Suppressive effect of antioxidants on intercellular adhesion molecule-1 (ICAM-1) expression in human epidermal keratinocytes.

Intercellular adhesion molecule-1 (ICAM-1) is strongly expressed by human epidermal keratinocytes during the course of inflammatory skin diseases. To test the possibility that reactive oxygen species produced in the skin during an inflammatory response affect ICAM-1 expression, cultured human epidermal keratinocytes were treated with H2O2 at concentrations that did not damage the cells, and cell-surface ICAM-1 expression was analyzed. Expression of ICAM-1 was induced on keratinocytes by treatment with 300 microM H2O2 for 1 h. The antioxidant N-acetyl-L-cysteine strongly inhibited H2O2-induced ICAM-1 expression, whereas the antioxidants pyrrolidine dithiocarbamate and alpha-tocopherol were less inhibitory. N-acetyl-L-cysteine also suppressed keratinocyte surface expression of ICAM-1 induced by the cytokines interferon-gamma (IFN-gamma) or tumor necrosis factor-alpha (TNF-alpha), whereas pyrrolidine dithiocarbamate and alpha-tocopherol suppressed IFN-gamma-induced surface expression but not TNF-alpha-induced expression. We found that N-acetyl-L-cysteine treatment reduced ICAM-1 mRNA levels when keratinocytes were stimulated with either IFN-gamma or TNF-alpha; however, pyrrolidine dithiocarbamate and alpha-tocopherol had no effect on either IFN-gamma- or TNF-alpha-induced ICAM-1 mRNA levels. Our results indicate that reactive oxygen species may be involved in the skin inflammatory process by increasing epidermal ICAM-1 expression and that some antioxidants may be effective in suppressing the epidermal ICAM-1 expression induced by reactive oxygen species and cytokines in inflammatory skin diseases.

Disruption of Plasmodium falciparum-infected erythrocyte cytoadherence to human melanoma cells with inhibitors of glycoprotein processing.

Adherence of Plasmodium falciparum-infected erythrocytes (IE) to the venular endothelium in brain and other organs is characteristic of cerebral malaria, an often fatal complication in infected individuals. It has been shown that cytoadherence may be mediated through interaction of IE with glycoproteins on host target cell surfaces, including CD36 (GPIV), intercellular adhesion molecule-1 (ICAM-1), and thrombospondin. Inhibitors of glycoprotein synthesis and processing were tested for their abilities to decrease IE adherence to C32 human melanoma cells. The alpha-glucosidase inhibitor, castanospermine, was effective in disrupting cytoadherence in vitro when incubated with C32 cells (IC50 = 600-700 microM). Castanospermine-6-butyrate was even more effective than the parent compound (IC50 = 9 microM) in disrupting cytoadherence. The mannosidase inhibitors, swainsonine and deoxymannojirimycin, had no effect on cytoadherence at concentrations up to 2 mM. No effect on cytoadherence was observed when the glucosidase and mannosidase inhibitors were incubated with IE rather than the C32 cell cultures. The level of CD36 on the C32 cell surface was decreased as measured by fluorescence-activated cell sorting (FACS) analysis with the same inhibitors which inhibited cytoadherence. Cells labeled with fluorescein isothiocyanate (FITC) OKM5 monoclonal antibody, which recognizes CD36 and disrupts cytoadherence, showed decreased fluorescence when treated with tunicamycin and castanospermine-6-butyrate but not when treated with swainsonine or deoxymannojirimycin. ICAM-1 levels, as measured by surface labeling of C32 cells with FITC CD54 monoclonal antibody, were decreased in cells treated with tunicamycin. However, incubation of cells with castanospermine-6-butyrate or deoxymannojirimycin decreased cell surface ICAM-1 levels only slightly. These findings suggest that (1) in C32 cells, levels of cell surface CD36, and not ICAM-1, change proportionally to the level of cytoadherence; (2) drugs which can affect the carbohydrate moiety of cellular glycoproteins decrease cytoadherence of IE to C32 cells; and (3) protection against the development of cerebral malaria may be possible with inhibitors of glycoprotein biosynthesis.

Apoptotic DNA fragmentation in the rat cerebral cortex induced by permanent middle cerebral artery occlusion.

Recent investigations have demonstrated internucleosomal DNA fragmentation in ischemic neuronal tissue. This type of fragmentation is characteristic of programmed cell death or apoptosis and suggests that neuronal death in stroke may be more complex than simple necrotic death. The present experiments provide a detailed examination of the regional localization and time course for apoptotic DNA fragmentation in the cerebral cortex following focal cerebral ischemia. Spontaneously hypertensive rats were subjected to permanent right middle cerebral artery occlusion and the cerebral cortices were examined for evidence of DNA fragmentation using electrophoretic, flow cytometric, and histological approaches. An electrophoretic examination of cortical DNA at 24 h after the occlusion indicated that the majority of nucleosomal ladders were in the transition zone or penumbra and the core of the infarction, with no fragmentation apparent in the contralateral normal cortex. A flow cytometric analysis of DNA fragmentation in intact cells revealed a similar pattern, with increased fragmentation observed in ischemic cortex vs. the contralateral cortex. Saggital sections taken 1.5 mm lateral to midline were collected from animals at 1, 4, and 24 h after the infarction and DNA fragmentation was examined histologically by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL) staining. Quantitative analysis of these sections indicated that DNA fragmentation can be observed in the anterior and central area of the infarctions as soon as 1 h after the occlusion and that the extent and magnitude of the fragmentation increases at 4 and 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Radiosensitivity of polyamine-depleted HeLa cells and modulation by the aminothiol WR-1065.

The radiosensitivity of cultured HeLa cells was increased upon depletion of the natural cellular polyamines putrescine, spermidine and spermine through treatment of cultures with inhibitors of polyamine biosynthesis. This increased radiosensitivity was manifested as a decrease in the D0 and by the absence of a shoulder in the survival curves. However, our previous studies have shown that the initial yield of X-ray-induced DNA damage did not appear to be elevated in polyamine-depleted cells. In addition, polyamine-depleted cells exhibited markedly altered X-ray-induced changes in the distribution of cells in the phases of the cell cycle characterized by increased time of onset and lengthened duration of G2-phase delay. Addition of polyamines to cultures for short periods prior to irradiation restored normal radioresistance and reversed the anomalous features of the G2-phase delay profile. Polyamine supplementation experiments as well as studies in which combinations of inhibitors were employed to modulate specific polyamine levels suggest that spermidine may play a primary role in governing cellular radioresponsiveness. The radioprotective aminothiol WR-1065 protected normal and polyamine-depleted cells to a proportionately similar extent (protection factor of 2.4 and 2.8, respectively) but had no apparent ability to restore the shoulder or alter the G2-phase delay markedly in polyamine-depleted cells. The findings reported here extend our previous observations that polyamine depletion results in a compromised ability to respond to X irradiation and suggest that a defect in repair and/or the G2-phase delay response may be the determining factors.

Identification of a specific receptor for interleukin-1 in vascular smooth muscle cells: regulation by interleukin-1 and interleukin-6.

A fluorescently labeled ligand was utilized to establish the existence of an interleukin-1 (IL-1) receptor in vascular smooth muscle. The binding of the phycoerythrin-labeled IL-1 beta to the murine T cell line, EL-4, was examined as a positive control. The phycoerythrin-labeled IL-1 beta identified a specific IL-1 receptor in the EL-4 cells. Vascular smooth muscle cells were also positively stained by the fluorescent ligand. The binding of phycoerythrin-labeled IL-1 beta to these cells was saturable and reversed by 100-fold excess unlabeled IL-1 beta. Incubation of the vascular smooth muscle cells with IL-1 beta (25 ng/ml) or IL-6 (250 ng/ml) for 18 h increased and decreased, respectively, the percentage of cells positively stained by phycoerythrin-labeled IL-1 beta which suggests these cytokines regulate IL-1 receptor expression in these cells. These data indicate a specific receptor for IL-1 exists in vascular smooth muscle cells.

Factors influencing selected heterosexual male college students' condom use.

This study assessed selected heterosexual male college students' use of condoms, their reasons for using condoms, and their attitudes toward sexuality and condoms. Three hundred five male subjects completed a questionnaire that assessed class standing, marital status, reasons for using condoms, number of recent sexual partners, intention to use condoms, and attitudes toward sexuality and condoms. Although no relationship between attitudes toward sexuality and attitudes toward condoms was noted, a negative correlation (-.42) was found between attitude toward condoms and intention to use condoms within the next month if the subject were to have intercourse during that time. Recommendations for increasing condom use are presented.

PIP: The prevalence of condom use, the reasons condoms are selected, and attitudes toward condoms and sexuality were investigated in a sample of 305 heterosexual male students at Virginia Polytechnic Institute. 72% of respondents reported that they had used a condom at least once. Of the 219 students reporting a history of condom use, 27 (12%) did so primarily to prevent the spread of sexually transmitted diseases and 164 (75%) were mainly concerned with preventing pregnancy. The subjects' mean score on a attitudes toward condoms scale was 67.89 (the scale ranged from 21-105), suggesting that the overall attitude toward condoms was neither positive nor negative. The lowest score on the scale was for the statement, "condoms are pleasant to use," while the highest score was for the item, "I would have no objection if my partner suggested that we use a condom." On the other hand, the statement, "I would avoid using condoms if at all possible," received widespread acceptance. The only significant correlation between attitudinal items and condom use was that males who intended to use condoms within the next month were more likely to have a positive attitude toward condom use. 2 of the findings of this study have implications for contraceptive education programs among young men. 1st, there is a need for greater emphasis on the prophylactic function of condom use. 2nd, given the finding that most students were willing to use condoms if pressured by their female sexual partners, programs should be designed to encourage women students to suggest or even provide condoms to their male partners.

Swainsonine, an inhibitor of mannosidase II during glycoprotein processing, enhances concanavalin A-induced T cell proliferation and interleukin 2 receptor expression exclusively via the T cell receptor complex.

The T cell receptor (TCR) is a disulfide-linked heterodimer consisting of both complex and high-mannose types of N-linked oligosaccharides. The objective of the present investigation was to examine the effect of altered oligosaccharide structure on the expression and function of the TCR. Human mononuclear lymphocytes (MNL) were treated with castanospermine (CAST) or swainsonine (SW), inhibitors of glucosidase I or mannosidase II, respectively. Treatment with these inhibitors does not prevent glycosylation, but results in synthesis of glycoproteins with high-mannose or hybrid types of oligosaccharides. Treatment of MNL with CAST (1000-10 microM) or SW (100-1 microM) for up to 72 hr had no effect on cell surface expression of of the TCR. SW potentiated Con A-induced T cell proliferation without effecting anti-CD3 (OKT3) or alloantigen-induced proliferation. CAST had no effect on Con A, anti-CD3, or alloantigen-induced T cell proliferation. The T cell proliferative response to Con A in the presence of SW was completely eliminated in the presence of monoclonal anti-TCR antibodies. Monoclonal anti-CD2, -CD3, -CD4, -CD8, or isotypic control monoclonal antibodies had no effect on SW enhancement of T cell proliferation. SW treatment potentiated Con A-induced MNL expression of both the alpha and beta subunits of the IL 2R. This effect was also specifically blocked by anti-TCR monoclonal antibodies. These results demonstrate that selective changes in the glycosylation state of the TCR complex can alter mitogen recognition and subsequent cellular activation.

Methyl-acetylenicputrescine (MAP), an inhibitor of polyamine biosynthesis, reduces the frequency and cytolytic activity of alloantigen-induced LyT 2.2 positive lymphocytes in vivo.

The objective of the present investigation was to examine the effect of (2R,5R)-6-heptyne-2,5-diamine (methyl-acetylenicputrescine; MAP), an irreversible inhibitor of ODC, on the induction of alloreactivity in vivo. Treatment of mice with MAP (0.5-0.01% in drinking water) inhibited CTL induction in a dose-dependent manner with an IC50 of approximately 144 mg/kg/day. MAP treatment reduced the frequency of LyT2+ (cytolytic/suppressor) splenic lymphocytes by greater than 75%. In contrast, MAP did not alter the number of L3T4+ (helper/inducer) lymphocytes. MAP treatment reduced lymphocyte putrescine and spermidine levels by 61 and 40%, respectively. The inhibitory effect of MAP on CTL induction could be reversed by simultaneous administration of putrescine (500 mg/kg). These data indicate that the observed inhibitory effect of MAP on CTL induction is mediated through inhibition of polyamine biosynthesis. Furthermore, results of the present investigation suggest that inhibition of polyamine biosynthesis may provide a unique target for immunosuppression.

Human polymorphonuclear leukocyte interaction with cyclosporine A.

The effects of cyclosporin A (cyA) on human polymorphonuclear leukocyte function, including phagocytosis, its associated metabolic burst, bacterial killing, and chemotaxis, were evaluated. Both Pseudomonas aeruginosa and Staphylococcus aureus were used as test particles. Polymorphonuclear leukocytes incubated in 10 and 50 micrograms of cyA per ml behaved normally with respect to phagocytosis and hexose monophosphate shunt activity at both high (10:1) and low (2:1) S. aureus/leukocyte ratios. With a small bacterial inoculum, killing of S. aureus was slightly impaired at early times only in the presence of 50 micrograms of cyA per ml. Phagocytosis and killing of P. aeruginosa with both large and small bacterial inocula were unaffected by cyA. Chemotaxis was within normal limits under all conditions. In addition, polymorphonuclear leukocytes from four renal transplant recipients receiving both cyA and prednisone demonstrated normal metabolic bursts and bacterial killing with both small and large inocula of S. aureus.

Demographic and practice characteristics of chiropractors.

The purpose of this study was to determine the demographic and practice characteristics of chiropractors. A four-part survey questionnaire was developed and mailed to a national stratified (by state) random sample of chiropractors. The analysis of data revealed that most chiropractors hold an undergraduate degree, practice in an urban area, are licensed to practice in only one state, belong to their state's chiropractic association, and are active in continuing education. Other data analyzed related to income, sources of new patients and average fees charged.

Human aortic endothelial cells express the type I but not the type II receptor for interleukin-1 (IL-1).

Endothelial cells (EC) are very responsive to the proinflammatory cytokine interleukin-1 (IL-1). EC are induced by IL-1 to secrete chemotactic factors and to increase expression of cell surface adhesion molecules leading to increased leukocyte adhesion. Activated EC further contribute to the inflammatory response by secreting additional cytokines. IL-1 interacts with EC through high-affinity cell-surface receptors. However, the low number of receptors present on EC has made characterization difficult. Further, recent evidence has suggested diversity in the responses of EC from different regions of the vascular system. Interested in the effect of IL-1 on early atherosclerotic lesion formation, we have characterized the IL-1 receptors on human aortic endothelial cells (HAEC). Using a direct binding assay, we found that HAEC have 1,000-3,000 IL-1 receptors per cell and bind IL-1 alpha with a Kd of 3.5 x 10(-10) M. We found that a monoclonal antibody specific for the type I receptor completely blocks IL-1 alpha binding. The blocking antibody also completely inhibits the IL-1 induced increase in intracellular adhesion molecule 1 (ICAM-1) expression by HAEC. Using solution hybridization and ribonuclease protection with an antisense probe, a sensitive method for detection of low abundance mRNA species we found that HAEC as well as human umbilical vein EC (HUVEC) have significant levels of mRNA for the type I IL-1 receptor. To test whether HAEC might also contain transcripts for the type II IL-1 receptor, we compared levels of mRNAs by polymerase chain reaction (PCR) amplification of cDNAs reverse-transcribed from total RNA. We found only transcripts for the type I receptor and not the type II receptor in HAEC. Based on this data, we conclude that aortic endothelial cells respond to IL-1 through the type I receptor.