Leon A. Rozenszajn

The Esterase Activity in Megaloblasts, Leukaemic and Normal Haemopoietic Cells

Summary. Esterase activity was investigated in blood cells of peripheral blood and bone marrow in healthy subjects and in various pathological conditions. As substrates were used: α‐naphthyl acetate, naphthol AS‐D acetate and naphthol AS‐D chloroacetate. Fast blue B and fast garnet G. B. C. were used as coupling agents.

Ultrasound inhibits the adhesion and migration of smooth muscle cells in vitro

This study investigated in vitro the effect of therapeutic ultrasound (ULS) on smooth muscle cell (SMC) function as adhesion, migration and proliferation. Experiments were conducted on aortic SMC in culture. The LD50 was established (1.5 W for 15 s at a frequency of 20 kHz) and used as standard dose in all experiments. Control SMC and viable sonicated SMC were compared in each experiment. Migratory capacity decreased 2.4-fold after sonication and stayed reduced for up to 24 h. Adhesion capacity decreased 5.5-fold after ULS. The proliferative capacity was similar to that of nonsonicated SMC. Sonication was accompanied by the disorganization of alpha-SM actin fibers and diminished distribution of vinculin; tyrosinated alpha tubulin and vimentin appeared unaffected. These changes might be responsible for the observed inhibition of SMC adhesion and migration. Sonicated cells exhibited less lamellipodia, membrane collapse and bleb formation. The signal transduction cascade, which involves activation of the phospholipase-C pathway, was unaffected by ULS.

ADHESION MOLECULES INVOLVED IN THE INTERACTIONS BETWEEN EARLY T CELLS AND MESENCHYMAL BONE MARROW STROMAL CELLS

We previously reported that among the various thymic lymphocyte subpopulations, the immature T cells preferentially adhere to mesenchymal bone marrow stroma. In the present study we examined the interactions between phenotypically defined populations of early T cells and stromal cell lines. The immature T cells segregated into two subpopulations according to their adhesive capacity. Whereas the majority of the adherent CD4-CD8- T cells were devoid of CD3/TCRalphabeta, most of the nonadherent CD4-CD8- T cells expressed this receptor complex. The adhesion of T cells to bone marrow stroma almost entirely was accounted for by CD49d and CD90, whereas that of adherent CD4-CD8- cells also was dependent on CD44, CD62L, and CD117 receptor. Blocking antibody combinations failed to reduce the adherence of these early T cells to less than 50% that of the control. On the other hand, the adhesion of unselected thymocytes to the stroma was reduced by 80%, using the same blocking antibodies. Therefore, the participation of additional molecules in the adhesion of early T cells to mesenchymal stroma is implicated. Comparison between the interaction of T cells with bone marrow mesenchymal or with thymus-derived epithelial stroma indicated that T cells utilize a selected set of adhesion molecules under each situation. Although CD49d and CD90 participated in both cases, CD11a, CD18, and CD2 receptors played a dominant role in the adhesion of T cells to thymic epithelium only. This study may point to a role of mesenchymal stroma in the regulation of early T-cell lymphopoiesis in the bone marrow.

Stimulation and inhibition of human T-lymphocyte colony cell proliferation by hemopoietic cell factors.

Abstract Human lymphocytes, isolated from peripheral blood and stimulated with phytohemagglutinin M (PHA) prior to being seeded on a two-layer medium of soft agar which contained the mitogen, developed into colonies 3–4 days after seeding in the culture system. The cloning potential of PHA-treated lymphocytes is significantly enhanced by adding, to the soft agar culture, culture fluid (CF) obtained from mitogen-treated lymphocytes or a feeder layer (FL) prepared either from lymphocytes isolated from peripheral blood or from T-cell enriched populations. PHA seems to stimulate the release of lymphocyte colony enhancing factor (LCEF) from the T-sensitized lymphocytes. The addition of CF or FL to the culture medium appears to increase the amount of LCEF, resulting in enhancement of the number and size of lymphocyte colonies. When CF derived from spleen cells or from the peripheral blood adherent-cell population was added to the lower layer of the soft agar culture, the growth and development of lymphocyte colonies was inhibited. This suggests that monocyte-macrophages release a lymphocyte colony inhibiting factor (LCIF) into the CF. The extent of inhibition or stimulation of colony formation is a function of the number and type of cells used to prepare the CF or FL and the concentration of CF in the culture medium. The presence of FL or CF derived from spleen non-adherent cells, white blood cells, bone marrow cells, or a B-cell enriched population had no effect on colonies growing in the culture. This may possibly be due to the paucity of T lymphocytes and monocyte-macrophages present in these materials. A control system in which LCIF, produced by monocyte-macrophages, and LCEF, produced by T lymphocytes, participate in the regulation of lymphocyte production is postulated.

Induction of Apoptosis by Ultrasound Application in Human Malignant Lymphoid Cells

Abstract: In the present study, we have focused on the specific question of whether ultrasound application (ULS) delivered with optimized parameters for cavitation generation can stimulate apoptosis in lymphoid cell lines. Suspended T and B lymphoid cell lines (Jurkat and Raji, respectively) were exposed to low frequency ULS (750 KHz) at an intensity level of 54.6 W/cm2 spatial peak temporal average (SPTA) at focal area, which was found to be the optimal physical parameter to induce apoptosis in these malignant cell lines. Unsonicated cells and cells exposed to γ‐radiation (20 Gy) using 137Cs source were used as control. Apoptosis was evaluated by cell morphology changes, cell‐cycle analysis, and phosphatidylserine exposure. Fraction of cells with low mitochondria membrane potential was observed 1 h after sonication, accompanied by cytochrome c release from mitochondria to the cytosol and caspase‐3 activation. Here we present evidence that ULS exposure with cavitation formation on malignant lymphoid cell lines differs from γ‐radiation and is associated with time‐dependent apoptosis, which is mitochondria‐caspase dependent.

Adhesion of Thymocytes to Bone Marrow Stromal Cells: Regulation by bFGF and IFN-γ

We recently reported on selective interactions between immature T cell subpopulations and bone marrow (BM) stromal cells. To further study this process, we first examined the efficacy of methods estimating cell‐cell adhesion and then investigated the effects of cytokines on thymocyte‐stroma associations. Techniques based on the use of the fluorochromes calcein‐acetomethylester (calcein‐AM) and fluorescein diacetate (FDA) were studied and compared to regular cell counting methods. With calcein‐AM labeling, the retention time was relatively long, while with FDA labeling, there was a rapid cellular efflux. Using calcein‐AM, we developed an accurate quantitative fluorometric assay for determining the adherence of thymocytes to a BM stromal cell line (MBA‐13). A maximal fraction of about 29% thymocytes was found to adhere to confluent MBA‐13 cell layers after four to six h of coculture. Whereas interleukin 1 did not change the rate of adhesion of thymocytes to the stroma, interferon‐γ (IFN‐γ) significantly increased adhesion. Basic fibroblast growth factor (bFGF) had a dose‐dependent biphasic effect on thymocyte adhesion, and a greater fraction of double negative thymocytes adhered to stroma pretreated with bFGF. Taken together, these results suggest that IFN‐γ and bFGF modulate T cells‐BM stromal cell adhesion.

T‐Lymphocyte Colony Growth in vitro: Factors Modulating Clonal Expansion

The community of ceils termed thymus-derived (T) lymphocytes comprise several functionally heterogeneous cell populations. These populations are of paramount importance in the numerous and variegated immune responses which characterize cell-mediated immunity. Investigations concerned with the properties, functions and interrelations of the cells of the immune system and their products have broadened and deepened our understanding of the regulating roles these cells play in immunoiogical phenomena. The use of recently developed in vitro techniques and assays for growing different classes of hemopoietic precursor cells led to the accumulation of much information and permitted insight into the mechanism and control of hemopoiesis and cellular kinetics. Studies on T-cell populations forged ahead through the development of short-term cell culture systems and the use of polyclonal T-cell activators such as Concanavalin A (Con A) and particularly phytohemagglutinin (PHA) (Nowell 1960, Stoho & Paul 1973). These mitogens provide a nonspecific stimulus to T-cell populations: the lymphocytes are triggered into an activated state which is manifested by morphological changes that are easily recognized blast transformation and mitosis (Yoffet et al. 1965). The stimulation of lymphocytes by PHA and Con A causes the activation of soluble, cyclic AMP-dependent protein kinase: this may be one of the early events linked to and regulating lymphocyte mitogenesis (Klimpel et al. 1979). Additional parameters frequently used to assay the proliferative responses of T lymphocytes to the lectins, in appropriate concentrations and under suitable suspension culture conditions, are increments in the number

Regulatory effects of macrophage-secreted factors on T-lymphocyte colony growth☆

Abstract Colonies of mouse T lymphocytes developed when lymph node cells, presensitized with PHA in liquid culture, were seeded in a two-layer soft agar culture system containing the mitogen in the lower layer. The clonal proliferation was suppressed when culture fluid supernatants from macrophages (adherent peritoneal cells or spleen cells) from a number of mouse strains were incorporated in the agar culture system. The magnitude of the suppression was a function of the number of cells used to prepare the culture fluid and the amount of supernatant added to the culture. The inhibitory effect disappeared with dialysis and the dialyzed culture fluid exhibited an enhancing effect on T-lymphocyte colony proliferation. Diaflo ultrafiltration of culture fluid supernatant separated two substances, lymphocyte colony-inhibitory factor (LCIF), concentrated mainly in the fraction with molecular weight of less than 1000, and lymphocyte colony-enhancing factor (LCEF), located chiefly in the fraction with molecular weight of 10,000 to 30,000. The inhibitory factor was heat stable, while the enhancing factor was heat sensitive. LCIF was not species specific. It appears that adherent cells-marcophages release two biologic substances able to influence the cloning of T lymphocytes, lymphocyte colony-inhibitory factor and lymphocyte colony-enhancement factor. Unless the two are separated, the mild activity of LCEF is completely masked by the relatively strong action of LCIF.

Characterization of the interference of T cell activation by reserpine

It has been suggested that reserpine blocks expression of delayed hypersensitivity (DH) reactions by depleting tissue mast cells of serotonin, thereby preventing a T cell-dependent release of mast cell serotonin necessary to localize and to amplify the DH response. However, reserpine blocks expression of DH in mast cell-deficient mice. Recently, we showed that the ability of reserpine to interfere with the expression of contact sensitivity was independent of an effect on mast cells, but reflected an effort of the drug on effector T cell function. In the present study we evaluated the mechanisms by which reserpine abrogates the expression of T cell functions. By using human peripheral blood mononuclear cells or enriched T cell populations we found that the drug inhibited, in a dose-dependent fashion, the proliferation of T cells after mitogen stimulation. Reserpine also interfered with the mitogen-induced IL-2 production by these cells, but the IL-2 receptor expression, as measured by immunofluorescence, was unaffected. Despite this, in the continuous presence of reserpine, exogenous IL-2 did not bypass reserpine inhibition of PHA-induced proliferation. By using the fluorescent indicator quin-2 we have demonstrated that preincubation with reserpine prevented the increase of cytosolic free calcium, which accompanies PHA-induced proliferative responses of human T lymphocytes. These results identify the sites of action of reserpine in human T lymphocytes and are sufficient to explain its ability to block cell-mediated immune responses in vitro and in vivo.

Colony Growth of T Lymphocytes in vitro

Human lymphocytes stimulated with PHA in liquid phase (step 1) and then seeded in a two-layer soft agar system (step 2) grew and developed into T cell colonies. Colony formation was enhanced when the