5′-Nucleotidase in lymphocytes from various clinical disorders

The enzyme 5′-nucleotidase (5′-N) was demonstrated cytochemically in blood cells using a modification of the Wachstein and Meisel technique [30]. In peripheral blood the activity was found to be localised mainly in the cell membrane of lymphocytes. A semiquantitative score of 5′N positivity, was assessed in lymphocytes from eight normal donors and 60 patients with various types of leukaemia and lymphoma. The lymphocyte score of 5′-N was slightly reduced in CML and AML but markedly reduced in most lymphoproliferative states tested. The only supranormal activity was found in two of 18 cases of CLL.     

Hairy-cell leukaemia: A B-cell neoplasm with a severe deficiency of circulating normal B lymphocytes

A series of 10 patients with hairy-cell leukaemia (HCL) was studied. The hairy cells (HCs) of all the patients studied possessed surface membrane immunoglobulin of a single light chain type (Smlg) not acquired from the serum. Combined Smlg, esterase cytochemical, ultrastructural, surface marker and serological studies showed that many more cells were involved in the B-cell neoplastic proliferation than was apparent by simple morphological examination. In addition, the virtual absence of cells rosetting with yeast-C3b indicator particles, and of cells displaying a B-cell esterase pattern or possessing the non-predominant light chain Smlg, indicated a severe deficiency of circulating normal B lymphocytes in HCL.     

Production of stem cell proliferation regulators by fractionated haemopoietic cell suspensions.

Fractionated cell populations from haemopoietic tissues have been examined for their yield of stem cell proliferation inhibitory or stimulatory activities. These activities show marked concentration differences depending on the proliferative state of the stem cells in the haemopoietic tissues investigated. However, irrespective of whether haemopoietic tissue contains stem cells which are actively or minimally proliferating, inhibitory activity is produced by cells with a density of 1.052-1.060 gm/cm³, whilst cells with density 1.064-1.072 gm/cm³ elaborate stimulatory material. From inhibitor/stimulator dose response studies, it is concluded that the control of stem cell proliferation is mediated by an appropriate balance of stimulatory and inhibitory factors which, however, are unlikely to be produced by the stem cells themselves.     

DNA strand breakage and ADP-ribosyl transferase mediated DNA ligation during stimulation of human bone marrow cells by granulocyte-macrophage colony stimulating activity

ADP-ribosyl transferase (ADP-RT) is a chromatin-bound nuclear enzyme catalysing the transfer of ADP-ribose from NAD+ to chromatin proteins. The enzyme is activated by DNA strand breaks and has been suggested to have roles in both DNA repair (via its effect on DNA ligase II) and in differentiation. We recently demonstrated that specific inhibitors of ADP-RT preferentially inhibit differentiation of human granulocyte-macrophage progenitor cells to the macrophage lineage and that the specific proliferation/differentiation stimulus granulocyte-macrophage colony stimulating activity (GM-CSA) activates ADP-RT in human marrow cells within 3 h of exposure. The purpose of this study was to investigate the role of ADP-RT in monocyte-macrophage differentiation. By altering the time of addition of ADP-RT inhibitor it was demonstrated that maximal inhibition of macrophage differentiation only occurs when the inhibitor is added within the first 24 h of culture. This suggests that it is an early event during the induced differentiation of granulocyte-macrophage progenitor cells which requires ADP-RT. Fluorometric assay of the level of DNA strand breaks showed that GM-CSA induces DNA strand breaks which are rapidly ligated only if ADP-RT is available. These data and those of our earlier studies suggest that DNA rearrangement may be involved in differentiation of granulocyte-macrophage progenitors to the monocyte-macrophage pathway. Such a DNA rearrangement could provide a molecular basis for commitment of multipotent progenitors to a single lineage.     

An inhibitor of murine stem cell proliferation produced by normal human bone marrow

Media conditioned by normal human bone marrow cells contain a specific inhibitor of haemopoietic stem cell proliferation. Molecular ultrafiltration and dose response studies indicate that it is similar to a previously described factor obtained from freshly isolated or long-term cultured murine bone marrow cells. It is suggested that the mechanisms involved in the control of murine and human stem cell proliferation may be essentially identical.     

Blastic transformation of a well-differentiated monocytic leukemia: Changes in cytochemical and cell surface markers

The clinical course of a patient with a well-differentiated monocytic leukemia which later underwent blastic transformation is described. Cytochemical, ultrastructural and cell surface analysis data were obtained at periods throughout her illness and correlated with the blastic transformation. Although surface markers characteristic of monocytic leukemia persisted, a deficiency of peroxidase in the granules of this patient's monocytes was observed as well as loss of α-naphthyl butyrate esterase staining during transformation.     

Regulators of stem cell proliferation in the haemopoietic tissues of W/Wv and SI/SId mice

The presence of CFU-S proliferation stimulatory and inhibitory activities in the bone marrow and spleens of normal mice (+/+) and mice with mutations affecting the proliferative behaviour of stem cells (S1/S1^d and W/W^v) has been investigated.S1/S1^d and +/+ bone marrow and spleen contain virtually no detectable stimulator but the corresponding tissues from W/W^v mice are both strongly stimulatory. SI/SI^d marrow in particular, but also +/+ marrow are strongly inhibitory whilst +/+ spleen, S1/SI^d spleen, W/W^v marrow and W/W^v spleen all contain inhibitory activity but at a lower specific activity.The data are compatible with studies of cell and tissue grafts that have indicated an intrinsic haemopoietic stem cell defect in W/W^v mice and an extrinsic, microenvironmental defect in SI/SI^d mice. It is suggested that they are also compatible with defective regulatory interactions between stem cells and regulator-producing cells and that the W and S1 loci may code for products involved in the production of, or response to, CFU-S proliferation regulators.     

Fractionation and characterization of the blast cell population of the mouse thymus by physical cell separation methods and ultrastructural analysis.

Sedimentation at unit gravity and discontinous density gradients were used either separately or sequentially in an attempt to isolate cell preparations that are significantly enriched in one of the several subtypes of thymocytes present in the C57BL mouse thymus. Electron microscopy analysis of the cellular content of fractions obtained by such methods demonstrates that fractionation by 1g sedimentation allows the isolation of either pure preparations of small cortical lymphocytes or of fractions that are significantly enriched in blast cells, whereas density gradient centrifugation yields to the selection of the low to medium density lymphoblasts. A sequential separation procedure using both sedimentation at unit gravity and density gradient centrifugation was found to be the method of choice to isolate pure preparations of medullary lymphoid cells or to obtain fractions devoid of small lymphocytes and highly enriched in blast cells of the X subtype. These methods may thus serve as a basis for the selective isolation of putative target cells which are believed to be present in the thymus and to play a role in the pathogenesis of thymic lymphomas in C57BL mice.     

Purine metabolism in relation to leukemia and lymphoid cell differentiation

A number of inborn errors of purine metabolism have been associated with immunodeficiency diseases. From studies to the possible mechanism(s) leading to the defects in the immune system, it appeared that the accumulation of deoxyATP and deoxyGTP and the subsequent inhibition of ribonucleotide reductase played an important role. The inhibition of methylation pathways through the accumulation of s-adenosylmethionine seems to be a second valid concept. The amount to which certain subtypes of lymphoid cells were affected by the enzyme deficiencies was strongly related to the enzymatic make-up of the cells. Lymphoid cells from different maturation stages could be affected in a specific way, depending on the different enzyme activities of these cells. Studies on human lymphoblastic leukemias showed that, related to the immunological subtype, the different leukemias could be characterized by a different enzymatic make-up. In this paper we discuss the possibilities for a specific enzyme directed chemotherapy, directed against specific subtypes of human lymphoblastic leukemias. Experimental evidence indicates that for example the adenosine deaminase inhibitor 2'deoxycoformycin can be used as a specific drug against acute lymphoblastic leukemia with the T cell phenotype.