Paratrygon aiereba irradiated anti-mucus serum reduce edematogenic activity induced in experimental model.

Accidents by freshwater stingrays are common in northern Brazil, there is no specific therapy for high morbidity and local tissue destruction. The irradiation of venoms and toxins by ionizing radiation has been used to produce appropriate immunogens for the production of antisera. We planned to study the efficacy of stinging mucus irradiation in the production of antisera, with serum neutralization assays of edematogenic activity and quantification of cytokines performed in animal models of immunization with native and irradiated mucus of Paratrygon aiereba, a large freshwater stingray. Antiserum potency and its cross-reactivity with mucus from other freshwater stingrays were detected by ELISA. Immunization models demonstrated the ability to stimulate a strong humoral response with elevated levels of serum IgG detectable by ELISA, and both native and irradiated mucus were immunogenic and capable of recognizing mucus proteins from other freshwater neotropical stingrays. Mucus P. aiereba causes cellular and humoral adaptive immune responses in cells of immunized mice producing antibodies and cytokines such as TNF-α, IL-6 and IL-17. Rabbit antisera immunized with mucus from P. aiereba irradiated at 2 kGy showed a significant reduction of mucus-induced edematogenic activity in mice. Our data suggest that the use of antisera against freshwater stingray mucus show the possibility of specific therapy for these accidents.

Pharmacokinetics of neutron-irradiated meglumine antimoniate in Leishmania amazonensis-infected BALB/c mice.

BACKGROUND: Cutaneous leishmaniasis (CL) is a parasitic disease caused by the protozoan Leishmania spp. Pentavalent antimonial agents have been used as an effective therapy, despite their side effects and resistant cases. Their pharmacokinetics remain largely unexplored. This study aimed to investigate the pharmacokinetic profile of meglumine antimoniate in a murine model of cutaneous leishmaniasis using a radiotracer approach. METHODS: Meglumine antimoniate was neutron-irradiated inside a nuclear reactor and was administered once intraperitoneally to uninfected and L. amazonensis-infected BALB/c mice. Different organs and tissues were collected and the total antimony was measured. RESULTS: Higher antimony levels were found in infected than uninfected footpad (0.29% IA vs. 0.14% IA, p = 0.0057) and maintained the concentration. The animals accumulated and retained antimony in the liver, which cleared slowly. The kidney and intestinal uptake data support the hypothesis that antimony has two elimination pathways, first through renal excretion, followed by biliary excretion. Both processes demonstrated a biphasic elimination profile classified as fast and slow. In the blood, antimony followed a biexponential open model. Infected mice showed a lower maximum concentration (6.2% IA/mL vs. 11.8% IA/mL, p = 0.0001), a 2.5-fold smaller area under the curve, a 2.7-fold reduction in the mean residence time, and a 2.5-fold higher clearance rate when compared to the uninfected mice. CONCLUSIONS: neutron-irradiated meglumine antimoniate concentrates in infected footpad, while the infection affects antimony pharmacokinetics.

Gamma irradiation of Toxoplasma gondii protein extract improve immune response and protection in mice models.

Gamma radiation induces protein changes that enhance immunogenicity for venoms, used in antivenin production. Coccidian parasites exposed to gamma radiation elicit immune response with protection in mice and man, but without studies on the effect of gamma radiation in soluble acellular extracts or isolated proteins. Toxoplasmosis is a highly prevalent coccidian disease with only one vaccine for veterinary use but with remaining tissue cysts. Total parasite extracts or recombinant proteins used as immunogen induce usually low protection. Here, we study gamma radiation effect on T. gondii extracts proteins (STAG) and its induced immunity in experimental mice models. By SDS-PAGE, protein degradation is seen at high radiation doses, but at ideal dose (1500 Gy), there are preservation of the antigenicity and immunogenicity, detected by specific antibody recognition or production after mice immunization. Immunization with STAG irradiated at 1500 Gy induced significant protection in mice immunized and challenged with distinct T. gondii strains. In their blood, higher levels of specific CD19+, CD3+CD4+ and CD3+CD8+ activated cells were found when compared to mice immunized with STAG. Irradiated T. gondii tachyzoites extracts induce immune response and protection in mice in addition, could be a feasible alternative for Toxoplasma vaccine.

Pharmacokinetic of meglumine antimoniate encapsulated in phosphatidylserine-liposomes in mice model: A candidate formulation for visceral leishmaniasis.

Visceral leishmaniasis (VL) is a fatal parasitic disease caused by the protozoan Leishmania spp. Meglumine antimoniate (MA) is the main treatment and has demonstrated a promising efficacy in a VL-model when encapsulated into negatively charged liposomes. Considering the current concept for the evaluation of pharmacokinetic parameters at early phases of drug discovery, we developed a formulation of MA-encapsulated into phosphatidylserine liposomes (MA-LP) and analyzed the in vitro antileishmanial activity, physicochemical properties, and pharmacokinetic profile in a mice model. The liposomal formulation had an internal mean diameter of 114 nm and a high stability in plasma. MA-LP was 23-fold more in vitro effective against Leishmania infantum-infected macrophages than the free drug, with a selectivity index higher than 220. The pharmacokinetic studies demonstrated that the liposomes increased the uptake of the drug by the liver and spleen and promoted sustained levels. MA-LP was first eliminated through renal excretion, followed by biliary excretion. In the blood, MA-LP followed a biexponential open model. This work emphasizes the importance of liposomes as potential drug delivery systems for visceral leishmaniasis.

Immunity in the spleen and blood of mice immunized with irradiated Toxoplasma gondii tachyzoites.

Toxoplasma gondii infection induces a strong and long-lasting immune response that is able to prevent most reinfections but allows tissue cysts. Irradiated, sterilized T. gondii tachyzoites are an interesting vaccine, and they induce immunity that is similar to infection, but without cysts. In this study, we evaluated the cellular immune response in the blood and spleen of mice immunized with this preparation by mouth (v.o.) or intraperitoneally (i.p.) and analyzed the protection after challenge with viable parasites. BALB/c mice were immunized with three i.p. or v.o. doses of irradiated T. gondii tachyzoites. Oral challenge with ten cysts of the ME-49 or VEG strain at 90 days after the last dose resulted in high levels of protection with low parasite burden in the immunized animals. There were higher levels of specific IgG, IgA and IgM antibodies in the serum, and the i.p. immunized mice had higher levels of the high-affinity IgG and IgM antibodies than the orally immunized mice, which had more high-affinity IgA antibodies. B cells (CD19(+)), plasma cells (CD138(+)) and the CD4(+) and CD8(+) T cell populations were increased in both the blood and spleen. Cells from the spleen of the i.p. immunized mice also showed antigen-induced production of interleukin-10 (IL-10), interferon gamma (IFN-γ) and interleukin 4 (IL-4). The CD4(+) T cells, B cells and likely CD8(+) T cells from the spleens of the i.p. immunized mice proliferated with a specific antigen. The protection was correlated with the spleen and blood CD8(+) T cell, high-affinity IgG and IgM and antigen-induced IL-10 and IL-4 production. Immunization with irradiated T. gondii tachyzoites induces an immune response that is mediated by B cells and CD4(+) and CD8(+) T cells, with increased humoral and cellular immune responses that are necessary for host protection after infection. The vaccine is similar to natural infection, but free of tissue cysts; this immunity restrains infection at challenge and can be an attractive and efficient model for vaccine development in toxoplasmosis.

Uptake and antileishmanial activity of meglumine antimoniate-containing liposomes in Leishmania (Leishmania) major-infected macrophages.

Leishmaniasis is a parasitic disease caused by the intramacrophage protozoa Leishmania spp. and may be fatal if left untreated. Although pentavalent antimonials are toxic and their mechanism of action is unclear, they remain the first-line drugs for treatment of leishmaniasis. An effective therapy could be achieved by delivering antileishmanial drugs to the site of infection. Compared with free drugs, antileishmanial agent-containing liposomes are more effective, less toxic and have fewer adverse side effects. The aim of this study was to develop novel meglumine antimoniate (MA)-containing liposome formulations and to analyse their antileishmanial activity and uptake by macrophages. Determination of the 50% inhibitory concentration (IC(50)) values showed that MA-containing liposomes were ≥10-fold more effective than the free drug, with a 5-fold increase in selectivity index, higher activity and reduced macrophage toxicity. The concentration required to kill 100% of intracellular amastigotes was ≥40-fold lower when MA was encapsulated in liposomes containing phosphatidylserine compared with the free drug. Fluorescence microscopy analysis revealed increased uptake of fluorescent liposomes in infected macrophages after short incubation times compared with non-infected macrophages. In conclusion, these data suggest that MA encapsulated in liposome formulations is more effective against Leishmania-infected macrophages than the non-liposomal drug. Development of liposome formulations is a valuable approach to the treatment of infectious diseases involving the mononuclear phagocyte system.

Humoral responses and immune protection in mice immunized with irradiated T. gondii tachyzoites and challenged with three genetically distinct strains of T. gondii.

Toxoplasma gondii is an obligate intracellular parasite that infects a variety of mammals and birds. T. gondii also causes human toxoplasmosis; although toxoplasmosis is generally a benign disease, ocular, congenital or reactivated disease is associated with high numbers of disabled people. Infection occurs orally through the ingestion of meat containing cysts or by the intake of food or water contaminated with oocysts. Although the immune system responds to acute infection and mediates the clearance of tachyzoites, parasite cysts persist for the lifetime of the host in tissues such as the eye, muscle, and CNS. However, T. gondii RH strain tachyzoites irradiated with 255Gy do not cause residual infection and induce the same immunity as a natural infection. To assess the humoral response in BALB/c and C57BL/6J mice immunized with irradiated tachyzoites either by oral gavage (p.o.) or intraperitoneal (i.p.) injection, we analyzed total and high-affinity IgG and IgA antibodies in the serum. High levels of antigen-specific IgG were detected in the serum of parenterally immunized mice, with lower levels in mice immunized via the oral route. However, most serum antibodies exhibited low affinity for antigen in both mice strain. We also found antigen specific IgA antibodies in the stools of the mice, especially in orally immunized BALB/c mice. Examination of bone marrow and spleen cells demonstrated that both groups of immunized mice clearly produced specific IgG, at levels comparable to chronic infection, suggesting the generation of IgG specific memory. Next, we challenged i.p. or p.o. immunized mice with cysts from ME49, VEG or P strains of T. gondii. Oral immunization resulted in partial protection as compared to challenged naive mice; these findings were more evident in highly pathogenic ME49 strain challenge. Additionally, we found that while mucosal IgA was important for protection against infection, antigen-specific IgG antibodies were involved with protection against disease and disease pathogenesis. Most antigen responsive cells in culture produced specific high-affinity IgG after immunization, diverse of the findings in serum IgG or from cells after infection, which produced low proportion of high-avidity IgG.

200 Gy sterilised Toxoplasma gondii tachyzoites maintain metabolic functions and mammalian cell invasion, eliciting cellular immunity and cytokine response similar to natural infection in mice.

200Gy gamma-irradiated Toxoplasma gondii RH tachyzoites failed to reproduce in vitro and in vivo. In short-term cultures, these parasites maintained a respiratory response, the ability to invade cells and preserved protein and nucleic acid synthesis. ELISA and Western blotting techniques demonstrated the similarity in humoral response between mice infected with gamma-irradiated tachyzoites and animals infected with naive parasites and treated with sulfadoxine, higher than mice immunised with formaldehyde-killed tachyzoites. Splenocyte stimulation by T. gondii antigen produced lymphoproliferative response and cytokine profile (IL-10, IL-12, IFN-gamma and TNF-alpha) similar to those produced by chronic natural infection. Mice immunised with irradiated tachyzoites had extended survival times after subsequent tachyzoite challenge, and displayed minimal cerebral pathology after cyst challenge. Irradiated tachyzoites lose their reproductive ability whilst maintaining metabolic function and may provide a novel tool for the study of toxoplasmosis and vaccine development.