IgE Assays Research Articles

Predicting the outcome of oral food challenges with hen's egg through skin test end‐point titration

Oral food challenge (OFC) is the diagnostic 'gold standard' of food allergies but it is laborious and time consuming. Attempts to predict a positive OFC through specific IgE assays or conventional skin tests so far gave suboptimal results. To test whether skin test with titration curves predict with enough confidence the outcome of an oral food challenge. Children (n=47; mean age 6.2 +/- 4.2 years) with suspected and diagnosed allergic reactions to hen's egg (HE) were examined through clinical history, physical examination, oral food challenge, conventional and end-point titrated skin tests with HE white extract and determination of serum specific IgE against HE white. Predictive decision points for a positive outcome of food challenges were calculated through receiver operating characteristic (ROC) analysis for HE white using IgE concentration, weal size and end-point titration (EPT). OFC was positive (Sampson's score >or=3) in 20/47 children (42.5%). The area under the ROC curve obtained with the EPT method was significantly bigger than the one obtained by measuring IgE-specific antibodies (0.99 vs. 0.83, P<0.05) and weal size (0.99 vs. 0.88, P<0.05). The extract's dilution that successfully discriminated a positive from a negative OFC (sensitivity 95%, specificity 100%) was 1 : 256, corresponding to a concentration of 5.9 microg/mL of ovotransferrin, 22.2 microg/mL of ovalbumin, and 1.4 microg/mL of lysozyme. EPT is a promising approach to optimize the use of skin prick tests and to predict the outcome of OFC with HE in children. Further studies are needed to test whether this encouraging finding can be extended to other populations and food allergens.

Comparison and Evaluation of Several Dermatophagoides Pteronyssinus Allergen Extracts for Skin Prick Test

To evaluate the significance of several Dermatophagoides pteronyssinus allergen extracts for skin prick test (SPT) in patients allergic to Dermatophagoides pteronyssinus. Two hundred and nineteen patients enrolled in Peking Union Medical College Hospital underwent SPT and serum specific IgE assay to detect the Dermatophagoides pteronyssinus allergen. Three kinds of house dust mite allergen extracts were used for SPT, including the Dermatophagoides pteronyssinus extract prepared by our laboratory (group A), standardized Dermatophagoides pteronyssinus extract (group B), and mixed extracts of Dermatophagoides pteronyssinus and Dermatophagoides farinae (group C). Human serum specific IgE result was regarded as the reference standard for diagnosis of Dermatophagoides pteronyssinus allergy. The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic performance of SPT with the extracts of three groups. SPT results showed that the median wheal diameter of group A, group B, and group C was 0.43, 0.35, and 0.28 cm, respectively, with significant difference among three groups (P<0.05). The difference was significant between group A and B (P<0.01) as well as group A and C (P<0.01), but not between group B and C (P>0.05). There was no local urticaria or systemic allergic reactions following the procedure of SPT. Local reaction was observed in 5 patients and delayed reaction was in 2 patients of group A. As for group B and C, local reaction occurred in 3 cases and delayed reaction in 2 cases in each group. The area under ROC curve of SPT with extract in group A, group B, and group C was 0.765, 0.801, and 0.782, respectively. Based on the detection results of serum specific IgE, the sensitivity of SPT in diagnosis of Dermatophagoides pteronyssinus allergy with extract of group A, group B, and group C was 92.4%, 87.0%, and 81.5%, and the specificity was 60.6%, 73.2%, and 74.8%, respectively. The Dermatophagoides pteronyssinus extract for SPT prepared by our laboratory offers good sensitivity and specificity comparable to commercially available allergen extracts, and it may be an appropriate candidate for clinical screening and diagnosis of Dermatophagoides pteronyssinus allergy.

Influence of total serum IgE levels on the in vitro detection of β‐lactams‐specific IgE antibodies

Allergic reactions to beta-lactams are a frequent cause of adverse drug reactions; the diagnosis is based on history, clinical examination, skin testing (prick and intradermal) and demonstration of serum-specific IgE antibodies (Abs). We compared the diagnostic performance of the Phadia CAP system for the detection of IgE to beta-lactams carried out using the new test with cut-off limits of 0.10 kUA/L and the old test with cut-off limits of 0.35 kUA/L for positive results; subsequently, we analysed the effect of total serum IgE values and of atopic phenotype on the diagnostic performance of the tests. The study comprised a total of 34 patients with a history of immediate adverse reactions to beta-lactams, which were confirmed by positive skin testing, and 115 control subjects with tolerance to beta-lactams over the last year. The Phadia CAP System was used for the determination of serum total and specific IgE Abs towards penicilloyl G (c1), penicilloyl V (c2), ampicilloyl (c5) and amoxicilloyl (c6). The overall diagnostic performance was assessed as a diagnostic odds ratio (DOR). The new test showed a higher sensitivity (85% vs. 44%) than the old test and a lower specificity (54% vs. 80%) but the overall diagnostic performance was poor (DOR 6.78 vs. 3.16, P = 0.333) in both tests. The total IgE value influences the DOR of both tests; DOR was better for values under 200 kU/L [DOR = 66; 95% confidence interval (CI): 11.3-384.1] or 500 kU/L (DOR = 45.7; 95% CI: 5.3-394.4) for the new and old tests, respectively. The reduction in the positive cut off value has not significantly improved the overall diagnostic performance of the beta-lactams-specific IgE assay. Because of the influence of serum total IgE on the detection of beta-lactam-specific IgE Abs, the combination of both tests is mandatory in the in vitro diagnostic approach of beta-lactam allergy.

The importance of nasal provocation test in the diagnosis of natural rubber latex allergy

Most studies regarding natural rubber latex (NRL) allergy have concentrated on the prevalence using skin prick test (SPT) and specific IgE assay. The objective of this study is to examine the target organ (skin, nasal mucosa) responses in patients with positive SPT to NRL using the nasal provocation test (NPT) and glove use test (GUT). Four thousand four hundred and twenty patients presented to our polyclinic between July 2003 and January 2007 were evaluated. One thousand six hundred and ninety-nine patients had positive SPT to one or more allergens (NRL and other inhaler allergens). Twenty-nine patients with positive SPT to NRL comprised the NRL sensitive group (group 1). Thirty-five randomized patients with positive SPT to an inhaler allergen other than NRL and negative NRL-specific IgE comprised atopic control group (group 2). Thirty healthy individuals who had no allergic diseases and had negative SPT and NRL-specific IgE comprised the healthy control group (group 3). The lowest NRL allergen concentration leading to NPT positiveness was 0.05 microg/mL. NPT was negative in groups 2 and 3. NPT was found to have a sensitivity of 96%, specificity of 100%, negative predictive value of 98% and positive predictive value of 100%. GUT was found to have a sensitivity of 81%, specificity of 90%, negative predictive value of 75% and positive predictive value of 93%. Nasal provocation test was successfully used for the first time in the diagnosis of NRL allergy. NPT is a more sensitive method as compared to GUT.

Enzyme-Linked Immunosorbent Assays with High Sensitivity for Antigen-Specific and Total Murine IgE: A Useful Tool for the Study of Allergies in Mouse Models

In studies on allergies in mouse models, IgE production is an essential parameter to be evaluated. Here, we examine the effect of commercially available immunoreaction enhancer solutions and different blocking reagents in enzyme-linked immunosorbent assay (ELISA) for total or antigen-specific murine IgE in order to improve the assays. Sera from mice immunized with recombinant house dust mite major allergens, Der f 1 and Der p 1, were used for the assays. Total IgE was measured by sandwich ELISA using monoclonal antibodies against murine IgE. Antigen-specific IgE was assayed using allergen-coated plates. Sensitivity or signal intensity in ELISA was compared among conditions differing in the use of enhancer solutions, blocking reagents, or monoclonal antibodies, and incubation time. Use of enhancer solutions improved the sensitivity of ELISA for total IgE by approximately 30-fold of that using a conventional buffer. A blocking reagent caused more unwanted enhancement of the background signal in blank wells in ELISA for total IgE compared with another blocking reagent, however, improved signal intensity in ELISA for antigen-specific ELISA without significant enhancement of the background signal. Optimal assay conditions were determined. Enhancer solutions are effective in improving ELISAs for total and antigen-specific murine IgE. Selection of blocking reagents was important to decrease unwanted enhancement of background signals and was effective in enhancing signals for positive samples. The ELISAs improved in this study are useful for the study of allergies in mouse models.

Acute urticaria caused by the injection of goat-derived hyaluronidase

Hyaluronidase is a goat testicular protein that hydrolyzes hyaluronic acid, a structural component of the intercellular matrix. It is commonly used as a spreading factor to improve the diffusion of drugs, including local anesthetics and chemotherapeutics. We experienced a 55-yr-old female with generalized urticaria that developed within 1 hr after the epidural injection of hyaluronidase. She had a history of allergic rhinitis, and had suffered from post-herpetic neuralgia and a herniated disc for several years. To relieve her pain, she had been given epidural injections consisting of mepivacaine hydrochloride, triamcinolone acetonide, and morphine sulfate biweekly for one year. Hyaluronidase had been administered several times with these drugs before this episode of generalized urticaria. Skin prick testing showed a positive response to 1,500 IU/mL of hyaluronidase extract, as compared to histamine. The patient's serum hyaluronidase-specific IgE level, determined using an enzyme-linked immunosorbent assay (ELISA), was markedly elevated, as compared to unexposed healthy controls. An IgE immunoblot analysis using hyaluronidase extract and the patient's serum showed IgE binding components at 31 and 21 kDa, whereas no corresponding IgE binding component was found in healthy controls. An ELISA inhibition test showed significant, dose-dependent inhibition with the serial addition of hyaluronidase extract. This is the first case of an IgE-medicated allergic reaction to goat (Naemorhedus goral raddenus) hyaluronidase, demonstrated by skin testing and a specific IgE and immunoblot assay.

Allergie à Arundo donax. Une nouvelle observation chez un jeune clarinettiste

The authors report the case of a 10-year-old child with cheilitis due to a clarinet reed. It was a question of an allergy to giant reed ( Arundo donax), the antigen of which has recently been identified. Its molecular similarity to pollen allergens explains the association with pollinosis, as we observed in the present case. On the other hand, the onset of an allergy to a domestic wood does not have a clear explanation. The diagnosis was based on the history, prick tests, patch tests, serum-specific IgE assays and RAST inhibition. The lesion disappeared with avoidance.

Etiologia, manifestações clínicas e alterações presentes nas crianças respiradoras orais

OBJETIVO: Investigar a etiologia, as principais manifestacoes clinicas e as alteracoes presentes em criancas de 3 a 9 anos, respiradoras orais, residentes na regiao urbana de Abaete (MG). METODOS: Estudo com amostra aleatoria representativa da populacao do municipio de 23.596 habitantes. Clinicamente, foram consideradas respiradoras orais as criancas que roncavam, dormiam com a boca aberta, babavam no travesseiro e apresentavam queixas de obstrucao nasal frequente ou intermitente. As criancas com diagnostico clinico de respirador oral foram submetidas a endoscopia nasal, teste alergico cutâneo e raio X do cavum, hemograma, contagem de eosinofilos, dosagem de IgE total e parasitologico de fezes. Os dados foram analisados utilizando o programa SPSS® versao 10.5. RESULTADOS: As principais causas da respiracao oral foram: rinite alergica (81,4%), hipertrofia de adenoides (79,2%), hipertrofia de amigdalas (12,6%) e desvio obstrutivo do septo nasal (1,0%). As principais manifestacoes clinicas do respirador oral foram: dormir com a boca aberta (86%), roncar (79%), cocar o nariz (77%), babar no travesseiro (62%), dificuldade respiratoria noturna ou sono agitado (62%), obstrucao nasal (49%) e irritabilidade durante o dia (43%). CONCLUSAO: Algumas manifestacoes clinicas sao muito frequentes na crianca respiradora oral. Essas manifestacoes devem ser reconhecidas e consideradas no diagnostico clinico da respiracao oral.

Etiology, clinical manifestations and concurrent findings in mouth-breathing children

To investigate the etiology, main clinical manifestations and other concurrent findings in mouth-breathing children aged 3 to 9 years and resident in the urban area of Abaeté (MG), Brazil. This study was based on a representative random sample of the town population, of 23,596 inhabitants. Clinical diagnosis of mouth-breathing was defined as a combination of snoring, sleeping with mouth open, drooling on the pillow and frequent or intermittent nasal obstruction. Children with a clinical diagnosis of mouth-breathing underwent nasal endoscopy, allergy skin tests and X ray of the rhinopharynx, full blood tests, eosinophil counts, total IgE assay and fecal parasitology. Data were analyzed using SPSS version 10.5. The main causes of mouth-breathing were: allergic rhinitis (81.4%), enlarged adenoids (79.2%), enlarged tonsils (12.6%), and obstructive deviation of the nasal septum (1.0%). The main clinical manifestations of mouth breathers were: sleeping with mouth open (86%), snoring (79%), itchy nose (77%), drooling on the pillow (62%), nocturnal sleep problems or agitated sleep (62%), nasal obstruction (49%), and irritability during the day (43%). Certain clinical manifestations are very common among mouth-breathing children. These manifestations must be recognized and considered in the clinical diagnosis of mouth-breathing.

Diagnosing Hypersensitivity Reactions to Cephalosporins in Children

The goals were to evaluate the usefulness of skin tests, patch tests, serum specific IgE assays, and challenges in diagnosing hypersensitivity reactions to cephalosporins and to clarify the pathogenic mechanism of such reactions. Children with immediate manifestations (within 1 hour) underwent immediate-reading skin tests with penicillin reagents and any suspect cephalosporins, serum specific IgE assays, and challenges; some children underwent reevaluations. Children with nonimmediate manifestations (after >1 hour) were assessed with patch tests, delayed-reading skin tests, and challenges. We evaluated 148 children with hypersensitivity reactions to cephalosporins, mainly cefaclor and ceftriaxone; 105 had experienced nonimmediate manifestations (mostly urticarial eruptions and maculopapular rashes) and 43 immediate manifestations (anaphylactic shock, urticaria and/or angioedema, and erythema). None of the nonimmediate reactors demonstrated positive results in patch tests and/or delayed skin tests; only 1 subject displayed immediate positive responses to penicillin skin-test reagents. Among the 104 patients with negative results, 96 underwent challenges; 95 tolerated the challenges, and 1 reacted to the cefaclor pediatric suspension and tolerated the challenge with a cefaclor capsule. In the first allergologic evaluation, 33 of the 43 children with immediate reactions displayed skin-test positivity. Of the 10 patients with negative results, 7 underwent challenges, followed by therapeutic courses and reevaluations for 4. All challenges and therapeutic courses were tolerated; in the reevaluation, 1 girl demonstrated positive skin-test results for both the responsible cephalosporin and penicillin reagents. Overall, IgE-mediated hypersensitivity was diagnosed for 34 (79%) of 43 subjects. Extremely few nonimmediate manifestations associated with cephalosporin therapy are actually hypersensitivity reactions, whereas most immediate reactions to cephalosporins are IgE-mediated. Cephalosporin skin testing is a useful tool for evaluating such reactions.

Immediate allergy from valacyclovir

A 50-year-old woman was referred because of general malaise, diffuse pruritus, angio-oedema of hands and feet and lowered consciousness and faintness within 5 min of intake of a tablet of 500 mg valacyclovir (Zelitrex ; GSK, Genval, Belgium) for genital herpes simplex. History revealed no prior allergies. Flow cytometric analysis (FACScan; BD, Immunocytometry Systems, San Jose, CA, USA) of activated basophils was performed using quadruple labelling with Alexa 448-coupled anti-IgE (SigmaAldrich, Chemic GmBH, Steinheim, Germany), anti-phospho-p38 MAPK (T180/Y182) conjugated with Alexa Fluor 647 (BD Biosciences), anti-CD203c (kindly provided by HJ Buhring, Department of Internal medicine, University of Tubingen, Germany) and phycoerythrin-conjugated anti-CD63 antibodies [Pharmingen; BD Biosciences, Erembodegem, Belgium; for details: (1)]. The test included a negative control (stimulation buffer), a positive control (anti-IgE; Pharmingen) and stimulation with acyclovir (Zovirax ; GSK, dilution 1 up to 10 lg/ml). The test was also performed in a healthy control individual tolerant for acyclovir. Upon challenge with acyclovir, basophils of the patient showed a manifest dose-dependent phosphorylation of p38 MAPK from 0% to 78% and up-regulated CD63 and CD203c expression from 0% to 43% and 8% to 47%, respectively (Fig. 1). Phosphorylation of p38 MAPK and expression of CD63 and CD203c on basophils from the control individual remained unchanged. A lymphocyte transformation test (LTT) with acyclovir assessed by the incorporation of [methyl-H] thymidine (2) was borderline with a stimulation index (SI), i.e. counts per minute (cpm) of the acyclovir-challenged lymphocytes divided by the cpm cultured with serumfree medium of 2.1 for two consecutive stimulation concentrations of 10 and 100 lg/ml (normal 1.6). Skin prick tests for acyclovir were positive in the patient with a wheal and flare reaction of 5/27 mm for a 10 dilution but negative in an acyclovirtolerant control individual (up to neat solution). There is no serological assay for acyclovir-specific IgE that is readily available. Valacyclovir is the l-valine ester of acyclovir. After oral administration, valacyclovir is rapidly absorbed and extensively metabolized by gut hydroxylases into acyclovir and the essential amino acid l-valine (3). The mode of action and spectrum of anti-viral activity of valacyclovir are thus identical to acyclovir (4). In contrast to valacyclovir, acyclovir is available in a parenteral (intravenous) formulation that can be readily applied for in vitro and in vivo allergy-testing. Acyclovir and valacyclovir are considered effective and safe drugs (4, 5) and severe related immunologic adverse events remain anecdotal. However, systemic reactions, such as urticaria, angio-oedema and wide-spread erythematous dermatoses have been described (6–8). Note that several patients with systemic reactions had already presented an allergic contact dermatitis to topical application of acyclovir and that on several occasions crossreactivity with famcylovir that also contains a 2-aminopurine nucleus has been reported (7, 9, 10). Foscarnet and cidofovir do not contain that core, and might, therefore be considered as potential alternative antiviral drugs. We present a patient in whom the temporal relationship between intake and symptoms and results of additional tests are highly indicative of a valacyclovirrelated adverse event. Finally, our results endorse the basophil activation test (BAT) to be a potential additional tool to diagnose drug allergy [for review: (11)] AL LERGY 2 0 0 8 : 6 3 : 9 4 1 – 9 5 0 • a 2008 THE AUTHORS • JOURNAL COMPILAT ION a 2008 BLACKWELL MUNKSGAARD • CONTRIBUT IONS TO THIS SECT ION WILL NOT UNDERGO PEER REVIEW, BUT WILL BE REV IEWED BY THE ASSOCIATE EDITORS •

Occupational rhinitis and asthma due to crickets

Insects may cause airborne hypersensitivity reactions. However, few reports exist on specific allergy to crickets. To report a case of occupational rhinitis and bronchial asthma in a cricket farm worker. A 28-year-old woman developed rhinitis and bronchial asthma related to her job in a farm where she was exposed to crickets: Gryllus campestris, Gryllus bimaculatus, and Acheta domestica. Extracts were prepared from whole and crushed bodies and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Skin prick tests, specific IgE assays (enzyme allergosorbent test [EAST], immunoblotting, EAST inhibition assays), serial peak expiratory flow monitoring at work, and specific (A domestica) and nonspecific bronchial challenge tests were performed. Skin prick test results were positive for G campestris, G bimaculatus, and A domestica. Levels of specific IgE were 2.9, 2.4, and 5.4 kU/L, respectively. The total IgE level was 131 kU/L. Serial peak expiratory flow monitoring at work was consistent with occupational asthma. The result of a bronchial challenge test with A domestica was positive with a dual response and elicited an increase in nonspecific bronchial hyperresponsiveness. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting revealed a similar pattern of IgE-binding bands with the 3 cricket extracts (bands of 78 and 64 kDa appeared in nonreducing conditions, whereas bands of 107 to 80, 58, and 52 kDa appeared in reducing conditions). None of these bands was detected by control sera. EAST inhibition studies showed a high degree of cross-reactivity among the 3 species. Crickets are responsible for occupational rhinitis and asthma by an IgE mechanism. Cross-reactivity among the crickets tested in our study was found.

The need for a biological diagnosis of penicillin allergy

According to the Nomenclature Review Committee of the World Allergy Organization, drug allergies refer to drug hypersensitivity reactions where a definite immunological mechanism [1] can be demonstrated. The prevalence of self-reported penicillin allergy is high; indeed, a crosssectional survey in Portugal found a 4.5% adult population self-reported penicillin allergy [2]. The prevalence rate of true penicillin allergy is, however, much lower [3, 4], and therefore a firm diagnosis is needed [5]. The clinical picture of drug allergy is very heterogeneous, encompassing distinct diseases such as morbilliform or bullous exanthema, urticaria and angiooedema, pustular exanthemas, fixed drug eruptions, anaphylaxis, blood cell dyscrasia, fever, interstitial lung disease, hepatitis, nephritis, vasculitis, DRESS (drug reaction with eosinophilia and systemic symptoms) and various forms of autoimmune diseases. However, it is important to distinguish between immediate and non-immediate reactions. The former occur within the first hour after the last drug administration and are manifested clinically by urticaria, angiooedema, rhinitis, bronchospasm or anaphylactic shock. The diagnosis of drug allergy often relies on clinical histories, skin tests and a few validated in vitro tests, such as serum-specific IgE assays, which are available only for a few drugs. The sensitivity of these tests is not 100% and, in selected cases, provocation tests are therefore necessary. However, new diagnostic tools, such as the basophil activation test (BAT), have been developed and are still under validation. Their routine use could increase the sensitivity of diagnostic work-ups, thus reducing the need for drug provocation tests and filling the gap between the high number of non-allergic drug allergy suspicions and the lack of specialized centres in all countries, which would enable confirmation or elimination of the diagnosis. There is evidence now that the BAT can contribute to the diagnosis of anaphylactic reactions due to several drugs, particularly muscle relaxants and b-lactams [6, 7]. At present, the most commonly used antigens in BATs are CD63 and CD203c. CD63, the gp53 or lysosyme-associated membrane protein (LAMP)-3, is one of the most reported markers for flow cytometric determination of basophil activation. CD63 is deeply anchored into the basophilic intracytoplasmic granules and is generally weakly expressed on the surface of basophils, monocytes, macrophages and platelets. In cases of allergic inflammation, as a result of fusion between the granule and the plasma membrane, CD63 is expressed with a high density on activated basophils and mirrors histamine release [8]. This up-regulated expression of basophilic CD63 can be detected by multi-colour flow cytometry using specific monoclonal antibodies [9]. As far as b-lactams are concerned, the CD63-based BAT was first evaluated in two studies [7, 10] including, respectively, 58 and 70 patients with immediate reactions to these antibiotics. BAT sensitivity was about 50% and specificity was over 90%. However, unlike the study by Sanz et al. [7], the one by Torres et al. [10] assessed not only patients with positive histories and positive skin tests and/or CAP-FEIA but also patients negative to both these tests and positive to challenges. It is interesting to note that one of the seven patients of the latter group was BAT positive. This group of patients with true allergy and negative skin tests is of crucial importance. They account for up to 33% of b-lactam-allergic patients and are only diagnosed by potentially harmful and cumbersome oral provocation tests [11]. Another more basophil-specific marker, CD203c (neural cell surface differentiation antigen E-NPP3, ecto-nucleotide pyrophosphatase/phosphodiesterase 3), is a multifunctional ectoenzyme exclusively and constitutively expressed on the surface of basophils [12]. Five years Correspondence: Prof. Pascal Demoly, Allergy Department, Hopital Arnaud de Villeneuve, University Hospital of Montpellier and Inserm U657, 34295 Montpellier, Cedex 5, France. E-mail: pascal.demoly@inserm.fr doi: 10.1111/j.1365-2222.2008.02981.x Clinical and Experimental Allergy, 38, 869–871

Grass pollen immunotherapy: IL-10 induction and suppression of late responses precedes IgG4 inhibitory antibody activity

Grass pollen immunotherapy is an effective treatment for seasonal allergic rhinitis that provides the opportunity to study the induction and maintenance of allergen-specific immune tolerance. We investigated the relationship between clinical responsiveness, regulatory cytokine production, and antibody responses to allergen during 1 year of immunotherapy. Eighteen subjects with severe seasonal allergic rhinitis were randomized double-blind to receive active or placebo injections of an alum-adsorbed grass pollen vaccine (Alutard SQ). Subjects underwent repeated testing of early- and late-phase skin responses to intradermal allergen, and cellular responses to grass pollen allergen were tested. Sera were tested for allergen-specific IgG4, IgA, and inhibitory activity in biologic assays of IgE responses. Grass pollen immunotherapy was effective in reducing overall symptom scores (P < .05) and conjunctival reactivity (P < .05). In the active group significant IL-10 production occurred early at low allergen doses and at a similar time as inhibition of late skin responses at 2 to 4 weeks. Serum allergen-specific IgG4, IgA, and inhibitory antibody activity for basophil histamine release and IgE-facilitated allergen binding to B cells occurred later, at 6 to 12 weeks, at higher allergen doses and preceded inhibition of early skin responses. IL-10 responses occur early but at immunotherapy doses that are not clinically effective. Later induction of inhibitory antibodies, including IgG4 and IgA, might be required for efficacy through modulation of IgE-mediated events.

Proteomic analysis of wheat proteins recognized by IgE antibodies of allergic patients

Wheat belongs to six major food allergens inducing IgE-mediated hypersensitivity reaction manifesting as cutaneous, gastrointestinal, and respiratory symptoms. Although cereals are a staple food item in most diets, only a few wheat proteins causing hypersensitivity have been identified. To characterize wheat allergens, salt-soluble wheat extracts were separated by 1-DE and 2-DE and IgE-binding proteins were detected by immunoblotting using sera of patients with allergy to ingested wheat. Proteins, frequently recognized by IgE on 2-DE were analyzed by MALDI-TOF and QTOF and their spectrum was completed by 1-DE and LCQ(DECA) nLC-MS/MS IT technique. Using all three techniques we identified 19 potential wheat allergens such as alpha-amylase inhibitors, beta-amylase, profilin, serpin, beta-D-glucan exohydrolase, and 27K protein. Employing newly developed ELISA, levels of IgE Abs against Sulamit wheat extract and alpha-amylase inhibitors type 1 and 3 were quantified and shown to be significantly elevated in sera of allergic patients compared to those of healthy controls. The level of IgE Abs against alpha-amylase inhibitor type 3 was lower, slightly above the cut-off value in the majority of patients' sera. Our findings contribute to the identification of wheat allergens aimed to increase the specificity of serum IgE and cell activation diagnostic assays.

Optimizing allergen components in litchi and peach extracts for in vitro specific IgE assays

Optimizing allergen components in litchi and peach extracts for in vitro specific IgE assays

Anaphylactoid reactions in two patients after omalizumab administration after successful long-term therapy

Anti-IgE therapy with omalizumab, a recombinant humanized monoclonal antibody, has anti-inflammatory effects in allergic asthma and rhinitis. Although omalizumab has been exceptionally safe, reactions after its administration have been reported. The goal of this study was to assess two patients who experienced apparent anaphylaxis after omalizumab administration. Two cases of apparent anaphylaxis after omalizumab administration are reported with diagnostic evaluation using skin testing and unique IgE and IgG anti-omalizumab serological assays. At the time of evaluation, both atopic asthmatic patients had total (free and bound) serum IgE levels of 199 kIU/L (100% free) and 200 kIU/L (80% free and 20% bound), respectively. Epicutaneous skin tests to omalizumab were negative at 150 mg/mL of omalizumab in both subjects and the nonexposed negative control subject. Intradermal skin tests were positive at 0.15 mg/mL in subject 1 and negative at 1.5 mg/mL of omalizumab in subject 2 and the control subject. Intradermal testing to polysorbate produced significant wheal/flare reaction in subject 2 but not in the negative control subject. Serological assays for IgE or IgG antibodies reactive with omalizumab were negative. The in vitro and in vivo immunologic data support the conclusion that the adverse reactions experienced by two patients after omalizumab administration after more than a year of successful omalizumab therapy for asthma were likely anaphylactoid in nature. Polysorbate, an excipient in omalizumab, is known to cause similar reactivity to other medicines and is the most likely cause of these reactions.

Évaluation d'une nouvelle technique de dosage des immunoglobulines spécifiques E des protéines du lait de vache

Topics Diagnosis of cow's milk intolerance (CMI) is founded upon assay of specific IgE against cow's milk proteins whose ImmunoCap technic from pharmacia diagnostics (Phadia) allows to quantify from 0.1 KUA/l. We retrospectively studied the advantages of this extent of the scale of concentrations with regard to that traditionally scale restricted at 0.35 kUA/l and we verified the forecasted threshold of 0.1 kUA/l. Material and methods We selected 68 serums from 63 children, 32 with CMI confirmed, 12 with probable CMI and 19 controls. Anti alpha lactalbumin, beta lactoglobulin and casein IgE had been measured for every sample on UniCap 100, with a couple of dosage protocols: the usual one which allowed to quantify IgE from 0.35 to 100 kUA/l and the new one with a scale of calibration fit for quantify from 0.1 kUA/l. Results The threshold of this new protocol had been determined on 0.1 kUA/l for the three allergens. The two protocols were well correlated. For 21 among 68 samples (31%), the new calibration permitted to calculate concentrations with specific IgE but it was obtained < 0.35 KUA/l with the old one. These samples correspond with 10 confirmed CMI, 3 probable CMI, 7 controls, among which 5 isolated gastro oesophageal reflux GER. Conclusion Results of this study showed us that the new IgE dosage protocol can be a rearlier diagnosis tool of a cow's milk sensitization. Prospective study is needed to confirm. We asked about possible link between sensitization and GER.

The validity of the diagnosis of chronic obstructive pulmonary disease in general practice

To determine the validity of the diagnosis of chronic obstructive pulmonary disease (COPD) in general practice in patients given a diagnosis of COPD and treated with bronchodilators. From the medical records of eight Health Centres in Northern Greece, 319 subjects aged over 40 years and diagnosed as "COPD" were entered into the study. All filled in a special questionnaire and were subjected to spirometry, rhinomanometry and chest X-ray. One hundred and sixty patients (50.2%) met the GOLD criteria for COPD. Twenty-six of them were non-smokers and underwent further evaluation: blood eosinophil count, serum IgE assay, high resolution computed tomography (HRCT) scan of the chest, and echocardiography; 16 were given a different diagnosis. One hundred and fifty-nine subjects (49.8%) with an FEV1/ FVC ratio >0.7 did not meet the GOLD criteria for COPD; 71 suffered from nasal obstruction, 13 from asthma, six had restrictive pulmonary disease and 69 had no respiratory disease. Diagnostic errors in patients with respiratory symptoms in the primary healthcare setting are frequent. Patients suspected to have COPD should undergo spirometry testing after bronchodilation. An alternative diagnosis must be sought for non-smoking patients with irreversible airway obstruction.

Decreased T-bet expression and changes in chemokine levels in adults with asthma

T-bet is a novel transcription factor regulating lineage commitment of T helper (Th) lymphocytes to a predominant Th1 phenotype. Previous studies on T-bet and asthma focused mainly on bronchial biopsy specimens. This study assessed the relationship between T-bet expression and levels of selected chemokines in the peripheral blood of asthmatics. Blood was collected from 24 steroid-naive asthmatics, 39 asthmatics on inhaled corticosteroid and 32 age- and sex-matched controls for assay of T-bet expression, specific IgE and chemokines (interferon-gamma inducible protein-10 (IP-10/CXCL10), monokines induced by interferon-gamma (MIG/CXCL9), monocyte chemotactic protein-1 (MCP-1/CCL2), regulated upon activation normal T cell expressed and secreted (RANTES/CCL5) and interleukin-8 (IL-8/CXCL8) levels. T-bet mRNA expression was assessed by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). Chemokine levels were assessed by immunofluorescence flow cytometry. The mean (s.d.) age and forced expiratory volume in 1 s (FEV(1))% predicted of the asthmatics were 43 x 6 (14 x 6) years and 85 x 9 (20.0)%, respectively. The median (IQR) T-bet expression after normalization with beta-actin was suppressed in asthmatics versus controls [asthmatics 0 x 71 (0 x 59) versus controls 1 x 07 (1 x 14), P=0 x 03].The median (IQR) of plasma RANTES was elevated, whereas IP-10 was suppressed in asthmatics versus controls (RANTES: 13658 x 0 (13673 x 3) versus 6299 x 5 (19407 x 8) pg/ml, P=0 x 03; IP-10: 1047 x 6 (589 x 8) versus 1306 x 4 (759 x 9) pg/ml, P=0 x 001). There was a weak and negative correlation between T-bet expression and RANTES level in the asthmatics (r=-0 x 29, P=0 x 032). T-bet could be measured in peripheral blood and its expression was suppressed in asthmatics. This is in keeping with asthma being a predominantly Th2 disease and T-bet probably plays a role in the pathogenesis of asthma. Further studies are needed to explore the potential application of peripheral blood monitoring of T-bet.