Ahmed M. El-Waseef

Use of a Novel Enzyme Immunoassay Based on Detection of Circulating Antigen in Serum for Diagnosis of Helicobacter pylori Infection

ABSTRACT Recently, noninvasive diagnostic tests for Helicobacter pylori infection have gained in significance. We have developed a sensitive and specific noninvasive immunoassay based on the detection of an H. pylori circulating antigen (HpCA) in sera from H. pylori-infected individuals. Monospecific antibody and Western blot analyses were used to demonstrate the presence of the target antigen in H. pylori cell lysate and serum samples. A novel enzyme-linked immunosorbent assay (ELISA) was developed for the detection of HpCA in serum. Endoscopic biopsy specimens from the gastric antra of 221 individuals (143 males and 78 females) with dyspeptic symptoms were evaluated for H. pylori infection, with culture used as a “gold standard” for diagnosis. The target H. pylori antigen was identified at 58 kDa. HpCA has been detected by ELISA with high degrees of sensitivity, specificity, and efficiency (>90%), and ELISA results show no significant difference (P > 0.05) from results of H. pylori culture of gastric biopsy specimens. The tests positive and negative predictive values were also high (95 and 86%, respectively). In conclusion, a sensitive and specific immunoassay was developed for the detection of HpCA in human serum. This test can be applied for noninvasive laboratory and field diagnoses of H. pylori infection.

Approach for chemosensitization of cisplatin-resistant ovarian cancer by cucurbitacin B.

Ovarian cancer is the most deadly gynecological cancer. The first line in treatment is platinum-based drugs. However, most patients suffer from tumor recurrence, characterized by resistance to cisplatin. A plausible approach to address the tumor resistance is to co-administer the chemotherapeutic agents along with natural products to offer a synergistic effect and optimize the dosage regimen. Cucurbitacin B is a natural product and displays antitumor activity against a wide array of cancer cell lines. The aim of this work is to determine the antitumor activity against ovarian cancer cell line (A2780) and possible sensitization activity on cisplatin-resistant cell line (A2780CP) in 2D and 3D culture model. 3D spheroids were generated from A2780CP cell line. A2780, A2780CP, and the spheroids were treated with cucurbitacin B, cisplatin alone, or pretreated with cucurbitacin B followed by cisplatin. The viability, cell cycle, and apoptosis were analyzed. Level of ROS and total glutathione was measured. In this study, cucurbitacin B showed cytotoxicity against the ovarian cancer cell lines, and pretreatment of A2780CP cells leads to a significant increase in the cytotoxicity of cisplatin. The mechanism behind the sensitization effect was dependent in part on the depletion of the total glutathione, an increase in ROS through a decrease in the level of dual-specificity tyrosine-regulated kinase (Dyrk1B), decrease in pERK1/2 and pSTAT3 level. The viability of spheroids treated with a combination of cisplatin and cucurbitacin B were significantly decreased. The resulting data shows that cucurbitacin B is a promising chemosensitizer for the cisplatin-resistant ovarian cancer.

Immunochemical identification and detection of a 36-kDa Toxoplasma gondii circulating antigen in sera of infected women for laboratory diagnosis of toxoplasmosis.

Abstract The detection of Toxoplasma gondii circulating antigens has been indicated to be a reliable diagnostic approach of active human toxoplasmosis. However, few reports have appeared in the literature regarding the diagnostic potential of T. gondii circulating antigens. Here, a specific antibody and western blot analyses were used to demonstrate the presence of a highly reactive antigen of 36‐kDa, not only in the extract of T. gondii tachyzoites, but also in selected sera of women with confirmed laboratory and clinical signs of recent toxoplasmosis. The 36‐kDa Toxoplasma antigen was purified from T. gondii tachyzoites and human serum using electroelution from preparative polyacrylamide gels. The purified polypeptides showed a single peak at 10.9 min when analyzed by capillary zone electrophoresis. Based on the above encouraging results, we have developed an ELISA format for the detection of target Toxoplasma antigen (TAg‐ELISA) in human serum samples. The TAg‐ELISA detected the target antigen in 88% sera of acutely infected women and showed high degree of specificity (91%) among sera from non‐infected women. In conclusion, the detection of 36‐kDa Toxoplasma circulating antigen in human sera appears to be a promising alternative approach for laboratory diagnosis of active T. gondii infection.

Interferon-gamma is associated with hepatic dysfunction in fibrosis, cirrhosis, and hepatocellular carcinoma

ABSTRACT The relation between interferon–gamma (IFN-γ) levels and the severity of liver diseases through fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) has not been fully clarified. Thus, we aimed to characterize IFN-γ levels in liver-diseased patients. IFN-γ levels were determined by Western-blot and ELISA in sera from 30 healthy individuals, 53 patients with non-significant fibrosis (F0-F1), 47 with moderate/severe fibrosis (F2-F3), 44 cirrhotic patients (F4), and 50 with HCC. Enhanced levels of IFN-γ were associated with the progression of liver disease. The differences were statistically significant (P < 0.0001) when patients with F2-F3, F4, or HCC were compared with F0-F1 or healthy controls. The increase in IFN-γ was associated with HCC (OR = 0.98, 95% CI 0.97–0.99, P = 0.002). There was no statistically significant association between IFN-γ levels and HCV-RNA (IU/ml) (r = 0.1, P = 0.43) or HCV-NS4 (µg/mL) (r = 0.1, P = 0.17). There was significant (P < 0.0001) association between IFN-γ levels and the fibrosis stages and activity, albumin, platelet count, total bilirubin, and international normalized ratio (INR). In conclusion, elevated concentrations of IFN-γ represent a characteristic feature of liver disease severity regardless of underlying disease. Significant correlations with indices of hepatic dysfunction suggest that enhanced IFN-γ levels represent a consequence of liver dysfunction rather than of inflammatory disease.

Associations of human leucocyte antigen class II‐DQB1 alleles with hepatitis C virus infection in Egyptian population: a multicentre family‐based study

Hepatitis C infection is a global pandemic. HLA‐DQB1 alleles are believed to have an effective role in immune response against HCV including susceptibility to or protection from this infection. The aim of this study was to investigate the contribution of HLA‐DQB1 alleles in the outcome of HCV genotype‐4 infection through a family‐based association study. Egyptian families with HCV (324) were recruited for this study (324 index positive for RNA‐HCV, 225 positive relatives representing chronic hepatitis C cases and 582 family members negative for HCV‐RNA [control], 63 of whom spontaneously cleared the virus. All subjects were genotyped for HLA‐DQB1 alleles by sequence‐specific primers (SSP‐PCR) and sequence‐based typing (SBT) methods. The frequency of DQB1*02:01:01 carriage was significantly higher in infected patients when compared to controls and those who spontaneously cleared virus (OR=5.47, P<.0001 and OR= 6.5234, P<.0001, respectively), and the carriage of the DQB1*03:01:01:01 allele was significantly higher in those who cleared and controls when compared to the infected patients (OR=0.2889, P<.0001 and OR=0.3016, P<.0001, respectively). On the other hand, the frequency of DQB1*06:01:01 and QB1*05:01:01:01 alleles was not associated with infection (comparison of infected and cleared patients showed OR of 2.1598 [P<.01]), but it becomes nonsignificant after adjustments with the Bonferroni formula (PC>0.05) and OR= 1.3523, P>.05, respectively. This study shows that clearance of HCV is associated with DQB1*03:01:01:01 allele and chronicity of HCV infection associated with the risk allele: DQB1*02:01:01.

Combined use of epithelial membrane antigen and nuclear matrix protein 52 as sensitive biomarkers for detection of bladder cancer.

Background The advent of noninvasive urine-based markers as well as other novel modalities has yielded improved diagnostic accuracy. However, the new markers failed to reach higher sensitivity and specificity. We therefore evaluated the potential role of epithelial membrane antigen (EMA) and nuclear matrix protein 52 (NMP-52) singly and combined as noninvasive biomarkers for the detection of bladder cancer (BC). Methods A total of 160 individuals including 66 patients with BC, 54 patients with benign urologic disorders and 40 healthy volunteers were investigated. Urinary EMA at 130 kDa and NMP at 52 kDa were identified, purified and quantified by Western blot, electroelution and enzyme-linked immunosorbent assay (ELISA). The diagnostic performance of each biomarker and their combination were compared using area under receiver operating characteristic curves (AUC). Results Mean urinary EMA, 2.42 µg/mL, and NMP-52, 17.85 µg/mL, were significantly elevated in patients with BC compared to controls, 1.18 and 3.44 µg/mL, respectively (p<0.0001). The combined use of these markers yielded values which were increased 4.4- and 13.7-fold in the benign and malignant disease groups, respectively, with respect to the normal group. The values of EMA and NMP-52 were significantly higher in patients with higher-grade tumors than those with lower-grade tumors (p<0.0001). Moreover, this combination could predict all BC stages and grades with 0.91 AUC, 94% sensitivity and 80% specificity. Conclusions EMA and NMP-52 in combination could be promising noninvasive biomarkers for BC detection.

Protective effect of Spirulina against cyclophosphamide-induced urotoxicity in mice

Abstract Cyclophosphamide (CP) is an anti-neoplastic drug, which is widely used for treating cancer and non-malignant tumors. One of the major side effects of CP is hemorrhagic cystitis. Spirulina (Arthrospira platensis; Sp) is a blue-green algae with the ability to attenuate oxidative stress, which may be utilized for alleviating side effects of chemotherapeutic drugs in the clinic. The aim of the present study was to evaluate the ability of Sp, to protect mice from cyclophosphamide-induced urotoxicity and hemorrhagic cystitis due to its antioxidant properties. Adult female mice were orally administered Sp (600 g/kg body weight/day) over nine days as well as a single dose of CP (40 mg/kg body weight) intraperitoneally either four days previously (CP + Sp group) or four days after the start of Sp intake (Sp + CP group); two further groups were treated with either Sp or CP only, respectively. The results showed that CP induced hemorrhagic cystitis in mice, with levels of malondialdehyde (MAD) significantly increased and those of glutathione (GSH) decreased compared with the control group (P < 0.05), while the opposite effects were observed in the mice who received Sp only (P < 0.05). Furthermore, in the CP + Sp group, MAD and GSH levels were improved compared with those in the CP only group, and in the Sp + CP group, the effects of CP were reversed. In addition, histomorphological alterations of the urinary bladder were significantly lower than those in the CP group. In conclusion, pre-treatment with Sp protected mice from CP-induced urotoxicity, probably via its anti-oxidant and anti-apoptotic properties.