الفهرس 
INTRODUCTION
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Abstract
Hepatitis C virus (HCV) infection is gaining increasing attention as a global health crisis. Egypt reported the highest prevalence of HCV worldwide of genotype 4. HCV infected persons have three times higher death rates than those of age-matched general population. Approximately, 8–10 million people of Egyptians gave serological evidence of HCV infection. The current standard therapy achieves a sustained virological response (SVR) in approximately 55% of patients, with significant differences between genotypes. The management of patients with CHC is complex and challenging, due to the potential AEs of antiviral therapies and common co-morbidities often found in this group of patients. The use of soluble inflammatory markers (chemotactic cytokines) has been examined over the past ten years as predictors of both liver staging and response to therapy in CHC patients. IP-10/ CXCL10, is interferon-inducible protein of 10 kDa and the tenth member of the CXC family of small chemotactic cytokines. CXCL10 is produced by lymphoid cells, hepatocytes and endothelial cells in response to interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α) and to HCV proteins such as NS5A and core and it serves to recruit activated T-helper 1 (Th1), natural killer (NK) cells, macrophages, and dendritic cells cells for normal host defense against intracellular pathogens. Induction of CXCL10 mRNA in response to IFN-γ is seen as early as 30min, with peak levels seen after 5h. Signaling intermediates requisite to CXCL10 gene expression include Jak-2 and STAT1. It is expressed in liver tissue from patients with hepatitis C infection, and relates to hepatic infiltration with CXCR3-positive T cells within the hepatic lobules. Expression of CXCL10 is more specific for hepatic inflammation than that of other CXCR3 ligands, and is undetectable in normal liver tissue.
So, the present work was designed to study the serum level of CXCL10/IP-10 as a rapid and an early virological response predictor to combination therapy (PEG-IFN plus ribavirin) in chronic hepatitis C patients and its correlation with pretreatment histopathological grading and staging according to modified Ishak’s scoring system. Also, the present work was aiming to find technically feasible and financially reasonable serum biomarker, which could be used in the pretreatment assessment of candidate patients in conjunction with other predictors.
To achieve this goal, 30 chronic hepatitis C patients and 15 healthy subjects were included in the study. They all were candidate to receive anti HCV combination therapy. Patients who had received previous anti-viral therapy or systemic anti-cancer therapy or contraindications to interferon and ribavirin therapy before the study were excluded. None of the patients had concomitant schistosomiasis; history of alcohol consumption, obesity, bleeding diathesis, hepatic decompensation, chronic diseases such as diabetes mellitus or connective tissue diseases and malignancies.
All patients were evaluated clinically as regards age, sex, manifestations of advanced liver disease, liver and spleen size, systemic examination to exclude presence of any contraindications to combination therapy (head and neck examination for thyroid or lymph node swelling, cardiac and chest examination),and anthropometric measures (weight and height) to calculate body mass index (BMI).
Routine investigations were done including; complete blood picture, fasting and 2 hours post prandial blood sugar, blood urea level, serum creatinine, lipid profile (serum cholesterol and triglyceride level), thyroid function test [thyroid stimulating hormone (TSH), free triiodothyronin (T3), free tetraiodothyronin (T4)l and iver test profile [Serum aspartate and alanine aminotransferases (AST and ALT), serum albumin, serum bilirubin, serum gamma glutamyl transpeptidase (GGT) and prothrombin time. Also, viral Testing were done including (Anti-HCV antibodies, hepatitis B surface antigen (HBs Ag) and total hepatitis B core antibodies (HBc Ab total), anti-human immunodeficiency virus antibody (HIV Ab) using enzyme linked immunosorbant assay and HCV-RNA detection by quantitative polymerase chain reaction (PCR) technique a real-time PCR assay with a lower limit of detection of 10–20 IU/ml). All patients were subjected to measurement of serum levels of CXCL10/IP-10 three times during the study: before start of combination therapy (PEG-INF plus ribavirin), after 4 weeks for rapid virological response and after 12 weeks for early virological response. Abdominal Ultrasonography was done for all patients for assessment of liver (size, echogenicity, and cirrhosis), spleen, ascites, and to exclude associated illness. Also, liver biopsy was done for all patients as routine prerequisites for antiviral therapy to evaluate the degree of activity for chronic hepatitis (grading), and the presence or absence of fibrosis (staging) according to modified histological activity index (Modified HAI; Ishak scoring system).
Patients were monitored during therapy to assess the response to treatment and for the occurrence of side effects. The schedule will be monthly visits during the first 12 weeks of treatment. At each visit the patient will be: questioned regarding the presence of side effects, they were also be queried about adherence to treatment and laboratory monitoring was done including measurement of complete blood count, serum creatinine, ALT levels and quantitative HCV RNA at weeks 4 and 12. Thyroid function was monitored monitored at week 12th while on treatment.
Statistical analysis of the data was performed using Data were fed to the computer using the Predictive Analytics Software (PASW Statistics 18). Statistical significance was assessed at P < 0.05. All calculated P values were two-tailed. The sensitivity and specificity of serum IP-10/CXCL-10 in discriminating patients and controls, responders and non responders and the sensitivity and specificity of pretreatment serum IP-10/CXCL10 in discriminating RVR and EVR were assessed by plotting a receiver-operating characteristic (ROC) curve and determining its cut-off values.
The results from the present study are summarized as follows:
Ninteen (63.3%) patients were responders to antiviral therapy [10 (33.3%) RVR (4 males and 6 females) and 9 (30%) EVR (8 males and one female)], while eleveen (36.7%) patients were non responders (7 males and 4 females). There was no significant difference between the responders and non responders as regards sex (FEp = 1.000).
The mean age of responders was 34.89 ± 8.40 years (range: 22 to 51 years), while the mean age of non responders was 36.20 ± 9.18 years (range: 20 to 52 years). There was no significant difference between the responders and non responders as regards age (P = 0.314).
The liver was enlarged in 3 (10%) patients [non responders] while its size was average in 27 (90%) patients [19 responders and 8 non responders]. The liver size was greater in non responders than in responders and the difference is statistically significant (P = 0.041).
There was no significant difference between the mean spleen size in responders and non responders (11.43 ± 1.06 cm vs 11.47 ± 0.76 cm, P = 0.900).
The mean BMI was (24.37 ± 2.10 Kg/m2) in responders and (24.75 ± 2.49 Kg/m2) in non responders, with no significant difference between the two groups as regards BMI (P = 0.280).
The modified HAI grading was found to be minimal in 7 (23.3%) patients, mild in 14 (46.7%) patients, moderate in 8 (26.7%) patients and marked in 1 (3.3%) patient.
The modified HAI grading ranged 3-13 with a mean value 6.87 ± 2.79. The mean modified HAI grading was significantly lower in RVR, EVR and total responders than in non responders (4.70 ± 0.95, 5.44 ± 1.51 and 5.44 ± 1.51 vs 10.0 ± 1.61, P = < 0.001, < 0.001 and <0.001 respectively).
The modified HAI staging was found to be stage 2 in 13 (43.3%) patients, stage 3 in 12 (40.0%) patients and stage 4 in 5 (16.7%) patients.
The modified HAI staging ranged 2-4 with a mean value 2.73 ± 0.74. The mean modified HAI staging was significantly lower in RVR and EVR than in non responders (2.40 ± 0.52 and 2.44 ± 0.53 vs 3.27 ± 0.79, P = 0.013 and 0.004 respectively).
The mean serum levels of CXCL10 showed significant increase in patients compared with controls either pretreatment or at the 4th or the 12th week (361.63 ± 193.28 pg/mL, 318.90 ± 179.05 pg/mL and 276.37 ± 174.23 pg/mL vs 131.40 ± 61.91 pg/mL, P = <0.001, <0.001 and 0.008 respectively).
While, the mean serum levels of CXCL10 showed significant decrease in RVR, EVR and total responders compared with non responders, either pretreatment (147.70 ± 28.30 pg/mL, 341.56 ± 91.28 pg/mL and 239.53 ± 118.29 pg/mL vs 572.55 ± 76.71 pg/mL, P = <0.001, <0.001 and <0.001 respectively), at the 4th week (129.60 ± 30.50 pg/mL, 274.44 ± 51.37 pg/mL and 197.26 ± 83.71 pg/mL vs 529.0 ± 60.75 pg/mL, P = <0.001, <0.001 and <0.001 respectively) or at the 12th week (111.40 ± 27.43 pg/mL, 199.22 ± 54.12 pg/mL and 153.0 ± 60.89 pg/mL vs 489.45 ± 49.94 pg/mL, P = <0.001, <0.001 and <0.001 respectively).
There were no significant differences between serum levels of CXCL10 in patient groups [RVR, EVR, total responders and non responders] and controls as regards sex (P = 1.000, 0.846, 0.057, 0.570 and 0.354 respectively), age (P = 0.437, 0.589, 0.816, 0.292 and 0.783 respectively) and BMI (P = 0.522, 0.897, 0.583, 0.714 and 0.557 respectively).
No significant differences were found between patients with modified HAI stage 2 and 3 [either RVR, EVR, total responders or non responders] as regards serum levels of CXCL10 (P = 0.670, 0.461, 0.804 and 0.481 respectively), while a significant increase was found in the patients with stage 4 when compared with patients with stage 2 or 3 (P = 0.004 and 0.011 respectively).
The mean serum levels of CXCL10 showed significant increase in patients with moderate modified HAI grading as compared with patients with minimal or mild modified HAI grading (609.12 ± 51.47 pg/mL vs 188.14 ± 77.99 pg/mL and 300.28 ± 43.09 pg/mL, P = 0.001 and <0.001 respectively).
No significant correlation was found between CXCL10 and hepatic steatosis in the studied patients groups [RVR, total responders and non responders] (P = 0.909, 0.146 and 0.200 respectively).
By plotting a ROC curve, the sensitivity and specificity of pretreatment serum CXCL10 in discriminating patients and controls were found to be 66.67% and 100% respectively at a cut-off value of 250 pg/ml, and the sensitivity and specificity of pretreatment serum CXCL10 in discriminating responders and non responders were found to be 90.91% and 94.74% respectively at a cut-off value of 460 pg/ml, while the sensitivity and specificity of pretreatment serum CXCL10 in discriminating RVR and EVR were found to be 100% and 90% respectively at a cut-off value of 180 pg/ml.
Statistical correlations between serum levels of CXCL10 and pretreatment HCV-PCR, modified HAI (staging and grading) and other parameters showed the following results:
• There were no statistically significant correlations between age and BMI on one hand and the serum CXCL10 levels in patients and controls (r = 0.065, P = 0.670; r = 0.244, P = 0.381 and r = 0.160, P = 0.295; r = -0.034, P = 0.904 respectively) on the other hand.
• No statistically significant correlations were found between serum CXCL10 levels in patients and controls on one hand and hemoglobin (r = 0.109, P = 0.474 and r = 0.220, P = 0.432 respectively), ALP (r = -0.244, P = 0.193 and r = 0.145, P = 0.605 respectively), GGT (r = -0.266, P = 0.078 and r = -0.142, P = 0.613 respectively), bilirubin (r = 0.128, P = 0.402 and r = 0.088, P = 0.756 respectively), prothrombin activity (r = 0.075, P = 0.625 and r = 0.107, P = 0.704 respectively) and AFP (r = -0.073, P = 0.633 and r = 0.193, P = 0.491 respectively) on the other hand.
• A negative correlation was found between serum CXCL10 levels and albumin levels in controls (r = -0.521, P = 0.046).
• A negative correlation was found between serum CXCL10 levels on one hand and ALT and AST levels (r = -0.774, P = 0.005 and r = -0.715, P = 0.013 respectively) in non responders on the other hand, while a direct correlation was found between serum CXCL10 levels on one hand and AST levels (r = 0.697, P = 0.025) in RVR on the other hand.
• There were no statistically significant correlations between pretreatment CXCL10 levels and pretreatment HCV-PCR levels in different patient groups [RVR, EVR, total responders and non responders] (r = 0.079, P = 0.829; r = 0.469, P = 0.203; r = 0.348, P = 0.145 and r = -0.433, P = 0.184 respectively).
• A significant positive correlation was found between serum CXCL10 levels on one hand and modified HAI staging and grading of the patients (r = -0.433, P = 0.014 and r = 0.762, P = < 0.001 respectively) on the other hand (figure 22).