Dual immune effect of iNKT cells considering human cutaneous and visceral leishmaniasis: An example of cell plasticity according to different disease scenarios.

Although the semi-invariant natural killer T cells (iNKT) are a small subpopulation of cells in the peripheral blood, they are presumed to play a role in early stages of infection against various pathogens, including protozoa. This work investigates the activation status and cytokine profile of iNKT cells during human Leishmania infantum and Leishmania braziliensis infection. We studied iNKT cells in patients with symptomatic active visceral leishmaniasis (AVL) (n = 8), patients with symptomatic active cutaneous leishmaniasis (ACL) (n = 13), negative endemic controls (NEC) (n = 6) and non-endemic controls (NonEC) (n = 6), with and without total Leishmania antigen stimulus (TLA). The number of iNKT cells in the peripheral blood of patients with ACL and AVL unaltered in relation to control groups. Moreover, the iNKT cells from ACL showed a hyperactivation profile compared to patients with AVL. Additionally, TLA induced IFN-gamma production in iNKT cells from patients with ACL, while in iNKT of patients with AVL, TLA induced a decrease in this cytokine. Higher IL-17 and IL-10 production by iNKT cells from patients with ACL were also observed compared to all other groups. There were no changes in iNKT IL-10-producing cells in AVL after TLA stimulation. However, TLA induced increase in IL-10 in iNKT cells in patients with ACL. These findings suggest that, although iNKT cells showed distinct profiles in patients with ACL and AVL, they play a dual role in immune modulation in both Leishmania infections.

Factors affecting prevailing population levels of Brevipalpus yothersi (Acari: Tenuipalpidae) in citrus areas affected by citrus leprosis in the State of Sao Paulo, Brazil.

Brevipalpus yothersi Baker (Acari: Tenuipalpidae) was recently confirmed as one of the main vectors of citrus leprosis. Knowledge about this mite's biology is essential to understand its population dynamics, in order to solve management issues in citrus orchards and explain why citrus leprosis is more severe in some regions. This paper aimed to study biological factors affecting prevailing population levels of B. yothersi and incidence of citrus leprosis. Mites were sampled from orchards in the south, north and northwestern regions of São Paulo State, Brazil. We assessed duration of the developmental stages, oviposition, incubation period, egg viability, longevity, net reproductive rate (Ro), mean generation time (T), intrinsic rate of increase (rm) and finite rate of increase (λ). There were small differences in parameter values between the three populations. Our results indicated that the various measures adopted in the control of the three populations did not lead to major biological differences between populations for the evaluated parameters.

Clock genes expression and locomotor activity are altered along the light-dark cycle in transgenic zebrafish overexpressing growth hormone.

In the present work it was demonstrated that transgenic Danio rerio overexpressing growth hormone (GH-transgenic) present either altered gene expression at a determined time point, or different expression pattern along the LD cycle, when compared with non-transgenic (NT) animals, in the positive and negative loops of the circadian system. Gene expression of clock paralogs was reduced in GH fish at the beginning of the dark phase, leading to diminished expression amplitude along the LD cycle. Furthermore, although no differences were observed between NT and GH animals for bmal1a and cry2b expression at each time point, only GH fish presented amplitude along the LD cycle. Also, the locomotor activity behavior was evaluated for both groups. GH-transgenic animals presented higher locomotor activity along the whole LD cycle when compared with NT animals. These data suggest that alterations in the gene expression patterns along the LD cycle of the positive and negative loops of the circadian system, could lead to altered locomotor activity behavior in GH-transgenic fish, and GH overexpression could be responsible for these alterations, either affecting the pathways involved in the expression of genes from the circadian system or altering the metabolism.

Persistent effects of pre-Columbian plant domestication on Amazonian forest composition.

The extent to which pre-Columbian societies altered Amazonian landscapes is hotly debated. We performed a basin-wide analysis of pre-Columbian impacts on Amazonian forests by overlaying known archaeological sites in Amazonia with the distributions and abundances of 85 woody species domesticated by pre-Columbian peoples. Domesticated species are five times more likely than nondomesticated species to be hyperdominant. Across the basin, the relative abundance and richness of domesticated species increase in forests on and around archaeological sites. In southwestern and eastern Amazonia, distance to archaeological sites strongly influences the relative abundance and richness of domesticated species. Our analyses indicate that modern tree communities in Amazonia are structured to an important extent by a long history of plant domestication by Amazonian peoples.

New efficient DNA extraction method to access the microbiome of Ricinus communis seeds.

Ricinus communis (castor bean) seeds are used to produce an alcohol-soluble oil that is used in more than 400 industrial processes. Despite its economic importance, there has been little research on the endophytic microbiota of castor bean seeds. This microbiota is important for plant metabolic processes and may have considerable biotechnological potential, such as production of lipases and plant growth promoter agents. We evaluated several DNA extraction methodologies in order to access the microbial diversity of castor bean through a metagenomic approach. Based on our observations, we developed a new methodology that takes advantage of the low solubility of calcium phosphates and the high affinity of these phosphates for proteins and polysaccharides. The extracted DNA quality was evaluated by PCR, using a selective primer pair for bacterial and mitochondrial 16S rDNA genes (799F and 1492R). We found this methodology quantitatively and qualitatively more efficient than the other approaches. In evaluating this new extraction methodology, we found that the difficulties of DNA extraction from castor bean seeds, such as abundant oil, polysaccharides, phenolic compounds, and plant enzymes, could be overcome. The resulting extracts had high concentration and purity, and they were obtained faster than with previous methods. The samples contained virtually all of the DNA, including the microbial DNA; this was validated by PCR analysis.

Endothelialization of chitosan porous conduits via immobilization of a recombinant fibronectin fragment (rhFNIII7-10).

The present study aimed to develop a pre-endothelialized chitosan (CH) porous hollowed scaffold for application in spinal cord regenerative therapies. CH conduits with different degrees of acetylation (DA; 4% and 15%) were prepared, characterized (microstructure, porosity and water uptake) and functionalized with a recombinant fragment of human fibronectin (rhFNIII(7-10)). Immobilized rhFNIII(7-10) was characterized in terms of amount ((125)I-radiolabelling), exposure of cell-binding domains (immunofluorescence) and ability to mediate endothelial cell (EC) adhesion and cytoskeletal rearrangement. Functionalized conduits revealed a linear increase in immobilized rhFNIII(7-10) with rhFNIII(7-10) concentration, and, for the same concentration, higher amounts of rhFNIII(7-10) on DA 4% compared with DA 15%. Moreover, rhFNIII(7-10) concentrations as low as 5 and 20µg ml(-1) in the coupling reaction were shown to provide DA 4% and 15% scaffolds, respectively, with levels of exposed cell-binding domains exceeding those observed on the control (DA 4% scaffolds incubated in a 20µg ml(-1) human fibronectin solution). These grafting conditions proved to be effective in mediating EC adhesion/cytoskeletal organization on CH with DA 4% and 15%, without affecting the endothelial angiogenic potential. rhFNIII(7-10) grafting to CH could be a strategy of particular interest in tissue engineering applications requiring the use of endothelialized porous matrices with tunable degradation rates.

Beyond behavioural observations: a deeper view through the sensory reactions of children with profound intellectual and multiple disabilities.

BACKGROUND: The present study was aimed at assessing the behavioural and physiological responsiveness of three children with profound intellectual and multiple disabilities to a set of sensory stimuli. METHODS: Eighteen items, 'believed' by clinicians to be appropriate for routine interventions, were presented to the participants and responsiveness was assessed in terms of: (i) consistent behavioural and electrodermal reactions and (ii) biphasic changes in heart rate. RESULTS: Results were twofold. First, all participants showed frequent consistent physiological reactions but rare consistent behavioural ones. Second, all participants showed biphasic changes in heart rate, pointing to the possibility of different appraisals (novelty and pleasantness) of most of the presented items. CONCLUSIONS: Data here presented may have implications for the development and the emotional well-being of individuals with profound intellectual and multiple disabilities in that it suggests that reactions to, and potential appraisals of, sensory stimuli may occur despite the lack of consistent observable behaviours.

Herpes simplex 2 e citomegalovirus: efeitos citopáticos vs mecanismos de infecção: [revisão] / Herpes simplex 2 virus and cytomegalovirus cytophatic effects vs infection mechanics: [review]

O vírus, contrariamente a outras classes de agentes que afetam o organismo, não são observáveis em microscopia óptica, refletindo-se apenas microscopicamente em efeitos virais (ECPV), que permitem identificá-los por constituírem um conjunto de alterações patognomônicas a nível celular. O aumento nuclear, a variação da morfologia celular, a perda do padrão da cromatina, as inclusões intranucleares ou intracitoplasmáticas ou a multinucleação, são alguns dos ECPV mais comuns. Entre os vírus que afetam a mucosa genital na família Herpesviridae, inserem-se Herpes Simplex 2 e o Citomegalovirus, cujo diagnóstico deverá ter em conta a complexidade dos mecanismos de replicação. Assim, e apesar dos vários métodos para a detecção do vírus, a citologia continua a ser um método viável para o diagnóstico destas infecções virais, sendo que a compreensão do processo de infecção contribui para a correta avaliação microscópica. Este trabalho tem como finalidade efetuar uma revisão bibliográfica sobre o grupo Herpes, focando o Herpes Simplex 2 e o Citomegalovirus, assim como, explicar e relacionar os ECPV, associados ao grupo Herpes , tendo por base os mecanismos de infecção.

Solitary hemangioma of the small bowel disclosed by wireless capsule endoscopy.

A nine-year-old child presented with melena and anemia. She had similar symptoms five months earlier and had undergone an extensive workup with upper gastrointestinal endoscopy and colonoscopy, both normal and 99m-Tc-RBC-scintigraphy which was positive in the right lower quadrant. This time, capsule endoscopy was performed and disclosed an hemangioma with a dark spot suggesting recent bleeding in the ileum. The lesion was resected. Pathological examination revealed a transmural cavernous hemangioma. Small bowel hemangioma is a rare disease. Its diagnosis is extremely difficult and is usually obtained during surgery. Capsule endoscopy is an endoscopic technique that can improve preoperative diagnosis, as reported in the present case.

Surface characterization and cell response of a PLA/CaP glass biodegradable composite material.

Bioabsorbable materials are of great interest for bone regeneration applications, since they are able to degrade gradually as new tissue is formed. In this work, a fully biodegradable composite material containing polylactic acid (PLA) and calcium phosphate (CaP) soluble glass particles has been characterized in terms of surface properties and cell response. Cell cultures were performed in direct contact with the materials and also with their extracts, and were evaluated using the MTT assay, alkaline phosphatase activity, and osteocalcin measurements. The CaP glass and PLA were used as reference materials. No significant differences were observed in cell proliferation with the extracts containing the degradation by-products of the three materials studied. A relation between the materials wettability and the material-cell interactions at the initial stages of contact was observed. The most hydrophilic material (CaP glass) presented the highest cell adhesion values as well as an earlier differentiation, followed by the PLA/glass material. The incorporation of glass particles into the PLA matrix increased surface roughness. SEM images showed that the heterogeneity of the composite material induced morphological changes in the cells cytoskeleton.

Attachment, spreading and short-term proliferation of human osteoblastic cells cultured on chitosan films with different degrees of acetylation.

Chitosan (Ch) is being actively investigated as a non-protein template for the growth of an increasing number of anchorage-dependent cells, including chondrocytes and bone cells. In the present work, Ch films with degrees of N-acetylation (DAs) in the range of 4 to 49% were evaluated with respect to the attachment, spreading and short-term proliferation of osteoblasts, using human osteoblastic MG-63 cells. The films were characterized in terms of surface morphology and surface charge by atomic force microscopy and streaming potential measurements, respectively. Cell attachment was assessed after 3 and 24 h of cell culture. After 24 h of incubation, cell attachment was found to be dependent on the DA, lower DAs favouring cell adhesion. With time, cell spreading and cytoskeleton organization were only attained for DAs

The influence of ramp rate on VO2peak and "excess" VO2 during arm crank ergometry.

The principal aim of this study was to examine how different ramp rates influenced the attainment of peak physiological responses during incremental arm crank ergometry (ACE). Additionally, the study examined whether there was any evidence for the development of an "excess" VO (2) during ACE due to upward curvi-linearity in the VO (2)-work rate relationship, and whether this was influenced by the ramp rate. Sixteen physically active, though non-specifically trained, men (mean +/- S age 30 +/- 8 years; height 1.79 +/- 0.07 m; body mass 84.7 +/- 13.2 kg) volunteered to participate. Having completed a familiarisation test, all subjects returned to the laboratory to complete two ramp tests on an electrically-braked ergometer in a counter-balanced order. Both ramp tests started at 60 W with work rate subsequently incremented by either 6 or 12 W . min (-1). Pulmonary gas exchange was measured breath-by-breath throughout the tests. Subjects achieved a greater final work rate during the 12 W . min (-1) test compared to the 6 W . min (-1) test (168 +/- 28 vs. 149 +/- 26 W; p < 0.001). The VO (2peak) (3.06 +/- 0.65 vs. 2.96 +/- 0.48 L . min (-1); p = 0.27), HR (peak) (179 +/- 15 vs. 177 +/- 16 b . min (-1); p = 0.17) and V.E (peak) (112 +/- 22 vs. 105 +/- 16 L . min (-1); p = 0.09) were not different between the tests, but VCO (2peak) (3.54 +/- 0.64 vs. 3.27 +/- 0.46 L . min (-1); p = 0.01) RER (peak) (1.17 +/- 0.07 vs. 1.11 +/- 0.06; p < 0.001), and end-exercise blood (lactate) (11.9 +/- 2.1 vs. 10.8 +/- 2.6 mmol . L (-1); p = 0.005) were all higher in the 12 W . min (-1) test. An "excess" VO (2) was observed in 13 out of 16 tests at 12 W . min (-1) and in 15 out of 16 tests at 6 W . min (-1). Neither the magnitude of the "excess" VO (2) (0.42 +/- 0.41 vs. 0.37 +/- 0.18 L . min (-1); p = 0.66) nor the VO (2) at which the V.O (2)-work rate relationship departed from linearity (2.17 +/- 0.34 vs. 2.18 +/- 0.32 L . min (-1); p = 0.94) were significantly different between the two ramp tests. These data indicate that differences in ramp rate within the range of 6 - 12 W . min (-1) influence the peak values of work rate, VCO (2) and RER, but do not influence peak values of VO (2) or HR during ACE. The development of an "excess" VO (2) appears to be a common feature of ramp exercise in ACE, although the mechanistic basis for this effect is presently unclear.

Three-dimensional culture of human osteoblastic cells in chitosan sponges: the effect of the degree of acetylation.

In this investigation, the effect of the degree of acetylation (DA) of chitosan on the behavior of human osteoblastic MG-63 cells cultured in three-dimensional chitosan matrices was assessed. Chitosan sponges with DAs in the range of 4 to 49% were prepared and characterized in terms of microstructure, porosity, and pore size. Collagen sponges were used as 3D control. Cell proliferation was determined using the MTT assay while the retention of the osteoblastic phenotype was monitored by assaying alkaline phosphatase activity. Cell morphology, cytoskeletal organization, and viability were assessed using different microscopy techniques. Chitosan sponges showed a similar microstructure regardless the DA, except for the highest DA used, where a more heterogeneous pore distribution was observed. In terms of cell proliferation, alkaline phosphatase activity and cell viability, cells cultured in chitosan scaffolds performed as well as in the 3D control regardless the DA, except for the highest DA used, where an inhibitory effect on cell proliferation was found. However, while in sponges with DAs < or = 13% cells attached and spread displaying long cell filopodia and numerous cell-to-cell contacts, in sponges with higher DAs cells tended to remain spherical and grow into spheroid-like cellular aggregates. In the present study, the DA played a key role in determining the affinity of osteoblastic cells towards the substrates, possibly by influencing the nature of the initial adsorbed protein layer.

Chemical modification of chitosan by phosphorylation: an XPS, FT-IR and SEM study.

In the present work, the surface of chitosan membranes was modified using a phosphorylation method carried out at room temperature. Phosphorylation may be of particular interest in materials for orthopaedic applications, due to the cation-exchange properties of phosphate functionalities. Phosphate groups chelate calcium ions, thus inducing the deposition of an apatite-like layer known to improve the osteoconduction of polymer-based implants. Additionally, the negatively charged phosphate functionalities, together with the positively charged amine groups from chitosan, are expected to provide chitosan with an amphoteric character, which may be useful as a combinatorial therapeutic strategy, by simultaneously allowing the immobilization of signalling molecules like growth factors. Phosphorylation was carried out at room temperature using the H3PO4/Et3PO4/P2O5/butanol method. Surface characterization was performed by XPS, ATR-FT-IR, and SEM. Cross-sections were analyzed by SEM fitted with EDS. The phosphate content increased with the reaction time, as shown by XPS and ATR-FT-IR, a P/N atomic ratio of 0.73 being obtained after 48 h of treatment. High-resolution XPS spectra regarding C1s, O1s, N1s and P2p are discussed. The introduction of a neutralization step led to a reduction of P content, which pointed out to the presence of phosphates ionically bound to protonated amines, in addition to phosphate esters. EDS analysis of cross-sections revealed a gradual P reduction up to 50% towards the inner part of the membrane.

Rat bone marrow stromal cell osteogenic differentiation and fibronectin adsorption on chitosan membranes: the effect of the degree of acetylation.

Cell adhesion, migration, and proliferation of a few anchorage-dependent cells cultured on chitosan (Ch) matrices are influenced by the degree of N-acetylation (DA) of Ch. In the present work, we examined the influence of the DA on the attachment, spreading, proliferation, and osteogenic differentiation of rat bone marrow stromal cells (rBMSCs). Ch membranes were characterized in terms of surface morphology, roughness, and wettability, and in terms of adsorption of an adhesive protein, fibronectin (Fn). Chs with DAs in the range of 4 to 49% were used. Among the Ch samples, the DA of 4% led to the highest Fn surface concentration, both from single protein solution and from diluted serum. Furthermore, the levels of Fn adsorbed from serum found for this DA were threefold higher than for the tissue culture polystyrene control, indicating that in the presence of competitive proteins Ch is more specific toward Fn adsorption than tissue culture polystyrene. rBMSCs cultured on Ch carrying a DA of 4% were able to spread, proliferate, and differentiate, reaching a higher level of osteogenic differentiation than on the control, despite the lower cell attachment observed for all Ch samples. Because the Ch sample with a DA of 4% showed the highest Fn adsorption from serum, we suggest that cell adhesion, spreading, and osteogenic differentiation of rBMSCs on Ch may be mediated by the adsorbed layer of Fn.

Acute toxicity (LD50 and CD50) of lidocaine and prilocaine in combination with adrenaline and felypressin.

The toxicity of the combination of vasoconstrictors to local anaesthetic solutions has been debated since its first use in the beginning of this century. A combination of two vasoconstrictors to a local anaesthetic has been proposed by some researchers. In this study they were evaluated the acute toxicity (lethal dose 50%, convulsion dose 50%) and latency times of loss of righting reflex and convulsion as well as the duration of convulsion) of 2% lidocaine or 3% prilocaine, when administered in combination with adrenaline and felypressin at various concentrations. Lethal dose 50% studies showed that for both anaesthetics the solutions with higher concentrations of adrenaline were more toxic. The opposite was observed in the convulsion dose 50% studies. No alterations were observed in the control groups. All lidocaine solutions increased the latency of loss of righting reflex. The latency of convulsion was increased in some groups, but once the convulsion was achieved there was no difference in its duration. There was no statistical difference among prilocaine groups for any of the variables studied. Based on the experimental model studied, it was concluded that there is no advantage in the association of two vasoconstrictors concerning the toxicity of lidocaine and prilocaine solutions.

[The use and interpretation of tuberculin tests]. / Utilização e interpretação dos testes tuberculínicos.

The author reviews the difficulties in diagnosing tuberculosis infection with the current BCG National Vaccination Plan. The author points out some directions that, in her point of view, may provide a clearer interpretation of the tuberculin skin test, bearing in mind the child's clinical, epidemiologic and social background.