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

Metformin is an anti-diabetic drug with anorexigenic properties. The precise cellular mechanisms of its action are not entirely understood. Adipose tissue has recently been recognized as an important endocrine organ that is pivotal for the regulation of insulin resistance and energy homeostasis. Due to its thermogenic capacity brown adipose tissue contributes to the regulation of energy metabolism and is an attractive target tissue for pharmacological approaches to treating insulin resistance and obesity. Leptin is the prototypic adipocyte-derived hormone inducing a negative energy balance. We investigated effects of metformin on adipocyte metabolism,signalling,and leptin secretion in a brown adipocyte model. Metformin acutely stimulated p44/p42 mitogen-activated protein (MAP) kinase in a dose- (3.2-fold at 1 mmol/l,Ptextless 0.05) as well as time-dependent (3.8-fold at 5 min,Ptextless 0.05) manner. This stimulation was highly selective since phosphorylation of intermediates in the stress kinase,janus kinase (JAK)-signal transducer and activator of transcription (STAT),and phosphatidylinositol (PI) 3-kinase signalling pathways such as p38 MAP kinase,STAT3,and Akt was unaltered. Furthermore,chronic metformin treatment for 12 days dose-dependently inhibited leptin secretion by 35% and 75% at 500 mumol/l and 1 mmol/l metformin respectively (Ptextless 0.01). This reduction was not caused by alterations in adipocyte differentiation. Moreover,the impairment in leptin secretion by metformin was reversible within 48 h after removal of the drug. Pharmacological inhibition of p44/p42 MAP kinase prevented the metformin-induced negative effect on leptin secretion. Taken together,our data demonstrate direct acute effects of metformin on adipocyte signalling and endocrine function with robust inhibition of leptin secretion. They suggest a selective molecular mechanism that may contribute to the anorexigenic effect of this antidiabetic compound. View Publication

Because osteoblasts and marrow adipocytes are derived from a common mesenchymal progenitor,increased adipogenesis may occur at the expense of osteoblasts,leading to bone loss. Our previous in vitro studies indicated that activation of the proadipogenic transcription factor peroxisome proliferator-activated receptor isoform gamma 2 with rosiglitazone suppressed osteoblast differentiation. Here,we show that 5-month-old Swiss-Webster mice receiving rosiglitazone for 28 d exhibited bone loss associated with an increase in marrow adipocytes,a decrease in the ratio of osteoblasts to osteoclasts,a reduction in bone formation rate,and a reduction in wall width--an index of the amount of bone formed by each team of osteoblasts. Rosiglitazone had no effect on the number of early osteoblast or osteoclast progenitors,or on osteoblast life span,but decreased the expression of the key osteoblastogenic transcription factors Runx2 and Osterix in cultures of marrow-derived mesenchymal progenitors. These effects were associated with diversion of bipotential progenitors from the osteoblast to the adipocyte lineage,and suppression of the differentiation of monopotential osteoblast progenitors. However,rosiglitazone had no effect on osteoblastic cells at later stages of differentiation. Hence,rosiglitazone attenuates osteoblast differentiation and thereby reduces bone formation rate in vivo,leading to bone loss. These findings provide a mechanistic explanation for the recent evidence that peroxisome proliferator-activated receptor isoform gamma activation is a negative regulator of bone mass and suggest that the increased production of oxidized fatty acids with age may indeed be an important mechanism for age-related osteoporosis in humans. View Publication

The transcription factor p53 coordinates cell cycle arrest and apoptosis in response to DNA damage. Pifithrin-alpha (PFT-alpha) is a small molecule inhibitor of p53 activity that is frequently used in cell culture studies of p53 function. Here we report an investigation of the stability of this compound. PFT-alpha rapidly converts to a planar tricyclic derivative,with a half-life of 4.2 h under physiological conditions. This spontaneous conversion greatly alters the structural and physicochemical properties of the drug. PFT-alpha has a pKa of 9.11 and is an ionic species in physiological medium,whereas the tricyclic derivative has a pKa of 4.36 and exists as the neutral free base at pH 7. The tricyclic derivative is very hydrophobic,with a log P of 4.26. Although PFT-alpha is generally used at 10-30 microM concentration,the aqueous solubility of its derivative is only 0.2 microM,and it can form a visible precipitate under conditions of typical use. The conversion of PFT-alpha proceeds via an intramolecular cyclization reaction involving the imine and carbonyl groups. Modification of the carbonyl function creates a stable analogue of PFT-alpha that remains soluble indefinitely. These results provide a strategy for the rational design of PFT-alpha analogues that exhibit predictable stability,hydrophobicity,and aqueous solubility. View Publication

Numerous proteins undergo modification by palmitic acid (S-acylation) for their biological functions including signal transduction,vesicular transport and maintenance of cellular architecture. Although palmitoylation is an essential modification,these proteins must also undergo depalmitoylation for their degradation by lysosomal proteases. Palmitoyl-protein thioesterase-1 (PPT1),a lysosomal enzyme,cleaves thioester linkages in S-acylated proteins and removes palmitate residues facilitating the degradation of these proteins. Thus,inactivating mutations in the PPT1 gene cause infantile neuronal ceroid lipofuscinosis (INCL),a devastating neurodegenerative storage disorder of childhood. Although rapidly progressing brain atrophy is the most dramatic pathological manifestation of INCL,the molecular mechanism(s) remains unclear. Using PPT1-knockout (PPT1-KO) mice that mimic human INCL,we report here that the endoplasmic reticulum (ER) in the brain cells of these mice is structurally abnormal. Further,we demonstrate that the level of growth-associated protein-43 (GAP-43),a palmitoylated neuronal protein,is elevated in the brains of PPT1-KO mice. Moreover,forced expression of GAP-43 in PPT1-deficient cells results in the abnormal accumulation of this protein in the ER. Consistent with these results,we found evidence for the activation of unfolded protein response (UPR) marked by elevated levels of phosphorylated translation initiation factor,eIF2alpha,increased expression of chaperone proteins such as glucose-regulated protein-78 and activation of caspase-12,a cysteine proteinase in the ER,mediating caspase-3 activation and apoptosis. Our results,for the first time,link PPT1 deficiency with the activation of UPR,apoptosis and neurodegeneration in INCL and identify potential targets for therapeutic intervention in this uniformly fatal disease. View Publication

Angiotensin II (Ang II) type 2 (AT2) receptors are abundantly expressed not only in the fetal brain where they probably contribute to brain development,but also in pathological conditions to protect the brain against stroke; however,the detailed mechanisms are unclear. Here,we demonstrated that AT2 receptor signaling induced neural differentiation via an increase in MMS2,one of the ubiquitin-conjugating enzyme variants. The AT2 receptor,MMS2,Src homology 2 domain-containing protein-tyrosine phosphatase 1 (SHP-1),and newly cloned AT2 receptor-interacting protein (ATIP) were highly expressed in fetal rat neurons and declined after birth. Ang II induced MMS2 expression in a dose-dependent manner,reaching a peak after 4 h of stimulation,and this effect was enhanced with AT1 receptor blocker,valsartan,but inhibited by AT2 receptor blocker PD123319. Moreover,we observed that an AT2 receptor agonist,CGP42112A,alone enhanced MMS2 expression. Neurons treated with small interfering RNA of MMS2 failed to exhibit neurite outgrowth and synapse formation. Moreover,the increase in AT2 receptor-induced MMS2 mRNA expression was enhanced by overexpression of ATIP but inhibited by small interfering RNA of SHP-1 and overexpression of catalytically dominant-negative SHP-1 or a tyrosine phosphatase inhibitor,sodium orthovanadate. After AT2 receptor stimulation,ATIP and SHP-1 were translocated into the nucleus after formation of their complex. Furthermore,increased MMS2 expression mediates the inhibitor of DNA binding 1 proteolysis and promotes DNA repair. These results provide a new insight into the contribution of AT2 receptor stimulation to neural differentiation via transactivation of MMS2 expression involving the association of ATIP and SHP-1. 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

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

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

B cell aggregates in the central nervous system (CNS) have been associated with rapid disease progression in patients with multiple sclerosis (MS). Here we demonstrate a key role of carcinoembryogenic antigen-related cell adhesion molecule1 (CEACAM1) in B cell aggregate formation in MS patients and a B cell-dependent mouse model of MS. CEACAM1 expression was increased on peripheral blood B cells and CEACAM1(+) B cells were present in brain infiltrates of MS patients. Administration of the anti-CEACAM1 antibody T84.1 was efficient in blocking aggregation of B cells derived from MS patients. Along these lines,application of the monoclonal anti-CEACAM1 antibody mCC1 was able to inhibit CNS B cell aggregate formation and significantly attenuated established MS-like disease in mice in the absence of any adverse effects. CEACAM1 was co-expressed with the regulator molecule T cell immunoglobulin and mucin domain -3 (TIM-3) on B cells,a novel molecule that has recently been described to induce anergy in T cells. Interestingly,elevated coexpression on B cells coincided with an autoreactive T helper cell phenotype in MS patients. Overall,these data identify CEACAM1 as a clinically highly interesting target in MS pathogenesis and open new therapeutic avenues for the treatment of the disease. View Publication

Spatial organization of the genome plays a central role in gene expression,DNA replication,and repair. But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization (ATAC-see),a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ,cell sorting,and deep sequencing to reveal the identity of the imaged elements. ATAC-see revealed the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion,termed NETosis. Integration of ATAC-see with flow cytometry enables automated quantitation and prospective cell isolation as a function of chromatin accessibility,and it reveals a cell-cycle dependence of chromatin accessibility that is especially dynamic in G1 phase. The integration of imaging and epigenomics provides a general and scalable approach for deciphering the spatiotemporal architecture of gene control. View Publication

Tissue-specific control of gene expression is an invaluable tool for studying various biological processes and medical applications. Efficient regulatory systems have been utilized to control transgene expression in various types of DNA viral or integrating viral vectors. However,existing regulatory systems are difficult to transfer into negative-strand RNA virus vector platforms because of significant differences in their transcriptional machineries. In this study,we developed a novel strategy for regulating transgene expression mediated by a cytoplasmic RNA vector based on a replication-defective and persistent Sendai virus (SeVdp). Because of the capacity of Sendai virus (SeV) nonstructural C proteins to specifically inhibit viral RNA synthesis,overexpression of C protein significantly reduced transgene expression mediated by SeVdp vectors. We found that SeV C overexpression concomitantly reduced SeVdp mRNA levels and genomic RNA synthesis. To control C expression,target sequences for an endogenous microRNA were incorporated into the 3' untranslated region of the C genes. Incorporation of target sequences for miR-21 into the SeVdp vector restored transgene expression in HeLa cells by decreasing C expression. Furthermore,the SeVdp vector containing target sequences for let-7a enabled cell-specific control of transgene expression in human fibroblasts and induced pluripotent stem cells. Our findings demonstrate that SeV C can be used as an effective regulator for controlling transgene expression. This strategy will contribute to efficient and less toxic SeVdp-mediated gene transfer in various biological applications. View Publication