Abundância de microartrópodes do solo em fragmentos de mata com araucária no sul do Brasil

The abundance of soil microarthropods from seven fragments of Araucaria Forest, Muitos Capões, Rio Grande do Sul, Brazil, was compared. The size of the fragments ranged from 0.25 ha to 35 ha, the two largest fragments are situated within the Aracuri Ecological Station and the remaining five are situated in a cattle ranching farm. In June 2000, three plots (10 m x 10 m) were established in the central area of each patch, and three soil cores (7 cm diameter x 6 cm deep) were taken per plot. The abundance of microarthropods in the upper six centimeters (soil + litter) varied between 63209 and 102704 ind.m-2, with oribatid mites (Acari, Cryptostigmata) being dominant in all fragments (between 46.9 % and 61.3 % of total individuals). Most microarthropod groups presented a decrease in abundance with decreasing fragment area, with a statistically significant difference between smaller and larger fragments. The proportion of oribatids also decreased with decreasing fragment area. The results suggest that the growing fragmentation process of Araucaria forests in southern Brazil, associated to a tendency for reducing the size of remnant fragments, can affect the abundance of soil microarthropods, and therefore, the quality and health of this ecosystem.

Levantamento populacional do cervo-do-pantanal Blastocerus dichotomus (Mammalia, Cervidae) no Parque Nacional de Ilha Grande e entorno: implicações para a conservação

Nós conduzimos um levantamento populacional aéreo do cervo-do-pantanal, Blastocerus dichotomus (Illiger, 1815), no Parque Nacional de Ilha Grande e seu entorno, entre os estados do Paraná e Mato Grosso do Sul, Brasil, na estação seca. Utilizamos a técnica da contagem dupla, e a população foi estimada em 1.079 ± 207 cervos em uma área amostral de 1.081 km², corresponde a uma densidade de 0,998 ± 0,192 ind/km². A população mostrou-se mais concentrada no interior do parque, ocupando também várzeas alteradas fora dos limites do parque. Estes resultados devem subsidiar medidas de manejo e conservação da população no parque nacional e seu entorno, devido à enorme pressão antrópica sobre a população de cervos na região.

Population dynamics of the pea crab Austinixa aidae (Brachyura, Pinnotheridae): a symbiotic of the ghost shrimp Callichirus major (Thalassinidea, Callianassidae) from the southwestern Atlantic

The Pinnotheridae family is one of the most diverse and complex groups of brachyuran crabs, many of them symbionts of a wide variety of invertebrates. The present study describes the population dynamics of the pea crab Austinixa aidae (Righi, 1967), a symbiont associated with the burrows of the ghost shrimp Callichirus major (Say, 1818). Individuals (n = 588) were collected bimonthly from May, 2005 to September, 2006 along a sandy beach in the southwestern Atlantic, state of São Paulo, Brazil. Our data indicated that the population demography of A. aidae was characterized by a bimodal size-frequency distribution (between 2.0 and 4.0 mm and between 8.0 and 9.0 mm CW) that remained similar throughout the study period. Sex ratio does not differ significantly from 1:1 (p > 0.05), which confirms the pattern observed in other symbiontic pinnotherids. Density values (1.72 ± 1.34 ind. • ap.-1) are in agreement with those found for other species of the genus. The mean symbiosis incidence (75.6%) was one of the highest among species of the Pinnotheridae family, but it was the lowest among the three studied species of the genus. Recruitment pattern was annual, beginning in May and peaking in July, in both years, after the peak of ovigerous females in the population (from March to May). Our findings describe ecological and biological aspects of A. aidae similar to those of other species of this genus, even from different geographic localities.

Densidade, tamanho populacional e conservação de primatas em fragmento de Mata Atlântica no sul do Estado de Minas Gerais, Brasil

Este trabalho teve como objetivo estimar a densidade e o tamanho populacional de quatro espécies de primatas [Alouatta clamitans Cabrera, 1940; Callicebus nigrifrons (Spix, 1823); Callithrix aurita (É. Geoffroy, 1812); Cebus nigritus (Goldfuss, 1809)] que ocorrem em um fragmento de Mata Atlântica de aproximadamente 350 ha, localizado no município de Pouso Alegre, estado de Minas Gerais e reunir subsídios para a conservação dessas espécies na região. O levantamento populacional foi realizado através do método de amostragem de distâncias em transecções lineares (Distance Sampling). Os dados foram coletados entre os meses de abril e agosto de 2008 a partir de quatro transecções implantadas na área de estudo. Os cálculos de densidade e tamanho populacional foram realizados empregando-se o programa Distance 5.0. As densidades foram estimadas em 23,83 ± 9,78 ind./km² para Callicebus nigrifrons, 14,76 ± 5,92 ind./km² para Callithrix aurita e 7,71 ± 2,13 ind./km² para Cebus nigritus. O tamanho populacional foi estimado em 83,0 ± 34,0 indivíduos para C. nigrifrons, 52,0 ± 20,8 indivíduos para Callithrix aurita e 27,0 ± 7,4 indivíduos para Cebus nigritus. Com relação ao bugio (A. guariba clamitans), constatou-se que apenas um grupo com seis indivíduos sobrevive na área. Conclui-se que, no caso de continuarem isoladas, essas populações têm poucas chances de sobrevivência no futuro frente aos riscos de eventos estocásticos. A criação de corredores ecológicos conectando a área de estudo aos outros fragmentos em seu entorno e a translocação de indivíduos de outras áreas da Mata Atlântica para esta região poderão constituir alternativas para garantir a viabilidade dessas populações em longo prazo. Para tanto, é necessário que se consolide uma política pública no município de Pouso Alegre voltada à criação, ampliação e gestão de Unidades de Conservação, e ao incentivo para a adoção de práticas produtivas sob critérios de sustentabilidade no entorno dessas áreas de interesse ecológico.

Space-time variation of the relative abundance of Limnoperna fortunei in deep zones of São Gonçalo Channel, Rio Grande do Sul, Brazil

This work describes the spatial-temporal variation of the relative abundance and size of Limnoperna fortunei (Dunker, 1857) collected in São Gonçalo Channel through bottom trawl with a 0.5 cm mesh, at depths between 3 and 6 m. The estimative of mean relative abundance (CPUE) ranged from 2,425.3 individuals per drag (ind./drag) in the spring to 21,715.0 ind./drag in the fall, with an average of 9,515.3 ind./drag throughout the year. The estimated mean density of L. fortunei for the deep region of São Gonçalo Channel ranged from 1.2 to 10.3 ind./m², and it was recorded a maximum density of 84.9 ind./m² in the fall of 2008. The method of sampling using bottom trawl enabled the capture of L. fortunei under the soft muddy bottom of the channel, in different sizes ranging from 0.4 to 3.2 cm. This shows that the structure of the L. fortunei adult population under the bottom of the São Gonçalo Channel is composed mostly of small individuals (<1.4 cm), which represent up to 74% of the population collected.

Variação sazonal no recrutamento de Phragmatopoma caudata (Polychaeta, Sabellariidae) na costa sudeste do Brasil: biometria e validação de metodologia para categorização de classes etárias

Este estudo explora a maturação de gametas e biometria de Phragmatopoma caudata Krøyer in Mörch, 1863 para endossar uma metodologia e oferecer uma técnica adequada para estudos que objetivam avaliar a ecologia populacional. A análise de correlação de Pearson confirmou a relação positiva (r = 0,90, P <0,0001) entre o comprimento do corpo e o comprimento da coroa opercular. Indivíduos com opercular crown < 0,9 mm podem ser considerados como juvenil devido à ausência de gametas. Portanto, utilizando-se o método aprovado para separar as classes de tamanho, a população dos recifes de P. caudata no Parque Estadual Xixová-Japuí (PEJX) na Baía de Santos, Estado de São Paulo, foi examinada durante dois anos, com o objetivo de analisar a densidade populacional e o padrão sazonal da classe juvenil. Em período de elevadas taxas de juvenis, a densidade populacional atingiu 128.115 ind./m², porém, a média foi 65.090±22.033 ind./m². As análises estatísticas (Kruskal-Wallis H = 18,475, p < 0,01) revelaram existir variação significativa na composição juvenil entre as estações chuvosa e seca. Apesar da presença de juvenis em meses de seca, as estações chuvosas contemplaram 92,1% dos juvenis amostrados. O padrão de juvenis observado pode estar relacionado com fatores biológicos (e.g. gametogênese e ciclo de vida) e abióticos (e.g. suprimento alimentar e correntes marinhas). Estes resultados destacam a necessidade de programas de monitoramento de longo prazo que integrem elementos ecológicos e abióticos, a fim de obter uma compreensão mais completa da ecologia desse poliqueta e ajudar a gerenciar a biodiversidade marinha do PEJX.

Occurrence of Hirudinea species in a post urban reach of a Patagonian mountain stream

Temporal (May 2005 to February 2006) and habitat distribution (pools and riffles) of Hirudinea species was analyzed at a post urban reach from Esquel stream (Chubut province, Patagonia, Argentina). Site was located 5.7 km downstream a Waste Treatment Plant. Mean values of nutrients: ammonia, nitrates and soluble reactive phosphate, as well water conductivity, turbidity and total suspended solids indicated physical and organic pollution. Leeches assemblage was composed by the glossiphonids: Helobdella scutifera Blanchard, 1900, H. michaelseni (Blanchard, 1900), H. simplex (Moore, 1911), Helobdella sp., H. hyalina Ringuelet, 1942, H. obscura Ringuelet, 1942 and the semiscolecid Patagoniobdella variabilis (Blanchard, 1900). From these H. hyalina and H. obscura are new records for Chubut province. Helobdella hyalina (810 ind.m-2) and H. simplex (465 ind. m-2) clearly dominated the assemblage at the reach. Only H. simplex displayed a spatial preference being significantly more abundant in pools than in riffle habitats (p<0.001). Species recruitment occurred mostly at September, December and March when juveniles were very abundant. Although several species of Helobdella were able to live in the disturbed section of the stream, only H. simplex and H. hyalina sustained large populations at the site and can be considered as tolerant to organic enrichment. This information is valuable to future studies on stream condition assessment in mountainous areas in Patagonia, and in other areas in which these species are present.

Pathogenia da anemia na Ancylostomose: II - causas determinantes dos phenomenos regenerativos e degenerativos nessa anemia e contribuições para elucidar o seu mechanismo intimo

Estudamos 25 casos escolhidos de Ancylostomose, procurando determinar quaes os factores que provocam a regeneração hematica, procurando o mechanismo desta regeneração e o desenvolver do processo anemiante. Preferimos seleccionar um numero pequeno de casos e acompanhal-o durante um largo tempo, observando alguns delles durante 1 a 1 anno e meio. A parte hematologica constou, além de 568 exames rotineiros (numeração das hematias, dosagem de Hb., determinação do hematocrito, e na maioria das vezes, determinação da taxa de reticulocytose verificação do aspecto morphologico do sangue em esfregaços), de pesquisas sobre a resistencia globular, sobre a taxa de proteinas no sôro, e em alguns casos, contagem de plaquetas, determinação da viscosidade, etc. Empregamos para estas pesquizas os methodos usuaes, a não ser na determinação da resistencia globular, para a qual descrevemos detalhadamente um processo pouco usado. Durante todo o transcorrer das observações, a actividade biologica dos helminthos parasitos foi controlada por numerosos exames de fezes, que constavam da pesquiza de ovos eliminados, de reacções chimicas que demonstrassem a presença de sangue. Exames de urina foram feitos periodicamente e outros exames foram praticados quando sobrevinha uma complicação ou qualquer facto inesperado. Observamos nos casos graves da doença, uma anemia de caracteristicas fixas. E' uma anemia hypochromica, microcytica, fracamente regenerativa. Verificamos que a degeneração dos indices hematicos, isto é, que o grau de hypochromia e micocytose, não se apresentam nestes casos extremos de modo variavel, mas sim, de maneira particularmente constante; e verificamos mais que estes indices assim degenerados (Ind. Vol. 53 uc., Ind. Hb. 13 yy, Ind. Sat. 23%) tambem são encontrados segundo observações de outros autores, em varias anemias hypochromicas humanas ou de outros mammiferos. No exame de esfregaços, são extremamente raros ou mesmo ausentes, os normoblastos, as hematias com restos nucleares e as hematias polychromaticas. A reticulose oscilla em torno de 3%. Este aspecto é constante nos casos graves, sendo entretanto variavel na unidade de volume o numero destas hematias acima caracterisadas. A média dessa ultima cifra em numerosos casos é de 2,50 M., o que determina uma taxa de Hb. egual a 23%. Ao analysarmos certas observações, que descrevem aspectos hematologicos differentes daquelles aqui descriptos, concluimos pela existencia nesses casos anomalos, de superposições de outras doenças, cuja etio-pathogenia nada tem a ver com os factores que determinam o apparecimento da anemia ancylostomotica. A regeneração hematica processa-se na Ancylostomose unicamente após a administração de ferro em dóses variaveis, conforme o sal empregado. Mostraram-se inactivas nos casos aqui observados, as administrações de figado crú, triptophano, hystidina, lecithina, Vit. B, saes de arsenico, manganez, cobalto, cobre e a alimentação com dietas ricas em ferro. Em virtude dos resultados que obtivemos com a administração isolada de ferro, concluimos pela inefficiencia da administração de substancias pyrrholicas, da fracção hepatica sensivel nas anemias hypochromicas, e da associação ao ferro de acido chlorhydrico. Todas essas substancias, e tambem a elimanação simples dos helminthos parasitos, não só mostraram-se isoladamente sem acção sobre o sangue, como não auxiliaram a regeneração provocada pelo ferro...

Control biológico de patógenos fúngicos en precosecha de frutas: mejora de la tolerancia de agentes de biocontrol al estrés hídrico y térmico

El control biológico ha demostrado ser una posible alternativa a los productos químicos de síntesis en el control de plagas y enfermedades. Sin embargo, esta técnica se ve limitada en muchos casos por las condiciones fluctuantes del medio y por el estrecho margen de condiciones ambientales bajo las cuales los agentes de biocontrol son capaces de establecerse y controlar de forma efectiva, así como por la dificultad de obtener un producto formulado final con viabilidad y vida útil adecuadas. El presente proyecto se planteaba como objetivo principal el estudio de los mecanismos de supervivencia en condiciones de estrés ambiental de dos agentes de biocontrol (Candida sake CPA-1 y Pantoea agglomerans CPA-2), con la finalidad de mejorar su competencia ecológica y su efectividad, mediante manipulación fisiológica. El enfoque era de gran novedad en el campo del biocontrol y no obstante los resultados obtenidos han sido muchos y muy satisfactorios y suponen una vía abierta para poder hacer del control biológico una estrategia competitiva y comparable a los productos químicos de síntesis. A continuación se describen los resultados obtenidos en cada uno de los objetivos para los dos agentes de biocontrol objeto de estudio por separado.

HARNESSING iNKT CELLS WITH RECOMBINANT CD1d FUSION PROTEINS TO REDIRECT INNATE AND ADAPTIVE IMMUNITY TO THE TUMOR

Les thérapies du cancer, comme la radiothérapie et la chimiothérapie, sont couramment utilisées mais ont de nombreux effets secondaires. Ces thérapies invasives pour le patient nécessitent d'être améliorées et de nombreuses avancées ont été faites afin d'adapter et de personnaliser le traitement du cancer. L'immunothérapie a pour but de renforcer le système immunitaire du patient et de le rediriger de manière spécifique contre la tumeur. Dans notre projet, nous activons les lymphocytes Invariant Natural Killer T (iNKT) afin de mettre en place une immunothérapie innovatrice contre le cancer. Les cellules iNKT sont une unique sous-population de lymphocytes T qui ont la particularité de réunir les propriétés de l'immunité innée ainsi qu'adaptative. En effet, les cellules iNKT expriment à leur surface des molécules présentes aussi sur les cellules tueuses NK, caractéristique de l'immunité innée, ainsi qu'un récepteur de cellules T (TCR) qui représente l'immunité adaptative. Les cellules iNKT reconnaissent avec leur TCR des antigènes présentés par la molécule CD1d. Les antigènes sont des protéines, des polysaccharides ou des lipides reconnus par les cellules du système immunitaire ou les anticorps pour engendrer une réponse immunitaire. Dans le cas des cellules iNKT, l'alpha-galactosylceramide (αGC) est un antigène lipidique fréquemment utilisé dans les études cliniques comme puissant activateur. Après l'activation des cellules iNKT avec l'αGC, celles-ci produisent abondamment et rapidement des cytokines. Ces cytokines sont des molécules agissant comme des signaux activateurs d'autres cellules du système immunitaire telles que les cellules NK et les lymphocytes T. Cependant, les cellules iNKT deviennent anergiques après un seul traitement avec l'αGC c'est à dire qu'elles ne peuvent plus être réactivées, ce qui limite leur utilisation dans l'immunothérapie du cancer. Dans notre groupe, Stirnemann et al ont publié une molécule recombinante innovante, composée de la molécule CD1d soluble et chargée avec le ligand αGC (αGC/sCD1d). Cette protéine est capable d'activer les cellules iNKT tout en évitant l'anergie. Dans le système immunitaire, les anticorps sont indispensables pour combattre une infection bactérienne ou virale. En effet, les anticorps ont la capacité de reconnaître et lier spécifiquement un antigène et permettent l'élimination de la cellule qui exprime cet antigène. Dans le domaine de l'immunothérapie, les anticorps sont utilisés afin de cibler des antigènes présentés seulement par la tumeur. Ce procédé permet de réduire efficacement les effets secondaires lors du traitement du cancer. Nous avons donc fusionné la protéine recombinante αGC/CD1d à un fragment d'anticorps qui reconnaît un antigène spécifique des cellules tumorales. Dans une étude préclinique, nous avons démontré que la protéine αGC/sCD1d avec un fragment d'anticorps dirigé contre la tumeur engendre une meilleure activation des cellules iNKT et entraîne un effet anti-tumeur prolongé. Cet effet anti-tumeur est augmenté comparé à une protéine αGC/CD1d qui ne cible pas la tumeur. Nous avons aussi montré que l'activation des cellules iNKT avec la protéine αGC/sCD1d-anti-tumeur améliore l'effet anti- tumoral d'un vaccin pour le cancer. Lors d'expériences in vitro, la protéine αGC/sCD1d-anti- tumeur permet aussi d'activer les cellules humaines iNKT et ainsi tuer spécifiquement les cellules tumorales humaines. La protéine αGC/sCD1d-anti-tumeur représente une alternative thérapeutique prometteuse dans l'immunothérapie du cancer. - Les cellules Invariant Natural Killer T (iNKT), dont les effets anti-tumoraux ont été démontrés, sont de puissants activateurs des cellules Natural Killer (NK), des cellules dendritiques (DC) et des lymphocytes T. Cependant, une seule injection du ligand de haute affinité alpha-galactosylceramide (αGC) n'induit une forte activation des cellules iNKT que durant une courte période. Celle-ci est alors suivie d'une longue phase d'anergie, limitant ainsi leur utilisation pour la thérapie. Comme alternative prometteuse, nous avons montré que des injections répétées d'αGC chargé sur une protéine recombinante de CD1d soluble (αGC/sCD1d) chez la souris entraînent une activation prolongée des cellules iNKT, associée à une production continue de cytokine. De plus, le maintien de la réactivité des cellules iNKT permet de prolonger l'activité anti-tumorale lorsque la protéine αGC/sCD1d est fusionnée à un fragment d'anticorps (scFv) dirigé contre la tumeur. L'inhibition de la croissance tumorale n'est optimale que lorsque les souris sont traitées avec la protéine αGC/sCD1d-scFv ciblant la tumeur, la protéine αGC/sCD1d-scFv non-appropriée étant moins efficace. Dans le système humain, les protéines recombinantes αGC/sCD1d-anti-HER2 et anti-CEA sont capables d'activer et de faire proliférer des cellules iNKT à partir de PBMCs issues de donneurs sains. De plus, la protéine αGC/sCD1d-scFv a la capacité d'activer directement des clones iNKT humains en l'absence de cellules présentatrices d'antigènes (CPA), contrairement au ligand αGC libre. Mais surtout, la lyse des cellules tumorales par les iNKT humaines n'est obtenue que lorsqu'elles sont incubées avec la protéine αGC/sCD1d-scFv anti- tumeur. En outre, la redirection de la cytotoxicité des cellules iNKT vers la tumeur est supérieure à celle obtenue avec une stimulation par des CPA chargées avec l'αGC. Afin d'augmenter les effets anti-tumoraux, nous avons exploité la capacité des cellules iNKT à activer l'immunité adaptive. Pour ce faire, nous avons combiné l'immunothérapie NKT/CD1d avec un vaccin anti-tumoral composé d'un peptide OVA. Des effets synergiques ont été obtenus lorsque les traitements avec la protéine αGC/sCD1d-anti-HER2 étaient associés avec le CpG ODN comme adjuvant pour la vaccination avec le peptide OVA. Ces effets ont été observés à travers l'activation de nombreux lymphocytes T CD8+ spécifique de la tumeur, ainsi que par la forte expansion des cellules NK. Les réponses, innée et adaptive, élevées après le traitement avec la protéine αGC/sCD1d-anti-HER2 combinée au vaccin OVA/CpG ODN étaient associées à un fort ralentissement de la croissance des tumeurs B16- OVA-HER2. Cet effet anti-tumoral corrèle avec l'enrichissement des lymphocytes T CD8+ spécifiques observé à la tumeur. Afin d'étendre l'application des protéines αGC/sCD1d et d'améliorer leur efficacité, nous avons développé des fusions CD1d alternatives. Premièrement, une protéine αGC/sCD1d dimérique, qui permet d'augmenter l'avidité de la molécule CD1d pour les cellules iNKT. Dans un deuxième temps, nous avons fusionné la protéine αGC/sCD1d avec un scFv dirigé contre le récepteur 3 du facteur de croissance pour l'endothélium vasculaire (VEGFR-3), afin de cibler l'environnement de la tumeur. Dans l'ensemble, ces résultats démontrent que la thérapie médiée par la protéine recombinante αGC/sCD1d-scFv est une approche prometteuse pour rediriger l'immunité innée et adaptive vers le site tumoral. - Invariant Natural Killer T cells (iNKT) are potent activators of Natural Killer (NK), dendritic cells (DC) and T lymphocytes, and their anti-tumor activities have been well demonstrated. However, a single injection of the high affinity CD1d ligand alpha-galactosylceramide (αGC) leads to a strong but short-lived iNKT cell activation followed by a phase of long-term anergy, limiting the therapeutic use of this ligand. As a promising alternative, we have demonstrated that when αGC is loaded on recombinant soluble CD1d molecules (αGC/sCD1d), repeated injections in mice led to the sustained iNKT cell activation associated with continued cytokine secretion. Importantly, the retained reactivity of iNKT cell led to prolonged antitumor activity when the αGC/sCD1d was fused to an anti-tumor scFv fragments. Optimal inhibition of tumor growth was obtained only when mice were treated with the tumor-targeted αGC/CD1d-scFv fusion, whereas the irrelevant αGC/CD1d-scFv fusion was less efficient. When tested in a human system, the recombinant αGC/sCD1d-anti-HER2 and -anti-CEA fusion proteins were able to expand iNKT cells from PBMCs of healthy donors. Furthermore, the αGC/sCD1d-scFv fusion had the capacity to directly activate human iNKT cells clones without the presence of antigen-presenting cells (APCs), in contrast to the free αGC ligand. Most importantly, tumor cell killing by human iNKT cells was obtained only when co- incubated with the tumor targeted sCD1d-antitumor scFv, and their direct tumor cytotoxicity was superior to the bystander killing obtained with αGC-loaded APCs stimulation. To further enhance the anti-tumor effects, we exploited the ability of iNKT cells to transactivate the adaptive immunity, by combining the NKT/CD1d immunotherapy with a peptide cancer vaccine. Interestingly, synergistic effects were obtained when the αGC/sCD1d- anti-HER2 fusion treatment was combined with CpG ODN as adjuvant for the OVA peptide vaccine, as seen by higher numbers of activated antigen-specific CD8 T cells and NK cells, as compared to each regimen alone. The increased innate and adaptive immune responses upon combined tumor targeted sCD1d-scFv treatment and OVA/CpG vaccine were associated with a strong delay in B16-OVA-HER2 melanoma tumor growth, which correlated with an enrichment of antigen-specific CD8 cells at the tumor site. In order to extend the application of the CD1d fusion, we designed alternative CD1d fusion proteins. First, a dimeric αGC/sCD1d-Fc fusion, which permits to augment the avidity of the CD1d for iNKT cells and second, an αGC/sCD1d fused to an anti vascular endothelial growth factor receptor-3 (VEGFR-3) scFv, in order to target tumor stroma environment. Altogether, these results demonstrate that the iNKT-mediated immunotherapy via recombinant αGC/sCD1d-scFv fusion is a promising approach to redirect the innate and adaptive antitumor immune response to the tumor site.

Estudio de la patogenia de clostridium perfringens tipo b y d en pequeños rumiantes

Proyecto de investigación realizado a partir de una estancia en la University of California, Davis, Estados Unidos, entre octubre y desembre del 2007. Clostridium perfringens (C. perfringens) tipo C causa enteritis necrotizante en humanos y enterotoxemias en animales domésticos. Esta bacteria produce beta toxina (CPB), alfa toxina (CPA) y perfringolisina (PFO) durante la fase logarítimca de crecimiento. En nuestro estudio se evaluó la relación entre CPB y la virulencia del aislamiento CN3685 de Cl. perfringens tipo C en un modelo caprino con inoculación intraduodenal. De manera similar a la infección natural por C. perfringens tipo C, el cultivo vegetativo del tipo salvaje de CN3685 provocó dolor abdominal, diarrea hemorrágica, enteritis necrotizante, colitis, edema pulmonar, hidropericardio y muerte en 2 cabritos, a las 24 horas postinoculación. Por otro lado, mediante tecnología Targe Tron® se prepararon mutantes isogénicos carentes de toxina CPB, los cuales fueron inoculados siguiendo el modelo anteriormente descrito. Los resultados mostraron que estos mutantes carecían de todo tipo de virulencia, ya que no se observaron signos clínicos durante las primeras 24 h postinoculación ni tampoco lesiones macroscópicas ni histopatológicas. Posteriormente se desarrolló un modelo experimental similar a los anteriores, en los que se había repuesto la capacidad de producción de CPB en los mutantes. Los dos animales inoculados con estos mutantes complementarios presentaron signos clínicos y lesiones similares a las observadas en el caso del tipo salvaje. Estos resultados muestran que la toxina CPB es necesaria y suficiente para inducir la enfermedad causada por CN3685. Esto a su vez, demuestra la importancia de este tipo de toxina en la patogénesis de C. perfringems tìpo C.

A protective cocktail vaccine against murine cutaneous leishmaniasis with DNA encoding cysteine proteinases of Leishmania major.

The protection elicited by the intramuscular injection of two plasmid DNAs encoding Leishmania major cysteine proteinase type I (CPb) and type II (CPa) was evaluated in a murine model of experimental cutaneous leishmaniasis. BALB/c mice were immunized either separately or with a cocktail of the two plasmids expressing CPa or CPb. It was only when the cpa and cpb genes were co-injected that long lasting protection against parasite challenge was achieved. Similar protection was also observed when animals were first immunized with cpa/cpb DNA followed by recombinant CPa/CPb boost. Analysis of the immune response showed that protected animals developed a specific Th1 immune response, which was associated with an increase of IFN-gamma production. This is the first report demonstrating that co-injection of two genes expressing different antigens induces a long lasting protective response, whereas the separate injection of cysteine proteases genes is not protective.

Leishmania cysteine proteinases: from gene to subunit vaccine.

Whole genome sequences of microbial pathogens present new opportunities for clinical application. Presently, genome sequencing of the human protozoan parasite Leishmania major is in progress. The driving forces behind the genome project are to identify genes with key cellular functions and new drug targets, to increase knowledge on mechanisms of drug resistance and to favor technology transfer to scientists from endemic countries. Sequencing of the genome is also aimed at the identification of genes that are expressed in the infectious stages of the parasite and in particular in the intracellular form of the parasite. Several protective antigens of Leishmania have been identified. In addition to these antigens, lysosomal cysteine proteinases (CPs) have been characterized in different strains of Leishmania and Trypanosoma, as new target molecules. Recently, we have isolated and characterized Type I (CPB) and Type II (CPA) cysteine proteinase encoding genes from L. major. The exact function of cysteine proteinases of Leishmania is not completely understood, although there are a few reports describing their role as virulence factors. One specific feature of CPB in Leishmania and other trypanosomatids, is the presence of a Cterminal extension (CTE) which is possibly indicative of conserved structure and function. Recently, we demonstrated that DNA immunization of genetically susceptible BALB / c mice, using a cocktail of CPB and CPA genes, induced long lasting protection against L. major infection. This review intends to give an overview of the current knowledge on genetic vaccination used against leishmaniasis and the importance of CP genes for such an approach.

The Irish Study of Sexual Health and Relationships

This study was commissioned by the Department of Health and Children and the Crisis Pregnancy Agency (CPA) in response to a recommendation of the National AIDS Strategy Committee. The findings of this report will be considered by various organisations and will inform the future development and strategic direction of the Crisis Pregnancy Agency’s work in reducing the number of crisis pregnancies. Read the report (PDF, 2.2mb) View the summary report (PDF, 859KB) View the survey questionnaire (PDF, 235kb)

New imidazolidinic bioisosters: potential candidates for antischistosomal drugs

The emergence of strains of Schistosoma resistant to praziquantel has drawn attention to the search for new schistosomacide drugs. Imidazolidinic derivatives have performed outstandingly against adult S. mansoni worms when evaluated in vitro. The molecular modification of imidazolidine by way of bioisosteric replacement gives rise to variations in its biological response. This study verifies the potential of substituent groups in the derivatives (Z)3-benzyl-5-(2-fluoro-benzylidene)-imidazolidine-2,4-dione NE4, 3-benzyl-5-(4-chloro-arylazo)-4-thioxo-imidazolidin -2-ona PT5, 3-benzyl-5-(3-fluoro-benzylidene)-1-methyl-2-thioxo-imidazolidin-4-one JT53; 3-benzyl-1-methyl-5-(4-methyl-benzylidene)-2-thioxo-imidazolidin-4-one JT63; 3-benzyl-1-methyl-5-(4-methoxi-benzylidene)-2-thioxo -imidazolidin-4-one JT68; 3-(4-chloro-benzyl)-1-methyl-5-(4-methoxi-benzylidene)-2-thioxo-imidazolidin-4-one JT69; 3-(4-phenyl-benzyl)-1-methyl-5-(4-methoxi-benzylidene)-2-thioxo-imidazolidin-4-one JT72 by determining the viability in vitro of adult S. mansoni worms in the presence of these derivatives. The susceptibility of the worms obtained from mice and kept in culture in the presence of different concentrations was determined by way of schistosomacide kinetic, observed every 24 h over a period of eight days. The results show that the worms were more sensitive to the PT5 derivative at a concentration of 58 µM which killed 100% of the worms after 24 h of contact, also giving rise to alterations in the tegument surface of the worms with the formation of bubbles and peeling. These observations suggest a strong electronic contribution of the arylazo grouping in the biological response.