RESUMO
We have previously reported that there are differences in the number of predominant amoebic antigens recognized by serum and small intestinal antibodies induced after local and systemic immunization with glutarldehyde-fixed Entamoeba histolytica trophozoites (GFT) in BALB/c mice, by an immunoblot analysis. Moreover, by enzyme-linked immunosorbent assay (ELISA) analysis, we found differences in the antiamoebic antibody isotype patterns elicited at the large and small intestines. To further characterize the antiamoebic immune response induced in BALB/c mice, after local (oral and rectal) and systemic (intraperitoneal and intramuscular) immunization with GFT, we performed an immunoblot analysis of the amoebic proteins predominantly recognized by immunoglobulins (Ig)G, IgA and IgM in the serum and in the small and large intestines. The present work shows differences between the large and small intestine in the IgG- and IgA-antibody recognition pattern of amoebic proteins, thus confirming and extending our previous findings supporting the compartmentalization of the intestinal immune response. Furthermore, our reported observation that there are differences in the amoebic proteins predominantly recognized by antibodies of different isotypes was extended to the intestines, as some proteins with relative molecular weights of 24-25, 66, 140 kDa are strongly recognized by IgG but not by other antibody isotypes.
Assuntos
Entamoeba histolytica/imunologia , Entamebíase/imunologia , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Intestino Grosso/imunologia , Intestino Delgado/imunologia , Animais , Anticorpos Antiprotozoários/biossíntese , Anticorpos Antiprotozoários/imunologia , Especificidade de Anticorpos , Antígenos de Protozoários/imunologia , Entamebíase/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Imunidade nas Mucosas/imunologia , Imunização/métodos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Lectinas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB CAssuntos
Antiprotozoários/farmacologia , Entamoeba histolytica/efeitos dos fármacos , Intestino Grosso/patologia , Intestino Delgado/patologia , Mebendazol/farmacologia , Metronidazol/farmacologia , Vacinas Protozoárias/administração & dosagem , Animais , Entamoeba histolytica/fisiologia , Intestino Grosso/parasitologia , Intestino Delgado/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Cavidade PeritonealRESUMO
Systemic and mucosal and immune responses can be manipulated with immunomodulators. Here we show the modulatory effects of cholera toxin (CT) and beta-1,3-glucan (GLU) on the rat antiamebic serum and fecal antibody responses to one or four intraperitoneal (IP) or intragastric (IG) doses of glutaraldehyde-fixed Entamoeba histolytica trophozoites (GFT). One IP dose of GFT maximized serum IgM and IgG antiamebic antibodies on days 4 and 9, respectively; CT coadministration increased IgM antibodies, whereas IgG titers increased with CT or GLU; coproantibodies were undetectable after GFT alone or coadministered with GLU, whereas CT coadministration maximized fecal IgA antibodies on day 6. One IG dose of GFT alone increased serum IgM and IgG antibodies 2.5 times and no further increases were detected using GLU, whereas CT doubled serum IgG antibodies; GFT did not affect the coproantibody responses, whereas GLU coadministration maximized IgG coproantibody levels on day 6 and CT increased IgG and IgA coproantibody levels on the same day. On the other hand, four IG doses of GFT alone or with GLU induced tolerance, whereas GFT alone via the IP route increased serum antibodies slightly and GLU coadministration increased serum IgG antibody titers 300-fold. CT coadministration by both routes increased IgA coproantibodies, and simultaneous CT+GLU coadministration induced lower responses than either CT or GLU. Different antiamebic immune responses might therefore be attained through the use of different immunization routes and immunomodulators to induce protective immunity against intestinal or extraintestinal amebiasis.
Assuntos
Adjuvantes Imunológicos/farmacologia , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Antígenos de Superfície/imunologia , Toxina da Cólera/farmacologia , Entamoeba histolytica/imunologia , Glucanos/farmacologia , Mucosa Intestinal/imunologia , beta-Glucanas , Animais , Anticorpos Antiprotozoários/administração & dosagem , Formação de Anticorpos/efeitos dos fármacos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Infusões Parenterais , Injeções Intraperitoneais , Ratos , Fatores de TempoRESUMO
Recently we discovered that the Cry1Ac protoxin of Bacillus thuringiensis administered to Balb/c mice intraperitoneally (i.p.) or intragastrically is a systemic and intestinal immunogen as potent as cholera toxin. To further characterize the mucosal immunogenicity of Cry1Ac we additionally tried the intranasal (i.n.) and rectal routes and used enzyme-linked immunoassays to determine anti-Cry1Ac antibody responses in the serum as well as in vaginal and tracheobronchial washes and in the fluids of the large and the small intestine. Immunization by the i.p., i.n. and rectal routes induced IgM, IgG and IgA antibodies in all the mucosal surfaces analyzed, but the magnitude and predominant isotype of each response depended on the route used and the mucosal site analyzed. These data extend our findings on the striking mucosal immunogencity of Cry1Ac and provide additional evidence on the compartmentalization of the mucosal immune system.
Assuntos
Bacillus thuringiensis/imunologia , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Endotoxinas/imunologia , Precursores de Proteínas/imunologia , Administração Intranasal , Animais , Anticorpos Antibacterianos/sangue , Toxinas de Bacillus thuringiensis , Feminino , Proteínas Hemolisinas , Imunidade nas Mucosas , Injeções Intraperitoneais , Intestino Grosso/imunologia , Intestino Delgado/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Reto , Sistema Respiratório/imunologia , Vacinação/métodos , Vagina/imunologiaRESUMO
Bacillus thuringiensis (Bt), considered a safe insecticide, produces insecticidal proteins named Cry during sporulation, which possess exceptional immunological properties. In this work using an immunohistochemical test we demonstrated that Cry1Ac protoxin (pCry1Ac) binds to the mucosal surface of the mouse small intestine. Ligand blot assay allowed us to detect, under denaturing conditions, six pCry1Ac-binding polypeptides present in brush border membrane vesicles isolated from the small intestine. Moreover, this protein induced in situ temporal changes in the electrophysiological properties of the mouse jejunum. The data obtained indicate a possible interaction in vivo of Cry proteins with the animal bowel which could induce changes in the physiological status of the intestine.
Assuntos
Bacillus thuringiensis/química , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas , Endotoxinas/metabolismo , Intestino Delgado/metabolismo , Intestino Delgado/microbiologia , Proteínas de Membrana/metabolismo , Animais , Toxinas de Bacillus thuringiensis , Eletrofisiologia , Escherichia coli/metabolismo , Imunofluorescência , Proteínas Hemolisinas , Jejuno/metabolismo , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microvilosidades/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Ligação Proteica , Proteínas Recombinantes/metabolismo , Fatores de TempoRESUMO
The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice. The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route. The antibody fraction in serum and intestinal fluids consisted mainly of IgG1. In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyer's patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT). Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected. The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins.
Assuntos
Anticorpos Antibacterianos/biossíntese , Bacillus thuringiensis/imunologia , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Endotoxinas/imunologia , Imunoglobulina G/biossíntese , Mucosa Intestinal/imunologia , Animais , Anticorpos Antibacterianos/sangue , Toxinas de Bacillus thuringiensis , Ensaio de Imunoadsorção Enzimática , Feminino , Proteínas Hemolisinas , Imunoglobulina G/sangue , Imunoglobulina M/biossíntese , Imunoglobulina M/sangue , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The present paper describes important features of the immune response induced by the Cry1Ac protein from Bacillus thuringiensis in mice. The kinetics of induction of serum and mucosal antibodies showed an immediate production of anti-Cry1Ac IgM and IgG antibodies in serum after the first immunization with the protoxin by either the intraperitoneal or intragastric route. The antibody fraction in serum and intestinal fluids consisted mainly of IgG1. In addition, plasma cells producing anti-Cry1Ac IgG antibodies in Peyer's patches were observed using the solid-phase enzyme-linked immunospot (ELISPOT). Cry1Ac toxin administration induced a strong immune response in serum but in the small intestinal fluids only anti-Cry1Ac IgA antibodies were detected. The data obtained in the present study confirm that the Cry1Ac protoxin is a potent immunogen able to induce a specific immune response in the mucosal tissue, which has not been observed in response to most other proteins
Assuntos
Animais , Feminino , Anticorpos Antibacterianos/biossíntese , Bacillus thuringiensis/imunologia , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Imunoglobulina G/biossíntese , Mucosa Intestinal/imunologia , Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/administração & dosagem , Toxinas Bacterianas/administração & dosagem , Ensaio de Imunoadsorção Enzimática , Imunização , Imunoglobulina G/sangue , Imunoglobulina M/biossíntese , Imunoglobulina M/sangue , Mucosa Intestinal/metabolismo , Camundongos Endogâmicos BALB CRESUMO
The spore-forming soil bacterium Bacillus thuringiensis produces parasporal inclusion bodies composed by delta-endotoxins also known as Cry proteins, whose resistance to proteolysis, stability in highly alkaline pH and innocuity to vertebrates make them an interesting candidate to carrier of relevant epitopes in vaccines. The purpose of this study was to determine the mucosal and systemic immunogenicity in mice of Cry1Ac protoxin from B. thuringiensis HD73. Crystalline and soluble forms of the protoxin were administered by intraperitoneal or intragastric route and anti-Cry1Ac antibodies of the major isotypes were determined in serum and intestinal fluids. The two forms of Cry1Ac protoxin administered by intraperitoneal route induced a high systemic antibody response, however, only soluble Cry1Ac induced a mucosal response via intragastric. Serum antibody levels were higher than those induced by cholera toxin. Systemic immune responses were attained with doses of soluble Cry1Ac ranging from 0.1 to 100 microg by both routes, and the maximal effect was obtained with the highest doses. High anti-Cry1Ac IgG antibody levels were detected in the large and small intestine fluids from mice receiving the antigen via i.p. These data indicate that Cry1Ac is a potent systemic and mucosal immunogen.
Assuntos
Anticorpos Antibacterianos/biossíntese , Bacillus thuringiensis/imunologia , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Endotoxinas/imunologia , Mucosa Intestinal/imunologia , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/administração & dosagem , Toxina da Cólera/imunologia , Relação Dose-Resposta Imunológica , Endotoxinas/administração & dosagem , Fezes/microbiologia , Feminino , Proteínas Hemolisinas , Imunização , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Recently we demonstrated that recombinant Cry1Ac protoxin from Bacillus thuringiensis is a potent systemic and mucosal immunogen. In this study we compared the adjuvant effects of Cry1Ac and cholera toxin (CT) for the hepatitis B surface antigen (HBsAg) and bovine serum albumin (BSA). The antibody responses of intestinal secretions and serum were determined by ELISA in Balb/c mice immunized through the intragastric (IG) or intraperitoneal (IP) routes. When HBsAg was administered via IG, the anti-HBsAg intestinal response was not enhanced by either Cry1Ac or CT, whereas via IP Cry1Ac increased the anti-HBsAg intestinal immunoglobulin (Ig)G response and CT increased the intestinal IgA and IgM responses. Serum anti-BSA antibodies increased when BSA was co-administered with CT or Cry1Ac by both routes. Cholera toxin and Cry1Ac co-administered via IP increased the IgG anti-BSA response in fluid of the large intestine and CT also increased the IgA and IgM responses slightly. When co-administered via IP, CT and Cry1Ac did not affect the IgG anti-BSA response of the small intestine significantly. We conclude that Cry1Ac is a mucosal and systemic adjuvant as potent as CT which enhances mostly serum and intestinal IgG antibody responses, especially at the large intestine, and its effects depend on the route and antigen used. These features make Cry1Ac of potential use as carrier and/or adjuvant in mucosal and parenteral vaccines.