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

Regions of the normal arterial intima predisposed to atherosclerosis are sites of ongoing monocyte trafficking and also contain resident myeloid cells with features of dendritic cells. However,the pathophysiological roles of these cells are poorly understood. Here we found that intimal myeloid cells underwent reverse transendothelial migration (RTM) into the arterial circulation after systemic stimulation of pattern-recognition receptors (PRRs). This process was dependent on expression of the chemokine receptor CCR7 and its ligand CCL19 by intimal myeloid cells. In mice infected with the intracellular pathogen Chlamydia muridarum,blood monocytes disseminated infection to the intima. Subsequent CCL19-CCR7-dependent RTM was critical for the clearance of intimal C. muridarum. This process was inhibited by hypercholesterolemia. Thus,RTM protects the normal arterial intima,and compromised RTM during atherogenesis might contribute to the intracellular retention of pathogens in atherosclerotic lesions. View Publication

T cell dysfunction is crucial to the pathogenesis of systemic lupus erythematosus (SLE); however,the molecular mechanisms involved in the deficient expression of the T cell receptor-associated CD3zeta chain in SLE are not clear. SLE T cells express abnormally increased levels of an alternatively spliced isoform of CD3zeta that lacks a 562-bp region in its 3'-untranslated region (UTR). We showed previously that two adenosine/uridine-rich elements (ARE) in this splice-deleted region of CD3zeta transcript are critical for the mRNA stability and protein expression of CD3zeta. In this study we show for the first time that the mRNA-stabilizing protein HuR binds to these two ARE bearing regions of CD3zeta 3'-UTR. Knockdown of HuR resulted in decreased expression of the CD3zeta chain,whereas overexpression led to the increase of CD3zeta chain levels. Additionally,overexpression of HuR in human T cells resulted in increased mRNA stability of CD3zeta. Our results identify the 3'-UTR of CD3zeta as a novel target for the mRNA-stabilizing protein HuR. Thus,the absence of two critical AREs in the alternatively spliced CD3zeta 3'-UTR found in SLE T cells may result in decreased HuR binding,representing a possible molecular mechanism contributing to the reduced stability and expression of CD3zeta in SLE. View Publication

Human immunodeficiency virus type 1 (HIV-1) isolates from 20 chronically infected patients who participated in a structured treatment interruption (STI) trial were studied to determine whether viral fitness influences reestablishment of viremia. Viruses derived from individuals who spontaneously controlled viremia had significantly lower in vitro replication capacities than viruses derived from individuals that did not control viremia after interruption of antiretroviral therapy (ART),and replication capacities correlated with pre-ART and post-STI viral set points. Of note,no clinically relevant improvement of viral loads upon STI occurred. Virus isolates from controlling and noncontrolling patients were indistinguishable in terms of coreceptor usage,genetic subtype,and sensitivity to neutralizing antibodies. In contrast,viruses from controlling patients exhibited increased sensitivity to inhibition by chemokines. Sensitivity to inhibition by RANTES correlated strongly with slower replication kinetics of the virus isolates,suggesting a marked dependency of these virus isolates on high coreceptor densities on the target cells. In summary,our data indicate that viral fitness is a driving factor in determining the magnitude of viral rebound and viral set point in chronic HIV-1 infection,and thus fitness should be considered as a parameter influencing the outcome of therapeutic intervention in chronic infection. View Publication

Two distinct dendritic cell (DC) subpopulations have been evidenced in mice on the basis of their differential CD8alpha expression and their localization in lymphoid organs. Several reports suggest that CD8alpha(+) and CD8alpha(-) DC subsets could be functionally different. In this study,using a panel of MHC class I- and/or class II-restricted peptides,we analyzed CD4(+) and CD8(+) T cell responses obtained after i.v. injection of freshly purified peptide-pulsed DC subsets. First,we showed that both DC subsets efficiently induce specific CTL responses and Th1 cytokine production in the absence of CD4(+) T cell priming. Second,we showed that in vivo activation of CD4(+) T cells by CD8alpha(+) or CD8alpha(-) DC,injected i.v.,leads to a nonpolarized Th response with production of both Th1 and Th2 cytokines. The CD8alpha(-) subset induced a higher production of Th2 cytokines such as IL-4 and IL-10 than the CD8alpha(+) subset. However,IL-5 was produced by CD4(+) T cells activated by both DC subsets. When both CD4(+) and CD8(+) T cells were primed by DC injected i.v.,a similar pattern of cytokines was observed,but,under these conditions,Th1 cytokines were mainly produced by CD8(+) T cells,while Th2 cytokines were produced by CD4(+) T cells. Thus,this study clearly shows that CD4(+) T cell responses do not influence the development of specific CD8(+) T cell cytotoxic responses induced either by CD8alpha(+) or CD8alpha(-) DC subsets. View Publication

In humans the majority of endothelial cells (EC) constitutively express MHC class II Ags. We know that in vitro ECs can activate CD45RO(+) B7-independent CD4(+) T cells to proliferate and produce IL-2. The in vivo correlate of this T cell response is not known,and here we have explored whether endothelial expression of MHC class II Ags affects the transendothelial migration of alloreactive CD4(+) CD45RO(+) B7-independent T cells. Alloreactive CD4(+) T cell clones and lines were generated against HLA-DR11,DR13,DR4,and DR1 MHC Ags,and their rates of migration across untreated EC line Eahy.926 (MHC class II negative) or Eahy.926 transfected with CIITA (EahyCIITA) to express DR11 and DR13 were investigated. The migrations of EahyCIITA-specific T cell clones and lines were retarded in a DR-specific manner,and retardation was reversed in the presence of mAb to DR Ag. When investigating the ability of T cells to proliferate in response to EahyCIITA before and after transmigration,migrated cells were still able to proliferate,but the frequency of EahyCIITA-specific cells was much reduced compared with that of nonmigrated cells. The use of fluorescently labeled T cells revealed that specific cells become trapped within the endothelial monolayer. Pretreatment of EahyCIITA with IFN-gamma restored the ability of DR11- or DR13-specific T cells to transmigrate and proliferate,thus abrogating DR-specific retardation. We conclude that cognate interaction between T cells and endothelial MHC class II initiates a stop signal possibly similar to an immunological synapse,but this is overcome in an inflammatory milieu. View Publication

The maintenance of homeostasis in the gut is a major challenge for the immune system. Here we demonstrate that the transcription factor MAF plays a central role in T cells for the prevention of gastro-intestinal inflammation. Conditional knock out mice lacking Maf in all T cells developed spontaneous late-onset colitis,correlating with a decrease of FOXP3+RORgammat+ T cells proportion,dampened IL-10 production in the colon and an increase of inflammatory TH17 cells. Strikingly,FOXP3+ specific conditional knock out mice for MAF did not develop colitis and demonstrated normal levels of IL-10 in their colon,despite the incapacity of regulatory T cells lacking MAF to suppress colon inflammation in Rag1-/- mice transferred with na{\{i}}ve CD4+ T cells. We showed that one of the cellular sources of IL-10 in the colon of these mice are TH17 cells. Thus MAF is critically involved in the maintenance of the gut homeostasis by regulating the balance between Treg and TH17 cells either at the level of their differentiation or through the modulation of their functions." View Publication

The role of human NK cells in viral infections is poorly understood. We used a cytokine flow-cytometry assay to simultaneously investigate the IFN-gamma response of NK and T lymphocytes to influenza A virus (fluA). When PBMCs from fluA-immune adult donors were incubated with fluA,IFN-gamma was produced by both CD56(dim) and CD56(bright) subsets of NK cells,as well as by fluA-specific T cells. Purified NK cells did not produce IFN-gamma in response to fluA,while depletion of T lymphocytes reduced to background levels the fluA-induced IFN-gamma production by NK cells,which indicates that T cells are required for the IFN-gamma response of NK cells. The fluA-induced IFN-gamma production of NK cells was suppressed by anti-IL-2 Ab,while recombinant IL-2 replaced the helper function of T cells for IFN-gamma production by NK cells. This indicates that IL-2 produced by fluA-specific T cells is involved in the T cell-dependent IFN-gamma response of NK cells to fluA. Taken together,these results suggest that at an early stage of recurrent viral infection,NK-mediated innate immunity to the virus is enhanced by preexisting virus-specific T cells. View Publication

Group 2 innate lymphoid cells (ILC2s) in the lung are stimulated by inhaled allergens. ILC2s do not directly recognize allergens but they are stimulated by cytokines including interleukin (IL)-33 released by damaged epithelium. In response to allergens,lung ILC2s produce T helper 2 cell type cytokines inducing T cell-independent allergic lung inflammation. Here we examined the fate of lung ILC2s upon allergen challenges. ILC2s proliferated and secreted cytokines upon initial stimulation with allergen or IL-33,and this phase was followed by a contraction phase as cytokine production ceased. Some ILC2s persisted long after the resolution of the inflammation as allergen-experienced ILC2s and responded to unrelated allergens more potently than naive ILC2s,mediating severe allergic inflammation. The allergen-experienced ILC2s exhibited a gene expression profile similar to that of memory T cells. The memory-like properties of allergen-experienced ILC2s may explain why asthma patients are often sensitized to multiple allergens. View Publication

Recent work investigating exercise induced changes in immunocompetence suggests that some of the ambiguity in the literature is resultant from different cell isolation protocols and mitogen selection. To understand this effect,we compared post-exercise measures of T cell activation and proliferation using two different stimulation methods (costimulation through CD28 or stimulation with phytohaemagglutinin [PHA]). Further,we investigated whether exercise induced changes are maintained when T cell isolation from whole blood is delayed overnight in either a room temperature or chilled (4°C) environment. As expected,an increased proliferation response was observed post-exercise in T cells isolated from whole blood of previously trained individuals immediately after blood collection. Also,cells stimulated with PHA after resting overnight in whole blood were not adversely impacted by the storage conditions. In contrast,allowing cells to rest overnight in whole blood prior to stimulation through CD28,lessened the proliferation observed by cells following exercise rendering both the room temperature and chilled samples closer to the results seen in the control condition. Changes in early markers of activation (CD25),followed a similar pattern,with activation in PHA stimulated cells remaining fairly robust after overnight storage; whereas cell activation following stimulation through CD3+CD28 was disproportionately decreased by the influence of overnight storage. These findings indicate that decisions regarding cell stimulation methods need to be paired with the timeline for T cell isolation from whole blood. These considerations will be especially important for field based studies of immunocompetence where there is a delay in getting whole blood samples to a lab for processing as well as clinical applications where a failure to isolate T cells in a timely manner may result in loss of the response of interest. View Publication

The antitumor activity of LPS was first described by Dr. William Coley. However,its role in lung cancer remains unclear. The aim of our study was to elucidate the dose-dependent effects of LPS (0.1-10 μg/mouse) in a mouse model of B16-F10-induced metastatic lung cancer. Lung tumor growth increased at 3 and 7 d after the administration of low-dose LPS (0.1 μg/mouse) compared with control mice. This was associated with an influx of plasmacytoid dendritic cells (pDCs),regulatory T cells,myeloid-derived suppressor cells,and CD8(+) regulatory T cells. In contrast,high-dose LPS (10 μg/mouse) reduced lung tumor burden and was associated with a greater influx of pDCs,as well as a stronger Th1 and Th17 polarization. Depletion of pDCs during low-dose LPS administration resulted in a decreased lung tumor burden. Depletion of pDCs during high-dose LPS treatment resulted in an increased tumor burden. The dichotomy in LPS effects was due to the phenotype of pDCs,which were immunosuppressive after the low-dose LPS,and Th1- and T cytotoxic-polarizing cells after the high-dose LPS. Adoptive transfer of T cells into nude mice demonstrated that CD8(+) T cells were responsible for pDC recruitment following low-dose LPS administration,whereas CD4(+) T cells were required for pDC influx after the high-dose LPS. In conclusion,our data suggest differential effects of low-dose versus high-dose LPS on pDC phenotype and tumor progression or regression in the lungs of mice. View Publication

HIV-1 infection leads to numerous B cell abnormalities,including hypergammaglobulinemia,nonspecific B cell activation,nonspecific class switching,increased cell turnover,breakage of tolerance,increased immature/transitional B cells,B cell malignancies,as well as a loss of capacity to generate and maintain memory,all of which contribute to a global impairment of the immune humoral compartment. Several cytokines and soluble factors,which are increased in sera of HIV-1-infected individuals,have been suggested to directly or indirectly contribute to these B cell dysfunctions,and one of these is the B cell-activating factor (BAFF). We report in this study that HIV-1 (X4- and R5-tropic) upregulates BAFF expression and secretion by human monocytes. Moreover,we show that the virus-mediated production of BAFF by monocytes relies on a type I IFN response by a small percentage of plasmacytoid dendritic cells (pDCs) present in the monocyte cultures. HIV-1-induced type I IFN by pDCs triggers BAFF production in both classical and intermediate monocytes,but not in nonclassical monocytes,which nonetheless display a very strong basal BAFF production. We report also that basal BAFF secretion was higher in monocytes obtained from females compared with those from male donors. This study provides a novel mechanistic explanation for the increased BAFF levels observed during HIV-1 infection and highlights the importance of pDC/monocyte crosstalk to drive BAFF secretion. View Publication