搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

PURPOSE: Immunotherapy holds promise as a new strategy for the eradication of residual cells in acute myeloid leukaemia (AML). Leukaemic antigen presenting cells (APCs) combining optimal antigen presentation and tumour antigenicity could be used as potent T cell activators. For clinical purposes it is desirable to culture APCs under serum-free conditions. Therefore,we compared morphological,immunophenotypical and functional outcome of the serum-free culture of AML-APCs to their serum-enriched culture. METHODS: AML blasts (n=19) were cultured in the presence of either a cytokine mix or calcium ionophore (CI) for 14 and 2 days,respectively,in FCS-containing medium (FCS),StemSpan serum-free medium (SP) and CellGro serum-free medium (CG). After culture relative yields were calculated and immunophenotypic analysis of APC markers was performed. The mixed leukocyte reaction (MLR) was used to determine T cell stimulating capacity. RESULTS: Serum-free culture of AML-APCs resulted in comparable morphology,relative yields and immunophenotype to serum-enriched culture. By comparing both serum-free media we observed a trend towards a more mature phenotype of CI-cultured AML-APCs in SP. MLR showed that serum-free cultured cells have equal T cell stimulatory capacity in comparison with serum-enriched culture. CONCLUSION: These data show that the serum-free culture of AML-APCs is feasible and that these APCs are comparable to serum-enriched cultured AML-APCs with regard to morphological,immunophenotypical and functional characteristics. These AML-APCs are suitable for the development of active specific immunisation protocols which meet the criteria for good clinical practise (GCP). View Publication

Multi-parameter flow cytometry analysis of T regulatory (Treg) cells is a widely used approach in basic and translational research studies. This approach has been complicated by a lack of specific markers for Treg cells and lack of uniformity in the quantification of Treg cells. Given the central role of Treg cells in the inception and perpetuation of diverse immune responses as well as its target as a therapeutic,it is imperative to have established methodologies for Treg cell analysis that are robust and usable for studies with multiple subjects as well as multicenter studies. In this study,we describe an optimized multi-parameter flow cytometry protocol for the quantification of human Treg cells from freshly obtained and viably frozen samples and correlations with epigenetic Treg cell analysis (TSDR demethylation). We apply these two methodologies to characterize Treg cell differences between cord blood and adult peripheral blood. In summary,the optimized protocol appears to be robust for Treg cell quantification from freshly isolated or viably frozen cells and the multi-parameter flow cytometry findings are strongly positively correlated with TSDR demethylation thus providing several options for the characterization of Treg cell frequency and function in large translational or clinical studies. View Publication

In systemic lupus erythematosus,defective clearance of apoptotic debris and activation of innate cells result in a chronically activated type 1 IFN response,which can be measured in PBMCs of most patients. Metformin,a widely used prescription drug for Type 2 diabetes,has a therapeutic effect in several mouse models of lupus through mechanisms involving inhibition of oxidative phosphorylation and a decrease in CD4+ T cell activation. In this study,we report that in CD4+ T cells from human healthy controls and human systemic lupus erythematosus patients,metformin inhibits the transcription of IFN-stimulated genes (ISGs) after IFN-alpha treatment. Accordingly,metformin inhibited the phosphorylation of pSTAT1 (Y701) and its binding to IFN-stimulated response elements that control ISG expression. These effects were independent of AMPK activation or mTORC1 inhibition but were replicated using inhibitors of the electron transport chain respiratory complexes I,III,and IV. This indicates that mitochondrial respiration is required for ISG expression in CD4+ T cells and provides a novel mechanism by which metformin may exert a therapeutic effect in autoimmune diseases. View Publication

Injury induces the recruitment of bone marrow-derived cells (BMDCs) that contribute to the repair and regeneration process. The behavior of BMDCs in injured tissue has a profound effect on repair,but the regulation of BMDC behavior is poorly understood. Aberrant recruitment/retention of these cells in wounds of diabetic patients and animal models is associated with chronic inflammation and impaired healing. BMD Gr-1(+)CD11b(+) cells function as immune suppressor cells and contribute significantly to tumor-induced neovascularization. Here we report that Gr-1(+)CD11b(+) cells also contribute to injury-induced neovascularization,but show altered recruitment/retention kinetics in the diabetic environment. Moreover,diabetic-derived Gr-1(+)CD11b(+) cells fail to stimulate neovascularization in vivo and have aberrant proliferative,chemotaxis,adhesion,and differentiation potential. Previously we demonstrated that gene transfer of HOXA3 to wounds of diabetic mice is taken up by and expressed by recruited BMDCs. This is associated with a suppressed inflammatory response,enhanced neovascularization,and accelerated wound healing. Here we show that sustained expression of Hoxa3 in diabetic-derived BMD Gr-1(+)CD11b(+) cells reverses their diabetic phenotype. These findings demonstrate that manipulation of adult stem/progenitor cells ex vivo could be used as a potential therapy in patients with impaired wound healing. View Publication

Dyskeratosis congenita (DC) is an inherited bone marrow (BM) failure syndrome associated with mutations in telomerase genes and the acquisition of shortened telomeres in blood cells. To investigate the basis of the compromised hematopoiesis seen in DC,we analyzed cells from granulocyte colony-stimulating factor mobilized peripheral blood (mPB) collections from 5 members of a family with autosomal dominant DC with a hTERC mutation. Premobilization BM samples were hypocellular,and percentages of CD34(+) cells in marrow and mPB collections were significantly below values for age-matched controls in 4 DC subjects. Directly clonogenic cells,although present at normal frequencies within the CD34(+) subset,were therefore absolutely decreased. In contrast,even the frequency of long-term culture-initiating cells within the CD34(+) DC mPB cells was decreased,and the telomere lengths of these cells were also markedly reduced. Nevertheless,the different lineages of mature cells were produced in normal numbers in vitro. These results suggest that marrow failure in DC is caused by a reduction in the ability of hematopoietic stem cells to sustain their numbers due to telomere impairment rather than a qualitative defect in their commitment to specific lineages or in the ability of their lineage-restricted progeny to execute normal differentiation programs. View Publication

Regulated adhesion of T cells by the integrins LFA-1 (lymphocyte function-associated antigen-1) and VLA-4 (very late antigen-4) is essential for T-cell trafficking. The small GTPase Rap1 is a critical activator of both integrins in murine lymphocytes and T-cell lines. Here we examined the contribution of the Rap1 regulatory pathway in integrin activation in primary CD3(+) human T cells. We demonstrate that inactivation of Rap1 GTPase in human T cells by expression of SPA1 or Rap1GAP blocked stromal cell-derived factor-1alpha (SDF-1alpha)-stimulated LFA-1-ICAM-1 (intercellular adhesion molecule-1) interactions and LFA-1 affinity modulation but unexpectedly did not significantly affect binding of VLA-4 to its ligand VCAM-1 (vascular cell adhesion molecule 1). Importantly,silencing of the Rap1 guanine exchange factor CalDAG-GEFI inhibited SDF-1alpha- and phorbol 12-myristate 13-acetate (PMA)-induced adhesion to ICAM-1 while having no effect on adhesion to VCAM-1. Pharmacologic inhibition of Phospholipase C (PLC) blocked Rap1 activation and inhibited cell adhesion and polarization on ICAM-1 and VCAM-1. Protein kinase C (PKC) inhibition led to enhanced levels of active Rap1 concomitantly with increased T-cell binding to ICAM-1,whereas adhesion to VCAM-1 was reduced. Thus,PLC/CalDAG-GEFI regulation of Rap1 is selectively required for chemokine- and PMA-induced LFA-1 activation in human T cells,whereas alternate PLC- and PKC-dependent mechanisms are involved in the regulation of VLA-4. View Publication

Efficient gene transfer into human hematopoietic stem cells (HSCs) is an important goal in the study of the hematopoietic system as well as for gene therapy of hematopoietic disorders. A lentiviral vector based on the human immunodeficiency virus (HIV) was able to transduce human CD34+ cells capable of stable,long-term reconstitution of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. High-efficiency transduction occurred in the absence of cytokine stimulation and resulted in transgene expression in multiple lineages of human hematopoietic cells for up to 22 weeks after transplantation. View Publication

The lipocalin mouse 24p3 has been implicated in diverse physiological processes,including apoptosis,iron trafficking,development and innate immunity. Studies from our laboratory as well as others demonstrated the proapoptotic activity of 24p3 in a variety of cultured models. However,a general role for the lipocalin 24p3 in the hematopoietic system has not been tested in vivo. To study the role of 24p3,we derived 24p3 null mice and back-crossed them onto C57BL/6 and 129/SVE backgrounds. Homozygous 24p3(-/-) mice developed a progressive accumulation of lymphoid,myeloid,and erythroid cells,which was not due to enhanced hematopoiesis because competitive repopulation and recovery from myelosuppression were the same as for wild type. Instead,apoptotic defects were unique to many mature hematopoietic cell types,including neutrophils,cytokine-dependent mast cells,thymocytes,and erythroid cells. Thymocytes isolated from 24p3 null mice also displayed resistance to apoptosis-induced by dexamethasone. Bim response to various apoptotic stimuli was attenuated in 24p3(-/-) cells,thus explaining their resistance to the ensuing cell death. The results of these studies,in conjunction with those of previous studies,reveal 24p3 as a regulator of the hematopoietic compartment with important roles in normal physiology and disease progression. Interestingly,these functions are limited to relatively mature blood cell compartments. View Publication

TGF-beta can be a potent suppressor of lymphocyte effector cell functions and can mediate these effects via distinct molecular pathways. The role of TGF-beta in regulating CD16-mediated NK cell IFN-gamma production and antibody-dependent cellular cytotoxicity (ADCC) is unclear,as are the signaling pathways that may be utilized. Treatment of primary human NK cells with TGF-beta inhibited IFN-gamma production induced by CD16 activation with or without IL-12 or IL-2,and it did so without affecting the phosphorylation/activation of MAP kinases ERK and p38,as well as STAT4. TGF-beta treatment induced SMAD3 phosphorylation,and ectopic overexpression of SMAD3 resulted in a significant decrease in IFN-gamma gene expression following CD16 activation with or without IL-12 or IL-2. Likewise,NK cells obtained from smad3(-/-) mice produced more IFN-gamma in response to CD16 activation plus IL-12 when compared with NK cells obtained from wild-type mice. Coactivation of human NK cells via CD16 and IL-12 induced expression of T-BET,the positive regulator of IFN-gamma,and T-BET was suppressed by TGF-beta and by SMAD3 overexpression. An extended treatment of primary NK cells with TGF-beta was required to inhibit ADCC,and it did so by inhibiting granzyme A and granzyme B expression. This effect was accentuated in cells overexpressing SMAD3. Collectively,our results indicate that TGF-beta inhibits CD16-mediated human NK cell IFN-gamma production and ADCC,and these effects are mediated via SMAD3. View Publication

Redirecting Ag specificity by transfer of TCR genes into PBLs is an attractive method to generate large numbers of cytotoxic T cells for immunotherapy of cancer and viral diseases. However,transferred TCR chains can pair with endogenous TCR chains,resulting in the formation of mispaired TCR dimers and decreased or unspecific reactivity. TCR gene transfer into hematopoietic stem cells (HSCs) is an alternative to create T cells with desired Ag specificity,because in this case expression of endogenous TCR chains is then less likely owing to allelic exclusion. We generated TCR-transduced T cells from peripheral T cells using the lymphocytic choriomeningitis virus-specific P14 TCR. After transfer of the P14 TCR genes into HSCs and subsequent reconstitution of irradiated mice,TCR-engineered HSC-derived T cells were produced. We then compared the Ag-specific T cell populations with P14 TCR-transgenic T cells for their therapeutic efficiency in three in vivo models. In this study,we demonstrate that TCR-transduced T cells and TCR-engineered HSC-derived T cells are comparable in controlling lymphocytic choriomeningitis virus infection in mice and suppress growth of B16 tumor cells expressing the cognate Ag in a comparable manner. View Publication