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Interleukin (IL)-13 has recently been shown to play important and unique roles in asthma,parasite immunity,and tumor recurrence. At least two distinct receptor components,IL-4 receptor (R)alpha and IL-13Ralpha1,mediate the diverse actions of IL-13. We have recently described an additional high affinity receptor for IL-13,IL-13Ralpha2,whose function in IL-13 signaling is unknown. To better appreciate the functional importance of IL-13Ralpha2,mice deficient in IL-13Ralpha2 were generated by gene targeting. Serum immunoglobulin E levels were increased in IL-13Ralpha2-/- mice despite the fact that serum IL-13 was absent and immune interferon gamma production increased compared with wild-type mice. IL-13Ralpha2-deficient mice display increased bone marrow macrophage progenitor frequency and decreased tissue macrophage nitric oxide and IL-12 production in response to lipopolysaccharide. These results are consistent with a phenotype of enhanced IL-13 responsiveness and demonstrate a role for endogenous IL-13 and IL-13Ralpha2 in regulating immune responses in wild-type mice. View Publication

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The Fas receptor and its ligand have been implicated in mediating the bone marrow (BM) suppression observed in graft-versus-host disease and a number of other BM-failure syndromes. However,previous studies have suggested that Fas is probably not expressed on human hematopoietic stem cells (HSCs),but up-regulated as a consequence of their commitment and differentiation,suggesting that progenitors or differentiated blood cells,rather than HSCs,are the targets of Fas-mediated suppression. The present studies confirm that candidate HSCs in human cord blood and BM lack constitutive expression of Fas,but demonstrate that Fas expression on CD34+ progenitor and stem cells is correlated to their cell cycle and activation status. With the use of recently developed in vitro conditions promoting HSC self-renewing divisions,Fas was up-regulated on virtually all HSCs capable of multilineage reconstituting nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice in vivo,as well as on long-term culture-initiating cells (LTC-ICs). Similarly,in vivo cycling of NOD-SCID repopulating cells upon transplantation,resulted in up-regulation of Fas expression. However,repopulating HSCs expressing high levels of Fas remained highly resistant to Fas-mediated suppression,and HSC function was compromised only upon coactivation with tumor necrosis factor. Thus,reconstituting human HSCs up-regulate Fas expression upon active cycling,demonstrating that HSCs could be targets for Fas-mediated BM suppression. View Publication

Hematologic stem cell rescue after high-dose cytotoxic therapy is extensively used for the treatment of many hematopoietic and solid cancers. Gene marking studies suggest that occult tumor cells within the autograft may contribute to clinical relapse. To date purging of autografts contaminated with cancer cells has been unsuccessful. The selective oncolytic property of reovirus against myriad malignant histologies in in vitro,in vivo,and ex vivo systems has been previously demonstrated. In the present study we have shown that reovirus can successfully purge cancer cells within autografts. Human monocytic and myeloma cell lines as well as enriched ex vivo lymphoma,myeloma,and Waldenström macroglobulinemia patient tumor specimens were used in an experimental purging model. Viability of the cell lines or purified ex vivo tumor cells of diffuse large B-cell lymphoma,chronic lymphocytic leukemia,Waldenström macroglobulinemia,and small lymphocytic lymphoma was significantly reduced after reovirus treatment. Further,[35S]-methionine labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of cellular proteins demonstrated reovirus protein synthesis and disruption of host cell protein synthesis as early as 24 hours. Admixtures of apheresis product with the abovementioned tumor cells and cell lines treated with reovirus showed complete purging of disease. In contrast,reovirus purging of enriched ex vivo multiple myeloma,Burkitt lymphoma,and follicular lymphoma was incomplete. The oncolytic action of reovirus did not affect CD34+ stem cells or their long-term colony-forming assays even after granulocyte colony-stimulating factor (G-CSF) stimulation. Our results indicate the ex vivo use of an unattenuated oncolytic virus as an attractive purging strategy for autologous stem cell transplantations. View Publication

Human stem cells derived from human fertilized oocytes,fetal primordial germ cells,umbilical cord blood,and adult tissues provide potential cell-based therapies for repair of degenerating or damaged tissues. However,the diversity of major histocompatibility complex (MHC) antigens in the general population and the resultant risk of immune-mediated rejection complicates the allogenic use of established stem cells. We assessed an alternative approach,employing chemical activation of nonfertilized metaphase II oocytes for producing stem cells homozygous for MHC. By using F1 hybrid mice (H-2-B/D),we established stem cell lines homozygous for H-2-B and H-2-D,respectively. The undifferentiated cells retained a normal karyotype,expressed stage-specific embryonic antigen-1 and Oct4,and were positive for alkaline phosphatase and telomerase. Teratomatous growth of these cells displayed the development of a variety of tissue types encompassing all three germ layers. In addition,these cells demonstrated the potential for in vitro differentiation into endoderm,neuronal,and hematopoietic lineages. We also evaluated this homozygous stem cell approach in human tissue. Five unfertilized blastocysts were derived from a total of 25 human oocytes,and cells from one of the five hatched blastocysts proliferated and survived beyond two passages. Our studies demonstrate a plausible homozygous stem cell" approach for deriving pluripotent stem cells that can overcome the immune-mediated rejection response common in allotransplantation View Publication

Cord blood (CB) cells are a useful source of hematopoietic cells for transplantation. The hematopoietic activities of CB cells are different from those of bone marrow and peripheral blood (PB) cells. Platelet recovery is significantly slower after transplantation with CB cells than with cells from other sources. However,the cellular mechanisms underlying these differences have not been elucidated. We compared the surface marker expression profiles of PB and CB hematopoietic cells. We focused on two surface markers of hematopoietic cell immaturity,i.e.,CD34 and AC133. In addition to differences in surface marker expression,the PB and CB cells showed nonidentical differentiation pathways from AC133(+)CD34(+) (immature) hematopoietic cells to terminally differentiated cells. The majority of the AC133(+)CD34(+) PB cells initially lost AC133 expression and eventually became AC133(-)CD34(-) cells. In contrast,the AC133(+)CD34(+) CB cells did not go through the intermediate AC133(-)CD34(+) stage and lost both markers simultaneously. Meanwhile,the vast majority of megakaryocyte progenitors were of the AC133(-)CD34(+) phenotype. We conclude that the delayed recovery of platelets after CB transplantation is due to both subpopulation distribution and the process of differentiation from AC133(+)CD34(+) cells. View Publication

Human immunodeficiency virus type 1 (HIV-1) impacts the activation state of multiple lineages of hematopoietic cells. Chronic HIV-1 infection among individuals with progressive disease can be associated with increased levels of activated signal transducers and activators of transcription (STATs) in peripheral blood mononuclear cells. To investigate interactions between HIV-1 and CD4(+) cells,activated,phosphorylated STAT proteins in nuclear extracts from lymphocytic and promonocytic cell lines as well as primary monocyte-derived macrophages were measured. Levels of activated STATs increased six- to tenfold in HUT78 and U937 cells within 2 h following exposure to virions. The response to virus was dose-dependent,but kinetics of activation was delayed relative to interleukin-2 or interferon-gamma. Activation of STAT1,STAT3,and STAT5 occurred with diverse viral envelope proteins,independent of coreceptor use or viral replication. Envelope-deficient virions had no effect on STAT activation. Monoclonal antibody engagement of CD4 identified a novel role for CD4 as a mediator in the activation of multiple STATs. Results provide a model for HIV-1 pathogenesis in infected and noninfected hematopoietic cells. View Publication

In humans the majority of endothelial cells (EC) constitutively express MHC class II Ags. We know that in vitro ECs can activate CD45RO(+) B7-independent CD4(+) T cells to proliferate and produce IL-2. The in vivo correlate of this T cell response is not known,and here we have explored whether endothelial expression of MHC class II Ags affects the transendothelial migration of alloreactive CD4(+) CD45RO(+) B7-independent T cells. Alloreactive CD4(+) T cell clones and lines were generated against HLA-DR11,DR13,DR4,and DR1 MHC Ags,and their rates of migration across untreated EC line Eahy.926 (MHC class II negative) or Eahy.926 transfected with CIITA (EahyCIITA) to express DR11 and DR13 were investigated. The migrations of EahyCIITA-specific T cell clones and lines were retarded in a DR-specific manner,and retardation was reversed in the presence of mAb to DR Ag. When investigating the ability of T cells to proliferate in response to EahyCIITA before and after transmigration,migrated cells were still able to proliferate,but the frequency of EahyCIITA-specific cells was much reduced compared with that of nonmigrated cells. The use of fluorescently labeled T cells revealed that specific cells become trapped within the endothelial monolayer. Pretreatment of EahyCIITA with IFN-gamma restored the ability of DR11- or DR13-specific T cells to transmigrate and proliferate,thus abrogating DR-specific retardation. We conclude that cognate interaction between T cells and endothelial MHC class II initiates a stop signal possibly similar to an immunological synapse,but this is overcome in an inflammatory milieu. View Publication

Insulin resistance plays a central role in the development of type 2 diabetes,but the precise defects in insulin action remain to be elucidated. Glycogen synthase kinase 3 (GSK-3) can negatively regulate several aspects of insulin signaling,and elevated levels of GSK-3 have been reported in skeletal muscle from diabetic rodents and humans. A limited amount of information is available regarding the utility of highly selective inhibitors of GSK-3 for the modification of insulin action under conditions of insulin resistance. In the present investigation,we describe novel substituted aminopyrimidine derivatives that inhibit human GSK-3 potently (K(i) textless 10 nmol/l) with at least 500-fold selectivity against 20 other protein kinases. These low molecular weight compounds activated glycogen synthase at approximately 100 nmol/l in cultured CHO cells transfected with the insulin receptor and in primary hepatocytes isolated from Sprague-Dawley rats,and at 500 nmol/l in isolated type 1 skeletal muscle of both lean Zucker and ZDF rats. It is interesting that these GSK-3 inhibitors enhanced insulin-stimulated glucose transport in type 1 skeletal muscle from the insulin-resistant ZDF rats but not from insulin-sensitive lean Zucker rats. Single oral or subcutaneous doses of the inhibitors (30-48 mg/kg) rapidly lowered blood glucose levels and improved glucose disposal after oral or intravenous glucose challenges in ZDF rats and db/db mice,without causing hypoglycemia or markedly elevating insulin. Collectively,our results suggest that these selective GSK-3 inhibitors may be useful as acute-acting therapeutics for the treatment of the insulin resistance of type 2 diabetes. View Publication

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),an endocrine disrupting chemical (EDC),can cause carcinogenesis,immunosuppression,and teratogenesis,through a ligand-activated transcription factor,the aryl hydrocarbon receptor (AhR). Despite remarkable recent advances in stem cell biology,the influence of TCDD on hematopoietic stem cells (HSCs),which possess the ability to reconstitute long-term multilineage hematopoiesis,has not been well investigated. In this study we examined the influence of TCDD on HSCs enriched for CD34(-),c-kit(+),Sca-1(+),lineage negative (CD34-KSL) cells. The number of the CD34-KSL cells was found to be increased about four-fold upon a single oral administration of TCDD (40 micro g/kg body weight). Surprisingly,we found that these TCDD-treated cells almost lost long-term reconstitution activity. This defect was not present in AhR(-/-) mice. These findings suggest that modulation of AhR/ARNT system activity may have an effect on HSC function or survival. View Publication

The generation and maintenance of virus-specific CD4(+) T cells in humans are not well understood. We used short in vitro stimulation assays followed by intracellular cytokine staining to characterize the timing,magnitude,and Ag specificity of CD4(+) T cells over the course of primary EBV infection. Lytic and latent protein-specific CD4(+) T cells were readily detected at presentation with acute infectious mononucleosis and declined rapidly thereafter. Responses to BZLF-1,BMLF-1,and Epstein-Barr nuclear Ag-3A were more commonly detected than responses to Epstein-Barr nuclear Ag-1. Concurrent analyses of BZLF-1-specific CD4(+) and CD8(+) T cells revealed differences in the expansion,specificity,and stability of CD4(+) and CD8(+) T cell-mediated responses over time. Peripheral blood EBV load directly correlated with the frequency of EBV-specific CD4(+) T cell responses at presentation and over time,suggesting that EBV-specific CD4(+) T cell responses are Ag-driven. View Publication

Embryonic stem cells are pluripotent cells derived from the inner cell mass of mouse blastocysts that have been shown to differentiate spontaneously into cell types representing all three germ layers. This study shows that ES cells were induced to differentiate in vitro into mineralized osteoblasts under the influence of ascorbic acid,beta-glycerophosphate and 1alpha,25-OH vitamin D3. The activity of alkaline phosphatase,an early osteoblast marker,was found to be increased around day 12 of culture. Mineralized cells were clearly identified by histochemical staining,which detects mineralized calcium. The major noncollagenous component of bone matrix,osteocalcin,was localized to the mineralized cells by immunofluorescence. The expression of bone-specific genes was analyzed by real-time quantitative PCR. Osteocalcin and bone sialoprotein (BSP) were identified as early as in the fourth week of embryonic stem cell culture,both being characteristic for late stages of osteoblastic differentiation,indicating that at this time of culture the identified cells represent mature" osteoblasts. The osteoblast-specific transcription factor Cbfa1 was induced a few days earlier. The expression of osteopontin and osteonectin View Publication