poster presentations - poster session i

34th Annual Meeting: ISEH - Abstracts / Experimental Hematology 33 (2005) 39-143 73

Abstracts

POSTER PRESENTATIONS - POSTER SESSION I

135INTERNAL TANDEM DUPLICATION AND ASP835 MUTATION OF THE FMS-LIKE TYROSINE KINASE 3 (FLT3) GENE IN CHILDREN WITH ACUTE PROMYELOCYTIC LEUKEMIAY. Hoshi1*, T. Rikiishi1, K. Fujii1, N. Suwabe1, M. Imaizumi1, K. Iinuma1, M. Tsurusawa2, K. Horibe3, J. Hara4, I. Tsukimoto51Tohoku University School of Medicine, Sendai, Japan2Aichi Medical University, Aichi, Japan3National Nagoya Hospital, Nagoya, Japan4Osaka University, Osaka, Japan5Toho University, Tokyo, Japan

[Objective] FLT3 regulates cell growth and is frequently mutated inacute promyelocytic leukemia (APL). Although FLT3 mutation is thoughtas a risk factor for adult patients, its clinical significance remains unclearfor children. In this study, we investigated clinical relevance of FLT3mutation in children with APL.

[Patients and Methods] 48 children (30 boys, 18 girls; age <16y) withAPL were studied. With cDNA prepared from marrow cells of patients, thePML-RARalpha and FLT3 mutation were analyzed as described (Tohoku JExp Med 193:127, 2001; Blood 103:1085, 2004, respectively). Briefly,RT-PCR amplified DNA fragments of FLT3 containing regions of exons14/15 for internal tandem duplication (ITD) or Asp835 mutation weresequenced or digested with EcoRV.

[Results] Of 48, six patients (12.5 %) had ITD and six (12.5 %) hadAsp835 mutation. The average age of patients with ITD or Asp835mutation (10.8y) was significantly higher than that of patients withoutmutation (8.1y) (P<0.05). As compared to average white blood cell countat onset in patients without mutation (6575/ul), that of patients with ITDwas significantly higher (34498/ul) (p=0.05), whereas that of patients withAsp835 mutation (24650/ul) was not significantly different. Moreover, thefrequency of ITD or Asp835 mutation in patients with S-form PML-RARalpha (46 %) was significantly higher than that in patients with L-form (15 %) (p<0.05).

[Discussion] In childhood APL, FLT3 was mutated in older children atfrequency of 25 % with no difference between ITD and Asp835 mutation,suggesting a lower frequency of FLT3 mutation in children than thatreported in adult APL. Moreover, a significant association of FLT3mutation with hyerleukocytosis or S-form PML-RARalpha may suggestFLT3 mutation to be a risk factor for children with APL as well. However,a longer period of observation was needed to clarify whether FLT3mutation would be associated with poor outcome.

136THE POTENTIAL OF ACHIEVING A REMISSION CAN BE IDENTIFIED USING GENE EXPRESSION ANALYSIS AT DIAGNOSISA. F. Gilkes, K. I. Mills*, A. K. BurnettWales College of Medicine, Cardiff University, Cardiff, UK

Acute Myeloid Leukaemia (AML) is a heterogeneous disease as can beseen at the morphological, cytochemical, immunological, cytogenetic andmolecular level. Cytogenetics has been established as a major predictor ofresponse to treatment and risk of relapse, and are the basis of the currentpatient treatment schemes. Patients are stratified, by cytogenetics, atdiagnosis 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 typesof abnormalities] risk categories. Factors, such as age and presenting whitecell count, are also indicative of prognosis; an internal tandem duplication(ITD) mutation of the FLT3 receptor (~30% of cases) is highly predictiveof risk of relapse. Considerable variation in response to treatment andoverall survival are observed, even within the well-defined, favourable andadverse, risk groups indicating that refinement of prognostic prediction isneeded.

In Cardiff, we have analysed 221 diagnostic AML samples usingAffymetrix U133A GeneChips, the patient cohort included all majorcytogenetic groups and had been analysed for FLT3 mutations. The aim ofthis study was to identify genes differential expressed between patientswho achieved a remission and those who failed. Complete clinical data wasavailable from 149 samples, at the time of analysis.

Using SAM and ANOVA analyses, we identified a series of genesspecific for each of the cytogenetic based prognostic risk groupsdifferentially expressed between responders and non-responders. Littleoverlap between the gene lists was seen and there was no obviouscorrelation with the ability to achieve remission and the presence of aFLT3 mutation.

This study further reinforces the molecular heterogeneity of AML, butalso offers the possibility of refining prognostic groups and identifyingpatients for specific therapies based on their potential to achieve aremission status based on current therapeutic options.

137DEVELOPMENT OF A QUANTITATIVE REAL-TIME PCR METHOD FOR MONITORING CEBPA MUTATIONS IN NORMAL KARYOTYPE ACUTE MYELOID LEUKAEMIA PATIENT SAMPLESL-L. Smith1,2*, M. Smith2, L. Goff2, M. Jenner2, A. Lister2, J. Fitzgibbon2, D. Bonnet11Cancer Research UK, LRI, Haematopoietic Stem Cell Laboratory, London, UK2Cancer Research UK, Medical Oncology Unit, St Bartholomew's Hospital, Medical College, London, UK

Association of long-term clinical remission and molecular disease-eradication is well established in acute myeloid leukaemia (AML) patientswith 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/intermediatekaryotype. In these patients specific mutations have therefore beeninvestigated as one approach to quantify disease burden. Mutations inCEBPA, the gene encoding the granulocytic transcription factor C/EBPalpha, are present in approximately 20% of normal karyotype AMLand are concordant between presentation and relapse. In this study we setout to develop mutation specific quantitative real-time PCR assays forCEBPA mutations. Five assays were developed for normal karyotype M1patients via mutation specific DNA amplification. Four insertionmutations, ranging from 3 to 57 base pairs in length, cluster togetherallowing the use of a common probe and reverse primer, resulting in anamplicon of approximately 160 base pairs. Specificity was achieved bymaking use of the unique sequence generated by the insertion mutations.Serial 10-fold dilutions were preformed in duplicate using DNAconcentration equivalent to a range of cells from 125000 to 4. Sensitivity ofthe assays was approximately 5x10-5. To test specificity patient DNA wasserially diluted in wild type DNA, maintaining an overall DNAconcentration equivalent to 10000 cells in each reaction. Cell number ineach reaction was monitored by amplification of beta-2 microglobulin.These assays demonstrate a high degree of specificity and sensitivitysuggesting that mutation based MRD may have a complimentary role withflow sorting cells, translocation and WT1 based approaches currently usedto quantify MRD in AML.

138THYROID RECEPTOR INTERACTING PROTEIN 15 (TRIP15) IN ACUTE MYELOID LEUKAEMIA (AML)F. L. Khanim1*, R. E. Hayden1, Q. L. Trong2, M. T. Drayson3, C. M. Bunce11School of Biosciences, The University of Birmingham, UK2Dept of Hematology and Oncology, Cedars-Sinai, UCLA, LA, USA3Div of Immunity and Infection, The University of Birmingham, UK

Our studies indicate that thyroid hormone signaling is dysregulated inacute myeloid leukaemia (AML). Our studies have identified that TRbeta1is either lost or expressed at very low levels in approximately 50% ofAMLs. Furthermore, re-expression of TRbeta1 resulted in cell death inTRbeta1-negative KG1a AML cells but not K562 CML cells which,already expresses TRbeta1.

These data indicated that suppression of TRbeta1 signaling maycontribute to the pathology of AML. We therefore reasoned that in theremaining 50% of TRbeta1-positive AMLs, thyroid hormone action couldbe dysregulated through other mechanisms. Thyroid Receptor InteractingProtein 15 (TRIP15; also known as CSN2, SGN2) acts as a TRbeta1-selective nuclear co-repressor. We have used quantitative real-time RT-

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74 34th Annual Meeting: ISEH - Abstracts / Experimental Hematology 33 (2005) 39-143

PCR to measure TRIP15 mRNA levels in a large panel of primary AMLsamples (MRC AML12 trial) and AML cell lines. These studiesdemonstrated that AML cell lines and greater than 90% of primary AMLsamples expressed significantly higher levels of TRIP15 than normalresting or proliferating myeloid blast cells, thereby implicating TRIP15deregulation in the pathogenesis of AML.

In the light of our earlier observations it is tempting to speculate TRIP15contributes to the progression of AML by suppression of TRbeta1signaling. However, we observed TRIP15 overexpression in TRbeta1negative AMLs indicating that additional mechanisms may be involved. Inthis regard it is known that TRIP15 also acts a subunit of the signalosomeand thereby regulates the modification and subsequent proteasomaldegradation of key regulatory proteins including p53, Ikappa-B andinterferon regulatory factor 8 (IRF8). Ongoing experiments usinginterfering RNA and transfection approaches to modulate TRIP15expression in both TRbeta1-positive and -negative cell lines and primarymyeloid cells will be used to elucidate which of the TRIP15 pathwayspromote the malignant behaviour of AML cells.

139CONSTITUTIVE SERINE-585 PHOSPHORYLATION OF THE GM-CSF RECEPTOR AND THE DEREGULATION OF SURVIVAL SIGNALLING IN AML BLASTSJ. A. Powell1*, D. Thomas2, S. H. Lee2, A. F. Lopez1, M. A. Guthridge11Cytokine 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 thederegulation of the signals that promote both cell proliferation and cellsurvival. We have previously identified a novel tyrosine/serine bidentatemotif in the GM-CSF receptor that independently regulates cell survivaland proliferation. Signalling occurs through either Ser585 at lowercytokine concentrations leading to cell survival or through Tyr577 athigher cytokine concentrations leading to cell survival as well asproliferation, differentiation or functional activation. In the current study,we have shown that the phosphoserine-585 survival pathway is deregulatedin most AML patients analysed. GM-CSF titration experiments on primaryAML blasts revealed constitutive phosphoserine-585 survival signallingthrough the 14-3-3, PI-3K, Akt pathway. However, these same cellsexhibited normal phosphotyrosine mediated signalling, inducible at highcytokine concentrations. All AML blasts examined exhibited factor-independent survival in vitro, most of which were susceptible to inhibitorsof PKA. Analysis of primary mononuclear cells (MNC) revealed normalbi-phasic regulation of serine-585, and normal phosphotyrosine mediatedsignalling. Treatment of normal primary MNC with PKA inhibitorsreduced GM-CSF induced serine-585 phosphorylation, whereas forskolininduced phosphorylation of serine-585. We therefore propose thatconstitutive Ser585 survival signalling in AML blasts is mediated by aPKA, phosphoserine-585, Akt signalling pathway.

140EXPRESSION OF VEGF, ITS RECEPTORS AND HYPOXIA INDUCIBLE FACTORS IN ACUTE MYELOBLASTIC LEUKAEMIAY. Y. Zhang1,2*, K. C. Gatter1,2, F. Pezzella2, G. Pillai2, H. Turley2, A. Peniket2, G. G. Zhou1,2, S. M. Watt1,21National Blood Service, Oxford, UK2University of Oxford, Oxford, UK

Here, we have analysed 144 archival cases of leukaemia and non-neoplastic myeloproliferative disorders and 22 reactive control bonemarrows. We have studied the expression and cellular localisation ofangiogenic cytokines such as vascular endothelial growth factor (VEGF)and its tyrosine kinase receptor VEGFR-2. Using a novel antibody to thephosphorylated form of VEGFR-2 (pVEGFR-2), developed in ourlaboratory, we have shown that microvessel density (MVD) and theexpression of both pVEGFR-2 and its ligand VEGF are increased in acutemyeloid (AML) and lymphoblastic (ALL) leukaemias. VEGFR-2 andVEGF are found mainly in neoplastic cells, indicating that VEGF/VEGFR-2 signalling may play an important role in the proliferation and survival ofblast cells. We further analysed the expression of hyopxia induciblereceptors, VHL and their downstream targets. Our studies demonstrateincreased 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-2alpha is normally regulated by the tumor suppressor protein VHL. VHLtargets the HIFs to the proteosome in normal conditions. Under low oxygentensions, HIF proteins are stabilised and translocated to the nucleus. Apositive correlation between HIFs and CXCR4 in AMLs was seen. Wehave also observed a negative correlation of expression between VHL andHIFs and also VHL and CXCR4 suggesting that the exponential growth ofblast cells in haematological malignancies may be regulated by hypoxia orby mutations in genes involved in oxygen sensing and signalling.

141TRICHOSTATIN A ANALOGUE, A POTENT INHIBITOR OF HISTONE DEACETYLASE, INHIBITS CELL PROLIFERATION AND INDUCES APOPTOSIS IN HUMAN LEUKEMIA CELLSB. D. Chen*, X. LiDivision of Hematology-Oncology, Karmanos Cancer Institute, Wayne State University, Detroit, MI, USA

Histone acetylation plays an important role in the silencing andactivation of genes involved in tumorigenesis. Trichostatin A, a knownanti-fungal drug, is a potent inhibitor of histone deacetylase (HDAC) withpotential anti-tumor activity. In this study, we investigated the effect ofHDAC inhibitor III, an amide analogues of trichostatin A, on THP-1human leukemia cells. We showed that low dose HDAC inhibitor III (<0.2microM) inhibits the growth of THP-1 cells at G1 phase with low cytotoxiceffect. Growth arrest induced by HDAC inhibitor III is associated withincreased levels of cyclin–dependent protein kinase inhibitor p21 andcyclin E, in agreement with G1 phase arrest. Low dose of HDAC inhibitorIII also exerted some differentiating effect on THP-1 cells as judged by theexpression of c-fms proto-oncogene (M-CSF receptor). At higher doses (2microM), HDAC inhibitor III induces THP-1 cells to undergo apoptosis,which was associated with the cleavage of PARP, cytochrome c releaseand 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 inhibitorIII is a potent activator of NF-kappaB transcription factor. RT-PCR assayshowed that the inhibitor could transiently increase IL-1 expression yetmarkedly decreased TNF-alpha expression. Prolonged treatment withHDACI III at high doses was accompanied by an increased level of iNOSmRNA. Yet, aminoguanidine failed to overcome HDAC inhibitor III–induced apoptosis, suggesting that NO-mediated signal pathway is notinvolved in the induction of apoptosis. Our results show that HDACinhibitor III is a potent growth inhibitor and inducer of apoptosis in humanleukemia cells and suggest potential therapeutic strategies of HDACinhibitors for patients with leukemias.

142KG1A PROVIDES A MODEL FOR THE COMMITMENT OF PRIMATIVE HAEMOPOIETIC CELLS TO MYELOID DIFFERENTIATIONK. S. Mathias1*, R. E. Hayden1, F. L. Khanim1, M. T. Drayson2, N. J. Davies1, C. M. Bunce11School of Biosciences, University of Birmingham, Birmingham, UK2Division of Immunity and Infection, University of Birmingham, Birmingham, UK

KG1a cells were derived from an erythroleukaemia in myeloblasticrelapse but express low level transcripts of genes associated with immatureT and B cells. This suggested that KG1a represent primitive haemopoieticcells and a model for early events associated with lineage restriction/commitment. However, KG1a have proven resistant to inducers ofdifferentiation and consequently have fallen from common use.Conversely, we demonstrate here that the culture of KG1a in a chemicallydefined 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 theabsence of CD117. However, when transferred to ITS+ supplementedmedium KG1a progressively lost CD34 and acquired CD117 expression,becoming CD34+ve/CD117+ve (days 1-3) and CD34-ve/CD117+ve (by day4). Having lost CD34 expression, the cells went on to increase theirexpression of the myeloid commitment marker CD33 (beginning aroundday 7). These changes in antigen expression faithfully recapitulate thosethat occur during the normal commitment of haemopoietic stem cells to



Abstracts

myeloid cell differentiation. These observations reinstate KG1a as a potentmodel for studies of genomic, proteomic and metabolomic eventsassociated with cell commitment. They also highlight that serum containssignals that block the maturation of AML cells. Importantly, we haverecently demonstrated that serum represses HL-60 cell differentiation.Culture of HL-60 cells in medium supplemented with ITS+, resulted inslowing of cell growth, increased CD11b expression and the acquisition ofmorphological features consistent with neutrophil differentiation.However, serum-free differentiation of HL-60 did not occur in the presenceof 1nM triodothyronine (thyroid hormone; T3), indicating that serum-borneT3 suppresses HL-60 differentiation. In contrast T3 was unable to counterKG1a serum-free differentiation. Thus the mechanisms by which serumrepresses KG1a and HL-60 cell differentiation are likely to be distinct.

143A SULFUR-SUBSTITUTED FATTY ACID ANALOG IMPROVES SURVIVAL AND IMPAIRS METASTASIS IN RAT MODELS OF ACUTE LEUKEMIAP. O. Iversen1*, D. R. Sørensen2, R. K. Berge3, A. C. Rustan4, C. A. Drevon11Department of Nutrition, IMB, University of Oslo, Norway2Department of Comparative Medicine, the National Hospital, Oslo, Norway3Institute of Medicine, Section of Medical Biochemistry, University of Bergen, Norway4Pharmaceutical Biology, University of Oslo, Norway

Background: Polyunsaturated fatty acids (PUFA) may cause apoptosisin leukemic cell lines (1). Recent data suggest that a sulfur-substituted fattyacid (tetradecylthioacetic acid; TTA) can induce apoptosis in apromyelocytic leukemic cell line (2). However, whether PUFA and/orTTA can modify leukemogenesis in the intact organism, is unknown.

Aim: We examined whether dietary intake of n-3 PUFA or TTA couldimprove outcome in rats with either acute myeloid leukemia (AML) oracute lymphoid leukemia (ALL).

Methods: We studied rats harbouring either an acute promyelocyticleukemia (AML, ref. 3), or an acute T-cell leukemia (ALL, ref. 4). The ratswere randomized to isoenergetic diets with either standard pellets(Control), n-3 PUFA or TTA (10 rats/diet). The main endpoint wassurvival, while bone marrow (BM) leukemic cellularity and extramedullardissemination (spleen weight and plasma activity of matrixmetalloproteinases, MMP) served as secondary endpoints.

Results: The rats were killed when they showed signs of disease, or after35 days. The main results are summarized in the table.

Conclusion: Dietary intake of TTA, but not of n-3 PUFA, in rats withacute leukemia, prolonged their survival, and TTA intake was associatedwith reduced leukemic cell burden as well as diminished extramedullardissemination.

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.

144IMATINIB MESYLATE FOR REFRACTORY ACUTE MYELOBLASTIC LEUKEMIA HARBORING INV(16) AND C-KIT EXON 8 MUTATIONN. Asou1*, T. Nanri1, N. Matsuno1, T. Kawakita1, H. Suzushima2, H. Mitsuya11Kumamoto University School of Medicine, Kumamoto, Japan2NTT West Kyushu General Hospital, Kumamoto, Japan

Heterodimeric transcription factor AML1(RUNX1)/PEBP2beta(CBFbeta)is a most common chromosomal translocation target in leukemia. Theinv(16) generates the PEBP2beta-MYH11 fusion protein that is predictedto interfere with the function of AML1/PEBP2beta in a dominant-negativemanner and contributes to impaired differentiation of hematopoietic cells.However, studies using knock-in mice with PEBP2beta-MYH11 haverevealed that the fusion protein is critical for leukemogenesis but thatadditional genetic events are required for the development of acutemyeloblastic leukemia (AML). Recently, mutations in the C-KIT genewere frequently found in patients with inv(16) AML. Of 15 patients withinv(16) AML in our hospital, 3 and 2 had mutations in the C-KIT and FLT3genes, respectively. These mutations could represent potential therapeutictargets for specific tyrosine kinase inhibitors. An approximately half ofAML patients harboring inv(16) generally have a favorable prognosis,however, the rest of such patients are known to have a relatively poorprognosis. In this study, we present a case of AML with M4Eo harboringinv(16) and a C-KIT exon 8 mutation who was successfully treated withimatinib mesylate. No autophosphorylation of the C-KIT exon 8 mutantprotein in primary AML cells was observed with western blot analysis,while the stem cell factor (Kirin Brewery Co. Ltd) markedly elicited C-KITmutant phosphorylation. Furthermore, imatinib (Novartis Pharma AG)treatment of primary AML cells strongly down-regulated phosphorylationof the C-KIT mutant. A 58-year-old man with second relapsed AML wastreated with high-dose cytarabine and mitoxantrone but onlyunsuccessfully. However, the following treatment with 400 mg of imatinibmesylate for 2 weeks in combination with low-dose cytarabine, aclarubicinand granulocyte colony-stimulating factor plus vincrisitine andprednisolone brought about complete hematological remission. Thisobservation indicates that imatinib can be an alternative treatment modalityfor AML with the C-KIT exon 8 mutation.

145GENERATION OF AKR1C3 KNOCK-DOWN HAEMOPOIETIC CELL LINESJ. Birtwhistle1*, F. L. Khanim1, R. E. Hayden1, H. Schrewe2, J. K. Chipman1, C. M. Bunce11School of Biosicences, University of Birmingham, Birmingham, UK2Department of Developmental Genetics, Max-Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany

We have shown that inhibitors of the aldo-keto reductase AKR1C3promote myeloid cell differentiation whereas over-expression of theenzyme suppresses differentiation. Based on these studies we haveproposed AKR1C3 as a novel regulator of haemopoiesis and as a target inthe treatment of acute myeloid leukaemia (AML). To this end we arecurrently conducting a phase II trial of the AKR1C3 inhibitormedroxyprogesterone acetate in elderly and relapsed AML. However, allthe known AKR1C3 inhibitors have AKR1C3-idependent activities and itis therefore unclear to what extent their actions are mediated solely viaAKR1C3.

There is compelling evidence that AKR1C3 is capable of activatingpolycyclic aromatic hydrocarbon (PAH) carcinogens. Expression of theenzyme by haemopoietic stem and progenitor cells may therefore representan 'Achilles heel' rendering them susceptible for oncogenesis. However, tofully elucidate the role of AKR1C3 in either myelopoiesis or carcinogenactivation needs more precise intervention than the use of so called 'dirty'inhibitors. We have therefore used short hairpin interfering RNA (sh-iRNA) to generate cell lines in which AKR1C3 is specifically knockeddown. For these studies we elected to use the KG1a myeloid and the K562CML blast crisis cell lines. These lines have good transfection efficienciesand real-time RT-PCR analyses identified that they also had high initialexpression of AKR1C3. Stable transfectant AKR1C3 knock-down celllines have been produced and confirmed by the presence of the transfectedneomycin resistance gene along with growth through G418 selection andreduced levels of the AKR1C3 expression as measured by real-time RT-

Diet-parameter AML-Control

AML-PUFA

AML-TTA

ALL-Control

ALL-PUFA

ALL-TTA

Mean survival (days) 30 31 >35* 16 16 20*BM leuk.cells (%) 93±11 95±16 58±9* 92±11 96±14 45±8*Spleen weight (g) 4.5±0.7 4.3±0.8 2.2±0.4* 3.5±0.4 3.7±0.6 2.1±0.4*MMP activity(% of Control)

100 98±8 60±4* 100 98±7 33±5*

* denotes P<0.05 compared with Control/PUFA



PCR. These lines will now be used in assays of myeloid and erythroiddifferentiation and susceptibility to environmemtal carcinogens. The latterapproach will use the Comet Assay which can be used to detect both singleDNA strand breaks and DNA oxidative damage in cells exposed topotentially genotoxic AKR1C3 substrates.

146CELLULAR IMMUNE STATUS IN PATIENTS WITH ACUTE PROMYELOCYTIC LEUKEMIAY. Li1*, L. Yang1, S. Chen1, Q. Yin1, J. Tang1, G. Przybylski2, C. A. Schmidt21Institute of Hematology, Medical College, Jinan University, Guangzhou 510632, China2Dept. of Hematology and Oncology, Ernst-Moritz-Arndt University Greifswald, Greifswald 17487, Germany

Recent thymic output function, T cell receptor (TCR) repertoire andantigen specific T cell expansion would be the important markers forevaluation the cellular immune status in patients. Defects of cellularimmunity may play a role in hematologic malignancies. Little is knownabout the feature of T-cell immune state in leukemia. In order to evaluatethe cellular immune status in patients with APL, signal joint T-cell receptorrearrangement excision circles (TRECs) level and TCR V beta repertoireusage and clonality were analyzed. 17 cases with APL were selected for theresearch. Quantitative detection of TRECs in DNA of peripheral bloodmononuclear cells (PBMC) was preformed by real-time PCR, the TRECs-number was related to the number of T-cells by determination of thenumber of CD3-positive cells. The expression and cloanlity analysis weredetected by RT-PCR and genescan technique. 14 normal individuals servedas controls. In compare with normal individuals (6.36±5.28 copies /1000CD3+cells), a dramatic reduction of TRECs values in APL (0.77±1.60copies /1000 CD3+cells, P=0.005) could be found. The number ofexpressed V beta subfamilies (from 2 to 21 subfamilies) was varied indifferent patients with APL. The frequent expression V beta subfamilieswere Vbeta2 (64.7%), Vbeta15 (58.8%), Vbeta3 and Vbeta5 (47.1%).Clonal expanded T-cells in some V beta subfamilies could be identified onalmost all patients, predominant in Vbeta10, Vbeta23, Vbeta3 andVbeta21. This is the first description of TRECs level in APL patients. Theresult showed a prominent reduction of thymic output naive T cellsfunction in APL. The predominant usage and clonal expansion of TCR Vbeta subfamily T cells could be identified in APL patients, indicating thehost could have the ability for specific immune response to leukemiaassociated antigen, in despite of T cell immunodeficiency was showed inpatients.

147THE VALUE OF ORAL CYTARABINE OCFOSFATE AND ETOPOSIDE IN THE TREATMENT OF PATIENTS WITH REFRACTORY AML AND AML IN ELDERLY PATIENTSA. Horikoshi*, K. Takei, Y. Nakagawa, Y. Hosokawa, S. SawadaNihon 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 150to 300 mg/m2/d was pharmacokinetically concluded to be comparable tothe continuous infusion of ara-c at 20 mg/m2/d. To evaluate the toxicityand antileukemic efficacy of SPAC combined with oral etoposide (EP), atotal of 21 refractory cases (10 men/11 women, median age 69 years; range33-86 years), with 11 de novo AML and 10 AML evolving frommyelodysplastic syndromes basically received induction therapy withSPAC at 300 mg/d and EP at 50 mg/d for 14 days. Thirteen patients hadpreviously been untreated. Seventeen patients had abnormal karyotypesincluding 8 complex abnormalities. Patients had complications thatincluded hypertension, congestive heart failure, old myocard infarction,aneurysm, dibetes mellitus, hyper lipidemia, pneumonia and phlegmon.Seven patients had performance status 3~4. Seven patients achievedcomplete remission(CR), and 2 patients achieved partial remission(PR).The nadir of leukocyte and platelet counts occurred at 3~31 days from thestart of therapy. The durations of leukocytopenia(<1000) andthrombocytopenia(<20000) were not prolonged, but some CR and PRpatients showed a long duration of leukopenia(23~51days). No severeadverse events were observed. Two out of 12 patients in the non-respondergroup achieved CR and PR after additional induction therapy. The durationof survival was 1.5~16 months. The ara-c and EP concentrations in

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 inindividual cases. We concluded that SPAC with EP can be administratedsafely and has potential for the treatment of high-risk and AML patients inpoor condition.

148CONTROL OF CELL DEATH LEVELS USING TH9402-BASED PDT TREATMENT ON FRESH PBMC, AND POTENTIAL APPLICATION TO PATIENTS WITH CGVHDA. Lévesque1, A. L. Savard1, D. C. Roy2, F. Foss3, C. Scotto1*1Celmed BioSciences Inc, Saint-Laurent, Quebec, Canada2Maisonneuve Rosemont Hospital, Montreal, Quebec, Canada3Tufts New England Medical Center, Boston, MA, USA

Recently, extracorporeal photopheresis (ECP) has shown interestingclinical activity for the treatment of drug-refractory chronic graft-vs-hostdisease (cGvHD), inducing Th1/Th2 immunomodulation to restoreimmune tolerance. Several studies indicate that target cell apoptosis mayhave a role in the control of cGVHD, and increasing apoptotic levels mayfavor immune modulation. We have developed an approach to eliminateimmunoreactive cells using the TheraluxTM photodynamic cell therapy(PDT) system based on the use of the rhodamine-derivative TH9402illuminated ex vivo with a visible light source (514nm wavelength). Thecapacity of TH9402 PDT to induce increasing levels of T cell apoptosis hasnot been investigated.

The induction of apoptotic cell death was studied using peripheral bloodmononuclear cells (PBMCs) isolated from healthy volunteers (HV) andcGvHD patients. We found a good correlation between PDT conditionsand levels of cell death induced. In comparison to controls showing 8±4percent (%) of apoptosis, as measured by TUNEL assay on cells harvested3 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- andinter-donor variability in TH9402 incorporation are very low (~5% and10%, respectively). To ensure that these findings could also be applied to aclinical setting, PBMC from cGvHD patients were treated with 0.33 and1.32 micromolar of TH9402 to trigger either low or high levels ofapoptosis. PBMC from cGvHD patients showed a sensitivity similar to thatof PBMC from HV, with 38±9% and 73±13% of apoptosis when treatedwith 0.33 and 1.32 micromolar TH9402, respectively.

Based on these data, we intend to begin a pilot clinical study evaluatingtwo controlled PDT conditions inducing low and high levels of apoptosisin order to assess their efficacy and biological effect to treat cGVHD inhumans.

149THE ARTERIAL AND VENOUS POTENTIAL OF HUMAN MAPC AND AC133 DERIVED ENDOTHELIAL CELLS IS MODULATED BY ACTIVATION OF NOTCH AND PATCHED LIGANDSX. L. Aranguren1*, C. Clavel1, A. Luttun2, M. Barajas1, C. Moreno1, G. Abizanda1, A. Echavarri1, M. Araña1, E. J. Andreu1, F. Prósper11Foundation for Applied Medical Research, Division of Cancer, Area of Cell Therapy and Hematology, Clinica Universitaria, Pamplona, Spain2Stem Cell Institute, University of Minnesota Medical School, Minneapolis, MN 55455, USA

We have analyzed the endothelial potential from different populations ofadult stem cells, human MAPC and cord blood derived AC133 cells toestablish the arterial and venous potential of each population and thesignaling pathways implicated in their differentiation. Both populationsdifferentiate into mature/functional endothelium in vitro as determined byexpression of markers such as von Willebrand factor, VE-cadherin,VEGFR1 and 2, Tie1 and Tie2, CD105 and CD51/CD61, matrigel tubeformation and ac-LDL-Dil uptake. However, while AC133 cellsdifferentiate mostly to microvascular and venous endothelium, MAPCdifferentiate to both arterial and venous endothelium, as determined byexpression of specific markers (Hey-2, Ephrin B1, Ephrin B2, Dll-4, EphB4). Analysis of molecules involved in arterial development pathways(notch and patched) have shown clear differences between bothpopulations of stem cells. Modulation of these pathways in vitro modifiesthe endothelial fate of AC133 and MAPC. Addition of shh and Dll-4enhance arterial potential of MAPC derived endothelium. In vitro 3Dmatrigel differentiation assay show that with Shh and Dll-4, number of



Abstracts

vessels and their size is higher than VEGF and bFGF alone. In vivomatrigel assay show that addition of Shh and Dll4 enhance vascularstructures because the higher collagen deposition and smooth muscle cells.Synergist effect has shown between media and cells. Light polarized showthat blood vessels are better in cells with artery media (VEGF, bFGF, Shhand dll-4), than in other groups. MAPCs are be able in vivo to differentiateto mature endothelial cells by expression of UEA lectin. Conclusions:MAPCs are a promise cells for treatment of ischemic tissue because theyhave both in vitro and in vivo potential to differentiated toward endothelialcells, and they have high potential to differentiate to artery phenotypeendothelial cells. Again, These potential can be modulate with notch andsonic pathways.

150DEVELOPMENT OF NOVEL DRUGS DERIVED FROM RING-SUBSTITUTED 9-ARYLIDENEANTHRONES AS POTENTIAL AGENTS FOR LEUKEMIA THERAPYE. Levy1, Z. Rappaport2, D. Arad3, O. Sphilberg4, I. Levy1, I. Nathan1*1Ben Gurion University of the Negev and Soroka University Medical Center, Israel2Hebrew University, Jerusalem, Israel3MND Diagnostic Ltd., Israel4Rabin Medical Center, Petah-Tiqva, Israel

In a search for novel derivatives, which can serve as antileukemic agentswe have screened and tested a large series of vinylic compounds on humanleukemic cell lines: HL-60 promyelocytic leukemia, CCRF-CEM Tlymphoblastic leukemia and U-937 monocytic leukemia. The ring-substituted 9-benzylideneanthrones had the strongest antileukemic effectwith LC50 <0.1 micrg/ml but had no effect on lymphocytes from healthydonors. These substituted 9-arylideneanthrones killed the leukemic cells byinduction of apoptosis as measured by acridine orange/ ethidium bromidestaining and phosphatidyl serine exposure using annexin V-FITC. Thesecompounds also caused G1 arrest in CCRF-CEM cell line. The signalingpathway leading to cell death were studied. The results showed aninvolvment of protein kinase C-epsilon (PKC-epsilon) but not PKC-alphain substituted 9- benzylideneanthrones activity. PKC-epsilon activationwas manifested by translocation of the enzyme from the cytosol to themembrane, as assessed by western blotting. In addition, the production ofreactive oxygen species (ROS) was observed as determined by thefluorescent probes specific to H2O2 and O2- DCFH-DA and hydroethidine,respectively. The lipophilic antioxidant vitamin E was able to abolish theROS generation and the apoptotic activity of these 9-arylidene anthrones.Primary cells obtained from CLL patients underwent apoptosis in responseto treatment with 9-arylideneanthrones. The results imply the antileukemicmechanism of these arylideneanthrones and their potential as antileukemicagents.

151LYMPHOID-PRIMED MULTIPOTENT PROGENITOR CELLS EFFICIENTLY AND DURABLY RECONSTITUTE THE IMMUNE SYSTEM IN X-SCID MICE TRANSPLANTED IN UTEROK. Liuba1,3*, S. Stott2, G. Lingman1, S. E. W. Jacobsen31Department of Obstetrics and Gynaecology, Institution for Clinical Sciences, Lund University, Sweden2Wallenberg Neuroscience Center, Section of Neurobiology, and Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund University, Sweden3Hematopoietic 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) ischaracterized by lack of functional T and NK cells, resulting in enhancedsusceptibility to infections and death in early life. Although conventionalbone marrow transplantation (BMT) can cure X-SCID, there isconsiderable mortality and morbidity associated with BMT. In uterotransplantation of hematopoietic stem cells (HSCs) has been reported to besuccessful. Although lacking evidence for donor-derived myeloid cells, itis believed that sustainable immune reconstitution in X-SCID patients isdependent on low-level HSC engraftment, with preferential lymphoidreconstitution. We have recently identified in normal mouse BM, aprimitive Lin- Sca1+kit+CD34+flt3+ lymphoid –primed multipotentprogenitor (LMPP), representing the earliest lineage-restricted stem/progenitor cell in the hematopoietic system (Adolfssson et al, Cell in

press). Herein, we compared the potential of purified adult wild type Lin-

Sca1+kit+CD34- flt3- long-term (LT) HSCs and the LMPPs to reconstituteand correct the immune deficiency in X-SCID fetal mice. Notably, LMPPsreconstituted T, B and NK cells more rapidly but with similar long-termefficiency as LT-HSCs, and neither of the two populations gave detectablereconstitution of the myeloid lineages or the Lin-Sca1+kit+ HSCcompartment cell. Importantly, although transplanted with congenic cells,the immune reconstitution with LMPPs was considerable better in fetalthan young adult X-SCID recipients, supporting that the fetalmicroenvironment is more permissive to donor-derived cell replacementtherapy. Thus, representing a considerable larger population of BMprogenitors than LT-HSCs, we propose that LMPPs are likely to be the besttarget cells for cell replacement and gene therapy of inborn immune-deficiencies.

152ONE-STEP NON-MAGNETIC NEGATIVE SELECTION OF HIGHLY PURIFIED LYMPHOCYTE SUBSETS FROM PREVIOUSLY FROZEN HUMAN CELL SAMPLESK. A. Ross1, A. Wong1, A. Lopez1, A. C. Eaves2, T. E. Thomas1, C. E. Peters1*1StemCell Technologies Inc, Vancouver, BC, Canada2Terry Fox Laboratory, Vancouver, BC, Canada

We have developed a rapid, one-step procedure (SpinSepTM) for theisolation of highly purified human cell subsets by negative selectionwithout the need for magnets, columns, or other specialized equipment.This procedure couples the specificity of monoclonal antibodies with theease of buoyant density centrifugation. A cocktail of bispecific antibodyreagents ('TACs') selectively couples cell surface antigens on unwantedcells in a mixed cell suspension to specially designed, densemicroparticles, similar to red blood cells and their use in RosetteSepTM.The cell suspension is centrifuged over a buoyant density medium. Theunwanted cells pellet; the desired, unlabeled cells are recovered in the layerabove the buoyant density medium. The specificity of the antibodiesincorporated into the TACs determines which cells will be targeted fordepletion and consequently which cells will be enriched. Lymphocytesubsets were enriched from previously frozen peripheral bloodmononuclear cell samples within one hour using this method. The purityachieved with this new centrifugation-based procedure is comparable tothat obtained with more complicated, multi-step immunomagnetic negativeselection techniques, and the viability of the enriched cells exceeds 90%.Desired cells are not labeled with antibody, thus avoiding perturbations offunction that may occur due to antibody binding. In summary, SpinSepTM

is a simple and robust way to rapidly obtain cell suspensions with highpurity and recovery from previously frozen cell samples without the use ofany specialized equipment.

153COMPARISON OF BONE MARROW AND UMBILICAL CORD BLOOD AS A SOURCE OF LIVER STEM CELLSK. H. Ryu1*, E. S. Yoo1, Y. J. Jung2, S. Y. Woo2, J. Y. Seoh2, H. S. Han31Dept of Pediatrics, Ewha Womans University College of Medicine, Seoul, Korea2Dept of Microbiology, Ewha Womans University College of Medicine, Seoul, Korea3Dept of Surgery, Seoul National University College of Medicine, Seoul, Korea

Objective: The purpose of this study was to suggest human umbilicalcord blood (UCB) and bone marrow (BM) as the source of liver stem cellsand to generate mature hepatocyte lineage cells.

Methods: Mononuclear cells from UCB and BM were cultured withfibroblast growth factor (FGF)-1, FGF-2, stem cell factor (SCF), andhepatocyte growth factor. (HGF) Cultured cells were monitored in invertedlight microscopy and analyzed by reverse transcription-polymerase chain

Desired Cells n % Viability(mean±SD)

% Purity(mean±SD)

% Recovery(mean±SD)

CD3+ T cells 5 97±2 99±1 58±17CD4+ T cells 5 98±1 95±2 61±12CD8+ T cells 8 94±5 91±5 43±14B cells 13 94±7 93±6 47±15



reaction (RT-PCR), immunofluorescent (IF) staining analysis for detectinghepatocyte lineage markers and by flow cytometry for demonstratinghepatocyte lineage differentiation rather than hematopoietic one.

Results: Cell growth in given culture condition supplemented with FGF-1, FGF-2, SCF, and HGF yielded large sized and oval shaped cells whichwere adherent to culture dishes. mRNA of ALB, CK18 and alphafetoprotein were detected from day 7 in both UCB and BM derived cells byRT-PCR. In IF analysis, these cells expressed albumin (ALB),cytokeratin(CK)-19 from 7-day of culture and some of the ALB producingcells contained proliferating cell nuclear antigen. Cellular differentiationexpressing CK-18 molecules was observed by flow cytomery analysis.

Conclusions: Liver stem cells may exist in UCB as well as BM and canproliferate into hepatocyte lineage cells in primary culture system in vitroand may serve cell sources for cell therapy of hepatic tissues.

154ADOPTIVE IMMUNOTHERAPY IN ACUTE MYELOGENOUS LEUKEMIA: A NOVEL APPROACHT. A. Lane1*, R. K. Zhong1, M. R. Loken2, E. D. Ball11University of California, San Diego, School of Medicine; San Diego, CA, USA2Hematologics, 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 myeloidleukemia (AML; not M3) for adoptive immunotherapy. PBMNC from 12AML pts with >50% blasts were cultured in 10% serum with 50 ng/mlGM-CSF, 1000 IU/ml IL-4 and 5 mcg/ml anti-CTLA4 on days 0-8 toinduce 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. Tcells 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% wereCD4+IFN-gamma+CD69+ and 9.8±4.1% were CD8+IFN-gamma+CD69+ by ELISPOT. CTL produced 42±23% lysis of autologousAML 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 assesssafety we investigated post culture AML blast colony growth and cellphenotype by multiparameter flow cytometry. Pre-culture there were 28,50, and 295 blast colonies/50,000 MNC (n=3), but no colonies grew frompost-culture samples plated at 0.5-1x106 cells/dish (n=6). Pre-cultureAML blasts (CD34+) represented 70, 76, 89 and 96% of MNC (n=4); butthe post-culture samples revealed no blasts in 400,000 to 1,600,000 cellscounted. Experiments in a closed bag system indicated that >109 CD3+cells were generated from <50 ml peripheral blood. Conclusion: The novelAML-CTL culture generated CTL that were highly reactive to autologousAML blasts without requiring the identification of patient-specific antigentargets and may facilitate adoptive immunotherapy for pts with AML.

155PHASE 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 CIRRHOSISL. Catani1*, S. Lorenzini2, A. Isidori1, S. Talarico1, E. Loggi2, A. Gramenzi2, F. Bonifazi1, P. Andreone2, M. Baccarani1, R. M. Lemoli11Institute of Hematology and Medical Oncology L. A. Seràgnoli, Bologna, Italy2Department of Hepatology, Cardioangiology and Internal Medicine, Bologna, Italy

Bone marrow-derived hematopoietic stem cells (HSCs) contribute totissue regeneration after acute or chronic liver damage. In this study, weassessed whether Granulocyte-colony stimulating factor can be safelyadministered to patients with liver cirrhosis in order to expand and tomobilize HSCs. Seventeen patients with advanced liver disease [16 male,mean age: 59 years; 12 HCV, 1 HBV, 1 HDV, 3 alcohol abuse; medianCTP score: 7 (range 5-9), median MELD score: 11.5 (range 7-17)] wereconsecutively treated with rhu-Granulocyte-colony stimulating factor(Lenograstim). Increasing doses of Granulocyte-colony stimulating factorwere administered subcutaneously for 7 consecutive days to five cohorts of

three patients each, starting from 2 microg/kilog/daily. The dose ofGranulocyte-colony stimulating factor was escalated according toFibonacci's increment rule until the successful mobilization of 10CD34+cells/microl in at least 2/3 patients. Fifteen patients were enrolled inthe phase I study. Granulocyte-colony stimulating factor mobilizing dosewas found at 15 microg/Kilog/day when mobilization of HSCs occurred in2/3 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 thephase II study with the mobilizing dose, and all of them showed asuccessful mobilization and collection of HSCs. Overall, the median peakvalues of CD34+ and CD133+ cells in mobilizing patients were 41.5±13.5cells/microl and 26.5±3.2 cells/microl, respectively. No severe adverseevents were observed at any dosage. From the 6.6 microg/kilog dose ofGranulocyte-colony stimulating factor or higher, the side effects observedwere bone pain (10/17), fever (4/17) and alkaline phosphatase increase (10/17). Up to 30 days after treatment with Granulocyte-colony stimulatingfactor, we observed a significant decrease of alpha-foetoprotein in all butone patients and the decrease of transaminases in mobilizing patients. Inconclusion, the administration of Granulocyte-colony stimulating factor topatients with liver cirrhosis is safe and feasible and is capable ofmobilizing HSCs at the dosage of 15 microg/kilog/day.

156TYPE I METHEMOGLOBINEMIA ARISING DUE TO A CHANGE IN COFACTOR SPECIFICITY OF CYTOCHROME B5 REDUCTASEM. 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. Lappin31Belfast City Hospital, Belfast, UK2Biochemistry and Molecular Biology, University of South Florida College of Medicine, Tampa, Florida, USA3Haematology, Queen's University, Belfast, Northern Ireland4National Centre for Hereditary Coagulation Disorders, St. James's Hospital, Dublin, Ireland5Paediatrics, Altnagelvin Hospital, Londonderry, Northern Ireland

The soluble form of NADH-cytochrome b5 reductase (cytb5r, EC1.6.2.2) is present exclusively in the cytoplasm of erythrocytes andcatalyses the reduction of methemoglobin. This reaction utilizescytochrome b5 and NADH co-factor as the source of H+ ions. Spontaneousand continual oxidation of hemoglobin gives rise to the non-functional metform but the accumulation of methemoglobin is prevented by the constantaction of cytb5r. Deficiency of the soluble enzyme results in cyanosis ortype I recessive congenital methemoglobinemia (RCM), but a combineddeficiency of both soluble and membrane bound forms results in severeneurological impairment or type II RCM. Screening Irish type I RCMpatients identified a novel mutation, D239G, present in the NADH-bindinglobe of cytb5r. To date we have found in total three such novel mutationsand a heterologous expression system was chosen to study the effect of theD239G, and the previously detected E255- and G291D mutations on cytb5rfunction. All variants were successfully expressed enabling their enzymekinetic properties, protein stability and cofactor oxidation-reductionpotential measurements to be compared. The D239G mutation was foundto have no adverse impact on protein stability, unlike the E255- and G291Dvariants. In addition, it exhibited marginally reduced catalytic activity of94%, while the E255- and G291D variants retained only 38% and 58% ofwild type activity respectively. Measuring the affinity of the D239Gvariant for co-factors NADH and NADPH indicated that loss of asparticacid 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 convertingmethemoglobin to its functional less oxidized state thus causing the build-up of methemoglobin, resulting in type I RCM.



Abstracts

157NATURAL REGRESSION OF PRIMITIVE HEMATOPOIESIS IN ZEBRAFISH EMBRYOS IS MEDIATED BY A DEATH RECEPTOR GENET. T. Kwan1, R. Liang1, S. Lin2, S. C. Ekker3, C. M. Verfaillie3, A. Y. Leung1*1Department of Medicine, University of Hong Kong, Hong Kong2Department of Molecular, Cellular, and Developmental Biology, University of California Los Angeles, Los Angeles, USA3University 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 ofprimitive hematopoiesis during late embryonic development. Morpholinos(MO) against genes encoding for zDR and chordin (zChd), as well as arandom sequence (RS) MO (control) were injected into wild-type (WT)embryos at 1-4 cell stage. Injected embryos were maintained at 280C untilanalyzed.

At 24 hpf (hours post-fertilization), zDR mRNA expression is restrictedto the ICM (intermediate cell mass, site of primitive hematopoiesis inzebrafish) of WT embryos and is upregulated in embryos injected withzChd-MO (CM) embryos. The latter is characterized by increased ventralmesoderm specification. WT embryos injected with zDR-MO (zDRmo

embryos) demonstrated increased hemoglobin (Hb) staining (shown by O-dianisidine) in the axial circulation and duct of Curvier (DC) at 48 hpf. InCM embryos, Hb staining is significantly increased in the ICM and DC,and is further enhanced in embryos co-injected with zDR-MO. This isconfirmed quantitatively with a modified cyanomethemoglobin method.Injection of RS-MO into WT or CM embryos had no effects on Hb. Therewas little apoptosis (shown by TUNEL assay) in the ICM of WT andzDRmo embryos at both 24 and 48 hpfs. Apoptosis is increased in the CMembryos. At 24 hpf, it occurs predominantly on the surface of the embryos.At 48 hpf, it is abundant in the expanded ICM. Apoptotic signals arereduced in embryos co-injected with zDR-MO.

In conclusion, knock-down of zDR in WT and CM embryos led to anincrease in Hb content and in the latter, attenuation of apoptosis. Theseresults suggested that zDR may mediate the natural regression of primitivehematopoiesis during zebrafish development by apoptosis. Whether similarmechanisms might operate in the mammalian system would have to befurther studied.

158INSULIN-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 BLOODT. Nagatomo1, K. Muta2, S. Ohga1, M. Ochiai1, S. Hikino1, T. Hara1*1Department of Pediatrics, Graduate School of Medical Sciences, Kyushu University, Japan2Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Japan

To reveal the novel function of erythroid progenitor cells in fetalerythropoiesis, the gene expression pattern in umbilical cord blood (CB)derived CD36+ erythroid progenitor cells (EPCs) was analyzed usingcDNA microarray containing 240 cytokine and growth factor relatedgenes. Among the genes analyzed, insulin-like growth factor II (IGF-II)gene showed a 124-fold higher expression in CB-EPCs, compared to thatseen in phytohemagglutinin (PHA)-stimulated adult peripheral bloodmononuclear cells (PBMCs). Real-time PCR revealed that the IGF-IImRNA levels in CB-EPCs were higher than those seen in blood cells suchas PB-CD4+ cells, PB-CD8+ cells, CB-CD4+ cells, CB-CD8+ cells andCB-CD14+ cells. When CB-EPCs were cultured with erythropoietin (EPO)in serum-free medium, the number of erythroid colonies was decreased inthe presence of IGF-II-neutralizing antibody. To assess the role of IGF-IIin erythropoiesis further, we purified erythroid colony-forming cells(ECFCs) from human umbilical cord blood by magnetic selection andliquid culture with IL-3, stem cell factor and EPO. The expression levels ofIGF-II, type 1 or 2 IGF receptor in the mature ECFCs were higher thanthose in the immature ECFCs. Addition of IGF-II-neutralizing antibodydecreased the number of ECFCs in liquid culture with EPO. The anti-proliferative effect of IGF-II-neutralizing antibody was evident in bothdimethylthiazole tetrazolium bromide (MTT) and bromodeoxyuridine(BrdU) incorporation assays. When apoptosis of cells was examined using

Annexin V, the addition of IGF-II-neutralizing antibody increasedapoptosis, thereby indicating the anti-apoptotic effects of IGF-II secretedfrom erythroid cells. Furthermore, ECFCs failed to undergo normalerythroid maturation in the presence of IGF-II-neutralizing antibody, asassessed by flowcytometric analysis and morphology. These findingsindicate that IGF-II, which is produced by erythroid progenitor cellsthemselves, has crucial roles in controlling erythropoiesis by modulatingapoptosis, proliferation and maturation via an autocrine mechanism.

159ERYTHROPOIESIS IN VITRO IN NORM AND PATHOLOGICAL STATEST. Saralidze1*, M. Sheklashvili1, L. Mohchevishvili2, T. Shvelidze1, M. Bogvelishvili1, G. Saralidze31Institute of Hematology and Transfusiology, Tbilisi, Georgia2Children's Hospital, Tbilisi, Georgia3Sport Department Dispanser, Tbilisi, Georgia

Blood and bone marrow cell cultures were used for investigation oferythropoiesis and macrophage-lymphocyte rosettes (MLRos) formation in20 donors and in 64 patients with different pathological states involvingerythropoiesis: 30 patients with iron deficient anemia (IDA), 10 -autoimmune hemolytic anemia (AIHA), 9 –refractory anemia (RA), 5-aplastic anemia (AA), 7- chronic erythromyelosis (CE), 3- polycytemiavera (PV).

In bone marrow cultures prolongation of erythropoiesis up to 3 days andmaintenance of differentiation and maturation of erythrocaryocytes(oxyphillic normoblasts exceeds polychromatophilic and basophilic forms)is observed in IDA and AIHA, while in donors and in AAerythrocaryocytes are observed only on first day and rarely on second dayof cultivation. In RA patients diserythropoiesis and prevalence ofpolychromatophilic and basophilic forms over oxyphillic normoblasts isobserved on 1-2 days of cultivation. Notable that in 3 patients with RAproliferation of blast cells in vitro revealed impending acute leukemia. InCE patients ineffective erythropoiesis with abundance of basophilic andpolychromatophillic normoblasts and figures of asymmetric mitosis of redblood are revealed up to 16 days of cultivation, whereas in cases ofpolycytemia vera effective erythropoiesis is maintained up to 12 daysreflecting malignancy of disease. Study of MLRos amount in bloodleukocyte cultures showed decreased immune reactivity of organism inIDA and AA patients and high sensitivity of immunocompetent cells inAIHA. Increase of these rosettes in some cases of RA revealed immunegenesis of anemia and therefore necessity of immunosuppressive therapy.Our data shows that in vitro studies are useful for estimation oferythropoiesis in different states. Prolongation of erythropoiesis more than4-5 days reveals increased proliferative activity of erythrocaryocytes andpoints to malignant nature of disease. Increase of MLRos amount in vitroshows sensibilisation of immunocompetent cells and reveals immuneconflict in the pathogenesis of disease.

160IN VITRO ERYTHROPOIESIS INDUCED FROM HUMAN CD34 POSITIVE CELLSI. Dorn*, J. Ribbat, S. Boie, H. Kirchner, P. SchlenkeInstitute of Immunology and Transfusion Medicine, University of Luebeck, Germany

Purpose: The regulation of human erythropoiesis is only partlyunderstood. Thus, an in vitro erythropoiesis model might be very useful toinvestigate, which factors and molecular mechanisms are involved inmaturation of human erythroid precursor cells. Therefore, two differentculture techniques were tested for optimal proliferation and differentiationof CD34 positive cells to reticulocytes.

Methods: Purified CD34 positive cells from leukapheresis were culturedover 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). Cellgrowth and differentiation was evaluated by flow cytometry usingfluorescent microparticles and antibodies against CD34, 36, 71 andglycophorin A (GPA). Cytospin preparations were made and stained byPappenheim and neutrale benzidine.

Results: The initial purity of CD34 positive cells was always greater 95percent. During the two-phase model cell numbers increased up to 50fold.On d16 more than 95 percent expressed GPA and were strongly

iseh05.book 9 Tuesday, May 10, 2005 10:22 AM


haemoglobin positive. Cells showed morphological characteristics ofnormoblasts and about 40 percent enucleated reticulocytes. In the three-phase model, a greater proliferation (up to 260fold) could be observed. Ond21 more than 90 percent were GPA positive, most of them normoblastsand 50 percent reticulocytes.

Conclusions: Both models were able to show different stages of humanerythropoiesis, including terminal enucleation. Although proliferation wasgreater in the three-phase model, no differences in maturation andenucleation could be observed. On the last culture days more than 90percent of cells were GPA positive and consisted mainly of normoblastsand reticulocytes. By varying culture conditions or inhibiting signalpathways, both models might be a tool for further investigations in the fieldof erythropoiesis. Our present studies investigate the influence of theoxygen content on proliferation and differentiation of erythroidprogenitors.

161G PROTEIN-COUPLED RECEPTORS IN HYDROXYUREA ACTIVATION OF NITRIC OXIDE/CGMP PATHWAYS AND GAMMA-GLOBIN INDUCTIONV. P. Cokic1*, A. P. Economou2, L. M. Wahl3, P. Milenkovic1, A. N. Schechter41Institute for Medical Research, Belgrade, Serbia and Montenegro2University of Geneva, Faculty of Medicine, Geneva, Switzerland3NIDCR, National Institutes of Health, Bethesda, USA4NIDDK, National Institutes of Health, Bethesda, USA

We have shown previously that nitric oxide (NO) and the solubleguanylyl cyclase (sGC) pathways are involved in hydroxyurea-inducedactivation of fetal hemoglobin in human erythroid cells. It was alreadyreported that hydroxyurea activates p38 MAP kinase and induces c-junexpression in human erythroleukemic cells. Since all of these pathways arealso activated by G protein-coupled receptors (GPCR) in this study, weanalyzed hydroxyurea (40 microM) activation of GPCR genes in humanerythroid progenitor cells. We found increases in the beta 2 adrenergicreceptor (3.8 fold) and dopamine receptors D1 and D2 (3 and 2.2 fold), inthe 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 theMAP kinase pathway: SSI-1 (2.7 fold); and NFkB pathway: IL-8 andhelix-loop-helix zipper protein (3.7 and 4.3 fold). We have also found thathydroxyurea (30 microM) increases intracellular cGMP levels (60-90 fmol/1x105 cells, about 2 fold induction) as well as cAMP levels (1000-1800fmol/1x105 cells, 2-5 fold induction) during 4 hours of incubation ofhuman erythroid progenitor cells. Further, hydroxyurea (10-50 microM)induced NO production in human and mouse macrophages and thatinduction demonstrated NO synthase (NOS)-dependence. Co-culturestudies of mouse macrophages and erythroid cells in the presence ofhydroxyurea (10-50 microM) induced gamma-globin mRNA expression oferythroid progenitor cells, after 24 hours of incubation. Furthermore,hydroxyurea induced gamma-globin mRNA expression, with dosedependent effects, in co-culture studies of human macrophages anderythroid cells, with major effects after 48 hours of incubation.Macrophages, as part of stromal microenvironment, enriched gamma-globin induction during hydroxyurea treatment in comparison withindependent erythroid cultures. Macrophages supported hydroxyureainduction of NO and consequently amplified gamma-globin induction viaGPCR-dependent NO/cGMP pathway.

162GENERATION OF FUNCTIONAL HEMOGLOBIN-SYNTHESIZING ERYTHROID CELLS FROM HUMAN EMBRYONIC STEM CELLF. Ma1,3*, D. Wang1, S. Hanada1, K. Ogami2, K. Umeda3, T. Hekei3, H. Sakai4, E. Tsuchida4, T. Nakahata3, K. Tsuji11Division of Cellular Therapy, Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, Japan2Department of Transfusion, The Institute of Medical Science, The University of Tokyo, Japan3Department of Pediatrics, Kyoto University Graduate School of Medicine, Japan

4Advanced Research Institute for Science and Engineering, Waseda University, Tokyo, Japan

Human embryonic stem (HES) cell provides a unique tool to study earlyevents occurring in the development of human tissues and specific celllineage, such as hematopoiesis. Because of its titopotent property, thepossibility of HES cells to produce supplementary tissues unfold an almostunlimited potential of its use in regenerative medicine. By using an in vitroco-culture system with murine fetal liver derived stromal cells, wesuccessfully induced differentiation of HES cells (H1) into hematopoieticprogenitor cells. These HES-derived progenitors were within cobble stone-like cells in the co-culture and highly enriched in a CD34+ fraction. Inclonal culture with a cytokine combination (SCF, IL-3, IL-6, TPO, EPOand G-CSF), they gave rise to various types of hematopoietic colonies,including myeloid, erythroid and multi-lineage colonies. Immunochemicalanalysis showed that all of multi-lineage colonies examined containederythroid cells possessing both alpha and beta globins (100 and 70%,respectively), indicating that they have already synthesized adult-typehemoglobin, which exhibited the oxygen affinity in vitro. Interestingly, inadditive suspension culture for 1week, the erythroid cells in day-15colonies not only further proliferated, but also matured, as they had 100%adult-type hemoglobin and 4 to 11% enucleated. We also determined theblood type of H1-derived red blood cells. Our study provides a possibilityto use HES-derived red blood cells as a substitution of blood transfusionand should be benefit for future clinical use.

163THE ROLE OF THE NOVEL GENE JUNE-1 IN ERYTHROID DEVELOPMENT AND IN EPO-SENSITIVE CELLSS. Rainey*, V. Smyth, Y. Zhengqiang, E. Dunlop, T. R. J. LappinDept Haematology, Centre for Cancer Research and Cell Biology, Queen's University Belfast, UK

The hormone erythropoietin (EPO) leads to red blood cell developmentafter binding to EPO receptors (EPO-Rs) on erythroid progenitors.However, little is known about the genes that are regulated by EPO. EPO-Rs have now also been found on a variety of non-hematopoietic tissues,including some tumors; an important finding since rHuEPO is often used totreat anemia in cancer patients. It is therefore important to study thedownstream effects of EPO in order to understand the role of this hormonein cellular proliferation, growth, and differentiation in both normal anddisease processes.

We have identified a novel gene that is upregulated in the presence ofEPO, named JUNE-1. Cloning and sequencing reveal that this 5 exon, 1.2kb transcript produces a protein of 44 kDa, which is highly conservedacross species. The identification of a nuclear localization signal and PHDmotif (zinc finger), suggest that JUNE-1 may be involved in chromatinremodelling or transcriptional regulation. RT-PCR studies show expressionin many tissue types, including a variety of leukemia and lymphoma celllines.

Studies are underway to determine the function of JUNE-1, usingnormal and leukemic cells, including a murine erythroleukemia (MEL) cellsystem, useful for studying erythroid differentiation in vitro. Antibodies toJUNE-1 are in development, and will be used in protein expression studiesacross panels of mouse and human tissues, and in specific subcellularcompartments. GST tagged JUNE-1 constructs are being expressed inbacterial systems, to identify JUNE-1 protein binding partners by affinitychromatography, co-immunoprecipitation, and mass spectrometry. Studiesare also underway using RNAi to identify phenotypes associated withJUNE-1 knockdown. Since this gene may be a transcription factor, it willalso be important to determine what genes are affected downstream ofJUNE-1; changes in gene expression will be examined using gene chipmicroarrays.

164ARSENIC TRIOXIDE INHIBITS TNF-ALPHA INDUCED SUPPRESSION OF ERYTHROID DIFFERENTIATION OF HUMAN CORD BLOOD CD34+ CELLSJ. 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. ParkSoon 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 ofproinflammatory cytokines (such as cancer)- is one of the most common

iseh05.book 0 Tuesday, May 10, 2005 10:22 AM


Abstracts

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 traditionalChinese medicine. Our recent studies suggest that low dose of arsenictrioxide induces erythroid differentiation of K562 human leukemic cellsand high dose of arsenic trioxide induce apoptosis. In this study, we haveinvestigated in vitro effect of arsenic trioxide on the erythroiddifferentiation and it could inhibit TNF-alpha induced suppression oferythroid differentiation of human cord blood CD34+ cells. Expression ofglycophorin A was 35.94±7.94% after 7 days culture of human cord bloodCD34+ cells and was decreased to 17.63±7.33% when culture of humancord blood CD34+ cells with 100ng/mL of TNF-alpha. Expression ofglycophorin A was increased in dose dependent manner after 7 daystreatment with arsenic trioxide and arsenic trioxide increased percentage ofglycophorin A in culture with TNF-alpha compared to TNF-alpha alone.The results of colony assay of CFU-MIX and BFU-E after culture withvarious conditions revealed similar patterns with expression of glycophorinA. These results suggest that arsenic trioxide induces erythroiddifferentiation of human cord blood CD34+ cells and can reverse TNF-alpha induced suppression of erythroid differentiation of human cord bloodCD34+ cells.

165INFLUENCE OF ADHERENT CELLS ON CYTOKINE PRODUCTION BY NORMAL BONE MARROW ERITHROID CELLSV. V. Denisova*, I. A. Lisukov, A. D. Kulagin, I. V. Kruchkova, S. A. Sizikova, S. V. Sennikov, V. A. KozlovInstitute of Clinical Immunology, SB RAMS, Novosibirsk, Russia

Background: As shown earlier, marrow erythroid nuclear cells produceof IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, IL-17, TNF-alpha, IFN-gamma, G-CSF, GM-CSF, MCP-1, MIP-1beta. Adherent marrow cellsfraction is consist of any immunoregulatory cells: monocyte/macrophagues, mature lymphocytes, mesenchymal cells and etc.

The aim: We investigate regulatory influence of adherent cells (AC) andtheir products on cytokine production by marrow Glycophorine A + ENCin healthy individuals.

Methods: GlA+ ENC and AC were obtained from bone marrow biopsyspecimens from 22 healthy individuals. Positive selection of GlA+ ENCfrom total MNC population performed with the help of specific antibodiesto Gl A by panning procedure. Controls of purity were performed using thesmears staining of hemoglobin-containing cells by benzidine. Adherentcells were collected by adhesion to plastic and cultivated for 24 hours forsupernatant obtaining. ENC (1 million cell/ml) were cocultured in differentratio: 20, 30, 40% of AC presence or after 30% AC supernatant addition.After 24 hours cultivation the concentration of IL-6 and TNF-alpha inmarrow ENCs culture media was measured by electrochemiluminescencemethod (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-6production by ENC was revealed.

Conclusion: We suggest, adherent cells fraction have ability to suppresscytokine production by erythroid marrow nuclear cells as cells-to-cellscontact either by means of humoral factors.

166IL-7 ENGINEERED STROMAL CELLS EXERT A DOSE DEPENDENT EFFECT ON NAïVE T CELLSP. Sportoletti*, B. Del Papa, M. Di Ianni, M. De Ioanni, E. Bonifacio, V. Lanterna, L. Moretti, T. Zei, F. Falzetti, A. TabilioDepartment 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 apotential therapeutic agent which could replenish depleted T cell numbersin several clinical conditions.

To investigate the effects of different amounts of IL-7 on naïve T cellpopulation we constructed an in vitro system using transducedmesenchymal cells to deliver IL-7 and compared their impact on naive Tcells.

Normal immunoselected naive T cells, were cultured with mesenchymalcells producing either 16 or 400 picog/ml IL-7. After 7 days'culture at thelower dosage CD45RO+ and CD45RA+ expression did not differsignificantly from the starting fraction (12% vs 6.5% and 58% vs 60%

respectively). The apoptosis rate remained stably compared with culture ofnaïve T cells alone (14% vs 14.9%). Only 6.9% cells entered phase S.Similar expansion rates in the CD4+ and CD8+ subpopulations wereobserved (19% and 26% respectively). A low profile of proliferation wasmaintained as evaluated with CD 25 and CD71 expression (9.3% and 10%respectively).

At the higher dosage CD45RO+ expression increased from the startingfraction of 6.5% to 62%, the number of CD45RA+ cells decreased from60% to 50% and the apoptosis rate, as evaluated with annexin V, increasedto 21%. Cell cycle progression was induced with 15% cells entering phaseS. CD8 expression rose from the starting fraction 19.3% to 40%, theproliferation marker CD71 was upregulated fom 8.7% to 31.2% and theactivation marker CD25 rose from 9.7% to 17.8%.

We have demonstrated that IL-7 transduced stromal cells can modulatenaïve T cells priming in a dose dependent way. Because of their differentcytokine production, they might be the ideal vehicle to safely addresslymphopoiesis in T cell deficient host.

167SUCCESSFUL RETROVIRAL GENE TRANSFER TO HEMATOPOIETIC STEM CELLS HIGHLY EXPANDED BY FIBROBLAST GROWTH FACTOR -1A. Crcareva1,2*, T. Saito1,3, A. Kunisato1,4, M. Sakata-Yanagimoto1, S. Chiba11Departments of Hematology/Oncology and Cell Therapy/Transplantation Medicine, University of Tokyo Graduate School of Medicine and Hospital, Japan2Deptartment of Stem Cell Biology, University of Groningen, The Netherlands3Transplantation Biology Research Center, BMT Section, Massachusetts General Hospital East, Boston, MA, USA4Kirin Brewery Co. Ltd Pharmaceutical Research Laboratories, Japan

The two major prerequisites for successful retrovirus infection are toenrich the hematopoietic stem cells (HSCs) and to have them enter the cellcycle. To this point, in mouse HSC transduction protocols, treatment ofdonor mice with 5-FU and/or cell sorting with subsequent cytokinestimulation during the infection period have been major elements.Regarding the starting cell population, freshly isolated bone marrow cellsare thought to be the best target cells for retrovirus-mediated gene transferbecause of their high stem cell potential.

Here, we present a method for retrovirus transduction into mouse long-term reconstituting cells pre-cultured in vitro with fibroblast growth factor-1 (FGF-1) as a single cytokine. Whole bone marrow cells were cultured inthe presence of FGF-1 and heparin for 3 weeks. Both non-adherent andattached cells were transferred into retronectin-coated dishes and infectedwith virus supernatant from MP34 cells stably transduced with pMY/GFPretrovirus. During the 3-day transduction period, SCF and TPO were addedto the FGF1-stimulated cultures. The retrovirus-transduced cells weresorted and transplanted into lethally irradiated recipient mice with orwithout competitor cells.

The experiments illustrated that the number of bone marrow-derivedcompetitive repopulation units (CRUs) was increased from 600 to 9,200per mouse after a 3-week culture period with FGF-1. Following retroviraltransduction of the expanded cells, the absolute number of sortedretrovirus-transduced CRUs was 7,200. Using the retrovirus-transducedcells in non-competitive reconstitution assay, we achieved radiationprotection and long-term bone marrow reconstitution in 100% recipientswith average myeloid and lymphoid chimerisms of 70% and 50%,respectively. This protocol allows the transplantation of 150 recipients withcells derived from a single donor mouse.

In conclusion, FGF-1-expanded bone marrow cells constitute excellentsource of stem cells that could be used in a range of gene deliveryprotocols.



168EFFICIENT TRANSDUCTION OF HUMAN CD34+ CELLS USING SAIMIRIINE HERPESVIRUS 2G. M. Doody1, J. P. Leek1, A. K. Bali2, A. F. Markham1, E. A. de Wynter1*1University of Leeds, Leeds, UK2University Erlangen-Nurnberg, Erlangen, Germany

Saimiriine herpesvirus 2 (HVS)-based gene therapy vectors may havepotential clinical applications. In this study we used a cosmid-generatedHVS vector incorporating enhanced green fluorescent protein (HVS-EGFP) to transduce CD34+ cells isolated from cord blood. The extent ofgene transfer into the committed progenitors was assessed in the CFU-GEMM assay. Our results showed there was no significant difference inthe colony numbers or colony morphology between transduced and mock-tranduced cultures (p=0.85). However, when the colonies were examinedunder fluorescence microscopy, about 70% of the erythroid colonies fromHVS-EGFP transduced cultures expressed EGFP (EGFP+). More than90% of the cells from the colonies were Glycophorin A (Glyco A) positivewith the majority expressing both EGFP and Glyco A. Next, we evaluatedthe effect of the virus on erythroid expansion in liquid culturessupplemented with SCF, EPO and TPO for 14 days. The transducedCD34+ cord blood cells gave rise to EGFP-expressing erythroid cellsthough the level of EGFP expression was lower than that observed in theclonogenic assays. The presence of the transgene did not alter the erythroiddifferentiation potential but there was a striking difference in the number ofcells generated. At day 14, cell numbers in the transduced cultures hadincreased on average 520-fold compared to 220-fold in mock-transducedcultures suggesting the virus may influence the rate of proliferation. Themorphology of the cells from both cultures was indistinguishable.Maintenance of the episome was confirmed by FISH and Gardella gelelectrophoresis. In summary, this is the first demonstration that the HVS-EGFP vector can infect primitive human hematopoietic progenitor cellsand that expression of the transgene is maintained during proliferation anddifferentiation. This may have particular relevance to disorders affectingthe erythroid lineage. Studies are ongoing to examine transductionefficiency in stem cell populations and other hematopoietic cell lineages.

169EFFICIENT NON-VIRAL TRANSFECTION OF MOUSE AND HUMAN EMBRYONIC STEM CELLSZ. N. Berneman1*, V. F. I. Van Tendeloo1, D. R. Van Bockstaele1, P. G. Jorens2, P. B. Singh3, P. Ponsaerts11Experimental Hematology, Antwerp University Hospital, Edegem, Belgium2Clinical Pharmacotherapy, Antwerp University Hospital, Edegem, Belgium3Research Center Borstel, Borstel, Germany

BACKGROUND: Development of efficient non-viral gene transfertechnologies for embryonic stem (ES) cells is urgently needed for variousexisting and new ES cell-based research strategies. In this study weinvestigated mRNA electroporation as a tool for short-term gene transfer inboth mouse and human ES cells.

METHODS: Culture and mRNA electroporation conditions for feeder-free cultured mouse and human ES cells were optimised on three mouse EScell lines (E14, R1 and HM-1) and one human ES cell line (H9). Afterelectroporation with EGFP mRNA, transfected ES cell populations wereanalysed by FACS for EGFP expression, viability and phenotype.

RESULTS: (A) Electroporation of EGFP mRNA in mouse ES cellsresulted in high level transgene expression (>90% EGFP positive cells)combined with low electroporation-induced cell mortality (>90% viablecells). Moreover, the electroporation procedure did not have influence onES cell phenotype and further cell culture of undifferentiated ES cellpopulations. (B) Although human ES cells are much more sensitive ascompared to mouse ES cells, we were able to develop improved cultureand electroporation conditions for feeder-free maintained H9 human EScells, which resulted in high level transgene expression (>90% EGFP+cells) combined with high cell viability (>90% viable cells) after EGFPmRNA electroporation.

CONCLUSIONS: RNA electroporation is a highly efficient method forshort-term genetic loading of both mouse and human ES cells. Ongoingresearch 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 cell-fate within in vitro cultured embryoid bodies.

170MULTI-GENE TARGETING WITH ANTISENSE OLIGODEOXYNUCLEOTIDES: AN EXPLORATORY STUDY EMPLOYING PRIMARY HUMAN LEUKEMIA CELLSJ. B. Opalinska1*, B. Machalinski1, J. Ratajczak2, M. Z. Ratajczak2, A. M. Gewirtz31Pommeranian Medical University, Szczecin, Poland2University of Louisville, Louisville, Kentucky, USA3University of Pennsylvania School of Medicine, Philadelphia, PA, USA

We previously reported that the Myb and Vav protooncogenes areamenable to silencing with antisense oligodeoxynucleotides (AS ODN)and that inhibition of either impairs leukemic cell growth. Since theexpression of these genes is not known to be linked, we sought todetermine the therapeutic value of silencing both genes simultaneously inK562, and primary patient (n=9) chronic myelogenous leukemia (CML),cells. K562, and primary CML cells were exposed to AS ODN, alone or incombination, for 24 or 72 hours and then cloned in methylcellulosecultures. Effects on K562 cluster, and BFU-E and CFU-GM colonies weredetermined, and correlated with the ability to downregulate the targetedmRNA. After 24 hours' exposure, K562 cell growth was inhibited in asequence specific, dose responsive manner with either Myb or Vav ASODN. Exposure to both ODN simultaneously considerably enhancedgrowth inhibition and accelerated apoptosis. Primary cell results were morecomplex. After 24 and 72 hour exposures to either anti-vav, or anti-mybAS ODN, equivalent colony forming unit (CFU) inhibition was observed.Exposing cells to both AS ODN simultaneously for 24 hours did not resultin additional inhibition of colony formation. However, after 72 hoursincubation with both ODN, colony formation was diminished significantlywhen compared to either ODN alone (from ~30% to ~78% for CFU-GM;~50% to ~80% for BFU-E). We hypothesize that exposing primaryleukemic cells to ASODN targeted to two, or possibly more, genes mightsignificantly augment the therapeutic utility of these molecules.

171EPIGENETIC REGULATION OF MOUSE CD19 GENE IN B CELL DEVELOPMENTH. Tagoh*, C. BoniferMolecular Medicine Unit, University of Leeds, St James's University Hospital, Leeds LS9 7TF

CD19 is a B cell co-receptor expressed throughout B cell developmentexcept plasma cells. A lack of CD19 expression in B cells causes a defectin their antibody responses as well as a decrease in the number of mature Bcells. In contrast, over-expression of CD19 results in hyperactive B cellsand loss of tolerance, suggesting the importance of properly regulatedCD19 expression. Although aberrant expression of CD19 expression isfound in some myeloid leukaemias, the cause of this lineage promiscuousexpression is unknown. In order to address this question, we examined thechromatin structure of the mouse CD19 locus to understand themechanisms that regulate tissue and stage restricted CD19 expression.

A B cell specific transcription factor, Pax5, is necessary for CD19expression and binds to the promoter. We found that chromatin at themouse CD19 promoter is DNase I hypersensitive in a B cell specific andPax5 dependent fashion. However, as seen with the human CD19 gene, themouse CD19 promoter alone is not tissue-specifically active. We searchedfor other elements regulating CD19 expression and we have identified aDNase I hyper-sensitive site (DHS) at about 2kb upstream of the promoter(DHS-2kb). DHS-2kb showed B cell specific enhancer activity andharbours putative E2A/Tal-a, AML1, and ets-1 sites. We show by in vivofootprinting that these sequence motifs are occupied in B cells.Interestingly, DNaseI hyper-sensitivity and protein occupancy at DHS-2kbare found even in the absence of Pax5. In vitro induction of Pax5 induced aDHS at the promoter and transcription of CD19. These findings suggestthat the DHS-2kb element has already been reorganised at the level ofchromatin structure and Pax5 is required to activate CD19 expression atpro-B cell stage. We are investigating the order of events of CD19 geneactivation occurring in B cell differentiation.



Abstracts

172GENE EXPRESSION PROFILING REVEALS FUNDAMENTAL DIFFERENCES BETWEEN LEUKEMIC STEM CELLS (LIN-CD34+CD38-) AND MATURE BLASTS (LIN-CD34+CD38+) OF ACUTE MYELOID LEUKAEMIA (AML) PATIENTSK. Zibara1,2*, D. Pearce1, D. Taussig1,2, E. Bureau1, S. Skoulakis2, S. Tomlinson3, B. Young1,21Cancer Research UK, LRI, Haematopoietic Stem Cell Laboratory, London, UK2Cancer Research UK, Medical Oncology Unit, St Bartholomew's Hospital, Medical College, London, UK3Cancer Research UK, LRI, Computational Genome Analysis Unit, London, UK

The identification of LSC has important implications for future researchas well as for the development of novel therapies. The phenotypicdescription of LSC now enables their purification and should facilitate theidentification of genes that are preferentially expressed in these cellscompared to normal HSC. However, gene-expression profiling is usuallyconducted on mononuclear cells of AML patients from either peripheralblood and/or bone marrow. The aim of this study was to compare the geneexpression profile of highly purified LSC versus leukemic blasts in order toidentify genes that might have important roles in driving the leukemia. Forthis purpose, we analyzed the gene expression profiles of highly purifiedLSCs (Lin-CD34+CD38-) and more mature blast cells (Lin-CD34+CD38+) isolated from 7 adult AML patients. Affymetrixmicroarrays (U133A chip), containing 22,283 genes, were used for theanalysis. Comparison of Lin-CD34+CD38- cell population to the Lin-CD34+CD38+ cell fraction showed 5421 genes to be expressed in bothfractions. Comparative analysis of gene-expression profiles showedstatistically significant differential expression of 133 genes between the 2cell populations. Gene ontology was used to determine the categories of theup-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 thegenes showing the highest differences in expression levels were thefollowing: ribonucleotide reductase M2 polypeptide, thymidylatesynthetase, ZW10 interactor, cathepsin G, azurocidin 1, topoisomerase II,CDC20, nucleolar and spindle associated protein 1, Rac GTPase activatingprotein 1, leukocyte immunoglobulin-like receptor, proliferating cellnuclear antigen, myeloperoxidase, cyclin A1. Some transcripts detectedhave not been implicated in HSC functions, and others have unknownfunction so far. This work identifies new genes that might play a role inleukemogenesis and cancer stem cells. In addition to being a more efficientway to further understand the biology of LSC, this should also provide amore efficient way of identifying new therapeutics and diagnostic targets.

1735-AZA-2'-DEOXYCYTIDINE AND VALPROIC ACID INDUCE CHANGES IN MULTI-DRUG RESISTANCE PROFILES IN LEUKAEMIA CELL LINESH. M. Martin1*, L. J. Hale2, P. R. Kearns21Dept. of Pathology and Microbiology, University of Bristol, UK2Dept. of Clinical Sciences, University of Bristol, UK

Current evidence from pre-clinical studies and early clinical trialssuggests global demethylating agents, (5-aza-2'-deoxycytidine) and histonedeacetylase inhibitors (valproic acid), have promising anti-leukaemiaactivity. However, their inherent lack of specificity may result in inducedexpression of genes which alter the phenotypic behaviour of leukaemicblasts. For example, expression of the multi-drug resistance gene MDR1 isknown to be under epigenetic control. Demethylation can induceexpression of MDR1 causing resistance to anthracyclines and vincaalkaloids, both important therapeutic agents in the treatment of leukaemia.This study investigated the effect of 5-aza-2'-deoxycytidine and valproicacid on the expression and methylation status of a group of membranebound drug efflux pumps in a panel of leukaemia cell lines, with particularemphasis on associated changes in chemosensitivity to conventionalcytotoxic drugs.

The 9 leukaemia cell lines were exposed to either 5-aza-2'-deoxycytidine, valproic acid or both agents for a continuous period of 120hours. Chemosensitivity to doxorubicin, vincristine, prednisolone anddexamethasone was measured pre- and post exposure to 5-aza-2'-deoxycytidine and/or valproic acid using the SRB assay. RNA and DNA

were extracted from pre and post drug exposed cells, and the geneexpression and promoter methylation status of 6 membrane bound drugefflux pumps were analysed using RT-PCR, combined bisulphiterestriction analysis (COBRA) and methylation specific PCR.

5-aza-2'-deoxycytidine exposure, alone and in combination withvalproic acid, was shown to induce chemo-resistance in the majority ofleukaemia cell lines. Interestingly, in some cases, a synergistic effect onglucocorticoid resistance was demonstrated. Increased chemo-resistancecorrelated with increased gene expression and demethylation of one ormore of the studied drug efflux pumps in all cases. These findings mayhave important implications for the scheduling of combination studies of 5-aza-2'-deoxycytidine and valproic acid with conventional cytotoxic drugs.

174GENE EXPRESSION PROFILING OF SINGLE HAEMATOPOIETIC PRECURSOR CELLSE. Sakhinia1*, A. Bashein1, A. M. Buckle2, R. Byers1, G. Brady31The University of Manchester, Faculty of Medical and Human Sciences, School of Medicine Division of Laboratory and Regenerative Medicine, Manchester, UK2The University of Manchester, Faculty of Life Sciences, Manchester, UK3Epistem Ltd., Incubator Bldg., Grafton Street, Manchester, UK

Expression profiling of haematopoietic cells is hampered by theheterogeneous nature of haematopoietic tissues and the absolute rarity ofearly-unrestricted progenitors. To overcome these restrictions we havedeveloped a quantitative gene expression-profiling method, which we haveused to compare expression of lymphoid and myeloid lineage markers inmouse bone marrow single haematopoietic precursors, fractionated BMand differentiating FDCP mix cells.

Global amplification of cDNA was carried out in all samples, yieldingrepresentative PolyA cDNAs pools. Gene specific RT-PCR was thenapplied to cDNAs and the expression profile of the lymphoid and myeloidassociated genes (LEF-1, Ikaros, Mb-1, EBF, CD19, Sox-4, B29, CD45, c-fms, Lysozyme, PU.1 and CD5) measured by real-time PCR. The resultsdemonstrate that both conventional PCR and TaqMan analysis can beapplied to PolyA cDNA derived from single cells and the expressionpattern for GAP, c-fms, lysozyme and CD45 determined by TaqMananalysis was in good agreement with previous Southern hybridisationstudies. TaqMan analysis of 40 mouse myeloid hematopoietic precursorsfailed to detect any expression of LEF-1, EBF, CD19, Ikaros, and CD5(despite efficient detection in lymphoid cells). Surprisingly, the lymphoidassociated genes Sox-4 and B29 were detected with a peak of expression ingranulocyte/macrophage precursors. Sox-4 and B29 expression was alsofound in multipotent FDCP mix cells with a pattern consistent with a peakof expression in granulocyte/macrophage precursors. Although there are nopublished accounts of Sox-4 expression in myeloid cells the B29 data isconsistent with previous observations that B29 is expressed in early foetalbipotent B cell/macrophage progenitors and in a progranulocyte/macrophage cell line. Our findings suggest a potential role for Sox-4 and/orB29 in early myelopoiesis and preliminary results indicate that single cellcluster analysis may provide a means of identifying gene hierarchies andcharacterising individual cells on the basis of their expression profiles.

175CYTOKINE-ASSOCIATED GENE EXPRESSION IN DENDRITIC CELLS (DCS): COMPARISON BETWEEN ADULT PERIPHERAL BLOOD- DERIVED DCS AND UMBILICAL CORD BLOOD-DERIVED DCS BY CDNA MICROARRAYY. Koga1, A. Matsuzaki1,2, A. Suminoe1, H. Hattori1, T. Hara1*1Department of Pediatrics, Graduate School of Medical Sciences,Kyushu University, Fukuoka, Japan2Division of Child Health, School of Health Sciences, Kyushu University, Fukuoka, Japan

Background: The immaturity and dysregulation of neonatal immunesystem may cause an increased susceptibility to infectious morbidity andmortality 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 orderto elucidate the difference in DC function between newborns and adults.

Materials and Methods: Immature DCs were obtained from UCB andAPB of healthy human donors as a mononuclear cell population withHLA-DR+, TCR-alpha/beta-, CD14-, CD56- and CD19-. Granulocyte-



macrophage colony-stimulating factor, interleukin-3, and human TNF-alpha were added to generate mature DCs. Total RNA was extracted, andgene expression was compared by using cDNA microarray containing 553cytokine-associated genes between UCB- and APB-derived DCs.

Results: In immature DCs, a total of 83 genes showed higher expressionin UCB-derived DCs than in APB-derived DCs, which included calciumbinding 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. Inmature DCs, on the other hand, seven genes, all of which were alsodetected in immature DCs, showed higher expression in UCB-derived DCsthan in APB-derived DCs, while 48 genes exhibited decreased expressionin UCB-derived DCs, which included chemokine genes associated withdifferentiation and IL-12 production of T cells.

Conclusions: The expression levels of cytokine-associated genes weresignificantly different between UCB- and APB-derived DCs. Thisdifference may result in a decreased reactivity of DC-mediated immunityin newborns.

176TRANSCRIPTIONAL PROFILING OF MONOCYTE-DERIVED PROGENITOR CELLSL. H. Weng1*, R. Parwaresch1, M. Walter3, M. Bonin3, T. Thurau2, M. Kleine2, D. C. Cai41Institut für Hämatopathologie und Lymphknotenregister des Universitätsklinikums Schleswig-Holstein, Germany2Planton GmbH, am Kiel-Kanal 44, D-24106 Kiel, Germany3Medizinische Genetik, Universitästklinik Tübingen, Calwerstr.7, D-72076 Tübingen, Germany4Institut für Pflanzenbau und Züchtung Christian-Albrechts-Universität zu Kiel, Olshausenstr. 40, D-24118, Germany

Human peripheral blood monocytes are able to be induced into novelpluripotent progenitor cells, having great potential in both reproducible andtherapeutic application. For understanding the molecular mechanismunderlying, transcriptional profiles of monocyte-derived progenitor cellswere generated and analysed. Monocytes were cultivated in adifferentiation medium containing various effectors. Six days afterstimulation, differentiated cells (M6) were harvested and used fortranscription profiling experiments (Affymetrix, GeneChip U133 plus 2) inwhich non-stimulated monocytes (M0) served as control. As a result, about39% of whole human transcripts could be detected in both M6 and M0samples. Compared to M0, 1795 transcripts were found to be differentiallyexpressed in M6, from which 853 transcripts were up-regulated and 942transcripts were down-regulated (p<0.05, change fold >2.5). The changefolds of the transcripts varied from +241 to –219. Database analysisrevealed that about 53% of differentially expressed transcripts aremolecules with defined functions showing binding, catalytic andtranscription-regulator activities. In contrast, approx. 35% of thedifferentially expressed genes remain functionally unknown andunclassified in now database. We found that most of the up-regulated genesare involved in pathways such as cell cycle, electron transport chain,complement-activation-classical and biosynthesis whereas the down-regulated genes were mainly found in apoptosis, G-protein signalling,GPCRs and TGF-beta pathways. These results provide an insight intomolecular characteristics of early differentiation of monocytes into theprogenitor cells. Molecular and functional characterization of candidategenes are in progress.

177DAILY EXPRESSION OF CLOCK GENE EXPRESSION RHYTHM WAS ABOLISHED IN PERIPHERAL BLOOD CELLS OF CHRONIC MYELOID LEUKEMIAS. F. Lin1*, M. Y. Yang1, T. C. Liu1, J. G. Chang21Kaohsiung Medical University, Kaohsiung, Taiwan2China Medical University, Taichung, Taiwan

Increasing amounts of data have indicated the physiological significanceof circadium clock gene regulation in various peripheral cells. Circadiumclock gene Per1,Per2,Per3 are expressed in a circadian manner in humanperipheral mononuclear cells, with the peak level occurring during thehabitual time of activity, has been reported. The present study investigatedwhether circadium clock gene function in PB mononuclear cells of chronicmyelogenous leukemia.

We examined expression of the human homology of Per1, Per2, Per3,Cry1, Cry2, Clock, Ck1e, BMAL1 and Tim in PB mononuclear cells from14 CML patients and 30 healthy control by real time quantitative reversetranscriptase polymerase chain reaction. The daily variation of clock geneexpression was examined at 2am. 8am. 2pm. and 8pm. Significant dailyvariation in Per2, CK1e and BMAL1 gene expression were observed inhealthy individuals, while CML patients exhibited no daily variation inthese clock genes. The expression level also showed significantly lowerthan the healthy controls. The role of functional circadium machinery inCML patients needed for further investigation.

178THE '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. GauglerInstitut Paoli-Calmettes, Marseille, France

In this report, we investigated the 'cytokine storm' in 113 patients whoreceived a RIC regimen before allo-SCT from an HLA-identical sibling. 10different 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 plasmasamples collected from patients before allo-SCT, at day 0 and 1, 2 and 3months 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 lymphoidmalignancy. The remaining 26 patients (23%) were treated for metastaticnon-hematological malignancies. The majority of patients (n=90, 80%)had an advanced disease. In addition to fludarabine, the RIC regimenincluded busulfan and ATG in 80 patients (71%) and low dose irradiationin 33 (29%) patients. In this cohort, the overall cumulative incidence ofgrade 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 significantlyassociated with the development of severe grade 2-4 aGVHD. Of note, tothe contrary of myeloablative allo-SCT, TNF-alpha levels did not show asignificant correlation with the risk of aGVHD. IL-12 levels weresignificantly 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 4615pg/mL (P<10e-4). Interestingly, IL-12 levels significantly decreased afterappropriate aGVHD treatment. In multivariate analysis, IL-12 levelmeasured around the first month after RIC allo-SCT was the strongestpredictive factor for aGVHD development and severity (P<10e-4;RR=10.8). In all, results from this analysis suggest that monitoring of IL-12, a key cytokine in the induction of Th1 and cytotoxic immuneresponses, appears to be a useful indicator of aGVHD after RIC allo-SCT,and anti-IL-12-based therapy might represent a potential tool forcontrolling alloreactivity.

179HALOFUGINONE INHIBITS T CELL ACTIVATION: POSSIBLE IMPLICATIONS FOR GRAFT VERSUS HOST DISEASE (GVHD)M. Leiba1*, L. Cahalon2, A. Shimoni1, M. Pines3, O. Lider2, A. Zanin-Zhorov2, I. Hecht2, U. Sela2, A. Nagler11Hematology and BMT, Chaim Sheba Medical Center, Tel Hashomer, Israel2Immunology Dept., Weizmann Institute of Science, Rehovot, Israel3Institute of Animal Science, Bet Dagan, Israel

Introduction: Halofuginone, a low molecular weight plant alkaloid, wasshown to inhibit collagen alpha1(I) gene expression in several animalmodels and patients with fibrotic diseases including scleroderma andGVHD (Nagler et.al, Transplantation, 1999, BBMT 2004). In addition,studies now show that halofuginone inhibits transforming growth factorbeta, a potent fibrogenic factor, but also a novel immunomodulator. Theaim, of the present study was to investigate halofuginone effects onactivated T cells, to better understand the mechanism of action, andconsequently the therapeutic potential in GVHD.

Methods: Peripheral blood T cells were activated by anti-CD3 McAbs inthe presence or absence of halofuginone (5-40 ng\ml) and assessed for:NFkappaB activity, cytokine production, including tumor necrosis factoralpha and interferon gamma, T cell apoptosis, chemotaxis andphosphorylation of P38 Mitogen Activated Protein Kinase (MAPK).



Abstracts

Results: Pre-incubation of the peripheral blood activated T cells withhalofuginone resulted in a significantly decrease level of NFkappaBactivity, in a dose dependent manner (NFkappaB activity index wasreduced by 75%, p=0.005). Additionally, halofuginone inhibited thesecretion of the proinflammatory cytokines: tumor necrosis factor alphaand interferon gamma (55% reduction, p=0.005). Similarly, halofuginonewas able to inhibit apoptosis in the activated T cells (50% reduction,p=0.005). Finally, halofuginone also significantly inhibitedphosphorylation of P38 MAPK in activated T cells (phosphorylation wasreduced by 60%, p=0.0001). In contrast, T cell chemotaxis was notaffected by halofuginone.

Conclusion: Halofuginone inhibits activated peripheral blood T cellfunctions, and production of proinflammatory cytokines throughNFkappaB activation, and P38 MAPK phosphorylation, making it anattractive immunomodulator and an anti-inflammatory agent. Our findingsthus suggest that halofuginone may have a therapeutic role in GVHD, andfurther improve our understanding of T cell signaling pathways in normaland pathological conditions.

180PERSISTENT MIXED CHIMERISM IN PLASMA CELLS FOLLOWING ALLOGENEIC STEM-CELL TRANSPLANTATION (ALLOSCT) IN PATIENTS WITH ACUTE LEUKEMIA IS A SURROGATE MARKER FOR DISEASE RELAPSEA. Shimoni*, L. Trachenbrot, G. Ishoev, I. Hardan, N. Shem-Tov, A. NaglerChaim Sheba Medical Center, Tel-Hashomer, Israel

Chimerism within cellular subsets following alloSCT has beenextensively studied, however, there is only limited data on chimerismkinetics within the plasma cell population (PC) and its prognosticsignificance. We studied 45 patients with acute leukemia, at serial time-points, following myeloablative (51%) or reduced-intensity conditioning(49%) and peripheral-blood SCT from sex-mismatched donors. BM smearswere evaluated by Duet combined cytogenetic/morphologic analysissystem. This system scans BM smears, saves cell coordinates, the stain isthan removed, and FISH for X and Y markers is applied to the same slide.Recipient cells are identified by their gender and their morphologyrecovered. 30/45 patients (67%) had recipient PC detected during the first 3months after SCT, constituting 0.01-1.6% of BM cells. This was oftenassociated 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). BMtests, 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 inlymphoid or other lineages at this stage. However, outcome after alloSCTwas significantly different. Among the 12 patients with recipient PC, 8relapsed, while among the 12 patients with no recipient PC, only 1relapsed, and one died of infection. 2-year disease-free survival was 83%,and 27 %, respectively (p=0.05). In conclusion, recipient PC may persistfor long duration after alloSCT and are relatively resistant to conditioningand allogeneic responses. Moreover, persistence of host PC beyond 6months is a surrogate marker for ineffective GVL, even in patients withGVHD, therefore associated with increased risk for disease relapse.

181THE ENHANCING EFFECT OF IMMUNOSUPPRESSIVE AGENT ON THE CYTOLYTIC ACTIVITY OF INHIBITORY NK CELL RECEPTOR (CD94/NKG2A)-EXPRESSING CD8 T CELLSJ. Tanaka1*, N. Iwao1, T. Toubai1, N. Kato1, Y. Miura1, S. Ota2, M. Asaka2, M. Imamura11Hematology and Oncolog, Hokkaido University Graduate School of Medicine, Japan23rd 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 ontumor and leukemic cells through the recognition of HLA-E and inducecytolytic attack without an inhibitory signal against HLA class I-decreasedtarget cells. Tacrolimus (FK506) is a potent immunosuppressive agent that

inhibits transcription of cytokines such as IL-2 in T cells. We found thatthere was no effect of FK506 (0.3 ng/mL) on the expansion of CD94/NKG2A-expressing T cells from granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (G-PBMCs) by thestimulation using immobilized OKT3 and IL-15 for 7 days. There was aslight decrease in the absolute numbers of CD94/NKG2A-expressing Tcells in G-PBMCs culture with FK506 compared with those in culturewithout FK506. However, the proportions of activating NK cell receptorNKG2D-expressing T cells and intracellular granzyme A expression inCD94/NKG2A-expressing cells were much more increased in G-PBMCscultured with FK506. The cytolytic activity levels of purified CD94-expressing cells from 7-day cultures with FK506 tested against 51Cr-labeled K562 cells by standard 4-hour 51

Cr release assays without prior sensitization were much higher thanthose 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 datasuggested that FK506 did not inhibit cytolytic activities of inhibitory NKR-expressing T cells and that there was a possibility of cytolytic activitiesbeing enhanced through the induction of cytotoxic molecules such asNKG2D and granzyme. Therefore, FK506 can strongly inhibit antigen-specific T cell immune functions but might spare antitumor activitymediated by NK cells and also inhibitory NKR-expressing T cells. Theeffect of FK506 on T cells and inhibitory NKR-expressing T cells mighthave some roles on the regulation of delicate balance between GVHD andGVL.

182CHARACTERISTICS OF NK SUBSETS AND INHIBITORY C-TYPE LECTINS IN AML PATIENTS AND THEIR HLA-MATCHED ALLOGENEIC DONORSH. 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. KimCatholic Hemopoietic Stem Cell Transplantation Center, St Mary's Hospital, Seoul, South Korea

Objective: The ability to distinguish harmful target cells or infectiousmicrobes by NK cells from healthy individual is definitely dependent onthe level of both inhibitory and activating receptors. The C-type lectinsCD94/NKG2A is considered that it perform a surveillance function for theabsence of self-identifying classical class I MHC or even HLA-Emolecules. However, we have not noticed any report to direct a guidelinein the clinic, specifically for the treatment of AML patients based on thisclue.

Materials and Methods: Blood samples were obtained from adultpatients(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 amulticolor flow cytometry. The cells were identified using the anti-CD159a Ab, anti-CD16/56 Ab, together with the anti-CD3 Ab. Finally werecruited the whole clinical data for the patients enrolled in this study.

Results: The expression levels of NKG2A in the normal donorpopulation were higher than those of AML patients who were either incomplete remission or not. Although age was not a major factor, male sexmight be another factor to determine the expressions of NKG2A accordingto this analysis. Interestingly, patients who showed poor response toinduction chemotherapy revealed a tendency of lower expressions ofNKG2A (P<0.05). Further, high levels of donor CD16/56+CD3+ cellsshowed more favourable outcomes. However, expression levels of donorNKG2A in association with the disease-free survival were not statisticallysignificant in a multivariate regression analysis (P=0.09).

Conclusion: Based on this study, we could suggest a certain role ofspecific inhibitory receptor NKG2A against AML even in the allogeneicsibling transplantation setting.



183DENDRITIC CELL-DIRECTED CYTOKINES INDUCED A GVL EFFECT IN PATIENTS WITH ACUTE LEUKEMIA WHO RELAPSED AFTER ALLOGENEIC TRANSPLANTE. K. Waller1,2, M. Arellano1,2*1Winship Cancer Institute, Atlanta, GA, USA2Emory University Hospital, Atlanta, GA, USA

Background: Relapsed acute leukemia following allogeneichematopoietic progenitor cell transplantation (HPCT) has a dismalprognosis. An attractive approach would be to 'break tolerance', inducing agraft versus leukemia effect (GvL). GM-CSF and Interferon-alpha (IFN)induce a dendritic cell-like differentiation of leukemia cells, and activateallo-reactive T-cells in vitro.

Methods: A retrospective study of 105 patients with AML or ALL whorelapsed after allogeneic HPCT. Overall survival was compared amongpatients treated with cytokines, second transplant, DLI, and supportivecare.

Results: Median post-relapse survival for all patients was 61 days.Palliative chemotherapy or supportive care (N=75) resulted in a mediansurvival of 50 days. Donor lymphocytes (N=13) resulted in a mediansurvival of 158 days. 12 patients underwent a second allogeneic HPCTwith a median survival of 419 days, but no long-term survivors. 5 patientsreceived cytokine immunotherapy with GM-CSF and/or interferon-alphawith a median survival of 427+ days with 2/5 patients remaining alive at amedian follow-up of >2 years. Median doses of 500 ug GM-CSF weregiven 3X/week (median 8 doses) and 3 million units IFN 1-3 times perweek (median of 5 doses). 4/5 patients achieved a pathologic orhematologic remission with an average response of 3.3 mos. (6 wks. to >2years). Cytokine toxicities including malaise, myalgias, rash, and feverwere not dose-limiting. GVHD occurred in 4/5 patients, at a median of 6weeks. One patient remains without evidence of disease on noimmunosuppressive drugs. Cytokine-treated patients had better survivalcompared 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 withrelapsed ALL/AML may induce allo-reactive T-cells with GvL activity.Toxicities are acceptable and long-term survival is not worse than othertherapies, warranting further study.

184SUCCESSFUL TREATMENT WITH AN INDUCTION OF GVHD IN RELAPSED ATL AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATIONT. Kamimura1*, T. Miyamoto2, N. Kawano3, H. Henzan1, S. Hayashi11Division of Hematology, Harasanshin Hospital, Fukuoka, Japan2Center for Cellular and Molecular Medicine, Kyushu University Hospital, Japan3Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Japan

Adult T cell leukemia/lymphoma (ATL) is known to be one of the mostaggressive hematological malignancies, however, successful allogeneichematopoietic stem cell transplantation (allo-HSCT) have been reported.We describe two cases of relapsed ATL after allo-HSCT, who had beensuccessfully treated with an induction of graft-versus host disease(GVHD). A 52-year-old woman, who had been diagnosed as acute ATLwith multiple subcutaneous tumors, received allogeneic peripheral bloodstem cell transplantation from HLA-identical sibling donor. Although acomplete remission (CR) was achieved early after transplantation,subcutaneous ATL relapse developed on day 92. She had received donorlymphocyte infusion (DLI) on day 115 and achieved CR with developmentof chronic GVHD, and has remained CR for 36 months after DLI. Theother, a 56-year-old man with acute ATL, received allogeneic bonemarrow transplantation from HLA-identical unrelated donor. On day 52,ATL was relapsed with multiple nodular skin infiltration when acuteGVHD was not evident. Skin biopsies performed on day 54 showed aprominent infiltration of ATL cells and the small-sized lymphocytessurrounding ATL cells in the dermis. Subcutaneous tumors disappearedalong withdrawal of the immunosuppressant, while acute GVHDdeveloped on day 75. Immunohistochemistry of the skin lesions on day 77presented a relative decrease of ATL cells but a dominant increase of thecytotoxic T-cells, which were positive for CD8 and T-cell-restrictedintracellular 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 forpatients with ATL.

185PERICARDIUM INVOLVEMENT IN ACUTE GRAFT-VERSUS-HOST DISEASE FOLLOWING ALLOGENEIC STEM CELL TRANSPLANTATIONY. Kikushige*, K. Nagafuji, K. Takase, A. Numata, T. Miyamoto, M. HaradaMedicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Japan

Several factors are responsible for cardiac complications after allogeneicstem cell transplantation (allo-SCT). These factors include cardiotoxiceffects such as chemotherapy, radiation, immunosuppressive drugs, andinfectious events. We describe two cases with pericardial effusion as amanifestation of acute GVHD after allo-SCT. One male patient with adultT-cell leukemia/lymphoma (ATL) underwent allo-BMT from the unrelateddonor mismatched at one HLA antigen after the myeloablativeconditioning (busulfan and cyclophosphamide) followed by GVHDprophylaxis with tacrolimus and methotrexate. On day 13, he complainedof orthopnea, and echocardiography showed marked pericardial effusion.Treatment such as diuretics, vasodilator and cathecolamine was given witha minimal improvement. Pericardial fluid was exudative and contained amajority of donor-derived CD8+HLA-DR+ T-cells. Examinations ofcardiac fluid revealed negativity for viral, bacterial, fungal infections, andrelapse of ATL. Subsequently, he developed acute GVHD (skin), and anadditional immunosuppressant, prednisolone (PSL) started for treatment ofGVHD. Along with regression of skin GVHD, cardiac effusion graduallydecreased. The other with AML, received cord blood transplantationmismatched 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 andechocardiography showed massive pericardial effusion. Cardiac fluid wassterile and contained the donor-derived T cells. On day 9, the level ofbilirubin 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. Thelevel of bilirubin gradually decreased in parallel with the improvement ofpericardial effusion. These observations indicate that pericardium may beconsidered as one of potential target organs of acute GVHD. Thus, for thepatients with serosal effusions early after allo-SCT, it will be important tomake a correct diagnosis and treatment since pericardial effusionassociated with GVHD would be fatal.

186HEDGEHOG PATHWAY ELEMENTS IN HEMATOPOIETIC DIFFERENTIATIONK. Detmer1*, A. N. Walker1, R. K. Humphries21Mercer University School of Medicine, Macon, GA, USA2Terry Fox Laboratories, Vancouver, BC, Canada

Hedgehog (Hh) signaling has been shown to be a potent regulator of cellfate determination, proliferation and differentiation in non-hematopoieticsystems. Expression of Hh pathway genes is found in hematopoietic stemand progenitor cells but their roles are largely unknown. Hh signaltransduction acts on the Gli family of transcription factors abolishingtranscription repression by Gli3 and inducing transcription activation byGli1 and Gli2. To examine the role of the Hh pathway in hematopoietichomeostasis, deregulation of the pathway was achieved through murineretroviral transduction and bone marrow transplantation using the GLI1gene or the gene for a constitutively active Hh-receptor mutant, SmoM2.

Dramatic perturbations in hematopoiesis were evident upon examinationof bone marrow cells where there was an inversion of the erythroid/myeloid ratio. At 16 weeks post-transplant, GLI1-transduced cells were60% Gr1/Mac1 positive versus 30% for controls and 35% ter119 positiveversus 58% for controls (p<0.05). By contrast, SmoM2 increased theproportion of ter119 positive cells. Wright-stained cytospin preparations ofmarrow showed morphology consistent with the changes in the erythroid/myeloid ratio. The erythroid/myeloid distribution of untransduced cellspresent was altered suggesting that Hh signaling affects hematopoieticdifferentiation through cell autonomous and non-autonomous mechanisms.



Abstracts

While peripheral blood counts were in general normal suggestinghomeostatic regulatory mechanisms could overcome the bone marroweffect, reconstitution of the T lineage was delayed. At 3-4 weeks post-transplant, the population of CD4+ and CD8+ lymphocytes was 64% and41% (p<0.05) of control, respectively, in SmoM2-expressing mice but notGLI1-expressing mice. These results were consistent with a model of T-cell ontogeny in which down regulation of Hh signaling promotes T-cellmaturation, and together with the bone marrow study, indicate that theeffects of Hh signaling in the hematopoietic system are mediated by theactions of different members of the Gli protein family acting in concert.

187ACTIVATION OF SPHINGOSINE KINASE MEDIATES SUPPRESSIVE EFFECT OF INTERLEUKIN-6 ON HUMAN MULTIPLE MYELOMA CELL APOPTOSISQ. 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 multiplemyeloma (MM) cells via activation of multiple signaling cascades.Sphingosine kinase (SPK), the enzyme that phosphorylates sphingosine toform sphingosine-1-phosphate (S1P), has been identified as a signalmolecule and plays important roles in the regulation of cell proliferationand apoptosis. However, whether SPK activation is involved in the IL-6signal of multiple myeloma cells and what are its roles in thepathologenesis of MM remain unclear. In this report, we show that IL-6activates SPK in MM cells and SPK activation mediates suppressiveeffects of IL-6 on MM cell apoptosis. Both MM cell lines and primary MMcells constitutively express SPK. Treatment of MM cells with IL-6 resultedin activation of SPK in a concentration-dependent manner. Either Ly294002 or PD98059, specific inhibitors for the PI3K and ERK/MAPKpathways, respectively, blocked the IL-6-induced activation of SPK. Wefurther demonstrated that IL-6-induced activation of SPK inhibited Dex-induced apoptosis of multiple myeloma cells. IL-6 stimulation or retroviralmediated overexpression of SPK gene in MM cells resulted in increasedintracellular SPK activity and up regulation of mcl-1, leading to increasedcell proliferation and survival. Conversely, inhibition of SPK by isRNAreduces IL-6-induced up regulation of mcl-1 and blocked the suppressiveeffect of IL-6. Taken together, these results delineate a key role for SPKactivation in IL-6-induced proliferation and survival of multiple myelomacells. And SPK is identified as a potentially new therapeutic target inmultiple myeloma.

188REGULATION OF ADIPOCYTE DIFFERENTIATION IN MOUSE MARROW STROMAL CELLS BY LEVEL OF ANTI-OXIDANT RESERVESJ. Glowacki1*, S. Lechpammer1, M. W. Epperly2, S. Zhou1, S. Nie2, J. S. Greenberger21Brigham and Women's Hospital, Boston, MA, USA2University of Pittsburgh Cancer Institute, Pittsburgh, PA, USA

Mice deficient in manganese-superoxide dismutase (Sod2-/-) die afterbirth with a massive accumulation of lipid in liver and skeletal muscle.Recently, we reported that long-term bone marrow cultures establishedfrom neonatal Smad3-/- mice showed 16.5-fold more adipocytes andprolonged hematopoiesis, compared with Smad3+/+. Here we investigatedconstitutive adipocytogenesis in bone marrow stromal cells from Sod2-/-and Smad3-/- mice, and tested the hypothesis that adipocytogenesis islinked with antioxidant pool sizes.

Bone marrow stromal cells from Sod2-/-, Smad3-/-, and wildtype micewere cultured±SD adipocytogenic supplements (10 microg/ml insulin, 1microM dexamethasone, 100 mM indomethacin). Adipocytogenesis wasassessed by enumeration of cells stained with 3% oil red-O dye and by RT-PCR for peroxisome proliferator-activated receptor-gamma (PPARgamma)and lipoprotein lipase (LPL). Antioxidant pool size was established bymeasurement of glutathione levels (GSH) and glutathione peroxidaseactivity (GPX).

In basal medium, Sod2-/- demonstrated constitutive adipocytogenesis,and exhibited increased lipid production in adipocytogenic medium.Treatment with 4 mM amifostine (WR2721), significantly decreased lipidaccumulation by Sod2-/- cells in basal (p=0.037) and adipocytogenic(p=0.021) media, with undetectable expression of PPARgamma and LPL.

In contrast, Sod2+/+ cells required adipocytogenic medium; WR2721decreased their adipocytogenesis (p=0.001). The antioxidant pool size inSod2-/- cells under basal conditions was significantly lower then in Sod2+/+ cells, as evidenced by measurements of GSH (78.6%; p=0.0089) andGSX (52.7%; p<0.001). Treatment with WR2721 significantly increasedboth GSH and GPX levels in Sod2-/- and +/+ cells cultured in basal andadipocytogenic media (all p<0.001). The differentiation of adipocytes inSod2-/-cells was inversely correlated with the level of GSH (Spearman r=-0.9429, p=0.0167).

In conclusion, restoring antioxidant pool size with WR2721 reducedconstitutive adipocytogenesis and inhibited induced adipocytogenesis.These data provide evidence for the involvement of the cellular redoxpathway in adipocyte differentiation of bone marrow stromal cells.

189SIGNAL TRANSDUCTION CHANGES INDUCED BY CONSTITUTIVE MUTANTS OF THE GRANULOCYTE MACROPHAGE COLONY-STIMULATING FACTOR RECEPTOR: UNDERSTANDING THE LEUKEMOGENIC POTENTIAL OF MUTANTS OF THE COMMON BETA SUBUNITM. Perugini1,2*, A. Brown1, T. J. Gonda3, R. J. D'Andrea1,2,41Child Health Research Institute, Adelaide, Australia2Department of Paediatrics, University of Adelaide, Australia3Centre for Immunology and Cancer Research, Brisbane, Australia4Queen Elizabeth Hospital, Adelaide, Australia

Granulocyte macrophage colony-stimulating factor (GM-CSF),Interleukin-3 (IL-3) and Interleukin-5 (IL-5) have overlapping andpleiotropic effects on haemopoietic cells, including mitogenesis,differentiation and functional activation. The receptors for these cytokinesare comprised of a common beta subunit and a ligand-specific alphasubunit. Constitutively activated mutants of the common beta subunit thatare able to confer factor-independent growth and differentiation in anumber of haemopoietic cell lines have been identified by our laboratory.In vivo, a transmembrane mutation can induce acute myeloid leukaemiawhereas extracellular mutations can induce myeloproliferative disorders.To establish the nature of signalling from these mutants, a number of signaltransduction pathways have been examined in an immature myeloid cellline, FDB1, which can switch between proliferation (in IL-3) anddifferentiation (in GM-CSF). We have uncovered a JAK2/STAT5-independent signal sufficient for granulocyte/macrophage differentiationand generated by the extracellular FIdelta mutation. As the activity of thismutant is dependent on the GM-CSF receptor alpha subunit, this JAK-independent signal may rely on interactions of other tyrosine kinases withthe GM-CSF receptor alpha subunit. A putative SH3 domain bindingmotif, SBP, in the cytoplasmic portion of the GM-CSF receptor alphasubunit is essential for receptor signalling and may serve as a docking sitefor SH3 domain-containing molecules such as Src Family Kinase (SFK)members. To date we and others have identified numerous interactions ofsignalling molecules with the GM-CSF receptor alpha subunit and we arenow using a number of approaches to identify which of these require theSH3 domain binding site and are required for the FIdelta signalling.

190HYDROXYUREA INDUCES ENOS ACTIVITY AND PROTEIN LEVELS IN ENDOTHELIAL CELLSV. P. Cokic1*, B. B. Beleslin-Cokic2, C. T. Noguchi3, A. N. Schechter31Laboratory of Experimental Hematology, Institute for Medical Research, Belgrade, Serbia and Montenegro2Institute of Endocrinology, Diabetes and Metabolism, School of Medicine, University Clinical Center, Belgrade, Serbia and Montenegro3Molecular Medicine Branch, NIDDK, National Institutes of Health, Bethesda, Maryland, USA

Hydroxyurea is a cell cycle-specific drug, which has been used to treatmyeloproliferative diseases and sickle cell anemia. We have recentlyshown that hydroxyurea, like nitric oxide (NO)-donor compounds,increased cGMP levels in human erythroid cells. We show now thathydroxyurea increases NO production in primary human umbilical veinendothelial cells (HUVEC) and a human bone marrow endothelial cell line.Hydroxyurea induction of NO is blocked by competitive inhibitors of NOsynthase (NOS) such as NG-nitro-L-arginine-methyl ester (L-NAME) andN-nitro-L-arginine. In addition, hydroxyurea-induced cGMP levels also



demonstrated NOS dependence in HUVEC. The hydroxyurea-induced risein intracellular calcium is followed by an increase in cAMP. We find thathydroxyurea dose- and time-dependently induced rapid and transientphosphorylation of eNOS at Ser-1177 in a preferentially cAMP-dependentprotein kinase (PKA)-dependent manner, whereas NO production isregulated by the mechanisms dependent on both PKA and Akt in HUVEC.We observe that hydroxyurea as well as specific and non-specificinhibitors of the proteasome increase eNOS protein levels in HUVEC, afterprolonged treatment. Analogously, NO production in HUVEC and humanbone marrow endothelial cell line is also stimulated by specific and non-specific proteasome inhibitors. Besides short term induction of eNOSactivity, hydroxyurea's endothelial cell effects appear to be mediated bylong term posttranscriptional augmentation in eNOS levels via inhibitionof the proteasome activity. These experiments demonstrate a mechanismby which hydroxyurea may affect NO levels in large and small bloodvessels, accounting for its inhibition of endothelial adherence of normaland sickle red blood cells.

191INTER-STRAIN VARIATION IN MOBLIZATION PROFICIENCY IS NOT REGULATED BY A GENETIC VARIATION ON THE SDF-1/CXCR4 AXISZ. 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 andhuman. HSPC mobilization is a dynamic and complex process. Stem cellshave to exit their micro-environmental niche in the BM, migrate throughthe BM-blood vascular barrier, and circulate in the blood withoutimmediately homing back to BM or any other organ. The interplaybetween cell adhesion and cell migration in mobilization of HSPCs is stillnot 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 tochromosome 11, and we will present new data on our positional cloningapproach to identify the phenotype-conferring gene in the interval.Measuring the ability of HSPCS from PB or BM from either mobilized ornon-mobilized mice to migrate across and endothelial cell layer in responseto SDF-1, we detected no significant differences between the two inbredstrains in the frequency of migrating progenitor cells. In addition, wedetected no inter-strain variation in the expression of the receptor for SDF-1, CXCR4 on HSPCs from the two strains. Surprisingly, mobilized PBprogenitor cells showed a significant, strain-independent 2-fold increase inthe ability to cross an endothelial cell monolayer in the absence of SDF-1compared to an experimental set-up without an endothelial barrier. Theimplications of this finding are not clear and warrant further investigation.Thus, the inter-strain variation in mobilization proficiency, although atleast in part being progenitor cell intrinsic, is most likely not due to agenetic variation on the SDF-1 CXCR4 axis. We are in the process ofinvestigating the adhesion properties of HSPCs from the two strains uponstem cell mobilization.

192EFFECT OF A NOVEL NF-KAPPA B INHIBITOR, IMD-0354, ON HUMAN HEMATOPOIETIC CELL MALIGNANCIESA. Tanaka1*, M. Konno1, T. Hebishima1, S. Muto2, A. Itai2, H. Matsuda11Tokyo University of Agriculture and Technology, Tokyo, Japan2Institute of Medicinal Molecular Design Inc., Tokyo, Japan

Constitutive activation of NF-kappaB is one of common features inmalignancies of hematopoietic cells, including lymphoma, leukemia, andmast cell tumors. Here we clearly demonstrated that a novel NF-kappaBinhibitor, IMD-0354, restrained factor-independent proliferation of mastcells with c-kit mutations but not of normal mast cells. In HMC-1 cellswith the Asp816Val and Val560Gly mutations, we found that NF-kappaBwas constitutively activated without any exogenous stimulation. When theDNA binding activity of NF-kappaB was inhibited by treatment with IMD-0354, cell proliferation was completely suppressed. We detected theexpression of cyclin D2, D3, and E in HMC-1 cells and observed thatcyclin D3 expression was dramatically decreased by treatment with IMD-0354. These findings indicated that auto-phosphorylated c-kit receptors

induced NF-kappaB activation, resulting in up-regulation of cyclin D3expression and cell cycle progression. Since contribution of NF-kappaB inprogression of various lymphoma and leukemia has been demonstrated, weexamined effect of IMD-0354 on some other hematopoietic cellmalignancies. IMD-0354 suppressed proliferation of several cell lines ofhuman lymphoma, leukemia, and multiple myeloma. The observationsfrom the current study suggest a therapeutic potential for compounds thatinterfere with NF-kappaB signaling in malignancies of hematopoietic celllinage, including lymphoma and mast cell tumors.

193DOES THE BRAIN STEM CONTROL RENAL EPO SYNTHESIS?U. von Wussow*, J. Klaus, U. Frackowski, H. PagelUniversity of Luebeck, Luebeck, Germany

Current data from our laboratory suggest that the renal Epo synthesis isnot only defined by the renal oxygen supply. In the ongoing study hypoxicconditions in the brain stem of rats were achieved by cisternal puncture andinsufflation of artificial cerebrospinal fluid (CSF). The intracranialpressure (ICP) was raised up to 100 mmHg for 10 min. Three hours laterplasma Epo concentrations were measured by ELISA. The results aresummerized in the table.

Elevation of ICP and therefore, the decrease of the oxygen partialpressure in the CSF led to an increase of Epo in the plasma. Bilateralnephrectomy (NX) or hypophysectomy (HX) eliminated this effect.Unspecific stressors, like the restriction of the mobility (ROM), were notcapable 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 oranemic hypoxia, these sensors lead to an increased production of anhumoral factor, which initiates Epo synthesis in the kidney.

194INVESTIGATING THE BINDING INTERACTION BETWEEN GRANULOCYTE MACROPHAGE-COLONY STIMULATING FACTOR AND THE SOLUBLE ALPHA SUBUNIT OF ITS RECEPTOR THROUGH HYDROGEN/DEUTERIUM EXCHANGE MASS SPECTROMETRYJ. L. Harris*, D. C. Schriemer, C. B. BrownUniversity of Calgary, Calgary, Alberta, Canada

Juvenile myelomonocytic leukemia (JMML) is a life-threateningdisorder in young children in which conventional chemotherapy is highlyineffective and transplant patients commonly relapse. The survival andcontinued proliferation of JMML cells is dependent upon granulocyte-macrophage-colony-stimulating-factor (GM-CSF) which must bind to themulti-subunit (alpha and beta) GM-CSF receptor (GMR) to initiatebiological response. An effective therapeutic avenue for JMML couldtherefore involve the development of interaction inhibitors to the GM-CSF:GMR complex. In this project we apply a novel tool in structuralbiology, hydrogen/deuterium exchange mass spectrometry (HDX MS), tocarry out drug target analysis as a means of developing novel cancertherapeutics. A better understanding of the protein interactions betweenGM-CSF and GMR, and identification of the critical residues involved inthe complex formation will ultimately lead to improved therapeutics. HDXMS is an emerging technology enabling exploration of protein bindingsites as well as the resulting, induced conformational changes. Successfuldigestion is essential to HDX MS in order to localize sites of deuteration tothe 3D structure. We believe that HDX MS will offer unique insight intothe binding interaction between GM-CSF and a soluble isoform ofGMRalpha (sGMRalpha). To date reliable digestion and deuteration ofGM-CSF and sGMRalpha individually has been accomplished.Preliminary data for the digestion of the GM-CSF:sGMRalpha complexhas demonstrated positive results and deuteration has been observed.Comparison of the rate of deuterium incorporation in the labeled complexversus the individual labeled proteins will allow us to determine the effectsof complex formation and thus identify the binding sites. Using HDX MS

controls ICP ICP/NX ICP/HX ROMEpo (mU/ml) 5.2±0.9 14.1±5.3* 2.6±0.5 ns 4.7±2.1 ns 5.9±1.7 nsICP: 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



Abstracts

to obtain new structural knowledge about the GM-CSF:sGMRalphacomplex will assist with the targeting of therapeutic drugs towards JMML.It may be applied to other biological complexes and provide new treatmentoptions for other types of cancer.

195GENETIC EVIDENCE OF A NOVEL BLOOD DIFFERENTIATION PATHWAY FROM LYMPHOMYELOID HEMATOPOIETIC STEM/PROGENITOR CELLS, INDEPENDENT OF COMMON MYELOID PROGENITORSA. Hultquist*, R. Månsson, L. Yang, L. Thorén, H. Qian, K. Liuba, M. Sigvardsson, S. E. JacobsenLund Stemcell Center, Lund, Sweden

We have recently identified three novel subsets of multipotenthematopoietic stem/progenitor cells (HSCs) in the Lin-Sca-1+c-Kithi

(LSK) compartment of adult murine bone marrow based on differentialexpression of CD34 and the tyrosine kinase receptor flt3. Long-term HSCs(LT-HSCs) lack CD34 and flt3 expression (LSKCD34-flt3-), whereasshort-term HSCs (ST-HSCs) are LSKCD34+flt3-. A third LSK populationis characterized by co-expression of CD34 and flt3 (LSKCD34+flt3+) andpossess a combined myeloid (granulocytic/monocytic) and lymphoid (Band T cell) differentiation potential, but surprisingly lack megakaryocytic(Mk) and erythroid (E) potential (Adolfsson et al, Cell in press). Thesefindings implicate an alternative road map for blood lineage developmentdistinct from the classical model in which the first lineage commitmentstep of HSCs is thought to result in a strict separation into myelopoiesisand lymphopoiesis. In the current study we sought genetic evidence insupport of this new model through genetic profiling of these three HSCsubpopulations in adult and fetal mice, using affymetrix chips, quantitative(Q)-PCR and single cell PCR. In contrast to pluripotent LT-HSCs and ST-HSCs, LSKCD34+flt3+ cells downregulated or turned of several genescritically involved in promoting Mk and E lineage development, such asthe Epo and Tpo receptors as well as the transcription factor GATA-1. Incontrast, genes specific for lymphoid development, such as Rag-1, sterileIg and IL-7Ralfa, were upregulated in LSKflt3+ cells, but absent in LT-HSCs and ST-HSCs. However, in agreement with their sustained ability toproduce granulocytes and monocytes, G-CSFR and PU.1 expression weresustained from LT-HSCs throughout the LSKCD34+flt3+ stage.Particularly noteworthy, single cell PCR demonstrated that a fraction ofsingle LSKCD34+flt3+ cells upregulating lymphoid specific genes also co-expressed G-CSFR. Thus, genetic profiling at the single cell level providefurther and compelling evidence for a novel road map for blood lineagedevelopment, independent of the common myeloid progenitor.

196HERITABLE BONE MARROW TRANSPLANTATION DEFECTS IN ENU-GENERATED MOUSE PEDIGREESJ. Antonchuk*, W. Alexander, D. HiltonWalter & Eliza Hall Institute, Parkville, Victoria, Australia

We have used a forward genetic approach to identify potentially novelregulatory mechanisms governing hematopoietic stem cell (HSC) function.The chemical mutagen N-ethyl-N-nitrosourea (ENU) was used to introducerandom point mutations into the germline of mice, the progeny of whichwere then screened for bone marrow transplantation defects. We screenedfor exacerbation of the Mpl-/- HSC defect by the ability of mice to beengrafted in a non-myeloablative transplantation setting with 1,000,000wildtype (Ly5.1+) bone marrow cells, and selected mice with >3% Ly5.1+blood cells at 4 weeks post-transplant. We originally screened 2,200 1stgeneration (G1) progeny of ENU-treated Mpl-/- males. Of these, 41 micewere selected which had significant Ly5.1 engraftment. Selected G1 micewere then backcrossed to Mpl-/-, and their progeny were screened forheritability of the phenotype. Of these 41 selected G1s, 3 produced affectedoffspring and were named NMT1, NMT2, and NMT3. The proportions ofaffected offspring from affected backcrosses were: 28% (74/264) forNMT1; 19% (12/64) for NMT2; and 33% (14/43) for NMT3. We alsocarried out a 3rd generation screen, to identify recessively inheritedmutations. A total of 2,460 mice were screened as above, comprising 140independent pedigrees (average 18 mice/pedigree). Of these, 4 pedigreeswere selected in which multiple mice were significantly engrafted, thesepedigrees were renamed NMT4 - NMT7. Frequencies of affected micewere 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

within each pedigree (NMT1-NMT7), to generate homozygous inbredlines carrying the respective mutations. These will then be used for geneticmapping of the 7 mutant alleles. This forward genetic approach has thusgenerated 7 novel pedigrees with defective HSC competitive ability, andshould enhance our understanding of the underlying genetic regulatorymechanisms controlling HSCs.

197DIFFERENTIAL LOSS OF ALDEHYDE DEHYDROGENASE ACTIVITY DURING EARLY STAGES OF HUMAN LYMPHOID AND MYELOID LINEAGE RESTRICTIONK. Luecke*, O. Christ, S. Imren, C. Smith, K. Leung, M. Hamilton, A. Eaves, C. EavesTerry Fox Laboratory, BC Cancer Agency, Vancouver, BC, Canada

Hematopoietic cells are typically viewed as a hierarchy defined by theco-ordinately timed differentiation steps they undergo over a series ofmany cell cycles. Aldehyde dehydrogenase (ALDH) confers selectiveresistance to alkylating agents and, when cells expressing ALDH areexposed to BODIPY-Fl1-labeled amino-acetaldehyde (BAAA), theyconvert BAAA into BODIPY-Fl1-aminoacetate (BAA). If retained withinthe cell, the generated BAA allows ALDH+ cells to be specificallydetected by multi-parameter flow cytometry. It has been known for sometime that primitive human hematopoietic cells are ALDH+, but the relativelevels of ALDH activity in different types of human repopulating cells hadnot been previously delineated. Here we show that human cord blood cellswith short term myeloid-restricted (3 weeks), or either short term (6-12weeks), or sustained (>5 months) lympho-myeloid repopulating activity innon-obese diabetic-scid/scid (NOD/SCID)-ß2-microglobulin-/- mice alldisplay the same very high levels of ALDH activity. Further, removal ofcells expressing CD2, CD3, CD7, CD14, CD16, CD24, CD36, CD38,CD56, CD66b, and/or glycophorin from low density (LD) ALDH+ humancord 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 bylimiting dilution assays in NOD/SCID mice. Nevertheless, a significantproportion of the CD34+CD38- cells LD SSClo cord blood cells werefound to have an ALDHlo-neg phenotype. Transplantation experimentswith these cells revealed they contained a previously undetected class ofprogenitors that display short term repopulating ability with restriction toB-lymphoid differentiation that was greater in NOD/SCID-ß2-microglobulin-/- mice than in NOD/SCID mice. These findings suggestthat loss of ALDH activity during human hematopoietic differentiationmay accompany lineage restriction to lymphopoiesis within theCD34+CD38- population while persisting in CD34+CD38+ myeloid-restricted cells with short term repopulating activity.

198CRITICAL ROLE OF C-KIT IN MAINTENANCE OF HEMATOPOIETIC STEM CELLSL. Thoren*, K. Liuba, C. Jensen, S. W. JacobsenLund University, Lund, Sweden

The proto-oncogene c-kit is a tyrosine kinase receptor highly expressedon hematopoietic stem cells (HSCs) and early progenitors. However, therole of c-kit in regulation of HSCs remains unclear. Several spontaneous c-kit mutations of different severity have been identified in mice, butprevious studies have suggested that these mice have surprisingly mildHSC phenotypes, with only marginally reduced HSC numbers. Herein weinvestigated in more detail the HSC phenotype in White Spotting 41 (W41)mice, in which a point mutation at kinase position 831 generates a weakkinase signal down-steam of the receptor. Phenotypic analysisdemonstrated that Lin-Sca1+kit+flt3- HSCs in fetal liver and 10-12 weekold mice were only 2-fold reduced. However, transplantation of limitingnumbers of whole BM cells into lethally irradiated recipients demonstratedthat HSCs capable of sustaining hematopoiesis long-term were reduced atleast ten-fold in w41 mice, and c-kit deficient HSCs capable of secondaryreconstitution were reduced to almost undetectable levels. Kinetic analysisof competitively transplanted recipients demonstrated that the ability ofkit-deficient HSCs to multilineage reconstitute was progressively lost withtime, suggesting that c-kit plays an essential role in HSC maintenance andself-renewal. Thus, c-kit is not important for generation and initialexpansion of HSCs, but appears to be essential for sustained self-renewalof HSCs.



199G0 HEMATOPOIETIC STEM CELLS DO NOT EXIST IN MOUSE FETAL LIVER AND THOSE IN S/G2/M DISPLAY A PRONOUNCED BUT REVERSIBLE ENGRAFTMENT DEFECTM. Bowie1,2*, L. McCaffrey1, C. Eaves1,21BC Cancer Agency, Vancouver, Canada2Genetics 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 purifiedday 14.5 mouse fetal liver HSCs (11% pure) in single-cell culturescontaining 300 ng/ml Steel factor (SF) and 20 ng/ml IL-11 supported theprevailing assumption that HSC turnover is higher in fetal liver than inadult marrow. To address this question directly, we have now performed 3types of experiments, all of which show that all day 14.5 mouse fetal liverHSCs are in cycle. In a first series of experiments (n=3), in vivo limitingdilution assays of HSCs were performed on livers of fetuses removed fromday 14.5 pregnant mice injected intravenously with 100 mg/kg 5-fluorouracil (5-FU), or saline, 24 hr previously. The results show a >100-fold reduction in HSCs in the 5-FU-treated group (i.e., to <1 HSC per fetalliver). Similar assays of HSCs in Ter-119- day 14.5 fetal liver cells culturedovernight in 50 ng/ml SF showed a 140-fold decrease in HSCssimultaneously exposed to high specific activity 3H-thymidine (relative tocontrols cultured in medium and SF alone, 5 experiments). Finally, assaysof freshly isolated Ter-119- fetal liver cells separated by FACS into G0, G1and S/G2/M fractions after staining with Hoechst 33342 and Pyronin Yshowed HSC activity was detected exclusively in the G1 fraction even afterintra-femoral injections, although equivalent activity was recovered fromS/G2/M cells if these were cultured for 6 hours in 100 ng/mL SF prior totransplantation (intravenously). Thus, fetal liver HSCs lack a G0 fractionand show the same reversible engraftment defect as proliferating adultHSCs transiting S/G2/M. These findings also indicate that measured fetalliver HSC frequencies represent an ~2-fold underestimate. Thus currentlyattainable purities should allow meaningful analysis of moleculardifferences that distinguish fetal and adult HSC properties.

200ISOLATION AND FUNCTIONAL CHARACTERIZATION OF CORD BLOOD CD34+ CELLS WITH TELOMERASE REVERSE TRANSCRIPTASE (HTERT) EXPRESSIONM. Järås*, A. Edqvist, J. Rebetz, B. Widegren, X. FanLund University, Sweden

Telomerase activity has been demonstrated in the bulk human CD34+progenitor cell population, but it is unclear whether repopulatinghematopoietic cells have telomerase activity. We therefore asked, whetherthere is heterogeneity in hTERT expression among CD34+ cells and ifCD34+ cells expressing hTERT have NOD/SCIDBeta2m-/- micerepopulating capacity. In order to functionally characterize hTERT-expressing cord blood (CB) CD34+ cells, we isolated such cells at thesingle cell level by using an adenoviral vector (cTERT-dGFP), encodingdestabilized GFP under the control of the hTERT promoter. Two dayspost-transduction of CD34+ cells with the cTERT-dGFP vector or thecontrol vector encoding EGFP under the control of the PGK-1 promoter atan MOI of 100, the percentage of GFP+ cells were 17±4.3% and47±6.7%, respectively; indicating a heterogeneity in telomerase activityamong CD34+ cells. At this time-point, GFP+ cells were sorted by flowcytometry into a hTERT and a control vector sorted fraction. Staining forthe Ki-67 antigen and 7-AAD revealed that in comparison to the controlvector sorted cells, the hTERT sorted cells had a greater proportion of cellsin 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-sortedcells were similar. Interestingly, hTERT sorted cells were devoid ofsecondary NOD/SCIDBeta2m-/- mice (n=8) repopulating capacity,whereas 3 out of 5 mice in the mock transduced group and 3 out of 7 micein the control vector transduced group were positive for human cellengraftment. In summary, these data suggest that the quiescent humanhematopoietic stem cells (HSCs) have low to undetectable levels oftelomerase activity, which is upregulated in proliferating cells, colonyforming progenitors, and short-term repopulating stem cells.

201AGE-DEPENDENT CHANGES IN HEMATOPOIETIC STEM AND PROGENITOR CELLSD. J. Pearce1*, D. Taussig1,2, A. Eddaoudi3, D. Bonnet11Hematopoietic Stem Cell Laboratory, Cancer Research UK, London, UK2Cancer Research UK Department of Medical Oncology, St. Bartholomew's Hospital, London, UK3FACS Laboratory, Cancer Research UK, London, UK

It is thought that as we age, accumulated damage causes stem cells to dieby apoptosis. In the murine hematopoietic system however, the number ofmurine C57Bl6 stem/progenitor cells is actually thought to increase withage and this has been attributed to the inbred nature of this strain.

Consistent with previous reports, we found that the percentage ofphenotypically-defined stem/progenitor cell subsets (lineage negative, c-kit+, Sca-1+ (KLS)) increases during the murine lifespan. However, boththe absolute number of SP cells and the proportion of the KLS cellpopulation that is an SP stem cell, increases dramatically with age. SP cellsfrom older mice have a reduced (p<0.01) 4-month competitiverepopulation potential when compared to SP cells from younger mice buthave the expected low progenitor activity (1 in 8.5 single-sorted cellsproduced colonies). Furthermore, SP cells from old mice possess a similarcell cycle profile (89.5% G0/G1) to that previously reported for adult SPcells (Goodell et al, 1996).

Hence, our observed increase of SP with age is not due to the acquisitionof the SP phenotype by progenitors or committed cells; rather thisdifference in SP distribution reflects an age-dependent change inhematopoietic stem cell biology. Specifically, as the number of stem andprogenitor cells increases with age, the proportion that is a stem cellincreases but these have a reduced potential when competed with cellsfrom younger mice. We are currently investigating engraftment variablesthat may explain the reduced stem cell function of SP cells from oldermice.

202Withdrawn

.

Age (days) SP % of total KLS % of total Sp % of KLS CRU fold enrichment49 0.016 0.29 1.37 Not Done61 0.04 0.49 4.4 905±326221 0.054 0.19 21.2 Not done414 0.16 0.42 32.4 123±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.

iseh05.book Page 90 Friday, May 13, 2005 5:05 PM


Abstracts

203THE G PROTEIN-COUPLED RECEPTOR CYSLT1 SIGNALS VIA ERK/MAP-KINASE IN CD34+ PROGENITOR CELLS AND STIMULATES PROLIFERATION SYNERGISTICALLY WITH INTERLEUKIN-3A. M. Boehmler*, G. Seitz, T. Wiesner, L. Kanz, R. MöhleUniversity of Tübingen, Tübingen, Germany

We have previously shown that the G protein-coupled receptor (GPCR)cysLT1 is preferentially expressed in immature CD34+ hematopoieticprogenitor cells (HPC). In the present study, we analyzed the signaltransduction pathways in mobilized CD34+ HPC activated by the cysLT1ligand LTD4, an inflammatory lipid mediator (leukotriene), which is alsoproduced by bone marrow stromal and endothelial cells when deprived ofhematopoietic cells. Of note, the effects of LTD4 on intracellular calciumfluxes, a typical early signaling step of Gi-coupled GPCR, weresubstantially stronger than those observed after stimulation of the HPC-associated chemokine receptor CXCR4 with optimal doses of thecorresponding ligand stromal cell-derived factor-1 (SDF-1). Similarly toSDF-1, signal transductions pathways leading to cell locomotion such aspolymerization of filamentous actin were significantly increased inresponse 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 wheninterleukin-3 was included in the cytokine cocktail. In these cultures, theeffects of LTD4 was comparable to the increase of the cell number inducedby addition of FLT3-ligand. As analyzed by Western blot, LTD4 induced arapid phosphorylation of ERK/MAP-kinase in CD34+ cells, whilephosphorylation of NF-kappa B, representing a second pathway of GPCRsignaling associated with cell proliferation, was unaffected. These resultssuggest that in contrast to the chemokine receptor CXCR4, activation ofthe ERK/MAP-kinase signaling pathway by cysLT1 results in an effect oncell proliferation which is comparable to that induced by hematopoieticgrowth factors.

204ENHANCEMENT OF CORD BLOOD LONG TERM CULTURE-INITIATING CELL MAINTENANCE BY O-SULFATED HEPARAN SULFATES AND BONE MORPHOGENIC PROTEIN 4 OR WNT-3AL. E. Colvin Wanshura1, E. J. Stephenson1, M. S. Nelson1, P. Gupta1,2*1VA Medical Center, Minneapolis, MN, USA2Hematology-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 thebone morphogenic protein (BMP) family of proteins and related factors arecritical for hematopoietic stem cell development, survival andproliferation. Since HS modulate the biological activity of the BMP andWnt pathways, we examined how combinations of HS, BMPs, BMPinhibitors and Wnt-3a influence primitive umbilical cord blood (UCB)progenitors in vitro. CD34+/CD38- UCB cells were cultured for 2 or 5weeks in presence of Flt3 ligand + thrombopoietin + OS-HS (basalmedium), with or without additional factors. The progeny was evaluatedfor LTC-IC maintenance using a limiting dilution assay. Addition of BMP-4 improved 5-week LTC-IC maintenance to 179±31% compared to basalmedium (P=0.03). Sonic hedgehog (SHH), which upregulates BMPsignaling, achieved similar results (5-week LTC-IC maintenance184±34% compared to basal medium; P=0.01). The BMP inhibitorsChordin, Caronte or Follistatin each reduced 5-week LTC-IC maintenanceto about 50% compared to basal medium. Addition of Wnt-3a improved 5-week 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 5weeks. Thus, 2-week LTC-IC maintenance was reduced by BMP-4 or SHHto about 70%, whereas Chordin increased it to 412±122%, compared tobasal medium. These data indicate that factors whose activities are knownto be modulated by HS, including BMPs and their inhibitors, SHH andWnts, significantly influence LTC-IC maintenance. Further, our datasuggest that these factors may be used to selectively achieve either short-term or long-term LTC-IC maintenance. Current studies are investigatingthe mechanistic interactions of these proteins with heparan sulfates.

205NEUROTROPHIC FACTORS, BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF) AND NEUROTROPHIN-3 (NT-3) SUPPORT EX VIVO EXPANSION OF MULTI-LINEAGE HEMATOPOIETIC STEM AND PROGENITOR CELLSL. 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 Kong2Department of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Hong Kong

Our previous study demonstrated that neural progenitor cell line C17.2supports ex vivo expansion of cord blood CD34+ cells in a non-contactculture (Leukemia, 19, 91-97, 2005). C17.2 cells also expressed variousneurotrophic factors including BDNF and NT-3. This study furtherinvestigated effects of BDNF (5, 10, 50 and 100ng/ml) and NT-3 (10 and100ng/ml) on hematopoiesis. Enriched CD34+ cells (MACS beads) werecultured in methylcellulose with or without BDNF or NT-3, in addition to acocktail of cytokines including erythropoietin (3IU/ml), granulocytemacrophage-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 by100±57.7%, 22.3±2.27%, and 47.0±17.4%, respectively, when comparedto control cultures (N=8, p<0.05). BDNF at 10ng/ml significantlyenhanced the proliferation of BFU/CFU-E and CFU-GEMM by17.0±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, anda cytokine combination containing thrombopoietin (T, 50ng/ml), S (50ng/ml) and flt-3-ligand (F, 80ng/ml) in QBSF-60 serum-free medium for 8days. In control TSF cultures, the number of CD34+ cells, CD34+CD38-cells, total nucleated cells, CFU-GM, BFU/CFU-E, CFU-GEMM andCFU-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 theexpansion 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 ofCD34+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 thatneurotrophic factors BDNF and NT-3 support ex vivo expansion of CD34+stem and progenitor cells, and indicated cross-talks between hematopoiesisand neuropoiesis.

206INHIBITION OF ABCG2 ON CHRONIC MYELOID LEUKAEMIA (CML) CELLS DEPLETES CD34+ STEM CELLSN. E. Jordanides1, D. Gilmour2, T. L. Holyoake1,2, J. C. Mountford1,2*1University of Glasgow, Glasgow, UK2Scottish National Blood Transfusion Service

We have described a population of quiescent stem cells (qSC) in patientswith chronic phase (CP) CML at diagnosis. In vitro studies have proventhis population to be highly insensitive to IM, and worryingly shown thatqSC are accumulated after CML CD34+ cells are treated with IM. Oneexplanation for this is the presence on these cells of multi-drug resistance(ABC) pumps. We previously showed that ABCG2, a stem cell-associatedpump, is expressed on these cells, that it is functional and interacts withIM. We therefore investigated the effect of ABCG2 inhibition on CMLCD34+ cells. Using CFSE to track cell division we treated CD34+enriched CML samples with 5microM IM±the ABCG2 inhibitorfumitremorgin C (FTC) or with 10microM FTC alone for 3d. Incomparison to untreated controls 5microM IM reduced the total number ofcells 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. Incontrast, 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) andno accumulation of qSC (104.6±33.8%). We saw no additive effect whenIM and FTC were given concurrently. Therefore, we suggest FTC may beused to deplete CD34+ stem cells from CML. Longer-term differentiationassays have demonstrated no increase in mature markers on FTC treatedcells despite the significant reduction in CD34 (n=4). Hence, this depletionis not by the induction of differentiation. We propose that ABCG2 isaberrantly expressed on a much higher % of CD34+ cells in CML and is



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

207REGULATION OF MURINE HEMATOPOIETIC STEM AND PROGENITOR CELLS THROUGH THEIR SURFACE EXPRESSION OF CD1DH. E. Broxmeyer1,2*, K. Christopherson1,2, G. Hangoc1,2, S. Cooper1,2, C. Mantel1,2, A. Bendelac3, R. Brutkiewicz1,21Department of Microbiology and Immunology, and Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN, USA2Walther Cancer Institute, Indianapolis, IN, USA3Department of Pathology, University of Chicago, Chicago, IL, USA

CD1 molecules, involved in immune cell interactions, are similar toMHC class I antigens. We found that 99.8% of Sca1+c-kit+Lin- bonemarrow cells express CD1d. While competitive repopulating ability ofCD1d -/- and +/+ hematopoietic stem cells (HSC) were similar in lethallyirradiated primary mice, there was a significant decrease in repopulation ofCD1d -/-, compared to +/+ cells in secondary recipients. Stimulation of +/+HSC in vitro with anti-CD1d enhanced their in vivo competitiverepopulating capacity. Most hematopoietic progenitor cells (HPC) sortedinto the CD1d+ cell population. Ratios of CD1+:CD1- CFU-GM, BFU-Eand CFU-GEMM were respectively 28:1, 163:1 and 160:1. Absolutenumbers of CFU-GM, BFU-E and CFU-GEMM in marrow and spleenwere significantly enhanced 2-3 fold in CD1d -/- mice, and % marrow HPCin S-phase of cell cycle was increased ~2 fold, and HPC in spleen, in aslow or non-cycling state in +/+ mice were in rapid cell cycle (~60% in S-phase) in CD1d -/- mice. Pretreatment in vitro of +/+ HPC in unseparatedor Sca1+Lin- marrow with different CD1d antibodies greatly enhancednumbers of immature CFU-GM, BFU-E, and CFU-GEMM stimulated bycombinations of cytokines, as well as mature populations of CFU-GM andCFU-M, respectively stimulated by GM-CSF and M-CSF. This increasewas not noted with CD1d -/-. The cytoplasmic tail of CD1d was requiredfor anti-CD1d enhancement of HPC proliferation in vitro; HPC fromtruncated CD1d cytoplasmic tail knock-in mice did not respond to anti-CD1d. Without cytokine addition, anti-CD1d did not stimulate colonyformation of +/+ cells. CD1d was necessary for Flt3-ligand and SCFenhancement of cytokine-stimulated HPC colony formation, and forinhibition by myelosuppressive chemokines, but not by TNF-alpha or IFN-gamma. These results implicate CD1d on HSC and HPC with theirregulation, a heretofore unknown function for CD1d.

208SMAD4 DEFICIENT HEMATOPOIETIC STEM CELLS HAVE IMPAIRED REPOPULATION CAPACITYG. Karlsson*, U. Blank, J. L. Moody, S. Singbrant, T. Utsugisawa, S. KarlssonMolecular 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, bonemorphogenetic proteins, and activins affect proliferation and maintenanceof HSCs. However, redundancy between different members of the familywithhold important information making earlier studies targeting singleTGF-b superfamily members limited. The TGF-b family receptors signal tothe nucleus through Smad proteins. We have investigated the role of Smadsignaling in hematopoiesis by means of Smad4 deficient mice, using theinducible MxCre-loxP system. Smad4 is the common Smad for all Smadsignaling pathways and Smad4 deficiency will therefore be expected toeliminate all Smad-specific signaling in hematopoietic stem cells (HSCs).Three to five weeks after induction, the Smad4 deficient mice rapidly turnsick and succumb to anemia. Additionally, these mice experienceabnormalities in the colon mucosa with an increased lamina propria andenlarged crypts infiltrated with inflammatory cells. Lethally irradiatedrecipients reconstituted with bone marrow from Smad4 deficient mice donot show any signs of disease, demonstrating that the Smad4 deficientHSCs exhibit repopulating capacity and that the disease phenotypeobserved in mice totally deficient in Smad4 is of extra hematopoieticorigin. However, when performing competitive transplantation assays,Smad4 deficient bone marrow cells had a significantly lower repopulationcapacity at 16 weeks compared to controls (11.4±8.6 % vs. 40.2±19.6 %,

respectively. p=0.01). This phenotype is maintained in secondaryrecipients at 16 weeks (0.2±0.1 % vs. 5.1±2.2 %), indicating that theSmad4 deficiency causes aberrant function of long term HSCs. Lineagedistribution in peripheral blood, as well as homing efficiency and apoptosislevels in primitive hematopoietic cells, were normal after transplantation.Thus, our findings demonstrate that Smad4 deficiency reduces thereconstitution ability of HSCs and demonstrate an important role for Smadsignaling to determine fate options and function of HSCs.

209LIN-SCA+ SORTED MURINE BONE MARROW: FLUCTUATIONS IN PHENOTYPE EXPRESSION THROUGH CELL CYCLEG. J. Dooner*, M. S. Dooner, G. A. Colvin, P. J. QuesenberryCenter for Stem Cell Biology, Roger Williams Medical Center, Providence, RI, USA

Previous work in our laboratory reveals that marrow stem cellsdemonstrate changes in engraftment (Habibian, et al J Ex. Hem, 188:393-398, 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), mRNAand surface expression of adhesion proteins (Becker et al., Exp Hematol27:533-541, 1999). We hypothesize a model in which progenitor/stem cellsare on a continuum, rather than in a hierarchy (Quesenberry, et al., Blood,100:4266-4271, 2002; Quesenberry, et al., Stem Cell Reviews, 2005 inpress), and primitive marrow stem cells continually and reversibly changephenotype, fluctuating between the phenotype of a typical progenitor andthat of a relatively mature hematopoietic cell. These fluctuations appear tobe related to cell cycle status. This theory is based on studies with purifiedLin-Ho dull/Rh dull (LRH) murine marrow cells cultured inthrombopoietin, 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, andsteel factor.

Marked and reversible shifts in stem cell surface marker, differentiationmarker, and transcription factor expression in LRH and Lin-Sca-1+ cyclingcells were demonstrated by quantitative reverse transcription-polymerasechain reaction (Real Time RT-PCR). Fog exhibits,decreased expressionover time (0, 24 and 48h in Lin-Sca-1+), whereas expression varies withCD4, CD34, Sca-1 and CD45R (0, 32, 40, 48hours in LRH cells). Relativeto time zero (Time0=1), Sca-1 shows a 17 fold increase at 32h, with a 5-fold and 9-fold increases in expression at 40h and 48h respectively. Thesedata show that primitive marrow stem cells express a variety ofhematopoietic genes, expression modulates with cell cycle transit andperhaps most importantly that observed changes in gene expression can bereversible. This is consistent with the continuum theory of stem cellregulation.

210PHAGE DIRECTED HOMING INHIBITION IN SKELETAL MUSCLED. 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. AbediRoger Williams Medical Center, Providence, RI, USA

Recent literature has demonstrated the presence of marrow derived stemcells in skeletal muscle. This population has strong hematopoietic andprobably skeletal muscle regenerative potentials. Our experiments showthat lineage negative and lineage negative Sca+ bone marrow cells arecapable of homing to skeletal muscle tissue 3 hours after intravenousinfusion. Cells can be identified after 7 days and one month, indicatingresidency of the homed cells. Phage display technology was used toidentify the adhesion molecules and receptors involved in homing.Selected plaques were amplified for DNA sequencing. Forty non-injuriesderived and sixty injury derived plaques were sequenced. The non-injuryplaques had two predominant sequences and the injury plaques had threedominant sequences. Purified phage from the five dominant sequences wasused in a phage homing inhibition study. The mice selected to receive theinjury derived phage were injured with cardiotoxin the day before thephage were injected. Mice were injected intravenously with 1013 of theirrespective phage and then injected fifteen minutes later with 2x106 CFSElabeled lineage negative cells. There were sacrificed at 3 hour and 24 hourtime points to determine inhibition of homing by phage. The collectedtissue was digested in 0.2% collagenase and analyzed by flow cytometry.Results indicate that the predominant injury derived phage were successfulin blocking homing to injured muscle tissue by a range of 62-71 % when



Abstracts

compared to injured tissue receiving just lineage negative cells. The non-injury derived phage did not significantly block homing to skeletal muscle.These results demonstrate the feasibility of using phage display to identifyfunctionally significant homing peptides and adhesion molecules that canbe used for future enhancement of marrow cells homing to other tissueswith possible clinical application in regenerative medicine.

211HAEMOPOIESIS IN THE APC MIN/+ MOUSEE. A. de Wynter*, A. F. Markham, P. L. ColettaUniversity of Leeds, Leeds, UK

The Adenomatous polyposis coli (APC) tumour suppressor gene ismutated in a hereditary form of intestinal tumorigenesis in both mice andman. Apc Min/+ mice carry a heterozygous germ line mutation in Apc andtumorigenesis proceeds following somatic loss of the second Apc alleleresulting 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 andprogenitor B cells in the bone marrow (BM). BM transplantationexperiments showed that development of the transplanted Apc Min/+ BMin wild-type animals appeared normal. In contrast, the Apc Min/+ mouseBM environment only supported short-term reconstitution of haemopoiesiswith wild-type BM cells. In addition, the number of CFU-F in Apc Min/+BM was significantly lower than that of wild-type controls. Takentogether, 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, weinvestigated haemopoiesis in vitro. Long-term bone marrow cultures fromApc Min/+ and wild-type animals of different ages were established usingthe Dexter system. Each week the non-adherent cells in cultures werecounted and analyzed in CFU-GEMM assays. Our results showed that inmice less than 80 days old, there was little difference in the cell productionof 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 proliferationin Apc Min/+ BM which may be masked in vivo by secondary phenotypicfeedback. We are currently investigating the role of Wnt signalling in thissystem.

212HIGH CORRELATION OF 6FUCT ACTIVITY WITH THE EXPRESSION OF GPIIB/IIIA COMPLEX DURING MEGAKARYOCYTIC DIFFERENTIATIONJ. Kaminska1*, U. Bany-Laszewicz1, E. Klimczak-Jajor1, D. Smolka1, J. Szczepanowska2, M. Majewski1, R. Mollicone3, J. Koscielak11Institute of Hematology and Blood Transfusion, Warsaw. Poland2The Nencki Institute of Experimental Biology, Warsaw, Poland3Universite de Paris, Villejuif Cedex, France

Megakaryocytopoiesis is a complex multistep process involving therelease of platelets into the blood circulation. The expansion ofmegakaryocyte (Mk) progenitors ex vivo requires the identification of theirlineage-restricted proteins and glicoproteins. The earliest marker ofmegakaryocytopoiesis, appearing as early as the CFU-Mk stage, is theGpIIb/IIIa complex (CD41a). In platelets, this complex functions as areceptor for fibrinogen, von Willebrand factor, fibronectin and vibronectin.Two of the enzymes involved in GpIIb/IIIa synthesis are alpha-1,6-fucosyltransferase (6FucT) and beta-4-galactosyltransferase1 (GalT1).

In our study the activity of 6FucT and GalT1 in cord blood CD34+ cellscultured in medium promoting megakaryocytopoiesis was determined. Theanalysis of phenotype and morphology of cultured cells confirmed thedifferentiation and maturation into Mk progenitors. The activity of 6FucTand GalT1 in cultured cells increased with a simultaneous increasedexpression of the CD41a antigens. High correlation of 6FucT activity(correlation factor=0.78) and low correlation of GalT1 activity (correlationfactor=0.3) with the expression of GpIIb/IIIa during megakaryocytopoiesiswas observed. We also found that the increase in the activity of 6FucT incultured cord blood CD34+ cells was almost 4-fold higher than that of the

ubiquitous GalT1, additionally confirming the special status of 6FucT inmegakaryocytopoiesis. The estimation of the mRNA expression for 6FucTwill confirm our observation on the molecular level.

In conclusion, the activity of 6FucT should be a useful marker of theearly commitment of cultured hematopoietic stem cells into themegakaryocytic lineage.

213PHOSPHATIDYLINOSITOL-3-KINASE-DEPENDENT ACTIVATION OF ERK IN PRIMITIVE BONE MARROW CD34+ CELLSP. Aravena1, F. Tong1,3, D. Headley1,2,3, D. R. Sutherland1*1Department of Pathology, Princess Margaret Hospital, Toronto, Canada2Medical Oncology and Hematology, Princess Margaret Hospital, Toronto, Canada3Division of Applied Molecular Oncology, Ontario Cancer Insitute, Toronto, Canada

Activation of ERK (Extracellular signaling Regulated Kinase) signalingby growth factors normally proceeds through the ras to raf to MEK to ERKpathway, but an additional requirement for PI3-kinase activation wasrecently identified in undifferentiated murine hematopoietic stem cellsstimulated by the c-kit ligand Stem Cell Factor (Wandzioch et al., Blood2004;104:51-57). To assess if this atypical activation pathway also occursin normal candidate human hematopoietic stem cells, we developed amulticolor flow cytometry technique that combines a standardized cellsurface staining methodology (to identify primitive CD34+ subsets)followed by SCF activation and subsequent fixation and permeabilizationto detect intracellular phosphorylation of ERK. Experiments wereperformed 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+ andCD34bright/CD117+ phenotypes phosphorylate ERK in response to SCFstimulation. Wortmannin blocked the phosphorylation of ERK in thesesubsets, but not in less primitive CD34+ cell fractions. These resultsidentify a novel signaling mechanism in primitive/uncommitted humanbone marrow CD34+ stem cells that involves an additional level of controlcompared to the classical ERK pathway.

214C-KIT EXPRESSION AND SIGNALING IN THE REGULATION OF HEMATOPOIETIC STEM CELL PROLIFERATION AND SENESCENCEJ. C. Chen*, A. L. Smith, F. M. Ellison, N. S. YoungNational Heart, Lung and Blood Institute, Bethesda, MD, USA

We tested c-Kit expression and c-Kit mediated cell expansion in vitro aswell as cell engraftment in vivo to examine the role of c-Kit signaling in theregulation of hematopoietic cell proliferation and senescence. Proportionand total number of Lin-Kit+CD34- cells, cells that contain long-termhematopoietic stem cells (HSCs), reduced significantly from young to oldage in BALB mice. In contrast, Lin-CD34-CD117+ BM cell percentageand total cell number increased from young to old age in normal B6 miceas well as in B6-Hbd/Hbd mice that carry a spontaneous hemoglobin-deficient mutation. Surprisingly, when sorted Lin-Kit+CD34- BM cellswere cultured in vitro for 7 days in the presence of 25 ng/mL stem cellfactor (SCF), cell expansion reduced with donor age in BALB as well as inB6 and B6-Hbd/Hbd mice. Our results indicate that proliferative ability ofindividual Lin-Kit+CD34- cells declined with age in B6 and B6-Hbd/Hbdmice as have been observed in BALB mice, and that aged Lin-Kit+CD34-BM cells from all three genotypes had a reduced ability to respond to thesame level of SCF stimulation in comparison to young cells of the samegenotypes. In a competitive repopulation assay, engraftment of sorted Lin-Kit+CD34- cells from old B6 and B6-Hbd/Hbd donors were lower incomparison to sorted Lin-Kit+CD34- cells from young B6 and B6-Hbd/Hbd donors. Thus, functional senescence occurs to all HSCs. The highoverall BM cell engraftment ability seen in old B6 donors may reflect anincrease in the number of functioning cells, not a gain of functionality percell.



215BONE MARROW-DERIVED PROGENITORS IN HEMOPHILIC SYNOVITISS. A. Acharya*, D. D. MacDonald, I. Goldberg, D. M. DiMichele, D. C. Lyden, M. W. HilgartnerDepartment 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 hemarthrosesleading to synovial proliferation, hypertrophy, and bony arthropathysimilar to rheumatoid arthritis and osteoarthritis. Because the proliferatingsynovial pannus in hemophilic joint disease (HJD) demands increasedoxygen and nutrients, de novo blood vessel formation is a critical part ofthe pathological process. As such, angiogenesis, and the concomitantrecruitment of bone marrow-derived (BMD) hematopoietic and endothelialprogenitor cells (HPCs and EPCs) have been implicated in the progressionof proliferative joint disease.

We have observed the presence of these progenitors, as well as potentpro-angiogenic and BMD progenitor mobilizing factors (vascularendothelial growth factor (VEGF) and stromal-derived factor-1 (SDF-1)),in histological sections of HJD synovium. Furthermore, HJD subjectsshowed a statistically significant increase in circulating cells expressingfunctional markers of HPCs, such as VEGF receptor-1 (VEGFR1) andCXCR4 via flow cytometric analysis, compared to hemophilic subjectswithout JD and healthy controls. Quantitative RT-PCR revealed thatVEGFR1, VEGFR2, and CXCR4 mRNA expression were similarlyincreased. Correspondingly, peripheral blood levels of HPC and EPCmobilization and chemotaxis mediators (VEGF, SDF-1, and MMP-9) werealso significantly elevated in subjects with HJD.

Ongoing experiments indicate the increased presence of circulatingvascular progenitors in HJD subjects. We will therefore quantify andcharacterized these BMD progenitors as early (VEGFR1+/AC133+,VEGFR2+/AC133+, CXCR4/AC133+) and late (VEGFR1+/ CD11b+,VEGFR2+/CD11b+) in these subjects. In addition, preliminary datasuggest differential capacities of peripheral blood from HJD patients andcontrol groups to form early outgrowth colony forming unit-endothelialcells (CFU-ECs), thereupon quantifying functional EPCs. The aboveprogenitors, in combination with various angiogenic growth factor levels,may serve as surrogate biologic markers to provide effective means ofdetecting early synovial proliferation and identifying candidates for earlyintervention. Ultimately, anti-angiogenic agents may provide a novelpreventative treatment for hemophilic arthropathy.

216LIPID RAFTS PLAY A ROLE IN THE FORMATION OF AN ADHESIVE JUNCTION BETWEEN HOMOTYPICALLY AGGREGATED CD34-POSITIVE CELLST. E. Bullock*, S. B. Marley, M. Y. GordonImperial College, London, UK

The function of CD34, a marker of primitive haemopoietic stem andprogenitor cells, remains unclear although studies have suggested roles inheterotypic and homotypic cell adhesion and signalling pathways,including those involved in cell cycle regulation and progenitor cell self-renewal. Homotypic aggregation between CD34-positive cells has beenproposed to be a mechanism for preventing inappropriate proliferation invivo. CD34 localises at the contact point between cells induced toaggregate with the anti-CD34 antibody, QBEND10, and between multi-cellular aggregates isolated from normal bone marrow. Adhesionmolecules such as LFA-1 and the actin cytoskeleton also localise to thiscontact point suggesting the existence of an adhesive junction betweenCD34-positive cells which can be likened to the specific arrangement ofreceptor segregation, known as the immunological synapse, between a Tcell and antigen-presenting cell. Lipid rafts are distinct plasma membranedomains that are rich in cholesterol and glycosphingolipids. They areproposed to act as platforms to transport and organise the T cell receptor,co-stimulators, signal transduction molecules and the actin cytoskeleton atthe immunological synapse. The aim of this study was to determine the roleof lipid rafts in CD34-positive cell aggregation.

Using immunofluorescence we have demonstrated that lipid raftslocalise to the contact point between CD34-mediated homotypicallyaggregated cells. Disrupting lipid rafts with the cholesterol-depletingagent, methyl-beta-cyclodextrin, significantly reduced aggregation in both

KG1a and primary CD34-positive cells. Isolation and analysis of lipid raftsfrom aggregated KG1a cells using Brij 35 reveals that a minor proportionof the total cellular CD34 is present within lipid rafts.

As in immunological synapse formation, lipid rafts play a key role in theformation of an adhesive junction between homotypically aggregatedCD34-positive cells and may act as platforms for the transport of adhesionand signalling molecules to the contact point, thereby facilitating CD34-mediated adhesion and associated signalling.

217EVIDENCE OF INTERCONVERSION WITHIN HEMATOPOIETIC LINEAGE COMMITTED CELLSL. 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 humanbone marrow CD133+ stem cells can be induced to convert to myeloidcells, and vice versa, in vitro. We subsequently employed rapid analysis ofgene expression and two-dimensional gel electrophoresis techniques inorder to study the gene and protein expression patterns of hematopoieticlineage differentiation and interconversion, in five cytokine-inducederythroid and myeloid populations from human CD133+ bone marrowstem cells. We identified and categorized the mRNA expression patterns of266 gene-specific fragments into 3 groups – genes expressed in 1, 2 or allpopulations. Protein expression profiles of the cells were consistent withthat of the expressed genes. To better define the characteristics of cells thatpossess this ability to interconvert, we analyzed the cells for theirexpression of CD133 antigen for stem cells, CD36 for erythroid, and CD13for myeloid cells by three-color flow cytometry. Enriched CD133+ cells onday 0 of culture displayed a progressive decline in antigen expression to0.06%±0.02% and 0.1%±0.05% cells on day 14 with EPO and G-CSFinduction, respectively. We then sorted the CD36+CD13+ subpopulationsfrom both the EPO and G-CSF 14-day-induced cells and re-cultured themfor another 14 days in EPO, G-CSF, or cytokine-free medium.Surprisingly, up to 80% of the double positive cells of EPO inductionbecame triple positive with EPO re-stimulation, and 29.8% CD13+appeared with G-CSF re-culture, while no significant numbers of singleCD133+ or CD36+ cells were detected. Control cells exhibited nosignificant changes. Our data suggest that hematopoietic lineageinterconversion may be a characteristic of normal hematopoiesis arisingfrom early primitive precursor cells that maintain the ability to co-expressmyeloid and erythroid markers. Further analysis of the genes and proteinproducts identified within these 'differentiated' populations may provideinsights into the mechanisms underlying hematopoietic lineageinterconversion.

218UNIQUELY PROTRACTED G1 TRANSIT OF EX VIVO SELF RENEWING HEMATOPOIETIC STEM CELLSJ. M. Nygren*, D. Bryder, S. E. W. JacobsenHematopoietic 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 withconsiderable biological and clinical implications, in particular for the failedattempts to successfully ex vivo expand HSCs for transplantation. Althoughbased on extensive evidence that HSCs, in steady state and during activeproliferation, reside predominantly in G0/G1, direct evidence for HSCfunction fluctuating with cell cycle position is still lacking. Herein, usingviable cell cycle and cell division tracking, we demonstrate that ex vivoexpanded and self-renewing HSCs in S/G2/M are not compromised inrepopulating activity. Rather, their enrichment in G1 results from HSCshaving a protracted G1 transit (without entering G0). This unique cell cycletransit of HSCs and its regulation are likely to be critical for maintenanceof the indispensable HSC pool.



Abstracts

219PERIPHERAL BLOOD DERIVED EPCS ARE MACROPHAGE COLONIES WITH THE CAPACITY TO ENHANCE ENDOTHELIAL GROWTH BY SOLUBLE FACTORSX. L. Aranguren1*, C. Moreno1, M. Soriano2, G. Abizanda1, E. J. Andreu1, J. M. Garcia-Verdugo2, F. Prósper11Foundation for Applied Medical Research, Division of Cancer, Area of Cell Therapy and Hematology Service, Clínica Universitaria, Universidad de Navarra, Spain22Department of Cell Biology, Instituto Cavanilles, University of Valencia, Valencia, Spain

Several populations of endothelial progenitor cells have been describedincluding AC133 cells from the BM, MAPCs or SVF derived cells andhave been characterized by flow citometry, culture techniques and in vivoexperiments. Levels of culture defined peripheral blood EPC have beenshown to have a negative correlation with cardiovascular diseases. Wehave analyzed the frequency of BM, CB and PB mononuclear cell derivedEPC using the techniques described by Hill (N Engl J Med 2003; 348:593-600) and characterized their endothelial phenotype and functionality. Incomparison with AC133 derived EPC, mononuclear cell derived EPCsretain hematopoietic markers, like CD45, CD14 and CD15 and lackendothelial markers like von willebrand factor and VE-cadherin. Thesecells are be able to take up ac-LDL, property associated to hepatocyte,endothelial and macrophage cells and express both endothelial andmacrophage markers such as CD31. Electronic microscopy showed thatperipheral blood derived colonies are really macrophage forming colonies(CFU). Mononuclear cell derived EPCs express pro-angiogenic cytokinesincluding VEGF, angiopoietin, HGF, PLGF and GM-CSF while EPCconditioned media stimulated proliferation of AC133 derived endothelialcells. Using an in vivo matrigel assay in scid beige mice we coulddemonstrate that while AC133 cells are able to give rise human endothelialcells (UEA positive cells), mononuclear cell derived EPCs could notdifferentiate into human endothelial cells. In conclussion, mononuclear cellderived EPC are be able to induce angiogenesis by the release of growthfactors but not by their endothelial differentiation capacity.

220TELOMERASE INHIBITION BY ANTISENSE TREATMENT INDUCES THE DIFFERENTIATION OF A HUMAN EMBRYONIC STEM CELL LINE, SNU-3 TO HEMATOPOIETIC STEM CELLSS. 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. Kim11Department of Internal Medicine, Division of Oncology and Hematology, Korea University Medical Center, Seoul, Korea2Institute of Stem Cell Research, Korea University College of Medicine, Seoul, Korea

Background: This study was tried to evaluate the effect of adenovirus-mediated telomerase antisense (TA) expression on cellular proliferationand apoptosis of the ex-vivo culture from human embryonic stem cells tohematopoietic stem cells.

Methods: Cultured embryonic stem lines (SNU-3 cell line) tohemopoietic stem cells were classified to control and study groups. Controlgroup was that of which embryonic body were cultured to LTC-IC assaynot treated with telomerase antisense oligonucleotides (TA). Study groupwas that of which embryonic body were cultured to LTC-IC assay treatedwith TA. We evaluated CD34+ cell number and viable cell percentage andthe degree of apoptosis of each samples at 5 days after LTC-IC assay andthe results were compared between each groups. These studies were triedfor 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 0.6-2.9(median: 1.6x103). Viable cell percentage of control and study groupswere 76-89(median: 83) % and 78-90(median: 85) %, respectively, withoutsignificant difference. And there was no difference of apoptosis degree(absorbance, A405nm – A490nm) between control and studygroups(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 CFU-GM in control and study group was 0.1-0.5(medium:0.3)x103 and 0.2-0.5(medium:0.3)x103, respectively without no significant difference.

Conclusion: We found the phenomenon that adenovirus-mediatedantisense expression of telomerase RNA doesn't interrupt thedifferentiation of embryonic stem cells to hematopoietic stem cells withoutsignificant effect on the apoptosis degree. Further studies will be warrantedto confirm these results and verify the mechanism of these phenomena.

221ENGINEERING NICHES FOR THE IN VITRO MANIPULATION OF HEMATOPOIETIC STEM CELL FATEM. P. Lutolf, R. Doyonnas*, H. M. BlauBaxter 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 ofdifferentiation. In these niches the stem cells are exposed to a complexinterplay of soluble and insoluble, short- and long-range signals expressedby supportive cells in close proximity. Outside of the niche, stem cellsrapidly lose their differentiation potential. In the last few years, substantialprogress has been made in understanding hematopoietic stem cell (HSC)regulation by the niche. Several signaling pathways that appear to governHSC self-renewal have been identified, paving the way for efficient in vitroHSC expansion. Yet, the HSC niche itself remains poorly defined andknowledge regarding the relevant signaling pathways has not yet beentranslated into reliable culture methodologies. We are engineering artificialniches for the ex vivo manipulation of HSC function. Syntheticpolyethylene glycol hydrogel networks serve as well-defined displayplatforms for studying the interplay of previously identified HSCregulatory proteins in diverse combinations. Such engineered niches mayserve as novel in vitro model systems for elucidating the molecularmechanisms that regulate HSC fate and may also have therapeutic use infacilitating the expansion of HSCs in tissue culture.

222MHC CLASS I-RESTRICTED CD4+ HELPER T CELLS AUGMENT CD8+ CYTOTOXIC T CELL MEDIATED ANTI-TUMOUR IMMUNITYA. Tsallios1,2*, H. J. Stauss2, E. C. Morris1,21Imperial College, London, UK2Royal Free and University College Medical School, London, UK

CD4+ T cells are critical for the generation of anti-tumour immuneresponses via cytokine secretion and enhancement of CTL priming andeffector function. However, very few class II-binding tumour antigenshave been identified limiting the isolation and adoptive transfer of tumour-specific CD4+ cells.

We have used retroviral-mediated transfer of TCR alpha and beta chaingenes to target CD4+ T cells against an MHC class I-presented modeltumour antigen. The F5 TCR recognises the influenza virus Anucleoprotein peptide (NP366-379) presented by murine Db MHC class I.Splenocytes from C57Bl/6 mice (H2b) were transduced with the pMX-F5TCRalpha-IRES-TCRbeta retroviral vector and further expanded in thepresence of IL-2. Prior to functional assays cells were sorted into TCR-tdCD4+ and TCR-td CD8+ populations. In some experiments, the TCR andCD8alpha genes were co-transferred into helper T cells to produce TCR-tdCD4+8+ cells.

Both purified TCR-td CD8+ and CD4+ cells secreted IFNgamma in anantigen-specific manner when stimulated with peptide loaded DCs. Peptiderequirements were lower for CD8+ cells (10pM) than CD4+ cells (100pM).However, when stimulated with NP-expressing tumour cells, the TCR-tdCD4+ cells only secreted IFNgamma when co-expressing CD8 (CD4+8+cells) or in the presence of additional DCs. Surprisingly, the proliferativecapacity of TCR-td CD8+, CD4+ and CD4+8+ cells correlated only withIL-2 production but not IFNgamma secretion.

Further experiments showed that IFNgamma-producing CD4+8+ T cellswhich proliferated poorly and produced little IL-2 were unable to expandin vivo or provide help for tumour rejection. In contrast, CD4+ T cellsproducing high levels of IL-2 expanded, provided help for CTL-mediatedtumour rejection and developed long term T cell memory.

The data demonstrates both the anti-tumour efficacy of TCR-td T cellsand the in vivo synergy between TCR-transduced CD4+ and CD8+ T cellsspecific for the same epitope resulting in long-term tumour protection.



223GENERATION OF CYTOMEGALOVIRUS-SPECIFIC T CELLS FROM SEROPOSITIVE SUBJECTS FOR ADOPTIVE IMMUNOTHERAPY USING PP65P. Diamanti1,2*, R. J. Garland2, D. H. Pamphilon1, C. G. Steward2,3, A. Blair1,21Bristol Institute of Transfusion Sciences, UK2Department of Pathology and Microbiology, University of Bristol, UK3Department of Paediatric Oncology and Haematology, Royal Hospital for Children, Bristol, UK

It has long been acknowledged that the transfer of cellular immuneresponses from a Cytomegalovirus (CMV) seropositive donor to arecipient that reactivates CMV post stem cell transplantation (SCT) resultsin quicker immune reconstitution. It has been demonstrated that CMV-specific CD4+ and CD8+ cytotoxic T lymphocytes from the donor areimportant in resolving CMV infection and protecting recipients fromdeveloping CMV disease post SCT. Recently CMV recombinant proteinpp65, an immunodominant target for CD4+ and CD8+ T cell responses, hasbeen developed for the stimulation and isolation of donor CMV-specific Tcells. The aim of this study was to investigate the potential of pp65 togenerate CMV-specific CD8+ and CD4+ T cells from seropositive subjectsfor use as adoptive immunotherapy after SCT. Peripheral bloodmononuclear cells were isolated from single unit CMV-positive buffy-coats and were stimulated with pp65 for 16 hours. CMV-specific T cellsthat produced IFNgamma were captured using the IFNgamma-secretionassay and were enriched using the MiniMacs system. The phenotype of thecells, both pre and post enrichment was evaluated by flow cytometry usingmonoclonal antibodies against CD3, CD4 and IFNgamma. Experiments todate have shown that pp65 is capable of stimulating both CD4+ and CD8+

T cells to secrete IFNgamma. Flow cytometric analysis revealed that priorto 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+ cellsincreased significantly (192-fold and 117-fold) to 84.4% and 94.9% in theCD3+/CD4+ and CD3+/CD4- subfractions, respectively. Large scale CMV-specific T cell capture experiments, performed under GMP conditions, areongoing. The cytotoxicity and lack of alloreactivity of these cells will beassessed to determine their suitability for immunotherapeutic use postSCT.

224EFFECT OF INTERLEUKIN 15 SUPPLEMENTATION ON PEPTIDE-SPECIFIC EXPANSION OF HUMAN T CELLSD. P. Hart*, E. C. Morris, H. J. StaussDepartment of Immunology, Imperial College, London, UK

The in vitro generation of peptide specific T cells requires cytokinesupplementation to support proliferation and survival. Interleukins 2 and 7have commonly been used. Recently, an increasing interest in generatingtherapeutic peptide specific cytotoxic T lymphocytes in vitro for adoptivetransfer 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 cellhomeostasis and inducer of antigen independent expansion of CD8 T cellsmaking it an attractive candidate cytokine for stimulation protocols.

The effect of IL15 or IL2 on recall and naive T cell responses wasexamined during the stimulation of human PBMC with CMV tegumentderived peptide, pp65, (NLVPMVATV) and melanocyte differentiationantigen, Melan A (ELAGIGILTV) respectively. All peptides were HLA-A2 restricted. Phycoerythrin (PE) labelled tetramer staining and FACSanalysis was performed at 7 and 14 days post stimulation. Priming andsubsequent expansion of naive T cells specific for Melan A was moreefficient in the presence of IL-15 compared with IL2 (28.5% vs 1.1% after2 weeks). However, stimulation and expansion of memory T cells specificfor 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 inthe presence of IL2 abrogated the beneficial effect of IL2 and CTLexpansion was reduced to the level seen with IL15 alone (pp65 tetramerpositive cells IL2 11.7%; IL15 1.1%; IL2+15 1.3%). PBMC cultured in thepresence of IL15 or IL2 show marked differences in cytokine secretionprofiles, however these are independent of the stimulating peptide.

These data suggest IL15 supplementation used in peptide-specificstimulation exerts a proliferative effect favouring expansion of naive T cellresponse, while IL2 leads to preferential expansion of antigen-specificmemory T cells.

225CYTOTOXIC T CELLS SPECIFIC FOR THE WILMS' TUMOR PROTEIN ARE PRESENT IN PATIENTS WITH BREAST CANCER AND ARE OF SUFFICIENT AVIDITY TO KILL BREAST CANCER CELLSR. Gillmore1*, S. Xue1, A. Holler1, J. Kaeda2, D. Hadjiminas3, V. Healy4, Y. Ghani1, R. C. Coombes5, J. Waxman5, H. J. Stauss11Department of Immunology, Imperial College, London, UK2Department of Investigative Sciences, Hammersmith Hospital, London, UK3Department of Surgery, St Mary's NHS Trust, London, UK4Department of Histopathology, St Mary's NHS Trust, London, UK5Department of Cancer Medicine, Imperial College, London, UK

The Wilms' tumor protein, WT1, is over-expressed in the vast majorityof acute leukemias and currently trials are taking place investigating thepotential benefit of WT1-based immunotherapy in these patients. The aimof our research has been to validate the targeting of WT1 in breast cancer.Having determined the incidence of WT1 mRNA expression in primarybreast tumours (~90%) we analysed the immune responses to WT1.Patients with stage I/II breast cancer were studied since vaccinationstrategies, to boost any pre-existing immunity, are more likely to besuccessful 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 HLA-A2 positive by flow cytometry. Of these, 5 yielded samples sufficient forexpansion and tetramer analysis. Fluorescent HLA-A2/WT1 tetramerswere used to quantify WT1-specific T cells immediately ex vivo andfollowing repeated antigen-specific in vitro stimulation. The functionalcapability of the tetramer-positive cells was assessed using 51Cr-releasekilling assays and intracellular cytokine staining, to identify interferon-gamma producing cells.

RESULTS: WT1-specific T cells were detectable in all draining LNs butnone of the PB samples. LN samples from 3 patients underwent repeated invitro stimulations until the frequency of tetramer-positive cells peaked at0.16%, 4.04% and 21.3% of viable CD3+ lymphocytes. These T cellsdisplayed cytotoxicity and IFN-gamma production in response to target T2cells pulsed with the relevant WT1 peptide epitope but not when T2 cellswere pulsed with an irrelevant peptide. Importantly, these T cells alsokilled HLA-A2 positive WT1 mRNA expressing breast cancer cell linesfollowing treatment with interferon-gamma.

CONCLUSION: Patients with early breast cancer possess WT1-specificT cells of sufficient avidity to recognise and kill breast cancer cells. Thesedata provide strong support for the initiation of vaccination trials in thispatient group.

226IMMUNOGENICITY OF THE TUMOUR ASSOCIATED ANTIGEN WT1F. Ramirez*, Y. Ghani, H. StaussImperial College London, London, UK

Wilm's tumour antigen 1 is a tumour-associated antigen over expressedin leukemia and various solid tumours. We are studying WT1immunogenicity in the mouse to explore its suitability for vaccinationbased tumour immunotherapy. WT1 expression is restricted tohaematopoietic stem cells, kidney cells and few others. This restrictedpattern of expression suggests that the immune system may not becompletely tolerant to WT1. However, mRNA-WT1 has been detected inmouse lymphoid organs, including the thymus, although it is unclear whichcells are responsible.

Two MHC class I-restricted epitopes have been previously characterisedin mouse by searching for class I binding motifs and in vitro generation ofallo-MHC-restricted CTL. The two epitopes are presented by WT1-expressing tumour cells. We have explored different vaccination protocolsto elicit self-MHC-restricted WT1-specific CTL responses in C57BL/6mice.



Abstracts

Our results show that peptide vaccination with incomplete Freund'sadjuvant was inefficient in eliciting WT1-specific CTL responses. Somemice generated a low response while in others the response wasundetectable. Several boosting with peptides did not enhance the response.Contrary to similar models, co-stimulation with peptides that stimulateCD4 T cells or anti-CD40 monoclonal antibody injection did not increasethe CTL response. However, peptide-loaded dendritic cells consistentlyinduced strong WT1-specific CTL responses.

Mice immunised with peptide-loaded dendritic cells were challengedwith tumour cell lines expressing WT1 and very low or no protection wasobserved. This correlates with our inability to detect high-avidity WT1-specific CTL responses after expansion of WT1-specific T cell lines invitro from immunised mice.

In summary our data show that only after optimal vaccination withdendritic cells it was possible to generate WT1-specific CTL responses.However, DC vaccination seems to stimulate low avidity CTL responsesunable to confer protection against tumour challenge. These data suggestthat mice are substantially tolerant to the two WT1 epitopes used in thisstudy.

227PHASE I CLINICAL TRIAL OF CT-011, A HUMANIZED MONOCLONAL ANTIBODY DIRECTED AGAINST A B7 FAMILY-ASSOCIATED PROTEIN, IN PATIENTS WITH ADVANCED HEMATOLOGICAL MALIGNANCIESA. Nagler1*, A. Kneller1, A. Avigdor1, M. Leiba1, R. Koren2, L. N. Klapper2, M. Schickler2, A. Shimoni11Hematology, BMT and CBB, Chaim Sheba Medical Center,Tel Hashomer, Israel2CureTech Ltd., Yavne, Israel

Objective: CT-011, a humanized monoclonal antibody that is directedagainst a B7 family-associated protein, was previously shown to efficientlyelicit anti-cancer immune response against a wide range of murine andhuman tumor models (Hardy et al. PNAS 94:5756-5760, 1997). A Phase Iclinical study was set to evaluate the safety and determine the MTD of CT-011 in patients with advanced stage hematological malignancies.

Methods: 15 patients with advanced hematological malignancies (AML-7, NHL-4, CLL-3, HD-1) participated in the study. All pts failed severallines 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 inescalating 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-relatedtoxicities. Common adverse events included minimal allergic reactions andlow grade fever. No single dose MTD was found in this study. One AMLpatient with resistant leukemia that was platelet-dependent with platelets<10x109/L is currently 7 months post CT-011 infusion in PR and is platelettransfusion-independent. Two additional patients (NHL-1, HD-1) remainwith SD for over 6 months. Five other patients are alive with active diseasewith a median follow up of 3 (1-6) months, while seven patients died fromtheir advanced resistant disease. Accrual to this study as well as patientfollow up continues.

Conclusions: A single administration of CT-011 is safe and welltolerated in patients with advanced hematological malignancies. Theobserved anti-tumor activity may be related to CT-011 interaction with theB7 receptor family-associated protein resulting in enhancement of tumor-specific immune response. Future studies will evaluate the combination ofdonor lymphocyte infusion and CT-011 for patients with hematologicalmalignancies having minimal residual disease after stem celltransplantation.

228DOES FETAL-MATERNAL MICROCHIMERISM CONFER IMMUNOLOGIC TOLERANCE OF FETAL CELLS?G. L. Gilmore*, M. Holm, J. Lister, R. K. ShadduckWestern Pennsylvania Cancer Institute, Pittsburgh, PA USA

Fetal-maternal microchimerism [MC] is a benign condition that occursduring gestation, due to trafficking of cells from the fetus to the maternalcirculation (and vice versa) via the placenta. MC can persist for decades.The incidence of MC has been reported to be 33% in normal parouswomen, but the MC status of cancer patients is unknown. To study this, weobtained blood samples from 169 normal parous women, and 26 parouscancer patients. 33 non-parous women served as controls. We detected

male DNA by two rounds of PCR with nested primer sets specific for the Ychromosome. The sensitivity of the assay is 1 male cell/1 million femalecells, validated with mixtures of male and female cell lines. We found thefrequency of MC depends on the amount of DNA assayed: using 1 ugDNA [~300,000 genomes], 18% of normal parous females were MCpositive %. 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 5ug DNA was tested, but this rose to 76% when >25 ug DNA wasexamined. The lower frequency of MC among cancer patients at 5 ug DNAis presumably due to prior chemotherapy. Suprisingly, we found 2 normalnon-parous donors were MC; this number did not increase when moreDNA was analyzed. The reason for MC among non-parous females isunknown.

Our results reveal MC is more frequent among parous women thanpreviously reported, and imply a long-lasting tolerance of fetal tissueantigens, since fetal cells can be detected in the maternal circulationdecades after birth. We are currently assessing whether MC translates intoimmunological tolerance to male offspring leukocytes. If this proves to bethe case, this suggests that son-to-mother cellular therapy may be a feasibleapproach to treat malignancy.

229A TELOMERASE-DERIVED PEPTIDE ANTIGEN GENERATES ANTILEUKEMIC T CELLS FROM B-CLL PATIENTS IN VITROA. Choudhury*, M. Palma, P. Kokhaei, L. Hanson, A. Osterborg, H. MellstedtKarolinska Institute, Stockholm, Sweden

Telomerase is up regulated in the vast majority of human malignanciesand therefore may be considered as a ubiquitous tumor antigen. Hightelomerase activity has been previously reported in patients with B-cellchronic lymphocytic leukemia (B-CLL). In the present study, the presenceof naturally occurring, telomerase-reactive, T-cells in the peripheral bloodof B-CLL patients were examined. We have also attempted to expand invitro T-cells reactive to a telomerase-derived peptide and assess theirability to lyse autologous B-CLL cells.

19/25 B-CLL patients tested demonstrated telomerase positivity by RT-PCR. Of these, PBMC of 5 positive and 3 negative patients were furtherutilized for generating telomerase-reactive T-cells. Monocyte-deriveddendritic cells (DC) were pulsed with a 16 aa long, telomerase peptide or a17 aa Ras peptide and used to stimulate autologous T- cells. In 3 of the 5patients a significantly higher proliferative response was noted against thetelomerase peptide compared to Ras. No significant telomerase-specificproliferation was observed with T-cells of telomerase-negative patients.Antibodies against both MHC class I and class II had an inhibitory effecton T-cell proliferation. T-cells were put through two rounds of stimulationand expansion with telomerase- or Ras peptide-pulsed DC and examinedfor their ability to lyse autologous leukemia cells in vitro. The cytotoxicityof telomerase- stimulated T-cells was approximately 53% at an effector:target ratio of 50:1. In comparison the cytotoxicity of Ras-stimulated T-cells was 14%. The cytotoxicity was blocked with antibodies against MHCclass I but not with anti-class II antibodies. Our data demonstrates thatsome B-CLL patients have naturally occurring T-cells against telomeraseand boosting anti-telomerase immunity may be an approach toimmunotherapy of B-CLL

230INDUCTION AND IDENTIFICATION OF MYELOMA-SPECIFIC CYTOTOXIC T CELLSL. Zahradova1*, D. Ocadlikova1, L. Kovarova1, M. Penka3, R. Hajek1, J. Michalek1,21Laboratory of Experimental Hematology and Cell Immunotherapy, University Hospital, Brno, Czech Republic2Cancer Immunobiology Center, University of Texas, Southwestern Medical Center, Dallas, Texas, USA3Department of Clinical Hematology, University Hospital, Brno, Czech Republic

Multiple myeloma (MM) was considered as low immunogenic incurabledisease. The attempts were made to invert the immune status to recognizemyeloma cells by cells of the immune system. Here we studied biology ofmyeloma-specific T cells in vitro.



Irradiated myeloma cell line ARH 77 was used as tumor antigen tostimulate peripheral blood mononuclear cells (PBMC) of 8 healthyvolunteers. Dendritic Cells loaded by irradiated autologous MM cells wereused to stimulate PBMC of 10 MM patients. Activated responder T cellswere immunomagnetically separated based on surface expression ofinterferon gamma and expanded in 5 cases by phytohemaglutinin andrepeated high-doses of interleukin 2. Cytotoxicity against the originalmyeloma cells was tested after the expansion using propidium iodide or 7-amino actinomycin D. Responder T cells were labeled by CFSE todistinguish them from myeloma cells. Third-party PBMC and interferongamma negative fraction of T cells served as controls.

The percentage of interferon gamma positive cells in healthy donors wasenriched from 2.8±0.9 and 2.6±0.8 to 48.6±23.4 and 73.2±25.9 ofCD3+CD4+ and CD3+CD8+ T cells, respectively, by immunomagneticseparation. Interferon gamma positive Tcells were further expanded invitro from 0.54x106±0.05x106 to 214.00x106±103.46x106 within 4weeks. A specific cytotoxicity was tested after expansion. The killing ofmyeloma cells by expanded interferon gamma positive T cells reached68.1±14.2%, while interferon gamma negative fraction killed only0.8±0.3% of myeloma cells. As another control, killing of third-partyPBMC by expanded interferon gamma positive T cells was 6.9±2.5%.Similar observations were made in MM patients and will be presented onISEH meeting.

Data demonstrate a specific cytotoxic effect of expanded interferongamma positive T cells against myeloma cell line ARH 77 and open thepossibility for clinical use of tumor-specific T cells in cancerimmunotherapy.

Supported by grant IGA MZCR 7475-3

231EX VIVO EXPANSION OF CMV LYSATE STIMULATED CD4+T CELLS FOR ADOPTIVE IMMUNOTHERAPYY. Heike*, M. Hosokawa, Y. Morita, K. Tobinai, Y. TakaueNational Cancer Center, Tokyo, Japan

Cytomegalovirus (CMV) reactivation/disease was one of the seriouscomplications after allogeneic stem cell transplantation. Although anti-virus drugs have been developed, they caused other complications leadingto the failure of transplantation. CMV specific T cells worked an importantrole in preventing and treating CMV reactivation/disease. Adoptivetransfer of dendritic cells or T cells cultured with CMV antigen peptidesare another therapeutic options and several groups reported the usefulnessof them.

In these years, our group monitored the recovery of anti-CMV T cells bymethods of ELISPOT analysis and Tetramer analysis using HLA-A02 and–A24 associated antigen peptides. Those results suggested detection rate ofCMV specific T cells in HLA-A24 cases were lower than that of HLA-A02cases, which suggested the therapeutic strategy using A24 related CMVantigen peptide was not practical for HLA-A24 patient, which was a majorpopulation in Japan. Upon these backgrounds, we are planning theadoptive transfers of CMV lysate stimulated CD4 T cells against CMVreactivation/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 healthyvolunteers after written informed consents. PBMNCs were cultured with10 microg/ml of CMV lysate and irradiated autologus MNCs, used asfeeder cells, during first 9 days for selection, followed by additional 4 daysculture with lysate, irradiated autologus MNCs and 50 U/ml IL-2.

Result and Discussion: Cultured MNCs showed the followingcharacters. 1) Total cell numbers on day 13 were 10- 30 times higher thaninitial cell numbers on day 0. 2) 90 % of CD3 T cells expressed CD4antigens on day 13. 3) 99% of CD3+ cells showed CD45RO antigen. 4)35% of CD4+ cells showed CD25 antigen. 4) Cultured CD4+T cellsshowed IFN-gamma production. We are transferring our culture systemfrom a culture dish/flask system to a closed bag system for future clinicalapplication. In our presentation, we will show our pre-clinical data anddiscuss our future immunotherapy

232DOCOSAHEXANOIC ACID (DHA) EXERTS DIRECT CYTOTOXICITY ON SUSCEPTIBLE LEUKEMIC CELL LINEST. Yamagami*, C. Porada, R. Pardini, E. D. Zanjani, G. Almeida-PoradaUniversity of Nevada, Reno, NV, USA

Studies have shown that Docosahexanoic acid, one of the maincomponents of fish oil, helps inhibit the promotion and progression ofbreast, prostate and colon cancer. DHA has also been shown to increase thetherapeutic effects of doxorubicin, mitomycin C and cyclophospahmide,while enhancing arsenic trioxide-mediated apoptosis in arsenic trioxide-resistant leukemia cells. We have previously demonstrated that 150microMolar DHA does not adversely affect human bone marrow (BM)derived-CD34+ cells in their ability to differentiate and proliferate in CFCassays, but induces apoptosis in vitro on the undifferentiated subtype ofacute myeloid leukemia cell line KG1a. In the present studies, in additionto KG1a, we used K562, HL-60, and MOLT-4 leukemic cell lines toelucidate possible mechanisms involved in DHA-induced apoptosis.Leukemia cell lines were cultured for 4 days in the presence/absence ofDHA (150 microMolar). Viability and percentage of apoptotic cells in eachculture system were determined by flow cytometry using PI andAnnexinV. Progressive loss of viability and an increase in AnnexinVexpression was observed on DHA treated KG1a and MOLT-4, but not onK562 and HL-60 cell lines. In DHA-susceptible cell lines, quantification ofbcl-2 and Bax proteins demonstrated an increased expression of bax withtime in culture, as well as an increased bax/bcl-2 ratio. In contrast, theinverse effect was observed in DHA-resistant cell lines. Furthermore, cellcycle analysis demonstrated that DHA induced G0/G1-cell cycle arrest inKG1a and Molt-4, while in DHA-resistance cell lines (K562 and HL-60),no significant change in cell cycle was observed. Our study shows thatDHA may be able to exert direct cytotoxicity on certain types of leukemiccells, and could therefore be more broadly applied in the treatment ofleukemias.

233COMPARISON OF SHORT-TERM DENDRITIC CELLS CULTURES STIMULATED BY CALCIUM IONOPHORE AND CD40L IN HEALTHY VOLUNTEERSL. Kovarova1*, J. Michalek1,2, R. Hajek1,31Dept. of Clinical Hematology, Masaryk University Hospital, Brno, Czech Republic2Pediatric Dept., Masaryk University Hospital, Brno, Czech Republic3Dept. of Internal Medicine - Hematooncology, Masaryk University Hospital, Brno, Czech Republic

Objectives: Dendritic cells (DC) are antigen-presenting cells that canactivate naive T lymphocytes and initiate a primary immune response. DCoriginate from bone marrow and alternatively they can be generated frommononuclear cells of peripheral blood. In this study, we analyzed therelative number of activated DC and expression of costimulation markersin healthy volunteers after three types of short-term cultivation withdifferent stimulating agens as calcium ionophore (CI) and CD40Ligand(CD40L).

Methods: 6 healthy volunteers were recruited from TransfusionDepartment of our hospital. Peripheral blood mononuclear (PBMNC) cellswere separated and three types of DC cultures were initiated. Full PBMNCwere cultured in RPMI-1640 supplemented with FCS, adherent PBMNCwere acquired after 2h of culture in RPMI-1640 with or without FCS, thencultured in RPMI-1640 with FCS. CI or CD40L were added and thephenotype (CD80, CD83, CD86, HLA-DR, lineage mixture, CD3, CD14,CD19) was analyzed after 24 hours by multicolor flow cytometry. Aliquotsof PBMNC were analyzed as controls.

Results: The expression of the CD83 antigen was low on freshly isolatedPBMNC (0,3%) and it was upregulated after 24h culture on DC. Thehighest 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 andCD40L stimulation (46%; 42%), but expression of CD80 was higher afterstimulation with CI then CD40L (29,3%; 17,4%).

Conclusion: There was an upregulation of costimulatory moleculesCD80, CD86 and also HLA-DR on DC when CI or CD40L were used, butthese stimulating agens unaffected the expression of CD83. Thesepreliminary results were used to find optimal cultural strategy of DC.

Supported by grant IGA MZCR NR 8081-3.



Abstracts

234HUMAN MYELOMA CELLS EXPRESS RUNX2 GENE THAT REGULATES OSTEOPONTIN PRODUCTION: ROLE IN MYELOMA-INDUCED ANGIOGENESISN. Giuliani1*, S. Colla1, F. Morandi1, M. Lazzaretti2, C. Mancini2, S. Bonomini1, R. Rizzato1, G. Sammarelli1, V. Rizzoli11Hematology, University of Parma, Italy2Pathology, University of Parma, Italy

Osteopontin (OPN) is involved in angiogenesis, cell survival and tumorprogression. OPN gene expression by human cells is mainly regulated bythe bone specific trascription factor Runx2 namely also CBFA1 (Runx2/Cbfa1) even if other transcription factors may be also involved in theproduction of OPN by cancer cells as the CCAAT/enhancer-bindingprotein alpha (C/EBPa) and AML-1.

In this study we show that human myeloma cell lines (HMCLs) but notnormal CD19+ cells directly produce OPN and express its major regulatinggene Runx2/Cbfa1. The activity of Runx2/Cbfa1 in HMCLs has been alsodemonstrated by a gel mobility shift assay. On the other hand we found thatall human myeloma cell lines (HMCLs) tested were negative for C/EBPamRNA, whereas AML-1A and AML-1B mRNA were expressed in allHMCLs even if any correlation has not been found between OPNexpression and AML-1A/AML-1B ratio. In addition we found that OPNproduction by myeloma was up-regulated at both mRNA and protein levelby IL-6 and IGF-I through a Runx2/Cbfa1 mediated mechanism; in turnrecombinant human OPN was able to increase myeloma cells proliferation.The potential role of OPN in MM-induced angiogenesis has been alsoinvestigated in an experimental model of angiogenesis. rhOPN treatmentstimulated vessel formation and the conditioned medium (CM) of HMCLssignificantly increased vessel formation. On the contrary OPN-immunodepleted CM of HMCLs had not a stimulatory effect on vesselformation and the presence of anti OPN Ab inhibited vessel formationinduced by HMCLs.

The expression of OPN by purified bone marrow (BM) CD138+ cellshas been also investigated in 60 newly diagnosed multiple myeloma (MM)patients, finding that 40% of MM patients tested expressed OPN. Asignificant higher microvascular density and number of microvessels perfield were observed in the group of patients positive for OPN, incomparison with OPN negative ones.

In conclusion our data highlight the direct ectopic production of OPN byhuman myeloma cells with a Runx2/CBFA1 mediated mechanism and thecapacity of myeloma-derived OPN to stimulate angiogenesis.

235THE VARIABLE POTENCY OF HISTONE DEACETYLASE (HDAC) INHIBITORS TO KILL LEUKAEMIA CELLS IS NOT DEFINED BY THE HDAC EXPRESSION PROFILE OF THE TARGET CELLSF. L. Khanim1*, C. Pearce1, R. E. Hayden1, B. M. Turner2, C. Craddock3, G. Pratt4, C. M. Bunce11School of Biosciences, The University of Birmingham, UK2Div of Immunity and Infection, The University of Birmingham, UK3CRUK Institute of Cancer Research, The University of Birmingham, UK4Dept of Haematology, Heartlands Hospital, Birmingham, UK

Tissue homeostasis, requires the co-ordinated expression of genesregulating cell proliferation, differentiation and apoptosis genes.Modification of core histone proteins by histone deacetylases (HDACs) isone mechanism regulating this process. It has become evident that HDACactivity is deregulated in many cancers, including leukaemias, leading tointense interest in the possible therapeutic benefits of HDAC-inhibitors(HDIs). Although HDI trials are underway in a wide range of tumours, it isnot yet known whether different cancers deregulate different HDACs andwhether, as a consequence, they show selective sensitivities to separateHDIs.

Quantitative real-time PCR was used to compare the mRNA levels ofthe 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-celltumours with a generalized over-expression of all HDACs with the soleexception of HDAC11 which was under-expressed. This result was distinctfrom that seen in AMLs in which overall expression was variable but withconsistent downregulation of HDAC5, RBBP4, SIRT4 and upregulation ofRBBP7, HDAC2 and MLL.

To determine relative HDI sensitivities, AML, BL and MM cell lineswere exposed for 72 hours to increasing concentrations of three differentHDIs, trichostatin A (TSA), sodium valproate (SV) and suberoylanilidehydroxamic acid (SAHA) and cell survival measured. A wide spectrum ofsensitivities were observed however the sensitivities of MM, BL and AMLcell lines to HDIs, did not correlate with their differing patterns of HDACexpression. These data implicate that underlying mechanisms, other thanHDAC expression alone dictate sensitivity to HDIs. These findings haveimportant implications for the understanding of the role of HDACs incancer and for the future therapeutic use of their inhibitors

236EFFECTS OF SPHINGOSINE 1-PHOSPHATE (S1P) AND EXPRESSION OF S1P RECEPTORS IN CHRONIC LYMPHOCYTIC LEUKEMIA (B-CLL): POTENTIAL ROLE IN CELL TRAFFICKING AND SURVIVALG. Seitz*, S. Yildirim, A. M. Boehmler, L. Kanz, R. MöhleUniversity of Tübingen, Tübingen, Germany

Recent studies suggest that presence of the lipid mediator sphingosine 1-phosphate (S1P) in the peripheral blood and expression of S1P receptors(i.e., S1P1) is a prerequisite for the egress of lymphocytes (particularly Tcells) from lymphoid organs into the circulation. As circulatinglymphocytic lymphoma cells are a hallmark of chronic lymphocyticleukemia, we analyzed expression of different S1P receptors and theeffects of S1P on CLL cells. By qualitative and quantitative (TaqMan) RT-PCR, mRNA expression of S1P1 and S1P4 was found in CLL cell lines(EHEB, MEC-1) and in most samples of primary CD19+ cells isolatedfrom the peripheral blood of untreated B-CLL patients. mRNA of otherS1P receptors (S1P2, S1P3, S1P5) was less consistently found. Normal,nonmalignant B cells were strongly positive for S1P1, while other S1Preceptors were weakly expressed or negative. S1P induced typical effectsof chemotactic G protein-coupled receptors, such as actin polymerizationand chemotaxis (modified Boyden chamber assay) in CLL cell lines. Alsoprimary CLL cells responded to S1P with increased actin polymerizationas a first step of cell locomotion. Of note, the S1P1/5 superagonist(antagonist) FTY720 (1 uM) induced apoptosis in primary CLL cells asmeasured by MTT-test and staining with Annexin-FITC, suggesting thatalso cell proliferation and survival might be regulated by S1P receptors.We conclude that sphingosine 1-phosphate, ubiquitously present inconsiderable amounts in the peripheral blood, may contribute to thetrafficking of B-CLL cells, and their prolonged survival.

237A NOD/SCID XENOGRAPH MODEL OF DIFFUSE LARGE CELL LYMPHOMAR. M. Rossi*, R. A. Conkling, S. H. Bernstein, C. J. JordanJames P. Wilmot Cancer Center, University of Rochester, Rochester, NY, USA.

The objective of this study was to establish animal models of lymphomafor use in testing the efficacy of chemotherapy agents and variousexperimental drugs. To achieve this goal, 8 Diffuse Large Cell Lymphoma(DLCL) cell lines were chosen for analysis, consisting of both the activatedB 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 intoimmune deficient NOD/SCID mice, without prior conditioning, and testedusing both subcutaneous (SQ) and intravenous (IV) delivery. In addition,total cell number and injection volume were varied. The growth rates of thetumors in vivo were determined and tissue samples were obtained to assesstumor pathology and molecular endpoints. Of the 8 lines tested, 4consistently developed tumors (OCI-Ly10, OCI-Ly7, OCI-Ly19, andFarage), with kinetics ranging from 10-60 days post-transplant. Theremaining four lines failed to form tumors at 6 months post injection.Analysis of subcutaneous tumors showed marked vascularization withrobust localized growth. Intravenous injection of the OCI-Ly19 and to alesser extent the OCI-Ly10 lines generated disseminated disease withengraftment of marrow, spleen, liver and some lymph node tissues. Splenichistology indicated disruption of the red and white pulp areas. In addition,preliminary data indicate CNS infiltration for OCI-Ly19 tumors. To permitreal time in vivo imaging studies, both OCI-Ly10 and 19 were modified toexpress luciferase and shown to be detectable in vitro at cell concentrationsas low as 50,000. Further, in vivo analyses indicated luciferase expressionin multiple tissues and tumor masses. In summary, we have generated



versatile and reproducible DLCL xenograph models. Currently, studies areunderway to determine the in vivo effects of several novel therapeuticagents, including proteasome inhibitors, PKC-beta inhibitors, and the noveltriterpeniod CDDO.

238EXPRESSION OF CD133 IN T-ALL: MALIGNANT TRANSFORMATION OF A PRIMITIVE PROGENITOR CELL?C. V. Cox1*, R. S. Evely2, A. Blair1,31Bristol Institute for Transfusion Sciences, Bristol, UK2Bristol Haematology and Oncology Centre, Bristol, UK3University of Bristol, Bristol, UK

Treatment of children with T-ALL has improved considerably over thelast decade. However, a significant proportion ~30% remain refractory totreatment. The disease may be maintained by a subpopulation of cells thatare resistant to drug regimens designed to target the bulk population. Wehave previously shown that T-ALL cells capable of long-term proliferationin vitro and in vivo are CD34+/CD4- or CD7-. In this study we haveattempted to further characterise these putative T-ALL stem cells byinvestigating the expression of CD133 on these cells. T-ALL cells from 5patients for were sorted for expression of CD133 and CD7 and growth wasevaluated in long term culture and in the NOD/SCID model. In this groupof 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 proliferatingcells were derived from the CD133+/CD7- subfraction and this trendcontinued with >58±25% of the proliferating cells derived from thissubfraction at week 6. Morphological analyses confirmed the cultured cellshad blast morphology. Results from the NOD/SCID assay in 3 cases todate, demonstrate that engraftment was achieved using 105-107 unsortedcells (21-98% CD45+) and with the CD133+/CD7- subfraction only (0.5-54.4% CD45+) using as few as with 1.4x103-5x103 cells. There was noengraftment with the other subfractions despite injecting 10 to 1000 foldmore cells. The engrafted cells expressed high levels of CD34, CD2, CD4and CD7 and very low levels of CD133. The phenotype of the engraftedcells was similar to that of the patients at diagnosis, implying they haddifferentiated in vivo. These data add to the evidence that a cell with aprimitive phenotype is the target for transformation in T-ALL. These cellsmay be the most relevant targets for emerging therapeutic strategies.

239CLINICAL USEFULNESS OF FREE LIGHT CHAIN CONCENTRATION AS A TUMOR MARKER IN MULTIPLE MYELOMAS. Y. Kang1, J. T. Suh1, H. J. Yoon2*, W. I. Lee11Department of Laboratory Medicine, KyungHee University School of Medicine, Seoul, Korea2Department 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 immunofixationelectrophoresis (IFE). However, PEP and IFE are not satisfactory insensitivity, objectivity and facility. Recently, a highly sensitive, automatedimmunoassay for measurement of free light chain (FLC) concentrations inserum and urine has been developed for the identification and monitoringof patients with monoclonal gammopathy. To explore the clinicalusefulness of measurement of FLC concentrations, we measured thekappa(K) and lambda(L) FLC concentrations and calculated the K/L FLCratios for three groups (multiple myeloma (MM), other diseases, andcontrol) and compared the results of the FLC assay with the results of PEPor IFE. The concentrations of serum K and L FLCs and the K/L FLC ratiosfor 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 andIFE, but FLC assay showed abnormal concentrations of FLCs andabnormal K/L FLC ratios in most cases. As compared with the PEP, the K/L FLC ratio revealed higher sensitivity in all diagnostic ranges withdifferent cutoff values. Particularly, when the cutoff value 2.0 for K/L FLCratio was used, specificity and PPV were largely improved than when thecutoff values 1.2 and 1.5 were used. These findings indicated that FLCassay enables to detect myeloma patients with very low M protein due toearly stage or after therapy and to distinguish patients with monoclonalincrease 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/LFLC ratios with FLC concentrations is useful in detection of M protein,particularly with negative serum or urine IFE results, and differentiation ofmonoclonal gammopathies from patients with polyclonal increase of FLCdue to other conditions.

240BEZAFIBRATE AND MEDROXYPROGESTERONE ACETATE AS NOVEL THERAPIES IN B-CELL CLLR. E. Hayden1, F. L. Khanim1, N. J. Davies1, G. Pratt2, M. T. Drayson3, P. A. H. Moss4, C. M. Bunce1*1School of Biosciences, University of Birmingham, UK2Department of Haematology, Heartlands Hospital, Birmingham, UK3Division of Immunity and Infection, University of Birmingham, Birmingham, UK4CRUK Institute of Cancer Research, University of Birmingham, Birmingham, UK

We have previously demonstrated that combined bezafibrate (bez) andmedroxyprogesterone acetate (MPA) provide a powerful pro-apoptoticsignal against Burkitts lymphoma (BL) cell lines and primary BL cells.Here, we demonstrate similar killing of B-cell CLL cells. Primary CLLperipheral blood mononuclear cell preparations were cultured in threeculture conditions: on irradiated stromal cultures that either express or donot express CD40-ligand and in the presence of IL4, or in mediumsupplemented with the commercial serum replacement ITS+ and in theabsence of stroma and Il-4. These culture conditions were selected toprovide a variable state of activation, reflecting the in vivo status of CLLcells. Cell survival in all cultures was high. Thymidine incorporation andMTT assays demonstrated that all CD40-ligand stimulated CLL cells weredriven into cell cycle, whereas in the other culture systems the cellsremained non-proliferate. Both bez and MPA reduced thymidineincorporation in all CD40-ligand exposed CLLs and markedly so whenused in combination (bez/MPA). However, cell killing by bez/MPA wasnot restricted to cycling CLL cells as demonstrated by both MTT assaysand 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 cultureswere more variable.

We have also previously reported the potentiation of AML celldifferentiation and apoptosis by CA/MPA. Based on these studies we haveinstigated a phase II trial of Bezalip (Bez) and provera (MPA) in elderlyand relapsed AML (acronym BAP). The drugs have been well toleratedand have in vivo activity as measured by haematological responses andchanges in transfusion dependency. The data described here indicate that asimilar approach may provide benefit in CLL.

241BOTH CASPASE-DEPENDENT AND -NDEPENDENT DEATH PATHWAYS ARE INDUCED BY PHOTODYNAMIC THERAPY USING TH9402Q. Y. Dai1*, J. Filep1, P. Dubé1, C. Scotto2, D. C. Roy11Division of Hematology-Oncology, Hospital Maisonneuve-Rosemont, Montreal, Canada.2Celmed BioSciences, Montreal, Canada.

Purpose: Photodynamic therapy (PDT) is a powerful strategy toeliminate a variety of tumors and alloreactive T cells. While the cytotoxiceffect of the novel rhodamine-derived TH9402 photosensitizer has beenattributed to the generation of radical oxygen species, the cell deathpathways involved have not been investigated. In this study, we evaluatedthe ability of PDT to induce apoptosis and necrosis, and assessed theunderlying molecular mechanisms.

Results: We found a direct correlation between TH9402 concentration(0-10 microM) and intensity of light exposure (0-10J/cm2) with the extentand rapidity of elimination of malignant lymphoma EL-4 and mastocytomaP815 cells. Increasing the intensity of PDT shifted the ratio frompredominantly apoptotic to predominantly necrotic cell death as measuredby Annexin-V/7AAD at 2-4h after PDT. To study the induction ofapoptosis, we measured the impact of PDT (10 microM and 5J/cm2) onmitochondrial transmembrane potential using DiOC6(3) and detectedmarked decreases as early as 2h after PDT. This was associated withrelease of cytochrome C from the mitochondria. Investigation of the



Abstracts

caspase-dependent (classical) apoptotic pathway revealed activation ofcaspase 9 and 3, as early as 15 minutes after PDT. TH9402 also evokedcytosolic release of AIF, suggesting involvement of a caspase-independent(non-classical) pathway. This was accompanied by decreased presence ofBax protein in the cytosol. Finally, caspase inhibition with ZVAD-FMKand DEVD-CHO failed to alter the percentage of PDT-induced AnnV+/7AAD- cells, indicating involvement of a caspase-independent deathpathway.

Conclusion: Using conditions that induce apoptosis, PDT causesactivation of both caspase-dependent and -independent death pathways.The observation that the TH9402 photosensitizer mediates cytotoxicitythrough activation of a number of death pathways suggests that it couldeffectively limit the capacity of tumor cells to become resistant to TH9402.

242RAPID GENOTYPING FOR THIOPURINE METHYLTRANSFERASE POLYMORPHISMS IN PATIENTS WITH ACUTE LEUKAEMIA USING LIGHTCYCLER PCRM. A. Catherwood1*, J. E. A. Davison1, M. F. McMullin1,21Haematology Department, Belfast City Hospital, Belfast, UK2Department of Haematology, Queen's University of Belfast, Belfast, UK

Introduction: Patients with acute lymphoblastic leukaemia (ALL) aretreated with thiopurines. Thiopurine methyltransferase (TPMT) catalysesthe S-methylation of thiopurines and more than 11% of the Caucasianpopulation are heterozygous or homozygous carriers for TPMTpolymorphisms. TPMT deficient patients accumulate excessivethioguanine nucleotides in haemopoietic tissue leading to severehaematological toxicity. Therefore, screening for TPMT polymorphisms inpatients prior to treatment with thiopurines is advantageous. The aim ofthis study was to develop a rapid genotyping method for the detection ofTPMT polymorphisms suitable for high throughput in a clinical setting andcompare this with standard methods in a group of patients with acuteleukaemia.

Materials and Methods: TPMT genotype was determined for the TPMTalleles (TPMT *2, *3A, *3B & *3C) in a group of 55 patients with ALLusing LightCycler PCR.

Results: All 55 ALL patients were homozygous for the wildtype alleleof the polymorphism G238C in exon 5. Three Patients were heterozygousfor the G460A mutation in exon 7 by standard PCR with restrictionfragment length polymorphism (RFLP) and four were heterozygous byLightCycler PCR, which were confirmed by sequencing.

Three patients were heterozygous for the A719G mutation in exon 10using LightCycler PCR, which was confirmed by sequencing. Of thesethree patients, one was found to also have the G460A mutation.

Conclusion: We describe a rapid assay for the genotyping of TPMTpolymorphism using real-time fluorescence PCR to facilitate rapidprocessing of samples in a clinical setting. This strategy is superior tostandard PCR-RFLP genotyping methods which in our study has producedfalse-positive results. This rapid diagnostic assay provides informative dataon TPMT polymorphisms in ALL patients prior to treatment withthiopurines.

243NEW CHEMOTHERAPY PROTOCOL (P-CAN) FOR TREATMENT OF AGGRESSIVE NON-HODGKIN'S LYMPHOMAK. Rasul1*, A. Awidi1, M. Kelta1, A. Mubarak1, U. Al-Homsi1, A. Al-Hassan1, A. Chung-Lopez2, A. Bener31Department of Medicine, Hematology/Oncology Section, Hamad Medical Corporation, Doha, Qatar2Department of Laboratory Medicine and Pathology, Hamad Medical Corporation, Doha, Qatar3Department of Medical Statistics, Hamad Medical Corporation, Doha, Qatar

Background: This work aims at determining the efficacy of modifiedCHOP combination in which Vinorelbine (Navelbine) replaces Vincristinefor the treatment of aggressive Non-Hodgkin's Lymphomas (NHL).

Patients and methods: this open label pilot study included 19 patientswith aggressive NHL and one patient with low grade NHL who weretreated with the new combination which we abbreviated as P-CAN(Prednisolone 100mg/day P.O day 1-5, Cyclophosphamide 750mg/m2 i.vday 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, Internationalprognostic index (IPI) 0-3. Seven patients stage I, one patient stage II, eightpatients stage III and 3 patients in stage IV. 14 patients with nodal diseaseand five patients with extra-nodal disease. They received total of 97 cyclesof the chemotherapy (3-7 cycles).

Results: 18 out of 19 patients achieved complete response (CR). In onepatient the response could not be assessed, one patient progressed while ontreatment. 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 inchemo-naive aggressive NHL. The addition of Vinorelbine to steroid,Adriamycin, and Cyclophosphamide seems improve the response. Furtherlarger trials are needed to study this combination and its impact on longeroverall survival.

244THE COMPARATIVE ANALYSIS OF SERUM PROTEOMES FOR THE DISCOVERY OF BIOMARKERS FOR HODGKIN'S DISEASEJ. Y. Kwak1*, N. R. Lee1, E. K. Song1, C. Y. Yim1, Y. G. Kwak21Internal Medicine, Chonbuk National University Hospital, Jeonju, South Korea2Pharmacology, Chonbuk National University Medical School, Jeonju, South Korea

Backgrounds and Purpose: Hodgkin's disease(HD) belongs to lymphoidmalignancies of B cell origin and it's etiology is known to be related toEBV infection. The histological characteristic of HD is the presence ofReed-Sternberg(RS) cells and the diagnosis is established by confirmingthe histologic findings of the tissues from involved organs. In order to findless invasive, easier and simpler diagnostic methods for HD, we analyzedserum proteomes of HD patients.

Materials and Methods: We compared the two dimensionalelectrophoresis patterns of bone marrow sera of six patients with HD andthose of six normal subjects. The differentially expressed spots between thetwo groups were identified by matrix-assisted laser desorption/ionization-time-of-flight(MALDI-TOF) and electrospray ionization quadupole time-of-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 twospots, ALB protein and chain A of human serum albumin mutant R218hcomplexed with thyroxine were down-regulated in the sera of HD groupcompared with control group.

Conclusion: Our current study suggests that if further studies are carriedout, the differentiated expressed serum proteins might be used as simplerdiagnostic and follow-up markers for HD.

245CHINESE HERBS DECOCTION BEVERAGE INDUCING SIGNIFICANT RESPONSE IN PROGRESSIVE B-CLL AND IN VITRO APOPTOSIS ON B CLL CELLSA. Berrebi*, L. Bassous, L. ShvidelKaplan Medical Center, Rehovot, Israel

An article published in Leukemia Research (2003, 27) reportedremission in a CLL patient treated only by Chinese herbs with a significantapoptosis of B-CLL cells in vitro.

An 80-year-old biologist was followed in our Institute for several yearsbecause of B-CLL that progressed slowly to stage Binet B, including highlymphocytosis (80x109/l) and splenomegaly. This patient who by himselfread this article, asked the article authors to refer him to this Chinesemedicine, unsuccessfully. Then he asked a well-known Israeli herbalist toprescribe him Chinese herbs. These herbs mixture had to be decocted inwater and the extract was recommended to be taken every day at least 0.6liter per day. We should emphasize that our Institute was not involved inthe decision of the patient to take this medication. A slowly decrease of thelymphocyte 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 inculture for 20 hours to 5 days using annexin kit for flow cytometry. Weexamined samples from 9 advanced B CLL patients. In five cases asignificant apoptosis (30%-100%) was noted after 20 hours, and in 4 othercases up to 90% of apoptosis was obtained after 3 to 5 days by adding theherbal extract every day. The optimal effect was equally with 1:10 and 1:20



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

246DOUBLING TIME OF SOLUBLE CD23 IS AN ADDITIONAL MARKER IN CLL PATIENTS EXPRESSING OR NOT ZAP-70N. Meuleman*, L. Lagneaux, M. Dejeneffe, A. Delforge, D. BronJules Bordet Institute, Brussels, Belgium

Background: Chronic lymphocytic leukemia (CLL) is a disease withunpredictable natural history in stage A patients (pts). Recently, It has beendemonstrated that ZAP-70 and IgVH mutational status are powerfulbiological prognostic factors and we previously reported that the doublingtime of soluble CD23 (sCD23DT) is a prognostic factor for progression ofdisease in untreated stage A CLL. Therefore we evaluated prospectivelyZAP-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 enzyme-linked immunosorbent assay (ELISA).ZAP-70 expression is determined inleukemic 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, themedian age was 62 years. The median follow-up was 55 months. Atpresent, results of 31 pts are available for analysis and an update will bemade 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 inferiorto one year. Among ZAP-70+ and sCD23DT<1 year pts, 83% required atreatment. For pts with positive ZAP-70 and a sCD23DT superior to oneyear, 80% of pts do not need a treatment. In the group of good prognosispts with negative ZAP-70, 40% of the pts required a treatment. in the ZAP-70 negative pts population, 4/20 have a sCD23DT<1 year and all these ptsrequired a treatment for disease progression. All the pts with indolentprogression, negative ZAP-70 and who do not need a treatment have asCD23DT superior to one year

In conclusion, 1) sCD23DT remains a prognostic marker for diseaseprogression even in ZAP-70 negative patients. 2) In ZAP-70 positive pts,the sCD23DT allows to better identify the pts requiring a treatment.

247BLOOD DENDRITIC CELLS IN PATIENTS WITH HEMATOLOGICAL MALIGNANCIESJ. Weclawek-Tompol, E. Grotthus, A. Chybicka*, B. Rybka, R. Ryczan, D. Noworolska-SaurenDepartment of Children Bone Marrow Transplantation, Oncology and Hematology Medical University of Wroclaw, Poland

Dendritic cells (DCs) constitute heterogeneous leukocyte populationwith various functional and phenotypic characteristics. They playfundamental role in induction and control of primary as well as secondaryimmune response and may be also important for the induction ofimmunologic 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 betweennumber of BDC and activation of T-lymphocytes.

The total number of 82 patients: 36 females and 46 males, aged from 15months to 18 years (median 9 years) treated in the Department of ChildrenBone Marrow Transplantation, Oncology and Hematology, MedicalUniversity of Wroclaw, Poland (ALL - 45, AML – 15, HD – 10, NHL-B –8, LCAC - 4) were examined. Control group contained of 21 healthychildren – potential donors to allogenic BMT.

Peripheral blood was collected at time of diagnosis, duringchemotherapy, at time of relapse or progression. The populations of BDCand activated lymphocytes CD3+CD26+ were analyzed by flow cytometry.The subpopulations of BDC were defined on the basis of expression ofspecific markers for distinct blood dendritic cell populations: BDCA-1,BDCA-2, BDCA-3. The activated BDC were identified when antigen CD83, characteristic for their activation, was expressed.

We observed increased percentage and absolute count of BDC beforestart of treatment in children with ALL or AML, compared to controlgroup. Increased percentage of BDC was present during whole therapy.Those yields explained positive correlation with value of activated Tlymphocytes.

Increased value of BDC in children with hematological malignancies, aswell as in children 1-2 months after cessation of treatment, may indicate anactivation of immune system. This fact may play important role in controlof residual disease.

248RIN3, 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 TRANSPORTA. D. Clark1*, L. Telfer1,2, J. Baird1, G. J. Graham1,21Cancer Research Beatson Laboratories, Glasgow, UK2Division of Immunology, Infection and Inflammation, Glasgow University, Glasgow, UK

Chronic lymphocytic leukaemia is the commonest leukaemia in thewestern world. Here we describe exciting data supporting a role for RIN3as a ras effector linking intracellular signalling and vesicle transport inthese cells. We initially isolated murine RIN3 (muRIN3) from embryoidbodies as a proportion of the constituent ES cells transited a haemopoieticstem cell (HSC) phenotype. Full length muRIN3 was obtained by libraryscreening and RACE PCR technology. The human homologue (huRIN3)was found to map to chromosome 14q32. Using full length muRIN3 toscreen 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 underwentmyeloid differentiation but increased expression was seen duringlymphopoiesis. High expression levels were confirmed in human andmurine secondary lymphoid tissue by northern blotting and real time PCR.HuRIN3 was then found to be markedly overexpressed in human ChronicLymphocytic Leukaemia compared to normal lymphocytes. Interestingly,no expression was seen in Burkitt's lymphoma which is associated with at(8:14q32) translocation. Functionally, domain homology data suggestedthat RIN3 acted as a Rab5GEF. Confocal microscopy confirmedinteraction between RIN3 and Rab5 in early endosomes where RIN3 alsoco-localised with MIP1 alpha in dramatically enlarged endosomal vesicleswhen internalised via the D6 receptor. RIN3 interacted specifically withGTP bound Ras, in pull down assays and yeast two hybrid binding partnersincluded Abi1/E3B1, Diacylglycerol kinase alpha, TGF beta stimulatedclone 22, and a novel Rho GEF homologous to the Vav proteins, allcomponents of signalling cascades. In summary, these data suggest thatRIN3 is involved in linking intracellular signalling and vesicle transport,possibly by specifically activating or sequestering downstream Raseffectors in macromolecular complexes or modulating signal transductionby influencing vesicle movement.

249SUPPRESSION OF B LYMPHOPOIESIS BY NEOPTERIN IS BASED ON STROMAL CELL-DERIVED CYTOKINES, AND FACILITATES A CAPACITY OF MYELOID CELL PROLIFERATION DURING INFLAMMATORY PROCESSI. Tsuboi1*, S. Aizawa1, T. Harada1, M. Hiramoto1, Y. Hirabayashi2, J. Kanno2, T. Inoue31Deapartment of Anatomy, Nihon University School of Medicine, Tokyo, Japan2Division of Cellular and Molecular Toxicology, National Institute of Health Sciences, Tokyo, Japan3Biological Safety and Resarch Center, National Institute of H Health Sciences, Tokyo, Japan

Neopterin (NP) is produced by monocytes and is known to be a usefulbiomarker for inflammatory immunological activation. We previouslyfound that NP enhances in vivo and in vitro granulopoiesis by inducingstromal cell production of cytokines in mice. In the senescence accelerated(SA) mice, because of their known stromal cell impairment after 30 weeksold, mice show simultaneous down-regulation of IL-7, a positive regulatorfor B lymphopoiesis, as well as TGF-b, a negative regulator, resulting in



Abstracts

their senescent suppression of B lymphopoiesis. Thus, the model may beuseful tool to contribute to elucidate possible interaction of NP and stromalcells on the B lymphopoiesis. In young control mice, NP decreased thenumber of colonies in semi-solid medium derived by pre-B cell progenitorcells (CFU-pre-B) from whole bone marrow cells, but not those from a pro-B/pre-B-enriched cell population, possibly due to lack of the stromal cellscomponent. Intraperitoneal injection of graded doses of NP into controlmice resulted in a profound and dose-dependent reduction of femoral CFU-Pre-B colonies within 1 day. Gene expression of negative regulators for Blymphopoiesis, such as TGF-b, TNFa and IL-6 in the bone marrow were upregulated within 6 h after NP treatment. However, in the case of elderly SAmice, the in vivo and in vitro responses of B lymphopoiesis after NPtreatment as well as the up-regulation of negative regulators in the bonemarrow was less-significant than in young SA mice. These resultsdemonstrated that NP can not enhance but suppress B lymphopoiesis byinducing stromal cell derived cytokines and that the response was muchmilder in the elderly SA mice than in young SA control mice due todeterioration of stromal cell function with age.

250ACCLERATED TELOMERE SHORTENING IN GPI(-) GRANULOCYTES FROM PATIENTS WITH PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH) DETECTED BY NEWLY DEVELOPED PROAEROLYSIN FLOW-FISHF. Beier1*, S. Balabanov1,2, T. Buckley3, K. Dietz4, U. Hartmann1, M. Rojewski5, L. Kanz1, H. Schrezenmeier5, T. H. Brümmendorf1,21Division of Hematology, Oncology and Immunology, University of Tübingen, Germany2Deptartement of Hematology and Oncology, University Hospital Eppendorf, Hamburg, Germany3Department of Biochemistry and Microbiology, University of Victoria, Victoria, B.C., Canada4Department of Medical Biometry, University of Tübingen, Germany5Department of Transfusion Medicine, University of Ulm, and Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, Germany

Paroxysmal nocturnal haemoglobinuria (PNH) is caused by a somaticmutation in the X-linked PIG-A gene resulting in a deficiency of GPI-linked proteins on the cell surface. Accelerated telomere shortening hasbeen linked to disease stage and degree of (pan-) cytopenia in patients withbone marrow failure syndromes. The aim of the current study was toanalyze the impact of replicative stress on telomere length in residualGPI(+) vs. GPI(-) hematopoiesis in patients with PNH.

For this purpose, we developed Proaerolysin flow-FISH, a novelmethodology which combines Proaerolysin staining (for GPI expression)with flow-FISH (for telomere length measurement). Peripheral bloodgranulocytes from 16 patients with PNH and 22 healthy individuals wereanalyzed. 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) aswell 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 PNHassuming that damage to the GPI(+) HSC compartment leads tocompensatory hyperproliferation of residual GPI(-) HSC.


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