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

Unconjugated mAbs have emerged as useful cancer therapeutics. Ab-dependent cellular cytotoxicity (ADCC) is believed to be a major antitumor mechanism of some anticancer Abs. However,the factors that regulate the magnitude of ADCC are incompletely understood. In this study,we described the relationship between Ab affinity and ADCC. A series of human IgG1 isotype Abs was created from the anti-HER2/neu (also named c-erbB2) C6.5 single-chain Fv (scFv) and its affinity mutants. The scFv affinities range from 10(-7) to 10(-11) M,and the IgG Abs retain the affinities of the scFv from which they were derived. The apparent affinity of the Abs ranged from nearly 10(-10) M (the lowest affinity variant) to almost 10(-11) M (the other variants). The IgG molecules were tested for their ability to elicit ADCC in vitro against three tumor cell lines with differing levels of HER2/neu expression using unactivated human PBMC from healthy donors as the effector cells. The results demonstrated that both the apparent affinity and intrinsic affinity of the Abs studied regulate ADCC. High-affinity tumor Ag binding by the IgGs led to the most efficient and powerful ADCC. Tumor cells expressing high levels of HER2/neu are more susceptible to the ADCC triggered by Abs than the cells expressing lower amounts of HER2/neu. These findings justify the examination of high affinity Abs for ADCC promotion. Because high affinity may impair in vivo tumor targeting,a careful examination of Ab structure to function relationships is required to develop optimized therapeutic unconjugated Abs. View Publication

TLR4 is a unique TLR because downstream signaling occurs via two separate pathways,as follows: MyD88 and Toll IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-beta (TRIF). In this study,we compared and contrasted the interplay of these pathways between murine dendritic cells (DCs) and macrophages during LPS stimulation. During TLR4 activation,neither pathway on its own was critical for up-regulation of costimulatory molecules in DCs,whereas the up-regulation of costimulatory molecules was largely TRIF dependent in macrophages. LPS-induced secreted factors,of which type I IFNs were one of the active components,played a larger role in promoting the up-regulation of costimulatory molecules in macrophages than DCs. In both cell types,MyD88 and TRIF pathways together accounted for the inflammatory response to LPS activation. Furthermore,signaling of both adaptors allowed maximal T cell priming by LPS-matured DCs,with MyD88 playing a larger role than TRIF. In sum,in our experimental systems,TRIF signaling plays a more important role in LPS-induced macrophage activation than in DC activation. View Publication

During pregnancy the uterine decidua is populated by large numbers of natural killer (NK) cells with a phenotype CD56(superbright)CD16(-)CD9(+)KIR(+) distinct from both subsets of peripheral blood NK cells. Culture of highly purified CD16(+)CD9(-) peripheral blood NK cells in medium containing TGFbeta1 resulted in a transition to CD16(-)CD9(+) NK cells resembling decidual NK cells. Decidual stromal cells,when isolated and cultured in vitro,were found to produce TGFbeta1. Incubation of peripheral blood NK cells with conditioned medium from decidual stromal cells mirrored the effects of TGFbeta1. Similar changes may occur upon NK cell entry into the decidua or other tissues expressing substantial TGFbeta. In addition,Lin(-)CD34(+)CD45(+) hematopoietic stem/progenitor cells could be isolated from decidual tissue. These progenitors also produced NK cells when cultured in conditioned medium from decidual stromal cells supplemented with IL-15 and stem cell factor. View Publication

We have generated mice null for IFN-beta and report the diverse consequences of IFN-beta for both the innate and adaptive arms of immunity. Despite no abnormalities in the proportional balance of CD4 and CD8 T cell populations in the peripheral blood,thymus,and spleen of IFN-beta-/- mice,activated lymph node and splenic T lymphocytes exhibit enhanced T cell proliferation and decreased tumor necrosis factor alpha production,relative to IFN-beta+/+ mice. Notably,constitutive and induced expression of tumor necrosis factor alpha is reduced in the spleen and bone marrow (BM) macrophages,respectively,of IFN-beta-/- mice. We also observe an altered splenic architecture in IFN-beta-/- mice and a reduction in resident macrophages. We identify a potential defect in B cell maturation in IFN-beta-/- mice,associated with a decrease in B220+ve/high/CD43-ve BM-derived cells and a reduction in BP-1,IgM,and CD23 expression. Circulating IgM-,Mac-1-,and Gr-1-positive cells are also substantially decreased in IFN-beta-/- mice. The decrease in the numbers of circulating macrophages and granulocytes likely reflects defective maturation of primitive BM hematopoiesis in mice,shown by the reduction of colony-forming units,granulocyte-macrophage. We proceeded to evaluate the in vivo growth of malignant cells in the IFN-beta-/- background and give evidence that Lewis lung carcinoma-specific tumor growth is more aggressive in IFN-beta-/- mice. Taken altogether,our data suggest that,in addition to the direct growth-inhibitory effects on tumor cells,IFN-beta is required during different stages of maturation in the development of the immune system. View Publication

The impact of shipping temperatures and preservation media used during transport of either peripheral blood mononuclear cells (PBMCs) or Jurkat cells was assessed,in view of implementing of a proficiency testing scheme on mononuclear cell viability. Samples were analyzed before and after shipment at different temperatures (ambient temperature,dry ice,and liquid nitrogen) and in different preservation media (serum with cryoprotectant,commercial cryopreservation solution,and room temperature transport medium). Sample quality was assessed by viability assays (Trypan Blue dye exclusion,flow cytometry,Cell Analysis System cell counting (CASY)),and by ELISpot functional assay. The liquid nitrogen storage and shipment were found to be the most stable conditions to preserve cell viability and functionality. However,we show that alternative high quality shipment conditions for viable cells are dry ice shipment and commercial cryopreservation solution. These were also cost-efficient shipment conditions,satisfying the requirements of a proficiency testing scheme for viable mononuclear cells. Room temperature transport medium dramatically and adversely affected the integrity of mononuclear cells. View Publication

HIV-infected slow progressors (SP) represent a heterogeneous group of subjects who spontaneously control HIV infection without treatment for several years while showing moderate signs of disease progression. Under conditions that remain poorly understood,a subgroup of these subjects experience failure of spontaneous immunological and virological control. Here we determined the frequency of SP subjects who showed loss of HIV control within our Canadian Cohort of HIV(+) Slow Progressors and identified the proinflammatory cytokine IL-32 as a robust biomarker for control failure. Plasmatic levels of the proinflammatory isoforms of IL-32 (mainly β and γ) at earlier clinic visits positively correlated with the decline of CD4 T-cell counts,increased viral load,lower CD4/CD8 ratio and levels of inflammatory markers (sCD14 and IL-6) at later clinic visits. We present here a proof-of-concept for the use of IL-32 as a predictive biomarker for disease progression in SP subjects and identify IL-32 as a potential therapeutic target. View Publication

Innate Defense Regulators (IDRs) are short synthetic peptides that target the host innate immune system via an intracellular adaptor protein which functions at key signaling nodes. In this work,further details of the mechanism of action of IDRs have been discovered. The studies reported here show that the lead clinical IDR,SGX94,has broad-spectrum activity against Gram-negative and Gram-positive bacterial infections caused by intracellular or extracellular bacteria and also complements the actions of standard of care antibiotics. Based on in vivo and primary cell culture studies,this activity is shown to result from the primary action of SGX94 on tissue-resident cells and subsequent secondary signaling to activate myeloid-derived cells,resulting in enhanced bacterial clearance and increased survival. Data from non-clinical and clinical studies also show that SGX94 treatment modulates pro-inflammatory and anti-inflammatory cytokine levels,thereby mitigating the deleterious inflammatory consequences of innate immune activation. Since they act through host pathways to provide both broad-spectrum anti-infective capability as well as control of inflammation,IDRs are unlikely to be impacted by resistance mechanisms and offer potential clinical advantages in the fight against emerging and antibiotic resistant bacterial infections. View Publication

Mucosa-associated invariant T (MAIT) cells are a unique innate T cell subset that is necessary for rapid recruitment of activated CD4(+) T cells to the lungs after pulmonary F. tularensis LVS infection. Here,we investigated the mechanisms behind this effect. We provide evidence to show that MAIT cells promote early differentiation of CCR2-dependent monocytes into monocyte-derived DCs (Mo-DCs) in the lungs after F. tularensis LVS pulmonary infection. Adoptive transfer of Mo-DCs to MAIT cell-deficient mice (MR1(-/-) mice) rescued their defect in the recruitment of activated CD4(+) T cells to the lungs. We further demonstrate that MAIT cell-dependent GM-CSF production stimulated monocyte differentiation in vitro,and that in vivo production of GM-CSF was delayed in the lungs of MR1(-/-) mice. Finally,GM-CSF-deficient mice exhibited a defect in monocyte differentiation into Mo-DCs that was phenotypically similar to MR1(-/-) mice. Overall,our data demonstrate that MAIT cells promote early pulmonary GM-CSF production,which drives the differentiation of inflammatory monocytes into Mo-DCs. Further,this delayed differentiation of Mo-DCs in MR1(-/-) mice was responsible for the delayed recruitment of activated CD4(+) T cells to the lungs. These findings establish a novel mechanism by which MAIT cells function to promote both innate and adaptive immune responses. View Publication

Acute coronary syndromes (ACS) are precipitated by a rupture of the atherosclerotic plaque,often at the site of T cell and macrophage infiltration. Here,we show that plaque-infiltrating CD4 T cells effectively kill vascular smooth muscle cells (VSMC). VSMCs sensitive to T cell-mediated killing express the death receptor DR5 (TNF-related apoptosis-inducing ligand [TRAIL] receptor 2),and anti-TRAIL and anti-DR5 antibodies block T cell-mediated apoptosis. CD4 T cells that express TRAIL upon stimulation are expanded in patients with ACS and more effectively induce VSMC apoptosis. Adoptive transfer of plaque-derived CD4 T cells into immunodeficient mice that are engrafted with human atherosclerotic plaque results in apoptosis of VSMCs,which was prevented by coadministration of anti-TRAIL antibody. These data identify that the death pathway is triggered by TRAIL-producing CD4 T cells as a direct mechanism of VSMC apoptosis,a process which may lead to plaque destabilization. View Publication

Langerhans cells (LCs) constitute a subset of DCs that initiate immune responses in skin. Using leprosy as a model,we investigated whether expression of CD1a and langerin,an LC-specific C-type lectin,imparts a specific functional role to LCs. LC-like DCs and freshly isolated epidermal LCs presented nonpeptide antigens of Mycobacterium leprae to T cell clones derived from a leprosy patient in a CD1a-restricted and langerin-dependent manner. LC-like DCs were more efficient at CD1a-restricted antigen presentation than monocyte-derived DCs. LCs in leprosy lesions coexpress CD1a and langerin,placing LCs in position to efficiently present a subset of antigens to T cells as part of the host response to human infectious disease. View Publication