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The effect of low dose (10−12–10−7M) ARA-C on differentiation and proliferation in liquid and semisolid culture of marrow cells from 13 patients with myelodysplastic syndrome (MDS) were studied following incubation in liquid culture with low dose ARA-C. In six of ten patients an increasing number of myeloid cells acquired the morphologic appearance of mature monocyte-macrophages. Increasing number of cells reacted positively to fluoride sensitive naphthyl acetate esterase and specifically bound MY4 monoclonal antibody. Phagocytosis and killing of Candida albicans by monocyte-macrophages incubated with low dose ARA-C was normal and similar to that of the untreated cells. All MDS patients showed reduced myeloid colony and increased cluster formation. Low dose ARA-C had slight but non-significant inhibitory effects on myeloid colony growth. The results indicated that the differentiation pattern of myeloid precursor cells form a subset of MDS patients was altered by exposure to low dose ARA-C in vitro.

The antileukemic activity of murine recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) and a combination of rGM-CSF and recombinant interleukin-3 (rIL-3) was examined by using a murine model of spontaneous B-cell leukemia (BCL1) in BALB/c mice. All untreated mice inoculated with 2 x 10(2) BCL1 cells developed leukemia within 4 weeks, with extreme lymphocytosis and a massive increase in both spleen weight and cell number while the number of myeloid progenitors (CFU-C) per spleen was decreased. In contrast, rGM-CSF-or rGM-CSF- and rIL-3- treated recipients did not show any evidence of leukemia or splenomegaly at 4 weeks and showed a significant increase in CFU-C per spleen. Hematologic parameters in the peripheral blood of untreated mice showed anemia and thrombocytopenia. Significant elevations in these parameters were recorded in mice treated with either protocol of CSF. Treatment of recipient mice with either rGM-CSF or rGM-CSF and rIL- 3 prolonged their median survival from 6 weeks in untreated controls (range, 5 to 9 weeks) up to the time they were killed at 105 days. Adoptive transfer of spleen cells obtained from mice treated with rGM- CSF, mice treated with a combination of rGM-CSF and rIL-3, and untreated controls, into normal secondary recipients indicated improved survival in recipients inoculated with rGM-CSF. These data indicate that CSFs may inhibit in vivo expansion of leukemic cells of lymphoid origin.

The active metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), has been shown to enhance the growth of human granulocyte/macrophage haemopoietic progenitors in vitro and to induce these cells to differentiate along the monocyte/macrophage pathway. In order to evaluate the relationship between specific receptors for 1,25(OH)2D3 and the role of 1,25(OH2D3 in the regulation of haemopoietic cell differentiation, we examined the effect of haemopoietic cell differentiation, we examined the effect of 1,25(OH)2D3 on the in vitro growth and differentiation patterns of marrow myeloid progenitor cells from two patients with 1,25(OH)2D3 resistant rickets, resulting from defective receptors to vitamin D. A significant rise in the frequency of myeloid colonies in control marrow cell cultures was induced by 2 X 10(-9) to 2 X 10(-7)M 1,25(OH)2D3. This rise reached a plateau at 2 X 10(-9)_2 X 10(-8) M 1,25(OH)2D3, resulting in a maximal 54 +/- 9% increase in colony numbers. In contrast, no stimulatory effect could be detected when 1,25(OH)2D3 was added to cultured marrow cells from the patients with 1,25(OH)2D3 resistance. Analysis of colony composition revealed that 2 X 10(-8) and 2 X 10(-7) M, 1,25(OH)2D3 induced a 50 +/- 26% increase in the frequency of colonies composed only of monocytes/macrophages in control, but not in the patients' marrow cell cultures. The effect of 2 X 10(-8) and 2 X 10(-7) M 1,25(OH)2D3 on progenitor cell differentiation towards monocytes/macrophages was also observed in marrow cell suspension cultures. Whereas 1,25(OH)2D3 induced a 81-136% increase in the frequency of monocytes in control marrow cells, no effect could be detected on the generation of mature monocytes in marrow cells of the 1,25(OH)2D3 resistant patients. Our results show that marrow granulocyte/macrophage progenitor cells from patients with 1,25(OH)2D3 resistance fail to respond to 1,25(OH)2D3. We thus demonstrate that the effect of 1,25(OH)2D3 on the proliferation and differentiation of haemopoietic progenitor cells is mediated through its binding to specific cytoplasmic receptors.

Heparin uptake by cultured macrophages was investigated from the standpoint of: (1) whether the increased uptake in the presence of polycations is due to charge neutralization, and (2) whether the heparin becomes internalized. Regarding the first point, our results are compatible with the notion that charge neutralization is mainly responsible for the enhanced uptake of heparin in the presence of protamine, histone, poly(DL-lysine) and poly(L-ornithine). As for the second point, chasing experiments at low and high temperatures strongly suggest that while heparin binds onto the cell membrane at both 4 degrees C and 37 degrees C, it undergoes internalization only at 37 degrees C.

The mechanism of heparin rebound is still unclear and the present study reports on its successful reproduction under in vitro conditions. By measuring clotting time, by the use of electrophoretic techniques and with the employ of protease inhibitors it was found that protamine is proteolytically degraded on incubation in plasma. The obtained results suggest that heparin rebound entails the degradation of protamine and the release of the previously bound heparin into the circulation.

Summary. Peripheral blood cells of 17 patients with myeloproliferative disorders were grown in vitro to form mixed colonies (CFU-mix). CFU-mix were found in four of nine patients with chronic phase chronic myelocytic leukaemia (CML) at levels 28- to 132-fold higher than the upper limits of normal subjects. These patients also had higher than normal levels of circulating granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitors. Development of blastic crisis was associated with disappearance of CFU-mix from the blood. CFU-mix was detected in the blood of three of five patients with polycythaemia vera (PV) and two patients with myelofibrosis with myeloid metaplasia (MMM) at levels slightly higher than those of normal subjects. Our results are consistent with a clonal expansion of multipotent haematopoietic precursor cells in CML and abnormal kinetics of this cell in CML, PV and MMM.

Summary. Haemopoietic cells with pluripotent capabilities, were grown in soft-gel cultures from the peripheral blood of a glucose-6-phosphate dehydrogenase (G6PD) electrophoretic A,B heterozygous female with chronic myelocytic leukaemia (CML). The cells produced mixed colonies composed of erythrocytic and granulocytic progeny. Only type B isoenzyme of G6PD was found in mixed colonies, in mature neutrophils, erythrocytes, and committed granulocyte and erythrocyte stem cells. The fibroblasts of the patient contained both A and B isoenzymes of G6PD. This study provides direct evidence that a pluripotent haemopoietic stem cell is involved in the malignant clone in CML.

Mouse macrophages contain monoamine oxidase (MAO) A activity and traces of MAO B, as judged by a strong deamination of 5-hydroxytryptamine and tyramine and a marginal one of benzylamine. Significant inhibition of MAO activity occurred in the presence of the specific inhibitors clorgyline and deprenyl. MAO A activity was considerably depressed in phagocytizing cells.

The separate effects of vitamin D3 (1,25(OH)2D3) and 13-cis retinoic acid on the differentiation in liquid culture of marrow cells from seven patients with myelodysplastic syndrome (MDS) were studied. Following incubation with 1,25(OH)2D3, an increasing number of myeloid cells acquired the morphological appearance of mature monocyte-macrophages and reacted positively to fluoride-sensitive naphthyl acetate esterase and specifically bound My4 monoclonal antibody (McAb). Incubation of bone marrow cells with 13-cis retinoic acid enhanced the number of cells with the morphological appearance of metamyelocytes and mature granulocytes as well as those that reacted positively with AS-D naphthol chloroacetate esterase. The results suggest that the differentiation pattern of myeloid precursor cells from MDS patients can be modulated by 1,25(OH)2D3 and 13-cis retinoic acid.

The proliferation and differentiation effects of the synthetic retinoid TTNPB and of 13- cis retinoic acid (RA) on hemopoietic progenitors from bone marrow of myelodysplastic syndrome (MDS) patients were compared. The addition of TTNPB or RA to culture plates containing MDS patient's marrow cells stimulated myeloid colony (CFU-C) growth and caused a significant increase in granulocytic colonies (CFU-G). In the presence of RA the increase in CFU-G was statistically insignificant. Cellular differentiation studies in liquid suspension culture revealed that the two retinoic acid analogues cause a marked decrease in immature granulocytes and an increase in mature granulocytes. There was further an increase in the number of cells that reacted positively with monoclonal antibodies (McAb) binding specifically to granulocytes (B4,3, B13,9 and Leu M 4 ) and a decrease in the percentage of cells reacting with the McAb against Ia-like determinants. These findings indicate that TTNPB is as active as RA in stimulating the growth of hemopoietic progenitors from MDS patients and in enhancing granulocytic differentiation in liquid culture.

In previous studies we have shown that the synthetic retinoid (E)-4[2-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl-1- propenyl]benzoic acid (TTNPB) stimulates the growth of myeloid progenitors from normal and myelodysplastic patients. In the present study we compared TTNPB with 13-cis-retinoic acid (RA) in its potential to inhibit cell growth and to induce morphological differentiation and functional activity in two cell lines established in vitro from either acute promyelocytic (HL-60) or acute myelomonocytic (LK) patients. Both agents, 10(-6) M, were found to effectively inhibit cell growth and cause a significant decrease in number of immature granulocytes in both cell lines. However, while in HL-60 cells this decrease was associated with a concomitant increase in fully mature granulocytes (neutrophil-like cells) the maturation of LK cells was blocked at the metamyelocyte stage. Study of the functional activity of the induced cells revealed that the rate of superoxide (O-2) production, as assayed by superoxide dismutase-inhibitable ferricytochrome c reduction, was faster in RA treated HL-60 cells than in TTNPB treated cells (0.41 vs. 0.25 nmol. O2-/10(6) cells/60 min). Superoxide production by LK cells treated by either TTNPB or RA was negligible. The percentage of O2(-)-producing cells was determined cytochemically by their ability to reduce the dye nitroblue tetrazolium (NBT). The results showed that production of O2-by LK cells exposed to TTNPB or RA was negligible by this method as well. A higher percentage of HL-60 cells reduced NBT following incubation with RA than with TTNPB (93 +/- 4% vs 26 +/- 2%), but neither of the two retinoids affected the ability of LK cells to reduce NBT. TTNPB thus proved less effective than RA in inducing morphological and functional differentiation in HL-60 cells, whereas in LK cells both agents inhibited cell growth but induced only partial cell differentiation.

Heparin uptake and desulphation by cultured macrophages were investigated. Histones, polyamino-acids, protamine and eosinophil-basic protein stimulated both heparin uptake and desulphation, processes found to be non-related. Poly- l -ornithine and poly- dl -lysine increased the heparin uptake by about 33-fold, and histone produced up to 7.5-fold increase in the desulphation. The same polycations inhibited heparin desulphation by macrophage extracts.

Medium conditioned by pokeweed-mitogen-activated human spleen cells enhances the survival of human multipotent stem cells (CFU-mix), erythroid progenitors (BFU-E), and myeloid progenitors (CFU-C) in liquid culture. The effect of adenosine on bone marrow cell survival in liquid culture in the presence of 2′deoxycoformycin and pokeweed-mitogen spleen-conditioned medium was studied. The combination of 2′deoxycoformycin and adenosine was found to have opposite effects on T lymphocytes and hemopoietic progenitors, i.e., the latter were preserved whereas the growth of T lymphocytes was inhibited following prolonged (4–7 days) incubation in suspension culture. The combination of 2′deoxycoformycin and adenosine removed not only all antigenically detectable T cells, but also functionally detectable cells, as judged by response to mitogens. The results suggest that in vitro culture of bone marrow cells in the presence of 2′deoxycoformycin and adenosine might be considered as an alternative way for the removal of T lymphocytes for allogeneic bone marrow transplantation.

The modulatory effect of 13- cis retinoic acid (RA) on the growth, differentiation and function of hematopoietic cells in human long-term cultures was studied. RA (5 × 10 −8 M) induced enhancement of myeloid progenitor cell growth in the non-adherent layer throughout 6 weeks of incubation while it did not affect the number of myeloid progenitors in the adherent layer. The vitamin did not alter the differentiation pattern of colony forming unit-culture (CFU-C). The addition of RA to cultures for 5 weeks did not alter the cellular composition of the adherent layer. Prolonged exposure of hematopoietic cells to RA did not affect the functional activity of neutrophils and macrophages, i.e. the cells were active in phagocytosing Candida albicans (CA).

Due to its potent anticoagulant effect, heparin is widely used both in the prevention and treatment of thrombosis (6). Since heparin is rapidly removed from the blood, continuous intravenous infusion of this material is required to maintain therapeutic blood levels.

Comparison of myelopoiesis in continuous human bone marrow and cord blood cultures was performed. The presence of major histocompatibility complex (MHC) antigens on myeloid precursors (CFU-GM) from cord blood was confirmed using monoclonal antibodies (MoAb) defining class I (i.e., anti- β 2 -microglobulin (B 2 , MG 1 ) anti-HLA-A,B heavy chain (W6/32), and anti-HLA locus B(4E), and class II (HLA-DR (7,2)) antigens. The effect of long-term culture on the expression of these antigens was investigated. Our studies indicate that following prolonged in vitro culture there is a decrease in the expression of MHC antigens on CFU-GM. Our observations provide a rationale for considering the use of cord blood leukocytes subjected to long-term culture as an alternative to allogeneic hematopoietic cell transplantation.

We studied the ability of the human hemopoietic growth factors, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) to activate polymorphonuclear neutrophils (PMN) for increased phagocytosis of opsonized Candida albicans and enhanced degranulation. Exposure of neutrophils to these two growth factors resulted in an increased number of Candida phagocytosed. Pretreatment of the neutrophils with the monoclonal antibody anti-Mol abrogated the enhanced phagocytosis associated with GM-CSF priming but not that of G-CSF primed PMN. In examining the effect of these two colony-stimulating factors (CSFs) on neutrophil degranulation we found that GM-CSF induced enhanced release of lysozyme from cytochalasin-treated PMN in the presence of Candida; however, G-CSF did not. The effect of GM-CSF on lysozyme release was abrogated by anti-Mol antibody. These data suggest that GM-CSF and G-CSF prime PMN for certain enhanced functional activities by distinct mechanisms. The differential effect of the CSFs on neutrophil degranulation may relate to the more common inflammatory symptoms seen when GM-CSF is used clinically as compared to the experience with G-CSF.

// Ido Didi Fabian 1 , Mordechai Rosner 1 , Ina Fabian 2 , Vicktoria Vishnevskia-Dai 1 , Ofira Zloto 1 , Elena Shinderman Maman 3,4 , Keren Cohen 3,4 , Martin Ellis 4 , Hung-Yun Lin 5,6 , Aleck Hercbergs 7 , Paul J. Davis 6,8 and Osnat Ashur-Fabian 3,4 1 Goldschleger Eye Institute, Sheba Medical Center, Tel Hashomer, affiliated to Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 2 Department of Cell and Developmental Biology, the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 3 Department of Human Molecular Genetics and Biochemistry, the Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 4 Translational Hemato-Oncology Laboratory, The Hematology Institute and Blood Bank, Meir Medical Center, Kfar-Saba, affiliated to Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel 5 Institute of Cancer Biology and Drug Discovery, School of Medical Technology, Taipei Medical University, Taipei, Taiwan 6 Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Albany, NY, USA 7 Department of Radiation Oncology, Cleveland Clinic, Cleveland, OH, USA 8 Department of Medicine, Albany Medical College, Albany, NY, USA Correspondence to: Ido Didi Fabian, email: // Keywords : integrin, uveal melanoma, thyroid Received : Spetember 23, 2014 Accepted : February 22, 2015 Published : March 14, 2015 Abstract Uveal melanoma is highly metastatic, prognosis is poor and there are no effective treatments to extend survival. Accumulating evidence suggests that thyroid hormones have a mitogenic effect via binding to αvβ3 integrin. We aimed to examine the impact of thyroid status on survival in a murine B16F10 model for ocular melanoma, highly expressing the integrin. In two independent experiments oral propylthiouracil (PTU) was used to induce hypothyroidism (n=9), thyroxine to induce hyperthyroidism (n=11) and mice given plain water served as control (n=8). At day 21, the subretinal space was inoculated with 10 2 B16F10 cells. In non-inoculated mice (n=6 of each group) serum free T 4 (FT 4 ) levels were measured and additional non-inoculated mice (3 given PTU and 4 given thyroxine or water) served as internal control to demonstrate the impact of the dissolved substance. The PTU-inoculated mice showed clinical evidence of intraocular tumor growth significantly later than the thyroxine mice ( P =0.003) and survival time was significantly longer ( P