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OBJECTIVE To investigate the long-term effect of cryopreservation on hepatocyte function,as well as attempt to improve cell viability and function through the utilization of the hypothermic preservation solution,HypoThermosol (HTS),as the carrier solution. SUMMARY BACKGROUND DATA Advances in the field of bioartificial liver support have led to an increasing demand for successful,efficient means of cryopreservation of hepatocytes. METHODS Fresh rat hepatocytes were cryopreserved in suspension in culture media (Media-cryo group) or HTS (HTS-cryo group),both supplemented with 10% DMSO. Following storage up to 2 months in liquid nitrogen,cells were thawed and maintained in a double collagen gel culture for 14 days. Hepatocyte yield and viability were assessed up to 14 days postthaw. Serial measurements of albumin secretion,urea synthesis,deethylation of ethoxyresorufin (CYT P450 activity),and responsiveness to stimulation with interleukin-6 (IL-6) were performed. RESULTS Immediate postthaw viability was 60% in Media-cryo and 79% in HTS-cryo,in comparison with control (90%). Albumin secretion,urea synthesis and CYT P450 activity yielded 33%,55%,and 59% in Media-cryo and 71%,80%,and 88% in HTS-cryo,respectively,compared with control (100%). Assessment of cellular response to IL-6 following cryopreservation revealed a similar pattern of up-regulation in fibrinogen production and suppression of albumin secretion compared with nonfrozen controls. CONCLUSIONS This study demonstrates that isolated rat hepatocytes cryopreserved using HTS showed high viability,long-term hepatospecific function,and response to cytokine challenge. These results may represent an important step forward to the utilization of cryopreserved isolated hepatocytes in bioartificial liver devices. View Publication

A series of substituted 2-(aminopyridyl)- and 2-(aminopyrimidinyl)thiazole-5-carboxamides was identified as potent Src/Abl kinase inhibitors with excellent antiproliferative activity against hematological and solid tumor cell lines. Compound 13 was orally active in a K562 xenograft model of chronic myelogenous leukemia (CML),demonstrating complete tumor regressions and low toxicity at multiple dose levels. On the basis of its robust in vivo activity and favorable pharmacokinetic profile,13 was selected for additional characterization for oncology indications. View Publication

In the present study,we assessed the susceptibility of freshly isolated neuroblastoma cells to killing mediated by normal human natural killer (NK) cells and analyzed the receptor-ligand interactions that regulate this event. We show that killing of freshly isolated neuroblasts,similar to neuroblastoma cell lines,involves NKp46 and NKp30 (natural cytotoxicity receptors). However,freshly isolated neuroblasts were generally more resistant to NK-mediated lysis than conventional neuroblastoma cell lines. Moreover,a significant heterogeneity in susceptibility to lysis existed among neuroblastomas derived from different patients. Remarkably,susceptibility to lysis directly correlated with the surface expression,on neuroblasts,of poliovirus receptor [PVR (CD155)],a ligand for the DNAX accessory molecule-1 [DNAM-1 (CD226)] triggering receptor expressed by NK cells. Indeed,PVR-expressing neuroblastomas were efficiently killed by NK cells. Moreover,monoclonal antibody-mediated masking of either DNAM-1 (on NK cells) or PVR (on neuroblasts) resulted in strong inhibition of tumor cell lysis. Thus,assessment of the PVR surface levels may represent a novel useful criterion to predict the susceptibility/resistance of neuroblastomas to NK-mediated killing. View Publication

Monocyte cytokines (ie,monokines) induce natural killer (NK) cells to produce interferon-gamma (IFN-gamma),which is critical for monocyte clearance of infectious pathogens and tumor surveillance. Human CD56bright NK cells produce far more IFN-gamma in response to monokines than do CD56dim NK cells. The kinases and phosphatases involved in regulating IFN-gamma production by monokine-activated NK cells are not clearly identified. SHIP1 is a 5' inositol phosphatase that dephosphorylates the phosphatidylinositol-3 kinase (PI-3K) product PI3,4,5P3. Here,we show that constitutive expression of SHIP1 is distinctly lower in CD56bright NK cells compared with CD56dim NK cells,suggesting it could be an important negative regulator of IFN-gamma production in monokine-activated NK cells. Indeed,overexpression of SHIP1 in CD56bright NK cells followed by monokine activation substantially lowered IFN-gamma production. This effect was not seen when NK cells were infected with a SHIP1 mutant containing an inactive catalytic domain. Finally,NK cells in SHIP1-/- mice produced more IFN-gamma in response to monokines in vivo than did NK cells from wild-type mice. Collectively,these results demonstrate that SHIP1 negatively regulates monokine-induced NK cell IFN-gamma production in vitro and in vivo and provide the first molecular explanation for an important functional distinction observed between CD56bright and CD56dim human NK subsets. View Publication

The role of human NK cells in viral infections is poorly understood. We used a cytokine flow-cytometry assay to simultaneously investigate the IFN-gamma response of NK and T lymphocytes to influenza A virus (fluA). When PBMCs from fluA-immune adult donors were incubated with fluA,IFN-gamma was produced by both CD56(dim) and CD56(bright) subsets of NK cells,as well as by fluA-specific T cells. Purified NK cells did not produce IFN-gamma in response to fluA,while depletion of T lymphocytes reduced to background levels the fluA-induced IFN-gamma production by NK cells,which indicates that T cells are required for the IFN-gamma response of NK cells. The fluA-induced IFN-gamma production of NK cells was suppressed by anti-IL-2 Ab,while recombinant IL-2 replaced the helper function of T cells for IFN-gamma production by NK cells. This indicates that IL-2 produced by fluA-specific T cells is involved in the T cell-dependent IFN-gamma response of NK cells to fluA. Taken together,these results suggest that at an early stage of recurrent viral infection,NK-mediated innate immunity to the virus is enhanced by preexisting virus-specific T cells. View Publication

Overwhelming evidence from leukemia research has shown that the clonal population of neoplastic cells exhibits marked heterogeneity with respect to proliferation and differentiation. There are rare stem cells within the leukemic population that possess extensive proliferation and self-renewal capacity not found in the majority of the leukemic cells. These leukemic stem cells are necessary and sufficient to maintain the leukemia. Interestingly,the BCR/ABL fusion gene,which is present in chronic myelogenous leukemia (CML),was also detected in the endothelial cells of patients with CML,suggesting that CML might originate from hemangioblastic progenitor cells that can give rise to both blood cells and endothelial cells. Here we isolated fetal liver kinase-1-positive (Flk1+) cells carrying the BCR/ABL fusion gene from the bone marrow of 17 Philadelphia chromosome-positive (Ph+) patients with CML and found that these cells could differentiate into malignant blood cells and phenotypically defined endothelial cells at the single-cell level. These findings provide direct evidence for the first time that rearrangement of the BCR/ABL gene might happen at or even before the level of hemangioblastic progenitor cells,thus resulting in detection of the BCR/ABL fusion gene in both blood and endothelial cells. View Publication

There are several different technical approaches to the isolation of hematopoietic stem cells (HSCs) with long-term repopulating ability,but these have problems in terms of yield,complexity,or cell viability. Simpler strategies for HSC isolation are needed. We have enriched primitive hematopoietic progenitors from murine bone marrow of mice from different genetic backgrounds by lineage depletion followed by selection of cells with high aldehyde dehydrogenase activity using the Aldefluor reagent (BD Biosciences,Oxford,U.K.). Lin- ALDH(bright) cells comprised 26.8 +/- 1.0% of the total Lin- population of C57BL6 mice,and 23.5 +/- 1.0% of the Lin- population of BALB/c mice expressed certain cell-surface markers typical of primitive hematopoietic progenitors. In vitro hematopoietic progenitor function was substantially higher in the Lin- ALDH(bright) population compared with the Lin- ALDH(low) cells. These cells have higher telomerase activity and the lowest percentage of cells in S phase. These data strongly suggest that progenitor enrichment from Lin- cells on the basis of ALDH is a valid method whose simplicity of application makes it advantageous over conventional separations. View Publication

Hematopoietic cells (HCs) promote blood vessel formation by producing various proangiogenic cytokines and chemokines and matrix metalloproteinases. We injected mouse colon26 colon cancer cells or human PC3 prostate adenocarcinoma cells into mice and studied the localization of HCs during tumor development. HCs were distributed in the inner tumor mass in all of the tumor tissues examined; however,the localization of HCs in the tumor tissue differed depending on the tumor cell type. In the case of colon26 tumors,as the tumor grew,many mature HCs migrated into the tumor mass before fine capillary formation was observed. On the other hand,although very few HCs migrated into PC3 tumor tissue,c-Kit+ hematopoietic stem/progenitor cells accumulated around the edge of the tumor. Bone marrow suppression induced by injection of anti-c-Kit neutralizing antibody suppressed tumor angiogenesis by different mechanisms according to the tumor cell type: bone marrow suppression inhibited the initiation of sprouting angiogenesis in colon26 tumors,while it suppressed an increase in the caliber of newly developed blood vessels at the tumor edge in PC3 tumors. Our findings suggest that HCs are involved in tumor angiogenesis and regulate the angiogenic switch during tumorigenesis. View Publication

Although osteoporosis is a relatively common complication after allogeneic stem cell transplantation,the role of bisphosphonates in its management has not yet been completely established. Thirty-two patients who underwent allogeneic stem cell transplantation were prospectively evaluated for bone mineral density (BMD) at the lumbar spine (LS) and femoral neck (FN) after a median period of 12.2 months. Then,15 of the patients with osteoporosis or rapidly progressing osteopenia (bone loss textgreater 5%/yr) received three monthly doses of 4 mg zoledronic acid iv. Fifteen patients were followed up without treatment,and all 30 patients were reevaluated after 12 months for BMD and bone turnover markers. By using enriched mesenchymal stem cells in the colony-forming units fibroblast (CFU-F) assay,we evaluated the osteogenic stromal lineage. This procedure was performed in both groups of patients at study entry and after 12 months. The average BMD loss was 3.42% at LS and 3.8% at FN during a 1-yr longitudinal evaluation in 32 patients. Subsequently,BMD increased at both LS and FN (9.8 and 6.4%,respectively) in the zoledronic acid-treated cohort. Hydroxyproline excretion decreased,and serum bone-specific alkaline phosphatase increased significantly,whereas serum osteocalcin increase did not reach the limit of significance. A significant increase in CFU-F growth in vitro was induced by in vivo zoledronic acid administration. In the untreated group,no significant change was observed in bone turnover markers,LS BMD (-2.1%),FN BMD (-2.3%),and CFU-F colony number. In conclusion,short-term zoledronic acid treatment consistently improved both LS and FN BMD in transplanted patients who were at high risk for fast and/or persistent bone loss,partly by increasing the osteogenic progenitors in the stromal cell compartment. View Publication

Recent reports have indicated that human immunodeficiency virus (HIV) and murine leukemia virus (MLV) vectors preferentially integrate into active genes. Here,we used a novel approach based on genetic trapping to rapidly score several thousand integration sites and found that MLV vectors trapped cellular promoters more efficiently than HIV vectors. Remarkably,1 in 5 MLV integrations trapped an active promoter in different cell lines and primary hematopoietic cells. Such frequency was even higher in growth-stimulated lymphocytes. We show that the different behavior of MLV and HIV vectors was dependent on a different integration pattern within transcribed genes. Whereas MLV-based traps showed a strong bias for promoter-proximal integration leading to efficient reporter expression,HIV-based traps integrated throughout transcriptional units and were limited for expression by the distance from the promoter and the reading frame of the targeted gene. Our results indicate a strong propensity of MLV to establish transcriptional interactions with cellular promoters,a behavior that may have evolved to enhance proviral expression and may increase the insertional mutagenesis risk. Promoter trapping efficiency provides a convenient readout to assess transcriptional interactions between the vector and its flanking genes at the integration site and to compare integration site selection among different cell types and in different growth conditions. View Publication

SU5416 is a multi-targeted kinase inhibitor that potentially has the ability to directly block tumor growth by inhibiting Kit signaling,as well as blocking angiogenesis by inhibiting vascular endothelial growth factor receptor (VEGFR) signaling. Previous work has demonstrated that SU5416 efficiently blocks Kit-mediated growth of small cell lung cancer (SCLC) in vitro. To determine the drug's effect on in vivo growth of SCLC,we studied its activity,alone and in combination with carboplatin,in chemotherapy-resistant H526,and chemotherapy-sensitive H209 murine xenograft models. SU5416 efficiently inhibited Kit activity in vivo when administered on a twice-weekly schedule. When administered over a 3-week period to animals bearing established tumors,it inhibited growth by at least 70%. It was at least as effective as carboplatin in suppressing growth of H526 xenografts. However,the combination with carboplatin was not superior to the most active single agent in either xenograft model at the doses and schedule utilized. SU5416 clearly inhibited growth in part by inhibiting angiogenesis,with microvessel density dropping by approximately 50% in treated xenografts. In addition to the recognized mechanism of inhibition of VEGFR,we uncovered a novel mechanism of angiogenesis suppression by demonstrating reduced VEGF expression in SU5416-treated xenografts. In vitro,stem cell factor treatment of the H526 cell line enhanced expression of VEGF,which was efficiently blocked with SU5416. Thus,we have demonstrated that SU5416 can inhibit SCLC growth by directly inhibiting tumor cell proliferation and by inhibiting angiogenesis,in part by inhibiting Kit-mediated VEGF expression. These data suggest that kinase inhibitors that target both Kit and VEGFR could play an important role in the treatment of SCLC,as well as other malignancies that express Kit. View Publication

It has become apparent that chromatin modification plays a critical role in the regulation of cell-type-specific gene expression. Here,we show that an inhibitor of histone deacetylase,valproic acid (VPA),induced neuronal differentiation of adult hippocampal neural progenitors. In addition,VPA inhibited astrocyte and oligodendrocyte differentiation,even in conditions that favored lineage-specific differentiation. Among the VPA-up-regulated,neuron-specific genes,a neurogenic basic helix-loop-helix transcription factor,NeuroD,was identified. Overexpression of NeuroD resulted in the induction and suppression of neuronal and glial differentiation,respectively. These results suggest that VPA promotes neuronal fate and inhibits glial fate simultaneously through the induction of neurogenic transcription factors including NeuroD. View Publication