Antiviral activity of chitosan nanoparticles encapsulating silymarin (Sil-CNPs) against SARS-CoV-2 (in silico and in vitro study).

To develop a specific treatment against COVID-19, we investigated silymarin-chitosan nanoparticles (Sil-CNPs) as an antiviral agent against SARS-CoV-2 using in silico and in vitro approaches. Docking of Sil and CNPs was carried out against SARS-CoV-2 spike protein using AutoDock Vina. CNPs and Sil-CNPs were prepared by the ionic gelation method and characterized by TEM, FT-IR, zeta analysis, and the membrane diffusion method to determine the drug release profile. Cytotoxicity was tested on both Vero and Vero E6 cell lines using the MTT assay. Minimum binding energies with spike protein and ACE2 were -6.6, and -8.0 kcal mol-1 for CNPs, and -8.9, and -9.7 kcal mol-1 for Sil, respectively, compared to -6.6 and -8.4 kcal mol-1 respectively for remdesivir (RMV). CNPs and Sil-CNPs were prepared at sizes of 29 nm and 82 nm. The CC50 was 135, 35, and 110 µg mL-1 for CNPs, Sil, and Sil-CNPs, respectively, on Vero E6. The IC50 was determined at concentrations of 0.9, 12 and 0.8 µg mL-1 in virucidal/replication assays for CNPs, Sil, and Sil-CNPs respectively using crystal violet. These results indicate antiviral activity of Sil-CNPs against SARS-CoV-2.

New insight into HCV E1/E2 region of genotype 4a.

INTRODUCTION: Hepatitis C virus (HCV) genome contains two envelope proteins (E1 and E2) responsible for the virus entry into the cell. There is a substantial lack of sequences covering the full length of E1/E2 region for genotype 4. Our study aims at providing new sequences as well as characterizing the genetic divergence of the E1/E2 region of HCV 4a using our new sequences along with all publicly available datasets. METHODS: The genomic segments covering the whole E1/E2 region were isolated from Egyptian HCV patients and sequenced. The resulting 36 sequences 36 were analyzed using sequence analysis techniques to study variability within and among hosts in the same time point. Furthermore, previously published HCV E1/E2 sequence datasets for genotype 4a were retrieved and categorized according to the geographical location and date of isolation and were used for further analysis of variability among Egyptian over a period of 15 years, also compared with non-Egyptian sequences to figure out region-specific variability. RESULTS: Phylogenetic analysis of the new sequences has shown variability within the host and among different individuals in the same time point. Analysis of the 36 sequences along with the Egyptian sequences (254 sequences in E1 in the period from 1997 to 2010 and 8 E2 sequences in the period from 2006 to 2010) has shown temporal change over time. Analysis of the new HCV sequences with the non-Egyptian sequences (182 sequences in E1 and 155 sequences in the E2) has shown region specific variability. The molecular clock rate of E1 was estimated to be 5E-3 per site per year for Egyptian and 5.38E-3 for non-Egyptian. The clock rate of E2 was estimated to be 8.48E per site per year for Egyptian and 6.3E-3 for non-Egyptian. CONCLUSION: The results of this study support the high rate of evolution of the Egyptian HCV genotype 4a. It has also revealed significant level of genetic variability among sequences from different regions in the world.

Dynamic interplay between CXCL levels in chronic hepatitis C patients treated by interferon.

BACKGROUND: Combined pegylated interferon-α and ribavirin therapy has sustained virological response (SVR) rates of 54% to 61%. Pretreatment predictors of SVR to interferon therapy have not been fully investigated yet. The current study assesses a group of chemokines that may predict treatment response in Egyptian patients with chronic HCV infection. PATIENTS AND METHODS: CXCL5, CXCL9, CXCL11, CXCL12, CXCL 13, CXCL 16 chemokines and E-Cadherin were assayed in 57 chronic HCV patients' sera using quantitative ELISA plate method. All studied patients were scheduled for combined pegylated interferon alpha and ribavirin therapy (32 patients received pegylated interferon α 2b, and 25 patients received pegylated interferon α 2a). Quantitative hepatitis C virus RNA was done by real time RT-PCR and HCV genotyping by INNOLIPAII. RESULTS: There was no significant difference (p > 0.05) in baseline HCV RNA levels between responders and non-responders to interferon. A statistically significant difference in CXCL13 (p = 0.017) and E-Cadherin levels (P = 0.041) was reported between responders and nonresponders at week 12. Significant correlations were found between changes in the CXCL13 levels and CXCL9, CXCL16, E-cadherin levels as well as between changes in E-cadherin levels and both CXCL16 and ALT levels that were maintained during follow up. Also, significant changes have been found in the serum levels of CXCL5, CXCL13, and CXCL16 with time (before pegylated interferon α 2 a and α 2 b therapy, and at weeks 12 and 24) with no significant difference in relation to interferon type and response to treatment. CONCLUSION: Serum levels of CXCL13 and E-Cadherin could be used as surrogate markers to predict response of combined PEG IFN-α/RBV therapy, especially at week 12. However, an extended study including larger number of patients is needed for validation of these findings. CLINICAL TRIAL NO: NCT01758939.

Evaluation of noninvasive versus invasive techniques for the diagnosis of Helicobacter pylori infection.

BACKGROUND: Helicobacter pylori is one of the most common bacterial strains causing chronic infections, affecting over one half of the world's population. There is increasing interest in noninvasive methods for diagnosing H. pylori infection. The aim of the study was to evaluate 3 different noninvasive methods of diagnosis: the stool antigen test (HpSA), the serum antibody test, and the stool-polymerase chain reaction (PCR) test as against invasive methods based on histopathologic diagnosis. MATERIALS AND METHODS: Gastric biopsies were obtained during endoscopy. Sections were stained with hematoxylin and eosin and Giemsa stain. Serum samples were tested for H. pylori antibody using an enzyme-linked immnunosorbent assay kit for the semiquantitative determination of IgG antibodies; stool samples were tested for H. pylori antigen using polyclonal enzyme-linked immnunosorbent assay kits. DNA samples from stool specimens were extracted, followed by PCR for the detection of H. pylori UreA. RESULTS: The results revealed that 18/19 (94.7%) patients were positive for H. pylori infection as detected by Giemsa stain, and 84.2% were positive on the basis of hematoxylin and eosin stain, with a sensitivity and specificity of 88.9% and 100%, respectively. Diagnosis by noninvasive methods, including the serum antibody test, revealed a sensitivity and positive predictive value of 88.9% and 94.2%, respectively, whereas the stool antigen test recorded a sensitivity and positive predictive value of 72.2% and 92.9%, respectively. The stool-PCR test recorded a sensitivity of 72.2% and specificity of 100%. CONCLUSIONS: Among the noninvasive methods for diagnosis of H. pylori infection, the 3 methods used in this study recorded promising results, including good sensitivity, which was the highest in the serum antibody test, whereas the stool-PCR test recorded excellent specificity.

Serum levels of ß-catenin as a potential marker for genotype 4/hepatitis C-associated hepatocellular carcinoma.

The global rising incidence of hepatocellular carcinoma (HCC), which parallels the increase of hepatitis C virus (HCV) prevalence, has sparked a renewed interest in discovering additional HCC serum markers. In this study, we investigated the clinical use of serum E-cadherin, ICAM, MMP-2, VEGF, OPN and ß-catenin as potential diagnostic makers for HCV/genotype 4-associated HCC. Twenty cases of healthy subjects, 11 cases with asymptomatic HCV/genotype 4 carriers (ASC), 28 chronic hepatitis (CH) cases and 32 patients with HCC were enrolled in this study. Serum levels of proteins were measured by a sandwich-enzyme-linked (ELISA) assay. The diagnostic accuracy of each candidate marker was evaluated using receiver-operating characteristic (ROC) curve analysis, reporting the area under the curve (AUC) and its 95% confidence interval (CI). We demonstrated that serum ß-catenin levels were significantly elevated in patients with HCC compared to those with CH, ASC and healthy controls. Among the six studied markers, ß-catenin was also found to be the only marker that can significantly discriminate between patients with HCC and those with CH; therefore, ß-catenin could be considered as a potential marker for early diagnosis of HCV-associated HCC in patients infected with HCV genotype 4.

Serum levels of soluble Fas, soluble tumor necrosis factor-receptor II, interleukin-2 receptor and interleukin-8 as early predictors of hepatocellular carcinoma in Egyptian patients with hepatitis C virus genotype-4.

BACKGROUND: Liver disease progression from chronic hepatitis C virus (HCV) infection to hepatocellular carcinoma (HCC) is associated with an imbalance between T-helper 1 and T-helper 2 cytokines. Evaluation of cytokines as possible candidate biomarkers for prediction of HCC was performed using soluble Fas(sFas), soluble tumor necrosis factor receptor-II (sTNFR-II), interleukin-2 receptor (IL-2R) and interleukin-8 (IL-8). RESULTS: The following patients were recruited: 79 with HCV infection, 30 with HCC, 32 with chronic liver disease associated with elevated liver enzyme levels (with or without cirrhosis) in addition to 17 with chronic HCV with persistent normal alanine aminotransferase levels (PNALT). Nine normal persons negative either for HCV or for hepatitis B virus were included as a control group. All persons were tested for sFas, sTNFR-II, IL-2R and IL-8 in their serum by quantitative ELISA. HCC patients had higher levels of liver enzymes but lower log-HCV titer when compared to the other groups. HCC patients had also significantly higher levels of sFas, sTNFR-II and IL-2R and significantly lower levels of IL-8 when compared to the other groups. Exclusion of HCC among patients having PNALT could be predicted with 90 % sensitivity and 70.6 % specificity when sTNFR-II is [greater than or equal to] 389 pg/ml or IL-8 is < 290 pg/ml. CONCLUSIONS: Serum TNFR-II, IL-2Ralpha and IL-8, may be used as combined markers in HCV-infected cases for patients at high risk of developing HCC; further studies, however, are mandatory to check these findings before their application at the population level.

Disease progression from chronic hepatitis C to cirrhosis and hepatocellular carcinoma is associated with repression of interferon regulatory factor-1.

BACKGROUND/AIM: Infection with hepatitis C virus (HCV) frequently results in a persistent infection, suggesting that it has evolved efficient mechanism(s) for blocking the host cell's innate antiviral response. The immune response to virus infection results in activation or direct induction of the interferon regulatory factors (IRFs), which are a family of proteins involved in the regulation of interferon (IFN) and IFN inducible genes. IRF-3 and IRF-7 have been shown to play an essential role in virus-dependent signaling, whereas IRF-1 is critical for proper IFN-dependent gene expression. This study has been performed to show the expression profile of IRF-1, IRF-3, and IRF-7 in Egyptian patients with HCV-related liver diseases and hepatocellular carcinoma (HCC). MATERIALS AND METHODS: This study included 90 patients, who were positive for HCV infection by reverse transcription PCR, divided into three groups: group I (Gr I) included 30 patients with chronic hepatitis C, group II (Gr II) included 30 patients with liver cirrhosis in addition to group III (Gr III) of 30 patients with HCC. Reverse transcription PCR analysis was performed to determine the expression profile of IRF-1, IRF-3, and IRF-7 genes extracted from the peripheral blood mononuclear cells of those patients. RESULTS: IRF-1expression was significantly higher (P<0.001) in patients of Gr I (86.6%) compared with those in Gr II (46.7%) and Gr III (36.7%), whereas IRF-3 expression was significantly higher (P<0.005) among patients of Gr II (73.3%) in comparison with that in Gr I (50%) and Gr III (36.7%). In contrast, although expression of IRF-7 was higher in Gr II than in the other groups, there was no statistically significant difference (P > 0.05). CONCLUSION: Alterations in IRFs expression might be considered as markers associated with a higher risk of cirrhosis in patients with chronic HCV infection. Expression of IRF-1 and IRF-3 were more prevalent in patients with chronic HCV and cirrhosis, respectively, in comparison with HCC patients. Thus, IRF-1 could be nominated as one of the tumor suppressor factors and could aid in the early detection of HCC.

Impact of SEN virus on immunoblot INNO-LIA HCV reactivity pattern and disease outcome in Egyptian hepatocellular carcinoma patients.

Recently, a novel virus designated SEN virus (SENV), which is thought to be related to posttransfusion hepatitis, was discovered. The aim of the present study was to investigate the prevalence and clinical significance of 2 SENV variants (SENV-D and SENV-H) in patients with hepatocellular carcinoma (HCC) and healthy adults. Also, to investigate the possible effect of SEN virus on the humoral immune response against different proteins of HCV through analyzing reactivity patterns of the confirmatory INNO-LIA HCV Ab III update in relation to SEN viremia. We investigated SEN virus infection in 41 patients with HCC (25 males and 16 females) and twenty healthy blood donors (12 males and 8 females). All samples were taken from the National Cancer Institute, Cairo University. We used semi nested polymerase chain reaction (PCR) amplification to detect SENV-D and SENV-H strains in serum. All patients were tested against HCV antibody by ELISA and HCV viremia by RT-PCR. Furthermore, nineteen patients positive for HCV antibody by EIA (10 positive for SEN DNA and 9 non viremic for SEN) were confirmed in the immunoblot assay. SENV DNA was detected in 68 % (28 of 41) of patients with HCC and in 64 % (21 of 33) HCV-related HCC, in comparison to 5% (1 of 20) healthy blood donor populations. The blood biochemical parameters, and performance status did not differ significantly between the SENV DNA-positive and- negative patients. However, the overall survival rate was 50 % after two years follow up in SENV DNA-positive and 14 % in SENV DNA-negative HCC patients. Reactivity to NS5 and E2 were less (22 % and 44 % of cases) in SENV negative cases, than in SENV positive cases (70 % and 80 % of cases, respectively). In conclusion, SENV DNA seems to be highly prevalent among Egyptian HCC patients. Cross reactivity between SENV proteins and HCV NS5, E2 or the increased immune response in SENV positive cases and consequently the increased reactivity to HCV NS5 and E2 proteins could not be ruled out. Although there was no apparent effect of SENV on biochemical tests, survival rates of SENV DNA-positive HCC patients were higher thannegative cases, which might be due to other factors affecting survival in our Egyptian HCC patients.

Seroprevalence of herpes simplex virus types 1 and 2, Epstein-Barr virus, and cytomegalovirus in children with acute lymphoblastic leukemia in Egypt.

OBJECTIVE: Viral infection, especially caused by herpes viruses, is now recognized as an important cause of morbidity and mortality in immunocompromised cancer patients. This study aimed at studying seroprevalence of 3 herpes viruses Herpes simplex virus types 1 and 2 (HSV 1 and 2), Epstein-Barr virus (EBV), and cytomegalovirus (CMV) in children with acute lymphoblastic leukemia (ALL). METHODS: We conducted this study on 68 newly diagnosed pediatric patients with ALL presented to the Pediatric Oncology Service of National Cancer Institute, Cairo University, Egypt from November 2001 to June 2003. We used enzyme-linked immunosorbent assay in detecting HSV 1 and 2, CMV, EBV antibodies of both types immunoglobulin (Ig) M and IgG detection of DNA for both CMV and EBV by polymerase chain reaction was carried out. RESULTS: High seroprevalence of HSV-1 and 2, CMV and EBV IgG antibodies in both leukemic children and their control was observed (69%, 100%, 83%) and (80%, 100%, 95%). Significantly higher percentage of HSV-1 and 2 IgM or reactivated infection was found among leukemic children 17/68 (25%) compared with normal control 0%. Analysis showed that prevalence of HSV 1 and 2 IgG increased from 18/33 (54%) in children <5 years to 11/13 (77%) in children >10 years, and reactivation of HSV-1 and 2 increased with increasing age from 1/33 (3%) in children <5 years to 4/13 (30%) in children >10 year. This was in contrast to seroprevalence of CMV and EBV IgG which were 100% and 83% in children <5 years. No difference in seroprevalence was found among both gender, and no difference was found in leukemic patients with granulocytopenia. CONCLUSION: The data show a higher exposure to HSV-1 and 2 both primary infections and reactivation among ALL children. Therefore, acyclovir prophylaxis could be highly effective for seropositive leukemic patients who are undergoing induction chemotherapy.

The role of cyclins and cyclins inhibitors in the multistep process of HPV-associated cervical carcinoma.

BACKGROUND: Human papillomavirus (HPV) types 16 and 18 are associated with cervical carcinogenesis. This is possibly achieved through an interaction between HPV oncogenic proteins and some cell cycle regulatory genes. However, the exact pathogenetic mechanisms are not well defined yet. METHODS: We investigated 110 subjects (43 invasive squamous cell carcinoma (ISCC), 38 CIN III, 11 CIN II, 18 CIN I) confirmed to be positive for HPV16 and/or 18 as well as 20 normal cervical tissue (NCT) samples for abnormal expression of cyclin D1, cyclin E, CDK4, cyclin inhibitors (p21 (waf), p27, p16 (INK4A)) and Ki-67 using immunohistochemistry and differential PCR techniques. RESULTS: There was a significant increase in the expression of Ki-67, cyclin E, CDK4, p16 (INK4A) (p=0.003, 0.001, 0.001) and a significant decrease in p27 (Kip1) from NCT to ISCC (p=0.003). There was a significant correlation between altered expression of p27 (KIP1) and p16(INK4A) (p<0.001), cyclin D1 and CDK4 (p=0.001), cyclin E and p27 (Kip1) (p=0.011) in all studied groups. In ISCC, there was significant relationship between standard clinicopathological prognostic factors and high Ki-67 index , increased cyclin D1 and cyclin E, reduced p27 (Kip1) and p21 (waf). CONCLUSION: 1) Aberrations involving p27 (KIP1), cyclin E, CDK4 and p16 (INK4A) are considered early events in HPV 16 and 18-associated cervical carcinogenesis (CINI & II), whereas cyclin D1 aberrations are late events (CINIII & ISCC) 2) Immunohistochemical tests for p16 (INK4A) and cyclin E could help in early diagnosis of cervical carcinoma 3) Only FIGO stage, cyclin D1, p27 (Kip1) and Ki-67 are independent prognostic factors that might help in predicting outcome of cervical cancer patients.

Role of human papilloma virus (HPV) in common and genital warts and its relation to P53 expression.

BACKGROUND AND AIM: Human papilloma viruses (HPVs) are small DNA tumor viruses that infect epithelial tissues and cause warts. One of the viral genes responsible for HPV's oncogenic activity is E6 which is known to inactivate the cellular p53 tumor suppressor gene. We aim to detect the presence of HPV infection and its different types in human warts, and to identify the relation between HPV and p53 expression in skin and genital lesions. PATIENTS AND METHODS: We studied markers of HPV infection in overall of 30 patients (20 with common warts, and 10 with genital warts). Also, 30 normal skin samples were taken from each patient as a normal control. Detection of HPV was done using polymerase chain reaction (PCR), and HPV typing was performed using LiPA (Line immuno Probe Assay). In addition, all skin lesions were examined by immunohistochemistry for p53 expression. RESULTS: In patients with common warts, HPV DNA was found in 4/20 (20%) of cases which was of HPV types 11, 31, 6, 33 (p=0.28). Also, P53 expression was found in 4/20 (20%) of cases (p=0.26). No single patient showed reactivity of both HPV and p53 expression. In patients with genital warts, however, HPV DNA was found in 6/10 (60%) of cases. Of these, 5 cases were positive for HPV type 6 and one case had HPV type 11. Three patients (30%) were positive for p53, and two of them (66%) were positive for both HPV and p53. In the normal skin control, 2/30 (6.6%) were positive for HPV DNA which were of types 5, and 31. CONCLUSIONS: We conclude that; (1) Prevalence rate of HPV infection in warts is higher than those of normal control group, and Egyptian patients with genital warts had higher prevalence rate of HPV than those with common warts, (2) In Egypt, HPV types 6, and 11 are the most prevalent genotypes associated with genital warts and HPV types 6, 11, 31, and 33 are associated with common warts, (3) There was no definite relation between p53 expression and HPV detection, (4) Also, there was no association between the different HPV types and p53 detection in these non-cancerous lesions.

p53 mutation in HCV-genotype-4 associated hepatocellular carcinoma in Egyptian patients.

HCV-associated hepatocellular carcinoma (HCC) is a common neoplasm in Egypt where genotype-4 is prevalent. In the present study the incidence and pattern of p53 mutations was assessed in relation to HCV-genotype- 4 in Egyptian HCC patients. We investigated 25 HCV positive HCCs for p53 mutations/overexpression in relation to HCV-NS3 by immunohistochemistry, SSCP and sequencing. Genotyping was done using LiPA-II and TRUGENE 5' NC' sequencing kit. Results were correlated to standard clinicopathologic prognostic factors for HCC. Thirteen cases showed p53 overexpression, and 10 showed p53 mutation (13 mutations) by sequencing (72% concordance). The highest mutation rate was in exons 6 and 7 (30%) followed by exons 5 and 8 (20%). Mutations included 3 transitions, 5 transversions, 3 deletions, and 2 insertions. All exon 7 mutations were at codon 249 specific for AFB1 (AGG-->AGT, Arg-->Ser) and codon 248 specific for vinyl chloride contamination (CGG-->TGG, Arg-->Trp). Other mutations reported are novel. Immunostaining for HCV NS3 was detected in 19 cases independent of p53 mutation. p53 aberrations were significantly associated with poor prognostic factors for HCC. However, no specific pattern for p53 mutations was observed in HCV genotype 4-associated HCC and no significant relation between p53 mutations, HCV-NS3 expressions or any HCV sub-genotype-4 sequence.

Cytokine profile in Egyptian hepatitis C virus genotype-4 in relation to liver disease progression.

AIM: To observe the imbalance between T helper cell Th1 and Th2 cytokines in several chronic hepatitis disease at different stages of disease progression. METHODS: We measured the cytokine levels of Th1 (IL-2 and IL-2R), Th2 (IL-10) and the pro-inflammatory cytokines (IL-6 and IL-6R and TNF and TNF-RI and II) by the ELISA technique in the sera of 33 hepatocellular carcinoma (HCC) patients and 20 chronic liver disease (CLD) patients. In addition, 20 asymptomatic hepatitis C virus carriers and 20 healthy subjects negative for hepatitis C virus(HCV) markers served as controls. RESULTS: Anti-HCV antibodies were found to be positive in 94% of HCC cases and 75% of CLD cases. On the other hand, HCV viremia was detected using RT-PCR in 67% of HCC cases and 65% of CLD cases. HBsAg was positive in 9% of HCC cases and 30% of CLD cases. Also bilharzial-Ab was positive in 55% of HCC cases, 65% of CLD cases and in 70% of asymptomatic carriers (ASC). HCC patients had significantly higher values of IL-2R, TNF-RII (P<0.001), and TNF-RI (P>0.05), but lower TNFalpha (P<0.001) and IL-6 (P = 0.032) in comparison to ASC. But, in comparison to non-cancer controls, HCC patients had higher values of IL-2R, IL-6R, TNF-RI and TNF-RII, but lower TNF-alpha (P<0.001). CLD patients had higher IL-2R, TNF-RI, and TNF-RII (P<0.001) than ASC. But, in comparison to non-cancer controls, CLD patients had higher values of IL-2R, TNF-RI and TNF-RII, but lower TNF-alpha (P<0.001). IL-10 was higher (though not significantly) in HCC and CLD patients than in symptomatic carriers and non-cancer controls. CONCLUSION: Liver disease progression from CLD to HCC due to HCV genotype-4 infection is associated with an imbalance between Th1 and Th2 cytokines. IL-2R, TNF-RI, and TNF-RII could be used as potential markers.