Search PubMed⌕ Search

Biomedical subjects

R N Poston

Publications and source records attributed to R N Poston.

At least 19 recordsLinked to original sources
PubMed2001

Cell biotinylation provides a sensitive and effective detection technique for cellular adhesion assays: comparison with existing methods.

A simple, sensitive, colorimetric labelling method was devised to quantify cell adhesion, based on labelling the cell plasma membrane with biotin. This method was applied in adhesion assays, which involved the adherence of biotin-labelled, PMA-stimulated, U937 cells. These cells resemble monocytes, and were bound onto fibronectin-coated wells and to an ECV304 cell monolayer. The adherent U937 cells were detected by the addition of a peroxidase-conjugated anti-biotin antibody and a soluble colorimetric substrate. This assay is convenient, fast and sensitive, and able to detect 320-1000 U937 cells under the conditions described. This study has used titration assays to compare the biotinylation method with the existing cell quantification approaches of 51Cr radiolabelling and antibody dependent ELISA. Chromium labelling was the most sensitive technique, but we found the biotinylation method to be more convenient than radioactive labelling and more sensitive than conventional ELISA. Biotinylated cells were also used very effectively in a Stamper-Woodruff adhesion assay with U937 cells binding to histological sections of atherosclerotic plaques. The selective detection of the bound cells permitted automated quantitation by image analysis. Whole cell biotinylation may have wider applications in biological research.

Antibodies, Monoclonal↗
PubMed2000

ICAM-1 deficiency reduces atherosclerotic lesions in double-knockout mice (ApoE(-/-)/ICAM-1(-/-)) fed a fat or a chow diet.

Intercellular adhesion molecule (ICAM)-1, a major adhesion molecule, plays a critical role in the homing of leukocytes to sites of atherosclerotic lesions. However, very little is known on the role of ICAM-1 in initiating and perpetuating vascular lesions in ApoE(-/-) mice fed a chow or a fat diet. This study has investigated the mean aortic lesions in mice (C57BL6 background) with a single-knockout (ApoE(-/-)) or double-knockout (DKO; ApoE(-/-), ICAM-1(-/-)) fed a chow or a fat diet over a period of 3, 6, 15, and 20 weeks. A 3-fold reduction in lesion size was observed at all time points in DKO mice fed a chow diet. However, in DKO mice fed a fat diet, a marked reduction in the aortic lesion was observed at 3 and 15 weeks, which did not reach a significant level at 6 and 20 weeks. This study shows in essence that DKO mice are protected from developing significant lesions for up to 6 weeks when fed a chow diet and from 3 to 6 weeks when fed a fat diet. After 6 weeks, the lesion size of the DKO mice follows that of the single-knockout mice when fed a chow diet and gets to the same level in mice fed a fat diet. Plasma cholesterol levels were not altered as a result of ICAM-1 deficiency. These studies show that ICAM-1 is implicated in the formation and progression of atherosclerotic lesions.

Animals↗
PubMed2000

Plasma concentrations of reputed tumor-associated soluble CD44 isoforms (v5 and v6) in smokers are dose related and decline on smoking cessation.

There is some evidence to suggest that smoking may affect circulating levels of CD44 (sCD44) molecules. Therefore, we investigated the effect of smoking on the circulating level of sCD44 by comparing the change in total sCD44, sCD44v5, and sCD44v6 concentrations over 1 year in a group of people who quit smoking (n = 30) and a control group of people who continued to smoke (n = 30). Smoking status and compliance were monitored by analysis of plasma cotinine and expired CO levels and also by self-reported tobacco use. We show a dose-dependent relationship between smoke intake and baseline plasma concentrations of reputed tumor-associated CD44 variant isoforms (sCD44v5 and sCD44v6) in smokers (n = 60). There was a significant decline in the level of both sCD44v5 and sCD44v6 in quitters as compared with continuing smokers [-13.2 (95% confidence interval, -7.6 to -18.8; P < 0.001) and -62.2 ng/ml (95% confidence interval, -33.9 to -90.6; P < 0.001), respectively], but not in the total sCD44 concentration. These results show that the increased concentrations of sCD44v5 and sCD44v6 in smokers are dose related and reversible and suggest that the attributed diagnostic specificity and prognostic value of sCD44 molecules in malignant and inflammatory disease may be affected by smoking status.

Adult↗
PubMed1999

Potential mechanisms of susceptibility to periodontitis in tobacco smokers.

Tobacco smoking is probably the most important, controllable environmental risk factor in periodontitis. It results in changes in the vascular, inflammatory, immune and healing responses. The degree of exposure to tobacco smoking can be measured in pack years or by measuring serum cotinine and nicotine levels. In a previous paper we reported elevated levels of serum soluble intercellular adhesion molecule-1 (sICAM-1) in smokers, regardless of periodontal status. Elevated sICAM-1 has been found to be a risk marker for cardiovascular disease. In the present paper we report the short-term effects of an episode of smoking on blood flow and levels of sICAM-1. Human volunteers included non-smokers, light smokers and heavy smokers. Relative blood flow was monitored in the gingivae and forehead skin using a laser Doppler flowmeter and serum levels of sICAM-1, cotinine and nicotine measured before during and up to 60 min following an episode of smoking. We could not provide evidence to support the theory that there is localized vasoconstriction within the gingival tissues. In contrast, there was a significant increase in blood flow in the forehead skin of light smokers which was not observed in non-smoking controls or in heavy smokers, suggesting a long-term tolerance in this latter group. The level of sICAM-1 remained unchanged during this episode, further suggesting a long-term effect. In a parallel group of subjects, we were able to demonstrate a direct significant correlation between sICAM and serum cotinine levels. These observations may be relevant to aetiological mechanisms in periodontitis and other smoking-associated diseases.

Cotinine↗
PubMed1997

Effect of inhaled glucocorticoids on IL-1 beta and IL-1 receptor antagonist (IL-1 ra) expression in asthmatic bronchial epithelium.

BACKGROUND: Accumulating evidence suggests that the cytokine network is central to the immunopathology of bronchial asthma and the existence of naturally occurring cytokine antagonists has added to this complexity. Upregulation of both interleukin 1 beta (IL-1 beta) and its naturally occurring receptor antagonist, interleukin 1 receptor antagonist (IL-1ra), has previously been observed on asthmatic bronchial epithelium compared with normal airways. METHODS: The effect of inhaled beclomethasone dipropionate (BDP) on asthmatic bronchial epithelial expression of IL-1 beta and IL-1ra was studied. Frozen bronchial biopsy specimens from nine asthmatic subjects receiving 1000 micrograms BDP daily for eight weeks and from six asthmatic subjects receiving matching placebo were stained with anti-IL-1 beta and anti-IL-1ra antibodies. Hue-saturation-intensity (HSI) colour image analysis was used to quantify the brown immunoperoxidase reaction colour present on the bronchial epithelium. RESULTS: There was a significant twofold decrease in the epithelial expression of IL-1 beta after treatment with BDP but no significant change was seen in IL-1ra (P = 0.175). CONCLUSION: The selective inhibition of IL-1 beta, without effect on IL-1ra, provides a novel mechanism for the anti-inflammatory action of glucocorticosteroids.

Adult↗
PubMed1996

The effects of prednisolone on the cutaneous tuberculin response in patients with corticosteroid-resistant bronchial asthma.

We have used the classical tuberculin cutaneous delayed hypersensitivity immune response to investigate the effects of corticosteroids on mononuclear cell function in nine subjects with corticosteroid-resistant asthma (CRA) and six subjects with corticosteroid-sensitive asthma (CSA), who demonstrated sensitivity to intradermal purified protein derivative (PPD) of Mycobacterium tuberculosis. In a double-blind, crossover, placebo-controlled study, patients were given oral prednisolone or placebo, starting on day 0, and a predetermined intradermal dose of PPD on day 7. On day 9 the site of the induration was measured and biopsied for immunohistochemical analysis. There was no difference in skin induration between the CSA and CRA groups during the placebo arm of the study (p = 0.38). Prednisolone significantly suppressed the cutaneous induration (p, 0.003) in the CSA group but not in the CRA group. As compared with placebo, the number of macrophages (p = 0.018), eosinophils (p = 0.009), and T memory cells (p = 0.009) was suppressed by prednisolone in the CSA group but not in the CRA group. No significant suppression of the number of neutrophils or monocytes/immature macrophages was induced by prednisolone in either group. There was no difference in intracellular adhesion molecule-1, vascular cell adhesion molecule-1, and endothelial leukocyte adhesion molecule-1 expression in blood vessels or epidermis between the CSA and CRA groups, with no suppression by prednisolone in either group. These findings suggest a defect in the responsiveness of cellular immune mechanisms to the suppressive effects of corticosteroids in steroid-resistant asthma. The differential suppressive effects of corticosteroids on cellular recruitment in the PPD response between the individuals with CSA and those with CRA are not due to modulation of expression of endothelial adhesion molecules.

Adrenal Cortex Hormones↗
PubMed1996

Expression of interleukin-1 beta (IL-1beta) and interleukin-1 receptor antagonist (IL-1ra) on asthmatic bronchial epithelium.

Accumulating evidence suggests that the cytokine network is central to the immunopathology of bronchial asthma and recent findings have suggested that naturally occurring cytokine antagonists may also be involved. In this study we looked at the expression of interleukin-1 beta (IL-1beta) and its naturally occurring receptor antagonist, IL-1ra, in the normal and asthmatic bronchial wall. Frozen bronchial biopsies from 12 normal and 18 asthmatic individuals were double stained with EBM11 (a CD68 macrophage marker) and either a rabbit anti-IL-1beta or a rabbit anti-IL-1ra. Hue-saturation-intensity color image analysis (HSI) was used to quantify the brown immunoperoxidase reaction product present on the bronchial epithelium. There was an increased expression of both IL-1beta and IL-1ra in the asthmatic bronchial epithelium, p < 0.0002 and p < 0.0001, respectively. Additionally, the numbers of macrophages, of IL-1beta producing cells, and the percentage of macrophages producing IL-1beta were significantly increased in the asthmatic submucosa (p < 0.004, p < 0.002, and p < 0.008, respectively).

Adult↗
PubMed1996

Localized adhesion of monocytes to human atherosclerotic plaques demonstrated in vitro: implications for atherogenesis.

Blood-derived macrophages in the arterial intima are a characteristic feature of active atherosclerotic plaques. Adherent monocytes on the luminal surface and increased adhesion molecules on the endothelium have suggested that specific molecular mechanisms are involved in monocyte/macrophage traffic into the arterial wall. Adhesion of human monocytes and related cell lines was therefore studied in vitro to histological sections of human plaques. At 37 degrees C, these cells bound selectively to the plaques. Binding to the endothelium occurred and was also present extensively in the diseased intima. Inhibition studies showed that the endothelial and general intimal binding had largely similar molecular properties. Strong inhibition was produced by antibodies to the monocyte-specific adhesion molecule CD14, to beta2 integrins, and to ICAM-1. Likewise, a peptide containing the Arg-Gly-Asp sequence was strongly inhibitory, suggesting that binding of leukocyte integrins to arterial extracellular matrix was synergistic with cell-cell interactions. A P-selectin antibody was exceptional in giving selective inhibition of endothelial adhesion, which correlates with the specific endothelial localization of this adhesion molecule. These results show that monocytes adhere to atherosclerotic plaques through the focal activation of multiple arterial wall adhesion molecules, confirming the adhesion hypothesis. A positive feedback theory for the pathogenesis of atherosclerosis can be suggested, based on the ability of macrophages in the wall to activate the endothelium, induce adhesion molecules, and facilitate additional monocyte entry. The adhesion assay provides a means for the identification of adhesion inhibitors with therapeutic potential.

Antibodies, Monoclonal↗
PubMed1994

Intramuscular gold decreases cytokine expression and macrophage numbers in the rheumatoid synovial membrane.

OBJECTIVES: Cytokines, released from mononuclear cells (MNC) are mediators of joint destruction in rheumatoid arthritis (RA). The mechanisms of action of gold salts used in the treatment of RA are unknown. The aim of this study was to investigate cytokine expression and intensity of MNC infiltrate in the RA synovial membrane (SM) following treatment with sodium aurothiomalate (SAT). METHODS: Sequential blind needle biopsies were obtained at entry into the study and at two and 12 weeks after the start of SAT therapy in 10 patients with active RA. SMs were stained with a panel of monoclonal antibodies to assess cytokine expression (IL-1 alpha, IL-1 beta, TNF-alpha, IL-6, and GM-CSF). RESULTS: There was a significant decrease in IL-1 alpha, IL-1 beta, IL-6 and TNF-alpha expression 12 weeks after treatment (p < 0.004, p < 0.002, p < 0.009 and p < 0.004 respectively). This was noted in the lining layer, the perivascular aggregates and the connective tissue areas. Detailed examination of the MNC infiltrate showed a significant reduction in inflammatory monocytes (MONO) in the lining layer at two weeks (p < 0.03). A decrease in the number of CD68+ macrophages (MAC) was noted in the perivascular and connective tissue areas at 12 weeks. No significant changes were observed in the number of T and B cells and blood vessels. CONCLUSION: The results suggest that gold may suppress RA disease activity by diminishing MONO and MAC numbers and consequently monokine production in the SM.

Adult↗
PubMed1994

Increased expression of the monocyte chemoattractant protein-1 in bronchial tissue from asthmatic subjects.

The expression of the monocyte chemoattractant protein (MCP-1), a member of the chemokine family of low molecular weight cytokines, was assessed by immunohistochemistry in bronchial biopsies from 12 asthmatic and 12 normal subjects. Both a monoclonal antibody (F9) and a polyclonal antibody were employed to detect MCP-1, while the mouse myeloma protein (MOPC21) was used as a negative control. Strong positive reactions for MCP-1 were seen in the bronchial epithelium. Subepithelial macrophages, blood vessels, and bronchial smooth muscle were also stained. Hue-saturation-intensity color image analysis was used to quantify reactions of the monoclonal antibody in the epithelial and subepithelial layers. With the monoclonal antibody, asthmatic biopsies showed 51.8 +/- 3.7% (mean +/- SEM) of the epithelium staining positively, whereas normal subjects reacted much less, with 6.4 +/- 1.9% of the epithelium staining (P < 0.0001); there was no overlap between the two groups. Likewise, staining was increased in the subepithelium of asthmatic airway biopsies, with 11.5 +/- 3.1% and 2.0 +/- 1.0% staining positively in asthmatic and normal subepithelium, respectively, (P < 0.002). There was a significant correlation between staining of the epithelium and subepithelium (r = 0.77, P < 0.001). The polyclonal anti-MCP-1 antibody also gave strong reactions in the epithelium and subepithelium, with 34.0 +/- 7.8% of the asthmatic and 1.6 +/- 1.0% of the normal bronchial epithelium staining positively (P < 0.0001). These increased levels of MCP-1 in the asthmatic airways suggest that they may play a role in macrophage recruitment and activation and thereby contribute to the inflammatory pathology of bronchial asthma.

Adult↗
PubMed1994

Increase in the adhesion molecule P-selectin in endothelium overlying atherosclerotic plaques. Coexpression with intercellular adhesion molecule-1.

P-selectin (GMP-140) is an adhesion molecule present within endothelial cells that is rapidly translocated to the cell membrane upon activation, where it mediates endothelial-leukocyte interactions. Immunohistochemical analysis of human atherosclerotic plaques has shown strong expression of P-selectin by the endothelium overlying active atherosclerotic plaques. P-selectin is not, however, detected in normal arterial endothelium or in endothelium overlying inactive fibrous plaques. Color image analysis was used to quantitate the degree of P-selectin expression in the endothelium and demonstrates a statistically significant increase in P-selectin expression by atherosclerotic endothelial cells. Double immunofluorescence shows that some of this P-selectin is expressed on the luminal surface of the endothelial cells. Previous work has demonstrated a significant up-regulation in the expression of the intercellular adhesion molecule-1 in atherosclerotic endothelium and a study on the expression of intercellular adhesion molecule-1 and P-selectin in atherosclerosis shows a highly positive correlation. These results suggest that the selective and cooperative expression of P-selectin and intercellular adhesion molecule-1 may be involved in the recruitment of monocytes into sites of atherosclerosis.

Arteriosclerosis↗
PubMed1993

Detection of GM-CSF in asthmatic bronchial epithelium and decrease by inhaled corticosteroids.

The presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) in airway epithelial cells in vivo was assessed in 15 asthmatic and 9 normal subjects. GM-CSF was analyzed using immunohistochemistry with a polyclonal and a monoclonal antibody. Hue saturation intensity color image analysis was used to quantify staining. Asthmatic airway epithelial cells stained significantly more with anti-GM-CSF than those from normal subjects (p = 0.0013 and p = 0.0003 for the polyclonal and monoclonal antibodies, respectively). Additionally, 8 asthmatic individuals inhaled 1,000 micrograms beclomethasone diproprionate per day for 8 wk and 6 asthmatic patients inhaled matching placebo. There was a significant reduction of GM-CSF in the epithelium in the patients who were given corticosteroids (p = 0.014), whereas the group of subjects who were given placebo showed no significant change in GM-CSF staining. There was a correlation between the percentage suppression of GM-CSF staining by inhaled corticosteroids and the percentage increase in FEV1 (r = 0.61, p < 0.05) and percentage decrease in carbachol responsiveness (r = 0.80, p < 0.01). These findings suggest that GM-CSF may play a role in the inflammatory processes of bronchial asthma and that the epithelial cell may be a target cell for drug action.

Administration, Inhalation↗
PubMed1993

The immunohistochemical heterogeneity of atheroma macrophages: comparison with lymphoid tissues suggests that recently blood-derived macrophages can be distinguished from longer-resident cells.

We studied the antigenic markers of macrophages (Mphs) in atherosclerotic human arteries by immunohistochemistry and compared them with the patterns in Mph subpopulations of tonsil and lymph node, which are also described. The staining of atheroma intimal Mphs was assessed semiquantitatively in the subendothelial, mid, and outer intima. Three patterns of reactivity with Mph antibodies were recognized. (a) Pan-Mph (antibodies HAM56, EBM11, and CD14 group). Staining was maximal in the mid-intimal zone. (b) Subendothelial Mphs (anti-muramidase, anti-alpha-1-antitrypsin and MAC387). In lymphoid tissue, sinusoidal Mphs and a few inflammatory Mphs were stained, as well as blood monocytes. This group of antibodies recognizes Mphs that are likely to be recently blood-derived (RBD-Mphs). (c) Antibodies reactive with various histiocyte populations in lymphoid tissues (anti-Factor XIII; anti-HLA Class II and LN2) also gave maximal staining in the mid-intimal zone, but differences between lesion types suggest that they are recognizing heterogeneous subpopulations of Mphs. These observations demonstrate the heterogeneity of tissue Mphs and suggest that an insight into the dynamics of tissue Mphs can be obtained from the cell phenotype. They indicate that all stages of atherosclerosis can have an outward traffic of Mphs from the blood through the arterial intima.

Antibodies, Monoclonal↗
PubMed1993

Expression of vascular cell adhesion molecule-1 in normal and inflamed synovium.

BACKGROUND: The intercellular adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) has been implicated in a number of interactions between leukocytes and cells of lymphoid and connective tissue, including endothelial cells. Such interactions within synovial tissue may be important in the pathogenesis of rheumatoid arthritis. EXPERIMENTAL DESIGN: The expression of VCAM-1 on specific cell populations in normal and inflamed synovium was investigated using a range of double-labeling techniques. RESULTS: The strongest VCAM-1 staining was found to be confined to four nonmacrophage populations (as judged in terms of CD68 expression, nonspecific esterase activity, content of prolyl hydroxylase and activity of uridine diphosphoglucose dehydrogenase): (i) type B synoviocytes, (ii) vascular wall cells outside the endothelial layer, (iii) scattered stromal cells with cytoplasmic processes, and (iv) cells resembling follicular dendritic reticulum cells in lymphoid aggregates with germinal centers (in the three samples of rheumatoid arthritic tissue where these were present). Some macrophages of the synovial intima showed weak VCAM-1 staining. Endothelial cell staining was seen but it was consistently weaker than the staining of cells of group ii. CONCLUSIONS: The four cell populations described as showing bright staining for VCAM-1 may all be involved in local interactions with leukocytes of the macrophage or lymphoid series subsequent to initial leukocyte entry into the tissue. Expression of VCAM-1 by these cells may play a role in such interactions.

Arthritis, Rheumatoid↗