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

Macrophages have a central role in the pathogenesis of cryptococcosis since they are an important line of defense,serve as a site for fungal replication,and also can contribute to tissue damage. The objective of this study was to investigate the interaction of macrophages with cells from smooth-colony variants (SM) and mucoid-colony variants (MC) arising from phenotypic switching of Cryptococcus neoformans. Alveolar macrophages (AMs) isolated from SM- and MC-infected mice exhibited differences in gene and surface expression of PD-L1,PD-L2,and major histocompatibility class II (MHC-II). PD-L1 and PD-L2 are the ligands for PD1 and are differentially regulated in Th1- and Th2-type cells. In addition,macrophage activation in SM- and MC-infected mice was characterized as alternatively activated. Flow cytometric and cytokine analysis demonstrated that MC infection was associated with the emergence of Th17 cells and higher levels of interleukin-17 (IL-17) in lung tissue,which were reduced by AM depletion. In conclusion,our results indicate that macrophages play a significant role in maintaining damage-promoting inflammation in the lung during MC infection,which ultimately results in death. View Publication

Systemic lupus erythematosus (SLE) is characterized by increased vascular risk due to premature atherosclerosis independent of traditional risk factors. We previously proposed that IFN-α plays a crucial role in premature vascular damage in SLE. IFN-α alters the balance between endothelial cell apoptosis and vascular repair mediated by endothelial progenitor cells (EPCs) and myeloid circulating angiogenic cells (CACs). In this study,we demonstrate that IFN-α promotes an antiangiogenic signature in SLE and control EPCs/CACs,characterized by transcriptional repression of IL-1α and β,IL-1R1,and vascular endothelial growth factor A,and upregulation of IL-1R antagonist and the decoy receptor IL-1R2. IL-1β promotes significant improvement in the functional capacity of lupus EPCs/CACs,therefore abrogating the deleterious effects of IFN-α. The beneficial effects from IL-1 are mediated,at least in part,by increases in EPC/CAC proliferation,by decreases in EPC/CAC apoptosis,and by preventing the skewing of CACs toward nonangiogenic pathways. IFN-α induces STAT2 and 6 phosphorylation in EPCs/CACs,and JAK inhibition abrogates the transcriptional antiangiogenic changes induced by IFN-α in these cells. Immunohistochemistry of renal biopsies from patients with lupus nephritis,but not anti-neutrophil cytoplasmic Ab-positive vasculitis,showed this pathway to be operational in vivo,with increased IL-1R antagonist,downregulation of vascular endothelial growth factor A,and glomerular and blood vessel decreased capillary density,compared with controls. Our study introduces a novel putative pathway by which type I IFNs may interfere with vascular repair in SLE through repression of IL-1-dependent pathways. This could promote atherosclerosis and loss of renal function in this disease. View Publication

Phagocytic receptors must diffuse laterally to become activated upon clustering by multivalent targets. Receptor diffusion,however,can be obstructed by transmembrane proteins (pickets") that are immobilized by interacting with the cortical cytoskeleton. The molecular identity of these pickets and their role in phagocytosis have not been defined. We used single-molecule tracking to study the interaction between Fcγ receptors and CD44 an abundant transmembrane protein capable of indirect association with F-actin hence likely to serve as a picket. CD44 tethers reversibly to formin-induced actin filaments curtailing receptor diffusion. Such linear filaments predominate in the trailing end of polarized macrophages where receptor mobility was minimal. Conversely receptors were most mobile at the leading edge where Arp2/3-driven actin branching predominates. CD44 binds hyaluronan anchoring a pericellular coat that also limits receptor displacement and obstructs access to phagocytic targets. Force must be applied to traverse the pericellular barrier enabling receptors to engage their targets. View Publication

Viral infection triggers induction of antiviral cytokines and effectors,which are critical mediators of innate antiviral immune response. It has been shown that the processing body-associated protein LSm14A is involved in the induction of antiviral cytokines in cell lines but in vivo evidence is lacking. By generating LSm14A-deficient mice,in this study,we show that LSm14A plays a critical and specific role in the induction of antiviral cytokines in dendritic cells (DCs) but not in macrophages and fibroblasts. Induction of antiviral cytokines triggered by the DNA viruses HSV-1 and murid herpesvirus 68 and the RNA virus vesicular stomatitis virus but not Sendai virus was impaired in Lsm14a(-/-) DCs,which is correlated to the functions of the adaptor protein MITA/STING in the antiviral signaling pathways. LSm14A deficiency specifically downregulated MITA/STING level in DCs by impairing its nuclear mRNA precursor processing and subsequently impaired antiviral innate and adaptive immune responses. Our findings reveal a nuclear mRNA precursor processing and cell-specific regulatory mechanism of antiviral immune responses. View Publication

SAMHD1 restricts human immunodeficiency virus type 1 (HIV-1) replication in myeloid cells and CD4(+) T cells,while Vpx can mediate SAMHD1 degradation to promote HIV-1 replication. Although the restriction mechanisms of SAMHD1 have been well-described,SAMHD1 expression and Vpx-mediated SAMHD1 degradation during chronic HIV-1 infection were poorly understood. Flow cytometric analysis was used to directly visualize ex vivo,and after in vitro SIV-Vpx treatment,SAMHD1 expression in CD4(+) T cells and monocytes. Here we report activated CD4(+) T cells without SAMHD1 expression were severely reduced,and SAMHD1 in CD4(+) T cells became susceptible to SIV-Vpx mediated degradation during chronic HIV-1 infection,which was absent from uninfected donors. These alterations were irreversible,even after long-term fully suppressive antiretroviral treatment. Although SAMHD1 expression in CD4(+) T cells and monocytes was not found to correlate with plasma viral load,Vpx-mediated SAMHD1 degradation was associated with indicators of immune activation. In vitro assays further revealed that T-cell activation and an upregulated IFN-I pathway contributed to these altered SAMHD1 properties. These findings provide insight into how immune activation during HIV-1 infection leads to irreparable aberrations in restriction factors and in subsequent viral evasion from host antiviral defenses. View Publication

We have recently shown that proteasome inhibitor PS-341 induces apoptosis in drug-resistant multiple myeloma (MM) cells,inhibits binding of MM cells in the bone marrow microenvironment,and inhibits cytokines mediating MM cell growth,survival,drug resistance,and migration in vitro. PS-341 also inhibits human MM cell growth and prolongs survival in a SCID mouse model. Importantly,PS-341 has achieved remarkable clinical responses in patients with refractory relapsed MM. We here demonstrate molecular mechanisms whereby PS-341 mediates anti-MM activity by inducing p53 and MDM2 protein expression; inducing the phosphorylation (Ser15) of p53 protein; activating c-Jun NH(2)-terminal kinase (JNK),caspase-8,and caspase-3; and cleaving the DNA protein kinase catalytic subunit,ATM,and MDM2. Inhibition of JNK activity abrogates PS-341-induced MM cell death. These studies identify molecular targets of PS-341 and provide the rationale for the development of second-generation,more targeted therapies. View Publication

Depletion of CD4+CD25+ regulatory T cells (Treg) by treatment with alphaCD25 antibody synergizes with vaccination protocols to engender protective immunity in mice. The effectiveness of targeting CD25 to eliminate Treg is limited by the fact that CD25,the low-affinity interleukin-2 receptor,is up-regulated on conventional T cells. At present,foxp3 is the only product known to be exclusively expressed in Treg of mice. However,foxp3 is not expressed on the cell surface and hence cannot be targeted with antibodies. In this study,we tested the hypothesis that vaccination of mice against foxp3,a self-antigen expressed also in the thymus,is capable of stimulating foxp3-specific CTL that will cause the depletion of Treg and enhanced antitumor immunity. Vaccination of mice with foxp3 mRNA-transfected dendritic cells elicited a robust foxp3-specific CTL response and potentiated vaccine-induced protective immunity comparably with that of alphaCD25 antibody administration. In contrast to alphaCD25 antibody treatment,repeated foxp3 vaccination did not interfere with vaccine-induced protective immunity. Importantly,foxp3 vaccination led to the preferential depletion of foxp3-expressing Treg in the tumor but not in the periphery,whereas alphaCD25 antibody treatment led to depletion of Treg in both the tumor and the periphery. Targeting foxp3 by vaccination offers a specific and simpler protocol for the prolonged control of Treg that may be associated with reduced risk of autoimmunity,introducing an approach whereby specific depletion of cells is not limited to targeting products expressed on the cell surface. View Publication

Downregulation of host gene expression is one of the many strategies employed by intracellular pathogens such as Mycobacterium tuberculosis (MTB) to survive inside the macrophages and cause disease. The underlying molecular mechanism behind the downregulation of host defense gene expression is largely unknown. In this study we explored the role of histone deacetylation in macrophages in response to infection by virulent MTB H37Rv in manipulating host gene expression. We show a significant increase in the levels of HDAC1 with a concomitant and marked reduction in the levels of histone H3-acetylation in macrophages containing live,but not killed,virulent MTB. Additionally,we show that HDAC1 is recruited to the promoter of IL-12B in macrophages infected with live,virulent MTB,and the subsequent hypoacetylation of histone H3 suppresses the expression of this gene which plays a key role in initiating Th1 responses. By inhibiting immunologically relevant kinases,and by knockdown of crucial transcriptional regulators,we demonstrate that protein kinase-A (PKA),CREB,and c-Jun play an important role in regulating HDAC1 level in live MTB-infected macrophages. By chromatin immunoprecipitation (ChIP) analysis,we prove that HDAC1 expression is positively regulated by the recruitment of c-Jun to its promoter. Knockdown of HDAC1 in macrophages significantly reduced the survival of intracellular MTB. These observations indicate a novel HDAC1-mediated epigenetic modification induced by live,virulent MTB to subvert the immune system to survive and replicate in the host. View Publication

The p110δ isoform of PI3K is known to play an important role in immunity,yet its contribution to CTL responses has not been fully elucidated. Using murine p110δ-deficient CD8(+) T cells,we demonstrated a critical role for the p110δ subunit in the generation of optimal primary and memory CD8(+) T cell responses. This was demonstrated in both acute viral and intracellular bacterial infections in mice. We show that p110δ signaling is required for CD8(+) T cell activation,proliferation and effector cytokine production. We provide evidence that the effects of p110δ signaling are mediated via Akt activation and through the regulation of TCR-activated oxidative phosphorylation and aerobic glycolysis. In light of recent clinical trials that employ drugs targeting p110δ in certain cancers and other diseases,our study suggests caution in using these drugs in patients,as they could potentially increase susceptibility to infectious diseases. These studies therefore reveal a novel and direct role for p110δ signaling in in vivo CD8(+) T cell immunity to microbial pathogens. View Publication

Human melanoma cells express various tumour antigens that are recognized by CD8(+) cytotoxic T lymphocytes (CTLs) and elicit tumour-specific responses in vivo. However,natural and therapeutically enhanced CTL responses in melanoma patients are of limited efficacy. The mechanisms underlying CTL effector phase failure when facing melanomas are still largely elusive. Here we show that,on conjugation with CTL,human melanoma cells undergo an active late endosome/lysosome trafficking,which is intensified at the lytic synapse and is paralleled by cathepsin-mediated perforin degradation and deficient granzyme B penetration. Abortion of SNAP-23-dependent lysosomal trafficking,pH perturbation or impairment of lysosomal proteolytic activity restores susceptibility to CTL attack. Inside the arsenal of melanoma cell strategies to escape immune surveillance,we identify a self-defence mechanism based on exacerbated lysosome secretion and perforin degradation at the lytic synapse. Interfering with this synaptic self-defence mechanism might be useful in potentiating CTL-mediated therapies in melanoma patients. View Publication

Acute myeloid leukemia (AML) is the most common type of acute leukemia affecting older individuals at a median age of 67 years. Resistance to intensive induction chemotherapy is the major cause of death in elderly AML; hence novel treatment strategies are warranted. CD33-directed antibody-drug conjugates (Gemtuzumab ozogamicin) have been shown to improve overall survival,validating CD33 as a target for antibody-based therapy of AML. Here we report the in vitro efficacy of BI 836858,a fully human,Fc-engineered,anti-CD33 antibody using AML cell lines and primary AML blasts as targets. BI 836858-opsonized AML cells significantly induced both autologous and allogeneic natural killer (NK)-cell degranulation and NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). In vitro treatment of AML blasts with decitabine (DAC) or 5-azacytidine,two hypomethylating agents that show efficacy in older patients,did not compromise BI 836858-induced NK cell-mediated ADCC. Evaluation of BI 836858-mediated ADCC in serial marrow AML aspirates in patients who received a ten-day course of DAC (pre-DAC,days 4,11 and 28 post-DAC) revealed significantly higher ADCC in samples at day 28 post-DAC when compared to pre-DAC treatment. Analysis of ligands (L) to activating receptors (NKG2D showed significantly increased NKG2DL expression in day 28 post-DAC samples compared to pre-DAC samples; when NKG2DL receptor was blocked using antibodies,BI 836858-mediated ADCC was significantly decreased,suggesting that DAC enhances AML blast susceptibility to BI 836858 by upregulating NKG2DL. These data provide a rationale for combination therapy of Fc-engineered antibodies such as BI 836858 with azanucleosides in elderly patients with AML. View Publication

The development of effective cancer vaccines remains an urgent,but as yet unmet,clinical need. This deficiency is in part due to an incomplete understanding of how to best invoke dendritic cells (DC) that are crucial for the induction of tumor-specific CD8(+) T cells capable of mediating durable protective immunity. In this regard,elevated expression of the transcription factor X box-binding protein 1 (XBP1) in DC appears to play a decisive role in promoting the ability of DC to cross-present Ags to CD8(+) T cells in the therapeutic setting. Delivery of DNA vaccines encoding XBP1 and tumor Ag to skin DC resulted in increased IFN-? production by plasmacytoid DC (pDC) from skin/tumor draining lymph nodes and the cross-priming of Ag-specific CD8(+) T cell responses associated with therapeutic benefit. Antitumor protection was dependent on cross-presenting Batf3(+) DC,pDC,and CD8(+) T cells. CD103(+) DC from the skin/tumor draining lymph nodes of the immunized mice appeared responsible for activation of Ag-specific naive CD8(+) T cells,but were dependent on pDC for optimal effectiveness. Similarly,human XBP1 improved the capacity of human blood- and skin-derived DC to activate human T cells. These data support an important intrinsic role for XBP1 in DC for effective cross-priming and orchestration of Batf3(+) DC-pDC interactions,thereby enabling effective vaccine induction of protective antitumor immunity. View Publication