presents a milder disease than patients with chronic hepatitis C. The low number of infected hepatocytes found in patients with occult HCV infection may be related with less liver damage. Furthermore, immune response of these patients may be finetuned better than that of patients with chronic hepatitis C, leading to a more effective control of the infection (6).
The aim of this work is to study the Th1/Th2 cytokine profiles in patients with occult HCV infection versus patients with chronic hepatitis C.

Samples:
1. Cytokines assay: From each patient two ml venous blood was collected into clean dry plastic tube and allowed to clot, the yielded serum was used for quantitative detection of IL-2, IL-4 and IFN-γ serum levels which were measured with enzyme-linked immunosorbent assay (ELISA) kits (Bender Med Systems, Vienna, Austria). 5

Liver biopsy
The patients underwent an ultrasound-guided liver biopsy (using Tru-cut needles) for diagnostic purposes. Liver biopsy specimens were divided into 2 portions. One portion was fixed in 10% formalin and was paraffin embedded for routine histological diagnosis. Histological evaluation was performed by a pathologist who was blinded with respect to the HCV RNA status of the liver biopsy specimens. Necroinflammatory activity and fibrosis were scored according to the METAVIR score system (18,19

Total RNA extraction and purification
Lysate (600 µl) with ethanol from the previous step was applied to the assembled column and collection tube and centrifuged for 1 minute. The flowthrough was discarded and the spin column and collection tube was reassembled and the process was repeated until the lysate finishes. After this 400 µl of wash solution was added to the column and centrifuged for one minute, the flowthrough was discarded and the process repeated twice. Furthermore, several negative controls (no-RNA) were included in each PCR step, to assure the specificity of the results.

Statistical analysis:
The statistical analysis of data done by using excel program and SPSS program statistical package for social science version 10. The description of the data done in form of mean (+/-) SD for quantitative data and 8 frequency & proportion for qualitative data. The analysis of the data was done to test statistical significant difference between groups. For quantitative date student t-test was used to compared between 2 gp paired sample t-test was used to compare one gp at different measurements. A 2-tailed P value <0.05 was considered to denote statistical significance.

Discussion:
Several studies have suggested that T-cell immunoregulatory cytokines play a key role in both HCV viral persistence and in the extent of liver damage, while some cytokines may exert a pro-inflammatory activity, such as IFN-α, IL-8, TNF-α and IL-2, which can prime T-cells towards a Th-1 type immunity, others have a predominantly anti-inflammatory activity, as is the case for IL-4 and IL-10, which are involved in Th-2 immunity.
The aims of the present study were (1)