Faten Nagy

Circulating miRNAs as Predictor Markers for Activation of Hepatic Stellate Cells and Progression of HCV-Induced Liver Fibrosis.

Introduction Liver fibrosis is the excessive accumulation of extracellular matrix that occurs by activation of hepatic stellate cells (HSCs), which has been identified as the major driver of liver fibrosis. Several studies confirmed that miRNAs have regulatory effects on the activation of HSCs by affecting the signaling pathways. The aim of this study was to develop non-invasive diagnostic markers by measuring different circulating miRNAs in serum as predictor markers for early diagnosis of liver fibrosis and its progression. Methods In this case-control study, we enrolled 66 subjects with chronic hepatitis C (CHC) with early stage of fibrosis and 65 subjects with CHC with late-stage fibrosis. Also, 40 subjects were included as normal controls. The six main miRNAs, i.e., miR-138, miR-140, miR-143, miR-325, miR-328, and miR-349, were measured using the reverse transcription-polymerase chain reaction. Results In the cases of CHC both with early and late stage of fibrosis, the circulating levels of the six main miRNAs were significantly higher than the levels in the control group. ROC analysis indicated that the sensitivity and specificity of miR-138 were 89.3% and 71.43%, respectively, in the early stage of fibrosis. In the late stage, the sensitivity and specificity of miR-138 were 89.3 and 93.02%, respectively, whereas, for miR-143, they were 75.0 and 88.4%, respectively. Conclusions Circulating miR-138 could serve as a non-invasive biomarker for the detection of early fibrosis. Also, miR-138 and miR-143 could be specific biomarkers for indicating the late stage of liver fibrosis.

OL-037 Effect of treatment with antifibrotic drugs in combination with PZQ in immunized Schistosoma mansoni infected murine model

The main problem in schistosomal hepatic morbidity is fibrosis and extensive scarring induced by living eggs. In this study, we tried to study the effect of treatment using antihelminthic drug (PZQ) and/or antifibrotic drugs (PTX and silymarin) in combination with immunization. The parasitological parameters, the dynamics of serum-specific immunoglobulins and splenic cytokines associated with changes in granuloma diameter were assessed. Naive mice were immunized intravenously with 10 ug of SEA in three doses at 2 days intervals 6 weeks before infection. Animals were infected by tail immersion with 100 cercariae and divided into several groups. Three groups were treated with PZQ, PTX or silymarin administered alone. Another two groups were treated with PZQ combined with PTX or silymarin. All treated animals and respective controls were sacrificed 12 weeks post infection. Immunization did not affect worm reduction , but slight decrease in granuloma diameter, increase in immunoglobulins and cytokines was observed . Reduction in worm burden was associated with reduction in ova count and changes in oogram pattern which were mainly due to PZQ treatment. Increasing reduction in granuloma diameter, elevation of immunogloulins and cytokines levels were observed in the groups treated with PZQ alone or cmbined with PTX or silymarin. In conclusion, in this study, treatment with PZQ complemented with immunization resulted in significant reduction of parasitological parameters and rise of specific Igs. Addition of antifibrotic drugs PTX or silymarin to PZQ, potentiated an antipathology effect which minimized and ameliorated liver fibrosis by inhibition of HSC activation and accentuation of the effect of suppressor Treg cells. (Journal of American Science 2010; 6(5):208-216). (ISSN: 1545-1003). Key word: Schistosoma mansoni, Praziquantel, Pentoxifyllin, silymarin.

Protective Role of Purified Cysteine Proteinases against Fasciola gigantica Infection in Experimental Animals

Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by Fasciola gigantica play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in Freunds adjuvant followed by 3 booster doses 1 mg each at 4 week intervals) were injected intramuscularly into sheep 1 week prior to infect orally with 300 F. gigantica metacercariae. All the sheep were humanely slaughtered 12 weeks after the first immunization. Changes in the worm burden, ova count, and humoral and cellular responses were evaluated. Significant reduction was observed in the worm burden (56.9%), bile egg count (70.7%), and fecel egg count (75.2%). Immunization with CP was also found to be associated with increases of total IgG, IgG1, and IgG2 (P<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-γ, and TNF-α, revealed significant decreases (P<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-β, and IL-6, showed significant increases (P<0.05). In conclusion, it has been found that CP released by F. gigantica are highly important candidates for a vaccine antigen because of their role in the fluke biology and host-parasite relationships.

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. Naïve 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-γ, IL-4, and TNF-α, 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.

Serum Markers of Epithelial Mesenchymal Transition as Predictors of HCV-induced Liver Fibrosis, Cirrhosis and Hepatocellular Carcinoma

Introduction Hepatitis C virus (HCV) is a major cause of chronic liver disease in Egypt, leading to hepatic fibrosis, liver cirrhosis (LC), and hepatocellular carcinoma (HCC). Liver fibrosis is characterized by excessive deposition of extracellular matrix (ECM). Newly-recognized pathogenic mechanisms point to the epithelial-mesenchymal transition (EMT) of hepatocytes to matrix synthesizing (myo-) fibroblasts. Transforming growth factor-beta (TGF-β1), bone morphogenic protein (BMP)-7, and connective tissue growth factor (CTGF) are biomarkers reflecting the EMT process. YKL-40 is a glycoprotein member of ECM and plays a role in cancer cell proliferation. The purpose of this study was to determine the serum biomarkers of EMT and its impact on the fibrogenic process and tumorigenesis in HCV-genotype 4 patients. Methods In this case-control study that was conducted in 2013–2014, 97 HCV-infected patients were subjected to clinical examination, laboratory investigations, and liver biopsy. According to the histopathologic examination, they were classified to F0 (14 cases), F1 (17 cases), F2 (15 cases), F3 (18 cases), F4 (22 cases), and HCC (11 cases). Fifteen age- and gender-matched subjects were included as normal controls. Serum levels of TGF-β1, BMP-7, CTGF, YKL-40 were assessed, and the TGF-β1/BMP-7 ratios were calculated. The data were analyzed by plotting the receiver operating characteristic curve (ROC), Pearson product-moment correlation coefficient, and Spearman’s rank correlation coefficient (Spearman’s rho). Results Serum levels of TGF-β1, BMP-7, CTGF, and YKL-40 were significantly increased in all patient groups compared to controls (p < 0.001). LC exhibited the highest CTGF level and YKL-40 was highest in HCC. The TGF-β1/ BMP-7 ratios reflected the progression of EMT from CHC to LC, however, there was no significant difference between LC and HCC. TGF-β1/ BMP-7 ratio is considered to reflect positive correlation with CTGF in LC group (r = 0.629; p < 0.03) and YKL-40 in HCC group (r = 0.504; p < 0.04). Conclusion Increased TGF-β1/BMP-7 ratio and CTGF levels reflect the rate of EMT and provide information about fibrogenic activity. Also, this ratio, in association with YKL-40, can be used to predict malignant transformation in HCV-genotype 4 Egyptian patients.

PP-198 Comparison between different immunological techniques for detection of circulating Fasciola antigen in sheep

The detection of Fasciola antigen in serum or stool could be more valuable in diagnosis, hence early treatment before irreparable damage. In this study, fresh adult Fasciola gigantica worms were collected, then incubation in culture medium and collected medium was used to extract crude excretory-secretory (E/S) antigen. E/S was used to immunize rabbits to raise specific antibodies against Fasciola spp. Purified antibodies are further used as primary capture to coat ELISA plates. The secondary capture of antibodies was by conjugation with horse reddish peroxidase. Sandwich ELISA and DOT-ELISA were performed to detect Fasciola antigens in both serum and stool samples collected from a total of 152 sheep. After slaughtering, gross inspection of liver and parasitological stool examination, sheep were divided into Fasciola positive group (97 sheep), other helminthic infection group (30 sheep) and healthy control group (30 sheep). Fasciola antigen detected in serum of sheep by ELISA showed 94.8 % sensitivity and 95 % specificity. Copro-antigen detected by ELISA showed 96.9% sensitivity and 96.7% specificity. The sensitivity and specificity of coproantigen by ELISA in stool sample were higher than that recorded by Sandwich ELISA for serum. Dot ELISA sensitivity was found to be 98.9% and specificity 98.3%. In conclusion the Dot ELISA gives better sensitivity and specificity than sandwich ELISA for serum and coproantigen in stool by ELISA. (New York Science Journal 2010;3(7):34-39). (ISSN: 1554-0200).

The Role of MicroRNAs in Response to Interferon Treatment of Chronic Hepatitis C patients.

Introduction Treatment of HCV using a combination of pegylated interferon (PEG-IFN) and ribavirin fails in about 40% of the patients with HCV genotype 4 infections, and it is physically and economically demanding. Thus, it is highly important to identify factors that can help to predict the likelihood that a patient will respond to this treatment. Methods In this study, five miRNAs, i.e., miRNA-122, miRNA-199, miRNA-192, miRNA-30, and miRNA-128, were selected according to previous studies that demonstrated their noticeable functions in viral replication, indicating that they potentially could be used by host cells to control viral infections. The five miRNAs were measured using real-time, reverse transcription-polymerase chain reactions. The data were analyzed using the t-test and chi-squared test. Results We found that the expression level of miRNA-122 was significantly increased in the responders’ group (p < 0.01) over that in the non-responders’ groups before and after treatment; both increased significantly (p < 0.01) compared with the normal control group. Conclusion miR-122 might be a useful predictor for virological responses to treatment with PEG-interferon plus ribavirin therapy in patients with HCV.