The safety of immunotherapy.
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Desensitization, Immunologic↗
Biomedical subjects
M T Krishna
Publications and source records attributed to M T Krishna.
Diagnostic role of tests for T cell receptor (TCR) genes.
Rapid advances in molecular biological techniques have made it possible to study disease pathogenesis at a genomic level. T cell receptor (TCR) gene rearrangement is an important event in T cell ontogeny that enables T cells to recognise antigens specifically, and any dysregulation in this complex yet highly regulated process may result in disease. Using techniques such as Southern blot hybridisation, polymerase chain reaction, and flow cytometry it has been possible to characterise T cell proliferations in malignancy and in diseases where T cells have been implicated in the pathogenesis. The main aim of this article is to discuss briefly the process of TCR gene rearrangement and highlight the disorders in which expansions or clonal proliferations of T cells have been recognised. It will also describe various methods that are currently used to study T cell populations in body fluids and tissue, their diagnostic role, and current limitations of the methodology.
Blotting, Southern↗
Repeated daily exposure to 2 ppm nitrogen dioxide upregulates the expression of IL-5, IL-10, IL-13, and ICAM-1 in the bronchial epithelium of healthy human airways.
BACKGROUND: Repeated daily exposure of healthy human subjects to NO2 induces an acute airway inflammatory response characterised by neutrophil influx in the bronchial mucosa AIMS: To assess the expression of NF-kappaB, cytokines, and ICAM-1 in the bronchial epithelium. METHODS: Twelve healthy, young non-smoking volunteers were exposed to 2 ppm of NO2/filtered air (four hours/day) for four successive days on separate occasions. Fibreoptic bronchoscopy was performed one hour after air and final NO2 exposures. Bronchial biopsy specimens were immunostained for NF-kappaB, TNF-alpha, eotaxin, Gro-alpha, GM-CSF, IL-5, -6, -8, -10, -13, and ICAM-1 and their expression was quantified using computerised image analysis. RESULTS: Expression of IL-5, IL-10, IL-13, and ICAM-1 increased following NO2 exposure. CONCLUSION: Upregulation of the Th2 cytokines suggests that repeated exposure to NO2 has the potential to exert a "pro-allergic" effect on the bronchial epithelium. Upregulation of ICAM-1 highlights an underlying mechanism for leucocyte influx, and could also explain the predisposition to respiratory tract viral infections following NO2 exposure since ICAM-1 is a major receptor for rhino and respiratory syncytial viruses.
Adult↗
Effect of ozone on bronchial mucosal inflammation in asthmatic and healthy subjects.
Epidemiological studies suggestthat asthmatics are more affected by ozone than healthy people. This study tested three hypotheses (1) that short-term exposure to ozone induces inflammatory cell increases and up-regulation of vascular adhesion molecules in airway lavages and bronchial tissue 6 h after ozone exposure in healthy subjects; (2) these responses are exaggerated in subjects with mild allergic asthma; (3) ozone exacerbates pre-existent allergic airways inflammation. We exposed 15 mild asthmatic and 15 healthy subjects to 0.2 ppm of ozone or filtered air for 2 h on two separate occasions. Airway lavages and bronchial biopsies were obtained 6 h post-challenge. We found that ozone induced similar increases in bronchial wash neutrophils in both groups, although the neutrophil increase in the asthmatic group was on top of an elevated baseline. In healthy subjects, ozone exposure increased the expression of the vascular endothelial adhesion molecules P-selectin and ICAM- 1, as well as increasing tissue neutrophil and mast cell numbers. The asthmatics showed allergic airways inflammation at baseline but ozone did not aggravate this at the investigated time point. At 6 h post-ozone-exposure, we found no evidence that mild asthmatics were more responsive than healthy to ozone in terms of exaggerated neutrophil recruitment or exacerbation of pre-existing allergic inflammation. Further work is needed to assess the possibility of a difference in time kinetics between healthy and asthmatic subjects in their response to ozone.
Adult↗
What is the association between air pollution and allergic diseases?
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Air Pollutants↗
Hymenoptera stings and serum tryptase.
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Anaphylaxis↗
Inhibition of mast cell tryptase by inhaled APC 366 attenuates allergen-induced late-phase airway obstruction in asthma.
BACKGROUND: APC 366, a selective inhibitor of mast cell tryptase, has been shown to inhibit antigen-induced early asthmatic response (EAR), late asthmatic response (LAR), and bronchial hyperresponsiveness (BHR) in a sheep model of allergic asthma. OBJECTIVE: The purpose of this study was to investigate the effects of APC 366 on antigen-induced EAR, LAR, and BHR in mild atopic asthmatics not on any anti-inflammatory therapy. METHODS: Sixteen mild atopic asthmatics, each with a demonstrable antigen-induced EAR, LAR, and BHR to histamine, were recruited into this randomized, double-blinded, crossover study. APC 366 (5 mg)/placebo was administered by aerosol inhalation 3 times per day on treatment days 1 through 4. Allergen challenge was carried out on day 4. Histamine challenge was performed the following morning, 1 hour after final dosing. RESULTS: Subjects were shown to have a significantly smaller overall mean area under the curve for the LAR (P =.012) and mean maximum fall in FEV(1) for the LAR (P =.007) after pretreatment with APC 366 in comparison with placebo. No significant effects on BHR were demonstrable. Although the EAR was reduced by 18% after treatment with APC 366 in comparison with placebo, this was not statistically significant. CONCLUSION: Short-term repeated administration of APC 366 significantly reduced the magnitude of antigen-induced LAR in atopic asthmatics, which supports the role of mast cell tryptase in the pathophysiology of the LAR.
Administration, Inhalation↗
The anti-inflammatory effects of leukotriene-modifying drugs and their use in asthma.
Asthma is a chronic inflammatory disease of the airways. Anti-inflammatory drug therapy, primarily using corticosteroids, is now considered the first-line treatment in the management of all grades of asthma severity. Although corticosteroids are believed to be the most potent anti-inflammatory agents available, they do not suppress all inflammatory mediators involved in the asthmatic response. Leukotrienes, which are lipid mediators generated from the metabolism of arachidonic acid, play an important role in the pathogenesis of asthma. They produce bronchospasm, increase bronchial hyperresponsiveness, mucus production, and mucosal edema, and enhance airway smooth muscle cell proliferation and eosinophil recruitment into the airways, and their synthesis or release is unaffected by corticosteroid administration. The use of leukotriene synthesis inhibitors or leukotriene receptor antagonists as anti-inflammatory therapies in asthma has therefore been investigated. Beneficial effects of leukotriene-modifying drugs have been demonstrated in the management of all grades of asthma severity, and there is evidence that certain patient groups (such as those with exercise-induced asthma or aspirin-induced asthma) may be particularly suitable for such therapy.
Adrenal Cortex Hormones↗
Involvement of the epidermal growth factor receptor in epithelial repair in asthma.
Epithelial damage and airway remodeling are consistent features of bronchial asthma and are correlated with disease chronicity, severity, and bronchial hyperreactivity. To examine the mechanisms that control bronchial epithelial repair, we investigated expression of the epidermal growth factor receptor (c-erbB1, EGFR) in asthmatic bronchial mucosa and studied repair responses in vitro. In biopsies from asthmatic subjects, areas of epithelial damage were frequently observed and exhibited strong EGFR immunostaining. EGFR expression was also high in morphologically intact asthmatic epithelium. Using image analysis, EGFR immunoreactivity (% of total epithelial area, median (range) was found to increase from 9.4 (4.1-20.4) in normal subjects (n=10) to 18.4 (9.3-28.9) in mild asthmatics (P<0.01, n=13) and 25.4 (15.4-31.8) in severe asthmatics (P<0.00, n=5). Epithelial EGFR immunoreactivity remained elevated in patients treated with corticosteroids and was positively correlated with subepithelial reticular membrane thickening. Using 16HBE 14o- bronchial epithelial cells, we found that EGF accelerated repair of scrape-wounded monolayers and that the EGFR-selective inhibitor, tyrphostin AG1478, inhibited both EGF-stimulated and basal wound closure whereas dexamethasone was without effect. Intrinsic activation of the EGFR was confirmed by analysis of tyrosine phosphorylated proteins, which revealed a rapid, damage-induced phosphorylation of the EGFR, irrespective of the presence of exogenous EGF. To assess the relationship between EGFR-mediated repair and tissue remodeling, release of the profibrogenic mediator TGF-beta2 was also measured. Scrape wounding increased release of TGF-beta2 from epithelial monolayers and EGF had no additional stimulatory effect. However, when repair was retarded with AG1478, the amount of TGF-beta2 increased significantly. These data indicate that the EGFR may play an important role in bronchial epithelial repair in asthma and that impairment of this function may augment airway remodeling.
Adult↗
Effects of short-term exposure to 0.2 ppm ozone on biomarkers of inflammation in sputum, exhaled nitric oxide, and lung function in subjects with mild atopic asthma.
To gain further insight into the kinetics of airway inflammatory response and explore the possibility of nitric oxide as a surrogate marker of the lower airway inflammatory response to ozone, nine subjects with mild atopic asthma were exposed to filtered air or 0.2 ppm ozone for 2 hours with intermittent exercise. Lung function was measured at baseline and immediately after exposures. Sputum induction was performed at 6 hours and at 24 hours after exposures, and exhaled nitric oxide levels were measured at baseline, immediately, 6, and 24 hours after both exposures. A significant decline in forced expiratory volume in one second and inspiratory capacity was detectable following exposure to ozone. In addition, a 2-fold increase was observed in the percentage of polymorphonuclear leukocytes 6 hours after exposure to ozone, with no changes in other biomarkers at this time point. By 24 hours after ozone exposure, the neutrophilia had subsided but there was an increase in albumin, total protein, myeloperoxidase, and eosinophil cationic protein. Exhaled nitric oxide levels, histamine, interleukin-8, and growth-related oncogene-alpha in sputum did not change significantly following ozone exposure. It was concluded that short-term ozone exposure induces an acute inflammatory response in asthmatic airways, characterized by early polymorphonuclear leukocyte influx followed by plasma extravasation and activation of eosinophils and neutrophils. Exhaled nitric oxide is not a useful marker for detecting inflammatory response to ozone in persons with mild asthma.
Administration, Inhalation↗
Inflammatory mechanisms underlying potentiation of effects of inhaled aeroallergens in response to nitrogen dioxide in allergic airways disease.
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Allergens↗
Compromised concentrations of ascorbate in fluid lining the respiratory tract in human subjects after exposure to ozone.
OBJECTIVES: Ozone (O3) imposes an oxidative burden on the lung in two ways. Firstly, directly as a consequence of its oxidising character during exposure, and secondly, indirectly by engendering inflammation. In this study the second pathway was considered by ascertaining the impact of O3 on the redox state of the fluid lining the respiratory tract 6 hours after challenge. METHODS: Nine subjects were exposed in a double blind crossover control trial to air and 200 ppb O3 for 2 hours with an intermittent exercise and rest protocol. Blood samples were obtained and lung function (forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1)) assessed before, immediately after, and 6 hours after exposure. Bronchoalveolar lavage (BAL) was performed 6 hours after challenge. Inflammation was assessed in BAL fluid (total and differential cell counts, plus myeloperoxidase concentrations), and plasma and BAL fluid redox state were determined by measuring concentrations of antioxidants and markers of oxidative damage. RESULTS: Neutrophil numbers in BAL fluid increased 2.2-fold (p = 0.07) 6 hours after exposure and this was accompanied by increased myeloperoxidase concentrations in BAL fluid (p = 0.08). On the other hand, BAL fluid macrophage and lymphocyte numbers decreased 2.5-fold (p = 0.08) and 3.1-fold (p = 0.08), respectively at this time. Of the antioxidants examined, only ascorbate in BAL fluid was affected by O3, falling in all subjects relative to air values (0.1 (0.0-0.3) v 0.3 (0.2-1.2) mumol/l (p = 0.008)). A marginal decrease in plasma ascorbate was also detected at this time (p < 0.05). Although the decrease in macrophage numbers seemed to be causally related to the increase in neutrophils (R = -0.79), myeloperoxidase concentrations (R = -0.93) and ascorbate concentrations (R = 0.6), no clear associations were apparent between ascorbate changes and neutrophils or myeloperoxidase concentration after O3. CONCLUSIONS: Ascorbate in the fluid lining the respiratory tract is depleted as a consequence of O3 exposure at 6 hours after exposure. This was contemporaneous with, although not quantitatively related to the increase in neutrophil numbers and myeloperoxidase concentrations. Decreased macrophage numbers 6 hours after O3 related to the degree of neutrophilic inflammation with populations conserved where ascorbate concentration in the fluid lining the respiratory tract were high after exposure. These results imply that ascorbate has a critical protective role against inflammatory oxidative stress induced by O3.
Adult↗
Persistent airway inflammation but accommodated antioxidant and lung function responses after repeated daily exposure to nitrogen dioxide.
Nitrogen dioxide (NO2) is a common indoor and outdoor air pollutant that may induce deterioration of respiratory health. In this study the effects of repeated daily exposure to NO2 on airway antioxidant status, inflammatory cell and mediator responses, and lung function were examined. Healthy nonsmoking subjects were exposed under controlled conditions to air (once) and to 2 ppm of NO2 for 4 h on four consecutive days. Lung function measurements were made before and immediately after the end of each exposure. Bronchoscopy with endobronchial biopsies, bronchial wash (BW), and bronchoalveolar lavage (BAL) was carried out 1.5 h after the air exposure and after the last exposure to NO2. Repeated NO2 exposure resulted in a decrease in neutrophil numbers in the bronchial epithelium. The BW revealed a twofold increase in content of neutrophils (p < 0.05) and a 1.5-fold increase in myeloperoxidase (MPO) (p < 0.01) indicative of both migration and activation of neutrophils in the airways. After the fourth NO2 exposure, antioxidant status of the airway fluid was unchanged. Significant decrements in FEV1 and FVC were found after the first exposure to NO2, but these attenuated with repeated exposures. Together, these data indicate that four sequential exposures to NO2 result in a persistent neutrophilic inflammation in the airways, whereas changes in pulmonary function and airway antioxidants are resolved. We conclude that NO2 is a proinflammatory air pollutant under conditions of repeated exposure.
Adult↗
T cell receptor (TCR) Vbeta gene usage in bronchoalveolar lavage and peripheral blood T cells from asthmatic and normal subjects.
T cells are thought to play an important regulatory role in asthma, but little is known about the T cell repertoire of the human lung or whether asthma is associated with any specific repertoire changes. Flow cytometry and MoAbs to TCR VB (TCRBV) families were used to quantify bronchoalveolar lavage (BAL) and blood T cells from normal and atopic individuals. Clonality was then assessed by polymerase chain reaction (PCR) amplification of cDNA and gene scanning using consensus and family-specific TCRBV primers and confirmed by sequence analysis. In addition, blood and BAL T cell populations were studied pre- and post-allergen challenge in four patients with allergic asthma. The majority of TCRBV families detected in blood by MoAb staining were also represented in BAL. While differences between BAL and blood populations were evident in each individual studied, these differences were not consistent between individuals or between CD4+ and CD8+ T cell subpopulations. These results are in broad agreement with other published studies, but in contrast to previous work we found a consistent difference between TCRBV7 family usage in blood and BAL in all individuals studied, and a consistently increased proportion of CD4+ BAL T cells bearing BV5S2/3 in asthmatics only. After allergen challenge, the pattern of TCRBV gene usage was largely unchanged as judged by flow cytometry. Gene scanning of PCR products generated from consensus VB primers revealed polyclonal lymphocyte populations in blood and BAL from all seven atopic individuals: in one normal tested polyclonal populations were found in blood and oligoclonal populations in BAL. Selected families amplified with family-specific primers BV5S2/3, BV6 and BV7 (chosen because of their predominance in BAL compared with blood) were more variable and revealed predominant polyclonal populations in blood and polyclonal or oligoclonal populations in BAL. In one asthmatic patient a clonal BV5S2 family was found in BAL. Following allergen challenge there were no significant changes in polyclonality/oligoclonality/clonality in three cases, but in one case a clonal BV5S2 population was found after challenge, that had not been evident beforehand. The lung T cell repertoire is thus broadly representative of blood T cells, but shows population differences that may result from response to persistent exposure to airborne antigens common to normal and atopic individuals. Oligoclonal TCRBV family expansion appears to be primarily lung-specific but independent of atopic asthma, although our challenge data in one case support the concept that clonal populations may follow local allergen challenge. These data are consistent with selection and amplification of specific T cell families in the lung in response to local antigenic exposure.
Adult↗
Effects of 0.2 ppm ozone on biomarkers of inflammation in bronchoalveolar lavage fluid and bronchial mucosa of healthy subjects.
Short-term exposure to ozone at peak ambient levels induces neutrophil influx and impairs lung function in healthy humans. In order to investigate the mechanisms contributing to neutrophil recruitment and to examine the role of T-cells in the acute inflammatory response, we exposed 12 healthy humans to 0.2 parts per million (ppm) of ozone and filtered air on two separate occasions for 2 h with intermittent periods of rest and exercise (minute ventilation = 30 L x min(-1)). Fibreoptic bronchoscopy was performed 6 h after the end of exposures. Total protein, tryptase, histamine, myeloperoxidase, interleukin (IL)-8 and growth-related oncogene-alpha (Gro-alpha) were measured and total and differential cell counts were performed in bronchoalveolar lavage (BAL) fluid. Flow cytometry was performed on BAL cells to study total T-cells, T-cell receptors (alphabeta and gammadelta), T-cell subsets (CD4+ and CD8+ cells) and activated T-cell subsets (CD25+). Using immunohistochemistry, neutrophils, mast cells, total T-cell numbers, T-cell subsets, CD25+ T-cells and leukocyte endothelial adhesion molecules including P-selectin, E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular adhesion molecule (VCAM)-1 were quantified in the bronchial biopsies. Paired samples were available from nine subjects. Following ozone exposure there was a threefold increase in the proportion of polymorphonuclear neutrophils (PMNs) (p=0.07) and epithelial cells (p=0.05) in BAL fluid. This was accompanied by increased concentrations of IL-8 (p=0.01), Gro-alpha (p=0.05) and total protein (p=0.058). A significant positive correlation was demonstrated between the two chemokines and proportion of PMNs in BAL fluid. After ozone exposure there was a significant decrease in the CD4/CD8 ratio (p=0.05) and the proportion of activated CD4+ (p=0.01) and CD8+ T-cells (p=0.04). However, no significant changes were demonstrable in any of the inflammatory markers studied in the biopsies. Short-term exposure of healthy humans to 0.2 ppm ozone induced a neutrophil influx in peripheral airways at 6 h post exposure, but no apparent inflammatory response in proximal airways. This response seems to be mediated at least in part by interleukin-8 and growth-related oncogene-alpha.
Adult↗
Short-term ozone exposure upregulates P-selectin in normal human airways.
Short-term exposure to ambient levels of ozone induces neutrophilic bronchitis. To investigate the early events contributing to inflammatory cell recruitment in the airways we exposed 12 healthy nonsmoking volunteers to 0.12 ppm ozone or filtered air for 2 h on two separate occasions. Spirometry and fiberoptic bronchoscopy were performed immediately and at 1.5 h after the two exposures, respectively. Total protein, albumin, and total and differential cell counts were performed on the bronchial wash and BAL fluid. Bronchial biopsies were embedded in glycol methacrylate and immunostained for inflammatory cells, including neutrophils, mast cells, total T-cells (CD3), T-cell subset CD8, and leukocyte endothelial adhesion molecules, including VCAM-1, ICAM-1, E-selectin, and P-selectin. No significant changes were observed in FEV1, FVC, or any inflammatory indices in the bronchial wash and BAL fluid. In addition, no significant differences were seen in inflammatory cell numbers or percentages of vessels expressing VCAM-1, E-selectin, or ICAM-1 in the biopsies. The percentage of vessels expressing P-selectin increased significantly after ozone exposure: p = 0.016; median (IQR), 28.76 (26.36-36.94) versus 47.06 (38.14-56.86)%. The upregulation of P-selectin could signify an early inflammatory response to ozone such as margination and rolling of the neutrophils on the vessel wall prior to transendothelial migration.
Adult↗
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↗
Effects of ozone on epithelium and sensory nerves in the bronchial mucosa of healthy humans.
Neuropeptides released from sensory nerves during inflammation have potent effects on bronchomotor tone, airway secretion, and inflammatory cells. We investigated the effects of ozone on sensory nerves by exposing 12 healthy, nonsmoking subjects to 0.2 ppm ozone and filtered air (FA) for 2 h on separate occasions, with intermittent exercise and rest. Spirometry was performed at baseline and 15 min after exposures, and bronchoscopy (bronchial biopsy and bronchoalveolar lavage [BAL]) was done 6 h after exposure. Frozen sections were immunostained for the anatomic neural marker protein gene peptide (PGP) 9.5 and the sensory neutropeptides substance P (SP) and calcitonin-gene-related peptide (CGRP). Nerves in the submucosa were quantified by image analysis. A trend toward an increase in the levels of polymorphonuclear leukocytes (PMNs) (air versus ozone, median [interquartile range]: 3.5 [2 to 5.3%] versus 9.8 [4.2 to 16.3%], p = 0.07) and ciliated epithelial cells (median [interquartile range]: 1.6 [1.3 to 3.4%] versus 5 [2.2 to 9.8%], p = 0.05) was observed in the BAL fluid (BALF). There was a significant decrease in SP immunoreactivity following ozone exposure (median [interquartile range]: 0.6 [0.05 to 1.2] versus 0.15 [0.08 to 0.18], p < 0.05). A significant inverse correlation was observed between SP immunoreactivity and: (1) percent PMNs and ciliated epithelial cells in the BALF; and (2) percent change in FEV1 following exposure to ozone. These findings indicate that short-term exposure to 0.2 ppm ozone causes epithelial shedding and stimulates subepithelial sensory nerves to release SP into the airways. The release of SP could contribute to bronchoconstriction and subsequent neutrophil infiltration into the airways.
Adult↗