MicroRNAs expression profiling in Egyptian colorectal cancer patients.

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

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

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 É£ 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.

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

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.

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

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.

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.

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.

Antiviral activity of virocidal peptide derived from NS5A against two different HCV genotypes: an in vitro study.

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

Mouse monoclonal antibody towards e1 specific epitope blocks viral entry and intracellular viral replication in vitro.

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

Prevalence of viral hepatitis (A, B, C, and E) infection and co-infection among hospitalized children in Cairo, Egypt.

Viral hepatitis is considered a public health issue facing the entire world. The World Health Organization encouraged all countries to work together to eliminate this fatal infection and achieve the 2030 agenda. The present study aimed to investigate the silent infection of viral hepatitis (A, B, C, and E) among hospitalized children in Cairo, Egypt, to control and avoid chronic infection early on. This cross-sectional study included 184 randomly selected hospitalized children from three different hospitals in Cairo, Egypt. They were children aged between a few months to 15 years to determine viral hepatitis infection and co-infection. Antibodies to hepatitis A virus (HAV IgM), hepatitis E virus (HEV IgM), hepatitis C virus (HCV Ab), and hepatitis B virus surface antigen (HBs Ag) were performed by ELISA. If the ELISA results were positive, the viral load was quantified by real-time polymerase chain reaction (RT-PCR). Other laboratory investigations included alanine aminotransferase, aspartate aminotransferase, albumin, and complete blood count. Only five children (2.71%) had HCV Ab positive with no other viral (A, B, and E) co-infections as determined by ELISA. Also, the RT-PCR detected HCV RNA in these ELISA positive children. The remaining children (179/184) were all negative for all hepatitis viruses' markers (HAV IgM, HEV IgM, HBs Ag, and HCV Ab). In conclusion, this study documented that, Cairo hospitals serving Egyptian children had a low prevalence of viral hepatitis (A, B, C, and E). More research with larger sample sizes from hospitals across Egypt is needed.

Immunogenicity study of a Novel DNA-Based HCV vaccine candidate.

In this study, we aimed to evaluate the immunogenic profile of a chimeric DNA-based hepatitis C virus (HCV) vaccine candidate encoding the full-length viral core-E1-E2 (HCV-CE) fragment. The vaccine candidate was designed to uniformly express the HCV genotype 4 core-E1-E2 protein. The recombinant HCV-CE protein was bacterially expressed in C41 (DE3) cells, and then BALB/c mice were immunized with different combinations of DNA/DNA or DNA/protein prime/boost immunizations. The proper construction of our vaccine candidate was confirmed by specific amplification of the encoded fragments and basic local alignment search tool (BLAST) results of the nucleotide sequence, which revealed a high degree of similarity with several HCV serotypes/genotypes. The platform for bacterial expression was optimized to maximize the yield of the purified recombinant HCV-CE protein. The recombinant protein showed high specific antigenicity against the sera of HCV-infected patients according to the ELISA and western blot results. The predicted B- and T-cell epitopes showed high antigenic and interferon-γ (IFN-γ) induction potential, in addition to cross-genotype conservation and population coverage. The mice antisera further demonstrated a remarkable ability to capture 100% of the native viral antigens circulating in the sera of HCV patients, with no cross-reactivity detected in control sera. In conclusion, the proposed HCV vaccination strategy demonstrated promising potential regarding its safety, immunogenicity, and population coverage.

Harnessing immunoinformatics for developing a multiple-epitope peptide-based vaccination approach against SARS-CoV-2 spike protein.

COVID-19 has spread to over 200 countries with variable severity and mortality rates. Computational analysis is a valuable tool for developing B-cell and T-cell epitope-based vaccines. In this study, by harnessing immunoinformatics tools, we designed a multiple-epitope vaccine to protect against COVID-19. The candidate epitopes were designed from highly conserved regions of the SARS-CoV-2 spike (S) glycoprotein. The consensus amino acids sequence of ten SARS-CoV-2 variants including Gamma, Beta, Epsilon, Delta, Alpha, Kappa, Iota, Lambda, Mu, and Omicron was involved. Applying the multiple sequence alignment plugin and the antigenic prediction tools of Geneious prime 2021, ten predicted variants were identified and consensus S-protein sequences were used to predict the antigenic part. According to ElliPro analysis of S-protein B-cell prediction, we explored 22 continuous linear epitopes with high scores ranging from 0.879 to 0.522. First, we reported five promising epitopes: BE1 1115-1192, BE2 481-563, BE3 287-313, BE4 62-75, and BE5 112-131 with antigenicity scores of 0.879, 0.86, 0.813, 0.779, and 0.765, respectively, while only nine discontinuous epitopes scored between 0.971 and 0.511. Next, we identified 194 Major Histocompatibility Complex (MHC) - I and 156 MHC - II epitopes with antigenic characteristics. These spike-specific peptide-epitopes with characteristically high immunogenic and antigenic scores have the potential as a SARS-CoV-2 multiple-epitope peptide-based vaccination strategy. Nevertheless, further experimental investigations are needed to test for the vaccine efficacy and efficiency.

Overexpression of miRNA 26a and 26b with MMP-9 are valuable diagnostic biomarkers for colorectal cancer patients.

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

Molecules called miRNAs, found in blood, have a key role in colon cancer progression. This evidence highlights the importance of studying the role of some miRNAs in colorectal cancer (CRC) patients. miRNAs are one of the most recent targets for CRC screening and prognosis. miR-26a and -26b showed high overexpression in CRC patients. Results showed that both miR-26a and -26b had high diagnostic efficacy for CRC.

Polyphenol Content, Antioxidant, Cytotoxic, and Genotoxic Activities of Bombax ceiba Flowers in Liver Cancer Cells Huh7.

OBJECTIVE: Bombax ceiba (red Silk cotton tree) has great ethnopharmacological significance due to its widespread use to treat various diseases such as dysentery, inflammation, and tuberculosis. Despite decades of research, the studies on the in vitro anticancer/genotoxic activity of B. ceiba flower remains restricted. Thus, the present research explored the effect of ethanol extract from B. ceiba flowers on three human cancer cells, including lung A549 and liver HepG2 and Huh7 cell lines. METHODS: Cytotoxic and genotoxic activity of B. ceiba extract was examined by MTT and comet assay, respectively. Further, B. ceiba extract was analysed to determine total polyphenol content and DPPH antiradical scavenging activity. RESULTS: ethanol extract from B. ceiba flowers had a high polyphenols content with very potent antioxidant activity. Further, B. ceiba extract displayed moderate cytotoxicity against Huh7 cells and no cytotoxicity against HepG2 and A549 cells. The comet assay findings showed that Huh7 cells treated with four concentrations of B. ceiba extract (» IC50, ½ IC50, IC50, and double IC50) increased the comet tail formation within 48 h in a concentration-dependent manner. CONCLUSION: ethanol extract from B. ceiba flowers exhibited its cytotoxicity through induction of DNA fragmentation in Huh7 cells.

Neutralizing activities of caprine antibodies towards conserved regions of the HCV envelope glycoprotein E2.

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

Circulating macrophage inflammatory protein-1ß/IL-12p40 ratio predicts sofosbuvir-based treatment outcome in HCV- genotype 4 patients.

This study aimed to evaluate the prognostic value of baseline macrophage inflammatory protein (MIP)-1ß/IL12p40 ratio for antiviral treatment outcome in HCV genotype 4 patients. METHODS: Sera of 450 treatment-naïve chronic HCV patients and 50 healthy individuals were collected. Liver transaminases, total bilirubin and albumin were biochemically tested, viral RNA was quantified, and circulating MIP-1ß and IL-12p40 were estimated using human anti-MIP-1ß and IL-12p40 antibodies in Sandwich ELISA. RESULTS: No difference was observed in the baseline chemokines levels between responders and relapsers, but the later had a significantly higher MIP-1ß/IL-12p40 ratio (P< 0.0001). Multivariate regression analysis of baseline characteristics showed that gender, age, viral load, albumin level and chemokine ratios can significantly predict treatment outcome (P= 0.0114, 0.0095, 0.042, 0.0004 and < 0.0001; respectively). Accordingly, a predictive threshold of baseline chemokine ratio was calculated and it showed an AUC of 0.6917 (P= 0.0108; 95% CI: 0.5566 to 0.8268). The calculated threshold for predicting virologic response was 8.245, with positive and negative predictive values of 92.98% and 100%; respectively. The chemokine ratios had significant correlations with liver transaminases in treated groups whether pre or post-treatment. CONCLUSION: Baseline MIP-1ß/IL-12p40 ratio represents a non-invasive prognostic biomarker that would provide shorter treatment duration and minimizes the emergence of drug-resistant variants in HCV genotype 4-patients.

Conserved peptides within the E2 region of Hepatitis C virus induce humoral and cellular responses in goats.

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

Serum inducible protein-10 chemokine as a biomarker for clearance of HCV with and without treatment in Egyptian patients.

BACKGROUND AND AIMS: Assessment of the potential predictive value of serum inducible protein-10 chemokine (IP-10) in the clearance of HCV in Egyptian patients with and without treatment. MATERIALS AND METHODS: Ninety Egyptian individuals were involved in the current study where, 20 patients (23%) were chronic HCV (positive HCV antibodies and positive HCV RNA without treatment, 20 (22%) were healthy individuals (negative for both HCV antibodies and HCV RNA, 20 (22%) were natural clearance (positive HCV antibodies and negative for HCV RNA without treatment), 20 (22%) were achieved SVR after treatment (responders group, HCV positive and negative for HCV RNA after treatment) and 10 (11%) were non responders (positive HCV antibodies and still positive HCV RNA after treatment). HCV RNA was quantitated by real time PCR and serum IP10 level was measured by commercial ELISA kit. All biochemical and hematological examinations included liver function, CBC and alphefeto protein were assessed. RESULTS: The mean serum levels of IP-10 were significantly higher (p< 0.001) in CHC patients (345.4 ± 100) pg/ml compared with healthy control group (101.5 ± 31.4) and natural clearance group (103.2 ± 40.7). Also serum levels of IP-10 was significantly elevated in non-responders group (257.4 ± 52.5) compared with each of SVR group (103.5 ± 43.5) (p< 0.001) and healthy group (101.5 ± 31.4), (p< 0.001). Prediction of a clinical response based on a IP10 chemokine revealed high sensitivity (93%), specificity (96%), negative predictive value (96%), and area under curve is (1.00). Moreover, there is no correlation ((R= 0.05), P value p< 0.795) between serum level of IP-10 and HCV viral load. CONCLUSION: IP10 is a useful non-invasive biomarker for viral clearance and might be used to apply patients according to the predictable treatment outcome. Accordingly, patients who are unlikely to respond to treatment would avoid unnecessary exposure to medication that is related with high morbidity.

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

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).

Part III: Novel checkpoint kinase 2 (Chk2) inhibitors; design, synthesis and biological evaluation of pyrimidine-benzimidazole conjugates.

Recently a dramatic development of the cancer drug discovery has been shown in the field of targeted cancer therapy. Checkpoint kinase 2 (Chk2) inhibitors offer a promising approach to enhance the effectiveness of cancer chemotherapy. Accordingly, in this study many pyrimidine-benzimidazole conjugates were designed and twelve feasible derivatives were selected to be synthesized to investigate their activity against Chk2 and subjected to study their antitumor activity alone and in combination with the genotoxic anticancer drugs cisplatin and doxorubicin on breast carcinoma, (ER+) cell line (MCF-7). The results indicated that the studied compounds inhibited Chk2 activity with high potency (IC50 = 5.56 nM - 46.20 nM). The studied candidates exhibited remarkable antitumor activity against MCF-7 (IG50 = 6.6  µM - 24.9 µM). Compounds 10a-c, 14 and 15 significantly potentiated the activity of the studied genotoxic drugs, whereas, compounds 9b and 20-23 antagonized their activity. Moreover, the combination of compound 10b with cisplatin revealed the best apoptotic effect as well as combination of compound 10b with doxorubicin led to complete arrest of the cell cycle at S phase where more than 40% of cells are in the S phase with no cells at G2/M. Structure-activity relationship was discussed on the basis of molecular modeling study using Molecular modeling Environment program (MOE).

Part II: New candidates of pyrazole-benzimidazole conjugates as checkpoint kinase 2 (Chk2) inhibitors.

The development of checkpoint kinase 2 (Chk2) inhibitors for the treatment of cancer has been an ongoing and attractive objective in drug discovery. In this study, twenty-one feasible pyrazole-benzimidazole conjugates were synthesized to study their effect against Chk2 activity using Checkpoint Kinase Assay. The antitumor activity of these compounds was investigated using SRB assay. A potentiation effect of the synthesized Chk2 inhibitors was also investigated using the genotoxic anticancer drugs cisplatin and doxorubicin on breast carcinoma, (ER+) cell line (MCF-7). In vivo Chk2 and antitumor activities of 8d as a single-agent, and in combination with doxorubicin, were evaluated in breast cancer bearing animals induced by N-methylnitrosourea. The effect of 8d alone and in combination with doxorubicin was also studied on cell-cycle phases of MCF-7 cells using flow cytometry analysis. The results revealed their potencies as Chk2 inhibitors with IC50 ranges from 9.95 to 65.07 nM. Generally the effect of cisplatin or doxorubicin was potentiated by the effect of most of the compounds that were studied. The in vivo results indicated that the combination of 8d and doxorubicin inhibited checkpoint kinase activity more than either doxorubicin or 8d alone. There was a positive correlation between checkpoint kinase inhibition and the improvement observed in histopathological features. Single dose treatment with doxorubicin or 8d produced S phase cell cycle arrest whereas their combination created cell cycle arrest at G2/M from 8% in case of doxorubicin to 51% in combination. Gold molecular modelling studies displayed a high correlation to the biological results.

The Synergistic Effect of TNFα -308 G/A and TGFß1 -509 C/T Polymorphisms on Hepatic Fibrosis Progression in Hepatitis C Virus Genotype 4 Patients.

Tumor necrosis factor-alpha (TNFα) and transforming growth factor-beta (TGFß1) 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 TGFß1 -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 TGFß1 -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 TGFß1 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 TGFß1 -509 TT genotype than early fibrosis patients (p = 0.016, 0.028, respectively). Moreover, the TNFα and TGFß1 serum levels were significantly higher in HCV patients with TNFα A containing genotypes (GA+AA) (p = 0.004) and patients with TGFß1 T containing genotypes (CT+TT) (p = 0.001), respectively. The combined unfavorable TNFα (GA/AA) and TGFß1 (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 TGFß1 -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 TGFß1 -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.