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We evaluated the feasibility and efficacy of a reduced-intensity conditioning (RIC) regimen of fludarabine and melphalan to achieve rapid complete donor chimerism after allogeneic stem cell transplantation (SCT) in patients with metastatic solid tumors. Between January 1999 and January 2003,8 patients with metastatic breast cancer (BC) and 15 with metastatic renal cell carcinoma (RCC) underwent allogeneic SCT after an RIC regimen of 5 days of fludarabine and 2 days of melphalan. Filgrastim-mobilized stem cells from HLA-identical related or unrelated donors were infused. Prophylaxis for graft-versus-host disease (GVHD) consisted of tacrolimus and methotrexate. All 22 evaluable patients had 100% donor chimerism at day 30 and at all measurement times thereafter. One patient died 19 days after SCT. Nine patients (39%) had grades II to IV acute GVHD and 10 patients (43%) had chronic GVHD. Five patients (22%) died of nonrelapse treatment-related complications. Treatment-related disease response was seen in 10 patients (45%),with 3 complete responses,2 partial responses,and 5 minor responses. Fludarabine-melphalan is a feasible and effective RIC regimen for allogeneic SCT in metastatic BC and RCC. It induces rapid complete donor chimerism without the need for donor lymphocyte infusion. Tumor regression associated with GVHD is consistent with graft-versus-tumor effect. View Publication

The hemopoietic stem cell (HSC) compartment encompasses cell subsets with heterogeneous proliferative and developmental potential. Numerous CD34(-) cell subsets that might reside at an earlier stage of differentiation than CD34(+) HSCs have been described and characterized within human umbilical cord blood (UCB). We identified a novel subpopulation of CD34(-)CD133(-)CD7(-)CD45(dim)lineage (lin)(-) HSCs contained within human UCB that were endowed with low but measurable extended long-term culture-initiating cell activity. Exposure of CD34(-)CD133(-)CD7(-)CD45(dim)lin(-) HSCs to stem cell factor preserved cell viability and was associated with the following: 1) concordant expression of the stem cell-associated Ags CD34 and CD133,2) generation of CFU-granulocyte-macrophage,burst-forming unit erythroid,and megakaryocytic aggregates,3) significant extended long-term culture-initiating cell activity,and 4) up-regulation of mRNA signals for myeloperoxidase. At variance with CD34(+)lin(-) cells,CD34(-)CD133(-)CD7(-)CD45(dim)lin(-) HSCs maintained with IL-15,but not with IL-2 or IL-7,proliferated vigorously and differentiated into a homogeneous population of CD7(+)CD45(bright)CD25(+)CD44(+) lymphoid progenitors with high expression of the T cell-associated transcription factor GATA-3. Although they harbored nonclonally rearranged TCRgamma genes,IL-15-primed CD34(-)CD133(-)CD7(-)CD45(dim)lin(-) HSCs failed to achieve full maturation,as manifested in their CD3(-)TCRalphabeta(-)gammadelta(-) phenotype. Conversely,culture on stromal cells supplemented with IL-15 was associated with the acquisition of phenotypic and functional features of NK cells. Collectively,CD34(-)CD133(-)CD7(-)CD45(dim)lin(-) HSCs from human UCB displayed an exquisite sensitivity to IL-15 and differentiated into lymphoid/NK cells. Whether the transplantation of CD34(-)lin(-) HSCs possessing T/NK cell differentiation potential may impact on immunological reconstitution and control of minimal residual disease after HSC transplantation for autoimmune or malignant diseases remains to be determined. View Publication

Mice deficient in early B cell factor (EBF) are blocked at the progenitor B cell stage prior to immunoglobulin gene rearrangement. The EBF-dependent block in B cell development occurs near the onset of B-lineage commitment,which raises the possibility that EBF may act instructively to specify the B cell fate from uncommitted,multipotential progenitor cells. To test this hypothesis,we transduced enriched hematopoietic progenitor cells with a retroviral vector that coexpressed EBF and the green fluorescent protein (GFP). Mice reconstituted with EBF-expressing cells showed a near complete absence of T lymphocytes. Spleen and peripheral blood samples were textgreater95 and 90% GFP+EBF+ mature B cells,respectively. Both NK and lymphoid-derived dendritic cells were also significantly reduced compared with control-transplanted mice. These data suggest that EBF can restrict lymphopoiesis to the B cell lineage by blocking development of other lymphoid-derived cell pathways. View Publication

1. The mechanism of Ca2+ release from intracellular stores was studied in defolliculated Xenopus laevis oocytes by measuring whole-cell currents using the two-electrode voltage-clamp method. 2. The extracellular application of ionomycin,a selective Ca2+ ionophore,evoked an inward current consisting of a spike-like fast component followed by a long-lasting slow component with few superimposed current oscillations (fluctuations). The ionomycin response occurred in a dose-dependent manner and was dependent on Cl-. 3. No apparent refractory period was observed for repetitively evoked small ionomycin responses when the concentration of ionomycin was low (0.1 microM). In contrast,a larger ionomycin response (1 microM),consisting of fast and slow components,was followed by refractory period. Washing for 50-90 min was necessary for full recovery of the ionomycin response. 4. The response to ionomycin was suppressed by the extracellular application of acetoxymethyl ester of bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA AM,1-10 microM),a membrane-permeable intracellular Ca2+ chelator. 5. The ionomycin response was not affected by pertussis toxin (PTX,0.3-2.0 microgram/ml),a blocker of guanine nucleotide-binding regulatory proteins (G proteins). In contrast,the response to acetylcholine (ACh),which is known to occur via a G protein,was suppressed by PTX. 6. The fast component was not affected by removing Ca2+ from the bathing medium or by replacing extracellular Ca2+ with Ba2+ or Mn2+ (all of these solutions were supplemented with 2 mM EGTA),whereas the slow component was suppressed. 7. Injection of inositol 1,4,5-trisphosphate (IP3) following a response to extra-cellularly applied ionomycin did not evoke an appreciable membrane current. In contrast,ionomycin evoked a small inward current when it was applied after an inward-current response evoked by IP3 injection,whereas a second injection of IP3 did not evoke any appreciable current. 8. The results indicate that (a) ionomycin releases Ca2+ from its intracellular stores without the involvement of G proteins,resulting in activation of Ca(2+)-activated Cl- channels,(b) ionomycin mainly acts on the same intracellular Ca2+ stores as IP3,and (c) entry of Ca2+ from outside the cell considerably contributes to the slow component of the ionomycin response,whereas its fast component is predominantly dependent on the release of Ca2+ from the intracellular stores. View Publication

Retinoic acid (RA) exerts its pleiotropic effects on cell growth and differentiation through the activation of a family of transcription factors-the RA receptors (RARs). Three subtypes of these receptors exist,RAR alpha,RAR beta,and RAR gamma. The receptors are differentially expressed in different cell types and stages of development,suggesting that they may regulate different sets of genes. We have identified a synthetic retinoid with the characteristics of a selective RAR alpha antagonist. This antagonist counteracts RA effects on HL-60 cell differentiation and on B-lymphocyte polyclonal activation. Beyond its potential practical relevance,this and other specific antagonists will be useful to dissect the RAR system and to assign to one given receptor each of the many RA-regulated functions. View Publication

Optimal induction of clonal expansion by normal CD4 T cells requires a ligand that can engage the T cell receptor as well as functionally defined costimulatory activity on the same antigen-presenting cell surface. While the presence of effective costimulation induces proliferation,T cell receptor ligation in its absence renders T cells inactive or anergic. The molecular basis of this costimulatory activity remains to be defined. Here we describe a monoclonal antibody that can block the costimulatory activity of splenic accessory cells. Treatment with this antibody not only blocks the proliferation of CD4 T cells to a T cell receptor ligand,but also induces T cell nonresponsiveness to subsequent stimulation. Sequence analysis of the antigen recognized by this antibody indicates that it recognizes a protein that is identical to heat-stable antigen. Gene transfer experiments directly demonstrate that this protein has costimulatory activity. Thus,heat-stable antigen meets the criteria for a costimulator of T cell clonal expansion. View Publication

Embryonic stem (ES) cells are permanent pluripotent stem cell lines established from pre-implantation mouse embryos. There is currently great interest in the potential therapeutic applications of analogous cells derived from human embryos. The isolation of ES cells is commonly presented as a straightforward transfer of cells in the early embryo into culture. In reality,however,continuous expansion of pluripotent cells does not occur in vivo,and in vitro is the exception rather than the norm. Both genetic and epigenetic factors influence the ability to derive ES cells. We have tracked the expression of a key marker and determinant of pluripotency,the transcription factor Oct-4,in primary cultures of mouse epiblasts and used this to assay the effect of experimental manipulations on the maintenance of a pluripotent cell compartment. We find that expression of Oct-4 is often lost prior to overt cytodifferentiation of the epiblast. The rate and extent of Oct-4 extinction varies with genetic background. We report that treatment with the MAP kinase/ERK kinase inhibitor PD98059,which suppresses activation of the mitogen-activated protein kinases Erk1 and Erk2,results in increased persistence of Oct-4-expressing cells. Oct-4 expression is also relatively sustained in cultures of diapause embryos and of isolated inner cell masses. Combination of all three conditions allowed the derivation of germline-competent ES cells from the normally refractory CBA mouse strain. These findings suggest that the genesis of an ES cell is a relatively complex process requiring epigenetic modulation of key gene expression over a brief time-window. Procedures that extend this time-window and/or directly regulate the critical genes should increase the efficiency of ES cell derivation. View Publication

Sir2 and Hst1 are NAD+-dependent deacetylases involved in transcriptional repression in yeast. The two enzymes are highly homologous yet have different sensitivity to the small-molecule inhibitor splitomicin (compound 1) (Bedalov,A.,Gatbonton,T.,Irvine,W. P.,Gottschling,D. E.,and Simon,J. A. (2001) Proc. Natl. Acad. Sci. U. S. A. 98,15113-15118). We have now defined a critical amino acid residue within a small helical module of Hst1 that confers relative resistance to splitomicin. Parallel cell-based screens of 100 splitomicin analogues led to the identification of compounds that exhibit a higher degree of selectivity toward Sir2 or Hst1. A series of compounds based on a splitomicin derivative,dehydrosplitomicin (compound 2),effectively phenocopied a yeast strain that lacked Hst1 deacetylase while having no effect on the silencing activities of Sir2. In addition,we identified a compound with improved selectivity for Sir2. Selectivity was affirmed using whole-genome DNA microarray analysis. This study underscores the power of phenotypic screens in the development and characterization of selective inhibitors of enzyme functions. View Publication

SU9516 is a 3-substituted indolinone compound with demonstrated potent and selective inhibition toward cyclin dependent kinases (cdks). Here,we describe the kinetic characterization of this inhibition with respect to cdk2,1,and 4,along with the crystal structure in complex with cdk2. The molecule is competitive with respect to ATP for cdk2/cyclin A,with a K(i) value of 0.031 microM. Similarly,SU9516 inhibits cdk2/cyclin E and cdk1/cyclin B1 in an ATP-competitive manner,although at a 2- to 8-fold reduced potency. In contrast,the compound exhibited non-competitive inhibition with respect to ATP toward cdk4/cyclin D1,with a 45-fold reduced potency. The X-ray crystal structure of SU9516 bound to cdk2 revealed interactions between the molecule and Leu83 and Glu81 of the kinase. This study should aid in the development of more potent and selective cdk inhibitors for potential therapeutic agents. View Publication

IL-3,IL-5,and GM-CSF are related hematopoietic cytoines that are important for allergic inflammation. The receptors for human IL-5,IL-3,and GM-CSF are members of the hematopoietin receptor superfamily and are comprised of a cytokine-specific alpha chain and the common beta chain that is shared among these cytokines for signaling. Each of these cytokines contributes to the differentiation and function of leukocyte subpopulations and have clinical importance in protective immunity and in the pathophysiology of a spectrum of immunologic diseases that are as diverse as allergy and asthma,pulmonary alveolar proteinosis,neurodegenerative diseases,and malignancies. Delineating the biology of these cytokines is enabling the development of new strategies for diagnosing and treating these diseases and modulating immune responses. View Publication

We previously found that some myeloma cell lines express the heparin-binding epidermal growth factor-like growth factor (HB-EGF) gene. As the proteoglycan syndecan-1 is an HB-EGF coreceptor as well as a hallmark of plasma cell differentiation and a marker of myeloma cells,we studied the role of HB-EGF on myeloma cell growth. The HB-EGF gene was expressed by bone marrow mononuclear cells in 8 of 8 patients with myeloma,particularly by monocytes and stromal cells,but not by purified primary myeloma cells. Six of 9 myeloma cell lines and 9 of 9 purified primary myeloma cells expressed ErbB1 or ErbB4 genes coding for HB-EGF receptor. In the presence of a low interleukin-6 (IL-6) concentration,HB-EGF stimulated the proliferation of the 6 ErbB1+ or ErbB4+ cell lines,through the phosphatidylinositol 3-kinase/AKT (PI-3K/AKT) pathway. A pan-ErbB inhibitor blocked the myeloma cell growth factor activity and the signaling induced by HB-EGF. This inhibitor induced apoptosis of patients'myeloma cells cultured with their tumor environment. It also increased patients' myeloma cell apoptosis induced by an anti-IL-6 antibody or dexamethasone. The ErbB inhibitor had no effect on the interaction between multiple myeloma cells and stromal cells. It was not toxic for nonmyeloma cells present in patients' bone marrow cultures or for the growth of hematopoietic progenitors. Altogether,these data identify ErbB receptors as putative therapeutic targets in multiple myeloma. View Publication

The long-term culture-initiating cell (LTC-IC) assay is a physiological approach to the quantitation of primitive human hematopoietic cells. The readout using identification of cobblestone area-forming cells (CAFC) has gained popularity over the LTC-IC readout where cells are subcultured in a colony-forming cell assay. However,comparing the two assays,cord blood (CB) mononuclear cell (MNC) samples were found to contain a higher frequency of CAFC than LTC-IC (126 +/- 83 versus 40 +/- 31 per 10(5) cells,p = 0.0001). Overall,60% of week-5 cobblestones produced by CB MNC were not functional LTC-IC and were classified as false." Separation of CB MNC using immunomagnetic columns showed that false cobblestones were CD34(-)/lineage(+). Purified CD34(+) cells� View Publication