Characterization and in vitro activities of cell-free antigens from Histoplasma capsulatum-loaded biodegradable microspheres

In the last decades, the incidence of histoplasmosis, a pulmonary fungal disease caused by Histoplasma capsulatum, has increased worldwide. In this context, vaccines for the prevention of this infection or therapies are necessary. Cell-free antigens (CFAgs) from H. capsulatum when administered for murine immunization purposes are able to confer protection and control of the infection, since they activate cellular immunity. However the most of vaccination procedures need several anti, gens administrations and immunoadjuvants, which are not approved for use in humans. The aim of this study was to develop and characterize a vaccination approach using biodegradable PLGA microspheres (MS) that could allow the controlled and/or sustained release of the encapsulated antigens from H. capsulatum. CFAgs-loaded MS presented a size less than 10 mu m, were marked engulfed by bone marrow-derived macrophages (BMDM phi) and induced the nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by these cells. Our data show that CFAgs-loaded MS induce cell activation, suggesting an immunostimulant effect to be further investigated during immunization procedures. CFAgs-loaded MS present potential to be used as vaccine in order to confer protection against H. capsulatum infection. (C) 2009 Elsevier B.V. All rights reserved.

PREDICTION OF FAT-FREE MASS BY BIOELECTRICAL IMPEDANCE ANALYSIS IN OLDER ADULTS FROM DEVELOPING COUNTRIES: A CROSS-VALIDATION STUDY USING THE DEUTERIUM DILUTION METHOD

Objective: Several limitations of published bioelectrical impedance analysis (BIA) equations have been reported. The aims were to develop in a multiethnic, elderly population a new prediction equation and cross-validate it along with some published BIA equations for estimating fat-free mass using deuterium oxide dilution as the reference method. Design and setting: Cross-sectional study of elderly from five developing countries. Methods: Total body water (TBW) measured by deuterium dilution was used to determine fat-free mass (FFM) in 383 subjects. Anthropometric and BIA variables were also measured. Only 377 subjects were included for the analysis, randomly divided into development and cross-validation groups after stratified by gender. Stepwise model selection was used to generate the model and Bland Altman analysis was used to test agreement. Results: FFM = 2.95 - 3.89 (Gender) + 0.514 (Ht(2)/Z) + 0.090 (Waist) + 0.156 (Body weight). The model fit parameters were an R(2), total F-Ratio, and the SEE of 0.88, 314.3, and 3.3, respectively. None of the published BIA equations met the criteria for agreement. The new BIA equation underestimated FFM by just 0.3 kg in the cross-validation sample. The mean of the difference between FFM by TBW and the new BIA equation were not significantly different; 95% of the differences were between the limits of agreement of -6.3 to 6.9 kg of FFM. There was no significant association between the mean of the differences and their averages (r = 0.008 and p = 0.2). Conclusions: This new BIA equation offers a valid option compared with some of the current published BIA equations to estimate FFM in elderly subjects from five developing countries.

Affected and non-affected skin fibroblasts from systemic sclerosis patients share a gene expression profile deviated from the one observed in healthy individuals

Objectives To evaluate the gene expression profile of fibroblasts from affected and non-affected skin of systemic sclerosis (SSc) patients and from controls. Materials and methods Labeled cDNA from fibroblast cultures from forearm (affected) and axillary (non-affected) skin from six diffuse SSc patients, from three normal controls, and from MOLT-4/HEp-2/normal fibroblasts (reference pool) was probed in microarrays generated with 4193 human cDNAs from the IMAGE Consortium. Microarray images were converted into numerical data and gene expression was calculated as the ratio between fibroblast cDNA (Cy5) and reference pool cDNA (Cy3) data and analyzed by R environment/Aroma, Cluster, Tree View, and SAM softwares. Differential expression was confirmed by real time PCR for a set of selected genes. Results Eighty-eight genes were up- and 241 genes down-regulated in SSc fibroblasts. Gene expression correlation was strong between affected and non-affected fibroblast samples from the same patient (r>0.8), moderate among fibroblasts from all patients (r=0.72) and among fibroblasts from all controls (r=0.70), and modest among fibroblasts from patients and controls (r=0.55). The differential expression was confirmed by real time PCR for all selected genes. Conclusions Fibroblasts from affected and non-affected skin of SSc patients shared a similar abnormal gene expression profile, suggesting that the widespread molecular disturbance in SSc fibroblasts is more sensitive than histological and clinical alterations. Novel molecular elements potentially involved in SSc pathogenesis were identified.

Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and gene-expression profile with CD146(+) perivascular cells and fibroblasts

Objective. The relationship of multipotent mesenchymal stromal cells (MSC) with pericytes and fibroblasts has not been established thus far, although they share many markers of primitive marrow stromal cells and the osteogenic, adipogenic, and chondrogenic differentiation potentials. Materials and Methods. We compared MSCs from adult or fetal tissues, MSC differentiated in vitro, fibroblasts and cultures of retinal pericytes obtained either by separation with anti-CD146 or adhesion. The characterizations included morphological, immunophenotypic, gene-expression profile, and differentiation potential. Results. Osteogenic, adipocytic, and chondrocytic differentiation was demonstrated for MSC, retinal perivascular cells, and fibroblasts. Cell morphology and the phenotypes defined by 22 markers were very similar. Analysis of the global gene expression obtained by serial analysis of gene expression for 17 libraries and by reverse transcription polymerase chain reaction of 39 selected genes from 31 different cell cultures, revealed similarities among MSC, retinal perivascular cells, and hepatic stellate cells. Despite this overall similarity, there was a heterogeneous expression of genes related to angiogenesis, in MSC derived from veins, artery, perivascular cells, and fibroblasts. Evaluation of typical pericyte and MSC transcripts, such as NG2, CD146, CD271, and CD140B on CD146 selected perivascular cells and MSC by real-time polymerase chain reaction confirm the relationship between these two cell types. Furthermore, the inverse correlation between fibroblast-specific protein-1 and CD146 transcripts observed on pericytes, MSC, and fibroblasts highlight their potential use as markers of this differentiation pathway. Conclusion. Our results indicate that human MSC and pericytes are similar cells located in the wall of the vasculature, where they function as cell sources for repair and tissue maintenance, whereas fibroblasts are more differentiated cells with more restricted differentiation potential. (C) 2008 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Under-reporting of food intake is frequent among Brazilian free-living older persons: a doubly labelled water study

The assessment of food intake is essential for the development of dietetic interventions. Accuracy is low when intake is assessed by questionnaires, the under-reporting of food intake being frequent. Most such studies, however, were performed in developed countries and there is little data about the older population of developing nations. This study aimed to verify the total energy expenditure (TEE) of independent older Brazilians living in an urban area, through the doubly labelled water (DLW) method and to compare it with the reported energy intake obtained through the application of a food frequency questionnaire (FFQ). Initially, 100 volunteers aged from 60 to 75 years had their body composition determined by dual-energy X-ray absorptiometry (DEXA). Five volunteers of each quartile of body fat percentage had their energy expenditure determined by DLW. The mean age of the subjects included in this phase of the study was 66.4 +/- 3.5 years, and ten of the subjects were men. The mean TEE was 2565 +/- 614 and 2154 +/- 339 kcal.day(-1) for men and women, respectively. The Physical Activity Level (PAL) was 1.58 +/- 0.31 and 1.52 +/- 0.22, respectively. Under-reporting of food intake was highly prevalent, with a mean percentage of reported intake in relation to measured TEE of -17.7%. Thus, under-reporting of food intake is highly prevalent among Brazilian independent older persons. The DLW method is an important tool in nutritional studies and its use is to be recommended in developing countries. Copyright (C) 2010 John Wiley & Sons, Ltd.