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

Elotuzumab is a monoclonal antibody in development for multiple myeloma (MM) that targets CS1,a cell surface glycoprotein expressed on MM cells. In preclinical models,elotuzumab exerts anti-MM efficacy via natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC). CS1 is also expressed at lower levels on NK cells where it acts as an activating receptor. We hypothesized that elotuzumab may have additional mechanisms of action via ligation of CS1 on NK cells that complement ADCC activity. Herein,we show that elotuzumab appears to induce activation of NK cells by binding to NK cell CS1 which promotes cytotoxicity against CS1(+) MM cells but not against autologous CS1(+) NK cells. Elotuzumab may also promote CS1-CS1 interactions between NK cells and CS1(+) target cells to enhance cytotoxicity in a manner independent of ADCC. NK cell activation appears dependent on differential expression of the signaling intermediary EAT-2 which is present in NK cells but absent in primary,human MM cells. Taken together,these data suggest elotuzumab may enhance NK cell function directly and confer anti-MM efficacy by means beyond ADCC alone. View Publication

Neuropilin-1 (Nrp1) is a multifunctional protein,identified principally as a receptor for the class 3 semaphorins and members of the vascular endothelial growth factor (VEGF) family,but it is capable of other interactions. It is a marker of regulatory T cells (Tr),which often carry Nrp1 and latency-associated peptide (LAP)-TGF-beta1 (the latent form). The signaling TGF-beta1 receptors bind only active TGF-beta1,and we hypothesized that Nrp1 binds the latent form. Indeed,we found that Nrp1 is a high-affinity receptor for latent and active TGF-beta1. Free LAP,LAP-TGF-beta1,and active TGF-beta1 all competed with VEGF165 for binding to Nrp1. LAP has a basic,arginine-rich C-terminal motif similar to VEGF and peptides that bind to the b1 domain of Nrp1. A C-terminal LAP peptide (QSSRHRR) bound to Nrp1 and inhibited the binding of VEGF and LAP-TGF-beta1. We also analyzed the effects of Nrp1/LAP-TGF-beta1 coexpression on T cell function. Compared with Nrp1(-) cells,sorted Nrp1+ T cells had a much greater capacity to capture LAP-TGF-beta1. Sorted Nrp1(-) T cells captured soluble Nrp1-Fc,and this increased their ability to capture LAP-TGF-beta1. Conventional CD4+CD25(-)Nrp1(-) T cells coated with Nrp1-Fc/LAP-TGF-beta1 acquired strong Tr activity. Moreover,LAP-TGF-beta was activated by Nrp1-Fc and also by a peptide of the b2 domain of Nrp1 (RKFK; similar to a thrombospondin-1 peptide). Breast cancer cells,which express Nrp1,also captured and activated LAP-TGF-beta1 in a Nrp1-dependent manner. Thus,Nrp1 is a receptor for TGF-beta1,activates its latent form,and is relevant to Tr activity and tumor biology. View Publication

Pharmacological inhibitors of glycogen synthase kinase-3 (GSK-3) and cyclin-dependent kinases have a promising potential for applications against several neurodegenerative diseases such as Alzheimer's disease. Indirubins,a family of bis-indoles isolated from various natural sources,are potent inhibitors of several kinases,including GSK-3. Using the cocrystal structures of various indirubins with GSK-3beta,CDK2 and CDK5/p25,we have modeled the binding of indirubins within the ATP-binding pocket of these kinases. This modeling approach provided some insight into the molecular basis of indirubins' action and selectivity and allowed us to forecast some improvements of this family of bis-indoles as kinase inhibitors. Predicted molecules,including 6-substituted and 5,6-disubstituted indirubins,were synthesized and evaluated as CDK and GSK-3 inhibitors. Control,kinase-inactive indirubins were obtained by introduction of a methyl substitution on N1. View Publication

PURPOSE: The propensity of tumor cells to escape immune elimination could limit,if not defeat,the long-term benefits of effective immunotherapeutic protocols. Immunologic targeting of tumor stroma could significantly reduce the ability of tumors to evade immune elimination. Murine studies have shown that inducing immunity against angiogenesis-associated products engenders potent antitumor immunity without significant pathology. It is,however,not known whether T cells corresponding to stromal products are present in humans. In this study,we describe a method to screen for human stromal products that have not triggered significant tolerance and could therefore serve as candidate antigens for cancer immunotherapy. EXPERIMENTAL DESIGN: To identify candidates for human stromal antigens,we used an in vitro-screening method to determine whether dendritic cells transfected with mRNA encoding products,which are overexpressed in the tumor stroma,are capable of stimulating cytotoxic CD8(+) (CTL) responses from human peripheral blood mononuclear cells. RESULTS: CTL responses could be consistently generated against fibroblast activation protein (FAP) but not against matrix metalloproteinase-9 (MMP-9) or MMP-14. To enhance the immunogenicity of the mRNA-translated FAP product,a lysosomal targeting signal derived from lysosome-associated membrane protein-1 (LAMP-1) was fused to the COOH terminus of FAP to redirect the translated product into the class II presentation pathway. Dendritic cells transfected with mRNA encoding the FAP-LAMP fusion product stimulated enhanced CD4(+) and CD8(+) T-cell responses. CONCLUSION: This study identifies FAP,a protease preferentially expressed in tumor-associated fibroblasts,as a candidate human stromal antigen to target in the setting of cancer immunotherapy,and shows that differential expression of stromal products is not a sufficient criteria to indicate its immunogenicity in a vaccination setting. View Publication

Recent advances in immunometabolism link metabolic changes in stimulated macrophages to production of IL-1β,a crucial cytokine in the innate immune response to Mycobacterium tuberculosis. To investigate this pathway in the host response to M. tuberculosis,we performed metabolic and functional studies on human alveolar macrophages,human monocyte-derived macrophages,and murine bone marrow-derived macrophages following infection with the bacillus in vitro. M. tuberculosis infection induced a shift from oxidative phosphorylation to aerobic glycolysis in macrophages. Inhibition of this shift resulted in decreased levels of proinflammatory IL-1β and decreased transcription of PTGS2,increased levels of anti-inflammatory IL-10,and increased intracellular bacillary survival. Blockade or absence of IL-1R negated the impact of aerobic glycolysis on intracellular bacillary survival,demonstrating that infection-induced glycolysis limits M. tuberculosis survival in macrophages through induction of IL-1β. Drugs that manipulate host metabolism may be exploited as adjuvants for future therapeutic and vaccination strategies. View Publication

Human immunodeficiency virus type 1 (HIV-1) R5 isolates that predominantly use CCR5 as a coreceptor are frequently described as macrophage tropic. Here,we compare macrophage tropism conferred by HIV-1 R5 envelopes that were derived directly by PCR from patient tissue. This approach avoids potentially selective culture protocols used in virus isolation. Envelopes were amplified (i) from blood and semen of adult patients and (ii) from plasma of pediatric patients. The phenotypes of these envelopes were compared to those conferred by an extended panel of envelopes derived from brain and lymph node that we reported previously. Our results show that R5 envelopes vary by up to 1,000-fold in their capacity to confer infection of primary macrophages. Highly macrophage-tropic envelopes were predominate in brain but were infrequent in semen,blood,and lymph node samples. We also confirmed that the presence of N283 in the C2 CD4 binding site of gp120 is associated with HIV-1 envelopes from the brain but absent from macrophage-tropic envelopes amplified from blood and semen. Finally,we compared infection of macrophages,CD4(+) T cells,and peripheral blood mononuclear cells (PBMCs) conferred by macrophage-tropic and non-macrophage-tropic envelopes in the context of full-length replication competent viral clones. Non-macrophage-tropic envelopes conferred low-level infection of macrophages yet infected CD4(+) T cells and PBMCs as efficiently as highly macrophage-tropic brain envelopes. The lack of macrophage tropism for the majority of the envelopes amplified from lymph node,blood,and semen is striking and contrasts with the current consensus that R5 primary isolates are generally macrophage tropic. The extensive variation in R5 tropism reported here is likely to have an important impact on pathogenesis and on the capacity of HIV-1 to transmit. View Publication

We examined the interaction between OSU-03012 (also called AR-12) with phosphodiesterase 5 (PDE5) inhibitors to determine the role of the chaperone glucose-regulated protein (GRP78)/BiP/HSPA5 in the cellular response. Sildenafil (Viagra) interacted in a greater than additive fashion with OSU-03012 to kill stem-like GBM cells. Treatment of cells with OSU-03012/sildenafil: abolished the expression of multiple oncogenic growth factor receptors and plasma membrane drug efflux pumps and caused a rapid degradation of GRP78 and other HSP70 and HSP90 family chaperone proteins. Decreased expression of plasma membrane receptors and drug efflux pumps was dependent upon enhanced PERK-eIF2α-ATF4-CHOP signaling and was blocked by GRP78 over-expression. In vivo OSU-03012/sildenafil was more efficacious than treatment with celecoxib and sildenafil at killing tumor cells without damaging normal tissues and in parallel reduced expression of ABCB1 and ABCG2 in the normal brain. The combination of OSU-03012/sildenafil synergized with low concentrations of sorafenib to kill tumor cells,and with lapatinib to kill ERBB1 over-expressing tumor cells. In multiplex assays on plasma and human tumor tissue from an OSU-03012/sildenafil treated mouse,we noted a profound reduction in uPA signaling and identified FGF and JAK1/2 as response biomarkers for potentially suppressing the killing response. Inhibition of FGFR signaling and to a lesser extent JAK1/2 signaling profoundly enhanced OSU-03012/sildenafil lethality. View Publication

BACKGROUND Differentiation of pluripotent human embryonic stem cells (hESCs) to the cardiac lineage represents a potentially unlimited source of ventricular cardiomyocytes (VCMs),but hESC-VCMs are developmentally immature. Previous attempts to profile hESC-VCMs primarily relied on transcriptomic approaches,but the global proteome has not been examined. Furthermore,most hESC-CM studies focus on pathways important for cardiac differentiation,rather than regulatory mechanisms for CM maturation. We hypothesized that gene products and pathways crucial for maturation can be identified by comparing the proteomes of hESCs,hESC-derived VCMs,human fetal and human adult ventricular and atrial CMs. METHODS AND RESULTS Using two-dimensional-differential-in-gel electrophoresis,121 differentially expressed (textgreater1.5-fold; Ptextless0.05) proteins were detected. The data set implicated a role of the peroxisome proliferator-activated receptor $\$ in cardiac maturation. Consistently,WY-14643,a peroxisome proliferator-activated receptor $\$,increased fatty oxidative enzyme level,hyperpolarized mitochondrial membrane potential and induced a more organized morphology. Along this line,treatment with the thyroid hormone triiodothyronine increased the dynamic tension developed in engineered human ventricular cardiac microtissue by 3-fold,signifying their maturation. CONCLUSIONS We conclude that the peroxisome proliferator-activated receptor $\$ thyroid hormone pathways modulate the metabolism and maturation of hESC-VCMs and their engineered tissue constructs. These results may lead to mechanism-based methods for deriving mature chamber-specific CMs. View Publication

Human complement receptor type 2 (CR2/CD21) is a B lymphocyte membrane glycoprotein that plays a central role in the immune responses to foreign Ags as well as the development of autoimmunity to nuclear Ags in systemic lupus erythematosus. In addition to these three well-characterized ligands,C3d/iC3b,EBV-gp350,and CD23,a previous study has identified CR2 as a potential receptor for IFN-alpha. IFN-alpha,a multifunctional cytokine important in the innate immune system,has recently been proposed to play a major pathogenic role in the development of systemic lupus erythematosus in humans and mice. In this study,we have shown using surface plasmon resonance and ELISA approaches that CR2 will bind IFN-alpha in the same affinity range as the other three well-characterized ligands studied in parallel. In addition,we show that IFN-alpha interacts with short consensus repeat domains 1 and 2 in a region that serves as the ligand binding site for C3d/iC3b,EBV-gp350,and CD23. Finally,we show that treatment of purified human peripheral blood B cells with the inhibitory anti-CR2 mAb 171 diminishes the induction of IFN-alpha-responsive genes. Thus,IFN-alpha represents a fourth class of extracellular ligands for CR2 and interacts with the same domain as the other three ligands. Defining the role of CR2 as compared with the well-characterized type 1 IFN-alpha receptor 1 and 2 in mediating innate immune and autoimmune roles of this cytokine should provide additional insights into the biologic roles of this interaction. View Publication

Current evidence supports a model in which the low-affinity state of the platelet integrin alphaIIbbeta3 results from alphaIIbbeta3 adopting a bent conformation. To assess alphaIIbbeta3 biogenesis and how alphaIIbbeta3 initially adopts the bent conformation,we mapped the conformational states occupied by alphaIIb and beta3 during biogenesis using conformation-specific monoclonal antibodies (mAbs). We found that alphaIIbbeta3 complex formation was not limited by the availability of either free pro-alphaIIb or free beta3,suggesting that other molecules,perhaps chaperones,control complex formation. Five beta3-specific,ligand-induced binding site (LIBS) mAbs reacted with much or all free beta3 but not with beta3 when in complex with mature alphaIIb,suggesting that beta3 adopts its mature conformation only after complex formation. Conversely,2 alphaIIb-specific LIBS mAbs directed against the alphaIIb Calf-2 region adjacent to the membrane reacted with only minor fractions of free pro-alphaIIb,raising the possibility that pro-alphaIIb adopts a bent conformation early in biogenesis. Our data suggest a working model in which pro-alphaIIb adopts a bent conformation soon after synthesis,and then beta3 assumes its bent conformation by virtue of its interaction with the bent pro-alphaIIb. View Publication

Prostaglandin F2alpha inhibits adipose differentiation of primary culture of adipocyte precursors and of the adipogenic cell line 1246 in defined medium. In the present paper,we investigated the effect of FP receptor agonists cloprostenol and fluprostenol on the differentiation of newborn rat adipocyte precursors in primary culture. The results show that cloprostenol and fluprostenol are very potent inhibitors of adipose differentiation. Dose response studies indicate that both agonists are more potent than PGF2alpha in inhibiting adipocyte precursors differentiation. 50% inhibition of adipose differentiation was observed at a concentration of 3 x 10(-12) M for cloprostenol and 3 to 10 x 10(-11) M for fluprostenol respectively whereas the PGF2alpha concentration required to elicit the same effect was 10(-8) M. In contrast compounds structurally related to PGE2 such as 17-phenyl trinor PGE2 had no effect on adipose differentiation except when added at a 10,000-fold higher concentration. View Publication

Breast Cancer Stem (BCS) cells play critical roles in self-renewal,Multi Drug Resistance (MDR),differentiation and generation of secondary tumors. Conventional chemotherapy may efficiently kill the bulk of differentiated drug sensitive breast cancer cells,but not the MDR self-renewable BCS cells,leading to enrichment of the MDR BCS cells. In order to target the MDR BCS cells,we have isolated: 1) BCS cells from either breast cancer cell lines or fresh breast cancer specimens; 2) ATP binding cassette (ABC) transporter group G number 2 (ABCG2)-specific aptamers; and 3) BCS cell-binding aptamers. Interestingly,ABCG2-specific aptamers labeled the membrane surface of the ABCG2-expressing baby hamster kidney (BHK) cells,but stained whole cells of the BCS cells derived from mammospheres,implying that BCS cells might have much higher rate of endocytosis than the ABCG2-expressing BHK cells. In addition,5D3,a monoclonal antibody that recognizes the extracellular loops of ABCG2 protein,also stained whole BCS cells. Furthermore,BCS cell-binding aptamers stained whole BCS cells,but not the differentiated breast cancer MCF-7 cells. All these results support above conclusion that BCS cells might have high rate of endocytosis. Further experiments performed with aptamers and human transferrin or lactosylceramide showed that BCS cells do have much higher endocytosis rate than the differentiated breast cancer cells. Interestingly,clathrin dependent endocytosis inhibitors,such as monodansylcadaverine or sucrose,or caveolin-dependent endocytosis inhibitors,such as methyl-$$-cyclodextrin or genistein,can inhibit the internalization of transferrin or lactosylceramide into the differentiated breast cancer cells,but cannot block the internalization of these compounds into the BCS cells,suggesting that BCS cells undergo clathrin-independent and caveolin-independent endocytosis. Taken together,our data suggest that BCS cells have high rate of endocytosis and open the possibilities for delivering therapeutic agents directly into the MDR BCS cells with aptamer-coated liposomes. View Publication