Robert Schaer

Experimental Corynebacterium pseudotuberculosis primary infection in goats: kinetics of IgG and interferon-γ production, IgG avidity and antigen recognition by Western blotting

Corynebacterium pseudotuberculosis is the cause of caseous lymphadenitis (CLA) in small ruminants, a chronic granulomatous disease that provokes significant zootechnics losses to ovine and goat breeders in northern Brazil. The present work was conducted to analyse aspects of humoral and cellular immune responses after experimental infection. Eight goats were infected intradermally with a single dose of virulent C. pseudotuberculosis strain and specific IgG, interferon-gamma (IFN-gamma) production as well as IgG avidity and antigens pattern recognition dynamics against an excreted-secreted antigen were recorded during 20 weeks. At the end of the follow-up, animals were slaughtered and necropsied. Although no animals showed apparent clinical signs of infection at the end of the trial, IFN-gamma response, even more so than the humoral response, differentiated animals into two groups of high or medium/low response. The time-course of IFN-gamma production presented a short-lived primary response on day 5 after infection of animals of both groups, and a strong and long lasting secondary response starting on day 16 after infection in the high response group. The indirect ELISA used was able to detect a positive antibody titre between 6 and 11 days after infection in the two groups. IgG avidity index oscillated initially between 15 and 45%, and showed approximately 5% units increment during the 20 follow-up weeks. With only one individual exception, the qualitative antigens pattern recognition showed on day 11 after infection remained constant through the experiment. IgG avidity is highly correlated with IgG production, but could not be related with specific immunodominant bands. Both humoral and cellular responses kinetics presented a similar pattern of activation/deactivation but necropsy results suggested that the IFN-gamma test would be a very specific marker of CLA status.

Evaluation of the humoral and cellular immune response to different antigens of Corynebacterium pseudotuberculosis in Caninde´ goats and their potential protection against caseous lymphadenitis

Corynebacterium pseudotuberculosis is the etiologic agent of caseous lymphadenitis, a disease that affects goats and sheep, and can cause severe economic losses. In this study, four different antigenic extracts were obtained from the attenuated strain T1, which was isolated in the state of Bahia (Brazil). Forty-four Canindé breed goats were divided in five groups, each receiving a different antigen solution and saline buffer as a control. The humoral response was monitored through the identification of specific IgG by indirect ELISA and Western Blotting, and the production of IFN-gamma was followed in order to observe the activation of cellular response. After twelve weeks of antigen inoculation, the animals were challenged with 2 x 10(5)CFU of a wild strain, also isolated in Bahia, and necropsy was performed on all animals twelve weeks afterwards. It was observed that the attenuated bacteria gave a protection of 33.3%, in addition to the weak humoral response elicited. Animals inoculated with secreted antigen associated with Freunds incomplete adjuvant and oligodeoxynucleotide containing unmethylated CpG dinucleotides (CpG ODN) showed a strong humoral response, but this inoculation could not prevent the spread of challenge bacteria in the majority of animals. These results demonstrate the immunogenic potential of the attenuated T1 strain in the development of a vaccine against caseous lymphadenitis in goats.

Flavonoids inhibit angiogenic cytokine production by human glioma cells

VEGF and TGF‐β1 are cytokines that stimulate tissue invasion and angiogenesis. These factors are considered as molecular targets for the therapy of glioblastoma. Bevacizumab, a recombinant humanized monoclonal antibody developed against VEGF, inhibits endothelial cell proliferation and vessel formation. Flavonoids obtained from Dimorphandra mollis and Croton betulaster have been described as proliferation inhibitors of a human glioblastoma derived cell line. VEGF and TGF‐β1 levels were dosed by ELISA in a GL‐15 cell line treated with bevacizumab and also with the flavonoids rutin, 5‐hydroxy‐7,4′‐dimethoxyflavone, casticin, apigenin and penduletin. Rutin reduced the VEGF and TGF‐β1 levels after 24 h but not after 72 h. The other flavonoids significantly reduced TGF‐β1 production. Bevacizumab reduced only the VEGF levels in the supernatant from GL‐15 cultures. These results suggest that the flavonoids studied, and commonly used in popular medicine, present an interesting subject of study due to their potential effect as angiogenic factor inhibitors. Copyright

The Flavonoid Rutin but not the Alkaloid Arborinine Induces Apoptosis in a B-Cell Hybridoma Cell Line

The effects of arborinine, an alkaloid extracted from Erthela bahiensis and of rutin, a flavonoid obtained from Dimorphandra mollis (Benth.), Brazilian medicinal plants, on the viability and function of a murine B-cell hybridoma as a tumor model were investigated. The flavonoid rutin at 50 microM induced an increase in the number of apoptotic cells of one- to fivefold and reductions in cellular proliferation and monoclonal antibody production. Less but still significant necrosis was also induced by rutin under the same experimental conditions. On the other hand, the alkaloid arborinine exerted no significant effects on the studied parameters.

Flavonoid Rutin Alters the Viability and Function of Mitogen-Stimulated Splenocytes and Thymocytes Compared with Non Stimulated Cells

Rutin is a flavonoid obtained from Dimorphandra mollis (Benth.), a medicinal Brazilian plant used as antioxidative, antihemorrhagic, and blood vessel protector. The present study has examined its effects on the viability and function of immune system cells in vitro. Rat spleen and thymus cells were cultured with 10 nM, 1 μM, and 10 μM of the drug in the presence or absence of PWM, LPS, or ConA mitogens. Cellular proliferation was analyzed by H3-thymidin uptake and IFN-γ and IL-10 were measured by ELISA after 48 and 72 hr. Viability was measured by flow cytometry using Annexin V and PI after 24 and 48 hr. The flavonoid rutin inhibited splenocytes and thymocytes proliferation under ConA stimulation observed by an increase on apoptosis levels of thymocytes stimulated with PWM in 24 hr and on splenocytes stimulated with PWM in 48 hr. Function studies showed a decrease on IFN-γ production by splenocytes and thymocytes stimulated with PWM or ConA. Spleen cells cultured with LPS and rutin showed a decrease on apoptosis after 24 hr and an increase on the IL-10 levels after 48 hr. There was no significant variation on the necrosis rate, viability, and function of cells treated with rutin in the absence of mitogenic stimulus.

MAPK involvement in cytokine production in response to Corynebacterium pseudotuberculosis infection

BackgroundCaseous 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).ResultsThe 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.ConclusionThe 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.

Different Effects of Arborinine Alkaloid Obtained from Brazilian Erthela baihensis on Spleen and Thymus Cells Stimulated in Vitro with Different Mitogens

The present study has examined the effects of arborinine, an alkaloid obtained from Erthela bahiensis, a Brazilian plant popularly used as diuretic, antidiabetic, antithermic and expectorant, on the viability and function of immune system cells in vitro using a murine model. Rat spleen and thymus cells were cultured with 10nM, 1µM e 10µM of the drug in the presence or absence of pokeweed (PWM), lipopolysaccharide (LPS), or concanavallin (ConA) mitogens. Cellular proliferation was analyzed by H3‐thymidin uptake after 48 and 72 hr. Our results showed an inhibitory effect of arborinine on splenocytes proliferation under ConA or PWM stimulation and increased apoptosis on splenocytes and thymocytes stimulated with PWM in 24 hr. A decrease was observed on Interferon gamma (IFN-γ) production by ConA- or LPS-stimulated splenocytes in 48 hr and 72 hr and ConA- or PWM-stimulated thymocytes in 72 hr. In contrast, an increase on lymphoproliferation was observed on LPS-stimulated splenocytes and ConA- or PWM-stimulated thymocytes in 48 hr. On this period, apoptosis decreased on LPS- or PWM-stimulated splenocytes and IFN-γ production increased in PWM stimulated thymocytes. Arborinine also induced a decrease on Interleukin-10 production by splenocytes and thymocytes stimulated with ConA or PWM. There was no significant variation on the necrosis rate of the cells treated with arborinine or any change on their viability or function values in the absence of mitogenic stimulus.

Validation of an immunochromatographic assay for the multiple detection of specific antibodies against HIV, HBV, and HCV.

The manuscript published ahead of print as doi: [10.1128/CVI.00485-12][1] has been retracted. Although the authors maintain that the test procedures and results in the manuscript are valid and that they were not bound by a written contract with the product manufacturer, the authors have requested

Produção de sonda de DNA diretamente biotinilada com N-hidroxisuccinimida biotina ester (BNHS)

A utilizacao de sondas de DNA para analise genomica em fase solida tem se expandido e ultrapassado as areas da pesquisa, estendendo-se ao terreno do diagnostico de rotina nas areas de medicina humana e animal, bem como nas ciencias forenses. Estas sondas tem sido utilizadas desde o surgimento da primeira tecnica de hibridizacao descrita com o southern blotting, que envolve a imobilizacao, em filtros de papel, de fragmentos de DNA separados eletroforeticamente. Outra tecnica descrita como o dot-hibridizacao e menos complexa do que o southern convencional por nao requerer o passo previo de eletroforese e por ser menos exigente quanto a qualidade do DNA, ja que dispensa a digestao com enzima de restricao. A marcacao mais comum para estas sondas e o fosforo radioisotopo (32P), que, apesar de sua alta sensibilidade, apresenta os inconvenientes de risco fisico, necessidade de autorizacao para manipulacao de radioisotopos, alto custo e uma vida media muito curta. Uma serie de alternativas para o 32P tem sido utilizadas, com marcas diretas ou secundarias, entre elas a biotina, que tem mostrado grandes vantagens e tem sido usada tambem em laboratorios especializados e bem equipados. Este trabalho descreve uma modificacao do metodo classico descrito para sondas de DNA acopladas por biotina hidrazida. Utilizamos a N-hidroxisuccinimida biotina (BNHS) em dimetilformamida, para marcar residuos de citosina disponiveis em segmentos de fita simples de DNA extraido de Corynebacterium pseudotuberculosis. A variacao da proporcao de biotina e de DNA, bem como sua especificidade e sensibilidade, foram cuidadosamente testadas e criteriosamente analisadas atraves de ensaios de hibridizacao, adsorvendo-se DNA cromossomico do C. pseudotuberculosis e outros DNAs em papel de nitrocelulose, seguindo-se a incubacao com as sondas biotiniladas descritas,avidina-peroxidase e revelacao com 4-cloro-1-naftol. Os resultados mostraram que a sonda de DNA produzida tem as caracteristicas favoraveis esperadas. Desde que nao e necessario o nick translation nem a utilizacao de material radioativo ou mesmo de hidrazida, sondas deste tipo podem ser utilizadas no auxilio do diagnostico de infeccoes por C. pseudotuberculosis e outros microorganismos (AU)