Background/Aim: It has been reported that adipose tissue contain progenitor cells with angiogenic potential and that therapy based on adipose tissuederived progenitor cells administration may constitute a promising cell therapy in patients with ischemic disease. In this study we evaluated the effect of culture-expanded mesenchymal stem cells (MSC) derived from adipose tissue on neovascularization and blood flow in an animal model of limb ischemia in immunodeficient mice. Methods: MSC were cultured from human adipose tissue by collagenase digestion. Hindlimb ischemia was created by ligating the proximal femoral artery of male nude mice. Human adipose tissue stromal cells (hADSC) were transplanted one day or 7 days after ligation. Results: During culture expansion of hADSC CD34 expression was downregulated. The laser Doppler perfusion index was significantly higher in the CD34(-), Flk-1(-), CD31(-) ADSC-transplanted group than in the control group, even when cells were transplanted 7days after hindlimb ischemia. Histological examination showed that hADSC transplantation recovered muscle injury and increased vascular density, compared with the control group. The effect of hADSC was correlated with the number of transplanted cells, but not with the ratio of CD34 expression. In vitro, hADSC can form vessel-like structure and express von Willibrand Factor. Conditioned media from hADSC increased proliferation and inhibited apoptotic cell death in of human aortic endothelial cells. Conclusion: This study showed that hADSC can be an ideal source for therapeutic angiogenesis in ischemic disease.

Background/Aim: Although transplantation of MSC derived from bone marrow or adipose tissues has been shown in proangiogenic action in hindlimb ischemia model of nude mice, little information is available regarding comparison of the angiogenic potency between human adipose stromal cells (hADSC) and bone marrow stromal cells (hBMSC). We compared their therapeutic potential by transplantation of equal numbers of hADSC or hBMSC in a nude mice model of hindlimb ischemia.

Tumor necrosis factor-alpha (TNF-alpha) is a skeletal catabolic agent that stimulates osteoclastogenesis and inhibits osteoblast function. Although TNF-alpha inhibits the mineralization of osteoblasts, the effect of TNF-alpha on mesenchymal stem cells (MSC) is not clear. In this study, we determined the effect of TNF-alpha on osteogenic differentiation of stromal cells derived from human adipose tissue (hADSC) and the role of NF-kappaB activation on TNF-alpha activity. TNF-alpha treatment dose-dependently increased osteogenic differentiation over the first 3 days of treatment. TNF-alpha activated ERK and increased NF-kappaB promoter activity. PDTC, an NF-kappaB inhibitor, blocked the osteogenic differentiation induced by TNF-alpha and TLR-ligands, but U102, an ERK inhibitor, did not. Overexpression of miR-146a induced the inhibition of IRAK1 expression and inhibited basal and TNF-alpha- and TLR ligand-induced osteogenic differentiation. TNF-alpha and TLR ligands increased the expression of transcriptional coactivator with PDZ-binding motif (TAZ), which was inhibited by the addition of PDTC. A ChIP assay showed that p65 was bound to the TAZ promoter. TNF-alpha also increased osteogenic differentiation of human gastroepiploic artery smooth muscle cells. Our data indicate that TNF-alpha enhances osteogenic differentiation of hADSC via the activation of NF-kappaB and a subsequent increase of TAZ expression.

Invariant NKT (iNKT) cells are a distinct subset of T lymphocytes that recognize glycolipid Ags. Upon TCR stimulation, iNKT cells promptly secrete a wide range of cytokines and therefore have been investigated as a target for immunotherapy. However, after primary activation, iNKT cells become hyporesponsive toward their ligand (anergy). The further mechanism behind iNKT cell anergy is poorly understood. We found that a low level of programmed death-1 (PD-1) was constitutively expressed on iNKT cells and that PD-1 expression was increased after stimulation and lasted at least 2 mo. Moreover, not only did blocking of the PD-1/PD ligand 1 (PD-L1) pathway prevent the induction of anergy in iNKT cells, but anergic iNKT cells also recovered responsiveness and these "rescued" cells efficiently mediated antitumor immunity. Our findings suggest that the PD-1/ PD-L1 interaction is essential for the induction and maintenance of iNKT cell anergy. The Journal of Immunology, 2008, 181: 6707-6710. N atural killer T (NKT)3 cells are a distinctive population of T lymphocytes that can recognize glycolipids presented by CD1d, an MHC class I like-molecule (1). A major subset of NKT cells, called type I NKT cells or invariant NKT (iNKT) cells, express an invariant TCR composed of V␣14-J␣18 chains in mice (V␣24-J␣18 in humans). Upon TCR stimulation with a ligand such as ␣-galactosylceramide (␣GC), iNKT cells rapidly produce a wide range of cytokines including IL-4, IFN-␥, and IL-12 (1, 2). This response enables iNKT cells to enhance or regulate the activity of various immune cells in innate and acquired immunity (3). These immunomodulatory roles of iNKT cells are found in diverse diseases, promoting tumor rejection or regulating autoimmune disorders (4 -6).Another unique feature of iNKT cells is that they become unresponsive after stimulation with their ligands. For instance, iNKT cells that have been stimulated with ␣GC have reduced proliferation and cytokine production upon secondary stimulation with the same ligand (7,8). This iNKT cell anergy is a major obstacle in immunotherapeutic trials targeting iNKT cells; however, the mechanism behind the anergy is not clear. A classic concept of anergy in conventional T cells is that the cells become anergic when they receive a TCR signal with insufficient costimulatory signals. In contrast, it has recently been suggested that coinhibitory molecules may actively anergize or tolerize T cells by delivering inhibitory signals into TCR-stimulated T cells (9). Moreover, in cases of chronic viral infection, blockade of the programmed death-1 (PD-1) signal can reverse the anergic phenotype of CD8 T cells (10, 11).PD-1 is well known as a coinhibitory molecule on T cells. In conventional T cells, it is not expressed on naive T cells but is inducibly expressed after T cell activation. The interactions of PD-1 with the PD ligands (PD-L1 and PD-L2) can transduce inhibitory or costimulatory signals into the T cells (12). It is well established that PD-1 plays a critical role in the regulation of immune...

Her-2/neu is a well-characterized tumor-associated antigen overexpressed in human carcinomas such as breast cancer. Because Her-2/neu is a self-antigen with poor immunogenicity due to immunologic tolerance, active immunotherapy targeting Her-2/neu should incorporate methods to overcome immunologic tolerance to self-proteins. In this study, we developed a tolerogenic tumor model in mice using mouse Her-2/neu as self-antigen and investigated whether genetic vaccination with DNA plasmid and/or adenoviral vector expressing the extracellular and transmembrane domain of syngeneic mouse Her-2/neu or xenogenic human Her-2/neu could induce mouse Her-2/neu-specific CTL responses. Interestingly, adenoviral vectors expressing xenogenic human Her-2/neu (AdhHM) proved capable of breaking immune tolerance and of thereby inducing self-reactive CTL and antibodies, but not to the degree required to induce therapeutic antitumor immunity. In attempting to generate therapeutic antitumor immunity against established tumors, we adopted several approaches. Treatment with agonistic anti-glucocorticoid-induced TNFR family-related receptor (GITR) antibody plus AdhHM immunization significantly increased self-reactive CTL responses, and A-galactosylceramide (AGalCer)-loaded dendritic cells (DC) transduced with AdhHM were shown to break self-tolerance in a tolerogenic murine tumor model. Furthermore, gemcitabine treatment together with either AdhHM plus agonistic anti-GITR antibody administration or AGalCer-loaded DC transduced with AdhHM showed potent therapeutic antitumor immunity and perfect protection against preexisting tumors. Gemcitabine treatment attenuated the tumor-suppressive environment by eliminating CD11b + /Gr-1 + myeloid-derived suppressor cells. When combined with immunotherapies, gemcitabine offers a promising strategy for the Ag-specific treatment of human cancer. [Cancer Res 2007;67(15):7477-86]