Influence of meteorological variables and air pollutants on measurements from automatic pollen sampling devices.

This study examines the influence of meteorological factors and air pollutants on the performance of automatic pollen monitoring devices, as part of the EUMETNET Autopollen COST ADOPT-intercomparison campaign held in Munich, Germany, during the 2021 pollen season. The campaign offered a unique opportunity to compare all automatic monitors available at the time, a Plair Rapid-E, a Hund-Wetzlar BAA500, an OPC Alphasense, a KH-3000 Yamatronics, three Swisens Polenos, a PollenSense APS, a FLIR IBAC2, a DMT WIBS-5, an Aerotape Sextant, to the average of four manual Hirst traps, under the same environmental conditions. The investigation aimed to elucidate how meteorological factors and air pollution impact particle capture and identification efficiency. The analysis showed coherent results for most devices regarding the correlation between environmental conditions and pollen concentrations. This reflects on one hand, a significant correlation between weather and airborne pollen concentration, and on the other hand the capability of devices to provide meaningful data under the conditions under which measurements were taken. However, correlation strength varied among devices, reflecting differences in design, algorithms, or sensors used. Additionally, it was observed that different algorithms applied to the same dataset resulted in different concentration outputs, highlighting the role of algorithm design in these systems (monitor + algorithm). Notably, no significant influence from air pollutants on the pollen concentrations was observed, suggesting that any potential difference in effect on the systems might require higher air pollution concentrations or more complex interactions. However, results from some monitors were affected to a minor degree by specific weather variables. Our findings suggest that the application of real-time devices in urban environments should focus on the associated algorithm that classifies pollen taxa. The impact of air pollution, although not to be excluded, is of secondary concern as long as the pollution levels are similar to a large European city like Munich.

Spatial transcriptomics landscape of lesions from non-communicable inflammatory skin diseases.

Abundant heterogeneous immune cells infiltrate lesions in chronic inflammatory diseases and characterization of these cells is needed to distinguish disease-promoting from bystander immune cells. Here, we investigate the landscape of non-communicable inflammatory skin diseases (ncISD) by spatial transcriptomics resulting in a large repository of 62,000 spatially defined human cutaneous transcriptomes from 31 patients. Despite the expected immune cell infiltration, we observe rather low numbers of pathogenic disease promoting cytokine transcripts (IFNG, IL13 and IL17A), i.e. >125 times less compared to the mean expression of all other genes over lesional skin sections. Nevertheless, cytokine expression is limited to lesional skin and presented in a disease-specific pattern. Leveraging a density-based spatial clustering method, we identify specific responder gene signatures in direct proximity of cytokines, and confirm that detected cytokine transcripts initiate amplification cascades of up to thousands of specific responder transcripts forming localized epidermal clusters. Thus, within the abundant and heterogeneous infiltrates of ncISD, only a low number of cytokine transcripts and their translated proteins promote disease by initiating an inflammatory amplification cascade in their local microenvironment.

Microbial dysbiosis in a mouse model of atopic dermatitis mimics shifts in human microbiome and correlates with the key pro-inflammatory cytokines IL-4, IL-33 and TSLP.

BACKGROUND: Cutaneous bacterial dysbiosis is a characteristic hallmark of atopic dermatitis (AD), and it decisively influences the severity of the disease. Despite this, frequently used murine models of AD have not been characterized regarding the changes in skin microbiome communities. OBJECTIVE: To analyse the skin microbiome of two frequently used murine models for AD for assessing their applicability in translational research. METHODS: AD was induced in mice by topical application of calcipotriol or oxazolone. Following comparable elicitation of AD-like dermatitis, including IgE induction, the skin microbial communities were analysed and compared with human AD. RESULTS: We detected critical differences in the microbiota composition of diseased skin. In contrast to calcipotriol treatment, application of oxazolone induced significant changes in the cutaneous microbiota and a drastic drop of bacterial richness. Furthermore, an expansion of Staphylococci, particularly S. xylosus, was observed in the oxazolone group, also displaying positive correlations with AD key markers including pH, TEWL, IL-4, TSLP and IL-33. CONCLUSIONS: In this article, we show that (a) the model of choice to investigate AD needs to be characterized for the cutaneous microbiota if applicable and (b) the oxazolone-mediated mixed Th1-Th2 immune response triggers microbiota-induced alterations which share similarities to dysbiosis in human AD and represents therefore a suitable model for translational research on AD if alterations of the microbiome are in the focus of the investigation.

IL-4 receptor α blockade prevents sensitization and alters acute and long-lasting effects of allergen-specific immunotherapy of murine allergic asthma.

BACKGROUND: Allergen-specific immunotherapy (AIT) is the only causal treatment for allergy. However, success rates vary depending on the type of allergy and disease background of the patient. Hence, strategies targeting an increased therapeutic efficacy are urgently needed. Here, the effects of blockade of IL-4 and IL-13 signaling on different phases of AIT were addressed. METHODS: The impact of the recombinantly produced IL-4 and IL-13 antagonist IL-4 mutein (IL-4M) on allergic sensitization and AIT outcome in experimental allergic asthma were analyzed in a murine model. The effects of IL-4M administration were assessed prior/during sensitization, immediately after AIT under allergen challenge, and two weeks post-treatment. RESULTS: Intervention with IL-4M prior/during sensitization led to strong induction of IgG1, IgG2a, IgG2b, and IgG3, decrease of specific and total IgE, as well as of IL-5 in serum. Similar effects on the serum immunoglobulin levels were observed immediately after IL4M-supplemented AIT during allergen challenge. Additionally, IL4M markedly suppressed type-2 cytokine secretion of splenocytes beyond the effect of AIT alone. These effects were equaled to those of AIT alone two weeks post-treatment. Intriguingly, here, IL-4M induced a sustained decrease of Th2-biased Tregs (ST2+ FOXP3+ GATA3intermediate ). CONCLUSIONS: IL-4 and IL-13 blockade during experimental AIT demonstrates beneficial effects on immunological key parameters such as immunoglobulin and cytokine secretion immediately after AIT. Although two weeks later these effects were dropped to those of AIT alone, the number of potentially disease-triggering Th2-biased Tregs was further significantly decreased by IL-4M treatment. Hence, IL-4/IL13-targeting therapies prime the immune memory in therapy success-favoring manner.

Is the humoral immunity dispensable for the pathogenesis of psoriasis?

BACKGROUND: Imbalances of T-cell subsets are hallmarks of disease-specific inflammation in psoriasis. However, the relevance of B cells for psoriasis remains poorly investigated. OBJECTIVE: To analyse the role of B cells and immunoglobulins for the disease-specific immunology of psoriasis. METHODS: We characterized B-cell subsets and immunoglobulin levels in untreated psoriasis patients (n = 37) and compared them to healthy controls (n = 20) as well as to psoriasis patients under disease-controlling systemic treatment (n = 28). B-cell subsets were analysed following the flow cytometric gating strategy based on the surface markers CD24, CD38 and CD138. Moreover, immunofluorescence stainings were used to detect IgA in psoriatic skin. RESULTS: We found significantly increased levels of IgA in the serum of treatment-naïve psoriasis patients correlating with disease score. However, IgA was only observed in dermal vessels of skin sections. Concerning B-cell subsets, we only found a moderately positive correlation of CD138+ plasma cells with IgA levels and disease score in treatment-naïve psoriasis patients. Confirming our hypothesis that psoriasis can develop in the absence of functional humoral immunity, we investigated a patient who suffered concomitantly from both psoriasis and a hereditary common variable immune defect (CVID) characterized by a lack of B cells and immunoglobulins. We detected variants in three of the 13 described genes of CVID and a so far undescribed variant in the ligand of the TNFRSF13B receptor leading to disturbed B-cell maturation and antibody production. However, this patient showed typical psoriasis regarding clinical presentation, histology or T-cell infiltrate. Finally, in a group of psoriasis patients under systemic treatment, neither did IgA levels drop nor did plasma cells correlate with IgA levels and disease score. CONCLUSION: B-cell alterations might rather be an epiphenomenal finding in psoriasis with a clear dominance of T cells over shifts in B-cell subsets.

EAACI Guidelines on Allergen Immunotherapy: Allergic rhinoconjunctivitis.

Allergic rhinoconjunctivitis (AR) is an allergic disorder of the nose and eyes affecting about a fifth of the general population. Symptoms of AR can be controlled with allergen avoidance measures and pharmacotherapy. However, many patients continue to have ongoing symptoms and an impaired quality of life; pharmacotherapy may also induce some side-effects. Allergen immunotherapy (AIT) represents the only currently available treatment that targets the underlying pathophysiology, and it may have a disease-modifying effect. Either the subcutaneous (SCIT) or sublingual (SLIT) routes may be used. This Guideline has been prepared by the European Academy of Allergy and Clinical Immunology's (EAACI) Taskforce on AIT for AR and is part of the EAACI presidential project "EAACI Guidelines on Allergen Immunotherapy." It aims to provide evidence-based clinical recommendations and has been informed by a formal systematic review and meta-analysis. Its generation has followed the Appraisal of Guidelines for Research and Evaluation (AGREE II) approach. The process included involvement of the full range of stakeholders. In general, broad evidence for the clinical efficacy of AIT for AR exists but a product-specific evaluation of evidence is recommended. In general, SCIT and SLIT are recommended for both seasonal and perennial AR for its short-term benefit. The strongest evidence for long-term benefit is documented for grass AIT (especially for the grass tablets) where long-term benefit is seen. To achieve long-term efficacy, it is recommended that a minimum of 3 years of therapy is used. Many gaps in the evidence base exist, particularly around long-term benefit and use in children.

Sebocytes contribute to skin inflammation by promoting the differentiation of T helper 17 cells.

BACKGROUND: The main function of sebocytes is considered to be the production of lipids to moisturize the skin. However, it recently became apparent that sebocytes release chemokines and cytokines and respond to proinflammatory stimuli as well as the presence of bacteria. OBJECTIVES: To analyse the functional communication between human sebocytes and T cells. METHODS: Immunofluorescence stainings for CD4 and interleukin (IL)-17 were performed on acne sections and healthy skin. Migration assays and T-cell-stimulation cultures were performed with supernatants derived from unstimulated or prestimulated SZ95 sebocytes. Dendritic cells were generated in the presence of SZ95 supernatant and subsequently used in mixed leucocyte reactions. RESULTS: We showed that CD4+ IL-17+ T cells accumulate around the pilosebaceous unit and are in close contact with sebocytes in acne lesions. By using SZ95 sebocyte supernatant, we demonstrate a chemotactic effect of sebocytes on neutrophils, monocytes and T cells in a CXCL8-dependent manner. Furthermore, sebocyte supernatant induces the differentiation of CD4+ CD45RA+ naive T cells into T helper (Th)17 cells via the secretion of IL-6, transforming growth factor-ß and, most importantly, IL-1ß. No direct effects of sebocytes on the function of CD4+ CD45RO+ memory T cells were detected. Moreover, sebocytes functionally interact with Propionibacterium acnes in the maturation of dendritic cells, leading to antigen-presenting cells that preferentially prime Th17 cells. CONCLUSIONS: Our study provides evidence that human sebocytes actively participate in inflammatory processes in the skin by recruiting and communicating with immune cells. This interaction leads to the generation of Th17 cells, which might contribute to the pathogenesis not only of acne vulgaris, but also of several inflammatory skin diseases.

Perspectives in allergen immunotherapy: 2017 and beyond.

The Future of the Allergists and Specific Immunotherapy (FASIT) workshop provides a regular platform for global experts from academia, allergy clinics, regulatory authorities and industry to review developments in the field of allergen immunotherapy (AIT). The most recent meeting, held in February 2017, had two main themes: advances in AIT and hot topics in AIT from the regulatory point of view. The first theme covered opportunities for personalized AIT, advances in adjuvants and delivery systems, and the development of new molecules and future vaccines for AIT. Key topics in the second part of the meeting were the effects of the enactment of European Directive 2001/83 on the availability of allergens for therapy and diagnosis across the EU, the challenges of conducting Phase 3 studies in the field, the future role of allergen exposure chambers in AIT studies and specific considerations in performing AIT studies in the paediatric population. Finally, the group highlighted the forthcoming EAACI guidelines and their particular importance for the standardization of practice in the treatment of allergies. This review presents a comprehensive insight into those panel discussions and highlights unmet needs and also possible solutions to them for the future.

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.

Novel key cytokines in allergy: IL-17, IL-22.

The biology of the T cell cytokines Interleukin (IL-)17 and IL-22 has been a main focus in the field of clinical immunology in the last decade. This intensive interest in both cytokines has resulted in almost 5,000 scientific publications (www.pubmed.com) dealing with the molecular structure, extra- and intracellular signaling pathways, specific transcription factors and the function of IL-17 and IL-22. This review article highlights the main findings concerning IL-17 and IL-22 in the last years.

Application of recombinant antigen 5 allergens from seven allergy-relevant Hymenoptera species in diagnostics.

BACKGROUND: Hymenoptera stings can cause severe anaphylaxis in untreated venom-allergic patients. A correct diagnosis regarding the relevant species for immunotherapy is often hampered by clinically irrelevant cross-reactivity. In vespid venom allergy, cross-reactivity between venoms of different species can be a diagnostic challenge. To address immunological IgE cross-reactivity on molecular level, seven recombinant antigens 5 of the most important Vespoidea groups were assessed by different diagnostic setups. METHODS: The antigens 5 of yellow jackets, hornets, European and American paper wasps, fire ants, white-faced hornets, and Polybia wasps were recombinantly produced in insect cells, immunologically and structurally characterized, and their sIgE reactivity assessed by ImmunoCAP, ELISA, cross-inhibition, and basophil activation test (BAT) in patients with yellow jacket or Polistes venom allergy of two European geographical areas. RESULTS: All recombinant allergens were correctly folded and structural models and patient reactivity profiles suggested the presence of conserved and unique B-cell epitopes. All antigens 5 showed extensive cross-reactivity in sIgE analyses, inhibition assays, and BAT. This cross-reactivity was more pronounced in ImmunoCAP measurements with venom extracts than in sIgE analyses with recombinant antigens 5. Dose-response curves with the allergens in BAT allowed a differentiated individual dissection of relevant sensitization. CONCLUSIONS: Due to extensive cross-reactivity in various diagnostic settings, antigens 5 are inappropriate markers for differential sIgE diagnostics in vespid venom allergy. However, the newly available antigens 5 from further vespid species and the combination of recombinant allergen-based sIgE measurements with BAT represents a practicable way to diagnose clinically relevant sensitization in vespid venom allergy.

Added sensitivity of component-resolved diagnosis in hymenoptera venom-allergic patients with elevated serum tryptase and/or mastocytosis.

BACKGROUND: Anaphylaxis caused by hymenoptera venom allergy is associated with elevation of baseline serum tryptase (sBT) and/or mastocytosis in about 5% of patients. Up to now, no information has become available on single venom allergen sIgE reactivity and the usefulness of component-resolved approaches to diagnose this high-risk patient group. To address the component-resolved sIgE sensitization pattern and diagnostic sensitivity in hymenoptera venom-allergic patients with elevated sBT levels and/or mastocytosis, a panel of yellow jacket and honeybee venom allergens was applied on a widely used IgE immunoassay platform. METHODS: Fifty-three patients with mastocytosis and/or elevated sBT tryptase level and systemic reactions to hymenoptera venoms were analyzed for their IgE reactivity to recombinant yellow jacket and honeybee venom allergens by Immulite3 g. RESULTS: sIgE reactivity to Ves v 1, Ves v 5, Api m 1 to Api m 4 and Api m 10 was found at a similar frequency in hymenoptera venom-allergic patients with and without elevated sBT levels and/or mastocytosis. However, the use of the recombinant allergens and a diagnostic cutoff of 0.1 kUA /L allowed the diagnosis of patients with otherwise undetectable IgE to venom extract. The diagnostic sensitivity of yellow jacket venom allergy using the combination of Ves v 1 and Ves v 5 was 100%. CONCLUSIONS: In high-risk patients with elevated sBT levels and/or mastocytosis, the use of molecular components and decreasing the threshold sIgE level to 0.1 kUA /L may be needed to avoid otherwise undetectable IgE to hymenoptera venom extracts in about 8% of such patients.

Interleukin-4 and interferon-γ orchestrate an epithelial polarization in the airways.

Interferon-γ (IFN-γ) and interleukin-4 (IL-4) are key effector cytokines for the differentiation of T helper type 1 and 2 (Th1 and Th2) cells. Both cytokines induce fate-decisive transcription factors such as GATA3 and TBX21 that antagonize the polarized development of opposite phenotypes by direct regulation of each other's expression along with many other target genes. Although it is well established that mesenchymal cells directly respond to Th1 and Th2 cytokines, the nature of antagonistic differentiation programs in airway epithelial cells is only partially understood. In this study, primary normal human bronchial epithelial cells (NHBEs) were exposed to IL-4, IFN-γ, or both and genome-wide transcriptome analysis was performed. The study uncovers an antagonistic regulation pattern of IL-4 and IFN-γ in NHBEs, translating the Th1/Th2 antagonism directly in epithelial gene regulation. IL-4- and IFN-γ-induced transcription factor hubs form clusters, present in antagonistically and polarized gene regulation networks. Furthermore, the IL-4-dependent induction of IL-24 observed in rhinitis patients was downregulated by IFN-γ, and therefore IL-24 represents a potential biomarker of allergic inflammation and a Th2 polarized condition of the epithelium.

Current and future biomarkers in allergic asthma.

Diagnosis early in life, sensitization, asthma endotypes, monitoring of disease and treatment progression are key motivations for the exploration of biomarkers for allergic rhinitis and allergic asthma. The number of genes related to allergic rhinitis and allergic asthma increases steadily; however, prognostic genes have not yet entered clinical application. We hypothesize that the combination of multiple genes may generate biomarkers with prognostic potential. The current review attempts to group more than 161 different potential biomarkers involved in respiratory inflammation to pave the way for future classifiers. The potential biomarkers are categorized into either epithelial or infiltrate-derived or mixed origin, epithelial biomarkers. Furthermore, surface markers were grouped into cell-type-specific categories. The current literature provides multiple biomarkers for potential asthma endotypes that are related to T-cell phenotypes such as Th1, Th2, Th9, Th17, Th22 and Tregs and their lead cytokines. Eosinophilic and neutrophilic asthma endotypes are also classified by epithelium-derived CCL-26 and osteopontin, respectively. There are currently about 20 epithelium-derived biomarkers exclusively derived from epithelium, which are likely to innovate biomarker panels as they are easy to sample. This article systematically reviews and categorizes genes and collects current evidence that may promote these biomarkers to become part of allergic rhinitis or allergic asthma classifiers with high prognostic value.

Decreased FOXP3 protein expression in patients with asthma.

BACKGROUND: T-regulatory cells (T(reg)) are important in balancing immune responses and maintaining peripheral tolerance. Current evidence suggests that asthma is characterized by a relative deficiency in T(reg), allowing T helper 2 cells to expand. In this study, we aimed to evaluate circulating T(reg), defined by the protein FOXP3, in both control subjects and patients with stable asthma. METHODS: Peripheral blood mononuclear cells (PBMC) of control (n = 14) and asthmatic patients (n = 29) were labeled for CD4, CD25, and intracellular FOXP3 and analyzed using flow cytometry. In CD3/CD28 stimulated PBMC, the effects of dexamethasone on the transcription factors T-bet, GATA-3, FOXP3, and RORc2 and representative cytokines were studied. RESULTS: In control subjects and asthmatic patients, numbers of peripheral blood CD4(+)CD25(high) and CD4(+)CD25(high)FOXP3(+) T-cells were similar. However, FOXP3 protein expression within CD4(+)CD25(high) T-cells was significantly decreased in asthmatic patients. There was a tendency for increased FOXP3 expression within CD4(+)CD25(high) T-cells in glucocorticosteroid-treated patients when compared to steroid-naive asthmatic patients. In stimulated PBMC, dexamethasone treatment increased the anti-/proinflammatory transcription ratios of FOXP3/GATA-3, FOXP3/T-bet, and FOXP3/RORc2. CONCLUSION: Asthmatic patients have decreased FOXP3 protein expression within their CD4(+)CD25(high) T(reg). Our findings also suggest that treatment with inhaled glucocorticosteroids in asthmatics might increase this FOXP3 protein expression within the CD4(+)CD25(high) T-cell population.

Cyclooxygenase-2 in mucosal DC mediates induction of regulatory T cells in the intestine through suppression of IL-4.

Oral intake of protein leads to tolerance through the induction of regulatory T cells (Tr cells) in mesenteric lymph nodes (MLNs). Here we show that the inhibition of cyclooxygenase-2 (COX-2) in vivo suppressed oral tolerance and was associated with enhanced differentiation of interleukin (IL)-4-producing T cells and reduced Foxp3(+) Tr-cell differentiation in MLN. As a result, the functional suppressive capacity of these differentiated mucosal T cells was lost. IL-4 was causally related to loss of tolerance as treatment of mice with anti-IL-4 antibodies during COX-2 inhibition restored tolerance. Dendritic cells (DCs) in the MLN differentially expressed COX-2 and reductionist experiments revealed that selective inhibition of the enzyme in these cells inhibited Foxp3(+) Tr-cell differentiation in vitro. Importantly, the inhibition of COX-2 in MLN-DC caused increased GATA-3 expression and enhanced IL-4 release by T cells, which was directly related to impaired Tr-cell differentiation. These data provide crucial insights into the mechanisms driving de novo Tr-cell induction and tolerance in the intestine.

High-altitude climate therapy reduces local airway inflammation and modulates lymphocyte activation.

High-altitude climate therapy is a well-established therapeutic option, which improves clinical symptoms in asthma. However, little is known about the underlying immunological mechanisms. The study investigates the influence of high-altitude climate therapy on airway inflammation and cellular components of specific and unspecific immune response. Exhaled NO significantly decreased within 3 weeks of therapy in patients with allergic and intrinsic, moderate and severe asthma. Interleukin-10 (IL-10)-secreting peripheral blood mononuclear cells (PBMC) increased within 3 weeks of therapy in six of 11 patients, whereas transforming growth factor-beta(1)-secreting PBMC remained stable. Furthermore, monocyte activation, assessed by CD80 expression significantly decreased during therapy. The frequency of CRTH2-expressing T cells decreased, while regulatory T cells (T(reg)) remained stable. FOXP3 and GATA-3 mRNA expression in CD4(+) T cells did not change, while interferon-gamma and IL-13 mRNA expression decreased in eight of 10 patients. The current data demonstrate that high-altitude climate therapy reduces local airway inflammation. Furthermore, monocytes switch towards a tolerogenic phenotype under high-altitude climate therapy. The T(reg)/Th2 ratio increases; however, because of the absence of antigens/allergens, no de novo differentiation of Th2 nor T(reg) cells is observed. The high-altitude climate therapy therefore may form the immunological basis for the endogenous control of allergen-driven diseases.

Distinct leucocyte redistribution after glucocorticoid treatment among difficult-to-treat asthmatic patients.

Difficult-to-treat asthma (DTA) represents a heterogeneous subgroup of asthma. Up to now, the lack of specific diagnosis not only complicates appropriate specification and control of asthma, but also makes targeted research difficult. The aim of this study is to categorize this heterogeneous group of DTA patients (n=27; referring to the GINA guidelines) based on the distinct leucocyte redistribution (LR) after glucocorticoid (GC) treatment. Furthermore, the effect of adjuvant therapies was investigated for its impact on LR. The frequency of CD3+, CD4+, CD8+, CD14+, CD19+ and NK cells was analysed in peripheral blood before and 3 h after systemic GC treatment, along with the markers of activation HLA-DR and CD25. Within 3 h of GC administration, a significant average decrease of 16% in CD3+CD4+ (P < or = 0.001) and a 12% increase in NK-cell frequency (P < or = 0.001) clearly distinguished two groups of patients: LR-responsive and LR-unresponsive patients. The CD3+CD8+ T-cell number and activation marker remained unchanged. Patients who received adjuvant therapy, such as methotrexate or interferon-alpha, because of poor clinical response to GC showed an LR similar to that showed by responsive patients. DTA patients comprise at least two immunologically distinct groups: patients showing an immediate decrease in CD3+CD4+ T cells and an increase in NK cells following GC administration and patients lacking an immediate change. Analysis of LR not only may allow the identification of immunologic steroid resistance, but also may be of value for immunologic determination of effective steroid doses.