A recombinant chimeric protein composed of human and mice-specific CD4+ and CD8+ T-cell epitopes protects against visceral leishmaniasis.

In this study, a recombinant chimeric protein (RCP), which was composed of specific CD4+ and CD8+ T-cell epitopes to murine and human haplotypes, was evaluated as an immunogen against Leishmania infantum infection in a murine model. BALB/c mice received saline were immunized with saponin or with RCP with or without an adjuvant. The results showed that RCP/saponin-vaccinated mice presented significantly higher levels of antileishmanial IFN-γ, IL-12 and GM-CSF before and after challenge, which were associated with the reduction of IL-4 and IL-10 mediated responses. These animals showed significant reductions in the parasite burden in all evaluated organs, when both limiting dilution and quantitative real-time PCR techniques were used. In addition, the protected animals presented higher levels of parasite-specific nitrite, as well as the presence of anti-Leishmania IgG2a isotype antibodies. In conclusion, the RCP/saponin vaccine could be considered as a prophylactic alternative to prevent against VL.

Cross-protective efficacy from a immunogen firstly identified in Leishmania infantum against tegumentary leishmaniasis.

Experimental vaccine candidates have been evaluated to prevent leishmaniasis, but no commercial vaccine has been proved to be effective against more than one parasite species. LiHyT is a Leishmania-specific protein that was firstly identified as protective against Leishmania infantum. In this study, LiHyT was evaluated as a vaccine to against two Leishmania species causing tegumentary leishmaniasis (TL): Leishmania major and Leishmania braziliensis. BALB/c mice were immunized with rLiHyT plus saponin and lately challenged with promastigotes of the two parasite species. The immune response generated was evaluated before and 10 weeks after infection, as well as the parasite burden at this time after infection. The vaccination induced a Th1 response, which was characterized by the production of IFN-γ, IL-12 and GM-CSF, as well as by high levels of IgG2a antibodies, after in vitro stimulation using both the protein and parasite extracts. After challenge, vaccinated mice showed significant reductions in their infected footpads, as well as in the parasite burden in the tissue and organs evaluated, when compared to the control groups. The anti-Leishmania Th1 response was maintained after infection, being the IFN-γ production based mainly on CD4(+) T cells. We described one conserved Leishmania-specific protein that could compose a pan-Leishmania vaccine.

Endophytic fungi from the genus Colletotrichum are abundant in the Phaseolus vulgaris and have high genetic diversity.

AIMS: To evaluate the diversity of endophytic fungi from the leaves of the common bean and the genetic diversity of endophytic fungi from the genus Colletotrichum using IRAP (inter-retrotransposon amplified polymorphism) and REMAP (retrotransposon-microsatellite amplified polymorphism) analyses. METHODS AND RESULTS: The fungi were isolated by tissue fragmentation and identified by analysing the morphological features and sequencing the internal transcribed spacer (ITS) regions and the rDNA large subunit (LSU). Twenty-seven different taxa were identified. Colletotrichum was the most commonly isolated genera from the common bean (32.69% and 24.29% of the total isolates from the Ouro Negro and Talismã varieties, respectively). The IRAP and REMAP analyses revealed a high genetic diversity in the Colletotrichum endophytic isolates and were able to discriminate these isolates from the phytopathogen Colletotrichum lindemuthianum. CONCLUSIONS: Fungi from the genus Colletotrichum are abundant in the Phaseolus vulgaris endophytic community, and the IRAP and REMAP markers can be used to rapidly distinguish between C. lindemuthianum and other Colletotrichum members that are frequently found as endophytes. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the diversity of endophytic fungi present in the common bean and the use of IRAP and REMAP markers to assess the genetic diversity of endophytic fungi from the genus Colletotrichum.

Short communication: Multidrug-resistant Acinetobacter baumannii-calcoaceticus complex isolated from infant milk formula and utensils in a nursery in Rio de Janeiro, Brazil.

Infant milk formulas are not sterile products, and pathogenic bacteria can survive and multiply in these products. This study was performed, initially, to detect the presence of Salmonella spp. in reconstituted infant milk formula and on utensils previously sanitized used in their preparation or distribution in a nursery of a public hospital in Rio de Janeiro. None of the samples tested carried Salmonellaspp. However, further identification of colonies growing on the selective media revealed the presence of several other gram-negative bacteria. Seventeen isolates were identified as belonging to Acinetobacter baumannii-calcoaceticus complex. Fourteen isolates presented a multidrug-resistance profile, by disc diffusion assays, and one of them--JE4--was also resistant to imipenem. The detection of Acinetobacter isolates in this work demonstrates inadequate hygiene practices in the preparation or distribution of infant milk formula.

Immunocytochemical evaluation of aquaporins and cell wall components and their influence on foliar water uptake in Andean Melastomataceae.

Andean ecosystems are characterized by high humidity, mainly from rain and fog events. Because of differences in altitude two Andean ecosystems - sub-Andean forest and Páramo -face different environmental pressures that affect leaf anatomy and cell wall composition and, consequently, species foliar water uptake (FWU) capacity. Here, FWU capacity of eight species in the Melastomataceae was evaluated and found to be related to proportions of cell wall components and aquaporins in the two ecosystems. Cellulose was labelled with Calcofluor white, and aquaporin and pectins were labelled with monoclonal antibodies. There were differences in plant FWU capacity in both ecosystems, with higher FWU capacity in sub-Andean forest species than in Páramo forest species. Cell wall components were positively related to FWU, with increased FWU related to pectin and aquaporin content of the plasma membrane. Differences in water availability in the two analysed environments led to differences in FWU capacity that are associated with leaf anatomical traits and cell wall composition. In these two environments, plants with similar traits are selected to respond to given environmental pressures. Traits that favour FWU in sub-Andean forest species may lead to further advances of these species in this environments.

Differences in mitochondrial function between brain and heart of senile rats exposed to acute hypobaric hypoxia. Role of nitric oxide.

Rat brain and heart display different endogenous protective responses against hypobaric hypoxia in an age-dependent way. The aim of the present work was to evaluate the effects of acute hypobaric hypoxia (HH, 48 h) on brain and heart mitochondrial function as well as the participation of nitric oxide (NO) in old rats (22-month old). Cortical mitochondria from rats exposed to HH decreased respiratory rates (37 %, state 3) and membrane potential (20 %), but NO and H2O2 production increased by 48 %, and 23 %, respectively. Hippocampal mitochondria preserved O2 consumption and H2O2 production, decreased membrane potential (18 %) and increased NO production (46 %). By contrast, HH decreased NO production (53 %) in mitochondria from left heart ventricles associated with increased cytochrome oxidase activity (39 %) and decreased NADPH oxidase activity (31 %). Also, a tendency to increase complex I-III (24 %) and complex II-III (65 %) activity was observed. In conclusion, after HH hippocampal and cortical mitochondria showed mild uncoupling and increased NO production. However, only the hippocampus preserved O2 consumption and H2O2 levels. Interestingly, heart mitochondria showed a decreased ROS production through increased cytochrome oxidase activity associated with a decrease in NO production. This may be interpreted as a self-protective mechanism against hypoxia.

Altitudinal gradient drives regional and local diversity and composition patterns of epiphyllous bryophytes in ecological refuges.

'Brejos de altitude' is an ecosystem that has been subjected to severe exploitation, leading to an intense reduction in Brazil. Understanding the patterns of bryophyte diversity and composition, particularly of specialized species such as the epiphylls, to anthropic and abiotic variables is crucial for implementing protection measures. We investigated the relationship between composition and diversity of epiphyllous bryophytes and anthropic (edge effects) and abiotic (altitude) variables at local (within each 'Brejo') and regional (set of three 'Brejos') scale. Abiotic and anthropic variables were correlated with alpha and beta diversity (decomposed into species replacement and richness differences) using GLM. Their effects on species composition were evaluated using PERMANOVA. The localities at higher elevations harboured a richer bryoflora and overall beta diversity patterns were associated with altitude and locality, which acted over different scales. Regionally, the contribution of richness differences was limited with increasing altitude. Yet, dissimilarities among localities were associated with richness differences and replacement, denoting the importance of local factors. The composition was explained by local factors interacting with the regional altitudinal gradient. Anthropic activity was significant only when interacting with local factors and altitudinal gradient. Environmental filtering associated with altitude played a more important role in shaping the diversity and composition of epiphyllous bryophytes, at both regional and local scales. Each 'Brejo' acts as an ecological refuge, harbouring part of the whole environmental gradient, and presents a unique floristic composition. Moreover, partitioning beta diversity highlighted the interplay of local and regional forces shaping diversity patterns.

Interactions of gall-formers and leaf-chewers on a tropical tree fern: evidence for non-repulsion and co-occurrence between insect guilds.

Host plant selection by herbivores is driven by a complex array of cues, including leaf traits and previous leaf damage. Herbivore-associated cues to host selection at the plant and leaf scale aid understanding of mechanisms responsible for host preference that might translate into increased performance, as well as processes structuring herbivore populations mediated by interactions. We investigated how changes induced by a galling insect in the tropical fern Cyathea phalerata act as repellent or attractant cues for sawfly feeding and the effects of leaf size on herbivory levels. We recorded gall abundance, damage by chewers, leaf size, plant nutritional quality, phenolic concentration and leaf anatomical traits between galled and non-galled leaf samples. Galled samples contained less N, higher levels of phenolics and higher C/N ratio. However, leaf-chewing damage did not differ between galled and non-galled leaves. The gall structure was avoided by chewers, as it had high concentrations of phenolics, lignification and suberization. Larger leaves sustained higher gall abundance, but leaf size did not have a significant effect on chewer damage. A co-occurrence index calculated for both guilds indicated that galls and chewers exhibited a distribution that did not differ from random, reinforcing that the two guilds on C. phalerata do not show patterns of repulsion such as those maintained by interspecific competition. Sawflies dismissing chemical cues indicate that the increase in phenolics caused by galling insects does not generate increased protection of the galled pinnules. Our results highlight ferns as key resources for herbivores and as a potential plant group to study new research avenues on plant-insect interactions.

Abiotic factors drives floristic variations of fern's metacommunity in an Atlantic Forest remnant.

We analyzed floristic variations in fern's metacommunity at the local scale and their relationship with abiotic factors in an Atlantic Forest remnant of northeastern Brazil. Floristic and environmental variations were accessed on ten plots of 10 × 20 m. We performed cluster analyses, based on Bray-Curtis dissimilarity index to establish the floristic relationship. The influence of abiotic factors: luminosity, temperature, relative air humidity and relative soil moisture was evaluated from a redundancy analysis. We found 24 species belonging to 20 genera and 12 families. The fern's flora showed high floristic heterogeneity (>75% for most of the plot's associations). The fern's metacommunity was structured along an abiotic gradient modulated by temperature, luminosity, and relative soil moisture.

Proton stoichiometry of electron transport in rodent tumor mitoplasts.

The mechanistic vectorial H+/O translocation ratios characteristic of energy-conserving sites 2 + 3 and site 3 of the respiratory chain of two tumor cell lines were determined using succinate and ferrocytochrome c, respectively, as electron donors. The measurements were carried out on mitoplasts in order to allow ferrocytochrome c free access to its binding site on the inner mitochondrial membrane. The tumor cell lines used were Ehrlich ascites tumor and the AS30-D ascites tumor. K+ was used as charge-compensating cation in the presence of valinomycin. The O2 uptake rate measurements were made with a fast-responding membrane-less electrode whose response time was closely matched with that of a pH electrode. The rates of O2 uptake and H+ ejection during the apparent zero-order rate phase of respiration, analyzed by computer, were extrapolated to zero time. The observed H+/O ratios for succinate oxidation in both tumors exceeded 7 and approached 8 and the H+/O ratios for the cytochrome oxidase reaction closely approached 4.0, in agreement with data or normal mitochondria. However, the rates of H+ back decay in the tumor mitochondria are relatively high and may influence the net efficiency of oxidative phosphorylation under intracellular conditions.

Mitochondrial function in rat cerebral cortex and hippocampus after short- and long-term hypobaric hypoxia.

Taking into account the importance of aerobic metabolism in brain, the aim of the present work was to evaluate mitochondrial function in cerebral cortex and hippocampus in a model of sustained hypobaric hypoxia (5000 m simulated altitude) during a short (1 mo) and a long (7 mo) term period, in order to precise the mechanisms involved in hypoxia acclimatization. Hippocampal mitochondria from rats exposed to short-term hypobaric hypoxia showed lower respiratory rates than controls in both states 4 (45%) and 3 (41%), and increased NO production (1.3 fold) as well as eNOS and nNOS expression associated to mitochondrial membranes, whereas mitochondrial membrane potential decreased (7%). No significant changes were observed in cortical mitochondria after 1 mo hypobaric hypoxia in any of the mitochondrial functionality parameters evaluated. After 7 mo hypobaric hypoxia, oxygen consumption was unchanged as compared with control animals both in hippocampal and cortical mitochondria, but mitochondrial membrane potential decreased by 16% and 8% in hippocampus and cortex respectively. Also, long-term hypobaric hypoxia induced an increase in hippocampal NO production (0.7 fold) and in eNOS expression. A clear tendency to decrease in H2O2 production was observed in both tissues. Results suggest that after exposure to hypobaric hypoxia, hippocampal mitochondria display different responses than cortical mitochondria. Also, the mechanisms responsible for acclimatization to hypoxia would be time-dependent, according to the physiological functions of the brain studied areas. Nitric oxide metabolism and membrane potential changes would be involved as self-protective mechanisms in high altitude environment.

Regulation of mitochondrial respiration by oxygen and nitric oxide.

Although the regulation of mitochondrial respiration and energy production in mammalian tissues has been exhaustively studied and extensively reviewed, a clear understanding of the regulation of cellular respiration has not yet been achieved. In particular, the role of tissue pO2 as a factor regulating cellular respiration remains controversial. The concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers has evolved in the last few years and is still being researched. A recent concept that regulation of cellular respiration is regulated by ADP, O2 and NO preserves the notion that energy demands drive respiration but places the kinetic control of both respiration and energy supply in the availability of ADP to F1-ATPase and of O2 and NO to cytochrome oxidase. In addition, recent research indicates that NO participates in redox reactions in the mitochondrial matrix that regulate the intramitochondrial steady state concentration of NO itself and other reactive species such as superoxide radical (O2-) and peroxynitrite (ONOO-). In this way, NO acquires an essential role as a mitochondrial regulatory metabolite. No exhibits a rich biochemistry and a high reactivity and plays an important role as intercellular messenger in diverse physiological processes, such as regulation of blood flow, neurotransmission, platelet aggregation and immune cytotoxic response.

A comparative computational study of blood flow through prosthetic heart valves using the finite element method.

The steady flow of blood through three common types of prosthetic heart valves was simulated numerically using the finite element method. The velocity, pressure and stress fields were obtained for the disk-type, tilting-disk and ball-type prosthetic heart valves in aortic position, for increasing Reynolds numbers up to 900, 1500 and 2000 respectively. Computer graphics of calculated velocities are presented, showing in detail the accelerated flow, recirculation and stagnation areas developed in the prosthesis. Maximum wall shear stresses were found at 0.5, 1.4, and 1.2 diameters from the sewing ring downstream for the disk, tilting-disk and ball valves being the values of 55, 18 and 33 dyn cm-2 respectively. In the vicinity of the occluder, maximum shear stresses of 38, 30 and 47 dyn cm-2 respectively were computed. The flow characteristics and performance for each valve are compared, the results are presented in terms of energy loss and maximum shear stress. The velocity and stress fields are compared with in vitro evaluations found in the literature.

The occlusal wafer: simple technique for construction and its use in maxillofacial surgery.

The acrylic occlusal wafer is a thin piece of plastic which fits between and indexes the occlusal surfaces of the maxillary and mandibular teeth. This appliance has three functions: it positions the jaws relative to one another; it prevents sliding movement during intermaxillary fixation; and it prevents unwanted orthodontic movement during intermaxillary fixation from the pressure of intermaxillary wires or rubber bands on unsupported teeth. The role of the occlusal wafer is well established in orthognathic surgery where it is used as a key for setting the jaws when osteotomies are performed. It has been underutilized in trauma due to the time and technical expertise required for fabrication. This paper describes a technique which can result in occlusal wafers of surgical quality in about 20 minutes, requires minimal laboratory equipment, and can be mastered by clinical personnel. Four cases are presented which illustrate the use of occlusal wafers and the indications for the device are discussed.

Ureteral segment replacement using a circumferential small-intestinal submucosa xenogenic graft.

We wished to determine whether small-intestinal submucosa (SIS) will epithelialize when used as a ureteral replacement material. An 11-mm segment of native ureter was excised from eight New Zealand White rabbits and replaced with an 11-mm porcine SIS graft, which was circumferentially wrapped around a ureteral stent. The SIS ureteral grafts were harvested at 11 days or 35 days postimplantation and examined grossly and by standard light microscopy techniques. Partial epithelialization with the ingrowth of urothelium, smooth muscle cells, and blood vessels was observed in the grafts harvested at 11 days postimplantation. The SIS ureteral grafts examined at 35 days postimplantation showed additional restructuring of the smooth muscle cell layer and more organized epithelialization in comparison to the SIS graft examined at 11 days. After 35 days of regenerative healing, elements of all three layers of the native ureter were observed within the collagen matrix of the SIS graft. No significant complications were observed, but all subjects (8/8) demonstrated mild intra-abdominal adhesions. Mild collecting system dilatations were observed in 4/4 (100%) of the animals harvested at 35 days and in 0/4 (0%) of the animals harvested at 11 days. We have this demonstrated in this preliminary study that SIS xenografts will epithelialize when used as a ureteral replacement material. The repair mechanism of these ureteral grafts occurred through a regenerative healing process rather than by scar formation. With further studies, this material may prove to be a useful treatment option in patients with ureteral injuries.

An alternative to synthetic aortic grafts using jejunum.

Synthetic materials currently used for arterial grafts can provoke serious complications such as infection, rejection, and incomplete reendothelialization. Porcine small intestinal submucosa (SIS) allografts (homografts) may not share these disadvantages yet still may provide an effective graft alternative. This study was designed to provide additional information concerning the performance of this material when used as an allo-patch graft. A porcine model was selected to enable hemodynamic comparisons to the adult human physiology. Slaughterhouse small intestine was used to prepare the SIS graft material. A well-vascularized section ofjejunum was resected, rinsed, and maintained in 0.9% NaCl solution containing 10% gentamicin. The graft was prepared by removing the serosal layer, inverting the material, and discarding the mucosal surface in a similar manner. The remaining submucosa and stratum compactum constituted the graft material and was subdivided into sizes from 1.5 to 3.0 cm2 tacked to foil for manageability, and was returned to the gentamicin solution. The surgery was initiated with a femoral arterial line in the anesthetized swine, followed by isolation and preparation of the infrarenal aorta for grafting. Cross-clamps were applied and a 1.0 x 1.5-cm section of aortic wall was resected. Two sides of allograft were freed from the foil and sewn into the aorta. Then the remaining sides were removed from the foil and final graft placement was completed. The graft was harvested at postoperative day 28 and presented for microscopic examination. Results confirmed that reendothelialization with neovascularization had occurred during this interval.

[Effect of chronic hypobaric hypoxia on water distribution in the rat]. / Efecto de la hipoxia hipobárica crónica sobre la distribución del agua en la rata.

Rats of both sexes chronically submitted to a simulated sltitude of 4700 m (440 mm Hg) in a hypopressure chamber show a different behavior in relation to total body water. The aim of the present work was to analyze the behavior of body fluids in these conditions in order to precise whether or not the observed change in total body water is accompanied by a difference in its distribution. Male, female and castrated female rats, submitted to 440 mm Hg during nine months and their respective controls maintained at ambient pressure, were used. At the end of that period, and previous insertion of a carotid cannula, total water, extracellular fluid and blood volume were determinated by isotopic dilution techniques (Fig. 1), using tritiated water, radiosulfate and 131I human serum albumin, respectively. Blood samples were obtained without interruption of the hypopressure. Immediately after the experiments, the animals were sacrificed and total body fat was determined. Results, expressed in percentage of lean body mass (Table 1), showed that in intact female rats the increase in total body water would be due to an increase in interstitial water volume and that there was a significant decrease in plasma water volume. On the other hand, in castrated females, exposure to hypoxia produced dehydration, by a decrease in intraellular water, without significant changes in plasma water volume. The results in males showed a tendency in the same direction, but the differences were not statistically significant. The difference found in water distribution can be adscribed to an effect of estrogens, which may be direct or mediated by other endocrine factors.

Abiotic factors drives floristic variations of fern's metacommunity in an Atlantic Forest remnant / Fatores abióticos regulam as variações florísticas na metacomunidade de samambaias em um remanescente de Floresta Atlântica

Abstract We analyzed floristic variations in fern's metacommunity at the local scale and their relationship with abiotic factors in an Atlantic Forest remnant of northeastern Brazil. Floristic and environmental variations were accessed on ten plots of 10 × 20 m. We performed cluster analyses, based on Bray-Curtis dissimilarity index to establish the floristic relationship. The influence of abiotic factors: luminosity, temperature, relative air humidity and relative soil moisture was evaluated from a redundancy analysis. We found 24 species belonging to 20 genera and 12 families. The fern's flora showed high floristic heterogeneity (>75% for most of the plot's associations). The fern's metacommunity was structured along an abiotic gradient modulated by temperature, luminosity, and relative soil moisture.

Resumo Analisamos as variações florísticas na metacomunidade de samambaias em escala local e sua relação com fatores abióticos em um remanescente de Floresta Atlântica no Nordeste do Brasil. Variações florísticas e ambientais foram acessadas a partir de dez parcelas de 10 × 20 m. Realizamos análises de cluster, baseado no índice de similaridade de Bray-Curtis para estabelecer relações florísticas. A influência de fatores abióticos: luminosidade, temperatura, umidade relativa do ar e umidade relativa do solo foram avaliadas a partir da análise de redundância. Encontramos 24 espécies pertencentes a 20 gêneros e 12 famílias. A flora de samambaias exibiu uma elevada heterogeneidade florística (>75% para a maioria das associações entre plots). Observou-se que a metacomunidade de samambaias estava estruturada ao longo de um gradiente abiótico modulado pela temperatura, luminosidade e umidade relativa do solo.