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There is an enormous diversity of life forms present in the extremely intricate marine environment. The growth and development of seaweeds in this particular environment are controlled by the bacteria that settle on their surfaces and generate a diverse range of inorganic and organic chemicals. The purpose of this work was to identify epiphytic and endophytic bacterial populations associated with ten common marine macroalgae from various areas along the Mediterranean Sea coast in Alexandria. This was done to target their distribution and possible functional aspects. Examine the effects of the algal habitat on the counting and phenotypic characterization of bacteria, which involves grouping bacteria based on characteristics such as shape, colour, mucoid nature, type of Gram stain, and their ability to generate spores. Furthermore, studying the physiological traits of the isolates under exploration provides insight into the optimum environmental circumstances for bacteria associated with the formation of algae. The majority of the bacterial isolates exhibited a wide range of enzyme activities, with cellulase, alginase, and caseinase being the most prevalent, according to the data. Nevertheless, 26% of the isolates displayed amylolytic activity, while certain isolates from Miami, Eastern Harbor, and Montaza lacked catalase activity. Geographical variations with the addition of algal extract may impact on the enumeration of the bacterial population, and this might have a relationship with host phylogeny. The most significant observation was that endophytic bacteria associated with green algae increased in all sites, while those associated with red algae increased in Abu Qir and Miami sites and decreased in Eastern Harbor. At the species level, the addition of algal extract led to a ninefold increase in the estimated number of epiphytic bacteria for Cladophora pellucida in Montaza. Notably, after adding algal extract, the number of presented endophytic bacteria associated with Codium sp. increased in Abu Qir while decreasing with the same species in Montaza. In addition to having the most different varieties of algae, Abu Qir has the most different bacterial isolates., (© 2024. The Author(s).)

Hepatitis C virus (HCV) infection is a global health concern affecting millions worldwide. Chronic HCV infection often leads to liver inflammation and can progress to cirrhosis and hepatocellular carcinoma. Inflammatory cytokines are crucial in modulating the immune response during HCV infection. This review aims to investigate the impact of different inflammatory cytokines on HCV infection and associated immune responses. This review was conducted to identify relevant studies on the interplay between inflammatory cytokines and HCV infection. The analysis focused on the effects of key inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-1 (IL-1), and interferon-gamma (IFN-γ), on HCV replication, immune cell activation, and liver inflammation. The findings reveal that these inflammatory cytokines can significantly influence HCV infection and the subsequent immune response. TNF-α, IL-6, and IL-1 have been shown to enhance HCV replication, while IFN-γ exerts antiviral effects by inhibiting viral replication and promoting immune cell-mediated clearance of infected hepatocytes. Moreover, these cytokines contribute to the recruitment and activation of immune cells, such as natural killer cells, T cells, and macrophages, which play critical roles in controlling HCV infection. Understanding the precise mechanisms by which inflammatory cytokines impact HCV infection is crucial for developing more targeted therapeutic strategies. Modulating the levels or activity of specific cytokines may provide opportunities to attenuate HCV replication, reduce liver inflammation, and improve treatment outcomes. In conclusion, this review highlights the significance of inflammatory cytokines in influencing HCV infection and associated immune responses.

Background: The key role of miRNA expression in incidence and progression of colorectal cancer (CLC) have been developed over the last decade. Materials & methods: A total of 153 subjects were enrolled into two phases: 14 selected miRNAs were first evaluated in 50 subjects, then miR-26a and miR-26b relative expression were further evaluated in 103 subjects and their target protein MMP-9 was measured. Results: miR-26a and -26b showed highly significant overexpression. Both miR-26a and -26b (p < 0.001) had high diagnostic efficacy for CRC. There was a significant increase in serum MMP-9 protein in CRC patients with positive correlation with miR-26a and -26b expression levels (p < 0.001). Conclusion: miRNA 26a and 26b with MMP-9 can be used as diagnostic biomarker for CRC patients.

Objectives: In this study, we investigated the association between the IFN-λ3 rs12979860 single nucleotide polymorphism (SNP) and the transition from late fibrosis to HCC in Egyptian HCV-chronically infected patients., Methods: The rs12979860 SNP was genotyped using real-time PCR in DNA from the whole blood of healthy subjects (n=60) and HCV patient   s (n=342). We stratified the patients into (1) treatment-naïve patients (n=218) with advanced fibrosis (F2-F4, n=123) and HCC (n=95 Treatment-experienced patients (n=124)  who received SOF-based therapy for 12 weeks and achieved SVR (SVR12). DAA-treated patients were divided into 2 groups: group I (n=63) included patients with advanced hepatic fibrosis (F2-F4) who did not develop HCC within a year after treatment, and group II (n=61) included patients who were free of focal hepatic lesions before starting DAA therapy but developed HCC within a year., Results: Our results demonstrated that treatment-naïve patients with the CT/TT genotypes and the T allele were more likely to have HCC (odds ratio 3.1, 95% CI 1.44-6.71, P = 0.003 and odds ratio 1.89, 95% CI 1.28-2.76, P = 0.001, respectively). Binary regression analysis defined 3 independent predictors associated with HCC development: age (odds ratio 1.039, 95% CI 1.004-1.076, P = 0.028), alanine aminotransferase (odds ratio 1.008, 95% CI 1.002-1.015, P = 0.010), and rs12979860 (odds ratio 3.65, 95% CI 1.484-8.969, P = 0.005)., Conclusions: However, the rs12979860 SNP did not show any correlation with the progression of HCC post-treatment. Despite the debate on the contribution of IFN-λ3 rs12979860 to susceptibility to HCV-related HCC, our data confirm the role of this SNP in this context.

Objective: This study aimed at exploring the potential role of a panel of serum micro-RNA (miRNA) markers in liver fibrosis and hepatocellular carcinoma (HCC) diagnosis in patients with chronic hepatitis C virus (HCV) infection., Methods: The study included 157 chronic HCV patients and 62 HCC patients who presented to the Cairo University Center for Hepatic Fibrosis, Endemic Medicine Department, from 2015 to 2017. Relevant clinical and laboratory data were collected and sera were subjected to miRNA expression profiling. Eleven miRNA markers were studied and receiver operating characteristic curves were constructed to investigate the best cutoff values of the miRNAs that showed altered expression in HCC compared to HCV-associated advanced fibrosis., Results: miRNA expression profiling revealed 5 miRNAs (miR-124, miR-141, miR-205, miR-208a, miR-499a) were significantly upregulated and 2 miRNAs were significantly downregulated (miR-103a, miR-15a) in HCC compared to advanced fibrosis patients. No significant difference was observed in miRNA expression between advanced fibrosis and early hepatic fibrosis apart from a significant downregulation of miR-155-5p in advanced fibrosis., Conclusion: Serum miRNAs could serve as potential diagnostic tools for the diagnosis of HCC., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society for Clinical Pathology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Hepatitis C virus (HCV) is the leading cause of liver diseases worldwide. At present, combinations of different classes of direct-acting antiviral agents (DAAs) are used as treatment options for HCV, in which sofosbuvir (SOF) is the common DAA among different therapeutic regimes. In Egypt, SOF plus daclatasvir (DCV) is the widely used anti-HCV treatment protocol. Herein, we aimed to assess the association between 3 single-nucleotide polymorphisms (SNPs) at the genes coding for 2 SOF metabolizing enzymes: histidine triad nucleotide-binding protein 1 (HINT1) rs4696/rs7728773 and nucleoside diphosphate kinase 1 (NME1) rs3760468, together with the most potent anti-HCV innate molecule, i.e., interferon lambda 3 (IFNL3) rs12979860 and the response to SOF/DCV in Egyptian patients chronically infected with genotype 4 (GT4). SNPs were genotyped using real-time PCR in DNA from patients who achieved sustained virological response (SVR) at 12 weeks post-SOF/DCV treatment (i.e., responders; n = 188), patients who failed to achieve SVR12 (i.e., non-responders; n = 109), and healthy controls (n = 62). Our results demonstrated that patients bearing HINT1 rs7728773 CT/TT (odds ratio 2.119, 95% CI 1.263-3.559, p = 0.005) and IFNL3 rs12979860 CC (odds ratio 3.995, 95% CI 2.126-7.740, p = 0.0001) were more likely to achieve SVR12. However, neither HINT1 rs4696 nor NME1 rs3760468 seems to contribute to the responsiveness to SOF/DCV. Binary regression analysis defined 5 predictor factors independently associated with SVR12: age, bilirubin, hemoglobin, early stages of fibrosis, and combined HINT1 rs7728773 and IFNL3 rs12979860 favorable and mixed genotypes (odds ratio 3.134, 95% CI 1.518-6.47, p = 0.002), and that was confirmed by the combined ROC curve for the 5 predictor factors (AUC = 0.91, 95% CI 0.869-0.95, P = 0.0001). In conclusion, these data suggest that the two SNPs have the potential in predicting the response rate to SOF/DCV treatment in patients infected with HCV GT4. This study is the first to investigate the pharmacogenetics of SOF metabolizing enzyme and introduce HINT1 rs7728773 as a novel SNP that predicts the treatment efficacy., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

This study aims at controlling excess nutrients, including nitrogen and phosphorus, and harmful algae that pollute the marine environment using biosynthesized iron oxide nanoparticles. Aqueous extracts of brown seaweeds named Petalonia fascia , Colpomenia sinuosa, and Padina pavonica were selected for iron oxide nanoparticles' biosynthesis. The extracts were used as reductants of ferric chloride, and dark brown colors due to Fe 3 O 4 -NPs biosynthesis were observed. Iron signals were recorded in EDX spectra. FTIR analyses showed that the three algae extracts contained proteins and lipids that have the ability to proceed dual functions of bioreduction and stabilization of Fe 3 O 4 -NPs, suggesting that proteins in P. fascia , C. sinuosa, and P. pavonica extracts reduced FeCl 3 , and the aromatic compounds stabilized the biogenic Fe 3 O 4 -NPs. The biosynthesized Fe 3 O 4 -NPs via P. pavonica had the best nitrogen reduction percentage, followed by C. sinuosa and P. fascia for both concentrations 2 and 4 µg mL -1 . The best results of phosphorus removal and Chlorophyll a (Chl a ) reduction percentages using the Fe 3 O 4 -NPs concentrations were 2 and 4 µg mL -1 with nanoparticles synthesized via P. fascia and P. pavonica, respectively. The highest reduction in optical density for both 2 and 4 µg mL -1 was achieved by the nanoparticles synthesized by P. fascia. Novelty Statement Application of nanoparticles using seaweeds extracts could be alternative safe bioremediation of wastewaters. Currently, iron oxide nanoparticles are used to reduce nitrogen and phosphorus and reduce the blooming of harmful algae; little information about this issue has been reported. Such study also presented the anti-algal impacts of Fe 3 O 4 -NPs by tracing optical density and Chl a concentrations in the examined real seawater samples. Modern biotechnology to develop phytoremediation and seaweeds to enhance these remediation methods can be adopted.

Objectives: Colorectal cancer (CRC) is the third lethal malignancy worldwide. Dysregulation of microRNAs (miRNAs) mediates several growth factors signaling pathways and induces abnormal genes expression, which leads to colorectal carcinogenesis. We aimed to comprehensively assess the expression of miRNA-200c, miRNA-203a, miRNA-223 in Egyptian CRC tissue and their corresponding serum samples and to explore if they have any potential prognostic or diagnostic value for CRC patients., Methods: A total of 195 subjects (120 CRC patients and 75 healthy controls) participated in exploration and validation sets. The relative expression of miRNA-200c, miRNA-203a, and miRNA-223 was measured in both CRC tissue and serum samples, and the expressed miRNAs were compared in different CRC grades and types and the prognostic value was evaluated., Results: The expression levels of miRNA-200c and miRNA-203a were reduced in CRC tissue samples than adjacent noncancerous tissues. miRNA-223 level was significantly upregulated in both CRC tissue and serum samples with a positive association between them (r = 0.85, P = 0.001). The miRNA-223 can effectively discriminate CRC patients from controls and can significantly differentiate between colon and rectal cancer patients. The association between serum miRNA-223 expression and CRC development was validated in the second set and the ROC curve showed highly significant prognostic value with 90.1% sensitivity, 87% specificity, and area under the curve of 0.914 (95% confidence interval: 0.830-0.978, P = 0.0001). These results showed the association between miRNA-223 upregulation and the CRC carcinogenesis., Conclusion: Circulating miRNA-223 can be a potential noninvasive prognostic biomarker for Egyptian CRC patients.

Background: Colorectal cancer (CRC) rates are affected by genetics, ethnicity, and environmental factors; it is considered one of the most aggressive human malignancies with high mortality and morbidity rates worldwide due, in part, to its asymptomatic nature during the early stages of disease. Objective: Owing to the impact of microRNA (miRNA) dysregulation on CRC development and progression, this study was conducted to explore the expression levels of mir-21, -23a, and -27a in the sera and tissues of Egyptian CRC patients and to evaluate their diagnostic efficacy based on circulating levels. Methods: In the test phase, the relative expression levels of the studied miRNAs were evaluated in the sera of 70 participants (35 CRC patients and 35 healthy controls) using quantitative real-time-polymerase chain reaction and to verify their diagnostic value. The exploratory phase was designed to validate the tumor-derived trait by comparing the miRNA levels in the cancerous and adjacent noncancerous tissues. Results: The relative expression levels of the studied miRNAs were significantly upregulated in both serum and tumor tissues of the patients compared to their corresponding controls. In addition, significant positive correlations were found between the relative expression levels of the studied miRNAs in serum samples and their levels in the matched CRC tissues. The serum expression levels of mir-21 and -23a were more predictive of CRC than mir-27a. Conclusion: Circulating mir-21, -23a, and -27a expression levels appear to be valuable diagnostic biomarkers for CRC, especially when combined.

Egypt has increased incidence and high rate of early onset colorectal cancer (CRC). This study aimed to profile the expression levels of 84 circulating microRNAs (miRNAs) in Egyptian CRC patients and to evaluate the diagnostic accuracy of some selected miRNAs as diagnostic biomarkers for CRC patients. A total of 129 subjects (84 CRC patients and 45 healthy controls) were enrolled in two independent sample sets: the screening set (39 subjects) and the validation set (90 subjects). The expression profiles of 84 miRNAs were studied by miRNA PCR array in the screening set. Then four miRNAs (let-7c, 21, 26a, 146a) were selected to be studied by quantitative real-time PCR in the validation set. The PCR array results revealed significant up regulation of 20 miRNAs and downregulation of two miRNAs in CRC patients compared to the healthy subjects. Moreover, the expression levels of the four selected miRNAs were significantly higher in CRC serum samples than controls. The ROC analysis revealed that miRNAs (let-7c, 21, 26a and 146a) can effectively discriminate between CRC patients and the controls. The combination of the four miRNAs showed AUC of 0.950 (95% CI [0.898-1.002], p = .001). However, the combination of miR-21 and miR-26a showed the best diagnostic accuracy with AUC of 0.953 (95% CI [0.908-0.999], p = .001). The current data suggest that miRNAs (let-7c, 21, 26a, 146a) could play an important role in CRC development and they can be used as diagnostic biomarkers for CRC., (© 2019 International Union of Biochemistry and Molecular Biology.)

Background: Although DAAs hold promise to significantly reduce rates of chronic HCV infections, its eradication still requires development of an effective vaccine. Prolonged T cell responses and cross neutralizing antibodies are ideal for vaccination against the infection. We aimed to design and synthesize a 6 multi epitope peptide vaccine candidate and provide evidence for production of extended cellular and neutralizing Abs in mice., Methods: Six peptides derived from conserved epitopes in E1, E2 (n = 2),NS4B, NS5A and NS5B were designed, synthesized in a multiple antigenic peptide (MAP) form and administered w/o adjuvant to BALB/c mice as HCVp6-MAP at doses ranging from 800 ng to 16 μg. Humoral responses to structural epitopes were assayed by ELISA at different times after injection. ELISpot assay was used to evaluate IFN ɣ producing CD4 + / CD8 + T- lymphocytes at extended durations i.e. > 20 weeks. Viral neutralization by mice sera was tested for genotypes 2a (JFH1) and a chimeric 2a/4a virus (ED43/JFH1) in HCVcc culture., Results: HCVp6-MAP confers potent viral neutralization and specific cellular responses at > 1600 ng/ animal for at least 20 weeks., Conclusion: We report on a promising anti HCV vaccine for future studies on permissive hosts and in clinical trials.

Aims: To screen the newly recorded alga Grateloupia doryphora in the Egyptian Mediterranean Sea for antimicrobial activity., Methods and Results: Algal samples were collected from the Eastern Harbor, Alexandria, Egypt during summer (2016 and 2017). The dry and fresh methanolic, ethanolic and ethyl acetate algal extracts were tested against Bacillus subtilis, Enterococcus faecalis and Staphylococcus aureus as Gram-positive bacteria, Escherichia coli, Pseudomonas aeruginosa as Gram-negative bacteria and one yeast strain Candida albicans. A three-way analysis of variance showed significant differences based on the algal form, type of solvents and microbial species. The fresh methanolic and ethyl acetate extracts had equal effects on the tested pathogens with means of 14·44 and 15·16 respectively. However, the fresh algal extract was more effective on all the tested organisms than the dried material, except for ethyl acetate and ethanol extract on Bacillus subtilis (31 and 23 mm) respectively. In fact, P. aeruginosa was the most susceptible organism to the fresh algal extract, with much higher inhibition zones (30, 27 and 28 mm) for mehanolic, ethyl acetate and ethanolic extracts, respectively, compared with the dried ones. Furthermore, C. albicans was sensitive to the fresh algal extracts only. Compared with the commercial antibiotics piperacillin, rifampicin, cephalexin, metronidazole and fusidic acid tested against the same microbes, the results showed in general lower or comparable inhibition zones than algal extracts. The GC-MS fresh methanolic and ethyl acetate algal extracts revealed the presence of many potent compounds, with palmitic acid and 2-tetradecyloxirane as the common constituents in both extracts. The commercial palmitic acid was tested against the same microbes with successful elimination of all pathogens., Conclusions: Grateloupia doryphora showed broad spectrum of antimicrobial activity against the tested microbial species in this study., Significance and Impact of the Study: The alga can be useful in industry of pharmaceutical products. On the other hand, its usage in this domain can be a suitable solution to its introduction in our coasts., (© 2018 The Society for Applied Microbiology.)

Background: Liver fibrosis results from a wound healing response to chronic injury, which leads to excessive matrix deposition. Genome wide association studies have showen transcriptional dysregulation in mild and severe liver fibrosis. Recent studies suggested that genetic markers may be able to define the exact stage of liver fibrosis., Aim: To define genes or genetic pathways that could serve as markers for staging or as therapeutic targets to halt progression of liver fibrosis., Methods: The study was performed on 105 treatment naïve HCV genotype 4 infected patients [F0-F2, n = 56; F3-F4, n = 49] and 16 healthy subjects. The study included PCR array on 84 fibrosis related genes followed by customization of a smaller array consisting of 11 genes that were designed on the bases of results obtained from the larger array. Genes that displayed significant dysregulation at mRNA levels were validated at protein levels., Results and Discussion: Two major pathways exhibited high dysregulation in early fibrosis as compared with controls or when compared with late fibrosis, these were the TGFβ - related pathway genes and Matrix - deposition associated genes. Hepatic stellate cell (HSC) activators i.e. TGFβ pathway genes [TGFβ1, 2 and 3, their receptors TGFβR1 and 2, signaling molecules SMAD genes and PDGF growth factors] were considerably over-expressed at transcriptional levels as early as F0, whereas expression of their inhibitor TGIF1 was simultaneously down regulated. Matrix proteins including collagen and MMPs were upregulated in early fibrosis whereas tissue inhibitors TIMPs 1 and 2 began over expression in late fibrosis. Expression at protein levels was concordant with RNA data excluding dysregulation at post transcriptional levels., Conclusion: Since these 2 gene sets are closely interrelated regarding HSC activation and proliferation, we assume that the current findings suggest that they are favorable targets to further search for stage specific markers., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Herein, we examined the association between cytomegalovirus (CMV) coinfection and the progression of liver fibrosis in hepatitis C virus (HCV) infection, and investigated the effect of CMV coinfection on JAK-STAT pathway. CMV DNAemia was detected by PCR in DNA from controls (n = 120), and HCV patients with early (F0-F1, n = 131) and late (F2-F4, n = 179) liver fibrosis. By quantitative real time PCR (qRT-PCR), we examined the profile of 8 JAK-STAT transcripts in PBMCs RNA from 90 HCV patients (39 CMV positive and 51 CMV negative), 4 CMV mono-infected patients, and 15 controls. Our results demonstrated higher incidence of CMV in F2-F4 group than in control (OR 5.479, 95% CI 3.033-9.895, p < 0.0001) or F0-F1 groups (OR 2, 95% CI 1.238-3.181, p = 0.005). qRT-PCR showed downregulation of STAT2 (p = 0.006) and IRF7 (p = 0.02) in CMV positive group compared to CMV negative one. The downregulation of STAT2 and IRF7 was mainly in CMV positive patients with late fibrosis compared to CMV negative patients (p = 0.0007 for IRF7 and p = 0.01 for STAT2). Our results are the first to report that CMV coinfection is a possible risk factor for the progression of HCV-induced liver fibrosis, and thereby CMV screening and treatment are important for HCV patients.

The major complication of hepatitis C virus (HCV) infection is the induction of hepatic fibrosis. In this study, we investigated the correlation between the expression level of vascular endothelial growth factor (VEGFA) at mRNA and protein levels and the progression of HCV-related liver fibrosis. One hundred twenty subjects were selected for this study: 15 controls and 105 chronic HCV patients with different fibrosis grades (44 F0-F1 and 61 F2-F4). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure VEGFA mRNA in peripheral blood mononuclear cells, while enzyme-linked immunosorbent assay (ELISA) was used to measure the secreted VEGFA protein in serum. Both qRT-PCR and ELISA results showed that HCV patients have significantly higher VEGFA expression than that of controls (P = 0.036 and 0.043, respectively). Moreover, patients with late fibrotic stages (F2-F4) exhibited the highest levels of VEGFA mRNA and protein (P = 0.008 and 0.041, respectively) when compared with controls. An area under the receiver operating characteristic curve (AUC of the ROC) for the circulatory VEGFA protein between HCV patients with fibrosis and healthy controls was 0.92 (P = 0.043). Our data suggest that VEGFA protein is a promising noninvasively diagnostic biomarker for HCV-induced liver fibrosis.

Synthetic peptides are one of the hepatitis C virus (HCV)-specific small molecules that have antiviral activity and represent a target for HCV vaccine. This study aims to determine the lowest concentration of adjuvanted and non-adjuvanted (multiple antigenic peptide [MAP]) form of three conserved HCV envelope peptides that can induce murine immunogenic responses and evaluate the neutralization capacities of the generated antibodies (Abs) against HCV in cultured Huh7.5 cells. In this study, three HCV synthetic peptides, E1 peptide (a.a 315-323) and E2 peptides (a.a 412-419 and a.a 516-531) were synthesized. Female Balb/c mice were immunized with different concentration of either adjuvanted linear peptides or nonadjuvanted MAP peptides to determine the lowest dose that generates Ab responses enough to confer viral neutralization in vitro. The humoral responses targeting these peptides in immunized mice sera were measured by enzyme-linked immunosorbent assay (ELISA). Viral neutralization capacities of the generated mice Abs were assessed using Huh7.5 cells infected with the HCVcc infectious system (J6/JFH-1). The results of this study showed that the MAPs induce higher Ab titers than adjuvanted linear peptides after 4 weeks of immunization (p = 0.003). The viral neutralization experiments showed that the immunized mice sera contain anti E1/E2 Abs that blocked HCVcc (J6/JFH-1) entry into Huh7.5 cells. In conclusion, the three HCV envelope MAP peptides are more immunogenic and produce higher neutralizing Abs than linear peptides; therefore, they can be essential components for HCV vaccine.

Tumor necrosis factor-alpha (TNFα) and transforming growth factor-beta (TGFβ1) cytokines are highly implicated in liver fibrosis. Polymorphisms in these cytokines affect their expression, secretion, and activity. This study aimed to evaluate the influence of TNFα -308 G/A and TGFβ1 -509 C/T polymorphism on hepatic fibrosis progression in Egyptian patients with hepatitis C virus (HCV) genotype 4. Genotyping of TNFα -308 G/A and TGFβ1 -509 C/T was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in 122 subjects (50 healthy controls and 72 HCV patients). Also, serum TNFα and TGFβ1 levels were detected by enzyme-linked immunosorbent assay (ELISA). The genotyping results of early (F0-F1, n = 36) and late (F2-F4, n = 36) HCV fibrosis patients showed that late fibrosis patients had higher TNFα -308 AA genotype and TGFβ1 -509 TT genotype than early fibrosis patients (p = 0.016, 0.028, respectively). Moreover, the TNFα and TGFβ1 serum levels were significantly higher in HCV patients with TNFα A containing genotypes (GA+AA) (p = 0.004) and patients with TGFβ1 T containing genotypes (CT+TT) (p = 0.001), respectively. The combined unfavorable TNFα (GA/AA) and TGFβ1 (CT/TT) genotypes were highly associated with abnormal liver function parameters and were significantly higher in high activity (A2-A3) and late fibrosis (F2-F4) HCV patients (p = 0.023, 0.029). The multivariate analysis results confirmed that the combined TNFα-308 (AA) and TGFβ1 -509 (TT) unfavorable genotypes increased the risk of hepatic fibrosis progression by 6.4-fold than combined favorable genotypes (odds ratio: 6.417, 95% confidence interval [1.490-27.641], p = 0.013). In conclusion, both TNFα -308 G/A and TGFβ1 -509 C/T polymorphisms synergistically influence the hepatic fibrosis progression and can be used as potential biomarkers to predict hepatic disease progression in chronic hepatitis C patients.

Hepatitis C virus (HCV) is the leading cause of liver fibrosis and hepatocellular carcinoma (HCC). At present, there is no predictive biomarker for the patients at high risk of developing HCC. In this study, we examined the association between single-nucleotide polymorphisms (SNPs) in 3 innate immunity genes [2'-5'oligoadenylate synthetase 1 (OAS1) rs10774671, interleukin 28B (IL28B) rs12979860, and low molecular mass polypeptide 7 (LMP-7) at codon 49] besides cytomegalovirus (CMV) coinfection and susceptibility to HCC in genotype 4 (GT4) chronically infected Egyptian patients. SNPs were determined using restriction fragment length polymorphism analysis in DNA from HCC patients (n = 34) and compared with either controls (n = 70) or patients with early grades of liver fibrosis (n = 49). Our results demonstrated that patients bearing the genetic combination consisting of LMP-7 CA/AA [OR 4.75, 95% confidence interval (CI) 1.443-15.631, P = 0.007] and IL28B rs12979860 CT/TT (OR 6.00, 95% CI 1.603-22.455, P = 0.004) and positive for CMV viremia (OR 3.11, 95% CI 1.151-8.412, P = 0.02) were more likely to have HCC. However, OAS1 rs10774671 does not seem to contribute to the development of HCC. Binary regression analysis indicated that HCC risk significantly increases with the presence of each unfavorable genotype (LMP-7 CA/AA, IL28B rs12979860 CT/TT), when accompanied by the existence of CMV coinfection (probability of HCC risk is 0.8 for combined factors versus 0.14, 0.07, and 0.07 for individual factor IL28B, LMP-7, and CMV; respectively). These data suggest that the 2 SNPs and the coinfection in concert have potential in predicting the risk of HCC development in patients infected with HCV GT4.

Aim: To investigate the association of tumor necrosis factor alpha (TNFα) -G308A polymorphism with different liver pathological changes in treatment-naïve Egyptian patients infected with hepatitis C virus (HCV) genotype 4., Methods: This study included 180 subjects, composed of 120 treatment-naïve chronic HCV patients with different fibrosis grades (F0-F4) and 60 healthy controls. The TNFα -G308A region was amplified by PCR and the different genotypes were detected by restriction fragment length polymorphism analysis. The TNFα protein was detected by enzyme-linked immunosorbent assay. The influence of different TNFα -G308A genotypes on TNFα expression and liver disease progression were statistically analyzed. The OR and 95%CI were calculated to assess the relative risk confidence., Results: Current data showed that the TNFα -G308A SNP frequency was significantly different between controls and HCV infected patients (P = 0.001). Both the AA genotype and A allele were significantly higher in late fibrosis patients (F2-F4, n = 60) than in early fibrosis patients (F0-F1, n = 60) (P = 0.05, 0.04 respectively). Moreover, the GA or AA genotypes increased the TNFα serum level greater than the GG genotype (P = 0.002). The results showed a clear association between severe liver pathological conditions (inflammation, steatosis and fibrosis) and (GA + AA) genotypes (P = 0.035, 0.03, 0.04 respectively). The stepwise logistic regression analysis showed that the TNFα genotypes (GA + AA) were significantly associated with liver inflammation (OR = 3.776, 95%CI: 1.399-10.194, P = 0.009), severe steatosis (OR = 4.49, 95%CI: 1.441-14.0, P = 0.010) and fibrosis progression (OR = 2.84, 95%CI: 1.080-7.472, P = 0.034). Also, the A allele was an independent risk factor for liver inflammation (P = 0.003), steatosis (P = 0.003) and fibrosis (P = 0.014)., Conclusion: TNFα SNP at nucleotide -308 represents an important genetic marker that can be used for the prognosis of different liver pathological changes in HCV infected patients., Competing Interests: Conflict-of-interest statement: None.

IFN orchestrates the expression of various genes to halt hepatitis C virus (HCV) replication with the possibility of either reduced or increased liver fibrosis; due to controlled viral replication or overproduction of inflammatory mediators, repectively. In this study, we examined the transcriptional profiling of type I IFN related genes in HCV-chronically infected patients with varying degrees of liver fibrosis. PCR array was used to examine the expression of 84 type I IFN related genes in peripheral blood mononuclear cells (PBMCs) RNA from 12 treatment-naïve chronic HCV patients (5 F0-F1 and 7 F2-F4) and 5 healthy subjects. We further validated our results by quantitative real time PCR (qRT-PCR) in 103 treatment-naïve chronic HCV patients (43 F0-F1 and 60 F2-F4) and 15 controls. PCR array data revealed dysregulation in TLR7 pathway. The expression of TLR7 was decreased by 4 folds and MyD88 was increased by 3 folds in PBMCs of F2-F4 patients when compared to the healthy volunteers (p = 0.03 and 0.002, respectively). In addition, IRF7 and TLR7 showed dramatic downregulation (6 and 8 folds, respectively) in F2-F4 patients when compared to F0-F1 ones. qRT-PCR confirmed the altered expression patterns of TLR7 and MyD88 in F2-F4 patients when compared to either controls or F0-F1 patients. However, by qRT-PCR, IRF7 and NF-κB1 (TLR7 pathway transcription factors) exhibited similar mRNA abundance among F2-F4 and F0-F1 patients. These results suggest that TLR7 and MyD88 are possible candidates as biomarkers for the progression of HCV-induced liver fibrosis and/ or targets for therapeutic intervention.

The aim of this study was to assess the impact of genetic variants of oligoadenylate synthetase 1 (OAS1) single-nucleotide polymorphism (SNP) rs10774671 at the exon 7 splice acceptor site on liver fibrosis progression and hepatitis C virus (HCV) outcome in Egyptian HCV genotype 4 patients. In this study, 195 subjects were enrolled; 60 controls and 135 chronic HCV genotype 4 patients with different fibrosis grades. All subjects were genotyped for OAS1 SNP rs10774671 polymorphism by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. There was an increasing trend of liver fibrosis progression as 52.9% GG, 73.6% GA, and 83.3% AA genotypes were detected in late fibrosis patients (p = 0.025). The AA genotype was higher in the late fibrosis group than in the early fibrosis group (83.3% vs. 16.7%) (p = 0.001). The A allele was significantly affecting the liver fibrosis progression rate, more than the G allele (p = 0.001). The multivariate analysis showed that the OAS1 GA and AA genotypes were independent factors associated with liver progression (p = 0.009, odds ratio [OR] 3.467, 95% confidence interval [CI] 1.273-7.584). In addition, the A allele was associated with liver fibrosis progression (p = 0.014, OR 2.525, 95% CI 1.157-4.545). The polymorphism at OAS1 exon 7 rs3741981 might be a potential genetic marker and can be useful in the assessment of liver fibrosis progression and disease outcome in HCV-infected patients.

Background: Conventional diagnosis of infective endocarditis (IE) is based mainly on culture-dependent methods that may fail because of antibiotic therapy or fastidious microorganisms., Objectives: We aimed to evaluate the added values of serological and molecular methods for diagnosis of infective endocarditis., Patients and Methods: One hundred and fifty-six cases of suspected endocarditis were enrolled in the study. For each patient, three sets of blood culture were withdrawn and serum sample was collected for Brucella, Bartonella and Coxiella burnetii antibody testing. Galactomannan antigen was added if fungal endocarditis was suspected. Broad range PCR targeting bacterial and fungal pathogens were done on blood culture bottles followed by sequencing. Culture and molecular studies were done on excised valve tissue when available., Results: One hundred and thirty-two cases were diagnosed as definite IE. Causative organisms were detected by blood cultures in 40 (30.3 %) of cases. Blood culture-negative endocarditis (BCNE) represented 69.7 %. Of these cases, PCR followed by sequencing on blood and valvular tissue could diagnose five cases of Aspergillus flavus. Eleven patients with BCNE (8.3 %) were diagnosed as zoonotic endocarditis by serology and PCR including five cases of Brucella spp, four cases of Bartonella spp and two cases of Coxiella burnetii. PCR detected three cases of Brucella spp and two cases of Bartonella spp, while cases of Coxiella burnetii were PCR negative. The results of all diagnostic tools decreased the percentage of non-identified cases of BCNE from 69.7 to 49.2 %., Conclusion: Our data underline the role of serologic and molecular tools for the diagnosis of blood culture-negative endocarditis.

Water samples were seasonally collected from 12 stations of the eastern coast of Suez Gulf during autumn of 2012 and winter, spring, and summer of 2013 in order to investigate phytoplankton community structure in relation to some physicochemical parameters. The study area harbored a diversified phytoplankton community (138 species), belonging to 67 genera. Four algal groups were represented and classified as Bacillariophyceae (90 species), Dinophyceae (28 species), Cyanophyceae (16 species), and Chlorophyceae (4 species). The results indicated a relative high occurrence of some species namely.; Pleurotaenium trabecula of green algae; Chaetoceros lorenzianus, Proboscia alata var. gracillima, Pseudosolenia calcar-avis, and Pseudo-nitzschia pungens of diatoms; Trichodesmium erythraeum and Pseudoanabaena limnetica of cyanophytes. Most of other algal species were fairly distributed at the selected stations of the study area. The total abundance of phytoplankton was relatively low (average of 2989 unit/L) in the eastern coast of Suez Gulf, as compared its western coast and the northern part of the Red Sea. The diversity of phytoplankton species was relatively high (2.35-3.82 nats) with an annual average of 3.22 nats in the present study. The results concluded that most of eastern coast of Suez Gulf is still healthy, relatively unpolluted, and oligotrophic area, which is clearly achieved by the low values of dissolved phosphate (0.025-0.3 μM), nitrate (0.18-1.26 μM), and dissolved ammonium (0.81-5.36 μM). Even if the occurrence of potentially harmful algae species was low, the study area should be monitored continuously. The dissolved oxygen ranged between 1.77 and 8.41 mg/L and pH values between 7.6 and 8.41. The multiple regression analysis showed that the dissolved nitrate and pH values were the most effective factors that controlled the seasonal fluctuations of phytoplankton along the eastern coast of Suez Gulf during 2012-2013.

To evaluate the frequency of single-nucleotide polymorphism at the -88 myxovirus resistance (MxA) gene promoter region in relation to the status of hepatitis C virus (HCV) progression and response to combined interferon (IFN) in chronic HCV Egyptian patients. One hundred ten subjects were enrolled in the study; 60 HCV genotype 4-infected patients who underwent combined IFN therapy and 50 healthy individuals. All subjects were genotyped for -88 MxA polymorphism by the restriction fragment length polymorphism technique. There was an increasing trend of response to combined IFN treatment as 34.9% of GG, 64.3% of GT, and 66.7% of TT genotypes were sustained responders (P=0.05). The T allele was significantly affecting the response rate more than G allele (P=0.032). Moreover, the hepatic fibrosis score and hepatitis activity were higher in GG genotypes compared with the GT and TT genotypes. The multivariate analysis showed that the MxA GG genotype was an independent factor increasing the no response to IFN therapy (P=0.04, odds ratio [OR] 3.822, 95% confidence interval [CI] 1.056-11.092), also MxA G allele (P=0.0372, OR 2.905, 95% CI 1.066-7.919). MxA -88 polymorphism might be a potential biomarker to predict response to IFN and disease progression in chronic HCV-infected patients.

Introduction: The variable number of tandem repeats (VNTR) of the dopamine receptor D4 (DRD4) gene among humans may elucidate individual differences in susceptibility to neuropsychiatric diseases. Dopamine dysfunction may be involved with Attention Deficit Hyperactivity Disorder (ADHD) symptoms. In this study, we report the association between the phenotype of ADHD, a condition characterized by inattentiveness, hyperactivity, and impulsiveness, and a 48-base pair VNTR in exon 3 of the DRD4 polymorphism., Subjects and Methods: We used a case control approach conducted on 29 ADHD and 31 ethnically matched control Egyptian children (ages 6-12 years). Cases were assessed by a psychiatric semi-structured interview and the Conners' Parent Rating Scale. VNTR polymorphisms of the DRD4 gene were done by touchdown PCR program using exon 3-specific primers followed by agarose gel electrophoresis., Results: We observed a significant association between the existence of D4.4 allele of DRD4 and ADHD (P, 0.002); 6.9% of cases showed a single D4.4 and 10.3% showed a double D4.4 as compared to controls in whom D4.4 has never been detected., Conclusion: Children with smaller number of repeat alleles (two to four repeats) of the DRD4 gene have higher possibility to develop ADHD in Egyptian children.

Background: Hepatitis C virus (HCV) infection is associated with the development of hepatocellular carcinoma (HCC). The molecular mechanisms of HCV-associated carcinogenesis are unknown. We aim to investigate the alteration of the total nuclear DNA content (ploidy) in different histopathological liver tissues infected with HCV and their relation to the seropositivity of HCV RNA., Methods: Blood and liver tissues were collected from 26 patients. Diagnosis was carried out according to clinical and pathological examinations by specialized physicians. HCV RNA was detected in patients' sera and tissue samples by RT-PCR. To examine nuclear DNA ploidy, liver tissues were stained with blue Fulgen using the image analysis techniques. Finally, the patients' DNA content was examined by histochemical analysis depending on the optical density of DNA from liver biopsies using the grey image menu in each specimen., Results: The HCV RT-PCR results demonstrated that 13/26 (50%) patients had detectable HCV RNA in their sera samples while 18/26 (69%) had detectable HCV RNA in liver tissues. The DNA content from those patients measured by image cytometry showed a high level of alteration of nuclear DNA ploidy and proliferation in liver tissues with HCC, less alteration of nuclear DNA ploidy in cirrhotic patients, and least proliferation nearly normal in liver fibrosis patients. Moreover, the results of histochemical analysis confirmed the DNA image cytometry results and showed that positive HCV RNA liver tissues had more DNA ploidy than negative HCV RNA liver tissues with statistical significance (p-value < 0.05)., Conclusions: HCV positive liver tissue had alterations in DNA content (ploidy) which may lead to liver disease progression, malignant transformation of the liver cells and development of hepatocellular carcinoma.

This study aimed at assessment of the antiviral activity of an amphipathic α-helical peptide derived from the hepatitis C virus NS5A known as C5A virocidal peptide against different HCV genotypes. Two sources of HCV virus for in vitro study: HCV genotype 4 sera samples and JFH-1 infectious culture system genotype 2a were used. Several virocidal peptide concentrations were tested to determine the concentration that inhibits HCV propagation in Huh 7.5 cells according to three different prortocols (pre-infection, coinfection, and post infection). The capacity of the virocidal peptide to block HCV in Huh7.5 cells infected with different 10 individual serum samples was evaluated. In the pre-infection protocol, virocidal concentration (20, 50, and 75 μM) showed no viral RNA. In the co-infection protocol, virocidal concentrations (10, 20, 50, 75 μM) showed no viral RNA while in post-infection protocol, 75 μM was the only concentration that blocked the HCV activity. Results of Huh7.5 cell line transfected with HCV cc J6/JFH and treated with virocidal peptide revealed that only the higher virocidal concentration (75 μM) showed no amplification. The percentage of virocidal blocking in the 10 HCV individual serum samples was 60%. In conclusion, the C5A virocidal peptide has potent antiviral activity against HCV.

Concentrations of Cu, Zn, Cd, Pb, Ni, Co, Fe, Mn, and Hg were measured successively in water, sediments, and six macroalgal species belonging to three algal classes during 3 years (2008-2010) from Abu Qir Bay, Alexandria, Egypt: Chlorophyceae (Enteromorpha compressa, Ulva fasciata), Phaeophyceae (Padina boryana), and Rhodophyceae (Jania rubens, Hypnea musciformis, Pterocladia capillacea). The study aimed to assess the bioaccumulation potential of the seaweeds, as well as to evaluate the extent of heavy metal contamination in the selected study site. Metals were analyzed using atomic absorption spectrophotometry coupled with MH-10 hydride system. The obtained data showed that the highest mean concentrations of Cu, Zn, Fe, and Mn were recorded in E. compressa; Cd, Ni, and Hg exhibited their highest mean concentrations in P. boryana, while Pb and Co were found in J. rubens. Abundance of the heavy metals in the algal species was as follow: Fe > Mn > Zn > Pb > Ni > Co > Cu > Cd > Hg. E. compressa showed the maximum metal pollution index (MPI) which was 11.55. Bioconcentration factor (BCF) for the metals in algae was relatively high with a maximum value for Mn. The Tomlinson pollution load index (PLI) values for the recorded algal species were low, which ranged between 1.00 in P. boryana and 2.72 in E. compressa. Enrichment factors for sediments were low fluctuating between 0.43 for Hg to 2.33 for Mn. Accordingly, the green alga E. compressa, brown alga P. boryana, and red alga J. rubens can be nominated as bioindicators. Based on MPI and PLI indices, Abu Qir Bay in the present study is considered as low-contaminated area.

This article aims at testing several in vitro systems with various viral sources and cell lines for propagation of HCV to evaluate goat antibodies raised against three E2 epitopes in viral neutralization experiments. Four human cell lines (Huh-7, Huh-7.5, HepG2, and CaCo2) were tested using two different HCV viral sources; Genotype 4 infected sera and J6/JFH HCV cc particles. Neutralization capacity of goat Abs against conserved E2 epitopes; p412 (a.a 412-419), p517 (a.a 517-531), and p430 (a.a 430-447) were examined in the above mentioned in vitro systems. Although infection with patients' sera seems to mimic the in vitro situation, it has limited replication rates as compared with HCV cc particularly in Huh7.5 cells. Non-HCV adapted Huh-7 cells were also found susceptible for transfection with J6/JFH virus but at much slower kinetics. The results of the neutralization assay showed that anti p412 and anti p517 were highly neutralizing to HCVcc. Our data demonstrate that antibodies directed against the viral surface glycoprotein E2 reduced the infectivity of the J6/JFH virus and are promising agents for immunotherapy and HCV vaccine development.

HCV recurrence represents a universal phenomenon after liver transplantation. In this study Fifty HCV patients who underwent living donor liver transplantation were enrolled and factors that may accelerate HCV reinfection of the allograft such as donor's age and degree of liver steatosis, recipient's age, gender, BMI, MELD score, liver functions, HCV viral load, type of immunosuppressive drug, and genetic polymorphisms of IL28B, OAS, and IL1B were studied. The results of disease-free survival (DFS) rates showed inverse correlation with the recipient's postoperative levels of ALT, AST, ALP (P < 0.001, <0.001, and 0.006 resp.) as well as pre- and postoperative titers of HCV RNA (P < 0.003 and <0.001 resp.). Recipient's IL28B SNP was a significant factor in predicting postoperative DFS (P < 0.025). However, SNPs in OAS and IL1B genes had no apparent correlation with DFS. Cox proportional hazards model revealed that patients with elevated levels of ALT, preoperative viral titers, IL28B CT, and IL28B TT were 8.28, 4.22, 3.35, and 1.36 times, respectively, more likely to develop recurrence. In conclusion IL28B SNP, ALT level, and preoperative HCV titer besides proper choice of immunosuppressant are helpful for predicting posttransplant HCV recurrence and DFS.

We characterized viral neutralization by a murine monoclonal antibody (mAb315) developed against conserved E1 specific epitope aa 315-323 at pre- and post-binding steps of infection into Huh7 cells. Detection of native virus in infected Huh7 cells by mAb315 were demonstrated by immunostaining. Inhibitions of viral entry by three different concentrations of mAb315 were measured by intracellular amplification of HCV RNA post infection. HCV RNA positive sera from 24 patients were used to infect Huh7 cell line in absence or presence of mouse monoclonal antibody produced in Balb/c mice or culture supernatant of mouse hybrid cells. Monoclonal Ab mAb315 could detect synthetic peptide p315 adsorbed on peripheral human lymphocytes by flow cytometry and showed high immuno reactivity to E1 viral antigen in infected Huh7 cells by immunostaining. Antibody-mediated neutralization assays demonstrated the ability of mAb315 to block HCV binding/entry to target cells at 0.73 mg/mL ascitic fluid or 250 µg/mL culture supernatant of mouse hybrid cells. Sixteen of 24 infected sera could infect Huh7 cells (67%). Binding/entry of HCV was completely blocked by mAb315 in 11/16 cases (69%). These findings suggest that mAb315 can induce HCV neutralization in vitro, which makes it a candidate for developing HCV therapeutic antibodies.

Background: Chronic hepatitis C virus (HCV) infection is a globally serious public health issue., Objectives: In this study, we investigated CC chemokine receptor 5 (CCR5-59029) polymorphism which is considered an important component of the immune system in determining the outcome of HCV infection. Its critical role as a marker in response to interferon therapy of HCV infection is also investigated besides its effect on other clinical patient factors., Patients and Methods: This study was conducted on 82 Egyptian patients with chronic Hepatitis C Virus (HCV) infection who received PEG-INF + Ribavirin treatment for 48 weeks. The study was also conducted on 50 healthy controls (with negative results for HCV antibody and RNA PCR). Full history of patients in this study was recorded. Clinical and histological examinations, qualitative HCV nested RT-PCR, quantitative real -time PCR, and genotyping of HCV RNA genome were performed. CCR5-59029 polymorphism with nucleotide substitution from G to A was amplified. The amplicons were digested with restriction endonuclease Bsp 1286I, and produced RFLPs of the CCR5 genotypes were determined., Results: The present study showed a significant association between the functional SNP of CCR5 gene and the viral response to interferon in chronic HCV Egyptian patients. It was shown that the higher fibrosis stages (F2-F4) had significant association with nonresponse to treatment compared to the lower fibrosis stages (F0-F1) (95% confidence: 5.497 - 55.074, P = 0.0001). In addition, worse liver activity grade (A2-A3) had a very highly significant association with non-responder HCV patients compared to those with better liver activity grade (A1) (95% confidence: 2.242 - 20.974, P = 0.0007). Most importantly HCV patients with G allele had a high significant association with nonresponse to treatment, higher fibrosis stages and worse liver activity grades, while the A allele had a high significant association with sustained response, low fibrosis stages and relatively better liver activity grade (95% confidence: 3.347 - 15.036, P = 0.0001)., Conclusions: SNPs within the CCR5 gene should be considered as an important factor used in combination with other host gene SNPs when developing a mathematical model for anticipating response to HCV therapy.

Aim: To test whether the status of positive cytomegalovirus (CMV) DNA detection adds to the predictive value of IL28B and to further categorize C/T allele carriers., Methods: This study included 166 chronic hepatitis C (CHC) patients who received combined interferon and ribavirin therapy for 48 wk, 84 spontaneous hepatitis C virus (HCV) resolvers who were positive for IgG anti-HCV antibody and negative for HCV RNA, and 100 healthy subjects who were negative for both HCV antibodies and RNA as controls. Genomic DNA from peripheral blood was used for IL28B rs.12979860 single nucleotide polymorphism (SNP) and CMV DNA detection. A 139 bp fragment containing IL28B SNP was amplified in all subjects by polymerase chain reaction using a specifically designed primer. Then the IL28B rs.12979860 SNP was detected by restriction fragment length polymorphism (RFLP) genotyping. The presence of CMV DNA was tested by amplification of the gB1 gene using nested polymerase chain reaction. The role of CMV and IL28B rs.12979860 SNP genotypes in determining the response rate to combined interferon therapy and clinical status of patients were statistically analyzed., Results: Current data showed that 67% of patients carrying the IL28B 12979860 C/C allele had a sustained viral response (SVR) while the genotypes C/T and TT were associated with lower SVR rates, 50% and 48%, respectively. SVR rates for the C/C allele were lower than other HCV genotypes and/or other populations. Genotype CC was associated with the response to interferon (P = 0.025). Genotype C/C was reduced from 48% in controls to 14% in CHC patients suggesting its protective role against progression to chronicity. The majority of spontaneously cleared subjects (86%) were C/C, confirming its protective role. The C/T allele was present in 71% of CHC patients compared with 38% of controls, so the use of IL28B SNP genotyping only in these patients may be of little value as a predictor of response. CMV reactivation occurred in 40% of CHC patients. Co-infection with CMV seriously diminished the response to interferon (IFN) therapy, with SVR rates in C/C genotypes 87.5% in CMV-negative patients and 12.5% in CMV-positive patients (P < 0.0001). SVR rates among C/T carriers were reduced to < 50% in patients with positive CMV DNA while the non-response rate doubled. These data indicate that a supplemental assay for CMV viremia adds to the prognostic value of IL28B genotyping., Conclusion: The results suggest that both genetic (i.e., spontaneous) and therapeutic (IFN-based therapy) arms are complementary in the battle against HCV. CMV DNA testing may be of value to better predict the response to IFN, particularly in IL28B C/T carriers.

We aimed to establish Human cell lines producing human monoclonal antibodies to the envelope E1/E2 protein of hepatitis C virus (HCV). Two protocols for EBV immortalization of CD22⁺ cells separated from HCV positive patients were used; 1) Immortalization with 100% EBV only, 2) immortalization by 30% EBV and CPG2006. Immortalization was checked microscopically and verified by screening the culture supernatant for antibody production using dot blot and ELISA analysis. ELISA plates were coated by HCV E1/E2 derived from cell lysate transfected by plasmid expressed HCV E1/E2. Also we tested the reactivity of human antibodies based on ELISA plates coating with one linear peptides derived from HCV E1 (a.a 315-319) and two peptides derived from HCV E2 (a.a 412-419) and (a.a 517-530). Neutralization activity was measured using H77C HCV retroviral pseudoparticles (HCVpp). Fifteen clones secreting human immunoglobulin G against HCV E1/E2 protein were isolated. Results of ELISA plates coated with HCV peptides showed that one antibody was binding to E2 peptide (a.a 517-530), and two antibodies binding to HCV E2 peptide (a.a 412-419). The three generated antibodies showed extremely neutralization activity against HCV pp. The three human antibodies were IgG3 and IgG2. These antibodies may be useful for passive immunotherapy of HCV infection.

Background: IL28B single nucleotide polymorphisms (SNPs) play important roles in the management of hepatitis C virus (HCV) infections and are strongly associated with spontaneous and treatment-induced HCV clearance., Objectives: In the present study, the association between IL28B variants and the progression of HCV infection in Egyptian patients infected with type 4a virus will be examined., Patients and Methods: Frequencies of the protective genotype C/C of SNP, rs12979860 were determined in healthy subjects, spontaneous resolvers, and chronic HCV type 4 patients with low F scores and in patients with end stage liver disease (ESLD). This study included a total of 404 subjects. Patients infected with HCV type 4a (n = 304) were divided into; chronic hepatitis C (CHC) with low F scores (CHC, n = 110), end stage liver disease (n = 110), liver cirrhosis (LC) (n = 35) and hepatocellular carcinoma (HCC) patients (n = 75), spontaneous resolvers of HCV infection (n = 84) were also included. A healthy group representing the Egyptian population (n = 100) was also included in the genotyping of IL28B. The later was typed via a polymerase chain reaction based restriction fragment length polymorphism (PCR-RFLP) assay analysis on purified genomic DNA extracted from all individuals., Results: A significant increase (P < 0.0005) was observed in frequencies of IL-28B rs12979860 C/C genotypes in the healthy population, than in the CHC, LC and HCC groups (C/C = 48%, 13%, 0%.and 0% respectively). On the other hand the C/C genotype was significantly higher (P < 0.0005) in spontaneous resolvers than in healthy subjects. A comparable significant increase in the frequency of C/T allele accompanied by mild elevation of T/T allele frequency, were detected along the progression towards ESLD., Conclusions: Genotype C/C is associated with viral clearance during acute infection. The sharp decline in the C/C genotype from healthy to CHC subjects and the total absence of the C/C genotype in ESLD suggests a central role of this genotype against HCV disease progression.

Anti HCV vaccine is not currently available and the present antiviral therapies fail to cure approximately half of the treated HCV patients. This study was designed to assess the immunogenic properties of genetically conserved peptides derived from the C-terminal region of HVR-1 and test their neutralizing activities in a step towards developing therapeutic and/or prophylactic immunogens against HCV infection. Antibodies were generated by vaccination of goats with synthetic peptides derived from HCV E2. Viral neutralizing capacity of the generated anti E2 antibodies was tested using in vitro assays. Goats immunized with E2 synthetic peptides termed p412 [a.a 412-419], p430 [a.a 430-447] and p517 [a.a 517-531] generated high titers of antibody responses 2 to 4.5 fold higher than comparable titers of antibodies to the same epitopes in chronic HCV patients. In post infection experiments of native HCV into cultured Huh7.5 cells anti p412 and anti p 517 were proven to be neutralizing to HCV genotype 4a from patients' sera (87.5% and 75% respectively). On the contrary anti p430 exhibited weak viral neutralization capacity on the same samples (31.25%). Furthermore Ab mixes containing anti p430 exhibited reduced viral neutralization properties. From these experiments one could predict that neutralization by Abs towards different E2-epitopes varies considerably and success in the enrichment of neutralization epitope-specific antibodies may be accompanied by favorable results in combating HCV infection. Also, E2 conserved peptides p517 and p412 represent potential components of a candidate peptide vaccine against HCV infection.

Human cytomegalovirus (HCMV) is the most common cause of severe morbidity and mortality in immune- compromised individuals. This study was conducted to determine the incidence of HCMV infection in HCV patients who either spontaneously cleared the virus or progressed to chronic HCV infection. The study included a total of eighty four cases (48 females and 36 males) that were referred to blood banks for blood donation with an age range of 18-64 years (mean age 37.62 ± 10.03 years). Hepatitis C virus RNA and HCMV DNA were detected in sera by RT-nested PCR and nested PCR respectively in all subjects. Immunoglobulin G levels for HCV and HCMV were determined. Besides, IgM antibodies for HCMV infection were also determined in subjects' sera. Fifty three out of 84 cases (63%) were positive for HCV-RNA while 31 (37%) cases had negative HCV RNA. Forty six (87%) and 13 (25%) cases out of 53 HCV RNA positive patients were positive for HCMV IgG and IgM antibodies respectively. While 20 of 53 cases (38%) had detectable HCMV DNA. To examine the role of HCMV infection in HCV spontaneous resolution, two groups of HCV patients, group 1) chronic HCV infection (positive HCV RNA and positive IgG antibodies) vs group 2) spontaneous resolution (negative HCV RNA and positive IgG antibodies) were compared. The percentages of positive CMV IgG and IgM results is higher in chronic HCV patient than those in spontaneously cleared HCV patients and the difference is highly statistically significant (P value < 0.001). Also, there is a general trend towards elevated levels of CMV IgG antibodies in HCV chronic patients than those in spontaneously cleared HCV patients (P value < 0.02). HCMV DNA detection in group 1 was more than twice the value observed in group 2 (38% vs 14.3%, P value < 0.001). Moreover, levels of liver enzymes were significantly higher in HCV RNA positive cases co-infected with HCMV DNA than HCMV negative cases (P value < 0.001). The results indicate the role of HCMV in the liver pathogenesis. We conclude that chronic HCV patients co-infected with HCMV infection can be regarded as high risk groups for liver disease progression where they should be monitored for the long term outcome of the disease.

Background and Aim: Response to interferon therapy and disease progression in hepatitis C virus (HCV) infected patients differs among individuals, suggesting a possibility of a contribution of host genetic factors. 2'-5'-oligoadenylate synthetase 1 (OAS1), an important component of the innate immune system with a proven antiviral function, may therefore have a relationship with the response to interferon therapy and clinical course of HCV disease. Our aim was to determine the frequency of single nucleotide polymorphism (SNP) at exon 7 splice acceptor site (SAS) of the OAS1 gene in relation to the interferon response and status of HCV infection., Methods: A 203 bp fragment containing exon 7 SAS was amplified in 70 HCV chronic patients and 50 healthy controls. SNP was examined using restriction fragment length polymorphism (RFLP) genotyping method. Correlations of SNP genotypes with response to interferon and clinical status of patients were statistically analyzed., Results: There was an increasing trend of response from AA to AG to GG genotypes (P = 0.007). Genotype AA was associated with non-response to interferon and higher degree of liver fibrosis (P = 0.05). Multivariate analysis showed this SNP as independent and a significant determinant of the outcome of interferon therapy (odds ratio 4.913 [95% confidence interval 1.365-8.2], P = 0.006)., Conclusions: This is the first study to show a significant association between the functional SNP at exon 7 SAS of OAS1 gene and the viral response to interferon in chronic HCV patients. Patients with AA genotype were associated with progressive HCV disease and viral resistance to interferon therapy. This OAS SNP is a potential bio-marker to predict IFN response in chronic hepatitis C patients., (© 2011 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd.)

Background and Aim: Cytomegalovirus (CMV) is a ubiquitous pathogen that infects the majority of humans. Co-infection of CMV and hepatitis C virus (HCV) may deteriorate the prognosis of HCV-infected patients. This study was conducted to examine the role of CMV reactivation in determining the response rate to treatment with interferon and ribavirin therapy in chronic HCV patients., Methods: Viral loads and genotyping were assessed using reverse transcription polymerase chain reaction and Innolipa systems, respectively. Reactivation of CMV in HCV patients who were all positive for CMV immunoglobulin G antibodies was tested by amplification of the gB1 gene using the end-point dilution quantitative-nested polymerase chain reaction method., Results: CMV DNA was detected in 89.7% of non-responders and in 34.6% of sustained virological responders. Patients with reactivated CMV had significantly higher fibrosis scores (72.7%) than those with undetectable CMV DNA (23.8%, P=0.002). Patients with positive CMV had higher rates of non-response and relapse (79.5%) than those with negative CMV DNA (19%). Chronic HCV patients with latent CMV had higher rates of response (81%) to treatment than those with reactivated CMV (20.5%, P<0.001). Therefore, HCV patients with reactivated CMV and advanced fibrosis were least likely to achieve a sustained virological response following interferon therapy. This possibility is reduced to 50% of its original value in patients with reactivated CMV without fibrosis., Conclusions: Besides the staging of liver fibrosis, CMV co-infection should be considered as an extremely important factor when designing predictive models for HCV response to interferon treatment., (© 2010 Journal of Gastroenterology and Hepatology Foundation and Blackwell Publishing Asia Pty Ltd.)

Introduction: The highest estimated prevalence of HCV infection has been reported in Egypt, nearly 12% mostly type 4. Currently, a commercial vaccine to protect this high risk population as well as global HCV infected patients is not available., Objectives: In the present study, we aim at: (1) examining the viral binding capacities of purified monospecific polyclonal murine antibodies raised against genetically conserved viral protein sequences, i.e. synthetic peptides derived from those sequences located within envelope proteins and (2) assessment of immunogenic properties and safety parameters of those peptides individually and in a vaccine format in mice., Methods: Purified IgG Abs from immunized mice were used in immunocapture RT-PCR experiments to test viral neutralization by Abs raised against each of 4 peptides termed p35 (E1), p36 (E2), p37 (E2) and p38 (E2). Swiss mice were immunized with each of the 3 peptides (p35, p37 and p38) which generated neutralizing antibodies in immunocapture experiments. Antibody responses to corresponding peptides were determined using different routes of administration, different adjuvants, different doses and at different time points post-injection. To explore the dose range for future pharmacological studies, three doses namely 50 ng, 10 μg and 50 μg/25 gm mouse body weight were tested for biochemical and histopathological changes in several organs., Results: Murine Abs against p35, p37 and p38 but not p36 showed HCV neutralization in immunocapture experiments. Subcutaneous injection of peptides elicited higher responses than i.m. and i.p. Immunization with Multiple Antigenic Peptide (MAP) form or coupled to Al PO4 elicited the highest Ab responses. Peptide doses of 50 ng/25 gm body weight or less were effective and safe, however dose assessment still requires further study. Histopathological changes were observed in animals that received doses ∼1000 times higher than the potential therapeutic dose., Conclusion: Exploration of humoral immunogenicity, neutralization capacity and safety suggested that the peptides presented herein are candidate vaccine components for further preclinical assessment., (Copyright © 2009 Elsevier Ltd. All rights reserved.)

The reason(s) why human antibodies raised against hepatitis C virus (HCV) E2 epitopes do not offer protection against multiple viral infections may be related to either genetic variations among viral strains particularly within the hypervariable region-1 (HVR-1), low titers of anti E2 antibodies or interference of non neutralizing antibodies with the function of neutralizing antibodies. This study was designed to assess the immunogenic properties of genetically conserved peptides derived from the C-terminal region of HVR-1 as potential therapeutic and/or prophylactic vaccines against HCV infection. Goats immunized with E2-conserved synthetic peptides termed p36 (a.a 430-446), p37(a.a 517-531) and p38 (a.a 412-419) generated high titers of anti-p36, anti-p37 and anti-P38 antibody responses of which only anti- p37 and anti- p38 were neutralizing to HCV particles in sera from patients infected predominantly with genotype 4a. On the other hand anti-p36 exhibited weak viral neutralization capacity on the same samples. Animals super-immunized with single epitopes generated 2 to 4.5 fold higher titers than similar antibodies produced in chronic HCV patients. Also the studied peptides elicited approximately 3 fold increase in cell proliferation of specific antibody-secreting peripheral blood mononuclear cells (PBMC) from immunized goats. These results indicate that, besides E1 derived peptide p35 (a.a 315-323) described previously by this laboratory, E2 conserved peptides p37 and p38 represent essential components of a candidate peptide vaccine against HCV infection.

The performance of polyclonal monospecific rabbit anti-sera raised against synthetic peptides derived from conserved HCV sequences of genotype 4 was evaluated for efficient detection of viral core and E1 antigens in circulating immune complexes (ICs) precipitated from 65 serum samples of HCV patients. The infection was established in those patients by the presence of HCV RNA in their sera. A novel enzyme-linked immunosorbent assay (ELISA) was developed for the detection of HCV core and E1 antigen in serum samples. Western blot analyses were used to demonstrate the presence of the core and E1 target antigen in serum samples. The mean OD readings of both core and E1 antigens were significantly higher (P < 0.05) among the viremic patients when compared to controls. Also a significant positive correlation (P < 0.05, r = 0.98) between the values of both core and E1 was recorded. Western blot analysis based on monospecific antibodies against core and E1 recognized the 38-kDa and 88 -kDa bands respectively in the sera of all infected patients. No specific reaction was observed with the sera from uninfected individuals. Interestingly the results of core and E1 antigen levels displayed no positive correlation with the HCV copy number as measured by bDNA. Liver enzymes (ALT and AST) showed a moderate positive correlation (r = 0.44 and 0.47 respectively) with the viral core antigens level. The same trend holds true for E1 (r = 0.43 and 0.64 for ALT and AST respectively). HCV load in infected patients revealed extremely poor correlation with serum ALT and AST levels (r = 0.022 and 0.002 respectively). In conclusion we present a new combination of serological tools correlating with liver enzyme levels that could be utilized as supplemental tests to viral load testing. Also, a sensitive and specific immunoassay was developed for the detection of HCV core and E1 in human serum. This test can be applied for laboratory diagnosis of HCV infection.

Aim: To establish a cell culture system with long-term replication of hepatitis C virus (HCV) genome and expression of viral antigens in vitro., Methods: HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect naive cells. The presence of minus (antisense) RNA strand, and the detection of core and E1 antigens in cells were examined by RT-PCR and immunological techniques (flow cytometry and Western blot) respectively., Results: The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies (anti-core, and anti-E1). Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells., Conclusion: HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells.

Background: Hepatitis C (HCV) viral infection is a serious medical problem in Egypt and it has a devastating impact on the Egyptian economy. It is estimated that over 15% of Egyptians are infected by the virus and thus finding a cure for this disease is of utmost importance. Current therapies for hepatitis C virus (HCV) genotype 4 with interferon/ribavirin have not been successful and thus the development of alternative therapy for this genotype is desperately needed., Results: Although previous studies utilizing viral subgenomic or full cDNA fragments linked to reporter genes transfected into adhered cells or in a cell free system showed promise, demonstration of efficient viral replication was lacking. Thus, we utilized HepG2 cells infected with native HCV RNA genomes in a replication competent system and used antisense phosphorothioate Oligonucleotides (S-ODN) against stem loop IIId and the AUG translation start site of the viral polyprotein precursor to monitor viral replication. We were able to show complete arrest of intracellular replication of HCV-4 at 1 uM S-ODN, thus providing a proof of concept for the potential antiviral activity of S-ODN on native genomic replication of HCV genotype 4., Conclusion: We have successfully demonstrated that by using two S-ODNs [(S-ODN1 (nt 326-348) and S-ODN-2 (nt 264-282)], we were able to completely inhibit viral replication in culture, thus confirming earlier reports on subgenomic constructs and suggesting a potential therapeutic value in HCV type 4.