The role of adjuvants in therapeutic protection against paracoccidioidomycosis after immunization with the P10 peptide

Paracoccidioidomycosis (PCM), a common chronic mycosis in Latin America, is a granulomatous systemic disease caused by the thermo-dimorphic fungus Paracoccidioides brasiliensis. The glycoprotein gp43 is the main antigen target of P. brasiliensis and a 15-mer internal peptide (QTLIAIHTLAIRYAN), known as P10, defines a major CD4+-specific T cell epitope. Previous results have indicated that, besides having a preventive role in conventional immunizations prior to challenge with the fungus, protective anti-fungal effects can be induced in P. brasiliensis-infected mice treated with P10 administered with complete Freund’s adjuvant (CFA). The peptide elicits an IFN-γ-dependent Th1 immune response and is the main candidate for effective immunotherapy of patients with PCM, as an adjunctive approach to conventional chemotherapy. In the present study we tested the therapeutic effects of P10 combined with different adjuvants [aluminum hydroxide, CFA, flagellin, and the cationic lipid dioctadecyl-dimethylammonium bromide (DODAB)] in BALB/c mice previously infected with the P. brasiliensis Pb18 strain. Significant reductions in the number of colony forming units of the fungus were detected in lungs of mice immunized with P10 associated with the different adjuvants 52 days after infection. Mice treated with DODAB and P10, followed by mice treated with P10 and flagellin, showed the most prominent effects as demonstrated by the lowest numbers of viable yeast cells as well as reductions in granuloma formation and fibrosis. Concomitantly, secretion of IFN-γ and TNF-α, in contrast to interleukin (IL)-4 and IL-10, was enhanced in the lungs of mice immunized with P10 in combination with the tested adjuvants, with the best results observed in mice treated with P10 and DODAB. In conclusion, the present results demonstrate that the co-administration of the synthetic P10 peptide with several adjuvants, particularly DODAB, have significant therapeutic effects in experimental PCM

Analysis of different adjuvants associated to P10 peptide for treatment of BALB/c infected mice with Paracoccidioides brasiliensis.

Paracoccidioidomicose (PCM) é uma doença sistêmica granulomatosa causada pelo fungo termo-dimórfico Paracoccidioides brasiliensis. A gp43 é o principal antígeno diagnóstico que contém o peptídeo de 15 aminoácidos designado como P10, que desenvolve a resposta imune do tipo Th1 dependente de IFN-g. No presente trabalho, comparamos a efetividade de diferentes adjuvantes em camundongos BALB/c infectados intratraquealmente com o isolado virulento Pb18. Alúmen, Adjuvante Completo e Incompleto de Freund, FliC flagelina e o lipídeo catiônico, foram testados em associação ou não com P10. Após 52 dias de infecção, o lipídeo catiônico mostrou os melhores resultados, com o menor número de UFCs, redução de fibrose e baixo número de células fúngicas no tecido pulmonar, além do incremento significativo de IFN-g e TNF-a e a redução dos níveis de IL-4 e IL-10. Estes resultados sugerem que a interação do peptídeo P10 com o lipídeo catiônico pode gerar uma melhor resposta imune mediada por células do tipo Th1, evitando a rápida disseminação da PCM experimental.Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. The gp43 is the major diagnostic antigen that contains the peptide P10. This peptide has 15 amino acids and develops a Th1- dependent IFN-g immune response. In this work, we compare the effectiveness of different adjuvants in BALB/c mice infected intratracheally with virulent isolate Pb18. Alum, Complete Adjuvant and Incomplete Freund\'s adjuvant, fliC flagellin from Salmonella enterica and cationic lipid were tested in combination or not with P10. After 52 days of infection, the cationic lipid showed the best results with the least number of colony forming units, reduction of fibrosis, low number of fungal cells in lung tissue and the significant increase of IFN-g and TNF-a with reduced levels of IL-4 and IL-10. These results suggest that, the interaction of peptide P10 with cationic lipids can lead to a better immune response mediated by Th1 type cells by avoiding the rapid spread of experimental PCM

Serotypes of listeria Monocytogenes isolated on food produced in Cautin province, Chile

En Chile se ha aislado Listeria monocytogenes en diversos alimentos. El objetivo de este estudio fue aportar información sobre la presencia de la bacteria en longanizas artesanales (embutidos madurados), leche cruda y hortalizas producidos en la Provincia de Cautín y determinar los serotipos. Las muestras correspondieron a longaniza común artesanal, leche cruda y hortalizas, colectadas durante 2003. Para el aislamiento e identificación de L. monocytogenes se usó la metodología aprobada por el Food and Drug Administration (FDA) con incubación en caldo selectivo preenrriquecimiento para Listeria (35°C), y posterior siembra en medios selectivos Oxford y Palcam (35-37°C). Las colonias típicas fueron replicadas en caldo tripticasa de soya (TSB) y sometidas a pruebas bioquímicas de identificación. La serotipificación se realizó sobre 53 cepas, utilizando un set de antisuero comercial. L. monocytogenes se presentó en el 61,1% de las longanizas, en el 0,0% de las muestras de leche cruda y en el 16,6% de las de hortalizas. Estos resultados podrían indicar fallas en la higiene de la manipulación, en el lavado y sanitización de superficies de contacto con los alimentos. Se determinó la presencia del serotipo 4e en 3 cepas provenientes de longanizas fabricadas en la ciudad de Temuco, y 1 cepa de cada uno de los serotipos virulentos 1/2a, 1/2b y 4b, los dos primeros provenientes de longanizas y el último desde hortalizas. La presencia de estos serotipos 1/2a, 1/2b y 4b, plantean una clara amenaza de un eventual brote, particularmente en consumidores susceptibles. Estos resultados representan un importante desafío de prevención y control para las autoridades sanitarias [email protected] has been reported in Chile that L. monocytogenes is present in differents kind of foods. The aim of this study was to obtain information of the occurrence of the bacteria in sausages, raw milk and vegetables produced in Cautín Province, and to know the L. monocytogenes serotypes involved. For this purpose sausages, raw milk and several vegetables samples, were collected on 2003. For the isolation and identification of L. monocytogenes, the samples were grown in tripticase soy broth (TSB) inoculated into a preenrichment broth for Listeria (35°C followed by an inoculation on selectives medias Oxford and Palcam (35-37°C). Biochemical test were performed to all the typical Listeria colonies according to the methodology recommended by Bacteriological Analytical Manual (FDA). Serotyping was carried out using commercial specific antisera on 53 bacterial strains. Results of the analysis indicated the presence of L. monocytogenes in 61.1% of sausages samples, 0.0% in raw milk samples and in 16.6% of vegetables samples. These results could indicate failure in the hygiene practices, like cleaning and sanitizing the surface in contact with the food. The presence of serotipe 4e in 3 strains were found in some sausages. There was a strain of virulent serotype 1/2a, 1/2b and 4b, the first and second resulted isolated from sausages and the third one resulted from vegetables. The presence of serotypes 1/2a, 1/2b and 4b, is a threat of an eventual out-break, especially in persons quite susceptive, that is why, there is a huge risk for pubic health in customers from this region. These results represent for chilean public´s health authorities an enormous challenge for controlling and prevention procedures

Serotypes of Listeria monocytogenes Isolated on Food Produced in Cautin Province, Chile

It has been reported in Chile that L. monocytogenes is present in differents kind of foods. The aim of this study was to obtain information of the occurrence of the bacteria in sausages, raw milk and vegetables produced in Cautin Province, and to know the L. monocyto genes serotypes involved. For this purpose sausages, raw milk and several vegetables samples, were collected on 2003. For the isolation and identification of L. monocytogenes, the samples were grown in tripticase soy broth (TSB) inoculated into a preenrichment broth for Listeria (35 degrees C followed by an inoculation on selectives medias Oxford and Palcam (35-37 degrees C). Biochemical test were performed to all the typical Listeria colonies according to the methodology recommended by Bacteriological Analytical Manual (FDA). Serotyping was carried out using commercial specific antisera on 53 bacterial strains. Results of the analysis indicated the presence of L. monocytogenes in 61.1% of sausages samples, 0.0% in raw milk samples and in 16.6% of vegetables samples. These results could indicate failure in the hygiene practices, like cleaning and sanitizing the surface in contact with the food. The presence of serotipe 4e in 3 strains were found in some sausages. There was a strain of virulent serotype 1/2a, 1/2b and 4b, the first and second resulted isolated from sausages and the third one resulted from vegetables. The presence of serotypes 1/2a, 1/2b and 4b, is a threat of an eventual out-break, especially in persons quite susceptive, that is why, there is a huge risk for pubic health in customers from this region. These results represent for chilean public's health authorities an enormous challenge for controlling and prevention procedures

Characterization of a Methanogenic Microbial Consortium from a Coal Mine in Bogotá Basin

The work studied the methanogenic microbial consortium in a coal mine from the Bogotá basin in Colombia. Ex situ coal-enrichment cultures were established for in vitro growth and de novo gas production. Biogenic gas produced by cultures was analyzed by gas chromatography using thermal conductivity and flame ionization detectors. Cultures were used to isolate microbial specimens and to generate 16S rRNA gene libraries employing bacterial and archaeal primer sets. The gas chromatographic analysis showed methane production at 37 oC, but not at 60 oC, where CO2 was the major component of the biogenic gas. 16S rRNA gene sequence analysis of microbial isolates and clone libraries established that the methanogenic microbial consortium was formed by bacteria species from Bacillus and Gracilibacter genera plus archaea from the Methanothermobacter genus. This methanogenic microbial consortium was potentially responsible for biogenic gas generation in La Ciscuda coal mine. The results suggested that these methanogens produced methane by hydrogenotrophic or CO2 reduction pathways