In previous studies, we have shown that the myelopoiesis dependent upon myelosupportive stroma required production of growth factors and heparan-sulphate proteoglycans, as well as generation of a negatively charged sialidase-sensitive intercellular environment between the stroma and the myeloid progenitors. In the present study, we have investigated the production, distribution and role of gangliosides in an experimental model of in vitro myelopoiesis dependent upon AFT-024 murine liver-derived stroma. We used the FDC-P1 cell line, which is dependent upon GM-CSF (granulocyte/macrophage colony-stimulating factor) for both survival and proliferation, as a reporter system to monitor bioavailability and local activity of GM-CSF. G(M3) was the major ganglioside produced by stroma, but not by myeloid cells, and it was required for optimal stroma myelosupportive function. It was released into the supernatant and selectively incorporated into the myeloid progenitor cells, where it segregated into rafts in which it co-localized with the GM-CSF-receptor alpha chain. This ganglioside was also metabolized further by myeloid cells into gangliosides of the a and b series, similar to endogenous G(M3). In these cells, G(M1) was the major ganglioside and it was segregated at the interface by stroma and myeloid cells, partially co-localizing with the GM-CSF-receptor alpha chain. We conclude that myelosupportive stroma cells produce and secrete the required growth factors, the cofactors such as heparan sulphate proteoglycans, and also supply gangliosides that are transferred from stroma to target cells, generating on the latter ones specific membrane domains with molecular complexes that include growth factor receptors.
Introduction: Molecular biology procedures to detect, genotype and quantify hepatitis C virus (HCV) RNA in clinical samples have been extensively described. Routine commercial methods for each specifi c purpose (detection, quantifi cation and genotyping) are also available, all of which are typically based on polymerase chain reaction (PCR) targeting the HCV 5' untranslated region (5'UTR). This study was performed to develop and validate a complete serial laboratory assay that combines real-time nested reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) techniques for the complete molecular analysis of HCV (detection, genotyping and viral load) in clinical samples. Methods: Published HCV sequences were compared to select specifi c primers, probe and restriction enzyme sites. An original real-time nested RT-PCR-RFLP assay was then developed and validated to detect, genotype and quantify HCV in plasma samples. Results: The real-time nested RT-PCR data were linear and reproducible for HCV analysis in clinical samples. High correlations (> 0.97) were observed between samples with different viral loads and the corresponding read cycle (Ct -Cycle threshold), and this part of the assay had a wide dynamic range of analysis. Additionally, HCV genotypes 1, 2 and 3 were successfully distinguished using the RFLP method. Conclusions: A complete serial molecular assay was developed and validated for HCV detection, quantifi cation and genotyping.
Introduction: Newborn screening allows the screening of diseases that are still in the asymptomatic period and whose early diagnosis and treatment are associated with reduced infant morbidity and mortality. The aim of this study was to evaluate the public National Newborn Screening Program in the municipality of Carazinho, state of Rio Grande do Sul (RS), Brazil. Methods: This was a population-based, retrospective, descriptive study. We collected and transcribed data from a database of the Carazinho municipal laboratory, which is affiliated with the referral center for newborn screening in RS. The records of all individuals undergoing newborn screening from 2005 to 2010 were reviewed, and information was collected on the program coverage, time elapsed between birth and screening (first collection), and test results. Results: The program had a coverage of 75.5%. One suspected case of phenylketonuria, three suspected cases of congenital hypothyroidism and no suspected cases of hemoglobinopathy were identified. In addition, there were 18 positive results for hemoglobin S heterozygosity, five for hemoglobin D heterozygosity, two for hemoglobin C heterozygosity, and one for a rare variant hemoglobin. When analyzing the newborn's age at the time of blood collection, it was observed that 63.1% were within the recommended age range. Conclusions: Our findings suggest the need for optimization of public newborn screening in the evaluated municipality. The strategies to be adopted should include education of the population and especially of managers and health professionals about the importance of newborn screening.
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