the number of tion of contact with lymphocytes, as well cells undergoing division directly properas on the developmental stage of the tional to the quantity of folinic acid added. T HE USE OF methotrexate (MTX, 4-amino, l0-pteroyl glutamic acid) in cancer chemotherapy was based on its strong inhibition of dihydrofolic acid dehydrogenase (7.8 dihydrofolate: NADP oxidoreductase 1.5,1.4)12 and the resulting failure to produce tetrahydrofolic acid (THFA). This inhibition interferes with transfer of one-carbon units, which, in turn, affects synthesis of purine and pyrimidine, as well as that of methionine, serine, and glycine. The interference with nucleic acid synthesis affects rapidly dividing tumor cells much more than normal body cells.3 Most of this work was done on cells of leukemic patients in whom a correlation was found to exist between activity of dihydrofolic acid dehydrogenase and response to MTX.�7 The inhibitory effect of MTX on dihydrofolic acid dehydrogenase has been examined in various biological systems.8’#{176}’4’5 The chemical reaction between the enzyme and MTX has been investigated as well.”3 On the other hand, folinic acid had the property of canceling the inhibitory effect produced by MTX.’2 Since nonspecific’ stimulation, such as addition of phytohemagglutinin (PHA), could transform cultured lymphocytes into blast-like forms and induce mitosis, we decided to study the effect of MTX on these processes in cultures of normal human blood lymphocytes. This system enabled us also to study the effect of MTX at various stages of cell development.

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.

None of the modalities currently employed to induce apoptosis involves the use of ultrasound energy. We have demonstrated (Cancer Res. 60: 1014, 2000) that selected physical parameters utilized in therapeutic ultrasound (ULS) application resulting in cavitation process induce an apoptotic cell death. The aim of the present study was to investigate ULS application with rational selected physical parameters to optimize ultrasound cavitation- induced cell death and to determine whether apoptosis is involved. High intensity focussed ULS sonication was delivered with an induction of transient cavitation and intensity of 54 W/cm2 to malignant T and B lymphocyte-derived cell lines expressing p53 as Raji, Jurkat and NALM-6 as well as myeloid leukemia cell lines p53 negative HL-60, K562, U937. Here we present evidence that much of the cell damage surviving ULS exposure appears to occur through an apoptotic mechanism. Morphological alterations of apoptotic cells involve nuclear fragmentation and apoptotic body formation. Positive identification of apoptotic cells was based also on the detection of nuclear DNA strand break and changes in the surface of treated cells undergoing apaptosis expressed by the breakup of phosphatidylserine from the inner to the outer side of the membrane layer. In summary, after ULS exposure malignant hemopoietic cell lines can be induced to undergo apoptosis by p53-dependent and p53-independent apoptotic pathways.

Therapeutic ultrasound (ULS) and the resulting cavitation process has been shown to induce irreversible cell damage. In this study, we wanted to further investigate the mechanism of ULS-induced cell death and to determine whether apoptosis is involved. High intensity focused pulsed ULS sonication at a frequency of 750 KHz was delivered to HL-60, K562, U937, and M1/2 leukemia cell line cultures. ULS exposure used with induction of transient cavitation in the focal area was delivered with an intensity level of 103.7 W/cm2 and 54.6 W/cm2 spatial-peak temporal-average intensity. As a control, ULS of lower intensity was delivered at 22.4 W/cm2 spatial-peak temporal-average intensity, presumably without generation of cavitation. Our results indicated that DNA damage induced by ULS cavitation did not involve generation of free radicals in the culture media. Morphological alterations observed in cells after exposure to ULS included: cell shrinkage, membrane blebbing, chromatin condensation, nuclear fragmentation, and apoptotic body formation. Apoptotic cells were evaluated by fluorescence microscopy and detected using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, which identifies DNA breaks, and by the leakage of phosphatidylserine from the inner to the outer side of the membrane layer of treated cells. Some bioeffects induced on sonicated HL-60 cells, such as inhibition of cell proliferation, DNA repair, and cell-dependent apoptosis, were found to be similar to those produced by gamma-irradiation. Thus, much of the cell damage induced by therapeutic ULS in leukemia cells surviving ULS exposure appears to occur through an apoptotic mechanism.

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/TCRαβ, 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.

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.

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-gamma (IFN-gamma) significantly increased adhesion. Basic fihroblast 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 bFGP. Taken together, these results suggest that IFN-gamma and bFGF modulate T cells-BM stromal cell adhesion.

We investigated the interactions between the bone marrow microenvironment and T cell populations at different stages of maturation. Thymocytes were seeded onto confluent layers of bone marrow stromal cell lines (MBA-13 or 14F1.1). Within a few hours two main thymocyte populations were observed; one remained in the liquid phase and the other adhered to the stromal cells. After 24 hours of culture, most of the adhering cells expressed the phenotype of the precursors, double negative (DN) CD4(-)CD8(-), or of immature thymocytes, double positive (DP) CD4(+)CD8(+). The number of adhering DN cells did not change during the time of the culture, whereas that of the DP declined. The CD4(+)CD8(-) or CD4(-)CD8(+) cells did not adhere to any significant extent. The expression of CD3 antigen on adherent thymocytes was lower than that on nonadherent ones. Sorted thymocytes at a high level of purification (>96%) were cultured over stromal layer and, after 24 hours, 60% of the DN or 22% of the DP cells were found to adhere to the stroma. The culture medium was replaced every 24 hours or after 48 hours; no significant change was noted in the number of adhering DN and DP cells. The reappearance of immature T cells in the liquid phase suggested proliferation of this cell type. Thus, early thymocytes, phenotypically characterized as DN and DP, preferentially adhere to bone marrow stromal cells. This in vitro phenomenon may represent the function of the BM stroma as an extrathymic site of T cell lymphopoiesis.

The purpose of this work was to examine in vivo the safety of sonication in the coronary arteries in a live animal model. In intact dogs (n = 8), balloon dilatation was performed on the proximal left anterior descending artery (LAD) followed by sonication to the left circumflex artery (LCX) in power levels found to be optimal for thrombus ablation. Post-dilatation and post-ultrasound coronary angiography, echocardiography, histopathology, CK-MB, indices of hemolysis, and coagulation were compared. Sonication did not induce changes in the ECG or blood pressure. Coronary angiography revealed no adverse side effects or change in arterial diameter (2.3 +/- 0.7 vs. 2.4 +/- 0.3 mm). Echocardiography showed transient opacification of the myocardium. Histopathology revealed a comparable minimal degree of endothelial denudation. After sonication there were no changes in the level of CK-MB (312 +/- 168 vs. 283 +/- 207 IU), hemoglobin (11.3 +/- 0.9 vs. 12.7 +/- 1.1 gr%), haptoglobin (479 +/- 136 vs. 451 +/- 121 mg/dL), fibrinogen (142 +/- 18 vs. 165 +/- 28 mg%), partial thromboplastin time (17.3 +/- 3.2 vs. 17.6 +/- 3.4 sec), prothrombin time (13.3 +/- 7.8 vs. 11.5 +/- 2.9 sec), and degree of platelet aggregation (55 +/- 17 vs. 62 +/- 8%). Thus, the data suggest that transluminal coronary sonication exerts no overt adverse effects in vivo.

American Journal of HematologyVolume 47, Issue 2 p. 145-146 Letter and Correspondence Possible presence of T-cell progenitors in a population of B-prolymphocytic leukemia cells J. Radnay, J. Radnay Laboratories of Hematology, Immunology and Molecular Biology. Sapir Medical Centre, Kfar Saba, Department of Life Sciences, Bar-Ilan University, Ramat-Gan, IsraelSearch for more papers by this authorV. Leytin, V. Leytin Laboratories of Hematology, Immunology and Molecular Biology. Sapir Medical Centre, Kfar Saba, Department of Life Sciences, Bar-Ilan University, Ramat-Gan, IsraelSearch for more papers by this authorA. Alter, A. Alter Laboratories of Hematology, Immunology and Molecular Biology. Sapir Medical Centre, Kfar Saba, Department of Life Sciences, Bar-Ilan University, Ramat-Gan, IsraelSearch for more papers by this authorT. Ben-Tovim, T. Ben-Tovim Laboratories of Hematology, Immunology and Molecular Biology. Sapir Medical Centre, Kfar Saba, Department of Life Sciences, Bar-Ilan University, Ramat-Gan, IsraelSearch for more papers by this authorL. A. Rozenszajn, L. A. Rozenszajn Laboratories of Hematology, Immunology and Molecular Biology. Sapir Medical Centre, Kfar Saba, Department of Life Sciences, Bar-Ilan University, Ramat-Gan, IsraelSearch for more papers by this authorA. Klein, A. Klein Laboratories of Hematology, Immunology and Molecular Biology. Sapir Medical Centre, Kfar Saba, Department of Life Sciences, Bar-Ilan University, Ramat-Gan, IsraelSearch for more papers by this authorY. Manor, Y. Manor Hematology Unit, Sapir Medical Centre, Kfar Saba, Sackler School of Medicine, Tel Aviv University, IsraelSearch for more papers by this author J. Radnay, J. Radnay Laboratories of Hematology, Immunology and Molecular Biology. Sapir Medical Centre, Kfar Saba, Department of Life Sciences, Bar-Ilan University, Ramat-Gan, IsraelSearch for more papers by this authorV. Leytin, V. Leytin Laboratories of Hematology, Immunology and Molecular Biology. Sapir Medical Centre, Kfar Saba, Department of Life Sciences, Bar-Ilan University, Ramat-Gan, IsraelSearch for more papers by this authorA. Alter, A. Alter Laboratories of Hematology, Immunology and Molecular Biology. Sapir Medical Centre, Kfar Saba, Department of Life Sciences, Bar-Ilan University, Ramat-Gan, IsraelSearch for more papers by this authorT. Ben-Tovim, T. Ben-Tovim Laboratories of Hematology, Immunology and Molecular Biology. Sapir Medical Centre, Kfar Saba, Department of Life Sciences, Bar-Ilan University, Ramat-Gan, IsraelSearch for more papers by this authorL. A. Rozenszajn, L. A. Rozenszajn Laboratories of Hematology, Immunology and Molecular Biology. Sapir Medical Centre, Kfar Saba, Department of Life Sciences, Bar-Ilan University, Ramat-Gan, IsraelSearch for more papers by this authorA. Klein, A. Klein Laboratories of Hematology, Immunology and Molecular Biology. Sapir Medical Centre, Kfar Saba, Department of Life Sciences, Bar-Ilan University, Ramat-Gan, IsraelSearch for more papers by this authorY. Manor, Y. Manor Hematology Unit, Sapir Medical Centre, Kfar Saba, Sackler School of Medicine, Tel Aviv University, IsraelSearch for more papers by this author First published: October 1994 https://doi.org/10.1002/ajh.2830470221AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume47, Issue2October 1994Pages 145-146 RelatedInformation