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Biomedical subjects

Peter Bradding

Publications and source records attributed to Peter Bradding.

At least 19 recordsLinked to original sources
PubMed2007

Leukotriene B4 production in healthy subjects carrying variants of the arachidonate 5-lipoxygenase-activating protein gene associated with a risk of myocardial infarction.

Leukotrienes are implicated in the pathogenesis of coronary artery disease. Recently two haplotypes (HapA and HapB) in the gene encoding ALOX5AP (arachidonate 5-lipoxygenase-activating protein), the main regulator of 5-lipoxygenase, have been associated with a doubling of the risk of myocardial infarction. Studies have also shown that treatment with a leukotriene inhibitor reduces biomarkers of coronary risk in patients carrying HapA, raising the possibility of developing genotype-specific therapy. In the present study, we examined whether carriage of HapA or HapB is associated with increased LTB(4) (leukotriene B(4)) production in healthy subjects. Age- and gender-matched healthy HapA carriers (n=21), HapB carriers (n=20) and non-A/non-B carriers (n=18), with no reported history of cardiovascular disease, were recruited following DNA screening of 1268 subjects from a population-based study. Blood neutrophils were isolated, and LTB(4) production was measured in response to stimulation with 1 mumol/l of the calcium ionophore A23187. There was no difference in the mean level for LTB(4) production in the three groups (non-A/non-B, 24.9+/-8.3 ng/10(6) cells; HapA, 22.2+/-11.9 ng/10(6) cells; HapB, 19.8+/-4.8 ng/10(6); P=0.14). The findings indicate that if either the HapA or the HapB haplotype of ALOX5AP indeed increases cardiovascular risk, then the mechanism is not simply due to a systematically observable effect of the haplotype on LTB(4) production in response to stimulation. The results suggest that knowledge of a patient's haplotype may not provide useful information on the probable clinical response to ALOX5AP inhibitors.

5-Lipoxygenase-Activating Proteins↗
PubMed2007

The reclassification of asthma based on subphenotypes.

PURPOSE OF REVIEW: Despite asthma being widely regarded as a heterogeneous disease, a consensus for an accurate system of classification has not been agreed. Recent studies have suggested that the recognition of subphenotypes of asthma based on the pattern of airway inflammation may be particularly useful in increasing our understanding of the disease. The present review discusses the important literature in this field, placing current work in its historical context. RECENT FINDINGS: The use of noninvasive markers of airway inflammation has suggested the presence of four distinct phenotypes: eosinophilic, neutrophilic, mixed inflammatory and paucigranulocytic asthma. Recent studies suggest that these subgroups may differ in their aetiology, immunopathology and response to treatment. Several studies have focused on refractory asthma as a distinct phenotype with evidence of a more distal pattern of airway inflammation and of upregulation of the tumour necrosis factor-alpha axis. Finally, novel treatment approaches targeted at specific patterns of airway inflammation are emerging, making an appreciation of subphenotypes particularly relevant. SUMMARY: The present review will discuss limitations to current classification systems, identify key current studies based on identifying inflammatory subphenotypes and provide suggestions for a novel approach that may further improve our understanding in this area.

Animals↗
PubMed2006

Airway smooth muscle and mast cell-derived CC chemokine ligand 19 mediate airway smooth muscle migration in asthma.

RATIONALE: Airway smooth muscle (ASM) hyperplasia is a feature of asthma, and increases with disease severity. We hypothesized that this results from migration of ASM or progenitors in response to chemokines derived from ASM or mast cells within the ASM bundle. OBJECTIVES: To examine expression of the chemokine receptor, CC chemokine receptor (CCR) 7, in vivo by ASM in patients with asthma and healthy control subjects, and by primary cultures of ASM and fibroblasts; to define expression of its ligands, CC chemokine ligand (CCL) 19 and CCL21, in bronchial biopsies, and primary cultures of ASM and mast cells; and to investigate CCR7's role in ASM migration and repair. METHODS: ASM was isolated from bronchoscopy and resection tissue. Receptor and chemokine expression was examined by immunohistochemistry, immunofluorescence, flow cytometry, ELISA, and reverse transcriptase-polymerase chain reaction. CCR7 function was examined by intracellular calcium measurements, chemotaxis, wound healing assays, and measurement of cell proliferation. MEASUREMENTS AND MAIN RESULTS: ASM, myofibroblasts, and fibroblasts expressed CCR7. CCL19, but not CCL21, was highly expressed in bronchial biopsies by mast cells and vessels in asthma of all severities, ASM in severe disease, and ex vivo ASM and mast cells. ASM CCR7 activation by CCL19-mediated intracellular calcium elevation and concentration-dependent migration, but not proliferation. Importantly, mast cell and ASM-derived CCL19 mediated ASM migration and repair. CONCLUSIONS: The CCL19/CCR7 axis may play an important role in the development of ASM hyperplasia in asthma.

Asthma↗
PubMed2006

Cooperative molecular and cellular networks regulate Toll-like receptor-dependent inflammatory responses.

Viral and bacterial pathogens cause inflammation via Toll-like receptor (TLR) signaling. We have shown that effective responses to LPS may depend on cooperative interactions between TLR-expressing leukocytes and TLR-negative tissue cells. The aim of this work was to determine the roles of such networks in response to agonists of TLRs associated with antiviral and autoimmune responses. The TLR3 agonist poly(I:C) activated epithelial cells, primary endothelial cells, and two types of primary human smooth muscle cells (airway [ASMC] and vascular) directly, while the TLR7/8 agonist R848 required the presence of leukocytes to activate ASMC. In keeping with these data, ASMC expressed TLR3 but not TLR7 or TLR8. Activation of ASMC by poly(I:C) induced a specific cytokine repertoire characterized by induction of CXCL10 generation and the potential to recruit mast cells. We subsequently explored the ability of TLR agonists to cooperate in the induction of inflammation. Dual stimulation with LPS and poly(I:C) caused enhanced cytokine generation from epithelial and smooth muscle cells when in the presence of leukocytes. Thus, inflammatory responses to pathogens are regulated by networks in which patterns of TLR expression and colocalization of tissue cells and leukocytes are critical.

Cells, Cultured↗
PubMed2006

The role of the mast cell in the pathophysiology of asthma.

There is compelling evidence that human mast cells contribute to the pathophysiology of asthma. Mast cells, but not T cells or eosinophils, localize within the bronchial smooth muscle bundles in patients with asthma but not in normal subjects or those with eosinophilic bronchitis, a factor likely to be important in determining the asthmatic phenotype. The mechanism of mast cell recruitment by asthmatic airway smooth muscle involves the CXCL10/CXCR3 axis, and several mast cell mediators have profound effects on airway smooth muscle function. The autacoids are established as potent bronchoconstrictors, whereas the proteases tryptase and chymase are being demonstrated to have a range of actions consistent with key roles in inflammation, tissue remodeling, and bronchial hyperresponsiveness. IL-4 and IL-13, known mast cell products, also induce bronchial hyperresponsiveness in the mouse independent of the inflammatory response and enhance the magnitude of agonist-induced intracellular Ca2+ responses in cultured human airway smooth muscle. There are therefore many pathways by which the close approximation of mast cells with airway smooth muscle cells might lead to disordered airway smooth muscle function. Mast cells also infiltrate the airway mucous glands in subjects with asthma, showing features of degranulation, and a positive correlation with the degree of mucus obstructing the airway lumen, suggesting that mast cells play an important role in regulating mucous gland secretion. The development of potent and specific inhibitors of mast cell secretion, which remain active when administered long-term to asthmatic airways, should offer a novel approach to the treatment of asthma.

Animals↗
PubMed2006

Evidence of a role of tumor necrosis factor alpha in refractory asthma.

BACKGROUND: The development of tumor necrosis factor alpha (TNF-alpha) antagonists has made it feasible to investigate the role of this cytokine in refractory asthma. METHODS: We measured markers of TNF-alpha activity on peripheral-blood monocytes in 10 patients with refractory asthma, 10 patients with mild-to-moderate asthma, and 10 control subjects. We also investigated the effects of treatment with the soluble TNF-alpha receptor etanercept (25 mg twice weekly) in the patients with refractory asthma in a placebo-controlled, double-blind, crossover pilot study. RESULTS: As compared with patients with mild-to-moderate asthma and controls, patients with refractory asthma had increased expression of membrane-bound TNF-alpha, TNF-alpha receptor 1, and TNF-alpha-converting enzyme by peripheral-blood monocytes. In the clinical trial, as compared with placebo, 10 weeks of treatment with etanercept was associated with a significant increase in the concentration of methacholine required to provoke a 20 percent decrease in the forced expiratory volume in one second (FEV1) (mean difference in doubling concentration changes between etanercept and placebo, 3.5; 95 percent confidence interval, 0.07 to 7.0; P=0.05), an improvement in the asthma-related quality-of-life score (by 0.85 point; 95 percent confidence interval, 0.16 to 1.54 on a 7-point scale; P=0.02), and a 0.32-liter increase in post-bronchodilator FEV1 (95 percent confidence interval, 0.08 to 0.55; P=0.01). CONCLUSIONS: Patients with refractory asthma have evidence of up-regulation of the TNF-alpha axis. (ClinicalTrials.gov number, NCT00276029.).

ADAM Proteins↗
PubMed2006

Human lung mast cells adhere to human airway smooth muscle, in part, via tumor suppressor in lung cancer-1.

Mast cells infiltrate the airway smooth muscle (ASM) of patients with asthma, an event which is likely to be a key factor in the development of this disease. Adhesion is a fundamental mechanism facilitating cellular cross-talk. We have examined whether human lung mast cells (HLMC) and ASM adhere, and have also examined the mechanism involved. Primary cultures of HLMC and confluent human ASM were cocultured for 30 min, then nonadherent HLMC were removed by centrifugation. HLMC adhered avidly to ASM monolayers (mean +/- SEM adhesion 43.2 +/- 1.2%, n = 41). Adhesion was increased to 58.8 +/- 2.7% by 1 mM Mn2+ (p = 0.015), and was reduced by EDTA and EGTA to 20.5 +/- 1.5% and 21.0 +/- 1.3%, respectively (p < 0.0001). Adhesion-blocking Abs for ICAM-1, VCAM-1, CD18, and the alpha4 and beta1 integrins had no effect on HLMC adhesion. HLMC expressed tumor suppressor in lung cancer-1 (TSLC-1) and blocking this reduced adhesion from 38.5 +/- 4.8% to 28.3 +/- 3.7% (p = 0.004, n = 7). ASM did not express TSLC-1, indicating that TSLC-1 acts as a heterophilic adhesion molecule. In summary, HLMC adhere avidly to ASM in part via TSLC-1 and in part via an as-yet-undefined Ca2+-dependent pathway. This supports the hypothesis that adhesion is important in the recruitment and retention of HLMC by the ASM in asthma, and for the functional interaction of these cells.

Asthma↗
PubMed2006

Chemokine concentrations and mast cell chemotactic activity in BAL fluid in patients with eosinophilic bronchitis and asthma, and in normal control subjects.

BACKGROUND: Asthma and eosinophilic bronchitis share many immunopathologic features including increased numbers of eosinophils and mast cells in the superficial airway. The mast cell chemotactic activity of airway secretions has not been assessed in patients with eosinophilic bronchitis. OBJECTIVES: To investigate the concentration of chemokines in bronchial wash samples and BAL fluid, and the mast cell chemotactic activity in BAL fluid from subjects with asthma and eosinophilic bronchitis, and from healthy control subjects. METHODS: We measured the concentrations of CCL11, CXCL8, and CXCL10 in bronchial wash samples and BAL fluid from 14 subjects with eosinophilic bronchitis, 14 subjects with asthma, and 15 healthy control subjects. Mast cell chemotaxis to BAL fluid from these subjects was examined using the human mast cell line HMC-1. RESULTS: The bronchial wash sample and BAL fluid concentrations of CXCL10 and CXCL8 was increased in subjects with eosinophilic bronchitis compared to those in subjects with asthma and healthy control subjects (p < 0.05). The CCL11 concentration was below the limit of detection in most subjects. BAL fluid from subjects with eosinophilic bronchitis was chemotactic for mast cells (1.4-fold migration compared to a control, 95% confidence interval, 1.1 to 1.9; p = 0.04) and was inhibited by blocking CXCR1 (45% inhibition; p = 0.002), CXCR3 (38% inhibition; p = 0.034), or both (65% inhibition; p = 0.01). BAL fluid from the subjects with asthma and healthy control subjects was not chemotactic for mast cells. Mast cell migration to BAL fluid was correlated with the concentration of CXCL8 (r = 0.42; p = 0.031) and CXCL10 (r = 0.52; p = 0.007). CONCLUSION: In subjects with eosinophilic bronchitis, CXCL8 and CXCL10 concentrations were elevated in airway secretions. These chemokines may play a key role in mast cell recruitment to the superficial airway in this condition.

Adult↗
PubMed2005

Macrophage and mast-cell invasion of tumor cell islets confers a marked survival advantage in non-small-cell lung cancer.

PURPOSE: The role played by the innate immune system in determining survival from non-small-cell lung cancer (NSCLC) is unclear. The aim of this study was to investigate the prognostic significance of macrophage and mast-cell infiltration in NSCLC. METHODS: We used immunohistochemistry to identify tryptase+ mast cells and CD68+ macrophages in the tumor stroma and tumor islets in 175 patients with surgically resected NSCLC. RESULTS: 5-year survival was 52.9% in patients with an islet macrophage density greater than the median versus 7.7% when less than the median (P < .0001). In the same groups, respectively, median survival was 2,244 versus 334 days (P < .0001). Patients with a high islet macrophage density but incomplete resection survived markedly longer than patients with a low islet macrophage density but complete resection. CONCLUSION: The tumor islet CD68+ macrophage density is a powerful independent predictor of survival from surgically resected NSCLC. The biologic explanation for this and its implications for the use of adjunctive treatment requires further study.

Adult↗
PubMed2005

Detection of an activating c-kit mutation by real-time PCR in patients with anaphylaxis.

A well-characterised gain-of-function point mutation within exon 17 of the c-kit proto-oncogene known as Asp816Val is present in patients with mastocytosis. Activation of mast cells through this receptor primes them for IgE-dependent activation, and patients with mastocytosis are at increased risk of anaphylaxis. We hypothesised that the Asp816Val mutation is associated with a history of anaphylaxis in the general population. A mismatch amplification real-time PCR assay was developed and validated to test for the Asp816Val mutation. Subjects were recruited to four subject groups: normal non-atopics, atopics without anaphylaxis, food-induced anaphylactics and non-food anaphylactics. Blood samples collected from forty subjects were tested for the presence of Asp816Val. Thirteen subjects were found to carry the mutation; normals (2/9), atopics (2/10), food anaphylactics (5/11) and non-food anaphylactics (4/10). Statistical analysis of the data determined that there was no significant difference between the numbers of subjects found to carry the Asp816Val mutation in each of the groups although a trend towards an increased occurrence in anaphylactics was observed. In summary, the hypothesis that the presence of the Asp816Val mutation is linked to the occurrence of anaphylaxis was not supported, but interestingly, we have shown for the first time Asp816Val may occur more frequently than previously reported within the general population.

Anaphylaxis↗
PubMed2005

Beta2-adrenoceptor regulation of the K+ channel iKCa1 in human mast cells.

Human mast cells express the intermediate conductance Ca2+-activated K+ channel iKCa1, which opens following IgE-dependent activation. This results in cell membrane hyperpolarization and potentiation of both Ca2+ influx and degranulation. Mast cell activation is attenuated following exposure to beta2-adrenoceptor agonists such as salbutamol, an effect postulated to operate via intracellular cyclic AMP. In this study, we show that salbutamol closes iKCa1 in mast cells derived from human lung and peripheral blood. Salbutamol (1-10 microM) inhibited iKCa1 currents following activation with both anti-IgE and the iKCa1 opener 1-EBIO, and was reversed by removing salbutamol or by the addition of the selective beta2-adrenoceptor antagonist and inverse agonist ICI 118551. Interestingly, ICI 118551 consistently opened iKCa1 in quiescent cells, suggesting that constitutive beta2-receptor signaling suppresses channel activity. Manipulation of intracellular cAMP, Galphai, and Galphas demonstrates that the beta2-adrenergic effects are consistent with a membrane-delimited mechanism involving Galphas. This is the first demonstration that gating of the iKCa1 channel is regulated by a G protein-coupled receptor and provides a clearly defined mechanism for the mast cell "stabilizing" effect of beta2-agonists. Furthermore, the degree of constitutive beta2-receptor "tone" may control the threshold for human mast cell activation through the regulation of iKCa1.

Adrenergic beta-Agonists↗
PubMed2005

The CXCL10/CXCR3 axis mediates human lung mast cell migration to asthmatic airway smooth muscle.

Mast cell microlocalization within the airway smooth muscle bundle is an important determinant of the asthmatic phenotype. We hypothesized that mast cells migrate toward airway smooth muscle in response to smooth muscle-derived chemokines. In this study, we investigated (1) chemokine receptor expression by mast cells in the airway smooth muscle bundle in bronchial biopsies from subjects with asthma using immunohistology, (2) the concentration of chemokines in supernatants from stimulated ex vivo airway smooth muscle cells from subjects with and without asthma measured by enzyme-linked immunosorbent assay, and (3) mast cell migration toward these supernatants using chemotaxis assays. We found that CXCR3 was the most abundantly expressed chemokine receptor on human lung mast cells in the airway smooth muscle in asthma and was expressed by 100% of these mast cells compared with 47% of mast cells in the submucosa. Human lung mast cell migration was induced by airway smooth muscle cultures predominantly through activation of CXCR3. Most importantly, CXCL10 was expressed preferentially by asthmatic airway smooth muscle in bronchial biopsies and ex vivo cells compared with those from healthy control subjects. These results suggest that inhibition of the CXCL10/CXCR3 axis offers a novel target for the treatment of asthma.

Asthma↗
PubMed2005

Differential expression of CCR3 and CXCR3 by human lung and bone marrow-derived mast cells: implications for tissue mast cell migration.

The selective microlocalization of mast cells within specific airway structures, such as the airway smooth muscle and submucosal glands, in asthma is important in the pathophysiology of inflammatory lung disease. Chemokines are likely candidates mediating mast cell migration into these tissue compartments. In this study, we have defined the chemokine receptor profile of human lung mast cells (HLMC) compared with mast cells derived from human bone marrow (BM) and the human mast cell line HMC-1. CXC chemokine receptor 3 (CXCR3) was the most highly expressed chemokine receptor on ex vivo HLMC analyzed by flow cytometry, and CXCR3 expression by mast cells in the bronchial mucosa was confirmed by immuno-histochemistry. CXCR3 was functional, inducing a rise in cytosolic-free Ca2+, actin reorganization, and chemotaxis in response to the CXC ligands CXCL9, -10, and -11. CXCR3 activation did not induce degranulation or cytokine synthesis. In addition, more than 10% of ex vivo HLMC expressed CC chemokine receptor 3, CXCR1, and CXCR4. It is interesting that CXCR3 was not expressed by human BM-derived mast cells, suggesting its expression is induced during tissue maturation. As CXCR3 ligands are elevated in many pulmonary diseases, CXCR3 may be important for determining the anatomical microlocalization of mast cells within the human lung.

Bone Marrow↗
PubMed2005

The re-emergence of the mast cell as a pivotal cell in asthma pathogenesis.

Asthma is characterized by variable airflow obstruction, airway hyperresponsiveness, and airway inflammation. Mast cells have long been thought to play a central role in asthma through their ability to release proinflammatory mediators, but this role has been questioned by the lack of efficacy of antihistamines and so-called mast cell-stabilizing drugs. Recent comparisons between the immunopathology of asthma and eosinophilic bronchitis have led to the re-emergence of the mast cell as a pivotal cell in asthma. Eosinophilic bronchitis is a condition in which patients present with chronic cough, and shares many of the inflammatory features associated with asthma, but without variable airflow obstruction or airway hyperresponsiveness. The only striking pathologic difference between these conditions is that, in asthma, the airway smooth muscle is infiltrated by mast cells. This suggests that interactions between mast cells and airway smooth muscle cells are critical for the development of the disordered airway physiology in asthma.

Anti-Inflammatory Agents↗
PubMed2005

Mast cell ion channels.

IgE-dependent activation of human mast cells is a central feature of allergy. However, alternative non-IgE-mediated signals derived from the complex inflammatory milieu are also important in driving chronic mast cell activation in many tissues. Irrespective of the point from where pathological secretion is sustained, all mechanisms will change the activity of the final 'effector' ion channels involved in normal stimulus-secretion coupling. IgE-dependent activation of human mast cells is characterised by an influx of extracellular Ca2+, which is essential for subsequent release of both preformed (granule-derived) mediators and newly generated autacoids and cytokines. In addition, flow of ions such as K+ and Cl- are likely to play an important role in mast cell activation through their effect on cell membrane potential and thus Ca2+ influx. Emerging evidence suggests that mast cells express a number of channels carrying K+, Cl- and Ca2+. With current techniques it will be possible to identify the molecular origin of these channels and define precisely their role in mast cell function. Mast cell ion channels offer a novel target for the attenuation of allergic disease.

Animals↗
PubMed2004

The K+ channel iKCA1 potentiates Ca2+ influx and degranulation in human lung mast cells.

BACKGROUND: Human lung and blood-derived mast cells express a Ca2+-activated K+ channel (KCA) that has electrophysiological properties resembling the intermediate conductance KCA (iKCA1). This channel is predicted to enhance IgE-dependent mast cell responses. OBJECTIVE: To confirm the identity of this channel as iKCA1 in human lung mast cells and to examine the effect of an iKCA1 opener, 1-ethyl-2-benzimidazolinone (1-EBIO), on Ca2+ influx and degranulation after IgE-dependent activation. METHODS: iKCA1 expression was examined by using RT-PCR. Ion currents were measured by using the patch clamp technique in human peripheral blood-derived mast cells, freshly isolated human lung mast cells (HLMCs), and long-term cultured HLMCs (LTHLMCs). Currents were manipulated with the specific iKCA1 opener 1-EBIO and the iKCA1 blockers clotrimazole and TRAM-34. Ratiometric Ca2+ imaging was performed on single fura-2-loaded cells, and histamine release was measured by radioenzymatic assay. RESULTS: Both fresh HLMCs and LTHLMCs expressed iKCA1 mRNA. The iKCA1 opener 1-EBIO induced iKCA1 currents in 89% of human peripheral blood-derived mast cells, 12% of fresh HLMCs, and 67% of LTHLMCs, which were blocked by the iKCA1 blockers clotrimazole and TRAM-34. After cell activation with a suboptimal concentration of anti-IgE, 1-EBIO enhanced the IgE-dependent rise in cytosolic-free Ca2+ and potentiated IgE-dependent histamine release. CONCLUSION: Opening of iKCA1 enhances IgE-dependent Ca2+ influx and histamine release in HLMCs. Inhibition of iKCA1 may provide a novel approach to the treatment of mast cell-mediated disease.

Benzimidazoles↗
PubMed2004

Sputum and bronchial submucosal IL-13 expression in asthma and eosinophilic bronchitis.

BACKGROUND: Nonasthmatic eosinophilic bronchitis is a condition characterized by the presence of eosinophilic airway inflammation in the absence of airflow obstruction or airway hyperresponsiveness. In asthma, the T H 2-type cytokine IL-13 has been implicated in the development of airway inflammation and hyperresponsiveness. Whether the expression of IL-13 is different between these 2 conditions is unknown. OBJECTIVE: We sought to investigate whether IL-13 expression is increased in asthma compared with eosinophilic bronchitis. METHODS: Sputum samples from subjects with mild asthma (n = 30) and eosinophilic bronchitis (n = 15) and normal controls (n = 16) were dialyzed, and IL-13 concentration was measured by ELISA. In a subgroup of these patients, IL-13 protein expression in bronchial biopsies was assessed by immunohistochemistry. RESULTS: The concentration of sputum IL-13 was higher in patients with mild asthma than in normal controls ( P = .03) and in patients with eosinophilic bronchitis ( P = .03). The median (interquartile range) number of IL-13 + cells/mm 2 submucosa was significantly higher in asthma 4 (8) than eosinophilic bronchitis 1.7 (1.9) and normal controls 0.5 (1.1; P = .004). Eighty-three percent of the cells expressing IL-13 in the submucosa were eosinophils, and 8% were mast cells. The median (interquartile range) proportion of eosinophils that expressed IL-13 was higher in the subjects with asthma, 16 (10)%, than those with eosinophilic bronchitis, 7 (3)% ( P = .02). CONCLUSION: The increased expression of IL-13 in asthma compared with eosinophilic bronchitis supports the concept that IL-13 may play a critical role in the pathophysiology of asthma.

Adult↗