搜索结果: 'gmp t cell activator'

Studies have suggested involvement of interleukin 17 (IL-17) in autoimmune diseases,although its effect on B cell biology has not been clearly established. Here we demonstrate that IL-17 alone or in combination with B cell-activating factor controlled the survival and proliferation of human B cells and their differentiation into immunoglobulin-secreting cells. This effect was mediated mainly through the nuclear factor-kappaB-regulated transcription factor Twist-1. In support of the relevance of our observations and the potential involvement of IL-17 in B cell biology,we found that the serum of patients with systemic lupus erythematosus had higher concentrations of IL-17 than did the serum of healthy people and that IL-17 abundance correlated with the disease severity of systemic lupus erythematosus. View Publication

BACKGROUND Autologous bone marrow mononuclear cell (ABMMNC) therapy has shown promise in patients with heart failure (HF). Cell function analysis may be important in interpreting trial results. METHODS In this prospective study,we evaluated the safety and efficacy of the transendocardial delivery of ABMMNCs in no-option patients with chronic HF. Efficacy was assessed by maximal myocardial oxygen consumption,single photon emission computed tomography,2-dimensional echocardiography,and quality-of-life assessment (Minnesota Living with Heart Failure and Short Form 36). We also characterized patients' bone marrow cells by flow cytometry,colony-forming unit,and proliferative assays. RESULTS Cell-treated (n = 20) and control patients (n = 10) were similar at baseline. The procedure was safe; adverse events were similar in both groups. Canadian Cardiovascular Society angina score improved significantly (P = .001) in cell-treated patients,but function was not affected. Quality-of-life scores improved significantly at 6 months (P = .009 Minnesota Living with Heart Failure and P = .002 physical component of Short Form 36) over baseline in cell-treated but not control patients. Single photon emission computed tomography data suggested a trend toward improved perfusion in cell-treated patients. The proportion of fixed defects significantly increased in control (P = .02) but not in treated patients (P = .16). Function of patients' bone marrow mononuclear cells was severely impaired. Stratifying cell results by age showed that younger patients (%60 years) had significantly more mesenchymal progenitor cells (colony-forming unit fibroblasts) than patients<60 years (20.16 ± 14.6 vs 10.92 ± 7.8,P = .04). Furthermore,cell-treated younger patients had significantly improved maximal myocardial oxygen consumption (15 ± 5.8,18.6 ± 2.7,and 17 ± 3.7 mL/kg per minute at baseline,3 months,and 6 months,respectively) compared with similarly aged control patients (14.3 ± 2.5,13.7 ± 3.7,and 14.6 ± 4.7 mL/kg per minute,P = .04). CONCLUSIONS ABMMNC therapy is safe and improves symptoms,quality of life,and possibly perfusion in patients with chronic HF. View Publication

The murine heat-stable antigen (HSA) is a glycosyl-phosphatidylinositol-linked cell surface protein which has been implicated in cellular adhesion processes,the co-stimulation of CD4+ T cells,and B cell memory. We have recently demonstrated a significant reduction in pro-B and pre-B lymphocytes in transgenic mice that overexpress HSA. We now report that cross-linking HSA with the M1/69 monoclonal antibody induces the apoptosis of cultured B cell precursors in a stomal cell and cytokine-independent manner and that sensitivity to HSA-mediated cell death increases with developmental maturity. The cross-linking of HSA does not induce apoptosis in mature splenic B cells,but instead inhibits their ability to proliferate in response to anti-CD40 + IL-4. Taken together,these data implicate HSA as a potent negative regulator of B cell development and activation. View Publication

The concept of exploiting tumor intrinsic deficiencies in DNA damage repair mechanisms by inhibiting compensatory DNA repair pathways is well established. For example,ATM-deficient cells show increased sensitivity to the ATR inhibitor ceralasertib. DNA damage response (DDR)-deficient cells are also more sensitive to DNA damaging agents like the DNA crosslinker pyrrolobenzodiazepine (PBD) SG-3199. However,additional antitumor benefits from targeting the DDR pathways,which could operate through the activation of the innate immune system are less well studied. DNA accumulation in the cytosol acts as an immunogenic danger signal,inducing the expression of type-I interferon (IFN) stimulated genes (ISGs) by the activation of the cGAS-STING pathway. Here,we demonstrate that ATM -/- FaDu tumor cells have higher basal expression of ISGs when compared to WT cells and respond to ceralasertib and PBD SG-3199 by inducing higher levels of ISGs in a cGAS-STING-dependent manner. We show that sensitive tumor cells treated with ceralasertib and PBD SG-3199 activate dendritic cells (DCs) via a type-I IFN-dependent mechanism. However,STING deficiency in tumor cells does not prevent DC activation,suggesting that transactivation of the STING pathway occurs within DCs. Furthermore,depletion of the cytosolic DNA exonuclease TREX1 in tumor cells increases DC activation in response to PBD SG-3199-treated tumor cells,indicating that an increase in tumor-derived cytosolic DNA may further enhance DC activation. In summary,in this study,we show that ceralasertib and PBD SG-3199 treatment not only intrinsically target tumor cells but also extrinsically increase tumor cell immunogenicity by inducing DC activation,which is enhanced in ATM-deficient cells. View Publication

Numerous publications have described the importance of bone morphogenetic protein (BMP) signaling in the specification of hematopoietic tissue in developing embryos. Here we investigate the full role of canonical BMP signaling in both adult and fetal liver hematopoiesis using conditional knockout strategies because conventional disruption of components of the BMP signaling pathway result in early death of the embryo. By targeting both Smad1 and Smad5,we have generated a double-knockout mouse with complete disruption of canonical BMP signaling. Interestingly,concurrent deletion of Smad1 and Smad5 results in death because of extrahematopoietic pathologic changes in the colon. However,Smad1/Smad5-deficient bone marrow cells can compete normally with wild-type cells and display unaffected self-renewal and differentiation capacity when transplanted into lethally irradiated recipients. Moreover,although BMP receptor expression is increased in fetal liver,fetal liver cells deficient in both Smad1 and Smad5 remain competent to long-term reconstitute lethally irradiated recipients in a multilineage manner. In conclusion,canonical BMP signaling is not required to maintain either adult or fetal liver hematopoiesis,despite its crucial role in the initial patterning of hematopoiesis in early embryonic development. View Publication

In recent years,mesenchymal stem cells (MSCs) have been shown to inhibit T-lymphocyte proliferation induced by alloantigens or mitogens. However,no substantial information is available regarding their effect on natural killer (NK) cells. Here we show that MSCs sharply inhibit IL-2-induced proliferation of resting NK cells,whereas they only partially affect the proliferation of activated NK cells. In addition,we show that IL-2-activated NK cells (but not freshly isolated NK cells) efficiently lyse autologous and allogeneic MSCs. The activating NK receptors NKp30,NKG2D,and DNAM-1 represented the major receptors responsible for the induction of NK-mediated cytotoxicity against MSCs. Accordingly,MSCs expressed the known ligands for these activating NK receptors-ULBPs,PVR,and Nectin-2. Moreover,NK-mediated lysis was inhibited when IFN-gamma-exposed MSCs were used as target cells as a consequence of the up-regulation of HLA class I molecules at the MSC surface. The interaction between NK cells and MSCs resulted not only in the lysis of MSCs but also in cytokine production by NK cells. These results should be taken into account when evaluating the possible use of MSCs in novel therapeutic strategies designed to improve engraftment or to suppress graft-versus-host disease (GVHD) in bone marrow transplantation. View Publication

Metabolic labeling with stable isotopes is a prominent technique for comparative quantitative proteomics,and stable isotope labeling with amino acids in cell culture (SILAC) is the most commonly used approach. SILAC is,however,traditionally limited to simple tissue culture regimens and only rarely employed in the context of complex culturing conditions as those required for human embryonic stem cells (hESCs). Classic hESC culture is based on the use of mouse embryonic fibroblasts (MEFs) as a feeder layer,and as a result,possible xenogeneic contamination,contribution of unlabeled amino acids by the feeders,interlaboratory variability of MEF preparation,and the overall complexity of the culture system are all of concern in conjunction with SILAC. We demonstrate a feeder-free SILAC culture system based on a customized version of a commonly used,chemically defined hESC medium developed by Ludwig et al. and commercially available as mTeSR1 [mTeSR1 is a trade mark of WiCell (Madison,WI) licensed to STEMCELL Technologies (Vancouver,Canada)]. This medium,together with adjustments to the culturing protocol,facilitates reproducible labeling that is easily scalable to the protein amounts required by proteomic work flows. It greatly enhances the usability of quantitative proteomics as a tool for the study of mechanisms underlying hESCs differentiation and self-renewal. Associated data have been deposited to the ProteomeXchange with the identifier PXD000151. View Publication

Peripheral blood was collected from a clinically characterized female Kleefstra syndrome patient with a heterozygous,de novo,premature termination codon (PTC) mutation (NM024757.4(EHMT1):c.3413GtextgreaterA; p.Trp1138Ter). Peripheral blood mononuclear cells (PBMCs) were reprogrammed with the human OSKM transcription factors using the Sendai-virus (SeV) delivery system. The pluripotency of transgene-free iPSC line was verified by the expression of pluripotency-associated markers and by in vitro spontaneous differentiation towards the 3 germ layers. Furthermore,the iPSC line showed normal karyotype. Our model might offer a good platform to study the pathomechanism of Kleefstra syndrome,also for drug testing,early biomarker discovery and gene therapy studies. View Publication

Human bone marrow (BM) or mobilized peripheral blood (mPB) CD34(+) cells have been shown to loose their stem cell quality during culture period more easily than those from cord blood (CB). We previously reported that human umbilical CB stem cells could effectively be expanded in the presence of human recombinant cytokines and a newly established murine bone marrow stromal cell line HESS-5. In this study we assessed the efficacy of this xenogeneic coculture system using human BM and mPB CD34(+) cells as materials. We measured the generation of CD34(+)CD38(-) cells and colony-forming units,and assessed severe-combined immunodeficient mouse-repopulating cell (SRC) activity using cells five days after serum-free cytokine-containing culture in the presence or the absence of a direct contact with HESS-5 cells. As compared with the stroma-free culture,the xenogeneic coculture was significantly superior on expansion of CD34(+)CD38(-) cells and colony-forming cells and on maintenance of SRC activity. The PKH26 study demonstrated that cell division was promoted faster in cells cocultured with HESS-5 cells than in cells cultured without HESS-5 cells. These results indicate that HESS-5 supports rapid generation of primitive progenitor cells (PPC) and maintains reconstituting ability of newly generated stem cells during ex vivo culture irrespective of the source of samples. This xenogeneic coculture system will be useful for ex vivo manipulation such as gene transduction to promote cell division and the generation of PPC and to prevent loss of stem cell quality. View Publication

Utilizing a fast 31P magnetic resonance spectroscopy (MRS) 2-dimensional chemical shift imaging (2D-CSI) method,this study examined the heterogeneity of creatine kinase (CK) forward flux rate of hearts with postinfarction left ventricular (LV) remodeling. Immunosuppressed Yorkshire pigs were assigned to 4 groups: 1) A sham-operated normal group (SHAM,n = 6); 2) A 60 minutes distal left anterior descending coronary artery ligation and reperfusion (MI,n = 6); 3) Open patch group; ligation injury plus open fibrin patch over the site of injury (Patch,n = 6); and 4) Cell group,hiPSCs-cardiomyocytes,-endothelial cells,and -smooth muscle cells (2 million,each) were injected into the injured myocardium pass through a fibrin patch (Cell+Patch,n = 5). At 4 weeks,the creatine phosphate (PCr)/ATP ratio,CK forward flux rate (Flux PCr→ATP),and k constant of CK forward flux rate (kPCr→ATP) were severely decreased at border zone myocardium (BZ) adjacent to MI. Cell treatment results in significantly increase of PCr/ATP ratio and improve the value of kPCr→ATP and Flux PCr→ATP in BZ myocardium. Moreover,the BZ myocardial CK total activity and protein expression of CK mitochondria isozyme and CK myocardial isozyme were significantly reduced,but recovered in response to cell treatment. Thus,cell therapy results in improvement of BZ bioenergetic abnormality in hearts with postinfarction LV remodeling,which is accompanied by significantly improvements in BZ CK activity and CK isozyme expression. The fast 2D 31P MR CSI mapping can reliably measure the heterogeneity of bioenergetics in hearts with post infarction LV remodeling. View Publication

Cardiac tissue engineering is a promising method for regenerative medicine. Although we have developed human cardiac cell sheets by integration of cell sheet-based tissue engineering and scalable bioreactor culture,the risk of contamination by induced pluripotent stem (iPS) cells in cardiac cell sheets remains unresolved. In the present study,we established a novel culture method to fabricate human cardiac cell sheets with a decreased risk of iPS cell contamination while maintaining viabilities of iPS cell-derived cells,including cardiomyocytes and fibroblasts,using a methionine-free culture condition. When cultured in the methionine-free condition,human iPS cells did not survive without feeder cells and could not proliferate or form colonies on feeder cells or in coculture with cells for cardiac cell sheet fabrication. When iPS cell-derived cells after the cardiac differentiation were transiently cultured in the methionine-free condition,gene expression of OCT3/4 and NANOG was downregulated significantly compared with that in the standard culture condition. Furthermore,in fabricated cardiac cell sheets,spontaneous and synchronous beating was observed in the whole area while maintaining or upregulating the expression of various cardiac and extracellular matrix genes. These findings suggest that human iPS cells are methionine dependent and a methionine-free culture condition for cardiac cell sheet fabrication might reduce the risk of iPS cell contamination. View Publication

Pluripotent human embryonic stem (hES) cells are an important experimental tool for basic and applied research,and a potential source of different tissues for transplantation. However,one important challenge for the clinical use of these cells is the issue of immunocompatibility,which may be dealt with by the establishment of hES cell banks to attend different populations. Here we describe the derivation and characterization of a line of hES cells from the Brazilian population,named BR-1,in commercial defined medium. In contrast to the other hES cell lines established in defined medium,BR-1 maintained a stable normal karyotype as determined by genomic array analysis after 6 months in continuous culture (passage 29). To our knowledge,this is the first reported line of hES cells derived in South America. We have determined its genomic ancestry and compared the HLA-profile of BR-1 and another 22 hES cell lines established elsewhere with those of the Brazilian population,finding they would match only 0.011% of those individuals. Our results highlight the challenges involved in hES cell banking for populations with a high degree of ethnic admixture. View Publication