Preformulation studies for the development of a microemulsion formulation from Ambrosia peruviana All, with anti-inflammatory effect

Abstract Natural products are considered an important source of the therapeutic arsenal currently available. Among these alternatives are the seeds of Ambrosia peruviana (altamisa), whose extract has shown an anti-inflammatory effect. The main objective of this work was to perform a preformulation study of Ambrosia peruviana seeds ethanolic extract, where the main factors that affect the physical, chemical, and pharmacological stability of the extract were evaluated, as well as a compatibility study by differential scanning calorimetry (DSC) analysis against different excipients. A dry extract was obtained by rotary evaporation of the seeds macerated with 96% ethanol. The anti-inflammatory activity was determined by measuring its effect on NO production in RAW 264.7 macrophages, stimulated with LPS. The results showed that the dry extract maintained its stability over time when stored at a temperature of 4 and 25ºC, demonstrating its biological activity, the content of phenolic compounds, and its physicochemical parameters remain practically invariable. However, when exposed to high temperatures (60 ºC) it was affected. The thermal analysis revelated that the behavior of most of the selected excipients and the dry extract was maintained, which indicates that it did not present incompatibilities, therefore they can be candidates for formulating a microemulsion.

At-a-glance - Pollens, climate and allergies: Quebec initiatives. / Aperçu - Pollens, climat et allergies : initiatives menées au Québec.

Pollen allergies are a major source of seasonal allergic rhinitis in North America. This type of rhinitis affects 17% of adults in Quebec, a marked increase in the last 30 years. Ragweed (Ambrosia artemisiifolia L.) pollens are responsible for 50% to 90% of rhinitis cases. Climate change has played a significant role in the increased prevalence of seasonal allergic rhinitis over the last few decades. In 2015, the Quebec government put in place a strategy to tackle this problem, the Stratégie québécoise de réduction de l'herbe à poux et des autres pollens allergènes [Quebec strategy to reduce ragweed and other allergenic pollens]. Based on solid evidence, the Strategy advocates for co-operation between stakeholders and the integration of control measures into the maintenance practices of municipalities and other large public and private landowners. This article presents the scientific data underpinning the Strategy and initial successes of the action taken under the Strategy.

Climate change increases the quantities and allergenic potential of ragweed pollen. Seasonal allergic rhinitis caused by these pollens has increased significantly in North America. Simple environmental control methods can clinically decrease the impact of ragweed pollen. A coordinated policy that incorporates these control measures in municipalities' groundskeeping practices is being implemented in Quebec.

Les changements climatiques augmentent les quantités et le potentiel allergène du pollen de l'herbe à poux (ambroisie). La rhinite allergique saisonnière liée à ces pollens est en augmentation marquée en Amérique du Nord. Des moyens de contrôle environnemental simples peuvent diminuer cliniquement l'impact du pollen de l'herbe à poux. Une politique concertée qui intègre des mesures de contrôle dans les pratiques d'entretien des terrains par les municipalités est en implantation au Québec.

Protostemonine attenuates alternatively activated macrophage and DRA-induced asthmatic inflammation.

Asthma is one of the most common pulmonary diseases that threatens human life because of lack of effective medicines. Protostemonine (PSN), an active alkaloid extracted from the roots of Stemona sesslifolia, has anti-inflammatory effects on acute lung injury and acute liver failure. However, it has not been defined whether PSN alleviates asthmatic inflammation. Here, we reported that PSN inhibits pulmonary eosinophil infiltration, goblet cell hyperplasia, mucus secretion, IgE and Th2 cytokine (IL-4, IL-5, IL-13 and IL-33) production by using DRA (dust mites, ragweed and aspergillus)-induced murine asthma model. Moreover, PSN also attenuated the expression of Arginase-1 (Arg-1), Ym-1 and Fizz-1, markers of AAM (alternatively activated macrophage) polarization, in lung tissues. In addition, PSN attenuated IL-4-induced expression of Arg-1, Ym-1 and Fizz-1 in bone marrow derived macrophages (BMDMs). Treatment with PSN decreased IL-4-induced STAT6 phosphorylation, KLF4 and IRF4 expression in BMDMs. Collectively, our results indicated that PSN ameliorates AAM polarization and asthmatic inflammation and might be a potential agent for treating asthma.

Sequence conservation predicts T cell reactivity against ragweed allergens.

BACKGROUND: Ragweed is a major cause of seasonal allergy, affecting millions of people worldwide. Several allergens have been defined based on IgE reactivity, but their relative immunogenicity in terms of T cell responses has not been studied. OBJECTIVE: We comprehensively characterized T cell responses from atopic, ragweed-allergic subjects to Amb a 1, Amb a 3, Amb a 4, Amb a 5, Amb a 6, Amb a 8, Amb a 9, Amb a 10, Amb a 11, and Amb p 5 and examined their correlation with serological reactivity and sequence conservation in other allergens. METHODS: Peripheral blood mononuclear cells (PBMCs) from donors positive for IgE towards ragweed extracts after in vitro expansion for secretion of IL-5 (a representative Th2 cytokine) and IFN-γ (Th1) in response to a panel of overlapping peptides spanning the above-listed allergens were assessed. RESULTS: Three previously identified dominant T cell epitopes (Amb a 1 176-191, 200-215, and 344-359) were confirmed, and three novel dominant epitopes (Amb a 1 280-295, 304-319, and 320-335) were identified. Amb a 1, the dominant IgE allergen, was also the dominant T cell allergen, but dominance patterns for T cell and IgE responses for the other ragweed allergens did not correlate. Dominance for T cell responses correlated with conservation of ragweed epitopes with sequences of other well-known allergens. CONCLUSIONS AND CLINICAL RELEVANCE: These results provide the first assessment of the hierarchy of T cell reactivity in ragweed allergens, which is distinct from that observed for IgE reactivity and influenced by T cell epitope sequence conservation. The results suggest that ragweed allergens associated with lesser IgE reactivity and significant T cell reactivity may be targeted for T cell immunotherapy, and further support the development of immunotherapies against epitopes conserved across species to generate broad reactivity against many common allergens.

Temporal Association Between Nonfatal Self-Directed Violence and Tree and Grass Pollen Counts.

OBJECTIVES: Prior research suggests a possible association between pollen and suicide. No studies have examined the relationship between pollen and attempted suicide. This study examines the temporal association between airborne pollen counts and nonfatal suicidal and nonsuicidal self-directed violence (SDV) requiring an emergency department visit. METHODS: Data on daily emergency department visits due to nonfatal SDV as identified by ICD-9 diagnosis criteria were extracted from emergency department medical records of Parkland Memorial Hospital in Dallas, Texas, between January 2000 and December 2003. Concurrent daily airborne tree, grass, and ragweed pollen data from the city of Dallas were extracted from the National Allergy Bureau online database. The data were analyzed using the time series method of generalized autoregressive conditional heteroskedasticity. RESULTS: There were statistically significant and positive temporal associations between tree pollen counts and the number of nonfatal SDV events among women (P = .04) and between grass pollen counts and number of nonfatal SDV events among both men (P = .03) and women (P < .0001). There was no significant temporal association found between ragweed pollen counts and number of nonfatal SDV events. CONCLUSIONS: The study findings suggest that an increase in nonfatal SDV is associated with changes in tree and grass pollen counts. This is the first study that has examined an association between seasonal variation in tree and grass pollen levels and nonfatal SDV event data. The study also used a narrowly defined geographic area and temporal window. The findings suggest that pollen count may be a factor influencing seasonal patterns in suicidal behavior.

Is There Any Evidence for Rapid, Genetically-Based, Climatic Niche Expansion in the Invasive Common Ragweed?

Climatic niche shifts have been documented in a number of invasive species by comparing the native and adventive climatic ranges in which they occur. However, these shifts likely represent changes in the realized climatic niches of invasive species, and may not necessarily be driven by genetic changes in climatic affinities. Until now the role of rapid niche evolution in the spread of invasive species remains a challenging issue with conflicting results. Here, we document a likely genetically-based climatic niche expansion of an annual plant invader, the common ragweed (Ambrosia artemisiifolia L.), a highly allergenic invasive species causing substantial public health issues. To do so, we looked for recent evolutionary change at the upward migration front of its adventive range in the French Alps. Based on species climatic niche models estimated at both global and regional scales we stratified our sampling design to adequately capture the species niche, and localized populations suspected of niche expansion. Using a combination of species niche modeling, landscape genetics models and common garden measurements, we then related the species genetic structure and its phenotypic architecture across the climatic niche. Our results strongly suggest that the common ragweed is rapidly adapting to local climatic conditions at its invasion front and that it currently expands its niche toward colder and formerly unsuitable climates in the French Alps (i.e. in sites where niche models would not predict its occurrence). Such results, showing that species climatic niches can evolve on very short time scales, have important implications for predictive models of biological invasions that do not account for evolutionary processes.

Increased specific immunoglobulin G4 antibodies induced by natural exposure to ambrosia pollen in patients with allergy.

BACKGROUND: Induction of specific immunoglobulin G4 (sIgG4) response (so-called blocking antibodies) in patients who receive specific immunotherapy (SIT) has been observed for many years. Although many other mechanisms have been identified as key regulators of immunologic processes in peripheral tolerance to allergens, the rise of sIgG4 during immunotherapy, together with the clinical improvement, is still believed to be one of the most important mechanisms through which SIT reaches its clinical efficacy. OBJECTIVE: The aim of this prospective study was to measure levels of IgG4 and sIgE in subjects allergic to Ambrosia elatior pollen (common ragweed), before and during natural exposure to A. elatior pollen. Healthy controls were also included. METHODS: Twenty-four patients with allergic diseases of the respiratory tract and 24 healthy controls entered the study. The sIgG4 and sIgE levels were measured by using the enzyme-linked immunosorbent assay method before and during A. elatior pollination season. RESULTS: A significant increase of the sIgG4 level in subjects with allergy during natural exposure to an allergen (0.676 increased to 0.937; p < 0.05) was shown. An increase in sIgE levels was also observed. Healthy subjects had comparable levels of sIgG4 as in subjects with allergy before the pollination season and no change in sIgG4 and sIgE levels during the pollination season. CONCLUSION: Increases of sIgG4 and sIgE levels were induced by natural allergen exposure in subjects with allergy but not in healthy individuals. This result support further reevaluation of the "blocking" antibodies concept as one concept responsible for allergen tolerance in patients who receive SIT.

Sublingual grass and ragweed immunotherapy: Clinical considerations-a PRACTALL consensus report.

Sublingual allergen immunotherapy provides a new option for patients with allergic rhinitis in the United States. The efficacy of these sublingual immunotherapy tablets in the treatment of allergic rhinitis has been firmly established in large multicenter clinical trials. In addition, the clinical benefits of sublingual immunotherapy might persist after treatment is discontinued. Local reactions, such as gastrointestinal or oropharyngeal symptoms, are common. However, severe anaphylaxis is rare, and therefore the immunotherapy tablets can be administered at home. Sublingual immunotherapy for allergic rhinitis has been used successfully for years in Europe, and these products might be appropriate for patients who do not do well with standard drug therapy or for those who prefer a disease-modifying approach.

Diesel exhaust particles exacerbate allergic rhinitis in mice by disrupting the nasal epithelial barrier.

BACKGROUND: Diesel exhaust particles (DEP), traffic-related air pollutants, are considered environmental factors that affect allergic diseases adversely. However, the exact effect of DEP on allergic rhinitis (AR) is unclear. OBJECTIVE: We thought to investigate the effect of DEP on seasonal AR using a mouse model. METHODS: Ragweed-pollen-sensitized mice were nasally challenged with ragweed pollen in the presence or absence of DEP. The frequency of sneezing was evaluated immediately after each nasal challenge. The expression of a tight junction (TJ) protein, zonula occludens-1 (ZO-1), was examined by immunohistochemistry in AR mice. RPMI 2650 cells were used for in vitro examination of paracellular permeability. RESULTS: Mice challenged with ragweed pollen plus DEP showed increased frequency of sneezing compared with mice challenged with pollen alone. Interestingly, intranasal DEP pretreatment before ragweed pollen challenge increased ragweed-pollen-induced sneezing to levels comparable with the co-administration group. In vitro examination revealed that DEP reduced ZO-1 expression in RPMI 2650 cells. In addition, intranasal administration of DEP, but not ragweed pollen, disrupted nasal mucosal TJs in vivo. The effect of a single DEP treatment on ragweed-induced sneezing and ZO-1 expression persisted for at least 4 days and was inversely correlated. Finally, an antioxidant substance, N-acetyl-L-cysteine, inhibited DEP-mediated TJ disruption and exacerbation of sneezing in AR. CONCLUSIONS AND CLINICAL RELEVANCE: DEP disrupts TJs by a reactive oxygen species-mediated pathway, leading to the increased permeability of nasal epithelial cells. This may result in the promotion of allergen delivery into subepithelial tissues contributing to the exacerbation of immediate allergic responses.

A comparison of fluticasone propionate nasal spray and cetirizine in ragweed fall seasonal allergic rhinitis.

BACKGROUND: Intranasal corticosteroids are generally considered the most effective medication class for controlling allergic rhinitis. Previous comparative studies with oral antihistamines have been only partially informative due to a variety of variables encountered during their execution. OBJECTIVE: To compare fluticasone propionate nasal spray (FPNS) with the second-generation antihistamine cetirizine (oral tablet) and with placebo in a head-to-head study in a 2-week treatment study during fall ragweed season. METHODS: A total of 978 subjects were screened for this study. Six hundred and eighty-two subjects were randomized into the study (170 subjects, FPNS 200 mcg once daily; 170, cetirizine 10 mg once daily; 171, FPNS placebo; 171, cetirizine placebo) and comprised the intent-to-treat population. A 1-week placebo run-in was followed by a 2-week active treatment period during which time a total nasal symptom score (TNSS), total ocular symptom score, and the Nocturnal Rhinoconjunctivitis Quality of Life Questionnaire were collected. RESULTS: The primary efficacy end point was the mean change from baseline over the entire treatment period in A.M. reflective TNSS. The TNSS was the sum of the four individual nasal congestion, nasal itching, rhinorrhea, and sneezing scores, in which each symptom was scored on a scale of 0 to 3. Both FPNS and cetirizine improved the primary end point when compared with placebo during the active treatment period. Although there was a trend that favored FPNS with regard to the primary and secondary end points, there was not a statistical difference between the two treatments. CONCLUSION: FPNS and cetirizine were equally effective in treating fall seasonal allergic rhinitis during a 2-week head-to-head treatment investigation. Clinical trial NCT01916226, www.clinicaltrials.gov.

Concomitant sensitization to ragweed and mugwort pollen: who is who in clinical allergy?

BACKGROUND: In many areas of Europe, double sensitization to ragweed and mugwort is common, and because of the overlapping flowering periods of the 2 plants, it is not possible to diagnose the primary sensitizing allergen source and hence to determine the proper immunotherapy. OBJECTIVES: To elucidate whether double-sensitized patients are cosensitized or cross-sensitized and, in the latter case, to define the primary sensitizer. METHODS: Serum samples from 34 patients with late summer respiratory allergy underwent skin prick testing with whole ragweed, and mugwort extracts were analyzed for their reactivity to recombinant Art v 1 and Amb a 1 by ImmunoCAP and then to Amb a 1, Art v 6, and Art v 1 isoforms by a proteomic approach. In double reactors, the primary sensitizing sources were detected by inhibition experiments. RESULTS: Serum samples from patients monosensitized to ragweed contained IgE to epitopes specific of all Amb a 1 isoforms. In contrast, serum samples from double reactors found to be primarily sensitized to mugwort reacted to Art v 1 and Art v 6 and cross-reacted to a few Amb a 1 isoforms. Finally, serum samples from double reactors found to be primarily sensitized to ragweed contained IgE reacting to all Amb a 1 isoforms, part of which cross-reacted to Art v 6. We did not find cosensitized patients. CONCLUSION: This study found that Art v 6 plays an important role in mugwort allergy and that the cross-reactivity between Art v 6 and Amb a 1 is frequent, bidirectional, and clinically relevant in the area of Milan.

Effect of Antiasthma Simplified Herbal Medicine Intervention on neutrophil predominant airway inflammation in a ragweed sensitized murine asthma model.

BACKGROUND: Neutrophil-predominant asthma is less responsive to steroids and associated with poorer disease control. The effects of Antiasthma Simplified Herbal Medicine Intervention (ASHMI), a traditional Chinese medicine formula reported to be efficacious in asthmatic patients and murine asthma models, on neutrophil predominant asthma are unknown. OBJECTIVE: To determine the effects of standard ASHMI and refined formula ASHMI (ASHMI(II)) in a neutrophil-predominant murine model of ragweed (RW) asthma and explore underlying mechanisms. METHODS: BALB/c mice were systemically sensitized, intranasally challenged with RW extract, and orally treated with ASHMI, ASHMI(II), or vehicle (water). In a separate experiment, some RW sensitized mice were treated with dexamethasone before challenge. After RW challenge, airway hyperreactivity (AHR), total and differential bronchoalveolar lavage fluid leukocyte counts, lung histologic features, and bronchoalveolar lavage fluid cytokine and chemokine levels were assessed. RW stimulation of the murine macrophage cell line RAW264.7 was used to determine effects of ASHMI active compound ganoderic acid C1 (GAC1) on tumor necrosis factor α (TNF-α) production and regulation of phosphorylated IκB and histone deacetylase 2 (HDAC2) levels. RESULTS: ASHMI and ASHMI(II) markedly reduced AHR, mucous production, neutrophilic inflammation, and TNF-α, interleukin 8, and interleukin 17 levels and decreased eosinophilic inflammation and TH2 responses in vivo (P < .01-.001 for all). GAC1 inhibited TNF-α production in RW-stimulated RAW264.7 cells in association with suppression of phosphorylated IκB and increased HDAC2 expression. Dexamethasone failed to reduce AHR and neutrophilic inflammation. CONCLUSION: ASHMI treatment was efficacious in a murine model of neutrophil-predominant asthma via modulation of innate chemokines, TH2 responses, nuclear factor-κB, and HDAC2. ASHMI, and/or its constituent GAC1, may be a valuable option for treating neutrophil-predominant asthma.

A proof-of-concept study of the effect of a novel H3-receptor antagonist in allergen-induced nasal congestion.

BACKGROUND: H1-receptor inverse agonists are used effectively for treating several symptoms of allergic rhinitis, including nasal itching, rhinorrhea, and sneezing, although most agents are not very effective in treating nasal congestion. OBJECTIVE: This study evaluated the relative efficacy of a novel selective H3-receptor antagonist, JNJ-39220675, in preventing nasal congestion induced by exposing participants with ragweed allergy to ragweed allergen in an environmental exposure chamber model. METHODS: In this single-dose, patient-blind, double-dummy, placebo- and active-controlled, phase IIa cross-over study, 53 participants were randomized to JNJ-39220675 plus placebo, placebo plus pseudoephedrine, or only placebo. The primary efficacy assessment was change in nasal patency assessed by measuring the minimal cross-sectional area of the nasal cavity by using acoustic rhinometry. Secondary assessment included total nasal symptom scores (TNSSs) over the 8-hour environmental exposure chamber exposure period. RESULTS: Smaller decreases in minimal cross-sectional area were observed after JNJ-39220675 (least square mean difference, -0.126; P = .06) and pseudoephedrine (least square mean difference, -0.195; P = .004) treatment compared with placebo. The means for the baseline-adjusted area under the curve of TNSSs were significantly smaller for JNJ-39220675 (P = .0003) and pseudoephedrine (P = .04) versus placebo. JNJ-39220675 was significantly effective in treating all 4 individual symptoms (P ≤ .05 for all scores) compared with placebo, whereas pseudoephedrine only showed a trend for improvement in individual symptom scores of the TNSS. Insomnia was the most frequent adverse event (17.3%) associated with JNJ-39220675 treatment. CONCLUSION: Prophylactic treatment with the H3-antagonist JNJ-39220675 relieved allergen-induced nasal congestion by using standard nasal symptom scoring; however, in contrast to pseudoephedrine, it only showed a trend for increasing nasal patency by using objective measures.

[Ambrosia pollinosis]. / Pollinoses dues aux ambroisies.

Pollinosis is now called seasonal allergic rhinitis by the international terminology but pollinosis includes many other symptoms and so we will use the term Ambrosia pollinosis in this article. The characteristics of ragweed pollinosis are: severity, duration from August to September and the presence of asthma and/or tracheitis in about 50% of cases. Ambrosia: phanerogam, dicotyledon, annual plant, monoic. In France, fields in the mid Rhône Valley are covered with Ambrosia artemisiifolia L. but Ambrosia trifida L. is rare. The French Foundation For Ragweed Study (AFEDA) reports here 30years of clinical and epidemiological studies, involving considerable expense, that describe the geographic distribution of this pollinosis in Europe, and applies a predictive model of Ambrosia pollination to an appropriate treatment thanks to a sensitive sensor (Cour pollen trap). The spreading of Ambrosia is partly due to the regulations of the Common Agricultural Policy. There are numerous allergens; recently the major allergen of mugwort has been identified in ragweed. Profilins cause hypersensitivity reactions to certain foods. Genetic predisposition to developing this pollinosis is discussed because sometimes: the disease starts late in life, no personal or family history of atopy is found, immunoglobulin levels are low. Some publications have discussed a genetic predisposition to allergies to Juniperus ashei (United-States) and Cryptomeria japonica (Japan). The clinical efficacy of sublingual specific immunotherapy is well established and well accepted by patients.

Randomized controlled trial of a ragweed allergy immunotherapy tablet in North American and European adults.

BACKGROUND: In North America and Europe, millions of patients experience symptoms of allergic rhinitis with or without conjunctivitis (AR/C) on exposure to ragweed pollen. The disease burden can be significant, with most patients relying on symptomatic medications without disease-modifying potential. However, novel sublingual immunomodulatory treatment options may potentially play an important role if efficacy and side effect profiles allow the convenience of self-administration. OBJECTIVES: This study evaluated an allergy immunotherapy tablet (AIT; SCH 39641/MK-3641) for treatment of ragweed-induced AR/C in the first large randomized, double-blind multinational trial of this therapeutic modality for ragweed allergy. METHODS: Adults (n = 784) with short ragweed-induced AR/C were randomly assigned to approximately 52 weeks of daily self-administered ragweed AIT of 1.5, 6, or 12 units of Ambrosia artemisiifolia major allergen 1 (Amb a 1-U) or placebo. Subjects could use as-needed allergy rescue medication. Symptoms and medications were recorded daily. The primary efficacy end point was total combined daily symptom/medication score (TCS) during peak ragweed season. Safety was monitored through adverse event diaries maintained through study duration. RESULTS: During peak ragweed season, ragweed AIT of 1.5, 6, and 12 Amb a 1-U reduced TCS by 9% (-0.76; P = .22), 19% (-1.58; P = .01), and 24% (-2.04; P = .002) compared with placebo. During the entire season, ragweed AIT of 1.5, 6, and 12 Amb a 1-U reduced TCS by 12% (-0.88; P = .09), 18% (-1.28; P = .01), and 27% (-1.92; P < .001) compared with placebo. Treatment was well tolerated; no systemic allergic reactions occurred. CONCLUSIONS: In this trial, ragweed AIT of 12 Amb a 1-U was effective and tolerable with a safety profile that permitted daily self-administration of ragweed allergen immunotherapy.

Analysis of high allergenicity airborne pollen dispersion: common ragweed study case in Lithuania.

The appearance of ragweed pollen in the air became more frequent in northerly countries. Attention of allergologists and aerobiologists in these countries is focused on the phenomenon that Ambrosia plants found relatively sporadic but the amount of pollen is high in particular days. Over the latter decade, a matter of particular concern has been Ambrosia pollen, whose appearance in the air is determined by the plants dispersing it and meteorological processes that alter pollen release, dissemination, transport or deposition on surfaces. Pollen data used in this study were collected in three pollen-trapping sites in Lithuania. The data corresponding to 2006-2011 years of pollen monitoring were documented graphically and evaluated statistically. Analysis of the pollen data suggests that although the number of ragweed plants identified has not increased over the latter decade, the total pollen count has been on the increase during the recent period. The highest atmospheric pollen load is established on the last days of August and first days of September. The estimated effect of meteorological parameters on pollen dispersal in the air showed that in Lithuania ragweed pollen is recorded when the relative air humidity is about 70%, and the minimal air temperature is not less than 12°C. Analysis of wind change effect on pollen count indicates that pollen is most often recorded in the air when the changes in wind speed are low (1-2 m/s). We have established a regularity exhibiting an increase in ragweed pollen count conditioned by south-eastern winds in Lithuania.

Association of allergic asthma emergency room visits with the main biological and chemical air pollutants.

Joint effect of biological (pollen) and chemical air pollutants on asthma emergency room (ER) visits was analyzed for Szeged region of Southern Hungary. Our database of a nine-year period (1999-2007) includes daily number of asthma emergency room (ER) visits, and daily mean concentrations of CO, PM(10), NO, NO(2), O(3) and SO(2), furthermore two pollen variables (Ambrosia and total pollen excluding Ambrosia), as well. The analysis was performed for ER visits of asthma bronchiale using two age groups (adults and the elderly) of males and females for three seasons. Factor analysis was performed in order to clarify the relative importance of the pollutant variables affecting asthma ER visits. Asthma ER visits denote notably stronger associations with the pollutants in adult male than in adult female patients both for the pollen season of Ambrosia and the pollen-free season. Furthermore, adults are substantially more sensitive to severe asthma attack than the elderly for the season of total pollen excluding Ambrosia pollen. The joint effect of the chemical and pollen variables is the highest for the asthma ER cases in the pollen season of Ambrosia, basically due to the extra impact of the total pollen excluding Ambrosia pollen and partly due to Ambrosia pollen. A nonparametric regression technique was applied to discriminate between events of ER visit-no ER visit using pollen and chemical pollutants as explaining variables. Based on multiple correlations, the strongest relationships between ER visits and pollutants are observed during the pollen-free season. The elderly group with asthma bronchiale is characterized by weaker relationships between ER visits and pollutants compared to adults. Ratio of the number of correct decisions on the events of ER visit-no ER visit is lowest for the season of total pollen excluding Ambrosia pollen. Otherwise, similar conclusions hold as those received by multiple correlations.