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

Accumulating evidence indicates that various oncogenic mutations interfere with normal myeloid differentiation of leukemogenic cells during the early process of acute myeloid leukemia (AML) development. Differentiation therapy is a therapeutic strategy capable of terminating leukemic expansion by reactivating the differentiation potential; however,the plasticity and instability of leukemia cells counteract the establishment of treatments aimed at irreversibly inducing and maintaining their differentiation states. On the basis of our previous observation that autophagy inhibitor treatment induces the accumulation of cytosolic DNA and activation of cytosolic DNA-sensor signaling selectively in leukemia cells,we herein examined the synergistic effect of cytosolic DNA-sensor signaling activation with conventional differentiation therapy on AML. The combined treatment succeeded in inducing irreversible differentiation in AML cell lines. Mechanistically,cytosolic DNA was sensed by absent in melanoma 2 (AIM2),a cytosolic DNA sensor. Activation of the AIM2 inflammasome resulted in the accumulation of p21 through the inhibition of its proteasomal degradation,thereby facilitating the myeloid differentiation. Importantly,the combined therapy dramatically reduced the total leukemia cell counts and proportion of blast cells in the spleens of AML mice. Collectively,these findings indicate that the autophagy inhibition-cytosolic DNA-sensor signaling axis can potentiate AML differentiation therapy. Clinical effects on AML therapy are closely associated with reactivating the normal myeloid differentiation potential in leukemia cells. This study shows that autophagosome formation inhibitors activate the cytosolic DNA-sensor signaling,thereby augmenting conventional differentiation therapy to induce irreversible differentiation and cell growth arrest in several types of AML cell lines. View Publication

Mycobacterium tuberculosis ( Mtb ),the causative agent of tuberculosis (TB),is the most common coinfection among people living with HIV-1. This coinfection is associated with accelerated HIV-1 disease progression and reduced survival. However,the impact of the HIV-1/TB coinfection on HIV-1 replication and latency in CD4 + T cells remains poorly studied. Using the acellular fraction of tuberculous pleural effusion (TB-PE),we investigated whether viral replication and HIV-1 latency in CD4 + T cells are affected by a TB-associated microenvironment. Our results revealed that TB-PE impaired T cell receptor-dependent cell activation and decreased HIV-1 replication in CD4 + T cells. Moreover,this immunosuppressive TB microenvironment promoted viral latency and inhibited HIV-1 reactivation. This study indicates that the TB-induced immune response may contribute to the persistence of the viral reservoir by silencing HIV-1 expression,allowing the virus to persist undetected by the immune system,and increasing the size of the latent HIV-1 reservoir. Subject areas: Immunology,Virology View Publication

Interleukin-17 (IL-17) plays a central role in the pathogenesis of various autoimmune diseases. Soluble CD14 (sCD14),a marker of innate immune activation,is elevated in several inflammatory conditions. However,its influence on IL-17 production and the differentiation of Th17 cells remains poorly understood. We found that sCD14 enhances Th17-associated cytokine production and up-regulates critical transcription factors such as STAT3 and RORC. Notably,sCD14's effect on Th17 polarization was mediated indirectly through autologous sCD14-treated peripheral blood mononuclear cell (PBMC) supernatant (sCD14-PBMC-Sup). Additionally,we identified a distinct cytokine profile enriched for pro-inflammatory cytokines and chemokines in sCD14-treated T cells,further reinforcing the Th17-promoting role of sCD14. Interestingly,gamma-aminobutyric acid (GABA),a metabolite elevated in sCD14-treated monocytes,was identified as a potential contributor to Th17 polarization. GABA supplementation in T-cell cultures enhanced IL-17A secretion,indicating its role as a signaling molecule in T-cell differentiation. Our findings also revealed the expansion of innate lymphoid cell (ILC)2/3-like cells in T-cell cultures exposed to sCD14-PBMC-Sup and GABA,highlighting the potential role of monocytes in Th17-mediated immunity. Furthermore,while sCD14 promoted Th17 polarization,it simultaneously impaired T-cell activation and proliferation,suggesting an immunosuppressive effect mediated by soluble factors released from monocytes. These results underscore the dual role of sCD14 in modulating T-cell responses,promoting Th17 differentiation while suppressing T-cell effector functions. This study identifies a previously unrecognized role for sCD14 in promoting Th17 induction,highlighting its contribution to immune regulation and its potential as a therapeutic target in Th17-driven autoimmune conditions. View Publication

Genomic abnormalities may accumulate in human embryonic stem cells (hESCs) during in vitro maintenance. Characterization of the mechanisms enabling survival and expansion of abnormal hESCs is important due to consequences of genetic changes for the therapeutic utilization of stem cells. Furthermore,these cells provide an excellent model to study transformation in vitro. We report here that the histone deacetylase proteins,HDAC1 and HDAC2,are increased in karyotypically abnormal hESCs when compared to their normal counterparts. Importantly,similar to many cancer cell lines,we found that HDAC inhibitors repress proliferation of the karyotypically abnormal hESCs,whereas normal cells are more resistant to the treatment. The decreased proliferation correlates with downregulation of HDAC1 and HDAC2 proteins,induction of the proliferation inhibitor,cyclin-dependent kinase inhibitor 1A (CDKN1A),and altered regulation of tumor suppressor protein Retinoblastoma 1 (RB1). Through genome-wide transcriptome analysis we have identified genes with altered expression and responsiveness to HDAC inhibition in abnormal cells. Most of these genes are linked to severe developmental and neurological diseases and cancers. Our results highlight the importance of epigenetic mechanisms in the regulation of genomic stability of hESCs,and provide valuable candidates for targeted and selective growth inhibition of karyotypically abnormal cells. textcopyright 2013 Elsevier B.V. View Publication

The intracellular expression of the programmed death receptor 1 (PD1) identifies a subset of naive T(reg) cells with enhanced suppressive ability; antigen stimulation results in the surface expression of PD1. Because the role of T(reg) impairments in multiple sclerosis (MS) is still contradictory,we analyzed naive PD1- and PD1+ T(reg) cells in peripheral blood and cerebrospinal fluid (CSF) of relapsing-remitting multiple sclerosis (RR-MS) patients and of healthy control subjects. Results showed that 1) CSF PD1- T(reg) cells were significantly augmented in MS patients; 2) PD1- T(reg) cells were significantly increased in the peripheral blood of patients with stable disease (SMS) compared to those with acute (AMS) disease,and in patients responding to glatiramer acetate (COPA) compared to AMS- and COPA-unresponsive patients; and 3) PD1+ T(reg) cells were similar in CSF and peripheral blood of all groups analyzed. PD1- T(reg) cells were not increased in the peripheral blood of interferon-beta (IFNbeta) -responsive patients,but the suppressive ability of T(reg) cells was significantly higher in SMS and in COPA- or IFNbeta-responsive compared to AMS- and COPA-unresponsive individuals. The data herein suggest that PD1- T(reg) cells play a pivotal role in MS and offer a biological explanation for disease relapse and for the mechanism associated with response to COPA and IFNbeta. View Publication

Bacillus anthracis secretes two bipartite toxins,edema toxin (ET) and lethal toxin (LT),which impair immune responses and contribute directly to the pathology associated with the disease anthrax. Edema factor,the catalytic subunit of ET,is an adenylate cyclase that impairs host defenses by raising cellular cyclic AMP (cAMP) levels. Synthetic cAMP analogues and compounds that raise intracellular cAMP levels lead to phenotypic and functional changes in dendritic cells (DCs). Here,we demonstrate that ET induces a maturation state in human monocyte-derived DCs (MDDCs) similar to that induced by lipopolysaccharide (LPS). ET treatment results in downregulation of DC-SIGN,a marker of immature DCs,and upregulation of DC maturation markers CD83 and CD86. Maturation of DCs by ET is accompanied by an increased ability to migrate toward the lymph node-homing chemokine macrophage inflammatory protein 3beta,like LPS-matured DCs. Interestingly,cotreating with LT differentially affects the ET-induced maturation of MDDCs while not inhibiting ET-induced migration. These findings reveal a mechanism by which ET impairs normal innate immune function and may explain the reported adjuvant effect of ET. View Publication

Amyotrophic lateral sclerosis (ALS) is an incurable neuromuscular disease that leads to a profound loss of life quality and premature death. Around 10% of the cases are inherited and ALS8 is an autosomal dominant form of familial ALS caused by mutations in the vamp-associated protein B/C (VAPB) gene. The VAPB protein is involved in many cellular processes and it likely contributes to the pathogenesis of other forms of ALS besides ALS8. A number of successful drug tests in ALS animal models could not be translated to humans underscoring the need for novel approaches. The induced pluripotent stem cells (iPSC) technology brings new hope,since it can be used to model and investigate diseases in vitro. Here we present an additional tool to study ALS based on ALS8-iPSC. Fibroblasts from ALS8 patients and their non-carrier siblings were successfully reprogrammed to a pluripotent state and differentiated into motor neurons. We show for the first time that VAPB protein levels are reduced in ALS8-derived motor neurons but,in contrast to over-expression systems,cytoplasmic aggregates could not be identified. Our results suggest that optimal levels of VAPB may play a central role in the pathogenesis of ALS8,in agreement with the observed reduction of VAPB in sporadic ALS. View Publication

Staphylococcus aureus (S. aureus) is a human pathogen as well as a frequent colonizer of skin and mucosa. This bacterium potently activates conventional T-cells through superantigens and it is suggested to induce T-cell cytokine-production as well as to promote a regulatory phenotype in T-cells in order to avoid clearance. This study aimed to investigate how S. aureus impacts the production of regulatory and pro-inflammatory cytokines and the expression of CD161 and HELIOS by peripheral CD4(+)FOXP3(+) T-cells. Stimulation of PBMC with S. aureus 161:2-cell free supernatant (CFS) induced expression of IL-10,IFN-γ and IL-17A in FOXP3(+) cells. Further,CD161 and HELIOS separated the FOXP3(+) cells into four distinct populations regarding cytokine-expression. Monocyte-depletion decreased S. aureus 161:2-induced activation of FOXP3(+) cells while pre-stimulation of purified monocytes with S. aureus 161:2-CFS and subsequent co-culture with autologous monocyte-depleted PBMC was sufficient to mediate activation of FOXP3(+) cells. Together,these data show that S. aureus potently induces FOXP3(+) cells and promotes a diverse phenotype with expression of regulatory and pro-inflammatory cytokines connected to increased CD161-expression. This could indicate potent regulation or a contribution of FOXP3(+) cells to inflammation and repression of immune-suppression upon encounter with S. aureus. View Publication

Many breast cancer deaths result from tumors acquiring resistance to available therapies. Thus,new therapeutic agents are needed for targeting drug-resistant breast cancers. Drug-refractory breast cancers include HER2+ tumors that have acquired resistance to HER2-targeted antibodies and kinase inhibitors,and Triple-Negative" Breast Cancers (TNBCs) that lack the therapeutic targets Estrogen Receptor� View Publication

To study the role of different cytokine combinations on the proliferation and differentiation of highly purified primitive progenitor cells,a serum-free liquid culture system was used in combination with phenotypic and functional analysis of the cells produced in culture. CD34+ CD45RAlo CD71lo cells,purified from umbilical cord blood by flow cytometry and cell sorting,were selected for this study because of their high content of clonogenic cells (34%),particularly multipotent progenitors (CFU-MIX,12% of all cells). Four cytokine combinations were tested: (1) mast cell growth factor (MGF; a c-kit ligand) and interleukin-6 (IL-6); (2) MGF,IL-6,IL-3,and erythropoietin (Epo); (3) MGF,IL-6,granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-3 fusion protein (FP),macrophage colony-stimulating factor (M-CSF),and granulocyte-CSF (G-CSF); and (4) MGF,IL-6,FP,M-CSF,G-CSF,and Epo. Maximum numbers of erythroid progenitors (BFU-E,up to 55-fold increase) and mature erythroid cells were observed in the presence of MGF,IL-6,IL-3,and Epo,whereas maximum levels of myeloid progenitors (CFU-C,up to 70-fold increase) and mature myeloid cells were found in cultures supplemented with MGF,IL-6,FP,M-CSF,and G-CSF. When MGF,IL-6,FP,M-CSF,G-CSF,and Epo were present,maximum levels of both erythroid and myeloid progenitors and their progeny were observed. These results indicate that specific cytokine combinations can act directly on primitive hematopoietic cells resulting in significant expansion of progenitor cell numbers and influencing their overall patterns of proliferation and differentiation. Furthermore,the observations presented in this study suggest that the cytokine combinations used were unable to bias lineage commitment of multipotent progenitors,but rather had a permissive effect on the development of lineage-restricted clonogenic cells. View Publication

The molecular mechanisms underlying the differentiation of neural progenitor cells (NPCs) remain poorly understood. In this study we investigated the role of Ca(2+) and cAMP (cyclic adenosine monophosphate) in the differentiation of NPCs extracted from the subventricular zone of E14.5 rat embryos. Patch clamp recordings revealed that increasing cAMP-signaling with Forskolin or IBMX (3-isobutyl-1-methylxantine) significantly facilitated neuronal functional maturation. A continuous application of IBMX to the differentiation medium substantially increased the functional expression of voltage-gated Na(+) and K(+) channels,as well as neuronal firing frequency. Furthermore,we observed an increase in the frequency of spontaneous synaptic currents and in the amplitude of evoked glutamatergic and GABAergic synaptic currents. The most prominent acute effect of applying IBMX was an increase in L-type Ca(2+)currents. Conversely,blocking L-type channels strongly inhibited dendritic outgrowth and synapse formation even in the presence of IBMX,indicating that voltage-gated Ca(2+) influx plays a major role in neuronal differentiation. Finally,we found that nifedipine completely blocks IBMX-induced CREB phosphorylation (cAMP-response-element-binding protein),indicating that the activity of this important transcription factor equally depends on both enhanced cAMP and voltage-gated Ca(2+)-signaling. Taken together,these data indicate that the up-regulation of voltage-gated L-type Ca(2+)-channels and early electrical excitability are critical steps in the cAMP-dependent differentiation of SVZ-derived NPCs into functional neurons. To our knowledge,this is the first demonstration of the acute effects of cAMP on voltage-gated Ca(+2)channels in NPC-derived developing neurons. View Publication

There is an urgent need for new and advanced approaches to modeling the pathological mechanisms of complex human neurological disorders. This is underscored by the decline in pharmaceutical research and development efficiency resulting in a relative decrease in new drug launches in the last several decades. Induced pluripotent stem cells represent a new tool to overcome many of the shortcomings of conventional methods,enabling live human neural cell modeling of complex conditions relating to aberrant neurodevelopment,such as schizophrenia,epilepsy and autism as well as age-associated neurodegeneration. This review considers the current status of induced pluripotent stem cell-based modeling of neurological disorders,canvassing proven and putative advantages,current constraints,and future prospects of next-generation culture systems for biomedical research and translation. View Publication