技术资料

Adult neural stem and progenitor cells (NSPCs) are usually defined retrospectively by their ability to proliferate in vivo (bromodeoxyuridine uptake) or to form neurospheres and to differentiate into neurons,astrocytes and oligodendrocytes in vitro. Additional strategies to identify and to isolate NSPCs are of great importance for the investigation of cell differentiation and fate specification. Using the cell surface molecules Prominin-1 and Lewis X and a metabolic marker,the aldehyde dehydrogenase activity,we isolated and characterized five main populations of NSPCs in the neurogenic subventricular zone (SVZ) and the non-neurogenic spinal cord (SC). We used clonal analysis to assess neurosphere formation and multipotency,BrdU retention to investigate in vivo proliferation activity and quantified the expression of NSPC associated genes. Surprisingly,we found many similarities in NSPC subpopulations derived from the SVZ and SC suggesting that subtypes with similar intrinsic potential exist in both regions. The marker defined classification of NSPCs will help to distinguish subpopulations of NSPCs and allows their prospective isolation using fluorescence activated cell sorting. View Publication

To advance T cell-based HIV vaccine development,it is necessary to evaluate the immune correlates of a protective CD8(+) T-cell response. We have developed an assay that assesses the capacity ex vivo of HIV-specific CD8(+) T cells to suppress HIV-1 infection of autologous CD4(+) T cells. This assay directly reflects the ultimate effector function of CD8(+) T cells,the elimination of infected cells,and accurately differentiates the effective CD8(+) T-cell response in spontaneous HIV controllers from ineffective responses in other patients. In this article,we describe all the steps from cell purification to assessment of viral replication by HIV-p24 ELISA and analysis,along with conditions for cell culturing,and how to choose the viral infectious dose that gives the most reliable results. We also depict the conditions of a rapid assay on the basis of flow cytometry analysis of intracellular HIV-Gag products. These procedures take 14-17 d when the p24 ELISA assay is used,or 6 d with the intracellular Gag assay. View Publication

BACKGROUND: CBL missense mutations have recently been associated with juvenile myelomonocytic leukaemia (JMML),an aggressive myeloproliferative and myelodysplastic neoplasm of early childhood characterised by excessive macrophage/monocyte proliferation. CBL,an E3 ubiquitin ligase and a multi-adaptor protein,controls proliferative signalling networks by downregulating the growth factor receptor signalling cascades in various cell types. METHODS AND RESULTS: CBL mutations were screened in 65 patients with JMML. A homozygous mutation of CBL was found in leukaemic cells of 4/65 (6%) patients. In all cases,copy neutral loss of heterozygosity of the 11q23 chromosomal region,encompassing the CBL locus,was demonstrated. Three of these four patients displayed additional features suggestive of an underlying developmental condition. A heterozygous germline CBL p.Y371H substitution was found in each of them and was inherited from the father in one patient. The germline mutation represents the first hit,with somatic loss of heterozygosity being the second hit positively selected in JMML cells. The three patients display a variable combination of dysmorphic features,hyperpigmented skin lesions and microcephaly that enable a 'CBL syndrome' to be tentatively delineated. Learning difficulties and postnatal growth retardation may be part of the phenotype. CONCLUSION: A report of germline mutations of CBL in three patients with JMML is presented here,confirming the existence of an unreported inheritable condition associated with a predisposition to JMML. View Publication

Autophagy is characterized by the sequestration of bulk cytoplasm,including damaged proteins and organelles,and delivery of the cargo to lysosomes for degradation. Although the autophagic pathway is also linked to tumour suppression activity,the mechanism is not yet clear. Here we report that cytosolic FoxO1,a forkhead O family protein,is a mediator of autophagy. Endogenous FoxO1 was required for autophagy in human cancer cell lines in response to oxidative stress or serum starvation,but this process was independent of the transcriptional activity of FoxO1. In response to stress,FoxO1 was acetylated by dissociation from sirtuin-2 (SIRT2),a NAD(+)-dependent histone deacetylase,and the acetylated FoxO1 bound to Atg7,an E1-like protein,to influence the autophagic process leading to cell death. This FoxO1-modulated cell death is associated with tumour suppressor activity in human colon tumours and a xenograft mouse model. Our finding links the anti-neoplastic activity of FoxO1 and the process of autophagy. View Publication

The contribution of B cells to the pathology of Omenn syndrome and leaky severe combined immunodeficiency (SCID) has not been previously investigated. We have studied a mut/mut mouse model of leaky SCID with a homozygous Rag1 S723C mutation that impairs,but does not abrogate,V(D)J recombination activity. In spite of a severe block at the pro-B cell stage and profound B cell lymphopenia,significant serum levels of immunoglobulin (Ig) G,IgM,IgA,and IgE and a high proportion of Ig-secreting cells were detected in mut/mut mice. Antibody responses to trinitrophenyl (TNP)-Ficoll and production of high-affinity antibodies to TNP-keyhole limpet hemocyanin were severely impaired,even after adoptive transfer of wild-type CD4(+) T cells. Mut/mut mice produced high amounts of low-affinity self-reactive antibodies and showed significant lymphocytic infiltrates in peripheral tissues. Autoantibody production was associated with impaired receptor editing and increased serum B cell-activating factor (BAFF) concentrations. Autoantibodies and elevated BAFF levels were also identified in patients with Omenn syndrome and leaky SCID as a result of hypomorphic RAG mutations. These data indicate that the stochastic generation of an autoreactive B cell repertoire,which is associated with defects in central and peripheral checkpoints of B cell tolerance,is an important,previously unrecognized,aspect of immunodeficiencies associated with hypomorphic RAG mutations. View Publication

Activation of a naive T cell is a highly energetic event,which requires a substantial increase in nutrient metabolism. Upon stimulation,T cells increase in size,rapidly proliferate,and differentiate,all of which lead to a high demand for energetic and biosynthetic precursors. Although amino acids are the basic building blocks of protein biosynthesis and contribute to many other metabolic processes,the role of amino acid metabolism in T cell activation has not been well characterized. We have found that glutamine in particular is required for T cell function. Depletion of glutamine blocks proliferation and cytokine production,and this cannot be rescued by supplying biosynthetic precursors of glutamine. Correlating with the absolute requirement for glutamine,T cell activation induces a large increase in glutamine import,but not glutamate import,and this increase is CD28-dependent. Activation coordinately enhances expression of glutamine transporters and activities of enzymes required to allow the use of glutamine as a Krebs cycle substrate in T cells. The induction of glutamine uptake and metabolism requires ERK function,providing a link to TCR signaling. Together,these data indicate that regulation of glutamine use is an important component of T cell activation. Thus,a better understanding of glutamine sensing and use in T cells may reveal novel targets for immunomodulation. View Publication

The 8p11 myeloproliferative syndrome (EMS),also referred to as stem cell leukemia/lymphoma,is a chronic myeloproliferative disorder that rapidly progresses into acute leukemia. Molecularly,EMS is characterized by fusion of various partner genes to the FGFR1 gene,resulting in constitutive activation of the tyrosine kinases in FGFR1. To date,no previous study has addressed the functional consequences of ectopic FGFR1 expression in the potentially most relevant cellular context,that of normal primary human hematopoietic cells. Herein,we report that expression of ZMYM2/FGFR1 (previously known as ZNF198/FGFR1) or BCR/FGFR1 in normal human CD34(+) cells from umbilical-cord blood leads to increased cellular proliferation and differentiation toward the erythroid lineage in vitro. In immunodeficient mice,expression of ZMYM2/FGFR1 or BCR/FGFR1 in human cells induces several features of human EMS,including expansion of several myeloid cell lineages and accumulation of blasts in bone marrow. Moreover,bone marrow fibrosis together with increased extramedullary hematopoiesis is observed. This study suggests that FGFR1 fusion oncogenes,by themselves,are capable of initiating an EMS-like disorder,and provides the first humanized model of a myeloproliferative disorder transforming into acute leukemia in mice. The established in vivo EMS model should provide a valuable tool for future studies of this disorder. View Publication

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the blastocyst. Because of their ability to differentiate into a variety of cell types,human embryonic stem cells (hESCs) provide an unlimited source of cells for clinical medicine and have begun to be used in clinical trials. Presently,although several hundred hESC lines are available in the word,only few have been widely used in basic and applied research. More and more hESC lines with differing genetic backgrounds are required for establishing a bank of hESCs. Here,we report the first Canadian hESC lines to be generated from cryopreserved embryos and we discuss how we navigated through the Canadian regulatory process. The cryopreserved human zygotes used in this study were cultured to the blastocyst stage,and used to isolate ICM via microsurgery. Unlike previous microsurgery methods,which use specialized glass or steel needles,our method conveniently uses syringe needles for the isolation of ICM and subsequent hESC lines. ICM were cultured on MEF feeders in medium containing FBS or serum replacer (SR). Resulting outgrowths were isolated,cut into several cell clumps,and transferred onto fresh feeders. After more than 30 passages,the two hESC lines established using this method exhibited normal morphology,karyotype,and growth rate. Moreover,they stained positively for a variety of pluripotency markers and could be differentiated both in vitro and in vivo. Both cell lines could be maintained under a variety of culture conditions,including xeno-free conditions we have previously described. We suggest that this microsurgical approach may be conducive to deriving xeno-free hESC lines when outgrown on xeno-free human foreskin fibroblast feeders. View Publication

Reactive oxygen species (ROS) damage brain lipids,carbohydrates,proteins,as well as DNA and may contribute to neurodegeneration. We previously reported that ER- and oxidative stress cause neuronal apoptosis in infantile neuronal ceroid lipofuscinosis (INCL),a lethal neurodegenerative storage disease,caused by palmitoyl-protein thioesterase-1 (PPT1) deficiency. Polyunsaturated fatty acids (PUFA) are essential components of cell membrane phospholipids in the brain and excessive ROS may cause oxidative damage of PUFA leading to neuronal death. Using cultured neurons and neuroprogenitor cells from mice lacking Ppt1,which mimic INCL,we demonstrate that Ppt1-deficient neurons and neuroprogenitor cells contain high levels of ROS,which may cause peroxidation of PUFA and render them incapable of providing protection against oxidative stress. We tested whether treatment of these cells with omega-3 or omega-6 PUFA protects the neurons and neuroprogenitor cells from oxidative stress and suppress apoptosis. We report here that both omega-3 and omega-6 fatty acids protect the Ppt1-deficient cells from ER- as well as oxidative stress and suppress apoptosis. Our results suggest that PUFA supplementation may have neuroprotective effects in INCL. View Publication

Patients with mantle cell lymphoma (MCL) typically respond to initial treatment but subsequently relapse. This pattern suggests that a population of MCL cells is both drug resistant and capable of clonogenic growth. The intracellular enzyme retinaldehyde dehydrogenase (ALDH) provides resistance to several toxic agents. ALDH can also identify stem cells in normal adult tissues and tumorigenic cancer stem cells in several human malignancies. We studied ALDH expression in MCL and found small populations of ALDH(+) cells that were highly clonogenic. Moreover,ALDH(+) MCL cells were relatively quiescent and resistant to a wide range of agents. Normal B cells can be activated by specific unmethylated cytosine-phosphate-guanosine (CpG) DNA motifs through toll-like receptor 9,and we found that the synthetic CpG oligonucleotide 2006 (CpG) reduced the frequency of quiescent ALDH(+) MCL cells,induced terminal plasma cell differentiation,and limited tumor formation in vitro and in vivo. Treatment with CpG also significantly enhanced the activity of the proteasome inhibitor bortezomib that was associated with induction of the unfolded protein response. Our data suggest that CpG may target clonogenic and resistant ALDH(+) cells as well as improve the activity of proteasome inhibitors in MCL. View Publication

CD44+/CD24-/low tumor cells or aldehyde dehydrogenase 1 (ALDH1) positive tumor cells are considered cancer stem cells (CSCs) that possess the properties of self-renewal and tumorigenicity. However,their clinical value and significance in breast cancer remain controversial. A meta-analysis based on published studies was performed with the aim of obtaining an accurate evaluation of the association between the presence of CSCs in clinical samples and clinical outcome. A total of 12 eligible studies with 898 cases and 1,853 controls were included. CSC positive breast cancers,in particular those positive for ALDH1,were significantly associated with high histological grade,estrogen receptor (ER) negativity,progesterone receptor (PR) negativity,and human epidermal growth factor receptor type 2 (HER2) positivity. However,the presence of cancer stem cells was not associated with tumor size or nodal status. ALDH1 positive (RR = 2.83,95% CI: 2.16-3.67,P textless 0.001) and CD44+/CD24-/low tumor cells (RR = 2.32,95% CI: 1.51-3.60,P textless 0.001) were significantly associated with poor overall survival (OS). The stem cell markers are prognostic factors in breast cancer. Larger clinical studies are required to further evaluate the role of these markers in clinical practice. View Publication

The receptor for advanced glycation end products (RAGE) is produced either as a transmembrane or soluble form (sRAGE). Substantial evidence supports a role for RAGE and its ligands in disease. sRAGE is reported to be a competitive,negative regulator of membrane RAGE activation,inhibiting ligand binding. However,some reports indicate that sRAGE is associated with inflammatory disease. We sought to define the biological function of sRAGE on inflammatory cell recruitment,survival,and differentiation in vivo and in vitro. To test the in vivo impact of sRAGE,the recombinant protein was intratracheally administered to mice,which demonstrated monocyte- and neutrophil-mediated lung inflammation. We also observed that sRAGE induced human monocyte and neutrophil migration in vitro. Human monocytes treated with sRAGE produced proinflammatory cytokines and chemokines. Our data demonstrated that sRAGE directly bound human monocytes and monocyte-derived macrophages. Binding of sRAGE to monocytes promoted their survival and differentiation to macrophages. Furthermore,sRAGE binding to cells increased during maturation,which was similar in freshly isolated mouse monocytes compared with mature tissue macrophages. Because sRAGE activated cell survival and differentiation,we examined intracellular pathways that were activated by sRAGE. In primary human monocytes and macrophages,sRAGE treatment activated Akt,Erk,and NF-kappaB,and their activation appeared to be critical for cell survival and differentiation. Our data suggest a novel role for sRAGE in monocyte- and neutrophil-mediated inflammation and mononuclear phagocyte survival and differentiation. View Publication