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INTRODUCTION: The assay of endogenous erythroid colony formation (EEC),a characteristic of polycythemia vera and essential thrombocythemia,is not standardized. In this multicentric study,we tested four semisolid,serum-free,cytokine-free media based on either methylcellulose (M1,M2) or collagen (C1,C2) commercialized for the EEC assay. MATERIALS AND METHODS: Bone marrow mononuclear cells (BMMC) from 73 individuals (62 patients with either polycythemia vera (26),essential thrombocythemia (19),secondary polyglobuly (17) or chronic myeloid leukemia (2) and 11 healthy donors) were grown in parallel in the four media without,or with 0.01 U/ml erythropoietin (EPo). RESULTS: In all four media EEC formation was specific,as it was not observed in cultures of patients with secondary polyglobuly or chronic myeloid leukemia,nor of healthy donors. Analysis of fresh or MGG-stained collagen gel cultures allowed detection of EEC formation significantly more frequently than methylcellulose-based media; addition of 0.01 U/ml of EPo had little or no effect on EEC formation. Collagen-based medium C1 gave better results than the other media tested: the 'C1' EEC assay was positive for 68.2% of polycythemia vera cultures with significantly higher median EEC numbers (6.5/10(5) BMMC for patients with one major criteria of polycythemia vera and 19 and 21/10(5) BMMC for patients with two or three major criteria,respectively). Medium C1 was also better for essential thrombocythemia cultures with 47.4% of positive results but with a low median EEC number (6.7/10(5) BMMC). When associated with the ELISA dosage of serum EPo,the 'C1' EEC assay allowed confirmation or elimination of the diagnosis of polycythemia vera for 91% (20/22) of polyglobulic patients. CONCLUSION: We propose that serum-free collagen-based culture systems be considered to standardize the EEC assay,now part of the new criteria of polycythemia vera. View Publication

Proteasomal activity is required for normal cellular functions including cell division,where entry and exit from mitosis is strictly regulated by cyclins and cyclin-dependent kinases which are among the important substrates of the proteasomal degradative machinery. Inhibitors of proteasomal activity have been shown to be effective inducers of apoptosis in tumor cells and may be useful as anticancer agents,either alone or in combination with other drugs. We have examined the effect of MG-132,a dipeptide proteasomal inhibitor,on various human cancer cell lines. We have also examined the effect of MG-132 on normal CD34+ enriched primary human peripheral blood stem cells. Our results indicate that MG-312 is a potent anticancer agent with cytotoxic effects on a variety of human cancer cell lines irrespective of their p53 status. MG-132 was found to be more effective in combination with drugs such as doxorubicin and etoposide that act in the S/G2-phase of the cell cycle via a mechanism that involves stabilization of cyclin B1 and increased expression of Bax. Further,MG-132 inhibits CFU-GM colony formation of the CD34+ enriched PBSC population and this inhibition correlates with release of cyt C into the cytosol. View Publication

The p38 MAP kinase plays a crucial role in regulating the production of proinflammatory cytokines,such as tumor necrosis factor and interleukin-1. Blocking this kinase may offer an effective therapy for treating many inflammatory diseases. Here we report a new allosteric binding site for a diaryl urea class of highly potent and selective inhibitors against human p38 MAP kinase. The formation of this binding site requires a large conformational change not observed previously for any of the protein Ser/Thr kinases. This change is in the highly conserved Asp-Phe-Gly motif within the active site of the kinase. Solution studies demonstrate that this class of compounds has slow binding kinetics,consistent with the requirement for conformational change. Improving interactions in this allosteric pocket,as well as establishing binding interactions in the ATP pocket,enhanced the affinity of the inhibitors by 12,000-fold. One of the most potent compounds in this series,BIRB 796,has picomolar affinity for the kinase and low nanomolar inhibitory activity in cell culture. View Publication

To propose candidates for the prevention or treatment of osteoporosis,we have screened compounds naturally in food for their ability to regulate the differentiation and function of osteoclasts. One of the major green tea flavonoids,(-)-epigallocatechin-3-gallate (EGCG),was found to induce apoptotic cell death of osteoclast-like multinucleated cells after 24 h treatment in a dose-dependent manner (25-100 microM),whereas osteoblasts were not affected. In the present study,we report for the first time a novel cell-death-inducing mechanism triggered by EGCG. The induction of apoptosis by EGCG was suppressed by pretreatment of catalase or calcitonin. It was also suppressed by Fe(III) and Fe(II) chelators. Furthermore,EGCG promoted the reduction of Fe(III) into Fe(II),and the combination of EGCG/Fe(III)/H(2)O(2) induced single-strand DNA breakage in a cell free system. These results indicate that the Fenton reaction is primarily involved in EGCG-induced osteoclastic cell death. View Publication

Bone marrow progenitor cells develop into mature tissue myeloid cells under the influence of colony-stimulating factors. Cytokines that are elevated post-thermal injury have been shown to influence this process. We hypothesize that thermal injury alters myelopoiesis at the level of the progenitor cell. These differences should be visible after in vitro cultures that include colony-stimulating factors. Prior to culture,bone marrow at postburn day 1 (PBD1) was assessed for cell surface markers and the levels of myeloid progenitors. After culture in granulocyte/macrophage-stimulating colony-stimulating factor,the cell surface markers of the cultured cells were determined. PBD1 marrow from thermally injured rats had more progenitor cells responsive to granulocyte/macrophage-stimulating colony-stimulating factor than did sham. Cultured PBD1 marrow produced more CD90(br) MY(br) CD45(dim) CD4(-) MHCII(-) CD11b(dim) eosinophils than did sham. Cultured bone marrow from thermally injured animals produces myeloid cells with an altered phenotype. Similar changes in myelopoiesis may take place in vivo. View Publication

We have previously shown that 4-anilinoquinazolines can be potent inhibitors of vascular endothelial growth factor (VEGF) receptor (Flt-1 and KDR) tyrosine kinase activity. A novel subseries of 4-anilinoquinazolines that possess basic side chains at the C-7 position of the quinazoline nucleus have been synthesized. This subseries contains potent,nanomolar inhibitors of KDR (median IC(50) 0.02 microM,range 0.001-0.04 microM),which are comparatively less potent vs Flt-1 tyrosine kinase (median IC(50) 0.55 microM,range 0.02-1.6 microM). The compounds also retain some inhibitory activity against the tyrosine kinase associated to the endothelial growth factor receptor (EGFR) (median IC(50) 0.2 microM,range 0.075-0.8 microM) but demonstrate selectivity vs that associated to the FGF receptor 1 (median IC(50) 2.5 microM,range 0.9-19 microM). This selectivity profile is also evident in a growth factor-stimulated human endothelial cell (HUVEC) proliferation assay (i.e.,inhibition of VEGF textgreater EGF textgreater FGF),with inhibition of VEGF-induced proliferation being achieved at nanomolar concentrations (median IC(50) 0.06 microM). Further examination of compound 2 (ZD6474) in recombinant enzyme assays revealed excellent selectivity for the inhibition of KDR tyrosine kinase (IC(50) 0.04 microM) vs the kinase activity of erbB2,MEK,CDK-2,Tie-2,IGFR-1R,PDK,PDGFRbeta,and AKT (IC(50) range: 1.1 to textgreater100 microM). Anilinoquinazolines possessing basic C-7 side chains exhibited markedly improved aqueous solubility over previously described anilinoquinazolines possessing neutral C-7 side chains (up to 500-fold improvement at pH 7.4). In addition,aqueous solubility of the neutral fraction present at pH 7.4 of the basic subseries of anilinoquinazoline proved to be higher than that of the neutral analogue 1 (ZD4190). Oral administration of representative compounds to mice (50 mg/kg) produced plasma levels between 0.2 and 3 microM at 24 h after dosing. Our development candidate 2 demonstrated a very attractive in vitro profile combined with excellent solubility (330 microM at pH 7.4) and good oral bioavailability in rat and dog (textgreater 80 and textgreater 50%,respectively). This compound demonstrated highly significant,dose-dependent,antitumor activity in athymic mice. Once daily oral administration of 100 mg/kg of compound 2 for 21 days inhibited the growth of established Calu-6 lung carcinoma xenografts by 79% (P textless 0.001,Mann Whitney rank sum test),and substantial inhibition (36%,P textless 0.02) was evident with 12.5 mg/kg/day. View Publication

Oncogenic activation of the RET receptor tyrosine kinase is common in different human cancers. We found that the pyrazolo-pyrimidine PP1 inhibited RET-derived oncoproteins with a half maximal inhibitor concentration of 80 nM. Furthermore,RET/PTC3-transformed cells treated with 5 microM of PP1 lost proliferative autonomy and showed morphological reversion. PP1 prevented the growth of two human papillary thyroid carcinoma cell lines that carry spontaneous RET/PTC1 rearrangements and blocked anchorage-independent growth and tumorigenicity in nude mice of NIH3T3 fibroblasts expressing the RET/PTC3 oncogene. These findings suggest targeting RET oncogenes with PP1 or related compounds as a novel treatment strategy for RET-associated neoplasms. View Publication

In early life,a high susceptibility to infectious diseases as well as a poor capacity to respond to vaccines are generally observed as compared with observations in adults. The mechanisms underlying immune immaturity have not been fully elucidated and could be due to the immaturity of the T/B cell responses and/or to a defect in the nature and quality of Ag presentation by the APC. This prompted us to phenotypically and functionally characterize early life murine dendritic cells (DC) purified from spleens of 7-day-old mice. We showed that neonatal CD11c(+) DC express levels of costimulatory molecules and MHC molecules similar to those of adult DC and are able to fully maturate after LPS activation. Furthermore,we demonstrated that neonatal DC can efficiently take up,process,and present Ag to T cells in vitro and induce specific CTL responses in vivo. Although a reduced number of these cells was observed in the spleen of neonatal mice as compared with adults,this study clearly shows that neonatal DC have full functional capacity and may well prime Ag-specific naive T cells in vivo. View Publication

Vitamin C (ascorbic acid (AA)) is very popular for its antioxidant properties. Consequently,many other important aspects of this multifaceted molecule are often underestimated or even ignored. In the present paper,we have tried to bring to the foreground some of these aspects,including the peculiarities of the AA biosynthetic pathway in different organisms,the remarkable function of AA as a co-substrate of many important dioxygenases,the role of AA-regenerating enzymes and the known pathways of AA catabolism,as well as the intriguing function of AA in gene expression. View Publication

We have investigated the effects of thermal injury upon myelopoiesis. IL-3,GM-CSF,and IL-5 were used to stimulate myeloid colony formation. IL-3 induces early myeloid progenitors and a more developed myeloid progenitor,the granulocyte-macrophage colony-forming unit (GM-CFU),to multiply and develop into mature myeloid cells. GM-CSF induces GM-CFU to become mature myeloid cells,while IL-5 induces eosinophil progenitors to become mature eosinophils. Stem Cell Factor (SCF) + IL-6 and FLT3 ligand,which have no effect on colony formation by themselves,were used to enhance the effects of IL-3 and GM-CSF,respectively. We found that thermal injury increased the number of early myeloid progenitors and GM-CFU in the spleen with either IL-3 or GM-CSF as a stimulant. Thermal injury increased the number of early myeloid progenitors in the bone marrow when GM-CSF,but not IL-3,was used to stimulate colony growth. Also,thermal injury increased the numbers of eosinophil progenitors in rat spleen and bone marrow and increased splenic levels of IL-5 mRNA. View Publication

Potential pharmacological applications in the areas of oncology,dermatology,diabetes,and atherosclerosis of synthetic analogs of retinoic acid that target a specific nuclear receptor and/or biological response have generated great interest in the development of new retinoid and rexinoid drugs. The pan-retinoic acid receptor antagonist AGN 193109 has been previously reported to elevate CYP1A1 levels,implicating the aryl hydrocarbon receptor (AhR) as an additional target for this retinoid. AhR is a cytosolic ligand-dependent transcription factor that,in conjunction with the AhR nuclear translocator (Arnt),binds to dioxin response elements (DREs) located in the promoter region of target genes,such as CYP1A1,and induces their transcription. The purpose of these studies was to determine whether additional synthetic retinoids were capable of elevating CYP1A1 levels and to examine the mechanism of this increase in CYP1A. Two additional retinoids,AGN 190730 and AGN 192837,were found to be potent inducers of DRE-driven transcriptional activity; AGN 190730 was the most potent. Moreover,electrophoretic mobility-shift assays demonstrate that AGN 190730 can transform AhR into its active DNA recognition form. In addition,trypsin digestion of AGN 190730-treated AhR reveals a conformational change in the protein similar to the conformational change of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-bound AhR. Finally,competitive binding studies demonstrate that AGN 190730 can inhibit the binding of TCDD to AhR. The sum of the data demonstrates that some synthetic retinoids in addition to activating the retinoic acid receptor/retinoid X receptor pathway are capable of binding to AhR and activating the AhR/Arnt pathway. View Publication

BACKGROUND: Ex vivo expansion of cord blood (CB) hematopoietic stem and progenitor cells increases cell dose and may reduce the severity and duration of neutropenia and thrombocytopenia after transplantation. This study's purpose was to establish a clinically applicable culture system by investigating the use of cytokines,serum-free media,and autologous plasma for the expansion of CB cells and the engraftment of expanded product in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. STUDY DESIGN AND METHODS: Enriched CB CD34+ cells were cultured in four media (Iscove's modified Dulbecco's medium with FCS,Gibco; X-Vivo-10,BioWhittaker; QBSF-60,Quality Biological; and StemSpan SFEM,Stem Cell Technologies) with four cytokine combinations (thrombopoietin [TPO],SCF,Flt-3 ligand [FL] with and without G-CSF,and/or IL-6). The effect of autologous CB plasma was also investigated. The read-out measures were evaluated on Days 8 and 12. After expansion at the optimized condition,cultured cells were transplanted into sublethally irradiated NOD/SCID mice. The engraftment of human CD45+ cells and subsets in the bone marrow,spleen,and peripheral blood was determined. RESULTS: QBSF-60 or StemSpan SFEM supported high yields of early progenitors (CD34+ cells,textlessor= 64.8-fold; CD34+CD38- cells,330-fold; CFU-granulocyte erythroid macrophage megakaryocyte [GEMM],248-fold) and CFUs of the myeloid (CFU-GM,407-fold) and erythroid (BFU/CFU-E,144-fold) lineages. The expansion of the megakaryocytic lineage was consistently higher in X-Vivo-10 (CFU-megakaryocyte,684-fold). Autologous plasma promoted colony formation but reduced CD34+ cells and CFU-GEMM. The addition of G-CSF or IL-6 improved cell yields; G-CSF was more effective for committed progenitors. Expansion products from cultures in QBSF-60 with the cytokines engrafted and differentiated into the myeloid and lymphoid lineages in NOD/SCID mice. CONCLUSION: The data supported the strategy of expansion. The optimized condition may be applicable to clinical expansion for the abrogation or reduction of posttransplant cytopenia. View Publication