Comparison of gene expression in CD34+ cells from bone marrow and G-CSF-mobilized peripheral blood by high-density oligonucleotide array analysis - Supplemental Materials Only.

A prospective randomized trial has shown that there is a survival advantage for allogeneic transplant recipients who received granulocyte colony-stimulating factor (G-CSF)-stimulated peripheral blood mononuclear cells (GPBMC) versus those who received bone marrow (BM) as a source of stem cells. The biological basis for this advantage is not clear and may be attributable to qualitative as well as quantitative differences in the CD34 cells, T cells, and/or the monocytes transplanted. To begin to address this issue, gene expression patterns in CD34 cells isolated from these 2 stem cell sources were compared to identify functional pathways that may distinguish these 2 populations. CD34 cells were isolated to purity from the BM and peripheral blood stem cells of multiple healthy donors. (The complete data set will be available at http://parma.fhcrc.org/lgraf upon publication.) Two separate RNA preparations from pooled samples from both sources were analyzed by Affymetrix Oligonucleotide Array chips for expression of over 6400 human genes. Comparative analyses among the samples showed that a small set of 28 sequences increased and 38 sequences decreased in expression more than 3-fold in both of the GPBMC samples compared to those in BM samples. More highly expressed genes include several for nuclear proteins and transcriptional factors. Functional categorization of the genes decreased in expression indicated sequences influential in cell cycle progression, in agreement with the recognized quiescence of circulating CD34 cells. Multiple transcriptional regulators and chemokines were also found to be decreased. These data emphasize that in addition to increased numbers of CD34 cells, G-CSF mobilization also results in significant qualitative changes. Whether they impact engraftment remains to be determined

Intracellular Disposition of Fludarabine Triphosphate in Human Natural Killer Cells

Purpose. Fludarabine is a key component of several reduced-intensity conditioning regimens for hematopoietic cell transplantation (HCT). Shortly after reduced-intensity conditioning, the percent of donor natural killer (NK) cells has been associated with progression-free survival. Insufficient suppression of the recipient’s NK cells by fludarabine may lead to lower donor chimerism; however, the effect of fludarabine upon NK cells is poorly understood. Thus, in purified human NK cells we evaluated the uptake and activation of fludarabine to its active metabolite, fludarabine triphosphate (F-ara-ATP), and assessed the degree of interindividual variability in F-ara-ATP accumulation. Methods. Intracellular F-ara-ATP was measured in purified NK cells isolated from healthy volunteers (n = 6) after ex vivo exposure to fludarabine. Gene expression levels of the relevant transporters and enzymes involved in fludarabine uptake and activation were also measured in these cells. Results. F-ara-ATP accumulation (mean ± s.d.) was 6.00 ± 3.67 pmol/1x106 cells/4 hours, comparable to average levels previously observed in CD4+ and CD8+ T-lymphocytes. We observed considerable variability in F-ara-ATP accumulation and mRNA expression of transporters and enzymes relevant to F-ara-ATP accumulation in NK cells from different healthy volunteers. Conclusions. Human NK cells have the ability to form F-ara-ATP intracellularly and large interindividual variability was observed in healthy volunteers. Further studies are needed to evaluate whether F-ara-ATP accumulation in NK cells are associated with apoptosis and clinical outcomes

Expression analysis of the TAB2 protein in adult mouse tissues

Background: The Interleukin-1 (IL-1) signaling component TAK1 binding protein 2 (TAB2) plays a role in activating the NFκB and JNK signaling pathways. Additionally, TAB2 functions in the nucleus as a repressor of NFκB-mediated gene regulation. Objective: To obtain insight into the function of TAB2 in the adult mouse, we analyzed the in vivo TAB2 expression pattern. Materials and methods: Cell lines and adult mouse tissues were analyzed for TAB2 protein expression and localization. Results: Immunohistochemical staining for TAB2 protein revealed expression in the vascular endothelium of most tissues, hematopoietic cells and brain cells. While TAB2 is localized in both the nucleus and the cytoplasm in cell lines, cytoplasmic localization predominates in hematopoietic tissues in vivo. Conclusions: The TAB2 expression pattern shows striking similarities with previously reported IL-1 receptor expression and NFκB activation patterns, suggesting that TAB2 in vivo is playing a role in these signaling pathways