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Background Chronic rhinosinusitis (CRS) current therapeutic approaches still fail in some patients with severe persistent symptoms and recurrences after surgery. We aimed to evaluate the master transcription factors gene expression levels of T cell subtypes in chronic rhinosinusitis with nasal polyps (CRSwNP) and chronic rhinosinusitis without nasal polyps (CRSsNP) that could represent new, up-stream targets for topical DNAzyme treatment. Patients and methods Twenty-two newly diagnosed CRS patients (14 CRSwNP and 8 CRSsNP) were prospectively biopsied and examined histopathologically. Gene expression levels of T-box transcription factor (T-bet, TBX21), GATA binding protein 3 (GATA3), Retinoic acid-related orphan receptor C (RORC) and Forkhead box P3 (FOXP3) were analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). Results Eosinophilic CRSwNP was characterized by higher level of GATA3 gene expression compared to noneosinophilic CRSwNP, whereas there was no difference in T-bet, RORC and FOXP3 between eosinophilic and noneosinophilic CRSwNP. In CRSsNP, we found simultaneous upregulation of T-bet, GATA3 and RORC gene expression levels in comparison to CRSwNP; meanwhile, there was no difference in FOXP3 gene expression between CRSwNP and CRSsNP. Conclusions In eosinophilic CRSwNP, we confirmed the type 2 inflammation by elevated GATA3 gene expression level. In CRSsNP, we unexpectedly found simultaneous upregulation of T-bet and GATA3 that is currently unexplained; however, it might originate from activated CD8+ cells, abundant in nasal mucosa of CRSsNP patients. The elevated RORC in CRSsNP could be part of homeostatic nasal immune response that might be better preserved in CRSsNP patients compared to CRSwNP patients. Further data on transcription factors expression rates in CRS phenotypes are needed.

BACKGROUND: Confirmation of the clinical relevance of sensitisation is important for the diagnosis of allergic rhinitis. OBJECTIVE: To investigate the usefulness of an in vitro basophil activation test and component-resolved diagnosis in distinguishing between symptomatic allergic rhinitis patients and asymptomatic sensitization to house dust mites (HDMs). METHODS: Thirty-six subjects with a positive skin prick test (SPT) for HDM were divided into a symptomatic (n = 17) and an asymptomatic (n = 19) group on the basis of their clinical history and a nasal provocation test. A basophil CD63 response to in vitro stimulation with Dermatophagoides pteronyssinus whole allergen extract and the IgE reactivity profiles for Der p 1, 2, 4, 5, 7, 10, 11, 14, 15, 18, 21, 23 were evaluated. Serum IgE and IgG specific to D pteronyssinus whole allergen extract and total IgE were measured. RESULTS: There were no statistically significant differences in the levels of IgE (IgE levels were higher in symptomatic patients with P = 0.055) and IgG specific to D pteronyssinus and total IgE. Symptomatic patients showed a lower threshold for in vitro basophil activation (3.33 ng/mL vs 33.3 ng/mL), a higher area under the curve (AUC) of basophil activation (171 vs 127) (P = 0.017), a higher response to positive control with anti-FcεRI stimulation (97% vs 79%) (P < 0.001), a recognition of more HDM allergens (4 vs 2) and more frequent sensitization to rDer p 7 (P = 0.016) and rDer p 23 compared to asymptomatic subjects (P = 0.018). There was a positive correlation (r = 0.63; P < 0.001) between the number of recognized allergens and the AUC of basophil activation. CONCLUSION AND CLINICAL RELEVANCE: In the subjects studied, the differences in the basophil response to D pteronyssinus allergen extract, number of recognized HDM allergens and reactivity to rDer p 7 and rDer p 23 distinguish symptomatic from asymptomatic HDM sensitisation better than SPT or allergen extract-specific IgE. Information regarding the clinical relevance of sensitization is important for the prescription of allergen-specific immunotherapy.

A biomarker that could identify individuals at high risk for severe honeybee sting allergic reaction and/or systemic adverse events (SAEs) during venom immunotherapy (VIT) would improve the management of patients with honeybee (HB) venom allergy.To identify biomarkers for risk of severe sting reactions or SAEs during VIT.We recruited 332 patients undergoing HB VIT. We ascertained predictors of the severity of the field-sting reaction and the severity and threshold of SAEs during VIT. We assessed the use of cardiovascular medications; baseline serum tryptase (BST) levels; specific IgEs to HB venom, rApi m 1, and rApi m 10; and basophil activation test (BAT) response.Significant and independent predictors of a severe HB field-sting reaction were age (P = .008), an absence of skin symptoms (P = .001), BST (P = .014), and BAT response at an HB venom concentration of 0.1 μg/mL (P = .001). Predictors of severe SAEs during HB VIT were age (P = .025), BST (P = .006), and BAT response (P = .001). BAT response was also an individual and significant predictor of any SAEs and SAEs at a low cumulative allergen dose (median, 55 μg) during VIT build-up (P.001). The use of β-blockers and angiotensin-converting-enzyme inhibitors and specific IgE levels were not associated with the severity of HB field-sting reactions or VIT SAEs.BST and basophil activation are independent risk factors for severe HB sting anaphylaxis and SAEs during HB VIT. BAT response was the best biomarker for any SAEs and a lower threshold of SAEs during HB VIT. These risk factors can help guide recommendations for VIT and overcome systemic reactions to HB VIT.

Increased mucosal double-negative (DN) CD3+CD4-CD8- T cells were found for the first time in CRS and were much more abundant in uncontrolled CRSwNP than in well-controlled CRSwNP.

Background: The environment in swimming pools, which contain chlorine, might interact with the airway epithelium, resulting in oxidative stress and/or inflammation during high intensity training periods. Methods: We evaluated pulmonary functional (metacholine challenge test, FEV1 and VC), cellular (eosinophils and neutrophils), inflammatory (FeNo, IL-5, IL-6, IL-8 and TNF-[alpha]), oxidative (8-isoprostanes) and angiogenesis factors (VEGF) in induced sputum and peripheral blood of 41 healthy non-asthmatic elite swimmers (median 16 years) during the period of high intensity training before a national championship. The second paired sampling was performed seven months later after training had been stopped for one month. Results: There was a ten-fold increase (median 82-924 pg/ml P < 0.001) in 8-isoprostanes in induced sputum and five-fold increase (median 82-924 pg/ml P < 0.001) in sera during training in comparison to the period of rest. However, there was no difference in FEV1 (113 vs 116%), VC (119 vs 118%), FeNo (median 34 vs 38 ppb), eosinophils (2.7 vs 2.9% in sputum 180 vs 165 cells/[micro]l in blood), neutrophils, different cytokines or VEGF in induced sputum or sera. The only exception was TNF-[alpha], which was moderately increased in sera (median 23 vs 40 pg/ml P=0.02) during the peak training period. Almost half (18 of 41) of swimmers showed bronchial hyperresponsiveness during the peak training period (PC20 cutoff was 4 mg/ml). There was no correlation between hyperresponsiveness and the markers of oxidative stress or inflammation. Conclusions: High intensity training in healthy, non-asthmatic competitive swimmers results in marked oxidative stress at the airway and systemic levels, but does not lead to airway inflammation. However, we could not confirm that oxidative stress is associated with bronchial hyperresponsiveness (AHR), which is often observed during the peak exercise training period.

Introduction. The aim of this study was to compare the diagnostic value of two imunoglobulin E assays for penicillin in a group of patients with a history compatible with hypersensitivity reaction during penicillin treatment who exhibited positive skin testing and/or a drug provocation testing. Material and Methods. In the first part of the study, penicillin G, V and/or amoxicillin specific immunoglobulin E, positive with ImmunoCAP system (Termo Fisher, Waltham, Massachusetts, USA), were selected from our biobank sera, and measured specific immunoglobulin E with Immulite system (Siemens, Munich, Germany). The second part of the study included skin testing, and if negative, drug provocation testing was done with the culprit penicillin in patients with a history compatible with hyper?sensitivity reaction during penicillin treatment. To check the impact of high values of total immunoglobulin E on performance of specific immunoglobulin E, we examined the specific immunoglobulin E to penicillin using both methods in sera of penicillin tolerant patients with total immunoglobulin E over 700 kIU/L. Results. Out of 2.521 in vitro specific immunoglobulin E penicillin tests, 58 were positive with ImmunoCAP. Of these tests, 52 were tested by the Immulite and they were all negative. Among 114 patients with a history compatible with hypersensitivity reac?tion during penicillin treatment, hypersensitivity was confirmed in 36 (31.6%). In 11 patients with positive immediate skin or drug provocation tests, ImmunoCAP was positive in 3 patients and Immulite in 1 patient. Sensitivity of ImmunoCAP and Immulite were 27.1% and 9.1%, respec?tively. Specificity of ImmunoCAP and Immulite were 96.1% and 100%, respectively. Specific immunoglobulin E to penicillins was detected with ImmunoCAP in 8/22 sera with total immunoglobulin E over 700 kIU/L. All sera were negative by the Immulite. Conclusion. Both the Immuno?CAP and Immulite specific immunoglobulin E assays have low sensitiv?ity when assessing patients with a history of immediate hypersensitivity to penicillin, positive immediate skin, or drug provocation tests.

Objective: Hereditary angioedema due to C1 inhibitor deficiency (C1-INH-HAE) is a rare autosomal dominant disease caused by mutations in the SERPING1 gene. It can affect many regions in the body, but potentially life-threatening laryngeal oedemas are of concern. Methods: Twenty-three subjects from two families were recruited for clinical data evaluation and molecular analysis at General Hospital Šibenik, Croatia. Results: Decreased levels of C1 inhibitor were detected in 12 adult patients and three young asymptomatic persons. The same novel deletion of two nucleotides on exon 3 (c.74_75delAT) was identified in all of them. A history of laryngeal oedema was present in 10 patients (83%), and all patients reported laryngeal attacks at least once a year. The delay in diagnosis decreased noticeably from the first to the last generation. Conclusions: We identified a novel causative mutation in SERPING1 in several affected members of two apparently unrelated families with a high frequency of laryngeal oedema. Molecular analysis of large C1-INH-HAE families will provide new insights on the genotype-phenotype relationship. Key messages Hereditary angioedema due to C1 inhibitor deficiency is a rare autosomal dominant disease caused by mutations in the SERPING1 gene, and laryngeal oedema is of concern because it can cause death by asphyxiation. A novel causative mutation in SERPING1, a deletion of two nucleotides on exon 3 (c.74_75delAT), was identified in several affected members of two apparently unrelated families with a high frequency of laryngeal oedema. Molecular analysis of large C1-INH-HAE families will provide new insights on the genotype-phenotype relationship because it appears that the mutation type may affect disease severity.

Background T cells play a major role in delayed-type hypersensitivity reactions. Their reactivity can be assessed by measuring the upregulation of the activation marker CD69, followed by assessment of proliferation and cytokine production. The aim of our study was to develop a novel, whole blood-based, quantitative, absolute count activation index (AI) for analysis of CD69 upregulation in various subsets of T cells in nickel-hypersensitive patients and compare it with previously reported approaches. Methods The study population comprised 10 patients with nickel allergy and 9 healthy controls. CD69 expression of CD3+, CD3+CD4+, and CD3+CD8+ T cells in heparinized blood was determined with flow cytometry after incubation with nickel sulfate for 48 hours. The absolute count of CD69+ cells was determined using microbeads. Production of the cytokines IL-2, IL-5, IL-13, and IFN-γ was determined after stimulation of peripheral blood mononuclear cells with nickel sulfate for 48 hours. Results We showed absolute AI to be the most sensitive approach. The index was calculated as the ratio of the absolute count of nickel-stimulated CD69-positive T cells to the absolute count of CD69-positive T cells in nonstimulated blood. This novel quantitative approach was more discriminative than previously reported approaches in which the T-cell CD69 percentage AI and cytokine production are measured. Conclusions Our results demonstrated that measuring the absolute CD69 AI is a novel and accurate approach for quantification of antigen-specific T cells in the blood of patients with hypersensitivity reactions to nickel. This approach may be useful for better in vitro assessment of patients with delayed-type hypersensitivity reactions.

BACKGROUND Adverse systemic reactions (SRs) are more common in honeybee venom immunotherapy (VIT) than in wasp VIT. Factors that might be associated with SRs during the honeybee VIT are poorly understood. OBJECTIVE Our aim was to evaluate risk factors for SRs during the build-up phase of honeybee venom immunotherapy. METHODS We included 93 patients who underwent ultra-rush honeybee VIT. The adverse SRs and their severity was compared to various immunological (sIgE, tIgE, basophil CD63 response, baseline tryptase, and skin tests), patient-specific (age, sex, cardiovascular conditions and medications, and other allergic diseases), and sting-specific factors (anaphylaxis severity, time interval to onset of symptoms, and absence of cutaneous symptoms). RESULTS Twenty-three patients (24.7%) experienced mild SRs and 13 patients (14%) severe SRs. In five patients with severe SRs, the build-up was stopped. High basophil allergen sensitivity, evaluated as dose-response curve metrics of EC15, EC50, CD-sens, AUC, or the response to submaximal 0.01 μg/mL of venom concentration, was the most significant risk factor and only independent predictor of severe SRs and/or build-up stop. Time interval of

Background An important advantage of allergen immunotherapy as compared to pharmacotherapy for allergic rhinitis is the long-term effect that persists after completing immunotherapy. The mechanism of the sustained effect of allergen immunotherapy is not completely understood. Methods We conducted a 7-year study of monitoring allergen-specific basophil response and serological markers in 20 subjects with moderate-to-severe grass pollen-allergic rhinitis just before beginning and after up-dosing of subcutaneous grass pollen immunotherapy, before the first pollen season, and 1–2 years after completion of 3–5 years of treatment. Comparable untreated rhinitis subjects were followed at the same time points. Clinical outcomes included assessment of symptoms, use of rescue medication, and quality of life. The basophil response was also monitored after removal of IgG antibodies. Results Basophil response assessed as area under the curve (AUC) halved during initiation of SCIT and was 55% lower 1–2 years after completing SCIT. In the untreated group, the basophil response remained comparable. Although immunotherapy-induced grass pollen-specific IgG4 levels decreased to near pre-immunotherapy levels after completing SCIT, the removal of IgG antibodies resulted in an increase in basophil response almost to the pre-immunotherapy levels. In untreated subjects, removal of IgG did not have any effect on basophil response. Conclusions Grass pollen immunotherapy induces sustained suppression of the allergen-specific basophil response that persists after completion of treatment and could account for long-term clinical tolerance. It also seems to be associated with persistent blocking activity of IgG antibodies.

Background The role of basophils in anaphylaxis is unclear. Objective We sought to investigate whether basophils have an important role in human anaphylaxis. Methods In an emergency department study we recruited 31 patients with acute anaphylaxis, predominantly to Hymenoptera venom. We measured expression of basophil activation markers (CD63 and CD203c); the absolute number of circulating basophils; whole-blood FCER1A, carboxypeptidase A3 (CPA3), and L-histidine decarboxylase (HDC) gene expression; and serum markers (CCL2, CCL5, CCL11, IL-3, and thymic stromal lymphopoietin) at 3 time points (ie, during the anaphylactic episode and in convalescent samples 7 and 30 days later). We recruited 134 patients with Hymenoptera allergy and 76 healthy control subjects for comparison. We then investigated whether the changes observed during venom-related anaphylaxis also occur during allergic reactions to food in 22 patients with peanut allergy undergoing double-blind, placebo-controlled food challenge to peanut. Results The number of circulating basophils was significantly lower during anaphylaxis (median, 3.5 cells/μL) than 7 and 30 days later (17.5 and 24.7 cells/μL, P

Histamine intolerance (HIT) is characterized by an imbalance between histamine intake and the capacity for histamine degradation. The main enzyme for metabolizing ingested histamine is diamine oxidase (DAO). Determining DAO activity in serum may be useful in diagnosing HIT.Over a period of 3.5 years we recruited 316 subjects with clinically suspected HIT and 55 healthy controls. Serum DAO activity was measured with a quantitative enzyme immunoassay. Twenty patients with highly reduced DAO activity went on a histamine-free diet for 6-12 months. Afterwards, their DAO activity was determined again.We found that DAO activity was significantly lower in patients than in healthy control subjects (P 0.0001). Furthermore, 54 patients had highly reduced serum DAO activity ( 40 HDU/ml). Their main symptoms involved the skin, gastrointestinal tract, respiratory system, and eyes. In all the 20 patients with highly reduced DAO activity, the main clinical symptoms typical of histamine intolerance disappeared after they adopted a histamine-free diet. Furthermore, the serum DAO activity values measured increased significantly (P 0.0001).Our results suggest that determining DAO activity in serum is a useful tool in diagnosing HIT. Furthermore, our results showed the benefit of a histamine-free diet because after the diet the majority of symptoms disappeared and the serum DAO activity significantly increased.

INTRODUCTION: Bronchial thermoplasty (BT) is an effective treatment for carefully selected uncontrolled asthma patients. The treatment has significant effect on the structural cells of the airway wall and offers long-term beneficial clinical effects with minor side effects. Reports of other possible mechanisms of BT action are only scant. AIMS: We focused on inflammatory response after BT with the aim to detect the possible changes in T cell populations in severe asthmatic airways after treatment with BT. METHODS: We included 4 patients with uncontrolled asthma who were treated with BT at our clinic from Nov 2013 till March 2014. Bronchoalveolar lavage (BAL) samples were collected from the right lower lobe prior to, 3 and 6 weeks post initial BT. Samples were analysed by flow cytometry to determine the subsets of BAL T cells and their expression of activation and differentiation markers; CD4, CD8, CCR4, CXCR3, CCR6, CD25, CD69, HLA DR, CD27, CD28. RESULTS: At baseline, more BAL T cells expressed CD3+8+ cytotoxic (Tc) than helper T cell markers. Most of T cells expressed early activation marker CD69 and majority of Tc expressed fully differentiated phenotype CD27-CD28-. Th17 cells presented 14%, and CD4+25+ regulatory T cells (Treg) 25% of studied lymphocyte population. At 3 and 6 weeks after treatment we detected increase in Treg population to more than 40%. Treated patients reported less severe exacerbations and less symptoms in a year following BT treatment. CONCLUSIONS: The substantial increase of BAL immunomodulatory regulatory T cell proportion after BT treatment indicates the possible influence of BT not only on structural changes but also on inflammatory response.

Shortly after the report of pandemic 2009 influenza A (H1N1), vaccine manufacturers, in conjunction with public agencies, started developing a H1N1 vaccine. In 2009, various approaches were implemented around the globe. The United States and Australia finally approved only non-adjuvanted H1N1 influenza vaccines, whereas Canada and the EU also approved adjuvanted vaccines. In 2010, seasonal influenza vaccine without adjuvant was again widely accepted in both hemispheres. The addition of adjuvant to the vaccine enhances the immunogenity of the vaccine in the presence of a relatively low amount of antigen. However, it might also induce undesirable non-specific immune response. For this reason, we conducted a prospective observational study to monitor T cell absolute count and H1N1-specific immunogenicity after 2009 and 2010 immunization. Fourteen healthy volunteers received the monovalent H1N1 AS03 adjuvanted influenza vaccine (3.5 μg of H1N1 and squalene-based adjuvant) in October 2009. The immunization was associated with a significant increase in T lymphocyte absolute count (P < 0.0001), reaching abnormal values in 57% of subjects. During this period, none of the subject showed any manifestation of severe viral infection or inflammation. Acute infection by CMV or EBV viruses was also excluded. In October 2010, the same subjects received a seasonal non-adjuvanted influenza vaccine (15 μg of each: H1N1, H3N2, and B-Brisbane). However, after 2010 immunization, no change in T lymphocyte absolute count was observed. H1N1-induced immunogenicity was good for both vaccines. Our results suggest a pronounced non-specific T cell response after AS03-adjuvanted 2009 H1N1 vaccination.

Asthma is a common chronic disease characterized by airway inflammation and structural remodeling. Vascular endothelial growth factor (VEGF), a major regulator of angiogenesis, is elevated in asthma patients. VEGF contributes to airway responsiveness and remodeling. It has been shown that treatment of asthma patients decreases VEGF levels, and inhibition of VEGF diminishes asthma symptoms in mice. Therefore, polymorphisms in the vascular endothelial growth factor A (VEGFA) gene might be associated with asthma treatment response.This study enrolled 131 children with asthma treated with different therapies - specifically, the inhaled corticosteroid (ICS) fluticasone propionate or the leukotriene receptor antagonist (LTRA) montelukast. We performed an association analysis between improvement of lung function - assessed by measurement of the percentage of the predicted forced expiratory volume in 1 second (%predicted FEV(1)), the ratio between the FEV(1) and the forced vital capacity (FEV(1)/FVC) after 6 and 12 months of treatment, and asthma control after 12 months of treatment - and two polymorphisms, rs2146323 and rs833058, in the VEGFA gene.Polymorphism rs2146323 AC in VEGFA was associated with response to ICS therapy. Asthma patients with the AA genotype had a greater improvement in the %predicted FEV(1) than those with the AC or CC genotype (p = 0.018). Conversely, the AA genotype in rs2146323 was associated with uncontrolled asthma in patients regularly receiving LTRA therapy (p = 0.020) and a worse FEV(1)/FVC ratio in patients who episodically used LTRA therapy (p = 0.044). Furthermore, polymorphism rs833058 CT was associated with treatment response to episodically used LTRA therapy. A subgroup of patients with the TT genotype had an improvement in the %predicted FEV(1), compared with no improvement in patients with the CT or CC genotype (p = 0.029).Our results showed that treatment response to commonly used asthma therapies (ICS or LTRA) is associated with polymorphisms rs2146323 and rs833058 in VEGFA. With additional replication of this preliminary study, our findings could contribute to the development of individualized asthma therapy.

To cite this article:Žitnik SEK, Vesel T, Avcin T, Silar M, Kosnik M, Korosec P. Monitoring honeybee venom immunotherapy in children with the basophil activation test. Pediatr Allergy Immunol 2011 Doi: 10.1111/j.1399-3038.2011.01233.x Abstract Background: New in vitro methods are essential for developing better follow-up criteria for venom immunotherapy (VIT). Methods: Thirty-one children with a history of honeybee venom–induced systemic anaphylaxis were included in this prospective, single-blinded study. The basophil CD63 activation test (BAT) was assessed before starting VIT, at the end of the build-up phase (day 5), 6 months later, and after 2–4 yr of VIT. Results: Basophil CD63 activation test allowed identification of the culprit insect in 74% of honeybee venom–allergic children. In comparison, IgE reactivity was single positive in only 52% of children. Five days after starting VIT, BAT was highly comparable to before VIT. However, after 6 months and further after 2–4 yr of VIT, a significant and approximately fourfold decrease was demonstrated in CD63 response at sub-maximal 0.1 μg/ml allergen concentration, which mainly represents cellular sensitivity. No such differences were found at a higher 1 μg/ml of allergen concentration. Person-to-person analyses showed that after 2–4 yr of VIT, a marked CD63 decrease was evident in 85% of children. In addition, elevated basophil sensitivity measured before VIT was associated with the appearance of side effects observed during the build-up phase of VIT. Conclusion: Basophil CD63 allergen–specific sensitivity seems to be a promising tool for monitoring protective immune response in honeybee VIT.

Summary Asthma is one of the most common chronic diseases in childhood. It is well known that genetic variability contributes to asthma risk. One of the most replicated asthma candidate genes is ORM1-like 3 (Saccharomyces cerevisiae) (ORMDL3), which has been associated with childhood asthma susceptibility. Another asthma candidate gene is signal transducer and activator of transcription 6 (STAT6), a regulator of IgE class switching. Gene coding thromboxane A2 receptor (TBXA2R), involved in chronic airway inflammation, has been associated with asthma in several genetic studies. We have studied the association of polymorphism rs4795405 in ORMDL3, rs324011 in STAT6 as well as rs8113232 and rs3786989 in TBXA2R with asthma risk, various asthma phenotypes and asthma-related symptoms. The study group consisted of 154 children with asthma, in whom clinical parameters were measured and whose asthma control and atopic status were determined. A control group comprised 71 healthy children. Genotyping was performed using an allelic discrimination assay. The ORMDL3 polymorphism rs4795405 was suggestively associated with asthma risk. Furthermore, it was significantly associated with nonatopic asthma and asthma without rhinitis. No association was detected between the STAT6 polymorphism rs324011 or the TBXA2R polymorphisms rs8113232 and rs3786989 and asthma susceptibility. However, an association between rs324011 in STAT6 with recurrent wheezing in early childhood and a suggestive association between rs8113232 in TBXA2R with rhinitis in children with asthma were observed. Our results confirmed ORMDL3 as a candidate gene for childhood asthma susceptibility. STAT6 and TBXA2R polymorphisms were not associated with asthma risk, but they were associated with asthma-related symptoms.

SummaryBackground The mechanisms responsible for the difference between clinically irrelevant IgE-sensitization and allergic rhinitis are not fully understood. Objective We evaluated the humoral and cellular mechanisms that may be associated with the presence of allergic rhinitis symptoms. Methods We selected 26 subjects with positive grass pollen skin tests and IgE antibodies to Timothy (g6) and the major grass allergens rPhl p 1, 5b. Fourteen of those patients reported a history of allergic rhinitis. During winter, we performed a grass pollen CD63 basophile activation test using four log allergen concentrations, followed by a grass nasal provocation test (NPT). We obtained symptom scores in the subsequent pollination season. Results We showed that subjects with a positive NPT have significantly higher CD63 basophile grass pollen responsiveness than NPT-negative subjects, preferably at submaximal allergen concentrations, which represent cellular sensitivity. Moreover, basophile sensitivity positively correlated with the size of the grass-specific IgE fraction in relation to total IgE, and it was highly predictive of allergic rhinitis symptoms in the following pollination season. Conclusion and Clinical Relevance Allergic rhinitis symptoms are significantly associated with allergen-specific basophile sensitivity. In vitro evaluation of basophile sensitivity should prove useful for distinguishing clinical phenotype of allergic sensitization.

The aim of this study was to investigate the mRNA expression levels of multidrug resistance-associated proteins in chemo-naïve metastatic lung cancer cells and to determine the correlation with response to chemotherapy and overall survival. Metastatic cells were obtained by transbronchial fine needle aspiration biopsy of enlarged mediastinal lymph nodes in 14 patients with small cell lung cancer (SCLC) and 7 patients with non-small cell lung cancer (NSCLC). After cytological confirmation of lung cancer type, total RNA was extracted from biopsy samples and reverse transcribed to cDNA, and real-time PCR for the genes of interest [P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), breast cancer resistance protein (BCRP), lung resistance protein (LRP) and topoisomerase IIα (TOPIIα)], was performed. We observed significantly decreased expression of BCRP and significantly increased expression of TOPIIα in metastatic SCLC cells compared to NSCLC. Furthermore, in SCLC high topoisomerase IIα and low BCRP expression levels positively correlated with longer overall survival. Our results showed higher expression levels of BCRP as well as lower levels of topoisomerase IIα in chemo-naïve metastatic cells in NSCLC than in SCLC. These results correlate with previous observations that metastatic SCLC cells at the beginning of chemotherapy are potentially more sensitive to chemotherapeutic agents while in metastatic NSCLC cells resistance is usually inherent. We also showed that altered levels of topoisomerase IIα and BCRP in SCLC are important factors that contribute to resistance to chemotherapeutics that interfere with the enzyme and/or DNA and are highly associated with overall survival.

Background: Exposure to acetylsalicylic acid (ASA) may exacerbate respiratory or skin diseases or induce anaphylactoid reactions in apparently healthy individuals. We wanted to evaluate specific responsiveness of basophils to ASA in correlation with the clinical picture. Methods: We performed a prospective single-blind study of 59 subjects involved in clinical evaluation and/or ASA provocation testing. Whole blood basophils were stained with anti-CD63/CD123/HLA-DR mAbs after stimulation with 0.25 or 1 mg/ml ASA. Results: We found that 40 subjects were ASA tolerant and 19 were ASA intolerant. Both groups had comparable manifestations of asthma and/or rhinitis (13 in the tolerant and 9 in the intolerant group). Intolerant subjects showed significantly higher basophil responsiveness to ASA in comparison to tolerant subjects, which was concentration-dependent in both groups. The ratio between responses at 1 mg/ml of ASA and at baseline (activation index) was analyzed according to the clinical picture. We demonstrate that the activation index was higher only in the intolerant subjects with anaphylactoid reactions, but not in a subgroup of subjects with asthma/rhinitis. The ROC calculations show that the optimal threshold activation index was more than 2.18. The sensitivity was 80% and the specificity was 83% in the subgroup with anaphylactoid reactions. In the asthma/rhinitis subgroup, the sensitivity was 78% and the specificity was 50%. Conclusions: Our study demonstrates that there is a significantly higher in vitro basophil response to ASA in intolerant as compared to tolerant subjects. ROC analyses suggest that this measurement might only have a diagnostic value in subjects without asthma and/or rhinitis.

Summary Background The effects of pollen immunotherapy on effector cells of allergic inflammation, such as mast cells and basophils, are poorly understood. Objective For this reason, we conducted an open study on basophil allergen threshold sensitivity during birch pollen immunotherapy. Methods Basophil sensitivity was measured by CD63 flow cytometry in 14 patients with moderate–severe intermittent birch pollen rhinitis using four log allergen concentrations. In nine patients, we analysed the basophil sensitivity before and during treatment with subcutaneous birch immunotherapy (perennial scheme with allergoid). We also included eight birch-allergic donor subjects for IgG inhibition experiments and eight control subjects. Results There was a decrease in basophil allergen threshold sensitivity after 2, 3, and 5 months of immunotherapy. This decrease was correlated with an improvement in patients' symptoms measured on a visual analogue scale. The serum obtained after immunotherapy induced a significant decrease in allergen threshold sensitivity in donor birch-allergic basophils. This inhibition was not observed after IgG depletion from the serum. Conclusions In this study, we showed that birch immunotherapy-induced IgG antibodies are associated with a reduction in basophil allergen threshold sensitivity. Further studies are needed to show whether the changes in basophil sensitivity are of clinical relevance in pollen immunotherapy. Cite this as: N. Lalek, M. Kosnik, M. Silar and P. Korosec, Clinical & Experimental Allergy, 2010 (40) 1186–1193.

Invariant natural killer T (NKT) cells might contribute to the amplified and prolonged T-cell immune response that characterizes sarcoidosis. Therefore, we want to investigate the frequency and distribution of pulmonary invariant NKT cells in corticosteroid-naïve patients with sarcoidosis. We used multi-parameter flow cytometry with antibodies against CD3, CD4, CD8, CD14, CD19, CD45, CD16/56, TCR Valpha24, and TCR Vbeta11, on bronchoalveolar lavage fluid (BALF), to examine the frequency and distribution of pulmonary invariant NKT cells in 47 newly diagnosed sarcoidosis patients and in 8 control subjects. The frequencies of BALF Valpha24 Vbeta11 invariant NKT cells were significantly lower in patients with sarcoidosis in comparison to control subjects. Moreover, lower invariant NKT cell frequencies in patients with sarcoidosis significantly correlated with exaggerated BALF lymphocytosis and CD4 T cell responses. This study demonstrated a pulmonary deficiency in the frequency of a subset of T cells with immunoregulatory function in patients with sarcoidosis.

Summary Background Current guidelines do not adequately address the question of how best to manage patients with a convincing history of insect allergy, but negative venom-specific IgE and skin test results. Methods Forty-seven patients out of a total of 1219 (4%), with a positive history of sting allergy, were recruited over a period of 4.5 years. All recruited patients had a convincing history of a severe or a life-threatening anaphylactic reaction of Mueller grade II–IV (median grade III) after Hymenoptera sting, but negative venom-specific IgE and skin prick test results. Diagnostic work-up was prospectively followed by the CD63 basophil activation test and by intradermal skin testing. A control group of 25 subjects was also assessed. Results Thirty-five out of 47 (75%) patients demonstrated a positive basophil CD63 response after stimulation with bee and/or wasp venom. Intradermal venom skin tests were performed for 37 patients, 17 (46%) of whom showed positive results. Out of 20 patients who demonstrated negative intradermal test results, 12 patients showed a positive CD63 response (60%). In contrast, out of 9 patients who showed a negative CD63 response, only one was detected by intradermal testing (11%). In the control group, only two out of 25 (4%) subjects displayed a positive basophil response and/or intradermal test. Conclusion Here we show that, in complex cases with inconclusive diagnostic results, the CD63 activation test could be particularly useful and more sensitive than intradermal skin testing.

BACKGROUND: Immediate allergic reactions to β-lactam antibiotics are mediated by specific IgE antibodies. The Phadia CAP System FEIA ® is a commercial method for quantification of specific IgE. We wished to determine anti-β-lactam IgE antibodies in patients without penicillin allergy but with high levels of total IgE. METHODS: Sera from 41 patients (31 with high total IgE, 10 with low total IgE) were analyzed for IgE antibodies specific to penicilloyl G, penicilloyl V, amoxicilloyl and ampicilloyl using the CAP FEIA® method that was available up to 2006. Seven sera that tested positive were rechecked in a new improved system available after 2006. RESULTS: In patients without a history of penicillin allergy, the specificities of commercial tests for anti-β-lactam IgE antibodies were 100%, 60%, 27% and 20% at total IgE levels of 8–263 kU/l, 500–664 kU/l, 1000–2000 kU/l and > 2000 kU/l, respectively. In seven retested sera, only 2 (28%) were still positive for penicillin-specific IgE antibody. CONCLUSION: Before 2006, tests for anti-β-lactam IgE antibody in patients with total IgE > 500 kU/l were probably often false positive. Patients who were diagnosed as penicillin allergic before 2006 solely on the basis of a positive CAP FEIA® test for specific IgE should be considered for diagnostic reevaluation.

BACKGROUND: Complement component 5a (C5a) might be involved in the formation of wheals in patients with chronic urticaria (CU). We sought to compare the in vitro responsiveness of basophils to C5a in patients with CU and in a control group. METHODS: Basophil surface expression of activation marker CD63 induced by C5a, anti-FceRI mAb or anti-IgE pAb was measured using flow cytometry in 17 patients with CU and in 10 healthy controls. RESULTS: Patients with CU showed significantly greater basophil CD63 surface expression induced by C5a (median [interquartile range]; 16.4% [13–25.1]; P = 0.011) than the group of healthy controls (10.7% [7.2–16.8]). In contrast, basophil CD63 response to anti-IgE and anti-FceRI was lower in the CU group (12.3% [6–36.3]; 25.9% [12.5–60.5]) than in the control group (51.7% [6.7–84.3]; 62.1% [9.7–89.2]), although not statistically significant. CONCLUSION: Results of this pilot study suggest that patients with CU might have an enhanced basophil response to stimulation with C5a, indicating that further studies in CU basophil responsiveness are needed.

BACKGROUND: Among patients with allergy to insect stings, double positivity in tests for IgE antibodies specific to honey bee and wasp venoms is a frequent diagnostic problem. True double sensitization and possible cross-reactivity of venom hyaluronidases and with carbohydrate determinants must be considered in such patients. We studied the frequency of sensitization to carbohydrate determinants and the role of these in double positivity in tests for specific IgE antibodies. MATERIALS AND METHODS: A group of 66 patients (41 men, 25 women; 16–66 years) with double positivity for wasp and bee venoms were tested in the FEIA inhibition test in order to distinguish true double sensitization from cross-reactivity. Patients were tested for the presence of IgE antibodies specific to oilseed rape (OSR) pollen and MUXF3 allergens, both of which are rich in cross-reacting carbohydrate epitopes. RESULTS: Inhibition tests revealed true double sensitization in 37 patients (56.1%) and cross-reactivity in 29. Among those showing cross-reactivity, five were sensitized to honey bee venom and 24 to wasp venom. The median value of IgE specific for OSR pollen in patients sensitized to honey bee venom was 4.350 IU/ml, in patients sensitized to wasp venom 0.61 IU/ml, and in patients with double sensitization 0.25 IU/ml (P = 0.028, Kruskal–Wallis test). Findings for IgE specific for MUXF3 were similar. Discordance between OSR pollen positivity and MUXF3 positivity was found in 11.1% of the patients. CONCLUSION: The values of IgE specific for OSR pollen and MUXF3 in patients with primary sensitization to either honey bee venom or wasp venom were significantly higher than in patients with double sensitization. These results confirm that IgE antibodies against carbohydrates epitopes are a frequent cause of double positivity in tests for anti-venom IgE antibodies.

Background: Treatment failure of venom immunotherapy (VIT) is not rare and the risk and pathogenic factors for those failures are so far poorly understood. For that reason we evaluated allergen-specific basophil sensitivity in patients who did not tolerate field re-stings after completed VIT treatment. Methods: Basophil responsiveness was evaluated by flow cytometry analyses of basophil CD63 surface expression induced by different concentrations of bee or wasp venom (1, 0.1 and 0.01 µg/ml) in 14 treated patients who had experienced systemic allergic reactions (Muller grades II–III) and 17 treated patients who had no reactions after the field re-stings. We also included a group of 28 Hymenoptera venom-allergic patients who had not received VIT. Results: In 14 patients who still reacted to bee or wasp sting, basophil response at a venom concentration of 0.1 µg/ml was significantly higher than in patients who tolerated field re-stings (p = 0.03; t test). Basophil response was also slightly higher at a concentration of 1 µg/ml, but not to statistical significance (p = 0.12; t test). There was no difference in the response to direct cross-linking of the IgE and in venom-specific IgE and IgG4 serum concentrations between those 2 groups (p > 0.8; Fisher’s exact test, t test). Patients who tolerated field re-stings have also significantly lower basophil response in comparison to patients who had not received VIT, both at 0.1 and 1 µg/ml of venom concentrations (p < 0.001; t test). Conclusions: The results suggest that basophil venom-specific sensitivity is associated with the efficiency of VIT.

Background: We sought to determine whether basophil-allergen sensitivity could be transferred to donor basophils by passive IgE sensitisation in allergic rhinitis and anaphylactic Hymenoptera venom hypersensitivity. Methods: We studied 15 wasp venom-, 19 grass pollen- and 2 house dust mite-allergic patients, 2 healthy donors, and 8 wasp venom-allergic donors. In all subjects, we first evaluated the initial basophil response to wasp venom, grass pollen, or house dust mite allergen. Donor basophils were then stripped, sensitised with the different patients' serum IgE, and challenged with the corresponding allergen. The CD63 response of donor basophils was then compared with initial basophil responses. Results: In wasp venom-allergic subjects, the IgE transfer did not reflect the initial basophil-allergen sensitivity, because the venom IgE of subjects with high or low basophil sensitivity induced comparable responsiveness in healthy donor basophils. Furthermore, vice versa, when we sensitised the donor basophils of wasp venom-allergic individuals with different wasp venom or house dust mite IgE, we demonstrated that their response was predictable by their initial basophil allergen sensitivity. In the rhinitis allergy model, the IgE transfer correlated with the patients' initial basophil responsiveness because the grass pollen IgE of the subjects with high basophil allergen sensitivity induced significantly higher responsiveness of donor basophils than the IgE of subjects with initially low basophil allergen sensitivity. Conclusions: Our results suggest that basophil allergen sensitivity evaluated by flow-cytometric CD63 analysis depends on two distinct contribution factors. In anaphylactic Hymenoptera allergy, the major factor was intrinsic cellular sensitivity, whereas in pollen allergy, the major factor was allergen-specific IgE on the cell surface.

Natural killer T (NKT) cells, a newly identified subgroup of T cells with immunoregulatory function, may be implicated in the pathogenesis of interstitial lung disease (ILD).We used multiparameter flow cytometry with antibodies to CD3, CD4, CD8, CD14, CD19, CD45, CD16/56, CD56, CD161, and Valpha24 invariant T-cell receptor (TCR) in BAL fluid (BALF) to examine the frequency and distribution of pulmonary NKT cells in several cases of ILD. We included 57 patients with sarcoidosis and 17 patients with hypersensitivity pneumonitis.We found significantly higher frequencies of pulmonary NKT cells in patients with hypersensitivity pneumonitis in comparison to the other study patients with ILD (median proportion of NKT cells, 11%; range, 3 to 38%; vs 3%; range, 0 to 16%; p0.0001). In contrast, there was no difference in the proportion of conventional natural killer cells. We found that a major subset of NKT cells in the BALF of patients with hypersensitivity pneumonitis was a CD8+CD56+ population that did not express the invariant TCR.These results suggest the involvement of NKT cells in the pathogenesis of hypersensitivity pneumonitis.

Cat allergy is one of the most prevalent allergies worldwide and can lead to the development of rhinitis and asthma. Thus far, only allergen extracts from natural sources have been used for allergen-specific immunotherapy. However, extracts and whole allergens in immunotherapy present an anaphylaxis risk. Identification of allergen epitopes or mimotopes has an important role in development of safe and effective allergen-specific immunotherapy. Moreover, with a suitable immunogenic carrier, the absence of sufficient immune response elicited by short peptides could be surmounted. In this study, we identified five structural mimotopes of the major cat allergen Fel d 1 by immunoscreening with random peptide phage libraries. The mimotopes were computationally mapped to the allergen surface, and their IgE reactivity was confirmed using sera from cat-allergic patients. Importantly, the mimotopes showed no basophil activation of the corresponding cat-allergic patients, which makes them good candidates for the development of hypoallergenic vaccine. As bacteriophage particles are becoming increasingly recognized as immunogenic carriers, we constructed bacteriophage particles displaying multiple copies of each selected mimotope on major phage coat protein. These constructed phages elicited T cell-mediated immune response, which was predominated by the type 1 T cell response. Mimotopes alone contributed to the type 1 T cell response by promoting IL-2 production. Fel d 1 mimotopes, as well as their filamentous phage immunogenic carriers, represent promising candidates in the development of hypoallergenic vaccine against cat allergy.

Introduction: Thymic stromal lymphopoietin (TSLP) is an epithelial-cell–derived cytokine that is produced in response to proinflammatory stimuli, leading to inflammation driven by type 2 helper T (Th2) cells. TSLP thus may contribute to the mechanisms of airway remodelling in asthma and represent a potential targets for biologic asthma treatment. Aims and objectives: The aim of our study was to find out whether there is a difference in serum concentrations of TSLP in asthma patients, COPD and healthy controls. Methods: We analysed serum TSLP concentrations in 41 stable asthmatic patients, 24 COPD (12 stable and 12 during exacerbation) and 14 healthy controls. We used ELISA test for TSLP analyse. Results: Asthma patients have significantly higher systemic TSLP concentrations (median 203 pg/ml) comparing to patients with COPD (median 34 pg/ml; p=0.0022) and comparing to healthy controls (median 44 pg/ml, p=0.0012). There was no difference between stable and exacerbated COPD patients. ![Figure][1] Conclusions: TSLP is an asthma biomarker and might represent an immunological tool in differential diagnosis in obstructive lung disease. Further studies are needed see if there are any differences between patient with different asthma severity and/or phenotypes. [1]: pending:yes

SummaryBackground No study has assessed the diagnostic sensitivity of rApi m 1 and rVes v 5 on Immulite testing system. Objective To compare the diagnostic sensitivity of commercially available venom recombinant allergens between the currently available immunoassays [ImmunoCAP (CAP) and Immulite (LITE)] and establish their correlation with the severity of the sting reaction. Methods This study evaluated 95 bee venom and 110 yellow jacket venom-allergic subjects. We measured the levels of sIgE to rApi m 1, rVes v 5 (LITE and CAP), rApi m 2 (LITE), rVes v 1 (CAP) and total IgE (CAP). Forty-nine healthy subjects served as controls. Results The diagnostic sensitivity of rApi m 1 and rVes v 5 was significantly higher with the LITE than with the CAP system (71% vs. 88% and 82% vs. 93%). The specificity of both assays for both allergens was between 94% and 98%. Twenty-nine patients that tested negative for rApi m 1 or rVes v 5 with CAP were positive with LITE, but none of the patients that tested negative with LITE were positive with CAP. The positive values of rApi m 1 and rVes v 5 were on average 2.7 and 2.3 times higher, with the LITE than with the CAP system. The combination of rApi m 1 and rApi m 2 (LITE) and the combination of rVes v 5 (LITE) and rVes v 1 (CAP) almost matched the sensitivity of native venoms (95% and 97%, respectively), whereas the diagnostic sensitivity of the combination of rVes v 5 and rVes v 1 (CAP) did not reach the sensitivity of rVes v 5 (LITE) alone (90% vs. 93%). IgE levels to venom recombinants and total IgE did not correlate with the severity of sting reaction. Conclusions & Clinical Relevance The use of rApi m 1 and rVes v 5 with the LITE system significantly enhanced diagnostic utility of venom recombinants and should improve the dissection of bee and yellow jacket venom allergy.

Background: Systemic side-effects of venom immunotherapy (VIT) represent a considerable problem in the treatment of patients allergic to Hymenoptera venom. We examined the hypothesis whether basophil responsiveness might be connected with the adverse reactions to VIT. Methods: Basophil surface expression of activation marker CD63 induced by different concentrations of honeybee and wasp venom (0.1 and 1 μg/ml) was measured by flow cytometry in 34 patients with history of systemic anaphylactic reactions to Hymenoptera sting just before rush honeybee or wasp VIT. Results: Eleven of 34 patients had systemic anaphylactic reaction (Mueller grades I–III) and one patient a large local reaction to VIT. In those 12 patients, median percentage of activated basophils after stimulation with VIT-specific venom in concentration of 0.1 μg/ml was 99% (range: 17–195) of value reached with stimulation with 1 μg/ml. Side-effects occurred in all patients with 0.1/1 ratios over 92% (eight of 12). In contrast, in 22 patients with no side-effects, the median 0.1/1 ratio was 25% (range: 2–92). These concentration-dependent activation ratios were significantly different between the groups with and without side reactions (P

Background We recently showed a desensitization of FceRI-mediated basophil response after short-term VIT. Our aim was to evaluate the allergen specificity of this desensitization. Methods In 11 Hymenoptera-venom double positive subjects, basophil threshold sensitivity (CD-sens) to anti-FceRI, honeybee, and Vespula venom was assessed at the beginning and just before the first maintenance dose (MD) of single ultra-rush VIT. In some patients we also monitored CD-sens to rApi m 1 and/or rVes v 5 or other co-sensitizations (i.e., grass pollen). In additional 7 patients, basophils were stripped and sensitized with house dust mite (HDM) IgEs at the same time points. Results We demonstrated a marked reduction of CD-sens to anti-FceRI and VIT-specific venom before the first MD in all 18 subjects included. Furthermore, in 10 out of 11 double positive subjects, a significant and comparable decrease before the first MD was also evident for non-VIT venom; this nonspecific decrease was further supported by the opposite recombinant species-specific major allergen. In one subject with additional grass pollen allergy, a decrease of CD-sens to grass allergen was also demonstrated. Similarly, in 7 cases of patients with passively HDM-sensitized basophils, a significant reduction of CD-sens was also evident to de novo sensitized HDM allergen. Conclusions Short-term VIT induced basophil desensitization to VIT-specific as well as to VIT-nonspecific venom. As opposed to long-term VIT, which induces venom-specific changes, the effect of short-term VIT seems to be venom-nonspecific.

Hereditary angioedema (HAE) is a rare autosomal dominant disease characterized by swelling of the face, lips, tongue, larynx, genitalia, or extremities, with abdominal pain caused by intra-abdominal edema. HAE is caused by mutations affecting the C1 inhibitor gene, SERPING1, resulting in low levels of C1 inhibitor (Type I HAE) or normal levels of ineffective C1 inhibitor (Type II HAE). A nationwide survey identified nine unrelated families with HAE in Slovenia, among whom 17 individuals from eight families were recruited for genetic analyses. A diagnosis of HAE was established in the presence of clinical and laboratory criteria (low C1 inhibitor antigenic levels and/or function), followed up by a positive family history. Genetic studies were carried out using PCR and sequencing to detect SERPING1 mutations in promoter, noncoding exon 1, the 7 coding exons, and exon-intron boundaries. Multiplex ligation-dependent probe amplification was performed in order to search for large deletions/duplications in SERPING1 gene. A mutation responsible for HAE was identified in patients from seven families with the disease. In HAE type I families, one previously reported substitution (Gln67Stop, c.265C>T) and four novel mutations were identified. The new mutations included two missense substitutions, Ser128Phe (c.449C>T), and Glu429Lys (c.1351G>A), together with two frameshift mutations, indel (c.49delGinsTT) and deletion (c.593_594delCT). Both families with HAE type II harbored the two well-known substitutions affecting the arginyl residue at the reactive center in exon 8, Arg444Cys (c.1396C>T) and Arg444His (c.1397G>A), respectively. In one patient only the homozygous variant g.566T>C (c.-21T>C) was identified. Our study identified four novel mutations in the Slovenian HAE population, highlighting the heterogeneity of mutations in the SERPING1 gene causing C1 inhibitor deficiency and HAE. In a single patient with HAE a homozygous variant g.566T>C (c.-21T>C) might be responsible for the disease.

Background: Peanut sensitization is common in children. However, it is difficult to assess which children will react mildly and which severely. This study evaluated the relevance of basophil allergen sensitivity testing to distinguish the severity of peanut allergy in children. Methods: Twenty-seven peanut-sensitized children with symptoms varying from mild symptoms to severe anaphylaxis underwent peanut CD63 dose-response curve analysis with the inclusion of basophil allergen sensitivity calculation (CD-sens) and peanut component immunoglobulin E (IgE) testing. Results: Eleven children who had experienced anaphylaxis to peanuts showed a markedly higher peanut CD63 response at submaximal allergen concentrations and CD-sens (median 1,667 vs. 0.5; p < 0.0001) than 16 children who experienced a milder reaction. Furthermore, a negative or low CD-sens to peanuts unambiguously excluded anaphylactic peanut allergy. Children with anaphylaxis have higher levels of Ara h 1, 2, 3 and 9 IgE, but comparable levels of IgE to Ara h 8 and whole-peanut extract. The diagnostic specificity calculated with a receiver operating characteristic analysis reached 100% for CD-sens and 73% for Ara h 2. Conclusions: We demonstrated that severe peanut allergy is significantly associated with higher basophil allergen sensitivity. This cellular test should facilitate a more accurate diagnosis of peanut allergy.

Background: Previous reports suggest the usefulness of basophil activation testing (BAT) in Hymenoptera-allergic patients with negative venom-specific IgE antibodies. We sought to evaluate the diagnostic utility of this testing in a routine clinical laboratory setting. Materials and Methods: Twenty-one patients with anaphylactic reactions to Hymenoptera sting (median grade III) and negative venom-specific IgE were routinely and prospectively tested with BAT. Results: We were able to diagnose 81% (17 of 21) of patients with BAT and 57% (12 of 21) with intradermal skin testing. Three wasp venom-allergic patients showed IgE positivity to rVes v 5. Four patients (19%) were negative for all tests. In the case of double-positive BAT, the culprit insect correlated with the venom that induced a significantly higher basophil response. Conclusions: BAT allows the identification of severe Hymenoptera-allergic patients with negative specific IgE and skin tests. The routine use of this cellular test should facilitate prescription of venom immunotherapy in complex cases with inconclusive diagnostic results.

Background The precise immunological mechanisms for the early clinical protection of venom immunotherapy (VIT) have not yet been explained. Our aim was to evaluate whether high-affinity IgE receptor (FceRI) and the related basophil function have a role in the induction of short-term VIT protection. Methods We included 60 adults and 48 children. Basophil threshold sensitivity (CD-sens) to anti-FceRI stimulation, and FceRI gene and cell-surface expression were assessed at the beginning and just before the first maintenance dose (MD) of 100 μg of ultra-rush VIT (day 5) and at the beginning, during buildup, and just before the first MD of 70 μg and of 100 μg of semi-rush VIT (weeks 1–2 and 5). Results We demonstrated a significant reduction in CD-sens to anti-FceRI stimulation before the first MD in both ultra-rush and semi-rush VIT in all included subjects. FceRI gene and/or cell-surface expression was decreased in 34–100% of subjects, with different dynamics between VIT protocols. Conclusion We found a marked desensitization of FceRI-activated basophils after short-term VIT. This suppression, which could be highly relevant for the development of early protective mechanisms, might be also related to the changes at the level of FceRI expression.

Background There is no in vitro test to predict the induction of long-term tolerance in patients treated with venom immunotherapy (VIT). The aim of this study was to investigate whether immunotherapy-induced changes in basophil responsiveness reflect a state of protection and the induction of a tolerance. Methods Twenty-three patients with allergic reaction after Hymenoptera sting (11 wasp and 12 honeybee) were treated with VIT. In all patients, a CD63 basophil activation test was performed before the beginning of immunotherapy, after 1 year and after completing 4–6.5 years of immunotherapy (approximately 1 year after stopping). The tolerance was then evaluated by a sting challenge test. The basophil activation test was repeated 3–6 months after the challenge. Results Twenty-two subjects showed a negative sting challenge, and one subject, a positive sting challenge. Allergen-specific basophil response remained unchanged after 1 year of immunotherapy. However, after immunotherapy, a significant and approximately fourfold decrease was demonstrated in all tolerant subjects mainly in response to submaximal 0.1 μg/ml allergen concentration. This depression was sustained and did not change with the sting challenge test. In a nontolerant patient with a positive sting challenge, basophil response did not change. Conclusions Our results suggest that the depression of allergen-specific basophil response seems to be associated with the induction of a tolerance after completing a course of VIT.

Background. Lung cancer is the leading cause of cancer deaths. Angiogenesis is crucial process in cancer growth and progression. This prospective study evaluated expression of two central regulatory molecules: angiogenin and vascular endothelial growth factor (VEGF) in patients with lung cancer. Patients and methods. Clinical data, blood samples and broncho-alveolar lavage (BAL) from 23 patients with primary lung carcinoma were collected. BAL fluid was taken from part of the lung with malignancy, and from corresponding healthy side of the lung. VEGF and angiogenin concentrations were analysed by an enzyme-linked immunosorbent assay. Dilution of bronchial secretions in the BAL fluid was calculated from urea concentration ratio between serum and BAL fluid. Results. We found no statistical correlation between angiogenin concentrations in serum and in bronchial secretions from both parts of the lung. VEGF concentrations were greater in bronchial secretions in the affected side of the lung than on healthy side. Both concentrations were greater than serum VEGF concentration. VEGF concentration in serum was in positive correlation with tumour size (p = 0,003) and with metastatic stage of disease (p = 0,041). There was correlation between VEGF and angiogenin concentrations in bronchial secretions from healthy side of the lung and between VEGF and angiogenin concentrations in bronchial secretions from part of the lung with malignancy. Conclusion. Angiogenin and VEGF concentrations in systemic, background and local samples of patients with lung cancer are affected by different mechanisms. Pro-angiogenic activity of lung cancer has an important influence on the levels of angiogenin and VEGF.