技术资料

HIV-1 infected cells are eliminated in infected individuals by a variety of cellular mechanisms,the best characterized of which are cytotoxic T cell and NK cell-mediated killing. An additional antiviral mechanism is antibody-dependent cellular cytotoxicity. Here we use primary CD4(+) T cells infected with the BaL clone of HIV-1 as target cells and autologous NK cells,monocytes,and neutrophils as effector cells,to quantify the cytotoxicity mediated by the different effectors. This was carried out in the presence or absence of HIV-1-specific antiserum to assess antibody-dependent cellular cytotoxicity. We show that at the same effector to target ratio,NK cells and monocytes mediate similar levels of both antibody-dependent and antibody-independent killing of HIV-1-infected T cells. Neutrophils mediated significant antibody-dependent killing of targets,but were less effective than monocytes or NK cells. These data have implications for acquisition and control of HIV-1 in natural infection and in the context of vaccination. View Publication

Oct4 is critical to maintain the pluripotency of human embryonic stem cells (hESCs); however,the underlying mechanism remains to be fully understood. Here,we report that silencing of Oct4 in hESCs leads to the activation of tumor suppressor p53,inducing the differentiation of hESCs since acute disruption of p53 in p53 conditional knockout (p53CKO) hESCs prevents the differentiation of hESCs after Oct4 depletion. We further discovered that the silencing of Oct4 significantly reduces the expression of Sirt1,a deacetylase known to inhibit p53 activity and the differentiation of ESCs,leading to increased acetylation of p53 at lysine 120 and 164. The importance of Sirt1 in mediating Oct4-dependent pluripotency is revealed by the finding that the ectopic expression of Sirt1 in Oct4-silenced hESCs prevents p53 activation and hESC differentiation. In addition,using knock-in approach,we revealed that the acetylation of p53 at lysine 120 and 164 is required for both stabilization and activity of p53 in hESCs. In summary,our findings reveal a novel role of Oct4 in maintaining the pluripotency of hESCs by suppressing pathways that induce differentiation. Considering that p53 suppresses pluripotency after DNA damage response in ESCs,our findings further underscore the stringent mechanism to coordinate DNA damage response pathways and pluripotency pathways in order to maintain the pluripotency and genomic stability of hESCs. View Publication

RATIONALE: Human embryonic stem cell (hESC) derivatives are attractive candidates for therapeutic use. The engraftment and survival of hESC derivatives as xenografts or allografts require effective immunosuppression to prevent immune cell infiltration and graft destruction.backslashnbackslashnOBJECTIVE: To test the hypothesis that a short-course,dual-agent regimen of two costimulation-adhesion blockade agents can induce better engraftment of hESC derivatives compared to current immunosuppressive agents.backslashnbackslashnMETHODS AND RESULTS: We transduced hESCs with a double fusion reporter gene construct expressing firefly luciferase (Fluc) and enhanced green fluorescent protein,and differentiated these cells to endothelial cells (hESC-ECs). Reporter gene expression enabled longitudinal assessment of cell engraftment by bioluminescence imaging. Costimulation-adhesion therapy resulted in superior hESC-EC and mouse EC engraftment compared to cyclosporine therapy in a hind limb model. Costimulation-adhesion therapy also promoted robust hESC-EC and hESC-derived cardiomyocyte survival in an ischemic myocardial injury model. Improved hESC-EC engraftment had a cardioprotective effect after myocardial injury,as assessed by magnetic resonance imaging. Mechanistically,costimulation-adhesion therapy is associated with systemic and intragraft upregulation of T-cell immunoglobulin and mucin domain 3 (TIM3) and a reduced proinflammatory cytokine profile.backslashnbackslashnCONCLUSIONS: Costimulation-adhesion therapy is a superior alternative to current clinical immunosuppressive strategies for preventing the post-transplant rejection of hESC derivatives. By extending the window for cellular engraftment,costimulation-adhesion therapy enhances functional preservation following ischemic injury. This regimen may function through a TIM3-dependent mechanism. View Publication

Long-term marrow cultures (LTMC) allow the proliferation and differentiation of primitive human hematopoietic progenitor cells to be maintained for many weeks in the absence of exogenously provided hematopoietic growth factors. Previous investigations focused on defining various types of cells that are present in this culture system and on measuring the cycling behavior of the different subpopulations of colony-forming cells maintained within it. These studies suggested that mesenchymal stromal elements derived from the input marrow play a key role in regulating the turnover of the most primitive,high-proliferative potential erythroid and granulopoietic colony-forming cells that are found almost exclusively in the adherent layer of LTMC. In this study we show that the re-entry into S-phase of these primitive hematopoietic progenitors that occurs after each weekly medium change is due to an as yet undefined constituent of horse serum,which is absent from fetal calf serum. However,this effect is not unique to the factor present in horse serum. It is also elicited by the addition to LTMC of several well-defined growth regulatory molecules,ie,platelet-derived growth factor (PDGF),interleukin-1 (IL-1),transforming growth factor alpha (TGF-alpha),and IL-2. None of these was able to stimulate hematopoietic colony-forming cells in methylcellulose assays,although all have known actions on mesenchymal cells including,in some cases,the ability to increase production of growth factors that can stimulate primitive high-proliferative potential hematopoietic progenitors in clonogenic assays. Interestingly,a stimulating effect was not obtained after addition of endotoxin to LTMC. TGF-beta,a direct-acting negative regulator that acts selectively on primitive hematopoietic progenitor cells if added to LTMC simultaneously with new medium or IL-1,blocked their stimulating activity. These results suggest a model in which indirect,local modulation of both positive and negative regulatory factors via effects on mesenchymal elements determines the rate of turnover of adjacent populations of very primitive hematopoietic cells that are normally maintained in a quiescent state in vivo. View Publication

BACKGROUND AND OBJECTIVE While pro-inflammatory monocyte trafficking to the intestine has been partially characterised,the molecules required for migration of tolerogenic mononuclear phagocytes (dendritic cells (DC) and macrophages) are unknown. We hypothesised that the gut-homing receptor integrin α4β7 is required for this process. METHODS We used a T cell-mediated colitis model to study the role of α4β7 in the innate immune compartment. We then performed competitive bone marrow (BM) reconstitution experiments to assess the requirement of α4β7 in the generation of intestinal retinoic acid (RA)-producing CD11c(hi) DC (ALDE(+)DC) and CD64 macrophages. Using mixed BM chimeras we also asked whether α4β7 is required to give rise to tolerogenic mononuclear phagocytes. RESULTS Lack of β7 integrins in the innate immune compartment (β7(-/-)RAG2(-/-) mice) markedly accelerated T cell-mediated colitis,which was correlated with lower numbers and frequencies of ALDE(+)DC in mesenteric lymph nodes. Consistent with a role of α4β7 in the generation of intestinal mononuclear phagocytes,BM cells from β7(-/-) mice poorly reconstituted small intestine ALDE(+)DC and Mφ when compared to their wild type counterparts. In addition,mice lacking β7 integrins in the CD11c(hi) compartment showed decreased ability to induce Foxp3(+) T(REG) and IL-10-producing T cells. CONCLUSIONS Mice lacking β7 integrins in the innate immune compartment are more susceptible to intestinal inflammation,which is correlated with a requirement of β7 integrins to reconstitute gut mononuclear phagocytes with tolerogenic potential. View Publication

BACKGROUND Nuclear protein 1 (Nupr1) is a major factor in the cell stress response required for Kras(G12D)-driven formation of pancreatic intraepithelial neoplastic lesions (PanINs). We evaluated the relevance of Nupr1 in the development of pancreatic cancer. METHODS We investigated the role of Nupr1 in pancreatic ductal adenocarcinoma (PDAC) progression beyond PanINs in Pdx1-cre;LSL-Kras(G12D);Ink4a/Arf(fl/fl)(KIC) mice. RESULTS Even in the context of the second tumorigenic hit of Ink4a/Arf deletion,Nupr1 deficiency led to suppression of malignant transformation involving caspase 3 activation in premalignant cells of KIC pancreas. Only half of Nupr1-deficient;KIC mice achieved PDAC development,and incident cases survived longer than Nupr1(wt);KIC mice. This was associated with the development of well-differentiated PDACs in Nupr1-deficient;KIC mice,which displayed enrichment of genes characteristic of the recently identified human classical PDAC subtype. Nupr1-deficient;KIC PDACs also shared with human classical PDACs the overexpression of the Kras-activation gene signature. In contrast,Nupr1(wt);KIC mice developed invasive PDACs with enriched gene signature of human quasi-mesenchymal (QM) PDACs. Cells derived from Nupr1-deficient;KIC PDACs growth in an anchorage-independent manner in vitro had higher aldehyde dehydrogenase activity and overexpressed nanog,Oct-4 and Sox2 transcripts compared with Nupr1(wt);KIC cells. Moreover,Nupr1-deficient and Nurpr1(wt);KIC cells differed in their sensitivity to the nucleoside analogues Ly101-4b and WJQ63. Together,these findings show the pivotal role of Nupr1 in both the initiation and late stages of PDAC in vivo,with a potential impact on PDAC cell stemness. CONCLUSIONS According to Nupr1 status,KIC mice develop tumours that phenocopy human classical or QM-PDAC,respectively,and present differential drug sensitivity,thus becoming attractive models for preclinical drug trials. View Publication

BACKGROUND Triple-negative breast cancer (TNBC) has significantly worse prognosis. Acquired chemoresistance remains the major cause of therapeutic failure of TNBC. In clinic,the relapsed TNBC is commonly pan-resistant to various drugs with completely different resistant mechanisms. Investigation of the mechanisms and development of new drugs to target pan-chemoresistance will potentially improve the therapeutic outcomes of TNBC patients. METHODS In this study,1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT),combination index (CI)-isobologram,western blot,ALDEFLUOR analysis,clonogenic assay and immunocytochemistry were used. RESULTS The chemoresistant MDA-MB-231PAC10 cells are highly cross-resistant to paclitaxel (PAC),cisplatin (CDDP),docetaxel and doxorubicin. The MDA-MB-231PAC10 cells are quiescent with significantly longer doubling time (64.9 vs 31.7 h). This may be caused by high expression of p21(Waf1). The MDA-MB-231PAC10 cells express high aldehyde dehydrogenase (ALDH) activity and a panel of embryonic stem cell-related proteins,for example,Oct4,Sox2,Nanog and nuclealisation of HIF2$$ and NF-$$Bp65. We have previously reported that disulfiram (DS),an antialcoholism drug,targets cancer stem cells (CSCs) and enhances cytotoxicity of anticancer drugs. Disulfiram abolished CSC characters and completely reversed PAC and CDDP resistance in MDA-MB-231PAC10 cells. CONCLUSION Cancer stem cells may be responsible for acquired pan-chemoresistance. As a drug used in clinic,DS may be repurposed as a CSC inhibitor to reverse the acquired pan-chemoresistance. View Publication

INTRODUCTION We hypothesized that cells present in normal tissue that bear cancer stem cell markers may represent a cancer cell of origin or a microenvironment primed for tumor development,and that their presence may correlate with the clinically defined subtypes of breast cancer that show increased tumorigenicity and stem cell features. methods: Normal tissues sampled at least 5 cm from primary tumors (normal adjacent tissue) were obtained from 61 chemotherapy-naive patients with breast cancer treated with mastectomy. Samples were stained simultaneously with immunofluorescence for CD44/CD49f/CD133/2 stem cell markers. We assessed the association between CD44+CD49f+CD133/2+ staining in normal adjacent tissue and breast cancer receptor subtype (defined by the expression of the estrogen (ER),progesterone (PR),or human epidermal growth factor-2 (Her2) receptors). We also examined the correlation between CD44+CD49f+CD133/2+ immunofluorescence and each of two previously published gene signatures,one derived from stem-cell enriched tissue and one from BRCA mutated tissue expected to have defective DNA repair. RESULTS Patients with triple negative breast cancer (ER–/PR–/HER2–) expressed CD44+CD49f+CD133/2+ in 9 of 9 normal adjacent tissue samples compared with 7 of 52 ER+ and/or Her2+ tumors (P textless 0.001). Further,expression of CD44+CD49f+CD133/2+ by normal adjacent tissue correlated positively with a stem cell-derived tumorigenic signature (P textless0.001) and inversely with a defective DNA-repair signature (P textless0.001). CONCLUSION Normal cells bearing cancer stem cell markers are associated with the triple negative receptor subtype of breast cancer. This study suggests stem cell staining and gene expression signatures from normal breast tissues represent novel tissue-based risk biomarkers for triple negative breast cancer. Validation of these results in additional studies of normal tissue from cancer-free women could lay the foundation for future targeted triple negative breast cancer prevention strategies. View Publication

A large number of biologic,technological,and clinical studies await the development of procedures that will allow totipotent hematopoietic stem cells to be expanded in vitro. Previous work has suggested that hematopoiesis can be reconstituted using transplants of cells from long-term marrow cultures. We have used retrovirus mediated gene transfer to demonstrate that marked totipotent hematopoietic stem cells are both maintained and can be amplified in such cultures,and then subsequently regenerate and sustain lympho-myeloid hematopoiesis in irradiated recipients. Marrow cells from 5-fluorouracil-treated male mice were infected with a recombinant virus carrying the neomycin resistence gene and seeded onto irradiated adherent layers of pre-established,long-term marrow cultures of female origin. At 4 weeks,cells from individual cultures were transplanted into single or multiple female recipients. Southern blot analysis of hematopoietic tissue 45 days posttransplantation showed retrovirally marked clones common to lymphoid and myeloid tissues in 14 of 23 mice examined. Strikingly,for 3 of 4 long-term cultures,multiple recipients of cells from a single flask showed marrow and thymus repopulation with the same unique retrovirally marked clone. These results establish the feasibility of retroviral-marking techniques to demonstrate the maintenance of totipotent lympho-myeloid stem cells for at least 4 weeks in the long-term marrow culture system and provide the first evidence of their proliferation in vitro. Therefore,such cultures may serve as a starting point for identifying factors that stimulate totipotent hematopoietic stem cell expansion. View Publication

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by the degeneration of motor neurons. Currently,there is no effective therapy for ALS. Stem cell transplantation is a potential therapeutic strategy for ALS,and the reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) represents a novel cell source. In this study,we isolated a specific neural stem cell (NSC) population from human iPSCs based on high aldehyde dehydrogenase activity,low side scatter and integrin VLA4 positivity. We assessed the therapeutic effects of these NSCs on the phenotype of ALS mice after intrathecal or intravenous injections. Transplanted NSCs migrated and engrafted into the central nervous system via both routes of injection. Compared with control ALS,treated ALS mice exhibited improved neuromuscular function and motor unit pathology and significantly increased life span,in particular with the systemic administration of NSCs (15%). These positive effects are linked to multiple mechanisms,including production of neurotrophic factors and reduction of micro- and macrogliosis. NSCs induced a decrease in astrocyte number through the activation of the vanilloid receptor TRPV1. We conclude that minimally invasive injections of iPSC-derived NSCs can exert a therapeutic effect in ALS. This study contributes to advancements in iPSC-mediated approaches for treating ALS and other neurodegenerative diseases. View Publication

Down syndrome (DS) is among the most frequent genetic causes of intellectual disability,and ameliorating this deficit is a major goal in support of people with trisomy 21. The Ts65Dn mouse recapitulates some major brain structural and behavioral phenotypes of DS,including reduced size and cellularity of the cerebellum and learning deficits associated with the hippocampus. We show that a single treatment of newborn mice with the Sonic hedgehog pathway agonist SAG 1.1 (SAG) results in normal cerebellar morphology in adults. Further,SAG treatment at birth rescued phenotypes associated with hippocampal deficits that occur in untreated adult Ts65Dn mice. This treatment resulted in behavioral improvements and normalized performance in the Morris water maze task for learning and memory. SAG treatment also produced physiological effects and partially rescued both N-methyl-d-aspartate (NMDA) receptor-dependent synaptic plasticity and NMDA/AMPA receptor ratio,physiological measures associated with memory. These outcomes confirm an important role for the hedgehog pathway in cerebellar development and raise the possibility for its direct influence in hippocampal function. The positive results from this approach suggest a possible direction for therapeutic intervention to improve cognitive function for this population. View Publication

Diblock copolymers consisting of poly(ethylene glycol)-block-poly(γ-4-(((2-(piperidin-1-yl)ethyl)amino)methyl)benzyl-l-glutamate) (PEG-b-PVBLG-8) were synthesized and evaluated for their ability to mediate gene delivery in hard-to-transfect cells like IMR-90 human fetal lung fibroblasts and human embryonic s View Publication