Expression of neurotrophins and their receptors in human CD34+ bone marrow cells.

Bone marrow (BM) CD34+ cells have the ability to secrete growth factors, cytokines, and chemotactic factors. We sought to better characterize this population and to investigate whether human BM CD34+ cells express neurotrophins (NTs) and their relevant receptors. We also compared their expression levels with BM nucleated cells (NCs). BM CD34+ cells were evaluated with respect to the expression levels of neurotrophins using qRT-PCR, immunofluorescent staining, and Western blotting. Next, the expression of specific (TrkA, TrkB, TrkC) and non-specific (p75NTR) neurotrophin receptors was detected by qRT-PCR and immunofluorescent staining in BM CD34+ cells. Using qRT- PCR, we show that even in the absence of inducing factors, CD34+ cells spontaneously express neurotrophins such as NGF, BDNF, NT-3, and NT-4. In addition, the NT expression levels in BM CD34+ cells are considerably higher than in NCs. Furthermore, we confirmed intracellular NT expression in BM CD34+ cells at the protein level using immunofluorescent staining and Western blotting. Using qRT-PCR, we found that immunomagnetically separated BM CD34+ cells spontaneously express high-affinity neurotrophin receptors (TrkA, TrkB, and TrkC) and the low-affinity receptor p75NTR at higher levels than NCs. Immunomagnetic CD34+ cell separation enables for the rapid and gentle sorting of stem/progenitor cells (SPCs) to prepare specific cell types for use in research and clinical applications. Our study suggests that BM CD34+ cells have the potential to support trophic factors for neural tissue and could contribute towards the protection and regeneration of neural cells.

Effects of growth hormone therapeutic supplementation on hematopoietic stem/progenitor cells in children with growth hormone deficiency: focus on proliferation and differentiation capabilities.

We investigated the direct effects of growth hormone (GH) replacement therapy (GH-RT) on hematopoiesis in children with GH deficiency (GHD) with the special emphasis on proliferation and cell cycle regulation. Peripheral blood (PB) was collected from sixty control individuals and forty GHD children before GH-RT and in 3rd and 6th month of GH-RT to measure hematological parameters and isolate CD34(+)-enriched hematopoietic progenitor cells (HPCs). Selected parameters of PB were analyzed by hematological analyzer. Moreover, collected HPCs were used to analyze GH receptor (GHR) and IGF1 expression, clonogenicity, and cell cycle activity. Finally, global gene expression profile of collected HPCs was analyzed using genome-wide RNA microarrays. GHD resulted in a decrease in several hematological parameters related to RBCs and significantly diminished clonogenicity of erythroid progenies. In contrast, GH-RT stimulated increases in clonogenic growth of erythroid lineage and RBC counts as well as significant up-regulation of cell cycle-propagating genes, including MAP2K1, cyclins D1/E1, PCNA, and IGF1. Likewise, GH-RT significantly modified GHR expression in isolated HPCs and augmented systemic IGF1 levels. Global gene expression analysis revealed significantly higher expression of genes associated with cell cycle, proliferation, and differentiation in HPCs from GH-treated subjects. (i) GH-RT significantly augments cell cycle progression in HPCs and increases clonogenicity of erythroid progenitors; (ii) GHR expression in HPCs is modulated by GH status; (iii) molecular mechanisms by which GH influences hematopoiesis might provide a basis for designing therapeutic interventions for hematological complications related to GHD.

Mixed chimerism induction influences cytokine release from chimeric mice cells.

Interest in mixed chimerism has evolved from its role in the induction of alloantigen tolerance. However, its precise impact on the host organism remains to be elucidated. In the present work, we analyzed cytokine secretion from chimeric mice cells to assess the influence of different mixed chimerism induction protocols on immune system function in recipient mice. To our knowledge, there have been no reports on using this parameter for the optimization of the mixed chimerism induction method. B6.SJL-PtprcaPep3b or C57BL/6J mice were used as recipients and Balb/c as donors. We utilized four protocols which consisted of: 3Gy total body irradiation (day -1), the injection of 20-30×10(6) bone marrow cells (day 0), and a combination of CD40L (days 0 and 4), CD8 (day -2), and NK1.1 (day -3) blocking antibodies and cyclophosphamide (175mg/kg - day 2). The concentrations of cytokines (IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-17A, and TNF) were evaluated in the supernatants of unstimulated or phytohemagglutinin-stimulated chimeric spleen, bone marrow and peripheral blood cells in the 8th week of experiment. The induction of tolerance to Balb/c mouse antigens was initially tested in chimeric mice by assessing the presence of Vß5 and Vß11 TCR-expressing lymphocytes. The cytokine production was considerably increased, especially in chimeric mice treated by cyclophosphamide. Also the mixed chimerism itself seems to affect IFN-γ, IL-2, IL-4, IL-6, IL-17A, and TNF secretion. Using the optimized induction protocol, we established that chimeric mice cells secreted lower IFN-γ, IL-2, IL-4 and higher IL-6, IL-17A, and TNF levels as compared to control animals. We found that both donor and recipient cells markedly participated in the cytokine production. In conclusion, our optimization study based on cytokine assessment contributes to establishing an effective protocol of mixed chimerism induction with no cyclophosphamide use and better understanding of the influence of this phenomenon on the recipient organism.

Transient chaos measurements using finite-time Lyapunov exponents.

The notion of finite-time Lyapunov exponent averaged over initial conditions is used for characterizing transient chaos observed in one-dimensional maps. A model of its dependence on time is verified by comparing theoretically predicted values with those obtained numerically. Finally, the same model is used for estimating duration of transient chaos (rambling time) for some maps from the logistic family.