Immunization with purine salvation pathway recombinant enzymes induces the production of anti- Schistosoma mansoni immunoglobulines

Schistosomiasis, a disease caused by the trematodes of the Schistosoma genus, affects about 240 million people worldwide. The Praziquantel (PZQ) is the drug used to treat this disease. However, reports of resistant strains reinforce the need to develop a new schistosomicidal drug. The study of new drugs and vaccines that can contribute to the control of this pathology becomes urgent. A new approach can be held by the study of the following Schistosoma mansoni enzymes: purine nucleoside phosphorilase 1 (PNP), hypoxanthine guanine phosphoribosyltransferase (HGPRT) and adenylate kinase 1 (ADK). The parasite, incapable of synthetizing purine nucleotides through the de novo pathway, has multiple mechanisms to incorporate purine bases through the purine salvage pathway. Our goal was to assess, through immunoenzimatic assay (ELISA-indirect), the production of total IgG, IgE and IgG2a in the plasma after immunization with the PNP, HGPRT and ADK enzymes, using the S. mansoni cercariae - infected murine model. Our results showed that the immunization in Balb/c mice with the enzymes mentioned above induced production of the immunoglobulines at the 48th and 85th days, post-infection. Thereby, new assays must be made for a better assessment on how these enzymes modulate an immune response.CNPqFAPES

Evaluation of immunization with purine salvation pathway recombinant enzymes in Schistosoma mansoni worms and eggs in murine schistosomiasis

According to the World Health Organization (WHO), on tropical and subtropical areas, schistosomiasis is the second parasitic disease of greater prevalence, in terms of morbidity and mortality, surpassed only by malaria. The Praziquantel (PZQ) is used for the treatment of this disease. However, reports of resistant strains reinforce the need to develop a new schistosomicidal drug. The infection by the parasite induces an inflammatory reaction of long duration due to the presence of adult worms living in the mesenteric venous system. The parasite lays eggs in small vessels of the submucosa of the intestines. These eggs are transported by the blood flow to the liver and they cause a granulomatous inflammatory reaction. A new approach can be held by the study of the following Schistosoma mansoni enzymes: purine nucleoside phosphorilase 1 (PNP), hypoxanthine guanine phosphoribosyltransferase (HGPRT) and adenylate kinase (ADK). The parasite, incapable of synthetizing purine nucleotides through the de novo pathway, has multiple mechanisms to incorporate purine bases through the purine salvage pathway. In our results, we suggest that the immunization in Balb/c mice with the mentioned recombinant enzymes was capable of inducing a specific immune response, favoring the reduction of both the parasite load and number of eggs per gram of feces. The acquired data show that these enzymes can be considered as new targets to immunotherapy against schistosomiasis mansoni.CNPqFAPES

Modulating effect of total extract of Paenibacillus polymyxa RNC-D on macrophages and Leishmania (Leishmania) amazonensis in vitro

Leishmaniasis is an infection caused by Leishmania gender protozoa. The treatment of the disease is generally problematic, as the drugs used on clinical practice are toxic. One of the paths in the search for new therapeutic targets is the study of endophytic microorganisms-produced substances. In this study, the Total Lyophilized Extract (TLE) of the endophytic Paenibacillus polymyxa RNC-D was used, which was isolated from the Cerrado of São Carlos, Brazil. The TLE was assessed in terms of citotoxicity, ON production and citokynes production on BALB/3T3 fibroblasts, J774A.1 macrophages, RAW 264.7 macrophages, as well as directly on promastigote and amastigote stages of Leishmania (L) amazonensis. Our results have shown that the mortality rate of 50% (EC50) of fibroblasts (BALB/3T3) was of 1,171 mg/mL and 0,956 mg/mL after 48 and 72 hours, respectively. For macrophages (J774A.1) the mortality rate of 50% took place on doses of 0,994 mg/mL and 0,945 mg/mL after 48 and 72 hours, respectively. Regarding cellular death, at the period of 24 hours, the extract induced apoptosis and necrosis from the concentration of 5mg/mL in fibroblast (BALB/3T3) and from ≈1 mg/mL in macrophage (J774A.1). The threatment with ≈1 mg/mL concentration of the ETL induced TNF-α, IFN-γ and IL-10 cytokine production at the 24 hours period. The IL-10 had its production detected up to the 48 hours period, whereas the IL-12 was detected only at this period. For the toxicity essay on L. amazonensis promastigotes, the values of EC50 obtained at the 24 and 48 hours period were of 0,624 mg/mL and 0,547 mg/mL, respectively. Regarding promastigotes death percentage, our result have shown that the 0,5 mg/mL concentration induced death of 31% at the 24 hours period and 35% at 48 hours. As for the concentration of 1mg/mL of the extract, the death percentage was of 59% and 62% at the periods of 24 and 48 hours, respectively. For the parasitary load essay, it was observed a percentage of 25% of infected cells with na average of 3 amastigotes/cell for the groups treated with 0,1; 0,5 and 1 mg/mL of the TLE at both periods. Regarding cytokine production on RAW 264.7 macrophage that were infected/treated with the extract, a significant increase of TNF-α was observed at both periods for all tested concentrations. Concerning IFN-γ cytokine, its significant production was only observed at the 48 hours period in the 1mg/mL concentration, whereas the IL-12 production was only significant in the 0,5 mg/mL at the same period. A significant decrease of IL-4 was observed in all treated groups with the TLE at the 24 hours period. The significant ON production by RAW 264.7 macrophages was observed in the groups treated with 0.5 mg/mL and 1 mg/mL of the extract on both periods of 48 and 72 hours. Due to its modulatory action of the immunological activity, it is suggested that the TLE of P. Polymyxa RNC-D is a promising target for the development of new immunotherapeutical drugs for the treatment of several diseases, including, but not limited to, leishmaniasis.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)A leishmaniose é uma infecção causada por protozoários do gênero Leishmania. O tratamento desta doença é usualmente problemático, uma vez que os medicamentos utilizados na prática clínica são tóxicos. Um dos caminhos na busca por novos alvos terapêuticos é o estudo de moléculas produzidas por microrganismos endofíticos. Neste estudo, utilizou-se o Extrato Total Liofilizado (ETL) do endofítico Paenibacillus polymyxa RNC-D, isolado do Cerrado de São Carlos - Brasil. O extrato foi avaliado em fibroblasto BALB/3T3, macrófago J774A.1, macrófago RAW 264.7 e direto em formas promastigota e amastigota de Leishmania (L) amazonensis. Dessa forma, foi avaliado nestas células a citotoxicidade, produção de óxido nítrico (NO) e produção de citocinas. Nossos resultados mostraram que a taxa de mortalidade de 50% (EC50) de fibroblasto (BALB/3T3) foi observada em 1,171 mg/mL e 0,956 mg/mL após 48 e 72 horas, respectivamente. Em macrófago (J774A.1) a EC50 foi de de 0,994 mg/mL e 0,945 mg/mL após 48 e 72 horas, respectivamente. Em relação à morte celular no período de 24 horas, o extrato induziu apoptose e necrose a partir da concentração de 5 mg/mL em fibroblasto (BALB/3T3) e de ≈1 mg/mL em macrófago (J774A.1). O tratamento com ≈1 mg/mL da concentração do extrato induziu a produção das citocinas TNF-α, IFN-γ e IL-10, em 24 horas. A IL-10 teve sua produção detectada até 48 horas e IL-12 foi detectada a partir deste período. Para o ensaio de toxicidade em promastigotas de L. amazonensis, os valores da EC50 obtidos nos períodos de 24 horas e de 48 horas foram de 0,624 mg/mL e de 0,547 mg/mL, respectivamente. Em relação à porcentagem de morte das promastigotas, nossos resultados mostraram que na concentração de 1 mg/mL do extrato, a porcentagem de morte no período de 24 horas foi de 59% e de 62% no período de 48 horas. Para o ensaio da carga parasitária, todos os grupos tratados (0,1; 0,5 e 1 mg/mL) do extrato apresentaram uma porcentagem de aproximadamente 25 % de células infectadas com média de 3 amastigotas/célula para ambos os períodos. Em relação à produção de citocinas em macrófagos RAW 264.7 infectados/tratados com o extrato, observou-se um aumento significativo de TNF-α, nos períodos de 24 e 48 horas, em todos os grupos tratados com o extrato. Quanto à citocina IFN-γ, só foi observada a sua produção significativa no período de 48 horas na concentração de 1 mg/mL, assim como a produção de IL-12, só foi significativa na concentração de 0,5 mg/mL no mesmo período. Observou-se uma diminuição significativa de IL-4 em todos os grupos tratados com o extrato no período de 24 horas. A produção significativa de NO por macrófago RAW 264.7 no período de 48 e 72 horas foi observada nos grupos tratados com 0,5 mg/mL e 1 mg/mL do extrato. Devido à sua ação modulatória da atividade imunológica, sugere-se que o extrato em questão seja um alvo promissor para o desenvolvimento de novos medicamentos imunoterapêuticos para o tratamento de diversas doenças, incluindo, mas não limitado a, a leishmaniose

Efeito da imunização com enzimas recombinantes do metabolismo de nucleotídeos de Schistosoma mansoni sobre o desenvolvimento da esquistossomose mansônica experimental

Schistosimiasis mansoni is a neglected chronic parasitic disease that affects thousands of people worldwide, caused by the trematode Schistosoma mansoni. In the infected host the disease is characterized by the presence of granuloma, imunnopathological response of the cellular infiltration against egg antigens. Thus, the host-parasite relation favors hepatosplenomegaly, acite and hepatic fibrosis. Current chemotherapy is based on the use of Praziquantel (PZQ), used against all species of Schistosoma spp for over 30 years. The main issue is that the PZQ is practically inactive against immature schistosomula and favors the resistance growth of the existent lineages, which makes the study for new drugs and vaccines that can contribute to the control of this disease even more urgent. One of the paths on the search for new therapeutic targets is the study of essential enzymes to the S. mansoni. In particular, it is known that the enzymes Adenylate Kinase 1 and 2 (ADK), Uridine cytidine Kinase 1 and 2 (UCK), Hypoxanthine guanine phosphoribosyltransferase (HGPRT) e Purine nucleoside phosphorilase 1 (PNP) are found on the metabolic pathways of the parasite s nucleotide, participating in the metabolism of purines and pyrimidines. Our goals in this study were to assess the immunization with these enzymes, using the S. mansoni cercariae infected murine model, and subsequently analyze the acting in oviposition and growth of adult worms. Our results showed that the immunization in Balb/c mice with the UCK enzyme was capable of inducing a specific immune response, which favored a significant reduction of the parasitic load (adult worms). However, it was not possible to observe significant reduction in the number of eliminated eggs. Regarding the immunization with PNP and HGPRT enzymes, the mice had a reduction in the number of eggs per gram of feces. The data obtained are considered interesting and can be new targets for immunotherapy against schistosomiasis mansoni. Thereby, new assays must be made with different dosages of the enzymes for a better assessment on how these enzymes modulate the parasitic load through the eggs reduction, reduction in the adult worms retrieving, as well as the antibody levels during the murine infection by the S.mansoni.Universidade Federal de Sao CarlosA esquistossomose mansônica é uma doença parasitária, crônica e negligenciada que afeta milhares de pessoas ao redor do mundo, causada pelo trematódeo Schistosoma mansoni. No hospedeiro infectado a doença é caracterizada pela presença do granuloma, resultado imunopatológico do infiltrado celular contra antígenos dos ovos A quimioterapia atual é baseada no uso do Praziquantel (PZQ), usado contra todas as espécies de Schistosoma spp há mais de 30 anos. O principal problema é que o PZQ é praticamente inativo contra esquistossomulos imaturos e favorece o desenvolvimento de resistência das linhagens existentes. Um dos caminhos na busca por novos alvos terapêuticos é o estudo de enzimas que são essenciais para o S. mansoni. Em especial, sabe-se que as enzimas Adenilato Quinase 1 e 2 (ADK), Uridina Citidina Quinase 1 e 2 (UCK), Hipoxantina-guanina fosforibosiltransferase (HGPRT) e Purina Nucleosídeo Fosforilase 1 (PNP) são encontradas nas vias metabólicas de nucleotídeos do parasito, participando do metabolismo de purinas e pirimidinas. A estratégia de utilizar enzimas do parasito na esquistossomose mansônica murina foi de avaliar uma resposta induzida por estas enzimas quando aplicadas em camundongos BALB/c e desafiados com cercarias de S. mansoni. Desta forma, avaliamos a fase crônica de camundongos imunizados e infectados com S. mansoni, onde foram analisadas amostras parasitológicas, hematológicas, sorológicas e fluidos da cavidade peritoneal. Nosso objetivo neste estudo foi avaliar a imunização com essas enzimas, usando o modelo murino infectado com cercarias de S. mansoni e posteriormente avaliar a ação na oviposição e desenvolvimento de vermes adultos. Nossos resultados demonstraram que a imunização em camundongos Balb∕c com a enzima UCK foi capaz de induzir uma resposta imune específica, a qual favoreceu a diminuição significativa da carga parasitária (vermes adultos). No entanto, não foi possível observar redução significativa no número de ovos eliminados. Em relação à imunização com as enzimas PNP e HGPRT os camundongos que receberam as imunizações com PNP e HGPRT tiveram redução no número de ovos por grama de fezes. Os dados obtidos são considerados interessantes e podem ser considerados novos alvos para a imunoterapia contra a esquistosomose mansônica. Dessa forma, novos ensaios deverão ser realizados com diferentes doses das enzimas para melhor avaliar como essas enzimas modulam a carga parasitária através da redução de ovos, diminuição na recuperação de vermes adultos, assim como os níveis de anticorpos durante a infecção murina pelo S. mansoni

In Vitro Modulator Effect of Total Extract from the Endophytic Paenibacillus polymyxa RNC-D in Leishmania (Leishmania) amazonensis and Macrophages

Leishmaniases are diseases with high epidemiological relevance and wide geographical distribution. In Brazil, Leishmania (Leishmania) amazonensis is related to the tegumentary form of leishmaniasis. The treatment for those diseases is problematic as the available drugs promote adverse effects in patients. Therefore, it is important to find new therapeutic targets. In this regard, one alternative is the study of biomolecules produced by endophytic microorganisms. In this study, the total extract produced by the endophytic Paenibacillus polymyxa RNC-D was used to evaluate the leishmanicidal, nitric oxide, and cytokines production using RAW 264.7 macrophages. The results showed that, in the leishmanicidal assay with L. amazonensis, EC50 values at the periods of 24 and 48 hours were 0.624 mg/mL and 0.547 mg/mL, respectively. Furthermore, the cells treated with the extract presented approximately 25% of infected cells with an average of 3 amastigotes/cell in the periods of 24 and 48 hours. Regarding the production of cytokines in RAW 264.7 macrophages infected/treated with the extract, a significant increase in TNF-α was observed at the periods of 24 and 48 hours in the treated cells. The concentrations of IFN-γ and IL-12 showed significant increase in 48 hours. A significant decrease in IL-4 was observed in all cells treated with the extract in 24 hours. It was observed in the treated cells that the NO production by RAW 264.7 macrophages increased between 48 and 72 hours. The endophytic Paenibacillus polymyxa RNC-D extract modulates the mediators of inflammation produced by RAW 264.7 macrophages promoting L. amazonensis death. The immunomodulatory effects might be a promising target to develop new immunotherapeutic and antileishmanial drugs

Robust Phenotypic Activation of Eosinophils during Experimental Toxocara canis Infection

Eosinophils are multifunctional cells that have cytotoxic proinflammatory activities and stimulate CD4+ T-cells in experimental models of allergy and parasitic infections. Eosinophils, when exposed to antigens, are activated, expressing the CD38/CD69 molecules and exhibited increased expression of major histocompatibility complex (MHC-II), CD80 and CD86, suggesting they play a role upon Toxocara canis antigen stimulation. In the present study, we evaluated the profile of eosinophils using conventional and image flow cytometry upon experimental T. canis infection. T. canis antigens induced a robust activation on this subset, contributing to the immune responses elicited in the experimental model for T. canis-associated visceral larva migrans syndrome. Data analysis demonstrated that, during murine T. canis infection, eosinophils from peripheral blood, spleen, and bone marrow presented upregulated expression of CD69/MHC-II/CD80/CD86. As opposed to splenic and bone marrow eosinophils, circulating eosinophils had increased expression of activation markers upon T. canis infection. The enhanced connectivity between eosinophils and T-cells in T. canis-infected mice in all three compartments (peripheral blood, spleen, and bone marrow) also supports the hypothesis that eosinophils may adopt a role during T. canis infection. Moreover, in vitro T. canis antigen stimulation resulted in activation and upregulation of co-stimulatory-related molecules by bone marrow-derived eosinophils. Our findings are evidence of activation and upregulation of important activation and co-stimulatory-related molecules in eosinophils and suggest a reshape of activation hierarchy toward eosinophils during experimental T. canis infection