Characterization of drug –resistance profiles to directly acting agents in hepatitis C virus naive patients

Background: The development of direct-acting antiviral agents [DAAs] has revolutionized the treatment of HCV infection. The main challenge to HCV effective treatment with daas is the emergence of HCV drug-resistant variants. Detection of resistance associated variants is of importance in clinical settings in order to optimize DAA regimens, maximize success rates and reduce the impact of treatment failure

Objectives: The prevalence of possible mutations expected to induce potential directly acting antiviral agent [DAA] resistance was investigated in twenty DAAs naïve HCV infected patients

Methodology: The twenty HCV isolates were genotyped using the full length NS3/4A, NS5B, and two third of the carboxy terminal region [including ISDR] of NS5A gene sequences

Results: Eighteen [90%] out of 20 strains were diagnosed as subgenotype 4a while 2 [10%] were of subgenotype 4n, Amino acid frequencies at each position in the NS3 protease sequence were determined with the VESPA software program. Twenty four Genotype4-specific amino acid signatures were present in almost all of our sequences, but were absent from all other genotypes. Among the twenty four amino acid signatures only one mutation at position 41 [T/S] reported to be associated with resistance to protease inhibitors. Compared to the wild type HCV GT-4; nine mutations were detected among our isolates at a frequency ranging from 27% to 100%. None of these mutations were associated with resistance to protease inhibitors. Forty three mutations were detected among our isolates at a much lower frequency ranging from 5.5% to 16.6%. Only 5 out of them were associated with protease inhibitor resistance. Amplification of domain II and III including the interferon sensitivity-determining region and the interferon/ribavirin resistance-determining region of the NS5a region showed a number of mutations exceeding 4 in all isolates and 82.3% of them had from 10-30 mutations. Thirty two Genotype 4-specific amino acid signatures were present in almost all of our sequences and absent from all other genotypes. The primary NS5B nucleoside polymerase inhibitors [NPIs] resistance variant 282T was not detected in our isolates

Conclusion: The large number of natural polymorphism of HCV 4 isolates as well as the large number of mutations detected in this study and different from those associated with DAA resistance makes it more practical to detect resistance associated mutations in DAA treatment failure then to look for these mutations in naïve patients

T Regulatory Cell Responses to Immunization with a Soluble Egg Antigen in Schistosoma mansoni-Infected Mice

The aim of the study is to characterize the phenotypes of CD4+ CD25+ T regulatory cells within the liver granulomas and association with both Foxp-3 gene expression and splenic cytokines. Naive C57BL/6 mice were intravenously injected with multiple doses of the soluble egg antigen (SEA) 7 days before cercarial infection. The immunized and infected control groups were sacrificed 8 and 16 weeks post-infection (PI). Histopathology, parasitological parameters, splenic phenotypes for T regulatory cells, the FOXP-3 expression in hepatic granuloma using real-time PCR, and the associated splenic cytokines were studied. Histopathological examination of the liver revealed remarkable increase in degenerated ova within hepatic granuloma which decreased in diameter at weeks 8 and 16 PI (P<0.01). The percentage of T regulatory cells (CD4+ CD25+) increased significantly (P<0.01) in the immunized group compared to the infected control at weeks 8 and 16 PI. The FOXP-3 expression in hepatic granulomas increased from 10 at week 8 to 30 fold at week 16 PI in the infected control group. However, its expression in the immunized group showed an increase from 30 at week 8 to 70 fold at week 16 PI. The splenic cytokine levels of pro-inflammatory cytokines, IFN-gamma, IL-4, and TNF-alpha, showed significant decreases (P<0.05) compared to the infected control group. In conclusion, the magnitude and phenotype of the egg-induced effects on T helper responses were found to be controlled by a parallel response within the T regulatory population which provides protection in worm parasite-induced immunopathology.

Early detection of hepatocellular carcinoma on top of liver cirrhosis: the fas receptor and ligand system

Hepatocyte aberrations, accumulation of chromosomal damage and possibly initiation of hepatic carcinogenesis is thought to be caused by the continued viral replication and the persistent attempt by a less than optimal immune response to eliminate hepatitis C virus [HCV] infected cells. The identification of the "death factors" including Fas and its Ligand [Fas-L] as a major regulator of both apoptosis and immune function has provided insight into an attractive mechanism of tumor escape from immune clearance. To assess the hepatic expression of Fas/Fas-L, the Fas receptor [Fas-R] expression on lymphocyte, and serum soluble Fas [sFas] in an attempt to analyze the role of Fas receptor/ligand system in the multistep process of fibrosis/carcinogenesis and the possible use of the serum marker as possible candidate biomarkers for an early detection of hepatocellular carcinoma [HCC]. The current study included 100 samples from cases at Theodor Bilharz Research Institute and Kasr Al Aini Hospital in Egypt. There were 90 cases of chronic hepatitis C [CHC] infection [and negative hepatitis B virus infection]. There were 30 cases without liver cirrhosis, 30 cases with liver cirrhosis and 30 cases with HCC. 10 liver biopsies were taken from healthy livers as normal controls. Histopathologic study and immunohistochemistry for detection of hepatic Fas and Fas-L expression were determined for all cases. Electron microscopy [EM] and immunoelectron microscopy [IEM] examination for detection of Fas-R expression on lymphocytes were also done. sFas, liver function tests, serologic markers for viral hepatitis, and serum alpha-fetoprotein level [alpha-FP] were done. The sFas in both HCC and CHC with cirrhosis patients were significantly higher than those of normal controls and CHC without cirrhosis [P<0.01], but there was no significant difference between the cirrhosis and HCC patients. Positive hepatic expression of both Fas and Fas-L were significantly increased in the diseased groups [p<0. 01] compared to the control specimens. A progressive Fas and FasL increase from CHC without cirrhosis to CHC with cirrhosis followed by a decline from the latter to HCC. Apoptotic Fas and Fas-L proteins expression was significantly increased with the necroinflammatory activity and the advancement of fibrosis. There was a non-significant negative correlation between sFas and hepatic Fas. In addition a significant over expression of Fas-R on separated lymphocytes was associated with a higher frequency of apoptotic cell death as detected by EM examination. Conclusion: The Fas receptor/ligand system was significantly involved in the process of liver cirrhosis converting into HCC. Down-regulation of Fas expression, up-regulation of Fas-L expression in hepatocytes and elevation of serum sFas level were important in tumor evasion from immune surveillance and in hepatic carcinogenesis

Histological, immunohistochemical and electron microscopic studies on the effect of melatonin on pituitary-testicular axis in aged male albino rat

Aging is a biological process affecting mammalian tissues and organs at different rates. The present work was done to study the effect of melatonin as anti-aging therapy on the structure of the testis and the pars distalis. A total number of 25 male albino rats were used. The animals were divided into three groups: group [I] served as a control adult group [6 months age], group [II] was the control aged group [20-months age] and group [III] was treated with melatonin. The specimens of the testis and pituitary glands were prepared for light and electron microscopy. Histological and immunohistochemical techniques were used to examine these organs. It was found that age-related changes occur in the histological immunohistochemistry and ultrastructure of the testis and pituitary gland. Also, treatment with melatonin as anti-oxidant and free radical scavenger caused improvement of these changes in the pituitary gland as well as the testis. The age-related changes that occurred in the testis included progressive involution and germ cells depletion in the seminiferous tubules which contained only Sertoli cells. Ultrastructural apoptotic changes of germ cells increased with aging and abnormal morphology of the resulting sperms were found. This might result in decapacitating sperm function increase in interstitial tissue. Leydig cells exhibited ultrastructural changes which might lead to a decrease in their activity and testosterone production. As regard the pituitary gland; different utrastructural changes occurred in the somatotrophs or GH cells. The ultrastructrual and immunohistochemical studies of gonadotrophs or LH/FSH cells showed marked deterioration with progression of age. Treatment with melatonin from the adult state improved the morphological and ultrastructural changes that occurred with aging