Poster Presentations - Poster Session I

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135 INTERNAL TANDEM DUPLICATION AND ASP835 MUTATION OF THE FMS-LIKE TYROSINE KINASE 3 (FLT3) GENE IN CHILDREN WITH ACUTE PROMYELOCYTIC LEUKEMIA Y. Hoshi1*, T. Rikiishi1, K. Fujii1, N. Suwabe1, M. Imaizumi1, K. Iinuma1, M. Tsurusawa2, K. Horibe3, J. Hara4, I. Tsukimoto5 1 Tohoku University School of Medicine, Sendai, Japan 2 Aichi Medical University, Aichi, Japan 3 National Nagoya Hospital, Nagoya, Japan 4Osaka University, Osaka, Japan 5 Toho University, Tokyo, Japan [Objective] FLT3 regulates cell growth and is frequently mutated in acute promyelocytic leukemia (APL). Although FLT3 mutation is thought as a risk factor for adult patients, its clinical significance remains unclear for children. In this study, we investigated clinical relevance of FLT3 mutation in children with APL. [Patients and Methods] 48 children (30 boys, 18 girls; age <16y) with APL were studied. With cDNA prepared from marrow cells of patients, the PML-RARalpha and FLT3 mutation were analyzed as described (Tohoku J Exp Med 193:127, 2001; Blood 103:1085, 2004, respectively). Briefly, RT-PCR amplified DNA fragments of FLT3 containing regions of exons 14/15 for internal tandem duplication (ITD) or Asp835 mutation were sequenced or digested with EcoRV. [Results] Of 48, six patients (12.5 %) had ITD and six (12.5 %) had Asp835 mutation. The average age of patients with ITD or Asp835 mutation (10.8y) was significantly higher than that of patients without mutation (8.1y) (P<0.05). As compared to average white blood cell count at onset in patients without mutation (6575/ul), that of patients with ITD was significantly higher (34498/ul) (p=0.05), whereas that of patients with Asp835 mutation (24650/ul) was not significantly different. Moreover, the frequency of ITD or Asp835 mutation in patients with S-form PMLRARalpha (46 %) was significantly higher than that in patients with Lform (15 %) (p<0.05). [Discussion] In childhood APL, FLT3 was mutated in older children at frequency of 25 % with no difference between ITD and Asp835 mutation, suggesting a lower frequency of FLT3 mutation in children than that reported in adult APL. Moreover, a significant association of FLT3 mutation with hyerleukocytosis or S-form PML-RARalpha may suggest FLT3 mutation to be a risk factor for children with APL as well. However, a longer period of observation was needed to clarify whether FLT3 mutation would be associated with poor outcome.

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137 DEVELOPMENT OF A QUANTITATIVE REAL-TIME PCR METHOD FOR MONITORING CEBPA MUTATIONS IN NORMAL KARYOTYPE ACUTE MYELOID LEUKAEMIA PATIENT SAMPLES L-L. Smith1,2*, M. Smith2, L. Goff2, M. Jenner2, A. Lister2, J. Fitzgibbon2, D. Bonnet1 1Cancer Research UK, LRI, Haematopoietic Stem Cell Laboratory, London, UK 2Cancer Research UK, Medical Oncology Unit, St Bartholomew's Hospital, Medical College, London, UK Association of long-term clinical remission and molecular diseaseeradication is well established in acute myeloid leukaemia (AML) patients with t(15;17) and other favourable risk group leukaemias. However, translocations are not available as targets for minimal residual disease (MRD) detection in the 40-60% patients with normal/intermediate karyotype. In these patients specific mutations have therefore been investigated as one approach to quantify disease burden. Mutations in CEBPA, the gene encoding the granulocytic transcription factor C/ EBPalpha, are present in approximately 20% of normal karyotype AML and are concordant between presentation and relapse. In this study we set out to develop mutation specific quantitative real-time PCR assays for CEBPA mutations. Five assays were developed for normal karyotype M1 patients via mutation specific DNA amplification. Four insertion mutations, ranging from 3 to 57 base pairs in length, cluster together allowing the use of a common probe and reverse primer, resulting in an amplicon of approximately 160 base pairs. Specificity was achieved by making use of the unique sequence generated by the insertion mutations. Serial 10-fold dilutions were preformed in duplicate using DNA concentration equivalent to a range of cells from 125000 to 4. Sensitivity of the assays was approximately 5x10-5. To test specificity patient DNA was serially diluted in wild type DNA, maintaining an overall DNA concentration equivalent to 10000 cells in each reaction. Cell number in each reaction was monitored by amplification of beta-2 microglobulin. These assays demonstrate a high degree of specificity and sensitivity suggesting that mutation based MRD may have a complimentary role with flow sorting cells, translocation and WT1 based approaches currently used to quantify MRD in AML.

138 THYROID RECEPTOR INTERACTING PROTEIN 15 (TRIP15) IN ACUTE MYELOID LEUKAEMIA (AML) F. L. Khanim1*, R. E. Hayden1, Q. L. Trong2, M. T. Drayson3, C. M. Bunce1 1School of Biosciences, The University of Birmingham, UK 2 Dept of Hematology and Oncology, Cedars-Sinai, UCLA, LA, USA 3 Div of Immunity and Infection, The University of Birmingham, UK Our studies indicate that thyroid hormone signaling is dysregulated in acute myeloid leukaemia (AML). Our studies have identified that TRbeta1 is either lost or expressed at very low levels in approximately 50% of AMLs. Furthermore, re-expression of TRbeta1 resulted in cell death in TRbeta1-negative KG1a AML cells but not K562 CML cells which, already expresses TRbeta1. These data indicated that suppression of TRbeta1 signaling may contribute to the pathology of AML. We therefore reasoned that in the remaining 50% of TRbeta1-positive AMLs, thyroid hormone action could be dysregulated through other mechanisms. Thyroid Receptor Interacting Protein 15 (TRIP15; also known as CSN2, SGN2) acts as a TRbeta1selective nuclear co-repressor. We have used quantitative real-time RT-

Abstracts

THE POTENTIAL OF ACHIEVING A REMISSION CAN BE IDENTIFIED USING GENE EXPRESSION ANALYSIS AT DIAGNOSIS A. F. Gilkes, K. I. Mills*, A. K. Burnett Wales College of Medicine, Cardiff University, Cardiff, UK Acute Myeloid Leukaemia (AML) is a heterogeneous disease as can be seen at the morphological, cytochemical, immunological, cytogenetic and molecular level. Cytogenetics has been established as a major predictor of response to treatment and risk of relapse, and are the basis of the current patient treatment schemes. Patients are stratified, by cytogenetics, at diagnosis into favourable [t(15;17), t(8;21), or inv(16)], adverse [–(7), -(5), del(5q), del(3p) or complex abnormalities] or intermediate [all other types of abnormalities] risk categories. Factors, such as age and presenting white cell count, are also indicative of prognosis; an internal tandem duplication (ITD) mutation of the FLT3 receptor (~30% of cases) is highly predictive of risk of relapse. Considerable variation in response to treatment and overall survival are observed, even within the well-defined, favourable and adverse, risk groups indicating that refinement of prognostic prediction is needed. In Cardiff, we have analysed 221 diagnostic AML samples using Affymetrix U133A GeneChips, the patient cohort included all major cytogenetic groups and had been analysed for FLT3 mutations. The aim of this study was to identify genes differential expressed between patients who achieved a remission and those who failed. Complete clinical data was available from 149 samples, at the time of analysis.

Using SAM and ANOVA analyses, we identified a series of genes specific for each of the cytogenetic based prognostic risk groups differentially expressed between responders and non-responders. Little overlap between the gene lists was seen and there was no obvious correlation with the ability to achieve remission and the presence of a FLT3 mutation. This study further reinforces the molecular heterogeneity of AML, but also offers the possibility of refining prognostic groups and identifying patients for specific therapies based on their potential to achieve a remission status based on current therapeutic options.

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PCR to measure TRIP15 mRNA levels in a large panel of primary AML samples (MRC AML12 trial) and AML cell lines. These studies demonstrated that AML cell lines and greater than 90% of primary AML samples expressed significantly higher levels of TRIP15 than normal resting or proliferating myeloid blast cells, thereby implicating TRIP15 deregulation in the pathogenesis of AML. In the light of our earlier observations it is tempting to speculate TRIP15 contributes to the progression of AML by suppression of TRbeta1 signaling. However, we observed TRIP15 overexpression in TRbeta1 negative AMLs indicating that additional mechanisms may be involved. In this regard it is known that TRIP15 also acts a subunit of the signalosome and thereby regulates the modification and subsequent proteasomal degradation of key regulatory proteins including p53, Ikappa-B and interferon regulatory factor 8 (IRF8). Ongoing experiments using interfering RNA and transfection approaches to modulate TRIP15 expression in both TRbeta1-positive and -negative cell lines and primary myeloid cells will be used to elucidate which of the TRIP15 pathways promote the malignant behaviour of AML cells.

139 CONSTITUTIVE SERINE-585 PHOSPHORYLATION OF THE GMCSF RECEPTOR AND THE DEREGULATION OF SURVIVAL SIGNALLING IN AML BLASTS J. A. Powell1*, D. Thomas2, S. H. Lee2, A. F. Lopez1, M. A. Guthridge1 1Cytokine Receptor Laboratory, Department of Human Immunology, Hanson Institute, I.M.V.S. Frome Rd. Adelaide, SA, Australia 5000. 2Department of Hematology, I.M.V.S. Frome Rd. Adelaide, SA, Australia 5000 Transformation of a normal cell into a cancer cell requires the deregulation of the signals that promote both cell proliferation and cell survival. We have previously identified a novel tyrosine/serine bidentate motif in the GM-CSF receptor that independently regulates cell survival and proliferation. Signalling occurs through either Ser585 at lower cytokine concentrations leading to cell survival or through Tyr577 at higher cytokine concentrations leading to cell survival as well as proliferation, differentiation or functional activation. In the current study, we have shown that the phosphoserine-585 survival pathway is deregulated in most AML patients analysed. GM-CSF titration experiments on primary AML blasts revealed constitutive phosphoserine-585 survival signalling through the 14-3-3, PI-3K, Akt pathway. However, these same cells exhibited normal phosphotyrosine mediated signalling, inducible at high cytokine concentrations. All AML blasts examined exhibited factorindependent survival in vitro, most of which were susceptible to inhibitors of PKA. Analysis of primary mononuclear cells (MNC) revealed normal bi-phasic regulation of serine-585, and normal phosphotyrosine mediated signalling. Treatment of normal primary MNC with PKA inhibitors reduced GM-CSF induced serine-585 phosphorylation, whereas forskolin induced phosphorylation of serine-585. We therefore propose that constitutive Ser585 survival signalling in AML blasts is mediated by a PKA, phosphoserine-585, Akt signalling pathway.

140 EXPRESSION OF VEGF, ITS RECEPTORS AND HYPOXIA INDUCIBLE FACTORS IN ACUTE MYELOBLASTIC LEUKAEMIA Y. Y. Zhang1,2*, K. C. Gatter1,2, F. Pezzella2, G. Pillai2, H. Turley2, A. Peniket2, G. G. Zhou1,2, S. M. Watt1,2 1National Blood Service, Oxford, UK 2University of Oxford, Oxford, UK Here, we have analysed 144 archival cases of leukaemia and nonneoplastic myeloproliferative disorders and 22 reactive control bone marrows. We have studied the expression and cellular localisation of angiogenic cytokines such as vascular endothelial growth factor (VEGF) and its tyrosine kinase receptor VEGFR-2. Using a novel antibody to the phosphorylated form of VEGFR-2 (pVEGFR-2), developed in our laboratory, we have shown that microvessel density (MVD) and the expression of both pVEGFR-2 and its ligand VEGF are increased in acute myeloid (AML) and lymphoblastic (ALL) leukaemias. VEGFR-2 and VEGF are found mainly in neoplastic cells, indicating that VEGF/VEGFR2 signalling may play an important role in the proliferation and survival of blast cells. We further analysed the expression of hyopxia inducible receptors, VHL and their downstream targets. Our studies demonstrate increased expression of HIF-1 alpha, HIF-2 alpha, stroma derived factor

(SDF-1) and its receptor CXCR-4 is also observed in AML cases, compared with control samples. The expression of HIF-1 alpha and HIF-2 alpha is normally regulated by the tumor suppressor protein VHL. VHL targets the HIFs to the proteosome in normal conditions. Under low oxygen tensions, HIF proteins are stabilised and translocated to the nucleus. A positive correlation between HIFs and CXCR4 in AMLs was seen. We have also observed a negative correlation of expression between VHL and HIFs and also VHL and CXCR4 suggesting that the exponential growth of blast cells in haematological malignancies may be regulated by hypoxia or by mutations in genes involved in oxygen sensing and signalling.

141 TRICHOSTATIN A ANALOGUE, A POTENT INHIBITOR OF HISTONE DEACETYLASE, INHIBITS CELL PROLIFERATION AND INDUCES APOPTOSIS IN HUMAN LEUKEMIA CELLS B. D. Chen*, X. Li Division of Hematology-Oncology, Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA Histone acetylation plays an important role in the silencing and activation of genes involved in tumorigenesis. Trichostatin A, a known anti-fungal drug, is a potent inhibitor of histone deacetylase (HDAC) with potential anti-tumor activity. In this study, we investigated the effect of HDAC inhibitor III, an amide analogues of trichostatin A, on THP-1 human leukemia cells. We showed that low dose HDAC inhibitor III (<0.2 microM) inhibits the growth of THP-1 cells at G1 phase with low cytotoxic effect. Growth arrest induced by HDAC inhibitor III is associated with increased levels of cyclin–dependent protein kinase inhibitor p21 and cyclin E, in agreement with G1 phase arrest. Low dose of HDAC inhibitor III also exerted some differentiating effect on THP-1 cells as judged by the expression of c-fms proto-oncogene (M-CSF receptor). At higher doses (2 microM), HDAC inhibitor III induces THP-1 cells to undergo apoptosis, which was associated with the cleavage of PARP, cytochrome c release and activation of both caspases-8, -9, followed by caspase 3. In addition, HDAC inhibitor III could increase the levels of pro-apoptotic protein Bax, but decreased the levels of anti-apoptotic protein XIAP. HDAC inhibitor III is a potent activator of NF-kappaB transcription factor. RT-PCR assay showed that the inhibitor could transiently increase IL-1 expression yet markedly decreased TNF-alpha expression. Prolonged treatment with HDACI III at high doses was accompanied by an increased level of iNOS mRNA. Yet, aminoguanidine failed to overcome HDAC inhibitor III– induced apoptosis, suggesting that NO-mediated signal pathway is not involved in the induction of apoptosis. Our results show that HDAC inhibitor III is a potent growth inhibitor and inducer of apoptosis in human leukemia cells and suggest potential therapeutic strategies of HDAC inhibitors for patients with leukemias.

142 KG1A PROVIDES A MODEL FOR THE COMMITMENT OF PRIMATIVE HAEMOPOIETIC CELLS TO MYELOID DIFFERENTIATION K. S. Mathias1*, R. E. Hayden1, F. L. Khanim1, M. T. Drayson2, N. J. Davies1, C. M. Bunce1 1School of Biosciences, University of Birmingham, Birmingham, UK 2Division of Immunity and Infection, University of Birmingham, Birmingham, UK KG1a cells were derived from an erythroleukaemia in myeloblastic relapse but express low level transcripts of genes associated with immature T and B cells. This suggested that KG1a represent primitive haemopoietic cells and a model for early events associated with lineage restriction/ commitment. However, KG1a have proven resistant to inducers of differentiation and consequently have fallen from common use. Conversely, we demonstrate here that the culture of KG1a in a chemically defined serum-free medium releases the cells from their maturation block, resulting in commitment towards differentiation. In keeping with primitive haemopoietic cells, KG1a express CD34 in the absence of CD117. However, when transferred to ITS+ supplemented medium KG1a progressively lost CD34 and acquired CD117 expression, becoming CD34+ve/CD117+ve (days 1-3) and CD34-ve/CD117+ve (by day 4). Having lost CD34 expression, the cells went on to increase their expression of the myeloid commitment marker CD33 (beginning around day 7). These changes in antigen expression faithfully recapitulate those that occur during the normal commitment of haemopoietic stem cells to

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34th Annual Meeting: ISEH - Abstracts / Experimental Hematology 33 (2005) 39-143 myeloid cell differentiation. These observations reinstate KG1a as a potent model for studies of genomic, proteomic and metabolomic events associated with cell commitment. They also highlight that serum contains signals that block the maturation of AML cells. Importantly, we have recently demonstrated that serum represses HL-60 cell differentiation. Culture of HL-60 cells in medium supplemented with ITS+, resulted in slowing of cell growth, increased CD11b expression and the acquisition of morphological features consistent with neutrophil differentiation. However, serum-free differentiation of HL-60 did not occur in the presence of 1nM triodothyronine (thyroid hormone; T3), indicating that serum-borne T3 suppresses HL-60 differentiation. In contrast T3 was unable to counter KG1a serum-free differentiation. Thus the mechanisms by which serum represses KG1a and HL-60 cell differentiation are likely to be distinct.

143 A SULFUR-SUBSTITUTED FATTY ACID ANALOG IMPROVES SURVIVAL AND IMPAIRS METASTASIS IN RAT MODELS OF ACUTE LEUKEMIA P. O. Iversen1*, D. R. Sørensen2, R. K. Berge3, A. C. Rustan4, C. A. Drevon1 1Department of Nutrition, IMB, University of Oslo, Norway 2Department of Comparative Medicine, the National Hospital, Oslo, Norway 3Institute of Medicine, Section of Medical Biochemistry, University of Bergen, Norway 4Pharmaceutical Biology, University of Oslo, Norway Background: Polyunsaturated fatty acids (PUFA) may cause apoptosis in leukemic cell lines (1). Recent data suggest that a sulfur-substituted fatty acid (tetradecylthioacetic acid; TTA) can induce apoptosis in a promyelocytic leukemic cell line (2). However, whether PUFA and/or TTA can modify leukemogenesis in the intact organism, is unknown. Aim: We examined whether dietary intake of n-3 PUFA or TTA could improve outcome in rats with either acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL). Methods: We studied rats harbouring either an acute promyelocytic leukemia (AML, ref. 3), or an acute T-cell leukemia (ALL, ref. 4). The rats were randomized to isoenergetic diets with either standard pellets (Control), n-3 PUFA or TTA (10 rats/diet). The main endpoint was survival, while bone marrow (BM) leukemic cellularity and extramedullar dissemination (spleen weight and plasma activity of matrix metalloproteinases, MMP) served as secondary endpoints. Results: The rats were killed when they showed signs of disease, or after 35 days. The main results are summarized in the table. Conclusion: Dietary intake of TTA, but not of n-3 PUFA, in rats with acute leukemia, prolonged their survival, and TTA intake was associated with reduced leukemic cell burden as well as diminished extramedullar dissemination. References: 1. Finstad HS et al. Leukemia, 12: 921-929, 1998. 2. Tronstad KJ et al. Chemistry & Biology, 10: 609-618, 2003. 3. Martens ACM et al. Leukemia, 4: 241-257, 1990. 4. Nestvold J et al. Scand J Immunol, 60: 153-158, 2004. AML- AMLControl PUFA

AMLTTA

Mean survival (days) 30 31 >35* BM leuk.cells (%) 93±11 95±16 58±9* Spleen weight (g) 4.5±0.7 4.3±0.8 2.2±0.4* MMP activity 100 98±8 60±4* (% of Control) * denotes P<0.05 compared with Control/PUFA

ALL- ALLControl PUFA

ALLTTA

16 92±11 3.5±0.4 100

20* 45±8* 2.1±0.4* 33±5*

16 96±14 3.7±0.6 98±7

144 IMATINIB MESYLATE FOR REFRACTORY ACUTE MYELOBLASTIC LEUKEMIA HARBORING INV(16) AND C-KIT EXON 8 MUTATION N. Asou1*, T. Nanri1, N. Matsuno1, T. Kawakita1, H. Suzushima2, H. Mitsuya1 1 Kumamoto University School of Medicine, Kumamoto, Japan 2 NTT West Kyushu General Hospital, Kumamoto, Japan Heterodimeric transcription factor AML1(RUNX1)/PEBP2beta(CBFbeta) is a most common chromosomal translocation target in leukemia. The inv(16) generates the PEBP2beta-MYH11 fusion protein that is predicted to interfere with the function of AML1/PEBP2beta in a dominant-negative manner and contributes to impaired differentiation of hematopoietic cells. However, studies using knock-in mice with PEBP2beta-MYH11 have revealed that the fusion protein is critical for leukemogenesis but that additional genetic events are required for the development of acute myeloblastic leukemia (AML). Recently, mutations in the C-KIT gene were frequently found in patients with inv(16) AML. Of 15 patients with inv(16) AML in our hospital, 3 and 2 had mutations in the C-KIT and FLT3 genes, respectively. These mutations could represent potential therapeutic targets for specific tyrosine kinase inhibitors. An approximately half of AML patients harboring inv(16) generally have a favorable prognosis, however, the rest of such patients are known to have a relatively poor prognosis. In this study, we present a case of AML with M4Eo harboring inv(16) and a C-KIT exon 8 mutation who was successfully treated with imatinib mesylate. No autophosphorylation of the C-KIT exon 8 mutant protein in primary AML cells was observed with western blot analysis, while the stem cell factor (Kirin Brewery Co. Ltd) markedly elicited C-KIT mutant phosphorylation. Furthermore, imatinib (Novartis Pharma AG) treatment of primary AML cells strongly down-regulated phosphorylation of the C-KIT mutant. A 58-year-old man with second relapsed AML was treated with high-dose cytarabine and mitoxantrone but only unsuccessfully. However, the following treatment with 400 mg of imatinib mesylate for 2 weeks in combination with low-dose cytarabine, aclarubicin and granulocyte colony-stimulating factor plus vincrisitine and prednisolone brought about complete hematological remission. This observation indicates that imatinib can be an alternative treatment modality for AML with the C-KIT exon 8 mutation.

145 GENERATION OF AKR1C3 KNOCK-DOWN HAEMOPOIETIC CELL LINES J. Birtwhistle1*, F. L. Khanim1, R. E. Hayden1, H. Schrewe2, J. K. Chipman1, C. M. Bunce1 1 School of Biosicences, University of Birmingham, Birmingham, UK 2Department of Developmental Genetics, Max-Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany We have shown that inhibitors of the aldo-keto reductase AKR1C3 promote myeloid cell differentiation whereas over-expression of the enzyme suppresses differentiation. Based on these studies we have proposed AKR1C3 as a novel regulator of haemopoiesis and as a target in the treatment of acute myeloid leukaemia (AML). To this end we are currently conducting a phase II trial of the AKR1C3 inhibitor medroxyprogesterone acetate in elderly and relapsed AML. However, all the known AKR1C3 inhibitors have AKR1C3-idependent activities and it is therefore unclear to what extent their actions are mediated solely via AKR1C3. There is compelling evidence that AKR1C3 is capable of activating polycyclic aromatic hydrocarbon (PAH) carcinogens. Expression of the enzyme by haemopoietic stem and progenitor cells may therefore represent an 'Achilles heel' rendering them susceptible for oncogenesis. However, to fully elucidate the role of AKR1C3 in either myelopoiesis or carcinogen activation needs more precise intervention than the use of so called 'dirty' inhibitors. We have therefore used short hairpin interfering RNA (shiRNA) to generate cell lines in which AKR1C3 is specifically knocked down. For these studies we elected to use the KG1a myeloid and the K562 CML blast crisis cell lines. These lines have good transfection efficiencies and real-time RT-PCR analyses identified that they also had high initial expression of AKR1C3. Stable transfectant AKR1C3 knock-down cell lines have been produced and confirmed by the presence of the transfected neomycin resistance gene along with growth through G418 selection and reduced levels of the AKR1C3 expression as measured by real-time RT-

Abstracts

Diet-parameter

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PCR. These lines will now be used in assays of myeloid and erythroid differentiation and susceptibility to environmemtal carcinogens. The latter approach will use the Comet Assay which can be used to detect both single DNA strand breaks and DNA oxidative damage in cells exposed to potentially genotoxic AKR1C3 substrates.

plasmas of 7 patients showed mjor differences(ara-c; 4.60~64.55 ng/ml, EP; 0.16~1.24 microg/ml), indicated the different metabolic ability in individual cases. We concluded that SPAC with EP can be administrated safely and has potential for the treatment of high-risk and AML patients in poor condition.

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CELLULAR IMMUNE STATUS IN PATIENTS WITH ACUTE PROMYELOCYTIC LEUKEMIA Y. Li1*, L. Yang1, S. Chen1, Q. Yin1, J. Tang1, G. Przybylski2, C. A. Schmidt2 1 Institute of Hematology, Medical College, Jinan University, Guangzhou 510632, China 2 Dept. of Hematology and Oncology, Ernst-Moritz-Arndt University Greifswald, Greifswald 17487, Germany Recent thymic output function, T cell receptor (TCR) repertoire and antigen specific T cell expansion would be the important markers for evaluation the cellular immune status in patients. Defects of cellular immunity may play a role in hematologic malignancies. Little is known about the feature of T-cell immune state in leukemia. In order to evaluate the cellular immune status in patients with APL, signal joint T-cell receptor rearrangement excision circles (TRECs) level and TCR V beta repertoire usage and clonality were analyzed. 17 cases with APL were selected for the research. Quantitative detection of TRECs in DNA of peripheral blood mononuclear cells (PBMC) was preformed by real-time PCR, the TRECsnumber was related to the number of T-cells by determination of the number of CD3-positive cells. The expression and cloanlity analysis were detected by RT-PCR and genescan technique. 14 normal individuals served as controls. In compare with normal individuals (6.36±5.28 copies /1000 CD3+cells), a dramatic reduction of TRECs values in APL (0.77±1.60 copies /1000 CD3+cells, P=0.005) could be found. The number of expressed V beta subfamilies (from 2 to 21 subfamilies) was varied in different patients with APL. The frequent expression V beta subfamilies were Vbeta2 (64.7%), Vbeta15 (58.8%), Vbeta3 and Vbeta5 (47.1%). Clonal expanded T-cells in some V beta subfamilies could be identified on almost all patients, predominant in Vbeta10, Vbeta23, Vbeta3 and Vbeta21. This is the first description of TRECs level in APL patients. The result showed a prominent reduction of thymic output naive T cells function in APL. The predominant usage and clonal expansion of TCR V beta subfamily T cells could be identified in APL patients, indicating the host could have the ability for specific immune response to leukemia associated antigen, in despite of T cell immunodeficiency was showed in patients.

CONTROL OF CELL DEATH LEVELS USING TH9402-BASED PDT TREATMENT ON FRESH PBMC, AND POTENTIAL APPLICATION TO PATIENTS WITH CGVHD A. Lévesque1, A. L. Savard1, D. C. Roy2, F. Foss3, C. Scotto1* 1 Celmed BioSciences Inc, Saint-Laurent, Quebec, Canada 2 Maisonneuve Rosemont Hospital, Montreal, Quebec, Canada 3 Tufts New England Medical Center, Boston, MA, USA Recently, extracorporeal photopheresis (ECP) has shown interesting clinical activity for the treatment of drug-refractory chronic graft-vs-host disease (cGvHD), inducing Th1/Th2 immunomodulation to restore immune tolerance. Several studies indicate that target cell apoptosis may have a role in the control of cGVHD, and increasing apoptotic levels may favor immune modulation. We have developed an approach to eliminate immunoreactive cells using the TheraluxTM photodynamic cell therapy (PDT) system based on the use of the rhodamine-derivative TH9402 illuminated ex vivo with a visible light source (514nm wavelength). The capacity of TH9402 PDT to induce increasing levels of T cell apoptosis has not been investigated. The induction of apoptotic cell death was studied using peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers (HV) and cGvHD patients. We found a good correlation between PDT conditions and levels of cell death induced. In comparison to controls showing 8±4 percent (%) of apoptosis, as measured by TUNEL assay on cells harvested 3 days post-treatment, PBMC from HV subjected to PDT using 0.33, 0.66, or 1.32 micromolar of TH9402 showed 28±16%, 49±17%, and 78±11% of apoptosis, respectively. These studies have shown that the intra- and inter-donor variability in TH9402 incorporation are very low (~5% and 10%, respectively). To ensure that these findings could also be applied to a clinical setting, PBMC from cGvHD patients were treated with 0.33 and 1.32 micromolar of TH9402 to trigger either low or high levels of apoptosis. PBMC from cGvHD patients showed a sensitivity similar to that of PBMC from HV, with 38±9% and 73±13% of apoptosis when treated with 0.33 and 1.32 micromolar TH9402, respectively. Based on these data, we intend to begin a pilot clinical study evaluating two controlled PDT conditions inducing low and high levels of apoptosis in order to assess their efficacy and biological effect to treat cGVHD in humans.

147 THE VALUE OF ORAL CYTARABINE OCFOSFATE AND ETOPOSIDE IN THE TREATMENT OF PATIENTS WITH REFRACTORY AML AND AML IN ELDERLY PATIENTS A. Horikoshi*, K. Takei, Y. Nakagawa, Y. Hosokawa, S. Sawada Nihon University Nerima-Hikarigaoka Hospital, Tokyo, Japan Cytarabine ocfosfate (SPAC) is rapidly transformed into cytarabine (ara-c) when orally administrated. The oral administration of SPAC at 150 to 300 mg/m2/d was pharmacokinetically concluded to be comparable to the continuous infusion of ara-c at 20 mg/m2/d. To evaluate the toxicity and antileukemic efficacy of SPAC combined with oral etoposide (EP), a total of 21 refractory cases (10 men/11 women, median age 69 years; range 33-86 years), with 11 de novo AML and 10 AML evolving from myelodysplastic syndromes basically received induction therapy with SPAC at 300 mg/d and EP at 50 mg/d for 14 days. Thirteen patients had previously been untreated. Seventeen patients had abnormal karyotypes including 8 complex abnormalities. Patients had complications that included hypertension, congestive heart failure, old myocard infarction, aneurysm, dibetes mellitus, hyper lipidemia, pneumonia and phlegmon. Seven patients had performance status 3~4. Seven patients achieved complete remission(CR), and 2 patients achieved partial remission(PR). The nadir of leukocyte and platelet counts occurred at 3~31 days from the start of therapy. The durations of leukocytopenia(<1000) and thrombocytopenia(<20000) were not prolonged, but some CR and PR patients showed a long duration of leukopenia(23~51days). No severe adverse events were observed. Two out of 12 patients in the non-responder group achieved CR and PR after additional induction therapy. The duration of survival was 1.5~16 months. The ara-c and EP concentrations in

149 THE ARTERIAL AND VENOUS POTENTIAL OF HUMAN MAPC AND AC133 DERIVED ENDOTHELIAL CELLS IS MODULATED BY ACTIVATION OF NOTCH AND PATCHED LIGANDS X. L. Aranguren1*, C. Clavel1, A. Luttun2, M. Barajas1, C. Moreno1, G. Abizanda1, A. Echavarri1, M. Araña1, E. J. Andreu1, F. Prósper1 1Foundation for Applied Medical Research, Division of Cancer, Area of Cell Therapy and Hematology, Clinica Universitaria, Pamplona, Spain 2Stem Cell Institute, University of Minnesota Medical School, Minneapolis, MN 55455, USA We have analyzed the endothelial potential from different populations of adult stem cells, human MAPC and cord blood derived AC133 cells to establish the arterial and venous potential of each population and the signaling pathways implicated in their differentiation. Both populations differentiate into mature/functional endothelium in vitro as determined by expression of markers such as von Willebrand factor, VE-cadherin, VEGFR1 and 2, Tie1 and Tie2, CD105 and CD51/CD61, matrigel tube formation and ac-LDL-Dil uptake. However, while AC133 cells differentiate mostly to microvascular and venous endothelium, MAPC differentiate to both arterial and venous endothelium, as determined by expression of specific markers (Hey-2, Ephrin B1, Ephrin B2, Dll-4, Eph B4). Analysis of molecules involved in arterial development pathways (notch and patched) have shown clear differences between both populations of stem cells. Modulation of these pathways in vitro modifies the endothelial fate of AC133 and MAPC. Addition of shh and Dll-4 enhance arterial potential of MAPC derived endothelium. In vitro 3D matrigel differentiation assay show that with Shh and Dll-4, number of

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34th Annual Meeting: ISEH - Abstracts / Experimental Hematology 33 (2005) 39-143 vessels and their size is higher than VEGF and bFGF alone. In vivo matrigel assay show that addition of Shh and Dll4 enhance vascular structures because the higher collagen deposition and smooth muscle cells. Synergist effect has shown between media and cells. Light polarized show that blood vessels are better in cells with artery media (VEGF, bFGF, Shh and dll-4), than in other groups. MAPCs are be able in vivo to differentiate to mature endothelial cells by expression of UEA lectin. Conclusions: MAPCs are a promise cells for treatment of ischemic tissue because they have both in vitro and in vivo potential to differentiated toward endothelial cells, and they have high potential to differentiate to artery phenotype endothelial cells. Again, These potential can be modulate with notch and sonic pathways.

150 DEVELOPMENT OF NOVEL DRUGS DERIVED FROM RINGSUBSTITUTED 9-ARYLIDENEANTHRONES AS POTENTIAL AGENTS FOR LEUKEMIA THERAPY E. Levy1, Z. Rappaport2, D. Arad3, O. Sphilberg4, I. Levy1, I. Nathan1* 1 Ben Gurion University of the Negev and Soroka University Medical Center, Israel 2 Hebrew University, Jerusalem, Israel 3 MND Diagnostic Ltd., Israel 4 Rabin Medical Center, Petah-Tiqva, Israel In a search for novel derivatives, which can serve as antileukemic agents we have screened and tested a large series of vinylic compounds on human leukemic cell lines: HL-60 promyelocytic leukemia, CCRF-CEM T lymphoblastic leukemia and U-937 monocytic leukemia. The ringsubstituted 9-benzylideneanthrones had the strongest antileukemic effect with LC50 <0.1 micrg/ml but had no effect on lymphocytes from healthy donors. These substituted 9-arylideneanthrones killed the leukemic cells by induction of apoptosis as measured by acridine orange/ ethidium bromide staining and phosphatidyl serine exposure using annexin V-FITC. These compounds also caused G1 arrest in CCRF-CEM cell line. The signaling pathway leading to cell death were studied. The results showed an involvment of protein kinase C-epsilon (PKC-epsilon) but not PKC-alpha in substituted 9- benzylideneanthrones activity. PKC-epsilon activation was manifested by translocation of the enzyme from the cytosol to the membrane, as assessed by western blotting. In addition, the production of reactive oxygen species (ROS) was observed as determined by the fluorescent probes specific to H2O2 and O2- DCFH-DA and hydroethidine, respectively. The lipophilic antioxidant vitamin E was able to abolish the ROS generation and the apoptotic activity of these 9-arylidene anthrones. Primary cells obtained from CLL patients underwent apoptosis in response to treatment with 9-arylideneanthrones. The results imply the antileukemic mechanism of these arylideneanthrones and their potential as antileukemic agents.

press). Herein, we compared the potential of purified adult wild type LinSca1+kit+CD34- flt3- long-term (LT) HSCs and the LMPPs to reconstitute and correct the immune deficiency in X-SCID fetal mice. Notably, LMPPs reconstituted T, B and NK cells more rapidly but with similar long-term efficiency as LT-HSCs, and neither of the two populations gave detectable reconstitution of the myeloid lineages or the Lin-Sca1+kit+ HSC compartment cell. Importantly, although transplanted with congenic cells, the immune reconstitution with LMPPs was considerable better in fetal than young adult X-SCID recipients, supporting that the fetal microenvironment is more permissive to donor-derived cell replacement therapy. Thus, representing a considerable larger population of BM progenitors than LT-HSCs, we propose that LMPPs are likely to be the best target cells for cell replacement and gene therapy of inborn immunedeficiencies.

152 ONE-STEP NON-MAGNETIC NEGATIVE SELECTION OF HIGHLY PURIFIED LYMPHOCYTE SUBSETS FROM PREVIOUSLY FROZEN HUMAN CELL SAMPLES K. A. Ross1, A. Wong1, A. Lopez1, A. C. Eaves2, T. E. Thomas1, C. E. Peters1* 1 StemCell Technologies Inc, Vancouver, BC, Canada 2 Terry Fox Laboratory, Vancouver, BC, Canada We have developed a rapid, one-step procedure (SpinSepTM) for the isolation of highly purified human cell subsets by negative selection without the need for magnets, columns, or other specialized equipment. This procedure couples the specificity of monoclonal antibodies with the ease of buoyant density centrifugation. A cocktail of bispecific antibody reagents ('TACs') selectively couples cell surface antigens on unwanted cells in a mixed cell suspension to specially designed, dense microparticles, similar to red blood cells and their use in RosetteSepTM. The cell suspension is centrifuged over a buoyant density medium. The unwanted cells pellet; the desired, unlabeled cells are recovered in the layer above the buoyant density medium. The specificity of the antibodies incorporated into the TACs determines which cells will be targeted for depletion and consequently which cells will be enriched. Lymphocyte subsets were enriched from previously frozen peripheral blood mononuclear cell samples within one hour using this method. The purity achieved with this new centrifugation-based procedure is comparable to that obtained with more complicated, multi-step immunomagnetic negative selection techniques, and the viability of the enriched cells exceeds 90%. Desired cells are not labeled with antibody, thus avoiding perturbations of function that may occur due to antibody binding. In summary, SpinSepTM is a simple and robust way to rapidly obtain cell suspensions with high purity and recovery from previously frozen cell samples without the use of any specialized equipment.

151

Desired Cells

n

% Viability % Purity % Recovery (mean±SD) (mean±SD) (mean±SD)

CD3+ T cells CD4+ T cells CD8+ T cells B cells

5 5 8 13

97±2 98±1 94±5 94±7

99±1 95±2 91±5 93±6

58±17 61±12 43±14 47±15

153 COMPARISON OF BONE MARROW AND UMBILICAL CORD BLOOD AS A SOURCE OF LIVER STEM CELLS K. H. Ryu1*, E. S. Yoo1, Y. J. Jung2, S. Y. Woo2, J. Y. Seoh2, H. S. Han3 1Dept of Pediatrics, Ewha Womans University College of Medicine, Seoul, Korea 2 Dept of Microbiology, Ewha Womans University College of Medicine, Seoul, Korea 3 Dept of Surgery, Seoul National University College of Medicine, Seoul, Korea Objective: The purpose of this study was to suggest human umbilical cord blood (UCB) and bone marrow (BM) as the source of liver stem cells and to generate mature hepatocyte lineage cells. Methods: Mononuclear cells from UCB and BM were cultured with fibroblast growth factor (FGF)-1, FGF-2, stem cell factor (SCF), and hepatocyte growth factor. (HGF) Cultured cells were monitored in inverted light microscopy and analyzed by reverse transcription-polymerase chain

Abstracts

LYMPHOID-PRIMED MULTIPOTENT PROGENITOR CELLS EFFICIENTLY AND DURABLY RECONSTITUTE THE IMMUNE SYSTEM IN X-SCID MICE TRANSPLANTED IN UTERO K. Liuba1,3*, S. Stott2, G. Lingman1, S. E. W. Jacobsen3 1 Department of Obstetrics and Gynaecology, Institution for Clinical Sciences, Lund University, Sweden 2 Wallenberg Neuroscience Center, Section of Neurobiology, and Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund University, Sweden 3 Hematopoietic stem cell laboratory, Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund University, Sweden X-linked severe combined immune deficiency (X-SCID) is characterized by lack of functional T and NK cells, resulting in enhanced susceptibility to infections and death in early life. Although conventional bone marrow transplantation (BMT) can cure X-SCID, there is considerable mortality and morbidity associated with BMT. In utero transplantation of hematopoietic stem cells (HSCs) has been reported to be successful. Although lacking evidence for donor-derived myeloid cells, it is believed that sustainable immune reconstitution in X-SCID patients is dependent on low-level HSC engraftment, with preferential lymphoid reconstitution. We have recently identified in normal mouse BM, a primitive Lin- Sca1+kit+CD34+flt3+ lymphoid –primed multipotent progenitor (LMPP), representing the earliest lineage-restricted stem/ progenitor cell in the hematopoietic system (Adolfssson et al, Cell in

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reaction (RT-PCR), immunofluorescent (IF) staining analysis for detecting hepatocyte lineage markers and by flow cytometry for demonstrating hepatocyte lineage differentiation rather than hematopoietic one. Results: Cell growth in given culture condition supplemented with FGF1, FGF-2, SCF, and HGF yielded large sized and oval shaped cells which were adherent to culture dishes. mRNA of ALB, CK18 and alphafeto protein were detected from day 7 in both UCB and BM derived cells by RT-PCR. In IF analysis, these cells expressed albumin (ALB), cytokeratin(CK)-19 from 7-day of culture and some of the ALB producing cells contained proliferating cell nuclear antigen. Cellular differentiation expressing CK-18 molecules was observed by flow cytomery analysis. Conclusions: Liver stem cells may exist in UCB as well as BM and can proliferate into hepatocyte lineage cells in primary culture system in vitro and may serve cell sources for cell therapy of hepatic tissues.

154 ADOPTIVE IMMUNOTHERAPY IN ACUTE MYELOGENOUS LEUKEMIA: A NOVEL APPROACH T. A. Lane1*, R. K. Zhong1, M. R. Loken2, E. D. Ball1 1 University of California, San Diego, School of Medicine; San Diego, CA, USA 2 Hematologics, Inc., Seattle, WA, USA We investigated a novel method to generate autologous cytolytic T cells (CTL) from the peripheral blood of patients (pts) with acute myeloid leukemia (AML; not M3) for adoptive immunotherapy. PBMNC from 12 AML pts with >50% blasts were cultured in 10% serum with 50 ng/ml GM-CSF, 1000 IU/ml IL-4 and 5 mcg/ml anti-CTLA4 on days 0-8 to induce a dendritic cell phenotype (CD80+/86+) and stimulate T cells. 20 U/ ml IL-2 and 10 ng/ml OK-T3 were added on days 8, 14 respectively. T cells were expanded with IL-2/OK-T3 until >99% CD3+ cells (1-3 wk), then 1000 U/ml IL-2 was added until day 40. Results: AML blasts (CD33+CD3-neg) decreased to 80±20% by d 8, 62±25% d 21, <1% d 35. By d 42, CD3+ T cells expanded by 354±182 fold, with >99% CD3+ (40.6±22.9% CD4+; 57.8±22.8% CD8+) of which 10.7±4.7% were CD4+IFN-gamma+CD69+ and 9.8±4.1% were CD8+IFNgamma+CD69+ by ELISPOT. CTL produced 42±23% lysis of autologous AML blasts at E:T=20:1; blocked by anti-MHC class-I but not class II, suggesting CD8+ CTL. There was <5% lysis of autologous PHA blasts. CTL inhibited AML colony growth >95% at E:T=2:1 (n=3). To assess safety we investigated post culture AML blast colony growth and cell phenotype by multiparameter flow cytometry. Pre-culture there were 28, 50, and 295 blast colonies/50,000 MNC (n=3), but no colonies grew from post-culture samples plated at 0.5-1x106 cells/dish (n=6). Pre-culture AML blasts (CD34+) represented 70, 76, 89 and 96% of MNC (n=4); but the post-culture samples revealed no blasts in 400,000 to 1,600,000 cells counted. Experiments in a closed bag system indicated that >109 CD3+ cells were generated from <50 ml peripheral blood. Conclusion: The novel AML-CTL culture generated CTL that were highly reactive to autologous AML blasts without requiring the identification of patient-specific antigen targets and may facilitate adoptive immunotherapy for pts with AML.

155 PHASE I-II STUDY TO EVALUATE THE SAFETY AND THE EFFICACY OF GRANULOCYTE COLONY STIMULATING FACTOR (G-CSF) IN MOBILIZING HEMATOPOIETIC STEM CELLS IN PATIENTS WITH ADVANCED LIVER CIRRHOSIS L. Catani1*, S. Lorenzini2, A. Isidori1, S. Talarico1, E. Loggi2, A. Gramenzi2, F. Bonifazi1, P. Andreone2, M. Baccarani1, R. M. Lemoli1 1Institute of Hematology and Medical Oncology L. A. Seràgnoli, Bologna, Italy 2Department of Hepatology, Cardioangiology and Internal Medicine, Bologna, Italy Bone marrow-derived hematopoietic stem cells (HSCs) contribute to tissue regeneration after acute or chronic liver damage. In this study, we assessed whether Granulocyte-colony stimulating factor can be safely administered to patients with liver cirrhosis in order to expand and to mobilize HSCs. Seventeen patients with advanced liver disease [16 male, mean age: 59 years; 12 HCV, 1 HBV, 1 HDV, 3 alcohol abuse; median CTP score: 7 (range 5-9), median MELD score: 11.5 (range 7-17)] were consecutively treated with rhu-Granulocyte-colony stimulating factor (Lenograstim). Increasing doses of Granulocyte-colony stimulating factor were administered subcutaneously for 7 consecutive days to five cohorts of

three patients each, starting from 2 microg/kilog/daily. The dose of Granulocyte-colony stimulating factor was escalated according to Fibonacci's increment rule until the successful mobilization of 10 CD34+cells/microl in at least 2/3 patients. Fifteen patients were enrolled in the phase I study. Granulocyte-colony stimulating factor mobilizing dose was found at 15 microg/Kilog/day when mobilization of HSCs occurred in 2/3 patients on day 5 from the first administration (mean CD34+cells/ microl: 49,2±2,7). Two out of 4 patients have been enrolled so far in the phase II study with the mobilizing dose, and all of them showed a successful mobilization and collection of HSCs. Overall, the median peak values of CD34+ and CD133+ cells in mobilizing patients were 41.5±13.5 cells/microl and 26.5±3.2 cells/microl, respectively. No severe adverse events were observed at any dosage. From the 6.6 microg/kilog dose of Granulocyte-colony stimulating factor or higher, the side effects observed were bone pain (10/17), fever (4/17) and alkaline phosphatase increase (10/ 17). Up to 30 days after treatment with Granulocyte-colony stimulating factor, we observed a significant decrease of alpha-foetoprotein in all but one patients and the decrease of transaminases in mobilizing patients. In conclusion, the administration of Granulocyte-colony stimulating factor to patients with liver cirrhosis is safe and feasible and is capable of mobilizing HSCs at the dosage of 15 microg/kilog/day.

156 TYPE I METHEMOGLOBINEMIA ARISING DUE TO A CHANGE IN COFACTOR SPECIFICITY OF CYTOCHROME B5 REDUCTASE M. J. Percy1*, L. J. Crowley2, C. A. Davis2, M. F. McMullin3, G. Savage1, C. McMahon4, R. J. M. Quinn5, O. Smith4, M. J. Barber2, T. R. J. Lappin3 1Belfast City Hospital, Belfast, UK 2 Biochemistry and Molecular Biology, University of South Florida College of Medicine, Tampa, Florida, USA 3 Haematology, Queen's University, Belfast, Northern Ireland 4 National Centre for Hereditary Coagulation Disorders, St. James's Hospital, Dublin, Ireland 5 Paediatrics, Altnagelvin Hospital, Londonderry, Northern Ireland The soluble form of NADH-cytochrome b5 reductase (cytb5r, EC 1.6.2.2) is present exclusively in the cytoplasm of erythrocytes and catalyses the reduction of methemoglobin. This reaction utilizes cytochrome b5 and NADH co-factor as the source of H+ ions. Spontaneous and continual oxidation of hemoglobin gives rise to the non-functional met form but the accumulation of methemoglobin is prevented by the constant action of cytb5r. Deficiency of the soluble enzyme results in cyanosis or type I recessive congenital methemoglobinemia (RCM), but a combined deficiency of both soluble and membrane bound forms results in severe neurological impairment or type II RCM. Screening Irish type I RCM patients identified a novel mutation, D239G, present in the NADH-binding lobe of cytb5r. To date we have found in total three such novel mutations and a heterologous expression system was chosen to study the effect of the D239G, and the previously detected E255- and G291D mutations on cytb5r function. All variants were successfully expressed enabling their enzyme kinetic properties, protein stability and cofactor oxidation-reduction potential measurements to be compared. The D239G mutation was found to have no adverse impact on protein stability, unlike the E255- and G291D variants. In addition, it exhibited marginally reduced catalytic activity of 94%, while the E255- and G291D variants retained only 38% and 58% of wild type activity respectively. Measuring the affinity of the D239G variant for co-factors NADH and NADPH indicated that loss of aspartic acid at amino acid 239 increased selection towards the NADPH co-factor. Methemoglobin reduction requires the reduced form of cytochrome b5, which is produced when NADH is utilised as the H+ ion donor. Consequently, the D239G variant would be less efficient at converting methemoglobin to its functional less oxidized state thus causing the buildup of methemoglobin, resulting in type I RCM.

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34th Annual Meeting: ISEH - Abstracts / Experimental Hematology 33 (2005) 39-143

157 NATURAL REGRESSION OF PRIMITIVE HEMATOPOIESIS IN ZEBRAFISH EMBRYOS IS MEDIATED BY A DEATH RECEPTOR GENE T. T. Kwan1, R. Liang1, S. Lin2, S. C. Ekker3, C. M. Verfaillie3, A. Y. Leung1* 1 Department of Medicine, University of Hong Kong, Hong Kong 2 Department of Molecular, Cellular, and Developmental Biology, University of California Los Angeles, Los Angeles, USA 3 University of Minnesota, Minneapolis, USA In the present study, we examine the role of a zebrafish (Danio rerio) apoptotic gene, the death receptor (zDR), in the natural regression of primitive hematopoiesis during late embryonic development. Morpholinos (MO) against genes encoding for zDR and chordin (zChd), as well as a random sequence (RS) MO (control) were injected into wild-type (WT) embryos at 1-4 cell stage. Injected embryos were maintained at 280C until analyzed. At 24 hpf (hours post-fertilization), zDR mRNA expression is restricted to the ICM (intermediate cell mass, site of primitive hematopoiesis in zebrafish) of WT embryos and is upregulated in embryos injected with zChd-MO (CM) embryos. The latter is characterized by increased ventral mesoderm specification. WT embryos injected with zDR-MO (zDRmo embryos) demonstrated increased hemoglobin (Hb) staining (shown by Odianisidine) in the axial circulation and duct of Curvier (DC) at 48 hpf. In CM embryos, Hb staining is significantly increased in the ICM and DC, and is further enhanced in embryos co-injected with zDR-MO. This is confirmed quantitatively with a modified cyanomethemoglobin method. Injection of RS-MO into WT or CM embryos had no effects on Hb. There was little apoptosis (shown by TUNEL assay) in the ICM of WT and zDRmo embryos at both 24 and 48 hpfs. Apoptosis is increased in the CM embryos. At 24 hpf, it occurs predominantly on the surface of the embryos. At 48 hpf, it is abundant in the expanded ICM. Apoptotic signals are reduced in embryos co-injected with zDR-MO. In conclusion, knock-down of zDR in WT and CM embryos led to an increase in Hb content and in the latter, attenuation of apoptosis. These results suggested that zDR may mediate the natural regression of primitive hematopoiesis during zebrafish development by apoptosis. Whether similar mechanisms might operate in the mammalian system would have to be further studied.

158

Annexin V, the addition of IGF-II-neutralizing antibody increased apoptosis, thereby indicating the anti-apoptotic effects of IGF-II secreted from erythroid cells. Furthermore, ECFCs failed to undergo normal erythroid maturation in the presence of IGF-II-neutralizing antibody, as assessed by flowcytometric analysis and morphology. These findings indicate that IGF-II, which is produced by erythroid progenitor cells themselves, has crucial roles in controlling erythropoiesis by modulating apoptosis, proliferation and maturation via an autocrine mechanism.

159 ERYTHROPOIESIS IN VITRO IN NORM AND PATHOLOGICAL STATES T. Saralidze1*, M. Sheklashvili1, L. Mohchevishvili2, T. Shvelidze1, M. Bogvelishvili1, G. Saralidze3 1 Institute of Hematology and Transfusiology, Tbilisi, Georgia 2 Children's Hospital, Tbilisi, Georgia 3 Sport Department Dispanser, Tbilisi, Georgia Blood and bone marrow cell cultures were used for investigation of erythropoiesis and macrophage-lymphocyte rosettes (MLRos) formation in 20 donors and in 64 patients with different pathological states involving erythropoiesis: 30 patients with iron deficient anemia (IDA), 10 autoimmune hemolytic anemia (AIHA), 9 –refractory anemia (RA), 5aplastic anemia (AA), 7- chronic erythromyelosis (CE), 3- polycytemia vera (PV). In bone marrow cultures prolongation of erythropoiesis up to 3 days and maintenance of differentiation and maturation of erythrocaryocytes (oxyphillic normoblasts exceeds polychromatophilic and basophilic forms) is observed in IDA and AIHA, while in donors and in AA erythrocaryocytes are observed only on first day and rarely on second day of cultivation. In RA patients diserythropoiesis and prevalence of polychromatophilic and basophilic forms over oxyphillic normoblasts is observed on 1-2 days of cultivation. Notable that in 3 patients with RA proliferation of blast cells in vitro revealed impending acute leukemia. In CE patients ineffective erythropoiesis with abundance of basophilic and polychromatophillic normoblasts and figures of asymmetric mitosis of red blood are revealed up to 16 days of cultivation, whereas in cases of polycytemia vera effective erythropoiesis is maintained up to 12 days reflecting malignancy of disease. Study of MLRos amount in blood leukocyte cultures showed decreased immune reactivity of organism in IDA and AA patients and high sensitivity of immunocompetent cells in AIHA. Increase of these rosettes in some cases of RA revealed immune genesis of anemia and therefore necessity of immunosuppressive therapy. Our data shows that in vitro studies are useful for estimation of erythropoiesis in different states. Prolongation of erythropoiesis more than 4-5 days reveals increased proliferative activity of erythrocaryocytes and points to malignant nature of disease. Increase of MLRos amount in vitro shows sensibilisation of immunocompetent cells and reveals immune conflict in the pathogenesis of disease.

160 IN VITRO ERYTHROPOIESIS INDUCED FROM HUMAN CD34 POSITIVE CELLS I. Dorn*, J. Ribbat, S. Boie, H. Kirchner, P. Schlenke Institute of Immunology and Transfusion Medicine, University of Luebeck, Germany Purpose: The regulation of human erythropoiesis is only partly understood. Thus, an in vitro erythropoiesis model might be very useful to investigate, which factors and molecular mechanisms are involved in maturation of human erythroid precursor cells. Therefore, two different culture techniques were tested for optimal proliferation and differentiation of CD34 positive cells to reticulocytes. Methods: Purified CD34 positive cells from leukapheresis were cultured over 16d in a two-phase liquid assay (d1-9 EPO, SCF, IGF1; d10-16 EPO, Insulin) or over 21d in a three-phase assay (d1-7 SCF, Flt3-L, TPO, Insulin; d8-14 SCF, EPO, IGF1, Insulin; d15-21 EPO, Insulin). Cell growth and differentiation was evaluated by flow cytometry using fluorescent microparticles and antibodies against CD34, 36, 71 and glycophorin A (GPA). Cytospin preparations were made and stained by Pappenheim and neutrale benzidine. Results: The initial purity of CD34 positive cells was always greater 95 percent. During the two-phase model cell numbers increased up to 50fold. On d16 more than 95 percent expressed GPA and were strongly

Abstracts

INSULIN-LIKE GROWTH FACTOR II IS AN AUTOCRINE GROWTH FACTOR TO PREVENT APOPTOSIS AS WELL AS TO ENHANCE PROLIFERATION AND MATURATION OF ERYTHROID PROGENITOR CELLS IN CORD BLOOD T. Nagatomo1, K. Muta2, S. Ohga1, M. Ochiai1, S. Hikino1, T. Hara1* 1Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Japan 2Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Japan To reveal the novel function of erythroid progenitor cells in fetal erythropoiesis, the gene expression pattern in umbilical cord blood (CB) derived CD36+ erythroid progenitor cells (EPCs) was analyzed using cDNA microarray containing 240 cytokine and growth factor related genes. Among the genes analyzed, insulin-like growth factor II (IGF-II) gene showed a 124-fold higher expression in CB-EPCs, compared to that seen in phytohemagglutinin (PHA)-stimulated adult peripheral blood mononuclear cells (PBMCs). Real-time PCR revealed that the IGF-II mRNA levels in CB-EPCs were higher than those seen in blood cells such as PB-CD4+ cells, PB-CD8+ cells, CB-CD4+ cells, CB-CD8+ cells and CB-CD14+ cells. When CB-EPCs were cultured with erythropoietin (EPO) in serum-free medium, the number of erythroid colonies was decreased in the presence of IGF-II-neutralizing antibody. To assess the role of IGF-II in erythropoiesis further, we purified erythroid colony-forming cells (ECFCs) from human umbilical cord blood by magnetic selection and liquid culture with IL-3, stem cell factor and EPO. The expression levels of IGF-II, type 1 or 2 IGF receptor in the mature ECFCs were higher than those in the immature ECFCs. Addition of IGF-II-neutralizing antibody decreased the number of ECFCs in liquid culture with EPO. The antiproliferative effect of IGF-II-neutralizing antibody was evident in both dimethylthiazole tetrazolium bromide (MTT) and bromodeoxyuridine (BrdU) incorporation assays. When apoptosis of cells was examined using

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haemoglobin positive. Cells showed morphological characteristics of normoblasts and about 40 percent enucleated reticulocytes. In the threephase model, a greater proliferation (up to 260fold) could be observed. On d21 more than 90 percent were GPA positive, most of them normoblasts and 50 percent reticulocytes. Conclusions: Both models were able to show different stages of human erythropoiesis, including terminal enucleation. Although proliferation was greater in the three-phase model, no differences in maturation and enucleation could be observed. On the last culture days more than 90 percent of cells were GPA positive and consisted mainly of normoblasts and reticulocytes. By varying culture conditions or inhibiting signal pathways, both models might be a tool for further investigations in the field of erythropoiesis. Our present studies investigate the influence of the oxygen content on proliferation and differentiation of erythroid progenitors.

161 G PROTEIN-COUPLED RECEPTORS IN HYDROXYUREA ACTIVATION OF NITRIC OXIDE/CGMP PATHWAYS AND GAMMA-GLOBIN INDUCTION V. P. Cokic1*, A. P. Economou2, L. M. Wahl3, P. Milenkovic1, A. N. Schechter4 1 Institute for Medical Research, Belgrade, Serbia and Montenegro 2 University of Geneva, Faculty of Medicine, Geneva, Switzerland 3NIDCR, National Institutes of Health, Bethesda, USA 4 NIDDK, National Institutes of Health, Bethesda, USA We have shown previously that nitric oxide (NO) and the soluble guanylyl cyclase (sGC) pathways are involved in hydroxyurea-induced activation of fetal hemoglobin in human erythroid cells. It was already reported that hydroxyurea activates p38 MAP kinase and induces c-jun expression in human erythroleukemic cells. Since all of these pathways are also activated by G protein-coupled receptors (GPCR) in this study, we analyzed hydroxyurea (40 microM) activation of GPCR genes in human erythroid progenitor cells. We found increases in the beta 2 adrenergic receptor (3.8 fold) and dopamine receptors D1 and D2 (3 and 2.2 fold), in the NO / cGMP pathway (FLJ10210, 2.6 fold), the cAMP / PKA pathway (APM1, 3.1 fold) and the PI-3 kinase pathway (PKB alpha, 1.6 fold). Hydroxyurea also induced expression of several genes involved in the MAP kinase pathway: SSI-1 (2.7 fold); and NFkB pathway: IL-8 and helix-loop-helix zipper protein (3.7 and 4.3 fold). We have also found that hydroxyurea (30 microM) increases intracellular cGMP levels (60-90 fmol/ 1x105 cells, about 2 fold induction) as well as cAMP levels (1000-1800 fmol/1x105 cells, 2-5 fold induction) during 4 hours of incubation of human erythroid progenitor cells. Further, hydroxyurea (10-50 microM) induced NO production in human and mouse macrophages and that induction demonstrated NO synthase (NOS)-dependence. Co-culture studies of mouse macrophages and erythroid cells in the presence of hydroxyurea (10-50 microM) induced gamma-globin mRNA expression of erythroid progenitor cells, after 24 hours of incubation. Furthermore, hydroxyurea induced gamma-globin mRNA expression, with dose dependent effects, in co-culture studies of human macrophages and erythroid cells, with major effects after 48 hours of incubation. Macrophages, as part of stromal microenvironment, enriched gammaglobin induction during hydroxyurea treatment in comparison with independent erythroid cultures. Macrophages supported hydroxyurea induction of NO and consequently amplified gamma-globin induction via GPCR-dependent NO/cGMP pathway.

162 GENERATION OF FUNCTIONAL HEMOGLOBIN-SYNTHESIZING ERYTHROID CELLS FROM HUMAN EMBRYONIC STEM CELL F. Ma1,3*, D. Wang1, S. Hanada1, K. Ogami2, K. Umeda3, T. Hekei3, H. Sakai4, E. Tsuchida4, T. Nakahata3, K. Tsuji1 1 Division of Cellular Therapy, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Japan 2 Department of Transfusion, The Institute of Medical Science, The University of Tokyo, Japan 3 Department of Pediatrics, Kyoto University Graduate School of Medicine, Japan

4 Advanced Research Institute for Science and Engineering, Waseda University, Tokyo, Japan Human embryonic stem (HES) cell provides a unique tool to study early events occurring in the development of human tissues and specific cell lineage, such as hematopoiesis. Because of its titopotent property, the possibility of HES cells to produce supplementary tissues unfold an almost unlimited potential of its use in regenerative medicine. By using an in vitro co-culture system with murine fetal liver derived stromal cells, we successfully induced differentiation of HES cells (H1) into hematopoietic progenitor cells. These HES-derived progenitors were within cobble stonelike cells in the co-culture and highly enriched in a CD34+ fraction. In clonal culture with a cytokine combination (SCF, IL-3, IL-6, TPO, EPO and G-CSF), they gave rise to various types of hematopoietic colonies, including myeloid, erythroid and multi-lineage colonies. Immunochemical analysis showed that all of multi-lineage colonies examined contained erythroid cells possessing both alpha and beta globins (100 and 70%, respectively), indicating that they have already synthesized adult-type hemoglobin, which exhibited the oxygen affinity in vitro. Interestingly, in additive suspension culture for 1week, the erythroid cells in day-15 colonies not only further proliferated, but also matured, as they had 100% adult-type hemoglobin and 4 to 11% enucleated. We also determined the blood type of H1-derived red blood cells. Our study provides a possibility to use HES-derived red blood cells as a substitution of blood transfusion and should be benefit for future clinical use.

163 THE ROLE OF THE NOVEL GENE JUNE-1 IN ERYTHROID DEVELOPMENT AND IN EPO-SENSITIVE CELLS S. Rainey*, V. Smyth, Y. Zhengqiang, E. Dunlop, T. R. J. Lappin Dept Haematology, Centre for Cancer Research and Cell Biology, Queen's University Belfast, UK The hormone erythropoietin (EPO) leads to red blood cell development after binding to EPO receptors (EPO-Rs) on erythroid progenitors. However, little is known about the genes that are regulated by EPO. EPORs have now also been found on a variety of non-hematopoietic tissues, including some tumors; an important finding since rHuEPO is often used to treat anemia in cancer patients. It is therefore important to study the downstream effects of EPO in order to understand the role of this hormone in cellular proliferation, growth, and differentiation in both normal and disease processes. We have identified a novel gene that is upregulated in the presence of EPO, named JUNE-1. Cloning and sequencing reveal that this 5 exon, 1.2 kb transcript produces a protein of 44 kDa, which is highly conserved across species. The identification of a nuclear localization signal and PHD motif (zinc finger), suggest that JUNE-1 may be involved in chromatin remodelling or transcriptional regulation. RT-PCR studies show expression in many tissue types, including a variety of leukemia and lymphoma cell lines. Studies are underway to determine the function of JUNE-1, using normal and leukemic cells, including a murine erythroleukemia (MEL) cell system, useful for studying erythroid differentiation in vitro. Antibodies to JUNE-1 are in development, and will be used in protein expression studies across panels of mouse and human tissues, and in specific subcellular compartments. GST tagged JUNE-1 constructs are being expressed in bacterial systems, to identify JUNE-1 protein binding partners by affinity chromatography, co-immunoprecipitation, and mass spectrometry. Studies are also underway using RNAi to identify phenotypes associated with JUNE-1 knockdown. Since this gene may be a transcription factor, it will also be important to determine what genes are affected downstream of JUNE-1; changes in gene expression will be examined using gene chip microarrays.

164 ARSENIC TRIOXIDE INHIBITS TNF-ALPHA INDUCED SUPPRESSION OF ERYTHROID DIFFERENTIATION OF HUMAN CORD BLOOD CD34+ CELLS J. H. Won*, H. J. Cheong, S. J. Kim, S. B. Bae, C. K. Kim, N. S. Lee, K. T. Lee, S. K. Park, D. S. Hong, H. S. Park Soon Chun Hyang University College of Medicine, Seoul, Korea The anemia of chronic disease-which encompasses inflammation, infection, tissue injury, and conditions associated with the release of proinflammatory cytokines (such as cancer)- is one of the most common

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34th Annual Meeting: ISEH - Abstracts / Experimental Hematology 33 (2005) 39-143 forms of anemia seen clinically. Symptomatic anemia requires treatment. The two major forms of treatment are transfusions and erythropoietin. Arsenic trioxide used to treat human diseases for centuries in traditional Chinese medicine. Our recent studies suggest that low dose of arsenic trioxide induces erythroid differentiation of K562 human leukemic cells and high dose of arsenic trioxide induce apoptosis. In this study, we have investigated in vitro effect of arsenic trioxide on the erythroid differentiation and it could inhibit TNF-alpha induced suppression of erythroid differentiation of human cord blood CD34+ cells. Expression of glycophorin A was 35.94±7.94% after 7 days culture of human cord blood CD34+ cells and was decreased to 17.63±7.33% when culture of human cord blood CD34+ cells with 100ng/mL of TNF-alpha. Expression of glycophorin A was increased in dose dependent manner after 7 days treatment with arsenic trioxide and arsenic trioxide increased percentage of glycophorin A in culture with TNF-alpha compared to TNF-alpha alone. The results of colony assay of CFU-MIX and BFU-E after culture with various conditions revealed similar patterns with expression of glycophorin A. These results suggest that arsenic trioxide induces erythroid differentiation of human cord blood CD34+ cells and can reverse TNFalpha induced suppression of erythroid differentiation of human cord blood CD34+ cells.

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166 IL-7 ENGINEERED STROMAL CELLS EXERT A DOSE DEPENDENT EFFECT ON NAïVE T CELLS P. Sportoletti*, B. Del Papa, M. Di Ianni, M. De Ioanni, E. Bonifacio, V. Lanterna, L. Moretti, T. Zei, F. Falzetti, A. Tabilio Department of Clinical and Experimental Medicine, Hematology and Clinical Immunology Section, University of Perugia, Perugia, Italy Interleukin 7 (IL-7), a crucial cytokine in early T-cell development, is a potential therapeutic agent which could replenish depleted T cell numbers in several clinical conditions. To investigate the effects of different amounts of IL-7 on naïve T cell population we constructed an in vitro system using transduced mesenchymal cells to deliver IL-7 and compared their impact on naive T cells. Normal immunoselected naive T cells, were cultured with mesenchymal cells producing either 16 or 400 picog/ml IL-7. After 7 days'culture at the lower dosage CD45RO+ and CD45RA+ expression did not differ significantly from the starting fraction (12% vs 6.5% and 58% vs 60%

respectively). The apoptosis rate remained stably compared with culture of naïve T cells alone (14% vs 14.9%). Only 6.9% cells entered phase S. Similar expansion rates in the CD4+ and CD8+ subpopulations were observed (19% and 26% respectively). A low profile of proliferation was maintained as evaluated with CD 25 and CD71 expression (9.3% and 10% respectively). At the higher dosage CD45RO+ expression increased from the starting fraction of 6.5% to 62%, the number of CD45RA+ cells decreased from 60% to 50% and the apoptosis rate, as evaluated with annexin V, increased to 21%. Cell cycle progression was induced with 15% cells entering phase S. CD8 expression rose from the starting fraction 19.3% to 40%, the proliferation marker CD71 was upregulated fom 8.7% to 31.2% and the activation marker CD25 rose from 9.7% to 17.8%. We have demonstrated that IL-7 transduced stromal cells can modulate naïve T cells priming in a dose dependent way. Because of their different cytokine production, they might be the ideal vehicle to safely address lymphopoiesis in T cell deficient host.

167 SUCCESSFUL RETROVIRAL GENE TRANSFER TO HEMATOPOIETIC STEM CELLS HIGHLY EXPANDED BY FIBROBLAST GROWTH FACTOR -1 A. Crcareva1,2*, T. Saito1,3, A. Kunisato1,4, M. Sakata-Yanagimoto1, S. Chiba1 1Departments of Hematology/Oncology and Cell Therapy/Transplantation Medicine, University of Tokyo Graduate School of Medicine and Hospital, Japan 2Deptartment of Stem Cell Biology, University of Groningen, The Netherlands 3Transplantation Biology Research Center, BMT Section, Massachusetts General Hospital East, Boston, MA, USA 4Kirin Brewery Co. Ltd Pharmaceutical Research Laboratories, Japan The two major prerequisites for successful retrovirus infection are to enrich the hematopoietic stem cells (HSCs) and to have them enter the cell cycle. To this point, in mouse HSC transduction protocols, treatment of donor mice with 5-FU and/or cell sorting with subsequent cytokine stimulation during the infection period have been major elements. Regarding the starting cell population, freshly isolated bone marrow cells are thought to be the best target cells for retrovirus-mediated gene transfer because of their high stem cell potential. Here, we present a method for retrovirus transduction into mouse longterm reconstituting cells pre-cultured in vitro with fibroblast growth factor -1 (FGF-1) as a single cytokine. Whole bone marrow cells were cultured in the presence of FGF-1 and heparin for 3 weeks. Both non-adherent and attached cells were transferred into retronectin-coated dishes and infected with virus supernatant from MP34 cells stably transduced with pMY/GFP retrovirus. During the 3-day transduction period, SCF and TPO were added to the FGF1-stimulated cultures. The retrovirus-transduced cells were sorted and transplanted into lethally irradiated recipient mice with or without competitor cells. The experiments illustrated that the number of bone marrow-derived competitive repopulation units (CRUs) was increased from 600 to 9,200 per mouse after a 3-week culture period with FGF-1. Following retroviral transduction of the expanded cells, the absolute number of sorted retrovirus-transduced CRUs was 7,200. Using the retrovirus-transduced cells in non-competitive reconstitution assay, we achieved radiation protection and long-term bone marrow reconstitution in 100% recipients with average myeloid and lymphoid chimerisms of 70% and 50%, respectively. This protocol allows the transplantation of 150 recipients with cells derived from a single donor mouse. In conclusion, FGF-1-expanded bone marrow cells constitute excellent source of stem cells that could be used in a range of gene delivery protocols.

Abstracts

INFLUENCE OF ADHERENT CELLS ON CYTOKINE PRODUCTION BY NORMAL BONE MARROW ERITHROID CELLS V. V. Denisova*, I. A. Lisukov, A. D. Kulagin, I. V. Kruchkova, S. A. Sizikova, S. V. Sennikov, V. A. Kozlov Institute of Clinical Immunology, SB RAMS, Novosibirsk, Russia Background: As shown earlier, marrow erythroid nuclear cells produce of IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, IL-17, TNF-alpha, IFNgamma, G-CSF, GM-CSF, MCP-1, MIP-1beta. Adherent marrow cells fraction is consist of any immunoregulatory cells: monocyte/ macrophagues, mature lymphocytes, mesenchymal cells and etc. The aim: We investigate regulatory influence of adherent cells (AC) and their products on cytokine production by marrow Glycophorine A + ENC in healthy individuals. Methods: GlA+ ENC and AC were obtained from bone marrow biopsy specimens from 22 healthy individuals. Positive selection of GlA+ ENC from total MNC population performed with the help of specific antibodies to Gl A by panning procedure. Controls of purity were performed using the smears staining of hemoglobin-containing cells by benzidine. Adherent cells were collected by adhesion to plastic and cultivated for 24 hours for supernatant obtaining. ENC (1 million cell/ml) were cocultured in different ratio: 20, 30, 40% of AC presence or after 30% AC supernatant addition. After 24 hours cultivation the concentration of IL-6 and TNF-alpha in marrow ENCs culture media was measured by electrochemiluminescence method (ORIGEN, IGEN, USA). Result: After AC and ENC cocultivation a dose-depended inhibition (pw<0.01) and ACs supernatant-induced suppress of TNF-alpha and IL-6 production by ENC was revealed. Conclusion: We suggest, adherent cells fraction have ability to suppress cytokine production by erythroid marrow nuclear cells as cells-to-cells contact either by means of humoral factors.

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168 EFFICIENT TRANSDUCTION OF HUMAN CD34+ CELLS USING SAIMIRIINE HERPESVIRUS 2 G. M. Doody1, J. P. Leek1, A. K. Bali2, A. F. Markham1, E. A. de Wynter1* 1 University of Leeds, Leeds, UK 2 University Erlangen-Nurnberg, Erlangen, Germany Saimiriine herpesvirus 2 (HVS)-based gene therapy vectors may have potential clinical applications. In this study we used a cosmid-generated HVS vector incorporating enhanced green fluorescent protein (HVSEGFP) to transduce CD34+ cells isolated from cord blood. The extent of gene transfer into the committed progenitors was assessed in the CFUGEMM assay. Our results showed there was no significant difference in the colony numbers or colony morphology between transduced and mocktranduced cultures (p=0.85). However, when the colonies were examined under fluorescence microscopy, about 70% of the erythroid colonies from HVS-EGFP transduced cultures expressed EGFP (EGFP+). More than 90% of the cells from the colonies were Glycophorin A (Glyco A) positive with the majority expressing both EGFP and Glyco A. Next, we evaluated the effect of the virus on erythroid expansion in liquid cultures supplemented with SCF, EPO and TPO for 14 days. The transduced CD34+ cord blood cells gave rise to EGFP-expressing erythroid cells though the level of EGFP expression was lower than that observed in the clonogenic assays. The presence of the transgene did not alter the erythroid differentiation potential but there was a striking difference in the number of cells generated. At day 14, cell numbers in the transduced cultures had increased on average 520-fold compared to 220-fold in mock-transduced cultures suggesting the virus may influence the rate of proliferation. The morphology of the cells from both cultures was indistinguishable. Maintenance of the episome was confirmed by FISH and Gardella gel electrophoresis. In summary, this is the first demonstration that the HVSEGFP vector can infect primitive human hematopoietic progenitor cells and that expression of the transgene is maintained during proliferation and differentiation. This may have particular relevance to disorders affecting the erythroid lineage. Studies are ongoing to examine transduction efficiency in stem cell populations and other hematopoietic cell lineages.

169 EFFICIENT NON-VIRAL TRANSFECTION OF MOUSE AND HUMAN EMBRYONIC STEM CELLS Z. N. Berneman1*, V. F. I. Van Tendeloo1, D. R. Van Bockstaele1, P. G. Jorens2, P. B. Singh3, P. Ponsaerts1 1 Experimental Hematology, Antwerp University Hospital, Edegem, Belgium 2 Clinical Pharmacotherapy, Antwerp University Hospital, Edegem, Belgium 3 Research Center Borstel, Borstel, Germany BACKGROUND: Development of efficient non-viral gene transfer technologies for embryonic stem (ES) cells is urgently needed for various existing and new ES cell-based research strategies. In this study we investigated mRNA electroporation as a tool for short-term gene transfer in both mouse and human ES cells. METHODS: Culture and mRNA electroporation conditions for feederfree cultured mouse and human ES cells were optimised on three mouse ES cell lines (E14, R1 and HM-1) and one human ES cell line (H9). After electroporation with EGFP mRNA, transfected ES cell populations were analysed by FACS for EGFP expression, viability and phenotype. RESULTS: (A) Electroporation of EGFP mRNA in mouse ES cells resulted in high level transgene expression (>90% EGFP positive cells) combined with low electroporation-induced cell mortality (>90% viable cells). Moreover, the electroporation procedure did not have influence on ES cell phenotype and further cell culture of undifferentiated ES cell populations. (B) Although human ES cells are much more sensitive as compared to mouse ES cells, we were able to develop improved culture and electroporation conditions for feeder-free maintained H9 human ES cells, which resulted in high level transgene expression (>90% EGFP+ cells) combined with high cell viability (>90% viable cells) after EGFP mRNA electroporation. CONCLUSIONS: RNA electroporation is a highly efficient method for short-term genetic loading of both mouse and human ES cells. Ongoing research now focuses on either short-term (via direct mRNA

electroporation) or sustained (via mRNA-based FLPe-recombination) expression of transcription factors in ES cells and their influence on cellfate within in vitro cultured embryoid bodies.

170 MULTI-GENE TARGETING WITH ANTISENSE OLIGODEOXYNUCLEOTIDES: AN EXPLORATORY STUDY EMPLOYING PRIMARY HUMAN LEUKEMIA CELLS J. B. Opalinska1*, B. Machalinski1, J. Ratajczak2, M. Z. Ratajczak2, A. M. Gewirtz3 1 Pommeranian Medical University, Szczecin, Poland 2 University of Louisville, Louisville, Kentucky, USA 3 University of Pennsylvania School of Medicine, Philadelphia, PA, USA We previously reported that the Myb and Vav protooncogenes are amenable to silencing with antisense oligodeoxynucleotides (AS ODN) and that inhibition of either impairs leukemic cell growth. Since the expression of these genes is not known to be linked, we sought to determine the therapeutic value of silencing both genes simultaneously in K562, and primary patient (n=9) chronic myelogenous leukemia (CML), cells. K562, and primary CML cells were exposed to AS ODN, alone or in combination, for 24 or 72 hours and then cloned in methylcellulose cultures. Effects on K562 cluster, and BFU-E and CFU-GM colonies were determined, and correlated with the ability to downregulate the targeted mRNA. After 24 hours' exposure, K562 cell growth was inhibited in a sequence specific, dose responsive manner with either Myb or Vav AS ODN. Exposure to both ODN simultaneously considerably enhanced growth inhibition and accelerated apoptosis. Primary cell results were more complex. After 24 and 72 hour exposures to either anti-vav, or anti-myb AS ODN, equivalent colony forming unit (CFU) inhibition was observed. Exposing cells to both AS ODN simultaneously for 24 hours did not result in additional inhibition of colony formation. However, after 72 hours incubation with both ODN, colony formation was diminished significantly when compared to either ODN alone (from ~30% to ~78% for CFU-GM; ~50% to ~80% for BFU-E). We hypothesize that exposing primary leukemic cells to ASODN targeted to two, or possibly more, genes might significantly augment the therapeutic utility of these molecules.

171 EPIGENETIC REGULATION OF MOUSE CD19 GENE IN B CELL DEVELOPMENT H. Tagoh*, C. Bonifer Molecular Medicine Unit, University of Leeds, St James's University Hospital, Leeds LS9 7TF CD19 is a B cell co-receptor expressed throughout B cell development except plasma cells. A lack of CD19 expression in B cells causes a defect in their antibody responses as well as a decrease in the number of mature B cells. In contrast, over-expression of CD19 results in hyperactive B cells and loss of tolerance, suggesting the importance of properly regulated CD19 expression. Although aberrant expression of CD19 expression is found in some myeloid leukaemias, the cause of this lineage promiscuous expression is unknown. In order to address this question, we examined the chromatin structure of the mouse CD19 locus to understand the mechanisms that regulate tissue and stage restricted CD19 expression. A B cell specific transcription factor, Pax5, is necessary for CD19 expression and binds to the promoter. We found that chromatin at the mouse CD19 promoter is DNase I hypersensitive in a B cell specific and Pax5 dependent fashion. However, as seen with the human CD19 gene, the mouse CD19 promoter alone is not tissue-specifically active. We searched for other elements regulating CD19 expression and we have identified a DNase I hyper-sensitive site (DHS) at about 2kb upstream of the promoter (DHS-2kb). DHS-2kb showed B cell specific enhancer activity and harbours putative E2A/Tal-a, AML1, and ets-1 sites. We show by in vivo footprinting that these sequence motifs are occupied in B cells. Interestingly, DNaseI hyper-sensitivity and protein occupancy at DHS-2kb are found even in the absence of Pax5. In vitro induction of Pax5 induced a DHS at the promoter and transcription of CD19. These findings suggest that the DHS-2kb element has already been reorganised at the level of chromatin structure and Pax5 is required to activate CD19 expression at pro-B cell stage. We are investigating the order of events of CD19 gene activation occurring in B cell differentiation.

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172 GENE EXPRESSION PROFILING REVEALS FUNDAMENTAL DIFFERENCES BETWEEN LEUKEMIC STEM CELLS (LINCD34+CD38-) AND MATURE BLASTS (LIN-CD34+CD38+) OF ACUTE MYELOID LEUKAEMIA (AML) PATIENTS K. Zibara1,2*, D. Pearce1, D. Taussig1,2, E. Bureau1, S. Skoulakis2, S. Tomlinson3, B. Young1,2 1Cancer Research UK, LRI, Haematopoietic Stem Cell Laboratory, London, UK 2 Cancer Research UK, Medical Oncology Unit, St Bartholomew's Hospital, Medical College, London, UK 3 Cancer Research UK, LRI, Computational Genome Analysis Unit, London, UK The identification of LSC has important implications for future research as well as for the development of novel therapies. The phenotypic description of LSC now enables their purification and should facilitate the identification of genes that are preferentially expressed in these cells compared to normal HSC. However, gene-expression profiling is usually conducted on mononuclear cells of AML patients from either peripheral blood and/or bone marrow. The aim of this study was to compare the gene expression profile of highly purified LSC versus leukemic blasts in order to identify genes that might have important roles in driving the leukemia. For this purpose, we analyzed the gene expression profiles of highly purified LSCs (Lin-CD34+CD38-) and more mature blast cells (LinCD34+CD38+) isolated from 7 adult AML patients. Affymetrix microarrays (U133A chip), containing 22,283 genes, were used for the analysis. Comparison of Lin-CD34+CD38- cell population to the LinCD34+CD38+ cell fraction showed 5421 genes to be expressed in both fractions. Comparative analysis of gene-expression profiles showed statistically significant differential expression of 133 genes between the 2 cell populations. Gene ontology was used to determine the categories of the up-regulated transcripts. These transcripts, which are selectively expressed, include a number of known genes (e.g., receptors, signalling genes, proliferation and cell cycle genes and transcription factors). Among the genes showing the highest differences in expression levels were the following: ribonucleotide reductase M2 polypeptide, thymidylate synthetase, ZW10 interactor, cathepsin G, azurocidin 1, topoisomerase II, CDC20, nucleolar and spindle associated protein 1, Rac GTPase activating protein 1, leukocyte immunoglobulin-like receptor, proliferating cell nuclear antigen, myeloperoxidase, cyclin A1. Some transcripts detected have not been implicated in HSC functions, and others have unknown function so far. This work identifies new genes that might play a role in leukemogenesis and cancer stem cells. In addition to being a more efficient way to further understand the biology of LSC, this should also provide a more efficient way of identifying new therapeutics and diagnostic targets.

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were extracted from pre and post drug exposed cells, and the gene expression and promoter methylation status of 6 membrane bound drug efflux pumps were analysed using RT-PCR, combined bisulphite restriction analysis (COBRA) and methylation specific PCR. 5-aza-2'-deoxycytidine exposure, alone and in combination with valproic acid, was shown to induce chemo-resistance in the majority of leukaemia cell lines. Interestingly, in some cases, a synergistic effect on glucocorticoid resistance was demonstrated. Increased chemo-resistance correlated with increased gene expression and demethylation of one or more of the studied drug efflux pumps in all cases. These findings may have important implications for the scheduling of combination studies of 5aza-2'-deoxycytidine and valproic acid with conventional cytotoxic drugs.

174 GENE EXPRESSION PROFILING OF SINGLE HAEMATOPOIETIC PRECURSOR CELLS E. Sakhinia1*, A. Bashein1, A. M. Buckle2, R. Byers1, G. Brady3 1 The University of Manchester, Faculty of Medical and Human Sciences, School of Medicine Division of Laboratory and Regenerative Medicine, Manchester, UK 2The University of Manchester, Faculty of Life Sciences, Manchester, UK 3Epistem Ltd., Incubator Bldg., Grafton Street, Manchester, UK Expression profiling of haematopoietic cells is hampered by the heterogeneous nature of haematopoietic tissues and the absolute rarity of early-unrestricted progenitors. To overcome these restrictions we have developed a quantitative gene expression-profiling method, which we have used to compare expression of lymphoid and myeloid lineage markers in mouse bone marrow single haematopoietic precursors, fractionated BM and differentiating FDCP mix cells. Global amplification of cDNA was carried out in all samples, yielding representative PolyA cDNAs pools. Gene specific RT-PCR was then applied to cDNAs and the expression profile of the lymphoid and myeloid associated genes (LEF-1, Ikaros, Mb-1, EBF, CD19, Sox-4, B29, CD45, cfms, Lysozyme, PU.1 and CD5) measured by real-time PCR. The results demonstrate that both conventional PCR and TaqMan analysis can be applied to PolyA cDNA derived from single cells and the expression pattern for GAP, c-fms, lysozyme and CD45 determined by TaqMan analysis was in good agreement with previous Southern hybridisation studies. TaqMan analysis of 40 mouse myeloid hematopoietic precursors failed to detect any expression of LEF-1, EBF, CD19, Ikaros, and CD5 (despite efficient detection in lymphoid cells). Surprisingly, the lymphoid associated genes Sox-4 and B29 were detected with a peak of expression in granulocyte/macrophage precursors. Sox-4 and B29 expression was also found in multipotent FDCP mix cells with a pattern consistent with a peak of expression in granulocyte/macrophage precursors. Although there are no published accounts of Sox-4 expression in myeloid cells the B29 data is consistent with previous observations that B29 is expressed in early foetal bipotent B cell/macrophage progenitors and in a progranulocyte/ macrophage cell line. Our findings suggest a potential role for Sox-4 and/or B29 in early myelopoiesis and preliminary results indicate that single cell cluster analysis may provide a means of identifying gene hierarchies and characterising individual cells on the basis of their expression profiles.

175 CYTOKINE-ASSOCIATED GENE EXPRESSION IN DENDRITIC CELLS (DCS): COMPARISON BETWEEN ADULT PERIPHERAL BLOOD- DERIVED DCS AND UMBILICAL CORD BLOODDERIVED DCS BY CDNA MICROARRAY Y. Koga1, A. Matsuzaki1,2, A. Suminoe1, H. Hattori1, T. Hara1* 1 Department of Pediatrics, Graduate School of Medical Sciences,Kyushu University, Fukuoka, Japan 2 Division of Child Health, School of Health Sciences, Kyushu University, Fukuoka, Japan Background: The immaturity and dysregulation of neonatal immune system may cause an increased susceptibility to infectious morbidity and mortality in newborns. In this study, the gene expression in dendritic cells (DCs) derived from umbilical cord blood (UCB) and adult peripheral blood (APB) was comprehensively examined by using cDNA microarray in order to elucidate the difference in DC function between newborns and adults. Materials and Methods: Immature DCs were obtained from UCB and APB of healthy human donors as a mononuclear cell population with HLA-DR+, TCR-alpha/beta-, CD14-, CD56- and CD19-. Granulocyte-

Abstracts

5-AZA-2'-DEOXYCYTIDINE AND VALPROIC ACID INDUCE CHANGES IN MULTI-DRUG RESISTANCE PROFILES IN LEUKAEMIA CELL LINES H. M. Martin1*, L. J. Hale2, P. R. Kearns2 1 Dept. of Pathology and Microbiology, University of Bristol, UK 2 Dept. of Clinical Sciences, University of Bristol, UK Current evidence from pre-clinical studies and early clinical trials suggests global demethylating agents, (5-aza-2'-deoxycytidine) and histone deacetylase inhibitors (valproic acid), have promising anti-leukaemia activity. However, their inherent lack of specificity may result in induced expression of genes which alter the phenotypic behaviour of leukaemic blasts. For example, expression of the multi-drug resistance gene MDR1 is known to be under epigenetic control. Demethylation can induce expression of MDR1 causing resistance to anthracyclines and vinca alkaloids, both important therapeutic agents in the treatment of leukaemia. This study investigated the effect of 5-aza-2'-deoxycytidine and valproic acid on the expression and methylation status of a group of membrane bound drug efflux pumps in a panel of leukaemia cell lines, with particular emphasis on associated changes in chemosensitivity to conventional cytotoxic drugs. The 9 leukaemia cell lines were exposed to either 5-aza-2'deoxycytidine, valproic acid or both agents for a continuous period of 120 hours. Chemosensitivity to doxorubicin, vincristine, prednisolone and dexamethasone was measured pre- and post exposure to 5-aza-2'deoxycytidine and/or valproic acid using the SRB assay. RNA and DNA

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macrophage colony-stimulating factor, interleukin-3, and human TNFalpha were added to generate mature DCs. Total RNA was extracted, and gene expression was compared by using cDNA microarray containing 553 cytokine-associated genes between UCB- and APB-derived DCs. Results: In immature DCs, a total of 83 genes showed higher expression in UCB-derived DCs than in APB-derived DCs, which included calcium binding protein, ribonuclease RNase family, cell cycle related genes, adhesion molecule associated genes, chemokines, and TNF-related genes. Only one gene was expressed at a lower level in UCB-derived DCs. In mature DCs, on the other hand, seven genes, all of which were also detected in immature DCs, showed higher expression in UCB-derived DCs than in APB-derived DCs, while 48 genes exhibited decreased expression in UCB-derived DCs, which included chemokine genes associated with differentiation and IL-12 production of T cells. Conclusions: The expression levels of cytokine-associated genes were significantly different between UCB- and APB-derived DCs. This difference may result in a decreased reactivity of DC-mediated immunity in newborns.

176 TRANSCRIPTIONAL PROFILING OF MONOCYTE-DERIVED PROGENITOR CELLS L. H. Weng1*, R. Parwaresch1, M. Walter3, M. Bonin3, T. Thurau2, M. Kleine2, D. C. Cai4 1Institut für Hämatopathologie und Lymphknotenregister des Universitätsklinikums Schleswig-Holstein, Germany 2Planton GmbH, am Kiel-Kanal 44, D-24106 Kiel, Germany 3Medizinische Genetik, Universitästklinik Tübingen, Calwerstr.7, D72076 Tübingen, Germany 4Institut für Pflanzenbau und Züchtung Christian-Albrechts-Universität zu Kiel, Olshausenstr. 40, D-24118, Germany Human peripheral blood monocytes are able to be induced into novel pluripotent progenitor cells, having great potential in both reproducible and therapeutic application. For understanding the molecular mechanism underlying, transcriptional profiles of monocyte-derived progenitor cells were generated and analysed. Monocytes were cultivated in a differentiation medium containing various effectors. Six days after stimulation, differentiated cells (M6) were harvested and used for transcription profiling experiments (Affymetrix, GeneChip U133 plus 2) in which non-stimulated monocytes (M0) served as control. As a result, about 39% of whole human transcripts could be detected in both M6 and M0 samples. Compared to M0, 1795 transcripts were found to be differentially expressed in M6, from which 853 transcripts were up-regulated and 942 transcripts were down-regulated (p<0.05, change fold >2.5). The change folds of the transcripts varied from +241 to –219. Database analysis revealed that about 53% of differentially expressed transcripts are molecules with defined functions showing binding, catalytic and transcription-regulator activities. In contrast, approx. 35% of the differentially expressed genes remain functionally unknown and unclassified in now database. We found that most of the up-regulated genes are involved in pathways such as cell cycle, electron transport chain, complement-activation-classical and biosynthesis whereas the downregulated genes were mainly found in apoptosis, G-protein signalling, GPCRs and TGF-beta pathways. These results provide an insight into molecular characteristics of early differentiation of monocytes into the progenitor cells. Molecular and functional characterization of candidate genes are in progress.

177 DAILY EXPRESSION OF CLOCK GENE EXPRESSION RHYTHM WAS ABOLISHED IN PERIPHERAL BLOOD CELLS OF CHRONIC MYELOID LEUKEMIA S. F. Lin1*, M. Y. Yang1, T. C. Liu1, J. G. Chang2 1 Kaohsiung Medical University, Kaohsiung, Taiwan 2 China Medical University, Taichung, Taiwan Increasing amounts of data have indicated the physiological significance of circadium clock gene regulation in various peripheral cells. Circadium clock gene Per1,Per2,Per3 are expressed in a circadian manner in human peripheral mononuclear cells, with the peak level occurring during the habitual time of activity, has been reported. The present study investigated whether circadium clock gene function in PB mononuclear cells of chronic myelogenous leukemia.

We examined expression of the human homology of Per1, Per2, Per3, Cry1, Cry2, Clock, Ck1e, BMAL1 and Tim in PB mononuclear cells from 14 CML patients and 30 healthy control by real time quantitative reverse transcriptase polymerase chain reaction. The daily variation of clock gene expression was examined at 2am. 8am. 2pm. and 8pm. Significant daily variation in Per2, CK1e and BMAL1 gene expression were observed in healthy individuals, while CML patients exhibited no daily variation in these clock genes. The expression level also showed significantly lower than the healthy controls. The role of functional circadium machinery in CML patients needed for further investigation.

178 THE 'CYTOKINE STORM' AT THE ERA OF REDUCED INTENSITY CONDITIONING (RIC) ALLOGENEIC STEM CELL TRANSPLANTATION (ALLO-SCT) M. Mohty*, D. Blaise, C. Faucher, N. Vey, A. M. Stoppa, D. Coso, P. Viens, J. A. Gastaut, D. Olive, B. Gaugler Institut Paoli-Calmettes, Marseille, France In this report, we investigated the 'cytokine storm' in 113 patients who received a RIC regimen before allo-SCT from an HLA-identical sibling. 10 different cytokines (IL-1beta, IL-6, IL-8, IL-10, IL-12, IL-18, TNF-alpha, IFN-alpha, IFN-gamma, and FasL) were serially assessed in plasma samples collected from patients before allo-SCT, at day 0 and 1, 2 and 3 months after RIC allo-SCT. Patients' characteristics were as follow: median age of recipients was 49 (range, 18-63) years. 42 patients (37%) had a myeloid malignancy, whereas 45 patients (40%) had a lymphoid malignancy. The remaining 26 patients (23%) were treated for metastatic non-hematological malignancies. The majority of patients (n=90, 80%) had an advanced disease. In addition to fludarabine, the RIC regimen included busulfan and ATG in 80 patients (71%) and low dose irradiation in 33 (29%) patients. In this cohort, the overall cumulative incidence of grade 2-4 aGVHD was 45% (95%CI, 36-54%). In a univariate analysis, advanced disease stage (P=0.04) and a high IL-12 level (P<10e-4) measured around the first month after RIC allo-SCT were significantly associated with the development of severe grade 2-4 aGVHD. Of note, to the contrary of myeloablative allo-SCT, TNF-alpha levels did not show a significant correlation with the risk of aGVHD. IL-12 levels were significantly correlated (r=0.69) to the severity of aGVHD: grade 0-1, median 468 pg/mL; grade 2, median 2538 pg/mL; grade 3-4, median 4615 pg/mL (P<10e-4). Interestingly, IL-12 levels significantly decreased after appropriate aGVHD treatment. In multivariate analysis, IL-12 level measured around the first month after RIC allo-SCT was the strongest predictive factor for aGVHD development and severity (P<10e-4; RR=10.8). In all, results from this analysis suggest that monitoring of IL12, a key cytokine in the induction of Th1 and cytotoxic immune responses, appears to be a useful indicator of aGVHD after RIC allo-SCT, and anti-IL-12-based therapy might represent a potential tool for controlling alloreactivity.

179 HALOFUGINONE INHIBITS T CELL ACTIVATION: POSSIBLE IMPLICATIONS FOR GRAFT VERSUS HOST DISEASE (GVHD) M. Leiba1*, L. Cahalon2, A. Shimoni1, M. Pines3, O. Lider2, A. ZaninZhorov2, I. Hecht2, U. Sela2, A. Nagler1 1Hematology and BMT, Chaim Sheba Medical Center, Tel Hashomer, Israel 2 Immunology Dept., Weizmann Institute of Science, Rehovot, Israel 3 Institute of Animal Science, Bet Dagan, Israel Introduction: Halofuginone, a low molecular weight plant alkaloid, was shown to inhibit collagen alpha1(I) gene expression in several animal models and patients with fibrotic diseases including scleroderma and GVHD (Nagler et.al, Transplantation, 1999, BBMT 2004). In addition, studies now show that halofuginone inhibits transforming growth factor beta, a potent fibrogenic factor, but also a novel immunomodulator. The aim, of the present study was to investigate halofuginone effects on activated T cells, to better understand the mechanism of action, and consequently the therapeutic potential in GVHD. Methods: Peripheral blood T cells were activated by anti-CD3 McAbs in the presence or absence of halofuginone (5-40 ng\ml) and assessed for: NFkappaB activity, cytokine production, including tumor necrosis factor alpha and interferon gamma, T cell apoptosis, chemotaxis and phosphorylation of P38 Mitogen Activated Protein Kinase (MAPK).

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34th Annual Meeting: ISEH - Abstracts / Experimental Hematology 33 (2005) 39-143 Results: Pre-incubation of the peripheral blood activated T cells with halofuginone resulted in a significantly decrease level of NFkappaB activity, in a dose dependent manner (NFkappaB activity index was reduced by 75%, p=0.005). Additionally, halofuginone inhibited the secretion of the proinflammatory cytokines: tumor necrosis factor alpha and interferon gamma (55% reduction, p=0.005). Similarly, halofuginone was able to inhibit apoptosis in the activated T cells (50% reduction, p=0.005). Finally, halofuginone also significantly inhibited phosphorylation of P38 MAPK in activated T cells (phosphorylation was reduced by 60%, p=0.0001). In contrast, T cell chemotaxis was not affected by halofuginone. Conclusion: Halofuginone inhibits activated peripheral blood T cell functions, and production of proinflammatory cytokines through NFkappaB activation, and P38 MAPK phosphorylation, making it an attractive immunomodulator and an anti-inflammatory agent. Our findings thus suggest that halofuginone may have a therapeutic role in GVHD, and further improve our understanding of T cell signaling pathways in normal and pathological conditions.

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181 THE ENHANCING EFFECT OF IMMUNOSUPPRESSIVE AGENT ON THE CYTOLYTIC ACTIVITY OF INHIBITORY NK CELL RECEPTOR (CD94/NKG2A)-EXPRESSING CD8 T CELLS J. Tanaka1*, N. Iwao1, T. Toubai1, N. Kato1, Y. Miura1, S. Ota2, M. Asaka2, M. Imamura1 1 Hematology and Oncolog, Hokkaido University Graduate School of Medicine, Japan 23rd Department of Internal Medicine, Hokkaido University Graduate School of Medicine, Japan The C-type lectin superfamily inhibitory NK cell receptor (NKR) CD94/ NKG2A-expressing cells can monitor the global status of HLA class I on tumor and leukemic cells through the recognition of HLA-E and induce cytolytic attack without an inhibitory signal against HLA class I-decreased target cells. Tacrolimus (FK506) is a potent immunosuppressive agent that

inhibits transcription of cytokines such as IL-2 in T cells. We found that there was no effect of FK506 (0.3 ng/mL) on the expansion of CD94/ NKG2A-expressing T cells from granulocyte colony-stimulating factor (GCSF)-mobilized peripheral blood mononuclear cells (G-PBMCs) by the stimulation using immobilized OKT3 and IL-15 for 7 days. There was a slight decrease in the absolute numbers of CD94/NKG2A-expressing T cells in G-PBMCs culture with FK506 compared with those in culture without FK506. However, the proportions of activating NK cell receptor NKG2D-expressing T cells and intracellular granzyme A expression in CD94/NKG2A-expressing cells were much more increased in G-PBMCs cultured with FK506. The cytolytic activity levels of purified CD94expressing cells from 7-day cultures with FK506 tested against 51Crlabeled K562 cells by standard 4-hour 51 Cr release assays without prior sensitization were much higher than those from 7-day cultures without FK506 (56.1±26.1 and 29.2±19.0, p<0.05, five separate experiments, effector target ratio, 10:1). These data suggested that FK506 did not inhibit cytolytic activities of inhibitory NKRexpressing T cells and that there was a possibility of cytolytic activities being enhanced through the induction of cytotoxic molecules such as NKG2D and granzyme. Therefore, FK506 can strongly inhibit antigenspecific T cell immune functions but might spare antitumor activity mediated by NK cells and also inhibitory NKR-expressing T cells. The effect of FK506 on T cells and inhibitory NKR-expressing T cells might have some roles on the regulation of delicate balance between GVHD and GVL.

182 CHARACTERISTICS OF NK SUBSETS AND INHIBITORY C-TYPE LECTINS IN AML PATIENTS AND THEIR HLA-MATCHED ALLOGENEIC DONORS H. J. Kim*, B. H. Park, Y. Choi, S. Y. Kim, Y. J. Kim, C. K. Min, D. W. Kim, J. W. Lee, W. S. Min, C. C. Kim Catholic Hemopoietic Stem Cell Transplantation Center, St Mary's Hospital, Seoul, South Korea Objective: The ability to distinguish harmful target cells or infectious microbes by NK cells from healthy individual is definitely dependent on the level of both inhibitory and activating receptors. The C-type lectins CD94/NKG2A is considered that it perform a surveillance function for the absence of self-identifying classical class I MHC or even HLA-E molecules. However, we have not noticed any report to direct a guideline in the clinic, specifically for the treatment of AML patients based on this clue. Materials and Methods: Blood samples were obtained from adult patients(n=31) with AML and their HLA-matched sibling donors(n=31) just before the conditioning regimen, and on the day of transplantation, respectively. NK cells were purified using a negative magnetic sorting kit (Miltenyi Biotech). The lymphocytes were gated and were analysed by a multicolor flow cytometry. The cells were identified using the antiCD159a Ab, anti-CD16/56 Ab, together with the anti-CD3 Ab. Finally we recruited the whole clinical data for the patients enrolled in this study. Results: The expression levels of NKG2A in the normal donor population were higher than those of AML patients who were either in complete remission or not. Although age was not a major factor, male sex might be another factor to determine the expressions of NKG2A according to this analysis. Interestingly, patients who showed poor response to induction chemotherapy revealed a tendency of lower expressions of NKG2A (P<0.05). Further, high levels of donor CD16/56+CD3+ cells showed more favourable outcomes. However, expression levels of donor NKG2A in association with the disease-free survival were not statistically significant in a multivariate regression analysis (P=0.09). Conclusion: Based on this study, we could suggest a certain role of specific inhibitory receptor NKG2A against AML even in the allogeneic sibling transplantation setting.

Abstracts

PERSISTENT MIXED CHIMERISM IN PLASMA CELLS FOLLOWING ALLOGENEIC STEM-CELL TRANSPLANTATION (ALLOSCT) IN PATIENTS WITH ACUTE LEUKEMIA IS A SURROGATE MARKER FOR DISEASE RELAPSE A. Shimoni*, L. Trachenbrot, G. Ishoev, I. Hardan, N. Shem-Tov, A. Nagler Chaim Sheba Medical Center, Tel-Hashomer, Israel Chimerism within cellular subsets following alloSCT has been extensively studied, however, there is only limited data on chimerism kinetics within the plasma cell population (PC) and its prognostic significance. We studied 45 patients with acute leukemia, at serial timepoints, following myeloablative (51%) or reduced-intensity conditioning (49%) and peripheral-blood SCT from sex-mismatched donors. BM smears were evaluated by Duet combined cytogenetic/morphologic analysis system. This system scans BM smears, saves cell coordinates, the stain is than removed, and FISH for X and Y markers is applied to the same slide. Recipient cells are identified by their gender and their morphology recovered. 30/45 patients (67%) had recipient PC detected during the first 3 months after SCT, constituting 0.01-1.6% of BM cells. This was often associated with low-level recipient chimerism (<1%) among lymphocytes. In 12 patients recipient PC persisted beyond 6 months (up to >18 months), in 10 they disappeared (8 died earlier or have insufficient follow-up). BM tests, beyond 6 months, were available in 24 patients; 12 with recipient PC, and 12 without. There was no difference between the two groups in age, gender, donor and conditioning type, prior GVHD or disease status. Persistence of recipient PC was not associated with mixed-chimerism in lymphoid or other lineages at this stage. However, outcome after alloSCT was significantly different. Among the 12 patients with recipient PC, 8 relapsed, while among the 12 patients with no recipient PC, only 1 relapsed, and one died of infection. 2-year disease-free survival was 83%, and 27 %, respectively (p=0.05). In conclusion, recipient PC may persist for long duration after alloSCT and are relatively resistant to conditioning and allogeneic responses. Moreover, persistence of host PC beyond 6 months is a surrogate marker for ineffective GVL, even in patients with GVHD, therefore associated with increased risk for disease relapse.

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183 DENDRITIC CELL-DIRECTED CYTOKINES INDUCED A GVL EFFECT IN PATIENTS WITH ACUTE LEUKEMIA WHO RELAPSED AFTER ALLOGENEIC TRANSPLANT E. K. Waller1,2, M. Arellano1,2* 1 Winship Cancer Institute, Atlanta, GA, USA 2 Emory University Hospital, Atlanta, GA, USA Background: Relapsed acute leukemia following allogeneic hematopoietic progenitor cell transplantation (HPCT) has a dismal prognosis. An attractive approach would be to 'break tolerance', inducing a graft versus leukemia effect (GvL). GM-CSF and Interferon-alpha (IFN) induce a dendritic cell-like differentiation of leukemia cells, and activate allo-reactive T-cells in vitro. Methods: A retrospective study of 105 patients with AML or ALL who relapsed after allogeneic HPCT. Overall survival was compared among patients treated with cytokines, second transplant, DLI, and supportive care. Results: Median post-relapse survival for all patients was 61 days. Palliative chemotherapy or supportive care (N=75) resulted in a median survival of 50 days. Donor lymphocytes (N=13) resulted in a median survival of 158 days. 12 patients underwent a second allogeneic HPCT with a median survival of 419 days, but no long-term survivors. 5 patients received cytokine immunotherapy with GM-CSF and/or interferon-alpha with a median survival of 427+ days with 2/5 patients remaining alive at a median follow-up of >2 years. Median doses of 500 ug GM-CSF were given 3X/week (median 8 doses) and 3 million units IFN 1-3 times per week (median of 5 doses). 4/5 patients achieved a pathologic or hematologic remission with an average response of 3.3 mos. (6 wks. to >2 years). Cytokine toxicities including malaise, myalgias, rash, and fever were not dose-limiting. GVHD occurred in 4/5 patients, at a median of 6 weeks. One patient remains without evidence of disease on no immunosuppressive drugs. Cytokine-treated patients had better survival compared to 100 non-cytokine-treated patients with relapsed ALL or AML (median survival of 54 days, one-year survival of 8%, p=0.02). Conclusions: Administration of GM-CSF and IFN to patients with relapsed ALL/AML may induce allo-reactive T-cells with GvL activity. Toxicities are acceptable and long-term survival is not worse than other therapies, warranting further study.

184 SUCCESSFUL TREATMENT WITH AN INDUCTION OF GVHD IN RELAPSED ATL AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION T. Kamimura1*, T. Miyamoto2, N. Kawano3, H. Henzan1, S. Hayashi1 1 Division of Hematology, Harasanshin Hospital, Fukuoka, Japan 2Center for Cellular and Molecular Medicine, Kyushu University Hospital, Japan 3Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Japan Adult T cell leukemia/lymphoma (ATL) is known to be one of the most aggressive hematological malignancies, however, successful allogeneic hematopoietic stem cell transplantation (allo-HSCT) have been reported. We describe two cases of relapsed ATL after allo-HSCT, who had been successfully treated with an induction of graft-versus host disease (GVHD). A 52-year-old woman, who had been diagnosed as acute ATL with multiple subcutaneous tumors, received allogeneic peripheral blood stem cell transplantation from HLA-identical sibling donor. Although a complete remission (CR) was achieved early after transplantation, subcutaneous ATL relapse developed on day 92. She had received donor lymphocyte infusion (DLI) on day 115 and achieved CR with development of chronic GVHD, and has remained CR for 36 months after DLI. The other, a 56-year-old man with acute ATL, received allogeneic bone marrow transplantation from HLA-identical unrelated donor. On day 52, ATL was relapsed with multiple nodular skin infiltration when acute GVHD was not evident. Skin biopsies performed on day 54 showed a prominent infiltration of ATL cells and the small-sized lymphocytes surrounding ATL cells in the dermis. Subcutaneous tumors disappeared along withdrawal of the immunosuppressant, while acute GVHD developed on day 75. Immunohistochemistry of the skin lesions on day 77 presented a relative decrease of ATL cells but a dominant increase of the cytotoxic T-cells, which were positive for CD8 and T-cell-restricted intracellular antigen (TIA-1), around ATL cells. Our cases suggest the

presence of a possible graft-versus-ATL effect in the setting of allo-HSCT, and an induction of GVHD may be a potentially curative treatment for patients with ATL.

185 PERICARDIUM INVOLVEMENT IN ACUTE GRAFT-VERSUS-HOST DISEASE FOLLOWING ALLOGENEIC STEM CELL TRANSPLANTATION Y. Kikushige*, K. Nagafuji, K. Takase, A. Numata, T. Miyamoto, M. Harada Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Japan Several factors are responsible for cardiac complications after allogeneic stem cell transplantation (allo-SCT). These factors include cardiotoxic effects such as chemotherapy, radiation, immunosuppressive drugs, and infectious events. We describe two cases with pericardial effusion as a manifestation of acute GVHD after allo-SCT. One male patient with adult T-cell leukemia/lymphoma (ATL) underwent allo-BMT from the unrelated donor mismatched at one HLA antigen after the myeloablative conditioning (busulfan and cyclophosphamide) followed by GVHD prophylaxis with tacrolimus and methotrexate. On day 13, he complained of orthopnea, and echocardiography showed marked pericardial effusion. Treatment such as diuretics, vasodilator and cathecolamine was given with a minimal improvement. Pericardial fluid was exudative and contained a majority of donor-derived CD8+HLA-DR+ T-cells. Examinations of cardiac fluid revealed negativity for viral, bacterial, fungal infections, and relapse of ATL. Subsequently, he developed acute GVHD (skin), and an additional immunosuppressant, prednisolone (PSL) started for treatment of GVHD. Along with regression of skin GVHD, cardiac effusion gradually decreased. The other with AML, received cord blood transplantation mismatched at 2 HLA antigens, following the reduced-intensity regimen (fludarabine, busulfan, TBI). GVHD prophylaxis included cyclosporine (CSP) only. On day 5, the patient developed a chest pain and echocardiography showed massive pericardial effusion. Cardiac fluid was sterile and contained the donor-derived T cells. On day 9, the level of bilirubin was increased up to 5.9 mg/dl, and a diagnosis of GVHD (liver) was made on. The patient was treated with PSL in addition to CSP. The level of bilirubin gradually decreased in parallel with the improvement of pericardial effusion. These observations indicate that pericardium may be considered as one of potential target organs of acute GVHD. Thus, for the patients with serosal effusions early after allo-SCT, it will be important to make a correct diagnosis and treatment since pericardial effusion associated with GVHD would be fatal.

186 HEDGEHOG PATHWAY ELEMENTS IN HEMATOPOIETIC DIFFERENTIATION K. Detmer1*, A. N. Walker1, R. K. Humphries2 1 Mercer University School of Medicine, Macon, GA, USA 2 Terry Fox Laboratories, Vancouver, BC, Canada Hedgehog (Hh) signaling has been shown to be a potent regulator of cell fate determination, proliferation and differentiation in non-hematopoietic systems. Expression of Hh pathway genes is found in hematopoietic stem and progenitor cells but their roles are largely unknown. Hh signal transduction acts on the Gli family of transcription factors abolishing transcription repression by Gli3 and inducing transcription activation by Gli1 and Gli2. To examine the role of the Hh pathway in hematopoietic homeostasis, deregulation of the pathway was achieved through murine retroviral transduction and bone marrow transplantation using the GLI1 gene or the gene for a constitutively active Hh-receptor mutant, SmoM2. Dramatic perturbations in hematopoiesis were evident upon examination of bone marrow cells where there was an inversion of the erythroid/ myeloid ratio. At 16 weeks post-transplant, GLI1-transduced cells were 60% Gr1/Mac1 positive versus 30% for controls and 35% ter119 positive versus 58% for controls (p<0.05). By contrast, SmoM2 increased the proportion of ter119 positive cells. Wright-stained cytospin preparations of marrow showed morphology consistent with the changes in the erythroid/ myeloid ratio. The erythroid/myeloid distribution of untransduced cells present was altered suggesting that Hh signaling affects hematopoietic differentiation through cell autonomous and non-autonomous mechanisms.

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34th Annual Meeting: ISEH - Abstracts / Experimental Hematology 33 (2005) 39-143 While peripheral blood counts were in general normal suggesting homeostatic regulatory mechanisms could overcome the bone marrow effect, reconstitution of the T lineage was delayed. At 3-4 weeks posttransplant, the population of CD4+ and CD8+ lymphocytes was 64% and 41% (p<0.05) of control, respectively, in SmoM2-expressing mice but not GLI1-expressing mice. These results were consistent with a model of Tcell ontogeny in which down regulation of Hh signaling promotes T-cell maturation, and together with the bone marrow study, indicate that the effects of Hh signaling in the hematopoietic system are mediated by the actions of different members of the Gli protein family acting in concert.

187 ACTIVATION OF SPHINGOSINE KINASE MEDIATES SUPPRESSIVE EFFECT OF INTERLEUKIN-6 ON HUMAN MULTIPLE MYELOMA CELL APOPTOSIS Q. F. Li, L. S. Wang, H. F. Duan, C. T. Wu* Department of Experimental Hematology, Beijing Institute of Radiation Medicine, Beijing, P.R.China Interleukin-6 (IL-6) is a major growth and survival factor for multiple myeloma (MM) cells via activation of multiple signaling cascades. Sphingosine kinase (SPK), the enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P), has been identified as a signal molecule and plays important roles in the regulation of cell proliferation and apoptosis. However, whether SPK activation is involved in the IL-6 signal of multiple myeloma cells and what are its roles in the pathologenesis of MM remain unclear. In this report, we show that IL-6 activates SPK in MM cells and SPK activation mediates suppressive effects of IL-6 on MM cell apoptosis. Both MM cell lines and primary MM cells constitutively express SPK. Treatment of MM cells with IL-6 resulted in activation of SPK in a concentration-dependent manner. Either Ly 294002 or PD98059, specific inhibitors for the PI3K and ERK/MAPK pathways, respectively, blocked the IL-6-induced activation of SPK. We further demonstrated that IL-6-induced activation of SPK inhibited Dexinduced apoptosis of multiple myeloma cells. IL-6 stimulation or retroviral mediated overexpression of SPK gene in MM cells resulted in increased intracellular SPK activity and up regulation of mcl-1, leading to increased cell proliferation and survival. Conversely, inhibition of SPK by isRNA reduces IL-6-induced up regulation of mcl-1 and blocked the suppressive effect of IL-6. Taken together, these results delineate a key role for SPK activation in IL-6-induced proliferation and survival of multiple myeloma cells. And SPK is identified as a potentially new therapeutic target in multiple myeloma.

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In contrast, Sod2+/+ cells required adipocytogenic medium; WR2721 decreased their adipocytogenesis (p=0.001). The antioxidant pool size in Sod2-/- cells under basal conditions was significantly lower then in Sod2+/ + cells, as evidenced by measurements of GSH (78.6%; p=0.0089) and GSX (52.7%; p<0.001). Treatment with WR2721 significantly increased both GSH and GPX levels in Sod2-/- and +/+ cells cultured in basal and adipocytogenic media (all p<0.001). The differentiation of adipocytes in Sod2-/-cells was inversely correlated with the level of GSH (Spearman r=0.9429, p=0.0167). In conclusion, restoring antioxidant pool size with WR2721 reduced constitutive adipocytogenesis and inhibited induced adipocytogenesis. These data provide evidence for the involvement of the cellular redox pathway in adipocyte differentiation of bone marrow stromal cells.

189 SIGNAL TRANSDUCTION CHANGES INDUCED BY CONSTITUTIVE MUTANTS OF THE GRANULOCYTE MACROPHAGE COLONY-STIMULATING FACTOR RECEPTOR: UNDERSTANDING THE LEUKEMOGENIC POTENTIAL OF MUTANTS OF THE COMMON BETA SUBUNIT M. Perugini1,2*, A. Brown1, T. J. Gonda3, R. J. D'Andrea1,2,4 1Child Health Research Institute, Adelaide, Australia 2Department of Paediatrics, University of Adelaide, Australia 3 Centre for Immunology and Cancer Research, Brisbane, Australia 4 Queen Elizabeth Hospital, Adelaide, Australia Granulocyte macrophage colony-stimulating factor (GM-CSF), Interleukin-3 (IL-3) and Interleukin-5 (IL-5) have overlapping and pleiotropic effects on haemopoietic cells, including mitogenesis, differentiation and functional activation. The receptors for these cytokines are comprised of a common beta subunit and a ligand-specific alpha subunit. Constitutively activated mutants of the common beta subunit that are able to confer factor-independent growth and differentiation in a number of haemopoietic cell lines have been identified by our laboratory. In vivo, a transmembrane mutation can induce acute myeloid leukaemia whereas extracellular mutations can induce myeloproliferative disorders. To establish the nature of signalling from these mutants, a number of signal transduction pathways have been examined in an immature myeloid cell line, FDB1, which can switch between proliferation (in IL-3) and differentiation (in GM-CSF). We have uncovered a JAK2/STAT5independent signal sufficient for granulocyte/macrophage differentiation and generated by the extracellular FIdelta mutation. As the activity of this mutant is dependent on the GM-CSF receptor alpha subunit, this JAKindependent signal may rely on interactions of other tyrosine kinases with the GM-CSF receptor alpha subunit. A putative SH3 domain binding motif, SBP, in the cytoplasmic portion of the GM-CSF receptor alpha subunit is essential for receptor signalling and may serve as a docking site for SH3 domain-containing molecules such as Src Family Kinase (SFK) members. To date we and others have identified numerous interactions of signalling molecules with the GM-CSF receptor alpha subunit and we are now using a number of approaches to identify which of these require the SH3 domain binding site and are required for the FIdelta signalling.

190 HYDROXYUREA INDUCES ENOS ACTIVITY AND PROTEIN LEVELS IN ENDOTHELIAL CELLS V. P. Cokic1*, B. B. Beleslin-Cokic2, C. T. Noguchi3, A. N. Schechter3 1 Laboratory of Experimental Hematology, Institute for Medical Research, Belgrade, Serbia and Montenegro 2 Institute of Endocrinology, Diabetes and Metabolism, School of Medicine, University Clinical Center, Belgrade, Serbia and Montenegro 3 Molecular Medicine Branch, NIDDK, National Institutes of Health, Bethesda, Maryland, USA Hydroxyurea is a cell cycle-specific drug, which has been used to treat myeloproliferative diseases and sickle cell anemia. We have recently shown that hydroxyurea, like nitric oxide (NO)-donor compounds, increased cGMP levels in human erythroid cells. We show now that hydroxyurea increases NO production in primary human umbilical vein endothelial cells (HUVEC) and a human bone marrow endothelial cell line. Hydroxyurea induction of NO is blocked by competitive inhibitors of NO synthase (NOS) such as NG-nitro-L-arginine-methyl ester (L-NAME) and N-nitro-L-arginine. In addition, hydroxyurea-induced cGMP levels also

Abstracts

REGULATION OF ADIPOCYTE DIFFERENTIATION IN MOUSE MARROW STROMAL CELLS BY LEVEL OF ANTI-OXIDANT RESERVES J. Glowacki1*, S. Lechpammer1, M. W. Epperly2, S. Zhou1, S. Nie2, J. S. Greenberger2 1Brigham and Women's Hospital, Boston, MA, USA 2University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA Mice deficient in manganese-superoxide dismutase (Sod2-/-) die after birth with a massive accumulation of lipid in liver and skeletal muscle. Recently, we reported that long-term bone marrow cultures established from neonatal Smad3-/- mice showed 16.5-fold more adipocytes and prolonged hematopoiesis, compared with Smad3+/+. Here we investigated constitutive adipocytogenesis in bone marrow stromal cells from Sod2-/and Smad3-/- mice, and tested the hypothesis that adipocytogenesis is linked with antioxidant pool sizes. Bone marrow stromal cells from Sod2-/-, Smad3-/-, and wildtype mice were cultured±SD adipocytogenic supplements (10 microg/ml insulin, 1 microM dexamethasone, 100 mM indomethacin). Adipocytogenesis was assessed by enumeration of cells stained with 3% oil red-O dye and by RTPCR for peroxisome proliferator-activated receptor-gamma (PPARgamma) and lipoprotein lipase (LPL). Antioxidant pool size was established by measurement of glutathione levels (GSH) and glutathione peroxidase activity (GPX). In basal medium, Sod2-/- demonstrated constitutive adipocytogenesis, and exhibited increased lipid production in adipocytogenic medium. Treatment with 4 mM amifostine (WR2721), significantly decreased lipid accumulation by Sod2-/- cells in basal (p=0.037) and adipocytogenic (p=0.021) media, with undetectable expression of PPARgamma and LPL.

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demonstrated NOS dependence in HUVEC. The hydroxyurea-induced rise in intracellular calcium is followed by an increase in cAMP. We find that hydroxyurea dose- and time-dependently induced rapid and transient phosphorylation of eNOS at Ser-1177 in a preferentially cAMP-dependent protein kinase (PKA)-dependent manner, whereas NO production is regulated by the mechanisms dependent on both PKA and Akt in HUVEC. We observe that hydroxyurea as well as specific and non-specific inhibitors of the proteasome increase eNOS protein levels in HUVEC, after prolonged treatment. Analogously, NO production in HUVEC and human bone marrow endothelial cell line is also stimulated by specific and nonspecific proteasome inhibitors. Besides short term induction of eNOS activity, hydroxyurea's endothelial cell effects appear to be mediated by long term posttranscriptional augmentation in eNOS levels via inhibition of the proteasome activity. These experiments demonstrate a mechanism by which hydroxyurea may affect NO levels in large and small blood vessels, accounting for its inhibition of endothelial adherence of normal and sickle red blood cells.

191 INTER-STRAIN VARIATION IN MOBLIZATION PROFICIENCY IS NOT REGULATED BY A GENETIC VARIATION ON THE SDF-1/ CXCR4 AXIS Z. Xing, H. Geiger* Division of Experimental Hematology, Cincinnati Children's Hospital Medical Center and the University of Cincinnati College of Medicine, Cincinnati OH, USA The ability to mobilize hematopoietic stem and progenitor cells (HSPCs) in response to distinct regimens is conserved between mouse and human. HSPC mobilization is a dynamic and complex process. Stem cells have to exit their micro-environmental niche in the BM, migrate through the BM-blood vascular barrier, and circulate in the blood without immediately homing back to BM or any other organ. The interplay between cell adhesion and cell migration in mobilization of HSPCs is still not well understood. Two strains of mice, C57BL/6 (B6) and DBA/2 (D2) show measurable differences in G-CSF induced mobilization proficiency, with B6 being a poor mobilizer. This strain difference is linked to chromosome 11, and we will present new data on our positional cloning approach to identify the phenotype-conferring gene in the interval. Measuring the ability of HSPCS from PB or BM from either mobilized or non-mobilized mice to migrate across and endothelial cell layer in response to SDF-1, we detected no significant differences between the two inbred strains in the frequency of migrating progenitor cells. In addition, we detected no inter-strain variation in the expression of the receptor for SDF1, CXCR4 on HSPCs from the two strains. Surprisingly, mobilized PB progenitor cells showed a significant, strain-independent 2-fold increase in the ability to cross an endothelial cell monolayer in the absence of SDF-1 compared to an experimental set-up without an endothelial barrier. The implications of this finding are not clear and warrant further investigation. Thus, the inter-strain variation in mobilization proficiency, although at least in part being progenitor cell intrinsic, is most likely not due to a genetic variation on the SDF-1 CXCR4 axis. We are in the process of investigating the adhesion properties of HSPCs from the two strains upon stem cell mobilization.

192 EFFECT OF A NOVEL NF-KAPPA B INHIBITOR, IMD-0354, ON HUMAN HEMATOPOIETIC CELL MALIGNANCIES A. Tanaka1*, M. Konno1, T. Hebishima1, S. Muto2, A. Itai2, H. Matsuda1 1 Tokyo University of Agriculture and Technology, Tokyo, Japan 2Institute of Medicinal Molecular Design Inc., Tokyo, Japan Constitutive activation of NF-kappaB is one of common features in malignancies of hematopoietic cells, including lymphoma, leukemia, and mast cell tumors. Here we clearly demonstrated that a novel NF-kappaB inhibitor, IMD-0354, restrained factor-independent proliferation of mast cells with c-kit mutations but not of normal mast cells. In HMC-1 cells with the Asp816Val and Val560Gly mutations, we found that NF-kappaB was constitutively activated without any exogenous stimulation. When the DNA binding activity of NF-kappaB was inhibited by treatment with IMD0354, cell proliferation was completely suppressed. We detected the expression of cyclin D2, D3, and E in HMC-1 cells and observed that cyclin D3 expression was dramatically decreased by treatment with IMD0354. These findings indicated that auto-phosphorylated c-kit receptors

induced NF-kappaB activation, resulting in up-regulation of cyclin D3 expression and cell cycle progression. Since contribution of NF-kappaB in progression of various lymphoma and leukemia has been demonstrated, we examined effect of IMD-0354 on some other hematopoietic cell malignancies. IMD-0354 suppressed proliferation of several cell lines of human lymphoma, leukemia, and multiple myeloma. The observations from the current study suggest a therapeutic potential for compounds that interfere with NF-kappaB signaling in malignancies of hematopoietic cell linage, including lymphoma and mast cell tumors.

193 DOES THE BRAIN STEM CONTROL RENAL EPO SYNTHESIS? U. von Wussow*, J. Klaus, U. Frackowski, H. Pagel University of Luebeck, Luebeck, Germany Current data from our laboratory suggest that the renal Epo synthesis is not only defined by the renal oxygen supply. In the ongoing study hypoxic conditions in the brain stem of rats were achieved by cisternal puncture and insufflation of artificial cerebrospinal fluid (CSF). The intracranial pressure (ICP) was raised up to 100 mmHg for 10 min. Three hours later plasma Epo concentrations were measured by ELISA. The results are summerized in the table. Elevation of ICP and therefore, the decrease of the oxygen partial pressure in the CSF led to an increase of Epo in the plasma. Bilateral nephrectomy (NX) or hypophysectomy (HX) eliminated this effect. Unspecific stressors, like the restriction of the mobility (ROM), were not capable to induce renal Epo synthesis. From here, we like to hypothesise: Extrarenal oxygen sensitive sensors, in all probability located in the brain stem-hypothalamic-hypophyseal axis, control - at least in part - the renal Epo synthesis. During hypobaric or anemic hypoxia, these sensors lead to an increased production of an humoral factor, which initiates Epo synthesis in the kidney. controls ICP

ICP/NX

ICP/HX

ROM

Epo (mU/ml) 5.2±0.9 14.1±5.3* 2.6±0.5 ns 4.7±2.1 ns 5.9±1.7 ns ICP: intracranial pressure 100mmHg, NX: ICP after bilateral nephrectomy, HX: ICP after hypophysectomy, ROM: restricting of the mobility; ns: P>0.05, *: P<0.05 (Dunnett test vs.controls); n=4-5

194 INVESTIGATING THE BINDING INTERACTION BETWEEN GRANULOCYTE MACROPHAGE-COLONY STIMULATING FACTOR AND THE SOLUBLE ALPHA SUBUNIT OF ITS RECEPTOR THROUGH HYDROGEN/DEUTERIUM EXCHANGE MASS SPECTROMETRY J. L. Harris*, D. C. Schriemer, C. B. Brown University of Calgary, Calgary, Alberta, Canada Juvenile myelomonocytic leukemia (JMML) is a life-threatening disorder in young children in which conventional chemotherapy is highly ineffective and transplant patients commonly relapse. The survival and continued proliferation of JMML cells is dependent upon granulocytemacrophage-colony-stimulating-factor (GM-CSF) which must bind to the multi-subunit (alpha and beta) GM-CSF receptor (GMR) to initiate biological response. An effective therapeutic avenue for JMML could therefore involve the development of interaction inhibitors to the GMCSF:GMR complex. In this project we apply a novel tool in structural biology, hydrogen/deuterium exchange mass spectrometry (HDX MS), to carry out drug target analysis as a means of developing novel cancer therapeutics. A better understanding of the protein interactions between GM-CSF and GMR, and identification of the critical residues involved in the complex formation will ultimately lead to improved therapeutics. HDX MS is an emerging technology enabling exploration of protein binding sites as well as the resulting, induced conformational changes. Successful digestion is essential to HDX MS in order to localize sites of deuteration to the 3D structure. We believe that HDX MS will offer unique insight into the binding interaction between GM-CSF and a soluble isoform of GMRalpha (sGMRalpha). To date reliable digestion and deuteration of GM-CSF and sGMRalpha individually has been accomplished. Preliminary data for the digestion of the GM-CSF:sGMRalpha complex has demonstrated positive results and deuteration has been observed. Comparison of the rate of deuterium incorporation in the labeled complex versus the individual labeled proteins will allow us to determine the effects of complex formation and thus identify the binding sites. Using HDX MS

iseh05.book Page 89 Tuesday, May 10, 2005 10:22 AM

34th Annual Meeting: ISEH - Abstracts / Experimental Hematology 33 (2005) 39-143 to obtain new structural knowledge about the GM-CSF:sGMRalpha complex will assist with the targeting of therapeutic drugs towards JMML. It may be applied to other biological complexes and provide new treatment options for other types of cancer.

195 GENETIC EVIDENCE OF A NOVEL BLOOD DIFFERENTIATION PATHWAY FROM LYMPHOMYELOID HEMATOPOIETIC STEM/ PROGENITOR CELLS, INDEPENDENT OF COMMON MYELOID PROGENITORS A. Hultquist*, R. Månsson, L. Yang, L. Thorén, H. Qian, K. Liuba, M. Sigvardsson, S. E. Jacobsen Lund Stemcell Center, Lund, Sweden We have recently identified three novel subsets of multipotent hematopoietic stem/progenitor cells (HSCs) in the Lin-Sca-1+c-Kithi (LSK) compartment of adult murine bone marrow based on differential expression of CD34 and the tyrosine kinase receptor flt3. Long-term HSCs (LT-HSCs) lack CD34 and flt3 expression (LSKCD34-flt3-), whereas short-term HSCs (ST-HSCs) are LSKCD34+flt3-. A third LSK population is characterized by co-expression of CD34 and flt3 (LSKCD34+flt3+) and possess a combined myeloid (granulocytic/monocytic) and lymphoid (B and T cell) differentiation potential, but surprisingly lack megakaryocytic (Mk) and erythroid (E) potential (Adolfsson et al, Cell in press). These findings implicate an alternative road map for blood lineage development distinct from the classical model in which the first lineage commitment step of HSCs is thought to result in a strict separation into myelopoiesis and lymphopoiesis. In the current study we sought genetic evidence in support of this new model through genetic profiling of these three HSC subpopulations in adult and fetal mice, using affymetrix chips, quantitative (Q)-PCR and single cell PCR. In contrast to pluripotent LT-HSCs and STHSCs, LSKCD34+flt3+ cells downregulated or turned of several genes critically involved in promoting Mk and E lineage development, such as the Epo and Tpo receptors as well as the transcription factor GATA-1. In contrast, genes specific for lymphoid development, such as Rag-1, sterile Ig and IL-7Ralfa, were upregulated in LSKflt3+ cells, but absent in LTHSCs and ST-HSCs. However, in agreement with their sustained ability to produce granulocytes and monocytes, G-CSFR and PU.1 expression were sustained from LT-HSCs throughout the LSKCD34+flt3+ stage. Particularly noteworthy, single cell PCR demonstrated that a fraction of single LSKCD34+flt3+ cells upregulating lymphoid specific genes also coexpressed G-CSFR. Thus, genetic profiling at the single cell level provide further and compelling evidence for a novel road map for blood lineage development, independent of the common myeloid progenitor.

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within each pedigree (NMT1-NMT7), to generate homozygous inbred lines carrying the respective mutations. These will then be used for genetic mapping of the 7 mutant alleles. This forward genetic approach has thus generated 7 novel pedigrees with defective HSC competitive ability, and should enhance our understanding of the underlying genetic regulatory mechanisms controlling HSCs.

197 DIFFERENTIAL LOSS OF ALDEHYDE DEHYDROGENASE ACTIVITY DURING EARLY STAGES OF HUMAN LYMPHOID AND MYELOID LINEAGE RESTRICTION K. Luecke*, O. Christ, S. Imren, C. Smith, K. Leung, M. Hamilton, A. Eaves, C. Eaves Terry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada Hematopoietic cells are typically viewed as a hierarchy defined by the co-ordinately timed differentiation steps they undergo over a series of many cell cycles. Aldehyde dehydrogenase (ALDH) confers selective resistance to alkylating agents and, when cells expressing ALDH are exposed to BODIPY-Fl1-labeled amino-acetaldehyde (BAAA), they convert BAAA into BODIPY-Fl1-aminoacetate (BAA). If retained within the cell, the generated BAA allows ALDH+ cells to be specifically detected by multi-parameter flow cytometry. It has been known for some time that primitive human hematopoietic cells are ALDH+, but the relative levels of ALDH activity in different types of human repopulating cells had not been previously delineated. Here we show that human cord blood cells with short term myeloid-restricted (3 weeks), or either short term (6-12 weeks), or sustained (>5 months) lympho-myeloid repopulating activity in non-obese diabetic-scid/scid (NOD/SCID)-ß2-microglobulin-/- mice all display the same very high levels of ALDH activity. Further, removal of cells expressing CD2, CD3, CD7, CD14, CD16, CD24, CD36, CD38, CD56, CD66b, and/or glycophorin from low density (LD) ALDH+ human cord blood cells with low light side-scattering properties (SSClo cells) allowed the isolation of a population containing longterm (5 months) lympho-myeloid repopulating cells at a frequency of 1/360, as shown by limiting dilution assays in NOD/SCID mice. Nevertheless, a significant proportion of the CD34+CD38- cells LD SSClo cord blood cells were found to have an ALDHlo-neg phenotype. Transplantation experiments with these cells revealed they contained a previously undetected class of progenitors that display short term repopulating ability with restriction to B-lymphoid differentiation that was greater in NOD/SCID-ß2microglobulin-/- mice than in NOD/SCID mice. These findings suggest that loss of ALDH activity during human hematopoietic differentiation may accompany lineage restriction to lymphopoiesis within the CD34+CD38- population while persisting in CD34+CD38+ myeloidrestricted cells with short term repopulating activity.

198 CRITICAL ROLE OF C-KIT IN MAINTENANCE OF HEMATOPOIETIC STEM CELLS L. Thoren*, K. Liuba, C. Jensen, S. W. Jacobsen Lund University, Lund, Sweden The proto-oncogene c-kit is a tyrosine kinase receptor highly expressed on hematopoietic stem cells (HSCs) and early progenitors. However, the role of c-kit in regulation of HSCs remains unclear. Several spontaneous ckit mutations of different severity have been identified in mice, but previous studies have suggested that these mice have surprisingly mild HSC phenotypes, with only marginally reduced HSC numbers. Herein we investigated in more detail the HSC phenotype in White Spotting 41 (W41) mice, in which a point mutation at kinase position 831 generates a weak kinase signal down-steam of the receptor. Phenotypic analysis demonstrated that Lin-Sca1+kit+flt3- HSCs in fetal liver and 10-12 week old mice were only 2-fold reduced. However, transplantation of limiting numbers of whole BM cells into lethally irradiated recipients demonstrated that HSCs capable of sustaining hematopoiesis long-term were reduced at least ten-fold in w41 mice, and c-kit deficient HSCs capable of secondary reconstitution were reduced to almost undetectable levels. Kinetic analysis of competitively transplanted recipients demonstrated that the ability of kit-deficient HSCs to multilineage reconstitute was progressively lost with time, suggesting that c-kit plays an essential role in HSC maintenance and self-renewal. Thus, c-kit is not important for generation and initial expansion of HSCs, but appears to be essential for sustained self-renewal of HSCs.

Abstracts

HERITABLE BONE MARROW TRANSPLANTATION DEFECTS IN ENU-GENERATED MOUSE PEDIGREES J. Antonchuk*, W. Alexander, D. Hilton Walter & Eliza Hall Institute, Parkville, Victoria, Australia We have used a forward genetic approach to identify potentially novel regulatory mechanisms governing hematopoietic stem cell (HSC) function. The chemical mutagen N-ethyl-N-nitrosourea (ENU) was used to introduce random point mutations into the germline of mice, the progeny of which were then screened for bone marrow transplantation defects. We screened for exacerbation of the Mpl-/- HSC defect by the ability of mice to be engrafted in a non-myeloablative transplantation setting with 1,000,000 wildtype (Ly5.1+) bone marrow cells, and selected mice with >3% Ly5.1+ blood cells at 4 weeks post-transplant. We originally screened 2,200 1st generation (G1) progeny of ENU-treated Mpl-/- males. Of these, 41 mice were selected which had significant Ly5.1 engraftment. Selected G1 mice were then backcrossed to Mpl-/-, and their progeny were screened for heritability of the phenotype. Of these 41 selected G1s, 3 produced affected offspring and were named NMT1, NMT2, and NMT3. The proportions of affected offspring from affected backcrosses were: 28% (74/264) for NMT1; 19% (12/64) for NMT2; and 33% (14/43) for NMT3. We also carried out a 3rd generation screen, to identify recessively inherited mutations. A total of 2,460 mice were screened as above, comprising 140 independent pedigrees (average 18 mice/pedigree). Of these, 4 pedigrees were selected in which multiple mice were significantly engrafted, these pedigrees were renamed NMT4 - NMT7. Frequencies of affected mice were 9% (4/44) for NMT4; 21% (8/38) for NMT5; 24% (4/17) for NMT6; and 50% (2/4) for NMT7. We are now mating affected mice together

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G0 HEMATOPOIETIC STEM CELLS DO NOT EXIST IN MOUSE FETAL LIVER AND THOSE IN S/G2/M DISPLAY A PRONOUNCED BUT REVERSIBLE ENGRAFTMENT DEFECT M. Bowie1,2*, L. McCaffrey1, C. Eaves1,2 1BC Cancer Agency, Vancouver, Canada 2 Genetics Program, University of British Columbia, Vancouver, Canada During ontogeny, many properties of hematopoietic stem cells (HSCs) change. Our previous studies of the cell cycle kinetics of highly purified day 14.5 mouse fetal liver HSCs (11% pure) in single-cell cultures containing 300 ng/ml Steel factor (SF) and 20 ng/ml IL-11 supported the prevailing assumption that HSC turnover is higher in fetal liver than in adult marrow. To address this question directly, we have now performed 3 types of experiments, all of which show that all day 14.5 mouse fetal liver HSCs are in cycle. In a first series of experiments (n=3), in vivo limiting dilution assays of HSCs were performed on livers of fetuses removed from day 14.5 pregnant mice injected intravenously with 100 mg/kg 5fluorouracil (5-FU), or saline, 24 hr previously. The results show a >100fold reduction in HSCs in the 5-FU-treated group (i.e., to <1 HSC per fetal liver). Similar assays of HSCs in Ter-119- day 14.5 fetal liver cells cultured overnight in 50 ng/ml SF showed a 140-fold decrease in HSCs simultaneously exposed to high specific activity 3H-thymidine (relative to controls cultured in medium and SF alone, 5 experiments). Finally, assays of freshly isolated Ter-119- fetal liver cells separated by FACS into G0, G1 and S/G2/M fractions after staining with Hoechst 33342 and Pyronin Y showed HSC activity was detected exclusively in the G1 fraction even after intra-femoral injections, although equivalent activity was recovered from S/G2/M cells if these were cultured for 6 hours in 100 ng/mL SF prior to transplantation (intravenously). Thus, fetal liver HSCs lack a G0 fraction and show the same reversible engraftment defect as proliferating adult HSCs transiting S/G2/M. These findings also indicate that measured fetal liver HSC frequencies represent an ~2-fold underestimate. Thus currently attainable purities should allow meaningful analysis of molecular differences that distinguish fetal and adult HSC properties.

AGE-DEPENDENT CHANGES IN HEMATOPOIETIC STEM AND PROGENITOR CELLS D. J. Pearce1*, D. Taussig1,2, A. Eddaoudi3, D. Bonnet1 1 Hematopoietic Stem Cell Laboratory, Cancer Research UK, London, UK 2 Cancer Research UK Department of Medical Oncology, St. Bartholomew's Hospital, London, UK 3FACS Laboratory, Cancer Research UK, London, UK It is thought that as we age, accumulated damage causes stem cells to die by apoptosis. In the murine hematopoietic system however, the number of murine C57Bl6 stem/progenitor cells is actually thought to increase with age and this has been attributed to the inbred nature of this strain. Consistent with previous reports, we found that the percentage of phenotypically-defined stem/progenitor cell subsets (lineage negative, ckit+, Sca-1+ (KLS)) increases during the murine lifespan. However, both the absolute number of SP cells and the proportion of the KLS cell population that is an SP stem cell, increases dramatically with age. SP cells from older mice have a reduced (p<0.01) 4-month competitive repopulation potential when compared to SP cells from younger mice but have the expected low progenitor activity (1 in 8.5 single-sorted cells produced colonies). Furthermore, SP cells from old mice possess a similar cell cycle profile (89.5% G0/G1) to that previously reported for adult SP cells (Goodell et al, 1996). Hence, our observed increase of SP with age is not due to the acquisition of the SP phenotype by progenitors or committed cells; rather this difference in SP distribution reflects an age-dependent change in hematopoietic stem cell biology. Specifically, as the number of stem and progenitor cells increases with age, the proportion that is a stem cell increases but these have a reduced potential when competed with cells from younger mice. We are currently investigating engraftment variables that may explain the reduced stem cell function of SP cells from older mice. Age (days) SP % of total KLS % of total Sp % of KLS CRU fold enrichment 49 61 221 414

200 ISOLATION AND FUNCTIONAL CHARACTERIZATION OF CORD BLOOD CD34+ CELLS WITH TELOMERASE REVERSE TRANSCRIPTASE (HTERT) EXPRESSION M. Järås*, A. Edqvist, J. Rebetz, B. Widegren, X. Fan Lund University, Sweden Telomerase activity has been demonstrated in the bulk human CD34+ progenitor cell population, but it is unclear whether repopulating hematopoietic cells have telomerase activity. We therefore asked, whether there is heterogeneity in hTERT expression among CD34+ cells and if CD34+ cells expressing hTERT have NOD/SCIDBeta2m-/- mice repopulating capacity. In order to functionally characterize hTERTexpressing cord blood (CB) CD34+ cells, we isolated such cells at the single cell level by using an adenoviral vector (cTERT-dGFP), encoding destabilized GFP under the control of the hTERT promoter. Two days post-transduction of CD34+ cells with the cTERT-dGFP vector or the control vector encoding EGFP under the control of the PGK-1 promoter at an MOI of 100, the percentage of GFP+ cells were 17±4.3% and 47±6.7%, respectively; indicating a heterogeneity in telomerase activity among CD34+ cells. At this time-point, GFP+ cells were sorted by flow cytometry into a hTERT and a control vector sorted fraction. Staining for the Ki-67 antigen and 7-AAD revealed that in comparison to the control vector sorted cells, the hTERT sorted cells had a greater proportion of cells in the S/G2/M phase of the cell cycle (55±1.2% versus 37±3.6%, p<0.01), and fewer cells in G0 phase (8.7±2.3% versus 21±3.7%, p<0.05). Furthermore, the colony forming capacity and short-term (6 weeks) NOD/ SCIDBeta2m-/- repopulating capacity of the hTERT and the control-sorted cells were similar. Interestingly, hTERT sorted cells were devoid of secondary NOD/SCIDBeta2m-/- mice (n=8) repopulating capacity, whereas 3 out of 5 mice in the mock transduced group and 3 out of 7 mice in the control vector transduced group were positive for human cell engraftment. In summary, these data suggest that the quiescent human hematopoietic stem cells (HSCs) have low to undetectable levels of telomerase activity, which is upregulated in proliferating cells, colony forming progenitors, and short-term repopulating stem cells.

0.016 0.04 0.054 0.16

0.29 0.49 0.19 0.42

1.37 4.4 21.2 32.4

Not Done 905±326 Not done 123±49

Cells were competed in 8-12 week recipients against 2 x 105 whole bone marrow cells taken from 8-12 week mice. CRU fold enrichment is given as comparison to this 8-12 week sourced marrow.

202 Withdrawn

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THE G PROTEIN-COUPLED RECEPTOR CYSLT1 SIGNALS VIA ERK/MAP-KINASE IN CD34+ PROGENITOR CELLS AND STIMULATES PROLIFERATION SYNERGISTICALLY WITH INTERLEUKIN-3 A. M. Boehmler*, G. Seitz, T. Wiesner, L. Kanz, R. Möhle University of Tübingen, Tübingen, Germany We have previously shown that the G protein-coupled receptor (GPCR) cysLT1 is preferentially expressed in immature CD34+ hematopoietic progenitor cells (HPC). In the present study, we analyzed the signal transduction pathways in mobilized CD34+ HPC activated by the cysLT1 ligand LTD4, an inflammatory lipid mediator (leukotriene), which is also produced by bone marrow stromal and endothelial cells when deprived of hematopoietic cells. Of note, the effects of LTD4 on intracellular calcium fluxes, a typical early signaling step of Gi-coupled GPCR, were substantially stronger than those observed after stimulation of the HPCassociated chemokine receptor CXCR4 with optimal doses of the corresponding ligand stromal cell-derived factor-1 (SDF-1). Similarly to SDF-1, signal transductions pathways leading to cell locomotion such as polymerization of filamentous actin were significantly increased in response to the lipid mediator LTD4. In contrast to SDF-1, however, addition of LTD4 was also able to significantly (approximately two-fold) increase the in vitro expansion of both, committed and immature CD34+ progenitors in cytokine-supplemented in vitro cultures, but only when interleukin-3 was included in the cytokine cocktail. In these cultures, the effects of LTD4 was comparable to the increase of the cell number induced by addition of FLT3-ligand. As analyzed by Western blot, LTD4 induced a rapid phosphorylation of ERK/MAP-kinase in CD34+ cells, while phosphorylation of NF-kappa B, representing a second pathway of GPCR signaling associated with cell proliferation, was unaffected. These results suggest that in contrast to the chemokine receptor CXCR4, activation of the ERK/MAP-kinase signaling pathway by cysLT1 results in an effect on cell proliferation which is comparable to that induced by hematopoietic growth factors.

NEUROTROPHIC FACTORS, BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF) AND NEUROTROPHIN-3 (NT-3) SUPPORT EX VIVO EXPANSION OF MULTI-LINEAGE HEMATOPOIETIC STEM AND PROGENITOR CELLS L. Y. W. Ng1, C. K. Y. Chuen1, S. M. Lee1, K. S. Tsang2, K. Li1* 1Department of Paediatrics, The Chinese University of Hong Kong, Hong Kong 2Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Hong Kong Our previous study demonstrated that neural progenitor cell line C17.2 supports ex vivo expansion of cord blood CD34+ cells in a non-contact culture (Leukemia, 19, 91-97, 2005). C17.2 cells also expressed various neurotrophic factors including BDNF and NT-3. This study further investigated effects of BDNF (5, 10, 50 and 100ng/ml) and NT-3 (10 and 100ng/ml) on hematopoiesis. Enriched CD34+ cells (MACS beads) were cultured in methylcellulose with or without BDNF or NT-3, in addition to a cocktail of cytokines including erythropoietin (3IU/ml), granulocyte macrophage-colony stimulating factor (10ng/ml), interleukin-6 (10ng/ml) and stem cell factor (S, 50ng/ml). Our results showed that NT-3 at 100ng/ ml significantly increased CFU-GM, BFU/CFU-E and CFU-GEMM by 100±57.7%, 22.3±2.27%, and 47.0±17.4%, respectively, when compared to control cultures (N=8, p<0.05). BDNF at 10ng/ml significantly enhanced the proliferation of BFU/CFU-E and CFU-GEMM by 17.0±7.19% and 24.8±9.51% (p<0.05). In an ex vivo expansion study, enriched CD34+ cells were cultured in the presence of BDNF or NT-3, and a cytokine combination containing thrombopoietin (T, 50ng/ml), S (50ng/ ml) and flt-3-ligand (F, 80ng/ml) in QBSF-60 serum-free medium for 8 days. In control TSF cultures, the number of CD34+ cells, CD34+CD38cells, total nucleated cells, CFU-GM, BFU/CFU-E, CFU-GEMM and CFU-MK were expanded by 5.15±0.96, 15.9±3.64, 59.7±10.7, 40.0±8.56, 28.3±5.05, 24.5±5.29, 67.2±4.06 and 29.9±5.33 fold, respectively. The addition of NT-3 (100ng/ml) significantly enhanced the expansion of these progenitor cells to 7.25±0.63, 29±4.62, 79.6±12.7, 66.9±14.7, 61.4±15, 56.4±11.1, 86.4±4.92 and 61.5±13.9 fold (N=6, p<0.05). BDNF at 10ng/ml significantly increased the number of CD34+CD38- cells and CFU-GEMM to 32.0±9.23 and 43.4±8.93 fold (p<0.05). It also increased the percentage of CD34+CD38- cells (1.62±0.40 vs 0.90±0.14%). Our data provided the first evidence that neurotrophic factors BDNF and NT-3 support ex vivo expansion of CD34+ stem and progenitor cells, and indicated cross-talks between hematopoiesis and neuropoiesis.

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206 INHIBITION OF ABCG2 ON CHRONIC MYELOID LEUKAEMIA (CML) CELLS DEPLETES CD34+ STEM CELLS N. E. Jordanides1, D. Gilmour2, T. L. Holyoake1,2, J. C. Mountford1,2* 1 University of Glasgow, Glasgow, UK 2 Scottish National Blood Transfusion Service We have described a population of quiescent stem cells (qSC) in patients with chronic phase (CP) CML at diagnosis. In vitro studies have proven this population to be highly insensitive to IM, and worryingly shown that qSC are accumulated after CML CD34+ cells are treated with IM. One explanation for this is the presence on these cells of multi-drug resistance (ABC) pumps. We previously showed that ABCG2, a stem cell-associated pump, is expressed on these cells, that it is functional and interacts with IM. We therefore investigated the effect of ABCG2 inhibition on CML CD34+ cells. Using CFSE to track cell division we treated CD34+ enriched CML samples with 5microM IM±the ABCG2 inhibitor fumitremorgin C (FTC) or with 10microM FTC alone for 3d. In comparison to untreated controls 5microM IM reduced the total number of cells to 31.9±9.2 % and the number of CD34+ cells to 43.2±17.6%. However, the qSC significantly increased to 318±75.8% of control. In contrast, FTC alone did not effect a reduction in total cells (99.5±11.9%) but gave a significant reduction of CD34+ cells (58.6±8.4%; p=0.02) and no accumulation of qSC (104.6±33.8%). We saw no additive effect when IM and FTC were given concurrently. Therefore, we suggest FTC may be used to deplete CD34+ stem cells from CML. Longer-term differentiation assays have demonstrated no increase in mature markers on FTC treated cells despite the significant reduction in CD34 (n=4). Hence, this depletion is not by the induction of differentiation. We propose that ABCG2 is aberrantly expressed on a much higher % of CD34+ cells in CML and is

Abstracts

ENHANCEMENT OF CORD BLOOD LONG TERM CULTUREINITIATING CELL MAINTENANCE BY O-SULFATED HEPARAN SULFATES AND BONE MORPHOGENIC PROTEIN 4 OR WNT-3A L. E. Colvin Wanshura1, E. J. Stephenson1, M. S. Nelson1, P. Gupta1,2* 1 VA Medical Center, Minneapolis, MN, USA 2Hematology-Oncology-Transplantation Division, Dept. of Medicine, University of Minnesota Medical School, Minneapolis, MN, USA We have previously shown that specific O-sulfated heparan sulfates (OS-HS) improve human long term culture-initiating cell (LTC-IC) maintenance for up to 5 weeks in vitro. Recent studies indicate that the bone morphogenic protein (BMP) family of proteins and related factors are critical for hematopoietic stem cell development, survival and proliferation. Since HS modulate the biological activity of the BMP and Wnt pathways, we examined how combinations of HS, BMPs, BMP inhibitors and Wnt-3a influence primitive umbilical cord blood (UCB) progenitors in vitro. CD34+/CD38- UCB cells were cultured for 2 or 5 weeks in presence of Flt3 ligand + thrombopoietin + OS-HS (basal medium), with or without additional factors. The progeny was evaluated for LTC-IC maintenance using a limiting dilution assay. Addition of BMP4 improved 5-week LTC-IC maintenance to 179±31% compared to basal medium (P=0.03). Sonic hedgehog (SHH), which upregulates BMP signaling, achieved similar results (5-week LTC-IC maintenance 184±34% compared to basal medium; P=0.01). The BMP inhibitors Chordin, Caronte or Follistatin each reduced 5-week LTC-IC maintenance to about 50% compared to basal medium. Addition of Wnt-3a improved 5week LTC-IC maintenance to 268±38% compared to basal medium (P=0.0003). LTC-IC maintenance at 2 weeks was opposite to that seen at 5 weeks. Thus, 2-week LTC-IC maintenance was reduced by BMP-4 or SHH to about 70%, whereas Chordin increased it to 412±122%, compared to basal medium. These data indicate that factors whose activities are known to be modulated by HS, including BMPs and their inhibitors, SHH and Wnts, significantly influence LTC-IC maintenance. Further, our data suggest that these factors may be used to selectively achieve either shortterm or long-term LTC-IC maintenance. Current studies are investigating the mechanistic interactions of these proteins with heparan sulfates.

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expressed further into the progenitor stage than on normal cells. The expression of ABCG2 may be clinically significant in CP CML and inhibition of this pump may result in improved therapy.

207 REGULATION OF MURINE HEMATOPOIETIC STEM AND PROGENITOR CELLS THROUGH THEIR SURFACE EXPRESSION OF CD1D H. E. Broxmeyer1,2*, K. Christopherson1,2, G. Hangoc1,2, S. Cooper1,2, C. Mantel1,2, A. Bendelac3, R. Brutkiewicz1,2 1 Department of Microbiology and Immunology, and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN, USA 2 Walther Cancer Institute, Indianapolis, IN, USA 3 Department of Pathology, University of Chicago, Chicago, IL, USA CD1 molecules, involved in immune cell interactions, are similar to MHC class I antigens. We found that 99.8% of Sca1+c-kit+Lin- bone marrow cells express CD1d. While competitive repopulating ability of CD1d -/- and +/+ hematopoietic stem cells (HSC) were similar in lethally irradiated primary mice, there was a significant decrease in repopulation of CD1d -/-, compared to +/+ cells in secondary recipients. Stimulation of +/+ HSC in vitro with anti-CD1d enhanced their in vivo competitive repopulating capacity. Most hematopoietic progenitor cells (HPC) sorted into the CD1d+ cell population. Ratios of CD1+:CD1- CFU-GM, BFU-E and CFU-GEMM were respectively 28:1, 163:1 and 160:1. Absolute numbers of CFU-GM, BFU-E and CFU-GEMM in marrow and spleen were significantly enhanced 2-3 fold in CD1d -/- mice, and % marrow HPC in S-phase of cell cycle was increased ~2 fold, and HPC in spleen, in a slow or non-cycling state in +/+ mice were in rapid cell cycle (~60% in Sphase) in CD1d -/- mice. Pretreatment in vitro of +/+ HPC in unseparated or Sca1+Lin- marrow with different CD1d antibodies greatly enhanced numbers of immature CFU-GM, BFU-E, and CFU-GEMM stimulated by combinations of cytokines, as well as mature populations of CFU-GM and CFU-M, respectively stimulated by GM-CSF and M-CSF. This increase was not noted with CD1d -/-. The cytoplasmic tail of CD1d was required for anti-CD1d enhancement of HPC proliferation in vitro; HPC from truncated CD1d cytoplasmic tail knock-in mice did not respond to antiCD1d. Without cytokine addition, anti-CD1d did not stimulate colony formation of +/+ cells. CD1d was necessary for Flt3-ligand and SCF enhancement of cytokine-stimulated HPC colony formation, and for inhibition by myelosuppressive chemokines, but not by TNF-alpha or IFNgamma. These results implicate CD1d on HSC and HPC with their regulation, a heretofore unknown function for CD1d.

208 SMAD4 DEFICIENT HEMATOPOIETIC STEM CELLS HAVE IMPAIRED REPOPULATION CAPACITY G. Karlsson*, U. Blank, J. L. Moody, S. Singbrant, T. Utsugisawa, S. Karlsson Molecular Medicine and Gene Therapy and Lund Stem Cell Center, Lund University Hospital, Sweden. It has been shown that members of the transforming growth factor beta (TGF-b) superfamily of cytokines, including the TGF-bs, bone morphogenetic proteins, and activins affect proliferation and maintenance of HSCs. However, redundancy between different members of the family withhold important information making earlier studies targeting single TGF-b superfamily members limited. The TGF-b family receptors signal to the nucleus through Smad proteins. We have investigated the role of Smad signaling in hematopoiesis by means of Smad4 deficient mice, using the inducible MxCre-loxP system. Smad4 is the common Smad for all Smad signaling pathways and Smad4 deficiency will therefore be expected to eliminate all Smad-specific signaling in hematopoietic stem cells (HSCs). Three to five weeks after induction, the Smad4 deficient mice rapidly turn sick and succumb to anemia. Additionally, these mice experience abnormalities in the colon mucosa with an increased lamina propria and enlarged crypts infiltrated with inflammatory cells. Lethally irradiated recipients reconstituted with bone marrow from Smad4 deficient mice do not show any signs of disease, demonstrating that the Smad4 deficient HSCs exhibit repopulating capacity and that the disease phenotype observed in mice totally deficient in Smad4 is of extra hematopoietic origin. However, when performing competitive transplantation assays, Smad4 deficient bone marrow cells had a significantly lower repopulation capacity at 16 weeks compared to controls (11.4±8.6 % vs. 40.2±19.6 %,

respectively. p=0.01). This phenotype is maintained in secondary recipients at 16 weeks (0.2±0.1 % vs. 5.1±2.2 %), indicating that the Smad4 deficiency causes aberrant function of long term HSCs. Lineage distribution in peripheral blood, as well as homing efficiency and apoptosis levels in primitive hematopoietic cells, were normal after transplantation. Thus, our findings demonstrate that Smad4 deficiency reduces the reconstitution ability of HSCs and demonstrate an important role for Smad signaling to determine fate options and function of HSCs.

209 LIN-SCA+ SORTED MURINE BONE MARROW: FLUCTUATIONS IN PHENOTYPE EXPRESSION THROUGH CELL CYCLE G. J. Dooner*, M. S. Dooner, G. A. Colvin, P. J. Quesenberry Center for Stem Cell Biology, Roger Williams Medical Center, Providence, RI, USA Previous work in our laboratory reveals that marrow stem cells demonstrate changes in engraftment (Habibian, et al J Ex. Hem, 188:393398, 1998), homing (Cerny et al., J Hematother Stem Cell Res 11:913-922, 2002), differentiation (Colvin et al., J Cell Phys 199:20-31, 2004), mRNA and surface expression of adhesion proteins (Becker et al., Exp Hematol 27:533-541, 1999). We hypothesize a model in which progenitor/stem cells are on a continuum, rather than in a hierarchy (Quesenberry, et al., Blood, 100:4266-4271, 2002; Quesenberry, et al., Stem Cell Reviews, 2005 in press), and primitive marrow stem cells continually and reversibly change phenotype, fluctuating between the phenotype of a typical progenitor and that of a relatively mature hematopoietic cell. These fluctuations appear to be related to cell cycle status. This theory is based on studies with purified Lin-Ho dull/Rh dull (LRH) murine marrow cells cultured in thrombopoietin, Flt3, and steel factor, and Lineage negative, Sca-1 positive (Lin-Sca-1+) murine marrow cells in interleukin-3 (IL-3), IL-6, IL-11, and steel factor. Marked and reversible shifts in stem cell surface marker, differentiation marker, and transcription factor expression in LRH and Lin-Sca-1+ cycling cells were demonstrated by quantitative reverse transcription-polymerase chain reaction (Real Time RT-PCR). Fog exhibits,decreased expression over time (0, 24 and 48h in Lin-Sca-1+), whereas expression varies with CD4, CD34, Sca-1 and CD45R (0, 32, 40, 48hours in LRH cells). Relative to time zero (Time0=1), Sca-1 shows a 17 fold increase at 32h, with a 5fold and 9-fold increases in expression at 40h and 48h respectively. These data show that primitive marrow stem cells express a variety of hematopoietic genes, expression modulates with cell cycle transit and perhaps most importantly that observed changes in gene expression can be reversible. This is consistent with the continuum theory of stem cell regulation.

210 PHAGE DIRECTED HOMING INHIBITION IN SKELETAL MUSCLE D. Greer*, A. Peterson, D. A. Demers, M. S. Doomer, B. M. Foster, G. A. Colvin, F. M. Sanchez-Guijo, J. Pimental, C. Milani, P. J. Quesenberry, M. Abedi Roger Williams Medical Center, Providence, RI, USA Recent literature has demonstrated the presence of marrow derived stem cells in skeletal muscle. This population has strong hematopoietic and probably skeletal muscle regenerative potentials. Our experiments show that lineage negative and lineage negative Sca+ bone marrow cells are capable of homing to skeletal muscle tissue 3 hours after intravenous infusion. Cells can be identified after 7 days and one month, indicating residency of the homed cells. Phage display technology was used to identify the adhesion molecules and receptors involved in homing. Selected plaques were amplified for DNA sequencing. Forty non-injuries derived and sixty injury derived plaques were sequenced. The non-injury plaques had two predominant sequences and the injury plaques had three dominant sequences. Purified phage from the five dominant sequences was used in a phage homing inhibition study. The mice selected to receive the injury derived phage were injured with cardiotoxin the day before the phage were injected. Mice were injected intravenously with 1013 of their respective phage and then injected fifteen minutes later with 2x106 CFSE labeled lineage negative cells. There were sacrificed at 3 hour and 24 hour time points to determine inhibition of homing by phage. The collected tissue was digested in 0.2% collagenase and analyzed by flow cytometry. Results indicate that the predominant injury derived phage were successful in blocking homing to injured muscle tissue by a range of 62-71 % when

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compared to injured tissue receiving just lineage negative cells. The noninjury derived phage did not significantly block homing to skeletal muscle. These results demonstrate the feasibility of using phage display to identify functionally significant homing peptides and adhesion molecules that can be used for future enhancement of marrow cells homing to other tissues with possible clinical application in regenerative medicine.

ubiquitous GalT1, additionally confirming the special status of 6FucT in megakaryocytopoiesis. The estimation of the mRNA expression for 6FucT will confirm our observation on the molecular level. In conclusion, the activity of 6FucT should be a useful marker of the early commitment of cultured hematopoietic stem cells into the megakaryocytic lineage.

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HAEMOPOIESIS IN THE APC MIN/+ MOUSE E. A. de Wynter*, A. F. Markham, P. L. Coletta University of Leeds, Leeds, UK The Adenomatous polyposis coli (APC) tumour suppressor gene is mutated in a hereditary form of intestinal tumorigenesis in both mice and man. Apc Min/+ mice carry a heterozygous germ line mutation in Apc and tumorigenesis proceeds following somatic loss of the second Apc allele resulting in dysregulated Wnt signalling. We have previously shown that in addition to polyposis in Apc Min/+ mice, there is a progressive lymphodepletion from around 80 days of age, of the thymocytes, splenic natural killer cells, immature B cells and progenitor B cells in the bone marrow (BM). BM transplantation experiments showed that development of the transplanted Apc Min/+ BM in wild-type animals appeared normal. In contrast, the Apc Min/+ mouse BM environment only supported short-term reconstitution of haemopoiesis with wild-type BM cells. In addition, the number of CFU-F in Apc Min/+ BM was significantly lower than that of wild-type controls. Taken together, these data suggest that the BM microenvironment in Apc Min/+ mice may be defective. To determine whether or not there is an intrinsic defect in the Apc Min/+ BM microenvironment independent of the tumour phenotype, we investigated haemopoiesis in vitro. Long-term bone marrow cultures from Apc Min/+ and wild-type animals of different ages were established using the Dexter system. Each week the non-adherent cells in cultures were counted and analyzed in CFU-GEMM assays. Our results showed that in mice less than 80 days old, there was little difference in the cell production of non-adherent cells in Apc Min/+ and wild-type littermates. However, long-term cultures from Apc Min/+ mice more than 80 days of age, exhibited significant hyperproliferation. These results suggest that there is altered haemopoietic cell proliferation in Apc Min/+ BM which may be masked in vivo by secondary phenotypic feedback. We are currently investigating the role of Wnt signalling in this system.

PHOSPHATIDYLINOSITOL-3-KINASE-DEPENDENT ACTIVATION OF ERK IN PRIMITIVE BONE MARROW CD34+ CELLS P. Aravena1, F. Tong1,3, D. Headley1,2,3, D. R. Sutherland1* 1Department of Pathology, Princess Margaret Hospital, Toronto, Canada 2Medical Oncology and Hematology, Princess Margaret Hospital, Toronto, Canada 3Division of Applied Molecular Oncology, Ontario Cancer Insitute, Toronto, Canada Activation of ERK (Extracellular signaling Regulated Kinase) signaling by growth factors normally proceeds through the ras to raf to MEK to ERK pathway, but an additional requirement for PI3-kinase activation was recently identified in undifferentiated murine hematopoietic stem cells stimulated by the c-kit ligand Stem Cell Factor (Wandzioch et al., Blood 2004;104:51-57). To assess if this atypical activation pathway also occurs in normal candidate human hematopoietic stem cells, we developed a multicolor flow cytometry technique that combines a standardized cell surface staining methodology (to identify primitive CD34+ subsets) followed by SCF activation and subsequent fixation and permeabilization to detect intracellular phosphorylation of ERK. Experiments were performed in the presence or absence of the PI3-kinase inhibitor, wortmannin. Results show that primitive subsets of bone marrow CD34+ cells with CD34bright/CD38dull/negative, CD34bright/CD90+ and CD34bright/CD117+ phenotypes phosphorylate ERK in response to SCF stimulation. Wortmannin blocked the phosphorylation of ERK in these subsets, but not in less primitive CD34+ cell fractions. These results identify a novel signaling mechanism in primitive/uncommitted human bone marrow CD34+ stem cells that involves an additional level of control compared to the classical ERK pathway.

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C-KIT EXPRESSION AND SIGNALING IN THE REGULATION OF HEMATOPOIETIC STEM CELL PROLIFERATION AND SENESCENCE J. C. Chen*, A. L. Smith, F. M. Ellison, N. S. Young National Heart, Lung and Blood Institute, Bethesda, MD, USA We tested c-Kit expression and c-Kit mediated cell expansion in vitro as well as cell engraftment in vivo to examine the role of c-Kit signaling in the regulation of hematopoietic cell proliferation and senescence. Proportion and total number of Lin-Kit+CD34- cells, cells that contain long-term hematopoietic stem cells (HSCs), reduced significantly from young to old age in BALB mice. In contrast, Lin-CD34-CD117+ BM cell percentage and total cell number increased from young to old age in normal B6 mice as well as in B6-Hbd/Hbd mice that carry a spontaneous hemoglobindeficient mutation. Surprisingly, when sorted Lin-Kit+CD34- BM cells were cultured in vitro for 7 days in the presence of 25 ng/mL stem cell factor (SCF), cell expansion reduced with donor age in BALB as well as in B6 and B6-Hbd/Hbd mice. Our results indicate that proliferative ability of individual Lin-Kit+CD34- cells declined with age in B6 and B6-Hbd/Hbd mice as have been observed in BALB mice, and that aged Lin-Kit+CD34BM cells from all three genotypes had a reduced ability to respond to the same level of SCF stimulation in comparison to young cells of the same genotypes. In a competitive repopulation assay, engraftment of sorted LinKit+CD34- cells from old B6 and B6-Hbd/Hbd donors were lower in comparison to sorted Lin-Kit+CD34- cells from young B6 and B6-Hbd/ Hbd donors. Thus, functional senescence occurs to all HSCs. The high overall BM cell engraftment ability seen in old B6 donors may reflect an increase in the number of functioning cells, not a gain of functionality per cell.

Abstracts

HIGH CORRELATION OF 6FUCT ACTIVITY WITH THE EXPRESSION OF GPIIB/IIIA COMPLEX DURING MEGAKARYOCYTIC DIFFERENTIATION J. Kaminska1*, U. Bany-Laszewicz1, E. Klimczak-Jajor1, D. Smolka1, J. Szczepanowska2, M. Majewski1, R. Mollicone3, J. Koscielak1 1Institute of Hematology and Blood Transfusion, Warsaw. Poland 2 The Nencki Institute of Experimental Biology, Warsaw, Poland 3 Universite de Paris, Villejuif Cedex, France Megakaryocytopoiesis is a complex multistep process involving the release of platelets into the blood circulation. The expansion of megakaryocyte (Mk) progenitors ex vivo requires the identification of their lineage-restricted proteins and glicoproteins. The earliest marker of megakaryocytopoiesis, appearing as early as the CFU-Mk stage, is the GpIIb/IIIa complex (CD41a). In platelets, this complex functions as a receptor for fibrinogen, von Willebrand factor, fibronectin and vibronectin. Two of the enzymes involved in GpIIb/IIIa synthesis are alpha-1,6fucosyltransferase (6FucT) and beta-4-galactosyltransferase1 (GalT1). In our study the activity of 6FucT and GalT1 in cord blood CD34+ cells cultured in medium promoting megakaryocytopoiesis was determined. The analysis of phenotype and morphology of cultured cells confirmed the differentiation and maturation into Mk progenitors. The activity of 6FucT and GalT1 in cultured cells increased with a simultaneous increased expression of the CD41a antigens. High correlation of 6FucT activity (correlation factor=0.78) and low correlation of GalT1 activity (correlation factor=0.3) with the expression of GpIIb/IIIa during megakaryocytopoiesis was observed. We also found that the increase in the activity of 6FucT in cultured cord blood CD34+ cells was almost 4-fold higher than that of the

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215 BONE MARROW-DERIVED PROGENITORS IN HEMOPHILIC SYNOVITIS S. A. Acharya*, D. D. MacDonald, I. Goldberg, D. M. DiMichele, D. C. Lyden, M. W. Hilgartner Department of Pediatrics and Children's Blood Foundation Laboratories, Weill Medical College of Cornell University, New York, NY, USA The clotting defect in hemophilia results in recurrent hemarthroses leading to synovial proliferation, hypertrophy, and bony arthropathy similar to rheumatoid arthritis and osteoarthritis. Because the proliferating synovial pannus in hemophilic joint disease (HJD) demands increased oxygen and nutrients, de novo blood vessel formation is a critical part of the pathological process. As such, angiogenesis, and the concomitant recruitment of bone marrow-derived (BMD) hematopoietic and endothelial progenitor cells (HPCs and EPCs) have been implicated in the progression of proliferative joint disease. We have observed the presence of these progenitors, as well as potent pro-angiogenic and BMD progenitor mobilizing factors (vascular endothelial growth factor (VEGF) and stromal-derived factor-1 (SDF-1)), in histological sections of HJD synovium. Furthermore, HJD subjects showed a statistically significant increase in circulating cells expressing functional markers of HPCs, such as VEGF receptor-1 (VEGFR1) and CXCR4 via flow cytometric analysis, compared to hemophilic subjects without JD and healthy controls. Quantitative RT-PCR revealed that VEGFR1, VEGFR2, and CXCR4 mRNA expression were similarly increased. Correspondingly, peripheral blood levels of HPC and EPC mobilization and chemotaxis mediators (VEGF, SDF-1, and MMP-9) were also significantly elevated in subjects with HJD. Ongoing experiments indicate the increased presence of circulating vascular progenitors in HJD subjects. We will therefore quantify and characterized these BMD progenitors as early (VEGFR1+/AC133+, VEGFR2+/AC133+, CXCR4/AC133+) and late (VEGFR1+/ CD11b+, VEGFR2+/CD11b+) in these subjects. In addition, preliminary data suggest differential capacities of peripheral blood from HJD patients and control groups to form early outgrowth colony forming unit-endothelial cells (CFU-ECs), thereupon quantifying functional EPCs. The above progenitors, in combination with various angiogenic growth factor levels, may serve as surrogate biologic markers to provide effective means of detecting early synovial proliferation and identifying candidates for early intervention. Ultimately, anti-angiogenic agents may provide a novel preventative treatment for hemophilic arthropathy.

216 LIPID RAFTS PLAY A ROLE IN THE FORMATION OF AN ADHESIVE JUNCTION BETWEEN HOMOTYPICALLY AGGREGATED CD34-POSITIVE CELLS T. E. Bullock*, S. B. Marley, M. Y. Gordon Imperial College, London, UK The function of CD34, a marker of primitive haemopoietic stem and progenitor cells, remains unclear although studies have suggested roles in heterotypic and homotypic cell adhesion and signalling pathways, including those involved in cell cycle regulation and progenitor cell selfrenewal. Homotypic aggregation between CD34-positive cells has been proposed to be a mechanism for preventing inappropriate proliferation in vivo. CD34 localises at the contact point between cells induced to aggregate with the anti-CD34 antibody, QBEND10, and between multicellular aggregates isolated from normal bone marrow. Adhesion molecules such as LFA-1 and the actin cytoskeleton also localise to this contact point suggesting the existence of an adhesive junction between CD34-positive cells which can be likened to the specific arrangement of receptor segregation, known as the immunological synapse, between a T cell and antigen-presenting cell. Lipid rafts are distinct plasma membrane domains that are rich in cholesterol and glycosphingolipids. They are proposed to act as platforms to transport and organise the T cell receptor, co-stimulators, signal transduction molecules and the actin cytoskeleton at the immunological synapse. The aim of this study was to determine the role of lipid rafts in CD34-positive cell aggregation. Using immunofluorescence we have demonstrated that lipid rafts localise to the contact point between CD34-mediated homotypically aggregated cells. Disrupting lipid rafts with the cholesterol-depleting agent, methyl-beta-cyclodextrin, significantly reduced aggregation in both

KG1a and primary CD34-positive cells. Isolation and analysis of lipid rafts from aggregated KG1a cells using Brij 35 reveals that a minor proportion of the total cellular CD34 is present within lipid rafts. As in immunological synapse formation, lipid rafts play a key role in the formation of an adhesive junction between homotypically aggregated CD34-positive cells and may act as platforms for the transport of adhesion and signalling molecules to the contact point, thereby facilitating CD34mediated adhesion and associated signalling.

217 EVIDENCE OF INTERCONVERSION WITHIN HEMATOPOIETIC LINEAGE COMMITTED CELLS L. Chen, Y. Shi, K. Chin, G. P. Rodgers* Molecular and Clinical Hematology Branch, NIDDK, National Institutes of Health, Bethesda, MD, USA We have previously shown that erythroid population cells of human bone marrow CD133+ stem cells can be induced to convert to myeloid cells, and vice versa, in vitro. We subsequently employed rapid analysis of gene expression and two-dimensional gel electrophoresis techniques in order to study the gene and protein expression patterns of hematopoietic lineage differentiation and interconversion, in five cytokine-induced erythroid and myeloid populations from human CD133+ bone marrow stem cells. We identified and categorized the mRNA expression patterns of 266 gene-specific fragments into 3 groups – genes expressed in 1, 2 or all populations. Protein expression profiles of the cells were consistent with that of the expressed genes. To better define the characteristics of cells that possess this ability to interconvert, we analyzed the cells for their expression of CD133 antigen for stem cells, CD36 for erythroid, and CD13 for myeloid cells by three-color flow cytometry. Enriched CD133+ cells on day 0 of culture displayed a progressive decline in antigen expression to 0.06%±0.02% and 0.1%±0.05% cells on day 14 with EPO and G-CSF induction, respectively. We then sorted the CD36+CD13+ subpopulations from both the EPO and G-CSF 14-day-induced cells and re-cultured them for another 14 days in EPO, G-CSF, or cytokine-free medium. Surprisingly, up to 80% of the double positive cells of EPO induction became triple positive with EPO re-stimulation, and 29.8% CD13+ appeared with G-CSF re-culture, while no significant numbers of single CD133+ or CD36+ cells were detected. Control cells exhibited no significant changes. Our data suggest that hematopoietic lineage interconversion may be a characteristic of normal hematopoiesis arising from early primitive precursor cells that maintain the ability to co-express myeloid and erythroid markers. Further analysis of the genes and protein products identified within these 'differentiated' populations may provide insights into the mechanisms underlying hematopoietic lineage interconversion.

218 UNIQUELY PROTRACTED G1 TRANSIT OF EX VIVO SELF RENEWING HEMATOPOIETIC STEM CELLS J. M. Nygren*, D. Bryder, S. E. W. Jacobsen Hematopoietic Stem Cell Laboratory, Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund University, Lund, Sweden It has become an established notion that hematopoietic stem cells (HSCs) are compromised in S/G2/M of the cell cycle, a finding with considerable biological and clinical implications, in particular for the failed attempts to successfully ex vivo expand HSCs for transplantation. Although based on extensive evidence that HSCs, in steady state and during active proliferation, reside predominantly in G0/G1, direct evidence for HSC function fluctuating with cell cycle position is still lacking. Herein, using viable cell cycle and cell division tracking, we demonstrate that ex vivo expanded and self-renewing HSCs in S/G2/M are not compromised in repopulating activity. Rather, their enrichment in G1 results from HSCs having a protracted G1 transit (without entering G0). This unique cell cycle transit of HSCs and its regulation are likely to be critical for maintenance of the indispensable HSC pool.

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219 PERIPHERAL BLOOD DERIVED EPCS ARE MACROPHAGE COLONIES WITH THE CAPACITY TO ENHANCE ENDOTHELIAL GROWTH BY SOLUBLE FACTORS X. L. Aranguren1*, C. Moreno1, M. Soriano2, G. Abizanda1, E. J. Andreu1, J. M. Garcia-Verdugo2, F. Prósper1 1 Foundation for Applied Medical Research, Division of Cancer, Area of Cell Therapy and Hematology Service, Clínica Universitaria, Universidad de Navarra, Spain 22Department of Cell Biology, Instituto Cavanilles, University of Valencia, Valencia, Spain Several populations of endothelial progenitor cells have been described including AC133 cells from the BM, MAPCs or SVF derived cells and have been characterized by flow citometry, culture techniques and in vivo experiments. Levels of culture defined peripheral blood EPC have been shown to have a negative correlation with cardiovascular diseases. We have analyzed the frequency of BM, CB and PB mononuclear cell derived EPC using the techniques described by Hill (N Engl J Med 2003; 348:593600) and characterized their endothelial phenotype and functionality. In comparison with AC133 derived EPC, mononuclear cell derived EPCs retain hematopoietic markers, like CD45, CD14 and CD15 and lack endothelial markers like von willebrand factor and VE-cadherin. These cells are be able to take up ac-LDL, property associated to hepatocyte, endothelial and macrophage cells and express both endothelial and macrophage markers such as CD31. Electronic microscopy showed that peripheral blood derived colonies are really macrophage forming colonies (CFU). Mononuclear cell derived EPCs express pro-angiogenic cytokines including VEGF, angiopoietin, HGF, PLGF and GM-CSF while EPC conditioned media stimulated proliferation of AC133 derived endothelial cells. Using an in vivo matrigel assay in scid beige mice we could demonstrate that while AC133 cells are able to give rise human endothelial cells (UEA positive cells), mononuclear cell derived EPCs could not differentiate into human endothelial cells. In conclussion, mononuclear cell derived EPC are be able to induce angiogenesis by the release of growth factors but not by their endothelial differentiation capacity.

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Conclusion: We found the phenomenon that adenovirus-mediated antisense expression of telomerase RNA doesn't interrupt the differentiation of embryonic stem cells to hematopoietic stem cells without significant effect on the apoptosis degree. Further studies will be warranted to confirm these results and verify the mechanism of these phenomena.

221 ENGINEERING NICHES FOR THE IN VITRO MANIPULATION OF HEMATOPOIETIC STEM CELL FATE M. P. Lutolf, R. Doyonnas*, H. M. Blau Baxter Laboratory in Genetic pharmacology, Stanford University, CA, USA Adult stem cells reside within tissue-specific microenvironments (niches) that regulate their fate, either self-renewal or a pathway of differentiation. In these niches the stem cells are exposed to a complex interplay of soluble and insoluble, short- and long-range signals expressed by supportive cells in close proximity. Outside of the niche, stem cells rapidly lose their differentiation potential. In the last few years, substantial progress has been made in understanding hematopoietic stem cell (HSC) regulation by the niche. Several signaling pathways that appear to govern HSC self-renewal have been identified, paving the way for efficient in vitro HSC expansion. Yet, the HSC niche itself remains poorly defined and knowledge regarding the relevant signaling pathways has not yet been translated into reliable culture methodologies. We are engineering artificial niches for the ex vivo manipulation of HSC function. Synthetic polyethylene glycol hydrogel networks serve as well-defined display platforms for studying the interplay of previously identified HSC regulatory proteins in diverse combinations. Such engineered niches may serve as novel in vitro model systems for elucidating the molecular mechanisms that regulate HSC fate and may also have therapeutic use in facilitating the expansion of HSCs in tissue culture.

222 MHC CLASS I-RESTRICTED CD4+ HELPER T CELLS AUGMENT CD8+ CYTOTOXIC T CELL MEDIATED ANTI-TUMOUR IMMUNITY A. Tsallios1,2*, H. J. Stauss2, E. C. Morris1,2 1Imperial College, London, UK 2 Royal Free and University College Medical School, London, UK CD4+ T cells are critical for the generation of anti-tumour immune responses via cytokine secretion and enhancement of CTL priming and effector function. However, very few class II-binding tumour antigens have been identified limiting the isolation and adoptive transfer of tumourspecific CD4+ cells. We have used retroviral-mediated transfer of TCR alpha and beta chain genes to target CD4+ T cells against an MHC class I-presented model tumour antigen. The F5 TCR recognises the influenza virus A nucleoprotein peptide (NP366-379) presented by murine Db MHC class I. Splenocytes from C57Bl/6 mice (H2b) were transduced with the pMXF5TCRalpha-IRES-TCRbeta retroviral vector and further expanded in the presence of IL-2. Prior to functional assays cells were sorted into TCR-td CD4+ and TCR-td CD8+ populations. In some experiments, the TCR and CD8alpha genes were co-transferred into helper T cells to produce TCR-td CD4+8+ cells. Both purified TCR-td CD8+ and CD4+ cells secreted IFNgamma in an antigen-specific manner when stimulated with peptide loaded DCs. Peptide requirements were lower for CD8+ cells (10pM) than CD4+ cells (100pM). However, when stimulated with NP-expressing tumour cells, the TCR-td CD4+ cells only secreted IFNgamma when co-expressing CD8 (CD4+8+ cells) or in the presence of additional DCs. Surprisingly, the proliferative capacity of TCR-td CD8+, CD4+ and CD4+8+ cells correlated only with IL-2 production but not IFNgamma secretion. Further experiments showed that IFNgamma-producing CD4+8+ T cells which proliferated poorly and produced little IL-2 were unable to expand in vivo or provide help for tumour rejection. In contrast, CD4+ T cells producing high levels of IL-2 expanded, provided help for CTL-mediated tumour rejection and developed long term T cell memory. The data demonstrates both the anti-tumour efficacy of TCR-td T cells and the in vivo synergy between TCR-transduced CD4+ and CD8+ T cells specific for the same epitope resulting in long-term tumour protection.

Abstracts

TELOMERASE INHIBITION BY ANTISENSE TREATMENT INDUCES THE DIFFERENTIATION OF A HUMAN EMBRYONIC STEM CELL LINE, SNU-3 TO HEMATOPOIETIC STEM CELLS S. J. Kim1,2*, B. S. Kim1,2, J. H. Oh2, J. H. Yoo2, C. H. Song2, S. H. Kim2, C. W. Choi1, S. W. Shin1, Y. H. Kim1, J. S. Kim1 1 Department of Internal Medicine, Division of Oncology and Hematology, Korea University Medical Center, Seoul, Korea 2Institute of Stem Cell Research, Korea University College of Medicine, Seoul, Korea Background: This study was tried to evaluate the effect of adenovirusmediated telomerase antisense (TA) expression on cellular proliferation and apoptosis of the ex-vivo culture from human embryonic stem cells to hematopoietic stem cells. Methods: Cultured embryonic stem lines (SNU-3 cell line) to hemopoietic stem cells were classified to control and study groups. Control group was that of which embryonic body were cultured to LTC-IC assay not treated with telomerase antisense oligonucleotides (TA). Study group was that of which embryonic body were cultured to LTC-IC assay treated with TA. We evaluated CD34+ cell number and viable cell percentage and the degree of apoptosis of each samples at 5 days after LTC-IC assay and the results were compared between each groups. These studies were tried for 17 times. Results: The number of CD34+ cells in study group 0.8-2.4(median: 1.5x103) showed no significant difference with that in control group 0.62.9(median: 1.6x103). Viable cell percentage of control and study groups were 76-89(median: 83) % and 78-90(median: 85) %, respectively, without significant difference. And there was no difference of apoptosis degree (absorbance, A405nm – A490nm) between control and study groups(0.092-0.134(median:0.129) vs 0.087-0.152(median 0.138), negative control: 0.020, positive control: 0.186). The colony count of CFUGM in control and study group was 0.1-0.5(medium:0.3)x103 and 0.20.5(medium:0.3)x103, respectively without no significant difference.

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223 GENERATION OF CYTOMEGALOVIRUS-SPECIFIC T CELLS FROM SEROPOSITIVE SUBJECTS FOR ADOPTIVE IMMUNOTHERAPY USING PP65 P. Diamanti1,2*, R. J. Garland2, D. H. Pamphilon1, C. G. Steward2,3, A. Blair1,2 1Bristol Institute of Transfusion Sciences, UK 2 Department of Pathology and Microbiology, University of Bristol, UK 3Department of Paediatric Oncology and Haematology, Royal Hospital for Children, Bristol, UK It has long been acknowledged that the transfer of cellular immune responses from a Cytomegalovirus (CMV) seropositive donor to a recipient that reactivates CMV post stem cell transplantation (SCT) results in quicker immune reconstitution. It has been demonstrated that CMVspecific CD4+ and CD8+ cytotoxic T lymphocytes from the donor are important in resolving CMV infection and protecting recipients from developing CMV disease post SCT. Recently CMV recombinant protein pp65, an immunodominant target for CD4+ and CD8+ T cell responses, has been developed for the stimulation and isolation of donor CMV-specific T cells. The aim of this study was to investigate the potential of pp65 to generate CMV-specific CD8+ and CD4+ T cells from seropositive subjects for use as adoptive immunotherapy after SCT. Peripheral blood mononuclear cells were isolated from single unit CMV-positive buffycoats and were stimulated with pp65 for 16 hours. CMV-specific T cells that produced IFNgamma were captured using the IFNgamma-secretion assay and were enriched using the MiniMacs system. The phenotype of the cells, both pre and post enrichment was evaluated by flow cytometry using monoclonal antibodies against CD3, CD4 and IFNgamma. Experiments to date have shown that pp65 is capable of stimulating both CD4+ and CD8+ T cells to secrete IFNgamma. Flow cytometric analysis revealed that prior to enrichment, IFNgamma+ cells represented 0.44% of the CD3+/CD4+ cell population and 0.81% of the CD3+/CD4- cell population. However, following magnetic enrichment, the proportion of IFNgamma+ cells increased significantly (192-fold and 117-fold) to 84.4% and 94.9% in the CD3+/CD4+ and CD3+/CD4- subfractions, respectively. Large scale CMVspecific T cell capture experiments, performed under GMP conditions, are ongoing. The cytotoxicity and lack of alloreactivity of these cells will be assessed to determine their suitability for immunotherapeutic use post SCT.

224 EFFECT OF INTERLEUKIN 15 SUPPLEMENTATION ON PEPTIDESPECIFIC EXPANSION OF HUMAN T CELLS D. P. Hart*, E. C. Morris, H. J. Stauss Department of Immunology, Imperial College, London, UK The in vitro generation of peptide specific T cells requires cytokine supplementation to support proliferation and survival. Interleukins 2 and 7 have commonly been used. Recently, an increasing interest in generating therapeutic peptide specific cytotoxic T lymphocytes in vitro for adoptive transfer has prompted reassessment of cytokine stimulation conditions. Interleukin 15 shares basic growth and survival characteristics with IL2, but additionally has been described as a regulator of CD8 memory T cell homeostasis and inducer of antigen independent expansion of CD8 T cells making it an attractive candidate cytokine for stimulation protocols. The effect of IL15 or IL2 on recall and naive T cell responses was examined during the stimulation of human PBMC with CMV tegument derived peptide, pp65, (NLVPMVATV) and melanocyte differentiation antigen, Melan A (ELAGIGILTV) respectively. All peptides were HLAA2 restricted. Phycoerythrin (PE) labelled tetramer staining and FACS analysis was performed at 7 and 14 days post stimulation. Priming and subsequent expansion of naive T cells specific for Melan A was more efficient in the presence of IL-15 compared with IL2 (28.5% vs 1.1% after 2 weeks). However, stimulation and expansion of memory T cells specific for pp65 was more efficient in the presence of IL-2 compared with IL15 (35.4% vs 1.3% after 2 weeks). Addition of IL15 to cultures stimulated in the presence of IL2 abrogated the beneficial effect of IL2 and CTL expansion was reduced to the level seen with IL15 alone (pp65 tetramer positive cells IL2 11.7%; IL15 1.1%; IL2+15 1.3%). PBMC cultured in the presence of IL15 or IL2 show marked differences in cytokine secretion profiles, however these are independent of the stimulating peptide.

These data suggest IL15 supplementation used in peptide-specific stimulation exerts a proliferative effect favouring expansion of naive T cell response, while IL2 leads to preferential expansion of antigen-specific memory T cells.

225 CYTOTOXIC T CELLS SPECIFIC FOR THE WILMS' TUMOR PROTEIN ARE PRESENT IN PATIENTS WITH BREAST CANCER AND ARE OF SUFFICIENT AVIDITY TO KILL BREAST CANCER CELLS R. Gillmore1*, S. Xue1, A. Holler1, J. Kaeda2, D. Hadjiminas3, V. Healy4, Y. Ghani1, R. C. Coombes5, J. Waxman5, H. J. Stauss1 1Department of Immunology, Imperial College, London, UK 2Department of Investigative Sciences, Hammersmith Hospital, London, UK 3 Department of Surgery, St Mary's NHS Trust, London, UK 4 Department of Histopathology, St Mary's NHS Trust, London, UK 5Department of Cancer Medicine, Imperial College, London, UK The Wilms' tumor protein, WT1, is over-expressed in the vast majority of acute leukemias and currently trials are taking place investigating the potential benefit of WT1-based immunotherapy in these patients. The aim of our research has been to validate the targeting of WT1 in breast cancer. Having determined the incidence of WT1 mRNA expression in primary breast tumours (~90%) we analysed the immune responses to WT1. Patients with stage I/II breast cancer were studied since vaccination strategies, to boost any pre-existing immunity, are more likely to be successful in the context of low tumor burden. METHODS: Paired draining lymph node (LN) and peripheral blood (PB) samples were collected from 14 patients of whom 8 were tested HLAA2 positive by flow cytometry. Of these, 5 yielded samples sufficient for expansion and tetramer analysis. Fluorescent HLA-A2/WT1 tetramers were used to quantify WT1-specific T cells immediately ex vivo and following repeated antigen-specific in vitro stimulation. The functional capability of the tetramer-positive cells was assessed using 51Cr-release killing assays and intracellular cytokine staining, to identify interferongamma producing cells. RESULTS: WT1-specific T cells were detectable in all draining LNs but none of the PB samples. LN samples from 3 patients underwent repeated in vitro stimulations until the frequency of tetramer-positive cells peaked at 0.16%, 4.04% and 21.3% of viable CD3+ lymphocytes. These T cells displayed cytotoxicity and IFN-gamma production in response to target T2 cells pulsed with the relevant WT1 peptide epitope but not when T2 cells were pulsed with an irrelevant peptide. Importantly, these T cells also killed HLA-A2 positive WT1 mRNA expressing breast cancer cell lines following treatment with interferon-gamma. CONCLUSION: Patients with early breast cancer possess WT1-specific T cells of sufficient avidity to recognise and kill breast cancer cells. These data provide strong support for the initiation of vaccination trials in this patient group.

226 IMMUNOGENICITY OF THE TUMOUR ASSOCIATED ANTIGEN WT1 F. Ramirez*, Y. Ghani, H. Stauss Imperial College London, London, UK Wilm's tumour antigen 1 is a tumour-associated antigen over expressed in leukemia and various solid tumours. We are studying WT1 immunogenicity in the mouse to explore its suitability for vaccination based tumour immunotherapy. WT1 expression is restricted to haematopoietic stem cells, kidney cells and few others. This restricted pattern of expression suggests that the immune system may not be completely tolerant to WT1. However, mRNA-WT1 has been detected in mouse lymphoid organs, including the thymus, although it is unclear which cells are responsible. Two MHC class I-restricted epitopes have been previously characterised in mouse by searching for class I binding motifs and in vitro generation of allo-MHC-restricted CTL. The two epitopes are presented by WT1expressing tumour cells. We have explored different vaccination protocols to elicit self-MHC-restricted WT1-specific CTL responses in C57BL/6 mice.

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34th Annual Meeting: ISEH - Abstracts / Experimental Hematology 33 (2005) 39-143 Our results show that peptide vaccination with incomplete Freund's adjuvant was inefficient in eliciting WT1-specific CTL responses. Some mice generated a low response while in others the response was undetectable. Several boosting with peptides did not enhance the response. Contrary to similar models, co-stimulation with peptides that stimulate CD4 T cells or anti-CD40 monoclonal antibody injection did not increase the CTL response. However, peptide-loaded dendritic cells consistently induced strong WT1-specific CTL responses. Mice immunised with peptide-loaded dendritic cells were challenged with tumour cell lines expressing WT1 and very low or no protection was observed. This correlates with our inability to detect high-avidity WT1specific CTL responses after expansion of WT1-specific T cell lines in vitro from immunised mice. In summary our data show that only after optimal vaccination with dendritic cells it was possible to generate WT1-specific CTL responses. However, DC vaccination seems to stimulate low avidity CTL responses unable to confer protection against tumour challenge. These data suggest that mice are substantially tolerant to the two WT1 epitopes used in this study.

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228 DOES FETAL-MATERNAL MICROCHIMERISM CONFER IMMUNOLOGIC TOLERANCE OF FETAL CELLS? G. L. Gilmore*, M. Holm, J. Lister, R. K. Shadduck Western Pennsylvania Cancer Institute, Pittsburgh, PA USA Fetal-maternal microchimerism [MC] is a benign condition that occurs during gestation, due to trafficking of cells from the fetus to the maternal circulation (and vice versa) via the placenta. MC can persist for decades. The incidence of MC has been reported to be 33% in normal parous women, but the MC status of cancer patients is unknown. To study this, we obtained blood samples from 169 normal parous women, and 26 parous cancer patients. 33 non-parous women served as controls. We detected

male DNA by two rounds of PCR with nested primer sets specific for the Y chromosome. The sensitivity of the assay is 1 male cell/1 million female cells, validated with mixtures of male and female cell lines. We found the frequency of MC depends on the amount of DNA assayed: using 1 ug DNA [~300,000 genomes], 18% of normal parous females were MC positive %. At 5 ug DNA [1.5 million genomes], the frequency was 46%, and using >25 ug DNA [7.5 million genomes], 66% were MC positive. When we examined parous cancer patients, 20% were MC positive when 5 ug DNA was tested, but this rose to 76% when >25 ug DNA was examined. The lower frequency of MC among cancer patients at 5 ug DNA is presumably due to prior chemotherapy. Suprisingly, we found 2 normal non-parous donors were MC; this number did not increase when more DNA was analyzed. The reason for MC among non-parous females is unknown. Our results reveal MC is more frequent among parous women than previously reported, and imply a long-lasting tolerance of fetal tissue antigens, since fetal cells can be detected in the maternal circulation decades after birth. We are currently assessing whether MC translates into immunological tolerance to male offspring leukocytes. If this proves to be the case, this suggests that son-to-mother cellular therapy may be a feasible approach to treat malignancy.

229 A TELOMERASE-DERIVED PEPTIDE ANTIGEN GENERATES ANTILEUKEMIC T CELLS FROM B-CLL PATIENTS IN VITRO A. Choudhury*, M. Palma, P. Kokhaei, L. Hanson, A. Osterborg, H. Mellstedt Karolinska Institute, Stockholm, Sweden Telomerase is up regulated in the vast majority of human malignancies and therefore may be considered as a ubiquitous tumor antigen. High telomerase activity has been previously reported in patients with B-cell chronic lymphocytic leukemia (B-CLL). In the present study, the presence of naturally occurring, telomerase-reactive, T-cells in the peripheral blood of B-CLL patients were examined. We have also attempted to expand in vitro T-cells reactive to a telomerase-derived peptide and assess their ability to lyse autologous B-CLL cells. 19/25 B-CLL patients tested demonstrated telomerase positivity by RTPCR. Of these, PBMC of 5 positive and 3 negative patients were further utilized for generating telomerase-reactive T-cells. Monocyte-derived dendritic cells (DC) were pulsed with a 16 aa long, telomerase peptide or a 17 aa Ras peptide and used to stimulate autologous T- cells. In 3 of the 5 patients a significantly higher proliferative response was noted against the telomerase peptide compared to Ras. No significant telomerase-specific proliferation was observed with T-cells of telomerase-negative patients. Antibodies against both MHC class I and class II had an inhibitory effect on T-cell proliferation. T-cells were put through two rounds of stimulation and expansion with telomerase- or Ras peptide-pulsed DC and examined for their ability to lyse autologous leukemia cells in vitro. The cytotoxicity of telomerase- stimulated T-cells was approximately 53% at an effector: target ratio of 50:1. In comparison the cytotoxicity of Ras-stimulated Tcells was 14%. The cytotoxicity was blocked with antibodies against MHC class I but not with anti-class II antibodies. Our data demonstrates that some B-CLL patients have naturally occurring T-cells against telomerase and boosting anti-telomerase immunity may be an approach to immunotherapy of B-CLL

230 INDUCTION AND IDENTIFICATION OF MYELOMA-SPECIFIC CYTOTOXIC T CELLS L. Zahradova1*, D. Ocadlikova1, L. Kovarova1, M. Penka3, R. Hajek1, J. Michalek1,2 1 Laboratory of Experimental Hematology and Cell Immunotherapy, University Hospital, Brno, Czech Republic 2 Cancer Immunobiology Center, University of Texas, Southwestern Medical Center, Dallas, Texas, USA 3 Department of Clinical Hematology, University Hospital, Brno, Czech Republic Multiple myeloma (MM) was considered as low immunogenic incurable disease. The attempts were made to invert the immune status to recognize myeloma cells by cells of the immune system. Here we studied biology of myeloma-specific T cells in vitro.

Abstracts

PHASE I CLINICAL TRIAL OF CT-011, A HUMANIZED MONOCLONAL ANTIBODY DIRECTED AGAINST A B7 FAMILYASSOCIATED PROTEIN, IN PATIENTS WITH ADVANCED HEMATOLOGICAL MALIGNANCIES A. Nagler1*, A. Kneller1, A. Avigdor1, M. Leiba1, R. Koren2, L. N. Klapper2, M. Schickler2, A. Shimoni1 1 Hematology, BMT and CBB, Chaim Sheba Medical Center,Tel Hashomer, Israel 2CureTech Ltd., Yavne, Israel Objective: CT-011, a humanized monoclonal antibody that is directed against a B7 family-associated protein, was previously shown to efficiently elicit anti-cancer immune response against a wide range of murine and human tumor models (Hardy et al. PNAS 94:5756-5760, 1997). A Phase I clinical study was set to evaluate the safety and determine the MTD of CT011 in patients with advanced stage hematological malignancies. Methods: 15 patients with advanced hematological malignancies (AML7, NHL-4, CLL-3, HD-1) participated in the study. All pts failed several lines of conventional chemotherapy and radiotherapy as well as allo (n=6) or auto SCT (n=3). CT-011 was given in a single 5 hour IV infusion in escalating doses starting at 0.2 mg/kg up to 6.0 mg/kg (3 patients per dose). Results: CT-011 was safe and well tolerated with no treatment-related toxicities. Common adverse events included minimal allergic reactions and low grade fever. No single dose MTD was found in this study. One AML patient with resistant leukemia that was platelet-dependent with platelets <10x109/L is currently 7 months post CT-011 infusion in PR and is platelet transfusion-independent. Two additional patients (NHL-1, HD-1) remain with SD for over 6 months. Five other patients are alive with active disease with a median follow up of 3 (1-6) months, while seven patients died from their advanced resistant disease. Accrual to this study as well as patient follow up continues. Conclusions: A single administration of CT-011 is safe and well tolerated in patients with advanced hematological malignancies. The observed anti-tumor activity may be related to CT-011 interaction with the B7 receptor family-associated protein resulting in enhancement of tumorspecific immune response. Future studies will evaluate the combination of donor lymphocyte infusion and CT-011 for patients with hematological malignancies having minimal residual disease after stem cell transplantation.

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Irradiated myeloma cell line ARH 77 was used as tumor antigen to stimulate peripheral blood mononuclear cells (PBMC) of 8 healthy volunteers. Dendritic Cells loaded by irradiated autologous MM cells were used to stimulate PBMC of 10 MM patients. Activated responder T cells were immunomagnetically separated based on surface expression of interferon gamma and expanded in 5 cases by phytohemaglutinin and repeated high-doses of interleukin 2. Cytotoxicity against the original myeloma cells was tested after the expansion using propidium iodide or 7amino actinomycin D. Responder T cells were labeled by CFSE to distinguish them from myeloma cells. Third-party PBMC and interferon gamma negative fraction of T cells served as controls. The percentage of interferon gamma positive cells in healthy donors was enriched from 2.8±0.9 and 2.6±0.8 to 48.6±23.4 and 73.2±25.9 of CD3+CD4+ and CD3+CD8+ T cells, respectively, by immunomagnetic separation. Interferon gamma positive Tcells were further expanded in vitro from 0.54x106±0.05x106 to 214.00x106±103.46x106 within 4 weeks. A specific cytotoxicity was tested after expansion. The killing of myeloma cells by expanded interferon gamma positive T cells reached 68.1±14.2%, while interferon gamma negative fraction killed only 0.8±0.3% of myeloma cells. As another control, killing of third-party PBMC by expanded interferon gamma positive T cells was 6.9±2.5%. Similar observations were made in MM patients and will be presented on ISEH meeting. Data demonstrate a specific cytotoxic effect of expanded interferon gamma positive T cells against myeloma cell line ARH 77 and open the possibility for clinical use of tumor-specific T cells in cancer immunotherapy. Supported by grant IGA MZCR 7475-3

231 EX VIVO EXPANSION OF CMV LYSATE STIMULATED CD4+T CELLS FOR ADOPTIVE IMMUNOTHERAPY Y. Heike*, M. Hosokawa, Y. Morita, K. Tobinai, Y. Takaue National Cancer Center, Tokyo, Japan Cytomegalovirus (CMV) reactivation/disease was one of the serious complications after allogeneic stem cell transplantation. Although antivirus drugs have been developed, they caused other complications leading to the failure of transplantation. CMV specific T cells worked an important role in preventing and treating CMV reactivation/disease. Adoptive transfer of dendritic cells or T cells cultured with CMV antigen peptides are another therapeutic options and several groups reported the usefulness of them. In these years, our group monitored the recovery of anti-CMV T cells by methods of ELISPOT analysis and Tetramer analysis using HLA-A02 and –A24 associated antigen peptides. Those results suggested detection rate of CMV specific T cells in HLA-A24 cases were lower than that of HLA-A02 cases, which suggested the therapeutic strategy using A24 related CMV antigen peptide was not practical for HLA-A24 patient, which was a major population in Japan. Upon these backgrounds, we are planning the adoptive transfers of CMV lysate stimulated CD4 T cells against CMV reactivation/disease, which are resistant to the current anti-viral therapies. Materials and Methods: Peripheral Blood Mononuclear cells (PBMNCs) were prepared from twenty ml of peripheral blood obtained from healthy volunteers after written informed consents. PBMNCs were cultured with 10 microg/ml of CMV lysate and irradiated autologus MNCs, used as feeder cells, during first 9 days for selection, followed by additional 4 days culture with lysate, irradiated autologus MNCs and 50 U/ml IL-2. Result and Discussion: Cultured MNCs showed the following characters. 1) Total cell numbers on day 13 were 10- 30 times higher than initial cell numbers on day 0. 2) 90 % of CD3 T cells expressed CD4 antigens on day 13. 3) 99% of CD3+ cells showed CD45RO antigen. 4) 35% of CD4+ cells showed CD25 antigen. 4) Cultured CD4+T cells showed IFN-gamma production. We are transferring our culture system from a culture dish/flask system to a closed bag system for future clinical application. In our presentation, we will show our pre-clinical data and discuss our future immunotherapy

232 DOCOSAHEXANOIC ACID (DHA) EXERTS DIRECT CYTOTOXICITY ON SUSCEPTIBLE LEUKEMIC CELL LINES T. Yamagami*, C. Porada, R. Pardini, E. D. Zanjani, G. Almeida-Porada University of Nevada, Reno, NV, USA Studies have shown that Docosahexanoic acid, one of the main components of fish oil, helps inhibit the promotion and progression of breast, prostate and colon cancer. DHA has also been shown to increase the therapeutic effects of doxorubicin, mitomycin C and cyclophospahmide, while enhancing arsenic trioxide-mediated apoptosis in arsenic trioxideresistant leukemia cells. We have previously demonstrated that 150 microMolar DHA does not adversely affect human bone marrow (BM) derived-CD34+ cells in their ability to differentiate and proliferate in CFC assays, but induces apoptosis in vitro on the undifferentiated subtype of acute myeloid leukemia cell line KG1a. In the present studies, in addition to KG1a, we used K562, HL-60, and MOLT-4 leukemic cell lines to elucidate possible mechanisms involved in DHA-induced apoptosis. Leukemia cell lines were cultured for 4 days in the presence/absence of DHA (150 microMolar). Viability and percentage of apoptotic cells in each culture system were determined by flow cytometry using PI and AnnexinV. Progressive loss of viability and an increase in AnnexinV expression was observed on DHA treated KG1a and MOLT-4, but not on K562 and HL-60 cell lines. In DHA-susceptible cell lines, quantification of bcl-2 and Bax proteins demonstrated an increased expression of bax with time in culture, as well as an increased bax/bcl-2 ratio. In contrast, the inverse effect was observed in DHA-resistant cell lines. Furthermore, cell cycle analysis demonstrated that DHA induced G0/G1-cell cycle arrest in KG1a and Molt-4, while in DHA-resistance cell lines (K562 and HL-60), no significant change in cell cycle was observed. Our study shows that DHA may be able to exert direct cytotoxicity on certain types of leukemic cells, and could therefore be more broadly applied in the treatment of leukemias.

233 COMPARISON OF SHORT-TERM DENDRITIC CELLS CULTURES STIMULATED BY CALCIUM IONOPHORE AND CD40L IN HEALTHY VOLUNTEERS L. Kovarova1*, J. Michalek1,2, R. Hajek1,3 1 Dept. of Clinical Hematology, Masaryk University Hospital, Brno, Czech Republic 2 Pediatric Dept., Masaryk University Hospital, Brno, Czech Republic 3 Dept. of Internal Medicine - Hematooncology, Masaryk University Hospital, Brno, Czech Republic Objectives: Dendritic cells (DC) are antigen-presenting cells that can activate naive T lymphocytes and initiate a primary immune response. DC originate from bone marrow and alternatively they can be generated from mononuclear cells of peripheral blood. In this study, we analyzed the relative number of activated DC and expression of costimulation markers in healthy volunteers after three types of short-term cultivation with different stimulating agens as calcium ionophore (CI) and CD40Ligand (CD40L). Methods: 6 healthy volunteers were recruited from Transfusion Department of our hospital. Peripheral blood mononuclear (PBMNC) cells were separated and three types of DC cultures were initiated. Full PBMNC were cultured in RPMI-1640 supplemented with FCS, adherent PBMNC were acquired after 2h of culture in RPMI-1640 with or without FCS, then cultured in RPMI-1640 with FCS. CI or CD40L were added and the phenotype (CD80, CD83, CD86, HLA-DR, lineage mixture, CD3, CD14, CD19) was analyzed after 24 hours by multicolor flow cytometry. Aliquots of PBMNC were analyzed as controls. Results: The expression of the CD83 antigen was low on freshly isolated PBMNC (0,3%) and it was upregulated after 24h culture on DC. The highest expression was found in stimulated cultures of adherent PBMNC (without FCS) when compared CI and CD40L (13,6%; 17,8% respectively). The expression of CD86 was nearly the same for CI and CD40L stimulation (46%; 42%), but expression of CD80 was higher after stimulation with CI then CD40L (29,3%; 17,4%). Conclusion: There was an upregulation of costimulatory molecules CD80, CD86 and also HLA-DR on DC when CI or CD40L were used, but these stimulating agens unaffected the expression of CD83. These preliminary results were used to find optimal cultural strategy of DC. Supported by grant IGA MZCR NR 8081-3.

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234 HUMAN MYELOMA CELLS EXPRESS RUNX2 GENE THAT REGULATES OSTEOPONTIN PRODUCTION: ROLE IN MYELOMAINDUCED ANGIOGENESIS N. Giuliani1*, S. Colla1, F. Morandi1, M. Lazzaretti2, C. Mancini2, S. Bonomini1, R. Rizzato1, G. Sammarelli1, V. Rizzoli1 1 Hematology, University of Parma, Italy 2 Pathology, University of Parma, Italy Osteopontin (OPN) is involved in angiogenesis, cell survival and tumor progression. OPN gene expression by human cells is mainly regulated by the bone specific trascription factor Runx2 namely also CBFA1 (Runx2/ Cbfa1) even if other transcription factors may be also involved in the production of OPN by cancer cells as the CCAAT/enhancer-binding protein alpha (C/EBPa) and AML-1. In this study we show that human myeloma cell lines (HMCLs) but not normal CD19+ cells directly produce OPN and express its major regulating gene Runx2/Cbfa1. The activity of Runx2/Cbfa1 in HMCLs has been also demonstrated by a gel mobility shift assay. On the other hand we found that all human myeloma cell lines (HMCLs) tested were negative for C/EBPa mRNA, whereas AML-1A and AML-1B mRNA were expressed in all HMCLs even if any correlation has not been found between OPN expression and AML-1A/AML-1B ratio. In addition we found that OPN production by myeloma was up-regulated at both mRNA and protein level by IL-6 and IGF-I through a Runx2/Cbfa1 mediated mechanism; in turn recombinant human OPN was able to increase myeloma cells proliferation. The potential role of OPN in MM-induced angiogenesis has been also investigated in an experimental model of angiogenesis. rhOPN treatment stimulated vessel formation and the conditioned medium (CM) of HMCLs significantly increased vessel formation. On the contrary OPNimmunodepleted CM of HMCLs had not a stimulatory effect on vessel formation and the presence of anti OPN Ab inhibited vessel formation induced by HMCLs. The expression of OPN by purified bone marrow (BM) CD138+ cells has been also investigated in 60 newly diagnosed multiple myeloma (MM) patients, finding that 40% of MM patients tested expressed OPN. A significant higher microvascular density and number of microvessels per field were observed in the group of patients positive for OPN, in comparison with OPN negative ones. In conclusion our data highlight the direct ectopic production of OPN by human myeloma cells with a Runx2/CBFA1 mediated mechanism and the capacity of myeloma-derived OPN to stimulate angiogenesis.

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To determine relative HDI sensitivities, AML, BL and MM cell lines were exposed for 72 hours to increasing concentrations of three different HDIs, trichostatin A (TSA), sodium valproate (SV) and suberoylanilide hydroxamic acid (SAHA) and cell survival measured. A wide spectrum of sensitivities were observed however the sensitivities of MM, BL and AML cell lines to HDIs, did not correlate with their differing patterns of HDAC expression. These data implicate that underlying mechanisms, other than HDAC expression alone dictate sensitivity to HDIs. These findings have important implications for the understanding of the role of HDACs in cancer and for the future therapeutic use of their inhibitors

236 EFFECTS OF SPHINGOSINE 1-PHOSPHATE (S1P) AND EXPRESSION OF S1P RECEPTORS IN CHRONIC LYMPHOCYTIC LEUKEMIA (B-CLL): POTENTIAL ROLE IN CELL TRAFFICKING AND SURVIVAL G. Seitz*, S. Yildirim, A. M. Boehmler, L. Kanz, R. Möhle University of Tübingen, Tübingen, Germany Recent studies suggest that presence of the lipid mediator sphingosine 1phosphate (S1P) in the peripheral blood and expression of S1P receptors (i.e., S1P1) is a prerequisite for the egress of lymphocytes (particularly T cells) from lymphoid organs into the circulation. As circulating lymphocytic lymphoma cells are a hallmark of chronic lymphocytic leukemia, we analyzed expression of different S1P receptors and the effects of S1P on CLL cells. By qualitative and quantitative (TaqMan) RTPCR, mRNA expression of S1P1 and S1P4 was found in CLL cell lines (EHEB, MEC-1) and in most samples of primary CD19+ cells isolated from the peripheral blood of untreated B-CLL patients. mRNA of other S1P receptors (S1P2, S1P3, S1P5) was less consistently found. Normal, nonmalignant B cells were strongly positive for S1P1, while other S1P receptors were weakly expressed or negative. S1P induced typical effects of chemotactic G protein-coupled receptors, such as actin polymerization and chemotaxis (modified Boyden chamber assay) in CLL cell lines. Also primary CLL cells responded to S1P with increased actin polymerization as a first step of cell locomotion. Of note, the S1P1/5 superagonist (antagonist) FTY720 (1 uM) induced apoptosis in primary CLL cells as measured by MTT-test and staining with Annexin-FITC, suggesting that also cell proliferation and survival might be regulated by S1P receptors. We conclude that sphingosine 1-phosphate, ubiquitously present in considerable amounts in the peripheral blood, may contribute to the trafficking of B-CLL cells, and their prolonged survival.

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THE VARIABLE POTENCY OF HISTONE DEACETYLASE (HDAC) INHIBITORS TO KILL LEUKAEMIA CELLS IS NOT DEFINED BY THE HDAC EXPRESSION PROFILE OF THE TARGET CELLS F. L. Khanim1*, C. Pearce1, R. E. Hayden1, B. M. Turner2, C. Craddock3, G. Pratt4, C. M. Bunce1 1 School of Biosciences, The University of Birmingham, UK 2 Div of Immunity and Infection, The University of Birmingham, UK 3 CRUK Institute of Cancer Research, The University of Birmingham, UK 4 Dept of Haematology, Heartlands Hospital, Birmingham, UK Tissue homeostasis, requires the co-ordinated expression of genes regulating cell proliferation, differentiation and apoptosis genes. Modification of core histone proteins by histone deacetylases (HDACs) is one mechanism regulating this process. It has become evident that HDAC activity is deregulated in many cancers, including leukaemias, leading to intense interest in the possible therapeutic benefits of HDAC-inhibitors (HDIs). Although HDI trials are underway in a wide range of tumours, it is not yet known whether different cancers deregulate different HDACs and whether, as a consequence, they show selective sensitivities to separate HDIs. Quantitative real-time PCR was used to compare the mRNA levels of the three classes of HDACs and other histone modifiers in B-cell tumours; including chronic lymphocytic leukaemia (CLL), Burkitt's lymphoma (BL) and multiple myeloma (MM), and in acute myeloid leukaemia (AML). Strikingly, similar expression patterns were observed between the B-cell tumours with a generalized over-expression of all HDACs with the sole exception of HDAC11 which was under-expressed. This result was distinct from that seen in AMLs in which overall expression was variable but with consistent downregulation of HDAC5, RBBP4, SIRT4 and upregulation of RBBP7, HDAC2 and MLL.

A NOD/SCID XENOGRAPH MODEL OF DIFFUSE LARGE CELL LYMPHOMA R. M. Rossi*, R. A. Conkling, S. H. Bernstein, C. J. Jordan James P. Wilmot Cancer Center, University of Rochester, Rochester, NY, USA. The objective of this study was to establish animal models of lymphoma for use in testing the efficacy of chemotherapy agents and various experimental drugs. To achieve this goal, 8 Diffuse Large Cell Lymphoma (DLCL) cell lines were chosen for analysis, consisting of both the activated B cell (OCI-Ly 3, 10) and germinal center (OCI-Ly 7, 19, Toledo, Farage, and SUDHL 4 and 6) types of DLCL. Each line was transplanted into immune deficient NOD/SCID mice, without prior conditioning, and tested using both subcutaneous (SQ) and intravenous (IV) delivery. In addition, total cell number and injection volume were varied. The growth rates of the tumors in vivo were determined and tissue samples were obtained to assess tumor pathology and molecular endpoints. Of the 8 lines tested, 4 consistently developed tumors (OCI-Ly10, OCI-Ly7, OCI-Ly19, and Farage), with kinetics ranging from 10-60 days post-transplant. The remaining four lines failed to form tumors at 6 months post injection. Analysis of subcutaneous tumors showed marked vascularization with robust localized growth. Intravenous injection of the OCI-Ly19 and to a lesser extent the OCI-Ly10 lines generated disseminated disease with engraftment of marrow, spleen, liver and some lymph node tissues. Splenic histology indicated disruption of the red and white pulp areas. In addition, preliminary data indicate CNS infiltration for OCI-Ly19 tumors. To permit real time in vivo imaging studies, both OCI-Ly10 and 19 were modified to express luciferase and shown to be detectable in vitro at cell concentrations as low as 50,000. Further, in vivo analyses indicated luciferase expression in multiple tissues and tumor masses. In summary, we have generated

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versatile and reproducible DLCL xenograph models. Currently, studies are underway to determine the in vivo effects of several novel therapeutic agents, including proteasome inhibitors, PKC-beta inhibitors, and the novel triterpeniod CDDO.

238 EXPRESSION OF CD133 IN T-ALL: MALIGNANT TRANSFORMATION OF A PRIMITIVE PROGENITOR CELL? C. V. Cox1*, R. S. Evely2, A. Blair1,3 1 Bristol Institute for Transfusion Sciences, Bristol, UK 2 Bristol Haematology and Oncology Centre, Bristol, UK 3 University of Bristol, Bristol, UK Treatment of children with T-ALL has improved considerably over the last decade. However, a significant proportion ~30% remain refractory to treatment. The disease may be maintained by a subpopulation of cells that are resistant to drug regimens designed to target the bulk population. We have previously shown that T-ALL cells capable of long-term proliferation in vitro and in vivo are CD34+/CD4- or CD7-. In this study we have attempted to further characterise these putative T-ALL stem cells by investigating the expression of CD133 on these cells. T-ALL cells from 5 patients for were sorted for expression of CD133 and CD7 and growth was evaluated in long term culture and in the NOD/SCID model. In this group of patients, the CD133+/CD7+ and CD133+/CD7- subfractions represented <1% of total nucleated cells at sorting, (0.45±0.3% & 0.35±0.2%, respectively). However, after 3 weeks in culture, 49±19% of proliferating cells were derived from the CD133+/CD7- subfraction and this trend continued with >58±25% of the proliferating cells derived from this subfraction at week 6. Morphological analyses confirmed the cultured cells had blast morphology. Results from the NOD/SCID assay in 3 cases to date, demonstrate that engraftment was achieved using 105-107 unsorted cells (21-98% CD45+) and with the CD133+/CD7- subfraction only (0.554.4% CD45+) using as few as with 1.4x103-5x103 cells. There was no engraftment with the other subfractions despite injecting 10 to 1000 fold more cells. The engrafted cells expressed high levels of CD34, CD2, CD4 and CD7 and very low levels of CD133. The phenotype of the engrafted cells was similar to that of the patients at diagnosis, implying they had differentiated in vivo. These data add to the evidence that a cell with a primitive phenotype is the target for transformation in T-ALL. These cells may be the most relevant targets for emerging therapeutic strategies.

239 CLINICAL USEFULNESS OF FREE LIGHT CHAIN CONCENTRATION AS A TUMOR MARKER IN MULTIPLE MYELOMA S. Y. Kang1, J. T. Suh1, H. J. Yoon2*, W. I. Lee1 1 Department of Laboratory Medicine, KyungHee University School of Medicine, Seoul, Korea 2Department of Hematology-Oncoloy, KyungHee University School of Medicine, Seoul, Korea Monoclonal immunoglobulin, as a marker for monoclonal gammopathy, is evaluated by protein electrophoresis (PEP) and immunofixation electrophoresis (IFE). However, PEP and IFE are not satisfactory in sensitivity, objectivity and facility. Recently, a highly sensitive, automated immunoassay for measurement of free light chain (FLC) concentrations in serum and urine has been developed for the identification and monitoring of patients with monoclonal gammopathy. To explore the clinical usefulness of measurement of FLC concentrations, we measured the kappa(K) and lambda(L) FLC concentrations and calculated the K/L FLC ratios for three groups (multiple myeloma (MM), other diseases, and control) and compared the results of the FLC assay with the results of PEP or IFE. The concentrations of serum K and L FLCs and the K/L FLC ratios for the MM group and non-MM groups were distinct. In the MM group, some sera and urine samples had no evidence of M protein on PEP and IFE, but FLC assay showed abnormal concentrations of FLCs and abnormal K/L FLC ratios in most cases. As compared with the PEP, the K/ L FLC ratio revealed higher sensitivity in all diagnostic ranges with different cutoff values. Particularly, when the cutoff value 2.0 for K/L FLC ratio was used, specificity and PPV were largely improved than when the cutoff values 1.2 and 1.5 were used. These findings indicated that FLC assay enables to detect myeloma patients with very low M protein due to early stage or after therapy and to distinguish patients with monoclonal increase of FLC from patients with polyclonal increase of FLC due to other

conditions, particularly using K/L FLC ratio 0.3-2.0 as a diagnostic range. Despite of some technical limitations of the assay, the incorporation of K/L FLC ratios with FLC concentrations is useful in detection of M protein, particularly with negative serum or urine IFE results, and differentiation of monoclonal gammopathies from patients with polyclonal increase of FLC due to other conditions.

240 BEZAFIBRATE AND MEDROXYPROGESTERONE ACETATE AS NOVEL THERAPIES IN B-CELL CLL R. E. Hayden1, F. L. Khanim1, N. J. Davies1, G. Pratt2, M. T. Drayson3, P. A. H. Moss4, C. M. Bunce1* 1 School of Biosciences, University of Birmingham, UK 2 Department of Haematology, Heartlands Hospital, Birmingham, UK 3 Division of Immunity and Infection, University of Birmingham, Birmingham, UK 4 CRUK Institute of Cancer Research, University of Birmingham, Birmingham, UK We have previously demonstrated that combined bezafibrate (bez) and medroxyprogesterone acetate (MPA) provide a powerful pro-apoptotic signal against Burkitts lymphoma (BL) cell lines and primary BL cells. Here, we demonstrate similar killing of B-cell CLL cells. Primary CLL peripheral blood mononuclear cell preparations were cultured in three culture conditions: on irradiated stromal cultures that either express or do not express CD40-ligand and in the presence of IL4, or in medium supplemented with the commercial serum replacement ITS+ and in the absence of stroma and Il-4. These culture conditions were selected to provide a variable state of activation, reflecting the in vivo status of CLL cells. Cell survival in all cultures was high. Thymidine incorporation and MTT assays demonstrated that all CD40-ligand stimulated CLL cells were driven into cell cycle, whereas in the other culture systems the cells remained non-proliferate. Both bez and MPA reduced thymidine incorporation in all CD40-ligand exposed CLLs and markedly so when used in combination (bez/MPA). However, cell killing by bez/MPA was not restricted to cycling CLL cells as demonstrated by both MTT assays and measures of apoptosis, including mitochondrial depolarization, annexin V staining and accumulation of sub-G1 DNA. Conversely, responses of individual CLL samples in non-CD40-ligand exposed cultures were more variable. We have also previously reported the potentiation of AML cell differentiation and apoptosis by CA/MPA. Based on these studies we have instigated a phase II trial of Bezalip (Bez) and provera (MPA) in elderly and relapsed AML (acronym BAP). The drugs have been well tolerated and have in vivo activity as measured by haematological responses and changes in transfusion dependency. The data described here indicate that a similar approach may provide benefit in CLL.

241 BOTH CASPASE-DEPENDENT AND -NDEPENDENT DEATH PATHWAYS ARE INDUCED BY PHOTODYNAMIC THERAPY USING TH9402 Q. Y. Dai1*, J. Filep1, P. Dubé1, C. Scotto2, D. C. Roy1 1 Division of Hematology-Oncology, Hospital Maisonneuve-Rosemont, Montreal, Canada. 2 Celmed BioSciences, Montreal, Canada. Purpose: Photodynamic therapy (PDT) is a powerful strategy to eliminate a variety of tumors and alloreactive T cells. While the cytotoxic effect of the novel rhodamine-derived TH9402 photosensitizer has been attributed to the generation of radical oxygen species, the cell death pathways involved have not been investigated. In this study, we evaluated the ability of PDT to induce apoptosis and necrosis, and assessed the underlying molecular mechanisms. Results: We found a direct correlation between TH9402 concentration (0-10 microM) and intensity of light exposure (0-10J/cm2) with the extent and rapidity of elimination of malignant lymphoma EL-4 and mastocytoma P815 cells. Increasing the intensity of PDT shifted the ratio from predominantly apoptotic to predominantly necrotic cell death as measured by Annexin-V/7AAD at 2-4h after PDT. To study the induction of apoptosis, we measured the impact of PDT (10 microM and 5J/cm2) on mitochondrial transmembrane potential using DiOC6(3) and detected marked decreases as early as 2h after PDT. This was associated with release of cytochrome C from the mitochondria. Investigation of the

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34th Annual Meeting: ISEH - Abstracts / Experimental Hematology 33 (2005) 39-143 caspase-dependent (classical) apoptotic pathway revealed activation of caspase 9 and 3, as early as 15 minutes after PDT. TH9402 also evoked cytosolic release of AIF, suggesting involvement of a caspase-independent (non-classical) pathway. This was accompanied by decreased presence of Bax protein in the cytosol. Finally, caspase inhibition with ZVAD-FMK and DEVD-CHO failed to alter the percentage of PDT-induced AnnV+/ 7AAD- cells, indicating involvement of a caspase-independent death pathway. Conclusion: Using conditions that induce apoptosis, PDT causes activation of both caspase-dependent and -independent death pathways. The observation that the TH9402 photosensitizer mediates cytotoxicity through activation of a number of death pathways suggests that it could effectively limit the capacity of tumor cells to become resistant to TH9402.

242 RAPID GENOTYPING FOR THIOPURINE METHYLTRANSFERASE POLYMORPHISMS IN PATIENTS WITH ACUTE LEUKAEMIA USING LIGHTCYCLER PCR M. A. Catherwood1*, J. E. A. Davison1, M. F. McMullin1,2 1 Haematology Department, Belfast City Hospital, Belfast, UK 2 Department of Haematology, Queen's University of Belfast, Belfast, UK Introduction: Patients with acute lymphoblastic leukaemia (ALL) are treated with thiopurines. Thiopurine methyltransferase (TPMT) catalyses the S-methylation of thiopurines and more than 11% of the Caucasian population are heterozygous or homozygous carriers for TPMT polymorphisms. TPMT deficient patients accumulate excessive thioguanine nucleotides in haemopoietic tissue leading to severe haematological toxicity. Therefore, screening for TPMT polymorphisms in patients prior to treatment with thiopurines is advantageous. The aim of this study was to develop a rapid genotyping method for the detection of TPMT polymorphisms suitable for high throughput in a clinical setting and compare this with standard methods in a group of patients with acute leukaemia. Materials and Methods: TPMT genotype was determined for the TPMT alleles (TPMT *2, *3A, *3B & *3C) in a group of 55 patients with ALL using LightCycler PCR. Results: All 55 ALL patients were homozygous for the wildtype allele of the polymorphism G238C in exon 5. Three Patients were heterozygous for the G460A mutation in exon 7 by standard PCR with restriction fragment length polymorphism (RFLP) and four were heterozygous by LightCycler PCR, which were confirmed by sequencing. Three patients were heterozygous for the A719G mutation in exon 10 using LightCycler PCR, which was confirmed by sequencing. Of these three patients, one was found to also have the G460A mutation. Conclusion: We describe a rapid assay for the genotyping of TPMT polymorphism using real-time fluorescence PCR to facilitate rapid processing of samples in a clinical setting. This strategy is superior to standard PCR-RFLP genotyping methods which in our study has produced false-positive results. This rapid diagnostic assay provides informative data on TPMT polymorphisms in ALL patients prior to treatment with thiopurines.

NEW CHEMOTHERAPY PROTOCOL (P-CAN) FOR TREATMENT OF AGGRESSIVE NON-HODGKIN'S LYMPHOMA K. Rasul1*, A. Awidi1, M. Kelta1, A. Mubarak1, U. Al-Homsi1, A. AlHassan1, A. Chung-Lopez2, A. Bener3 1Department of Medicine, Hematology/Oncology Section, Hamad Medical Corporation, Doha, Qatar 2Department of Laboratory Medicine and Pathology, Hamad Medical Corporation, Doha, Qatar 3Department of Medical Statistics, Hamad Medical Corporation, Doha, Qatar Background: This work aims at determining the efficacy of modified CHOP combination in which Vinorelbine (Navelbine) replaces Vincristine for the treatment of aggressive Non-Hodgkin's Lymphomas (NHL). Patients and methods: this open label pilot study included 19 patients with aggressive NHL and one patient with low grade NHL who were treated with the new combination which we abbreviated as P-CAN (Prednisolone 100mg/day P.O day 1-5, Cyclophosphamide 750mg/m2 i.v day 1, Adriamycin (Doxorubicin) 60mg/m2 i.v day 1, Navelbine (Vinorelbine) 30 mg/m2 i.v day). The patients were 13 males and 6

females, mean age 50 years (34-65), performance state 0-2, International prognostic index (IPI) 0-3. Seven patients stage I, one patient stage II, eight patients stage III and 3 patients in stage IV. 14 patients with nodal disease and five patients with extra-nodal disease. They received total of 97 cycles of the chemotherapy (3-7 cycles). Results: 18 out of 19 patients achieved complete response (CR). In one patient the response could not be assessed, one patient progressed while on treatment. Toxicity was mainly hematological. The 3 years overall survival (OS) anddisease free survival (DFS) were 83%. Conclusion: P-CAN is an effective, well tolerated combination in chemo-naive aggressive NHL. The addition of Vinorelbine to steroid, Adriamycin, and Cyclophosphamide seems improve the response. Further larger trials are needed to study this combination and its impact on longer overall survival.

244 THE COMPARATIVE ANALYSIS OF SERUM PROTEOMES FOR THE DISCOVERY OF BIOMARKERS FOR HODGKIN'S DISEASE J. Y. Kwak1*, N. R. Lee1, E. K. Song1, C. Y. Yim1, Y. G. Kwak2 1Internal Medicine, Chonbuk National University Hospital, Jeonju, South Korea 2 Pharmacology, Chonbuk National University Medical School, Jeonju, South Korea Backgrounds and Purpose: Hodgkin's disease(HD) belongs to lymphoid malignancies of B cell origin and it's etiology is known to be related to EBV infection. The histological characteristic of HD is the presence of Reed-Sternberg(RS) cells and the diagnosis is established by confirming the histologic findings of the tissues from involved organs. In order to find less invasive, easier and simpler diagnostic methods for HD, we analyzed serum proteomes of HD patients. Materials and Methods: We compared the two dimensional electrophoresis patterns of bone marrow sera of six patients with HD and those of six normal subjects. The differentially expressed spots between the two groups were identified by matrix-assisted laser desorption/ionizationtime-of-flight(MALDI-TOF) and electrospray ionization quadupole timeof-flight(ESI Q-TOF) mass spectrometries. Results: Seven spots, immunoglobulin heavy chain gene 1 protein, fibrinogen gamma chain precursor, complement component 3 precursor, alpha-1-antitrypsin precursor, alpha-1-B-glycoprotein, alpha-1-antitrypsin, and chain A of alpha-1-antichymotrypsin, were up-regulated, and two spots, ALB protein and chain A of human serum albumin mutant R218h complexed with thyroxine were down-regulated in the sera of HD group compared with control group. Conclusion: Our current study suggests that if further studies are carried out, the differentiated expressed serum proteins might be used as simpler diagnostic and follow-up markers for HD.

245 CHINESE HERBS DECOCTION BEVERAGE INDUCING SIGNIFICANT RESPONSE IN PROGRESSIVE B-CLL AND IN VITRO APOPTOSIS ON B CLL CELLS A. Berrebi*, L. Bassous, L. Shvidel Kaplan Medical Center, Rehovot, Israel An article published in Leukemia Research (2003, 27) reported remission in a CLL patient treated only by Chinese herbs with a significant apoptosis of B-CLL cells in vitro. An 80-year-old biologist was followed in our Institute for several years because of B-CLL that progressed slowly to stage Binet B, including high lymphocytosis (80x109/l) and splenomegaly. This patient who by himself read this article, asked the article authors to refer him to this Chinese medicine, unsuccessfully. Then he asked a well-known Israeli herbalist to prescribe him Chinese herbs. These herbs mixture had to be decocted in water and the extract was recommended to be taken every day at least 0.6 liter per day. We should emphasize that our Institute was not involved in the decision of the patient to take this medication. A slowly decrease of the lymphocyte count to 6x109/l after 6 months of this treatment was observed. We studied the effect of the extract on apoptosis in vitro on B CLL cells in culture for 20 hours to 5 days using annexin kit for flow cytometry. We examined samples from 9 advanced B CLL patients. In five cases a significant apoptosis (30%-100%) was noted after 20 hours, and in 4 other cases up to 90% of apoptosis was obtained after 3 to 5 days by adding the herbal extract every day. The optimal effect was equally with 1:10 and 1:20

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dilutions. In comparison, the control cultures of B CLL cells remained viable after 5 days. In conclusion, a Chinese herbal decoction beverage (which formula is not yet known) induced a significant response in a progressive B CLL patient and apoptosis in vitro on B CLL cells from patients with advanced disease. We believe that this outstanding response looks out of interest, and in the next steps we will focus on segregation of the potential effect of these herbs.

246 DOUBLING TIME OF SOLUBLE CD23 IS AN ADDITIONAL MARKER IN CLL PATIENTS EXPRESSING OR NOT ZAP-70 N. Meuleman*, L. Lagneaux, M. Dejeneffe, A. Delforge, D. Bron Jules Bordet Institute, Brussels, Belgium Background: Chronic lymphocytic leukemia (CLL) is a disease with unpredictable natural history in stage A patients (pts). Recently, It has been demonstrated that ZAP-70 and IgVH mutational status are powerful biological prognostic factors and we previously reported that the doubling time of soluble CD23 (sCD23DT) is a prognostic factor for progression of disease in untreated stage A CLL. Therefore we evaluated prospectively ZAP-70 expression and IgVH mutational status in untreated CLL patients (pts) and correlated these markers with sCD23DT. Methods: sCD23 level is evaluated by commercial available enzymelinked immunosorbent assay (ELISA).ZAP-70 expression is determined in leukemic cells by flow cytometry, and positive expression was defined as >20% positive cells. Results: 67 pts with 53 stage A, 9 Stage B, 5 stage C were evaluated, the median age was 62 years. The median follow-up was 55 months. At present, results of 31 pts are available for analysis and an update will be made for July 2005. In the group of pts with positive ZAP-70 expression (n=11), 55% (n=6) required a treatment and 83% had a sCD23DT inferior to one year. Among ZAP-70+ and sCD23DT<1 year pts, 83% required a treatment. For pts with positive ZAP-70 and a sCD23DT superior to one year, 80% of pts do not need a treatment. In the group of good prognosis pts with negative ZAP-70, 40% of the pts required a treatment. in the ZAP70 negative pts population, 4/20 have a sCD23DT<1 year and all these pts required a treatment for disease progression. All the pts with indolent progression, negative ZAP-70 and who do not need a treatment have a sCD23DT superior to one year In conclusion, 1) sCD23DT remains a prognostic marker for disease progression even in ZAP-70 negative patients. 2) In ZAP-70 positive pts, the sCD23DT allows to better identify the pts requiring a treatment.

247 BLOOD DENDRITIC CELLS IN PATIENTS WITH HEMATOLOGICAL MALIGNANCIES J. Weclawek-Tompol, E. Grotthus, A. Chybicka*, B. Rybka, R. Ryczan, D. Noworolska-Sauren Department of Children Bone Marrow Transplantation, Oncology and Hematology Medical University of Wroclaw, Poland Dendritic cells (DCs) constitute heterogeneous leukocyte population with various functional and phenotypic characteristics. They play fundamental role in induction and control of primary as well as secondary immune response and may be also important for the induction of immunologic tolerance. The aim of the study was to analyze subpopulations of blood DC (BDC) in patients with hematological malignancies, at various points of treatment, in comparison with control group, and to estimate the correlation between number of BDC and activation of T-lymphocytes. The total number of 82 patients: 36 females and 46 males, aged from 15 months to 18 years (median 9 years) treated in the Department of Children Bone Marrow Transplantation, Oncology and Hematology, Medical University of Wroclaw, Poland (ALL - 45, AML – 15, HD – 10, NHL-B – 8, LCAC - 4) were examined. Control group contained of 21 healthy children – potential donors to allogenic BMT. Peripheral blood was collected at time of diagnosis, during chemotherapy, at time of relapse or progression. The populations of BDC and activated lymphocytes CD3+CD26+ were analyzed by flow cytometry. The subpopulations of BDC were defined on the basis of expression of specific markers for distinct blood dendritic cell populations: BDCA-1, BDCA-2, BDCA-3. The activated BDC were identified when antigen CD 83, characteristic for their activation, was expressed.

We observed increased percentage and absolute count of BDC before start of treatment in children with ALL or AML, compared to control group. Increased percentage of BDC was present during whole therapy. Those yields explained positive correlation with value of activated T lymphocytes. Increased value of BDC in children with hematological malignancies, as well as in children 1-2 months after cessation of treatment, may indicate an activation of immune system. This fact may play important role in control of residual disease.

248 RIN3, A RAS INTERACTING RAB5 GEF, THAT IS HIGHLY EXPRESSED IN CHRONIC LYMPHOCYTIC LEUKAEMIA, FUNCTIONALLY ASSOCIATES WITH MIP1 ALPHA IN THE EARLY ENDOSOME AND LINKS INTRACELLULAR SIGNALLING AND VESICLE TRANSPORT A. D. Clark1*, L. Telfer1,2, J. Baird1, G. J. Graham1,2 1 Cancer Research Beatson Laboratories, Glasgow, UK 2Division of Immunology, Infection and Inflammation, Glasgow University, Glasgow, UK Chronic lymphocytic leukaemia is the commonest leukaemia in the western world. Here we describe exciting data supporting a role for RIN3 as a ras effector linking intracellular signalling and vesicle transport in these cells. We initially isolated murine RIN3 (muRIN3) from embryoid bodies as a proportion of the constituent ES cells transited a haemopoietic stem cell (HSC) phenotype. Full length muRIN3 was obtained by library screening and RACE PCR technology. The human homologue (huRIN3) was found to map to chromosome 14q32. Using full length muRIN3 to screen chromosome 14 DNA extended the published sequence of huRIN3, importantly defining an amino terminal SH2 domain. Expression levels, assessed by array screening and real time PCR, fell as stem cells underwent myeloid differentiation but increased expression was seen during lymphopoiesis. High expression levels were confirmed in human and murine secondary lymphoid tissue by northern blotting and real time PCR. HuRIN3 was then found to be markedly overexpressed in human Chronic Lymphocytic Leukaemia compared to normal lymphocytes. Interestingly, no expression was seen in Burkitt's lymphoma which is associated with a t(8:14q32) translocation. Functionally, domain homology data suggested that RIN3 acted as a Rab5GEF. Confocal microscopy confirmed interaction between RIN3 and Rab5 in early endosomes where RIN3 also co-localised with MIP1 alpha in dramatically enlarged endosomal vesicles when internalised via the D6 receptor. RIN3 interacted specifically with GTP bound Ras, in pull down assays and yeast two hybrid binding partners included Abi1/E3B1, Diacylglycerol kinase alpha, TGF beta stimulated clone 22, and a novel Rho GEF homologous to the Vav proteins, all components of signalling cascades. In summary, these data suggest that RIN3 is involved in linking intracellular signalling and vesicle transport, possibly by specifically activating or sequestering downstream Ras effectors in macromolecular complexes or modulating signal transduction by influencing vesicle movement.

249 SUPPRESSION OF B LYMPHOPOIESIS BY NEOPTERIN IS BASED ON STROMAL CELL-DERIVED CYTOKINES, AND FACILITATES A CAPACITY OF MYELOID CELL PROLIFERATION DURING INFLAMMATORY PROCESS I. Tsuboi1*, S. Aizawa1, T. Harada1, M. Hiramoto1, Y. Hirabayashi2, J. Kanno2, T. Inoue3 1Deapartment of Anatomy, Nihon University School of Medicine, Tokyo, Japan 2Division of Cellular and Molecular Toxicology, National Institute of Health Sciences, Tokyo, Japan 3 Biological Safety and Resarch Center, National Institute of H Health Sciences, Tokyo, Japan Neopterin (NP) is produced by monocytes and is known to be a useful biomarker for inflammatory immunological activation. We previously found that NP enhances in vivo and in vitro granulopoiesis by inducing stromal cell production of cytokines in mice. In the senescence accelerated (SA) mice, because of their known stromal cell impairment after 30 weeks old, mice show simultaneous down-regulation of IL-7, a positive regulator for B lymphopoiesis, as well as TGF-b, a negative regulator, resulting in

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their senescent suppression of B lymphopoiesis. Thus, the model may be useful tool to contribute to elucidate possible interaction of NP and stromal cells on the B lymphopoiesis. In young control mice, NP decreased the number of colonies in semi-solid medium derived by pre-B cell progenitor cells (CFU-pre-B) from whole bone marrow cells, but not those from a proB/pre-B-enriched cell population, possibly due to lack of the stromal cells component. Intraperitoneal injection of graded doses of NP into control mice resulted in a profound and dose-dependent reduction of femoral CFUPre-B colonies within 1 day. Gene expression of negative regulators for B lymphopoiesis, such as TGF-b, TNFa and IL-6 in the bone marrow were up regulated within 6 h after NP treatment. However, in the case of elderly SA mice, the in vivo and in vitro responses of B lymphopoiesis after NP treatment as well as the up-regulation of negative regulators in the bone marrow was less-significant than in young SA mice. These results demonstrated that NP can not enhance but suppress B lymphopoiesis by inducing stromal cell derived cytokines and that the response was much milder in the elderly SA mice than in young SA control mice due to deterioration of stromal cell function with age.

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Abstracts

ACCLERATED TELOMERE SHORTENING IN GPI(-) GRANULOCYTES FROM PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH) DETECTED BY NEWLY DEVELOPED PROAEROLYSIN FLOW-FISH F. Beier1*, S. Balabanov1,2, T. Buckley3, K. Dietz4, U. Hartmann1, M. Rojewski5, L. Kanz1, H. Schrezenmeier5, T. H. Brümmendorf1,2 1 Division of Hematology, Oncology and Immunology, University of Tübingen, Germany 2Deptartement of Hematology and Oncology, University Hospital Eppendorf, Hamburg, Germany 3Department of Biochemistry and Microbiology, University of Victoria, Victoria, B.C., Canada 4Department of Medical Biometry, University of Tübingen, Germany 5Department of Transfusion Medicine, University of Ulm, and Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, Germany Paroxysmal nocturnal haemoglobinuria (PNH) is caused by a somatic mutation in the X-linked PIG-A gene resulting in a deficiency of GPIlinked proteins on the cell surface. Accelerated telomere shortening has been linked to disease stage and degree of (pan-) cytopenia in patients with bone marrow failure syndromes. The aim of the current study was to analyze the impact of replicative stress on telomere length in residual GPI(+) vs. GPI(-) hematopoiesis in patients with PNH. For this purpose, we developed Proaerolysin flow-FISH, a novel methodology which combines Proaerolysin staining (for GPI expression) with flow-FISH (for telomere length measurement). Peripheral blood granulocytes from 16 patients with PNH and 22 healthy individuals were analyzed. We found significantly shortened telomeres in GPI(-) granulocytes (mean±SE: 6.26±0.27 telomere fluorescence units (TFU), both compared to their GPI(+) counterparts (6.88±0.38 TFU; p=0.03) as well as to age-matched healthy individuals (7.73±0.23 TFU; p<0.001). Our findings are in support of a selective growth advantage model of PNH assuming that damage to the GPI(+) HSC compartment leads to compensatory hyperproliferation of residual GPI(-) HSC.