Publications by authors named "Reham Dawood"

41 Publications

The Impact of Direct-Acting Antiviral Agents on Cytomegalovirus Reactivation in Chronic Hepatitis C Infection.

Asian Pac J Cancer Prev 2022 Apr 1;23(4):1365-1372. Epub 2022 Apr 1.

Department of Endemic Medicine and Hepatogastroenterology, Faculty of Medicine, Cairo University, Cairo, Egypt.

Objective: The co-infection of HCV/CMV may accelerate the progression of liver diseases and worsen responsiveness to IFN treatment. The Direct-acting antiviral agents (DAAs), currently approved therapy for HCV, may cause a transient change in immune status, favoring the reactivation of other viruses. The current study aims to evaluate the impact of DAAs treatment on the reactivation of latent CMV in HCV patients.

Methods: The serological IgG, IgM Abs against CMV were detected by ELISA on192 HCV patients. The seronegative CMV IgM patients received (sofosbuvir/daclatasvir) regimen, then the CMV reactivation was examined by measuring the CMV IgM by ELISA and CMV DNA by real-time PCR.

Results: The serological data revealed that all patients were positive for CMV IgG (100%) while (64%) patients were positive for CMV IgM. The seronegative CMV IgM (36%) received the DAAs protocol. The sustained virological response was monitored by measuring the HCV RNA viremia in the patient sera. The serological data revealed that 28.6% of patients had a reactivation of CMV, while 18.5% of patients had detectable CMV DNA viremia. Moreover, there was a significant improvement in liver function as well as a decrease in FIB-4 and APRI scores at EOT. SVR was reached 97.4% among the total studied patients (N= 192).

Conclusion: CMV co-infection has no impact on the response rate to DAAs. However, the CMV reactivation might have occurred after the complete eradication of HCV by DAAs.
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http://dx.doi.org/10.31557/APJCP.2022.23.4.1365DOI Listing
April 2022

Bioactive glass doped with noble metal nanoparticles for bone regeneration: kinetics and proliferative impact on human bone cell line.

RSC Adv 2021 Jul 25;11(41):25628-25638. Epub 2021 Jul 25.

Universite de Rennes 1, UMR CNRS 6226 263 Avenue du Général Leclerc 35042 Rennes Cedex France.

This work investigates the bioactivity of novel silver-doped (BG-Ag) and gold-doped (BG-Au) quaternary 46S6 bioactive glasses synthesized a semi-solid-state technique. A pseudo-second-order kinetic model successfully predicted the uptake kinetic profiles of the initial ion-exchange release of Ca in simulated body fluid, the subsequent Si release, and finally, the adsorption of Ca and P onto the bioactive glasses. Doping with silver nanoparticles enhanced the rate of P uptake by up to approximately 90%; whereas doping with gold nanoparticles improved Ca and P uptake rates by up to about 7 and 2 times, respectively; as well as Ca uptake capacity by up to about 19%. The results revealed that the combined effect of Ca and Si release, and possibly the release of silver and gold ions into solution, influenced apatite formation due to their effect on Ca and P uptake rate and capacity. In general, gold-doped bioactive glasses are favoured for enhancing Ca and P uptake rates in addition to Ca uptake capacity. However, silver-doped bioactive glasses being less expensive can be utilized for applications targeting rapid healing. studies showed that BG, BG-Ag and BG-Au had no cytotoxic effects on osteosarcoma MG-63 cells, while they exhibited a remarkable cell proliferation even at low concentration. The prepared bioactive glass doped with noble metal nanoparticles could be potentially used in bone regeneration applications.
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http://dx.doi.org/10.1039/d1ra03876aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9036971PMC
July 2021

Reduced fitness of the mosquito Culex pipiens (Diptera: Culicidae) after feeding on a blood meal with hepatitis C virus.

J Invertebr Pathol 2022 03 24;189:107719. Epub 2022 Jan 24.

Department of Zoology, Faculty of Science, Tanta University, Tanta 31527, Egypt.

Hepatitis C virus (HCV) is a blood-borne virus. Given that mosquitoes can take blood meals from HCV patients, we aimed to test whether HCV in the blood meal can induce alterations in the biology of Culex pipiens. To address this aim, Cx. pipiens females were fed HCV-negative blood from healthy individuals or HCV-positive fresh blood samples harvested from viremic HCV patients. Replication of HCV in mosquitoes was confirmed by negative strand-specific RT-PCR and sequencing of RNA extracted from the mosquito bodies 7 days post-feeding. In addition, several parameters that determine the fitness of the mosquitoes were measured. Virus acquisition was associated with alterations in the architecture of the gut microvilli and the immune response, indicated by an increase in phenol oxidase activity. Interestingly, the mosquitoes that were fed the HCV-positive blood meal showed shorter median longevity (8 days) and laid fewer eggs than the control mosquitoes. Furthermore, the offspring of females fed the HCV-positive blood meal demonstrated a lower emergence rate than the controls. In sum, the results indicate that feeding on HCV by Cx. pipiens decreases fitness, which may, in turn, affect its potential as a vector.
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http://dx.doi.org/10.1016/j.jip.2022.107719DOI Listing
March 2022

Evaluation of seven gene signature for predicting HCV recurrence post-liver transplantation.

J Genet Eng Biotechnol 2021 Nov 10;19(1):174. Epub 2021 Nov 10.

Department of Microbial Biotechnology, Genetic Engineering Division, National Research Centre, Dokki, P.O. 12622, Giza, Egypt.

Background: Orthotropic liver transplantation (OLT) offers a therapeutic choice for hepatocellular carcinoma (HCC) patients. The poor outcome of liver transplantation is HCV recurrence. Several genome-wide associated studies (GWAS) have reported many genetic variants to be associated with HCV recurrence. Seven gene polymorphisms formed a cirrhosis risk score (CRS) signature that could be used to distinguish chronic HCV patients at high risk from those at low risk for cirrhosis in non-transplant patients. This study aims to examine the association of CRS score and other clinical parameters with the probability for HCC emergence and/or the rate of HCV recurrence following liver transplantation.

Results: Seven gene polymorphisms, forming the CRS, were genotyped by real-time PCR using allelic discrimination protocol in 199 end-stage liver disease patients (79 child A, 43 child B, and 77child C), comprising 106 patients who encountered liver transplantation. Recipient CRS scores were correlated with HCV recurrence (HCV-Rec) at the end of the third year after OLT. Around 81% (39) recipients with low steatosis (LS; < 3.5%) donor percentage revealed no HCV recurrence (non-Rec) (p<0.001). CRS score could distinguish between child A, child B, and child C only at the low-risk group. Among the HCV Rec group 27% (8/30), 40% (12/30), and 33% (10/30) fell into the high, moderate, and low CRS risk groups, respectively. Stepwise logistic regression evinced two features more likely to be seen in HCV-Rec patients: abnormal ALT [OR, 1.1; 95% CI, 1.02-1.2] and donor steatosis >3.5% [OR, 46.07; 95% CI, 1.5-1407.8].

Conclusions: Accordingly, the CRS score seems to be less useful to predict HCV recurrence after OLT. ALT and donor steatosis (exceed 3.5%) can significantly promote the HCV recurrence post-OLT. Moreover, the combination of MMF and CNI positively heightens HCV recurrence.
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http://dx.doi.org/10.1186/s43141-021-00266-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8581076PMC
November 2021

Significance of Hereditary Hemochromatosis Gene (HFE) Mutations in Chronic Hepatitis C and Hepatocellular Carcinoma Patients in Egypt: A Pilot Study.

Asian Pac J Cancer Prev 2021 Sep 1;22(9):2837-2845. Epub 2021 Sep 1.

Department of Endemic Medicine, Faculty of Medicine, Cairo University, Cairo, Egypt.

Background: Hereditary hemochromatosis is a genetic disease defined by enhanced overloading of iron and associated with Chronic Hepatitis C (CHC). This study aims to evaluate the correlation of the HFE gene mutations on Egyptian CHC with liver disease progression and the risk of HCC development.

Methods: The HFE mutations (C282Y and H63D) were genotyped on 100 CHC patients and 50 healthy individuals by a hybridization assay. The serum iron content was also measured for all subjects.

Results: A significant elevation of the serum iron, ferritin, and TIBC in HCV-infected patients (p≤0.05). The H63D mutation was detected in 23% of the all studied samples. The serum iron and the H63D heterozygosity were correlated significantly between non-cirrhotic and cirrhotic without HCC patients.

Conclusion: The H63D heterozygosity might have a potential role in iron accumulation. Moreover, HFE mutations did not tend to be associated with an increased risk of HCC in cirrhotic patients.
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http://dx.doi.org/10.31557/APJCP.2021.22.9.2837DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8850884PMC
September 2021

Development of a gene signature for predicting cirrhosis risk score of chronic liver disease associated with HCV infection in Egyptians.

Microb Pathog 2021 Apr 18;153:104805. Epub 2021 Feb 18.

Department of Microbial Biotechnology, Genetic Engineering Division, National Research Centre, 33 EL Bohouth Street Dokki, Giza, 12622, Egypt.

Background: Complex diseases such as fibrosis are likely polygenic. Lately, cirrhosis risk score (CRS) clearly discriminated Chronic HCV patients with high-risk versus those with low-risk for cirrhosis better than clinical factors.

Methods: Herein, the CRS was assessed via genotyping by allelic discrimination assays in 243 HCV Egyptian patients categorized into 164 patients didn't develop HCC (93 mild, 71 advanced fibrosis); and 79 patients developed HCC. APRI and FIB-4 scores were calculated, compared with CRS and correlated with degree of fibrosis progression.

Results: Median of the three CRS, APRI and FIB-4 scores were significantly elevated in late fibrotic and HCC patients (p < 0.001); however CRS displayed proper discrimination (mild fibrosis (0.59; 0.4-0.75), advanced fibrosis (0.75; 0.7-0.86) and HCC (0.73; 0.57-0.77); (p < 0.001)). The ROC analysis of CRS score displayed modest accuracy to discriminate between mild and advanced fibrotic patient; AUC was 0.73; p < 0.0001), while AUC was only 0.57 (p = 0.05) for the discrimination between HCC and no HCC. Moreover, the combination of CRS, APRI and FIB4 lessened the power of correlation (AUC, 0.63 (p < 0.0001)) in fibrosis prognosis. In HCC prognosis, the combination of CRS, APRI and FIB4 in HCC patients showed modest accuracy with AUC, 0.59 (p = 0.0001).

Conclusion: The diagnostic accuracy of FIB-4 for predicting liver fibrosis was nearly identical to that of CRS, however the strength of CRS score stemmed from that it is built on 7 SNPs host genetic factor. Our study validates non invasive algorithms for fibrosis prognosis purposes which may aid in decision making for therapeutic intervention.
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http://dx.doi.org/10.1016/j.micpath.2021.104805DOI Listing
April 2021

Treatment of hepatitis C virus infection with direct-acting antivirals plus ribavirin eliminates viral RNA from peripheral blood mononuclear cells and reduces virologic relapse in diverse hepatic parenchymal changes.

Arch Virol 2021 Apr 3;166(4):1071-1081. Epub 2021 Feb 3.

Department of Microbial Biotechnology, National Research Center, Cairo, Egypt.

Elimination of hepatitis C virus (HCV) may fail, leading to a non-response outcome because of inappropriate testing for viral RNA in peripheral blood mononuclear cells (PBMCs). Sequelae of HCV genotype 4 therapy with sofosbuvir and daclatasvir ± ribavirin were assessed in our study at the 12 week after end of treatment (EOT) by screening for viral genomic RNA in serum and PBMCs with correlation to hepatic parenchymal changes. We recruited 102 out of 2165 patients who had received sofosbuvir/daclatasvir, either alone (n = 1573) or together with ribavirin (n = 592). Subjects were classified into three groups based on testing by single-step reverse transcription PCR: group I, HCV negative in both serum and PBMCs (n = 25); group II, HCV positive in PBMCs only (n = 52); and group III, HCV positive in both serum and PBMCs (n = 25). Groups I and II (n = 77) were selected out of 2102 (every 27 subject), while group III (n = 25) were selected from every second or third serologic relapse (n = 63). The pre-sampling population (n = 2165) showed sustained virologic response (SVR) in 33.21%; serologic relapse in 2.91%; HCV RNA only in PBMCs (66.79%) compared to serologic relapses and potential cure (P < 0.0001); higher serologic (38 out of 63, P = 0.03210) and cellular (36 out of 52, P = 0.0002) relapses in dual therapy than in triple therapy. The post-sampling population (n = 102) showed more HCV relapses in dual (50 out of 60) than in triple (27 out of 42) therapy (P = 0.0351); increased HCV antisense RNA strand in relapses compared to positive-sense strands alone (P < 0.001); and significant SVR events in undetectable (15 out of 31) compared to early (10 out of 55, P = 0.0058) and cirrhotic liver tissue changes (0 out of 16, P = 0.0006). In summary, HCV treatment with sofosbuvir/daclatasvir is followed by higher rates of serologic and intracellular viral RNA relapse than treatment with sofosbuvir/daclatasvir plus ribavirin. Cellular and serum viral RNA relapses are accompanied by HCV-induced hepatic pathology. An increased SVR with no detectable liver tissue changes was observed after triple therapy due to elimination of HCV RNA from PBMCs.
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http://dx.doi.org/10.1007/s00705-021-04969-4DOI Listing
April 2021

Bioinformatics prediction of B and T cell epitopes within the spike and nucleocapsid proteins of SARS-CoV2.

J Infect Public Health 2021 Feb 15;14(2):169-178. Epub 2020 Dec 15.

Department of Microbial Biotechnology, Genetic Engineering Division, National Research Centre, 33 EL Bohouth Street, Dokki, Giza 12622, Egypt. Electronic address:

Background: The striking difference in severity of SARS CoV2 infection among global population is partly attributed to viral factors. With the spike (S) and nucleocapsid (N) are the most immunogenic subunits, genetic diversity and antigenicity of S and N are key players in virulence and in vaccine development.

Aim: This paper aims at identifying immunogenic targets for better vaccine development and/or immunotherapy of COVID 19 pandemic.

Methods: 18 complete genomes of SARS CoV2 (n=14), SARS CoV (n=2) and MERS CoV (n=2) were examined. Bioinformatics of viral genetics and protein folding allowed functional tuning of NH2 Terminal Domain (NTD) of S protein and development of epitope maps for B and T cell responses.

Conclusion: A deletion of amino acid residues Y144 and G107 were discovered in NTD of S protein derived from Indian and French isolates resulting in altered pocket structure exclusively located in NTD and reduced affinity of NTD binding to endogenous nAbs and disrupted NTD mediated cell entry. We therefore, proposed a set of B and T cell epitopes based on Immune Epitope Database, homologous epitopes for nAbs in convalescent plasma post SARS CoV infection and functional domains of S (NTD, Receptor Binding domain and the unique polybasic Furin cleavage site at S1/S2 junction). Nevertheless, laboratory data are required to develop vaccine and immunotherapeutics.
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http://dx.doi.org/10.1016/j.jiph.2020.12.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7737509PMC
February 2021

Reactivation of human cytomegalovirus inhibits expression of liver fibrosis related cytokines in patients chronically infected with hepatitis C virus genotype 4a.

Microb Pathog 2021 Mar 28;152:104596. Epub 2020 Oct 28.

Microbial Biotechnology Department, Genetic Engineering Division, National Research Centre, 33 EL Bohouth St. (former El Tahrir St.), Dokki, Giza, 12622, Egypt. Electronic address:

Background: The impact of human cytomegalovirus (HCMV) reactivation on the expression pattern of matrix metalloproteinases, their inhibitors and related cytokines during HCV infection poorly understood.

Methods: Reactivation of CMV in 95 subjects (75 chronically infected HCV patients and 20 healthy subjects) was examined. All studied subjects had detectable IgG antibodies for CMV, but only 35/75 of HCV patients (46.7%) had detectable CMV DNA. The expressions of 11 fibrosis related genes by quantitative real-time PCR were analyzed in subjects' PBMCs. The serum levels of TGFβ2 and PDGFα have been measured by ELISA.

Results: Chronically infected HCV patients with reactivated CMV had less expression of TGF-β1, TGF-β2, PDGFα and STAT1 transcripts than HCV patients with latent CMV (p = 0.037, 0.006, 0.001 and 0.009; respectively) and normal controls (TGF-β2, p = 0.008). Moreover the expression of (TGFβ2 and PDGFα) genes decreased significantly in CMV-reactivated patients during the early stage of fibrosis relative to the comparable stage of HCV infection (p = 0.004 and 0.008; respectively). Besides, the mRNA abundance of STAT1 gene in CMV-reactivated patients decreased dramatically as compared to HCV infections during the late stage of fibrosis (p = 0.014). The TGFβ2 protein level has been declined dramatically in CMV-reactivated patients compared to HCV infected patients and control group (p = 0.001 and 0.033; respectively). Our results suggest that CMV reactivation disrupts the expression of several cytokines as compared to solitary infection with HCV. Noticeably, the expressions of matrix metalloproteinases genes and their inhibitors have not been significantly influenced by reactivation of CMV.

Conclusion: The current data reveal that reactivation of CMV partially blocks the upregulation of 2 important pro-inflammatory cytokines i.e. TGFβ 2 and PDGFα at early stages of fibrosis, moreover this CMV mediated blockage of the STAT1 shows statistical significance at late stage of fibrosis.
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http://dx.doi.org/10.1016/j.micpath.2020.104596DOI Listing
March 2021

Key Players of Hepatic Fibrosis.

J Interferon Cytokine Res 2020 10 25;40(10):472-489. Epub 2020 Aug 25.

Genetic Engineering Division, Department of Microbial Biotechnology, National Research Centre, Giza, Egypt.

Hepatic fibrosis is a complex mechanism defined by the net deposition of the extracellular matrix () owing to liver injury caused by multiple etiologies such as viral hepatitis and nonalcoholic fatty liver disease. Many cell types are implicated in liver fibrosis development and progression. In general, liver fibrosis starts with the recruitment of inflammatory immune cells to generate cytokines, growth factors, and other activator molecules. Such chemical mediators drive the hepatic stellate cells (HSCs) to activate the production of the component. The activation of HSC is thus a crucial event in the fibrosis initiation, and a significant contributor to collagen deposition (specifically type I). This review explores the causes and mechanisms of hepatic fibrosis and focuses on the roles of key molecules involved in liver fibro genesis, some of which are potential targets for therapeutics to hamper liver fibro genesis.
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http://dx.doi.org/10.1089/jir.2020.0059DOI Listing
October 2020

Recipient interleukin 6 gene polymorphism and expression predict HCV recurrence post liver transplantation.

Gene 2020 Sep 10;754:144887. Epub 2020 Jun 10.

Department of Microbial Biotechnology, Genetic Engineering Division, National Research Centre, 33 EL Bohouth Street Dokki, Giza 12622, Egypt.

Background: Liver transplantation (LTX)is a lifesaving- effective protocol for patients suffering end stage liver disease (ESLD) and its complications post HCV infection. Recurrence of disease is a frequent clinical complication that is observed in patients undergoing LTX. Cytokines play a central role in the immunological events occurring after the surgery.

Methods: Using Allelic Discrimination PCR, the allelic variation G174C of IL-6 gene was investigated. The abundance of IL6- mRNA and plasma IL6 cytokine levels were evaluated by using qRT-PCR in peripheral blood mononuclear cells (PBMCs) and enzyme-linked immunosorbent assay (ELISA) respectively in 76 liver transplant recipients recruited from Al Sahel teaching hospital, Ministry of Health and Population Cairo Egypt within the period between June 2015 and October 2017.

Results: The frequencies of IL-6 GG genotype and the G allele were significantly detected more in LTX recipients who experienced HCV recurrence versus those who did not suffer recurrence when compared to healthy controls (P = 0.001) and (P = 0.006), respectively. On the contrary, levels of IL-6 related transcripts in PBMC's of recurrent patients were indifferent from non-recurrent patients and healthy controls (P ≥ 0.124). Interestingly, the circulating IL-6 protein in plasma was significantly elevated in recurrent as compared to the non-recurrent recipients (P = 0.002).

Conclusion: HCV recurrence post liver transplantation occur more frequently in patients with -174 G/G IL-6 genotype and elevated plasma IL-6 levels.
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http://dx.doi.org/10.1016/j.gene.2020.144887DOI Listing
September 2020

A multiepitope peptide vaccine against HCV stimulates neutralizing humoral and persistent cellular responses in mice.

BMC Infect Dis 2019 Nov 5;19(1):932. Epub 2019 Nov 5.

Micrbial Biotechnology Department, National Research Center, 33 Tahrir street, Dokki, Cairo, 12622, Egypt.

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.
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http://dx.doi.org/10.1186/s12879-019-4571-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6833294PMC
November 2019

Correlation between IL28B/TLR4 genetic variants and HCC development with/without DAAs treatment in chronic HCV patients.

Genes Dis 2020 Sep 27;7(3):392-400. Epub 2019 May 27.

Department of Microbial Biotechnology, Genetic Engineering Division, National Research Centre, 33 EL Bohouth St.(former El Tahrir St.), Dokki, Giza, P.O. 12622, Egypt.

In Egypt, Sofosbuvir (SOF) in combination with Dataclasvir (DCV) is the broadly used DAAs with excellent therapeutic profile. This study is designed to explore the relation between IL28B/TLR4 genetic variants and each of the followings; HCC development post SOF/DCV treatment, progression to HCC in naïve patients and SOF/DCV therapy outcome. A total of 493 blood samples were collected (controls ( = 70); HCV patients treated with SOF/DCV ( = 252) of whom 65 patients developed HCC, 187 patients didn't develop HCC (125 responders, 62 relapsers); naïve HCV patients ( = 171) had early ( = 48), late liver fibrosis ( = 21) and HCC ( = 102)). Both SNPs were genotyped using a TaqMan 5' allelic discrimination assay. At IL28B rs12979860 SNP, the C allele was significantly correlating with the response rate more than T allele (OR 1.9, 95% CI 1.29-2.9,  = 0.004), while at TLR4 rs4986791 SNP, no association was found (OR 6.5, 95% 0.57-75.28,  = 0.09). Both SNPs couldn't detect the probability for HCC emergence after treatment. In naïve patients, the protective alleles were detected in their lowest frequency in HCC patients ( = 0.1, for rs12979860 and,  = 0.001 for rs4986791). SOF/DCV combination improved SVR rates in HCV genotype 4a infected patients regardless of IL28B genotype, with the best rates in those lacking the T allele.
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http://dx.doi.org/10.1016/j.gendis.2019.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452484PMC
September 2020

Establishment of serum derived infectivity coculture model for enhancement of hepatitis C virus replication in vitro.

Hum Antibodies 2019 ;27(3):185-191

Genetic Engineering Division, Department of Microbial Biotechnology, National Research Center, Giza, Egypt.

Background And Aims: Although HCV is one of the major health problems worldwide with the highest prevalence of genotype 4a in Egypt, it is poorly understood because of the limitations of having a robust in vitro model that allows the investigation and understanding of viral pathogenesis and life cycle. Genomic replicons for HCV are widely used and proved to have strong replication efficiency in cell culture, however, they are not able to produce infectious particles to enable the investigation of the whole viral life cycle and they mostly represent few sub-genomic classes for HCV. Hence, Genotype specific replication system is necessary to address specific sub-genomic phenotypes related to Hepatitis C pathogenicity.

Methods: In this study we attempt to develop a sustainable co-culture model, which potentially provides essential route of infection for HCV by using HCV-positive sera from infected patients. In this novel in vitro model, we tested the viral replication in co-cultured Huh 7.5 and HepG2 cells in order to sustain full viral replication cycle. We used high viral load serum of HCV-infected patients (10 × 106 to 20 × 106 IU/ml) as a source for HCV particles to infect co-cultured cells for 7 days.

Results And Conclusions: Viral replication capacity was increased 3-5 folds in the coculture condition compared to the individual cell lines, which indicates an improvement to viral infectivity in vitro.

Significance Statement: This novel coculture system represents a new in vitro model that will help study the underlying mechanisms of HCV pathogenicity.
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http://dx.doi.org/10.3233/HAB-190370DOI Listing
February 2020

Safety and tolerability of mice to repeated subcutaneous injections of a peptide mix as a potential vaccine against HCV infection.

Hum Antibodies 2019 ;27(2):105-110

Background And Aims: In this study, the safety and tolerability of new candidate HCV vaccine named Cenv6 were screened in mice. Cenv6 peptide is composed of 6 synthetic HCV peptides (3 structural and 3 nonstructural peptides).

Methods: Forty eight mice were enrolled in this study, 12 controls and 36 mice (the thirty-six mice were categorized into 3 groups according to administered doses (3 monthly doses of 800 ng, 1600 ng, and 16 μg/25 gm mouse body weight (bw))). Hematological, biochemical and histopathological changes were appraised.

Results: Our data indicated that the doses of 800 ng and 1600 ng of Cenv6 per 25 gm mouse body weight were safe as compared to the dose 16 μg/25 gm bw (10 times more than the potential therapeutic dose) for all examined tissues while the 16 μg Cenv6 dose provoked histopathological changes in kidneys, liver and lungs.

Conclusions: The extravagant histopathological changes in different organs have exiled the 16 μg dose out of acceptable range and validated that Cenv6 is safe and tolerable at the two lower doses (800 and 1600 ng/25 gm bw).
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http://dx.doi.org/10.3233/HAB-180354DOI Listing
August 2019

Extrahepatic Upregulation of Transforming Growth Factor Beta 2 in HCV Genotype 4-Induced Liver Fibrosis.

J Interferon Cytokine Res 2018 08;38(8):341-347

1 Genetic Engineering Division, Department of Microbial Biotechnology, National Research Centre , Giza, Egypt .

Elevated levels of transforming growth factor-β (TGF-β) family mediate myofibroblast generation and extracellular matrix deposition, thus making TGF-β recognized as major profibrogenic cytokines. In this article, we provide evidence that extrahepatic TGF-β2 expression at RNA and protein levels in peripheral leucocytes and serum, respectively, correlate with hepatic fibrogenesis. Current study includes a total of 110 subjects [89 naive hepatitis C virus (HCV)-infected patients (f0-f4) and 21 healthy controls]. Array profiling of 84 fibrosis-related transcripts revealed that TGF-β2 RNA was significantly upregulated compared with controls. Transcription results were confirmed by specific qRT-PCR on TGF-β2 RNA in peripheral leucocytes and TGF-β2 protein by ELISA in serum. PCR array and qRT-PCR for TGF-β2 RNA in peripheral leucocytes revealed that HCV-infected patients, regardless of the degree of fibrosis, had significantly elevated TGF-β2 RNA levels compared with controls (P = 0.018 and 0.047, respectively). This extrahepatic upregulation of TGF-β2 RNA was confirmed by elevated levels of secretory TGF-β2 protein in infected sera (P = 0.001). The Area Under the Curve of the receiver operating characteristic curve for the TGF-β2 protein between patients and controls was 0.80, a value that renders serum TGF-β2 protein a promising biomarker for liver fibrosis.
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http://dx.doi.org/10.1089/jir.2018.0045DOI Listing
August 2018

Hepatitis C virus serologic relapse after treatment with direct-acting antivirals is dependent on viral RNA levels in peripheral blood mononuclear cells and the grade of liver cirrhosis.

Arch Virol 2018 Oct 3;163(10):2765-2774. Epub 2018 Jul 3.

Ahmed Maher Teaching Hospital, 341 Port Said Street, Bab El Khalq, Cairo, 11638, Egypt.

The disappearance of hepatitis C virus (HCV) from serum and tissues for 12 weeks after the end of treatment (EOT) with direct-acting antivirals (DAAs) is known as a "sustained virologic response" (SVR) and occurs more frequently in non-cirrhotic patients than in cirrhotic patients. In this study, we evaluated the outcome of HCV treatment with sofosbuvir (SOF) plus ledipasvir (LDV) at both EOT and 12 weeks after EOT in patients with and without hepatic cirrhosis to address the relationship of serologic relapse to persistent infection of PBMCs and the frequency of hepatic encephalopathy and hepatocellular carcinoma (HCC) after treatment. Seventy-five patients with post-HCV liver cirrhosis were assigned to one of three groups (A, B, and C), each of which included 25 patients and corresponded to the patients' Child-Turcotte-Pugh (CTP) classification. All of the patients received a daily dose of SOF (400 mg) plus LDV (90 mg) for 24 weeks and were tested using HCV single-strand reverse transcription (SRT) and PCR analysis of PBMCs at both EOT and 12 weeks after EOT. Fourteen (18.7%) out of 75 patients (all study populations) had intra-PBMC HCV RNA, but only nine of them (64.3%) developed HCV RNA serum relapse (seroconversion) 12 weeks after EOT (P < 0.001). Encephalopathy was significantly higher in group C at EOT and 12 weeks after EOT (P < 0.05). Development of HCC was observed in decompensated patients of group C (2 out of 5 = 40.0%) 12 weeks post-EOT (P = 0.03). In conclusion, detection of HCV RNA within PBMCs at the EOT provides an indication of potential relapse after 12 weeks. Moreover, development of encephalopathy and HCC after HCV eradication by SOF plus LDV therapy is perhaps a future warning for post-treatment hepatic decompensation in cirrhotic patients.
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http://dx.doi.org/10.1007/s00705-018-3922-7DOI Listing
October 2018

HCV Therapy Follow-up Fractionation (CTF2) by Intra-PBMC Nested RNA PCR Recognizes Early Virologic Response and Relapse.

J Clin Transl Hepatol 2018 Jun 25;6(2):147-154. Epub 2018 Apr 25.

Department of Microbial Biotechnology, National Research Center, Cairo, Egypt.

Sustained virologic response is evaluated by single-step reverse transcription (SRT) PCR assay, which assesses hepatitis C virus (HCV) clearance from plasma but not from tissues such as peripheral blood mononuclear cells (PBMCs). Persistence of HCV RNA in PBMCs beyond end of treatment (EOT) is associated with nonresponse. Our goal was to measure intra-PBMC HCV RNA levels during oral antiviral therapy according to the HCV therapy follow-up fractionation (CTF2) protocol. Compensated chronic HCV patients ( = 2 78 SRT-PCR positive) were scheduled to receive oral antiviral therapy. Subjects were followed-up by SRT and intra-PBMCs HCV RNA PCR at the end of the 2, 6, 10, 14, 18 and 24 weeks to evaluate virus clearance from plasma and PBMCs, respectively. The CTF2 protocol evaluated SRT and PBMC PCR status at each follow-up point for determining therapy continuation or interruption to address cost effectiveness. All patients tested negative by SRT PCR after therapy for 2 weeks. Application of the CTF2 protocol revealed: a) increasing HCV clearance rate from 75.9% at the end of 10 week to 90.3% at the end of 24 week ( < 0.00001); b) faster clearance of HCV from plasma compared to PBMCs at each point of follow-up until the 18 week ( < 0.05); c) higher viral elimination rates diagnosed by PBMC HCV RNA PCR(-) compared to PBMC HCV RNA PCR(+) from the 6 to 24 week of treatment ( < 0.0001); d) higher over-time increase curve of combined plasma and PBMC HCV RNA determined negativity compared to the decline in positivity curves by PBMC PCR at the 6-18 week compared to the 24 week ( < 0.01)-these results validated treatment continuation; and e) solitary evaluation of EOT sustained HCV infection and relapses by PBMC HCV RNA ( < 0.001). Early elimination of serum and tissue (PBMC) HCV infection by oral antiviral therapy can be achieved and evaluated during a cost-effective CTF2 protocol application.
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http://dx.doi.org/10.14218/JCTH.2017.00077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6018311PMC
June 2018

Dysregulation of fibrosis related genes in HCV induced liver disease.

Gene 2018 Jul 21;664:58-69. Epub 2018 Apr 21.

Micrbial Biotechnology Department, National Research Center, Dokki, Cairo, 12622, Egypt.

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.
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http://dx.doi.org/10.1016/j.gene.2018.04.032DOI Listing
July 2018

Vascular Endothelial Growth Factor Expression in Hepatitis C Virus-Induced Liver Fibrosis: A Potential Biomarker.

J Interferon Cytokine Res 2017 07 4;37(7):310-316. Epub 2017 May 4.

1 Department of Microbial Biotechnology, Genetic Engineering Division, National Research Center, Giza, Egypt .

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.
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http://dx.doi.org/10.1089/jir.2016.0127DOI Listing
July 2017

Expression of Reactive Oxygen Species-Related Transcripts in Egyptian Children With Autism.

Biomark Insights 2017 23;12:1177271917691035. Epub 2017 Feb 23.

Microbial Biotechnology Department, Genetic Engineering Division, National Research Centre, Giza, Egypt.

The molecular basis of the pathophysiological role of oxidative stress in autism is understudied. Herein, we used polymerase chain reaction (PCR) array to analyze transcriptional pattern of 84 oxidative stress genes in peripheral blood mononuclear cell pools isolated from 32 autistic patients (16 mild/moderate and 16 severe) and 16 healthy subjects (each sample is a pool from 4 autistic patients or 4 controls). The PCR array data were further validated by quantitative real-time PCR in 80 autistic children (55 mild/moderate and 25 severe) and 60 healthy subjects. Our data revealed downregulation in , and transcripts (1.5, 3.8, 1.2, 1.7, and 2.2, respectively; < .05 for all) in autistic group compared with controls. In addition, and exhibited 1.4- and 1.7-fold downregulation, respectively, in severe autistic patients when compared with mild/moderate group ( = .005 and .0008, respectively). This study helps in a better understanding of the underlying biology and related genetic factors of autism, and most importantly, it presents suggested candidate biomarkers for diagnosis and prognosis purposes as well as targets for therapeutic intervention.
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http://dx.doi.org/10.1177/1177271917691035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5391985PMC
February 2017

Mice Antibody Response to Conserved Nonadjuvanted Multiple Antigenic Peptides Derived from E1/E2 Regions of Hepatitis C Virus.

Viral Immunol 2017 06 12;30(5):359-365. Epub 2017 Apr 12.

1 Department of Microbial Biotechnology, National Research Center , Dokki, Giza, Egypt .

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.
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http://dx.doi.org/10.1089/vim.2016.0123DOI Listing
June 2017

Methylene Tetrahydrofolate Reductase Gene Polymorphism is Associated with Severity of Liver Steatosis in Chronically Infected Patients with HCV Genotype 4.

Clin Lab 2017 Mar;63(3):419-426

Background: Methylene tetrahydrofolate reductase (MTHFR) C677T polymorphism was reported as a genetic variant in liver steatosis and fibrosis. This is a study of the association between MTHFR C677T polymorphism and HCV core with severity of steatosis in HCV GT4 patients.

Methods: 111 HCV patients and 112 control subjects were recruited. Polymorphism was detected by RFLP analysis, core Ag was detected by ELISA.

Results: Combined HCV infection and MTHFR C677T polymorphism increases the risk to develop steatosis by 3.63- and 5.21-fold in subjects with single (CT) and double (TT) substitutions, respectively. Patients with chronic HCV infection had a 2.88- and 8.57-fold higher risk to develop steatosis in CT and TT genotypes, respectively, than patients with the (CC) genotype. No significant difference in core Ag titers were observed.

Conclusions: MTHFR C677T polymorphism is a valuable genetic marker for steatosis, while HCV core Ag titer had no association with grades of steatosis in GT4 infections.
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http://dx.doi.org/10.7754/Clin.Lab.2016.160624DOI Listing
March 2017

Level of Human Antibodies Targeting HCV E1/E2 Peptides and Spontaneous Clearance of HCV in Blood Donors.

Clin Lab 2016 Oct;62(10):1879-1885

Background: Hepatitis C virus (HCV) is a major health problem worldwide particularly in Egypt. The humoral immune response has an important function in the control of HCV infection. The aim of this study was to investigate the role of neutralizing antibodies in Hepatitis C Virus (HCV) clearance in infected individuals.

Methods: This study was carried out on apparently healthy blood donors (n = 200). Detectable HCV antibodies were assessed by commercial ELISA and specific human immunoglobulins targeting peptides derived from HCV E1/E2 glycoproteins were measured in donors' blood using an in house optimized ELISA. Human IgG purification was carried out from positive HCV RNA and negative HCV RNA samples in order to evaluate its neutralizing activity in vitro using Huh 7 cells.

Results: The studied cohort included 96/200 subjects who tested positive for HCV antibodies, among which 56/96 (58%) samples were positive for HCV RNA (Group 1) and 40/96 (42%) samples had undetectable HCV RNA (Group 2). ELISA results showed that Human HCV immunoglobulin (HHI) targeting HCV E1 synthetic peptide (a.a. 315 - 323) was detectable in 63/96 (66%) and HHI targeting HCV E2 (a.a. 412 - 419) tested positive in 14/96 (15%) while 19/96 (20%) were positive for HCV E2 (a.a. 517 - 531). HHI higher than the cutoff level against peptide HCV E1 (a.a. 315 - 323) was detected in 22/63 (35%) in Group 2 and positive in 41/63 (65%) in Group 1. HHI against peptide HCV E2 (a.a. 412 - 419) was positive in 7 (50%) blood donors in Group 2 and also positive in 7 (50%) of Group 1. While HHI targeting HCV E2 (a.a. 517 - 531) was positive in 11 (60%) in Group 2 compared with 8 cases (40%) in Group 1. Purified human antibodies from cases positive for HCV antibodies and negative for HCV RNA showed in vitro neutralization at concentrations 30 and 10 µg/mL while the same concentration of purified human IgG from cases positive for HCV RNA showed no viral neutralization.

Conclusions: The tested epitope(s) derived from HCV envelope E1 and E2 are important for viral clearance and hence can be used for HCV vaccine development.
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http://dx.doi.org/10.7754/Clin.Lab.2016.160108DOI Listing
October 2016

The Synergistic Effect of TNFα -308 G/A and TGFβ1 -509 C/T Polymorphisms on Hepatic Fibrosis Progression in Hepatitis C Virus Genotype 4 Patients.

Viral Immunol 2017 03 2;30(2):127-135. Epub 2017 Feb 2.

1 Department of Microbial Biotechnology, National Research Centre , Giza, Egypt .

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.
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http://dx.doi.org/10.1089/vim.2016.0083DOI Listing
March 2017

Three Gene Signature for Predicting the Development of Hepatocellular Carcinoma in Chronically Infected Hepatitis C Virus Patients.

J Interferon Cytokine Res 2016 12 11;36(12):698-705. Epub 2016 Oct 11.

1 Genetic Engineering Division, Department of Microbial Biotechnology, National Research Centre , Dokki, Giza, Egypt .

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.
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http://dx.doi.org/10.1089/jir.2016.0042DOI Listing
December 2016

Tumor necrosis factor-α -G308A polymorphism is associated with liver pathological changes in hepatitis C virus patients.

World J Gastroenterol 2016 Sep;22(34):7767-77

Noha G Bader El Din, Sally Farouk, Reem El-Shenawy, Marwa K Ibrahim, Reham M Dawood, Mostafa K El Awady, Department of Microbial Biotechnology, National Research Centre, Dokki, Giza 12622, Egypt.

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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016377PMC
http://dx.doi.org/10.3748/wjg.v22.i34.7767DOI Listing
September 2016

Low Molecular Mass Polypeptide 7 Single Nucleotide Polymorphism is Associated with the Progression of Liver Fibrosis in Patients Infected with Hepatitis C Virus Genotype 4.

Clin Lab 2016 ;62(3):381-7

Background: The hepatitis C virus (HCV) is a leading cause of liver disease and the consequent complications of cirrhosis. However, there is no precise biomarker to predict the patients at high risk of developing progressive disease. We showed previously the implication of low molecular mass polypeptide-7 (LMP-7) single nucleotide varia- tions in the response to combined pegylated IFN and ribavirin therapy in patients infected with HCV genotype 4. In this study, we examined the possible relationship between LMP-7 genotypes and both the degree of liver fibrosis and the transition from end stage of fibrosis to hepatocellular carcinoma (HCC).

Methods: LMP-7 single nucleotide variation at codon 49 (substitution from A to C) was determined using restriction fragment length polymorphism analysis in leucocyte DNA from healthy subjects (n = 36) and HCV-chronically infected patients of genotype 4 either with different grades of liver fibrosis (n = 77) or with hepatocellular carci- noma (n = 25). Chronic HCV-infected patients having liver fibrosis were categorized into two groups based on the degree of fibrosis, early fibrosis (F0-F2, n = 37) and late fibrosis (F3-F4, n = 40).

Results: Our results demonstrated that patients with the LMP-7 CA/AA genotypes were more likely to have advanced fibrosis scores than those bearing the CC genotype, although LMP-7 polymorphism does not seem to contribute to the progression from the late stage of fibrosis to hepatocellular carcinoma.

Conclusions: These results suggest that LMP-7 polymorphism is a candidate prognostic marker in mathematical models designed for predicting the progression of HCV-related liver disease. Nevertheless, the mechanism whereby LMP-7 leads to the progression of liver fibrosis remains to be determined.
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http://dx.doi.org/10.7754/clin.lab.2015.150710DOI Listing
June 2016

Transcriptional Dysregulation of Upstream Signaling of IFN Pathway in Chronic HCV Type 4 Induced Liver Fibrosis.

PLoS One 2016 2;11(5):e0154512. Epub 2016 May 2.

Department of Microbial Biotechnology, Genetic Engineering Division, National Research Centre, Dokki, Giza, Egypt.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0154512PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4852926PMC
July 2017

Alteration of the Total Nuclear DNA Ploidy in Different Histopathological Liver Tissues Negative and Positive for HCV RNA.

Clin Lab 2015 ;61(9):1247-56

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.
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http://dx.doi.org/10.7754/clin.lab.2015.141249DOI Listing
December 2015
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