Apoptosis of peripheral blood cytotoxic T lymphocytes in chronic hepatitis C virus infection: relation to disease severity and response to therapy

More than 80% of HCV-infected individuals develop chronic disease, which can progress to liver cirrhosis and hepatocellular carcinoma. T cell-mediated protection against HCV depends on constantly activated effector CD8+ T cells that control emergence, spread and expansion of the virus. Why these cells fail to contain HCV replication in 70-80% of the individuals who develop persistent viremia is not clear. Fas receptors [APO-1 or CD95] and the Fas ligand system [Fas/FasL] have been implicated in the induction of apoptosis which is related with the pathogenesis of hepatitis C. Was to assess the expression of CD95 [Fas/APO-1] [as an apoptotic marker] on peripheral blood cytotoxic T lymphocytes from patients with chronic HCV infection and to correlate this with disease severity in the liver, response to antiviral therapy and extrahepatic autoimmune manifestations. Thirty patients with evidence of chronic HCV infection and 10 healthy age and sex matched volunteers with negative HCV antibodies sera serving as controls were enrolled into the present study. Our patients were classified into two main groups according to antiviral therapy, Group A: They were 17 patients not receiving treatment and Group B: They were 13 patients who received antiviral therapy for 24 weeks. Group A patients were further subdivided according to the presence or absence of extrahepatic manifestations into two groups: Group I: They were 11 patients not receiving therapy and had no extrahepatic manifestations. Group II: They were 6 patients not receiving therapy and had extrahepatic manifestations. Group I patients were also subdivided according to the necroinflammatory score in liver biopsy into three groups with: Mild, moderate and severe disease activity. They were also subdivided as regard fibrosis stage in liver biopsy into groups with: Moderate and severe fibrosis. Group B patients were further subdivided according to their response to antiviral therapy into: Group III: 6 patients received therapy and were non responders and Group IV: 7 patients received therapy and showed good response. After detailed history taking and thorough clinical examination, the following investigations were done: CBC, AST and ALT, serum cryoglobulins [for patients with extrahepatic disease] and flowcytometric estimation of the percentage of apoptotic peripheral blood cytotoxic T lymphocytes [CD8+CD95+] and non apoptotic cytotoxic T lymphocytes [CD8+ CD95-]. Current liver biopsy was performed in patients of Group I and examined histopathologically. There was highly statistically significant difference between HCV patients and controls as regard ALT, percentage of apoptotic cytotoxic T lymphocytes [CD8+ CD95+] and non apoptotic cytotoxic T lymphocytes [CD8+ CD95-] [p<0.001]. There was statistically significant difference between patients of Group I, II, III, IV and controls in percentage of CD8+CD95+ and CD8+CD95- cells. Comparison between Group I and II revealed non significant difference in percentage of CD8+CD95+ and CD8+CD95- cells or in percentage of CD8-CD95+ cells [apoptotic non cytotoxic T] [p>0.05]. Comparison between Group III and IV revealed statistically significant difference in percentage of CD8+CD95+ and CD8+CD95- cells [p<0.05]. In Group I the percentage of CD8+CD95+ tended to correlate with activity index of liver biopsy but significantly correlated with serum ALT in Group III. In Group II significant positive correlation between percentage of CD8+CD95+ and AST with tendency toward correlation with ALT was found. Also percentage of CD8+CD95+ correlated with purpura and arthritis but not with cryoglobulinemia. Percentage of CD8+CD95+ showed non statistically significant correlation except with serum prothrombin time in group IV. There was increase in the percentage of CD8+CD95+ cells in higher grade of necroin-flamatory activity and in higher stage of fibrosis in liver biopsy in group I. Our findings support the suggestion of major role of peripheral blood CD8+ T cells in elimination of HCV and suggest that cellular immune response plays a key role not only in viral elimination, but also in liver pathology associated with HCV-infection. Monitoring apoptosis of CD8+ T cells by measuring FAS expression is useful in follow-up of antiviral response in these patients. Finally, Fas/FasL pathway is critical in persistent HCV infection in humans and represents a potential target for restoring function of exhausted HCV-specific CTLs

DNA microarray for identification of fungal pathogens causing invasive fungal infections [IFIs] in neutropenic patients

Opportunistic invasive fungal infections [IFIs] remain as important cause of morbidity and mortality. Candida and Aspergillus species are the most common fungi that cause disease in immunocompromised patients and transplant recipients. This study was designed to identify Candida and AspergilIus spp. as possible causes of IFIs in neutropenic patients with different hematological diseases, using high multiplexing capacity of DNA microarray [species identification array]. Twenty eight patients admitted to Hematology unit-Ain Shams University Hospitals with provisional diagnosis of lFl were enrolled in this study. Venous blood samples were collected to detect Candida and Aspergillus spp. using DNA microarray. Nineteen out of 28 studied patients [67.9%] were infected with Candida and Aspergillus spp. Invasive aspergillosis constituted 13/19[68.4%] distributed as follows: A.fumigatus 6/19 [31.6%], A.flavus 4/19 [21%] and A.niger 3/19 [15.8%]. On the other hand, Fl with Candida spp. constituted 6/19 [31.6%] distributed as follows; C.glabrata 3/19 [15.8%], C.tropicalis 2/19 [10.5%] and C.albicans 1/19 [5.3%]. Duration of hospital stay [mean +/- SD = 30.8 +/- 4 days] was statistically significant among the infected group in comparison to other patients [mean +/- SD = 22.7 +/- 2.3 days]. Nineteen out of 28 studied patients [67.9%] were infected with Candida and Aspergillus spp. Invasive aspergillosis constituted 13/19 [68.4%], while Candidal infection constituted 6/19 [31.6%]. DNA microarray represents a reliable method of potential use in clinical laboratories for parallel one-shot detection and identification of the most common fungal pathogens at the species level for prompt management of infection with tailored antifungal treatments