Ex Vivo Expanded Donor Alloreactive Regulatory T Cells Lose Immunoregulatory, Proliferation, and Antiapoptotic Markers After Infusion Into ATG-lymphodepleted, Nonhuman Primate Heart Allograft Recipients.

BACKGROUND: Regulatory T cell (Treg) therapy is a promising approach to amelioration of allograft rejection and promotion of organ transplant tolerance. However, the fate of infused Treg, and how this relates to their therapeutic efficacy using different immunosuppressive regimens is poorly understood. Our aim was to analyze the tissue distribution, persistence, replicative activity and phenotypic stability of autologous, donor antigen alloreactive Treg (darTreg) in anti-thymocyte globulin (ATG)-lymphodepleted, heart-allografted cynomolgus monkeys. METHODS: darTreg were expanded ex vivo from flow-sorted, circulating Treg using activated donor B cells and infused posttransplant into recipients of major histocompatibility complex-mismatched heart allografts. Fluorochrome-labeled darTreg were identified and characterized in peripheral blood, lymphoid, and nonlymphoid tissues and the graft by flow cytometric analysis. RESULTS: darTreg selectively suppressed autologous T cell responses to donor antigens in vitro. However, following their adoptive transfer after transplantation, graft survival was not prolonged. Early (within 2 wk posttransplant; under ATG, tacrolimus, and anti-IL-6R) or delayed (6-8 wk posttransplant; under rapamycin) darTreg infusion resulted in a rapid decline in transferred darTreg in peripheral blood. Following their early or delayed infusion, labeled cells were evident in lymphoid and nonlymphoid organs and the graft at low percentages (<4% CD4+ T cells). Notably, infused darTreg showed reduced expression of immunoregulatory molecules (Foxp3 and CTLA4), Helios, the proliferative marker Ki67 and antiapoptotic Bcl2, compared with preinfusion darTreg and endogenous CD4+CD25hi Treg. CONCLUSIONS: Lack of therapeutic efficacy of infused darTreg in lymphodepleted heart graft recipients appears to reflect loss of a regulatory signature and proliferative and survival capacity shortly after infusion.

Combined effects of melatonin and FGF-2 on mouse preosteoblast behavior within interconnected porous hydroxyapatite ceramics - in vitro analysis.

Objective Biocompatible materials such as interconnected porous hydroxyapatite ceramics (IP-CHA) loaded with osteogenic cells and bioactive agents are part of an evolving concept for overcoming craniofacial defects by use of artificial bone tissue regeneration. Amongst the bioactive agents, melatonin (MEL) and basic fibroblast growth factor (FGF-2) have been independently reported to induce osteoblastic activity. The present in vitro study was undertaken to examine the relationship between these two bioactive agents and their combinatory effects on osteoblastic activity and mineralization in vitro. Material and Methods Mouse preosteoblast cells (MC3T3-E1) were seeded and cultured within cylindrical type of IP-CHA block (ø 4x7 mm) by vacuum-assisted method. The IP-CHA/MC3T3 composites were subjected to FGF-2 and/or MEL. The proliferation assay, alkaline phosphatase enzyme activity (ALP), mRNA expressions of late bone markers, namely Osteocalcin (OCN) and Osteopontin (OPN), and Alizarin Red staining were examined over a period of 7 days. Results FGF-2 mainly enhanced the proliferation of MC3T3-E1 cells within the IP-CHA constructs. MEL mainly induced the mRNA expression of late bone markers (OCN and OPN) and showed increased ALP activity of MC3T3 cells cultured within IP-CHA construct. Moreover, the combination of FGF-2 and MEL showed increased osteogenic activity within the IP-CHA construct in terms of cell proliferation, upregulated expressions of OCN and OPN, increased ALP activity and mineralization with Alizarin Red. The synergy of the proliferative potential of FGF-2 and the differentiation potential of MEL showed increased osteogenic activity in MC3T3-E1 cells cultured within IP-CHA constructs. Conclusion These findings indicate that the combination of FGF-2 and MEL may be utilized with biocompatible materials to attain augmented osteogenic activity and mineralization.

Combined effects of melatonin and FGF-2 on mouse preosteoblast behavior within interconnected porous hydroxyapatite ceramics - in vitro analysis

ABSTRACT Objective Biocompatible materials such as interconnected porous hydroxyapatite ceramics (IP-CHA) loaded with osteogenic cells and bioactive agents are part of an evolving concept for overcoming craniofacial defects by use of artificial bone tissue regeneration. Amongst the bioactive agents, melatonin (MEL) and basic fibroblast growth factor (FGF-2) have been independently reported to induce osteoblastic activity. The present in vitro study was undertaken to examine the relationship between these two bioactive agents and their combinatory effects on osteoblastic activity and mineralization in vitro. Material and Methods Mouse preosteoblast cells (MC3T3-E1) were seeded and cultured within cylindrical type of IP-CHA block (ø 4x7 mm) by vacuum-assisted method. The IP-CHA/MC3T3 composites were subjected to FGF-2 and/or MEL. The proliferation assay, alkaline phosphatase enzyme activity (ALP), mRNA expressions of late bone markers, namely Osteocalcin (OCN) and Osteopontin (OPN), and Alizarin Red staining were examined over a period of 7 days. Results FGF-2 mainly enhanced the proliferation of MC3T3-E1 cells within the IP-CHA constructs. MEL mainly induced the mRNA expression of late bone markers (OCN and OPN) and showed increased ALP activity of MC3T3 cells cultured within IP-CHA construct. Moreover, the combination of FGF-2 and MEL showed increased osteogenic activity within the IP-CHA construct in terms of cell proliferation, upregulated expressions of OCN and OPN, increased ALP activity and mineralization with Alizarin Red. The synergy of the proliferative potential of FGF-2 and the differentiation potential of MEL showed increased osteogenic activity in MC3T3-E1 cells cultured within IP-CHA constructs. Conclusion These findings indicate that the combination of FGF-2 and MEL may be utilized with biocompatible materials to attain augmented osteogenic activity and mineralization.