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AIM To investigate the role of autophagy in MTA-induced odontoblastic differentiation of human dental pulp cells (HDPCs). METHODOLOGY In MTA-treated HDPCs, odontoblastic differentiation was assessed based on expression levels of dentine sialophosphoprotein (DSPP) and dentine matrix protein 1 (DMP1), alkaline phosphatase activity (ALP) activity by ALP staining and the formation of mineralized nodule by Alizarin red S staining. Expression of microtubule-associated protein 1A/1B-light chain3 (LC3), adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signalling molecules and autophagy-related genes was analysed by Western blot analysis and Acridine orange staining was used to detect autophagic lysosome. For in vivo experiments, tooth cavity preparation models on rat molars were established and the expression of proteins-related odontogenesis and autophagy markers was observed by Immunohistochemistry and Western blot analysis. Kruskal-Wallis with Dunn's multiple comparison was used for statistical analysis. RESULTS Mineral trioxide aggregate (MTA) promoted odontoblastic differentiation of HDPCs, accompanied by autophagy induction, including formation of autophagic lysosome and cleavage of LC3 to LC3II (P

Type 1 diabetes (T1D) remains a major health problem worldwide, with a steadily rising incidence yet no cure. Phosphoinositide 3-kinase-γ (PI3Kγ), a member of a family of lipid kinases expressed primarily in leukocytes, has been the subject of substantial research for its role in inflammatory diseases. However, the role of PI3Kγ inhibition in suppressing autoimmune T1D remains to be explored. We tested the role of the PI3Kγ inhibitor AS605240 in preventing and reversing diabetes in NOD mice and assessed the mechanisms by which this inhibition abrogates T1D. Our data indicate that the PI3Kγ pathway is highly activated in T1D. In NOD mice, we found upregulated expression of phosphorylated Akt (PAkt) in splenocytes. Notably, T regulatory cells (Tregs) showed significantly lower expression of PAkt compared with effector T cells. Inhibition of the PI3Kγ pathway by AS605240 efficiently suppressed effector T cells and induced Treg expansion through the cAMP response element-binding pathway. AS605240 effectively prevented and reversed autoimmune diabetes in NOD mice and suppressed T-cell activation and the production of inflammatory cytokines by autoreactive T cells in vitro and in vivo. These studies demonstrate the key role of the PI3Kγ pathway in determining the balance of Tregs and autoreactive cells regulating autoimmune diabetes.

B7 ligands deliver both costimulatory and coinhibitory signals to the CD28 family of receptors on T lymphocytes, the balance between which determines the ultimate immune response. Although B7-H4, a recently discovered member of the B7 family, is known to negatively regulate T cell immunity in autoimmunity and cancer, its role in solid organ allograft rejection and tolerance has not been established. Targeting the B7-H4 molecule by a blocking antibody or use of B7-H4(-/-) mice as recipients of fully MHC-mismatched cardiac allografts did not affect graft survival. However, B7-H4 blockade resulted in accelerated allograft rejection in CD28-deficient recipients. B7-1/B7-2-double-deficient recipients are truly independent of CD28/CTLA-4:B7 signals and usually accept MHC-mismatched heart allografts. Blockade of B7-H4 in these mice also precipitated rejection, demonstrating regulatory function of this molecule independent of an intact CD28/CTLA-4:B7 costimulatory pathway. Accelerated allograft rejection was always accompanied by increased frequencies of alloreactive IFN-γ-, IL-4- and Granzyme B-producing splenocytes. Finally, intact recipient, but not donor, B7-H4 is essential for prolongation of allograft survival by blocking CD28/CTLA4:B7 pathway using CTLA4-Ig. These data are the first to provide evidence of the regulatory effects of B7-H4 in alloimmune responses in a murine model of solid organ transplantation.

OBJECTIVE A number of clinical trials are underway to test whether mesenchymal stem cells (MSCs) are effective in treating various diseases, including type 1 diabetes. Although this cell therapy holds great promise, the optimal source of MSCs has yet to be determined with respect to major histocompatibility complex matching. Here, we examine this question by testing the ability of congenic MSCs, obtained from the NOR mouse strain, to reverse recent-onset type 1 diabetes in NOD mice, as well as determine the immunomodulatory effects of NOR MSCs in vivo. RESEARCH DESIGN AND METHODS NOR MSCs were evaluated with regard to their in vitro immunomodulatory function in the context of autoreactive T-cell proliferation and dendritic cell (DC) generation. The in vivo effect of NOR MSC therapy on reversal of recent-onset hyperglycemia and on immunogenic cell subsets in NOD mice was also examined. RESULTS NOR MSCs were shown to suppress diabetogenic T-cell proliferation via PD-L1 and to suppress generation of myeloid/inflammatory DCs predominantly through an IL-6-dependent mechanism. NOR MSC treatment of experimental type 1 diabetes resulted in long-term reversal of hyperglycemia, and therapy was shown to alter diabetogenic cytokine profile, to diminish T-cell effector frequency in the pancreatic lymph nodes, to alter antigen-presenting cell frequencies, and to augment the frequency of the plasmacytoid subset of DCs. CONCLUSIONS These studies demonstrate the inimitable benefit of congenic MSC therapy in reversing experimental type 1 diabetes. These data should benefit future clinical trials using MSCs as treatment for type 1 diabetes.

The proinflammatory cytokine IL-6 plays an important role in controlling T-cell differentiation, especially the development of Th17 and regulatory T cells. To determine the function of IL-6 in regulating allograft rejection and tolerance, BALB/c cardiac grafts were transplanted into wild-type or IL-6-deficient C57BL/6 mice. We observed that production of IL-6 and IFN-γ was upregulated during allograft rejection in untreated wild-type mice. In IL-6-deficient mice, IFN-γ production was greater than that observed in wild-type controls, suggesting that IL-6 production affects Th1/Th2 balance during allograft rejection. CD28-B7 blockade by CTLA4-Ig inhibited IFN-γ production in C57BL/6 recipients, but had no effect on the production of IL-6. Although wild-type C57BL/6 recipients treated with CTLA4-Ig rejected fully MHC-mismatched BALB/c heart transplants, treatment of IL-6-deficient mice with CTLA4-Ig resulted in graft acceptance. Allograft acceptance appeared to result from the combined effect of costimulatory molecule blockade and IL-6-deficiency, which limited the differentiation of effector cells and promoted the migration of regulatory T cells into the grafts. These data suggest that the blockade of IL-6, or its signaling pathway, when combined with strategies that inhibit Th1 responses, has a synergistic effect on the promotion of allograft acceptance. Thus, targeting the effects of IL-6 production may represent an important part of costimulation blockade-based strategies to promote allograft acceptance and tolerance.

Ischemic reperfusion injury (IRI) enhances allograft immunogenicity, worsens transplantation outcome, and is the primary cause of activation of the recipient innate immune response, resulting in subsequent amplification of the alloimmune adaptive response. Here, we aimed at demonstrating that the link between innate injury and alloimmunity occurs predominantly through activation of allograft-derived dendritic cells (ADDC). Perfusion of MCI-186, a free radical scavenger, into donor cardiac allografts prior to transplantation resulted in prolongation of complete MHC-mismatched allograft survival in the absence of immunosuppression (MST of 8 vs. 26 days). This prolongation was associated with a reduction in trafficking of ADDC to recipient lymphoid tissue as well as a reduction in T cell priming. Depleting ADDC with diphtheria toxin (using DTR-GFP-DC mice as donors) 24 h prior to transplant resulted in abrogation of the prolongation observed with MCI-186 treatment, demonstrating that the beneficial effect of MCI-186 is mediated by ADDC. This donor-specific anti-ischemic regimen was also shown to reduce chronic rejection, which represents the primary obstacle to long-term allograft acceptance. These data for the first time establish a basis for donor anti-ischemic strategies, which in the ever-expanding marginal donor pools, can be instituted to promote engraftment.

Clinical trials using mesenchymal stem cells (MSCs) have been initiated worldwide. An improved understanding of the mechanisms by which allogeneic MSCs evade host immune responses is paramount to regulating their survival after administration. This study has focused on the novel role of serine protease inhibitor (SPI) in the escape of MSCs from host immunosurveillance through the inhibition of granzyme B (GrB). Our data indicate bone marrow–derived murine MSCs express SPI6 constitutively. MSCs from mice deficient for SPI6 (SPI6−/−) exhibited a 4-fold higher death rate by primed allogeneic cytotoxic T cells than did wild-type MSCs. A GrB inhibitor rescued SPI6−/− MSCs from cytotoxic T-cell killing. Transduction of wild-type MSCs with MigR1-SPI6 also protected MSCs from cytotoxic T cell–mediated death in vitro. In addition, SPI6−/− MSCs displayed a shorter lifespan than wild-type MSCs when injected into an allogeneic host. We conclude that SPI6 protects MSCs from GrB-mediated killing and plays a pivotal role in their survival in vivo. Our data could serve as a basis for future SPI-based strategies to regulate the survival and function of MSCs after administration and to enhance the efficacy of MSC-based therapy for diseases.