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Biomedical subjects

S T Holgate

Publications and source records attributed to S T Holgate.

At least 217 records · Page 12Linked to original sources
PubMed1997

Increased levels of IL-4 in CD8+ T cells in atopic asthma.

BACKGROUND: In view of reports that CD8+ T cells may produce T(H2)-type cytokines and our own finding that levels of intracellular IL-4 are higher in CD8+ than CD4+ T cells in healthy nonatopic subjects, we have hypothesized that the capacity of CD8+ T cells to produce IL-4 may be increased in atopic asthma, a disease characterized by high production of T(H2) cytokines. METHODS: Levels of IL-4 and interferon-gamma were measured by ELISA in cell lysates and in 20- and 48-hour cultures of concanavalin A-stimulated purified peripheral blood CD8+ T cells in seven patients with mild atopic asthma and seven healthy nonatopic subjects. RESULTS: Resting CD8+ T cells in patients with asthma contained significantly more IL-4 than those of healthy nonatopic subjects (median, 26 pg/10(6) cells; range, 17 to 84 pg/10(6) cells vs 16 pg/10(6) cells; 10 to 28 pg/10(6) cells), with no difference in intracellular interferon-gamma levels. In the healthy control subjects, but not in the patients with asthma, levels of intracellular IL-4 correlated negatively with levels of interferon-gamma in resting CD8+ T cells (r[s] = -0.9411, p = 0.005). Stimulation with concanavalin A produced a consistent and significant increase in secretion of interferon-gamma, but not IL-4, with no difference between the two groups of subjects. CONCLUSION: The results of this study suggest that CD8+ T cells from patients with asthma may be an important source of the T(H2)-type cytokine IL-4. This capacity appears to be acquired in vivo, possibly by conditioning by IL-4 produced in the inflamed airways.

Asthma↗
PubMed1997

A comparison of RT-PCR, in-situ hybridisation and in-situ RT-PCR for the detection of rhinovirus infection in paraffin sections.

We describe an in-situ RT-PCR method for the amplification of rhinovirus (RV) in fixed, paraffin-embedded HeLa cells employed as a model for human respiratory epithelium. HeLa cells were infected in-vitro with inocula of rhinovirus-16 ranging from 10(2) to 10(6) 50% tissue culture infective doses (TCID50), incubated for 18 h then fixed and processed into paraffin blocks. Sections of the cell preparation were subjected to standard RT-PCR, in-situ hybridisation (ISH) or in-situ RT-PCR using specific oligonucleotide primers or probes directed against the 5' non-coding region of RV RNA. RT-PCR was found to be capable of detecting RV16 RNA in one 8 microns-thick section of cells infected with the lowest virus titre. ISH using digoxigenin labelled oligonucleotide probes located RV16 signal in the majority of HeLa cells at the highest virus titre, but in few or no cells with the lowest virus titre. In contrast, in-situ RT-PCR detected RV16 in the majority of cells infected with this amount of RV16. There was a slight loss of morphology and fine localisation associated with the in-situ thermal cycling process. However, the sensitivity of in-situ RT-PCR is comparable to standard RT-PCR and greater than ISH for the detection of RV. In-situ RT-PCR has wide applications for sensitive localization of low copy viral and RNA sequences within cells to investigate the role of viruses in a variety of clinical conditions.

HeLa Cells↗
PubMed1997

Asthma: early predisposing factors.

The significance of factors affecting the development of asthma and atopy in children must be judged against their ability to contribute to the increase in childhood asthma. Although genetic factors are clearly important to the development of asthma and atopy, they can not explain the increased prevalence of these conditions.

Asthma↗
PubMed1997

Seasonal allergic conjunctivitis is accompanied by increased mast cell numbers in the absence of leucocyte infiltration.

BACKGROUND: Seasonal allergic conjunctivitis (SAC) is the most common allergic disease to affect the eye, occurring alone or in association with allergic rhinitis. Infiltration with mast cells and eosinophils is characteristic of the chronic forms of allergic conjunctivitis such as vernal and atopic keratoconjunctivitis, and these cell types also contribute significantly to allergic inflammation in the skin. Indirect evidence for a similar pattern of cellular events in SAC comes from studies which demonstrate raised eosinophil and neutrophil numbers in conjunctival scrapings and elevated levels of mast cell tryptase in tears following allergen challenge. OBJECTIVE: To directly characterize the inflammatory cell infiltrate in SAC and to determine its clinical relevance. METHODS: We employed specific immunohistochemical staining to count mast cells, eosinophils and neutrophils in the conjunctival epithelium and lamina propria of eight atopic patients with SAC in, and 12 SAC patients out of the hay fever season. Sixteen patients with no history of ocular allergy were used as control subjects. RESULTS: Mast cells were absent from normal epithelium. During the pollen season median mast cell numbers in the lamina propria were found to be increased by 61% in patients with SAC compared with normals (P=0.012). Eosinophils were found in the lamina propria in less than half of the symptomatic patients with SAC and in only three patients were eosinophils present in the epithelium. The neutrophil numbers in the lamina propria of patients with SAC tended to be higher than normals but these changes did not reach statistical significance. CONCLUSION: These data based on the direct assessment of conjunctival tissue provide evidence that symptoms occur in SAC in the absence of detectable recruitment of eosinophils or neutrophils. This suggests that this disorder is related to mast cell-mediated changes.

Adult↗
PubMed1997

Effects of high dose intravenous immunoglobulin in two severe corticosteroid insensitive asthmatic patients.

Preliminary observations of the clinical efficacy of intravenous immunoglobulin in two patients with severe corticosteroid insensitive asthma are reported. In both patients treatment with intravenous immunoglobulin resulted in clinical improvement and enabled a significant reduction in the dose of prednisolone. In one of the patients fibreoptic bronchoscopy with endobronchial biopsies was performed and peripheral blood was analysed by flow cytometry before and after treatment. Immunohistological analysis of the biopsy samples after treatment showed a decrease in the number of all cell types, especially CD3+ T cells, CD4+ T cells, and activated CD25+ T lymphocytes, which was associated with a reduction in peripheral blood T cell activation. Intravenous immunoglobulin may be a valid option for the treatment of corticosteroid insensitive asthma. To elucidate the role and mode of action of intravenous immunoglobulin further studies in larger groups of patients are needed.

Adolescent↗
PubMed1997

Activation of eosinophils with cytokines produced by lung mast cells.

Here we show that the supernatant from activated lung mast cells induced the release of eosinophil cationic protein (ECP) from eosinophils. Lung mast cells were purified using affinity magnetic selection with monoclonal antibody (mAb) YB5.B8 to achieve a final mast cell purity of 93-99%. Eosinophils were purified by immunomagnetic negative selection (>98.0% pure). The supernatant was obtained from lung mast cells activated for 24 h with 1 microg/ml anti-IgE and 50 ng/ml stem cell factor (SCF). Human eosinophils were incubated with various concentrations of the supernatants for 4 h and ECP released was measured by RIA. Using 4 different donors' supernatant from mast cells, each donor's supernatant caused a dose-dependent release of ECP from eosinophils. The dilutant of 1:2 (v/v) of the supernatant induced 657.5 +/- 55.6 ng/10(6) eosinophils of ECP which is statistically significant (p = 0.008, n = 4) compared with the culture medium alone. Anti-interleukin (IL-5 neutralizing mAb, 10 microg/ml, and anti-tumor necrosis factor-alpha (TNF alpha) neutralizing mAb, 10 microg/ml, significantly inhibited the supernatant-induced ECP release in 79.3 +/- 9.4 and 68.2 +/- 14.1% (mean +/- SEM, n = 6, p < 0.005), respectively. Anti-granulocyte/macrophage colony-stimulating factor (GM-CSF) neutralizing mAb, 50 microg/ml, caused 68.0 +/- 6.1% of inhibition (p = 0.002). The isotype negative control had no measurable inhibitory or stimulatory effect for the stimuli. We confirmed that mast cells produce IL-5, GM-CSF and TNF alpha in response to IgE-dependent stimulation by using RT-PCR, in situ hybridization, ELISA and immunocytochemistry. A million of lung mast cells generated 41.4 pg (7.0-273.6) (median with range) of TNF alpha, 252.6 pg (158.7-3,652) of GM-CSF and 735 pg (< 10-2,750) of IL-5 24 h after activation with SCF and anti-IgE. These findings indicate that the human mast cells may contribute to the chronicity of tissue inflammation.

Blood Proteins↗
PubMed1997

Role of nasal interleukin-8 in neutrophil recruitment and activation in children with virus-induced asthma.

Neutrophil infiltration is a major feature in the pathogenesis of the common cold, and respiratory viral infection is the major cause of asthma exacerbations. The factors regulating the neutrophil influx are unknown. Interleukin-8 (IL-8) is a potent neutrophil chemoattractant, which has been implicated in several inflammatory diseases. In this study, we investigated the presence of IL-8 chemokine in the nasal aspirates of asthmatic children (n = 12) in whom asthma was precipitated by proven viral infection. There were increased IL-8 levels in nasal aspirates from children during the virus-induced asthma exacerbations compared with samples from the same children when they had been asymptomatic for 2 wk (medians 863 and < 20 pg/ml, respectively, p < 0.01). Biological relevance was shown in that IL-8 levels correlate with increased nasal aspirate neutrophil myeloperoxidase levels and there was also a correlation between myeloperoxidase levels and upper respiratory symptom severity. Furthermore, we purified IL-8 from these samples, and demonstrated biological neutrophil chemotactic activity. These are the first in vivo data to suggest an important role for IL-8 in neutrophil influx in proven upper respiratory viral infection associated with asthma exacerbations. We suggest that IL-8 might provide a target for therapeutic intervention in virus-induced respiratory diseases.

Asthma↗
PubMed1997

Free and complexed interleukin-8 in blood and bronchial mucosa in asthma.

We have tested the hypothesis that the expression of interleukin-8 (IL-8) is increased in bronchial tissue and circulating leukocytes of atopic asthmatics, indicating a role for this chemokine in asthma. The concentration of IL-8 in its free form and complexed with IgG or IgA was measured by ELISA in bronchial tissue, serum, and lysates of freshly isolated peripheral blood mononuclear cells and granulocytes from subjects with mild or severe asthma and nonatopic nonasthmatic subjects. Serum ECP was measured by fluorescent enzyme immunoassay. Free IL-8 was detected in the sera (n = 44) and bronchial tissue (n = 9) of all subjects with severe atopic asthma, but it was undetectable in normal subjects and subjects with mild atopic asthma, suggesting that free IL-8 is a marker of severe asthma. A positive correlation between free IL-8 and serum ECP levels found in severe disease suggests that IL-8 is associated with eosinophil activation. Complexes of IL-8 with IgA and IgG were detected in all serum and tissue samples. However, the levels of the IL-8-IgA complex were increased in the bronchial mucosa in asthma, and in blood were related to disease activity. Together, these results point to upregulation of IL-8 production in asthma and the induction of IL-8 binding immunoglobulins of the IgA class in the inflamed mucosa. We suggest a proinflammatory role for these complexes in lung tissue.

Asthma↗
PubMed1997

Transforming growth factor-beta 1 in asthma. Measurement in bronchoalveolar lavage fluid.

Airway wall remodeling is an established pathological feature in asthma. Its causes are not well understood, but one mediator of potential relevance is transforming growth factor-beta 1 (TGF-beta 1). We have measured levels of immunoreactive TGF-beta 1 in bronchoalveolar lavage (BAL) fluid from clinically stable atopic asthmatics and healthy control subjects. We have also examined the influence of allergen exposure on TGF-beta 1 release in the airways using a segmental bronchoprovocation model, with BAL performed at two time points following endobronchial allergen and sham saline challenges. Basal concentrations of TGF-beta 1 were significantly higher in asthmatics than control subjects (median 8.0 versus 5.5 pg/ml, p = 0.027). Following segmental bronchoprovocation, concentrations of TGF-beta 1 at the allergen- and saline-challenged sites were not significantly different after 10 min, (31.3 versus 25.0 pg/ml, p = 0.78), but after 24 h there were significantly higher TGF-beta 1 concentrations at the allergen-challenged sites (46.0 versus 21.5 pg/ml, p = 0.017). We conclude that basal TGF-beta 1 levels in the airways are elevated in atopic asthma and that these levels increase further in response to allergen exposure. These findings are consistent with the hypothesis that TGF-beta 1 is implicated in airway wall remodeling in asthma.

Adult↗
PubMed1997

The inflammatory effects of 2 ppm NO2 on the airways of healthy subjects.

Nitrogen dioxide (NO2) is a free radical and a common oxidant in polluted air. Here we present data on the time course of inflammation after NO2 exposure, as reflected in bronchial biopsy and airway lavage specimens. Healthy, nonsmoking subjects were exposed to air or 2 ppm NO2 for 4 h in random order on separate occasions. Endobronchial biopsies, bronchial washing (BW), and bronchoalveolar lavage (BAL) were done at 1.5 h (n = 15) or 6 h (n = 15) after exposure. In BW, exposure to NO2 induced a 1.5-fold increase in interleukin-8 (IL-8) (p < 0.05) at 1.5 h and a 2.5-fold increase in neutrophils (p < 0.01) at 6 h. In BAL fluid (BALF), small increases were observed in CD45RO+ lymphocytes, B-cells, and natural killer (NK) cells only. Immunohistologic examination of bronchial biopsy specimens showed no signs of upregulation of adhesion molecules, and failed to reveal any significant changes in inflammatory cells at either time point after NO2 exposure. In summary, NO2 induced a neutrophilic inflammation in the airways that was detectable in BW at 6 h after NO2 exposure. The increase in neutrophils could be related to the enhanced IL-8 secretion observed at 1.5 h after exposure. The absence of adhesion-molecule upregulation or cellular inflammation in mucosal biopsy specimens indicates that the major site of inflammation following exposure to NO2 may be in the smaller airways and not in the alveoli.

Adult↗
PubMed1997

The candidate region approach to the genetics of asthma and allergy.

To date, the strongest linkage claims for genes underlying asthma and atopy have been for chromosomes 5 and 11. Chromosome 5q contains the cytokine cluster and the beta2 adrenoceptor, and the beta chain of the high-affinity IgE receptor (FCepsilonRI-beta) is encoded on chromosome 11q13. We have attempted to replicate these findings in two distinct sample populations from the United Kingdom. Allelic associations were identified in both regions, but there was no significant evidence for linkage. Although we could not substantiate the existence of the nucleotide changes reported within exon 6 of the FCepsilonRI-beta gene, an amino acid substitution in exon 7 was strongly linked to asthma and atopy. We have also identified positive linkage and allelic associations to several markers on chromosome 12q in both our UK populations. Independent evidence from another study also supports linkage to 12q, so although our data could not confirm linkage to chromosomes 5 or 11, we have identified an additional region of the genome that could be important for the genetic predisposition to asthma and atopy.

Asthma↗
PubMed1997

Release of RANTES, MIP-1 alpha, and MCP-1 into asthmatic airways following endobronchial allergen challenge.

We have investigated the presence of regulated on activation, normal T-cell expressed and probably secreted (RANTES), macrophage inflammatory peptide-1 alpha (MIP-1 alpha), and macrophage chemotactic peptide (MCP-1) in the bronchoalveolar lavage fluid (BALF) obtained from normal (n = 7) and stable asthmatic subjects (n = 8), and studied their kinetic release into asthmatic airways following endobronchial allergen challenge (n = 18). Measurements of RANTES, MIP-1 alpha, and MCP-1 in 10 times (10x) concentrated BALF showed that these three chemokines were present in both normal controls and stable asthmatic patients, but no significant difference between the two groups was found in the levels of the three chemokines. However, at 4 h after allergen challenge, BALF levels of RANTES, MIP-1 alpha, and MCP-1 were significantly increased in fluid obtained from the allergen-challenge site when compared with the saline-challenge control site (median: 175 pg/ml versus 11.5 pg/ml, 258 pg/ml versus 88 pg/ml, and 900 pg/ml versus 450 pg/ml, respectively). At 24 h, levels of the three chemokines returned to baseline values. To investigate whether cells in BALF obtained 4 h after allergen exposure release chemokines, they were cultured for 24 h. BALF cells from the allergen site released more RANTES and MCP-1 than those from the saline site, but released similar amounts of MIP-1 alpha. These findings suggest that RANTES, MIP-1 alpha, and MCP-1 may regulate cell trafficking in asthma in response to allergen exposure.

Adult↗
PubMed1997

Expression of CD44 isoforms is increased in the airway epithelium of asthmatic subjects.

Since shedding of columnar, but not basal, epithelial cells is common in asthma, cell adhesion molecules such as CD44, which are differentially expressed on these cell types, are likely to be important in this disease. In bronchial epithelium of asthmatic and nonasthmatic subjects, CD44 isoforms have been localized by light- and electron-microscopic immunocytochemistry. Immunoreactivity for total CD44 (mAb Hermes-3/mAb 25.32) and for isoforms containing CD44v9 (mAb 11.24), CD44v6 (mAb 11.9), and CD44v4 (mAb 11.10) have been compared. In nonasthmatic samples, CD44s and CD44v9 were seen on basal but not columnar epithelial cells. Weak CD44v6 immunoreactivity was found infrequently in the bronchus, whereas CD44v4 immunoreactivity was absent. This indicates the presence of a distinct population of basal cells that express CD44. No CD44 was detected in areas of close cell-cell or cell-matrix contact, thus precluding the involvement of CD44 in stable adhesion in these areas. CD44 immunoreactivity was locally increased in areas showing morphologic damage to the epithelium. In epithelium from asthmatic subjects, the mean level of CD44 immunoreactivity on basal-cell membranes was doubled (4.3 versus 2.0 gold particles/microns membrane) as compared with nonasthmatic subjects. Increased expression of CD44 in asthmatic subjects, suggests that it has a significant role in the pathobiology of this disease, whereas the restricted distribution of this increase supports an association with repair rather than with inflammatory processes.

Asthma↗
PubMed1997

The effect of dipyridamole and theophylline on hypercapnic ventilatory responses: the role of adenosine.

The purine nucleoside, adenosine, has been implicated as a neuromodulator in central respiratory depression during prolonged exposure to hypoxia. It may also be a mediator of hypoxic hyperpnoea, acting on the carotid bodies. As there may be adenosine-sensitive mechanisms of hypoxic respiratory control, we sought to determine whether adenosine might be involved as a respiratory modulator in another central but non-oxygen-related control mechanism, the ventilatory response to hyperoxic hypercapnia. Twelve normal subjects were studied following 3 days of oral treatment with placebo, dipyridamole (which potentiates adenosine effects by inhibiting cellular uptake), and theophylline (a specific adenosine antagonist of cell surface receptors). The drugs were given in a random order, double-blind fashion. Resting end-tidal carbon dioxide tension (PET,CO2) and the maximum rate of isometric inspiratory pressure change at the mouth ((dP/dt) max), an index of respiratory drive, were determined in all subjects on each treatment. Hyperoxic, hypercapnic ventilatory responses were determined in seven of these subjects using a rebreathing technique. For each hypercapnic response, minute ventilation (V1E) and (dP/dt) max were plotted against PET,CO2 breath-by-breath. Resting PET,CO2 breathing room air was lower with theophylline (5.47 (SD 0.21) kPa) than with placebo (5.74 (0.26) kPa) or dipyridamole (5.86 (0.34) kPa), with no significant drug differences in resting (dP/dt)max. However, neither the slope nor the PET, CO2 intercept of the relationship between ventilation or respiratory drive and PET, CO2 were altered by the study drugs under hyperoxic conditions. We conclude that endogenous adenosine-related mechanisms are unlikely to be involved in determining either the sensitivity or the threshold of the ventilatory response to carbon dioxide under hyperoxic conditions. However, in normoxia, a centrally-acting, tonic, adenosine-mediated, respiratory modulation is not ruled out.

Adenosine↗