RESUMO
Most food allergy cases are associated with a limited group of allergens. This could be attributed to an increased ability of some foods to sensitize and trigger allergic reactions. However, there are no validated animal models to evaluate the sensitizing or allergenic potentials of proteins. Our aim was to evaluate three protocols of adjuvant-free intraperitoneal sensitization that differ in the time points for sample collection (days 14, 28 and 35 from beginning of the sensitization) and also in the number of immunizations (2, 5 and 3, respectively). Ovalbumin (OVA; 0.05 mg), cow milk proteins (CMP; 0.025, 0.05 and 0.25 mg), and potato acid phosphatase (PAP; low allergenic protein; 250.0 mg) were administered intraperitoneally (ip) to BALB/c mice (n = 4â»6) and the protein-specific IgE and IgG antibody responses were evaluated using ELISA. Additional serum protein-specific IgE antibodies evaluations were carried out after IgG depletion. Anti-OVA IgE antibodies were detected in mice from all three protocols. The responses were higher in the group of mice that underwent the 28-day protocol than in those that underwent the 14- or 35-day protocols (p < 0.01 and p < 0.05, respectively). Anti-CMP IgE antibodies were detected in both the 14- and 28-day protocols, but the response was higher in the group that underwent the 28-day protocol (p < 0.001). The anti-CMP IgE antibody response detection was improved after serum IgG depletion (p < 0.001). Anti-PAP IgE antibodies were not detected. Mice with undetectable serum levels of protein-specific IgE triggered anti-OVA, -CMP, and -PAP IgG responses. An adjuvant-free 28-day protocol with five ip immunizations seems appropriate for evaluation of the inherent sensitizing or allergenic capacity of the studied proteins. Reproducible results were obtained utilizing the BALB/c mouse strain. Inter-laboratory studies including a larger number of proteins should be carried out to validate this model.
Assuntos
Fosfatase Ácida/imunologia , Hipersensibilidade Alimentar/imunologia , Hipersensibilidade a Leite/imunologia , Proteínas do Leite/imunologia , Ovalbumina/imunologia , Solanum tuberosum/imunologia , Fosfatase Ácida/administração & dosagem , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Hipersensibilidade Alimentar/sangue , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Injeções Intraperitoneais , Camundongos Endogâmicos BALB C , Hipersensibilidade a Leite/sangue , Proteínas do Leite/administração & dosagem , Ovalbumina/administração & dosagem , Raízes de Plantas/imunologia , Solanum tuberosum/enzimologia , Fatores de TempoRESUMO
Caseous lymphadenitis (CLA) is a chronic disease responsible for significant economic losses in sheep and goat breeding worldwide. The treatment for this disease is not effective, and an intense vaccination schedule would be the best control strategy. In this study, we evaluated the associations of rCP09720 or rCP01850 proteins from Corynebacterium pseudotuberculosis with recombinant exotoxin phospholipase D (rPLD) as subunit vaccines in mice. Four experimental groups (10 animals each) were immunized with a sterile 0.9% saline solution (G1), rPLD (G2), rPLDâ¯+â¯rCP09720 (G3), and rPLDâ¯+â¯rCP01850 (G4). The mice received two doses of each vaccine at a 21-day interval and were challenged 21â¯days after the last immunization. The animals were evaluated daily for 40â¯days after the challenge, and mortality rate was recorded. The total IgG production level increased significantly in the experimental groups on day 42 after the first vaccination. Similarly, higher levels of specific IgG2a were observed in experimental groups G2, G3, and G4 compared to the IgG1 levels on day 42. G4 showed a significant (pâ¯<â¯.05) humoral response against both antigens of the antigenic formulations. The cellular immune response induced by immunization was characterized by a significant (pâ¯<â¯.05) production of interferon-γ compared to that in the control, while the concentrations of interleukin (IL)-4 and IL-12 were not significant in any group. A significant increase of tumor necrosis factor was observed only in G4. The survival rates after the challenge were 30% (rPLD), 40% (rPLDâ¯+â¯rCP09720), and 50% (rPLDâ¯+â¯rCP01850). Thus, the association of rCP01850 with rPLD resulted in the best protection against the challenge with C. pseudotuberculosis and induced a more intense type 1 T-helper cell immune response.
Assuntos
Vacinas Bacterianas/imunologia , Infecções por Corynebacterium/prevenção & controle , Corynebacterium pseudotuberculosis/imunologia , Linfadenite/veterinária , Fosfolipase D/imunologia , Proteínas Recombinantes/imunologia , Fosfatase Ácida/administração & dosagem , Fosfatase Ácida/genética , Fosfatase Ácida/imunologia , Animais , Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/genética , Infecções por Corynebacterium/imunologia , Infecções por Corynebacterium/microbiologia , Corynebacterium pseudotuberculosis/química , Corynebacterium pseudotuberculosis/enzimologia , Corynebacterium pseudotuberculosis/genética , Esterases/administração & dosagem , Esterases/genética , Esterases/imunologia , Cabras/microbiologia , Imunidade Celular , Imunoglobulina G/sangue , Interferon gama/biossíntese , Interferon gama/imunologia , Linfadenite/imunologia , Linfadenite/microbiologia , Linfadenite/prevenção & controle , Camundongos , Fosfolipase D/administração & dosagem , Fosfolipase D/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Ovinos/microbiologia , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/microbiologia , Doenças dos Ovinos/prevenção & controle , Células Th1/imunologia , Vacinação/veterinária , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
AIM: Destructive periodontitis is associated with a Th1-Th17 immune response and activation of RANKL-induced osteoclasts. In addition, Porphyromonas gingivalis K1 and K2 serotypes induce a strong Th1-Th17 response. This study aimed to investigate whether these P. gingivalis serotypes induce higher osteoclasts activation, by increased Th17-associated RANKL production, and an antigen-specific memory T-lymphocyte response. MATERIAL AND METHODS: The RANKL production and TRAP(+) osteoclast induction were quantified on naïve T lymphocytes stimulated with dendritic cells primed with the P. gingivalis serotypes. The T-bet, GATA-3, RORC2 and Foxp3 expression was correlated with RANKL production. The frequency of proliferating memory T lymphocytes in response to P. gingivalis serotypes was determined in both periodontitis and healthy subjects. RESULTS: T lymphocytes stimulated by K1 or K2-primed dendritic cells elicited higher levels of RANKL and TRAP(+) osteoclasts than cells stimulated with the other serotypes. RANKL positively correlated with RORC2. Whereas periodontitis patients had a higher frequency of memory T lymphocytes responding to K1 or K2, healthy subjects had a higher frequency of memory T lymphocytes responding to K4 or K(-) . CONCLUSIONS: P. gingivalis serotypes K1 and K2, but not others, are associated with an increased production of the osteoclastogenesis-related factor RANKL. This important information suggests that these serotypes could elicit a greater bone resorption in vivo and have a role in the periodontitis pathogenesis.
Assuntos
Memória Imunológica/imunologia , Osteoclastos/imunologia , Porphyromonas gingivalis/imunologia , Ligante RANK/imunologia , Sorogrupo , Linfócitos T/imunologia , Fosfatase Ácida/análise , Fosfatase Ácida/imunologia , Animais , Antígenos de Bactérias/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Diferenciação Celular/imunologia , Linhagem Celular , Periodontite Crônica/imunologia , Seleção Clonal Mediada por Antígeno , Células Dendríticas/imunologia , Fatores de Transcrição Forkhead/análise , Fator de Transcrição GATA3/análise , Humanos , Isoenzimas/análise , Isoenzimas/imunologia , Macrófagos/imunologia , Camundongos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/análise , Osteoclastos/efeitos dos fármacos , Porphyromonas gingivalis/classificação , Ligante RANK/análise , Proteínas com Domínio T/análise , Linfócitos T/microbiologia , Fosfatase Ácida Resistente a Tartarato , Células Th1/imunologia , Células Th17/imunologiaRESUMO
Rosiglitazone (RGZ), an oral anti-hyperglycemic agent used for non-insulin-dependent diabetes mellitus, is a high-affinity synthetic agonist for peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Both in vitro and in vivo experiments have also revealed that RGZ possesses anti-inflammatory properties. Therefore, in the present study, we investigated the anti-inflammatory effects of RGZ in a rat model of periodontal disease induced by ligature placed around the mandible first molars of each animal. Male Wister rats were divided into four groups: 1) animals without ligature placement receiving administration of empty vehicle (control); 2) animals with ligature receiving administration of empty vehicle; 3) animals with ligature receiving administration with oral RGZ (10 mg/kg/day); and 4) animals with ligature receiving administration of subcutaneous RGZ (10 mg/kg/day). Thirty days after induction of periodontal disease, the animals were sacrificed, and mandibles and gingival tissues were removed for further analysis. An in vitro assay was also employed to test the inhibitory effects of RGZ on osteoclastogenesis. Histomorphological and immunohistochemical analyses of periodontal tissue demonstrated that RGZ-treated animals presented decreased bone resorption, along with reduced RANKL expression, compared to those animals with ligature, but treated with empty vehicle. Corresponding to such results obtained from in vivo experiments, RGZ also suppressed in vitro osteoclast differentiation in the presence of RANKL in MOCP-5 osteoclast precursor cells, along with the down-regulation of the expression of RANKL-induced TRAP mRNA. These data indicated that RGZ may suppress the bone resorption by inhibiting RANKL-mediated osteoclastogenesis elicited during the course of experimental periodontitis in rats.
Assuntos
Hipoglicemiantes/administração & dosagem , Osteoclastos/efeitos dos fármacos , Periodontite/tratamento farmacológico , Ligante RANK/metabolismo , Tiazolidinedionas/administração & dosagem , Fosfatase Ácida/genética , Fosfatase Ácida/imunologia , Fosfatase Ácida/metabolismo , Administração Oral , Perda do Osso Alveolar/prevenção & controle , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Hipoglicemiantes/farmacologia , Imuno-Histoquímica , Isoenzimas/genética , Isoenzimas/imunologia , Isoenzimas/metabolismo , Masculino , Osteoclastos/imunologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Osteogênese/efeitos dos fármacos , PPAR gama/agonistas , Periodontite/imunologia , Periodontite/patologia , Periodontite/fisiopatologia , Ligante RANK/genética , Ligante RANK/imunologia , Ratos , Ratos Wistar , Rosiglitazona , Fosfatase Ácida Resistente a Tartarato , Tiazolidinedionas/farmacologiaRESUMO
Paracoccidioides brasiliensis is a thermo-dimorphic fungus responsible for paracoccidioidomycosis (PCM), a systemic granulomatous mycosis prevalent in Latin America. The fungus releases many antigens which may be transiently bound to its cell surface. Some of them may show enzymatic functions essential for maintaining many cell processes and survival of the microorganism at different conditions. In this study, we report the characterization of a secreted 75kDa protein from P. brasiliensis with phosphatase activity. Biologic function of the molecule was demonstrated using two specific mAbs produced and characterized as IgM and IgG isotypes. Confocal microscopy and flow cytometry analysis demonstrated that both mAbs recognized the protein on the fungus surface, mainly in its budding sites. In vitro experiments showed that fungal growth was inhibited by blocking the protein with mAbs. In addition, opsonized yeast cells with both mAbs facilitated phagocytosis by murine peritoneal macrophages. Passive immunization using mAbs before P. brasiliensis mice infection reduced colony-forming units (CFU) in the lungs as compared with controls. Histopathology showed smaller inflammation, absence of yeast cells and no granuloma formation.
Assuntos
Fosfatase Ácida/imunologia , Anticorpos Monoclonais/imunologia , Proteínas Fúngicas/imunologia , Imunização Passiva , Proteínas de Membrana/imunologia , Paracoccidioides/crescimento & desenvolvimento , Paracoccidioidomicose/imunologia , Fosfatase Ácida/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/biossíntese , Antígenos de Fungos/imunologia , Antígenos de Fungos/metabolismo , Proteínas Fúngicas/metabolismo , Macrófagos Peritoneais/microbiologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Paracoccidioides/imunologia , Paracoccidioidomicose/microbiologia , Paracoccidioidomicose/prevenção & controle , FagocitoseRESUMO
As defined by the reaction with monoclonal antibodies, Leishmania mexicana promastigotes contain two acid phosphatases which together comprise about 90% of the cellular activity. A first enzyme recognized by monoclonal antibody AP4 is largely membrane-bound. The protein has an apparent molecular weight of 70,000-72,000, carries about seven N-linked glycan chains and is present in approximately 16,000 copies per cell. The protein is also expressed in the amastigote stage. A second enzyme reactive with monoclonal antibody AP3, that also recognizes lipophosphoglycan and a secreted acid phosphatase, is mainly found in the soluble fraction of promastigote lysates. It is suggested that this enzyme is the precursor of the secreted protein. The N-terminal sequences of the phosphatase recognized by AP4 and the secreted enzyme are similar but not identical. AP4 does not cross-react with phosphatase activity of Leishmania major or Leishmania donovani promastigotes, while AP3 recognizes part of the cellular and all of the secreted phosphatase activity of L. donovani promastigotes but not that of L. major which does not release an acid phosphatase into the culture medium.