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

Retinoic acid (RA),a vitamin A metabolite,modulates mucosal T helper cell responses. Here we examined the role of RA in regulating IL-22 production by γδ T cells and innate lymphoid cells in intestinal inflammation. RA significantly enhanced IL-22 production by γδ T cells stimulated in vitro with IL-1β or IL-18 and IL-23. In vivo RA attenuated colon inflammation induced by dextran sodium sulfate treatment or Citrobacter rodentium infection. This was associated with a significant increase in IL-22 secretion by γδ T cells and innate lymphoid cells. In addition,RA treatment enhanced production of the IL-22-responsive antimicrobial peptides Reg3β and Reg3γ in the colon. The attenuating effects of RA on colitis were reversed by treatment with an anti-IL-22 neutralizing antibody,demonstrating that RA mediates protection by enhancing IL-22 production. To define the molecular events involved,we used chromatin immunoprecipitation assays and found that RA promoted binding of RA receptor to the IL-22 promoter in γδ T cells. Our findings provide novel insights into the molecular events controlling IL-22 transcription and suggest that one key outcome of RA signaling may be to shape early intestinal immune responses by promoting IL-22 synthesis by γδ T cells and innate lymphoid cells. View Publication

The molecular and cellular requirements for the development of different populations of human dendritic cells (DC) were studied. Conditions were defined that support DC production from lymphoid progenitors but that fail to induce DC formation from peripheral monocytes. The production of these lymphoid-related DC was severely blocked when hematopoietic progenitors overexpressed Ik7,a mutant dominant-negative Ikaros protein. In contrast,Ik7 did not block the formation of DC in conditions supporting the development of monocyte-derived DC. Furthermore,Ik7 did not block the formation of monocyte/macrophages and enhanced granulopoiesis. One of the molecular mechanisms mediated by Ik7 appears to be down-regulation of the flt3-receptor mRNA. Thus,distinct signals control the formation of DC demonstrating that some aspects of DC diversity are determined in part by distinct molecular cues at the hematopoietic level. (Blood. 2000;95:128-137) View Publication

In congenital mitochondrial DNA (mtDNA) disorders,a mixture of normal and mutated mtDNA (termed heteroplasmy) exists at varying levels in different tissues,which determines the severity and phenotypic expression of disease. Pearson marrow pancreas syndrome (PS) is a congenital bone marrow failure disorder caused by heteroplasmic deletions in mtDNA. The cause of the hematopoietic failure in PS is unknown,and adequate cellular and animal models are lacking. Induced pluripotent stem (iPS) cells are particularly amenable for studying mtDNA disorders,as cytoplasmic genetic material is retained during direct reprogramming. Here,we derive and characterize iPS cells from a patient with PS. Taking advantage of the tendency for heteroplasmy to change with cell passage,we isolated isogenic PS-iPS cells without detectable levels of deleted mtDNA. We found that PS-iPS cells carrying a high burden of deleted mtDNA displayed differences in growth,mitochondrial function,and hematopoietic phenotype when differentiated in vitro,compared to isogenic iPS cells without deleted mtDNA. Our results demonstrate that reprogramming somatic cells from patients with mtDNA disorders can yield pluripotent stem cells with varying burdens of heteroplasmy that might be useful in the study and treatment of mitochondrial diseases. STEM CELLS2013;31:1287–1297 View Publication

Helminthic infections modulate host immunity and may protect people in less-developed countries from developing immunological diseases. In a murine colitis model,the helminth Heligmosomoides polygyrus bakeri prevents colitis via induction of regulatory dendritic cells (DCs). The mechanism driving the development of these regulatory DCs is unexplored. There is decreased expression of the intracellular signaling pathway spleen tyrosine kinase (Syk) in intestinal DCs from H. polygyrus bakeri-infected mice. To explore the importance of this observation,it was shown that intestinal DCs from DC-specific Syk(-/-) mice were powerful inhibitors of murine colitis,suggesting that loss of Syk was sufficient to convert these cells into their regulatory phenotype. DCs sense gut flora and damaged epithelium via expression of C-type lectin receptors,many of which signal through the Syk signaling pathway. It was observed that gut DCs express mRNA encoding for C-type lectin (CLEC) 7A,CLEC9A,CLEC12A,and CLEC4N. H. polygyrus bakeri infection downmodulated CLEC mRNA expression in these cells. Focusing on CLEC7A,which encodes for the dectin-1 receptor,flow analysis showed that H. polygyrus bakeri decreases dectin-1 expression on the intestinal DC subsets that drive Th1/Th17 development. DCs become unresponsive to the dectin-1 agonist curdlan and fail to phosphorylate Syk after agonist stimulation. Soluble worm products can block CLEC7A and Syk mRNA expression in gut DCs from uninfected mice after a brief in vitro exposure. Thus,downmodulation of Syk expression and phosphorylation in intestinal DCs could be important mechanisms through which helminths induce regulatory DCs that limit colitis. View Publication

Chronic hepatitis C virus (HCV) infection is characterized by diminished numbers and function of HCV-reactive T cells and impaired responses to immunization. Because host response to viral infection likely involves TLR signaling,we examined whether chronic HCV infection impairs APC response to TLR ligand and contributes to the origin of dysfunctional T cells. Freshly purified myeloid dendritic cells (MDC) and plasmacytoid DC (PDC) obtained from subjects with chronic HCV infection and healthy controls were exposed to TLR ligands (poly(I:C),R-848,or CpG),in the presence or absence of cytokine (TNF-alpha or IL-3),and examined for indices of maturation and for their ability to activate allogeneic naive CD4 T cells to proliferate and secrete IFN-gamma. TLR ligand was observed to enhance both MDC and PDC activation of naive CD4 T cells. Although there was increased CD83 and CD86 expression on MDC from HCV-infected persons,the ability of MDC to activate naive CD4 T cells in the presence or absence of poly(I:C) or TNF-alpha did not differ between HCV-infected and healthy control subjects. In contrast,PDC from HCV-infected persons had reduced activation marker (HLA-DR) and cytokine (IFN-alpha) expression upon R-848 stimulation,and these were associated with impaired activation of naive CD4 T cells. These data indicate that an impaired PDC responsiveness to TLR ligation may play an important role in the fundamental and unexplained failure to induce new T cell responses to HCV Ags and to other new Ags as a consequence of HCV infection. View Publication

Bone marrow mesenchymal stem/stromal cells (BM-MSCs) have immunoregulatory properties and have been used as immune regulators for the treatment of graft-versus-host disease (GVHD). Human dental tissue mesenchymal stem cells (DT-MSCs) constitute an attractive alternative to BM-MSCs for potential clinical applications because of their accessibility and easy preparation. The aim of this in vitro study was to compare MSCs from dental pulp (DP-MSCs),gingival tissue (G-MSCs),and periodontal ligament (PDL-MSCs) in terms of their immunosuppressive properties against lymphoid cell populations enriched for CD3+ T cells to determine which MSCs would be the most appropriate for in vivo immunoregulatory applications. BM-MSCs were included as the gold standard. Our results demonstrated,in vitro,that MSCs from DP,G,and PDL showed immunoregulatory properties similar to those from BM,in terms of the cellular proliferation inhibition of both CD4+- and CD8+-activated T-cells. This reduced proliferation in cell co-cultures correlated with the production of interferon-$\gamma$ and tumor necrosis factor alpha (TNF-$\alpha$) and the upregulation of programmed death ligand 1 (PD-L1) in MSCs and cytotoxic T-cell-associated Ag-4 (CTLA-4) in T-cells and increased interleukin-10 and prostaglandin E2 production. Interestingly,we observed differences in the production of cytokines and surface and secreted molecules that may participate in T-cell immunosuppression in co-cultures in the presence of DT-MSCs compared with BM-MSCs. Importantly,MSCs from four sources favored the generation of T-cell subsets displaying the regulatory phenotypes CD4+CD25+Foxp3+ and CD4+CD25+CTLA-4+. Our results in vitro indicate that,in addition to BM-MSCs,MSCs from all of the dental sources analyzed in this study might be candidates for future therapeutic applications. View Publication

Triple-negative breast cancer (TNBC) remains a significant challenge in oncology,contributing to a significant portion of cancer-related deaths among women. Current therapeutic options,including chemotherapy,surgery,radiation,and hormonal targeting therapies,exhibit limited efficacy,necessitating the exploration of innovative treatment modalities. The emergence of drug resistance and the persistence of cancer stem cells (CSCs) further emphasize the urgent need for novel therapeutic strategies. In this context,natural killer cell-derived extracellular vesicles (NK-EVs) have emerged as a promising cell-free therapeutic approach that exhibits high tumor infiltration and cytotoxicity against cancer cells and CSCs. This study aims to investigate the efficacy of NK-EVs as a therapeutic strategy for TNBC using various clinically relevant models,including patient-derived xenografts. Pathway analysis suggests strong activation of apoptosis via canonical caspase activation,as well as necrosis,thereby confirming the important cytotoxic effect of NK-EVs. Interestingly,NK-EVs were also found to suppress TNBC CSCs by disrupting their functionality and viability,and NK-EV treatment increased the expression of apoptosis markers in both CSCs and non-CSCs. By elucidating the therapeutic efficacy and translational potential of NK-EV-based interventions in TNBC,these findings offer critical insights for the development of future immunotherapeutic strategies against this aggressive subtype of breast cancer. View Publication

We studied the actions of geldanamycin (GA) and herbimycin A (HMA),inhibitors of the chaperone proteins Hsp90 and GRP94,on B chronic lymphocytic leukemia (CLL) cells in vitro. Both drugs induced apoptosis of the majority of CLL isolates studied. Whereas exposure to 4-hour pulses of 30 to 100 nM GA killed normal B lymphocytes and CLL cells with similar dose responses,T lymphocytes from healthy donors as well as those present in the CLL isolates were relatively resistant. GA,but not HMA,showed a modest cytoprotective effect toward CD34+ hematopoietic progenitors from normal bone marrow. The ability of bone marrow progenitors to form hematopoietic colonies was unaffected by pulse exposures to GA. Both GA and HMA synergized with chlorambucil and fludarabine in killing a subset of CLL isolates. GA- and HMA-induced apoptosis was preceded by the up-regulation of the stress-responsive chaperones Hsp70 and BiP. Both ansamycins also resulted in down-regulation of Akt protein kinase,a modulator of cell survival. The relative resistance of T lymphocytes and of CD34+ bone marrow progenitors to GA coupled with its ability to induce apoptosis following brief exposures and to synergize with cytotoxic drugs warrant further investigation of ansamycins as potential therapeutic agents in CLL. View Publication

IL-27 is formed by the association of a cytokine subunit,p28,with the soluble cytokine receptor EBV-induced gene 3 (EBI3). The IL-27R comprises gp130 and WSX-1. The marked difference between EBI3(-/-) and WSX-1(-/-) mice suggests that p28 has functions independent of EBI3. We have identified an alternative secreted complex formed by p28 and the soluble cytokine receptor cytokine-like factor 1 (CLF). Like IL-27,p28/CLF is produced by dendritic cells and is biologically active on human NK cells,increasing IL-12- and IL-2-induced IFN-gamma production and activation marker expression. Experiments with Ba/F3 transfectants indicate that p28/CLF activates cells expressing IL-6Ralpha in addition to the IL-27R subunits. When tested on CD4 and CD8 T cells,p28/CLF induces IL-6Ralpha-dependent STAT1 and STAT3 phosphorylation. Furthermore,p28/CLF inhibits CD4 T cell proliferation and induces IL-17 and IL-10 secretion. These results indicate that p28/CLF may participate in the regulation of NK and T cell functions by dendritic cells. The p28/CLF complex engages IL-6R and may therefore be useful for therapeutic applications targeting cells expressing this receptor. Blocking IL-6R using humanized mAbs such as tocilizumab has been shown to be beneficial in pathologies like rheumatoid arthritis and juvenile idiopathic arthritis. The identification of a new IL-6R ligand is therefore important for a complete understanding of the mechanism of action of this emerging class of immunosuppressors. View Publication

Previous studies from this laboratory have demonstrated that fibroblast growth factor 1 together with a number of co-activator molecules (dopamine,TPA,IBMX/forskolin),will induce the expression of the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) in 10% of human neurons (hNTs) derived from the NT2 cell line [10]. In the present study,we found that TH induction was increased to nearly 75% in hNTs when cells were permitted to age 2 weeks in culture prior to treatment with the differentiation cocktail. This high level of TH expression was sustained 7 days after removal of the differentiating agents from the media. Moreover,the induced TH present in these cells was enzymatically active,resulting in the production of low levels of dopamine (DA) and its metabolite DOPAC. These findings suggest that hNTs may provide an important tissue culture model for the study of factors regulating TH gene expression in human neurons. Moreover,hNTs may serve,in vivo,as a source of human DA neurons for use in transplantation therapies. View Publication

Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that can cause severe disease,including congenital birth defects during pregnancy(1). To develop candidate therapeutic agents against ZIKV,we isolated a panel of human monoclonal antibodies (mAbs) from subjects with prior ZIKV infection. A subset of mAbs recognized diverse epitopes on the envelope (E) protein and exhibited potently neutralizing activity. One of the most inhibitory mAbs,ZIKV-117,broadly neutralized infection of ZIKV strains corresponding to African,Asian,and American lineages. Epitope mapping studies revealed that ZIKV-117 recognized a unique quaternary epitope on the E protein dimer-dimer interface. We evaluated the therapeutic efficacy of ZIKV-117 in pregnant and non-pregnant mice. mAb treatment markedly reduced tissue pathology,placental and fetal infection,and mortality in mice. Thus,neutralizing human mAbs can protect against maternal-fetal transmission,infection and disease,and reveal important determinants for structure-based rational vaccine design efforts. View Publication

Mutation of highly conserved residues in transcription factors may affect protein-protein or protein-DNA interactions,leading to gene network dysregulation and human disease. Human mutations in GATA4,a cardiogenic transcription factor,cause cardiac septal defects and cardiomyopathy. Here,iPS-derived cardiomyocytes from subjects with a heterozygous GATA4-G296S missense mutation showed impaired contractility,calcium handling,and metabolic activity. In human cardiomyocytes,GATA4 broadly co-occupied cardiac enhancers with TBX5,another transcription factor that causes septal defects when mutated. The GATA4-G296S mutation disrupted TBX5 recruitment,particularly to cardiac super-enhancers,concomitant with dysregulation of genes related to the phenotypic abnormalities,including cardiac septation. Conversely,the GATA4-G296S mutation led to failure of GATA4 and TBX5-mediated repression at non-cardiac genes and enhanced open chromatin states at endothelial/endocardial promoters. These results reveal how disease-causing missense mutations can disrupt transcriptional cooperativity,leading to aberrant chromatin states and cellular dysfunction,including those related to morphogenetic defects. View Publication