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
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.
Assuntos
Adjuvantes Imunológicos , Bacillus thuringiensis , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Endotoxinas/imunologia , Imunidade/imunologia , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/farmacologia , Toxinas Bacterianas/farmacologia , Bovinos , Endotoxinas/farmacologia , Proteínas Hemolisinas , Antígenos de Superfície da Hepatite B/imunologia , Imunidade/efeitos dos fármacos , Imunidade nas Mucosas/efeitos dos fármacos , Imunidade nas Mucosas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Soroalbumina Bovina/imunologiaRESUMO
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
The pathogenic mechanisms of enteroaggregative Escherichia coli (EAggEC) infection are not fully elucidated. In this work we show that an ammonium sulfate precipitate of culture supernatant of EAggEC strain 049766 increased the potential difference (PD) and the short-circuit current (Isc) in rat jejunal preparations mounted in Ussing chambers. The precipitate contained two major proteins of 108 and 116 kDa, which were partially copurified by chromatography in DEAE-cellulose. This chromatographic fraction (peak I) increased jejunal PD and Isc in a dose-dependent manner, accompanied by a decrease in tissue electrical resistance. These effects were inhibited by incubation of peak I at 75 degreesC for 15 min or for 1 h with proteinase K at 37 degreesC. Rabbit polyclonal antibodies against peak I containing both the 108- and 116-kDa proteins inhibited the enterotoxic effect. Specific polyclonal antibodies raised against the 108-kDa but not against the 116-kDa protein inhibited the enterotoxic effect, suggesting that the 108-kDa protein is the active toxic species. Moreover, another EAggEC strain (065126) producing the 116-kDa protein but not the 108-kDa protein had no effect on rat jejunal mucosa in the Ussing chamber. The >100-kDa fraction derived from prototype EAggEC strain 042, which also expressed both 108- and 116-kDa proteins, also produced an enterotoxic effect on rat jejunal preparations in Ussing chambers; however, the same strain cured of its 65-MDa adherence plasmid did not. A subclone derived from the 65-MDa plasmid expressing the 108-kDa toxin (and not the 116-kDa protein) elicited rises in Isc. Tissue exposed to any preparation containing the 108-kDa toxin exhibited similar histopathologic changes, characterized by increased mucus release, exfoliation of cells, and development of crypt abscesses. Our data suggest that some EAggEC strains produce a ca. 108-kDa enterotoxin/cytotoxin which is encoded on the large virulence plasmid.
Assuntos
Toxinas Bacterianas/toxicidade , Enterotoxinas/toxicidade , Proteínas de Escherichia coli , Escherichia coli/patogenicidade , Animais , Toxinas Bacterianas/genética , Enterotoxinas/genética , Mucosa Intestinal/patologia , Masculino , Peso Molecular , Ratos , Ratos Sprague-DawleyRESUMO
We have investigated the role of IgE in the local immunity of intestinal amebiasis, a parasitic infection known to induce specific antibody-forming cells (AFC) and IgA antibodies in rodents and humans. We found that intragastric immunization of rats with glutaraldehyde-fixed Entamoeba histolytica trophozoites significantly increased antiameba AFC in the Peyer's patches and spleen and that the lamina propria of the cecum from immunized animals was infiltrated by eosinophils armed with IgE antibodies. Morphometric analysis showed that IgE-containing cells and eosinophils were nearly three times more abundant in the cecum of immunized rats. Antigenic challenge with amebal lysates provoked an increase in the short-circuit current and in the transepithelial potential difference in Ussing-chambered cecum preparations from immunized rats. Although eosinophilia and the increase of IgE are common consequences of infection by parasitic worms, our results indicate that local immunity in intestinal amebiasis also involves IgE deposition, eosinophil infiltration, and type I hypersensitivity, which may explain some symptoms of amebic dysentery such as colic, abdominal tension, tenesmus, and bloody stools.
Assuntos
Antígenos de Protozoários/imunologia , Entamoeba histolytica/imunologia , Eosinofilia/imunologia , Mucosa Gástrica/imunologia , Hipersensibilidade Imediata/imunologia , Imunoglobulina E/fisiologia , Amebíase/etiologia , Amebíase/imunologia , Animais , Células Produtoras de Anticorpos/citologia , Ceco/imunologia , Relação Dose-Resposta Imunológica , Eletrofisiologia , Imunização , Infusões Parenterais , Mucosa Intestinal/imunologia , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
One of the main drawbacks of experimental amebiasis is the lack of an adequate animal model for invasive intestinal lesions. Mongolian gerbils are useful because both intestinal and hepatic amebiasis can be produced experimentally with Entamoeba histolytica trophozoites. In this paper we show results obtained with in vivo and in vitro models of intestinal amebiasis in gerbils. We inoculated gerbils intracecally with monoxenic cultures of a highly virulent E. histolytica HM1:IMSS substrain. In the in vivo model an increase in mucus production was observed during the first 6 h of interaction. Microulcerative mucosal lesions appeared at 24-72 h postinoculation. Inflammatory infiltrate and edema of the lamina propria were associated with superficial foci of necrosis. At 96 h the cecal mucosa had an almost normal appearance and live amebas were no longer detected. In the in vitro model, early damage was detected in cecal strips mounted in Ussing chambers as a rapid fall in potential difference, short-circuit current, and transepithelial resistance that correlated with the extent of the microscopic lesions produced. The latter consisted of cellular edema and the appearance of small, translucent vacuoles at the base of epithelial cells. Further damage led to loss of intercellular junctions, destruction of interglandular epithelial cells, and edema of the lamina propria. The present results demonstrate that the gerbil is useful as an experimental model for the analysis of early stages of invasive intestinal amebiasis both in vivo and in vitro.
Assuntos
Disenteria Amebiana/patologia , Disenteria Amebiana/fisiopatologia , Entamoeba histolytica , Animais , Ceco/parasitologia , Ceco/patologia , Ceco/fisiopatologia , Modelos Animais de Doenças , Eletrofisiologia , Entamoeba histolytica/isolamento & purificação , Entamoeba histolytica/patogenicidade , Gerbillinae , Técnicas In Vitro , Mucosa Intestinal/parasitologia , Mucosa Intestinal/patologia , Masculino , Músculo Liso/parasitologia , Músculo Liso/patologia , Músculo Liso/fisiopatologia , VirulênciaRESUMO
We compared the enterotoxicity and cysteine proteinases (CP) of the low-virulence Entamoeba histolytica HM1 strain with the highly virulent 1659 clone, derived from HM1 by hamster liver passages. Enterotoxicity of 50,000 freeze-thawed trophozoites was determined on 0.28-cm2 intestinal segments mounted in Ussing chambers; CP activity of Nonidet-P40 amebal lysates was assayed by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and carbobenzoxy-L-arginine-L-arginyl-p-nitroaniline, a CP-specific substrate. Treatment of gerbil cecum segments with amebal lysates caused an immediate fall of their electrophysiologic properties (potential difference, short-circuit current, and transmural resistance) whose decay rates were clearly faster with 1659 than with HM1 lysates. Nonimmune and immune antiamebic human sera and the CP-specific inhibitor E-64 (trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane) prevented the fall of the electrophysiologic properties. Gelatinases, less active in HM1 than in 1659 trophozoites, were better preserved in lysates containing 10 mM p-hydroxymercuribenzoate (pHMB) to prevent autoproteolysis: in lysates without pHMB nearly no gelatinase bands were observed in HM1 samples, whereas intense 30K, 35K, 44K, and 75K bands were seen in 1659 samples; in lysates with pHMB only 53K and 75K bands were found that were much more intense in 1659 samples, 75K being barely visible in HM1 samples. The overall CP activity was 17 times higher in 1659 than in HM1 lysates, was inhibited by E-64 (mean inhibitory dose, 20 microM), was stimulated by 2-mercaptoethanol (ME) 3.7 times in HM1 and 2.4 times in 1659 lysates, and was reactivated by ME in lysates containing pHMB. Most of the CP activity in HM1 lysates sedimented at 15,600g but predominated in 1659 supernatants. The increase of E. histolytica virulence thus correlates with a remarkable increase both of in vitro enterotoxicity and of two CPs (53K and 75K), suggesting that these proteinases are significant pathogenicity factors.