Humoral and cellular immune responses in mice against secreted and somatic antigens from a Corynebacterium pseudotuberculosis attenuated strain: Immune response against a C. pseudotuberculosis strain.

BACKGROUND: Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis (CL), a chronic disease that affects goats and sheep. CL is characterized by the formation of granulomas in lymph nodes and other organs, such as the lungs and liver. Current knowledge of CL pathogenesis indicates that the induction of humoral and cellular immune responses are fundamental to disease control. The aim of this study was to evaluate the humoral and cellular immune responses in BALB/c mice inoculated with a C. pseudotuberculosis strain isolated in the state of Bahia, Brazil. RESULTS: The lymphocyte proliferation and in vitro production of IFN-γ, IL-4, IL-10, IL-12 and nitric oxide by spleen cells stimulated with secreted and somatic antigens from the studied strain were evaluated. IgG subclasses were also analyzed. Results showed a significant increase of Th1-profile cytokines after 60 days post-inoculation, as well as an important humoral response, represented by high levels of IgG2a and IgG1 against C. pseudotuberculosis. CONCLUSION: The T1 strain of C. pseudotuberculosis was shown to induce humoral and cellular immune responses in BALB/c mice, but, even at a dosage of 1x10(7) CFU, no signs of the disease were observed.

MAPK involvement in cytokine production in response to Corynebacterium pseudotuberculosis infection.

BACKGROUND: Caseous lymphadenitis (CL) is a contagious infectious disease of small ruminants caused by Corynebacterium pseudotuberculosis. Is characterized by the formation of abscesses in the lymph nodes and intestines of infected animals, induced by inflammatory cytokines. The production of cytokines, such as IL-10, TNF-α, IL-4 and IFN-γ, is regulated by mitogen-activated protein kinase (MAPK) pathway activation. The present study investigated the involvement of MAPK pathways (MAPK p38, ERK 1 and ERK 2) with respect to the production of cytokines induced by antigens secreted by C. pseudotuberculosis over a 60-day course of infection. CBA mice (n = 25) were divided into three groups and infected with 102 colony forming units (CFU) of attenuated strain T1, 102 CFU of virulent strain VD57 or sterile saline solution and euthanized after 30 or 60 days. Murine splenocytes were treated with specific inhibitors (MAPK p38 inhibitor, ERK 1/2 inhibitor or ERK 2 inhibitor) and cultured with secreted antigens obtained from pathogenic bacteria (SeT1 or SeVD57). RESULTS: The MAPK pathways evaluated were observed to be involved in the production of IL-10, under stimulation by secreted antigens, while the MAPK p38 and ERK 1 pathways were shown to be primarily involved in TNF-α production. By contrast, no involvement of the MAPK p38 and ERK 1 and 2 pathways was observed in IFN-γ production, while the ERK 2 pathway demonstrated involvement in IL-4 production only in the mouse splenocytes infected with VD57 under stimulation by SeT1. CONCLUSION: The authors hypothesize that MAPK p38 and ERK 1 pathways with respect to TNF-α production, as well as the MAPK p38 and ERK 1 and 2 pathways in relation to IL-10 production under infection by C. pseudotuberculosis are important regulators of cellular response. Additionally, the lack of the MAPK p38 and ERK 1/2 pathways in IFN-γ production in infected CBA murine cells stimulated with the two secreted/excreted antigens, in IL-4 production showing involvement only via the ERK 2 pathway under stimulation by SeT1 antigen during 60-day infection period with the virulent strain, suggests that these pathways regulated the production of pro-inflammatory and regulatory cytokines in the splenic cells of CBA mice.

Immunolocalization of FGF-2 and VEGF in rat periodontal ligament during experimental tooth movement.

OBJECTIVE: This article aimed at identifying the expression of fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) in the tension and pressure areas of rat periodontal ligament, in different periods of experimental orthodontic tooth movement. METHODS: An orthodontic force of 0.5 N was applied to the upper right first molar of 18 male Wistar rats for periods of 3 (group I), 7 (group II) and 14 days (group III). The counter-side first molar was used as a control. The animals were euthanized at the aforementioned time periods, and their maxillary bone was removed and fixed. After demineralization, the specimens were histologically processed and embedded in paraffin. FGF-2 and VEGF expressions were studied through immunohistochemistry and morphological analysis. RESULTS: The experimental side showed a higher expression of both FGF-2 and VEGF in all groups, when compared with the control side (P < 0.05). Statistically significant differences were also found between the tension and pressure areas in the experimental side. CONCLUSION: Both FGF-2 and VEGF are expressed in rat periodontal tissue. Additionally, these growth factors are upregulated when orthodontic forces are applied, thereby suggesting that they play an important role in changes that occur in periodontal tissue during orthodontic movement.

Immunolocalization of FGF-2 and VEGF in rat periodontal ligament during experimental tooth movement

OBJECTIVE: This article aimed at identifying the expression of fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) in the tension and pressure areas of rat periodontal ligament, in different periods of experimental orthodontic tooth movement. METHODS: An orthodontic force of 0.5 N was applied to the upper right first molar of 18 male Wistar rats for periods of 3 (group I), 7 (group II) and 14 days (group III). The counter-side first molar was used as a control. The animals were euthanized at the aforementioned time periods, and their maxillary bone was removed and fixed. After demineralization, the specimens were histologically processed and embedded in paraffin. FGF-2 and VEGF expressions were studied through immunohistochemistry and morphological analysis. RESULTS: The experimental side showed a higher expression of both FGF-2 and VEGF in all groups, when compared with the control side (P < 0.05). Statistically significant differences were also found between the tension and pressure areas in the experimental side. CONCLUSION: Both FGF-2 and VEGF are expressed in rat periodontal tissue. Additionally, these growth factors are upregulated when orthodontic forces are applied, thereby suggesting that they play an important role in changes that occur in periodontal tissue during orthodontic movement. .

OBJETIVO: o objetivo desse estudo foi identificar a expressão do fator de crescimento de fibroblastos 2 (FGF-2) e do fator de crescimento vascular endotelial (VEGF) nos lados de tensão e pressão do ligamento periodontal de ratos, durante movimento ortodôntico experimental, em diferentes períodos de tempo. MÉTODOS: uma força ortodôntica de 0,5N foi aplicada no primeiro molar superior direito de 18 ratos Wistar machos, por períodos de 3 (grupo I), 7 (grupo II) e 14 dias (grupo III). O primeiro molar do lado oposto foi utilizado como controle. Os animais foram sacrificados nos períodos de tempo mencionados, sendo a arcada superior removida e fixada. Após a desmineralização, os espécimes foram processados histologicamente e embebidos em parafina. A expressão do FGF-2 e do VEGF foram estudadas por meio de análise imuno-histoquímica. RESULTADOS: o ligamento periodontal dos dentes submetidos à movimentação ortodôntica mostraram maior expressão tanto de FGF-2 quanto de VEGF, em todos os grupos experimentais, quando comparados com os dentes do lado controle (p < 0,05). Diferenças estatisticamente significativas entre os lados de tensão e pressão também foram encontradas nos dentes submetidos à movimentação ortodôntica. CONCLUSÕES: tanto o FGF-2 quanto o VEGF são expressos no tecido periodontal de ratos, e esses fatores de crescimento são aumentados quando forças ortodônticas são aplicadas, sugerindo que esses desempenham um papel importante na reorganização do periodonto durante o movimento ortodôntico. .

Association between haptoglobin and IgM levels and the clinical progression of caseous lymphadenitis in sheep.

BACKGROUND: Sheep caseous lymphadenitis (CLA), caused by Corynebacterium pseudotuberculosis (Cp), is associated with direct economic losses and presents significant zoonotic potential. Despite the importance of the disease, a satisfactory vaccine model has not been developed. Thus, this study aimed to investigate the association between haptoglobin (Hp) and IgM levels and the clinical progression of CLA in primarily infected sheep and in sheep immunized with Cp- secreted antigens adjuvanted with Quillaja saponaria saponins. These animals were kept with CLA-positive sheep to simulate natural exposure that occurs in field conditions. During the experiment, the Hp and IgM levels were monitored for 21 days, and the development of internal CLA lesions was investigated through necropsies on day182 post-immunization. RESULTS: Primarily infected sheep in Group 2 (inoculated with 2x105 Cp virulent strain) had higher Hp values between the first and ninth days post inoculation (PI) than sheep in Group 1 (control; P < 0.05). Immunized animals in Group 3 had significantly higher Hp values between the third and seventh days PI, compared with the control group (P < 0.01). Binary logistic regression (BLR) analysis of primarily infected sheep indicated an association between Hp concentration and CLA clinical progression: animals with high Hp values had 99.9% less risk of having CLA abscesses than animals with low Hp levels (Odds ratio = 0.001, P < 0.05). Both experimental groups had significantly higher IgM titers than the control group around the ninth and eleventh days PI (P < 0.05). The BLR analysis for immunized sheep indicated an association between IgM levels and clinical progression: sheep with high IgM titers had 100.0% less risk of having CLA abscesses than animals with low IgM levels (Odds ratio = 0.000, P < 0.05). CONCLUSIONS: Resistance to C. pseudotuberculosis infection is supported by the early acute phase response, in which up-regulation of Hp and IgM were predictive of a lower risk of CLA lesion development. Because the immunogen used in this study induced a high production of both Hp and IgM, Q. saponaria saponin should be considered a promising candidate in vaccine formulations against sheep CLA.

Estudo das condições microbiológicas de farinhas de mandioca (Manihot esculenta Crantz) comercializadas no centro de abastecimento de Alagoinhas, Bahia / Study of the microbiological conditions of marketing of cassava (Manihot esculenta Crantz) flour in supply center of Alagoinhas, Bahia

No município de Alagoinhas, Bahia, e na maioria dos estados do Nordeste, a farinha de mandioca (Manihot esculenta Crantz) é produzida de forma artesanal em casas-de-farinha, na maioria das vezes localizadas no próprio local de plantio da mandioca. Este trabalho teve como objetivo avaliar as condições microbiológicas de farinhas de mandioca comercializadas no Centro de Abastecimento em Alagoinhas-Bahia. Foram coletadas 100 gramas de 30 amostras de farinha de mandioca, as quais foram acondicionadas em frascos estéreis, em recipiente isotérmico. As amostras foram submetidas à pesquisa de bactérias heterotróficas empregando-se o método de contagem padrão em placas, seguido da coloração de Gram, e a pesquisa para coliformes totais e termotolerantes foi realizada pela técnica do Número Mais Provável (NMP). Os resultados indicam que as amostras estão em conformidade com a legislação vigente em relação à análise de coliformes totais e termotolerantes (<3NMPg-1), na contagem de bactérias heterotróficas algumas amostras mostraram-se fora dos padrões aceitáveis. Na coloração de Gram foram observados bacilos, diplobacilos e estreptobacilos Gram positivos e Gram negativos e cocos, diplococos e estafilococos Gram positivos. A avaliação sanitário-ambiental revelou más condições de comercialização da farinha de mandioca no Centro de Abastecimento de Alagoinhas.

In the city of Alagoinhas, Bahia, and in most Northeastern states, the cassava (Manihot esculenta Crantz) flour is produced in a craft house, most often located in the place of production. This study aimed to evaluate the microbiological conditions of cassava flours marketed Supply Centre in Alagoinhas, Bahia. We collected 30 samples of cassava flour, 100 grams, which were placed in sterile, insulated container. The samples were tested for heterotrophic bacteria using the method of standard plate count, followed by Gram staining, the search for total and fecal coliforms was performed by the Most Probable Number (MPN). The results indicate that the samples that the samples are in accordance with current legislation regarding the analysis of total coliforms and thermotolerant (<3NMPg-1), the count of heterotrophic bacteria some samples were outside the acceptable standards. In Gram staining bacilli were observed, and diplobacillus estreptobacilos Gram positive and Gram negative cocci, diplococci and staphylococci Gram posotivos. The assessment of health and environmental conditions revealed but the marketing of cassava flour in the Central Supply Alagoinhas.

Antibody response to a chromatographic fraction of Porphyromonas gingivalis and its correlation with periodontal status

Porphyromonas gingivalis tem sido fortemente associado à gravidade das doenças periodontais e estimula respostas celulares e humorais no hospedeiro. Objetivo: Correlacionar os níveis de IgA, IgG e subclasses de IgG contra uma fração cromatogrßfica do extrato de Porphyromonas gingivalis ATCC33277 extract e descritores clínicos periodontais. Material e Métodos: Indivíduos com periodontite (29) e com saúde periodontal (26) foram avaliados de acordo com as medidas de profundidade de sondagem, sangramento à sondagem e nível de inserção clínica. O extrato de Porphyromonas gingivalis foi fracionado por cromatografia de troca iônica e a resposta humoral contra a fração IV foi avaliada por ôenzyme linked immunosorbent assayõ. Resultados e Conclusão: O percentual de sítios com sangramento à sondagem (critério 1) estava correlacionado significativamente com os níveis séricos de IgG2 (r = 0,385; p < 0,05). Os níveis de IgG total e IgG2 correlacionaram-se significativamente com o percentual de sítios com nível de inserção clínica (NIC) ≥ 3 mm (critério 2) (r = 0,428; p < 0,05 e r = 0,510; p < 0,01, respectivamente), NIC ≥ 5 mm (critérion 3) (r = 0,499 e r = 0,518, respectivamente; p < 0,01) e percentual de sítios com NIC ≥ 3 mm associado a profundidade de sondagem ≥ 4 mm e sangramento à sondagem no mesmo sítio (criterion 4) (r = 0,607; p < 0,001 e r = 0,487; p < 0,01, respectivamente). Foi observada uma correlação positiva estatisticamente significante entre os níveis de IgGA e IgG1 e o critério 4 (r = 0,339 e r = 0,345, respectivamente; p < 0,05), e entre os níveis de IgG3 e o critério 3 (p < 0,05; r = 0,370). Estes resultados indicam que quanto mais acurado for o critério de diagnóstico empregado para a determinação da doença periodontal, maiores os níveis séricos de imunoglobulinas.

SDS-PAGE and Western blot analysis of somatic and extracellular antigens of Corynebacterium pseudotuberculosis

Diferentes técnicas para isolar e descrever antígenos das frações secretadas de superfície e somáticas deCorynebacterium pseudotuberculosis foram estudadas por SDS-PAGE e imunoblot, utilizando-se pool de soros de cabras naturalmente infectadas. Os antígenos secretados foram obtidos do sobrenadante de cultura da bactéria cultivada em meio quimicamente definido ou em meio BHI (Brain Heart Infusion). A fração de superfície foi obtida por tratamento com NaCl 1M, e as frações somßticas foram obtidas por vários procedimentos (detergentes e ultra-som). Pela coloração por Coomassie blue, foram detectadas 20 bandas na fração secretada, 35 bandas no extrato de superfície e entre 40 e 50 bandas, a depender do processo de extração, na fração somßtica. Entre todas as frações estudadas, foram detectadas 16 proteínas imunorreativas. As bandas com pesos moleculares de 125, 108, 75, 68, 41, 40, 31 e 24 kDa foram reconhecidas commaior intensidade e todas elas foram encontradas na fração secretada. A utilização do meio quimicamente definido permitiu revelar, na fração secretada de C. pseudotuberculosis, a presença de proteínas de alto pesomolecular que não tinham sido previamente descritas.