RESUMO
The bioinsecticidal Cry1Ac proteins (protoxin and toxin) are potent immunogens that can activate macrophages by inducing upregulation of costimulatory molecules, pro-inflammatory cytokines, and mitogen-activated protein kinase (MAPK) signaling pathways. Besides, by the oral route, Cry1Ac toxin is mildly allergenic and induces intestinal lymphoid hyperplasia in mice. Given the potential utility of Cry1Ac protoxin as an adjuvant, as well as the human consumption of Cry1Ac toxin in transgenic crops, it is necessary to more deeply evaluate the toxicological potential of these proteins in mammalian immune cells. Here, were used in vitro evaluations in leukocyte and macrophage cell lines to test the potential toxicity of various doses of Cry1Ac proteins, by means of Alamar Blue, MTT, Annexin V, and JC1 assays. Our results indicated that neither Cry1Ac protoxin nor toxin elicited acute toxic effects, after monitoring the cell activity for 4, 8, 10, and 24 h of exposure. By flow cytometry and confocal microscopy analysis, it was observed that neither Cry1Ac toxin nor protoxin generated mitochondrial damage or depolarization or induced apoptosis or necrosis. In conclusion, despite their immunostimulatory effects, it was demonstrated that Cry1Ac proteins did not have cytotoxic effects, even at high concentrations, in primary leukocytes or macrophages or cell lines.
Assuntos
Toxinas de Bacillus thuringiensis/toxicidade , Toxinas Bacterianas/toxicidade , Endotoxinas/toxicidade , Proteínas Hemolisinas/toxicidade , Leucócitos/patologia , Macrófagos/patologia , Precursores de Proteínas/toxicidade , Animais , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Leucócitos/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Necrose , Células RAW 264.7 , Baço/patologia , Células THP-1 , Testes de Toxicidade Aguda , Testes de Toxicidade CrônicaRESUMO
OBJECTIVE AND DESIGN: Sodium caseinate (CasNa) induces differentiation and M-CSF production in mouse band granulocytes in vitro; however, it is not yet known if this molecule can also induce the proliferation and activation of the granulocyte lineage in vivo. In this work we evaluated the induction in vivo of granulopoiesis and the activation of granulocytes in mice treated with CasNa. MATERIAL OR SUBJECTS: BALB/c male mice 8-12 weeks old were used. TREATMENT: The animals were inoculated intraperitoneally with 1 ml of CasNa (10% in PBS p/v) four times (every 48 h). METHODS: Granulocyte proliferation was evaluated by flow cytometry; activation was evaluated by phagocytic indices. The cytokine was measured using an ELISA assay. RESULTS: We show that CasNa increased bone marrow granulopoiesis percentage (38.35 ± 10.88 vs. 64.94 ± 34.14 BrdU+/Gr-1+ cells) and the granulocytes generated presented increased phagocytic indices (0.3 ± 0.1 vs. 0.6 ± 0.11, p < 0.05). We also show that G-CSF (974 ± 411 vs. 3189 ± 350 pg/ml, p < 0.05) and GM-CSF increased in serum, but only G-CSF in bone marrow plasma. CONCLUSIONS: CasNa induces granulopoiesis with functional granulocytes, suggesting that this molecule could be an innate immune system activator.
Assuntos
Caseínas/farmacologia , Granulócitos/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Mielopoese/efeitos dos fármacos , Animais , Candida albicans , Proliferação de Células/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/sangue , Fator Estimulador de Colônias de Granulócitos e Macrófagos/sangue , Granulócitos/citologia , Granulócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose/efeitos dos fármacosRESUMO
Diabetes mellitus (DM) may alter bone remodeling, as osteopenia and osteoporosis are among the complications. Moreover, DM increases the risk and severity of chronic inflammatory periodontal disease, in which bone resorption occurs. Broad evidence suggests that chronic inflammation can contribute to the development of DM and its complications. Hyperglycemia is a hallmark of DM that may contribute to sustained inflammation by increasing proinflammatory cytokines, which are known to cause insulin resistance, via toll-like receptor (TLR)-4-mediated mechanisms. However, the mechanisms by which bone-related complications develop in DM are still unknown. Studies done on the effect of high glucose concentrations on osteoblast functions are contradictory because some suggest increases (although others suggest reductions) in the biomineralization process. Therefore, we evaluated the effect of high glucose levels on biomineralization and inflammation markers in a human osteoblastic cell line. Cells were treated with either physiological 5.5 mM or increasing concentrations of glucose up to 24 mM, and we determined the following: i) the quantity and quality of calcium-deposit crystals in culture and ii) the expression of the following: a) proteins associated with the process of biomineralization, b) the receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG), c) cytokines IL1, IL6, IL8, IL10, MCP-1 and TNF alpha, and d) TLR-2, -3, -4 and -9. Our results show that high glucose concentrations (12 mM and particularly 24 mM) alter the biomineralization process in osteoblastic cells and provoke the following: i) a rise in mineralization, ii) an increase in the mRNA expression of RANKL and a decrease of OPG, iii) an increase in the mRNA expression of osteocalcin, bone sialoprotein and the transcription factor Runx2, iv) a diminished quality of the mineral, and v) an increase in the expression of IL1beta, IL6, IL8, MCP-1 and IL10 mRNAs. In addition we found that both high glucose levels and hyperosmotic conditions provoked TLR-2, -3, -4 and -9 overexpression in osteoblastic cells, suggesting that they are susceptible to osmotic stress.
Assuntos
Calcificação Fisiológica/efeitos dos fármacos , Glucose/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Fosfatase Alcalina/metabolismo , Remodelação Óssea/efeitos dos fármacos , Calcificação Fisiológica/fisiologia , Cálcio/metabolismo , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Diabetes Mellitus/fisiopatologia , Humanos , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteoprotegerina/genética , Osteoprotegerina/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo , RNA Mensageiro/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
According to previous reports, intranasal administration of the Cry1Ac protein alone or with amoebic lysates increases protection against Naegleria fowleri meningoencephalitis in mice, apparently by eliciting IgA responses in the nasal mucosa. In the current study, we performed an immunohistochemical analysis of IgA in the nasal mucosa of mice immunized intranasally with Cry1Ac, and amoebic lysates or a combination of both. The animals were sacrificed 24 h after the last immunization or after an intranasal lethal challenge with N. fowleri. Our results indicate that all of the intranasal immunizations provoked an increase in areas with metaplasia in the olfactory epithelium, allowing for secretion of IgA. As a result, IgA antibodies were found interacting with trophozoites in the nasal lumen, and there was a marked increase of IgA in the metaplasic epithelium. On the other hand in nonimmunized mice trophozoites were observed invading the nasal mucosa, which was not the case for immunized mice. Our results suggest that intranasal immunization provokes cellular changes in the olfactory epithelium, leading to greater protection against N. fowleri that is probably caused by an increased secretion of IgA. The increased IgA response induced in the nasal mucosa by immunization probably impedes both amoebic adhesion and subsequent invasion of the parasite to the nasal epithelium.
Assuntos
Amebíase/imunologia , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Endotoxinas/imunologia , Proteínas Hemolisinas/imunologia , Imunização , Imunoglobulina A Secretora/análise , Meningoencefalite/imunologia , Naegleria fowleri/imunologia , Mucosa Olfatória/imunologia , Adjuvantes Imunológicos , Administração Intranasal , Amebíase/parasitologia , Animais , Anticorpos Antiprotozoários/análise , Antígenos de Protozoários/imunologia , Toxinas de Bacillus thuringiensis , Masculino , Meningoencefalite/parasitologia , Metaplasia , Camundongos , Camundongos Endogâmicos BALB C , Mucosa Nasal/imunologia , Mucosa Nasal/parasitologia , Proteínas Recombinantes/imunologiaRESUMO
The N-terminal half or toxic fragment of Bacillus thuringiensis Cry proteins is comprised of three structural domains. In a previous paper, we showed that this region plays an important role in the immunogenicity of the B. thuringiensis Cry proteins. Due to this ability and along with their stability it is worthy of investigating whether this region has carrier potential. To approach this, an eight amino acid hydrophobic motif in alpha-helix 7 of wild-type (WT) Cry1A toxins was exchanged for a diphtheria toxin epitope (DTB). The resultant recombinant toxins were tested for their ability to induce specific anti-Cry and anti-diphtheria toxin antibodies in mice after intraperitoneal and nasal immunization. We found that recombinant Cry1A toxins retained their ability to induce serum and mucosal anti-Cry Ab as well as IgG subclasses, although with a varied magnitude. By the systemic route, the effect of the amino acid substitution in the ratio of the IgG1/IgG2a Ab, leading in some sites toward IgG1 or IgG2a is more evident. Interestingly, mice produced specific anti-DTB IgG, and IgA after intranasal immunization. Together, our results support and show the immunogenic properties of the WT Cry1A toxins as well as its carrier potential for a DTB.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Proteínas de Transporte/imunologia , Toxina Diftérica/imunologia , Endotoxinas/genética , Endotoxinas/imunologia , Epitopos/genética , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/imunologia , Administração por Inalação , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/imunologia , Substituição de Aminoácidos/genética , Substituição de Aminoácidos/imunologia , Animais , Formação de Anticorpos , Toxinas de Bacillus thuringiensis , Proteínas de Transporte/genética , Toxina Diftérica/administração & dosagem , Toxina Diftérica/genética , Epitopos/imunologia , Imunização , Imunoglobulina A/biossíntese , Imunoglobulina G/biossíntese , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
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
Linfócitos B/imunologia , Entamoeba histolytica/imunologia , Glutaral/química , Imunoglobulinas/imunologia , Intestino Grosso/imunologia , Intestino Delgado/imunologia , Vacinas Protozoárias/administração & dosagem , Animais , Intestino Grosso/parasitologia , Intestino Delgado/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Vacinas Protozoárias/imunologiaAssuntos
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
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.