Nasal challenges with recombinant derivatives of the major birch pollen allergen Bet v 1 induce fewer symptoms and lower mediator release than rBet v 1 wild-type in patients with allergic rhinitis.

BACKGROUND: Genetic engineering of the major birch pollen allergen (Bet v 1) has led to the generation of recombinant Bet v 1 derivatives with markedly reduced IgE-binding capacity, but with retained T cell activating ability. OBJECTIVE: To compare the mucosal reactivity to rBet v 1 derivatives with rBet v 1 wild-type as basis for new therapeutic strategies for birch pollen allergy based on mucosal tolerance induction. METHODS: Outside the pollen season, 10 patients with birch pollen allergic rhinitis and mild asthma underwent four nasal challenge-sessions in a randomized, double-blind, and cross-over design, employing increasing doses of rBet v 1 fragment mix, rBet v 1 trimer, rBet v 1 wild-type and diluent (albumin). Nasal lavage fluids (NAL) were collected before the challenge-series as well as 10 min, 4 and 24 h thereafter. Nasal lavage fluid levels of tryptase as well as EPO and ECP were measured as indices of mast cell and eosinophil activity, respectively. RESULTS: All 10 patients tolerated the highest accumulated dose, 8.124 microg, when challenged with rBet v 1 trimer, eight with rBet v 1 fragments compared to one when challenged with rBet v 1 wild-type. No late phase reactions were observed. The change in tryptase levels (pre-challenge vs. 10 min) was significantly lower after challenges with rBet v 1 trimer and rBet v 1 fragments than with rBet v 1 wild-type. The change in EPO/ECP concentration pre-challenge versus 4 h post-challenge was lower for rBet v 1 trimer and the change was significantly lower when pre-challenge versus 24 h post-challenge to rBet v 1 fragments and rBet v 1 wild-type was examined. CONCLUSION: The derivatives induced significantly fewer symptoms and lower mast cell and eosinophil activation than rBet v 1 wild-type upon application to the nasal mucosa. They could in the future be candidates for immunotherapy based on mucosal tolerance induction.

The reproducibility and sample size requirements of exercise-induced bronchoconstriction measurements.

Dry air exercise challenges are frequently used to screen medications that have potential utility in the management of exercise-induced bronchoconstriction (EIB). The purpose of this study was to determine the reproducibility of three outcome measurements made using such challenges, and sample size requirements for drug evaluation studies based on these outcomes. Forty adult subjects with asthma, who tested positively on a screening exercise challenge, were subjected to two further identical challenges, separated by 1 to >35 days. Outcome measurements included the maximum per cent fall in forced expiratory volume in one second (FEV1), after exercise (% fallmax), and the area under the per cent fall in FEV1/time curve for 30 min (AUC30) and 60 min (AUC60) after exercise. The reproducibility of these outcomes, as assessed by intraclass correlation coefficients was 0.72, 0.53 and 0.35 for % fallmax, AUC30 and AUC60 measurements, respectively. The sample size requirements to demonstrate an attenuation of EIB equivalent to a 50% reduction in % fallmax was 9, 14 and 19 subjects for the % fallmax, AUC30 and AUC60 responses, respectively (90% power). It is concluded that the maximum percentage fall in forced expiratory volume in one second has greater reproducibility and results in greater power in clinical trials than area under the curve measurements. Sample size calculation curves are provided which may be used in study design and interpretation of published studies.

T cell receptor Vbeta expression in patients with allergic asthma before and after repeated low-dose allergen inhalation.

The objective of this study was to identify disease-associated T cell subsets by characterizing the lung and blood T cell receptor (TCR) repertoires in allergic asthmatics before and after repeated low-dose allergen challenge. Peripheral blood lymphocyte (PBL) and bronchoalveolar lavage (BAL) samples were obtained from eight patients with allergic asthma before and after a period of repeated low-dose allergen inhalations. RT-PCR followed by Southern blot allowed the quantification of relative Vbeta gene segment usage. Thirteen healthy individuals served as controls at PBL level. PBL as well as BAL T cells of asthmatics displayed a higher usage of Vbeta3, Vbeta5.2, and Vbeta6.1-3 and a lower usage of Vbeta16, Vbeta18, and Vbeta19 compared to PBL of healthy controls. Interestingly, TCR Vbeta7 and Vbeta9 usage was significantly higher in BAL than in PBL in asthmatics before as well as after challenge. TCR repertoire alterations after allergen challenge differed between individuals, with relatively mild changes.

Cytokine mRNA expression in patients with mild allergic asthma following low dose or cumulative dose allergen provocation.

BACKGROUND: Allergen provocation is a very useful way to study the inflammatory response in asthmatic patients. Although cumulative dose regimens are most often applied, another provocation model with repeated inhalations of low doses of allergens has recently come into use. OBJECTIVE: We were interested to compare these two allergen provocation models. To evaluate the inflammation induced by either model, we examined the mRNA expression of several cytokines that are implicated in the orchestration of the inflammatory response observed in asthma. METHODS: Interleukin (IL)-4, IL-5, IL-13 and interferon (IFN)-gamma mRNA expression was analysed in bronchoalveolar lavage (BAL) cells and peripheral blood (PB) CD4+ and PB CD8+ T cells following any of the two provocation regimens. IL-4 and IFN-gamma mRNA expression was analysed by a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) method, while IL-5 and IL-13 were analysed semiquantitatively, before and after allergen provocation with either model. RESULTS: After low dose provocations none of the cell populations studied showed a clear change in the pattern of IL-4 or IFN-gamma gene expression. In contrast, after cumulative dose provocation there was a clear tendency towards an increased IL-4 mRNA expression in BAL cells, correlating with a significant increase in IL-4 mRNA in PB CD4+ as well as in CD8+ T cells (P = 0.005 and P = 0.04, respectively). Regardless of the allergen provocation method used, in PB IL-4 mRNA was preferentially expressed by CD4+ cells while IFN-gamma was expressed more by CD8+ cells. IL-5 transcripts increased after low dose provocations in PB CD4+ T cells in six of eight patients, while after cumulative dose provocation IL-5 mRNA increased in BAL cells in six out of nine patients but decreased especially in PB CD8+ T cells in six out of eleven patients, suggesting an accumulation of IL-5 expressing cells to the lungs. CONCLUSION: Thus, the cumulative dose provocation regimen can induce a more pronounced Th2-like immune response in asthmatic patients than the low dose provocation model.

Increased interleukin-13 mRNA expression in bronchoalveolar lavage cells of atopic patients with mild asthma after repeated low-dose allergen provocations.

Immune and inflammatory responses mediated by cytokines are essential in the pathophysiology of asthma. The aim of this study was to analyse the cytokine mRNA profiles in bronchoalveolar lavage (BAL) cells of patients with mild atopic asthma, before and after induction of a subclinical allergic airway inflammation. For this purpose, eight patients with mild atopic asthma received low-dose allergen inhalations equivalent to 10% of a provocational dose causing a 20% fall in forced expiratory flow in 1 sec (PD20) for 7 weekdays. BAL was performed before and after low-dose provocations in patients, and without provocation in five healthy controls. Alveolar macrophages (AM) were enriched by negative selection, using magnetic beads, to enable separate studies of the BAL cells. Using a semiquantitative RT-PCR technique, the mRNA expression of macrophage-derived cytokines interleukin (IL)-1, IL-6, IL-8, IL-10, IL-12, IL-13, interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta was analysed. After low-dose provocations, we observed a significant increase in the expression of IL-13 mRNA (P = 0.01) in BAL cells enriched for AM of the asthmatic patients. The increased IL-13 mRNA positively correlated with the proportion of BAL fluid eosinophils (r = 0.7, P = 0.05). Moreover, a tendency was found towards an increased IL-1 and a reduced IL-6, IL-8, IFN-gamma and TNF-alpha expression by the BAL cells. Comparing asthmatic patients before low-dose provocations and healthy controls, a significantly higher expression of IL-6 (P<0.003), IL-10 (P<0.005) and TGF-beta (P<0.003) and a significantly lower expression of IL-8 (P<0.005) and TNF-alpha (P<0.01) was detected in the patients. In summary, repeated low-dose allergen provocations of asthmatic patients results in a modified BAL cell cytokine mRNA profile with increased production of IL-13, that may be of importance for the development of a Th2-like immune response. A possible source of the increased IL-13 mRNA is AM, which may have a more active function in the allergic inflammation than previously thought.

Decreased pulmonary levels of the anti-inflammatory Clara cell 16 kDa protein after induction of airway inflammation in asthmatics.

The Clara cell 16 kDa protein (CC16) maps to an atopy-associated region of chromosome 11 and has been ascribed an anti-inflammatory function. Using reverse-phase HPLC and Western blot analysis, we have evaluated the polypeptide pattern in bronchoalveolar lavage (BAL) fluid retrieved from asthmatics, before and after induction of airway inflammation by low-dose allergen inhalation challenge. A prominent decrease of CC16 was seen after induction of inflammation, and a further CC16 decrease was observed in lavage fluid where surfactant had been removed. Reduced levels of pulmonary CC16 may cause loss of anti-inflammatory activity in the airways and contribute to the development of airway inflammation in asthma.

Airway inflammation and altered alveolar macrophage phenotype pattern after repeated low-dose allergen exposure of atopic asthmatic subjects.

BACKGROUND: The alveolar macrophage (AM) constitutes an important link between pulmonary innate and adaptive immunity due to its antigen-presenting capacity and ability to express different immunomodulating mediators. The role of AMs in the pathogenesis of allergic inflammation has yet to be fully determined. OBJECTIVE: To investigate clinical effects and any change in the AM phenotype pattern after inhalation of sub-clinical doses of allergen by asthmatic patients. METHODS: Eight subjects with allergic asthma underwent repeated low-dose allergen provocations equivalent to 10% of PD20. AMs recovered with bronchoalveolar lavage (BAL) were characterized by flow cytometric analysis of adhesion molecules, co-stimulatory molecules and markers for AM population activation and heterogeneity. RESULTS: An allergic airway inflammation, sub-clinical in six out of eight subjects, was obtained after low-dose allergen provocations, as determined by increased airway methacholine reactivity, increased BAL fluid total cell and eosinophil counts and increased serum ECP levels. The AMs showed a post-challenge altered phenotype pattern with a decreased expression of CD11a, CD16, CD71 and HLA class I and an increased expression of CD11b and CD14. The AMs were positive for CD83 and a weak post-challenge increase in the CD83 expression was found. CONCLUSION: Repeated low-dose allergen exposure induces an allergic airway inflammation in asthmatic subjects. The inflammation is associated with an altered AM phenotype pattern, consistent with an influx of monocytes and a hypothetical increased accessory cell function in the airways, possibly contributing to the development and sustenance of airway inflammation in asthma.

Urinary excretion of inflammatory mediators during allergen-induced early and late phase asthmatic reactions.

BACKGROUND: It is generally accepted that the early asthmatic response to inhaled allergen is a result of IgE-mediated mast cell activation. In contrast, the underlying mechanism of the late asthmatic response is much less clear. OBJECTIVE: In order to investigate the pattern of mediator release during the early and late asthmatic responses to allergen, measurements of the urinary excretion of the mast cell markers 9alpha,11beta-PGF2 and Ntau-methylhistamine were made. In addition, urinary levels of eosinophil protein X (EPX) and leukotriene E4 (LTE4) were measured. METHODS: Twelve mild atopic asthmatics participated in the study. On the study day, pulmonary function was recorded at baseline and for 12 h after inhalation of allergen. Urine was collected prior to challenge and thereafter at 1 h intervals. Measurements of 9alpha, 11beta-PGF2 and LTE4 were made with enzyme-immunoassay, and levels of Ntau-methylhistamine and EPX were analysed with radioimmunoassay. RESULTS: All subjects developed both an early and late phase airway response. Within 1 h of the early peak airway response, there was a significant increase in the urinary concentrations (AUC/h) of 9alpha, 11beta-PGF2 (49.3 +/- 9.2 to 142.5 +/- 49.2; P < 0.001) Ntau-methylhistamine (10.4 +/- 1.4 to 19.5 +/- 1.4; P < 0.001) and LTE4 (43.7 +/- 5.9 to 105.9 +/- 21.3; P < 0.001). Levels of all three mediators were also significantly increased above baseline during the LAR to 79.4 +/- 9.5 (P < 0.01), 19.8 +/- 1.9 (P < 0.001) and 85.6 +/- 10.4 (P < 0.001), respectively. Levels of EPX remained unchanged during the early and late responses (39.2 +/- 10.2 to 37.5 +/- 18.5, 33.9 +/- 6.8). CONCLUSIONS: These results indicate that mast cell activation is a feature not only of the early but also the late asthmatic response. Finally, increased LTE4 supports the contribution of the leukotrienes to airway obstruction during both phases of the asthmatic response to allergen.

Evidence for mast cell activation during exercise-induced bronchoconstriction.

Controversy remains about the causative mediators in the bronchoconstrictive response to exercise in asthma. This study examined whether mast cell activation is a feature of exercise-induced bronchoconstriction by measuring urinary metabolites of mast cell mediators. Twelve nonsmoking subjects with mild asthma and a history of exercise-induced bronchoconstriction exercised on a stationary bicycle ergometer for 5 min at 80% maximum work load. Pulmonary function was monitored and urine was collected before and 30 and 90 min after the provocation. The urinary concentrations of the mast cell markers 9alpha,11beta-prostaglandin (PG)F2 and Ntau-methylhistamine, as well as leukotriene E4 (LTE4) were determined by immunoassay. Seven of the 12 subjects (responders) experienced bronchoconstriction (>15% fall in the forced expiratory volume in one second) following exercise, whereas the pulmonary function of the remaining five subjects (nonresponders) remained stable. The urinary excretion (mean+/-SE) of 9alpha,11beta-PGF2 in the responders increased significantly compared with the nonresponders at 30 (77.1+/-14.4 versus 37.2+/-5.6; p<0.05) and 90 min (79.3+/-8.6 versus 40.4+/-8.5, p<0.05) after exercise challenge. The urinary excretion of Ntau-methylhistamine and LTE4 was not significantly different between the two groups at 30 or 90 min after exercise. The findings represent the first documentation of increased urinary levels of 9alpha,11beta-prostaglandin F2 in adults following exercise challenge and provides clear evidence for mast cell activation during exercise-induced bronchoconstriction in asthmatics.

No signs of activity markers in peripheral blood despite increased bronchial reactivity after repeated low-dose allergen exposure.

The allergen inhalation test can be used as an experimental model to study pathophysiological events in allergic asthma. Repeated low-dose inhalations of allergen induce increased bronchial hyperresponsiveness (BHR) and resemble natural allergen exposure. The objective of the present study was to investigate whether eosinophil recruitment and activation in peripheral blood, differences in expression of lymphocyte surface antigens and increased bronchial responsiveness to histamine occur during and after repeated low-dose bronchial allergen challenge. Fourteen atopic asthmatic patients were challenged in a randomized double-blind manner for 7 days with either allergen in very low doses or placebo. We measured the concentration of eosinophils, eosinophil cationic protein (ECP) and the expression of the EG2-epitope on intracellular ECP in eosinophils and the expression of lymphocyte surface antigen markers in peripheral blood. The challenge period started and ended with a histamine provocation. The repeated low-dose allergen exposure resulted in a significant increase in BHR. No changes were seen in the placebo group. Concerning the inflammatory parameters in peripheral blood, no significant changes were seen during or after the week of low-dose allergen inhalations. Our results show that very low, repeated doses of allergen induce increased airway reactivity despite lack of evident clinical symptoms or signs of activation of inflammatory cells in peripheral blood.

Increased allergen-specific Th2 responses in vitro in atopic subjects receiving subclinical allergen challenge.

The study aimed to determine whether inhalation of subclinical allergen doses-leads to a shift in the balance between T helper (Th) 1 and Th2 cells in asthmatic patients. Elevated IgE requires allergen-specific T cells producing cytokines such as interleukin (IL)-4 or IL-13. Interferon-gamma (IFN-gamma) produced by Th1 cells counteracts the effects of IL-4. In nature, allergic persons are often exposed to low levels of allergen, leading to hyperreactivity, but not to acute allergic reactions. In this study, nine allergic persons inhaled low doses of allergen or placebo in a double-blind manner over seven consecutive weekdays. During the study, the bronchial responsiveness to histamine challenge increased, but no subject exhibited asthmatic symptoms. Blood was drawn on days 0, 1, 4, and 9, and the number of IL-4- and IFN-gamma-producing cells was measured by enzyme-linked immunospot (ELISPOT) assay after in vitro stimulation with a low-dose phytohemagglutinin (PHA) mixed with the relevant allergen or with PHA alone. In three of the four subjects receiving allergen, the IL-4/IFN-gamma ratio increased during the time of the study. No increase was seen in the placebo group. No increase was seen in serum IgE levels in any of the groups. We conclude that a shift in the balance between Th1 and Th2 cells can be detected in subjects exposed to subclinical allergen doses.

Combined antagonism of leukotrienes and histamine produces predominant inhibition of allergen-induced early and late phase airway obstruction in asthmatics.

We defined the contribution of histamine and leukotrienes to allergen-induced airway obstruction in asthmatics; 12 subjects with allergic asthma underwent identical allergen bronchoprovocations on four occasions. At a control session, all subjects displayed early (EAR) and late asthmatic (LAR) reactions. The mean (+/- SE) drop in FEV1 during EAR (0-2 h) and LAR (2-12 h) was 29 +/- 2% and 28 +/- 4%, respectively. Thereafter, the influence of 1 wk randomized pretreatment with the leukotriene receptor antagonist zafirlukast (Accolate) (80 mg twice daily), the antihistamine loratadine (10 mg twice daily), and the combination of both antagonists was assessed. Expressed as AUC FEV1 in percent of the control reaction, zafirlukast reduced the response during EAR and LAR by 62 +/- 11% and 55 +/- 12%, respectively (p < 0.05 versus control). Loratadine inhibited EAR and LAR by 25 +/- 14% and 40 +/- 16%, respectively (p < 0.05 versus control). Zafirlukast was significantly more effective than loratadine during EAR but not during LAR. The combination of zafirlukast and loratadine reduced the AUC FEV1 during EAR and LAR further, by 75 +/- 8% and 74 +/- 14%, respectively (p < 0.05 versus control). The combination was significantly (p < 0.05) more effective than either drug alone during the LAR. The findings indicate that leukotrienes and histamine together mediate the major part of both the EAR and the LAR following exposure of asthmatics to allergen. Combination of leukotriene antagonism and antihistamines may represent a new strategy for treatment of airway obstruction in asthma.

Eosinophil activity markers in peripheral blood have high predictive value for bronchial hyperreactivity in patients with suspected mild asthma.

The present study aimed to evaluate the predictive value of eosinophils and markers of their activity for bronchial hyperreactivity (BHR) in a population of patients with recently developed clinical symptoms of asthma. The activation of eosinophils was estimated by measuring eosinophil cationic protein (ECP) in serum. In addition, flow cytometry was used to measure the expression of the EG2-epitope on intracellular ECP in eosinophils from peripheral blood. Twenty-eight consecutive patients with clinical history of asthma were studied. Of the 28 patients, 18 had a positive bronchial challenge test measured as PD20 < or = 1600 micrograms histamine. A significantly higher concentration of eosinophils and a trend to higher ECP in the peripheral blood was found in the hyperreactive group than in the nonreactive group. However, the intracellular expression of ECP did not correlate with the PD20 value, and no significant difference between the groups was found. With one eosinophil activity marker, either serum ECP or EG2, BHR could be predicted in 70% of the patients. If we combined any two of the activity markers (serum ECP, EG2, or the percentage of eosinophils), the predictive value increased to 100%. We conclude that the blood eosinophil concentration, as well as, to some extent, serum ECP, has a high specificity for BHR in patients with recently developed clinical symptoms of asthma. Despite normal bronchial reactivity, some patients had signs of activated eosinophils, i.e., high serum ECP and increased EG2 expression. Thus, these markers may reflect early stages in the development of BHR. Our results also indicate that a combined evaluation of percentage of eosinophils and of eosinophil activity markers is of clinical value to predict BHR.

Anti-IgE-induced accumulation of leukocytes, mediators, and albumin in skin chamber fluid from healthy and atopic subjects.

The aim of this study was to examine potential differences between healthy and atopic subjects with regard to IgE-mediated cutaneous inflammation. For this purpose, we analyzed histamine, tryptase, leukotriene B4, albumin, eosinophils, and total leukocytes in skin chamber fluid after challenge with anti-human IgE. We also measured gross skin reactivity (wheal, flare, and late-phase reactions), circulating IgE, and eosinophils, as well as the state of eosinophil activation. It was found that despite having more circulating IgE, the skin responsiveness of the atopic subjects did not differ significantly from that of the nonatopic subjects with respect to mediator release, albumin extravasation, or total recruitment of leukocytes. Moreover, the sizes of anti-IgE-induced wheal, flare, and late-phase reactions were very similar in the two groups. On the other hand, significant recruitment of eosinophils during the IgE-mediated reaction was more or less restricted to the atopic group. Yet the recruited eosinophils, of which the majority was in an early state of activation before degranulation, did not seem to contribute significantly to the IgE-mediated delayed skin edema. Furthermore, the eosinophil count in anti-IgE chambers of the atopic subjects did not correlate with any of the other parameters monitored. Thus because the anti-IgE-induced recruitment of eosinophils appeared to be unrelated to factors such as the number of peripheral blood eosinophils, the degree of mast cell activation, the intensity of inflammatory skin changes, and the level of circulating IgE, it is apparent that the mechanisms for and pathophysiologic role of IgE-mediated dermal eosinophil accumulation in atopic subjects require further investigation.

Effects of loratadine on anti-IgE-induced inflammation, histamine release, and leukocyte recruitment in skin of atopics.

The aim of this study was to assess the ability of the H1-receptor antagonist loratadine to modify anti-IgE-induced cutaneous wheal-and-flare and late-phase reactions (WFR and LPR), as well as histamine release and leukocyte accumulation in skin chambers. For this purpose, 10 atopics with allergic rhinitis were entered into a double-blind crossover study in which they received either placebo or loratadine (20 mg/day orally) for 8 days separated by a 7-day washout period. Blisters were induced on both forearms on day 7 of each treatment period, and were unroofed on day 8 and covered with plastic skin chambers. Chamber fluids were collected during 7 h after 1-h incubation with anti-IgE or control IgG. Intradermal challenge with histamine and anti-IgE was performed at the same occasion. As compared to placebo treatment, loratadine inhibited the immediate WFRs to anti-IgE by 35% (wheal) and 65% (flare), respectively (P < 0.01), and corresponding reactions to histamine challenge by 50% and 70% (P < 0.001), respectively. Moreover, the initial phase (0-2 h) of the LPR induced by anti-IgE was attenuated by up to approximately 60% (P < 0.001) during loratadine treatment. Thereafter, no inhibition of the LPR was observed. The magnitude and time course of histamine release into skin chambers was virtually the same after loratadine and placebo treatment, with a peak during 0-1 h and a progressive decline during the following 2 h. Accumulation of alpha 2-macroglobulin, reflecting extravasation of large plasma proteins, also peaked during the first hour and was unaffected by loratadine during the 8-h observation period.(ABSTRACT TRUNCATED AT 250 WORDS)