Dog saliva - an important source of dog allergens.

BACKGROUND: Allergy to dog (Canis familiaris) is a worldwide common cause of asthma and allergic rhinitis. However, dander extract in routine diagnostics is not an optimal predictor of IgE-mediated dog allergy. Our objective was to evaluate saliva as an allergen source for improved diagnostics of allergy to dog. METHODS: IgE-binding proteins in dog saliva and dander extract were analysed by immunoblot and mass spectrometry (LC-MS/MS) using pooled or individual sera from dog-allergic patients (n = 13). Sera from 59 patients IgE positive to dander and 55 patients IgE negative to dander but with symptoms to dog were analysed for IgE against saliva and dander by ELISA. Basophil stimulation with dog saliva and dander extract was measured by flow cytometry among three dog-allergic patients. Additionally, IgE-binding protein profiles of saliva from different breeds were investigated by immunoblot. RESULTS: Greater number and diversity of IgE-binding proteins was found in saliva compared to dander extract and varied among dog breeds. In saliva, Can f 1, 2, 3 and 6 were identified but also four new saliva allergen candidates. The majority of the 59 dog dander-positive sera (n = 44) were IgE positive to dog saliva. Among patients IgE negative to dander, but with symptoms to dog, 20% were IgE positive to saliva. The biological activity of saliva was confirmed by basophil degranulation. CONCLUSIONS: Dog saliva is an allergen source for improved diagnostics of dog allergy. The IgE-binding protein profile of saliva from different dogs varies.

Bronchoalveolar lavage fluid exosomes contribute to cytokine and leukotriene production in allergic asthma.

BACKGROUND: Leukotrienes (LTs) are potent pro-inflammatory mediators involved in asthma. Exosomes, nanosized vesicles released from various cells, can stimulate or down-regulate immune responses, depending on the state and nature of the originating cell. We have recently shown an altered exosome profile in bronchoalveolar lavage fluid (BALF) of patients with sarcoidosis, but their role in asthma is unknown. Our aims were to investigate whether exosomes from BALF have LT biosynthetic capacity and to explore phenotypic and functional characteristics of BALF exosomes in asthma. METHODS: Bronchoalveolar lavage fluid exosomes were collected from healthy individuals (n = 13) and patients with mild allergic asthma to birch pollen (n = 12) before and after birch allergen provocation. Exosomes were characterized by flow cytometry and Western blot. Their capacity to induce IL-8 and LT production in the human bronchial epithelial cell (BEC) line 16HB14o- was measured by ELISA and reverse-phase HPLC, respectively. RESULTS: Compared to BALF exosomes from healthy individuals, BALF exosomes from asthmatics displayed higher levels of exosome-associated markers, such as the tetraspanins CD63 and CD81 and the scavenger receptor CD36. No major differences were observed between BALF exosomes from before and after allergen provocation. Furthermore, we show that BALF exosomes contain enzymes for LT biosynthesis. The effect of exosomes to promote LTC(4) and IL-8 release in BEC was significantly increased for exosomes from asthmatics, and the CysLT(1) receptor antagonist Montelukast reduced exosome-induced IL-8 secretion. CONCLUSIONS: Bronchoalveolar lavage fluid exosomes from asthmatic and healthy individuals exhibit distinct phenotypes and functions. BALF exosomes from asthmatics might contribute to subclinical inflammation by increasing cytokine and LTC(4) generation in airway epithelium.

Allergen provocation increases TH2-cytokines and FOXP3 expression in the asthmatic lung.

BACKGROUND: Allergic asthma is caused by allergen-specific IgE and T-helper cell (Th) type 2 responses towards airborne allergens. The objective of this study was to investigate local and systemic regulatory mechanisms in the early asthmatic response to bronchial allergen provocation. METHODS: Birch pollen-allergic patients with mild asthma (n = 13) and healthy nonallergic controls (n = 14) were subjected to bronchoalveolar lavage (BAL) and blood sampling. On patients BAL was performed twice: without preceding provocation ('before samples') and 24 h after bronchial provocation with birch pollen allergen. Lymphocytes in BAL and peripheral blood mononuclear cells (PBMCs) were phenotyped by multi-colour flow cytometry and cytokines measured by cytometric bead array. Proliferation and secreted cytokines were analysed in allergen-stimulated PBMCs, CD25(+) depleted PBMCs and PBMCs with IL-10 neutralizing antibodies. RESULTS: The numbers of CD69(+) and FOXP3(+) lymphocytes were higher in BAL after compared with before allergen provocation in asthmatic patients. Moreover, allergen provocation increased expression of FOXP3 in CD4(+)CD25(bright) cells. The cytokine profile in BAL fluid from asthmatics revealed higher levels of IL-5, compared with the controls, and an increase in IL-5, IL-6, IL-9 and IL-10 after allergen provocation. Pollen allergen stimulated PBMC cultures from asthmatic patients produced elevated levels of IL-5 and IL-13 compared with the controls, which were not affected by depletion of CD25(+) cells or IL-10 neutralization. CONCLUSION: Despite an increase in CD4(+)CD25(bright) cells expressing high levels of FOXP3 in response to bronchial allergen provocation, asthmatic patients exhibit enhanced levels of Th2 cytokines in the lung, which may indicate an inability among infiltrating cells to suppress Th2 responses.

The size of the disease relevant IgE antibody fraction in relation to 'total-IgE' predicts the efficacy of anti-IgE (Xolair) treatment.

BACKGROUND: Some patients with allergic asthma treated with anti-IgE (Xolair) do not become symptom free. Better criteria for response assessment than allergy skin tests or IgE determination are needed. The impact of the size of the disease relevant allergen-specific IgE antibody fraction, i.e. the percentage of IgE antibody of total IgE, was evaluated in cat allergic patients treated with the recommended doses of Xolair. Results were measured as changes in basophil allergen threshold sensitivity (CD-sens). METHODS: In a double-blind placebo controlled trial 20 patients with a high (>3.8%) and 18 with a low (<1%) percentage of IgE antibodies to cat were given Xolair for 16 weeks and the change in CD-sens was compared to 11 and 10 patients, respectively, in each group receiving placebo. RESULTS: The CD-sens dropped significantly in both the high (P < 0.001) and low (P < 0.001) group on Xolair but did not change significantly after placebo. For Xolair-treated patients, at the end of the trial there was a highly significant (P < 0.001) difference in CD-sens between the high group, where no patients, and the low group, where 13/18 patients, had become negative. CONCLUSIONS: The currently recommended doses of Xolair very efficiently eliminate IgE antibodies if the IgE antibody fraction is <1% of total IgE but has not enough effect on allergen sensitivity if the fraction is >3-4%. Further studies will show if increased doses of Xolair would help also these patients, who seem to represent about 1/3 of the patient population.

Clinical effects of immunotherapy with genetically modified recombinant birch pollen Bet v 1 derivatives.

BACKGROUND: Birch pollen and pollen from related trees of the Fagales order are a major cause of allergic rhinitis, conjunctivitis, and asthma through the spring season in northern and central Europe. OBJECTIVE: To investigate the clinical effects of injection immunotherapy with genetically modified derivatives of major birch pollen allergen Bet v 1 on pollen-induced allergic symptoms. METHODS: A three-arm double-blind placebo-controlled immunotherapy study was conducted with one pre-seasonal course of treatment using two derivatives of Bet v 1, namely a recombinant Bet v 1 trimer and an equimolar mixture of two recombinant Bet v 1 fragments together representing the whole protein sequence. Analysis of local and systemic adverse events was performed for 124 patients who had received at least one dose of medication. Clinical efficacy was monitored by symptom medication scores and interval scoring in the per protocol-treated population (n=84). In addition, skin and nasal provocation responses and allergen-specific antibodies were assessed. RESULTS: There were trends towards improvement in the subjects' well-being and clinical symptoms (nasal scores), although comparisons with a placebo group did not show statistical significance in the main end-point, the combined symptom medication score. Reductions in skin and nasal sensitivity were observed for some subjects with a trend for the Bet v 1 trimer to be more effective than the fragments. Treatment induced strong IgG1 and IgG4 allergen-specific antibody responses. Local injection-site reactions were most frequent in the trimer group affecting 59.5% of patients as opposed to 37.8% and 30.6% in the fragment and placebo groups, respectively. Systemic reactions were elicited more frequently by fragments. A large proportion of adverse side-effects appeared hours following injections, and might be attributable to concurrent exposure to related pollens. CONCLUSION: Single courses of injection immunotherapy with Bet v 1 allergen derivatives showed trends towards improved well-being and reduced reactivity to specific allergen provocation, but did not yield significant improvement in the combined symptom medication score in this study.

Higher immunoglobulin E antibody levels to recombinant Fel d 1 in cat-allergic children with asthma compared with rhinoconjunctivitis.

BACKGROUND: Current diagnosis of allergy and asthma to cat is confirmed using cat dander extract (CDE). We have previously engineered a recombinant major cat allergen, rFel d 1, with properties identical to the natural molecule. OBJECTIVE: The aim of the study was to evaluate IgE and IgG4 antibodies to rFel d 1 among sera from cat-allergic children and adults suffering from asthma and/or rhinoconjunctivitis (RC) in populations from Sweden and Austria. METHODS: Cat-allergic children and adults from Sweden (n=27 and 31, respectively) and Austria (n=41 and 41) with RC and/or asthma were selected. Sera were tested for IgE and IgG4 antibodies to CDE and rFel d 1 by CAP, and IgE to rFel d 1 by ELISA. Healthy subjects and non-cat-allergic patients (n=75) were included as controls. RESULTS: There was a high correlation between IgE responses to rFel d 1 and CDE among the 140 patients (r(s)=0.85, P<0.001); however, measured levels to rFel d 1 were on average 30% higher (P<0.0001). Ninety-eight percent of patients and none of the controls showed IgE to rFel d 1 and there was a threefold increased risk of asthma for half of the children with the highest IgE levels [odds ratio 3.23; 95% confidence interval (CI), 1.19-8.79] by ELISA. IgE responses to rFel d 1 among children with asthma were higher (median 19.4 kU/L) compared with children with RC (median 6.6 kU/L, P<0.05) and adults with asthma (median 3.0 kU/L, P<0.01). Furthermore, children with asthma displayed higher IgG4 levels than the asthmatic adults. CONCLUSION: A single recombinant molecule, rFel d 1, is at least as sensitive for in vitro diagnostics of cat allergy as the current extract-based test. Elevated IgE antibody levels to Fel d 1 are suggested to be a risk factor for asthma in cat-allergic children.

Immune regulation by CD4+CD25+ T cells and interleukin-10 in birch pollen-allergic patients and non-allergic controls.

BACKGROUND: CD4+CD25+ regulatory T (Treg) cells and the cytokines IL-10 or TGF-beta play key roles in the maintenance of T cell homeostasis and tolerance to infectious and non-infectious antigens such as allergens. OBJECTIVE: To investigate the regulation of immune responses to birch pollen allergen compared with influenza antigen by Treg cells obtained from birch pollen-allergic patients and non-allergic controls. METHODS: Peripheral blood was collected from 10 birch pollen-allergic patients and 10 non-allergic healthy controls. CD4+CD25+ and CD4+CD25- cells isolated by magnetic-activated cell sorting were co-cultured and stimulated with birch pollen extract or influenza vaccine in the absence or presence of anti-IL-10 or soluble TGF-betaRII. RESULTS: CD4+CD25+ cells from non-allergic controls were able to suppress influenza antigen and birch pollen stimulated effector cell proliferation, whereas CD4+CD25+ cells from allergic patients suppressed influenza antigen-, but not birch pollen-stimulated proliferation. The production of Th1 cytokines, but not Th2 cytokines, was suppressed by CD4+CD25+ cells from both allergic patients and controls, upon stimulation with birch pollen extract. Neutralization of IL-10 led to significantly increased production of IFN-gamma in cultures with CD4+CD25- T effector cells. In addition, six-fold higher concentrations of TNF-alpha were detected after neutralization of IL-10 in both CD4+CD25- and CD4+CD25+ cell cultures from allergic patients and controls. CONCLUSION: We demonstrate that the allergen-specific suppressive function of CD4+CD25+ cells from allergic patients is impaired compared with non-allergic controls. Moreover, neutralization of IL-10 enhances the production of TNF-alpha, suggesting counter-acting properties of IL-10 and TNF-alpha, where IL-10 promotes tolerance and suppression by Treg cells and TNF-alpha promotes inflammatory responses.

Changes in immune regulation in response to examination stress in atopic and healthy individuals.

BACKGROUND: Stress can aggravate the allergic inflammation, but determinants of disturbed immune regulation are largely unknown. OBJECTIVE: To determine systemic immunological, local inflammatory and functional airway responses to stress in healthy and atopic individuals. METHODS: Forty-one undergraduate students, 22 with allergy of whom 16 had asthma, and 19 healthy controls, were studied in a low-stress period and in association with a large exam. Subjects completed questionnaires on stress and health behaviours, underwent lung function tests, bronchial methacholine challenge, measurements of exhaled nitric oxide and urine cortisol. Blood cells were phenotyped, and cytokines from mononuclear blood cells were analysed. RESULTS: Perceived stress and anxiety increased in both groups during the exam period while cortisol increased only in the atopy group. Cytokine production decreased broadly in response to stress in both groups, which was paralleled by an increase in the proportion of regulatory T cells (CD4(+)CD45RO(+)CD25(bright)). Interestingly, atopic individuals, but not controls, reacted with a decreased T-helper type 1/T-helper type 2 (Th1/Th2) ratio and a decrease in natural killer (NK) cell numbers in response to stress. In control subjects only, exhaled nitric oxide decreased and forced expiratory volume in one second increased during stress. CONCLUSION: Atopic and non-atopic subjects shared some immune changes in response to stress, such as a dramatic decline in cytokines and an increase in the number of regulatory T cells in peripheral blood. However, other stress-induced immune changes were unique to atopic individuals, such as a skewed Th1/Th2 ratio and reduced NK cell numbers, indicating that some pathogenic mechanisms in atopics may be more strongly affected by stress than others.

Basophil allergen threshold sensitivity: a useful approach to anti-IgE treatment efficacy evaluation.

BACKGROUND: Monitoring of the allergen sensitivity of a patient is most important for optimal patient care and a basic prerequisite for immunomodulating treatment. The objective of this study was to investigate how basophil allergen sensitivity can be applied in the monitoring of anti-immunoglobulin E (IgE) treatment. METHODS: Basophils from timothy grass pollen allergic patients were, by flow cytometry, analysed for allergen threshold sensitivity (CD-sens) by measuring CD63 up-regulation on CD203c-identified basophils. The results were compared with maximal percentage CD63 up-regulation at one allergen dose (CD-max), skin prick test end-point allergen titration, (SPT-sens), nasal provocation titration tests (nasal provocation titre) and serum IgE and IgE antibody concentrations. RESULTS: There was a significant correlation (r = 0.50, P = 0.01) between CD-sens and SPT-sens, CD-sens and the IgE antibody concentration in percentage of 'total IgE' (relative IgE antibody concentration) (r = 0.72, P < 0.001) as well as between CD-sens and nasal provocation titre (r = 0.54, P < 0.05) but, in contrast, CD-max did not correlate with any of the sensitization parameters, i.e. SPT-sens, nasal provocation titre, absolute and relative IgE antibody concentration or CD-sens. CD-sens could be used to monitor omalizumab treatment efficacy while, based on CD-max, four of seven symptom-free patients on omalizumab would have been classified as having ongoing allergy. CONCLUSIONS: CD-sens seems to be very useful for the determination of a patient's allergen sensitivity and should be evaluated for the measurement and monitoring of anti-IgE treatment efficacy. CD-max, the conventional approach to basophil allergen challenge, which mirrors cell reactivity, gives incorrect information.

Antibody profiles and self-reported symptoms to pollen-related food allergens in grass pollen-allergic patients from northern Europe.

BACKGROUND: Most studies on pollen-related food allergy have so far focused on the association of birch/weed pollen allergens and plant food allergy. The aim of this study was to elucidate the allergen spectrum among a group of grass pollen-allergic patients from northern Europe and to relate the results to clinical histories of pollen-related food allergy. METHODS: Fifty-eight grass pollen-allergic patients answered a questionnaire regarding allergy to foods. Blood samples were taken to test IgE-reactivity to a large panel of pollen allergens and pollen- and nonpollen-related food allergens using crude allergen extracts and recombinant and native allergens. RESULTS: Three different groups of grass pollen-allergic patients were identified according to their IgE antibody profile: a grass pollen group only (19%), a grass and tree pollen group (29%) and a grass, tree and compositae (pan-) pollen group (48%). No sensitization to Bet v 1 as well as almost no IgE to plant food was observed in the grass pollen group. In contrast, nearly all patients in the two tree-related groups had IgE to Bet v 1, which reflected the high frequency of adverse reactions to typical birch-related food in these groups. Only four patients belonging to the pan-pollen group displayed IgE to profilin Phl p 12/Bet v 2. Patients in the pan-pollen group reported significantly more symptoms to food allergens compared with patients in the two other groups. The most frequently reported symptom was the oral allergy syndrome. CONCLUSIONS: Sensitization to grass pollen alone is rare among grass pollen-allergic patients from northern Europe. The majority of patients are in addition sensitized to birch (Bet v 1), which seems to be closely related to their pollen-derived food allergy. The study highlights the advantage of using well-defined allergen molecules for the diagnosis of cross-reactivity between pollen and food allergens.

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.

Lower apoptosis rate and higher CD69 expression in neutrophils from atopic individuals.

OBJECTIVE: In this study we aimed to examine the kinetics of CD69 expression and the susceptibility to apoptosis, in eosinophils and neutrophils, in the presence or absence of GM-CSF. We also addressed the question whether differences between atopic patients and healthy individuals exist in this respect. MATERIALS AND METHODS: Freshly isolated eosinophils and neutrophils from non-atopic and atopic donors were analysed by flow cytometry for Annexin/PI staining, caspase 3 activation and CD69 expression. RESULTS: We found a higher CD69 expression when atopic neutrophils were incubated with GM-CSF compared to non-atopic neutrophils, and that the kinetics of CD69-expression in neutrophils, but not in eosinophils, differed between non-atopic and atopic individuals (p < 0.004). We also found a higher viability in GM-CSF-stimulated neutrophils from non-atopic individuals as compared to neutrophils from atopic individuals (p < 0.05). CONCLUSION: These data suggest a potential role for neutrophils in the allergic inflammatory reaction through differences in apoptosis rates and CD69 expression between atopic and non-atopic individuals.

Sex dependent differences in physiological ageing in the immune system of lower airways in healthy non-smoking volunteers: study of lymphocyte subsets in bronchoalveolar lavage fluid and blood.

BACKGROUND: Age related changes in the immune system have been studied frequently but a possible relation to sex has not, to our knowledge, previously been examined. The effect of age and sex on the composition of lymphocyte subsets in bronchoalveolar lavage (BAL) fluid and peripheral blood was therefore examined. METHODS: Bronchoscopy with lavage was performed in 32 healthy non-atopic, non-smoking volunteers (16 women aged 26-63 years (mean 44) and 16 men aged 23-63 years (mean 39)). Cytospin preparations for differential counts of BAL fluid cells and surface antigen expression of lymphocytes from BAL fluid and blood were analysed by flow cytometry. RESULTS: Most parameters in the BAL fluid changed with age in women. The percentage of CD4+ lymphocytes increased with age from a mean of 48 (SD10)% in women aged < or =40 years to 69 (11)% in women aged >43 years (p=0.001). The percentage of CD8+ lymphocytes tended to decrease with age and the CD4/CD8 ratio was 5.8 (1.2) in women aged >43 years compared with 2.1 (0.7) in those aged < or =40 years (p<0.0001). Women aged >43 years differed from men aged >43 years as well as from younger subjects of both sexes with respect to CD4+ cells and CD4/CD8 ratio, and from younger women with respect to CD8+ cells. There was no age related change in the CD4/CD8 ratio in blood. No sex related differences were seen in the blood or BAL fluid of adults below the age of 40 years. CONCLUSIONS: The composition of lymphocytes with different phenotypes in the lower respiratory tract changes with age in women but not in men. This may have implications for some clinical conditions such as chronic dry cough which are observed predominantly in women.

Comparison of inflammatory responses to genetically engineered hypoallergenic derivatives of the major birch pollen allergen bet v 1 and to recombinant bet v 1 wild type in skin chamber fluids collected from birch pollen-allergic patients.

BACKGROUND: Nearly 60% of birch pollen-allergic patients react exclusively to Bet v 1. With use of the skin blister model, previously only established for installation of crude allergens, we have for the first time characterized the inflammatory response in vivo to recombinant birch pollen allergen, rBet v 1, molecules (rBet v 1 wild type, fragments and trimer). OBJECTIVE: Our purpose was to examine whether challenge with rBet v 1 derivatives (fragments and trimer) compared with rBet v 1 wild type differs with respect to influx of activated eosinophils and detectable levels of cytokines/chemokines related to allergic inflammation in skin chambers applied to birch pollen-allergic patients. METHODS: The skin blister chambers were filled for 2 hours with rBet v 1, the derivatives or PBS and heparin (negative control). The fluids were analyzed after 2 and 8 hours. The number of eosinophils was determined and EG2 and CD69 expression measured by flow cytometry. Cytokines and mediators were analyzed by ELISA and RIA techniques. RESULTS: Comparable numbers of eosinophils were recruited to the chambers challenged with rBet v 1 molecules, but the eosinophils from the rBet v 1 wild-type challenged chambers showed a significantly higher expression of CD69. The levels of eotaxin were similar in all 4 chambers, whereas rBet v 1 wild type induced significantly higher levels of histamine, eosinophil cationic protein, and GM-CSF than the derivatives did. Recombinant Bet v 1 trimer elicited significantly lower levels of IL-4 compared with rBet v1 wild type. CONCLUSION: Genetically engineered hypoallergenic rBet v 1 derivatives recruited eosinophils analogously with rBet v 1 wild type. However, the derivatives exhibited a lower capacity to activate eosinophils and to release proinflammatory mediators and T helper type 2-derived cytokines. The derivatives may therefore be candidate molecules for specific immunotherapy of birch pollen allergy with reduced risk of inducing allergenic or inflammatory side effects.

Impaired monocyte CD11b expression in interstitial inflammation in hemodialysis patients.

BACKGROUND: It is not known to what extent intravascular phenotypic alterations in adhesion molecule expression induced by hemodialysis influence the recruitment of monocytes and their ability to up-regulate CD11b at the local site of inflammation in the interstitium. Using a skin suction chamber technique, we addressed these issues in eight hemodialysis patients and in eight healthy subjects. METHODS: Two skin blisters were raised on the forearm of each individual and blister exudate collected. The blisters were then stimulated with autologous serum (active blister, intense inflammation) or buffer (control blister, intermediate inflammation), respectively. Thereafter the patients were treated with Cuprophan hemodialysis for four hours. After 10 hours, the exudate was aspirated from each chamber in all subjects. Monocyte count and expression of CD11b were analyzed in serum and blister fluid by flow cytometry. Then, monocytes from healthy blood donors were incubated in blister fluid from patients and healthy subjects in order to determine the local chemotactic activity in terms of CD11b up-regulation. Monocyte chemotactic protein-1 (MCP-1), a marker of systemic monocyte chemotactic activity, was also analyzed in serum at 0 and 10 hours in all individuals. RESULTS: The number of monocytes at the site of inflammation in the interstitium in hemodialysis patients correlated with the expression of CD11b on transmigrated cells (r = 0.78, P < 0.001). Monocytes collected in the active blister fluid of dialysis patients expressed equal levels of CD11b as cells collected from healthy subjects. By contrast, monocytes collected from the control blisters of patients expressed lower levels of CD11b than cells from healthy subjects (P < 0.01), despite equal interstitial biological activity of CD11b-mobilizing factors in blister fluid from patients and healthy subjects and the fact that patients had higher systemic chemotactic activity in terms of MCP-1 concentration in serum (P < 0.001). CONCLUSION: Monocytes from hemodialysis patients have the capacity to mobilize CD11b to the same extent as cells from healthy individuals at the inflammatory spot, but more intense stimuli are required for such actions, probably because of a transient refractoriness.

Skin test evaluation of genetically engineered hypoallergenic derivatives of the major birch pollen allergen, Bet v 1: results obtained with a mix of two recombinant Bet v 1 fragments and recombinant Bet v 1 trimer in a Swedish population before the birch pollen season.

BACKGROUND: More than 95% of birch pollen-allergic subjects react with the major birch pollen allergen, Bet v 1, and almost 60% of them are sensitized exclusively to this allergen. OBJECTIVE: The aim of this study was to compare the in vivo biologic activity of genetically engineered hypoallergenic derivatives of Bet v 1 (an equimolar mixture of 2 recombinant [r] Bet v 1 fragments and of rBet v 1 trimer) with that of rBet v 1 wild-type by skin prick and intradermal testing. METHODS: Birch pollen-allergic patients who had not received immunotherapy (n = 23), a group of allergic patients without birch pollen allergy (n = 12), and nonatopic persons (n = 8) from northern Europe (Sweden) underwent skin prick and intradermal testing with different concentrations of the recombinant allergens and commercial birch pollen extract before the birch pollen season. Immediate and late-phase reactions were recorded and allergen-specific IgE and IgG subclass responses were determined by CAP radioallergosorbent test and ELISA, respectively. RESULTS: Atopic persons without birch pollen allergy and nonatopic individuals did not have skin reactions to rBet v 1 wild-type and genetically engineered hypoallergenic derivatives. By intradermal testing, 8 of 23 and 13 of 23 birch pollen-allergic patients did not react with the highest concentration (1 microg/mL) of the rBet v 1 fragment mix and rBet v 1 trimer, respectively, compared with 1 with rBet v 1 wild type. Likewise, the highest concentration (100 microg/mL) of fragment mix or trimer failed to elicit a positive skin prick test in 18 of 23 and 15 of 23 patients in comparison with 0/23 with the monomer. No late reactions were observed. CONCLUSION: The recombinant hypoallergenic birch pollen allergens can probably be used for patient-tailored immunotherapy with a reduced risk to induce anaphylactic reactions.

Characterization of eosinophils and detection of eotaxin in skin chamber fluid after challenge with relevant allergen in patients with mild asthma.

BACKGROUND: A selective recruitment of eosinophils to sites of allergic inflammation is suggested to be controlled by regulation of cytokines, chemokines and adhesion molecules. OBJECTIVE: The aim of this study was to examine whether allergen challenge in skin chambers, applied on patients with allergic rhinitis and mild asthma, results in a selective influx of activated eosinophils and detectable levels of cytokines/chemokines related to eosinophil recruitment, such as interleukin (IL)-5 and eotaxin. METHODS: A skin blister was induced on the volar aspect of each forearm; one contained PBS-heparin buffer (control) and the other was challenged with relevant allergen. Peripheral blood was drawn before the allergen was applied to the skin chamber, and the expression of CD9, CD11b and EG2-epitope on intracellular eosinophil cationic protein (ECP) was analysed in eosinophils. Chamber fluid was collected 8 h after allergen application and analysed for differential cell counts, expression of eosinophil activity markers, the presence of ECP, eotaxin, and IL-5. RESULTS: The number of recruited leucocytes was equal in the allergen-challenged chambers and in controls. However, the number of eosinophils was significantly increased in the allergen-challenged chambers, and elevated levels of released ECP were measured. Moreover, the eosinophils recruited were activated, as shown by increased expression of EG2 and CD11b, and decreased expression of CD9, in comparison with blood eosinophils. In the skin chamber fluids, higher levels of eotaxin were detected in the allergen-challenged chambers than in controls, but there were no detectable levels of IL-5. CONCLUSION: We have demonstrated a selective recruitment of eosinophils, and higher levels of released ECP and eotaxin, in skin chambers stimulated with allergen, as compared with control chambers. Allergen challenge in skin chambers is a useful tool for studies of eosinophil recruitment, their state of activation, and their involvement in the allergic inflammatory response.

Allergen-induced accumulation of eosinophils and lymphocytes in skin chambers is associated with increased levels of interleukin-4 and sVCAM-1.

BACKGROUND: The aim of the study was to characterize the kinetic accumulation of various inflammatory mediators in allergen-challenged skin chambers applied on patients with pollen-related allergic rhinitis/mild asthma. METHODS: Skin blisters were induced on the forearms and challenged with allergen or phosphate-buffered saline (PBS). Peripheral blood was drawn before and 8 h after challenge for analysis of differential cell counts, sVCAM-1, and alpha2-macroglobulin. Chamber fluids, collected at 1, 4, and 8 h after allergen application, were analyzed for differential cell counts, histamine, interleukin (IL)-4, sVCAM-1, and alpha2-macroglobulin. RESULTS: The number of recruited leukocytes was equal in allergen and PBS chambers; however, the numbers of eosinophils and lymphocytes were significantly (P< or =0.05) elevated in allergen-challenged chambers at 8 h. Compared to PBS chambers, allergen chambers contained significantly (P<0.01-0.05) higher levels of histamine (at 1 and 4 h), IL-4 (at 4 and 8 h), alpha2-macroglobulin (at 1 and 8 h), and sVCAM-1 (at 1 and 8 h). In contrast to alpha2-macroglobulin, levels of sVCAM-1 in peripheral blood were significantly (P<0.05) increased at 8 h. CONCLUSIONS: Increased levels of sVCAM-1 and IL-4 in allergen-challenged chambers, in parallel with increased recruitment of eosinophils and lymphocytes, points to the participation of IL-4 and VCAM-1 in the development of the late-phase reaction. Increased levels of sVCAM-1 in allergen-challenged chambers probably reflects a combination of leakage and local production.

Increased levels of IL-5 positive peripheral blood eosinophils and lymphocytes in mild asthmatics after allergen inhalation provocation.

BACKGROUND: In allergic inflammation eosinophils and TH2-like lymphocytes are supposed to be the major effector cells and considered to contribute as cellular source of the key cytokine interleukin (IL)-5. OBJECTIVE: The purpose of this study was to enable detection of IL-5 containing leucocytes and to investigate whether the number of these cells in the blood circulation differed between healthy and asthmatics before and after allergen provocation. METHODS: The distribution of intracellular IL-5 in human peripheral blood eosinophils (PBE) and lymphocytes (PBL) has been investigated using fixation and cell membrane permeabilization with octyl-glucopyranoside, the FOG-method, and flow cytometry. The intracellular staining was performed on leucocytes without any prior purification and in vitro stimulation. The specificity of IL-5 binding to intracellular compartment of both PBE and PBL was confirmed by complete inhibition with human recombinant IL-5. RESULTS: Preformed intracellular IL-5 was detected in the main population of PBE (> 70%) in both healthy individuals and asymptomatic patients. Moreover, preformed intracellular IL-5 was also detected in 4.8% and 2.4% of PBL from healthy individuals and asymptomatic patients, respectively. There was a correlation between the absolute number of PBE and IL-5 positive PBE. In patients with pollen-related asthma, the number of IL-5 positive PBE and PBL increased significantly 24 h after an allergen inhalation provocation (P < 0.05). In the healthy control group no differences regarding IL-5 positive PBE and PBL were obtained pre- and post-allergen challenge. CONCLUSIONS: In patients with mild allergic asthma, but not in healthy individuals, allergen provocation induces an increased absolute number of IL-5 positive PBE and PBL. The reason for the relatively high number of IL-5 positive PBL is unclear, but a plausible explanation might be that other lymphocyte subsets besides CD4+ TH2 can produce IL-5. However, enumeration of IL-5 positive leucocytes may be used as an activity marker and also be a useful tool in monitoring the inflammation in asthma.