RESUMO
NTPDases are enzymes that hydrolyse diphosphate and triphosphate nucleosides, regulating purinergic signalling in many organisms. The Schistosoma mansoni NTPDases, SmATPDases 1 and 2, are antigenic proteins and display a significant homology with the isoforms found in mammalian cells. In this work, we investigated whether anti-SmATPDase antibodies from S. mansoni-infected mice sera show cross-reactivity with the NTPDase 1 isoform from macrophages and how this event affects the cell proliferation. By Western blot, anti-SmATPDase antibodies present in serum from infected mice recognized 2 bands with approximately 53 and 58 kDa, corresponding to NTPDase 1. Additionally, the enzyme was identified in macrophages by immunofluorescence and the anti-SmATPDase antibodies were able to reduce activity enzyme (22%). Macrophages incubated with commercial polyclonal antibodies reactive with NTPDase 1 (anti-CD39) showed a reduction of 40% of the enzyme activity. In proliferation assays, macrophage proliferation was inhibited 11% and 90% by pooled sera from infected animals and anti-CD39, respectively. The results suggest that inhibition of NTPDase 1 in macrophages by antibodies produced against the isoforms of the S. mansoni ATPDases could be a mechanism of regulation in the immune response during experimental schistosomiasis.
Assuntos
Anticorpos Antiprotozoários/imunologia , Antígenos CD/imunologia , Antígenos de Protozoários/imunologia , Apirase/imunologia , Reações Cruzadas/imunologia , Macrófagos/imunologia , Schistosoma mansoni/imunologia , Esquistossomose/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Western Blotting , Linhagem Celular , Proliferação de Células/fisiologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Células RAW 264.7 , Esquistossomose/parasitologia , Caramujos/parasitologiaRESUMO
Existem vários esforços para o desenvolvimento de produtos capazes de reduzir ou eliminar os microrganismos patogênicos presentes na cavidade oral. A literatura relata uma série de efeitos adversos associados ao uso contínuo destes produtos, dentre eles vômitos, diarreia e o escurecimento da dentina. A indução da resistência microbiana é um dos fatores de destaque relacionado ao uso destes produtos. Neste trabalho, o decocto de romã (Punica granatum L.), obtido a partir das cascas do fruto, foi utilizado para avaliação de seu potencial antimicrobiano sobre cepas de Pseudomonas aeruginosa, Candida albicans e Enterococcus faecalis, sendo ativos contra os dois primeiros microrganismos. A aplicação do decocto sobre os microrganismos presentes em amostras de saliva de crianças mostrou halos de inibição semelhantes ao obtido com a solução de clorexidina a 0,12%. A atividade antimicrobiana do decocto de romã aponta esta preparação como uma fonte em potencial para o desenvolvimento de produtos de uso oral...
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...
Assuntos
Humanos , Masculino , Feminino , Criança , Anti-Infecciosos , Plantas Medicinais , LythraceaeRESUMO
An antigenic conserved B domain was previously identified within nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. Now, the r-potDomain B, a 6× His-tag polypeptide belonging to the conserved B domain from the potato apyrase, and synthetic peptides LbB1LJ and LbB2LJ derived from the B domain from Leishmania NTPDase 1 were used as molecular tools for studies of the Leishmania amazonensis NTPDase 1. Widespread subcellular location of the specific NTPDase 1 was detected by Western blots of promastigote fractions and ultrastructural immunocytochemical microscopy using immune sera raised against these biomolecules. In addition, the L. amazonensis-infected BALB/c mice were evaluated at 12 to 120 days after infection, which progresses showing typical nodular lesion. High antibody reactivity with either r-potDomain B, LbB1LJ, or LbB2LJ was found in L. amazonensis-infected BALB/c mice indicating the antigenicity of the B domain from NTPDase 1 isoform. The IgG1 antibody reactivity significantly increased at 90-120 days postinfection, 18- to 24-fold when compared to the 12th day, and remained elevated even at 120th after infection, coinciding with the most active stage of the disease. In contrast, significantly higher IgG2a antibody reactivity with each biomolecule was observed at 40th day, about two- to fourfold higher than those found at 12th or 20th day, and decreased along 120-day period. Apparently, the conserved B domain is capable to induce IgG2a production in early disease stages. All together, these results suggest that r-potDomain B or synthetic peptides could be molecular starting points in experimental protocols of immunotherapy and/or vaccination for leishmaniasis.
Assuntos
Antígenos CD/metabolismo , Apirase/metabolismo , Leishmania/enzimologia , Leishmaniose/parasitologia , Sequência de Aminoácidos , Animais , Antígenos CD/genética , Antígenos de Protozoários , Apirase/genética , Regulação Enzimológica da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Anotação de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
Eremanthus erythropappus (DC) McLeisch, a plant popularly known as Candeia (Asteraceae), has high therapeutic potential. In this study, the in vitro schistosomicidal potentials of the ethanolic, dichloromethane and hexane extract of branches were evaluated. Couples of worms obtained from the infected mice were cultured in RPMI supplemented with foetal bovine serum and antibiotics. Four pairs of adult worms were exposed to increasing concentrations of each extract and examined by light microscope. The extracts at 100 and 200 µg mL(-1) had schistosomicidal activity, as demonstrated by the analysis of several aspects such as tegument darkening, absence of motility, incapacity of adhesion in culture plate and absence of egg in culture medium. At 50 and 75 µg mL(-1), the dichloromethane and hexane extracts were highly effective. The results suggest that these extracts could be useful in the development of new schistosomicidal drugs.
Assuntos
Asteraceae/química , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Esquistossomicidas/química , Esquistossomicidas/farmacologia , Animais , Camundongos , Schistosoma/efeitos dos fármacosRESUMO
An ATP diphosphohydrolase (EC 3.6.1.5) activity was identified in a Leishmania (Viannia) braziliensis promastigotes preparation (Lb). Ultrastructural cytochemical microscopy showed this protein on the parasite surface and also stained a possible similar protein at the mitochondrial membrane. Isolation of an active ATP diphosphohydrolase isoform from Lb was obtained by cross-immunoreactivity with polyclonal anti-potato apyrase antibodies. These antibodies, immobilized on Protein A-Sepharose, immunoprecipitated a polypeptide of approximately 48 kDa and, in lower amount, a polypeptide of approximately 43 kDa, and depleted 83% ATPase and 87% of the ADPase activities from detergent-homogenized Lb. Potato apyrase was recognized in Western blots by IgG antibody from American cutaneous leishmaniasis (ACL) patients, suggesting that the parasite and vegetable proteins share antigenic conserved epitopes. Significant IgG seropositivity in serum samples diluted 1:50 from ACL patients (n=20) for Lb (65%) and potato apyrase (90%) was observed by ELISA technique. Significant IgG antibody reactivity was also observed against synthetic peptides belonging to a conserved domain from L. braziliensis NDPase (80% seropositivity) and its potato apyrase counterpart (50% seropositivity), in accordance with the existence of shared antigenic epitopes and demonstrating that in leishmaniasis infection the domain r82-103 from L. braziliensis NDPase is a target for the human immune response.
Assuntos
Apirase/metabolismo , Leishmania braziliensis/enzimologia , Leishmaniose Cutânea/parasitologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/sangue , Apirase/genética , Apirase/imunologia , Western Blotting , Humanos , Imunoprecipitação , Isoenzimas , Leishmania braziliensis/imunologia , Leishmania braziliensis/ultraestrutura , Leishmaniose Cutânea/imunologia , Microscopia Eletrônica , Dados de Sequência MolecularRESUMO
Evolutionary and closer structural relationships are demonstrated by phylogenetic analysis, peptide prediction and molecular modelling between Solanum tuberosum apyrase, Schistosoma mansoni SmATPase 2 and Leishmania braziliensis NDPase. Specific protein domains are suggested to be potentially involved in the immune response, and also seem to be conserved during host and parasite co-evolution. Significant IgG antibody reactivity was observed in sera from patients with American cutaneous leishmaniasis (ACL) and schistosomiasis using potato apyrase as antigen in ELISA. S. mansoni adult worm or egg, L. braziliensis promastigote (Lb) and Trypanosoma cruzi epimastigote (EPI) have ATP diphosphohydrolases, and antigenic preparations of them were evaluated. In ACL patients, IgG seropositivity was about 43% and 90% for Lb and potato apyrase, respectively, while IgM was lower (40%) or IgG (100%) seropositivity for both soluble egg (SEA) and adult worm (SWAP) antigens was higher than that found for potato apyrase (IgM=10%; IgG=39%). In Chagas disease, IgG seropositivity for EPI and potato apyrase was 97% and 17%, respectively, while the IgM was low (3%) for both antigens. The study of the conserved domains from both parasite proteins and potato apyrase could lead to the development of new drug targets or molecular markers.
Assuntos
Apirase/imunologia , Sequência Conservada/imunologia , Mapeamento de Epitopos , Parasitos/enzimologia , Parasitos/imunologia , Solanum tuberosum/enzimologia , Sequência de Aminoácidos , Animais , Anticorpos Antiprotozoários/imunologia , Apirase/química , Doença de Chagas/sangue , Doença de Chagas/imunologia , Humanos , Leishmania braziliensis/enzimologia , Leishmania braziliensis/genética , Leishmania braziliensis/imunologia , Leishmaniose Cutânea/sangue , Leishmaniose Cutânea/imunologia , Dados de Sequência Molecular , Parasitos/genética , Filogenia , Estrutura Terciária de Proteína , Schistosoma mansoni/enzimologia , Schistosoma mansoni/genética , Schistosoma mansoni/imunologia , Esquistossomose/sangue , Esquistossomose/imunologia , Alinhamento de SequênciaRESUMO
A Leishmania (Leishmania) amazonensis ATP diphosphohydrolase isoform was partially purified from plasma membrane of promastigotes by preparative non-denaturing polyacrylamide gel electrophoresis. SDS-PAGE followed by Western blots developed with polyclonal anti-potato apyrase antibodies identified diffuse bands of about 58-63 kDa, possibly glycosylated forms of this protein. By ELISA technique, a significantly higher total IgG antibody level against potato apyrase was found in serum from promastigote-infected mice, as compared to the uninfected mice, confirming both the existence of shared epitopes between the parasite and vegetable proteins, and the parasite ATP diphosphohydrolase antigenicity. By Western blotting, serum from amastigote-infected BALB/c mice recognizes both potato apyrase and this antigenic ATP diphosphohydrolase isoform isolated from promastigotes, suggesting that it is also expressed in the amastigote stage. The infection monitored along a 90-day period in amastigote-infected mice showed reactivity of IgG2a antibody in early steps of infection, while the disappearance of the IgG2a response and elevation of IgG1 antibody serum levels against that shared epitopes were associated with the progression of experimental leishmaniasis. This is the first observation of the antigenicity of a L. (L.) amazonensis ATP diphosphohydrolase isoform, and of the ability of cross-immunoreactivity with potato apyrase to differentiate serologically stages of leishmaniasis in infected mice.
Assuntos
Apirase/imunologia , Leishmania mexicana/enzimologia , Leishmaniose Cutânea/diagnóstico , Solanum tuberosum/enzimologia , Animais , Variação Antigênica , Apirase/isolamento & purificação , Apirase/metabolismo , Western Blotting , Reações Cruzadas , Progressão da Doença , Eletroforese em Gel de Poliacrilamida , Epitopos , Feminino , Isoenzimas/imunologia , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Camundongos , Camundongos Endogâmicos BALB CRESUMO
We have previously showed that Schistosoma mansoni ATP-diphosphohydrolase and Solanum tuberosum potato apyrase share epitopes and the vegetable protein has immunostimulatory properties. Here, it was verified the in situ cross-immunoreactivity between mice NTPDases and anti-potato apyrase antibodies produced in rabbits, using confocal microscopy. Liver samples were taken from Swiss Webster mouse 8 weeks after infection with S. mansoni cercariae, and anti-potato apyrase and TRITC-conjugated anti-rabbit IgG antibody were tested on cryostat sections. The results showed that S. mansoni egg ATP diphosphohydrolase isoforms, developed by anti-potato apyrase, are expressed in miracidial and egg structures, and not in granulomatous cells and hepatic structures (hepatocytes, bile ducts, and blood vessels). Therefore, purified potato apyrase when inoculated in rabbit generates polyclonal sera containing anti-apyrase antibodies that are capable of recognizing specifically S. mansoni ATP diphosphohydrolase epitopes, but not proteins from mammalian tissues, suggesting that autoantibodies are not induced during potato apyrase immunization. A phylogenetic tree obtained for the NTPDase family showed that potato apyrase had lower homology with mammalian NTPDases 1-4, 7, and 8. Further analysis of potato apyrase epitopes could implement their potential use in schistosomiasis experimental models.
Assuntos
Animais , Masculino , Camundongos , Coelhos , Adenosina Trifosfatases/imunologia , Apirase/imunologia , Schistosoma mansoni/enzimologia , Esquistossomose mansoni/imunologia , Solanum tuberosum/enzimologia , Sequência de Aminoácidos , Adenosina Trifosfatases/metabolismo , Anticorpos Anti-Helmínticos/imunologia , Apirase/metabolismo , Reações Cruzadas , Modelos Animais de Doenças , Microscopia Confocal , Dados de Sequência Molecular , Schistosoma mansoni/imunologia , Schistosoma mansoni/metabolismoRESUMO
We have previously showed that Schistosoma mansoni ATP-diphosphohydrolase and Solanum tuberosum potato apyrase share epitopes and the vegetable protein has immunostimulatory properties. Here, it was verified the in situ cross-immunoreactivity between mice NTPDases and anti-potato apyrase antibodies produced in rabbits, using confocal microscopy. Liver samples were taken from Swiss Webster mouse 8 weeks after infection with S. mansoni cercariae, and anti-potato apyrase and TRITC-conjugated anti-rabbit IgG antibody were tested on cryostat sections. The results showed that S. mansoni egg ATP diphosphohydrolase isoforms, developed by anti-potato apyrase, are expressed in miracidial and egg structures, and not in granulomatous cells and hepatic structures (hepatocytes, bile ducts, and blood vessels). Therefore, purified potato apyrase when inoculated in rabbit generates polyclonal sera containing anti-apyrase antibodies that are capable of recognizing specifically S. mansoni ATP diphosphohydrolase epitopes, but not proteins from mammalian tissues, suggesting that autoantibodies are not induced during potato apyrase immunization. A phylogenetic tree obtained for the NTPDase family showed that potato apyrase had lower homology with mammalian NTPDases 1-4, 7, and 8. Further analysis of potato apyrase epitopes could implement their potential use in schistosomiasis experimental models.
Assuntos
Adenosina Trifosfatases/imunologia , Apirase/imunologia , Schistosoma mansoni/enzimologia , Esquistossomose mansoni/imunologia , Solanum tuberosum/enzimologia , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Anti-Helmínticos/imunologia , Apirase/metabolismo , Reações Cruzadas , Modelos Animais de Doenças , Masculino , Camundongos , Microscopia Confocal , Dados de Sequência Molecular , Coelhos , Schistosoma mansoni/imunologia , Schistosoma mansoni/metabolismoRESUMO
The fact that the Schistosoma mansoni egg has two ATP diphosphohydrolase (EC 3.6.1.5) isoforms with different net charges and an identical molecular weight of 63,000, identified by non-denaturing polyacrylamide gel electrophoresis and immunological cross-reactivity with potato apyrase antibodies, is shown. In soluble egg antigen (SEA), only the isoform with the lower net negative charge was detected and seemed to be the predominant species in this preparation. By confocal fluorescence microscopy, using anti-potato apyrase antibodies, the S. mansoni egg ATP diphosphohydrolase was detected on the external surface of miracidium and in von Lichtenberg's envelope. Intense fluorescence was also seen in the outer side of the egg-shell, entrapped by the surface microspines, suggesting that a soluble isoform is secreted. ATP diphosphohydrolase antigenicity was tested using the vegetable protein as antigen. The purified potato apyrase was recognized in Western blots by antibodies present in sera from experimentally S. mansoni-infected mice. In addition, high levels of IgG anti-ATP diphosphohydrolase antibodies were detected by ELISA in the same sera. This work represents the first demonstration of antigenic properties of S. mansoni ATP diphosphohydrolase and immunological cross-reactivity between potato apyrase and sera from infected individuals.