Endothelial precursor cells (EPCs) cultured from adult bone marrow (BM) have been shown to mediate neovasculogenesis in murine models of vascular injury. We sought to directly compare umbilical cord blood (UCB)- and BM-derived EPC surface phenotypes and in vivo functional capacity. UCB and BM EPCs derived from mononuclear cells (MNC) were phenotyped by surface staining for expression of stromal (Stro-1, CXCR4, CD105, and CD73), endothelial (CD31, CD146, and vascular endothelial [VE]-cadherin), stem cell (CD34 and CD133), and monocyte (CD14) surface markers and analyzed by flow cytometry. The nonobese diabetic/severe combined immunodeficiency murine model of hind-limb ischemia was used to analyze the potential of MNCs and culture-derived EPCs from UCB and BM to mediate neovasculogenesis. Histologic evaluation of the in vivo studies included capillary density as a measure of neovascularization. Surface CXCR4 expression was notably higher on UCB-derived EPCs (64.29%+/-7.41%) compared with BM (19.69%+/-5.49%; P=.021). Although the 2 sources of EPCs were comparable in expression of endothelial and monocyte markers, BM-derived EPCs contained higher proportions of cells expressing stromal cell markers (CD105 and CD73). Injection of UCB- or BM-derived EPCs resulted in significantly improved perfusion as measured by laser Doppler imaging at days 7 and 14 after femoral artery ligation in nonobese diabetic/severe combined immunodeficiency mice compared with controls (P<.05). Injection of uncultured MNCs from BM or UCB showed no significant difference from control mice (P=.119; P=.177). Tissue samples harvested from the lower calf muscle at day 28 demonstrated increased capillary densities in mice receiving BM- or UCB-derived EPCs. In conclusion, we found that UCB and BM-derived EPCs differ in CXCR4 expression and stromal surface markers but mediate equivalent neovasculogenesis in vivo as measured by Doppler flow and histologic analyses.

Long-term treatment with ibuprofen twice daily, at doses that achieve peak plasma concentration (C max ) Ͼ50 g/ml, slows progression of lung disease in patients with cystic fibrosis (CF). Previous data suggest that C max Ͼ50 g/ml is associated with a reduction in neutrophil (PMN) migration into the lung and that lower concentrations are associated with an increase in PMN migration. To estimate the threshold concentration at which ibuprofen is associated with a decrease in PMN migration in vivo, we measured the PMN content of oral mucosal washes in 35 healthy (age 19 -40 years) and 16 CF (age 18 -32 years) subjects who took ibuprofen twice daily for 10 days in doses that achieved C max 8 to 90 g/ml. C max Ͼ50 g/ml was associated with a 31 Ϯ 7% (mean Ϯ S.E.M.) reduction in PMNs in CF (n ϭ 11, p Ͻ 0.001) and 25 Ϯ 6% reduction in PMNs in healthy subjects (n ϭ 16, p Ͻ 0.001). Increasing concentrations above 50 g/ml was not associated with a greater decrease in PMNs. The reduction in PMN migration was consistently present 12 h after a dose, but not after 24 h. C max Ͻ50 g/ml was associated with an increase in PMNs of approximately 40%. These results suggest that C max Ͼ50 g/ml and twice daily dosing of ibuprofen are required to decrease PMN migration, and reinforce the current recommendation that pharmacokinetics should be performed in CF patients prescribed ibuprofen.

+ HLADR+ CD38 + (P = 0AE108) cell dose and event-free survival (EFS). Highresolution matching for HLA-class II (DRB1) resulted in improved EFS (P = 0AE02) and decreased risk for acute graft-versus-host disease (GVHD) (P = 0AE004). Early mortality (prior to post-transplant day +28) occurred in three patients, while 26 patients achieved myeloid engraftment. These results suggest that UCB graft matching at DRB1 is an important risk factor for acute GVHD and survival, while higher UCB graft cell doses of CD34 + , committed CD34 + progenitors and CD3 + T cells favourably influence UCB allogeneic engraftment.

On activation, umbilical cord blood (UCB) CD4þ T cells demonstrate reduced expression of tumor necrosis factor-a (TNF-a) and interferon-c (IFN-c), whereas maintaining equivalent interleukin-2 (IL-2) levels, as compared with adult peripheral blood (PB) CD4 þ T cells. Nuclear factor of activated T cells (NFAT1) protein, a transcription factor known to regulate the expression of IL-2, TNF-a and IFN-c, is reduced in resting and activated UCB CD4 þ T cells. In contrast, expression of Broad-complex-Tramtrack-Bric-a-Brac and Cap'n'collar homology 1 bZip transcription factor 2 (BACH2) was shown by gene array analyses to be increased in UCB CD4 þ T cells and was validated by qRT-PCR. Using chromatin immunoprecipitation, BACH2 was shown binding to the human IL-2 proximal promoter. Knockdown experiments of BACH2 by transient transfection of UCB CD4 þ T cells with BACH2 siRNA resulted in significant reductions in stimulated IL-2 production. Decreased IL-2 gene transcription in UCB CD4þ T cells transfected with BACH2 siRNA was confirmed by a human IL-2 luciferase assay. In summary, BACH2 maintains IL-2 expression in UCB CD4þ T cells at levels equivalent to adult PB CD4 þ T cells despite reduced NFAT1 protein expression. Thus, BACH2 expression is necessary to maintain IL-2 production when NFAT1 protein is reduced, potentially impacting UCB graft CD4þ T-cell allogeneic responses.

The key obstacle to clinical application of human inducible regulatory T cells (iTreg) as an adoptive cell therapy in autoimmune disorders is loss of FOXP3 expression in an inflammatory milieu. Here we report human iTreg co-cultured with bone marrow-derived mesenchymal stromal cells (MSCs) during short-term ex vivo expansion enhances the stability of iTreg FOXP3 expression and suppressive function in vitro and in vivo, and further that a key mechanism of action is MSC mitochondrial (mt) transfer via tunneling nanotubules (TNT). MSC mt transfer is driven by mitochondrial metabolic function (CD39/CD73 signaling) in proliferating iTreg and promotes iTreg expression of FOXP3 stabilizing factors BACH2 and SENP3. These results elucidate cellular and molecular mechanisms underlying human MSC mt transfer to proliferating cells. MSC mt transfer stabilizes FOXP3 expression in iTregs, thereby enhancing and sustaining their suppressive function in inflammatory conditions in vitro and in vivo.

Foxp3 is essential for T regulatory cell (Treg) function. Broad complex-Tramtrack-Bric-a-brac domain (BTB) and Cap'n'collar (CNC) homology 1, transcription factor 2 (BACH2) stabilizes Treg immune homeostasis in murine studies. However, little is known regarding what role, if any, BACH2 may have in Foxp3 regulation in human-induced Treg (iTreg). We examined Foxp3 expression and regulation comparing iTreg differentiated from umbilical cord blood (UCB) vs. adult blood (AB) naive CD4 T-cells. Foxp3 expression was higher in UCB vs. AB-derived iTreg, and was sustained during 21-day expansion in vitro. The number of Foxp3 iTreg generated from UCB vs. AB naive CD4 T-cells was higher in iTreg differentiation conditions. In addition, UCB iTreg were more potent in suppressing T-cell proliferation compared to AB iTreg. Naive UCB CD4 T-cells highly expressed BACH2 protein compared to AB. Putative transcriptional BACH2 binding sites were identified at the Foxp3 promoter, using BACH2 consensus sequence. Cross-linking chromatin immunoprecipitation (ChIP) showed that BACH2 binds to the Foxp3 proximal promoter in UCB iTreg, but not AB iTreg. BACH2 was transcriptionally active, as shRNA-mediated BACH2 knockdown resulted in reduction of Foxp3 gene transcription in UCB CD4 T-cells. In summary, BACH2 serves to stabilize robust Foxp3 expression in UCB CD4 T-cell-derived iTreg.

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