Session II-1: Cytokines, Growth Factors and Receptors I A PEPTIDE CLEAVED FROM THE STRESS ASSOCIATED VARIANT FORM OF ACETYLCHOLINESTERASE MODULATES HEMATOPOIESIS (2000)

s/Experimental Hematology 28 (2000) 31–131 117 ment in the BM under homeostasis, and (i.v.) probably enclose a morphogenetic organizer of HSC development. 269 Sunday, July 9, 2000 (14:15–16:00) Session II-1: Cytokines, Growth Factors and Receptors I A PEPTIDE CLEAVED FROM THE STRESS ASSOCIATED VARIANT FORM OF ACETYLCHOLINESTERASE MODULATES HEMATOPOIESIS V. Deutsch1, D. Grisaru1,2*, M. Shapira3*, M. Sternfeld3*, N. Melamed-Book3*, D. Kaufer3*, H. Soreq3*, and A. Eldor1* Depts. of Hematology1 and Obstetrics-Gynecology2, Tel Aviv Medical Center, Sackler Faculty of Medicine, Tel Aviv University, 3Dept. of Biological Chemistry, Hebrew University, Jerusalem, Israel Hematopoietic stress responses involve increases in white blood cells (WBC) and platelet counts, implying the existence of stress responsive factors that modulate hematopoiesis. Acetylcholinesterase AChE, which terminates neurotransmission in synapses, is also expressed in several hematopoietic lineages and has been postulated to play a role in hematopoiesis. In support of this notion we report that the extended promoter region of the ACHE gene contains both hematopoietic transcription factor binding sites and stress associated glucocorticoid response element (GRE) halfpalindromic sites. An intriguing stress-response candidate is the readthrough isoform AChE-R which is produced by alternative splicing of the ACHE gene. This soluble monomeric variant is expressed in embryonic and tumor cells, in mammalian hematopoietic cells and is induced in the brain under psychological, physical and chemical stress (Kaufer et al, Nature 393:373, 1998, Grisaru et al. FEBS 264:672, 1999). We found that following forces swim stress, a c-terminal peptide (ARP) is cleaved from ACHE-R which accumulates in mouse blood. Injection of synthetic ARP in mice augmented stress-associated blood cell expansion, while antisense suppression of AChE-R mRNA blocked stress-induced accumulation of ARP in mouse bone marrow (BM) and suppressed hematopoietic stress responses. In vitro, synthetic ARP (2 nM) induced survival and stimulated expansion of human CD34 cells, and early myeloid and megakaryocyte progenitors. Moreover, transgenic mice overexpressing AChE-R presented elevations in BM progenitors and WBC and platelet counts. Our findings present ARP, the C-terminal peptide of AChE-R, as a new hematopoietic growth factor which may promote myeloid and megakaryocytic expansion characteristic of stress. 270 Monday, July 10, 2000 (9:45–11:15) Session III-4: Molecular Regulation of Hematopoiesis I MEGAKARYOCYTE MATURATION IS ASSOCIATED WITH EXPRESSION OF THE CXC CHEMOKINE CONNECTIVE TISSUE ACTIVATING PEPTIDE CTAP III Varda Deutsch, Menachem Bitan*, Yael Friedman*, Amiram Eldor* and Israel Vlodavsky*1 The Hematology Institute, Tel Aviv Sourasky Medical Center, Tel Aviv and the Departments of Oncology and Hematology, Hadassah University Hospital, Jerusalem, Israel Connective tissue activating peptide III (CTAP III) is a CXC chemokine, derived from platelet basic protein (PBP), which has been identified in platelets, activated macrophages, neutrophils and in T lymphocytes. CTAP III can support stem cell derived hematopoiesis yet clearly inhibits the proliferation of committed megakaryocytes (MK) progenitors. This investigation was aimed at characterizing CTAP III in human MK and determining it association with proliferation and differentiation. We report high expression of CTAP III in mature MK from normal human bone marrow (BM) and from BM of patients with immune thrombocytopenic purpura (ITP) or chronic myeloid leukemia (CML) and in ex-vivo expanded MK. Immunostaining with anti-CTAPIII antibodies demonstrated its prominent presence in platelet producing like zones within the cytoplasm and intense staining around the periphery of the large mature MK. In logarithmically growing cell lines DAMI, CHRF-288 or MEG01 megakaryoblasts only 15% of the cells, which were primarily large cells (.30 mM), expressed CTAPIII. The addition of phorbol ester PMA, which causes proliferation arrest and differentiation induction, stimulated high levels of CTAPIII expression after 24 hours in more than 75% of the cells, being expressed in both the small cells and the large cells. RT-PCR reveled upregulation of CTAPIII mRNA after one hour of exposure to PMA which was sustained for 24 hours. While the biological function of CTAPIII in MK is not known, these studies demonstrate that molecular upregulation of CTAP III in MK is associated with maturation and like other chemokines may be involved in proliferation arrest and cellular interactions with extracellular matrix. 271 Monday, July 10, 2000 (16:00–17:00) Poster Session II: Progenitor Cell Processing, Mobilization and Purging KINETIC OF INTRAPROCEDURE CD341 CELL RELEASE DURING PBSC COLLECTION IN HEALTHY DONORS T. Bonfini*, P. Accorsi*, R. Giancola*, E. Liberatore*, M. Dell’Isola* , P. Di Bartolomeo*, G. Fioritoni* and A. Iacone* Dept. of Hematology and Transfusion Medicine, Civil Hospital, Pescara, Italy. Intro by K. Zuckerrman. Aim of our study was to evaluate the intraprocedure dynamic variables during PBSC collection in G-CSF mobilized healthy donors. Eleven donors aged 30 to 53 years were mobilized with Lenograstim 5 mg/kg twice and underwent to a median of 2 LKs/donor (range 1–4) starting from day 4. A total of 21 collections lasting 5.3 hours (4.3–6) were performed with Amicus-Baxter cell separator. Peripheral blood (PB) and separate collection bag samples were taken for each body volume processed (BVP) to analyze intraprocedure dynamic changes and CD341 cell release. A median of 62% (37–95) of initial CD34/ml values were found at end of procedures. CD341 yields were 163% (72–309) of initial circulating values. Amount of CD341 cell release during apheresis was 0.89 3 106 (0.19–3.15) per minute of procedure and 87.09 3 106 (17.05–292.49) per BVP. Intraprocedure data are shown in table. CD 341 cells bag 1 bag 2 bag 3 PB values 3 103/ml 40.44