Transmission electron microscopic observations on ultrastructural alterations in Schistosoma mansoni adult worms recovered from C57BL/6 mice treated with radiation-attenuated vaccine and/or praziquantel in addition to passive immunization with normal and vaccinated rabbit sera against infection.

Although the current treatment of schistosomiasis relies largely on praziquantel (PZQ), it has not been successful in significantly reducing the overall rate of disease cases, one of the suggested reasons being the inevitable resistance to PZQ. Previous studies showed that radiation-attenuated vaccine provides protection against Schistosoma mansoni in a host of various species. In the present study, we evaluated the effect of various vaccination strategies in C57BL/6 mice, including single or multiple vaccination strategy, subcurative dose (20 mg/kg) of PZQ, and a combination of single vaccination with subcurative dose of PZQ. Treatment either with subcurative dose of PZQ or with a single vaccination of attenuated cercariae (500 per mouse), caused significant reduction in total worm burden, hepatic, and intestinal ova counts of 43.03, 73.2, and 59.5 and 37.97, 52.02, and 26.3%, respectively. Furthermore, tegumental changes were observed. In multiple vaccinated group, there was an extensive lysis in tegumental layers. High deformations in gastrodermis, testis cells, vitelline cells, and oocytes were recorded. Also, this study is to explore the role of humoral immunity using highly resistant rabbits that had been exposed to three immunizations with ultraviolet (UV)-irradiated cercariae (8000 per rabbit in each immunization), and their sera were tested for their ability to transfer protection. The reduction in challenge worm burden had reached 32.76-43.64% when compared with recipients of normal serum or no serum. The reduction in hepatic and intestinal ova counts reached to 74.4 and 71.08% in group immunized with vaccinated rabbit sera. Swelling and extensive lysis of tegumental layers, gastrodermis lumen, spermatocytes, and deformation of oocytes were recorded with more severity than that recorded in normal rabbit sera group. Our findings recorded that multiple vaccination strategy is the most effective strategy then passive transfer of vaccinated rabbit. This gives guiding in the design the appropriate therapeutic strategy.

Chemoprevention of rat mammary carcinogenesis by spirulina.

Spirulina (SP) (Arthrospira platensis; previously Spirulina platensis) is a filamentous blue-green microalga (cyanobacterium) with potent dietary phytoantioxidant and anticancerous properties. We investigated the chemopreventive effect of SP against 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat breast carcinogenesis, and further studied its underlying mechanisms of action in vitro. Remarkably, SP cleared DMBA-induced rat mammary tumors, which was clearly confirmed by morphological and histological methods. SP supplementation reduced the incidence of breast tumors from 87% to 13%. At the molecular level, immunohistochemical analysis revealed that SP supplementation reduced expression of both Ki-67 and estrogen α. More interestingly, molecular analysis in the in vitro experiments indicated that SP treatment inhibited cell proliferation by 24 hours, which was accompanied by increased p53 expression, followed by increased expression of its downstream target gene, Cdkn1a (alias p21 or p21(Waf1/Cip1)). In addition, SP increased Bax and decreased Bcl-2 expression, indicating induction of apoptosis by 48 hours after SP treatment. To our knowledge, this is the first report of in vivo chemopreventive effect of SP against DMBA-induced breast carcinogenesis in rat, supporting its potential use in chemoprevention of cancer.

Ultrastructural changes of adult Schistosoma mansoni worms recovered from C57BL/6 mice passively immunized with normal and vaccinated rabbit sera in vivo.

The purpose of the study is to explore the role of humoral immunity against Schistosoma mansoni infection in C57BL/6 mice using highly resistant rabbits that had been exposed to three separate immunizations with ultraviolet (UV)-irradiated cercariae (8,000 attenuated cercariae/rabbit in each immunization), and their sera were tested for their ability to transfer protection against S. mansoni challenge. The present study showed the reduction in challenge worm burden had reached 32.76-43.64% when compared with recipients of normal serum or no serum. The surface topography of the worms collected from immunized mice with either normal rabbit sera (NRS) or vaccinated rabbit sera (VRS) revealed severe tegumental alterations, especially in the VRS group. Worms collected from groups that were immunized by NRS or VRS postinfection (200 normal cercariae/mouse) by day 42. Worms from group immunized with NRS showed damage in the tegument, characterized by severe swelling or erosion of tegumental folds, accompanied by changes in tubercles, swelling, shortening, and loss of spines in male worms. The alteration in female tegument was characterized by swelling of tegumental folds, atrophy of ventral sucker, damage of sensory papillae along all the body, severe peeling in some regions, and appearance of few small blebs. VRS induced more severe tegumental damage than NRS in both male and female worms. Severe shrunken vesicles were protruded from the surface between the two suckers. The tegument of the male showed a collapse of tubercles followed by the appearance of vesicles on their surfaces, fusion, erosion, and superficial focal peeling of tegumental folds. In the female worms, severe damage to the oral sucker, the surface between the two suckers, extensive peeling, severe swelling of the tegument, and damage of sensory papillae. In conclusion, the present study support the hypothesis that high levels of antibodies were developed in rabbit sera after multiple exposures to attenuated cercariae of S. mansoni. Furthermore, immunization might have transferred protection against the infection, indicated by severe morphological alterations, a sign of elimination of the worms. Further investigation is being carried out to reveal the molecular mechanisms underlying the transfer of protection.

Structural changes of Schistosoma mansoni adult worms recovered from C57BL/6 mice treated with radiation-attenuated vaccine and/or praziquantel against infection.

Although the current treatment of schistosomiasis relies largely on praziquantel (PZQ), it has not significantly reduced the overall number of disease cases, perhaps due to inevitable resistance to PZQ. Previous studies showed that radiation-attenuated vaccine gives protection levels for Schistosoma mansoni in host various species. In the present study, we evaluated the effect of various vaccination strategies in C57BL/6 mice, including single or multiple vaccination strategy, subcurative dose (20 mg/kg) of PZQ, and a combination of single vaccination with subcurative dose of PZQ. Groups of five mice were sacrificed postinfection in 42 days and schistosomes were collected by perfusion and examined by scanning electron microscopy. Treatment either with subcurative dose of PZQ or with a single vaccination of attenuated cercariae (500 per mouse), caused significant reduction in total worm burden, hepatic and intestinal ova counts 43.03%, 73.2%, 59.5% and 37.97%, 52.02%, 26.3%, respectively. Furthermore, tegumental changes were observed, including severe swelling, fusion of tegumental folds, vesicle formation, and loss or shortening of the spines on the tubercles. However, multiple vaccination strategy resulted in much higher reduction in total worm burden, hepatic and intestinal ova count. However, multiple vaccination strategy resulted in high reduction of worm burden, hepatic and intestinal ova counts 72.5%, 90.7%, 65.79%, respectively, and further causing swollen, disruption of tubercles teguments and erosion, extensive peeling, fusion of tegumental folds. Our findings suggest that multiple vaccination strategy is the most effective strategy to clear schistosomal infection, indicating its potential in guiding the design of appropriate therapeutic strategy against schistosomes.

Idiotype/anti-idiotype immunoregulatory network correlates with an improved clinical outcome of schistosomiasis mansoni in humans.

Anti-idiotypes (anti-Ids) have a potential role in the immune modulation of various diseases. To study the correlation of anti-Ids with schistosomiasis mansoni morbidity, ELISA using polyclonal idiotypes (Ids) was used to determine the presence of anti-Ids in sera of 69 patients susceptible and resistant to reinfection. Ids were purified against Soluble Worm Antigen (SWAP) from sera of New Zealand white rabbits immunized with SWAP. The results showed that anti-Ids were detected in 15 (40.5%) of susceptible and 21 (65.6%) of resistant patients. Correlation of intensity of infection with age revealed an inverse relationship in patients positive for anti-Ids (regression coefficient beta = -0.47, p < 0.05) and contrarily, a direct relationship in patients negative for anti-Ids (beta = 0.67, p < 0.001). In addition, there was a direct association between the presence of anti-Ids and the lack of schistosome-related symptoms (chi2 = 3.6, p < 0.05) and hepatomegaly (chi2 = 9.4, p < 0.01). Moreover, comparison of patients positive and negative for anti-Ids revealed that those negative for anti-Ids were more vulnerable to develop symptoms (3.7 times) and hepatomegaly (8.1 times). In conclusion, the study further confirms the role of Id/anti-Id regulatory network as an important participant in the assortment of an improved clinical outcome of schistosomiasis. This may help to formulate a better understanding of the mechanisms of protective immunity in humans and provide perspective for the development of a future vaccine.

Immune events associated with protection in C57BL/6 mice immunized with anti-idiotypic antibodies mimicking protective antigens shared between gamma-irradiated cercariae vaccine and human resistance model of Schistosoma haematobium.

Immunoregulation is central for successful manipulation of schistosomiasis. Unlike schistosome vaccine development strategies that relied on direct selection of antigens from crude responses leading to selection of mildly protective antigens, the present study tested the utility of selection of potentially protective antigens encompassed rounds of immunoregulation via idiotypic network. Anti-idiotypic antibodies (Ab2) were purified from sera of New Zealand white rabbits multiply immunized with gamma-irradiated cercariae of S. haematobium, using adult worm specific idiotypes (Ab1) purified from sera of subjects resistant to reinfection. Ab2 was used for immunization of C57BL/6 mice and consequences of immunization were monitored before and after challenge infection with S. haematobium. Results showed an increase of splenic T cell expression of intercellular adhesion molecule-1 (ICAM-1) and very late antigen-4 (VLA-4) upon immunization (average % stimulated cells 54.9 vs. 20.4, P < 0.05 for ICAM-1 and 31.1 vs. 6.6, P < 0.01 for VLA-4) and challenge, especially at day 6 (83.5 vs. 45.6, P < 0.01) for ICAM-1 and day 10 (50.4 vs. 20.8, P < 0.05) for VLA-4. Thereafter, both adhesion molecules declined at day 28 through 90. Similarly, lymphoproliferation was manifested upon immunization (OD570-630 0.27 vs. 0.09, P < 0.01) and challenge at day 6 (0.5 vs. 0.17, P < 0.01) through day 10 (0.49 vs.0.18, P < 0.05), then declined at day 28 through 90. Moreover, sera of Ab2-immunized mice exhibited an anti-anti-ids (Ab3) reactivity against antigens of approximate molecular weight 40, 80 and 160 kDa of adult worms, which were also recognized by Ab1. However, in contrast to Ab1, Ab3 showed no surface binding to 3 hr schistosomula. Strikingly, mice immunized with Ab2 showed strong resistance to challenge infection (approximately 82% reduction in worm burden, P < 0.001). Taking all, this alternative vaccine development strategy appears to filter out non-protective antigens. Indeed Ab3 recognizes much fewer numbers of antigens, which passed through two rounds of immune regulation. These antigens appear to represent a significant proportion of the protective response in the gamma-irradiated cercariae vaccine and human resistance model as well, providing the basis for an alternative vaccine for schistosomiasis.

Manipulation of the immune evasive properties of circulating cathodic antigen induces protective immunity against Schistosomiasis mansoni in C57BL/6 mice.

Schistosome circulating cathodic antigen (CCA) has been hypothesized to take part in immune evasion mechanisms that protect against host's immunity. To dissect its immunogenicity, lymphoproliferative responses of splenocytes were assessed in C57BL/6 mice immunized with recombinant plasmid constructs expressing full-length and truncated fragments of CCA cDNA, before and after challenge infection with S. mansoni cercariae. Prior to challenge, splenocytes of immunized mice showed low responses to phytohaemagglutinin (PHA) and high responses to native CCA, compared to controls. After challenge, PHA-responses increased on day 3 through day 7 and subsequently declined. On the other hand, CCA-induced responses increased on day 3 post-challenge then declined in mice immunized with CCA fragments lacking the N-terminus (-N CCA). Whereas, mice immunized with full-length or CCA fragment lacking the C-terminus (-C CCA) showed a delayed increase of CCA-induced responses that maximize on day 25. Interestingly, animals immunized with -N CCA showed a significant reduction in worm burden between 42-51%, while, mice immunized with full-length or -C CCA showed lower protection levels of about 15 and 37%, respectively. These findings suggest that CCA may contain immunosuppressive epitopes on the N-terminus. Abrogation of these epitopes could disrupt the immune evasion mechanism orchestrated by CCA, which could aid the development of an alternative vaccination approach.

Lymphoproliferative responses of splenocytes before and after challenge with Schistosoma haematobium in C57BL/6 mice vaccinated with human anti-Idiotypes.

Anti-idiotypic vaccines (anti-Id or antibody 2; Ab2) in experimental schistosomiasis engender varying degrees of resistance to challenge infection. To further characterize the mechanisms involved in the induction of protective immunity associated with such a vaccine model, spleen cells of mice vaccinated with human Ab2 (HAb2) were investigated for their lymphoproliferative responses before and after challenge infection with normal S. haematobium cercariae. HAb2 was purified from sera of chronically infected patients using protective rabbit antibodies (RAb1) isolated from sera of rabbits multiply immunized with UV-irradiated cercariae by affinity chromatography over soluble worm antigenic preparation (SWAP). Vaccination of C57BL/6 (C57) mice with HAb2 resulted in approximately 31% and approximately 36% protection in two experiments of resistance to infection. Splenocytes were collected prior to challenge at week 6-post initial immunization and after challenge at days 6, 10, 28 and 90. Prior to challenge, in vitro splenic responses of HAb2-vaccinated animals (HAb2-group) to phytohaemagglutinin (PHA) declined while both SWAP and HAb2-driven responses increased, all compared to naive control. After challenge, PHA responses increased in the two test groups on day 6 then significantly decreased to lower levels. On the other hand, SWAP- and HAb2-driven responses of HAb2 group increased by day 6 then declined while the same responses in infected control mice increased on days 10 through 28 and decreased by day 90. Generally, proliferation obtained following in vitro stimulation with HAb2 was greater than that with SWAP in the HAb2-group after challenge. These results suggested that human anti-Id antibodies could mimic at the T cell level the properties of a protective antigenic epitopes of the irradiated-cercariae vaccine.