HPLC-ESI-MS Characterization of Certain Polyphenolic Compounds of Carica papaya L. Fruit Extracts and Evaluation of Their Potential Against Murine Schistosomiasis mansoni.

The in vivo antischistosomal activities of Carica papaya L. extracts were evaluated and the characterization of the active secondary metabolites of the defatted methanolic extract was performed using HPLC-ESI-MS. The plant fruit powders were extracted with 85% methanol and fractionated using organic solvents. The in vivo antischistosomal effects of the methanolic extracts and its fractions, as well as the assessment of the relationship between the antischistosomal activity of these plant extracts and oxidative stress, was determined. In addition, the defatted methanolic extract was characterized by HPLC-ESI-MS analysis. The number of worms, ova, and the Oogram pattern displayed typical Schistosoma mansoni pathology 8 weeks after infection in mice. Treatment of the infected group with the defatted methanolic extracts significantly decreased worm burden, immature ova and mature ova, while increasing the percentage of dead ova in vivo. The butanol fraction was the most effective fraction reducing worm burden by 77%, ova count in the intestine by 76% and in the liver by 80%, and significantly decreased immature and mature ova (P<0.001) compared to the infected group. Additionally, the defatted methanolic extracts improved the reduced glutathione and malondialdehyde levels in hepatic tissues in the treated groups compared to the infected group. The HPLC-ESI-MS analysis of the Carica papaya defatted methanolic extract revealed the presence of several polyphenolic compounds. Carica papaya fruit extracts are rich with phenolic acids and flavonoids and show a significant effect against S. mansoni infections which may be used alternative to PZQ as anti-schistosomal drug against schistosomiasis.

BLOCKADE OF PGE2, PGD2 RECEPTORS CONFERS PROTECTION AGAINST PREPATENT SCHISTOSOMIASIS MANSONI IN MICE.

Schistosomiasis is a chronic disease with considerable social impact. Despite the availability of affordable chemotherapy, drug treatment has not significantly reduced the overall number of disease cases. Among other mechanisms, the parasite produces PGE2 and PGD2 to evade host immune defenses. To investigate the role of PGE2 and PGD2 in schistosomiasis, we evaluated the effects of L-161,982, Ah6809 (PGE2 receptor antagonists alone of combined with each other) and MK-0524 (PGD2 receptor antagonist) during prepatent Schistosoma mansoni infection. Drugs were administered intraperitoneally an hour before and 24 hours after infection of C57BL/6 mice with 100 Schistosoma mansoni cercariae. L-161,982, Ah6809, their combination and MK-0524 caused partial protection against pre-patent S. mansoni infection which was mediated by biasing the immune response towards Th1 phenotype. These results showed that blockade of PGE2 and PGD2 receptors confers partial protection against pre-patent S. mansoni infection in mice and that they may be useful as adjunctive therapy to current anti-schistosomal drugs or vaccines.

Monoclonal antibody-based dipstick assay: a reliable field applicable technique for diagnosis of Schistosoma mansoni infection using human serum and urine samples.

A field applicable diagnostic technique, the dipstick assay, was evaluated for its sensitivity and specificity in diagnosing human Schistosoma mansoni infection. A monoclonal antibody (mAb) against S. mansoni adult worm tegumental antigen (AWTA) was employed in dipstick and sandwich ELISA for detection of circulating schistosome antigen (CSA) in both serum and urine samples. Based on clinical and parasitological examinations, 60 S. mansoni-infected patients, 30 patients infected with parasites other than schistosomiasis, and 30 uninfected healthy individuals were selected. The sensitivity and specificity of dipstick assay in urine samples were 86.7% and 90.0%, respectively, compared to 90.0% sensitivity and 91.7% specificity of sandwich ELISA. In serum samples, the sensitivity and specificity were 88.3% and 91.7% for dipstick assay vs. 91.7% and 95.0% for sandwich ELISA, respectively. The diagnostic efficacy of dipstick assay in urine and serum samples was 88.3% and 90.0%, while it was 90.8% and 93.3% for sandwich ELISA, respectively. The diagnostic indices of dipstick assay and ELISA either in serum or in urine were statistically comparable (P>0.05). In conclusion, the dipstick assay offers an alternative simple, rapid, non-invasive technique in detecting CSA or complement to stool examinations especially in field studies.

Protective role of purified cysteine proteinases against Fasciola gigantica infection in experimental animals.

Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by Fasciola gigantica play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in Freund's adjuvant followed by 3 booster doses 1 mg each at 4 week intervals) were injected intramuscularly into sheep 1 week prior to infect orally with 300 F. gigantica metacercariae. All the sheep were humanely slaughtered 12 weeks after the first immunization. Changes in the worm burden, ova count, and humoral and cellular responses were evaluated. Significant reduction was observed in the worm burden (56.9%), bile egg count (70.7%), and fecel egg count (75.2%). Immunization with CP was also found to be associated with increases of total IgG, IgG(1), and IgG(2) (P<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-γ, and TNF-α, revealed significant decreases (P<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-ß, and IL-6, showed significant increases (P<0.05). In conclusion, it has been found that CP released by F. gigantica are highly important candidates for a vaccine antigen because of their role in the fluke biology and host-parasite relationships.

Saliva and sera IgA and IgG in Egyptian Giardia-infected children.

Giardiasis is a gastrointestinal infection of wide distribution that is more prevalent in childhood. Easy and rapid diagnosis of giardiasis is essential for reduction of this infection. This cross-sectional study included 62 children in which collection of saliva, stool and serum samples was performed. An enzyme-linked immunosorbent assay (ELISA) technique was evaluated to detect IgA and IgG responses in both saliva and serum samples. Twenty-two children were positive for Giardia duodenalis infection by direct examination of faecal specimens, 20 non-infected and 20 infected with other parasites. Salivary and serum IgA and IgG responses against G. duodenalis infection were significantly higher in Giardia parasitized than non-Giardia parasitized children (p < 0.001). This concludes that specific salivary IgA may serve as a diagnostic tool and specific salivary IgG as a screening tool in monitoring the exposure of various populations to Giardia duodenalis. The advantage of salivary assays over serum immunoglobulin assay is being easy and non-invasive in sampling technique which is important especially for young children.

Immunolocalization of Schistosoma mansoni and Schistosoma haematobium antigens reacting with their Egyptian snail vectors.

The reaction of the haemolymph and the tissue of infected intermediate hosts, Biomphalaria alexandrina and Bulinus truncatus to Schistosoma mansoni and S. haematobium antigens were investigated using the indirect immunoperoxidase technique. A new technique, Agarose cell block was used in collection of haemolymph which helped in collecting plenty of well formed cells in comparison to the ordinary one using the cytospin. Collected haemolymph and prepared tissues of uninfected and infected B. alexandria and B. truncatus were fixed and then reacted with anti-S. mansoni and anti-S. haematobium IgG polyclonal antibodies. The haemolymph and tissue of infected B. alexandrina and B. truncatus gave a positive peroxidase reaction represented by a brown colour. In haemolymph, the positive peroxidase reaction was detected mainly in the cytoplasm of the amoebocytes. In the tissue, it was detected in epithelial cells lining the tubules, male cells in the lumen of the tubules and in female oogonia cells along the periphery of the tubules. The similarity in the strength and distribution of positive reaction in B. alexandrina and B. truncates was observed as compared to control. Thus, the immunoperoxidase technique proved to be an effective indicator for the schistosome-antigen in the snails.

A Con A- purified hydatid glycoprotein fraction effectively diagnoses human hydatidosis.

Diagnosis and quantification of Echinococcus granulosus infection in man and animal hosts are centralized to feasible control. This study included 93 serum samples, 25 sure positive hydatid cases confirmed surgically, 7 suspected cases diagnosed by indirect haemagglutination IHA and 41 cases other parasitic infections (15 S. mansoni, 8 Fasciola, 7 Ascaris, 5 H. nana & 6 Ancylostoma) diagnosed by microscopic examination and were negative by ELISA and/or IHA for anti-hydatid antibody. Twenty negative serum samples served as healthy controls. Six types of hydatid fluid antigens (crude, host-free & Con-A purified) of human and camel origin were subjected to electrophoretic separation (SDS-PAGE) and immunoblotting (EITB). The anti-hydatid IgG was detected in sera of the different groups for evaluation of sensitivity, specificity and diagnostic efficacy of each type of antigens. Detection of circulating hydatid antigen (CAg) was performed using anti rabbit hyperimmune sera raised against Con-A purified either human or camel hydatid antigen. SDS-PAGE revealed several bands ranging from 55-185 kDa with 10 kDa band shared by all antigens. The specific bands revealed by EITB for Con-A purified camel and human antigens were at 80, 110 & 55, 110 kDa respectively. ELISA highest sensitivity (96.9%) was by using host-free Con-A purified glycoprotein fraction of human hydatid antigen. Highest specificity (98.4%) was recorded upon use of either Con-A purified camel or human antigen with 94.5% & 97.7% diagnostic efficacy respectively. Detection of circulating antigen by polyclonal antibodies against Con-A purified human hydatid antigen revealed 91.8% specificity.