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Effects of angiotensin (Ang)-(1-7),an AngII metabolite,on bone marrow-derived hematopoietic cells were studied. We identified Ang-(1-7) to stimulate proliferation of human CD34(+) and mononuclear cells in vitro. Under in vivo conditions,we monitored proliferation and differentiation of human cord blood mononuclear cells in NOD/SCID mice. Ang-(1-7) stimulated differentially human cells in bone marrow and accumulated them in the spleen. The number of HLA-I(+) and CD34(+) cells in the bone marrow was increased 42-fold and 600-fold,respectively. These results indicate a decisive impact of Ang-(1-7) on hematopoiesis and its promising therapeutic potential in diseases requiring progenitor stimulation. View Publication

Cyclophosphamide (CYC) can control diffuse proliferative lupus nephritis (DPLN) by potent immunosuppression but remains associated with serious and life-threatening complications. Drugs that specifically target mediators of DPLN may help to reduce CYC dose and side effects. Monocyte chemoattractant protein (MCP-1)/CCL2 mediates monocyte and T cell recruitment in DPLN and Ccl2-specific l-enantiomeric RNA Spiegelmer mNOX-E36 neutralizes the biological effects of murine Ccl2 in vitro and in vivo. We injected MRL(lpr/lpr) mice with DPLN from 14 weeks of age with vehicle,weekly 30 mg/kg CYC (full dose),monthly 30 mg/kg CYC (one-fourth full dose),pegylated control Spiegelmer,pegylated anti-Ccl2 Spiegelmer (3/week),pegylated anti-Ccl2 Spiegelmer plus CYC one-fourth full dose and mycophenolate mofetil. At week 24,DPLN and autoimmune lung injury were virtually abolished with CYC full dose but not with CYC one-fourth full dose. The CYC one-fourth full dose/Spiegelmer combination was equipotent to CYC full dose on kidney and lung injury. CD3(+)CD4(-)CD8(-) and CD3(+)CD4(+)CD25(+) T cells and serum interleukin-12p40 and tumor necrosis factor-alpha levels were all markedly affected by CYC full dose but not by CYC one-fourth full dose. No additive effects of anti-Ccl2 Spiegelmer were noted on bone marrow colony-forming unit-granulocyte macrophage counts and 7/4(high) monocyte counts,lymphoproliferation,and spleen T cell depletion. In summary,anti-Ccl2 Spiegelmer permits 75% dose reduction of CYC for controlling DPLN and pneumonitis in MRL-Fas(lpr) mice,sparing suppressive effects of full-dose CYC on myelosuppression and T cell depletion. We propose anti-Ccl2 Spiegelmer therapy as a novel strategy to reduce CYC toxicity in the treatment of severe lupus. View Publication

OBJECTIVES: In this study,we hypothesized that an antihuman-CD34 antibody immobilized on the surface of commercially available sirolimus-eluting stents (SES) could enhance re-endothelialization compared with SES alone. BACKGROUND: Previous experience with antihuman-CD34 antibody surface modified Genous stents (GS) (OrbusNeich Medical,Fort Lauderdale,Florida) has shown enhanced stent endothelialization in vivo. METHODS: In the phase 1 study,stents were deployed in 21 pig coronary arteries for single stenting (9 vessels: 3 GS,3 SES,and 3 bare-metal stents) and overlapping stenting with various combinations (12 vessels: 4 GS+GS,4 SES+SES,and 4 GS+SES) and harvested at 14 days for scanning electron and confocal microscopy. In phase 2,immobilized anti-CD34 antibody coating was applied on commercially available SES (SES-anti-CD34,n = 7) and compared with GS (n = 8) and SES (n = 7) and examined at 3 and 14 days by scanning electron/confocal microscopy analysis. RESULTS: In phase 1,single stent implantation showed greatest endothelialization in GS (99%) and in bare-metal stent (99%) compared with SES (55%,p = 0.048). In overlapping stents,endothelialization at the overlapping zone was significantly greater in GS+GS (95 +/- 6%) and GS+SES (79 +/- 5%) compared with the SES+SES (36 +/- 14%) group (p = 0.007). In phase 2,SES-anti-CD34 resulted in increased endothelialization compared with SES alone at 3 days (SES-anti-CD34 36 +/- 26%; SES 7 +/- 3%; and GS 76 +/- 8%; p = 0.01),and 14 days (SES-anti-CD34 82 +/- 8%; SES 53 +/- 20%; and GS 98 +/- 2%; p = 0.009). CONCLUSIONS: Immobilization of anti-CD34 antibody on SES enhances endothelialization and may potentially be an effective therapeutic alternative to improve currently available drug-eluting stents. View Publication

The CD45 antigen is present on all cells of the hematopoietic lineage. Using a murine model,we have determined whether a lytic CD45 monoclonal antibody can produce persistent aplasia and whether it could facilitate syngeneic or allogeneic stem cell engraftment. After its systemic administration,we found saturating quantities of the antibody on all cells expressing the CD45 antigen,both in marrow and in lymphoid organs. All leukocyte subsets in peripheral blood were markedly diminished during or soon after anti-CD45 treatment,but only the effect on the lymphoid compartment was sustained. In contrast to the prolonged depletion of T and B lymphocytes from the thymus and spleen,peripheral blood neutrophils began to recover within 24 hours after the first anti-CD45 injection and marrow progenitor cells were spared from destruction,despite being coated with saturating quantities of anti-CD45. Given the transient effects of the monoclonal antibody on myelopoiesis and the more persistent effects on lymphopoiesis,we asked whether this agent could contribute to donor hematopoietic engraftment following nonmyeloablative transplantation. Treatment with anti-CD45 alone did not enhance syngeneic engraftment,consistent with its inability to destroy progenitor cells and permit competitive repopulation with syngeneic donor stem cells. By contrast,the combination of anti-CD45 and an otherwise inactive dose of total-body irradiation allowed engraftment of H2 fully allogeneic donor stem cells. We attribute this result to the recipient immunosuppression produced by depletion of CD45(+) lymphocytes. Monoclonal antibodies of this type may therefore have an adjunctive role in nonmyeloablative conditioning regimens for allogeneic stem cell transplantation. View Publication

BACKGROUND Glycophorin C (GPC) is necessary in the maintenance of red blood cell structure. Severe autoimmune hemolytic anemia and hemolytic disease of the fetus and newborn (HDFN) have been associated with Gerbich (Ge) blood group system antigens expressed on GPC. Previous in vitro studies with cord blood progenitor cells have shown that anti-Ge suppresses erythropoiesis. STUDY DESIGN AND METHODS Here,we evaluated the K562 erythroleukemic cell line to study the cellular effects of a murine anti-GPC. Cell proliferation was evaluated after treatment with anti-GPC. Flow cytometry was used to evaluate exofacial phosphatidylserine (PS) expression and cell viability (propidium iodide binding). Cell morphology was evaluated under light microscopy with cytospin preparations stained with May-Grünwald Giemsa. RESULTS Anti-GPC dramatically inhibited K562 proliferation and increased PS expression,consistent with cytoplasmic blebbing,suggesting evidence of apoptosis. Z-VAD-FMK,an inhibitor of classical apoptosis,was unable to reverse the suppressive effect of anti-GPC. However,hemin was able to attenuate growth suppression. CONCLUSION Together,the data suggest that anti-GPC suppresses erythroid proliferation through the induction of nonclassical apoptosis. View Publication

An attractive target for therapeutic intervention is constitutively activated,mutant FLT3,which is expressed in a subpopulation of patients with acute myelocyic leukemia (AML) and is generally a poor prognostic indicator in patients under the age of 65 years. PKC412 is one of several mutant FLT3 inhibitors that is undergoing clinical testing,and which is currently in late-stage clinical trials. However,the discovery of drug-resistant leukemic blast cells in PKC412-treated patients with AML has prompted the search for novel,structurally diverse FLT3 inhibitors that could be alternatively used to override drug resistance. Here,we report the potent and selective antiproliferative effects of the novel mutant FLT3 inhibitor NVP-AST487 on primary patient cells and cell lines expressing FLT3-ITD or FLT3 kinase domain point mutants. NVP-AST487,which selectively targets mutant FLT3 protein kinase activity,is also shown to override PKC412 resistance in vitro,and has significant antileukemic activity in an in vivo model of FLT3-ITD(+) leukemia. Finally,the combination of NVP-AST487 with standard chemotherapeutic agents leads to enhanced inhibition of proliferation of mutant FLT3-expressing cells. Thus,we present a novel class of FLT3 inhibitors that displays high selectivity and potency toward FLT3 as a molecular target,and which could potentially be used to override drug resistance in AML. View Publication

Transcriptional profiling of differentiating human embryonic stem cells (hESCs) revealed that MIXL1-positive mesodermal precursors were enriched for transcripts encoding the G-protein-coupled APELIN receptor (APLNR). APLNR-positive cells,identified by binding of the fluoresceinated peptide ligand,APELIN (APLN),or an anti-APLNR mAb,were found in both posterior mesoderm and anterior mesendoderm populations and were enriched in hemangioblast colony-forming cells (Bl-CFC). The addition of APLN peptide to the media enhanced the growth of embryoid bodies (EBs),increased the expression of hematoendothelial genes in differentiating hESCs,and increased the frequency of Bl-CFCs by up to 10-fold. Furthermore,APLN peptide also synergized with VEGF to promote the growth of hESC-derived endothelial cells. These studies identified APLN as a novel growth factor for hESC-derived hematopoietic and endothelial cells. View Publication

Megakaryocytopoiesis gives rise to platelets by proliferation and differentiation of lineage-specific progenitors,identified in vitro as Colony Forming Unit-Megakaryocytes (CFU-Mk). The aim of this study was to refine and optimize the in vitro Standard Operating Procedure (SOP) of the CFU-Mk assay for detecting drug-induced thrombocytopenia and to prevalidate a model for predicting the acute exposure levels that cause maximum tolerated decreases in the platelets count,based on the correlation with the maximal plasma concentrations (C max) in vivo. The assay was linear under the SOP conditions,and the in vitro endpoints (percentage of colonies growing) were reproducible within and across laboratories. The protocol performance phase was carried out testing 10 drugs (selected on the base of their recognised or potential in vivo haematotoxicity,according to the literature). Results showed that a relationship can be established between the maximal concentration in plasma (C max) and the in vitro concentrations that inhibited the 10-50-90 percent of colonies growth (ICs). When C max is lower than IC10,it is possible to predict that the chemicals have no direct toxicity effect on CFU-Mk and could not induce thrombocytopenia due to bone marrow damage. When the C max is higher than IC90 and/or IC50,thrombocytopenia can occur due to direct toxicity of chemicals on CFU-Mk progenitors. View Publication

In a previous study of prevalidation,a standard operating procedure (SOP) for two independent in vitro tests (human and mouse) had been developed,to evaluate the potential hematotoxicity of xenobiotics from their direct and the adverse effects on granulocyte-macrophages (CFU-GM). A predictive model to calculate the human maximum tolerated dose (MTD) was set up,by adjusting a mouse-derived MTD for the differential interspecies sensitivity. In this paper,we describe an international blind trial designed to apply this model to the clinical neutropenia,by testing 20 drugs,including 14 antineoplastics (Cytosar-U,5-Fluorouracil,Myleran,Thioguanine,Fludarabine,Bleomycin,Methotrexate,Gemcitabine,Carmustine,Etoposide,Teniposide,Cytoxan,Taxol,Adriamycin); two antivirals (Retrovir,Zovirax,); three drugs for other therapeutic indications (Cyclosporin,Thorazine,Indocin); and one pesticide (Lindane). The results confirmed that the SOP developed generates reproducible IC90 values with both human and murine GM-CFU. For 10 drugs (Adriamycin,Bleomycin,Etoposide,Fludarabine,5-Fluorouracil,Myleran,Taxol,Teniposide,Thioguanine,and Thorazine),IC90 values were found within the range of the actual drug doses tested (defined as the actual IC90). For the other 10 drugs (Carmustine,Cyclosporin,Cytosar-U,Cytoxan,Gemcitabine,Indocin,Lindane,Methotrexate,Retrovir,and Zovirax) extrapolation on the regression curve out of the range of the actual doses tested was required to derive IC90 values (extrapolated IC90). The model correctly predicted the human MTD for 10 drugs out of 10 that had actual IC90 values" and 7 drugs out of 10 for those having only an extrapolated IC90. Two of the incorrect predictions (Gemcitabine and Zovirax) were within 6-fold of the correct MTD View Publication