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It is anticipated that gamma-secretase inhibitors (gamma-Sec-I) that modulate Notch processing will alter differentiation in tissues whose architecture is governed by Notch signaling. To explore this hypothesis,Han Wistar rats were dosed for up to 5 days with 10-100 micromol/kg b.i.d. gamma-Sec-I from three chemical series that inhibit Notch processing in vitro at various potencies (Notch IC(50)). These included an arylsulfonamide (AS) (142 nM),a dibenzazepine (DBZ) (1.7 nM),and a benzodiazepine (BZ) (2.2 nM). The DBZ and BZ caused dose-dependent intestinal goblet cell metaplasia. In contrast,the AS produced no detectable in vivo toxicity,despite higher exposure to free drug. In a time course using BZ,small intestinal crypt cell and large intestinal glandular cell epithelial apoptosis was observed on days 1-5,followed by goblet cell metaplasia on days 2-5 and crypt epithelial and glandular epithelial regenerative hyperplasia on days 4-5. Gene expression profiling of duodenal samples from BZ-dosed animals revealed significant time-dependent deregulation of mRNAs for various panendocrine,hormonal,and transcription factor genes. Somatostatin,secretin,mucin,CCK,and gastrin mRNAs were elevated twofold or more by day 2,and a number of candidate early-predictive" genes were altered on days 1-2� View Publication

PURPOSE: Allogeneic T lymphocytes can induce regression of metastatic breast cancer through an immune-mediated graft-versus-tumor (GVT) effect in murine models. To determine if a clinical GVT effect exists against metastatic breast cancer,allogeneic lymphocytes were used as adoptive cellular therapy after a reduced-intensity chemotherapy conditioning regimen and allogeneic hematopoietic stem-cell transplantation (HSCT) from human leukocyte antigen-matched siblings. PATIENTS AND METHODS: Sixteen patients with metastatic breast cancer that had progressed after treatment with anthracyclines,taxanes,hormonal agents,and trastuzumab,received allogeneic HSCT. The reduced-intensity transplant conditioning regimen consisted of cyclophosphamide and fludarabine. To distinguish an immunological GVT effect from any antitumor effect of cytotoxic chemotherapy in the transplant-conditioning regimen,allogeneic T lymphocytes were removed from the stem-cell graft and were subsequently administered late postallogeneic HSCT. Allogeneic lymphocytes containing 1 x 10(6),5 x 10(6),and 10 x 10(6) CD3(+) cells/kg were infused on days +42,+70,and +98 post-allogeneic HSCT,respectively. RESULTS: Objective tumor regressions occurred after day +28 post-allogeneic HSCT in six patients and were attributed to allogeneic lymphocyte infusions. Two of these responding patients had disease progression post-allogeneic HSCT before subsequent tumor regression. Tumor regressions occurred concomitantly with the establishment of complete donor T-lymphoid engraftment,were associated with the development of graft-versus-host disease (GVHD),and were abrogated by subsequent systemic immunosuppression for GVHD. CONCLUSION: Allogeneic lymphocytes can induce regression of advanced metastatic breast cancer. These results indicate that an immunological GVT effect from allogeneic lymphocytes exists against metastatic breast cancer and provide rationale for further development of allogeneic cellular therapy for this largely incurable disease. View Publication

The AML1/CBFbeta transcriptional complex is essential for the formation of definitive hematopoietic stem cells (HSCs). Moreover,development of the hematopoietic system is exquisitely sensitive to the level of this complex. To investigate the effect of AML1 dosage on adult hematopoiesis,we compared the hematopoietic systems of AML1+/- and AML1+/+ mice. Surprisingly,loss of a single AML1 allele resulted in a 50% reduction in long-term repopulating hematopoietic stem cells (LTR-HSCs). This decrease did not,however,extend to the next level of hematopoietic differentiation. Instead,AML1+/- mice had an increase in multilineage progenitors,an expansion that resulted in enhanced engraftment following transplantation. The expanded pool of AML1+/- progenitors remained responsive to homeostatic mechanisms and thus the number of mature cells in most lineages remained within normal limits. Two notable exceptions were a decrease in CD4(+) T cells,leading to an inversion of the CD4(+) to CD8(+) T-cell ratio and a decrease in circulating platelets. These data demonstrate a dosage-dependent role for AML1/CBFbeta in regulating the quantity of HSCs and their downstream committed progenitors,as well as a more restricted role in T cells and platelets. The latter defect mimics one of the key abnormalities in human patients with the familial platelet disorder resulting from AML1 haploinsufficiency. View Publication

Immunomodulatory derivative (IMiD) CC-4047,a new analog of thalidomide,directly inhibits growth of B-cell malignancies in vivo and in vitro and exhibits stronger antiangiogenic activity than thalidomide. However,there is little information on whether CC-4047 affects normal hematopoiesis. Here we investigated the effect of CC-4047 on lineage commitment and differentiation of hematopoietic stem cells. We found that CC-4047 effectively inhibits erythroid cell colony formation from CD34+ cells and increases the frequency of myeloid colonies. We also demonstrate that development of both erythropoietin-independent and erythropoietin-dependent red cell progenitors was strongly inhibited by CC-4047,while terminal red cell differentiation was unaffected. DNA microarray analysis revealed that red cell transcription factors,including GATA-1,GATA-2,erythroid Kruppel-like factor (EKLF),and growth factor independence-1B (Gfi-1b),were down-regulated in CC-4047-treated CD34+ cells,while myeloid transcription factors such as CCAAT/enhancer binding protein-alpha (C/EBPalpha),C/EBPdelta,and C/EBPepsilon were induced. Analysis of cytokine secretion indicated that CC-4047 induced secretion of cytokines that enhance myelopoiesis and inhibit erythropoiesis. In conclusion,these data indicate that CC-4047 might directly influence lineage commitment of hematopoietic cells by increasing the propensity of stem and/or progenitor cells to undergo myeloid cell development and concomitantly inhibiting red cell development. Therefore,CC-4047 provides a valuable tool to study the mechanisms underlying lineage commitment. View Publication

During ex vivo myoblast differentiation,a pool of quiescent mononucleated myoblasts,reserve cells,arise alongside myotubes. Insulin/insulin-like growth factor (IGF) and PKB/Akt-dependent phosphorylation activates skeletal muscle differentiation and hypertrophy. We have investigated the role of glycogen synthase kinase 3 (GSK-3) inhibition by protein kinase B (PKB)/Akt and Wnt/beta-catenin pathways in reserve cell activation during myoblast differentiation and myotube hypertrophy. Inhibition of GSK-3 by LiCl or SB216763,restored insulin-dependent differentiation of C2ind myoblasts in low serum,and cooperated with insulin in serum-free medium to induce MyoD and myogenin expression in C2ind myoblasts,quiescent C2 or primary human reserve cells. We show that LiCl treatment induced nuclear accumulation of beta-catenin in C2 myoblasts,thus mimicking activation of canonical Wnt signaling. Similarly to the effect of GSK-3 inhibitors with insulin,coculturing C2 reserve cells with Wnt1-expressing fibroblasts enhanced insulin-stimulated induction of MyoD and myogenin in reserve cells. A similar cooperative effect of LiCl or Wnt1 with insulin was observed during late ex vivo differentiation and promoted increased size and fusion of myotubes. We show that this synergistic effect on myotube hypertrophy involved an increased fusion of reserve cells into preexisting myotubes. These data reveal insulin and Wnt/beta-catenin pathways cooperate in muscle cell differentiation through activation and recruitment of satellite cell-like reserve myoblasts. View Publication

Evidence has emerged that mesenchymal stem cells (MSCs) represent a promising population for supporting new clinical concepts in cellular therapy. However,attempts to isolate MSCs from umbilical cord blood (UCB) of full-term deliveries have previously either failed or been characterized by a low yield. We investigated whether cells with MSC characteristics and multi-lineage differentiation potential can be cultivated from UCB of healthy newborns and whether yields might be maximized by optimal culture conditions or by defining UCB quality criteria. Using optimized isolation and culture conditions,in up to 63% of 59 low-volume UCB units,cells showing a characteristic mesenchymal morphology and immune phenotype (MSC-like cells) were isolated. These were similar to control MSCs from adult bone marrow (BM). The frequency of MSC-like cells ranged from 0 to 2.3 clones per 1 x 10(8) mononuclear cells (MNCs). The cell clones proliferated extensively with at least 20 population doublings within eight passages. In addition,osteogenic and chondrogenic differentiation demonstrated a multi-lineage capacity comparable with BM MSCs. However,in contrast to MSCs,MSC-like cells showed a reduced sensitivity to undergo adipogenic differentiation. Crucial points to isolate MSC-like cells from UCB were a time from collection to isolation of less than 15 hours,a net volume of more than 33 ml,and an MNC count of more than 1 x 10(8) MNCs. Because MSC-like cells can be isolated at high efficacy from full-term UCB donations,we regard UCB as an additional stem cell source for experimental and potentially clinical purposes. View Publication

Molecular mimicry between microbial and self-components is postulated as the mechanism that accounts for the antigen and tissue specificity of immune responses in postinfectious autoimmune diseases. Little direct evidence exists,and research in this area has focused principally on T cell-mediated,antipeptide responses,rather than on humoral responses to carbohydrate structures. Guillain-Barré syndrome,the most frequent cause of acute neuromuscular paralysis,occurs 1-2 wk after various infections,in particular,Campylobacter jejuni enteritis. Carbohydrate mimicry [Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-] between the bacterial lipooligosaccharide and human GM1 ganglioside is seen as having relevance to the pathogenesis of Guillain-Barré syndrome,and conclusive evidence is reported here. On sensitization with C. jejuni lipooligosaccharide,rabbits developed anti-GM1 IgG antibody and flaccid limb weakness. Paralyzed rabbits had pathological changes in their peripheral nerves identical with those present in Guillain-Barré syndrome. Immunization of mice with the lipooligosaccharide generated a mAb that reacted with GM1 and bound to human peripheral nerves. The mAb and anti-GM1 IgG from patients with Guillain-Barré syndrome did not induce paralysis but blocked muscle action potentials in a muscle-spinal cord coculture,indicating that anti-GM1 antibody can cause muscle weakness. These findings show that carbohydrate mimicry is an important cause of autoimmune neuropathy. View Publication

The present study demonstrates that CD4(+)CD25(+) T cells,expanded in peripheral blood of HIV-infected patients receiving highly active antiretroviral therapy (HAART),exhibit phenotypic,molecular,and functional characteristics of regulatory T cells. The majority of peripheral CD4(+)CD25(+) T cells from HIV-infected patients expressed a memory phenotype. They were found to constitutively express transcription factor forkhead box P3 (Foxp3) messengers. CD4(+)CD25(+) T cells weakly proliferated to immobilized anti-CD3 monoclonal antibody (mAb) and addition of soluble anti-CD28 mAb significantly increased proliferation. In contrast to CD4(+)CD25(-) T cells,CD4(+)CD25(+) T cells from HIV-infected patients did not proliferate in response to recall antigens and to p24 protein. The proliferative capacity of CD4 T cells to tuberculin,cytomegalovirus (CMV),and p24 significantly increased following depletion of CD4(+)CD25(+) T cells. Furthermore,addition of increasing numbers of CD4(+)CD25(+) T cells resulted in a dose-dependent inhibition of CD4(+)CD25(-) T-cell proliferation to tuberculin and p24. CD4(+)CD25(+) T cells responded specifically to p24 antigen stimulation by expressing transforming growth factor beta (TGF-beta) and interleukin 10 (IL-10),thus indicating the presence of p24-specific CD4(+) T cells among the CD4(+)CD25(+) T-cell subset. Suppressive activity was not dependent on the secretion of TGF-beta or IL-10. Taken together,our results suggest that persistence of HIV antigens might trigger the expansion of CD4(+)CD25(+) regulatory T cells,which might induce a tolerance to HIV in vivo. View Publication

Acute myelogenous leukemia 1 (AML1; runt-related transcription factor 1 [Runx1]) is a member of Runx transcription factors and is essential for definitive hematopoiesis. Although AML1 possesses several subdomains of defined biochemical functions,the physiologic relevance of each subdomain to hematopoietic development has been poorly understood. Recently,the consequence of carboxy-terminal truncation in AML1 was analyzed by the hematopoietic rescue assay of AML1-deficient mouse embryonic stem cells using the gene knock-in approach. Nonetheless,a role for specific internal domains,as well as for mutations found in a human disease,of AML1 remains to be elucidated. In this study,we established an experimental system to efficiently evaluate the hematopoietic potential of AML1 using a coculture system of the murine embryonic para-aortic splanchnopleural (P-Sp) region with a stromal cell line,OP9. In this system,the hematopoietic defect of AML1-deficient P-Sp can be rescued by expressing AML1 with retroviral infection. By analysis of AML1 mutants,we demonstrated that the hematopoietic potential of AML1 was closely related to its transcriptional activity. Furthermore,we showed that other Runx transcription factors,Runx2/AML3 or Runx3/AML2,could rescue the hematopoietic defect of AML1-deficient P-Sp. Thus,this experimental system will become a valuable tool to analyze the physiologic function and domain contribution of Runx proteins in hematopoiesis. View Publication

Tetraspanins are thought to facilitate the formation of multiprotein complexes at cell surfaces,but evidence illuminating the biologic importance of this role is sparse. Tetraspanin CD151 forms very stable laminin-binding complexes with integrins alpha3beta1 and alpha6beta1 in kidney and alpha3beta1 and alpha6beta4 in skin. It is encoded by a gene at the same position on chromosome 11p15.5 as the MER2 blood group gene. We show that CD151 expresses the MER2 blood group antigen and is located on erythrocytes. We examined CD151 in 3 MER2-negative patients (2 are sibs) of Indian Jewish origin with end-stage kidney disease. In addition to hereditary nephritis the sibs have sensorineural deafness,pretibial epidermolysis bullosa,and beta-thalassemia minor. The 3 patients are homozygous for a single nucleotide insertion (G383) in exon 5 of CD151,causing a frameshift and premature stop signal at codon 140. The resultant truncated protein would lack its integrin-binding domain. We conclude that CD151 is essential for the proper assembly of the glomerular and tubular basement membrane in kidney,has functional significance in the skin,is probably a component of the inner ear,and could play a role in erythropoiesis. View Publication

Here a new,intrinsically pluripotent,CD45-negative population from human cord blood,termed unrestricted somatic stem cells (USSCs) is described. This rare population grows adherently and can be expanded to 10(15) cells without losing pluripotency. In vitro USSCs showed homogeneous differentiation into osteoblasts,chondroblasts,adipocytes,and hematopoietic and neural cells including astrocytes and neurons that express neurofilament,sodium channel protein,and various neurotransmitter phenotypes. Stereotactic implantation of USSCs into intact adult rat brain revealed that human Tau-positive cells persisted for up to 3 mo and showed migratory activity and a typical neuron-like morphology. In vivo differentiation of USSCs along mesodermal and endodermal pathways was demonstrated in animal models. Bony reconstitution was observed after transplantation of USSC-loaded calcium phosphate cylinders in nude rat femurs. Chondrogenesis occurred after transplanting cell-loaded gelfoam sponges into nude mice. Transplantation of USSCs in a noninjury model,the preimmune fetal sheep,resulted in up to 5% human hematopoietic engraftment. More than 20% albumin-producing human parenchymal hepatic cells with absence of cell fusion and substantial numbers of human cardiomyocytes in both atria and ventricles of the sheep heart were detected many months after USSC transplantation. No tumor formation was observed in any of these animals. View Publication

Resistance to the ABL kinase inhibitor imatinib (STI571 or Gleevec) in chronic myeloid leukemia (CML) occurs through selection for tumor cells harboring BCR-ABL kinase domain point mutations that interfere with drug binding. Crystallographic studies predict that most imatinib-resistant mutants should remain sensitive to inhibitors that bind ABL with less stringent conformational requirements. BMS-354825 is an orally bioavailable ABL kinase inhibitor with two-log increased potency relative to imatinib that retains activity against 14 of 15 imatinib-resistant BCR-ABL mutants. BMS-354825 prolongs survival of mice with BCR-ABL-driven disease and inhibits proliferation of BCR-ABL-positive bone marrow progenitor cells from patients with imatinib-sensitive and imatinib-resistant CML. These data illustrate how molecular insight into kinase inhibitor resistance can guide the design of second-generation targeted therapies. View Publication