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]

Myeloid-derived suppressor cells (MDSCs), which are known to be accumulated in the blood, spleen, and bone marrow of tumor-bearing mice and cancer patients, were tested as APCs for a cellular vaccine because they have phenotypical similarity with inflammatory monocytes and may be differentiated from the same precursors as monocytes. Although MDSCs have immunosuppressive properties, in vivo transferred MDSCs, which present tumor Ag and NKT cell ligand (α-galactosylceramide), significantly prolonged survival time in metastatic tumor-bearing mice in a CD8+ cell-, NK cell-, and NKT cell-dependent manner vs a CD4+ T cell- and host dendritic cell-independent manner. Major concerns about using MDSCs as APCs in a vaccine are their suppression of CTLs and their induction of Foxp3+ regulatory T cells. However, α-galactosylceramide-loaded MDSCs did not suppress CD4+ and CD8+ T cells and allowed for the generation of Ag-specific CTL immunity without increasing the generation of regulatory T cells. Furthermore, stimulation with activated NKT cells induced changes on MDSCs in phenotypical or maturation markers, including CD11b, CD11c, and CD86. Taken together, these findings suggest that NKT cells facilitate the conversion of immunosuppressive MDSCs into immunogenic APCs, eliciting successful antitumor immunity and providing the basis for alternative cell-based vaccines.

Circulating tumor cells (CTCs) have gained increasing attention as physicians and scientists learn more about the role these extraordinarily rare cells play in metastatic cancer. In developing CTC technology, the critical criteria are high recovery rates and high purity. Current isolation methods suffer from an inherent trade-off between these two goals. Moreover, ensuring minimal cell stress and robust reproducibility is also important for the clinical application of CTCs. In this paper, we introduce a novel CTC isolation technology using selective size amplification (SSA) for target cells and a multi-obstacle architecture (MOA) filter to overcome this trade-off, improving both recovery rate and purity. We also demonstrate SSA-MOA's advantages in minimizing cell deformation during filter transit, resulting in more stable and robust CTC isolation. In this technique, polymer microbeads conjugated with anti-epithelial cell adhesion molecules (anti-EpCAM) were used to selectively size-amplify MCF-7 breast cancer cells, definitively differentiating from the white blood cells (WBCs) by avoiding the size overlap that compromises other size selection methods. 3 μm was determined to be the optimal microbead diameter, not only for size discrimination but also in maximizing CTC surface coverage. A multi-obstacle architecture filter was fabricated using silicon-on-glass (SOG) technology-a first such application of this fabrication technique-to create a precise microfilter structure with a high aspect ratio. The filter was designed to minimize cell deformation as simulation results predicted that cells captured via this MOA filter would experience 22% less moving force than with a single-obstacle architecture. This was verified by experiments, as we observed reliable cell capture and reduced cell deformation, with a 92% average recovery rate and 351 peripheral blood leukocytes (PBL) per millilitre (average). We expect the SSA-MOA platform to optimize CTC recovery rates, purity, and stability, increasing the sensitivity and reliability of such tests, thereby potentially expanding the utilization of CTC technologies in the clinic.

Formation of misfolded protein aggregates is a remarkable hallmark of various neurodegenerative diseases including Alzheimer disease, Parkinson disease, Huntington disease, prion encephalopathies, and amyotrophic lateral sclerosis (ALS). Superoxide dismutase 1 (SOD1) immunoreactive inclusions have been found in the spinal cord of ALS animal models and patients, implicating the close involvement of SOD1 aggregates in ALS pathogenesis. Here we examined the molecular mechanism of aggregate formation of ALS-related SOD1 mutants in vitro. We found that long-chain unsaturated fatty acids (FAs) promoted aggregate formation of SOD1 mutants in both dose-and time-dependent manners. Metal-deficient SOD1s, wild-type, and mutants were highly oligomerized compared with holo-SOD1s by incubation in the presence of unsaturated FAs. Oligomerization of SOD1 is closely associated with its structural instability. Heat-treated holo-SOD1 mutants were readily oligomerized by the addition of unsaturated FAs, whereas wild-type SOD1 was not. The monounsaturated FA, oleic acid, directly bound to SOD1 and was characterized by a solid-phase FA binding assay using oleate-Sepharose. The FA binding characteristics were closely correlated with the oligomerization propensity of SOD1 proteins, which indicates that FA binding may change SOD1 conformation in a way that favors the formation of aggregates. High molecular mass aggregates of SOD1 induced by FAs have a granular morphology and show significant cytotoxicity. These findings suggest that SOD1 mutants gain FA binding abilities based on their structural instability and form cytotoxic granular aggregates. Amyotrophic lateral sclerosis (ALS)1 is a progressive and fatal neurodegenerative disorder that mainly affects motor neurons in the brain stem and spinal cord. Approximately 10% of ALS patients are familial cases, with autosomal dominant inheritance. More than 90 different mutations in the gene coding for superoxide dismutase 1 (SOD1) have been identified in about 20% of familial ALS (FALS) families (1, 2). Although the molecular mechanisms of selective motor neuron degeneration by SOD1 mutants in FALS remain largely unknown, common pathological features of conformational diseases, as evidenced by SOD1 immunoreactive inclusions, are found in the spinal cord of ALS patients and in the SOD1 mutant FALS mouse model (3-8). The characteristics of FALS resemble those of many other neurodegenerative diseases in which a causative protein undergoes a conformational rearrangement, which endows it with a tendency to aggregate and form deposits within affected tissues.SOD1 is a 32-kDa homodimeric enzyme that decreases the intracellular concentration of superoxide radicals by catalyzing their dismutation to O 2 and H 2 O 2 . ALS-linked mutations of SOD1 are distributed throughout the primary and tertiary structures, and most mutations appear unrelated to the dismutase activity. Many biochemical and biophysical studies have reported that SOD1 mutants are structurally unstable compared with wild-type forms (...