技术资料

Drug abuse is a major comorbidity of HIV infection and cognitive disorders are often more severe in the drug abusing HIV infected population. CD14+CD16+ monocytes,a mature subpopulation of peripheral blood monocytes,are key mediators of HIV neuropathogenesis. Infected CD14+CD16+ monocyte transmigration across the blood brain barrier mediates HIV entry into the brain and establishes a viral reservoir within the CNS. Despite successful antiretroviral therapy,continued influx of CD14+CD16+ monocytes,both infected and uninfected,contributes to chronic neuroinflammation and the development of HIV associated neurocognitive disorders (HAND). Drug abuse increases extracellular dopamine in the CNS. Once in the brain,CD14+CD16+ monocytes can be exposed to extracellular dopamine due to drug abuse. The direct effects of dopamine on CD14+CD16+ monocytes and their contribution to HIV neuropathogenesis are not known. In this study,we showed that CD14+CD16+ monocytes express mRNA for all five dopamine receptors by qRT-PCR and D1R,D5R and D4R surface protein by flow cytometry. Dopamine and the D1-like dopamine receptor agonist,SKF38393,increased CD14+CD16+ monocyte migration that was characterized as chemokinesis. To determine whether dopamine affected cell motility and adhesion,live cell imaging was used to monitor the accumulation of CD14+CD16+ monocytes on the surface of a tissue culture dish. Dopamine increased the number and the rate at which CD14+CD16+ monocytes in suspension settled to the dish surface. In a spreading assay,dopamine increased the area of CD14+CD16+ monocytes during the early stages of cell adhesion. In addition,adhesion assays showed that the overall total number of adherent CD14+CD16+ monocytes increased in the presence of dopamine. These data suggest that elevated extracellular dopamine in the CNS of HIV infected drug abusers contributes to HIV neuropathogenesis by increasing the accumulation of CD14+CD16+ monocytes in dopamine rich brain regions. View Publication

Alcohol consumption has long been associated with a majority of liver diseases and has been found to influence both fetal and adult liver functions. In spite of being one of the major causes of morbidity and mortality in the world,currently,there are no effective strategies that can prevent or treat alcoholic liver disease (ALD),due to a lack of human-relevant research models. Recent success in generation of functionally active mature hepatocyte-like cells from human-induced pluripotent cells (iPSCs) enables us to better understand the effects of alcohol on liver functions. Here,we describe the method and effect of alcohol exposure on multistage hepatic cell types derived from human iPSCs,in an attempt to recapitulate the early stages of liver tissue injury associated with ALD. We exposed different stages of iPSC-induced hepatic cells to ethanol at a pathophysiological concentration. In addition to stage-specific molecular markers,we measured several key cellular parameters of hepatocyte injury,including apoptosis,proliferation,and lipid accumulation. View Publication

Receptor activator of nuclear factor kappa-B ligand (RANKL) is a critical osteoclastogenic factor expressed in bone marrow stromal/osteoblast lineage cells. Tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) levels are elevated in pathologic conditions such as multiple myeloma and inflammatory arthritis,and have been positively correlated with osteolytic markers. Osteoprotegerin (OPG) which inhibits osteoclastogenesis is a decoy receptor for RANKL and also known to interact with TRAIL. Herein,we show that TRAIL increases DR5 and DcR1 receptors but no change in the levels of DR4 and DcR2 expression in human bone marrow derived stromal/preosteoblast (SAKA-T) cell line. We further demonstrated that TRAIL treatment significantly decreased OPG mRNA expression. Interestingly,TRAIL treatment induced RANKL mRNA expression in these cells. In addition,TRAIL significantly increased NF-kB and c-Jun N-terminal kinase (JNK) activity. Human transcription factor array screening by real-time RT-PCR identified TRAIL up-regulation of the signal transducers and activators of the transcription (STAT)-6 expression in SAKA-T cells. TRAIL stimulation induced p-STAT-6 expression in human bone marrow derived primary stromal/preosteoblast cells. Confocal microscopy analysis further revealed p-STAT-6 nuclear localization in SAKA-T cells. Chromatin immunoprecipitation (ChIP) assay confirmed p-STAT-6 binding to the hRANKL gene distal promoter region. In addition,siRNA suppression of STAT-6 expression inhibits TRAIL increased hRANKL gene promoter activity. Thus,our results suggest that TRAIL induces RANKL expression through a STAT-6 dependent transcriptional regulatory mechanism in bone marrow stromal/preosteoblast cells. View Publication

Tendon-bone junctions (TBJs) are frequently injured,especially in athletic settings. Healing of TBJ injuries is slow and is often repaired with scar tissue formation that compromises normal function. This study explored the feasibility of using kartogenin (KGN),a biocompound,to enhance the healing of injured TBJs. We first determined the effects of KGN on the proliferation and chondrogenic differentiation of rabbit bone marrow stromal cells (BMSCs) and patellar tendon stem/progenitor cells (PTSCs) in vitro. KGN enhanced cell proliferation in both cell types in a concentration-dependent manner and induced chondrogenic differentiation of stem cells,as demonstrated by high expression levels of chondrogenic markers aggrecan,collagen II and Sox-9. Besides,KGN induced the formation of cartilage-like tissues in cell cultures,as observed through the staining of abundant proteoglycans,collagen II and osteocalcin. When injected into intact rat patellar tendons in vivo,KGN induced cartilage-like tissue formation in the injected area. Similarly,when KGN was injected into experimentally injured rat Achilles TBJs,wound healing in the TBJs was enhanced,as evidenced by the formation of extensive cartilage-like tissues. These results suggest that KGN may be used as an effective cell-free clinical therapy to enhance the healing of injured TBJs. View Publication

Without effective therapy,chronic-phase chronic myeloid leukemia (CP-CML) evolves into an acute leukemia (blast crisis [BC]) that displays either myeloid or B-lymphoid characteristics. This transition is often preceded by a clinically recognized,but biologically poorly characterized,accelerated phase (AP). Here,we report that IKAROS protein is absent or reduced in bone marrow blasts from most CML patients with advanced myeloid disease (AP or BC). This contrasts with primitive CP-CML cells and BCR-ABL1-negative acute myeloid leukemia blasts,which express readily detectable IKAROS. To investigate whether loss of IKAROS contributes to myeloid disease progression in CP-CML,we examined the effects of forced expression of a dominant-negative isoform of IKAROS (IK6) in CP-CML patients' CD34(+) cells. We confirmed that IK6 disrupts IKAROS activity in transduced CP-CML cells and showed that it confers on them features of AP-CML,including a prolonged increased output in vitro and in xenografted mice of primitive cells with an enhanced ability to differentiate into basophils. Expression of IK6 in CD34(+) CP-CML cells also led to activation of signal transducer and activator of transcription 5 and transcriptional repression of its negative regulators. These findings implicate loss of IKAROS as a frequent step and potential diagnostic harbinger of progressive myeloid disease in CML patients. View Publication

Tivantinib (ARQ197) was first reported as a highly selective inhibitor of c-MET and is currently being investigated in a phase III clinical trial. However,as recently reported by us and another group,tivantinib showed cytotoxic activity independent of cellular c-MET status and also disrupted microtubule dynamics. To investigate if tivantinib exerts its cytotoxic activity by disrupting microtubules,we quantified polymerized tubulin in cells and xenograft tumors after tivantinib treatment. Consistent with our previous report,tivantinib reduced tubulin polymerization in cells and in mouse xenograft tumors in vivo. To determine if tivantinib directly binds to tubulin,we performed an in vitro competition assay. Tivantinib competitively inhibited colchicine but not vincristine or vinblastine binding to purified tubulin. These results imply that tivantinib directly binds to the colchicine binding site of tubulin. To predict the binding mode of tivantinib with tubulin,we performed computer simulation of the docking pose of tivantinib with tubulin using GOLD docking program. Computer simulation predicts tivantinib fitted into the colchicine binding pocket of tubulin without steric hindrance. Furthermore,tivantinib showed similar IC50 values against parental and multidrug-resistant cells. In contrast,other microtubule-targeting drugs,such as vincristine,paclitaxel,and colchicine,could not suppress the growth of cells overexpressing ABC transporters. Moreover,the expression level of ABC transporters did not correlate with the apoptosis-inducing ability of tivantinib different from other microtubule inhibitor. These results suggest that tivantinib can overcome ABC transporter-mediated multidrug-resistant tumor cells and is potentially useful against various tumors. View Publication

Focal adhesion kinase (FAK) is up-regulated in thyroid cancer and small molecule FAK scaffolding inhibitor,Y15,was shown to decrease cancer growth in vitro and in vivo. We sought to test the effectiveness of Y15 in thyroid cancer cell lines,profile gene expression with Y15 compared with clinical trial FAK inhibitor PF-04554878,and use Y15 in novel drug combinations. Cell viability was decreased in a dose dependent manner in four thyroid cancer cell lines with Y15 and with higher doses in PF-04554878. Y397 FAK and total FAK were decreased with Y15 and decreased less with PF-04554878. Detachment and necrosis were increased in a dose-dependent manner in all cell lines with Y15. Clonogenicity was decreased in a dose-dependent manner for both Y15 and PF-04554878. We compared gene profiles between papillary thyroid cell lines,TPC1,BCPAP and K1,and 380,109,and 74 genes were significantly textgreater2-fold changed with Y15 treatment,respectively. Common up-regulated genes were involved in apoptosis,cell cycle,transcription and heat shock; down-regulated genes were involved in cell cycle,cell-to-cell interactions,and cancer stem cell markers. We also compared gene profiles of TT cells treated with Y15 versus PF-04554878. Y15 caused 144 genes to change over 4 fold and PF-04554878 caused 208 gene changes textgreater4-fold (ptextless0.05). Among genes changed 4 fold,11 were shared between the treatments,including those involved in metabolism,cell cycle,migration and transcription. Y15 demonstrated synergy with PF-04554878 in TT cells and also synergy with Cabozantinib,Sorafenib,Pazopanib,and strong synergy with Sunitinib in resistant K1 cells. This report revealed the biological effect of Y15 inhibitor,detected the unique and common gene signature profiles in response to Y15 in 4 different thyroid cancer cell lines,demonstrated differential response changes with Y15 and PF-04554878 treatment,and showed the synergy of Y15 with PF-04554878,Cabozantinib,Sorafenib,Pazopanib,and Sunitinib. View Publication

BACKGROUND Cardiovascular cell therapy represents a promising field,with several approaches currently being tested. The advanced therapy medicinal product (ATMP) for the ongoing METHOD clinical study (Bone marrow derived cell therapy in the stable phase of chronic ischemic heart disease") consists of fresh mononuclear cells (MNC) isolated from autologous bone marrow (BM) through density gradient centrifugation on standard Ficoll-Paque. Cells are tested for safety (sterility� View Publication

Differentiation of human stem cells to hepatocytes is crucial for industrial applications as well as to develop new therapeutic strategies for liver disease. The protocol described here,using sequentially growth factors known to play a role in liver embryonic development,efficiently differentiates human embryonic stem cells (hESC) as well as human-induced pluripotent stem cells (hiPSC) to hepatocytes by directing them through defined embryonic intermediates,namely,mesendoderm/definitive endoderm and hepatoblast and hepatocyte phenotype. After 28 days,the final differentiated progeny is a mixture of cells,comprising cells with characteristics of hepatoblasts and a smaller cell fraction with morphological and phenotypical features of mature hepatocytes. An extensive functional characterization of the stem cell progeny should be used to confirm that differentiated cells display functional characteristics of mature hepatocytes including albumin secretion,glycogen storage,and several detoxifying functions such as urea production,bilirubin conjugation,glutathione S-transferase activity,cytochrome activity and drug transporter activity. View Publication

AIM To enumerate and characterize mesenchymal stem cells (MSC) derived from human embryonic stem cells (hESC) for clinical application. MATERIALS & METHODS hESC were differentiated into hESC-MSC and characterized by the expression of surface markers using flow cytometry. hESC-MSC were evaluated with respect to growth kinetics,colony-forming potential,as well as osteogenic and adipogenic differentiation capacity. Immunosuppressive effects were assessed using peripheral blood mononuclear cell (PBMC) proliferation and cytotoxicity assays. RESULTS hESC-MSC showed similar morphology,and cell surface markers as adipose (AMSC) and bone marrow-derived MSC (BMSC). hESC-MSC exhibited a higher growth rate during early in vitro expansion and equivalent adipogenic and osteogenic differentiation and colony-forming potential as AMSC and BMSC. hESC-MSC demonstrated similar immunosuppressive effects as AMSC and BMSC. CONCLUSION hESC-MSC were comparable to BMSC and AMSC and hence can be used as an alternative source of MSC for clinical applications. View Publication

Pneumococcal colonization and disease is often associated with biofilm formation,in which the bacteria exhibit elevated resistance both to antibiotics and to host defense systems,often resulting in infections that are persistent and difficult to treat. We evaluated the effect of sinefungin,a nucleoside analogue of S-adenosylmethionine,on pneumococcal in vitro biofilm formation and in vivo colonization. Sinefungin is bacteriostatic to pneumococci and significantly decreased biofilm growth and inhibited proliferation and structure of actively growing biofilms but did not alter growth or the matrix structure of established biofilms. Sinefungin significantly reduced pneumococcal colonization in rat middle ear. The quorum sensing molecule (autoinducer-2) production was significantly reduced by 92% in sinefungin treated samples. The luxS,pfs,and speE genes were downregulated in biofilms grown in the presence of sinefungin. This study shows that sinefungin inhibits pneumococcal biofilm growth in vitro and colonization in vivo,decreases AI-2 production,and downregulates luxS,pfs,and speE gene expressions. Therefore,the S-adenosylmethionine (SAM) inhibitors could be used as lead compounds for the development of novel antibiofilm agents against pneumococci. View Publication

Declined production of collagen by fibroblasts is one of the major causes of aging appearance. However,only few of compounds found in cosmetic products are able to directly increase collagen synthesis. A novel small heterocyclic compound called kartogenin (KGN) was found to stimulate collagen synthesis of mesenchymal stem cells (MSCs). So,we hypothesized and tested that if KGN could be applied to stimulate the collagen synthesis of fibroblasts. Human dermal fibroblasts in vitro were treated with various concentrations of KGN,with dimethyl sulfoxide (DMSO) serving as the negative control. Real-time reverse-transcription polymerase chain reaction,Western blot,and immunofluorescence analyses were performed to examine the expression of collagen and transforming growth factor beta (TGF-β) signaling pathway. The production of collagen was also tested in vivo by Masson's trichrome stain and immunohistochemistry in the dermis of mice administrated with KGN. Results showed that without obvious influence on fibroblasts' apoptosis and viability,KGN stimulated type-I collagen synthesis of fibroblasts at the mRNA and protein levels in a time-dependent manner,but KGN did not induce expression of α-skeletal muscle actin (α-sma) or matrix metallopeptidase1 (MMP1),MMP9 in vitro. Smad4/smad5 of the TGF-β signaling pathway was activated by KGN while MAPK signaling pathway remained unchanged. KGN also increased type-I collagen synthesis in the dermis of BALB/C mice. Our results indicated that KGN promoted the type-I collagen synthesis of dermal fibroblasts in vitro and in the dermis of mice through activation of the smad4/smad5 pathway. This molecule could be used in wound healing,tissue engineering of fibroblasts,or aesthetic and reconstructive procedures. View Publication