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High molecular weight hyaluronic acid (HA) has been widely used with platelet-rich plasma (PRP) in regenerative medicine. This study aimed to investigate the in vitro association between HA crosslinked with 1,4-butanediol diglycidyl ether (BDDE), structured in microparticles and sponges, and PRP as composite scaffolds for growth factor release, proliferation of human adipose-derived mesenchymal stem cells and differentiation by the osteogenic marker alkaline phosphatase. The reason for using BDDE is to increase the residence time in vivo, improve the mechanical stability of free HA (fHA) and provide a larger surface area for cell proliferation and differentiation. PRP was obtained via controlled centrifugation of whole blood and activated with autologous serum and calcium. The results showed that structuration benefited the physicochemical and mechanical properties compared to fHA. Both structures were noncytotoxic. The release of growth factors from PRP was higher than fHA but similar to fibrin network. The effects on the proliferation of seeded mesenchymal cells were not intense. However, a remarkable activity of the osteogenic marker alkaline phosphatase was observed. We concluded that the structuration of HA provides injectable or solid formulations with better properties compared to fHA, which associated with PRP may be promising for regeneration of bone tissue. [ABSTRACT FROM AUTHOR]

Objectives: APAF-1 is a central component of the intrinsic pathway of apoptosis, where APAF-1 dysregulation results in the development of diverse human neoplasms. The aim of this study was to characterize the mRNA expression levels of APAF-1 transcripts in low-risk and high-risk MDS and to elucidate whether the expression levels of APAF-1 transcripts are modulated with increased apoptosis in CD34+ MDS cells undergoing erythroid differentiation. Methods: APAF-1 ( ) expression was verified, by quantitative RT-PCR, in bone marrow aspirates from 33 patients with myelodysplastic syndromes (MDS), at the time of diagnosis, and in erythroid differentiation cultures from CD34+ from normal donors and patients with MDS. Results: APAF-1 expression was significantly higher in low-risk, compared to high-risk MDS, according to IPSS ( P < 0.0001), FAB ( P = 0.0265), and cytogenetic risk ( P = 0.0134). Low-risk MDS-derived differentiated erythroid cells demonstrated an increased expression of APAF-1, compared with normal cells, accompanied by an augmented rate of apoptosis. Conclusions: Increased expression of APAF-1 in low-risk disease and its positive correlation with the apoptotic rate observed during the erythroblast differentiation of low-risk MDS cells may indicate that the modulation of APAF-1, at the transcriptional level, participates in the pathophysiology of MDS. [ABSTRACT FROM AUTHOR]

Platelet-rich plasma (PRP) associated with high molecular weight hyaluronic acid (HA) has been clinically used for tissue regeneration in orthopedics. Despite the recognized beneficial clinical outcomes (e.g., early pain control, improvement of patients' functional limitation and longer-term effectiveness compared to PRP and HA alone in mild and moderate osteoarthritis treatments), its use is still challenging and controversial due to lack of standardization of association practical protocols. Moreover, most studies neglect the matrix structure, that generates the ultimate properties of the association among platelets, fibrin network and the microparticles. In the present work, we aimed to analyze the influence of the PRP/HA association with a controlled matrix structure on the stability, rheological behavior, release of growth factors and in vitro proliferation of human adipose-derived mesenchymal cells (h-AdMSCs). The attenuation of the negative charge of HA was also evaluated. Pure PRP (P-PRP) (i.e., plasma enriched with platelets and poor in leukocytes) was prepared by centrifugation and activated with serum and calcium chloride (AP-PRP). Autocrosslinked hyaluronic acid (AHA) was prepared by organocatalyzed auto-esterification and structured in microparticles (MPAHA) by shearing. The attenuation of the negative charge of MPAHA was performed with chitosan (CHT) by polyelectrolyte complexation yielding MPAHA-CHT. The results showed that microparticles (MPs) have viscoelastic properties, extrusion force and swelling ratio appropriate for injectable applications. The association of AP-PRP with the controlled structure of MPAHA and MPAHA-CHT formed a matrix composed of platelets and of a fibrin network with fibers around 160 nm located preferably on the surface of the MPs with an average diameter of 250 μm. Moreover, AP-PRP/MPAHA and AP-PRP/MPAHA-CHT associations were non-toxic and supported controlled growth factor (PDGF-AB and TGF-β1) release and in vitro proliferation of h-AdMSC with a similar pattern to that of AP-PRP alone. The best h-AdMSC proliferation was obtained with the AP-PRP/MPAHA-CHT75:25 indicating that the charge attenuation improved the cell proliferation. Thus, the association of AP-PRP with the controlled structure of HA can be a valuable approach for orthopedic applications. [ABSTRACT FROM AUTHOR]

Apoptosis is dysregulated in patients with myelodysplastic syndrome (MDS) and acute myelogenous leukaemia (AML). FLIP (FLICE (FAS-associated death-domain-like IL-1 beta-converting enzyme)-inhibitory protein) has been described as an anti-apoptotic protein. Here, we characterize the expression level of FLIP(LONG) and FLIP(SHORT) mRNA in bone marrow aspirates from 61 patients diagnosed with MDS or AML. FLIP(SHORT) mRNA expression was significantly lower in low risk MDS, compared to high risk MDS, according to FAB classification (RA/RARS versus RAEB/RAEBt, P=0.0127) and IPSS (low risk/intermediate-1 versus intermediate-2/high risk, P=0.0345). Furthermore, FLIP(SHORT) mRNA expression was significantly lower in low risk MDS, compared to MDS-AML/AML de novo (P=0.0006), according to FAB classification. FLIP(LONG) expression did not differ between these groups. Increased levels of FLIP(SHORT) in RAEB and AML may be related to apoptosis resistance in these diseases and to MDS progression.

Steroids hormones modify the hematological features of homozygous sickle cell disease, including the levels of fetal hemoglobin. We used semi-quantitative RT-PCR analysis of GATA-1, GATA-2, NF-E2, and gamma-globin mRNA levels in a two-phase liquid culture system of human adult erythroid cells in order to assay the effect of progesterone upon gene expression. The levels of expression of GATA-1 and gamma-globin mRNA were significantly increased in cells treated with progesterone compared to untreated cells (1.7- to 2.0-fold). Progesterone treatment did not produce any stimulatory effect upon GATA-2 and NF-E2 mRNA expression. Differences in the synthesis of HbF protein could not be detected by flow cytometry, although we observed a small difference in mean intensity fluorescence between cells treated and cells untreated with progesterone on days 7 and 9. Using anti-transferrin receptor and anti-glycophorin A antibodies, we verified that addition of progesterone did not cause any change in erythroid proliferation and differentiation. In conclusion, it is possible that the increased expression of gamma-globin mRNA after progesterone treatment observed in this study may be related to the increased GATA-1 mRNA expression. Interactions of the steroid receptors with the basal transcriptional machinery and with transcription factors might mediate their transcriptional effects.