Escherichia coli vacuolating factor, involved in avian cellulitis, induces actin contraction and binds to cytoskeleton proteins in fibroblasts

Avian pathogenic Escherichia coli (APEC) isolated from avian cellulitis lesions produces a toxin, named Escherichia coli vacuolating factor (ECVF), that causes cell vacuolization and induces inflammatory response in broiler chicken. Methods We investigated the intracellular activities of ECVF in avian fibroblasts using fluorescence staining, electron microscopy, MTT and LDH measurements. As ECVF act specifically in avian cells, we performed blotting assay followed by mass spectrometry to better understand its initial intracellular protein recognition. Results ECVF induced actin contraction, mitochondrial damage and membrane permeability alterations. Ultrastructural analysis showed intracellular alterations, as nuclear lobulation and the presence of degraded structures inside the vacuoles. Moreover, ECVF induced cell death in fibroblasts. ECVF-biotin associates to at least two proteins only in avian cell lysates: alpha-actinin 4 and vinculin, both involved in cytoskeleton structure. Conclusion These findings demonstrated that ECVF plays an important role in avian cellulitis, markedly in initial steps of infection. Taken together, the results place this toxin as a target for drug and/or vaccine development, instead of the use of large amounts antibiotics.(AU)

Plesiomonas shigelloides exports a lethal cytotoxic-enterotoxin (LCE) by membrane vesicles

ABSTRACT Plesiomonas shigelloides isolated from water in Brazil was previously described as a hemorrhagic heat-labile cytotoxic-enterotoxin producer. We purified this toxin from culture supernatants using ion metallic affinity chromatography (IMAC) followed by molecular exclusion chromatography. The pure toxin presented molecular mass of 50 kDa and isoelectric point (pI) around 6.9 by 2D electrophoresis. When injected intravenously, the purified cytotoxic-enterotoxin induced also severe spasms followed by sudden death of mice. Hence, we entitled it as lethal cytotoxic-enterotoxin (LCE). The presence of membrane vesicles (MVs) on cell surfaces of P. shigelloides was observed by scan electron microscopy (SEM). From these MVs the LCE toxin was extracted and confirmed by biological and serological assays. These data suggest that P. shigelloides also exports this cytotoxic-enterotoxin by membrane vesicles, a different mechanism of delivering extra cellular virulence factors, so far not described in this bacterium.

HSP70 of Leishmania amazonensis alters resistance to different stresses and mitochondrial bioenergetics

The 70 kDa heat shock protein (HSP70) is a molecular chaperone that assists the parasite Leishmania in returning to homeostasis after being subjected to different types of stress during its life cycle. In the present study, we evaluated the effects of HSP70 transfection of L. amazonensis promastigotes (pTEX-HSP70) in terms of morphology, resistance, infectivity and mitochondrial bioenergetics. The pTEX-HSP70 promastigotes showed no ultrastructural morphological changes compared to control parasites. Interestingly, the pTEX-HSP70 promastigotes are resistant to heat shock, H2O2-induced oxidative stress and hyperbaric environments. Regarding the bioenergetics parameters, the pTEX-HSP70 parasites had higher respiratory rates and released less H2O2 than the control parasites. Nevertheless, the infectivity capacity of the parasites did not change, as verified by the infection of murine peritoneal macrophages and human macrophages, as well as the infection of BALB/c mice. Together, these results indicate that the overexpression of HSP70 protects L. amazonensis from stress, but does not interfere with its infective capacity.

Cultura primária de fibrocondrócitos de menisco de coelho / Primary culture of fibrocondrocytes from rabbit knee joint meniscus

Estudos envolvendo a obtenção de fibrocondrócitos em cultura no Brasil são escassos. Este trabalho descreve a cultura primária de fibrocondrócitos de menisco cultivados em alta densidade com algumas modificações nos métodos de extração já descritos na literatura, com o intuito de obter um maior número de células viáveis para cultivo celular. Foram utilizados meniscos de coelhos New Zealand com 120 dias. Os meniscos foram cortados e tratados com 2mg/ml de colagenase diluída em meio DMEM (meio de Eagle modificado por Dulbeccos) contendo 10% de SFB sob agitação durante três horas a 37°C. Osfibrocondrócitos foram cultivados em alta densidade (1x105/cm2) em frascos de cultura T25 em meio DMEM suplementado com 10% de SFB. As células atingiram a confluência celular após o 15° dia de cultivo e sintetizaram sua matrix extracelular evidenciada pela coloração com azul de toluidina. A curva de crescimento mostrou que os fibrocondrócitos duplicaram 2,5 vezes. A cinética de incorporação de sulfato radioativo nos glicosaminoglicanos sintetizados pelos fibrocondrócitos “in vitro” foi constante. Os fibrocondrócitos cultivados em alta densidade celular apresentaram aspectos ultra-estruturais semelhante as células “ in vivo”

Physicochemical and structural analysis of three regions of the deep digital flexor tendon of pigs

Few studies have discussed the relationship between the molecular organization and the physicochemical and biomechanical properties of pig tendons. In this work, we examined the extracellular matrix of the deep digital flexor tendon of pigs, which was subjected to tensional (proximal region) and compressive (distal and terminal regions) forces. The three regions of the tendon were used for swelling tests and their glycosaminoglycan content was determined. Longitudinal sections of the tendon were stained and observed using polarized light microscopy. The distal and terminal regions were swole more in water than the proximal region. After staining with toluidine blue the metachromasy was more intense in the distal and terminal regions, indicating an accumulation of proteoglycans in these regions. Analysis of the glycosaminoglycans by agarose gel electrophoresis showed that dermatan sulfate was present in all regions, whereas chondroitin sulfate occurred only in the regions of compression. The shape of the fibroblasts changed along the tendon: rounded cells occurred in regions under compression, while in the region under tension, elongated cells predominated. The organization and distribution of the collagen bundles were different for each region. Birefringence analysis revealed a more regular crimp pattern in the region under tension than in the regions under compressive forces. The elastic fibers also showed a different distribution in each region. These results indicate that the regional differences in the structure and composition of the deep digital flexor tendon of pigs are related to the biomechanical properties of the tendon.

Differential distribution of elastic system fibers in the pubic symphysis of mice during prefnancy, partum and post-partum

The expansion of the pubic symphysis during pregnancy in some rodet depends on the growth of the interpubic ligament, primarily through the biosynthesis of extracellular matrix. Although there is a significant metabolism of elastin in the female reproductive tract during pregnancy, little is known of the architectural and ultrastructural aspects of the elastic system fibers in the mouse pubic symphysis. In this study, the main characteristics of the elastic system of the mouse pubic symphysis during pregnancy, partum and post-partum were determined by light and electron microscopy. A distinct arrangement of microfibrils, elastin deposition and development of the extracellular matrix formed a supporting framework that uniformly distributed stress in order to provide adequate interpubic resilience during delivery. These changes support a role for elastic system fibers in symphyseal maturation and reconstruction during pregnancy and after delivery.

Ultrastructural characteristics of tensional regions in tendons from rats of different ages

The calcaneal tendon and the deep digital flexor tendon are collagen-rich structures which are adapted to resist tensile stress. Since during aging tendons undergo modifications in their mechanical properties and in collagen aggregation, an understanding of the structural changes involved is important. In this work, the structural organization of the tensile region of the calcaneal and deep difital flexor tendons was studied in male Wistar rats 30, 180 and 730 days old. Large quantities of rough endoplasmic reticulum and peripheral secretory microvesicles were observed in the calcaneal tendon of 30-day-old rats. In the case of the deep digital flexor tendon, this organelle remained well-developed up to 180 days. A marked decrease in rough endoplasmic reticulum was observed in both tendons in 730-day-old rats. Proteoglycans associated with collagen fibrils were visible in the two tendons of all age groups. The reduced amount of rough endoplasmic reticulum and secretory microvesicles may be correlated with the known lower turnover of extracellular matrix components during aging.

Diagnóstico rápido de vírus do grupo herpes por microscopia eletrônica: - observaçöes atípicas nas amostras de pacientes aidéticos / Rapid diagnostic of herpesvirus group by electron microscopy: atypical observations in the sample of patients with AIDS

A Seçäo de Microscopia Eletrônica do Instituto Adolfo Lutz recebeu no período de 1986-90, 129 amostras de materiais biológicos (líquido vesicular, raspado de lesäo genital, raspado de lesäo anal, saliva e urina) de pacientes aidéticos, com suspeita de infecçäo herpética, para diagnóstico rápido de vírus do grupo Herpes, por microscopia eletrônica. Dessas amostras, 25%(32/129) foram positivas para vírus Herpes, com algumas apresentando aspectos atípicos, tais como: a) grande quantidade de partículas virais; b) detecçäo de mais de um nucleocapsídeo num mesmo envoltório; e c) detecçäo de grandes agregados de nucleocapsídeos sem envoltórios. Estas observaçöes poderiam ser atribuídas às características de imunodepressäo desses pacientes e a sua divulgaçäo foi por nós considerada importante, pois seriam dados adicionais para o estudo de infecçöes virais, nesses pacientes imunodeprimidos