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

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

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

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

The significant advances in the differentiation of human pluripotent stem (hPS) cells into pancreatic endocrine cells,including functional ?-cells,have been based on a detailed understanding of the underlying developmental mechanisms. However,the final differentiation steps,leading from endocrine progenitors to mono-hormonal and mature pancreatic endocrine cells,remain to be fully understood and this is reflected in the remaining shortcomings of the hPS cell-derived islet cells (SC-islet cells),which include a lack of ?-cell maturation and variability among different cell lines. Additional signals and modifications of the final differentiation steps will have to be assessed in a combinatorial manner to address the remaining issues and appropriate reporter lines would be useful in this undertaking. Here we report the generation and functional validation of hPS cell reporter lines that can monitor the generation of INS+ and GCG+ cells and their resolution into mono-hormonal cells (INSeGFP,INSeGFP/GCGmCHERRY) as well as ?-cell maturation (INSeGFP/MAFAmCHERRY) and function (INSGCaMP6). The reporter hPS cell lines maintained strong and widespread expression of pluripotency markers and differentiated efficiently into definitive endoderm and pancreatic progenitor (PP) cells. PP cells from all lines differentiated efficiently into islet cell clusters that robustly expressed the corresponding reporters and contained glucose-responsive,insulin-producing cells. To demonstrate the applicability of these hPS cell reporter lines in a high-content live imaging approach for the identification of optimal differentiation conditions,we adapted our differentiation procedure to generate SC-islet clusters in microwells. This allowed the live confocal imaging of multiple SC-islets for a single condition and,using this approach,we found that the use of the N21 supplement in the last stage of the differentiation increased the number of monohormonal ?-cells without affecting the number of ?-cells in the SC-islets. The hPS cell reporter lines and the high-content live imaging approach described here will enable the efficient assessment of multiple conditions for the optimal differentiation and maturation of SC-islets. View Publication

The clinical success of the immune checkpoint inhibitor (ICI) targeting programmed cell death protein 1 (PD-1) has revolutionized cancer treatment. However,the full potential of PD-1 blockade therapy remains unrealized,as response rates are still low across many cancer types. Interleukin-2 (IL-2)-based immunotherapies hold promise,as they can stimulate robust T cell expansion and enhance effector function - activities that could synergize potently with PD-1 blockade. Yet,IL-2 therapies also carry a significant drawback: they can trigger severe systemic toxicities and induce immune suppression by expanding regulatory T cells. To overcome the challenges of PD-1 blockade and IL-2 therapies while enhancing safety and efficacy,we have engineered a novel fusion protein,AWT020,combining a humanized anti-PD-1 nanobody and an engineered IL-2 mutein (IL-2c). The IL-2c component of AWT020 has been engineered to exhibit no binding to the IL-2 receptor alpha (IL-2Rα) subunit and attenuated affinity for the IL-2 receptor beta and gamma (IL-2Rβγ) complex,aiming to reduce systemic immune cell activation,thereby mitigating the severe toxicity often associated with IL-2 therapies. The anti-PD-1 antibody portion of AWT020 serves a dual purpose: it precisely delivers the IL-2c payload to tumor-infiltrating T cells while blocking the immune-inhibitory signals mediated by the PD-1 pathway. AWT020 showed significantly enhanced pSTAT5 signaling in PD-1 expressing cells and promoted the proliferation of activated T cells over natural killer (NK) cells. In preclinical studies using both anti-PD-1-sensitive and -resistant mouse tumor models,the mouse surrogate of AWT020 (mAWT020) demonstrated markedly enhanced anti-tumor efficacy compared to an anti-PD-1 antibody,IL-2,or the combination of an anti-PD-1 antibody and IL-2. In addition,the mAWT020 treatment was well-tolerated,with minimal signs of toxicity. Immune profiling revealed that mAWT020 preferentially expands CD8 + T cells within tumors,sparing peripheral T and NK cells. Notably,this selective tumoral T-cell stimulation enables potent tumor-specific T-cell responses,underscoring the molecule’s enhanced efficacy and safety. The AWT020 fusion protein offers a promising novel immunotherapeutic strategy by integrating PD-1 blockade and IL-2 signaling,conferring enhanced anti-tumor activity with reduced toxicity. View Publication

Deregulation of apoptosis,the physiological form of cell death,is closely associated with immunological diseases and cancer. Apoptosis is activated either by death receptor-driven or mitochondrial pathways,both of which may provide potential targets for novel anticancer drugs. Although several ligands stimulating death receptors have been described,the actual molecular events triggering the mitochondrial pathway are largely unknown. Here,we show initiation of apoptosis by gradual depletion of the intracellular coenzyme NAD+. We identified the first low molecular weight compound,designated FK866,which induces apoptosis by highly specific,noncompetitive inhibition of nicotinamide phosphoribosyltransferase (NAPRT),a key enzyme in the regulation of NAD+ biosynthesis from the natural precursor nicotinamide. Interference with this enzyme does not primarily intoxicate cells because the mitochondrial respiratory activity and the NAD+ -dependent redox reactions involved remain unaffected as long as NAD+ is not effectively depleted by catabolic reactions. Certain tissues,however,have a high turnover of NAD+ through its cleavage by enzymes like poly(ADP-ribose) polymerase. Such cells often rely on the more readily available nicotinamide pathway for NAD+ synthesis and undergo apoptosis after inhibition of NAPRT,whereas cells effectively using the nicotinic acid pathway for NAD+ synthesis remain unaffected. In support of this concept,FK866 effectively induced delayed cell death by apoptosis in HepG2 human liver carcinoma cells with an IC(50) of approximately 1 nM,did not directly inhibit mitochondrial respiratory activity,but caused gradual NAD+ depletion through specific inhibition of NAPRT. This enzyme,when partially purified from K562 human leukemia cells,was noncompetitively inhibited by FK866,and the inhibitor constants were calculated to be 0.4 nM for the enzyme/substrate complex (K(i)) and 0.3 nM for the free enzyme (K(i)'),respectively. Nicotinic acid and nicotinamide were both found to have antidote potential for the cellular effects of FK866. FK866 may be used for treatment of diseases implicating deregulated apoptosis such as cancer for immunosuppression or as a sensitizer for genotoxic agents. Furthermore,it may provide an important tool for investigation of the molecular triggers of the mitochondrial pathway leading to apoptosis through enabling temporal separation of NAD+ decrease from ATP breakdown and apoptosis by several days. View Publication

Patient-derived pancreatic ductal adenocarcinoma (PDAC) organoid systems show great promise for understanding the biological underpinnings of disease and advancing therapeutic precision medicine. Despite the increased use of organoids,the fidelity of molecular features,genetic heterogeneity,and drug response to the tumor of origin remain important unanswered questions limiting their utility. To address this gap in knowledge,primary tumor- and patient-derived xenograft (PDX)-derived organoids,and 2D cultures for in-depth genomic and histopathologic comparisons with the primary tumor were created. Histopathologic features and PDAC representative protein markers (e.g.,claudin 4 and CA19-9) showed strong concordance. DNA- and RNA-sequencing (RNAseq) of single organoids revealed patient-specific genomic and transcriptomic consistency. Single-cell RNAseq demonstrated that organoids are primarily a clonal population. In drug response assays,organoids displayed patient-specific sensitivities. In addition,the in vivo PDX response to FOLFIRINOX and gemcitabine/abraxane treatments were examined,which was recapitulated in vitro with organoids. This study has demonstrated that organoids are potentially invaluable for precision medicine as well as preclinical drug treatment studies because they maintain distinct patient phenotypes and respond differently to drug combinations and dosage. IMPLICATIONS: The patient-specific molecular and histopathologic fidelity of organoids indicate that they can be used to understand the etiology of the patient's tumor and the differential response to therapies and suggests utility for predicting drug responses. View Publication

The monocyte population in blood is considered a possible source of endothelial precursors. Because endothelial-specific receptor tyrosine kinases act as regulators of endothelial cell function,we investigated whether expression of the vascular endothelial growth factor receptor-2 (VEGFR-2) on monocytes is important for their endothelial-like functional capacity. Peripheral-blood monocytes expressing vascular endothelial growth factor receptor-2 (VEGFR-2),or CD14+/VEGFR-2+,were isolated,and their phenotypic,morphologic,and functional capacities were compared with those of monocytes negative for this marker (CD14+/VEGFR-2-). CD14+/VEGFR-2+ cells constituted approximately 2% +/- 0.5% of the total population of monocytes and 0.08% +/- 0.04% of mononuclear cells in blood. CD14+/VEGFR-2+ cells exhibited the potential to differentiate in vitro into cells with endothelial characteristics. The cells were efficiently transduced by a lentiviral vector driving expression of the green fluorescence protein (GFP). Transplantation of GFP-transduced cells into balloon-injured femoral arteries of nude mice significantly contributed to efficient reendothelialization. CD14+/VEGFR-2- did not exhibit any of these characteristics. These data demonstrate that the expression of VEGFR-2 on peripheral blood monocytes is essential for their endothelial-like functional capacity and support the notion of a common precursor for monocytic and endothelial cell lineage. Our results help clarify which subpopulations may restore damaged endothelium and may participate in the maintenance of vascular homeostasis. View Publication

Under standard culture conditions,tumor cells are exposed to 20% O(2),whereas the mean tumor oxygen levels within the tumor are much lower. We demonstrate,using low-passaged human tumor cell cultures established from glioma,that a reduction in the oxygen level in these cell cultures dramatically increases the percentage of CD133 expressing cells. View Publication

Mature mammary epithelial cells are generated from undifferentiated precursors through a hierarchical process,but the molecular mechanisms involved,particularly in the human mammary gland,are poorly understood. To address this issue,we isolated highly purified subpopulations of primitive bipotent and committed luminal progenitor cells as well as mature luminal and myoepithelial cells from normal human mammary tissue and compared their transcriptomes obtained using three different methods. Elements unique to each subset of mammary cells were identified,and changes that accompany their differentiation in vivo were shown to be recapitulated in vitro. These include a stage-specific change in NOTCH pathway gene expression during the commitment of bipotent progenitors to the luminal lineage. Functional studies further showed NOTCH3 signaling to be critical for this differentiation event to occur in vitro. Taken together,these findings provide an initial foundation for future delineation of mechanisms that perturb primitive human mammary cell growth and differentiation. View Publication

Human skin-migratory dendritic cells (DCs) have the ability to prime and bias Th1 and Th2 CD4+ T lymphocytes. However,whether human cutaneous DCs are capable of initiating proinflammatory Th17 responses remains undetermined. We report that skin-migratory DCs stimulate allogeneic naive CD4+ T cells that differentiate simultaneously into two distinct effector Th17 and Th1 populations capable of homing to the skin,where they induce severe cutaneous damage. Skin-migratory Langerhans cells (smiLCs) were the main cutaneous DC subset capable of inducing Th17 responses dependent on the combined effects of IL-15 and stabilized IL-6,which resulted in IL-6 trans-signaling of naive CD4+ T cells. Different from smiLCs,purified skin-migratory dermal DCs did not synthesize IL-15 and were unable to bias Th17 responses. Nevertheless,these dermal DCs were capable of differentiating Th17 cells in mixed leukocyte cultures supplemented with IL-15 and stabilized IL-6. Overall,our data demonstrate that human epidermal smiLCs induce Th17 responses by mechanisms different from those previously described and highlight the need to target clinical treatments based on these variations. View Publication