|Year : 2014 | Volume
| Issue : 4 | Page : 780-784
Evaluation of CD95 in patients with chronic hepatitis C virus
Laila Mahmoud Montaser1, Imam Abd Elatif Waked2, Enas Said Essa1, Rasha Mohammed Abd Elhameed MBBCh 1
1 Department of Clinical Pathology, Faculty of Medicine, Menoufiya University, Menoufiya, Egypt
2 Department of Hepatology, National Liver Institute, Menoufiya University, Menoufiya, Egypt
|Date of Submission||10-Feb-2014|
|Date of Acceptance||10-Mar-2014|
|Date of Web Publication||22-Jan-2015|
Rasha Mohammed Abd Elhameed
Department of Clinical Pathology, Faculty of Medicine, Menoufia Governorate, Berket El Sab District
Source of Support: None, Conflict of Interest: None
The aim of this study was to assess CD95 expression on peripheral blood lymphocytes in patients with chronic hepatitis C virus (HCV) infection, both under interferon treatment and without interferon treatment, in comparison with a control group to assess its role in the pathogenesis of chronic hepatitis C disease.
Egypt has the highest prevalence of HCV in the world, estimated nationally at 14.7%. Fas (APO-1/CD95)-mediated apoptosis plays an important role in liver cell destruction in viral hepatitis.
Participants and methods
Peripheral blood lymphocytes were isolated from 15 patients with chronic HCV under interferon treatment and from 20 patients with HCV without interferon treatment, and CD95 expression was analyzed by flow cytometry. The results were compared with those of normal control volunteers.
The percentage of CD95 expression was significantly higher in patients with HCV without interferon treatment and in patients with HCV under interferon treatment compared with normal controls (27.09 ± 12.89 vs. 22.33 ± 11.42 vs. 11.09 ± 5.96, respectively). The percentage of CD95 expression did not differ significantly between patients with HCV without interferon treatment and patients with HCV under interferon treatment.
The increase in CD95 expression on PBMCs among HCV patients supports its role in the pathogenesis of chronic hepatitis C disease.
Keywords: CD95, flow cytometry, hepatitis C virus
|How to cite this article:|
Montaser LM, Waked IA, Essa ES, Abd Elhameed RM. Evaluation of CD95 in patients with chronic hepatitis C virus. Menoufia Med J 2014;27:780-4
|How to cite this URL:|
Montaser LM, Waked IA, Essa ES, Abd Elhameed RM. Evaluation of CD95 in patients with chronic hepatitis C virus. Menoufia Med J [serial online] 2014 [cited 2019 May 21];27:780-4. Available from: http://www.mmj.eg.net/text.asp?2014/27/4/780/149758
| Introduction|| |
Hepatitis related to hepatitis C virus (HCV) is a progressive disease that may result in chronic active hepatitis, cirrhosis, and hepatocellular carcinoma. It is estimated that over 200 million people are infected worldwide, and 80% develop a chronic form . It represents a global health problem as there is no vaccine available. The response to current standard-of-care therapy is limited, and liver failure related to chronic HCV infection is one of the most common reasons for liver transplants . Although liver biopsy represents the gold standard for evaluating the presence, type, and stage of liver fibrosis and for characterizing necroinflammation, it remains an expensive and invasive procedure with inherent risks. Thus, it cannot be performed frequently to monitor therapeutic outcomes ,.
As apoptotic cell death plays an important role in the development and homeostasis of multicellular organisms, the failure of cells to undergo apoptosis might be involved in the pathogenesis of a variety of human diseases, including autoimmune diseases, viral infections, and malignancies . Fas/APO-1 (CD95) is a type 1 transmembrane glycoprotein that signals sensitive cells to die by apoptosis upon ligation with anti-Fas/APO-1 MoAb  or the natural ligand for Fas, Fas ligand (FasL) . Much attention has recently been directed to the Fas/FasL system in the liver. Thus, the Fas/FasL system may be closely linked to the cell injury induced by hepatitis virus infection and the regulation of tumourigenesis in the liver .
| Participants and methods|| |
The study included 35 patients and 22 healthy volunteers divided into three groups. The first group comprised 22 apparently healthy volunteers as the control group, the second group included 20 patients with chronic HCV without interferon treatment, and the third group comprised 15 patients with chronic HCV under interferon treatment.
The patients were selected from the outpatient clinics of the National Liver Institute, Menoufia University.
All the required investigations were carried out for confirmation of the final diagnosis. The laboratory investigations included CBC and evaluation of alanine aminotransferase (ALT), AST, bilirubin, albumin, prothrombin, and CD95. Sampling was performed after informed consent was obtained from each patient included in the study to use the samples and clinical data for research purposes after being informed about the nature of the study.
Detection of FAS (CD95) was performed using monoclonal antibody (Dako, Denemark). This test depends on the ability of a monoclonal antibody to bind to the surface of the cells expressing CD95 lymphocytes and measured by flow cytometry.
Two volumes of EDTA blood was layered carefully on top of one volume of ficol and then centrifuged for 20 min at 1800 rpm. The mononuclear cells were deposited in a white band at the interference between plasma and ficol and then separation of mononuclear layer was carried out in a separate tube. It was washed with 3 ml of PBS and then centrifuged for 5 min at 3200 rpm. The supernatant was decanted and the sediment was resuspended in 500 ml of PBS.
For each sample two tubes were prepared: one for the test and the other for unstained control.
A volume of 10 ml of diluted monoclone was added to 100 ml of the previously prepared cell suspension, mixed well, and incubated at 2-8°c for 30 min; the cells were then washed in 2 ml of PBS.
Finally the cells were resuspended in 200 ml of PBS for final flow cytometric analysis.
All samples were analyzed using a flow cytometer (Becton Dickinson FACS Calibur).
Quantitative data were expressed as mean ± SD. Comparison of two groups was carried out with the Mann-Whitney U-test, whereas the analysis of variance test was performed to compare three or more groups; the Pearson correlation (correlation coefficient) was performed to study correlation between two normally distributed quantitative variables; P-values less than 0.05 were considered significant.
The c2 -test was used to study the association between two qualitative variables, and post-hoc tests were used at the second stage of the analysis of variance if the null hypothesis was rejected. At this stage the question of interest is which groups significantly differ from others with respect to the mean.
| Results|| |
The study population was classified into three groups. Group one included 22 apparently healthy volunteers as controls: 12 men and 10 women with a mean age of 44.55 ± 4.48 years. Group two comprised 20 patients with chronic HCV without interferon treatment: 15 men and five women with a mean age of 45.75 ± 10.82 years. The last group comprised 15 patients with chronic HCV under interferon treatment: eight men and seven women with a mean age of 42.07 ± 7.11 years. There was no significant difference between the three groups with respect to age and sex [Table 1].
|Table 1: Sociodemographic data of hepatitis C disease patients without treatment, hepatitis C disease patients under treatment, and controls|
Click here to view
The mean ± SD of the percentage of CD95t expression was 11.09 ± 5.96 versus 27.09 ± 12.89 versus 22.33 ± 11.42 in the control group, in patients with chronic HCV without interferon treatment, and in patients with chronic HCV under interferon treatment patients, respectively, and the difference was statistically significant (P < 0.001) between the control group and both HCV groups, and there was no significant difference in the percentage of CD95 expression between the two groups of HCV [Table 2]. The statistical analysis of data indicated that liver function tests including SGOT, SGPT, albumin, bilirubin, prothrombin concentration are statistically not correlated with CD95 in both HCV groups (P > 0.05 for each). A significant negative correlation between CD95 expression and albumin (r = −0.704, P = 0.003) was observed among HCV patients under interferon treatment. As regards blood picture, there was a significant negative correletion between CD95 expression and hemoglobin (r = −0.339) and total leukocytic count (r = −0.581) among HCV patients without interferon treatment; there was a significant negative correletion between CD95 expression and hemoglobin (r = −0.777) among HCV patients under interferon treatment [Table 3] and [Table 4].
|Table 2: Investigated laboratory criteria of hepatitis C disease patients without treatment, hepatitis C disease patients under treatment, HCC patients and controls|
Click here to view
|Table 3: Pearson's correlation between CD95 and laboratory criteria in hepatitis C disease group of patients without treatment|
Click here to view
|Table 4: Pearson's correlation between CD95 and laboratory criteria in hepatitis C disease under treatment group|
Click here to view
As regards viral load, there was no significant difference in CD95 expression between patients with low viremia and those with moderate viremia [Table 5].
|Table 5: Correlation between CD95 and viremia among hepatitis C disease patients group|
Click here to view
| Discussion|| |
In Egypt, HCV is a 'viral time bomb' as most patients do not realize that they are infected , 4.7% of chronic liver disease patients suffer from HCC. The development of HCC is mainly because of high rates of hepatitis B and C infections among Egyptian patients . Genotype 4 is the most common genotype of HCV in Egypt, and its response to treatment is still a controversy. The outcome of antiviral treatment seems to depend on many factors including the HCV genotype and the level of both viral replication and genetic complexity of the quasispecies population before the start of treatment .
An important insight into the mechanism of tumor escape from immune clearance has been provided by the identification of 'Death factors', including Fas/FasL as an important regulator of both apoptosis and immune function . Fas receptors are widely expressed in normal and diseased tissues. It has been implicated in tumor progression of several cancers .
In the present study, there was a statistically highly significant increase in CD95 expression on the peripheral mononuclear cells of patients with HCV without treatment and patients with HCV under treatment compared with the control group (27.09 ± 12.89, 22.33 ± 11.42, 11.09 ± 5.96, respectively), but there was no significant difference between the two HCV groups.
This is in agreement with the results of El Bassiouny et al. , who reported that the level of serum sFas in chronic active hepatitis was found to be significantly higher compared with that of healthy individuals (P < 0.01).
This is in agreement with the results of Toyoda et al. , who reported that serum sFas levels of CHC patients (2.78 ± 0.52 ng/ml, range 1.93-3.91 ng/ml) were significantly higher compared with those of healthy controls (1.86 ± 0.55 ng/ml, range 0.94-3.12 ng/ml) (P < 0.01).
Yoneyama et al.  reported that serum sFas concentration in patients with cirrhosis due to HCV was found to be higher compared with controls, and this elevation might be due to decreased clearance from the liver.
This study shows that there was no correlation between CD95 and liver enzymes among both HCV patients without treatment and HCV patients under treatment.
As regards viral load, this study shows that there was no significant difference between patients with low viremia and patients with moderate viremia.
This is in agreement with the results of Pelli et al. , who reported that in chronic hepatitis C patients, no correlation between sFas and both cytolysis (expressed as ALT levels) and HCV viremia was present.
This is in agreement with the results of Iio et al. , who reported a positive correlation of serum levels of sFas and Fas expression in the liver with levels of liver inflammation but not between the former and ALT levels.
Toyoda et al.  reported that serum levels of sFas had a positive correlation with histological activity and ALT levels and no correlation with HCV-RNA genotype, serum HCV-RNA load.
As regards CD95 expression on PBMNLs, the results of this study showed that there was no significant statistical difference between patients without treatment and patients under treatment.
Ohkawa et al.  reported higher sFas levels in nonresponders than in transient and sustained responders to interferon treatment.
Ozaslan et al.  measured the serum levels of sFas in patients at different degrees of HCV-induced liver disease and showed that serum sFas levels were significantly increased during chronic active hepatitis (6.4 ± 1.3) and cirrhosis (8.8 ± 3.8). However, in patients with milder HCV-induced liver disease (i.e. during interferon and ribavirin therapy and patients with viral replication but normal transaminase levels not under treatment) serum sFas levels were not significantly different compared with controls (3.3 ± 1.5 vs. 4.3 ± 1.7 vs. 4.7 ± 1.4, respectively) .
Our results may be illustrated by the fact that all of our HCV cases both under interferon therapy and without treatment were of Child A class - that is, with early-stage liver disease with expected lower CD95 expression. Further, patients under interferon therapy were recruited at earlier stages of treatment (12, 24 weeks).
| Acknowledgements|| |
Conflicts of interest
| References|| |
Lavanchy D. The global burden of hepatitis C. Liver Int 2009; 29
Ghany MG, Strader DB, Thomas DL, Seeff LBAmerican Association for the Study of Liver Diseases Diagnosis, management, and treatment of hepatitis C: an update. Hepatology 2009; 49
Bravo AA, Sheth SG, Chopra S. Liver biopsy. N Engl J Med 2001; 344
Thampanitchawong P, Piratvisuth T. Liver biopsy: complications and risk factors. World J Gastroenterol 1999; 5
Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995; 267
Oehm A, Behrmann I, Falk W, Pawlita M, Maier G, Klas C, et al
.. Purification and molecular cloning of the APO-1 cell surface antigen, a member of the tumor necrosis factor/nerve growth factor receptor superfamily. Sequence identity with the Fas antigen. J Biol Chem 1992; 267
Suda T, Takahashi T, Golstein P, Nagata S Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family. Cell 1993; 75
Jodo S, Kobayashi S, Nakajima Y, Matsunaga T, Nakayama N, Ogura N, et al
. Elevated serum levels of soluble Fas/APO-1 (CD95) in patients with hepatocellular carcinoma. Clin Exp Immunol 1998; 112
Montaser L, Abd Elmomen K, Abo Eleil D. Prevalence of HCV antibodies among blood donors in Menoufia University Hospital [thesis]; 2007; p. 104
Rahman El-Zayadi A, Abaza H, Shawky S, Mohamed MK, Selim OE, Badran HM. Prevalence and epidemiological features of hepatocellular carcinoma in Egypt - a single center experience. Hepatol Res 2001; 19
Zekri AR, Ashour MS, Hassan A, Alam El-Din HM, El-Shehaby AM, Abu-Shady MA Cytokine profile in Egyptian hepatitis C virus genotype-4 in relation to liver disease progression. World J Gastroenterol 2005; 11
Hammam O, Zahran M, Mahmoud O et al.
Early detection of hepatocellular carcinoma on top of liver cirrhosis: the Fas receptor and ligand system. Egypt J Hosp Med 2011; 45
Bebenek M, Du? D, KoŸlak J. Fas expression in primary breast cancer is related to neoplastic infiltration of perilymphatic fat. Adv Med Sci 2008; 53
El Bassiouny AE, El-Bassiouni NE, Nosseir MM, Zoheiry MM, El-Ahwany EG, Salah F, et al
. Circulating and hepatic Fas expression in HCV-induced chronic liver disease and hepatocellular carcinoma. Medscape J Med 2008; 10
Toyoda M, Kakizaki S, Horiguchi N, Sato K, Takayama H, Takagi H, et al
. Role of serum soluble Fas/soluble Fas ligand and TNF-alpha on response to interferon-alpha therapy in chronic hepatitis C. Liver 2000; 20
Yoneyama K, Goto T, Miura K, Mikami K, Ohshima S, Nakane K, et al
. The expression of Fas and Fas ligand, and the effects of interferon in chronic liver diseases with hepatitis C virus. Hepatol Res 2002; 24
Pelli N, Floreani A, Torre F, Delfino A, Baragiotta A, Contini P, et al
. Soluble apoptosis molecules in primary biliary cirrhosis: analysis and commitment of the Fas and tumour necrosis factor-related apoptosis-inducing ligand systems in comparison with chronic hepatitis C. Clin Exp Immunol 2007; 148
Iio S, Hayashi N, Mita E, Ueda K, Mochizuki K, Hiramatsu N, et al
. Serum levels of soluble Fas antigen in chronic hepatitis C patients. J Hepatol 1998; 29
Ohkawa K, Hiramatsu N, Mochizuki K, Mita E, Iio S, Yoshihara H, et al
. Significance of serum soluble Fas antigen level in chronic hepatitis C patients treated with interferon: relationship to the therapeutic response. J Gastroenterol Hepatol 2001; 16
Ozaslan E, Kiliçarslan A, Simºek H, Tatar G, Kirazli S Elevated serum soluble Fas levels in the various stages of hepatitis C virus-induced liver disease. J Int Med Res 2003; 31
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]