技术资料

Chronic obstructive pulmonary disease (COPD) is a highly prevalent,chronic inflammatory lung disease with limited existing therapeutic options. While modulation of peroxisome proliferator-activating receptor (PPAR)-$$ activity can modify inflammatory responses in several models of lung injury,the relevance of the PPARG pathway in COPD pathogenesis has not been previously explored. Mice lacking Pparg specifically in airway epithelial cells displayed increased susceptibility to chronic cigarette smoke (CS)-induced emphysema,with excessive macrophage accumulation associated with increased expression of chemokines,Ccl5,Cxcl10,and Cxcl15. Conversely,treatment of mice with a pharmacological PPAR$$ activator attenuated Cxcl10 and Cxcl15 expression and macrophage accumulation in response to CS. In vitro,CS increased lung epithelial cell chemokine expression in a PPAR$$ activation-dependent fashion. The ability of PPAR$$ to regulate CS-induced chemokine expression in vitro was not specifically associated with peroxisome proliferator response element (PPRE)-mediated transactivation activity but was correlated with PPAR$$-mediated transrepression of NF-$$B activity. Pharmacological or genetic activation of PPAR$$ activity abrogated CS-dependent induction of NF-$$B activity. Regulation of NF-$$B activity involved direct PPAR$$-NF-$$B interaction and PPAR$$-mediated effects on IKK activation,I$$B$$ degradation,and nuclear translocation of p65. Our data indicate that PPARG represents a disease-relevant pathophysiological and pharmacological target in COPD. Its activation state likely contributes to NF-$$B-dependent,CS-induced chemokine-mediated regulation of inflammatory cell accumulation. View Publication

Non-viral gene delivery into human embryonic stem cells (hESCs)is an important tool for controlling cell fate. However,the delivery efficiency remains low due in part to the tight colony structure of the cells which prevents effective exposure towards delivery vectors. We herein report a novel approach to enhance non-viral gene delivery to hESCs by transiently altering the cell and colony structure. (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide (Y-27632),a small molecule that inhibits the rho-associated protein kinase pathway,is utilized to induce transient colony spreading which leads to increased transfection efficiency by 1.5 to 2 folds in a spectrum of non-viral transfection reagents including Lipofectamine 2000 and Fugene HD. After removal of Y-27632 post-transfection,cells can revert back to its normal state and do not show alteration of pluripotency. This approach provides a simple,effective tool to enhance non-viral gene delivery into adherent hESCs for genetic manipulation. View Publication

The localization of memory T cells to human skin is essential for long-term immune surveillance and the maintenance of barrier integrity. The expression of CCR8 during naive T cell activation is controlled by skin-specific factors derived from epidermal keratinocytes and not by resident dendritic cells. In this study,we show that the CCR8-inducing factors are heat stable and protease resistant and include the vitamin D3 metabolite 1α,25-dihydroxyvitamin D3 and PGE2. The effect of either metabolite alone on CCR8 expression was weak,whereas their combination resulted in robust CCR8 expression. Elevation of intracellular cAMP was essential because PGE2 could be substituted with the adenylyl cyclase agonist forskolin,and CCR8 expression was sensitive to protein kinase A inhibition. For effective induction,exposure of naive T cells to these epidermal factors needed to occur either prior to or during T cell activation even though CCR8 was only detected 4-5 d later in proliferating T cells. The importance of tissue environments in maintaining cellular immune surveillance networks within distinct healthy tissues provides a paradigm shift in adaptive immunity. Epidermal-derived vitamin D3 metabolites and PGs provide an essential cue for the localization of CCR8(+) immune surveillance T cells within healthy human skin. View Publication

Here we introduce the representative method to culture HESCs under the feeder and feeder-free conditions,the former of which is used to maintain or expand undifferentiated HESCs,and the latter can be used for the preparation of pure HESCs RNA samples,or for screening factors influential on self-renewal of HESCs. We also describe a protocol and tips for conducting gene chip analysis focusing on widely used Affymetrix Microarrays. These techniques will provide us unprecedented scale of biological information that would illuminate a key to decipher complex signaling networks controlling pluripotency. View Publication

BACKGROUND: The unfolded protein response (UPR) is one of the pathways triggered to ensure quality control of the proteins assembled in the endoplasmic reticulum (ER) when cell homeostasis is compromised. This mechanism is primarily composed of three transmembrane proteins serving as stress sensors: PKR-like ER kinase (PERK),activating transcription factor 6 (ATF6),and inositol-requiring enzyme 1 (IRE1). These three proteins' synergic action elicits translation and transcriptional downstream pathways,leading to less protein production and activating genes that encode important proteins in folding processes,including chaperones. Previous reports showed that viruses have evolved mechanisms to curtail or customize this UPR signaling for their own benefit. However,HIV infection's effect on the UPR has scarcely been investigated. METHODS: This work investigated UPR modulation by HIV infection by assessing UPR-related protein expression under in vitro and in vivo conditions via Western blotting. Antiretroviral (ARV) drugs' influence on this stress response was also considered. RESULTS: In in vitro and in vivo analyses,our results confirm that HIV infection activates stress-response components and that ARV therapy contributes to changes in the UPR's activation profile. CONCLUSIONS: This is the first report showing UPR-related protein expression in HIV target cells derived directly from HIV-infected patients receiving different ARV therapies. Thus,two mechanisms may occur simultaneously: interference by HIV itself and the ARV drugs' pharmacological effects as UPR activators. New evidence of how HIV modulates the UPR to enhance its own replication and secure infection success is also presented. View Publication

Daratumumab is an anti-CD38 monoclonal antibody with lytic activity against multiple myeloma (MM) cells,including ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity). Owing to a marked heterogeneity of response to daratumumab therapy in MM,we investigated determinants of the sensitivity of MM cells toward daratumumab-mediated ADCC and CDC. In bone marrow samples from 144 MM patients,we observed no difference in daratumumab-mediated lysis between newly diagnosed or relapsed/refractory patients. However,we discovered,next to an expected effect of effector (natural killer cells/monocytes) to target (MM cells) ratio on ADCC,a significant association between CD38 expression and daratumumab-mediated ADCC (127 patients),as well as CDC (56 patients). Similarly,experiments with isogenic MM cell lines expressing different levels of CD38 revealed that the level of CD38 expression is an important determinant of daratumumab-mediated ADCC and CDC. Importantly,all-trans retinoic acid (ATRA) increased CD38 expression levels but also reduced expression of the complement-inhibitory proteins CD55 and CD59 in both cell lines and primary MM samples. This resulted in a significant enhancement of the activity of daratumumab in vitro and in a humanized MM mouse model as well. Our results provide the preclinical rationale for further evaluation of daratumumab combined with ATRA in MM patients. View Publication

Dysbindin is a schizophrenia susceptibility factor and subunit of the biogenesis of lysosome-related organelles complex 1 (BLOC-1) required for lysosome-related organelle biogenesis,and in neurons,synaptic vesicle assembly,neurotransmission,and plasticity. Protein networks,or interactomes,downstream of dysbindin/BLOC-1 remain partially explored despite their potential to illuminate neurodevelopmental disorder mechanisms. Here,we conducted a proteome-wide search for polypeptides whose cellular content is sensitive to dysbindin/BLOC-1 loss of function. We identified components of the vesicle fusion machinery as factors downregulated in dysbindin/BLOC-1 deficiency in neuroectodermal cells and iPSC-derived human neurons,among them the N-ethylmaleimide-sensitive factor (NSF). Human dysbindin/BLOC-1 coprecipitates with NSF and vice versa,and both proteins colocalized in a Drosophila model synapse. To test the hypothesis that NSF and dysbindin/BLOC-1 participate in a pathway-regulating synaptic function,we examined the role for NSF in dysbindin/BLOC-1-dependent synaptic homeostatic plasticity in Drosophila. As previously described,we found that mutations in dysbindin precluded homeostatic synaptic plasticity elicited by acute blockage of postsynaptic receptors. This dysbindin mutant phenotype is fully rescued by presynaptic expression of either dysbindin or Drosophila NSF. However,neither reduction of NSF alone or in combination with dysbindin haploinsufficiency impaired homeostatic synaptic plasticity. Our results demonstrate that dysbindin/BLOC-1 expression defects result in altered cellular content of proteins of the vesicle fusion apparatus and therefore influence synaptic plasticity. View Publication

Many acute and chronic anaemias,including haemolysis,sepsis and genetic bone marrow failure diseases such as Diamond-Blackfan anaemia,are not treatable with erythropoietin (Epo),because the colony-forming unit erythroid progenitors (CFU-Es) that respond to Epo are either too few in number or are not sensitive enough to Epo to maintain sufficient red blood cell production. Treatment of these anaemias requires a drug that acts at an earlier stage of red cell formation and enhances the formation of Epo-sensitive CFU-E progenitors. Recently,we showed that glucocorticoids specifically stimulate self-renewal of an early erythroid progenitor,burst-forming unit erythroid (BFU-E),and increase the production of terminally differentiated erythroid cells. Here we show that activation of the peroxisome proliferator-activated receptor α (PPAR-α) by the PPAR-α agonists GW7647 and fenofibrate synergizes with the glucocorticoid receptor (GR) to promote BFU-E self-renewal. Over time these agonists greatly increase production of mature red blood cells in cultures of both mouse fetal liver BFU-Es and mobilized human adult CD34(+) peripheral blood progenitors,with a new and effective culture system being used for the human cells that generates normal enucleated reticulocytes. Although Ppara(-/-) mice show no haematological difference from wild-type mice in both normal and phenylhydrazine (PHZ)-induced stress erythropoiesis,PPAR-α agonists facilitate recovery of wild-type but not Ppara(-/-) mice from PHZ-induced acute haemolytic anaemia. We also show that PPAR-α alleviates anaemia in a mouse model of chronic anaemia. Finally,both in control and corticosteroid-treated BFU-E cells,PPAR-α co-occupies many chromatin sites with GR; when activated by PPAR-α agonists,additional PPAR-α is recruited to GR-adjacent sites and presumably facilitates GR-dependent BFU-E self-renewal. Our discovery of the role of PPAR-α agonists in stimulating self-renewal of early erythroid progenitor cells suggests that the clinically tested PPAR-α agonists we used may improve the efficacy of corticosteroids in treating Epo-resistant anaemias. View Publication

Induced pluripotent stem (iPS) cells have an enormous potential for physiological studies. A novel protocol was developed combining the derivation of iPS from peripheral blood with an optimized directed differentiation to cardiomyocytes and a subsequent metabolic selection. The human iPS cells were retrovirally dedifferentiated from activated T cells. The subsequent optimized directed differentiation protocol yielded 30-45% cardiomyocytes at day 16 of differentiation. The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T,$\$-actinin and myosin light chain 2 (MLC2V). In a subsequent metabolic selection with lactate,the cardiomyocytes content could be increased to more than 90%. Loss of cardiomyocytes during metabolic selection were less than 50%,whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes. Electrophysiological characterization confirmed the typical cardiac features and the presence of ventricular,atrial and nodal-like action potentials within the derived cardiomyocyte population. Our combined and optimized protocol is highly robust and applicable for scalable cardiac differentiation. It provides a simple and cost-efficient method without expensive equipment for generating large numbers of highly purified,functional cardiomyocytes. It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling,drug discovery,and regenerative medicine. View Publication

Unintended exposure to teratogenic compounds can lead to various birth defects; however current animal-based testing is limited by time,cost and high inter-species variability. Here,we developed a human-relevant in vitro model,which recapitulated two cellular events characteristic of embryogenesis,to identify potentially teratogenic compounds. We spatially directed mesoendoderm differentiation,epithelial-mesenchymal transition and the ensuing cell migration in micropatterned human pluripotent stem cell (hPSC) colonies to collectively form an annular mesoendoderm pattern. Teratogens could disrupt the two cellular processes to alter the morphology of the mesoendoderm pattern. Image processing and statistical algorithms were developed to quantify and classify the compounds' teratogenic potential. We not only could measure dose-dependent effects but also correctly classify species-specific drug (Thalidomide) and false negative drug (D-penicillamine) in the conventional mouse embryonic stem cell test. This model offers a scalable screening platform to mitigate the risks of teratogen exposures in human. View Publication

Human peripheral blood and umbilical cord blood represent attractive sources of cells for reprogramming to induced pluripotent stem cells (iPSCs). However,to date,most of the blood-derived iPSCs were generated using either integrating methods or starting from T-lymphocytes that have genomic rearrangements thus bearing uncertain consequences when using iPSC-derived lineages for disease modeling and cell therapies. Recently,both peripheral blood and cord blood cells have been reprogrammed into transgene-free iPSC using the Sendai viral vector. Here we demonstrate that peripheral blood can be utilized for medium-throughput iPSC production without the need to maintain cell culture prior to reprogramming induction. Cell reprogramming can also be accomplished with as little as 3000 previously cryopreserved cord blood cells under feeder-free and chemically defined Xeno-free conditions that are compliant with standard Good Manufacturing Practice (GMP) regulations. The first iPSC colonies appear 2-3 weeks faster in comparison to previous reports. Notably,these peripheral blood- and cord blood-derived iPSCs are free of detectable immunoglobulin heavy chain (IGH) and T cell receptor (TCR) gene rearrangements,suggesting they did not originate from B- or T- lymphoid cells. The iPSCs are pluripotent as evaluated by the scorecard assay and in vitro multi lineage functional cell differentiation. Our data show that small volumes of cryopreserved peripheral blood or cord blood cells can be reprogrammed efficiently at a convenient,cost effective and scalable way. In summary,our method expands the reprogramming potential of limited or archived samples either stored at blood banks or obtained from pediatric populations that cannot easily provide large quantities of peripheral blood or a skin biopsy. View Publication

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