Imunolocalização da SmATPDase 2 de Schistosoma mansoni e estudo dos peptídeos sintéticos SmB1LJ e SmB2LJ derivados desta proteína na esquistossomose experimental

Schistosomiasis is a disease that affects approximately 200 million people worldwide causing great morbidity. Due to lack of vaccine for this disease, many antigens are searched. An antigenic domain B (r156-195) from the Schistosoma mansoni ATPDase 2 isoform was previously described, and two synthetic peptides (SmB1LJ, r155-176; SmB2LJ, r175-194) belonging to this domain were obtained. Inoculation of each peptide in healthy BALB/c mice induced significant immunostimulatory activity, increasing the IgG1 and IgG2a antibody levels, as detected by ELISA. The polyclonal anti-SmB1LJ and anti-SmB2LJ antibodies, when immobilized on Protein A-Sepharose, immunoprecipitated approximately 42-96% of the ADPase activity from the adult worm preparation. By Western blots of immunoprecipitated complex, three bands of 76, 63 and 55 kDa were identified by the two immune sera, confirming the ATPDase 2 identity, and suggesting that this protein is subject to several posttranslational processing, such as glycosylation and proteolysis, resulting finally in a secreted protein of 55 kDa. Additionally, polyclonal anti-SmB2LJ antibodies inhibited 62% and 37% of the ATPase and ADPase activities, respectively, whereas polyclonal anti-SmB1LJ antibodies inhibited ADPase (92%), but not ATPase activity, suggesting that the domain B from the S. mansoni ATPDase 2 is involved in enzyme catalytic regulation, being a new target for inhibitor design. Immunolabelled cryostat sections of infected mouse liver by the anti-SmB2LJ polyclonal antibodies revealed positive reactions on the external surface from the miracidium in the S. mansoni egg. Intense fluorescence was detected in the region between the miracidium and the inner side of the egg-shell, located in von Lichtenberg’s envelope, which is a region of production of immunogenic components. Fluorescence was also detected in the outer side of the egg-shell and entrapped by the surface microspines, confirming that this protein, like in the adult worm, is also secreted from the S. mansoni eggs. No reactivity was observed in the surrounding granumomatous tissues, which are rich en inflammatory cells. Serum samples from S. mansoni-infected Swiss mice were tested by ELISA using SmB1LJ or SmB2LJ as coating antigen. Significantly higher IgG, IgG1 or IgG2a antibody reactivity was detected against SmB1LJ, whereas only IgG1 antibody was highly reactive against SmB2LJ. The reactivity of plasma samples (diluted 1:200) from Swiss mice previously inoculated with potato apyrase or r-potDomain B, a 6xHis tag polypeptide belonging to the domain B (r78-117) from the potato apyrase, homologue to the domain B from the ATPDase 2, was also tested by ELISA using the peptides as coating antigens. Polyclonal IgG and IgG1 anti-potato apyrase antibodies significantly reacted with SmB1LJ, whereas only IgG1 reacted with SmB2LJ. Polyclonal anti-r-potDomain B antibodies did not significantly react with SmB2LJ. Compared to controls, plasma samples (diluted 1:200) from these animals pre-immunized with potato apyrase or r-potDomain B and challenged with S. mansoni did not react significantly with peptides. The results confirmed that the domain B from the ATPDase 2 is potentially involved in the host immune response, and peptides SmB1LJ and SmB2LJ can be used in schistosomiasis studies.A esquistossomose é uma doença que acomete cerca de 200 milhões de pessoas no mundo causando grande morbidade. Devido à falta de vacina para essa doença, muitos antígenos são pesquisados. Um domínio antigênico B (r156-195) da ATPDase 2 de Schistosoma mansoni foi previamente descrito, e dois peptídeos sintéticos (SmB1LJ, r155-176; SmB2LJ, r175-194) derivados deste domínio foram obtidos com o objetivo de avaliar a imunogenicidade dessa proteía. O inóculo de cada peptídeo em camundongos BALB/c induziu atividade imunoestimulatória significativa, elevando os níveis de anticorpos IgG1 e IgG2, como detectado por ELISA. Os anticorpos policlonais anti-SmB1LJ e anti-SmB2LJ, quando imobilizados em Proteína A-Sepharose, imunoprecipitaram aproximadamente 42-96% da atividade ADPásica da preparação de verme adulto. Por “Western blots” do complexo imunoprecipitado, três bandas de 76, 63 e 55 kDa foram identificadas pelos dois soros imunes, confirmando a identidade da ATPDase 2, e sugerindo que esta proteína está sujeita a vários processamentos pós-tradução, tais como glicosilação e proteólise, resultando finalmente em uma proteína secretada de 55 kDa. Adicionalmente, anticorpos policlonais anti-SmB2LJ inibiram 62% e 37% das atividades ATPásica e ADPásica, respectivamente, enquanto anticorpos policlonais anti-SmB1LJ inibiram ADPase (92%), mas não atividade ATPásica, sugerindo que o domínio B da ATPDase 2 do S. mansoni está envolvido na regulação catalítica da enzima, sendo um novo alvo para o desenho de inibidores. Imunomarcação com anticorpos policlonais anti-SmB2LJ de cortes de fígado de camundongo infectado revelaram reações positivas na superfície externa do miracídio no ovo de S. mansoni. Fluorescência intensa foi detectada na região entre o miracídio e o lado interno da casca do ovo, localizada no envelope de von Lichtenberg, a qual é uma região de produção de componentes imunogênicos. Fluorescência foi também detectada no lado de fora da casca do ovo, aprisionada pelos microespinhos de superfície, confirmando que esta proteína, como no verme adulto, é também secretada de ovos de S. mansoni. Nenhuma reatividade foi observada nos tecidos granulomatosos circundantes. Amostras de plasmas de camundongos Swiss infectados com S. mansoni foram testadas por ELISA usando SmB1LJ or SmB2LJ como antígeno. Reatividade significativamente maior de anticorpos IgG, IgG1 ou IgG2a foi detectada com SmB1LJ, enquanto somente anticorpo IgG1 foi altamente reativo com SmB2LJ. A reatividade de amostras de plasma (diluídas 1:200) de camundongos Swiss previamente inoculados com apirase de batata ou r-potDomínio B, um polipeptídio com cauda de hexahistidina derivado do domínio B (r78-117) da apirase de batata, e homólogo ao domínio B da ATPDase 2, foi também testada por ELISA usando os peptídeos como antígenos. Anticorpos policlonais IgG e IgG1 anti-apirase de batata reagiram significativamente com SmB1LJ, enquanto somente IgG1 reagiu com SmB2LJ. Anticorpos policlonais anti-r-potDomínio B não reagiram significativamente com SmB2LJ. Comparado aos controles, amostras de plasma (diluídas 1:200) destes animais pré-imunizados com apirase de batata ou r-potDomínio B e desafiados com S. mansoni não reagem significativamente com os peptídeos. Os resultados confirmaram que o domínio B da ATPDase 2 está potencialmente envolvido na resposta imune do hospedeiro, e os peptídeos SmB1LJ e SmB2LJ podem ser usados em estudos da esquistossomose.FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerai

Caracterização imunológica da apirase de batata e indução de anticorpos IgE por epitopos compartilhados entre as SmATPDases de Schistosoma mansoni e a apirase de Solanum tuberosum

Schistosomiasis is a neglected tropical disease that affects millions of people around the world. The lack of vaccine for this helminth disease prompts a search for antigenic molecules from the parasites of the genus Schistosoma. Among the molecules considered promising are an ATP diphosphohydrolase from Schistosoma mansoni. The parasite has two enzyme isoforms which are termed SmATPDases and are expressed in all as life cycle phases of this helminth. The potato apyrase is a plant protein belonging to the same family of parasite enzymes, shared epitopes with SmATPDase isoforms. Thus, the objective of this work was to perform an immunological characterization of potato apyrase in a model of experimental murine infection based on this protein homology. Plant protein was inoculated into C57BL / 6 mice that were infected with S. mansoni cercariae. Plant protein induced significant levels of IgG and IgG1 antibodies after immunization, which remained high for analysis 60 days after infection. The solenocytes culture of the immunized animals responded to the stimulus with a plant protein, inducing a significant production of IFN-γ, IL-10 and IL-5, but not IL-13. The immunization process did not provide protection, being unable to promote a reduction of the parasitic load. The immunomodulatory effect of immunization was observed through a reduction of the area affected by the hepatic granulomatous reaction in the immunized animals. Concerning the binding of IgE antibodies, potato apyrase showed significant reactivity with these antibodies present in plasma samples from patients with schistosomiasis. In Western blotting assays, IgE antibodies were able to recognize three non-homogenized languages of adult helminth worms at 91, 63 and 55 kDa. It was possible to infer that all the bands revealed correspond to SmAPTDase isoforms with various post-translational processing, such as glycosylation and proteolysis. (C57BL / 6) with a plant protein capable of inducing a significant production of IgE antibodies detected in ELISA and Western blotting techniques. As potato apyrase has an identity with a mammalian NTPDase 1 (CD39), we suggest that patients have an antibody against the NTPDases involved in maintaining the body's homeostasis. The obtained data show that the potato harvest has an immunoregulatory profile in schistosomiasis, and may contribute to the reduction of morbidity, inducing a production of IgE antibodies, considered as protectors in schistosomiasis.A esquistossomose é uma doença tropical negligenciada que acomete milhões de pessoas no mundo. A falta de vacina para essa doença helmíntica impulsiona a busca por moléculas antigênicas oriundas dos parasitos do gênero Schistosoma. Dentre as moléculas consideradas como promissoras, se encontra a ATP difosfohidrolase de Schistosoma mansoni. O parasito possui duas isoformas de desta enzima que são denominadas SmATPDases e são expressas em todos as fases do ciclo de vida deste helminto. A apirase de batata é uma proteína vegetal pertencente à mesma família de enzimas do parasito, compartilha epitopos com as isoformas de SmATPDases. Dessa forma, o objetivo deste trabalho foi realizar a caracterização imunológica da apirase de batata em modelo de infecção murina experimental com base nessa homologia protéica. A proteína vegetal foi inoculada em camundongos C57BL/6 que posteriormente foram infectados com cercárias de S. mansoni. A proteína vegetal induziu níveis significativos de anticorpos IgG e IgG1 após a imunização, os quais se mantiveram elevados na análise 60 dias após a infecção. A cultura de esplenócitos dos animais imunizados respondeu ao estímulo com a proteína vegetal, induzindo a produção de níveis significativos de IFN-γ, IL-10 e IL-5, mas não de IL-13. O processo de imunização não conferiu proteção, sendo incapaz de promover a redução da carga parasitária. O efeito imunomodulador da imunização foi observado através de uma redução da área afetada pela reação granulomatosa hepática nos animais imunizados. Em relação à ligação de anticorpos IgE, a apirase de batata mostrou reatividade significativa com estes anticorpos presentes em amostras do plasma de pacientes com esquistossomose. Em ensaios de Western blotting, os anticorpos IgE foram capazes de reconhecer três proteínas presentes no homogeneizado de vermes adultos do helminto, com 91, 63 e 55 kDa. Foi possível inferir que todas as bandas reveladas correspondem a isoformas de SmAPTDases com distintos processamentos pós-tradução, tais como glicosilação e proteólise. Comprovando esta reatividade, foi possível verificar que a imunização dos animais (C57BL/6) com a proteína vegetal foi capaz de induzir a produção significativa de anticorpos IgE detectáveis em técnicas de ELISA e Western blotting. Como a apirase de batata possui identidade com a NTPDase 1 (CD39) de mamíferos sugerimos que os pacientes possuam anticorpos naturais contra NTPDases envolvidos na manutenção da homeostase do organismo. O conjunto de dados obtidos mostra que a apirase de batata possui um perfil imunoregulatório na esquistossomose, podendo contribuir para a redução da morbidade, induzindo a produção de anticorpos IgE, tidos como protetores na esquistossomose

IgE antibodies from schistosomiasis patients to recognize epitopes in potato apyrase

Abstract INTRODUCTION: High percentages of structural identity and cross-immunoreactivity have been reported between potato apyrase and Schistosoma mansoni ATP diphosphohydrolase (SmATPDases) isoforms, showing the existence of particular epitopes shared between these proteins. METHODS: Potato apyrase was employed using ELISA, western blot, and mouse immunization methods to verify IgE reactivity. RESULTS: Most of the schistosomiasis patient’s (75%) serum was seropositive for potato apyrase and this protein was recognized using western blotting, suggesting that parasite and plant proteins share IgE-binding epitopes. C57BL/6 mice immunized with potato apyrase showed increased IgE antibody production. CONCLUSIONS: Potato apyrase and SmATPDases have IgE-binding epitopes

In vitro assessment of the antimicrobial effects of pomegranate (Punica granatum L.) peel decoction on saliva samples

Several products have been developed to eliminate or reduce potential pathogenic microorganisms of the oral microbiome. The continuous use of these synthetic products can result in side effects such as vomiting, diarrhea, darkening of the teeth and the induction of microbial resistance. Pomegranate (Punica granatum) peel decoction was tested to assess its antimicrobial activity. In vitro analysis showed the decoction had antimicrobial activity against strains of Pseudomonas aeruginosa and Candida albicans, but none was detected against Enterococcus faecalis. When tested on saliva samples from children, the decoction showed great potential in reducing the load of microorganisms, the inhibition haloes produced with saliva samples being similar to those of the antimicrobial control (0.12% chlorhexidine). The pomegranate peel decoction in water could thus provide a promising source for developing solutions for use against oral diseases

Immunostimulatory property of a synthetic peptide belonging to the soluble ATP diphosphohydro-lase isoform (SmATPDase 2) and immunolocalisation of this protein in the Schistosoma mansoni egg

A peptide (SmB2LJ; r175-194) that belongs to a conserved domain from Schistosoma mansoni SmATPDase 2 and is shared with potato apyrase, as predicted by in silico analysis as antigenic, was synthesised and its immunostimulatory property was analysed. When inoculated in BALB/c mice, this peptide induced high levels of SmB2LJ-specific IgG1 and IgG2a subtypes, as detected by enzyme linked immunosorbent assay. In addition, dot blots were found to be positive for immune sera against potato apyrase and SmB2LJ. These results suggest that the conserved domain r175-194 from the S. mansoni SmATPDase 2 is antigenic. Western blots were performed and the anti-SmB2LJ antibody recognised in adult worm (soluble worm antigen preparation) or soluble egg antigen antigenic preparations two bands of approximately 63 and 55 kDa, molecular masses similar to those predicted for adult worm SmATPDase 2. This finding strongly suggests the expression of this same isoform in S. mansoni eggs. To assess localisation of SmATPDase 2, confocal fluorescence microscopy was performed using cryostat sections of infected mouse liver and polyclonal antiserum against SmB2LJ. Positive reactions were identified on the external surface from the miracidium in von Lichtenberg's envelope and, in the outer side of the egg-shell, showing that this soluble isoform is secreted from the S. mansoni eggs