Birch pollen allergy in Europe.

Birch and other related trees of the families Betulaceae and Fagaceae (alder, hazel, oak, hornbeam, chestnut, and beech) constitute the birch homologous group. This grouping is primarily based on the extensive IgE cross-reactivity of allergen homologs to the major birch allergen Bet v 1. Birch pollen is the most dominant tree pollen in Northern and Central Europe and is a major cause of allergic rhinitis and, possibly, asthma symptoms. Over the last few decades, levels of birch pollen have risen and the period of exposure has increased due to climate changes. Subsequently, the prevalence of birch pollen sensitization has also increased. The cross-reactivity and sequential pollen seasons within the birch homologous group create a prolonged symptomatic allergy period beyond birch pollen alone. Furthermore, many plant food allergens contain homologs to Bet v 1, meaning that the majority of patients with birch pollen allergy suffer from secondary pollen food syndrome (PFS). As a result, the negative impact on health-related quality of life (HRQoL) in patients allergic to birch pollen is significant. The purpose of this manuscript was to narratively review topics of interest such as taxonomy, cross-reactivity, prevalence, clinical relevance, PFS, and HRQoL with regard to birch pollen allergy from a European perspective.

BSACI guideline for the diagnosis and management of peanut and tree nut allergy.

Peanut nut and tree nut allergy are characterised by IgE mediated reactions to nut proteins. Nut allergy is a global disease. Limited epidemiological data suggest varying prevalence in different geographical areas. Primary nut allergy affects over 2% of children and 0.5% of adults in the UK. Infants with severe eczema and/or egg allergy have a higher risk of peanut allergy. Primary nut allergy presents most commonly in the first five years of life, often after the first known ingestion with typical rapid onset IgE-mediated symptoms. The clinical diagnosis of primary nut allergy can be made by the combination of a typical clinical presentation and evidence of nut specifc IgE shown by a positive skin prick test (SPT) or specific IgE (sIgE) test. Pollen food syndrome is a distinct disorder, usually mild, with oral/pharyngeal symptoms, in the context of hay fever or pollen sensitisation, which can be triggered by nuts. It can usually be distinguish clinically from primary nut allergy. The magnitude of a SPT or sIgE relates to the probability of clinical allergy, but does not relate to clinical severity. SPT of ≥ 8 mm or sIgE ≥ 15 KU/L to peanut is highly predictive of clinical allergy. Cut off values are not available for tree nuts. Test results must be interpreted in the context of the clinical history. Diagnostic food challenges are usually not necessary but may be used to confirm or refute a conflicting history and test result. As nut allergy is likely to be a long-lived disease, nut avoidance advice is the cornerstone of management. Patients should be provided with a comprehensive management plan including avoidance advice, patient specific emergency medication and an emergency treatment plan and training in administration of emergency medication. Regular re-training is required.

Biomarkers for monitoring clinical efficacy of allergen immunotherapy for allergic rhinoconjunctivitis and allergic asthma: an EAACI Position Paper.

BACKGROUND: Allergen immunotherapy (AIT) is an effective treatment for allergic rhinoconjunctivitis (AR) with or without asthma. It is important to note that due to the complex interaction between patient, allergy triggers, symptomatology and vaccines used for AIT, some patients do not respond optimally to the treatment. Furthermore, there are no validated or generally accepted candidate biomarkers that are predictive of the clinical response to AIT. Clinical management of patients receiving AIT and efficacy in randomised controlled trials for drug development could be enhanced by predictive biomarkers. METHOD: The EAACI taskforce reviewed all candidate biomarkers used in clinical trials of AR patients with/without asthma in a literature review. Biomarkers were grouped into seven domains: (i) IgE (total IgE, specific IgE and sIgE/Total IgE ratio), (ii) IgG-subclasses (sIgG1, sIgG4 including SIgE/IgG4 ratio), (iii) Serum inhibitory activity for IgE (IgE-FAB and IgE-BF), (iv) Basophil activation, (v) Cytokines and Chemokines, (vi) Cellular markers (T regulatory cells, B regulatory cells and dendritic cells) and (vii) In vivo biomarkers (including provocation tests?). RESULTS: All biomarkers were reviewed in the light of their potential advantages as well as their respective drawbacks. Unmet needs and specific recommendations on all seven domains were addressed. CONCLUSIONS: It is recommended to explore the use of allergen-specific IgG4 as a biomarker for compliance. sIgE/tIgE and IgE-FAB are considered as potential surrogate candidate biomarkers. Cytokine/chemokines and cellular reponses provided insight into the mechanisms of AIT. More studies for confirmation and interpretation of the possible association with the clinical response to AIT are needed.

Lipid transfer protein sensitization: reactivity profiles and clinical risk assessment in an Italian cohort.

BACKGROUND: Nonspecific lipid transfer proteins (nsLTPs) represent a major cause of systemic food allergic reactions in the Mediterranean area. This study investigate hierarchical patterns and cluster relationships of IgE sensitization to different nsLTPs, and the relationship to clinical allergy in a large Italian cohort. METHODS: A total of 568 nsLTP-positive subjects after IgE ImmunoCAP-ISAC microarray analysis with Ara h 9, Art v 3, Cor a 8, Jug r 3, Pla a 3, Pru p 3 and Tri a 14 allergens were studied. IgE inhibition experiments were carried out with mugwort and plane tree pollen extracts. RESULTS: Eighty-two per cent of nsLTP-positive participants (94% if <6 years old) were Pru p 3(pos) , and 71% were Jug r 3(pos) . Participants who reacted to >5 nsLTPs reported a higher incidence of food-induced systemic reactions. Only Art v 3 and Pla a 3 (mugwort and plane tree nsLTPs, respectively) were associated with respiratory symptoms, and a correlation was observed between sensitization to pollen and plant food nsLTPs, particularly between Pla a 3 and tree nut/peanut nsLTPs. Co-sensitization to Par j 2 and PR-10 or profilin pan-allergens was associated with a lower prior prevalence of severe food-induced reactions. In inhibition assays, plane and mugwort pollen extracts inhibited 50-100% of IgE binding to food nsLTPs in microarrays. CONCLUSIONS: Testing IgE reactivity to a panel of nsLTP allergens unveils important associations between nsLTP sensitization profiles and clinical presentation and allows the identification of novel cluster patterns indicating likely cross-reactivities and highlighting potential allergens for nsLTP immunotherapy.

Parasitic worm therapy for allergy: is this incongruous or avant-garde medicine?

Helminth (worm) therapy has already been used in clinical trials associated with allergy. These were generally small scale, safety orientated trials of short duration, justified by epidemiological and experimental data indicating potentially beneficial immune modulation by some parasites. However, parasites by definition are disadvantageous to their hosts, and helminth infection in particular almost invariably induces an allergic phenotype, rendering this somewhat paradoxical therapeutic approach for allergy open to scrutiny. Is parasitic worm therapy for allergy incongruous medicine, or avant-garde medicine? In the present article, we assess the strength of evidence supporting the use of helminth therapy for allergy and critically appraise the trials already completed. Then, should this approach prove successful, we suggest strategies to improve the delivery of helminth therapy, and ways to discover immune response modifiers derived from worms.

Development and validation of a structured questionnaire for the diagnosis of oral allergy syndrome in subjects with seasonal allergic rhinitis during the UK birch pollen season.

BACKGROUND: Birch pollen-associated oral allergy syndrome, also known as pollen-food syndrome (PFS), is the most common food allergy in adults in the United Kingdom. Because of its characteristic rapid onset of oro-pharyngeal symptoms associated with specific plant foods, it was hypothesized that a history-based questionnaire could accurately diagnose PFS in subjects with rhino-conjunctivitis symptoms in the UK springtime. OBJECTIVE: In this study of diagnostic accuracy, we aimed to validate a simple PFS diagnostic questionnaire and algorithm against a reference diagnostic test method (RTM) comprising diagnosis by expert evaluation of clinical history, skin prick tests and oral food challenge, in subjects reporting allergic rhinitis (AR) in the UK birch pollen season from March to May. METHODS: Participants were UK adults reporting symptoms of spring time-AR (hayfever). They self-completed a diagnostic questionnaire in addition to undergoing an RTM comprising clinical history, skin prick testing to foods and pollens and oral food challenge. Subjects who reported anaphylaxis were excluded on the basis that they required specialist referral. RESULTS: One hundred and twenty three subjects took part in the study. Data from 110 participants were analysed; of the 13 exclusions, four provided insufficient data and nine reported anaphylaxis such that they warranted specialist assessment. Fifty-two participants (47%) were diagnosed with PFS by the RTM in comparison with 51 (46%) by a diagnostic questionnaire and algorithm (P=1.000, McNemar's test). The diagnostic questionnaire and algorithm had a sensitivity of 0.90 (0.78-0.96), a specificity of 0.93 (0.82-0.97), a positive predictive value of 0.92 (0.80-0.97) and a negative predictive value of 0.91 (0.80-0.96) when measured against the RTM. CONCLUSION AND CLINICAL RELEVANCE: The diagnostic questionnaire and algorithm is a practical and robust tool, which enables rapid identification, and therefore management, of individuals with PFS who experience rhino-conjunctivitis symptoms in the UK birch pollen season. Registered with CinicalTrials.Gov. registration number NCT00854958.

Immunotherapy for allergic rhinitis.

Allergic rhinitis (AR) affects more than 20% of the population in the United Kingdom and western Europe and represents a major cause of morbidity that includes interference with usual daily activities and impairment of sleep quality. This guidance prepared by the Standards of Care Committee (SOCC) of the British Society for Allergy and Clinical Immunology (BSACI) is for the management of AR in patients that have failed to achieve adequate relief of symptoms despite treatment with intranasal corticosteroids and/or antihistamines. The guideline is based on evidence and is for use by both adult physicians and paediatricians practising allergy. During the development of these guidelines, all BSACI members were included in the consultation process using a web-based system. Their comments and suggestions were carefully considered by the SOCC. Where evidence was lacking, consensus was reached by the experts on the committee. Included in this guideline are indications and contraindications for immunotherapy, criteria for patient selection, the evidence for short- and long-term efficacy of subcutaneous and sublingual immunotherapy, and discussion on safety and the different modes of immunotherapy including, pre-seasonal and co-seasonal treatments. There are sections on children, allergen standardization, vaccines used in the United Kingdom, oral allergy syndrome, cost effectiveness of immunotherapy and practical considerations of undertaking immunotherapy including recommendations on who should undertake immunotherapy and dosing schedules. Finally, there is discussion on potential biomarkers of response to immunotherapy, the use of component-resolved diagnostics, novel approaches, alternative routes and potential areas for future research.

CC chemokine receptor 4 (CCR4) in human allergen-induced late nasal responses.

BACKGROUND: CC Chemokine receptor 4 (CCR4) is preferentially expressed on Th2 lymphocytes. CCR4-mediated inflammation may be important in the pathology of allergic rhinitis. Disruption of CCR4 - ligand interaction may abrogate allergen-induced inflammation. METHODS: Sixteen allergic rhinitics and six nonatopic individuals underwent both allergen and control (diluent) nasal challenges. Symptom scores and peak nasal inspiratory flow were recorded. Nasal biopsies were taken at 8 h post challenge. Sections were immunostained and examined by light or dual immunofluorescence microscopy for eosinophils, T-lymphocytes, CCR4(+)CD3(+) and CXCR3(+)CD3(+) cells and examined by in situ hybridization for CCR4, IL-4 and IFN-gamma mRNA(+) cells. Peripheral blood mononuclear cells were obtained from peripheral blood of nine normal donors and the CCR4(+)CD4(+) cells assessed for actin polymerization in response to the CCR4 ligand macrophage-derived chemokine (MDC/CCL22) and the influence of a CCR4 antagonist tested. RESULTS: Allergic rhinitics had increased early and late phase symptoms after allergen challenge compared to diluent; nonatopics did not respond to either challenge. Eosinophils, but not total numbers of CD3(+) T cells, were increased in rhinitics following allergen challenge. In rhinitics, there was an increase in CCR4(+)CD3(+) protein-positive cells relative to CXCR3(+)CD3(+) cells; CCR4 mRNA+ cells were increased and IL-4 increased to a greater extent than IFN-gamma. CCR4(+)CD4(+) T cells responded to MDC in vitro, and this response was inhibited by the selective CCR4 antagonist. CONCLUSION: Lymphocyte CCR4 expression is closely associated with induction of human allergen-induced late nasal responses. Blocking CCR4-ligand interaction may provide a novel therapeutic approach in allergic disease.

Elevated CCR6+ CD4+ T lymphocytes in tissue compared with blood and induction of CCL20 during the asthmatic late response.

CCR6 is expressed by multiple leucocyte subsets, including peripheral blood memory T cells, and mouse models implicate a role for this receptor in diverse inflammatory responses that include allergic airway disorders, inflammatory bowel disease and autoimmune encephalitis. In order to study the role of CCR6 in humans, we have investigated the patterns of CCR6 expression and function on T cells from the peripheral blood, skin, nose and lung, in health and in allergic disease. Results show that CCR6 was expressed consistently on a higher proportion of tissue versus peripheral blood-derived CD4+ T cells (P < 0.01). CCR6 was expressed predominantly on CD4+ compared with CD8+ cells in both blood- and tissue-derived T cells (P < 0.001). The number of cells showing CCR6 expression was not proportionally greater in peripheral blood or nasal mucosal T cells of subjects with symptomatic allergic rhinitis. CCR6+ cells demonstrated enhanced functional responses to CCL20 and CCL20 was increased in bronchoalveolar lavage fluid of asthmatics following endobronchial allergen provocation (P < 0.05). Thus, CCR6 may be important in the regulation of T cell recruitment to tissue and up-regulation of CCL20 expression may contribute to the recruitment and/or retention of effector T cells in allergic asthma.

T lymphocytes expressing CCR3 are increased in allergic rhinitis compared with non-allergic controls and following allergen immunotherapy.

BACKGROUND: In T cell-associated allergic inflammation, homing of T-helper 2 (Th2) effector cells to mucosal sites may be influenced by chemokine receptor expression. Previous studies have identified CCR3 and CCR4 as putative markers of Th2 cells and CCR5 and CXCR3 as markers of Th1 cells. The aim of this study was to assess differential chemokine receptor expression from symptomatic atopic grass pollen-sensitive subjects, compared with patients on high-dose allergen injection immunotherapy (IT) and healthy controls. METHODS: We examined chemokine receptor expression (CCR1-7 and CXCR1-4) by flow cytometry of peripheral blood CD4+ and CD8+ T cells. We also depleted peripheral blood mononuclear cell (PBMC) populations of CCR3+ CD4+ cells by magnetic bead separation and cells were stimulated with grass pollen allergen for 6 days. Cytokine production was measured by enzyme-linked immunosorbent assay. RESULTS: On freshly isolated PBMC, atopic individuals exhibited increased numbers of CCR3+ CD4+ cells compared with normal controls (P < 0.01). CCR3 expression in IT patients was reduced compared with matched atopic rhinitic controls (P < 0.05) and comparable with that observed in normal subjects. Depletion of CCR3+ CD4+ cells from allergen-stimulated PBMC cultures resulted in decreased interleukin (IL)-5 production compared with whole CD4+ populations (P < 0.05). Freshly isolated CCR3+ CD4+ cells have significantly higher intracellular IL-4 and lower IFN-gamma levels than CCR3- CD4+ cells. CD4+ T cells cultured from both peripheral cells and nasal biopsies demonstrated increased expression of CCR3 in the presence of IL-4 (P < 0.05). CONCLUSION: CCR3+ CD4+ T cells are increased in allergic rhinitis, are reduced by allergen IT, have a Th2 phenotype and contribute to allergen-specific responses. Strategies against CCR3+ T cells may be effective in human allergic diseases.

Immunologic changes associated with allergen immunotherapy.

Specific allergen injection immunotherapy is highly effective in selected patients with IgE-mediated disease, including respiratory allergy and venom anaphylaxis. Research in this area provides insight into the immunologic basis of allergic disease and may assist in the development of more highly targeted treatment. Immunotherapy reduces immediate allergen-induced symptoms and concentrations of inflammatory mediators, including histamine and prostaglandin D2, in ragweed-sensitive patients. Similarly, nasal epithelial mast cell numbers are decreased. A characteristic feature of immunotherapy is its ability to inhibit late-phase responses. In the nose it is accompanied by a decrease in eosinophil numbers in lavage during late responses. Characteristic changes in serum immunoglobulins are found, with an initial increase in IgE followed by a blunting of seasonal increases in IgE in pollen-sensitive patients and a gradual decline in allergen-specific IgE levels over several years. This is accompanied by an increase in allergen-specific IgG (blocking antibodies), although neither appear to correlate closely with the clinical response to immunotherapy. One way in which immunotherapy may act is by modifying the T-lymphocyte response to subsequent natural allergen exposure. Studies in peripheral blood and within the target organ have demonstrated a shift in the balance of T-cell subsets away from TH2-type (producing particularly IL-4 and IL-5) in favor of a TH1-type T-lymphocyte response (with the preferential production of IFN-gamma). Studies of the nasal mucosa before and after immunotherapy have demonstrated suppression of the late nasal response and increases in the numbers of cells expressing mRNA for IFN-gamma. It is not clear whether this immune deviation is due to anergy of TH2/TH0 cells or increases in TH0/TH1 T-lymphocyte responses. An alternative may be amplification of suppressor CD8+ T cells, which may have a downregulatory effect. Novel approaches currently being explored include the use of T-cell reactive peptides, which might circumvent the risk of anaphylaxis, and the use of adjuvants such as IL-12 or mycobacterial vaccines to potentiate the effects of allergen in inducing immune deviation.

A simplified physical model of pressure wave dynamics and acoustic wave generation induced by laser absorption in the retina.

Shock waves have been proposed in the literature as a mechanism for retinal damage induced by ultra-short laser pulses. For a spherical absorber, we derive a set of linear equations describing the propagation of pressure waves. We show that the formation of shock fronts is due to the form of the absorber rather than the inclusion of nonlinear terms in the equations. The analytical technique used avoids the need for a Laplace transform approach and is easily applied to other absorber profiles. Our analysis suggests that the 'soft' nature of the membrane surrounding retinal melanosomes precludes shock waves as a mechanism for the retinal damage induced by ultra-short pulse lasers. The quantitative estimates of the pressure gradients induced by laser absorption which are made possible by this work, together with detailed meso-scale or molecular modelling, will allow alternative damage mechanisms to be identified.

T lymphocytes in asthma: bronchial versus peripheral responses.

Recent evidence points to the recruitment of T(H)2 cells, phenotype T lymphocytes, their activation, and the generation of T(H)2 cytokines, particularly IL-4 and IL-5, in both peripheral blood and bronchial mucosa of asthmatic patients, leading to local tissue eosinophilia and IgE-dependent mast-cell activation. Activation of T(H)2 T lymphocytes appears to be specific for asthma (as opposed to airway obstructive disease) and was shown to correlate with asthma severity as evidenced by the inverse correlation between CD25(+)/CD4(+) cells and peak expiratory flow rates. These findings support the fundamental importance of T-lymphocyte responses in bronchial asthma and delineate potential therapeutic strategies, such as broad-based immunosuppression versus a more selective approach targeted against CD4(+) T lymphocytes. The high efficacy of topical treatments (ie, inhalation) supports the notion that changes that are detectable in peripheral blood merely reflect a "spill-over" of local T-lymphocyte responses in the target organ. Conversely, the multiple systemic manifestations of allergy (such as allergic rhinitis and atopic dermatitis in atopic patients) support systemic therapeutic approaches.

CCR4 ligands are up-regulated in the airways of atopic asthmatics after segmental allergen challenge.

T-helper (Th) 2 cytokines are thought to mediate most features of allergic inflammation in atopic asthma. However, it remains unclear whether chemokine pathways direct selective recruitment of Th2 cells to the airways during human allergic responses. Bronchoalveolar lavage (BAL) was performed in 15 nonsmoking mild atopic asthmatics before and 24 h after a fibreoptic segmental allergen challenge, and chemokines related to T-cell recruitment were assayed by ELISA. The Th2-related C-C chemokine (CCR)4 ligands, macrophage-derived chemokine/C-C chemokine ligand (CCL)22 and thymus and activation-regulated chemokine/CCL17, were increased in BAL after challenge. These chemokines correlated significantly with lymphocyte numbers and with interleukin (IL)-5 and IL-13 in post-challenge BAL. In contrast, two out of three putative Th1-related chemokines did not change. There were no alterations in monokine induced by interferon (IFN)-gamma/CXC chemokine ligand (CXCL)9 or macrophage inflammatory protein-1alpha/CCL3; whereas a significant increase in IFN-induced protein-10kDa/CXCL10 was observed, which did not correlate with the T-cell influx. In peripheral mononuclear cells from atopic donors, CCL22 and CCL17 were induced by IL-4 and IL-13, further supporting the relationship between CCL22/CCL17 and Th2 cytokines. Finally, CCL22 was able to trigger actin polymerisation in peripheral CD4+ T-cells expressing CCR4. Thus, C-C chemokine receptor 4 ligands are up-regulated in the airways of atopic asthmatics following allergen exposure, contribute to the T-cell influx to the airways and are closely related to the Th2-cytokine response.

The topical glucocorticoids beclomethasone dipropionate and fluticasone propionate inhibit human T-cell allergen-induced production of IL-5, IL-3 and GM-CSF mRNA and protein.

BACKGROUND: T-cell production of eosinophil-active cytokines (IL-5, IL-3, GM-CSF) is thought to be fundamental to asthma pathogenesis. Inhaled aeroallergens may be one important stimulus for T-cell cytokine production in asthma. OBJECTIVE: To compare the potency and efficacy of the topical anti-asthma glucocorticoids beclomethasone dipropionate (BDP) and fluticasone propionate (FP) in inhibiting allergen-driven peripheral blood T-cell proliferation and production of IL-3, IL-5 and GM-CSF mRNA and protein. METHODS: Peripheral blood mononuclear cells from six atopic asthmatics sensitized to house dust mite (HDM) were cultured in the presence of HDM and serial dilutions of BDP or FP in vitro. Cellular proliferation (7 days) and culture supernatant cytokine concentrations (6 days) were measured by uptake of tritiated thymidine and ELISA, respectively. Cytokine mRNA expression (24 h) was measured in three subjects using a quantitative PCR technique. RESULTS: Both BDP and FP inhibited allergen-induced T-cell proliferation, expression of IL-3, IL-5 and GM-CSF mRNA, and secretion of the corresponding proteins in a concentration-dependent fashion. FP was considerably more potent, but not more efficacious, in exerting these actions. CONCLUSIONS: Both BDP and FP have the potential markedly to inhibit allergen-induced T-cell production of asthma-relevant cytokines. This activity is effected at the level of T-cell proliferation and cytokine gene transcription. These properties may be key features of the anti-asthma activity of these drugs. The greater potency of FP in vitro may be responsible for its greater clinical potency.

A new model for laser-induced thermal damage in the retina.

We describe a new model for laser-induced retinal damage. Our treatment is prompted by the failure of the traditional approach to accurately describe the image size dependence of laser-induced retinal injuries and by a recently reported study which demonstrated that laser injuries to the retina might not appear for up to 48 h post exposure. We propose that at threshold a short-duration, laser-induced, temperature rise melts the membrane of the melanosomes found in the pigmented retinal epithelial cells. This results in the generation of free radicals which initiate a slow chain reaction. If more than a critical number of radicals are generated then cell death may occur at a time much later than the return of the retina to body temperature. We show that the equations consequent upon this mechanism result in a good fit to the recent image size data although more detailed experimental data for rate constants of elementary reactions is still required. This paper contributes to the current understanding of damage mechanisms in the retina and may facilitate the development of new treatments to mitigate laser injuries to the eye. The work will also help minimize the need for further animal experimentation to set laser eye safety standards.

Evaluation of CD4+ T cells proliferating to grass pollen in seasonal allergic subjects by flow cytometry.

Our objective was to characterize T-cell responses to Phleum pratense in grass pollen allergic individuals and healthy controls using the fluorescent dye PKH26. Peripheral blood mononuclear cells were stimulated with P. pratense, or with recall antigens, and CD3+/CD4+ and CD3+/CD8+ T-cells that had proliferated were analysed by flow cytometry. In the presence of P. pratense CD4+/CD3+ T-cells proliferated more in grass pollen sensitive atopic patients than in nonallergic controls or in nongrass pollen sensitive atopic subjects. PPD and TT recall antigens elicited uniformly high proliferation in all T-cell subsets. Only half of pollen sensitive patients also had an increased proliferation of CD3+/CD8+ T-cells in response to P. pratense. We determined precursor frequency of CD4+ T cells in the original population that responded to P. pratense and found values ranging from 1 x 10-3 to 0.6 x 10-1, in the same range as those measured for PPD and TT. In conclusion, grass pollen sensitive atopic patients show enhanced CD4+ T-cell reactivity to P. pratense, and this could be related to the presence of elevated numbers of circulating allergen-specific CD4+ T cells. This flow cytometric method should allow the identification of other phenotypic markers such as intracellular cytokines in allergen specific responding CD4+ T cells.

Unstimulated basophils in atopic and nonatopic subjects express intracellular interleukin-4: detection by flow cytometry.

BACKGROUND: IgE-stimulated cultured basophils from atopic subjects are capable of secreting interleukin-4 (IL-4). We describe a flow-cytometric technique which identified intracellular IL-4 in unstimulated basophils unseparated from peripheral blood mononuclear cells (PBMC) in both atopic (AT) and nonatopic (NC) volunteers. METHODS: Freshly isolated PBMC were fixed in 4% paraformaldehyde (PFA). Surface staining with 22E7, a noncompetitive anti-FcepsilonRI-alpha antibody, allowed identification of basophils. Permeabilization by 0.1% saponin allowed staining of intracellular cytokines with specific monoclonal antibodies (mAbs). Two series of experiments utilizing different protocols and anticytokine mAbs were performed. The first protocol required a two-stage fluorochrome staining technique. The availability of fluorochrome-conjugated mAbs allowed a simpler, one-stage labelling procedure for the second protocol. RESULTS: With the first protocol, IL-4 (but not IFN-gamma), immunoreactivity was detectable in a majority (median 77%) of peripheral blood basophils from both AT and NC subjects (n=8). Basophil IL-4 immunoreactivity was again evident in experiment 2 but did not differ significantly between AT and NC subjects--either evaluated as percentage of IL-4+ basophils (AT median=66%, NC median=38.4%, P=0.41) or IL-4-specific mean fluorescence (AT median=0.85, NC median=0.3, P=0.07). CONCLUSIONS: This simple technique allowed the study of intracellular cytokine expression in unstimulated blood basophils. It demonstrated constitutive basophil expression of IL-4 (but not IFN-gamma) in all subjects, with no significant increases in atopics.

Relationship between IL-4 and IL-5 mRNA expression and disease severity in atopic asthma.

Atopic asthma is characterized by chronic inflammation of the bronchial mucosa in which eosinophil- and immunoglobulin E (IgE)-dependent mechanisms are believed to be prominent. Therefore, specific proeosinophilic mediators such as interleukin (IL)-5 and essential cofactors for IgE switching in B-lymphocytes such as IL-4 could play a pivotal role in asthma. However, the exact role that individual inflammatory mediators play in the development of the disease in humans is still unknown. Using semiquantitative reverse transcriptase-polymerase chain reaction amplification in bronchial biopsies from 10 atopic asthmatics, we have tested the hypothesis that IL-4 and IL-5 mRNA expression relative to beta-actin mRNA correlates with validated indicators of disease severity. IL-4 and IL-5 mRNA copies relative to beta-actin mRNA were detected in bronchial biopsies from atopic asthmatics. The numbers of IL-5 mRNA copies relative to beta-actin mRNA correlated with disease severity assessed by the Aas asthma score (r = 0.70, p = 0.01), baseline FEV1 (r = -0.94, p = 0.001), baseline peak expiratory flow rate (r = -0.77, p = 0.01), peak expiratory flow rate variability over 2 wk (r = 0.69, p = 0.028), and the histamine PC20 (r = -0.72, p = 0.018). Conversely, the numbers of IL-4 mRNA copies relative to beta-actin mRNA did not correlate with asthma severity, but they positively correlated with total serum IgE concentrations (r = -0.90, p = 0.001). Our present results support the concept that IL-5 may determine asthma clinical expression and severity, and by inference they support the development of IL-5 targeted therapies.

Costimulation through CD86 is involved in airway antigen-presenting cell and T cell responses to allergen in atopic asthmatics.

Atopic allergic asthma is characterized by activation of Th2-type T cells in the bronchial mucosa. Previous reports have suggested an important role for costimulation through the CD28/CTLA4-CD80/CD86 pathway in allergen activation of T cells in animal models of inhaled allergen challenge. However, human allergen-specific lines and clones were reported to be costimulation independent. We therefore examined CD80 and CD86 dependence of allergen-induced T cell proliferation and cytokine production in peripheral blood and bronchoalveolar lavage from atopic asthmatic subjects and controls. Both allergen-induced proliferation and IL-5 production from PBMC were inhibited by CTLA4-Ig fusion protein and anti-CD86, but not anti-CD80 mAbs. When allergen-specific CD4+ T cell lines from peripheral blood were examined, proliferation and cytokine production were found to be independent of CD80 or CD86 costimulation. However, when cells obtained directly from the airways were examined, allergen-induced proliferation of bronchoalveolar lavage T cells from atopic asthmatic subjects was inhibited by anti-CD86 but not anti-CD80. In addition, bronchoalveolar lavage-adherent cells from asthmatic, but not control subjects showed APC activity to autologous T cells. This was also inhibited by anti-CD86 but not anti-CD80. Thus allergen-induced T cell activation and IL-5 production in the airway in asthmatic subjects is susceptible to blockade by agents interfering with costimulation via CD86, and this may hold therapeutic potential in asthma.