Prova de absorção de gordura em crianças.

To overcome the difficulties observed with fecal fat estimations, we studied fat absorption in 4 m to 12 years old children, assessing increments of serum triglycerides after administration of corn oil. Eighteen well-nourished children without gastrointestinal symptoms or parasites, 31 children with protein-calorie malnutrition (PCM), with or without protracted diarrhea and/or intestinal parasites and nine children with specific malabsorptive entities were studied. Serum triglycerides (TG) were measured before, 2 and 4 hours after the administration of 2 g/kg of corn oil per os. For control infants below 2 years no significant differences between the fasting level and those after 2 and 4 hours were observed. For children above this age significant absorption occurred by 2 hours but more intensively after 4 hours, an increment of TG above 35 mg% being considered normal. A good discrimination between TG increments of controls and malabsorptive children over 2 years was observed. In conclusion, the oral fat loading test, is applicable for children over 2 year of age. For infants below this age other schedules should be tested.

Specific components found in circulating immune complexes (CIC) in paracoccidioidomycosis

Circulating immune complexes (CIC) from 15 paracoccidioidomycosis (PCM) patient sera and from 20 healthy control sera were analysed. After CIC precipitation, solubilization and acid treatment, only a little reactivity to P. brasiliensis antigens was found in the free antibodies from PCM-CIC. This result has suggested that there were antibodies with a high affinity bound to fungus components. Dissociated CIC were fractionated in a column of Sephacryl S300 and the fractions that probably contained antigens were pooled and applied to an affinity column, prepared with mouse anti-gp43 monoclonal antibody. Using ECL-Western blotting assay two polypeptide with apparent mass of 43 and 62 kDa were found.

Paracoccidioides brasiliensis-gp43 used as paracoccidioidin

A purified glycoprotein of 43 000 daltons from Paracoccidioides brasiliensis (gp43) was tested as paracoccidioidin in delayed-type hypersensitivity (DTH) tests in both experimental animals (guinea pig and mice) and patients with paracoccidioidomycosis (PCM). The gp43 paracoccidioidin was compared with the traditional Fava Netto antigen (AgFN). In guinea pigs, the intradermal injection of 2 μg of gp43 showed a similar response to those obtained with AgFN, showing in histological sections a population of lymphoid cells that participate in DTH. In mice, gp43 at a dose of 3.75μg showed positive DTH response. The use of gp43 as paracoccidioidin in humans showed that this molecule can be used to evaluate the DTH response in patients with PCM. Of 25 PCM patients studied, 48% were positive to gp43 while only 28% were positive to AgFN; 12 PCM patients were completely anergic to both antigens. Considering only those 13 PCM patients who were responsive to gp43 and/or to AgFN, 92.3% reacted against gp43 and 53.8% reacted against AgFN (P < 0.05). Gp43 skin test responses (13.67 ± 9.56 mm) were significantly larger than those obtained with AgFN (8.43 ±3.69 mm). Immunohistochemical study of the human skin showed a perivascular inflammatory response constituted predominantly by T lymphocytes, macrophages and polymorphonuclear leukocytes. © 1996 ISHAM.

A joint theoretical and kinetic investigation on the fragmentation of (N-halo)-2-amino cycloalkanecarboxylates

A combined theoretical and experimental study to elucidate the molecular mechanism for the Grob fragmentation of different (N-halo)-2-amino cyclocarboxylates with the nitrogen atom in exocyclic position: (N-Cl)-2-amino cyclopropanecarboxylate (1), (N-Cl)-2-amino cyclobutanecarboxylate (2), (N-Cl)-2-amino cyclopentanecarboxylate (3) and (N-Cl)-2-amino cyclohexanecarboxylate (4), and the corresponding acyclic compounds, (N-Cl)-2-amino isobutyric acid (A), (N-Cl)-2-amino butyric acid (B), has been carried out. The kinetics of decomposition for these compounds and related bromine derivatives were experimentally determined by conventional and stopped-flow UV spectrophotometry. The reaction products have been analyzed by GC and spectrophotometry. Theoretical analysis is based in the localization of stationary points (reactants and transition structures) on the potential energy surface. Calculations were carried out at B3LYP/6-31+G* and MP2/6-31+G* computing methods in the gas phase, while solvent effects have been included by means the self-consistent reaction field theory, PCM continuum model, at MP2/6-31+G* and MP4/6-31+G*//MP2/6-31+G* calculation levels. Based on both experimental and theoretical results, the different Grob fragmentation processes show a global synchronicity index close to 0.9, corresponding to a nearly concerted process. At the TSs, the N-Cl bond breaking is more advanced than the C-C cleavage process. An antiperiplanar configuration of these bonds is reached at the TSs, and this geometrical arrangement is the key factor governing the decomposition. In the case of 1 and 2 the ring strain prevents this spatial disposition, leading to a larger value of the activation barrier. Natural population analysis shows that the polarization of the N-Cl and C-C bonds along the bond-breaking process can be considered the driving force for the decomposition and that a negative charge flows from the carboxylate group to the chlorine atom to assist the reaction pathway. A comparison of theoretical and experimental results shows the relevance of calculation level and the inclusion of solvent effects for determining accurate unimolecular rate coefficients for the decomposition process. © 2002 Published by Elsevier Science B.V.

IL-12 and neutralization of endogenous IL-10 revert the in vitro antigen-specific cellular immunosuppression of paracoccidioidomycosis patients

Treatment of patients with paracoccidioidomycosis is still a challenge. Patients present defective lymphoproliferation and IFN-γ responses to the main Paracoccidioides brasiliensis antigen (gp43), which correlates with disease severity. Here, we demonstrated that the patients show also a defective synthesis of interleukin (IL)-12. Therefore, we attempted to revert this immune disfunction by adding IL-12 and neutralizing anti-IL-10 antibody to gp-43-stimulated peripheral blood mononuclear cell cultures. Both treatments increased IFN-γ secretion to levels observed with healthy sensitized individuals, but affected proliferation only modestly. When combined, the treatments further increased IFN-γ synthesis and cell proliferation. The addition of suboptimal concentrations of IL-2 also further increased the IL-12-mediated secretion of IFN-γ. Interestingly, the immune modulation was mostly antigen-specific, since the responses to Candida albicans' antigen were not affected. These results suggest that appropriate immune intervention with cytokines and/or anti-cytokines may help in the treatment of PCM. © 2002 Elsevier Science Ltd. All rights reserved.

Solubilization of proteins from human lymph node tissue and two-dimensional gel storage.

In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8 M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT (dithiothreitol) and 0.2% carrier ampholytes; (b) 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 40 mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.

Inhibition of human neutrophil apoptosis by Paracoccidioides brasiliensis: Role of interleukin-8

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidiodes brasiliensis that presents a wide spectrum of clinical manifestations. Because of the great number of neutrophils polymorphonuclear neutrophils (PMN) found in the P. brasiliensis granuloma, studies have been done to evaluate the role of these cells during the development of the infection. This fungus is found intracellularly in PMN and monocytes/macrophages, suggesting that it is capable of evading damage and surviving inside these cells. Thus, in the present study, we investigated whether P. brasiliensis can prolong the lifetime of PMN, and if this process would be related with IL-8 levels. PMN apoptosis and intracellular levels of IL-8 were analysed by flow cytometry and culture supernatants IL-8 levels were evaluated by enzyme-linked immunosorbent assay. We found that coincubation with P. brasiliensis yeast cells results in an inhibition of PMN apoptosis, which was associated with increase in IL-8 production by these cells. Cocultures treatment with monoclonal antibody anti-IL-8 reversed the inhibitory effect of P. brasiliensis on PMN apoptosis, besides to increase spontaneous apoptosis of these cells. These data show that, in contrast to other microbial pathogens that drive phagocytes into apoptosis to escape killing, P. brasiliensis can extend the lifetime of normal human PMN by inducing autocrine IL-8 production. © 2008 The Authors.

Dietary restriction abrogates antibody production induced by a DNA vaccine encoding the mycobacterial 65 kDa heat shock protein

Background: Protein-calorie malnutrition (PCM) is the most common type of malnutrition. PCM leads to immunodeficiency and consequent increased susceptibility to infectious agents. In addition, responses to prophylactic vaccines depend on nutritional status. This study aims to evaluate the ability of undernourished mice to mount an immune response to a genetic vaccine (pVAXhsp65) against tuberculosis, containing the gene coding for the heat shock protein 65 from mycobacteria. Methods: Young adult female BALB/c mice were fed ad libitum or with 80% of the amount of food consumed by a normal diet group. We initially characterized a mice model of dietary restriction by determining body and spleen weights, hematological parameters and histopathological changes in lymphoid organs. The ability of splenic cells to produce IFN-gamma and IL-4 upon in vitro stimulation with LPS or S. aureus and the serum titer of specific IgG1 and IgG2a anti-hsp65 antibodies after intramuscular immunization with pVAXhsp65 was then tested. Results: Dietary restriction significantly decreased body and spleen weights and also the total lymphocyte count in blood. This restriction also determined a striking atrophy in lymphoid organs as spleen, thymus and lymphoid tissue associated with the small intestine. Specific antibodies were not detected in mice submitted to dietary restriction whereas the well nourished animals produced significant levels of both, IgG1 and IgG2a anti-hsp65. Conclusion: 20% restriction in food intake deeply compromised humoral immunity induced by a genetic vaccine, alerting, therefore, for the relevance of the nutritional condition in vaccination programs based on these kinds of constructs. © 2009 Ishikawa et al; licensee BioMed Central Ltd.

Importance of xenarthrans in the eco-epidemiology of Paracoccidioides brasiliensis

Background. Several pathogens that cause important zoonotic diseases have been frequently associated with armadillos and other xenarthrans. This mammal group typically has evolved on the South American continent and many of its extant species are seriously threatened with extinction. Natural infection of armadillos with Paracoccidioides brasiliensis in hyperendemic areas has provided a valuable opportunity for understanding the role of this mammal in the eco-epidemiology of Paracoccidioidomycosis (PCM), one of the most important systemic mycoses in Latin America. Findings. This study aimed to detect P. brasiliensis in different xenarthran species (Dasypus novemcinctus, Cabassous spp., Euphractus sexcinctus, Tamandua tetradactyla and Myrmecophaga tridactyla), by molecular and mycological approaches, in samples obtained by one of the following strategies: i) from road-killed animals (n = 6); ii) from naturally dead animals (n = 8); iii) from animals that died in captivity (n = 9); and iv) from living animals captured from the wild (n = 2). Specific P. brasiliensis DNA was detected in several organs among 7/20 nine-banded armadillos (D. novemcinctus) and in 2/2 anteaters (M. tridactyla). The fungus was also cultured in tissue samples from one of two armadillos captured from the wild. Conclusion. Members of the Xenarthra Order, especially armadillos, have some characteristics, including a weak cellular immune response and low body temperature, which make them suitable models for studying host-pathogen interaction. P. brasiliensis infection in wild animals, from PCM endemic areas, may be more common than initially postulated and reinforces the use of these animals as sentinels for the pathogen in the environment. © 2009 Bagagli et al; licensee BioMed Central Ltd.

Use of algorithms for semi-automatic quantification of pulmonary fibrosis and emphysema

Obtaining a semi-automatic quantification of pathologies found in the lung, through images of high resolution computed tomography (HRCT), is of great importance to aid in medical diagnosis. Paraccocidioidomycosis (PCM) is a systemic disease that affects the lung and even after effective treatment leaves sequels such as pulmonary fibrosis and emphysema. It is very important to the area of tropical diseases that the lung injury be quantified more accurately. In this stud, we propose the development of algorithms in computational environment Matlab® able to objectively quantify lung diseases such as fibrosis and emphysema. The program consists in selecting the region of interest (ROI), and through the use of density masks and filters, obtaining the lesion area quantification in relation to the healthy area of the lung. The proposed method was tested on 15 exams of HRCT of patients with confirmed PCM. To prove the validity and effectiveness of the method, we used a virtual phantom, also developed in this research. © 2013 Springer-Verlag.

Intermolecular interactions of the malate synthase of Paracoccidioides spp

Background: The fungus Paracoccidioides spp is the agent of paracoccidioidomycosis (PCM), a pulmonary mycosis acquired by the inhalation of fungal propagules. Paracoccidioides malate synthase (PbMLS) is important in the infectious process of Paracoccidioides spp because the transcript is up-regulated during the transition from mycelium to yeast and in yeast cells during phagocytosis by murine macrophages. In addition, PbMLS acts as an adhesin in Paracoccidioides spp. The evidence for the multifunctionality of PbMLS indicates that it could interact with other proteins from the fungus and host. The objective of this study was to identify and analyze proteins that possibly bind to PbMLS (PbMLS-interacting proteins) because protein interactions are intrinsic to cell processes, and it might be possible to infer the function of a protein through the identification of its ligands. Results: The search for interactions was performed using an in vivo assay with a two-hybrid library constructed in S. cerevisiae; the transcripts were sequenced and identified. In addition, an in vitro assay using pull-down GST methodology with different protein extracts (yeast, mycelium, yeast-secreted proteins and macrophage) was performed, and the resulting interactions were identified by mass spectrometry (MS). Some of the protein interactions were confirmed by Far-Western blotting using specific antibodies, and the interaction of PbMLS with macrophages was validated by indirect immunofluorescence and confocal microscopy. In silico analysis using molecular modeling, dynamics and docking identified the amino acids that were involved in the interactions between PbMLS and PbMLS-interacting proteins. Finally, the interactions were visualized graphically using Osprey software. Conclusion: These observations indicate that PbMLS interacts with proteins that are in different functional categories, such as cellular transport, protein biosynthesis, modification and degradation of proteins and signal transduction. These data suggest that PbMLS could play different roles in the fungal cell. © 2013 de Oliveira et al.; licensee BioMed Central Ltd.

Early endosome antigen 1 (EEA1) decreases in macrophages infected with Paracoccidioides brasiliensis

Paracoccidioidomycosis (PCM) is a chronic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis, endemic in Latin America. P. brasiliensis has been observed in epithelial cells in vivo and in vitro, as well as within the macrophages. The identification of the mechanism by which it survives within the host cell is fertile ground for the discovery of its pathogenesis since this organism has the ability to induce its own endocytosis in epithelial cells and most likely in macrophages. The study of the expression of endocytic proteins pathway and co-localization of microorganisms enable detection of the mechanism by which microorganisms survive within the host cell. The aim of this study was to evaluate the expression of the endocytic protein EEA1 (early endosome antigen 1) in macrophages infected with P. brasiliensis. For detection of EEA1, three different techniques were employed: immunofluorescence, real-time polymerase chain reaction (PCR) and immunoblotting. In the present study, decreased expression of EEA1 as well as the rearrangement of the actin was observed when the fungus was internalized, confirming that the input mechanism of the fungus in macrophages occurs through phagocytosis. © 2013 ISHAM.

Expressão gênica das adesinas e fatores de virulência da fase miceliar e leveduriforme do complexo paracoccidioides e de sua interação com células epiteliais e fagócitos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Diagnóstico da paracoccidioidomicose em pacientes atendidos em serviços de rotina de hospital universitário

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Participação do gene Alc11a1 na infecção por Paracoccidioides brasiliensis em linhagens de camundongos selecionados segundo a alta ou baixa reatividade inflamatória aguda

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)