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BACKGROUND: Anaemia and microcytosis are common post kidney transplantation. The aim of this study was to evaluate the potential role of mammalian target of rapamycin (mTOR) inhibition in the development of anaemia and microcytosis in healthy animals and in human erythroid cultures in vitro. METHODS: Rats with normal kidney function were treated with sirolimus (n = 7) or vehicle (n = 8) for 15 weeks. Hemograms were determined thereafter. In the sirolimus withdrawal part of the study,rats received sirolimus (SRL) for 67 days (n = 4) 1 mg/kg three times per week or for 30 days (n = 4) and were observed until Day 120. Hemograms were performed regularly. Peripheral blood mononuclear cells from healthy controls (HC; n = 8),kidney transplant patients with sirolimus treatment with (SRL + MC; n = 8) or without microcytosis (SRL - MC; n = 8) were isolated and cultured in the absence or presence of SRL (5 ng/mL). RESULTS: SRL-treated animals had a reduced mean corpuscular volume (MCV) and elevated erythrocyte count compared with control animals after 15 weeks of treatment. This effect was evident as early as 4 weeks (MCV: 61.5 ± 1.8 versus 57 ± 1.7 fL; P = 0.0156; Red blood count 7.4 ± 0.3 × 10(9)/L versus 8.6 ± 0.5 × 10(9)/L; P = 0.0156) and was reversible 90 days after SRL withdrawal. SRL in the culture medium of erythroid cultures led to fewer colonies in cultures from HC as well as from kidney transplant patients (without SRL: 34.2 ± 11.4 versus with SRL: 27.5 ± 9.9 BFU-E-derived colonies P = 0.03),regardless if the cultures were derived from recipients with normocytic or with microcytic erythrocytes. The presence of tacrolimus in the culture medium had no influence on the number and size of colonies. CONCLUSION: mTOR inhibition induces microcytosis and polyglobulia,but not anaemia in healthy rats. This might be caused by growth inhibition of erythroid precursor cells. View Publication

Interleukin-6 (IL-6) and its soluble receptor (sIL-6R) are major factors for maintenance and expansion of hematopoietic stem cells (HSCs). Sensitivity of HSCs to IL-6 has been previously studied,in part by measuring the expression of IL-6R on the membrane (mIL-6R). Several studies have described the regulation of cell surface expression of IL-6R by several cytokines,but the role of glycoprotein 130 activation has not yet been investigated. In this study,CD133(+) cells were purified from adult peripheral blood and were precultured in the absence or presence of 5-fluorouracil (5-FU) for selection of quiescent HSCs. Cells were cultured with continuous or pulsed stimulations of an IL-6-sIL-6R fusion protein (hyperinterleukin-6 [HIL-6]) to 1) detect mIL-6R by flow cytometry,2) assess mIL-6R and sIL-6R RNAs by reverse transcription-polymerase chain reaction,3) measure sIL-6R in supernatants by enzyme-linked immunosorbent assay,4) analyze cell-cycle status,and 5) perform long-term culture-initiating cell assays. The level of mIL-6R(-) cells was preserved by 5-FU incubation. HIL-6 increased steady-state mIL-6R RNA and expression rate on HSCs,independently of treatment with 5-FU. Enhanced production of sIL-6R was observed with short pulses of HIL-6 on CD133(+) 5-FU-pretreated cells. This overproduction of sIL-6R was abrogated by tumor necrosis factor-alpha protease inhibitor-1,an inhibitor of a disintegrin and metalloprotease proteases,suggesting the shedding of mIL-6R. This phenomenon was mediated through the phosphatidylinositol-3'-kinase pathway and was involved in the maintenance of primitive HSCs. In conclusion,expression and production of IL-6R are tightly regulated and stage specific. We assume that sIL-6R produced by shedding should be involved in autocrine and paracrine loops in the HSC microenvironment. 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

The lipocalin mouse 24p3 has been implicated in diverse physiological processes,including apoptosis,iron trafficking,development and innate immunity. Studies from our laboratory as well as others demonstrated the proapoptotic activity of 24p3 in a variety of cultured models. However,a general role for the lipocalin 24p3 in the hematopoietic system has not been tested in vivo. To study the role of 24p3,we derived 24p3 null mice and back-crossed them onto C57BL/6 and 129/SVE backgrounds. Homozygous 24p3(-/-) mice developed a progressive accumulation of lymphoid,myeloid,and erythroid cells,which was not due to enhanced hematopoiesis because competitive repopulation and recovery from myelosuppression were the same as for wild type. Instead,apoptotic defects were unique to many mature hematopoietic cell types,including neutrophils,cytokine-dependent mast cells,thymocytes,and erythroid cells. Thymocytes isolated from 24p3 null mice also displayed resistance to apoptosis-induced by dexamethasone. Bim response to various apoptotic stimuli was attenuated in 24p3(-/-) cells,thus explaining their resistance to the ensuing cell death. The results of these studies,in conjunction with those of previous studies,reveal 24p3 as a regulator of the hematopoietic compartment with important roles in normal physiology and disease progression. Interestingly,these functions are limited to relatively mature blood cell compartments. View Publication

Ex vivo expansion of hematopoietic stem cells (HSCs) is important for many clinical applications,and knowledge of the surface phenotype of ex vivo-expanded HSCs will be critical to their purification and analysis. Here,we developed a simple culture system for bone marrow (BM) HSCs using low levels of stem cell factor (SCF),thrombopoietin (TPO),insulin-like growth factor 2 (IGF-2),and fibroblast growth factor-1 (FGF-1) in serum-free medium. As measured by competitive repopulation analyses,there was a more than 20-fold increase in numbers of long-term (LT)-HSCs after a 10-day culture of total BM cells. Culture of BM side population" (SP) cells� View Publication

Human bone marrow (BM) or mobilized peripheral blood (mPB) CD34(+) cells have been shown to loose their stem cell quality during culture period more easily than those from cord blood (CB). We previously reported that human umbilical CB stem cells could effectively be expanded in the presence of human recombinant cytokines and a newly established murine bone marrow stromal cell line HESS-5. In this study we assessed the efficacy of this xenogeneic coculture system using human BM and mPB CD34(+) cells as materials. We measured the generation of CD34(+)CD38(-) cells and colony-forming units,and assessed severe-combined immunodeficient mouse-repopulating cell (SRC) activity using cells five days after serum-free cytokine-containing culture in the presence or the absence of a direct contact with HESS-5 cells. As compared with the stroma-free culture,the xenogeneic coculture was significantly superior on expansion of CD34(+)CD38(-) cells and colony-forming cells and on maintenance of SRC activity. The PKH26 study demonstrated that cell division was promoted faster in cells cocultured with HESS-5 cells than in cells cultured without HESS-5 cells. These results indicate that HESS-5 supports rapid generation of primitive progenitor cells (PPC) and maintains reconstituting ability of newly generated stem cells during ex vivo culture irrespective of the source of samples. This xenogeneic coculture system will be useful for ex vivo manipulation such as gene transduction to promote cell division and the generation of PPC and to prevent loss of stem cell quality. View Publication

In up to one-third of patients with acute myeloid leukemia,a C-terminal frame-shift mutation results in abnormal and abundant cytoplasmic accumulation of the usually nucleoli-bound protein nucleophosmin (NPM),and this is thought to function in cancer pathogenesis. Here,we demonstrate a gain-of-function role for cytoplasmic NPM in the inhibition of caspase signaling. The NPM mutant specifically inhibits the activities of the cell-death proteases,caspase-6 and -8,through direct interaction with their cleaved,active forms,but not the immature procaspases. The cytoplasmic NPM mutant not only affords protection from death ligand-induced cell death but also suppresses caspase-6/-8-mediated myeloid differentiation. Our data hence provide a potential explanation for the myeloid-specific involvement of cytoplasmic NPM in the leukemogenesis of a large subset of acute myeloid leukemia. View Publication

A battery of clonal assays for myeloid progenitor cells (HPP-CFC,CFU-gemm,CFU-gm,CFU-g) was utilized to evaluate the myelotoxicity of a series of alkylating agents representing the spectrum of clinical times to nadir. Bone marrow aspirates from normal volunteers were incubated with mechlorethamine,busulfan,melphalan,carmustine or lomustine for 1 h and then cultured in methylcellulose with 30% serum and cytokines. There was a concentration-dependent inhibition of colony formation and often a differential toxicity to the myeloid progenitors with the alkylators tested. On a molar basis,mechlorethamine and melphalan were the most toxic of the alkylator drugs to the myeloid precursors. The most sensitive progenitor was CFU-gemm with the lowest inhibitory concentration IC(70) concentrations for mechlorethamine,melphalan,carmustine and lomustine. Generally,there was great similarity for drug effects between CFU-g and CFU-gm with overlapping inhibition curves. HPP-CFC proved to be the least sensitive of the progenitors to the toxic actions of the drugs. While there was no correlation between the time to clinical neutropenic nadir and the most sensitive progenitor in the clonal assays,the CFU-gm assay remains a suitable method for determining the myelotoxic potential of cytotoxic agents. View Publication

The developmental potential of hematopoietic progenitors is restricted early on to either the erythromyeloid or lymphoid lineages. The broad developmental potential of Pax5(-/-) pro-B cells is in apparent conflict with such a strict separation,although these progenitors realize the myeloid and erythroid potential with lower efficiency compared to the lymphoid cell fates. Here we demonstrate that ectopic expression of the transcription factors C/EBPalpha,GATA1,GATA2 and GATA3 strongly promoted in vitro macrophage differentiation and myeloid colony formation of Pax5(-/-) pro-B cells. GATA2 and GATA3 expression also resulted in efficient engraftment and myeloid development of Pax5(-/-) pro-B cells in vivo. The myeloid transdifferentiation of Pax5(-/-) pro-B cells was accompanied by the rapid activation of myeloid genes and concomitant repression of B-lymphoid genes by C/EBPalpha and GATA factors. These data identify the Pax5(-/-) pro-B cells as lymphoid progenitors with a latent myeloid potential that can be efficiently activated by myeloid transcription factors. The same regulators were unable to induce a myeloid lineage switch in Pax5(+/+) pro-B cells,indicating that Pax5 dominates over myeloid transcription factors in B-lymphocytes. View Publication

Mammalian presenilins (PS) consist of two highly homologous proteins,PS1 and PS2. Because of their indispensable activity in the gamma-secretase cleavage of amyloid precursor protein to generate Abeta peptides,inhibition of PS gamma-secretase activity is considered a potential therapy for Abeta blockage and Alzheimer's disease intervention. However,a variety of other substrates are also subject to PS-dependent processing,and it is thus imperative to understand the consequences of PS inactivation in vivo. Here we report a pivotal role of PS in hematopoiesis. Mice heterozygous for PS1 and homozygous for PS2 (PS1(+/)(-)PS2(-)(/)(-)) developed splenomegaly with severe granulocyte infiltration. This was preceded by an overrepresentation of granulocytic cells in the bone marrow and a greatly increased multipotent granulocyte-monocyte progenitor in the spleen. In contrast,hematopoietic stem cells and T- and B-lymphocytes were not affected. Importantly,treatment of wild-type splenocytes with a gamma-secretase inhibitor directly promoted the granulocyte-macrophage colony-forming unit (GM-CFU). These results establish a critical role of PS in myelopoiesis. Our finding that this activity can be directly modulated by its gamma-secretase activity has important safety implications concerning these inhibitors. View Publication

Expression of the constitutively activated TEL/PDGFbetaR fusion protein is associated with the t(5;12)(q33;p13) chromosomal translocation found in a subset of patients with chronic myelomonocytic leukemia. TEL/PDGFbetaR activates multiple signal transduction pathways in cell-culture systems,and expression of the TEL-PDGFRB fusion gene induces myeloproliferative disease (MPD) in mice. We used gene-targeted mice to characterize the contribution of signal transducer and activator of transcription (Stat) and Src family genes to TEL-PDGFRB-mediated transformation in methylcellulose colony and murine bone marrow transduction/transplantation assays. Fetal liver hematopoietic stem and progenitor cells harboring targeted deletion of both Stat5a and Stat5b (Stat5ab(null/null)) genes were refractory to transformation by TEL-PDGFRB in methylcellulose colony assays. Notably,these cell populations were maintained in Stat5ab(null/null) fetal livers and succumbed to transformation by c-Myc. Surprisingly,targeted disruption of either Stat5a or Stat5b alone also impaired TEL-PDGFRB-mediated transformation. Survival of TPiGFP--textgreaterStat5a(-/-) and TPiGFP--textgreaterStat5a(+/-) mice was significantly prolonged,demonstrating significant sensitivity of TEL-PDGFRB-induced MPD to the dosage of Stat5a. TEL-PDGFRB-mediated MPD was incompletely penetrant in TPiGFP--textgreaterStat5b(-/-) mice. In contrast,Src family kinases Lyn,Hck,and Fgr and the Stat family member Stat1 were dispensable for TEL-PDGFRB disease. Together,these data demonstrate that Stat5a and Stat5b are dose-limiting mediators of TEL-PDGFRB-induced myeloproliferation. View Publication