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 or 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.

T Regulatory Cell Responses to Immunization with a Soluble Egg Antigen in Schistosoma mansoni-Infected Mice

The aim of the study is to characterize the phenotypes of CD4+ CD25+ T regulatory cells within the liver granulomas and association with both Foxp-3 gene expression and splenic cytokines. Naive C57BL/6 mice were intravenously injected with multiple doses of the soluble egg antigen (SEA) 7 days before cercarial infection. The immunized and infected control groups were sacrificed 8 and 16 weeks post-infection (PI). Histopathology, parasitological parameters, splenic phenotypes for T regulatory cells, the FOXP-3 expression in hepatic granuloma using real-time PCR, and the associated splenic cytokines were studied. Histopathological examination of the liver revealed remarkable increase in degenerated ova within hepatic granuloma which decreased in diameter at weeks 8 and 16 PI (P<0.01). The percentage of T regulatory cells (CD4+ CD25+) increased significantly (P<0.01) in the immunized group compared to the infected control at weeks 8 and 16 PI. The FOXP-3 expression in hepatic granulomas increased from 10 at week 8 to 30 fold at week 16 PI in the infected control group. However, its expression in the immunized group showed an increase from 30 at week 8 to 70 fold at week 16 PI. The splenic cytokine levels of pro-inflammatory cytokines, IFN-gamma, IL-4, and TNF-alpha, showed significant decreases (P<0.05) compared to the infected control group. In conclusion, the magnitude and phenotype of the egg-induced effects on T helper responses were found to be controlled by a parallel response within the T regulatory population which provides protection in worm parasite-induced immunopathology.

Fasciola gigantica Fatty Acid Binding Protein (FABP) as a Prophylactic Agent against Schistosoma mansoni Infection in CD1 Mice

Although schistosomicidal drugs and other control measures exist, the advent of an efficacious vaccine remains the most potentially powerful means for controlling this disease. In this study, native fatty acid binding protein (FABP) from Fasciola gigantica was purified from the adult worm's crude extract by saturation with ammonium sulphate followed by separation on DEAE-Sephadex A-50 anion exchange chromatography and gel filtration using Sephacryl HR-100, respectively. CD1 mice were immunized with the purified, native F. gigantica FABP in Freund's adjuvant and challenged subcutaneously with 120 Schistosoma mansoni cercariae. Immunization of CD1 mice with F. gigantica FABP has induced heterologous protection against S. mansoni, evidenced by the significant reduction in mean worm burden (72.3%), liver and intestinal egg counts (81.3% and 80.8%, respectively), and hepatic granuloma counts (42%). Also, it elicited mixed IgG1/IgG2b immune responses with predominant IgG1 isotype, suggesting that native F. gigantica FABP is mediated by a mixed Th1/Th2 response. However, it failed to induce any significant differences in the oogram pattern or in the mean granuloma diameter. This indicated that native F. gigantica FABP could be a promising vaccine candidate against S. mansoni infection.

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, IgG1, and IgG2 (P<0.05). Data showed that the serum cytokine levels of pro-inflammatory cytokines, IL-12, IFN-gamma, and TNF-alpha, revealed significant decreases (P<0.05). However, the anti-inflammatory cytokine levels, IL-10, TGF-beta, 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.

Estimation of protease activity in Schistosoma mansoni life cycle stages and Biomphalaria Alexandria after cystein protease inhibitor

The efficacy of cysteine inhibitor on murine Schistosom mansoni was studied when the injection of inhibitor to infected mice for 10 successive days was either starting on day 1 [group 1] or on day 21 [group2] or on day 45 [group3] post infection. Eight weeks post infection animals were sacrificed and subjected for parasitological, histological and physiological assessments. Then levels of proteases activity were assayed in normal, infected and infected treated mice in serum, livers, intestine, as well as, in S. mansoni worms and ova collected from different groups under study. Also, B. alexandrina snails were exposed to LC5, LC10 and LC25 of each of bayluscide and A. arvensis then their hemolemph protease activity were assessed after 1 day, 3 days, 1 week, 2 weeks, 3 weeks and 4 weeks of exposure and compared with that assessed in hemolemph of control snails. Both the parasitological and physiological studies gave rise to the same conclusion denoting that treatment with cysteine protease inhibitor, phenyl vinyl sulfone, at 45 day post infection could reduce the worm burden, egg tissue load, granuloma diameter, ova hatchability, proteases activity in S. mansoni worms and ova. This treatment at the same time helps mice to attain approximately normal physiological state as expressed by their serum proteases activity. The findings representing toxicity and physiology studies gave rise to the same conclusion, emphasizing that the chemical molluscicides seriously affects snail physiology exerting irreversible alterations, while plant molluscicides has effect under which snails can acclimatize their physiology to survive

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

Role of secretory excretory products of Schistosoma mansoni eggs in modulating hepatic morbidity

In the present study the possible anti-morbidity effect of secretory excretory products [SEP] of Schistoma mansoni eggs [given to mice before infection] was investigated. Multiple small doses of SEP were injected intra-peritoneally into albino mice [100 micro g of purified SEP followed 2 weeks later by two booster doses of 50 micro g each at weekly intervals]. Data revealed reduction in CD4+ cells and increase in CD8+ cells of hepatic granuloma in SEP-immunized infected group, resulting in significant decrease in CD4+/CD8+ ratio, in comparison to infected control group. The serum cytokine level of both TNF--alpha and IFN-gamma were also significantly decreased. Histopathological examination of liver revealed remarkable increase in degenerated ova within hepatic granuloma which decreased in diameter [12%]. Significant reduction in worm burden [46%] and tissue egg loads [42.8% and 50% for hepatic and intestinal ova respectively] were observed. Mean while decreased percent of immature stages with increase in percent of dead ova in Oogram pattern was recorded. This work may help in decrease the severity of hepatic morbidity

Immunodiagnosis of human Wuchereria bancrofti infection using a pair of monoclonal antibodies against worm antigen

This study was designed to prepare monoclonal antibodies [MAbs] against filarial worm antigen [FWA] with immunodiagnostic potential for human filariasis. These MAbs were initially screened and then tested for their specificities against different parasite antigens [S.mansoni SEA, Echinococus granulosus and Fasciola hepatica] by ELISA. From a panel of anti-filarial antigen MAbs; a pair of MAbs [9F/10B and 5F1 611], highly reactive with filarial antigen and showing no cross reactivity against other parasites antigens were selected and characterized. The pair was found to be of IgG1 subclass. Identification of target antigens recognized by MAbs was performed using immunoelectrophoresis, SDS-polyacrylamid gel electrophoresis and enzyme-linked immunoelectrotransfer blot techniques. Both MAbs recognized one band with 88 kDa molecular weight by western blots. Chemical nature of MAbs target antigens was identified by periodate treatment method and proved to be glycoprotein in nature. The pair of MAbs was employed in sandwich ELISA for the defection of circulating filarial antigen [CFA]; one MAb [9F/10B] was used as antigen capturing antibody and the other [5F/6H] as peroxidase-conjugated antigen detecting antibody. The assay reached a lower detection limit of 125 ng/ml of filarial antigen. CFA levels were measured in serum samples from 71 filariasis patients [47 with microfilaraemia and 24 with elephantiasis], 45 patients with other parasites including schistosomiasis, fascioliasis and echinococcosis and 39 healthy individuals as negative control. CFA levels were detected in sera of 68 out of 71 filariasis patients showing an overall sensitivity of 95.8% [91.7% sensitivity for microfilaraemia group and 100% sensitivity for elephantiasis group]. All negative control sera were negative for CFA, while 3 patients out of the other parasite group were positive for CFA giving an overall specificity of 96.4%. These findings suggest that [9F/IOB] MAb and [5F/6H] MAb could be used as a reliable diagnostic indicator for the activity of human filariasis and as a cure monitor particularly in control programs for endemic areas

Intraperitoneal injection of echinostome antigens confers resistance to schistosomal infection in murine model

The effect of immunization protocol against S. mansoni infection with purified metacercarial antigen from E. liel alone or combined with BCG on resistance to S. mansoni infection and associated immunoparasitological changes in murine experimental model was studied. The results revealed highly significant reduction in worm burden and hepatic and intestinal tissue egg loads were observed on comparing the two immunized groups [purified metacercarial antigen alone or purified metacercarial antigen plus BCG] with the infected control group [p> 0.001]. Moreover the greatest reduction in the worm burden was observed in group injected with purified metacercarial antigen pluse BCG. A significant reduction [p> 0.001] was observed in granuloma diameter in the two immunized groups relative to infected control. However, the greatest reduction in granuloma diameter was noticed in group II [purified metacercarial antigen pluse BCG]. No significant difference was observed in granuloma diameter berween the two immunized groups. Anti-SEA serum specific immunoglobulins in group I and group II showed a significant increase in both IgG and IgM [p> 0.05] compared to the infected control group. A higher significant increase was found in the specific immunoglobulin isotype IgGI [p> 0.001] in both group I and II compared to infected control group. In conclusion, our findings showed that multiple intraperitoneal adminestration of purified metacercarial antigen induce highly significant reduction in worm burden, hepatic and intestinal tissue egg loads and granuloma size 8 weeks post infection

con A purified hydatid glycoprotein fraction effectively diagnoses human hydatidosis

Diagnosis and quantification of Echinococcus granulosus in-fection in man and animal hosts are centralized to feasible con-trol this study included 93 serum sample, 25 sure positive hydatid cases confirmed surgically, 7 suspected cases diagnosed by indirect haemagglutination IHA and 41 cases other parasitic infection [15 S. mansoni, 8 fasciola, 7 Ascaris, 5 H. nana and 6 Ancylostoma] diagnosed by microscopic examination and were negative by ELISA and/or IHA for anti-hydatid antibody. Twen-ty negative serum samples served as healthy controls. Six types of hydatid fluid antigens [crude, host-free and Con-A purified] of human and camel origin were subjected to electrophoretic separ-ation [SDS-PAGE] and immunoblotting [EITB]. The anti-hydat-id IgG was detected in sera of the different groups for evalua-tion of sensitivity, specificity and diagnostic efficacy each type of antigens. Detection of circulating hydatid antigen [CAg] was performed using anti rabbit hyperimmune sera raised again-st Con-A purified either human or camel hydatid antigen. SDS-PGE revealed several bands ranging from 55-185- kDa with 10kD 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 hydatied antigen. Highest specificity [98.4%] was reco-rded upon use of either Con-A purified camel or human antigen with 94.5% and 97.7 and diagnostic efficacy respectively. Detection of circulating antigen by polyclonal antibodies against Con-A purified human hydatid antigen revealed 91.8%specificity.