Induction of apoptosis by ultrasound application in human malignant lymphoid cells: role of mitochondria-caspase pathway activation.

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/cm(2) 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 gamma-radiation (20 Gy) using (137)Cs 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 gamma-radiation and is associated with time-dependent apoptosis, which is mitochondria-caspase dependent.

Apoptosis induction of human myeloid leukemic cells by ultrasound exposure.

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.

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.

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 of thymocytes to bone marrow stromal cells: regulation by bFGF and IFN-gamma.

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 investigating 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 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-gamma and bFGF modulate T cells-BM stromal cell adhesion.

Selective adhesion of immature thymocytes to bone marrow stromal cells: relevance to T cell lymphopoiesis.

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.

Safety of coronary ultrasound angioplasty: effects of sonication on intact canine coronary arteries.

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.

Streptokinase immunogenicity in thrombolytic therapy for acute myocardial infarction.

The major drawback in implementing thrombolytic therapy with streptokinase in cases of acute myocardial infarction (AMI) stems from its antigenicity. To evaluate the dimensions of this problem, the immune response following thrombolytic therapy with streptokinase was prospectively studied in 16 patients with AMI. Streptokinase was given to 12 patients once and to 4 patients twice within 4-14 days. No clinical immune responses were observed following treatment. The specific antistreptokinase immune responses subsequent to streptokinase administration were monitored. The cellular immune response, reflected by the 3H-thymidine uptake of streptokinase-stimulated peripheral blood mononuclear cells, was three times higher than that of the controls. The antistreptokinase circulating IgG antibody level, measured by an enzyme-linked immunosorbent assay, was 8 times higher than that of the controls. No antistreptokinase circulating IgE antibodies could be detected and no decrease in complement concentration was noted. The profile and magnitude of the immune response of patients who had received two treatments were similar to those of patients who had received a single treatment only. Our data suggest that thrombolytic therapy with streptokinase in AMI elicits a characteristic profile of antistreptokinase immune responses, in vitro, not necessarily associated with parallel clinical immune responses. An early repeat treatment with streptokinase was not accompanied by clinical or exacerbated in vitro immune responses.

Comparison of the Bactec and lysis concentration methods for recovery of Brucella species from clinical specimens.

In a comparison of the Bactec system and the lysis concentration procedure in the isolation of Brucella species in 54 patients the recovery rate was similar (60% and 55%, respectively). However, the recovery time was significantly shorter with the lysis concentration method than with the Bactec system (3.5 days versus 14 days). The lysis concentration procedure for the culture of Brucella is simple, inexpensive and reliable, and produces results for the clinician relatively quickly.

Ultrasonic angioplasty in totally occluded peripheral arteries. Initial clinical, histological, and angiographic results.

BACKGROUND: Ultrasonic angioplasty was recently shown to ablate thrombi and atherosclerotic plaques in vitro and to recanalize occluded arteries in experimental animal models. The goal of the present study was to examine the clinical feasibility of ultrasonic angioplasty. METHODS AND RESULTS: Intraoperative ultrasonic angioplasty was performed in vivo on totally occluded peripheral arteries (n = 7). The ultrasonic angioplasty device consists of a 1.6-mm diameter flexible wire attached to a piezoelectric crystal generating ultrasound at 20 kHz. The controls, totally occluded human atherosclerotic femoral arterial segments (n = 6), were crossed mechanically with the ultrasound wire ex vivo but without application of ultrasonic energy. Ultrasonic angioplasty achieved successful recanalization without perforation in all vessels. Angiograms of the treated arteries showed an average lumen patency of 82.5%. Histological examination of the recanalized arteries revealed that the recanalization had taken place through intima diffusely involved with complicated plaque. The treated arteries, compared with the controls, had greater area of recanalized lumen (5.9 +/- 1.8 versus 1.7 +/- 0.4 mm2, p less than 0.05) and more flow (49.3 +/- 16.0 versus 11.8 +/- 4.9 ml/min, p less than 0.03). The damage in treated and control arteries was similar. Size-distribution analysis of the plaque debris from the treated arteries showed that 41 +/- 5% of the debris was 0.2-8 microns, 48 +/- 8% was 8-30 microns, and the remainder was 30-100 microns. In the mechanically crossed arteries, there was a shift in the distribution to larger size debris with 47 +/- 1% greater than 100 microns (p less than 0.001). CONCLUSIONS: Ultrasonic angioplasty may be a useful clinical method for recanalization of total occlusions in patients with peripheral vascular disease. Ultrasonic energy appears to cause controlled injury to the atherosclerotic intima by selectively disrupting the ultrasound-sensitive occlusion.

Immunologic effects of AS101 in the treatment of cancer patients.

AS101 [ammonium trichloro(O,O'-dioxyethylene)tellurate] is a new immunomodulator previously shown to stimulate the production of different cytokines in vitro and in vivo. We report here our results of a phase I clinical trial conducted on 47 cancer patients with advanced malignancies. AS101 was administered intravenously at escalating doses from 1 to 10 mg/m2, twice or thrice a week. The maximal tolerated dose has not yet been determined. However, significant immunologic responses were noted at dose levels of 1-3 mg/m2 administered three times a week. At these doses statistically significant rises in gamma-interferon, natural killer cell activity, tumor necrosis factor and interleukin-2 (IL-2) levels as well as the expression of IL-2 receptors were noted. In most of the immunologic parameters the maximal response was seen at 3 mg/m2. Throughout the study toxicity was minimal. In view of these results phase II studies are currently being initiated.

Stimulation of human T cell colony growth by a lymphocyte colony enhancement factor derived from lymphocyte subpopulations.

Blood mononuclear cells (MNC) develop into T cell colonies when the cells are sensitized with PHA and seeded in a two-layer soft agar system. Conditioned medium (CM) derived from MNC enhanced lymphocyte colony formation when it was added to the culture system. CFU-TL appear to be stimulated into colony formation by molecules secreted by lymphocyte subpopulations contained in the seeded cells. In this study, human peripheral blood MNC were fractionated by a battery of techniques into adherent, E+, CD4+, CD8+, B and null cells. CM was prepared from each of the subpopulations and its effects on T cell colony growth assayed. All the lymphocyte subpopulations were found to generate lymphocyte colony enhancement factor (LCEF). After several purification procedures, CM prepared from CD4 and CD8+, displayed LCEF activity corresponding to proteins of molecular weight 30-40 and 100-140 kD.

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.

A new model of autoimmune disease. Experimental autoimmune uveoretinitis induced in mice with two different retinal antigens.

Experimental autoimmune uveoretinitis (EAU) is an organ-specific, T lymphocyte-mediated autoimmune disease, which serves as a model for several human ocular inflammations of an apparently autoimmune nature. EAU pathology in some rodents and in monkeys can readily be induced by immunization with several different retinal proteins; however, advancing research into the cellular mechanisms of this disease has raised the need for an EAU model in an immunologically and genetically well defined species. We report here the induction of EAU in the mouse, which has hitherto been considered a species refractory to EAU, with two retinal Ag, the retinal soluble Ag and the interphotoreceptor retinoid-binding protein. Although all the mouse strains tested exhibited lymphocyte responses and antibody titers to both retinal Ag, EAU was inducible in only some of the strains, and the uveitogenic responses to retinal soluble Ag and interphotoreceptor retinoid-binding protein appeared to be mutually exclusive. The EAU model in mice was found to differ in several respects from the EAU model in other rodent species. Induction of the disease was achieved with a relatively high dose of Ag and an intensified immunization protocol, and the onset of disease was later, the duration was longer, and the course was less acute. Anterior segment involvement was slight or nonexistent, and damage to the retina and uvea was of a focal rather than of a diffuse nature. Murine EAU appeared to approximate some types of human uveitis more closely than the EAU models described in other rodent species with respect to its pathologic manifestations as well as its more chronic course. The relatively longer duration of the active stage of disease in murine EAU should facilitate therapeutic intervention in established disease, which was not feasible in the more acute models of EAU. The extensive knowledge of the immunologic parameters of the mouse and the availability of genetically defined strains should be of great value in the study of cellular mechanisms and immunogenetics of ocular autoimmune disease.

Thymus-derived stromal cell lines.

We derived stromal cell lines from mouse thymus using methods previously established for bone marrow stroma. Two main morphologically distinct groups of cell strains emerged: epithelioid and mixed fibroblast-macrophage. Transmission electron microscopy revealed frequent junctional-complex formations between adjacent cells, a feature that characterized almost all of the thymus stromal lines, but was confined to only one of the five distinct subtypes of cell lines from bone marrow. In contrast to marrow stromal cells, the thymus-derived cell lines were all negative with fat-detecting reagents, had low acid phosphatase and no basic phosphatase activities and were unable to support the in vitro proliferation of myeloid progenitor cells (CFU-gm). Leukemia cell inhibitory activity (LCIA) was detected in one of the thymus stromal cell lines. The differences observed between cell lines derived from the stroma of the thymus and those from bone marrow may relate to the functional specificities of these organs.

Human muscle-derived, tissue specific, myocytotoxic T cell lines in dermatomyositis.

Mononuclear cells were isolated from the inflamed muscle tissue of a patient suffering from dermatomyositis (DM). These were expanded in long-term culture and maintained in the presence of IL-2 containing culture medium. Two cell lines were established, one of the helper/inducer (OKT4+) and the other of the suppressor/cytotoxic phenotype (OKT8+). The OKT4+ cell line exhibited a non HLA-restricted, tissue-specific, myocytotoxic effect on rat muscle cell culture. Its lymphoproliferative response to human muscle antigen was HLA-restricted. The OKT8+ cell line exhibited a non HLA-restricted, tissue-specific response to muscle antigens and no myocytotoxic activity in in vitro rat muscle cell culture. It is likely that clones of OKT4+ lymphocytes in patients suffering from DM are associated with the pathogenesis of the disease--they probably mediate the diffuse damage to skeletal muscle through their myocytotoxic activity.