Mona Zoheiry

Diagnostic efficacy of monoclonal antibody based sandwich enzyme linked immunosorbent assay (ELISA) for detection of Fasciola gigantica excretory/secretory antigens in both serum and stool

BackgroundThis research was carried out to develop a reliable monoclonal antibody (MoAb)-based sandwich enzyme linked immunosorbent assay (ELISA) for the diagnosis of active Fasciola gigantica infection in both serum and stool for comparative purposes.MethodsFrom a panel of MoAbs raised against F. gigantica excretory/secretory antigens (ES Ags), a pair (12B/11D/3F and 10A/9D/10G) was chosen due to its high reactivity and strict specificity to F. gigantica antigen by indirect ELISA.ResultsThe two MoAbs were of the IgG1 and IgG2a subclasses, respectively. Using SDS-PAGE and EITB, the selected MoAbs recognized 83, 64, 45 and 26 kDa bands of ES Ags. The lower detection limit of ELISA assay was 3 ng/ml. In stool, the sensitivity, specificity and diagnostic efficacy of ELISA was 96%, 98.2 and 97.1%; while in serum they were 94%, 94.6% and 94.3%, respectively. Moreover, a positive correlation was found between ova count in stool of F. gigantica infected patients and the OD readings of ELISA in both stool and serum samples (r = 0.730, p < 0.01 and r = 0.608; p < 0.01, respectively).ConclusionsThese data showed that the use of MoAb-based sandwich ELISA for the detection of F. gigantica coproantigens in stool specimens was superior to serum samples; it provides a highly efficient, non-invasive technique for the diagnosis of active F. gigantica infection.

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.

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.

The use of whey protein concentrate in management of chronic hepatitis C virus - a pilot study.

Introduction Whey protein contains biologically active ingredients that can prevent and attenuate disease besides being nutritive. The aim of the study was to clarify the effects of oral administration of whey protein on viral load and host defence mechanisms, in particular, phagocytic function of neutrophils, selected immunomodulatory cytokines and serum inflammatory markers, in compensated chronic hepatitis C virus (HCV) patients. Material and methods Twenty-seven HCV patients (20 males and 7 females) recruited from the hepatology clinic of the Theodor Bilharz Research Institute (TBRI) were given whey protein concentrate (WPC) twice daily for two months. In addition, 15 age and sex matched healthy participants were included in the study, as a control group. Neutrophil phagocytic activity, serum intercellular adhesion molecule (sICAM), interleukin-2 (IL-2), nitric oxide (NO), as well as HCV-RNA levels and routine investigations were determined for patients, before and after WPC supplementation and once for the control group. Results There was a significant decrease in viral load and markers of active inflammation, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), while serum albumin, total leucocyte counts and absolute neutrophil counts showed significant elevation accompanied by improvement of neutrophil phagocytic activity after WPC supplementation compared to pre-treated levels. The oral WPC supplementation was well tolerated without any serious adverse events. Conclusions Oral supplementation of WPC has promising results as a new therapeutic strategy against HCV and its sequelae by decreasing the viral load and active inflammation as well as improving the synthetic capacity of the liver and the phagocytic function of neutrophils, in these patients.

Expression analysis of liver-specific circulating micrornas in hcv-induced hepatocellular carcinoma in egyptian patients

ABSTRACT Objectives: Due to the absence of reliable and accurate biomarkers for the early detection of liver malignancy, circulating microRNAs have recently emerged as great candidates for prompt cancer identification. Therefore, the aim of this study was to investigate the potential of liver-specific circulating microRNAs as an accurate non-invasive diagnostic tool for early diagnosis of hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC). Methodology: A total of 165 patients were enrolled in this study and categorized into four main groups: 42 chronic hepatitis C (CHC) without cirrhosis, 45 CHC with cirrhosis (LC), 38 HCC with HCV patients, and 40 healthy controls. The expression profiles of seven miRNAs (miR-16, miR-34a, miR-125a, miR-139, miR-145, miR-199a, and miR-221) were analyzed using real-time PCR. Results: Serum levels of miRNA-125a, miRNA-139, miRNA-145, and miRNA199a were significantly lower (p < 0.01) in HCC than in both CHC and LC groups. On the other hand, no significant difference was shown in the expression of miR-16, miR-34a, and miR-221 between the CHC, LC, and HCC groups. MiR-16, miR-34a, and miR-221 were significantly elevated in the HCC group compared to the control group. MiR-34a showed the highest specificity and sensitivity. Conclusions: The results indicated that the measurement of serum levels of miR-125a, miR-139, miR-145, and miR-199a can help to differentiate HCC from CHC and LC. Also, miR-16, miR-34a, and miR-221 serum levels would have a prognostic value. MiR-34a had the highest specificity and sensitivity, indicating that it might serve as a novel and potential non-invasive biomarker for HCV-induced HCC.

Potential ultrastructure predicting factors for hepatocellular carcinoma in HCV infected patients

ABSTRACT Hepatitis C virus represents one of the rising causes of hepatocellular carcinoma (HCC). Although the early diagnosis of HCC is vital for successful curative treatment, the majority of lesions are diagnosed in an irredeemable phase. This work deals with a comparative ultrastructural study of experimentally gradually induced HCC, surgically resected HCC, and potential premalignant lesions from HCV-infected patients, with the prospect to detect cellular criteria denoting premalignant transformation. Among the main detected pathological changes which are postulated to precede frank HCC: failure of normal hepatocyte regeneration with star shape clonal fragmentation, frequent elucidation of hepatic progenitor cells and Hering canals, hepatocytes of different electron density loaded with small sized rounded monotonous mitochondria, increase junctional complexes bordering bile canaliculi and in between hepatocyte membranes, abundant cellular proteinaceous material with hypertrophied or vesiculated rough endoplasmic reticulum (RER), sequestrated nucleus with proteinaceous granular material or hypertrophied RER, formation of lipolysosomes, large autophagosomes, and micro-vesicular fat deposition. In conclusion, the present work has visualized new hepatocytic division or regenerative process that mimic splitting or clonal fragmentation that occurs in primitive creature. Also, new observations that may be of value or assist in predicting HCC and identifying the appropriate patient for surveillance have been reported. Moreover, it has pointed to the possible malignant potentiality of liver stem/progenitor cells. For reliability, the results can be subjected to cohort longitudinal study.

miRNA-221 and miRNA-222 are promising biomarkers for progression of liver fibrosis in HCV Egyptian patients

BACKGROUND Current methodologies used to determine the progression of hepatic fibrosis rely heavily on liver biopsy, a dangerous and invasive procedure, with semi-subjective analysis of the results of the biopsy. Thus, a new approach is immensely needed for monitoring the progression of liver fibrosis in Hepatitis C virus (HCV) patients. AIM OF WORK The purpose of this study was to find highly specific and sensitive miRNA biomarkers that can be used to detect different stages of liver fibrosis. METHODOLOGY The study consisted of 42 cases of chronic hepatitis C (CHC) with early-stage fibrosis, 45 cases of CHC with late-stage fibrosis, and 40 healthy subjects with no CHC or fibrosis as controls. Expression patterns of 5 miRNAs (miR-16, miR-146a, miR-214-5p, miR-221, and miR-222) were analyzed in each group using TaqMan real-time PCR. RESULTS Serum levels of miRNA-16, miRNA-146a, miRNA-221, and miRNA-222 were all significantly up-regulated in early and late stages of liver fibrosis. miRNA-222 had the highest sensitivity and specificity values in early and late fibrosis. miRNA-221 had the second highest sensitivity and specificity with the late-stage fibrosis group. Furthermore, miRNA-221 showed significant positive correlations with both miRNA-16 and miRNA-146a in the early- and late-stage fibrosis groups, with the early stage having a stronger correlation. CONCLUSIONS The results indicated that miRNA-16, miRNA-146a, miRNA-221, and miRNA-222 can be used to detect the presence of liver fibrosis. The high sensitivity and specificity of miRNA-222 and miRNA-221 in late-stage fibrosis indicate promising prognostic biomarkers for HCV-induced liver fibrosis.

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.

ROLE OF EPITHELIAL MESENCHYMAL TRANSITION IN HEPATIC FIBROGENESIS.

Liver fibrosis is a gradual process of increased secretion and decreased degradation of extra-cellular materials. Two cell types are now well recognized as being involved in liver fibrosis, i.e. hepatic stellate cells (HSCs) and portal mesenchymal cells. T iis process is initiated by the damage of hepatic cells, which leads to activation of hepatic stellate cells that differentiate into myofibroblasts leading to the formation of liver fibrosis. On the other hand, the epithelial-mesenchymal transition and mesenchymal-epithelial transition are crucial for the regulation of cellular plasticity during liver fibrosis. The EMT is a process in which molecular reprogramming leads epithelial cells to adopt a mesenchymal phenotype. During EMT, epithelial cells gain mesenchymal features which include changes in the expression of epithelial markers. The EMT process plays fundamental roles during embryogenesis, tissue fibrosis, and carcinogenesis. As multiple experimental studies of liver fibrosis have confirmed that established liver fibrosis is reversible upon cessation of the causative agent, modulation of the EMT markers could be promising as potential therapeutic agents. Better understanding of the molecular cascades of intracellular fibrogenic signaling and genetic factors that controlling the expression of EMT markers would be a powerful strategy for early diagnosis and treatment liver fibrosis at the genetic level. Activating or silencing of the responsible genes may be an efficient and more specific approach for treating liver fibrosis either through the arrest of EMT or the induction of MET.

701 SERUM MIRNAS AS A NOVEL NON-INVASIVE DIAGNOSTIC TOOL FOR HCV-INDUCED HEPATOCELLULAR CARCINOM

diagnosis across regions was A in 66–84% and B in 16–32%. Median time to systemic treatment for unresectable disease was 111 days in North America, 127 in Europe, and 41 in Asia. Approximately one third of patients across regions received systemic therapy as initial treatment; additional recorded treatments in this cohort included transplantation/resection (14–26%) and locoregional therapy (46–63%). Sorafenib was received by ~90% of systemically treated patients in North America and Europe, and 58% in Asia. These results will be updated with second interim data that will be available in March 2012. Conclusions: This interim analysis of the HCC BRIDGE study shows variations in the regional use of systemic treatments in patients with HCC, some of which may be related to differences in patient access to specific therapies. Insights derived from these data may prove helpful in informing efforts to improve the future management of patients with HCC.