Analysis of coagulation factors in angioedema/urticaria: increased values of D-dimer and fibrinogen in isolated angioedema.

INTRODUCTION: Recent research has shown that blood coagulation and the extrinsic coagulation cascade are involved in the pathogenesis of chronic spontaneous urticaria (CSU), but little is known about the coagulation factors in angioedema. METHODS: This study included 58 participants: 29 patients with chronic angioedema (14 with isolated angioedema and 15 with angioedema with wheals) and 29 healthy controls (HCs). We compared the values of coagulation factors in patients with isolated angioedema to those with wheals. Plasma levels of D-dimer, fibrinogen, and factor VII were measured by enzyme-linked immunosorbent assay (ELISA) for all participants. RESULTS: Significantly higher D-dimer (p = 0.016; ε² = 0.381) and fibrinogen (p = 0.044; ε² = 0.331) levels were recorded in patients with angioedema (both groups) than in the HCs, with higher levels for angioedema with wheals. Factor VII and fibrinogen levels did not differ significantly between the groups with angioedema, but coagulation factors were more often elevated in both angioedema groups than in HCs. CONCLUSIONS: One characteristic of angioedema is an elevated blood coagulation potential, which may help produce fibrin and may be important in controlling angioedema attacks.

Divergent Trends in the Prevalence of Children's Asthma, Rhinitis and Atopic Dermatitis and Environmental Influences in the Urban Setting of Zagreb, Croatia.

BACKGROUND: Previous studies have reported that the allergy epidemic in developed countries has reached its plateau, while a rise is expected in developing ones. Our aim was to compare the prevalence of allergic diseases among schoolchildren from the city of Zagreb, Croatia after sixteen years. METHODS: Symptoms of asthma, allergic rhinitis (AR) and atopic dermatitis (AD) and risk factors were assessed using the International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire. An allergic profile was determined by a skin prick test. RESULTS: The prevalence of current, ever-in-a-lifetime and diagnosed AR of 35.7%, 42.5% and 14.9% and AD of 18.1%, 37.1% and 31.1% demonstrated a significant increase. The asthma prevalence has remained unchanged. The allergen sensitivity rate has remained similar, but pollens have become dominant. Mould and dog exposure are risks for asthma (OR 14.505, OR 2.033). Exposure to cat allergens is protective in AR (OR 0.277). Parental history of allergies is a risk factor in all conditions. CONCLUSION: Over sixteen years, the prevalence of AR and AD, but not of asthma, have increased. The proportion of atopy has remained high. The AR/AD symptom rise is probably a consequence of increased pollen sensitisation united with high particulate matter concentrations. The stable asthma trend could be a result of decreasing exposures to indoor allergens.

Incidence of immediate allergic reactions to mRNA COVID-19 vaccines in adults with drug allergies and other allergic disorders.

Concerns have been raised about allergic reactions to messenger ribonucleic acid (mRNA) coronavirus disease 2019 (COVID-19) vaccines. A history of allergic reactions, including anaphylaxis to drugs, has been frequently reported in individuals with anaphylaxis to mRNA vaccines. To estimate the rate of immediate allergic reactions in patients with a history of drug allergy or other allergic disorders. We included adult patients who had received at least 1 dose of an mRNA COVID-19 vaccine at the Special Hospital for Pulmonary Diseases between March 1, 2021, and October 1, 2021, and who reported a history of drug allergy or other allergic diseases (asthma, allergic rhinitis, atopic dermatitis, food or insect venom allergy, mastocytosis, idiopathic anaphylaxis, acute or chronic urticaria, and/or angioedema). Immediate allergic reactions, including anaphylaxis, occurring within 4 hours of vaccination were recorded. Six immediate allergic reactions were noted in the cohort of 1679 patients (0.36%). One patient experienced anaphylaxis (0.06%), which resolved after epinephrine administration, and the other reactions were mild and easily treatable. Most patients with a history of allergies can safely receive an mRNA COVID-19 vaccine, providing adequate observation periods and preparedness to recognize and treat anaphylaxis.

Chronic spontaneous urticaria and angioedema in a patient with autoimmune thyroid disease resolved after thyroidectomy.

The link between chronic urticaria and accompanying thyroid disease is still not understood, with current treatment focusing on antihistamines and levothyroxine. A 35-year-old female patient presented with chronic idiopathic urticaria and facial angioedema for 9 months prior to evaluation. Oral corticosteroid therapy, antihistamines, leukotriene-antagonists, selenium, and omalizumab were all administered, with the disease relapsing within several days, accompanied with facial angioedema of varying severity. Laboratory results were negative for antinuclear antibodies (ANA) and cytoplasmic antineutrophil antibodies (ANCA). Immunoglobulins and complement levels were normal. Autologous serum testing, and skin-prick test for common inhalatory allergens were all normal. Levothyroxine was then administered with no effect on the symptoms. After considering all of the available treatment options, the patient decided to undergo total thyroidectomy. Urticaria and angioedema subsided on the third postoperative day, and she remains free of symptom recurrence during 10 months of postoperative follow-up. Her antiTPO titer decreased from > 1300 to 31.1 kIU/L and antiTG decreased from 272 to 4.9 kIU/L three months after the surgery. The most important element in this case report is an unexpected extra-thyroid presentation of an autoimmune thyroid disease, with a newly described association with facial angioedema. Additional important evidence may confirm the hypothesis that both conditions are indeed caused by a common immunological patohogenetic pathway that should be routinely evaluated in patients presenting with chronic idiopathic urticaria.

Chronic inducible urticaria: classification and prominent features of physical and non-physical types.

Chronic inducible urticaria (CIndU) is a common inflammatory skin condition characterized by the recurrence of itchy wheals and/or angioedema that lasts more than 6 weeks and is induced by specific physical or environmental stimuli (cold, heat, exercise, pressure, sunlight, vibration, water, etc.). According to the current international classification, it includes physical urticarias (dermographism, delayed-pressure urticaria, exercise-induced urticaria, cold urticaria, heat urticaria, solar urticaria, and vibratory urticaria) and non-physical urticarias caused by exposure to specific stimuli (cholinergic urticaria, contact urticaria, and aquagenic urticaria). In terms of frequency, more common types of CIndU are dermographism, cholinergic urticaria, and delayed-pressure urticaria. In clinical practice, it is often difficult to define the exact type of CIndU; management thus begins with accurate identification of a possible trigger and its avoidance. The definite diagnosis for CIndU requires obtaining a detailed medical history of a patient with comprehensive information about predisposing factors, physical examination, and provocation testing (challenge tests). It is always necessary to recognize the prophylactic options for all the types and to have access to different therapies (primarily second-generation H1 antihistamines, but also H2 antihistamines, hydroxyzine, doxepin, oral glucocorticoids, omalizumab/anti-IgE therapy, phototherapy, physical desensitization, immunomodulatory agents, etc.) individualized for each patient.

FasL (rs763110) gene polymorphism is not associated with susceptibility to rheumatoid arthritis in Croatian population.

AIM: To investigate the association of FasL gene polymorphism (rs763110) with rheumatoid arthritis occurrence, disease activity, and tumor necrosis factor-α (TNF-α) plasma concentration in Croatian patients, and to conduct an updated meta-analysis. METHODS: This cross-sectional study enrolled 81 patients with rheumatoid arthritis and 94 control patients. After the assessment of the Disease Activity Score (DAS)-28, blood was taken for analysis. DNA was isolated from the whole blood to determine FasL polymorphism (rs763110) by polymerase chain reaction. Protein levels of TNF-α were determined with ELISA. After a detailed literature search, we conducted an updated meta-analysis using the Review Manager 5 software. RESULTS: Rheumatoid arthritis patients had significantly higher TNF-α concentration in plasma (1.65 [1.2-2.42] pg/mL) than controls (0.99 [0.77-1.35] pg/mL, P<0.001). The FasL rs763110 polymorphism was not associated with rheumatoid arthritis occurrence in either codominant, dominant, recessive, overdominant, or log additive model. Furthermore, the rs763110 genotype was not associated with DAS 28 score or TNF-α concentration. After we added our results to an updated meta-analysis, the significant association previously reported for Western Eurasians was abolished. CONCLUSION: Our data suggest that the association between FasL rs763110 polymorphism and RA susceptibility in Western Eurasians observed in previous studies might be overestimated and should be limited to the population of Southwestern Asia until further investigations are performed.

Combined manual and automated immunophenotypisation identified disease-specific peripheral blood immune subpopulations in rheumatoid arthritis, ankylosing spondylitis and psoriatic arthritis.

OBJECTIVES: Rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA) are associated with abnormal immune cell functions. We combined manual and automated profiling in subpopulations of T-cells, B-cells and monocytes, in parallel to functional testing and clinical correlation. METHODS: Using flow cytometry, we analysed the expression of CCR4, CCR6 and CXCR5 on helper and cyotoxic T-cells, CD32B and CD86 on naïve and memory B-cells, and CCR1, CCR2, CCR4 and CXCR4 on monocytes in chronic high-disease activity patients to identify peripheral blood subpopulations. Cell activation, proliferative capability and osteoclastogenic effects were tested in vitro. Comparison with synovial compartment, clinical data and anti-TNF treatment were added to peripheral blood analysis. RESULTS: PsA had lower double-negative T-cell frequency, while RA had lower double-positive T-cell frequency and expanded Th1-like and cytotoxic T-cell subsets. CD32B expression was increased on naïve and memory B-cells in AS and associated with disease activity. CCR6+ and CXCR5+ cytotoxic T-cells and CD32B+ naïve and memory B-cells were highly enriched within the synovial compartment. T-cells and B-cells from AS exhibited enhanced activation and proliferation in vitro, whereas T-cell conditioned medium from RA produced an increased osteoclastogenic effect. CCR1 and CXCR4 were upregulated on osteoclastogenic monocyte subsets of RA, AS and PsA patients. Bioinformatic Citrus analysis identified additional T-cell, B-cell and monocyte clusters specifically associated with each disease. CONCLUSIONS: By combining manual and automated data analysis, our study revealed several disease-specific immune cell subpopulations, particularly cytotoxic T-cell subsets in RA and memory B-cell subsets in AS, which may serve as an indicator of active disease or possible therapeutic target.

JAK INHIBITOR CLINICAL RESPONSE IN POLYARTHRITIS: CASE REPORT.

The heterogeneity of rheumatoid arthritis (RA) presentation and molecular signature of RA subclasses in patients with early changes of small peripheral joints still remains a challenging problem. In clinical setting, classification of the disease subtypes is not possible and treatment adjustment is based on the continuous Disease Activity Score for disease severity recognition. A new approach in the treatment appears with the novel non biologic targeted synthetic disease-modifying antirheumatic drugs from the group of Janus kinase 1 and 3 (JAKI and JAK3), blocking interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15 and IL-21. We report a case of a 48-year-old patient who had suffered from polyarthritis from his age 40. Initial laboratory tests showed low inflammatory parameters and magnetic resonance imaging of both hands indicated an early stage of RA. Methylprednisolone and methotrexate therapy was initiated. The patient underwent additional tests, but there was not sufficient evidence for a precise diagnosis. According to the European League Against Rheumatism/American College of Rheumatology score-based algorithm, the patient was classified as seronegative RA based on joint involvement, duration of the disease, and synovitis not better explained by another disease. A partial clinical effect of the administered therapy (steroids as monotherapy and in combination, methotrexate and leflunomide) was noticed with the use of systemic steroids, but dramatic improvement was only achieved with a JAK inhibitor targeted therapy. Although the use of anti TNF-α blocker is a proposed procedure and the drug has not yet been registered in Europe, we took the opportunity to apply this new medication option. The patient, a construction worker, was treated for 20 months, which led to complete remission of the disease, without the need of basic or corticosteroid therapy. Full functional capacity necessary in his demanding job was also achieved. This result raised a question of timely introduction of immunomodulators in the polyarthritis treatment steps.

House dust mite-specific immunotherapy alters the basal expression of T regulatory and FcεRI pathway genes.

BACKGROUND: Regulatory T (Treg) cells and IgE-mediated signaling pathways could play important roles in the induction of allergen tolerance during house dust mite-specific subcutaneous immunotherapy (HDM-SCIT). Our aim was to compare the basal expression levels of Treg, T helper 1 (Th1) and Th2 transcription factors and components involved in IgE-mediated signaling in healthy subjects with those in HDM-allergic patients both untreated and successfully treated with HDM-SCIT. METHODS: Thirty-nine HDM-allergic patients who completed a 3- to 5-year course of mite extract SCIT, 20 mite-allergic controls and 25 healthy controls participated in this study. The efficacy of SCIT was monitored using skin-prick tests (SPTs), total immunoglobulin E (tIgE), specific IgE (sIgE), sIgG(4), nasal challenge and visual analog scale (VAS) scores at several time points. The mRNA levels of forkhead box protein 3 (FOXP3), T-BET, GATA-3, FcεRI, spleen tyrosine kinase (Syk), phosphatidylinositol 3 kinase (PI3K) and SH2 domain-containing inositol phosphatase (SHIP) were quantified by real-time RT-PCR using nonstimulated whole blood samples. RESULTS: Decreased wheal sizes and VAS scores, negative challenges and increased sIgG(4) levels indicated that SCIT was effective in the treated patients. Basal expression levels of FOXP3 and GATA-3 decreased and T-BET levels increased in both treated patients and in healthy controls compared to untreated patients. The IgE-mediated pathway kinases Syk and PI3K exhibited reduced expression, whereas SHIP phosphatase levels were elevated in both treated patients and healthy controls relative to untreated patients. The expression levels of FcεRI were not significantly altered. CONCLUSIONS: Immunotherapy using HDM extracts results in a modification of the basal expression levels of several IgE-related signaling factors and induces a highly significant upregulation of Th1-response and downregulation of Th2-response transcription factors. Interestingly, this therapy also appears to reduce the basal expression of FOXP3.

Effects of traffic air pollution and hornbeam pollen on adult asthma hospitalizations in Zagreb.

BACKGROUND: Environmental factors play an important role in asthma morbidity, although the contribution of individual pollutants or pollens in exacerbating asthma is not completely elucidated. Despite the evidence of importance of the hornbeam pollen in inducing allergic sensitization, its role in provoking asthma exacerbation has not been evaluated. The aim of the present study was to investigate effects of traffic pollutants on adult asthma hospitalization adjusting for pollens including hornbeam. METHODS: During a 3-year period, health and environmental data were collected and analyzed. Daily asthma hospitalizations were regressed on pollutants and potential confounding variables using an autoregressive Poisson model. RESULTS: The risk of asthma hospitalization was associated significantly with the 95th to 99th percentile increase in levels of nitrogen dioxide (RR = 1.22; 95% CI: 1.05-1.40), carbon monoxide (RR = 1.25; 95% CI: 1.01-1.55) and hornbeam pollen (RR = 1.21; 95% CI: 1.11-1.30). The effect of nitrogen dioxide was delayed by 5 days. No statistically significant increase in the risk of asthma hospitalization was found for PM(10) particles. A comparison among the standardized regression coefficients and their respective p values indicates that the most important risk factor for asthma hospitalization is associated with hornbeam pollen levels. No statistically significant interactions between pollutants and pollens were detected. CONCLUSIONS: The current results suggest that traffic-related air pollution is associated with increased risk of adult asthma hospitalization. Nonetheless, the most significant risk for asthma hospitalization is associated with hornbeam pollen levels in the city of Zagreb.

[Plant food allergy: apple as a model]. / Alergija na hranu biljnog podrijetla: jabuka kao model.

Allergic reactions to plant foodstuffs comprise a whole spectrum of clinical manifestations, from very mild ones, localized to oral mucosa, to the most severe including life-threatening anaphylactic shock. The reason is the presence of a few structurally and functionally different allergens in each foodstuff. On the other hand, the similarity between particular allergens in different foodstuffs and the presence of their homologues in other sources (pollens, insect venoms) give a lot of possibilities for cross-reactions during allergic sensitization. Apple has a significant place in human nutrition. However, it is also a fruit that most frequently causes allergic reactions. Apple allergens are well known and appropriately characterized, and their homologues are present in other vegetable foodstuffs. Thus, apple represents a good model of plant food hypersensitivity. The aim of this paper is to review the mechanisms of allergic sensitization, the types of clinical manifestations, the influence of allergen content variability, and the diagnostic and therapeutic issues connected with plant food allergy, using apple as a model.

Small and large bowel manifestation of leukocytoclastic vasculitis.

Leukocytoclastic vasculitis is a disease mostly limited to the skin. Extracutaneous manifestations that include visceral involvement are normally self-limiting and not life-threatening. We describe a 44-year-old man with palpable purpura, polyarthritis and microhematuria who developed severe vasculitis of the small and large bowel. Initial laboratory tests confirmed leukocytosis, slightly elevated C-reactive protein and mildly increased erythrocyte sedimentation rate. Skin biopsy revealed histological features typical of leukocytoclastic vasculitis. The search for trigger factors revealed urogenital infection with Ureaplasma urealyticum. Severe abdominal pain followed cutaneous symptoms eight days after admission. Abdominal x-ray showed several air-fluid levels in the lower right abdomen and an abdominal CT scan revealed thickening of the intestinal wall in several segments of jejunum, ileum and colon. C-reactive protein rose from 32 mg/l to 107 mg/l. Methylprednisolone pulse therapy rapidly improved gastrointestinal, cutaneous and articular symptoms. The aim of this report is to show the unpredictability of vasculitic disease and the difficulties in its classification. The report emphasizes the importance of adapting diagnosis and treatment according to disease severity rather than to the type of vasculitis. The specific etiological trigger remains unknown in this case, although a causal relationship with U. urealyticum infection is speculated.

Improving asthma control through telemedicine: a study of short-message service.

Home peak expiratory flow (PEF) measurement is recommended by asthma guidelines. In a 16-week randomized controlled study on 16 subjects with asthma (24.6 6.5 years old, asthma duration small ze, Cyrillic 6 months), we examined Global System for Mobile Communications (GSM) mobile telephone short-message service (SMS) as a novel means of telemedicine in PEF monitoring. All subjects received asthma education, self-management plan, and standard treatment. All measured PEF three times daily and kept a symptom diary. In the study group, therapy was adjusted weekly by an asthma specialist according to PEF values received daily from the patients. There was no significant difference between the groups in absolute PEF, but PEF variability was significantly smaller in the study group (16.12 +/- 6.93% vs. 27.24 +/- 10.01%, p = 0.049). forced expiratory flow in 1 second (FEV1; % predicted) in the study group was slightly but significantly increased (81.25 +/- 17.31 vs. 77.63 +/- 14.80, p = 0.014) and in the control group, unchanged (78.25 +/- 21.09 vs. 78.88 +/- 22.02, p = 0.497). Mean FEV1 was similar in the two groups both before and after the study. Controls had significantly higher scores for cough (1.85 +/- 0.43 vs. 1.42 +/- 0.28, p < 0.05) and night symptoms (1.22 +/- 0.23 vs. 0.85 +/- 0.32, p < 0.05). There was no significant difference between the groups in daily consumption of inhaled medicine, forced vital capacity, or compliance. Per patient, per week, the additional cost of follow-up by SMS was Euros 1.67 (equivalent to approximately $1.30 per 1 Euro), and SMS transmission required 11.5 minutes. Although a study group of 40 patients is needed for the follow-up study to achieve the power of 80% within the 95% confidence interval, we conclude that SMS is a convenient, reliable, affordable, and secure means of telemedicine that may improve asthma control when added to a written action plan and standard follow-up.

[Lymphangioleiomyomatosis]. / Limfangiolejomiomatoza.

Lymphangioleiomyomatosis (LAM) is a progressive and usually fatal interstitial lung disease characterized by an abnormal smooth-muscle proliferation in the lung and axial lymphatics. It affects almost exclusively young women of childbearing age. The presenting features most commonly include dyspnea, symptoms of pneumothorax and cough. Less commonly patients can present with chest pain, pleural or pericardial effusion and lymphedema. Our patient, a 41-year-old woman, complained mainly of fatigue that had lasted for 2 months and finally became febrile and dispneic, especially when lying down. Pulmonary diagnostic procedures revealed several multicystic destruction of lung parenchyma. There was also respiratory insufficiency with O2 saturation of 87% and lung diffusion capacity reduced to 48%. The retroperitoneum was filled with neoplastic mass as shown on an abdominal CT scan. Pathohistologic analysis of retroperitoneal mass together with the radiologic finding of the lungs correlated with the diagnosis of LAM. The patient was prescribed corticosteroid therapy, which led to rapid clinical improvement. After making a definite diagnosis, the patient was recommended further treatment with medroxyprogesterone. This case shows that LAM, although rare, can present a diagnostic problem to clinicians and should always be considered as one of the diagnostic possibilities in young women with nonspecific pulmonary symptoms.

Allergic diseases in relationship with environmental factors in a population of school children in Zagreb, Croatia.

Most scientists believe that increasing number of people with allergic diseases may be connected with some aspects of the "Western lifestyle". This paper discusses data obtained from questionnaires originally designed by the International Study of Asthma and Allergies in Childhood (ISAAC) Steering Committee concerning exposure to different environmental factors. The study included 1047 children. Allergic and non-allergic groups showed statistically significant differences in the attendance of kindergarten, vaccination against pertussis, pertussis infection, and parasite infestation. Exposure to tobacco smoke during pregnancy and exposure to dampness and moulds also entailed a risk for allergy. We speculate that changing some conditions, such as use of carpets and use of feather pillows, were connected with the expression of allergic diseases. Some correlations were consistent with earlier observations of other authors, while others differed and need further confirmation on a larger sample.

[Prevalence of symptoms of asthma, allergic rhinitis, conjunctivitis and atopic eczema: ISAAC (International Study of Asthma and Allergies in Childhood) in a population of schoolchildren in Zagreb]. / Prevalencija simptoma astme, alergijskog rinitisa i konjunktivitisa te atopijskog ekcema: ISAAC u populaciji skolske djece u Zagrebu.

OBJECTIVE: Numerous studies of the population prevalence of asthma, allergic rhinitis, and atopic eczema revealed some international differences. However, the International Study of Asthma and Allergies in Childhood (ISAAC) was the first one using a standardized methodology to evaluate the prevalence of these diseases, and to make comparisons within and between countries. The results showed marked variations in 12-month prevalence of asthma, allergic rhinoconjunctivitis, and atopic eczema symptoms with 20-fold (range 1.6-36.8%), 30-fold (range 1.4-39.7%), and 60-fold (range 0.3-20.5%) differences between the centres with the highest and the lowest prevalence, respectively. AIM: Our aim was to gain the insight into the prevalence of allergic diseases in Zagreb, Croatia by the methods of internationally standardized protocol, proposed by the ISAAC Steering Committee. METHODS: Original questionnaires, translated from English into Croatian, consisting of questions about the child's demographic characteristics, core modules on wheezing, rhinitis and eczema, and supplementary modules, were completed by parents of 10-year-old children (4th grade) attending 18 elementary schools in a city of Zagreb. Total of 1047 questionnaires were returned and analysed after the inconsistent responses were eliminated by phone calling. DISCUSSION: Phase one of the ISAAC study has shown a wide variation in the prevalence of asthma, allergic rhinoconjunctivitis, and atopic eczema symptoms throughout the world, with differing international patterns for the different disorders. Four prevalence ranges have been established for better illustration of the geographic distribution of asthma prevalence: (I) < 5%; (II) 5 to < 10%; (III) 10 to < 20%; (IV) > or = 20%. The highest 12-month prevalences of asthma symptoms were found in developed countries (UK, Australia, New Zealand, Republic of Ireland, and most centres in North, Central, and South America), being in prevalence range IV. The lowest prevalences (range I) were found in several Eastern European countries, Indonesia, Greece, China, Taiwan, Uzbekistan, India, and Ethiopia. According to the results of our study, a continental part of Croatia with a 12-month prevalence of wheezing of 6.02% corresponds to range II. Prevalence of asthma symptoms was greater in males, which is consistent with the results of the younger age group previously analysed. For allergic rhinoconjunctivitis and atopic eczema symptoms grouping of centres with a high prevalences into specific regions was less well defined than for asthma. Centres with the highest prevalences were scattered across the world. In contrast, centres with the lowest prevalences were similar to those for asthma symptoms. Our results of the 12-month prevalence of allergic rhinoconjunctivitis (12.13%), and atopic eczema (7.83%) symptoms were somewhere between the two extremes. As with asthma symptoms, the prevalence of rhinoconjunctivitis symptoms was greater in males. Contrary, the difference in prevalence of atopic eczema symptoms between the sex groups has not been found. The worldwide variations in prevalence of asthma, allergic rhinoconjunctivitis, and atopic eczema symptoms suggest that environmental factors may be critical to the development of these disorders in childhood. Furthermore, different patterns of geographical distribution of particular disorders suggest that major risk factors for them may be different or may involve different latency periods and time trends. Therefore, studies that include objective clinical assessment are required. CONCLUSION: According to our results, Zagreb is a city with relatively low prevalence of allergic diseases symptoms. Larger sample size of at least 3000 subjects is required to provide sufficient precision for estimates of symptom severity, and to generate adequate number of subjects with particular disorders for further analyses. Therefore, we recently increased our sample size to more than 3000 subjects, and started ISAAC Phase two (clinical examination, measures of bronchial hyperresponsiveness, measures of atopy, measures of environmental exposure to aeroallergens, and genetic analyses) in Zagreb, Croatia.