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

Avaliação da expressão das moléculas CD80, CD86 e MHCII em eosinófilos durante a síndrome da larva migrans visceral

Eosinophils are a hematopoietic cell originated from precursor cells found in bone marrow, whose differentiation and proliferation is regulated by cytokines such as GM-CSF, IL-3 and IL-5. When activated, eosinophils are capable of phagocytosis of small particles and bacteria, but their main form of activity in the inflammatory process is the release of toxic proteins, cytokines, enzymes, lipid mediators and reactive oxygen products. The increase in eosinophil is an important feature in many diseases such as allergy and parasitic infections. Provided APC (Antigen-Presenting Cells), eosinophils are considered similar to the CD (Dendritic Cells) in its potential to activate naïve T cells and may have potential as efficient as the CD in stimulating lung T cells in the upper airways in the model inflammation. The APC are defined by being able to take, processing and presenting antigen such as CD, macrophages, B lymphocytes and possibly eosinophils. The surface expression of APC is characterized by coestimatórias molecules CD80 (B7-1) and CD86 (B7-2) and also by MHCII. The proposed model for this evaluation was to Visceral Larva Migrans syndrome (VLMS) caused by Toxocara canis, one of the most frequent helminth in young dogs. One of the main consequences of this infection is the marked increase in circulating and tissue eosinophils. Eosinophilia has been associated with parasitic diseases particularly when the parasite invades or promotes tissue damage at mucosal surfaces In the present study we evaluated the expression of MHC II and CD80 and CD86 molecules coestimulatórias in eosinophils in VLMS. Our results showed that the molecules studied were expressed in eosinophils in the blood of mice infected with Toxocara canis compared with the control group. Correlating an intense eosinophil still during the course of the disease with increased IL-5 in the infected group. Suggests that during the course of Toxocara canis, eosinophils can exhibit behavior of an APC, increasing the expression of MHCII molecules coestimulatorias and possibily amplifyng the immune response in this model.O eosinófilo é uma célula hematopoiética, originada a partir de células precursoras presentes na medula óssea, cuja diferenciação e proliferação são reguladas por citocinas como GMCSF, IL-3 e IL-5. Quando ativados, os eosinófilos são capazes de realizar fagocitose de pequenas partículas e bactérias, mas sua principal forma de atuação no processo inflamatório consiste na liberação de proteínas tóxicas, citocinas, enzimas, mediadores lipídicos e produtos reativos de oxigênio. O aumento no número de eosinófilos é uma característica importante em diversas doenças como a alergia e as infecções parasitárias. Na condição de APC (Células Apresentadoras de Antígenos), os eosinófilos são considerados similares as CD (Células Dendríticas) em seu potencial para ativar células T naïve, podendo ter potencial tão eficiente quanto as CD pulmonares em estimular células T nas vias aéreas superiores no modelo da inflamação. As APC são definidas por serem capazes de ingerir, processar e apresentar o antígeno como: CD, macrófagos, linfócitos B e possivelmente os eosinófilos. A expressão na superfície da APC é caracterizada por moléculas coestimatórias CD80 (B7-1) e CD86 (B7-2) e ainda pelo MHCII. O modelo proposto para esta avaliação foi a Síndrome da Larva Migrans Visceral (SLMV) causada pelo Toxocara canis, um dos helmintos mais freqüentes em cães jovens. Uma das principais consequências desta infecção é o aumento marcante de eosinófilos circulantes e teciduais. A eosinofilia tem sido associada com doenças parasitárias particularmente quando o parasita invade os tecidos ou promove danos na superfície das mucosas. No presente estudo avaliamos a expressão de MHC II e moléculas coestimulatórias CD80 e CD86 em eosinófilos na SLMV. Nossos resultados mostraram que as moléculas analisadas foram expressas em eosinófilos no sangue de camundongos infectados com Toxocara canis quando comparado com o grupo controle. Correlacionando ainda uma intensa eosinofilia durante o curso da doença com o aumento de IL-5 no grupo infectado. Sugere que, durante o curso da infecção pelo Toxocara canis, eosinófilos podem apresentar comportamento de uma APC, aumentando a expressão de moléculas coestimulatórias e MHCII e possivelmente amplificando a resposta imune nesse modelo

Eosinophils as antigen presenting cells during toxocara canis experimental infection

Eosinophils are multifunctional cells that have pro-inflammatory cytotoxic activities and stimulate CD4 + T cells in experimental models of allergy and parasitic infections. Eosinophils, when exposed to antigens are activated and express CD38 / CD69 molecules and exhibit increased expression of the Major Histocompatibility Complex (MHC-II), CD80 and CD86, suggesting their role as atypical cells in the Presentation of Antigens. In the present study, we evaluated the hypothesis that, in addition to professional APCs (monocyte, dendritic and B cell), eosinophils are activated by Toxocara canis antigens and express a range of cell surface markers characteristic of antigen presenting cells. Thus, contributing to the immune responses induced in the experimental model by Visceral Migrans Larva syndrome associated with T. canis infection (VLMS). By means of flow cytometric assays the cell profiles of antigen-presenting cells were evaluated during T. canis experimental infection. Data analysis demonstrated that, during murine T. canis infection, peripheral blood, spleen and bone marrow eosinophils showed regulated expression of CD69 / MHC-II / CD80 / CD86, assuming a role as APC in comparison with the antigen presenting cells. Unlike eosinophils of the spleen and bone marrow, circulating eosinophils showed increased expression of activation markers along with APC-related molecules after T. canis infection. The same activation was not observed for professional APCs in the same compartments. Improved 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 play a role as PCA during T. canis infection. In addition, in vitro stimulation of T. canis antigen resulted in activation and elevation of regulation of APC-related molecules by eosinophils derived from bone marrow. These findings strongly suggest that T. canis infection reshapes eosinophils to act as APCs in this infection and not only as an effector cell in the control of parasitic infection.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Os eosinófilos são células multifuncionais que possuem atividades pró-inflamatórias citotóxicas e estimulam as células T CD4 + em modelos experimentais de alergia e infecções parasitárias. Os eosinófilos, quando expostos a antígenos são ativados e expressam as moléculas CD38/CD69 e exibiram uma expressão aumentada do Complexo de Histocompatibilidade Maior (MHC-II), CD80 e CD86, sugerindo seu papel como células atípicas na Apresentação de Antigênos. No presente estudo, avaliamos a hipótese que, além das APC profissionais (macrófagos monócitos, células dendríticas e células B), os eosinófilos são ativados por antígenos Toxocara canis e expressam uma gama de marcadores de superfície celular característicos das células apresentadoras de antígenos. E desse modo, contribuindo para as respostas imunes induzidas no modelo experimental pela síndrome da Larva Migrans Visceral associada a infecção com T. canis (VLMS). Por meio dos ensaios utilizando citometria de fluxo foram avaliados os perfis celulares das células que apresentam antigeno durante a infecção experimental pelo T. canis. A análise dos dados demonstrou que, durante a infecção murina de T. canis, os eosinófilos do sangue periférico, do baço e da medula óssea apresentaram a expressão regulada de CD69 / MHC-II / CD80 / CD86, assumindo um papel como APC em comparação com as células apresentadoras de antigenos profissionais. Ao contrário dos eosinófilos do baço e da medula óssea, os eosinófilos circulantes apresentaram aumento da expressão de marcadores de ativação juntamente com moléculas relacionadas à APC após a infecção por T. canis. A mesma ativação não foi observada para APCs profissionais nos mesmos compartimentos. A conectividade aprimorada entre eosinófilos e células T em camundongos infectados por T. canis em todos os três compartimentos (sangue periférico, baço e medula óssea) também suporta a hipótese de que os eosinófilos podem adotar um papel como APC durante a infecção por T. canis. Além disso, a estimulação in vitro de antígeno de T. canis resultou em ativação e elevação da regulação de moléculas relacionadas à APC por eosinófilos derivados da medula óssea. Essas descobertas sugerem fortemente que a infecção por T. canis remodela os eosinófilos para atuar como APCs nessa infecção e não somente como uma célula efetora no controle da infecção parasitária.FAPESP: 2012/08024-

Carbon Black CB-EDA Nanoparticles in Macrophages: Changes in the Oxidative Stress Pathway and in Apoptosis Signaling

The influence of black carbon nanoparticles on J774.A1 murine cells was investigated with the objective of exploring the cytotoxicity of black carbon functionalized with ethylenediamine CB-EDA. The results showed that CB-EDA has a cytotoxic profile for J774.A1 macrophages in a time- and dose-dependent manner. When phagocytosed by the macrophage, CB-EDA triggers a mechanism that leads to apoptosis. In this process, there is an increase in oxidative stress pathways due to the activation of nitric oxide and then ROS. This causes an imbalance in redox function and a disruption of membrane integrity that occurs due to high levels of LDH, in addition to favoring the release of the pro-inflammatory cytokines IL-6, IL-12, and tumor necrosis factor (TNF) in an attempt to modulate the cell. However, these stimuli are not sufficient to repair the cell and the level of mitochondrial integrity is affected, causing a decrease in cell viability. This mechanism may be correlated with the activation of the caspasse-3 pathway, which, when compromised, cleaves and induces cells death via apoptosis, either through early or late apoptosis. In view of this, the potential for cell damage was investigated by analyzing the oxidative and inflammatory profile in the macrophage lineage J774.A1 and identifying potential mechanisms and metabolic pathways connected to these processes when cells were exposed to NP CB-EDA for both 24 h and 48 h.The authors would like to thank Márcia Regina Cominetti (Departamento de Gerontologia, Universidade Federal de São Carlos, São Carlos, SP, Brazil) for availability of equipment. M.A. was supported by the Margarita Salas postdoctoral contract MGS/2021/21 (UP2021-021) financed by the European Union—Next Generation EU. The authors would also like to express their gratitude for the financial aid from the Fundação de Amparo à Pesquisa do Estado de São Paulo—FAPESP (2013/07296-2), the Financiadora de Estudos e Projetos—FINEP, the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—CAPES (Financial Code 001) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq

Efeito de bioterápico na eosinofilia durante a SLMV experimental

O Toxocara canis (Tc) é um parasito pertencente ao filo Nematódeo que possui como hospedeiro definitivo os cães, O homem é hospedeiro paratênico e contamina-se acidentalmente ao ingerir ovos contendo larvas infectantes (L3) do parasito, as quais são liberadas e atravessam a mucosa intestinal, atingem a circulação, Durante este processo migratório, antígenos de excreção e secreção (TES) são liberados provocando intensa reação inflamatória, do tipo Th2, caracterizando a síndrome, denominada Larva Migrans Visceral (SLMV), As principais características desta doença crônica são as eosinofilias sanguínea e tecidual persistentes, Desse modo, torna-se importante a busca por terapias que contribuam com a redução dos quadros inflamatórios com intensa eosinofilia, Assim, o uso deste bioterápico, produzido a partir do extrato antigênico de ovos e larvas de (Tc), e seu efeito no recrutamento de leucócitos totais, células mononucleares e eosinófilos no sangue, para o espaço broncoalveolar e para a cavidade peritoneal de camundongos infectados pelo (Tc) foi investigado, Foram utilizados camundongos fêmeas (Swiss), divididos nos grupos: Controle (C), Infectado (Tc), Imunizado (Im+Tc) e Tratado (Tc+Bio), Os animais Tc, Im+Tc e Tc+Bio receberam 500 ovos/animal por gavagem, Posteriormente, os animais foram eutanasiados no 18º dia da infecção e o número das células nos compartimentos foi determinado, Os resultados obtidos demonstraram que, Im+Tc, assim como nos Tc+Bio tiveram redução significativa dessas células nos compartimentos analisados quando comparados grupo Tc, Assim, sugeriu-se que a bioterapia modulou negativamente o recrutamento de células inflamatórias, principalmente eosinófilos no sangue, pulmão e intestino demonstrando um potencial anti-inflamatório desse bioterápico na SLMV experimental

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

HGPRT and PNP: Recombinant Enzymes from <i>Schistosoma mansoni</i> and Their Role in Immunotherapy during Experimental Murine Schistosomiasis

Schistosomiasis is a parasitic infection caused by trematode worms (also called blood flukes) of the genus Schistosoma sp., which affects over 230 million people worldwide, causing 200,000 deaths annually. There is no vaccine or new drugs available, which represents a worrying aspect, since there is loss of sensitivity of the parasite to the medication recommended by the World Health Organization, Praziquantel. The present study evaluated the effects of the recombinant enzymes of S. mansoni Hypoxanthine-Guanine Phosphoribosyltransferase (HGPRT), Purine Nucleoside Phosphorylase (PNP) and the MIX of both enzymes in the immunotherapy of schistosomiasis in murine model. These enzymes are part of the purine salvage pathway, the only metabolic pathway present in the parasite for this purpose, being essential for the synthesis of DNA and RNA. Female mice of Swiss and BALB/c strains were infected with cercariae and treated, intraperitoneally, with three doses of 100 µg of enzymes. After the immunotherapy, the eggs and adult worms were counted in the feces; the number of eosinophils from the fluid in the peritoneal cavity and peripheral blood was observed; and the quantification of the cytokine IL-4 and the production of antibodies IgE was analyzed. The evaluation of the number of granulomas and collagen deposition via histological slides of the liver was performed. The results demonstrate that immunotherapy with the enzyme HGPRT seems to stimulate the production of IL-4 and promoted a significant reduction of granulomas in the liver in treated animals. The treatment with the enzyme PNP and the MIX was able to reduce the number of worms in the liver and in the mesenteric vessels of the intestine, to reduce the number of eggs in the feces and to negatively modulate the number of eosinophils. Therefore, immunotherapy with the recombinant enzymes of S. mansoni HGPRT and PNP might contribute to the control and reduction of the pathophysiological aspects of schistosomiasis, helping to decrease the morbidity associated with the infection in murine model