182 J Contemp Med Sci | Vol. 3, No. 9, Winter 2017: 182–185 Research Assessment of Cox2 and CMV in patients with chronic HCV infection Heba F. Hassan,a Basim M. Khashman,b Omer A. Abdul Qader,c Alaa W. Izzatd aDepartment of Basic Sciences. College of Dentistry. University of Baghdad, Iraq. bIraqi national cancer research center (INCRC), University of Baghdad, Iraq. cKulliyyah of Dentistry oral medicine and oral pathology Department, International Islamic University Malaysia, Malaysia. dCollege of Dentistry, University of Baghdad, Iraq. Correspondence to Heba F. Hassan (email: heba_micro08@yahoo.com). (Submitted: 19 December 2017 – Revised version received: 11 January 2017 – Accepted: 20 February 2017 – Published online: 27 March 2017 ) Objective This study was established to determine the expression of COX-2 and CMV in patients with chronic C infection. Methods A total of 30 formalin-fixed of paraffin-embedded liver samples obtained from patients with chronic hepatitis C infection. In addition, 30 apparently normal liver autopsies were used as control group used for immunohistochemistry technique (IHC) to study the expression of cyclooxygenase (COX-2) and cytomegalovirus (CMV) in these samples. Results In current study, the results of expression of COX-2 were found in 25 cases (83.3%) of chronic HCV were strongly positive with a significant increase at P < 0.001, while the result of CMV expression was detected as brown cytoplasmic membranous staining of cells with positive CMV expression demonstrated in 21 (70.0%) out of 30 cases of chronic HCV infection cases. All control groups were negative for the expression of COX-2 and CMV. Conclusion This study concludes that COX-2 and CMV participates in the pathogenesis of chronic HCV infection. Keywords chronic HCV infection, cyclooxygenase (COX-2), cytomegalovirus (CMV) Introduction Hepatitis C virus (HCV) is a serious global health trouble that affects 130-170 million people worldwide.1,2 Data from WHO estimate that 3–4 million individuals are infected with HCV every year.3 Its complex disease persistent infection leading to cirrhosis, fibrosis, hepatocellular carcinoma (HCC) and liver transplantation.4 HCV is classified as Flaviviridae family and genus Hepacivirus. A single-stranded positive-sense RNA genome enveloped virus with a ~ 9.6 kb,5 There are seven gen- otypes of HCV, each with many subtypes depend on antigenic variability and geographical differences in distribution, transmission, disease progression and respond differently to treatment.6 HCV genome encodes large polyprotein of 3010 amino acids which is processed co-translationally into three structural and seven nonstructural (NS) polypeptides.7,8 Cyclooxygenase (COX) is the rate limiting enzymes in the production of prostanoids from arachidonic acid.9 There are two COX isoforms present in human that share more than 60% identity at the amino acid level identified. COX-1 is con- stitutively expressed both in normal cell types and in cirrhotic livers and is involved in the homeostatic functions of PGs.10 Whereas COX-2 is induced by different stimuli such as hor- mones, cytokines, growth factor and mitogens, COX-2 was not seen in normal liver, but showed de novo synthesis and pronounced upregulation in liver cirrhosis.11 High COX-2 expression has been reported to be relationship with liver damage and the liver cell pathologies such as the degree of inflammation, the viral infections, the development of fibrosis and HCC in human.12,13 This was illustrated by the effect of COX-2 on the secretion of matrix metalloproteinases (MMPs) by liver cells that be implicated in carcinogenesis and fibrino- genesis occurring in HCV-induced liver disease.14 CMV is the largest member of the virus family Herpesviridae belong to human herpes virus-5 and is ubiquitous virus that infects most all humans at some time in their lifetime,15 is a double-strand DNA virus 235-kb that encodes more than 200 proteins.16 Approximately70-100% of the world’s populations are carriers to this virus,17 the prevalence of infection are estimated 100% in Asia and Africa, and approximately 80% in USA and Europe depending on socioeconomic status.18 It has become the most common cause of severe disease with high morbidity and mortality in immune compromised individuals.19 Primary human CMV infection is occurred in early life and the virus persistence in a latent state, and reactivation may occur later in life.20 Therefore, reactivation of the virus is due to periods of down-regulation of the immune system, such as illness-related stress drug treatment, or during on-going activation of the immune system such as infection with other pathogens and inflammatory diseases.21 CMV can infect all organ and tissues, but manifestations of organ involvement generally include symptoms from the liver, the intestine, CNS and lung.22 Materials and Methods Patients and control samples: This study was carried on 30 liver biopsy specimens that diagnosed with chronic HCV infec- tion in 22 males and 8 females were collected at random from the pathology archive at the Liver and Digestive System Technical Hospital in Baghdad during the period from February 2010 to November 2012. The liver biopsy samples were estimated by a single pathologist, and the stage of fibrosis was depend on Ishak scores fibrosis from 0 to 6 where a score of 2 is defined as fibrous expansion of most portal areas, with or without short fibrous septa; three fibrous expansion of most portal areas with occasional por- tal-to-portal bridging; four fibrous expansion of most portal areas with marked bridging; five incomplete cirrhosis and 6 definite cirrhosis.23 All patients were confirmed to HCV infection by Elisa test. Control group of liver specimens were obtained from 30 autopsies (21 males and 9 females). From each tissue block, a series of 4 μm sections were cut and stained with hematoxylin and eosin for pathological evalua- tion and immune histochemistry. ISSN 2413-0516 Heba F. Hassan et al. 183J Contemp Med Sci | Vol. 3, No. 9, Winter 2017: 182–185 Research Assessment of cox2 and cmv in patients with chronic hcv infection Immunohistochemical Staining The antibodies used in this study included anti-COX-2 antibody and anti-CMV antibody. The sections were dewaxed in xylene then rehydrated in graded alcohol. Endogenous peroxidase activity and non-specific binding were blocked by incubation with 3% hydrogen peroxide and protein block, respectively. peroxidase blocking rea- gent for 10 min. Slides were pre-treated in microwave oven in citrate buffer (pH 6). Subsequently, slides were incubated with mouse monoclonal anti-COX-2 and anti-CMV anti- body and Slides were then incubated sequentially with pri- mary antibodies for 1hour at 37°C and secondary antibody for 10 minutes at room temperature followed by incubation with the Streptavidine-HRP antibodies for 10 minutes at 37°C. Diaminobenzidinehydrochloride (DAB) was used as the chromogen to visualize peroxidase activity. The sec- tions were counterstained with hematoxylin and overlaid with cover slips. Assessment of Immunohistochemistry Results Positive reading was detected when the cells display a brown cytoplasmic pigmentation of immunostaining, while negative reading was detected by the absence of immunostaining. Assess- ment of anti-Cox2 antibody and anti-CMV antibody Immuno- reactivity. The scoring system of immunostaining antibodies was assessed the positively stained cells, which counted at five repre- sentative random fields (40X) by using light microscope, the scoring of the antibodies with the criteria combined intensity with the rate of positive cell. The percentage of positive cells for the protein of interest were scored as 1 = (0–25%), 2 = (26–50%), 3 = (51–75%) and 4 = (76–100%)[8]. Statistical Analysis: Evaluation of the statistical significance of the data was performed by using the t-test and Chi-square test. Results Distribution of Patients and Control Group According to Gender Distribution of patients and control group according to gender was listed in Table 1. The results of this study showed that chronic HCV infection in the male was more predominance among patients, where 22 males (73.4%) and 8 females (26.6 %) out of total cases. there was no statistically significant differ- ence (p > 0.05) in gender between both studied groups, The mean age of patients was 44.9 years compared with control group was 46.9. Results of Immunohistochemical COX2 Expression The current study showed strong brown staining was seen in most cells with positive COX2 expression by Immunohisto- chemical (IHC) staining was found in 25 cases (83.3%) of chronic HCV were strongly positive with a significant increased at P < 0.001 (Table 2, Fig. 1). On the other hand there was no positive result among control group. CMV Expression The result of this study showed CMV expression was detected as brown cytoplasmic membranous staining of cells with Table 1. Distribution of patients and control group according to gender Studied groups Total Chronic HCV Control Gender type Male Count 22 21 43 % 73.4% 70.0% 71.7% Female Count 8 9 17 % 26.6 % 30.0% 28.3% Total Count 30 30 60 % 100.0% 100.0% 100.0% Mean of age 44.9 46.9 Range of age 14-65 29-68 Median of age 45.5 42.5 P value 0.984NS Table 2. Expression of COX2 in patients with chronic HCV infection and healthy control group Studied groups Chronic HCV Control COX2 Negative Count 5 30 % 16.7% 100.0% Positive Count 25 0 % 83.3 % 0.0% Total Count 30 10 % 100.0% 100.0% P value < 0.001** positive CMV expression by Immunohistochemical (IHC) staining demonstrated in 21 (70.0%) out of 30 cases of chronic HCV infection cases. There was highly significant statistically differences found between the patients and healthy control group at P < 0.001 (Table 3 and Fig. 2). Discussion The present study showed that high over expression of COX-2 (83.3%) in liver tissue in patients with chronic HCV infection with different stage of fibrosis. COX-2 is concerned in anti-ap- optosis, inflammation and carcinogenesis. This result agreed with Nunez et al.24 was found COX-2 is over expressed in the Fig 1. (A) Human liver tissue stained by immunohistochemistry for COX2 in patients with chronic HCV infection stained by DAB chromagen is showing as brown in positive cases at magnification 200X (B) Negative expression. A B 184 J Contemp Med Sci | Vol. 3, No. 9, Winter 2017: 182–185 Assessment of cox2 and cmv in patients with chronic hcv infection Research Heba F. Hassan et al. liver tissue in patients with chronic HCV infection and that the COX-2 hepatic upregulation was present especially at areas of active inflammation. These results were explained by the major action of COX-2 as factor in inflammation with the final induc- tion of COX-2 in necro-inflammatory injury of the liver25 Bassiouny et al.26 reported that the up-regulation of COX-2 expression was apparent in patients with chronic HCV infec- tion Regardless of the presence of cirrhosis while 80% of cir- rhotic cases showed marked COX-2 expression. Cheng and Hada27 was proved that up-regulation of COX-2 expression was present in cirrhotic tissues relative to HCC as well as in well-dif- ferentiated HCC. over-expression of COX-2 in cirrhosis was apparent in a study that assess in patients with cirrhosis due to HBV and HCV infection while our series inclusive HCV infec- tion patients Mohamed et al.28 Another study, we were able to express a significant relation found between overall COX-2 expression and viral load of the HCV-RNA.29 while Waris and Siddiqui30 was found the increase in Cox-2 mRNA expression in cells containing the HCV replicon, indicate that HCV gene expression control Cox-2 activity at the stage of transcription. Holt and Adams31 who indicated that COX-2-derived media- tors have special functions at various times in the pathogenesis of chronic hepatitis. Yosry et al.29 suggest that COX-2 expres- sion seems to be correlated between inflammatory activity of the liver of patients with chronic HCV infection and the response to antiviral treatment which was clear by the reduc- tion of COX-2 expression. Also Manning et al.32 indicated that reduction of liver Cox-2 level after giving INF as treatment. Gomaa et al.33 show upregulation of COX-2 in HCV infection, which is related with poor virological response. CMV is more widespread in developing countries and can affect 70–100% of the human population. In the current study showed high expression of CMV (70.0%) in the liver tissue in patients with HCV infection in different with stage of fibrosis. The infection by CMV was depended on the detection of CMV replication in the blood. reactivation of CMV infection was occurred in the absence of an effective immune response.34 CMV reactivated in immunosuppression35,36 and by directly increasing HCV repli- cation is unknown. This is the result of explosion of high levels of CMV replication.37 Bayram et al.38 was shown that CMV was diagnosed in HBV or HCV patients as a dual infection and that it can increase the risk of the disease. In Egypt, Tabll et al.39 were examined the potential role thatCMV plays in chronic HCV progression. Also Bader El din et al.,40 demonstrate higher rate of CMV co-infection in chronic HCV patients in Egypt than other patient populations. Whether CMV infection predisposes patients to HCV or HCV infection predisposes patients to CMV is not known. Various studies indicated that CMV causes hepatitis with fibrosis of liver cells and inflammation That means CMV influence the liver and overall immunological condition of the body.38,40,41 High level of liver enzymes and remarkable histological changes in the liver of CMV-HCV co-infected patients.38 The serum levels of AST and ALT enzymes showed elevated in CMV, thus indicating a role of CMV in pathogenesis of the liver. Rafael et al.,42 indicated that CMV infection interacted with HCV and raised the effect on the liver enzymes and cause hepatitis. Considering the fact that HCMV viruses contribute an immunomodulatory effect resulting in developed of immunosuppression43,44 and dysregu- lation of cytokine which could fast HCV pathogenesis of patients in critical condition.39 CMV infection promotes HCV pathogenesis by prohibition the normal mechanisms responsible for HCV elimination, thus playing vital role in HCV existence and pathogenicity.45 Conclusion COX-2 and CMV seem to be related to the inflammatory activity and participate in the pathogenesis in patient with chronic HCV infection. Acknowledgment I would like to express my thanks and my gratitude to all who contributed in this study. Conflicts of Interest There are no conflicts of interest. n Table 3. Expression of CMV in patients with chronic HCV infec- tion and healthy control group Studied groups Chronic HCV Control CMV Negative Count 9 30 % 30.0% 100.0% Positive Count 21 0 % 70.0 % 0.0% Total Count 30 32 % 100.0% 100.0% P value < 0.001** References 1. McCombs J, Matsuda T, Tonnu-Mihara I, et al. The risk of long-term morbidity and mortality in patients with chronic hepatitis C results from an analysis of data from a department of veterans affairs clinical registry. JAMA Intern Med. 2013;12505. 2. Hassan H: The Correlation of HCV, HLA-DR typing and endothelin level with Oral Lichen Planus, PhD thesis. Medical Microbiology. Al-Nahrain University, 2015. 3. World Health Organisation. Available at: http://www.who.int. Accessed September. 1,2012. 4. Kwon YC, Ray RB, Ray R: Hepatitis C virus infection: establishment of chronicity and liver disease progression. EXCLI J. 2014;13:977–996. 5. Leftkowitz E, King A, Adams M, et al. 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