Lysis and sonoporation of epidermoid and phagocytic monolayer cells by diagnostic ultrasound activation of contrast agent gas bodies.

Epidermoid A431 (human carcinoma) and phagocytic RAW-264.7 cells were grown as monolayers on 5-microm thick Mylar sheets by standard culture methods. The sheets formed one window of disk-shaped ultrasound (US) exposure chambers. A diagnostic US machine in spectral Doppler mode was used for exposure with a 3.5-MHz scanhead aimed upward at the chamber in a 37 degrees C water bath. Sonoporation and cell lysis were evaluated for assessment of cell membrane damage. For both epidermoid and RAW cells on the upper window with 1% Optison contrast agent, cell lysis was detectable in addition to sonoporation. The phagocytic cells tended to retain the gas bodies when incubated with contrast agent, and membrane damage occurred even for exposure on the bottom window. The effects were also seen for RAW cells incubated with 5% contrast agent for 15 min and then rinsed before exposure. Above a threshold range for lysis and sonoporation of 0.09 to 0.23 MPa, the counts of affected cells increased for both cell lines to about 20% at 0.83 MPa. These results indicate relatively low thresholds for membrane damage induced by diagnostic US activation of contrast agent gas bodies, with a potential for targeting of these effects to phagocytic cells.

Histology of herniations through the body wall and cuticle of zooplankton from the Laurentian Great Lakes.

Zooplankton of the Laurentian Great Lakes developed hernial protrusions whose gross appearance matches those on zooplankton described elsewhere in the world. We have carried out a histologic and cytologic analysis of the protrusions and found that they are composed of apparently degenerating or necrotic tissue(s) that has been expressed from the organism through the process of herniation. At their base the protrusions are continuous with viable tissue(s) within the organism through a fissure in the exoskeleton. Our observations lead us to suspect that these hernial protrusions are lethal. The development of such protrusions in zooplankton may be a worldwide phenomenon, but the cause of the herniation remains a mystery.

Functional expression of NAD(P)H oxidase p47 in lung microvascular endothelial cells.

Vascular endothelial cell superoxide (O(*)(2)) has an important role in intracellular signaling, in interaction with other reactive species such as nitric oxide, and in vascular dysfunction. Little is known regarding the source and function of O(*)(2) from microvascular endothelial cells from specific tissues. Mouse lung microvascular endothelial cells stimulated with phorbol ester (PMA) or NADPH generated significant O(*)(2), which was inhibited by diphenyleneiodonium (DPI) but not by allopurinol, rotenone, indomethacin, or quinacrine. Optimal O(*)(2) generation required cytosolic as well as particulate cell fractions of cells. In parallel studies, PMA induced increased expression of the p47 component of the NAD(P)H oxidase in the particulate fraction, which was inhibited by staurosporine and calphostin. These data demonstrate that NAD(P)H oxidase is an important source of O(*)(2) generation in lung microvascular endothelial cells.

Diagnostic ultrasound activation of contrast agent gas bodies induces capillary rupture in mice.

Interaction of diagnostic ultrasound with gas bodies produces a useful contrast effect in medical images, but the same interaction also represents a mechanism for bioeffects. Anesthetized hairless mice were scanned by using a 2.5-MHz transducer (610-ns pulses with 3.6-kHz repetition frequency and 61-Hz frame rate) after injection of Optison and Evans blue dye. Petechial hemorrhages (PHs) in intestine and abdominal muscle were counted 15 min after exposure to characterize capillary rupture, and Evans blue extravasation was evaluated in samples of muscle tissue. For 5 ml small middle dotkg(-1) contrast agent and exposure to 10 alternating 10-s on and off periods, PH counts in muscle were approximately proportional to the square of peak negative pressure amplitude and were statistically significant above 0.64 MPa. PH counts in intestine and Evans blue extravasation into muscle tissue were significant above 1. 0 MPa. The PH effect in muscle was proportional to contrast dose and was statistically significant for the lowest dose of 0.05 ml small middle dotkg(-1). The effects decreased nearly to sham levels if the exposure was delayed 5 min. The PH effect in abdominal muscle was significant and statistically indistinguishable for uninterrupted 100-s exposure, 10-s exposure, 100 scans repeated at 1 Hz, and even for a single scan. The results confirms a previous report of PH induction by diagnostic ultrasound with contrast agent in mammalian skeletal muscle [Skyba, D. M., Price, R. J., Linka, A. Z., Skalak, T. C. & Kaul, S. (1998) Circulation 98, 290-293].

Sonoporation of monolayer cells by diagnostic ultrasound activation of contrast-agent gas bodies.

Human (A431 epidermoid carcinoma) cells were grown as monolayers on 5 microm thick Mylar sheets, which formed the upper window for a 1-mm thick, 23-mm diameter disc-shaped exposure chamber. A 3.5-MHz curved linear-array transducer was aimed upward at the chamber, 7 cm away, in a 37 degrees C water bath. The chamber contained phosphate-buffered saline (PBS) with 10 mg/mL fluorescent dextran and 1% Optison ultrasound (US) contrast agent. Significant fluorescent cell counts, indicative of membrane damage (i.e., sonoporation), up to about 10% of cells within a 1-mm diameter field of view, were noted for spectral Doppler and two-dimensional (2-D) scan mode with or without a tissue-mimicking phantom. The effect was only weakly dependent on pulse-repetition frequency or exposure duration, but was strongly dependent on contrast agent concentration below 2%. Thus, diagnostic US activation of contrast-agent gas bodies can produce cell membrane damage.

Mechanism of drug-induced lupus. I. Cloned Th2 cells modified with DNA methylation inhibitors in vitro cause autoimmunity in vivo.

Treating activated CD4+ T cells with DNA methyltransferase inhibitors modifies gene expression and induces autoreactivity. Adoptive transfer of viable polyclonal autoreactive cells causes a lupus-like disease, most likely because of one or more effector functions expressed by the autoreactive cells. However, the number of potential effector mechanisms expressed by polyclonal cells is large. To more readily identify responsible mechanisms, we asked if autoimmunity can be induced by using the conalbumin-reactive, cloned Th2 cell line D10.G4.1, treated with 5-azacytidine (5-azaC) or procainamide (Pca). Treated, but not untreated, cells responded to syngeneic APCs without Ag, overexpressed LFA-1, spontaneously lysed syngeneic macrophages, and secreted relatively large amounts of IL-6, small amounts of IL-4, and no detectable IL-2 nor IFN-gamma. Adoptive transfer of treated, but not untreated, cells induced a severe immune complex glomerulonephritis, pulmonary alveolitis, central nervous system abnormalities including fibrinoid necrosis, karyorrhexis, and meningitis, and bile duct proliferation with periportal inflammatory cell infiltration resembling primary biliary cirrhosis. Anti-ssDNA, anti-dsDNA, and anti-histone Abs were also found. These experiments demonstrate that modification of this cloned T cell line with DNA methyltransferase inhibitors is sufficient to cause an autoimmune disease, with features of lupus as well as autoimmune liver disease. The results also raise the possibility that macrophage lysis, IL-6 secretion, and LFA-1 overexpression could contribute to the disease process. This system may be useful in testing the role of these and other pathologic mechanisms in the development of specific autoimmune lesions.

Anti-CD11a prevents deletion of self-reactive T cells in neonatal C57BR mice.

The process of thymic maturation permits development of T cells expressing receptors which recognize self-major histocompatibility complex (MHC) determinants, but deletes T cells recognizing self-MHC determinants with high affinity. This selection process is evolutionarily conserved, and presumably serves in part to prevent the release of autoreactive cells. However, the mechanisms involved in the selection process, and the molecules required are incompletely characterized. Lymphocyte function-associated antigen-1 (LFA-1) is an accessory molecule important in T-cell activation, is involved in thymocyte-epithelial cell binding, and contributes to the maturation of CD4-CD8- thymocytes to the CD4+CD8+ stage. In this report we have investigated whether LFA-1 also contributes to the thymic deletion of potentially self-reactive cells. Neonatal C57Br mice were injected with amounts of a monoclonal antibody to LFA-1 that saturated thymic binding sites, then splenocytes were examined for T cells expressing receptors normally deleted in the thymus. The results demonstrate that V beta 17a+ T cells, normally deleted in this strain, can be detected in the spleen following administration of anti-LFA-1, thus supporting the hypothesis that LFA-1 also contributes to negative selection. The potential significance of LFA-1 involvement in multiple thymic maturation events is discussed.

Treating activated CD4+ T cells with either of two distinct DNA methyltransferase inhibitors, 5-azacytidine or procainamide, is sufficient to cause a lupus-like disease in syngeneic mice.

Human antigen-specific CD4+ T cells become autoreactive after treatment with various DNA methylation inhibitors, including 5-azacytidine, procainamide, and hydralazine. This suggests a mechanism that could contribute to the development of some forms of autoimmunity. In this report we have asked whether T cells treated with DNA methylation inhibitors can induce autoimmunity. Murine CD4+ T cells were treated with 5-azacytidine or procainamide and were shown to respond to syngeneic antigen-presenting cells, similar to CD4+ human T cell clones treated with these drugs. Functional characterization demonstrated that cells treated with either drug spontaneously lysed syngeneic macrophages and secreted IL-4, IL-6, and IFN-gamma. Adoptive transfer of 5-azacytidine- or procainamide-treated cells into unirradiated syngeneic recipients induced an immune complex glomerulonephritis and IgG anti-DNA and antihistone antibodies. These experiments demonstrate that T cells treated with either of two distinct DNA methyltransferase inhibitors are sufficient to induce a lupus-like disease. It is possible that the lysis of macrophages, together with the release of cytokines promoting B cell differentiation, contributes to the autoantibody production and immune complex deposition. These results suggest that environmental agents that inhibit DNA methylation could interact with T cells in vivo to produce a lupus-like illness, a mechanism that could have relevance to drug-induced and idiopathic lupus.

Phenotypic and functional similarities between 5-azacytidine-treated T cells and a T cell subset in patients with active systemic lupus erythematosus.

OBJECTIVE: Antigen-specific CD4+ T cells treated with DNA methylation inhibitors become autoreactive, suggesting a novel mechanism for autoimmunity. To test whether this mechanism might be involved in systemic lupus erythematosus (SLE), phenotypic markers for the autoreactive cells were sought. METHODS: Cloned normal T cells were treated with the DNA methylation inhibitor 5-azacytidine (5-azaC) and studied for altered gene expression. T cells from patients with active SLE were then studied for a similar change in gene expression, and cells expressing the marker were tested for autoreactivity. RESULTS: 5-azaC-treated normal T cells had increased CD11a (leukocyte function-associated antigen 1 alpha) expression relative to other membrane molecules. A T cell subset with similar CD11a expression was found in patients with active SLE. This subset contained cells that spontaneously lysed autologous macrophages, with a specificity similar to that of 5-azaC-treated cells.

Effect of cyclosporin A, silica quartz dust, and protease inhibitors on virus-induced demyelination.

Treatment with cyclosporin A, from the time of virus infection, suppressed inflammation and demyelination in the spinal cord of SJL/J or ASW(H-2s) mice persistently infected with Theiler's murine encephalomyelitis virus. Demyelination was not decreased if treatment was given after inflammation was established. The decrease was independent of serum titers of immunoglobulin G to purified viral antigen but did correlate with decreased proliferation of T lymphocytes to virus and myelin antigens. Silica quartz dust, a direct toxin of macrophages, suppressed demyelination and inflammation if begun at time of virus infection. No therapeutic effect was seen with inhibitors of plasminogen activators or other neutral proteases found primarily in macrophages.