Development of an efficient in vivo cell-based assay system for monitoring hepatitis C virus genotype 4a NS3/4A protease activity.

BACKGROUND: Hepatitis C virus (HCV) represents a serious worldwide healthcare problem. No protective vaccines against HCV have been developed yet due to the fact that HCV is rapidly mutable, allowing the virus to escape from the neutralizing antibodies. Understanding of HCV was initially hampered by the inability to achieve viral replication in cell culture. Given its essential roles in viral polyprotein processing and immune evasion, HCV NS3/4A protease is a prime target for antiviral chemotherapy. We aimed to establish in vivo cell-based assay system for monitoring the activity of NS3/4A protease from HCV genotype 4a, the predominant genotype in Egypt, and the Middle East. Furthermore, the developed system was used to evaluate the inhibitory potency of a series of computer-designed chemically-synthesized compounds against NS3/4A protease from HCV genotype 4a. MATERIALS AND METHODS: Native as well as mutant cleavage sites to NS3/4A protease were cloned in frame into ß-galactosidase gene of TA cloning vector. The target specificity of HCV NS3/4A was evaluated by coexpression of ß-galactosidase containing the protease cleavage site with NS3/4A protease construct in bacterial cells. The activity of ß-galactosidase was colorimetrically estimated in the cell lysate using orthonitro phenyl ß-D-galactopyanoside (ONPG) as a substrate. RESULTS AND CONCLUSIONS: We successfully developed an efficient cell-based system based on the blue/white selection of bacterial cells that are able to express functional/nonfunctional ß-galactosidase enzyme.

Expression and immune recognition of polypeptides derived from Hepatitis C virus structural proteins.

BACKGROUND: Hepatitis C virus (HCV) is characterized by a high degree of nucleotide sequence variability between genotypes. This variability extends to functional and immunological determinants. Serological tests using antigenic segments derived from the HCV polyprotein have been used for the diagnosis of HCV infection. However, available diagnostic Kits do not necessarily take type variability into consideration and are not optimized for HCV genotype 4a (HCV4a), the predominant genotype in Egypt. AIM: The aim of this study was to express some HCV4a-derived polypeptides in order to identify those with immunodiagnostic utility. MATERIALS AND METHODS: Six sequential/overlapping genomic segments encoding 100-266 amino acid peptides from the core (peptide 1), envelope 1 (E1; peptide 2), envelope 2 (E2; peptides 4, 5, and 6), and E1/E2 (peptide 3) regions of the HCV4apolyprotein were selected for in vitro expression as glutathione S-transferase-fusion proteins. The immunoreactivity of the expressed peptides was evaluated against sera from HCV-infected/uninfected individuals using dot blot, western blot, and enzyme-linked immunosorbent assay. RESULTS: The expressed polypeptides were recognized by HCV-infected sera from 20 patients, while showing no immunoreactivity toward uninfected serum. Peptide 1 derived from the core protein was found to be the most immunoreactive. CONCLUSION: Expressed polypeptides hold good potential for use in the development of improved HCV immunodiagnostics.

The anti-apoptotic and anti-inflammatory properties of puerarin attenuate 3-nitropropionic-acid induced neurotoxicity in rats.

Puerarin (Pur), an isoflavonoid extracted from the dried roots of Pueraria lobata, has been reported to be useful in the treatment of various diseases. This study was designed to evaluate the anti-apoptotic and anti-inflammatory activities of Pur against 3-nitropropionic acid (3-NP) induced neurotoxicity. For 5 consecutive days, male Wistar rats were given Pur (200 mg/kg body mass) 30 min before treatment with 20 mg/kg body mass of 3-NP. The striata, hippocampi, and cortices of the 3-NP treated group showed apoptotic damage, inflammation, and energy deficit as well as histopathological lesions. The 3-NP-induced alteration in apoptotic biomarkers (caspase-3 activity/level, cytosolic cytochrome c, Bax/Bcl-2 levels) were significantly ameliorated by Pur treatment. Moreover, Pur pretreatment blocked 3-NP-induced inflammatory biomarkers (NF-κB, TNF-α, and iNOS) and prevented the energy deficit (ATP reduction). Nissl staining further confirmed Pur's neuroprotective effect. These results indicate that Pur may be a useful preventive approach to various neurodegenerative diseases with underlying apoptosis and neuroinflammation.

Puerarin ameliorates 3-nitropropionic acid-induced neurotoxicity in rats: possible neuromodulation and antioxidant mechanisms.

Puerarin (daidzein-8-C-glucoside), a major isoflavone glycoside purified from Pueraria lobata, is well reported to have a neuroprotective effect primarily by the antioxidant mechanisms. This investigation was designed to evaluate the efficacy of Puerarin (Pur) to offset 3-nitropropionic acid (3-NP) induced neurotoxicity. Male Wistar strain rats were given 3-NP (20 mg/kg, s.c.) over five consecutive days, whereas Pur (200 mg/kg, i.p.) was administrated 30 min before 3-NP. Rats treated with 3-NP exhibited significant weight loss, reduction of the prepulse inhibition, locomotor hypoactivity and hypothermia. The striata, hippocampi and cortices of the 3-NP treated rats showed abnormal levels of neurotransmitters, oxidative damage and characteristic histopathological lesions. Treatment with Pur ahead of 3-NP, significantly prevented weight loss, PPI deficit, locomotor hypoactivity and hypothermia. Pur treatment blocked the 3-NP-induced neurotransmitters abnormalities (GABA, DA, 5-HT and NE), and normalized the oxidative stress biomarkers (lipid peroxidation, reduced glutathione, glutathione peroxidase). Histopathological examination further affirmed Pur's neuroprotective effect against 3-NP-induced neurotoxicity. In conclusion, Pur protected the brain tissues from 3-NP induced neurotoxicity primarily by its neuromodulation and antioxidant effect.

Protective and anti-pathology effects of Sm fructose-1,6-bisphosphate aldolase-based DNA vaccine against schistosoma mansoni by changing route of injection.

This study aimed to evaluate the efficacy of fructose-1,6-bis phosphate aldolase (SMALDO) DNA vaccination against Schistosoma mansoni infection using different routes of injection. The SMALDO has been cloned into the eukaryotic expression vector pcDNA3.1/V5-His TOPO-TA and was used in injecting Swiss albino mice intramuscularly (IM), subcutaneously (SC), or intraperitoneally (IP) (50 µg/mouse). Mice vaccinated with non-recombinant pcDNA3.1 served as controls. Each group was immunized 4 times at weeks 0, 2, 4, and 6. Two weeks after the last booster dose, all mice groups were infected with 80 S. mansoni cercariae via tail immersion. At week 8 post-infection, animals were sacrificed for assessment of parasitological and histopathological parameters. High anti-SMALDO IgG antibody titers were detected in sera of all vaccinated groups (P<0.01) compared to the control group. Both the IP and SC vaccination routes resulted in a significant reduction in worm burden (46.2% and 28.9%, respectively, P<0.01). This was accompanied by a significant reduction in hepatic and intestinal egg counts (41.7% and 40.2%, respectively, P<0.01) in the IP group only. The number of dead eggs was significantly increased in both IP and IM groups (P<0.01). IP vaccination recorded the highest significant reduction in granuloma number and diameter (54.7% and 29.2%, respectively, P<0.01) and significant increase in dead miracidia (P<0.01). In conclusion, changing the injection route of SMALDO DNA vaccination significantly influenced the efficacy of vaccination. SMALDO DNA vaccination via IP route could be a promising protective and anti-pathology vaccine candidate against S. mansoni infection.

First insights into the metagenome of Egyptian mummies using next-generation sequencing.

We applied, for the first time, next-generation sequencing (NGS) technology on Egyptian mummies. Seven NGS datasets obtained from five randomly selected Third Intermediate to Graeco-Roman Egyptian mummies (806 BC-124AD) and two unearthed pre-contact Bolivian lowland skeletons were generated and characterised. The datasets were contrasted to three recently published NGS datasets obtained from cold-climate regions, i.e. the Saqqaq, the Denisova hominid and the Alpine Iceman. Analysis was done using one million reads of each newly generated or published dataset. Blastn and megablast results were analysed using MEGAN software. Distinct NGS results were replicated by specific and sensitive polymerase chain reaction (PCR) protocols in ancient DNA dedicated laboratories. Here, we provide unambiguous identification of authentic DNA in Egyptian mummies. The NGS datasets showed variable contents of endogenous DNA harboured in tissues. Three of five mummies displayed a human DNA proportion comparable to the human read count of the Saqqaq permafrost-preserved specimen. Furthermore, a metagenomic signature unique to mummies was displayed. By applying a "bacterial fingerprint", discrimination among mummies and other remains from warm areas outside Egypt was possible. Due to the absence of an adequate environment monitoring, a bacterial bloom was identified when analysing different biopsies from the same mummies taken after a lapse of time of 1.5 years. Plant kingdom representation in all mummy datasets was unique and could be partially associated with their use in embalming materials. Finally, NGS data showed the presence of Plasmodium falciparum and Toxoplasma gondii DNA sequences, indicating malaria and toxoplasmosis in these mummies. We demonstrate that endogenous ancient DNA can be extracted from mummies and serve as a proper template for the NGS technique, thus, opening new pathways of investigation for future genome sequencing of ancient Egyptian individuals.

The effect of Ginkgo biloba extract on 3-nitropropionic acid-induced neurotoxicity in rats.

3-Nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase enzyme (SDH), induces neurodegeneration similar to that observed in Huntington's disease (HD). Reduction of prepulse inhibition (PPI) of acoustic startle response, locomotor hypoactivity, bilateral striatal lesions as well as brain oxidative stress are major features of HD. The present study was designed to investigate neuroprotective effect of Ginkgo biloba extract (EGb 761) on 3-NP induced neurobehavioral changes and striatal lesions. Rats administered 3-NP (20mg/kg, s.c.) for five consecutive days exhibited PPI deficits and locomotor hypoactivity whereas, pretreatment of animals with EGb 761 (100mg/kg, i.p. for 15 days) ahead of and during the induction of HD by 3-NP (20mg/kg for 5 days starting at day 8) ameliorated 3-NP-induced neurobehavioral deficits. Administration of 3-NP increased the level of striatal malondialdehyde (MDA). This effect was prevented in animals pre-treated with EGb 761. Changes in the level of apoptotic regulatory gene expressions, following 3-NP treatment, were demonstrated as both an up-regulation and a down-regulation of the expression levels of striatal Bax and Bcl-xl genes, respectively. In addition, an up-regulation of the expression level of striatal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also observed. Pre-treatment with EGb 761 caused a down-regulation in striatal GAPDH and Bax together with an up-regulation of striatal Bcl-xl expression level as compared to the 3-NP treated group. Histochemical examination of striatal tissue showed that EGb 761 significantly prevented 3-NP induced inhibition of SDH activity. Histopathological examination further affirmed the neuroprotective effect of EGb 761 against 3-NP toxicity. Taken together, these results suggest that EGb 761 has a neuroprotective role in the current HD paradigm, which may be related to improvement of energy metabolism, antioxidant properties and antiapoptotic effects.

FHIT gene and flanking region on chromosome 3p are subjected to extensive allelic loss in Egyptian breast cancer patients.

The fragile histidine triad gene (FHIT) is a candidate tumor suppressor gene at chromosome 3p14.2. Deletions in FHIT gene were reported in different types of cancer including breast cancer. In this study, we investigated the loss of heterozygosity (LOH) incidence that target FHIT genomic structure and chromosome 3p in cancerous and pre-neoplastic lesions of Egyptian breast patients. Genomic DNA was isolated from tumor tissues and their normal counterparts of 55 Egyptian patients diagnosed with breast cancer and 11 patients diagnosed with preneoplastic breast lesions. LOH was detected in 51% of breast cancer cases in at least one microsatellite marker of the four investigated markers. While, none of the markers showed LOH among the pre-neoplastic breast lesions. We also observed a significant association between LOH and invasive ductal carcinoma (IDC) histopathological type while no association observed between LOH and patients' age, tumor grade, or lymph node involvement. We also investigated FHIT gene expression profiles in breast cancer using Oncomine database. We found that FHIT is significantly reduced in all investigated studies. We conclude that, FHIT is underexpressed in breast cancer tissues compared to their normal counterparts due to the extensive allelic loss that is observed in its gene structure.

Multiple Patterns of FHIT Gene Homozygous Deletion in Egyptian Breast Cancer Patients.

Fragile histidine triad (FHIT) gene encodes a putative tumour suppressor protein. Loss of Fhit protein in cancer is attributed to different genetic alterations that affect the FHIT gene structure. In this study, we investigated the pattern of homozygous deletion that target the FHIT gene exons 3 to 9 genomic structure in Egyptian breast cancer patients. We have found that 65% (40 out of 62) of the cases exhibited homozygous deletion in at least one FHIT exon. The incidence of homozygous deletion was not associated with patients' clinicopathological parameters including patients' age, tumour grade, tumour type, and lymph node involvement. Using correlation analysis, we have observed a strong correlation between homozygous deletions of exon 3 and exon 4 (P < 0.0001). Deletions in exon 5 were positively correlated with deletions in exon 7 (P < 0.0001), Exon 8 (P < 0.027), and exon 9 (P = 0.04). Additionally, a strong correlation was observed between exons 8 and exon 9 (P < 0.0001).We conclude that FHIT gene exons are homozygously deleted at high frequency in Egyptian women population diagnosed with breast cancer. Three different patterns of homozygous deletion were observed in this population indicating different mechanisms of targeting FHIT gene genomic structure.

Methylenetetrahydrofolate reductase gene polymorphisms and the risk of colorectal carcinoma in a sample of Egyptian individuals.

The study was planned as a pilot study to investigate two common polymorphisms in the MTHFR gene c.677C > T and c.1298A > C and their association with enhanced risk of colorectal cancer (CRC) in a sample of Egyptian individuals. Venous blood samples were withdrawn from 35 cases of CRC and 68 healthy controls. Specimens from colonic and rectal carcinoma tissues in addition to cancer free tissues were obtained from all cases. Frequencies of MTHFR677T and 1298C alleles were significantly higher among cases of CRC tumor tissues (50% and 56%, respectively) than germ line alleles in CRC patients (33% and 41%, respectively) and healthy controls (21% and 35%, respectively). Frequencies of heterozygous and homoyzgous polymorphisms of MTHFR at positions 677 and 1298 in carcinoma tissues were always the highest. At position 677, TT and CT genotype frequencies were 17% and 66% with an odds ratio {OR} of 11 [95% confidence interval {CI} 2.39-50.59] and OR 8.34 [95%CI 2.97-23.92], respectively, in carcinoma tissues. While in the germ line of patients the genotype frequencies of 677TT and CT were 6% and 54% with OR 1.57 [95%CI 0.26-9.51] and 2.99 [95%CI 1.25-7.12], respectively, compared to controls (6% and 29%, respectively). The combined genotype MTHFR 1298CC + AC frequencies were 86% with OR 3.71 [95%CI 1.28-10.78] in carcinoma tissues, 69% with OR 1.35 [95%CI 0.57-3.21] in germ line of patients and 62% in controls. The combined genotype 677CT plus any of the following genotypes 1298AA, AC or CC enhanced risk of CRC, when comparing germ line DNA polymorphism of patients versus peripheral blood DNA of control subjects with OR 4.5 [95%CI 0.94-21.56], OR 3.12 [95%CI 0.79-12.36] and OR 18 [95%CI 1.56-207.5], respectively, suggesting strong genetic predisposition of certain Egyptian population to CRC. These results suggested that at least one C to T polymorphism at 677MTHFR gene is required to significantly increase the risk for CRC development. Further large scale studies are required to confirm the present findings.

Lamivudine facilitates optimal chemotherapy in hepatitis B virus-infected children with hematological malignancies: a preliminary report.

Hepatitis B virus (HBV) reactivation is well documented in infected patients who have hematologic malignancies, precluding appropriate chemotherapy courses and, therefore, increasing the possibility of relapse of malignancies. The objective of this study was to evaluate lamivudine treatment to prevent hepatitis B reactivation in children with cancer who acquired infection with HBV and so allow completion of optimal chemotherapy. Ten children (7:3 M:F; median age: 9.8 years), undergoing chemotherapy for hematological malignancies and suffering from immunosuppressive-induced hepatitis B virus reactivation, were treated concurrently with lamivudine (3 mg/kg bw, od) for up to 18 months. All were HBsAg+ve, HBsAb-ve, HBV-DNA+ve. Serology markers (HBsAg/Ab, HBeAg/Ab, HBV-DNA) and ALT were tested 3 monthly. Histological assessments were performed pre- and 18 months post-lamivudine therapy. During lamivudine therapy chemotherapy courses were completed for all children, and none of the patients suffered reactivation of hepatitis. After a median follow-up of 10 months, remission of malignancy was maintained in 7/10 patients while 3 patients relapsed. HBeAg+ve seroconversion occurred in 4/9 HBeAg+ve children within 3 months. After 9 months of therapy, 8/10 were HBV-DNA-ve. Six out of 7 children with histological evidence of chronic hepatitis showed marked improvement post-therapy. Lamivudine therapy for up to 18 months in children receiving chemotherapy helped prevent recurrence of hepatitis B exacerbations and improved the underlying chronic hepatitis, while facilitating completion of appropriate chemotherapy regimens without compromise.

Severe liver disease is caused by HBV rather than HCV in children with hematological malignancies.

UNLABELLED: Patients receiving chemotherapy experience exacerbations of chronic hepatitis B (HBV) or hepatitis C (HCV) viral infections. We examined the pattern of liver disease induced by these infections in 92 children and adolescents with elevated transaminases (median age: 9 years). This included 76 with hematological malignancies (55 ALL, 15 NHL and six Hodgkin's disease) and 16 with thalassemia major. Liver disease was graded: A--occasional hypertransaminemia, B--persistent hypertransaminemia, C--severe hepatitis without encephalopathy, D--fulminant hepatic failure (FHF) and death. Screening included HBsAg, anti-HCV antibody, HBV-DNA and HCV-RNA: 26 had liver biopsies. A total of 60 (79%) patients with malignancies were HBsAg and/or HBV-DNA(+)(genotype D-E) and 47 (62%) were anti-HCV and/or HCV-RNA(+); 33 were coinfected with HBV and HCV. Grade A (n=24) included 16 with HCV and 12 with HBV (six coinfected); 18 with HBV and 11 with HCV (10 coinfected) were graded B (n=22). All grade C (n=25) had HBV with 16 HCV coinfected. FHF and death occurred in five HBV-DNA(+) patients, in four within a month of i.v. methotrexate. Patterns C and D were associated with HBsAg and HBV-DNA (P=0.001 and P<0.001, respectively). In all, 70% of HBV-infected children suffered chemotherapy-associated flares. None of the thalassemics had severe hepatitis exacerbations; 94% had HCV markers with none HBV-DNA(+). One died of progressive cirrhosis. CONCLUSIONS: Children with hematological malignancies have worse liver disease when associated with chronic HBV. FHF occurred in HBsAg/HBV-DNA(+) children following i.v. methotrexate. Early recognition of hepatic dysfunction in HBV carriers is essential in order to reduce incidence of life-threatening complications.

Analysis of Schistosoma mansoni genes using the expressed sequence tag approach.

Expressed sequence tags (ESTs) are partial cDNA sequences read from both ends of random expressed gene fragments used for discovering new genes. DNA libraries from four different developmental stages of Schistosoma mansoni used in this study generated 141 ESTs representing about 2.5% of S. mansoni sequences in dbEST. Sequencing was done by the dideoxy chain termination method. The sequences were submitted to GenBank for homology searching in nonredundant databases using Basic Local Alignment Search Tool for DNA (BLASTN) alignment and for protein (BLASTX) alignment at the National Center for Biotechnology Information (NCBI). Among submitted ESTs, 29 were derived from lambdagt11 sporocyst library, 70 from lambdaZap adult worm library, 31 from lambdaZap cercarial library, and 11 from lambdaZap female B worm library. Homology search revealed that eight (5.6%) ESTs shared homology to previously identified S.mansoni genes in dbEST, 15 (10.6%) are homologous to known genes in other organisms, 116 (81.7%) showed no significant sequence homology in the databases, and the remaining sequences (2.1%) showed low homologies to rRNA or mitochondrial DNA sequences. Thus, among the 141 ESTs studied, 116 sequences are derived from noval, uncharactarized S. mansoni genes. Those 116 ESTs are important for identification of coding regions in the sequences, helping in mapping of schistosome genome, and identifying genes of immunological and pharmacological significance.

Cloning and characterization of a Schistosoma mansoni 1H and 30S clones as two tegumental vaccine candidate antigens.

Two Schistosoma mansoni cDNA clones 30S and 1H were identified by immunoscreening of sporocyst lambdagt11 library and by random sequencing of clones from lambdaZap libraries, respectively. Clone 30S was one of 30 clones identified by an antibody raised against tegument of 3-h schistosomules. The clone was found to encode an 81 amino-acid protein fragment. It was expressed in Escherichia coli as a fusion protein of calculated molecular mass of about 35 kDa with C-terminus of Schistosoma japonicum glutathione-S-transferase (Sj26; about 26 kDa). The recombinant fusion protein was specifically recognized by serum of rabbits immunized with irradiated cercariae. Clone 1H is one of 76 expressed sequence tags derived from an adult worm library. It encodes the complete sequence of a tegumental membrane protein, Sm13. The 104 amino-acid open reading frame encodes a protein with a calculated molecular mass of about 11.9 kDa. Clone 1H was expressed in E. coli as an insoluble fusion protein with Sj26 of about 40 kDa. In Western blots, the fusion protein was recognized by serum from rabbits vaccinated with irradiated cercariae but not by preimmune rabbit sera. The cloning, characterization and expression of those proteins are therefore potentially usefull for vaccine development.

Cellular and humoral immune responses to recombinant Smp17.7 Schistosoma mansoni antigen.

To determine the immunological responses to S. mansoni antigen rSmp17.7, a total of 184 subjects, 174 patients from a schistosomiasis endemic area, and 10 controls were used. Proliferation, cytokine profile in culture supernatants from antigen-stimulated peripheral blood mononuclear cells and specific IgG1, IgG3, IgG4, IgA, IgM & IgE levels were assessed. The highest stimulation index to rSmp17.7 was detected in S. mansoni patients. The evaluation of the cytokine profile [IL-2, IL-4 & IFN-gamma] in response to this antigen showed a significant increase as demonstrated by ELISA. Specifically, IFN-gamma and IL-2 were significantly detected by flow cytometry. IgG1 and IgM were the only Igs which showed a significant increase. These results highlight the importance of rSmp17.7 as a candidate vaccine for schistosomiasis. The results pave the way to understand the mechanism of schistosome-vaccine efficacy.