Influenza a virus regulates interferon signaling and its associated genes; MxA and STAT3 by cellular miR-141 to ensure viral replication.

The antiviral response against influenza A virus (IAV) infection includes the induction of the interferon (IFN) signaling pathway, including activation of the STATs protein family. Subsequently, antiviral myxovirus resistance (MxA) protein and other interferon-stimulated genes control virus replication; however, the molecular interaction of viral-mediated IFN signaling needs more investigation. Host microRNAs (miRNAs) are small non-coding molecules that posttranscriptionally regulate gene expression. Here, we sought to investigate the possible involvement of miR-141 in IAV-mediated IFN signaling. Accordingly, the microarray analysis of A549 cells transfected with precursor miR-141 (pre-miR-141) was used to capture the potentially regulated genes in response to miR-141 overexpression independent of IAV infection. The downregulation of targeted genes by miR-141, in addition to viral gene expression, was investigated by quantitative real-time PCR, western blot analysis, and flow cytometric assay. Our findings showed a significant upregulation of miR-141 in infected A549 cells with different strains of IAV. Notably, IAV replication was firmly interrupted in cells transfected with the miR-141 inhibitor. While its replication significantly increased in cells transfected with pre-miR-141 confirming the crucial role of miRNA-141 in supporting virus replication. Interestingly, the microarray data of miR-141 transduced A549 cells showed many downregulated genes, including MxA, STAT3, IFI27, and LAMP3. The expression profile of MxA and STAT3 was significantly depleted in infected cells transfected with the pre-miR-141, while their expression was restored in infected cells transfected with the miR-141 inhibitor. Unlike interleukin 6 (IL-6), the production of IFN-ß markedly decreased in infected cells that transfected with pre-miR-141, while it significantly elevated in infected cells transfected with miR-141 inhibitor. These data provide evidence for the crucial role of miR-141 in regulating the antiviral gene expression induced by IFN and IL-6 signaling during IAV infection to ensure virus replication.

Crosstalk between miR-146a and pro-inflammatory cytokines in patients with systemic lupus erythematosus.

microRNA-146a (miR-146a) plays an essential role in immune anomalies and organ injury of systemic lupus erythematosus (SLE) by regulating the disease's inflammation and complications. Here, we analyzed the expression of miR-146a in SLE and a panel of pro-inflammatory cytokines (IL-1, IL-6, IL-8, IL-17, and TNF-α). Association between all measured parameters and the disease's clinical manifestation and response to treatment was monitored. Our study populations were 113 SLE patients and 104 healthy volunteers. miR-146a expression in peripheral blood mononuclear cells (PBMCs) was measured by quantitative real-time PCR (RT-qPCR). The content of the plasma cytokines (IL-1ß, IL-6, IL-8, IL-17, and TNF-α) was detected by enzyme-linked immunosorbent assay (ELISA). Compared with healthy controls, miR-146a expression was significantly increased (p < 0.05) in lupus patients. The analysis of the receiver operator characteristic curve (ROC) of miR-146a showed 91% sensitivity and 70% specificity. IL-1ß, IL-6, and IL-17 cytokines were significantly increased (p < 0.001), while IL-8 and TNF-α were significantly decreased (p < 0.001) in SLE patients against controls. The expression of miR-146a and TNF-α was upregulated considerably in SLE patients with severe disease activity. miR-146a expression was positively correlated with IL-6. Our results pointed to the elevation of miR-146a as a trade marker of SLE patients. Reduction of IL-8 and TNF-α in combination with an elevation of IL-1ß, IL-6, and IL-17 might refer to miR-146a's dual effect in controlling inflammation in lupus. Although we shed some light on the role of miR-146a in SLE, further study is recommended to improve our results.

mir-146a genetic polymorphisms in systemic lupus erythematosus patients: Correlation with disease manifestations.

This study aimed to investigate the genetic polymorphisms of miR-146a SNPs (rs2910164, rs57095329, and rs2431697) in systemic lupus erythematosus (SLE) patients and their association with clinical manifestations. The implication of SNPs on miR-146a expression level was also evaluated. SLE patients (113) and healthy controls (104) were registered in this study. The miR-146a SNPs were genotyped by polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP). Quantitative real-time PCR was used to measure the miR-146a expression in peripheral blood mononuclear cells (PBMCs). Our results showed that the genotype frequency of miR-146a SNPs didn't deviate significantly from the Hardy-Weinberg equilibrium (HWE). The AG genotype and G allele of miR-146a (rs57095329 A/G) might be considered a risk factor for the disease (OR = 2.27; CI: 0.78-6.57 and OR: 2.35; CI: 0.79-6.92 for AG genotype and G allele, respectively). Although, no statistical significance in the distribution of miR-146a SNPs (rs2910164, rs57095329, and rs2431697) was found, indicating the lack of association between the three SNPs and SLE susceptibility. Significantly, the higher frequency of the AA genotype of miR-146a (rs57095329) was associated with pancytopenia (P < 0.05), while the CT genotype of miR-146a (rs2431697) was associated (P < 0.05) with the antiphospholipid syndrome (APS). SLE patients had significantly higher levels of miR-146a compared to controls (P < 0.05). Elevation of miR-146a was independent of any SNP genotypes. In conclusion, this pilot study shows no association between miR-146a SNPs in our population group and susceptibility to lupus. Studies concerning other miRNAs in larger sample sizes are essential for a better understanding of their role in susceptibility to SLE disease.

Association of MTHFR and TYMS gene polymorphisms with the susceptibility to HCC in Egyptian HCV cirrhotic patients.

Identification of host genetic factors influencing the risk of developing hepatocellular carcinoma (HCC) in patients with chronic hepatitis C virus (HCV) infection may help to refine patients' selection to benefit from specific preventative measures and/or adapted screening policies. Thus, this study aimed to investigate the association of MTHFR c.677C > T and c.1298A > C in addition to TYMS 3'-UTR 6-bp ins/del polymorphisms with the susceptibility to HCV-related HCC in an Egyptian population. Polymerase chain reaction-restriction fragment length polymorphism was performed to genotype the polymorphisms in 194 HCV-infected patients subdivided into liver cirrhotic (LC, n = 104) and HCC (n = 90) patients as well as 100 healthy subjects. In healthy controls, the MTHFR c.677C > T polymorphism under the homozygous and recessive models (p = 0.005) and the c.1298A > C polymorphism under all the tested genetic models (p-values range from < 0.001 to 0.007) were associated with an increased risk of HCC. In LC patients, the MTHFR c.677C > T polymorphism under the homozygous, dominant, and recessive models (p-values range from 0.001 to 0.007), as well as MTHFR c.1298A > C under the homozygous model only (p = 0.014), increased the susceptibility to HCC. The C/C and T/C haplotypes of MTHFR c.677C > T and MTHFR c.1298A > C polymorphisms were contributed to an increased risk of healthy subjects to develop HCC (p-values range from < 0.001 to 0.015), while only the T/C haplotype was associated with the progression of HCC in LC patients (p = 0.001). In conclusion, MTHFR c.677C > T and c.1298A > C in addition to their haplotypes may contribute to the development of HCV-related HCC in an Egyptian population. These findings may aid in the early diagnosis and management of HCC.

Guava flavonoid glycosides prevent influenza A virus infection via rescue of P53 activity.

Influenza is a highly infectious disease caused by three types of viruses, including influenza A virus (IAV), influenza B virus, and, rarely, influenza C virus. IAV is a major, global public health threat, causing approximately 500 000 deaths per year worldwide. The new strains of IAV have emerged due to a mutation called antigenic shift, which results in a new subtype of the virus that shows resistance to common antiviral drugs. Here, guava and lemon extracts, including green leaves and flowers, were investigated for their activity against IAV replication in human A549 cells. Concomitantly, the cytotoxicity of a potent extract on host-cell multiplication was assessed. Our results reveal that guava extracts inhibit IAV replication, indicated by viral nucleoprotein expression profile and traditional plaque assay. Interestingly, treatment with guava extract inactivates Akt protein kinase and stimulates the pro-apoptotic protein P53, at early stages of infection. Furthermore, purified guava flavonoid glycosides (GFGs) show competitive inhibition of IAV-virus replication via early regulation of IL-1ß and IL-8 in association with P53 gene expression. The docking analysis of GFGs and the protein structure of upstream targets for the Akt signaling pathway indicates a sufficient interaction and stabilization with Gbr2 protein. These data indicate that treatment with GFGs disturbs IAV replication via activation of P53 and its apoptotic related factors after infection. Collectively, these data show that targeting of essential host kinases that are involved in the replication cycle of IAV and rescue of P53 activity by GFGs could represent a new strategy to eradicate IAV.

Prevalence and Prognostic Impact of CEBPA Gene Mutation (Simplified Assay Technique) in Egyptian Acute Myeloid Leukemia Patients with Normal Cytogenetics.

Mutations of the CCAAT/enhancer binding protein alpha (CEBPA) gene have been associated with a favorable outcome in patients with acute myeloid leukemia (AML), especially in those with a normal cytogenetics. However, few studies were done on Egyptian AML patients and none of them look for easier and less expensive method for CEBPA mutation screening. This study is aimed to investigate the prevalence of CEBPA mutations and its clinical and prognostic impact in Egyptian patients with cytogenetically normal AML (CN-AML). This was done using fragment analysis to assess this method as a cheaper and less laborious screening method compared to sequencing. Fluorescent PCR was done to amplify CEBPA gene in DNA extracted from 40 CN-AML patients. This was followed by fragment analysis of post-PCR products using GeneMapper software for detection of CEBPA mutations. CEBPA gene mutations were found in 7/40 CN-AML patients (17.5 %) and it was significantly associated with lower LDH levels (p = 0.039). All patients with CEBPA mutations achieved clinical remission and none of them showed refractoriness, relapsed, or died by the end of the 2 years study period. Furthermore, those patients demonstrate significantly longer overall and disease free survival than those with wild type CEBPA gene (p = 0.001 and 0.004 respectively). CEBPA mutation has a favorable prognostic impact in CN-AML. Fragment analysis is a good, lees laborious and cheaper method that can be used for CEBPA mutation screening in patients with CN-AML.

Circulating pro- and anti-angiogenic mediators in patients infected with hepatitis C at different stages of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a hypervascular tumor characterized by neovascularization. The objective of the current study was to determine circulating proangiogenic [vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), and tumor necrosis factor (TNF-α)] and antiangiogenic [IL-4, IL-12, interferon gamma-induced protein 10 (IP-10), and angiostatin] factors in Egyptian patients with different stages of HCC. Enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of these mediators in plasma of 135 HCC patients (57 Child-Pugh A, 24 Child-Pugh B, and 54 Child-Pugh C stage) and 50 healthy subjects. Results showed a significant increase in plasma levels of VEGF (P < 0.001), PDGF (P < 0.001), TNF-α (P < 0.01), angiostatin (P < 0.01), and IP-10 (P < 0.001) and a significant reduction in IL-12 (P < 0.001) in HCC patients in relation to normal controls. Classifying HCC patients based on their Child-Pugh's score revealed that the maximum production of proangiogenic mediators (VEGF and TNF-α) was present in HCC patients with Child-Pugh C score which coincides with maximum reduction in antiangiogenic mediators (IL-4, IL-12, and angiostatin). Taken together, these results indicated that the determination of these factors in different Child-Pugh's scores of HCC might be an important guide in clinical decision making regarding therapy and outcome.

Pattern of matrix metalloproteinases-9, P53 and BCL-2 proteins in Egyptian patients with pulmonary Mycobacterium tuberculosis.

Matrix metalloproteinases (MMPs) constitute a large family of enzymes that degrade extracellular matrix proteins (ECM). MMPs are implicated in different pathological conditions such as cancer. Bcl-2 and P53 are key controllers of programmed cell death (PCD) or apoptosis. The aim of the present study was to determine the MMP-9, P53 and Bcl-2 levels in Egyptian patients with Mycobacterium tuberculosis (MTB) (Group I) compared with healthy control individuals (Group II). The concentrations of serum MMP-9 were determined quantitatively using enzyme immunoassay (EIA). P53 and Bcl-2 levels were assayed by flow cytometric analysis using specific monoclones. MMP-9 level was significantly higher in MTB patients compared with healthy control. Similarly, P53 and Bcl-2 levels were increased in MTB patients compared with healthy ones. These data reflect the alteration of MMP-9 level during the course of MTB infection, accompanied with apparent dysregulation of cellular apoptosis as indicated by P53 and Bcl-2 over-expression.

Helicobacter pylori and Hepatitis C Virus Coinfection in Egyptian Patients.

INTRODUCTION: Chronic hepatitis C virus (HCV) infection is a leading cause of end-stage liver disease worldwide. It has been shown that Helicobacter pylori (H. pylori) plays an important role in chronic gastritis, peptic ulcer disease and gastric malignancies, and its eradication has been advocated. The association between H. pylori infection and liver cirrhosis in patients with hepatitis C virus has been documented in different parts of the world; nevertheless, no conclusive data is available in Egypt. MATERIALS AND METHODS: In the present study, the status of H. pylori infection was sought in 90 patients with chronic HCV infection and in 66 HCV-free healthy controls. RESULTS: The study showed that the H. pylori positivity was increased significantly (P = 0.03) in the HCV-infected patients when compared to that in healthy controls, where H. pylori infection was found in 50 (55.6%) out of 90 of the HCV-infected patients versus 26 (39.4%) out of 66 of the healthy controls. In HCV-infected patients, the prevalence of H. pylori infection was increased significantly (P = 0.04) from chronic active hepatitis to cirrhosis. H. pylori infection was present in 6/18 (33.3%), 10/21 (47.6%), 16/27 (59.3%), 18/24 (75.0%) patients with chronic active hepatitis, Child-Pugh score A, Child-Pugh score B and Child-Pugh score C, respectively. More importantly, the prevalence of H. pylori infection in HCV-infected patients was increased very significantly (P = 0.003) with increasing Meld (model for end-stage liver disease) score. The prevalence of H. pylori was documented in 9/28 (32.1%) patients with Meld score >10 and in 41/62 (66.1%) patients with Meld score >10. CONCLUSION: It may be stated that our results collectively reflect a remarkable increase in H. pylori prevalence with advancing hepatic lesions, and the eradication treatment may prove beneficial in those patients with chronic hepatitis C.

Sperm head defects and disturbances in spermatozoal chromatin and DNA integrities in idiopathic infertile subjects: association with cigarette smoking.

OBJECTIVES: To evaluate sperm chromatin and DNA integrities in idiopathic infertile men and determine the possible association(s) of cigarette smoking on oxidative stress markers, antioxidant capacity and semen quality. SUBJECTS AND METHODS: Semen samples from men referring to the andrology laboratory were categorized into 3 groups: fertile non-smokers (n=16), infertile non-smokers (n=36), and infertile smokers (n=34). Semen analysis was performed according to WHO criteria. The percentage of sperm DNA fragmentation index (%DFI) and the percentage of sperm with abnormally high DNA stainability (HDS%; immature spermatozoa) were determined by SCSA using the metachromatic properties of acridine orange. Lipid peroxidation, superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH) levels in seminal plasma and spermatozoa were measured by spectrophotometric assays. RESULTS: The classical semen parameters were negatively correlated with lipid peroxidation in spermatozoa; motility and morphology were negatively correlated with %DFI (p<0.05). HDS% was also negatively correlated with above markers except for morphology (r=-0.352, p=0.081). DFI% and HDS% were significantly higher in the infertile smokers group than in infertile non-smokers (p=0.032; p=0.001 respectively). Cigarette smoking was significantly associated with DFI%, HDS%, TBARS and the fraction of "round-headed" sperm (r=0.796, p=0.0001; r=0.371, p=0.033; r=0.606, r=0.591, p=0.001 respectively), and decreased SOD levels (r=-0.545). CONCLUSION: DFI%, HDS% and round-head sperms are increased in idiopathic infertile men; this increase is associated with cigarette smoking. These defects may be attributed to increased oxidative stress and insufficient scavenging antioxidant enzymes in the seminal fluid of infertile patients.

Value of serum anti-p53 antibodies as a prognostic factor in Egyptian patients with hepatocellular carcinoma.

OBJECTIVE: p53 antigen is an oncoprotective antigen and when damaged, leads to production of anti-p53 and also predisposes to various cancers, including hepatocellular carcinoma (HCC). Serum anti-p53 has been proven to have a prognostic value in patients with HCC. The objective of this study was to determine the prevalence and prognostic utility of serum anti-p53 in Egyptian patients with HCC. METHODS: Forty one patients with HCC, 26 patients with liver cirrhosis and 29 healthy controls were included in this study. For all the studied groups, we studied the clinical data, abdominal ultrasound (US) findings, biochemical liver function tests, serum alpha-fetoprotein (AFP) levels detected by enzyme immunoassay (EIA) kit and anti-p53 antibody levels by a modified enzyme-linked immunosorbent assay (ELISA). The severity of liver disease was assessed by Child-Pugh and MELD scores. Tumor characteristics were detected by (US) with or without computed tomography (CT) scan. These characteristics included tumor size, number and site. Tumor staging was done using Okuda, Cancer Liver Italian Program (CLIP) and Tokyo staging systems. Also, the overall survival of patients with HCC with reference to p53 antibody level was studied. RESULTS: The mean age of HCC patients was 57.95+/-8.41. There was a male predominance among HCC patients with male-to-female ratio of 3.6:1. Anti-p53 antibodies were detected in the sera of 68.3% of HCC patients, 50% of liver cirrhosis patients and 17.2% of healthy control subjects. The data showed that HCC patients had a significantly higher mean anti-p53 antibody values (p=0.0001), than both liver cirrhosis patients and healthy control groups. Our results revealed that anti-p53 has a positive significant correlation with AFP (p=0.002), severity of liver disease [Child Pugh score (p=0.02) and MELD score (p=0.0003)], tumor size (p<0.0001), tumor number (p=0.003) and tumor staging systems [Okuda (p=0.04), CLIP (p=0.006) and Tokyo (p<0.0001)]. Also, our results revealed that serum anti-p53 antibodies had a significant association with overall survival of patients with HCC (p=0.019) with a shorter survival time in anti-p53 positive status patients and with higher anti-p53 antibody levels within 19 months follow up. CONCLUSION: The detection of anti-p53 antibodies may be suitable for assessing the prognosis of HCC patients. The higher percentage of positivity of anti-p53 antibodies in Egyptian control subjects than reported elsewhere needs further thorough investigation.

Rapid and simple detection of a mycobacterium circulating antigen in serum of pulmonary tuberculosis patients by using a monoclonal antibody and Fast-Dot-ELISA.

OBJECTIVES: Several immunoassays have been established for detection of Mycobacterium tuberculosis (MTB) antigens in serum, sputum and cerebrospinal fluid of tuberculous patients using polyclonal or monoclonal antibodies raised against different mycobacterium antigens. Some of these assays display both high sensitivity and specificity for the detection of these antigens. However, these assays require special and highly expensive equipment and the procedures require long periods for their completion. Thus, the rationale of this study was to establish and evaluate Fast-Dot-Enzyme-Linked Immunosorbent Assay (FD-ELISA) as a fast, cheap and field applicable assay for detection of mycobacterium antigen in serum of patients with pulmonary TB. DESIGNS AND METHODS: This study included three groups: group I: 175 tuberculous patients with pulmonary TB proves with sputum Ziehl-Neelsen (ZN) for acid-fast bacilli and sputum culture (all cases were culture positive for MTB); Group II: 65 patients with diseases other than pulmonary TB as bronchial carcinoma (17 patients), bronchial asthma (29 patients) and chronic obstructive pulmonary disease (19 patients); group III: 50 healthy individuals. Groups II and III served as negative control groups. The target mycobacterium antigen was identified in both crude mycobacterium antigens extract and serum of patients with pulmonary TB, using western blotting technique and anti-TB monoclonal antibody (TB20-mAb) and then it was estimated in the serum samples of all studied groups as an index of tuberculosis, using a newly developed FD-ELISA. RESULTS: The target mycobacterium antigen was identified at 20 kDa molecular mass in crude mycobacterium antigens extract as well as in serum of patients with pulmonary TB. The developed FD-ELISA detected the mycobacterium antigen in the sera of 159 out of 175 pulmonary TB patients with a sensitivity of 90.8% and 93.0% positive predictive value (PPV). In addition, it identified 12 false weakly positive cases out of 115 samples of negative control groups (7 out of 65 non-TB patients and 5 out of 50 healthy individuals) with a specificity of 89.6% and 86.6% negative predictive value (NPV). Standardization of the FD-ELISA using a serial dilution of the purified mycobacterium antigen indicated that the assay was able to detect 1.8 microg/ml as a lowest detectable antigen concentration. CONCLUSIONS: The newly developed FD-ELISA is a simple, rapid and highly sensitive assay for detection of mycobacterium antigen in patients with pulmonary TB. Moreover, all steps were performed at room temperature and without the need to use expensive equipment, and this may enhance the application of this assay in tuberculosis screening programs. Further study is needed for confirmation of FD-ELISA reproducibility in light infected pulmonary TB patients and in a large population.

Pro-inflammatory and anti-inflammatory cytokines profile in rheumatoid arthritis patients.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by severe joint deformities due to bony erosions and tendon damage. Cytokines are protein mediators of inflammation and are produced as a result of the activation of various cellular reactions. They are the final mediators and/or regulators of the inflammatory process. Cytokines such as TNF-alpha and IL-6, play key roles in driving the inflammation and synovial cell proliferation that characterize rheumatoid arthritis and joint destruction. Sera from 58 RA patients were analyzed for TNF-alpha, IL-6, IL-10, TGF-beta, sTNF-R 1 and sTNF-R2 using ELISA. The proinflammatory cytokines TNF-alpha and IL-6 were significantly elevated in RA patients, while TGF-beta, an immunomodulatory cytokine, was elevated in control individuals. Assays of TNF receptors, sTNF-R1 and sTNF-R2, were noted to be significantly elevated in RA patients when compared to control. Our data indicate that local production of cytokine inhibitors is capable of diminishing cytokine and disease activity thereby may improve signs, symptoms and quality of life for patients with RA.

Utility of slot-blot-ELISA as a new, fast, and sensitive immunoassay for detection of carcinoembryonic antigen in the urine samples of patients with various gastrointestinal malignancies.

Carcinoembryonic antigen (CEA) is the most widely used clinical tumor marker. CEA immunoassay has found acceptance as a diagnostic adjunct in clinical diagnosis of gastrointestinal tumors (GIT). Several immunoassays have been established for detection of CEA in plasma, serum, tissue, feces, and urine of cancer patients using polyclonal or monoclonal antibodies raised against CEA. Some of these assays display both high sensitivity and specificity for the detection of CEA. However, these assays require special and highly expensive equipment and the procedures require long periods for their completion. In the present study, we established a Slot-Blot Enzyme Linked Immunosorbent Assay (SB-ELISA), based on anti-CEA monoclonal antibody (CEA-mAb), as a new, simple, fast, cheap, and non-invasive immunodiagnostic technique for detection of CEA in the urine of GIT patients. Urine and serum samples were collected from 248 GIT patients (58 with pancreatic cancer, 20 with hepatoma, 23 with ampullary carcinoma, 15 with hilar cholangiocarcinoma, 28 with gastric cancer, 14 with esophageal cancer, and 90 with colorectal cancer). Moreover, urine and serum samples were collected from 50 healthy individuals to serve as negative controls. The traditional ELISA technique was used for determination of CEA in the sera of GIT patients using anti-CEA monoclonal antibody. A comparison between the results of both techniques (ELISA and SB-ELISA) was carried out. The traditional ELISA detected CEA in the sera of 154 out of 248 GIT patients with a sensitivity of 59.8%, 51.7% positive predictive value (PPV) and 75.37% negative predictive value (NPV). In addition, it identified 15 false positive cases out of 50 healthy individuals with a specificity of 70%. The urinary CEA was identified by a Western blotting technique and CEA-mAb at a molecular mass of 180 Kda. The developed SB-ELISA showed higher sensitivity, specificity, PPV, and NPV (70.1%, 78%, 62.4%, and 82.13%, respectively) for detection of CEA in the urine of GIT patients. The semi-quantitative SB-ELISA showed a higher overall efficiency of 72.8% versus 63.4% in the case of the quantitative ELISA, for detection of CEA. In conclusion, SB-ELISA is more efficient for detection of CEA in gastrointestinal tumors. It is a simple, rapid, non-invasive, and sensitive assay. Moreover, all steps of the SB-ELISA are performed at room temperature, without the use of expensive equipment; this may enhance the application of this assay in field studies and mass screening programs.

Serum laminin assayed by Slot-Blot-ELISA in patients with combined viral hepatitis C and schistosomiasis.

OBJECTIVES: Hepatic schistosomiasis and chronic hepatitis C virus (HCV) are the most prevalent agents causing hepatic fibrosis in humans. Laminin (LA) has been related to liver fibrosis and subsequent development of portal hypertension in chronic liver disease. There are no available data describing the pattern of laminin in combined HCV and schistosoma-infected patients, thus the rationale of this study was to assess the serum LA as an index of liver fibrosis in patients with schistosomiasis and/or chronic viral hepatitis C and to evaluate a developed Slot-Blot Enzyme-Linked Immunosorbant Assay (Slot-Blot-ELISA) as a method of estimation. DESIGNS AND METHODS: This study included four groups: group I included 34 patients with schistosomiasis, group II included 58 patients infected with HCV, group III included 68 patients with combined chronic viral hepatitis C and schistosomiasis and group IV included 50 healthy individuals who served as a control group. Serum LA was measured in the different groups quantitatively by ELISA and semi-quantitatively by Slot-Blot-ELISA. RESULTS: Significantly higher serum LA concentrations measured by ELISA were found in patients with combined chronic viral hepatitis C and schistosomiasis than in patients with either chronic HCV (P = 0.005) or schistosomiasis (P < 0.001) alone. Serum LA was significantly higher in the patient groups than the control group (P < 0.001). Serum LA concentration was positively correlated with fibrosis grading scores. Semi-quantitative results of serum LA using the developed SB-ELISA were found to have approximately the same power of ELISA results in different groups. The overall sensitivity, specificity, positive predictive value, negative predictive value and efficiency of ELISA for estimation of serum LA were 85.6%, 84.0%, 94.5%, 64.6% and 90%, respectively and for SB-ELISA were 87.5%, 82.0%, 94%, 67.2% and 88%, respectively. CONCLUSIONS: Serum LA was significantly increased in patients coinfected with HCV and Schistosoma mansoni. The newly developed Slot-Blot-ELISA is a simple, rapid and highly sensitive assay for detection of LA in hepatic fibrosis. Moreover, all steps were performed at room temperature without the need to use expensive equipment, and this may enhance the application of this assay in screening programs. Further study is warranted for confirmation of SB-ELISA reproducibility in a large population.

Relationship of Helicobacter pylori to Bcl-2 family expression, DNA content, and pathological characteristics of gastric cancer.

BACKGROUND: Despite the fact that the association of Helicobacter pylori with an increased risk of gastric cancer has been well documented, the exact mechanisms of this association have not been fully elucidated. Scarce data on H. pylori infection and its relationship with the different pathological characteristics are available in Egypt. AIM OF THE STUDY: The rationale of the present study was to determine the prevalence of H. pylori in a group of gastric cancer patients and to analyze the relationship between H. pylori infection with the different pathological characteristics including the types of gastric cancer and tumor location within the stomach, in addition, to investigate the Bcl-2 and Bax expressions along with DNA flow cytometric analysis in the gastric cancer patients with and without H. pylori infection. METHODS: Samples were obtained from 66 consecutive patients with gastric cancer (46 males and 20 females). The youngest patient was 20 yr old, the oldest 76 yr with mean age of 52.8 yr. The samples were subjected for histopathological characterization, H. pylori detection, DNA flow cytometric analysis, and Bcl-2 and Bax expressions detection, in addition to apoptosis analysis. RESULTS: The obtained results showed that the H. pylori infection was found in 38/66 (57.6%) [Odds ratio=1.357 with 95% confidence interval (CI) 0.84-2.2]. There was a statistical significance for Bcl-2, Bax, and apoptosis with H. pylori status (p = 0.009, 0.008, 0.032, respectively). On the other hand, There was a statistical significance for H. pylori infection with the disease grade (p = 0.015) and lymph node metastasis (p = 0.05). No statistical significance was found between H. pylori status with the patients' age, gender, tumor site, tumor type, depth of invasion, and stromal reaction. CONCLUSIONS: These data may indicate that the H. pylori infection not only contributes in the disease formation through the apoptosis dysregulation but also takes a part in the disease dissemination and progression. In addition, it may reflect a biologic, pathogenic, and ethnic background affecting the relationship of H. pylori infection to gastric cancer in the Egyptian patients. A high rate of smoking in Egypt and the diet are important factors that may affect such background. Further studies are warranted.

Different cytokine patterns in patients coinfected with hepatitis C virus and Schistosoma mansoni.

Schistosoma mansoni (S. mansoni) and hepatitis C virus (HCV) coinfection is common in Egypt and other developing countries. Patients coinfected with HCV and schistosomiasis exhibit a unique clinical, virological and histological pattern manifested by viral persistence with high HCV RNA titers as well as higher necroinflammatory and fibrosis scores in their liver biopsy samples. Dual infections of schistosomiasis and viral infections display significant influences on host immune reactions including cytokine shift pattern alteration, cytotoxic T lymphocyte response and other impaired immunologic functions with diminished capacity to clear the virus. We investigated the cytokine pattern against HCV and S. mansoni antigens in patients coinfected with HCV and S. mansoni and compared them with responses in patients infected with HCV or S. mansoni alone. This study included 4 groups; (Gr I) included 20 patients infected with chronic HCV, their sera were reactive for anti-HCV antibodies, samples were verified for RNA detection to identify those who have viremia. (Gr II) included 15 patients infected with schistosomiasis alone, they were subjected to detection of S. mansoni ova in stool, rectal snip or serological test. (Gr III) included 20 patients with chronic HCV and schistosomiasis coinfection, which were diagnosed by the above-mentioned criteria. (Gr IV) included 15 healthy individuals, who were matched for age and sex and have no evidence of liver diseases served as control subjects. The results showed that a highly significant increase in serum IFN-gamma and IL-18 levels in patients infected with HCV alone compared with the other patient groups and control. On the other hand, a highly significant increase was found in serum IL-4 and IL-10 levels in coinfected patients and patients with schistosomiasis alone compared with the control but a significant increase was found in the two groups compared with HCV patients. A significant increase in serum IL-4 and IL-10 were also found in HCV patients compared with the control. In conclusions, our data showed that coinfected patients have dominant Th2 cytokine profile induced by S. mansoni and this Th2 antagonized and down-regulated the antiviral activities of Th1 cytokine profile in HCV infection that probably acquired after S. mansoni infection resulting in failing to mount significant HCV specific Th1 response and thereby fail to clear the virus in coinfected, compared with patients infected with HCV or schistosomiasis alone.

DNA strand scission by Fe(III) 2-methylaminopyridine complex and hydrogen peroxide.

Oxidative DNA damage is involved in mutagenesis, carcinogenesis, aging, radiation effects and also in the action of several anticancer drugs. Accumulated evidence indicates that iron may play an important role in these processes. The conversion of the closed circular double-stranded supercoiled plasmid pcDNA3 into the nicked circular and linear forms was used to investigate DNA nicking induced by the reactions of an iron complex of 2-methylaminopyridine (L), which exhibited a pronounced superoxide dismutase-mimetic activity and antitumour activity with H2O2. Hence the dose-response curve for the [FeL2Cl2]Cl.H2O-mediated H2O2-dependent DNA nicking was studied. For a fixed concentration of [FeL2Cl2]Cl.H2O (25 microM), the concentration of H2O2 producing a maximum extent of DNA nicking was 100 microM. The effects of these two constituents are synergistic. The biological antioxidants such as glycerol, sodium azide and superoxide dismutase significantly inhibited DNA breakage induced by [FeL2Cl2]Cl.H2O and hydrogen peroxide.