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Flow cytometric detection of hepatitis C virus antigens in infected peripheral blood leukocytes: Binding and entry

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World Journal of Gastroenterology 2005年第33期11卷 5203-5208页

作者： mostafa K el-awady Ashraf A Tabll el-Rashdy M Redwan Samar Youssef Moataza H Omran Fouad Thakeb Maha el-Demellawy Department of Biomedical Technology National Research Center Tahrir Street Dokki Cairo Egypt Genetic Engineering and Biotechnology Research Institute New Borg el-Arab City Alexandria Egypt Department of Gastroenterology-Hepatology Faculty of Medicine Cairo University Egypt Genetic Engineering and Biotechnology Research Institute New Borg el-Arab City Alexandria Egypt

AIM： We designed two synthetic-core-specific peptides core 1 （C1） and core 2 （C2）, and an E1-specific peptide （el）. We produced specific polyclonal antibodies against these peptides and used the antibodies for detection of HCV antigens on surface and within infected peripheral blood leukocytes. METHODS： Peripheral blood from a healthy individual who tested negative for HCV RNA was incubated with HCV type 4 infected serum for i h and 24 h at 37 ℃. Cells were stained by direct and indirect immunofluorescence and measured by flow cytometry. RESULTS： After 1 h of incubation, antibodies against C1, C2, and el detected HCV antigens on the surface of 27%, 26% and 73% of monocytes respectively, while 10%, 5% and 9% of lymphocytes were positive with anti-C1, anti-C2 and anti-E1 respectively. Only 1-3% of granulocytes showed positive staining with anti-C1, anti-C2 and anti E1 antibodies. After 24 h of incubation, we found no surface staining with anti-C1, anti-C2 or anti-E1. Direct immunostaining using anti-C2 could not detect intracellular HCV antigens, after 1 h of incubation with the virus, while after 24 h of incubation, 28% of infected cells showed positive staining. Only plus strand RNA was detectable intracellularly as early as 1 h after incubation, and remained detectable throughout 48 h post-infection. Interestingly, minus RNA strand could not be detected after 1 h, but became strongly detectable intracellularly after 24 h post-infection. CONCLUSION： Monocytes and lymphocytes are the preferred target cells for HCV infection in peripheral blood leukocytes. Our specific anti-core and anti-E1 antibodies are valuable reagents for demonstration of HCV cell cycle. Also, HCV is capable of infecting and replicating in peripheral blood mononuclear cells as confirmed by detection of minus strand HCV RNA as well as intracellular staining of core HCV antigen.

关键词： Flow cytometry Hepatitis C virus Envelope Core Antibodies Indirect immunofluorescence Minus and plus RNA strand Peripheral blood mononudear cells

Hepatitis C Virus RNA Strands Detection in Peripheral Blood Mononuclear Cells Legitimizes Virus Eradication in Negative Serum PCR Naive and Post-treatment Patients

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Journal of Clinical and Translational Hepatology 2017年第1期5卷 1-8页

作者： Mohamed Darwish Ahmed Abd Alla mostafa Kamel el awady Tropical Medicine Department Faculty of MedicineAl-Azhar UniversityCairoEgypt Department of Microbial Biotechnology National Research CenterCairoEgypt

Background and Aims:Hepatitis C virus(HCV)hepatotropism is associated with intra-peripheral blood mononuclear cell(PBMC)infection that causes post-treatment relapse in RNA seronegative *** understanding of the association of non-viremic hepatic fibrosis with positive anti-HCV IgG antibodies and active hepatocellular damage might be increased by PBMCs screening for intracellular ***,the goals of this study included evaluation of PBMCs PCR for diagnosing HCV infection,addressing PBMCs plus serum real-time(SRT)PCR benefits over SRT-PCR alone,studying intraPBMCs distribution of RNA sense and antisense strands,and identifying treatment feasibility in solitary intracellular ***:Enzyme-linked immunosorbent assay,SRT-PCR and PBMCs PCR were used to evaluate HCV infection in 401 *** patients were classified into groups of negative controls(n=30),positive controls(n=63),non-viremia post-treatment(experienced;n=166)and na(i)ve(n=49)cases,and non-viremia positive PBMCs PCR na(i)ve(n=35)and experienced(n=58)***:The diagnosis of true positive and negative by PBMCs PCR and SRT-PCR had 100%and 96.7%compatibility *** PCR detected intracellular HCV infection in 49 out of 215 non-viremia patients;among them,na(i)ve cirrhotics had significantly higher number of intracellular infection than the na(i)ve non-cirrhotic(Pely recognized in na(i)ve and experienced cases(P=0.01218).Intracellular HCV strands were detected in 18.02%of experienced *** of intracellular RNA strands showed significant decline in experienced compared to na(i)ve patients(Pellular HCV when SRT-PCR is *** and sense strands are respectively recognized more often in na(i)ve and experienced *** expected overall relapsing rate in our cohort was 18.02%.Int

关键词： HCV PBMCs RNA Strands

HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro

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World Journal of Gastroenterology 2006年第30期12卷 4836-4842页

作者： mostafa K el-awady Ashraf A Tabll Yasmine S el-Abd Mahmoud M Bahgat Hussein A Shoeb Samar S Youssef Noha G Bader el Din el-Rashdy M Redwan Maha el-Demellawy Moataza H Omran Wael T el-Garf Said A Goueli Department of Biomedical Technology National Research Center Tahrir Street PO 12622 Dokki Cairo Egypt Department of Medicinal Chemistry Na- tional Research Center Tahrir Street PO 12622 Dokki Cairo Egypt Department of Microbiology and Immunology Faculty of Pharmacy Cairo University Cairo Egypt Genetic Engineering and Biotechnology Research Institute New Borg El-Arab City Alexandria Egypt Department of Pathology School of Medicine University of Wisconsin Madison WI United States

AIM： TO establish a cell culture system with longterm 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 na＇ive 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.

关键词： Hepatitis C virus In vitro propagation Genomic replication Gene expression HepG2 cells

Molecular study on Y chromosome microdeletions in Egyptian males with idiopathic infertility

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Asian Journal of Andrology 2004年第1期6卷 53-57页

作者： mostafa K. el awady Sohair F. el Shater Ehab Ragaa Khaled Atef Ibrahim M. Shaheen Nagwa A. Megiud Department of Biomedical Technology Children with Special Needs National Research Center Dokki Cairo 12622 Egypt Department of Research on Children with Special Needs National Research Center Dokki Cairo 12622 Egypt Department of Dermatology Venereology and Andrology Faculty of Medicine Al-Azhar University Cairo Egypt

Aim: To determine the frequency of genetic deletions within the azoospermia factors in Egyptian infertile males. Methods: The Yq microdeletions in 33 infertile males with undetectable chromosomal anomalies were examined by mutiplex polymerase chain reaction (PCR). Deletions were confirmed using single PCR amplifications. Results: Four out of the total 33 (12 %) men had Yq11 microdeletions, thus supporting the average reported figures in other populations. Three of those 4 cases had single short tandem sequence deletions with discrete histological findings of their testes. Single sY272 deletion within AZFc was associated with Sertoli cell only syndrome, whereas a patient with isolated sY84 deletion within AZFa had immature testicular structure. The remaining case had a large deletion in AZFa-c and short stature. Conclusion: The present study supports the hypothesis that the Yqn encompasses genetic determinants of stature besides genes controlling spermatogenesis.

关键词： AZF Y microdeletions multiplex polymerase chain reaction male infertility azoospermia

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

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Genes & Diseases 2020年第3期7卷 392-400页

作者： Ghada M.Salum Reham M.Dawood Mai Abd el-Meguid Noha E.Ibrahim Ashraf O.Abdel Aziz mostafa K.el awady Department of Microbial Biotechnology Genetic Engineering DivisionNational Research Centre33 EL Bohouth St.(former El Tahrir St.)DokkiGizaP.O.12622Egypt Department of Tropical Medicine Faculty of MedicineCairo UniversityGizaP.O.12622Egypt

In Egypt,Sofosbuvir(SOF)in combination with Dataclasvir(DCV)is the broadly used DAAs with excellent therapeutic *** 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 naı¨ve patients and SOF/DCV therapy outcome.A total of 493 blood samples were collected(controls(n = 70);HCV patients treated with SOF/DCV(n = 252)of whom 65 patients developed HCC,187 patients didn’t develop HCC(125 responders,62 relapsers);naı¨ve HCV patients(n = 171)had early(n = 48),late liver fibrosis(n = 21)and HCC(n = 102)).Both SNPs were genotyped using a TaqMan 50 allelic discrimination *** IL28B rs12979860 SNP,the C allele was significantly correlating with the response rate more than T allele(OR 1.9,95%CI 1.29e2.9,p=0.004),while at TLR4 rs4986791 SNP,no association was found(OR 6.5,95%0.57e75.28,p = 0.09).Both SNPs couldn’t detect the probability for HCC emergence after *** naı¨ve patients,the protective alleles were detected in their lowest frequency in HCC patients(p = 0.1,for rs12979860 and,p = 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.

关键词： DAAs DCV HCV HCC IL28B SOF TLR4

Occult HCV Infection (OCI) Diagnosis in Cirrhotic and Non-cirrhotic Naive Patients by Intra-PBMC Nested Viral RNA PCR

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Journal of Clinical and Translational Hepatology 2017年第4期5卷 319-326页

作者： Mohamed Darwish Ahmed Abd Alla Saleh Ahmed elibiary George Y.Wu mostafa Kamel el-awady Tropical Medicine Department Faculty of MedicineAl-Azhar UniversityCairoEgypt Department of Medicine Division of Gastroenterology-HepatologyUniversity of Connecticut Health CenterFarmingtonCTUSA Department of Microbial Biotechnology National Research CenterCairoEgypt

Background and Aims:Occult HCV infections(OCIs)include IgG antibody seronegative cryptogenic(COCIs),as well as seropositive secondary na(i)ve(SNOCIs)and experienced(SEOCIs)*** used peripheral-blood-mononuclear-cell(PBMC)-PCR to evaluate COCIs and SNOCIs prevalence,serum HCV spontaneous disappearance(SCSD)in na(i)ve cirrhotics and non-cirrhotics,intra-PBMC HCV-RNA strands in relation to cirrhosis density in na(i)ve non-viremia cases,and HCV-RNA seroconversion after 1 year of solitary naive intra-PBMC ***:The anti-HCV IgG antibodypositive na(i)ve-patients(n=785)were classified into viremic(n=673)and non-viremic[n=112,including non-cirrhotics(n=55)and cirrhotics(n=57)],and 62 controls without evidence of *** and post-HCV non-viremia cases(n=62+112=174)were submitted to hepatic Fibroscanelastography *** subjects(n=847)were screened for intra-PBMC HCV-RNA sense and antisense strands by ***:Na(i)ve-OCI cases(4.84%)that were diagnosed by PBMC-PCR significantly raised the total numbers of HCV-infection to 714(p=0.01).The percent positivity of SNOCIs(34.82%)was significantly higher than for asymptomatic-COCIs(3.125%,p=0.0001).Comparing PBMC-PCR with single-step-reverse-transcription(SRT)-PCR for identification of SCSD in na(i)ve IgG antibody-positive nonviremia patients(n=112)revealed a decline in SCSD prevalence by PBMC-PCR(from 14.27%to 9.3%),regardless of presence of hepatic cirrhosis(p=0.03).SCSD was found to be higher by PBMC-PCR in non-cirrhotics compared to cirrhotics(p=0.0001),with an insignificant difference when using SRT-PCR(p=0.45).Intra-PBMC HCV-RNA infection was significantly more frequent in cirrhotics compared to both non-cirrhotics and controls(p<0.0005).An increased hepatic fibrosis density was recognized in intra-PBMC HCV-RNA infection with sense(p=0.0001)or antisense strand(p=0.003).HCV-RNA seroconversion was associated with intra-PBMC infection when both sense and antisense strands were dete

关键词： OCIs PBMCs Na(i)ve Cirrhosis

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

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Journal of Clinical and Translational Hepatology 2018年第2期6卷 147-154页

作者： Mohamed Darwish Ahmed Abd Alla Saleh Ahmed elibiary Ramy Hassan elshaboury George Y.Wu Reham M.Dawood mostafa Kamel el awady Tropical Medicine Department Faculty of MedicineAl-Azhar UniversityCairoEgypt Department of Pharmacy Massachusetts General HospitalBostonMAUSA Department of Medicine Division of Gastroenterology-HepatologyUniversity of Connecticut Health CenterHartfordCTUSA Department of Microbial Biotechnology National Research CenterCairoEgypt

Background and Aims:Sustained virologic response is evaluated by single-step reverse transcription(SRT)PCR as-say,which assesses hepatitis C virus(HCV)clearance from plasma but not from tissues such as peripheral blood mono-nuclear cells(PBMCs).Persistence of HCV RNA in PBMCs beyond end of treatment(EOT)is associated with *** goal was to measure intra-PBMC HCV RNA levels during oral antiviral therapy according to the HCV therapy follow-up fractionation(CTF2)***:Compen-sated chronic HCV patients(n=278 SRT-PCR positive)were scheduled to receive oral antiviral *** were followed-up by SRT and intra-PBMCs HCV RNA PCR at the end of the 2nd,6th,10th,14th,18th and 24th weeks to evaluate virus clearance from plasma and PBMCs,*** CTF2 protocol evaluated SRT and PBMC PCR status at each follow-up point for determining therapy continuation or inter-ruption to address cost ***:All patients tested negative by SRT PCR after therapy for 2 ***-cation of the CTF2 protocol revealed:a)increasing HCV clear-ance rate from 75.9%at the end of 10th week to 90.3%at the end of 24th week(pelimination rates diagnosed by PBMC HCV RNA PCR(?)compared to PBMC HCV RNA PCR(+)from the 6th to 24th week of treatment(pelapses by PBMC HCV RNA(pelimination of serum and tissue(PBMC)HCV infection by oral antiviral therapy can be achieved and evaluated during a cost-effective CTF2 pro-tocol application.

关键词： CTF2 protocol HCV PBMCs PCR Cost-effectiveness

Positional effect of mutations in 5'UTR of hepatitis C virus 4a on patients' response to therapy

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World Journal of Gastroenterology 2009年第12期15卷 1480-1486页

作者： mostafa K el awady Hassan M Azzazy Ahmed M Fahmy Sherif M Shawky Noha G Badreldin Samar S Yossef Moataza H Omran Abdel Rahman N Zekri Said A Goueli Department of Biomedical Technology National Research CenterDokkiCairo 12622Egypt Department of Chemistry and Yousef Jameel Science and Technology Research Center the American University in CairoCairo 11511Egypt Structural biology department Free University of AmsterdamDe Boelelaan 10851081 HV AmsterdamThe Netherlands National Cancer Institute Cairo 11796Egypt Department of Pathology and Laboratory Medicine University of WisconsinMadisonWI 53711United States

AIM： To investigate the effects of mutations in domain Ⅲ of the hepatitis C virus （HCV） internal ribosome entry sequences （IRES） on the response of chronic HCV genotype 4a patients to interferon therapy.METHODS： HCV RNA was extracted from 19 chronic HCV 4a patients receiving interferon/ribavirin therapy who showed dramatic differences in their response to combination therapy after initial viral clearance. IRES domain Ⅲ was cloned and 15 clones for each patient were sequenced. The obtained sequences were aligned with genotype 4a prototype using the ClustaIW program and mutations scored. Prediction of stem-loop secondary structure and thermodynamic stability of the major quasispecies in each patient was performed using the MFOLD 3.2 program with Turner energies and selected constraints on base pairing.RESULTS： Analysis of RNA secondary structure revealed that insertions in domain Ⅲ altered WatsonCrick base pairing of stems and reduced molecular stability of RNA, which may ultimately reduce binding affinity to ribosomal proteins. Insertion mutations in domain - were statistically more prevalent in sustained viral response patients （SVR, n = 14） as compared to breakthrough （BT, n = 5） patients.CONCLUSION： The influence of mutations within domain Ⅲ on the response of HCV patients to combination therapy depends primarily on the position, but not the frequency, of these mutations within IRES domain Ⅲ.

关键词： Hepatitis C virus Internal ribosome entrysequences Domain Ⅲ Genotype 4a Ribosomal sub-unit Interferon therapy RNA folding

Association between low molecular polypeptide 7 single nucleotide polymorphism and response to therapy in hepatitis C virus infection

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World Journal of Hepatology 2013年第3期5卷 97-103页

作者： Moataza H Omran Basma E Fotouh Samar S Youssef Noha E Ibrahim Wael Nabil el-Sayed M Mahdy Wafaa G Shosha mostafa K el-awady Microbial Biotechnology Department Genetic Engineering Division National Research Center Chemistry Department Faculty of Science Helwan University

AIM: To investigate the relationship between low molecular polypeptide-7 (LMP-7) gene polymorphism and response to interferon (IFN) therapy in chronic hepatitis C virus (HCV) patients. METHODS: LMP-7 polymorphism at codon 49 with nucleotide substitution from A to C was amplified in 104 chronic HCV patients of genotype 4. The amplicons were digested with restriction endonuclease Bsm I and the produced restriction fragment length polymorphism was analyzed. Patients received IFN + regional blood volume therapy for 48 wk and the frequency of thissingle nucleotide polymorphism (SNP) was statistically correlated with treatment response. The exclusion criteria for these patients were stated by the national health program for treating viral hepatitis. Main exclusion criteria included co-infection with hepatitis B virus or schistosomiasis, thyroid dysfunction, uncontrolled diabetes mellitus, history of long term drug or alcohol intake and autoimmune hepatitis. Multivariate analyses were done to correlate LMP-7 SNP plus several factors such as age, gender, weight, serum alpha-fetoprotein (AFP) and alanine aminotransferase levels, liver activity, fibrosis score and viral load with response to therapy. RESULTS: The data presented in this study clearly demonstrated statistically significant differences between sustained virological response (SVR) (defined as the absence of HCV RNA levels in the patient's sera at least 6 mo after discontinuation of treatment) and non-response (NR) (where HCV RNA levels in the patient's sera never become undetectable for 6 mo during or after treatment). Variables were described as odds ratio with 95%CI. The data were considered significant if P values were ≤ 0.05; highly significant if P ele were associated with SVR, while the other two genotypes C/A and A/A were associated with NR patients, 83.3% and 64.3% respectively, showing that g

关键词： Hepatitis C virus Interferon therapy Low molecular mass polypeptide Host gene Single nucleotide polymorphism