技术资料

Platelets are an abundant source of CD40 ligand (CD154),an immunomodulatory and proinflammatory molecule implicated in the onset and progression of several inflammatory diseases,including systemic lupus erythematosus (SLE),diabetes,and cardiovascular disease. Heretofore considered largely restricted to activated T cells,we initiated studies to investigate the source and regulation of platelet-associated CD154. We found that CD154 is abundantly expressed in platelet precursor cells,megakaryocytes. We show that CD154 is expressed in primary human CD34+ and murine hematopoietic precursor cells only after cytokine-driven megakaryocyte differentiation. Furthermore,using several established megakaryocyte-like cells lines,we performed promoter analysis of the CD154 gene and found that NFAT,a calcium-dependent transcriptional regulator associated with activated T cells,mediated both differentiation-dependent and inducible megakaryocyte-specific CD154 expression. Overall,these data represent the first investigation of the regulation of a novel source of CD154 and suggests that platelet-associated CD154 can be biochemically modulated. View Publication

Mef2c is a MADS (MCM1-agamous-deficient serum response factor) transcription factor best known for its role in muscle and cardiovascular development. A causal role of up-regulated MEF2C expression in myelomonocytic acute myeloid leukemia (AML) has recently been demonstrated. Due to the pronounced monocytic component observed in Mef2c-induced AML,this study was designed to assess the importance of Mef2c in normal myeloid differentiation. Analysis of bone marrow (BM) cells manipulated to constitutively express Mef2c demonstrated increased monopoiesis at the expense of granulopoiesis,whereas BM isolated from Mef2c(Delta/-) mice showed reduced levels of monocytic differentiation in response to cytokines. Mechanistic studies showed that loss of Mef2c expression correlated with reduced levels of transcripts encoding c-Jun,but not PU.1,C/EBPalpha,or JunB transcription factors. Inhibiting Jun expression by short-interfering RNA impaired Mef2c-mediated inhibition of granulocyte development. Moreover,retroviral expression of c-Jun in BM cells promoted monocytic differentiation. The ability of Mef2c to modulate cell-fate decisions between monocyte and granulocyte differentiation,coupled with its functional sensitivity to extracellular stimuli,demonstrate an important role in immunity--and,consistent with findings of other myeloid transcription factors,a target of oncogenic lesions in AML. View Publication

Chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene produce chimeric proteins that cause abnormal expression of a subset of HOX genes and leukemia development. Here,we show that MLL normally regulates expression of mir-196b,a hematopoietic microRNA located within the HoxA cluster,in a pattern similar to that of the surrounding 5' Hox genes,Hoxa9 and Hoxa10,during embryonic stem (ES) cell differentiation. Within the hematopoietic lineage,mir-196b is most abundant in short-term hematopoietic stem cells and is down-regulated in more differentiated hematopoietic cells. Leukemogenic MLL fusion proteins cause overexpression of mir-196b,while treatment of MLL-AF9 transformed bone marrow cells with mir-196-specific antagomir abrogates their replating potential in methylcellulose. This demonstrates that mir-196b function is necessary for MLL fusion-mediated immortalization. Furthermore,overexpression of mir-196b was found specifically in patients with MLL associated leukemias as determined from analysis of 55 primary leukemia samples. Overexpression of mir-196b in bone marrow progenitor cells leads to increased proliferative capacity and survival,as well as a partial block in differentiation. Our results suggest a mechanism whereby increased expression of mir-196b by MLL fusion proteins significantly contributes to leukemia development. View Publication

Tumors that recur following surgical resection of melanoma are typically metastatic and associated with poor prognosis. Using the murine B16F10 melanoma and a robust antimelanoma vaccine,we evaluated immunization as a tool to improve tumor-free survival following surgery. We investigated the utility of vaccination in both neoadjuvant and adjuvant settings. Surprisingly,neoadjuvant vaccination was far superior and provided approximately 100% protection against tumor relapse. Neoadjuvant vaccination was associated with enhanced frequencies of tumor-specific T cells within the tumor and the tumor-draining lymph nodes following resection. We also observed increased infiltration of antigen-specific T cells into the area of surgery. This method should be amenable to any vaccine platform and can be readily extended to the clinic. View Publication

BACKGROUND AIMS Peripheral blood stem cells (PBSC) have become the preferred stem cell source for autologous hematopoietic transplantation. A critical aspect of this treatment modality is cryopreservation of the stem cell products,which permits temporal separation of the PBSC mobilization/collection phase from the subsequent high-dose therapy. While controlled rate-freezing and liquid nitrogen storage have become 'routine' practice in many cell-processing facilities,there is clearly room for improvement as current cryopreservation media formulations still result in significant loss and damage to the stem/progenitor cell populations essential for engraftment,and can also expose the patients to relatively undefined serum components and larger volumes of dimethylsulfoxide (DMSO) that can contribute to the morbidity and mortality of the transplant therapy. METHODS This study compared cryopreservation of PBSC in a novel intracellular-like,fully defined,serum- and protein-free preservation solution,CryoStor (BioLife Solutions Inc.),with a standard formulation used by the Fred Hutchinson Cancer Research Center (FHCRC). Briefly,human PBSC apheresis specimens were collected and 5 x 10(7) cells/1 mL sample vial were prepared for cryopreservation in the following solutions: (a) FHCRC standard,Normosol-R,5% human serum albumin (HAS) and 10% DMSO; and (b) CryoStor CS10 (final diluted concentration of 5% DMSO). A standard controlled-rate freezing program was employed,and frozen vials were stored in the vapor phase of a liquid nitrogen freezer for a minimum of 1 week. Vials were then thawed and evaluated for total nucleated cell count (TNC),viability,CD34 and granulocytes by flow cytometry,along with colony-forming activity in methylcellulose. RESULTS The PBSC samples frozen in CryoStor CS10 yielded significantly improved post-thaw recoveries for total viable CD34(+),colony-forming units (CFU) and granulocytes. Specifically,relative to the FHCRC standard formulation,cryopreservation with CS10 resulted in an average 1.8-fold increased recovery of viable CD34(+) cells (P=0.005),a 1.5-fold increase in CFU-granulocyte-macrophage (GM) numbers (P=0.030) and a 2.3-fold increase in granulocyte recovery (P=0.045). CONCLUSIONS This study indicates that use of CryoStor for cryopreservation can yield significantly improved recovery and in vitro functionality of stem/progenitor cells in PBSC products. In addition,it is important to note that these improved recoveries were obtained while not introducing any extra serum or serum-derived proteins,and reducing the final concentration/volume of DMSO by half. Further in vitro and in vivo studies are clearly necessary; however,these findings imply use of CryoStor for cryopreservation could result in improved engraftment for those patients with a lower content of CD34(+) cells in their PBSC collections,along with reducing the requirement for additional apheresis collections and decreasing the risk of adverse infusion reactions associated with higher exposure to DMSO. View Publication

Stem cells are highly resistant to viral infection compared to their differentiated progeny; however,the mechanism is mysterious. Here,we analyzed gene expression in mammalian stem cells and cells at various stages of differentiation. We find that,conserved across species,stem cells express a subset of genes previously classified as interferon (IFN) stimulated genes (ISGs) but that expression is intrinsic,as stem cells are refractory to interferon. This intrinsic ISG expression varies in a cell-type-specific manner,and many ISGs decrease upon differentiation,at which time cells become IFN responsive,allowing induction of a broad spectrum of ISGs by IFN signaling. Importantly,we show that intrinsically expressed ISGs protect stem cells against viral infection. We demonstrate the in vivo importance of intrinsic ISG expression for protecting stem cells and their differentiation potential during viral infection. These findings have intriguing implications for understanding stem cell biology and the evolution of pathogen resistance. View Publication

Efficient gene transfer into human hematopoietic stem cells (HSCs) is an important goal in the study of the hematopoietic system as well as for gene therapy of hematopoietic disorders. A lentiviral vector based on the human immunodeficiency virus (HIV) was able to transduce human CD34+ cells capable of stable,long-term reconstitution of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. High-efficiency transduction occurred in the absence of cytokine stimulation and resulted in transgene expression in multiple lineages of human hematopoietic cells for up to 22 weeks after transplantation. View Publication