Effects of yuja peel extract and its flavanones on osteopenia in ovariectomized rats and osteoblast differentiation.

SCOPE: Yuja (Citrus junos Tanaka) possesses various health benefits, but its effects on bone health are unknown. In this study, the preventative effects of yuja peel ethanol extract (YPEE) on osteopenia were determined in ovariectomized (OVX) rats, and the mechanisms by which YPEE and its flavanones regulate osteoblastogenesis were examined in vitro. METHODS AND RESULTS: The effects of YPEE on osteoblastogenesis were investigated in MC3T3-E1 cells. YPEE promoted alkaline phosphatase (ALP) activity, mineralization, and the expression of osteoblast differentiation marker genes, such as ALP, runt-related transcription factor 2 (Runx2), and osteocalcin. YPEE and its flavanones promoted osteoblast differentiation via BMP-2-mediated p38 and the Smad1/5/8 signaling pathway. YPEE supplementation significantly decreased body weight and increased uterine weight and bone mineral density in OVX rats. Based on a micro-CT analysis of femurs, YPEE significantly attenuated osteopenia and increased trabecular volume fraction, trabecular separation, and trabecular number (p < 0.05). CONCLUSION: Dietary YPEE has a protective effect on OVX-induced osteopenia. YPEE and its flavanones promote osteoblastogenesis via the activation of the BMP/p38/Smad/Runx2 pathways. These results extend our knowledge of the beneficial effects of YPEE and provide a basis for the development of novel therapies for osteoporosis.

Combination cell therapy using mesenchymal stem cells and regulatory T-cells provides a synergistic immunomodulatory effect associated with reciprocal regulation of TH1/TH2 and th17/treg cells in a murine acute graft-versus-host disease model.

Mesenchymal stem cells (MSCs) have been considered to be an ideal cellular source for graft-versus-host disease (GVHD) treatment due to their unique properties, including tissue repair and major histocompatibility complex (MHC)-unmatched immunosuppression. However, preclinical and clinical data have suggested that the immunomodulatory activity of MSCs is not as effective as previously expected. This study was performed to investigate whether the immunomodulatory capacity of MSCs could be enhanced by combination infusion of regulatory T (Treg) cells to prevent acute GVHD (aGVHD) following MHC-mismatched bone marrow transplantation (BMT). For GVHD induction, lethally irradiated BALB/c (H-2(d)) mice were transplanted with bone marrow cells (BMCs) and spleen cells of C57BL/6 (H-2(b)) mice. Recipients were injected with cultured recipient-derived MSCs, Treg cells, or MSCs plus Treg cells (BMT + day 0, 4). Systemic infusion of MSCs plus Treg cells improved clinicopathological manifestations and survival in the aGVHD model. Culture of MSCs plus Treg cells increased the population of Foxp3(+) Treg cells and suppressed alloreactive T-cell proliferation in vitro. These therapeutic effects were associated with more rapid expansion of donor-type CD4(+)CD25(+)Foxp3(+) Treg cells and CD4(+)IL-4(+) type 2 T-helper (Th2) cells in the early posttransplant period. Furthermore, MSCs plus Treg cells regulated CD4(+)IL-17(+) Th17 cells, as well as CD4(+)IFN-γ(+) Th1 cells. These data suggest that the combination therapy with MSCs plus Treg cells may have cooperative effects in enhancing the immunomodulatory activity of MSCs and Treg cells in aGVHD. This may lead to development of new therapeutic approaches to clinical allogeneic hematopoietic cell transplantation.

The synergistic immunoregulatory effects of culture-expanded mesenchymal stromal cells and CD4(+)25(+)Foxp3+ regulatory T cells on skin allograft rejection.

Mesenchymal stromal cells (MSCs) are seen as an ideal source of cells to induce graft acceptance; however, some reports have shown that MSCs can be immunogenic rather than immunosuppressive. We speculate that the immunomodulatory effects of regulatory T cells (Tregs) can aid the maintenance of immunoregulatory functions of MSCs, and that a combinatorial approach to cell therapy can have synergistic immunomodulatory effects on allograft rejection. After preconditioning with Fludarabine, followed by total body irradiation and anti-asialo-GM-1(ASGM-1), tail skin grafts from C57BL/6 (H-2k(b)) mice were grafted onto the lateral thoracic wall of BALB/c (H-2k(d)) mice. Group A mice (control group, n = 9) did not receive any further treatment after preconditioning, whereas groups B and C (n = 9) received cell therapy with MSCs or Tregs, respectively, on days -1, +6 and +13 relative to the skin transplantation. Group D (n = 10) received cell therapy with MSCs and Tregs on days -1, +6 and +13. Cell suspensions were obtained from the spleens of five randomly chosen mice from each group on day +7, and the immunomodulatory effects of the cell therapy were evaluated by flow cytometry and real-time PCR. Our results show that allograft survival was significantly longer in group D compared to the control group (group A). Flow cytometric analysis and real-time PCR for splenocytes revealed that the Th2 subpopulation in group D increased significantly compared to the group B. Also, the expression of Foxp3 and STAT 5 increased significantly in group D compared to the conventional cell therapy groups (B and C). Taken together, these data suggest that a combined cell therapy approach with MSCs and Tregs has a synergistic effect on immunoregulatory function in vivo, and might provide a novel strategy for improving survival in allograft transplantation.

Mesenchymal stem cells for the treatment and prevention of graft-versus-host disease: experiments and practice.

Mesenchymal stem cells (MSCs) have emerged as a therapeutic approach in a range of medical fields, including regenerative medicine, cancer, autoimmune diseases, and inflammatory diseases, because of their unique properties of tissue repair and major histocompatibility complex-unmatched immunosuppression. Because both in vitro and in vivo findings demonstrate that MSCs possess potent immunoregulatory functions, there has been increasing interest in the role of MSCs in allogeneic hematopoietic stem cell transplantation, especially in the prevention and treatment of graft-versus-host disease (GVHD). GVHD is a major cause of transplantation-related mortality, and conventional immunosuppressants frequently fail to treat patients suffering from GVHD. Following Ringden's pilot study that used third-party MSCs to treat a steroid-refractory GVHD patient, MSCs have created growing interest as a therapeutic agent for GVHD. There have been further studies which demonstrated the potentials of MSC treatment in steroid-refractory GVHD, de novo GVHD, and also GVHD prevention. However, MSCs still present limitations. The need for MSCs to be "licensed" in a pro-inflammatory environment, especially in the presence of interferon gamma, allows only a narrow window for their administration. Thus, their effects have been less clear as a preventive measure before the inflammatory environment of GVHD is established and also when administered during a chronic setting where MSCs may be alternatively licensed. In this review, we focus on the immunomodulatory properties of MSCs and their effects in relation to GVHD. Given the efficacy of MSCs in murine models of GVHD and their safety in clinical trials, it is crucial that larger clinical trials are conducted and further modifications are investigated.

B cell-associated immune profiles in patients with end-stage renal disease (ESRD).

Most of the previous studies on immune dysregulation in end-stage renal disease (ESRD) have focused on T cell immunity. We investigated B cell subpopulations in ESRD patients and the effect of hemodialysis (HD) on B cell-associated immune profiles in these patients. Forty-four ESRD [maintenance HD patients (n = 27) and pre-dialysis patients (n = 17)] and 27 healthy volunteers were included in this study. We determined the percentage of B cell subtypes, such as mature and immature B cells, memory B cells, and interleukin (IL)-10+ cells, as well as B cell-producing cytokines (IL-10, IL-4 and IL-21) by florescent activated cell sorting (FACS). B cell-associated gene expression was examined using real-time PCR and B cell producing cytokines (IL-10, IL-4 and IL-21) were determined using an enzyme- linked immunosorbent assay (ELISA). The percentage of total B cells and mature B cells did not differ significantly among the three groups. The percentages of memory B cells were significantly higher in the pre-dialysis group than in the HD group (P < 0.01), but the percentage of immature B cells was significantly lower in the pre-dialysis group than in the other groups. The percentages of IL-10-expressing cells that were CD19+ or immature B cells did not differ significantly (P > 0.05) between the two subgroups within the ESRD group, but the serum IL-10 concentration was significantly lower in the pre-dialysis group (P < 0.01). The results of this study demonstrate significantly altered B cell-associated immunity. Specifically, an imbalance of immature and memory B cells in ESRD patients was observed, with this finding predominating in pre-dialysis patients.

Bone morphogenetic protein-2 stimulates Runx2 acetylation.

Runx2/Cbfa1/Pebp2aA is a global regulator of osteogenesis and is crucial for regulating the expression of bone-specific genes. Runx2 is a major target of the bone morphogenetic protein (BMP) pathway. Genetic analysis has revealed that Runx2 is degraded through a Smurf-mediated ubiquitination pathway, and its activity is inhibited by HDAC4. Here, we demonstrate the molecular link between Smurf, HDACs and Runx2, in BMP signaling. BMP-2 signaling stimulates p300-mediated Runx2 acetylation, increasing transactivation activity and inhibiting Smurf1-mediated degradation of Runx2. HDAC4 and HDAC5 dea-cetylate Runx2, allowing the protein to undergo Smurf-mediated degradation. Inhibition of HDAC increases Runx2 acetylation, and potentiates BMP-2-stimulated osteoblast differentiation and increases bone formation. These results demonstrate that the level of Runx2 is controlled by a dynamic equilibrium of acetylation, deacetylation, and ubiquitination. These findings have important medical implications because BMPs and Runx2 are of tremendous interest with regard to the development of therapeutic agents against bone diseases.

Transforming growth factor-beta stimulates p300-dependent RUNX3 acetylation, which inhibits ubiquitination-mediated degradation.

The Runt domain transcription factors (RUNXs) play essential roles in normal development and neoplasias. Genetic analyses of animals and humans have revealed the involvement of RUNX1 in hematopoiesis and leukemia, RUNX2 in osteogenesis and cleidocranial dysplasia, and RUNX3 in the development of T-cells and dorsal root ganglion neurons and in the genesis of gastric cancer. Here we report that RUNX3 is a target of the acetyltransferase activity of p300. The p300-dependent acetylation of three lysine residues protects RUNX3 from ubiquitin ligase Smurf-mediated degradation. The extent of the acetylation is up-regulated by the transforming growth factor-beta signaling pathway and down-regulated by histone deacetylase activities. Our findings demonstrate that the level of RUNX3 protein is controlled by the competitive acetylation and deacetylation of the three lysine residues, revealing a new mechanism for the posttranslational regulation of RUNX3 expression.