الفهرس 
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Abstract
Hepatitis C Virus (HCV) infection is a global health challenge with Egypt being one of the highly affected countries. HCV is a hepatotropic RNA virus of the family Flaviviridae whose genome exhibits significant genetic variability which lead to its classification into 7 major genotypes. Multiple systematic reviews have shown limited genotype diversity in Egypt with predominance of genotype 4 (G4) which accounts for more than 94% of HCV infected cases.
In 2018, a meta-analysis for HCV viremic rate among the Egyptian population indicated that HCV prevalence was estimated at 11.9% among the general population; 67% of those anti-HCV positive in Egypt are chronically infected and in need of treatment and highlighted that a declining trend for anti-HCV prevalence in Egypt suggesting a rapidly contracting HCV epidemic in this country.
Both arms of the immune system; innate and adaptive are essential for viral clearance. The innate response is the first line of defense during any infection. The hallmark of the innate response to HCV in the liver is the immediate induction of interferons (IFNs) and cytokines which creates an antiviral state in uninfected cells and inhibits the replication of HCV in infected cells. Adaptive immune response is composed of humoral immune response represented by antibody producing B cells and cellular immune responses presented by CD4+ T helper cells (Th) and CD8+ cytotoxic T lymphocytes (CTLs). HCV elimination is associated with strong and sustained T cell responses that target multiple epitopes within the different HCV proteins and that remain detectable long after resolution of infection.
Cytokines play an important role in viral clearance, inflammation, regeneration, and fibrosis. They can induce patterns of protective or immunopathological responses during the course of HCV infection. IL-10 and VEGF are two key players in the immune response against HCV infection. Both are seen elevated in patients with HCV, cirrhosis, and HCC and their concentrations are associated with disease progression and prognosis.
Interleukin-10 (IL-10) is an anti-inflammatory cytokine which can be produced virtually by all immune cells. It acts as an immunoregulator, inhibiting proinflammatory responses from innate and adaptive immunity and preventing tissue damage due to exacerbated adaptive immune response. Of further significance is the correlation between the HCV viral load and IL-10 production; high HCV replication is associated with high IL-10 levels. It has been postulated to have anti-fibrotic properties against HCV-induced liver inflammation and hepatocytes destruction, which may result in progression to fibrosis.
Vascular endothelial growth factor (VEGF) is a potent angiogenic factor playing a central role in many physiological as well as pathological angiogenesis. upregulation VEGF expression may facilitate the viral entry and play a role in viral persistence. Stimulation of angiogenesis during chronic liver disease causes structural and functional changes in the angioarchitecture which will accordingly be explained by the tissue hypoxia caused by the progressive inflammation and the increase in level of cytokines and proangiogenic factors during the healing process that follows the chronic inflammation. The increased hepatic angiogenesis in chronic HCV could provide the molecular basis for
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liver carcinogenesis and contribute to the increased risk of HCC accordingly the VEGF serum levels is suggested to be used as a prognostic factor of HCC.
Significant advances have been made in the screening and diagnosis of HCV which is a key point in preventing disease progression and viral spread. Screening using third generation serological assays has allowed the early detection with high sensitivity and specificity following the exposure to HCV with increase in the detection opportunities using the point-of-care testing. However, active HCV infection must be confirmed by direct molecular methods.
Egypt has addressed the HCV infection by setting a model of care (MOC) to control this epidemic and insure access to the proper treatment. In 2006, the Egyptian MOC was set by the National Committee for Control of Viral Hepatitis (NCCVH) which made a strategy that outlined the stepwise approach for patient care, prevention and management of HCV with a target to eliminate the disease (prevalence <1%) by 2030. The program is considered one of the most successful in the world in control of HCV with 1 million evaluated and more than 850, 000 treated under the umbrella of this program.
In 2011, the directly acting antiviral agents (DAAs) which are interferon free regimens that act directly against the viral replication has been introduced to replace the standard of care regimen (SOC) which depended on PEG-INF/RBV combination. The use of DAAs has lead to dramatic improvement in outcome of HCV infected patients with minimal side effects and cure rates of 85–95% across all patient populations.
The aim of the present work is to study the effect of directly acting agents (DAA) on the co-expression of vascular endothelial growth factor and interleukin-10 genes in chronic hepatitis C virus infected patients treated with these agents. After the approval of the Ethical Committee of the Medical Research Institute, twenty five chronic HCV infected patients were selected from the Department of Experimental and Clinical Internal Medicine, Medical Research Institute, Alexandria University.
After the consent data fulfillment, cases were subjected to detailed questionnaire, routine laboratory investigations as well as molecular studies including detection of the viral load and estimation of VEGF and IL-10 gene expression by real time PCR.
A total of 25 HCV infected patients were enrolled in the present study; all of which (100%) were treatment-Naïve cases. The females represented 52% (13/25) while the males were 48% (12/25) and their age ranged from 17 to 65 years.
Child Pugh score was calculated for all cases enrolled in the present study to estimate the degree of liver injury and the majority of the patients were categorized as 88% (22/25) child Pugh class (A) while class (B) were 12% (3/25) and none were classified as class (C).
All the cases enrolled in the present study performed liver function test including ALT, AST, Serum bilirubin (total and direct) and albumin as well as hematological analysis of platelets and prothrombin activity (INR) and HCV viral load before the initiation and after treatment with DAAs.
Before treatment, ALT activity ranged from 13-135 U/L with a mean of 46.40 U/L, AST activity ranged from 8-124 U/L with a mean of 43.96U/L, Albumin showed a range of 2.8-5.2 g/dl with a mean of 3.98 g/dl and finally total bilirubin showed a range from
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0.05-1.8 mg/dl with a mean of 0.64 mg/dl while direct bilirubin showed a range of 0.1-2.14 mg/dl with a mean of 0.35 mg/dl. As for the hematological parameters, platelets count showed a range of 40-304 X 103/μL with a mean of 178.08 x103/μL while the INR ranged between 1-1.9 with a mean of 1.18.
After treatment, ALT activity ranged from 4-35 U/L with a mean of 21.08 U/L. AST activity ranged from 16-35 U/L with a mean of 21.96 U/L, Albumin showed a range of 3.5-4.8 g/dl with a mean of 4.04 g/dl and finally total bilirubin showed a range from 0.3-1.2 mg/dl with a mean of 0.67 mg/dl while direct bilirubin showed a range of 0.1-0.6 mg/dl with a mean of 0.26 mg/dl. As for the hematological parameters, platelets count showed a range of 55-266 x 103/μL with a mean of 177.28 x 103/μL while the INR ranged between 1-1.2 with a mean of 1.08.
There is a statistically significant difference between the ALT and AST activity as well as prothrombin activity (INR) before and after treatment in the studied cases (p=0.0003, p= 0.0001, p=0.022 respectively).
As for the HCV viral load, the cases showed a range from 0.24x105 to 130 x105 IU/L mean 13.22 x 105 IU/L while all cases showed undetectable viral load (<25 IU/mL) at the end of antiviral therapy with a SVR of 100%.
APRI and FIB-4 scores were calculated for all cases in the present study as a non invasive marker for assessment of hepatic fibrosis or cirrhosis in chronic HCV patients. We observed a decrease in APRI (0.87 to 0.44) and FIB-4 (2.20 to 1.60) scores. This difference was found statistically significant (p=0.002 and p= 0.048 for APRI and FIB-4 respectively).
The main interest of the present study is to compare the serum mRNA expression of IL-10 and VEGF genes in CHCV patients before and DAAs treatment. The patients before treatment were considered the control group while after treatment was considered the target group. IL-10 expression was downregulated in 92% (23/25) of the cases ranging between -1.27 to -10.7 folds while 8% (2/25) of the cases were upregulated with a range of 3.94 – 7.35 folds. VEGF expression was heterogeneous showing spreading of values along a wide range showing 64% (16/25) of the cases being downregulated with a range of 1.1 – 3.872 while 36% (9/25) of the cases were upregulated with a range of 1.18 – 6.71 folds which can be explained by variability in the immune response among the studied patients.
Correlation between IL-10 and VEGF expression before treatment and the liver functions, hematological parameters and viral load shows a positive correlation with the viral load while other parameters showed no correlation while after treatment showed no correlation with any of the mentioned parameters.
The co- expression IL-10 and VEGF genes shows a positive correlation in the studied group which was statistically significant (r = 0.593, p=0.002) which can be explained by the similarities and overlap between IL-10 and VEGF in various function as the processes of inflammation and angiogenesis are intimately linked.