Autologous serum skin test reactions in chronic spontaneous urticaria differ from heterologous cell reactions.

BACKGROUND: Autoimmune chronic spontaneous urticaria (CSU) is due to mast cell (MC)-activating autoantibodies, which are screened for by the autologous serum skin test (ASST) and basophil tests (BTs). Many CSU patients are positive in only one of these tests. How often this occurs and why is currently unknown. OBJECTIVES: To characterize the prevalence of mismatched ASST and BTs in CSU patients, and to investigate possible reasons for these mismatches. METHODS: We determined the rates of ASST+/BT- and ASST-/BT+ mismatches in published CSU studies. We assessed sera from 48 CSU patients by ASST, two BTs (basophil histamine release assay, BHRA; basophil activation test, BAT), a MC histamine release assay (MCHRA) and by ex vivo skin microdialysis (SMD). RESULTS: The ASST/BT mismatch rate in published CSU studies was 31% (ASST+/BT-: 22%, ASST-/BT+: 9%). In our patients, the ASST/BHRA and ASST/BAT mismatch rate was 35.4% (ASST+/BHRA-: 18.8% and ASST-/BHRA+: 16.7%) and 31.3% (ASST+/BAT-: 6.3% and ASST-/BAT+: 25.0%), respectively, and the two BTs were significantly correlated (P = 0.0002). The use of heterologous MCs, in vitro and in situ, instead of basophils produced similar results (MCHRA mismatch: 47.9%, ASST+/MCHRA-: 18.8%, ASST-/MCHRA+: 29.2%; SMD mismatch: 40.0%, ASST+/SMD-: 10.0% and ASST-/SMD+: 30.0%), and the MCHRA was highly correlated with SMD results (P = 0.0002). CONCLUSIONS: The ASST and BTs show divergent results in a third of CSU patients. Mismatches cannot be explained by the choice of basophil assay, the type of heterologous cells exposed to CSU serum in vitro (basophils vs. mast cells), nor the experimental setting of heterologous skin mast cells (in vitro vs. in situ). Thus, serum-induced whealing, in CSU patients, seems to involve autologous skin signals modulating MC degranulation.

In chronic spontaneous urticaria, IgE against staphylococcal enterotoxins is common and functional.

BACKGROUND: Chronic spontaneous urticaria (CSU) is a frequent disorder with recurrent itchy wheals and/or angioedema. Despite the known effectiveness of omalizumab therapy, the relevant IgE antigens are largely unknown. Recently, increased rates of elevated levels of IgE towards Staphylococcus aureus enterotoxins (SEs) were described in CSU. AIM: To assess the prevalence and functional relevance of IgE to SEs in CSU. METHOD: We investigated serum levels of IgE against SEs in 49 CSU patients and in 15 CSU patients additional specific IgE to SE components and basophil histamine release (BHR). Sera of 15 healthy controls (HCs) served as control group. RESULTS: Twenty-five (51%) of the CSU patients had detectable levels of SE-IgE as compared to 5 (33%) of HCs. Specific IgE to one of the SEs, Staphylococcus enterotoxin B (SEB), was present in 5 (33%) of 15 randomly selected CSU patients vs 3 (20%) of HC. Total IgE serum levels in CSU patients were significantly correlated with SE-IgE (r = .52, P < .001) and SEB-IgE (r = .54, P = .04) serum concentrations. Interestingly, SEB-IgE levels were strongly correlated with disease activity (UASday) in CSU patients (r = .657, P = .01). Furthermore, BHR in response to SEB was significantly higher in basophils loaded with the serum of CSU patients compared to HC (P < .05) and was clinically correlated with duration of disease (r > .51, P < .05). DISCUSSION: IgE against SEs may contribute to the pathogenesis of CSU in a subpopulation of patients. Its role and relevance in the pathophysiology of CSU need to be further analysed.

MP29-02 reduces nasal hyperreactivity and nasal mediators in patients with house dust mite-allergic rhinitis.

BACKGROUND: Nasal hyperreactivity (NHR) is an important clinical feature of allergic rhinitis (AR). The efficacy of MP29-02 (azelastine hydrochloride (AZE) and fluticasone propionate [FP]) nasal spray on local inflammatory mediators and NHR in AR is unknown. We tested if MP29-02 decreases inflammatory mediators and NHR in AR and if this effect is due to restoration of nasal epithelial barrier function. METHODS: A 4-week double-blinded placebo-controlled trial with MP29-02 treatment was conducted in 28 patients with house dust mite (HDM) AR. The presence of NHR was evaluated by measuring reduction in nasal flow upon cold dry air exposure. The effects of AZE ± FP on barrier integrity and airway inflammation were studied in a murine model of HDM-induced NHR and on reduced activation of murine sensory neurons and human mast cells. RESULTS: MP29-02 but not placebo reduced NHR (P < .0001 vs P = .21), levels of substance P (P = .026 vs P = .941), and ß-hexosaminidase (P = .036 vs P = .632) in human nasal secretions. In wild-type C57BL6 mice, the reduction in ß-hexosaminidase levels (P < .0001) by AZE + FP treatment upon HDM challenge was found in parallel with a decreased transmucosal passage (P = .0012) and completely reversed eosinophilic inflammation (P = .0013). In vitro, repeated applications of AZE + FP desensitized sensory neurons expressing the transient receptor potential channels TRPA1 and TRPV1. AZE + FP reduced MC degranulation to the same extent as AZE alone. CONCLUSION: MP29-02 treatment reduces inflammatory mediators and NHR in AR. The effects of AZE + FP on MC degranulation, nasal epithelial barrier integrity, and TRP channels provide novel insights into the pathophysiology of allergic rhinitis.

Sugammadex hypersensitivity and underlying mechanisms: a randomised study of healthy non-anaesthetised volunteers.

BACKGROUND: We investigated potential for hypersensitivity reactions after repeated sugammadex administration and explored the mechanism of hypersensitivity. METHODS: In this double-blind, placebo-controlled study (NCT00988065), 448 healthy volunteers were randomised to one of three arms to receive three repeat i.v. administrations of either sugammadex 4 mg kg-1, 16 mg kg-1, or placebo. Primary endpoint was percentage of subjects with hypersensitivity (assessed by an independent adjudication committee). Secondary endpoint of anaphylaxis was classified per Sampson and Brighton criteria. Exploratory endpoints included skin testing, serum tryptase, anti-sugammadex antibodies [immunoglobulin (Ig) E/IgG], and other immunologic parameters. RESULTS: Hypersensitivity was adjudicated for 1/148 (0.7%), 7/150 (4.7%), and 0/150 (0.0%) subjects after sugammadex 4 mg kg-1, 16 mg kg-1, and placebo, respectively. After sugammadex 16 mg kg-1, one subject met Sampson criterion 1 and Brighton level 1 (highest certainty) anaphylaxis criteria; two met Brighton level 2 criteria. After database lock it was determined that certain protocol deviations could have introduced bias in the reporting of hypersensitivity signs/symptoms in a subject subset. Objective laboratory investigations indicated that potential underlying hypersensitivity mechanisms were unlikely to have been activated; the results suggest that most of the observed hypersensitivity reactions were unlikely IgE/IgG-mediated. CONCLUSION: Dose-dependent hypersensitivity or anaphylaxis reactions to sugammadex were observed when administered without prior neuromuscular blocking agent. Laboratory investigations do not suggest prevalent allergen-specific IgE/IgG-mediated immunologic hypersensitivity. Because it could not be fully excluded that estimates of hypersensitivity/anaphylaxis incidence were unbiased, an additional study was conducted to characterise the potential for hypersensitivity reactions and is described in a companion report. CLINICAL TRIAL REGISTRATION: http://www.clinicaltrials.gov NCT00988065; Protocol number P06042.

Basophil Histamine Release Induced by Amoxicilloyl-poly-L-lysine Compared With Amoxicillin in Patients With IgE-Mediated Allergic Reactions to Amoxicillin.

BACKGROUND: Amoxicillin (AX) is the ß-lactam most often involved in IgE-mediated reactions. Diagnosis is based mainly on skin testing, although sensitivity is not optimal. We produced a new AX derivative, amoxicilloyl-poly-L-lysine (APL), and analyzed its recognition of IgE using the passive histamine release test (pHRT). METHODS: The study population comprised patients (n=19) with confirmed AX allergy and specific IgE to AX and controls (n=10) with good tolerance to AX. pHRT was performed using "IgE-stripped" blood from a single donor that was sensitized in vitro by patient sera and incubated with AX or APL. Histamine release was determined and expressed as nanograms of histamine released per milliliter of blood. RESULTS: The clinical symptoms were anaphylaxis (n=9), urticaria (n=7), erythema (n=2), and nondefined immediate reactions (n=1). The median (IQR) time interval between reaction and study was 90 (60-240) days and between drug intake and development of symptoms 24 (10-60) minutes. The median sIgE level was 3.37 (0.95-5.89) kUA/L. The sensitivity of pHRT to APL was 79% and the specificity 100%, which were higher than data obtained with pHRT to AX (63% sensitivity and 90% specificity). There was a positive correlation between maximal histamine release levels obtained with AX and APL (r=0.63). CONCLUSIONS: In patients with immediate hypersensitivity reactions to AX, APL showed higher sensitivity and specificity than the culprit drug, AX, when tested in vitro by pHRT. This indicates that APL can improve the in vitro diagnostic accuracy of allergic reactions to AX. Further assessment of skin testing is necessary.

Clinical and diagnostic features of perioperative hypersensitivity to cefuroxime.

INTRODUCTION: The Danish Anaesthesia Allergy Centre (DAAC) investigated 89 adult patients with suspected perioperative cefuroxime-associated hypersensitivity reactions between 2004 and 2013. The goals were to determine whether the time to index reaction after cefuroxime exposure could be used to implicate cefuroxime as the cause of the reactions and explore different test modalities in diagnosing cefuroxime hypersensitivity. METHOD: Skin tests, in vitro tests, and titrated provocations were used to determine cefuroxime hypersensitivity. Patients were deemed cefuroxime positive on the basis of at least two positive tests and/or a positive provocation. RESULTS: One or more tests were positive for cefuroxime in 24 of 89 (27.0%) patients. One was only specific IgE positive and was deemed cefuroxime negative. Twenty-three (25.8%) were deemed cefuroxime positive. There were four specific IgE-, 4 histamine release test-, 13 skin test-, and 14 provocation positive patients. There were eight (34.8%) patients who were only provocation positive. Data on time to index reaction after cefuroxime exposure were available for 80 patients (22 in the positive group and 58 in the negative group), 22 of 22 (100%) of positive patients reacted in <15 min vs. only 38 of 58 (65.5%) of negative patients. CONCLUSION: All patients with confirmed hypersensitivity to cefuroxime reacted within 15 min of administration, but so did 65.5% of Cefuroxime negative patients, making timing of administration an unreliable predictor of causation in the perioperative setting. Provocations were always positive when carried out in skin test positive patients; however, eight patients had positive provocations only, highlighting the need for provocation in skin test negative patients.

Standardized testing with chlorhexidine in perioperative allergy--a large single-centre evaluation.

BACKGROUND: Perioperative allergic reactions to chlorhexidine are often severe and easily overlooked. Although rare, the prevalence remains unknown. Correct diagnosis is crucial, but no validated provocation model exists, and other diagnostic tests have never been evaluated. The aims were to estimate (i) the prevalence of chlorhexidine allergy in perioperative allergy and (ii) the specificity and sensitivity for diagnostic tests for chlorhexidine allergy. METHODS: We included all patients investigated for suspected perioperative allergic reactions in the Danish Anaesthesia Allergy Centre during 2004-2012. The following tests were performed: specific IgE (Immunocap® ; Phadia AB, Sweden), histamine release test (HR) (RefLab ApS, Denmark), skin prick test (SPT) and intradermal test (IDT). Positivity criteria were as follows: specific IgE >0.35 kUA/l; HR class 1-12; SPT mean wheal diameter ≥3 mm; IDT mean wheal diameter ≥ twice the diameter of negative control. Chlorhexidine allergy was post hoc defined as a relevant clinical reaction to chlorhexidine combined with two or more positive tests. Based on this definition, sensitivity and specificity were estimated for each test. RESULTS: In total, 22 of 228 patients (9.6%) met the definition of allergy to chlorhexidine. Estimated sensitivity and specificity were as follows: specific IgE (sensitivity 100% and specificity 97%), HR (sensitivity 55% and specificity 99%), SPT (sensitivity 95% and specificity 97%) and IDT (sensitivity 68% and specificity 100%). CONCLUSIONS: In patients investigated for suspected perioperative allergic reactions, 9.6% were diagnosed with allergy to chlorhexidine. Using our definition of chlorhexidine allergy, the highest combined estimated sensitivity and specificity was found for specific IgE and SPT.

Diagnosis of penicillin allergy revisited: the value of case history, skin testing, specific IgE and prolonged challenge.

BACKGROUND: Skin testing in duplicate, correlation between case history of immediate and nonimmediate reactions and challenge outcome and prolonged oral treatment with penicillin in the diagnostic evaluation of allergic reactions to ß-lactam antibiotics, mimicking real-life situations, have only been addressed in few studies. METHODS: A total of 342 patients suspected of having ß-lactam allergy were investigated according to the European Network for Drug Allergy (ENDA) guidelines and patients found to be negative in the ENDA program were supplemented with a 7-day oral treatment with penicillin. Skin testing with penicillins was performed in duplicate. Patients with case histories of reactions to other ß-lactams were also subsequently challenged with the culprit drug. RESULTS: Nineteen patients were IgE-sensitized to penicillin. Then, intracutaneous tests (ICTs) were performed, in which 35 patients tested positive for allergy, 21 with delayed and 14 with immediate reactions. Only three patients tested positive for the major (PPL) and/or minor (MDM) penicillin determinants, all being positive for penicillin G in ICT. The remaining 291 patients were challenged with penicillin: 10 tested positive in single-dose challenge and 23 tested positive in the 7-day challenge. A total of 17 of 78 patients with a negative penicillin challenge tested positive during challenges with other ß-lactams. We found no correlation between case histories of immediate and nonimmediate reactions and reaction time during challenge. CONCLUSION: The data suggest that case history is often insufficient to discriminate between immediate reactors and nonimmediate reactors. A 7-day challenge with the culprit ß-lactam may yield more positive reactions than the accepted one- or 2-day challenge. Interpretation of skin testing should be made with caution.

EAACI taskforce position paper: evidence for autoimmune urticaria and proposal for defining diagnostic criteria.

An autoimmune subset of chronic spontaneous urticaria is increasingly being recognized internationally, based on laboratory and clinical evidence that has accrued over the last 20 years. This evidence has been reviewed by a taskforce of the Dermatology section of the European Academy of Allergy and Clinical Immunology. Functional autoantibodies in chronic urticaria (CU) patient sera have been demonstrated against IgE and FcεRIα by basophil and mast cell histamine release assays and by basophil activation assays. Antibody specificity has been confirmed by immunoassay, but there is a poor correlation between functionality and immunoreactivity. Approximately 25% of CU patients have a positive basophil histamine release assay and show autoreactivity (a positive autologous serum skin test), whereas 50% are negative regarding both. Functionality of CU sera appears to be complement dependent on mast cells but not exclusively on basophils. Basophil activation by CU sera is predominantly restricted to IgG1 and IgG3 subclasses. Circumstantial evidence for CU being an autoimmune disease comes from an observed association with other autoimmune diseases, a strong association between serum functionality and HLA-DR4 haplotype and the good response of CU patients to immunotherapies. It was proposed that a study should be undertaken to prospectively validate potentially relevant clinical criteria (from the history, examination and routinely available clinical investigations) against a new 'gold standard' for the diagnosis of ACU (positive autoreactivity, functional bioassay and immunoassay) to define preliminary criteria sets for the diagnosis of ACU based on clinical and laboratory features with highest individual sensitivity and specificity.

IgE-mediated basophil tumour necrosis factor alpha induces matrix metalloproteinase-9 from monocytes.

BACKGROUND: IgE-mediated activation of mast cells has been reported to induce the release of tumour necrosis alpha (TNF-α), which may display autocrine effects on these cells by inducing the generation of the tissue remodelling protease matrix metalloproteinase-9 (MMP-9). While mast cells and basophils have been shown to express complementary and partially overlapping roles, it is not clear whether a similar IgE/TNF-α/MMP-9 axis exists in the human basophil. The purpose of this study was thus to investigate whether IgE-mediated activation of human basophils induces TNF-α and MMP-9 release. METHODS: Human peripheral blood mononuclear cells (PBMC), isolated basophils and monocytes were stimulated up to 21 h with anti-IgE. Mediator releases were assessed by ELISA, and surface expressions of mediators were detected by flow cytometry. Upregulation of cytokine production was detected by Western blot and polymerase chain reaction (PCR). RESULTS: IgE-mediated activation of basophils induced the synthesis and release of both TNF-α and MMP-9 from PBMC. In contrast, IgE-mediated activation of purified basophils induced the release and cellular expression of TNF-α but not MMP-9. Isolated monocytes did not release MMP-9 upon anti-IgE stimulation, but MMP-9 release was induced by stimulating monocytes with supernatants from activated basophils, and this release was inhibited by anti-TNF-α neutralizing antibodies. CONCLUSION: Our results strongly indicate that human basophils release TNF-α following IgE-dependent activation and that this cytokine subsequently stimulates MMP-9 release from monocytes. These findings support a direct involvement of basophils in inflammation as well as suggesting a role for the basophil in tissue remodelling.

Peanut cross-reacting allergens in seeds and sprouts of a range of legumes.

BACKGROUND: Recently, peanut-allergic patients have reported symptoms upon ingestion of bean sprouts produced from various legumes. OBJECTIVE: This study was designed to identify immunoreactivity to seeds and sprouts of legumes other than peanut in sera from peanut-allergic patients. METHODS: Crude protein extracts of seeds and sprouts (comprising cotelydons and hypocotyls/epicotyls) of peanut, soybean, green pea, blue lupine, mung bean, alfalfa, broad bean, and azuki bean were prepared. The reactivity of sera from 10 peanut-allergic patients to these extracts was analysed by indirect histamine release (HR), enzyme-allergosorbent test (EAST), EAST inhibition, and Western blots. Skin prick tests (SPTs) were performed on the patients with fresh legume seeds as well as four commercial legume sprouts, and food challenges with soybean, pea, and lupine were performed on a subgroup of the patients. RESULTS: All legume seeds and commercial sprouts induced positive SPTs in some of the patients. Indirect HR experiments indicated an extensive co-reactivity between peanut and the legumes, and cross-reactivity was observed for soybean, pea, and lupine seeds as well as lupine hypocotyls in EAST inhibition experiments. Of the 16 protein extracts, soybean, pea, and lupine seed extracts produced visible bands in Western blots. CONCLUSION: The symptoms reported by peanut-allergic patients after legume sprout intake might be caused by cross-reactivity of peanut-specific antibodies. The intake of raw legume sprouts might cause symptoms in peanut-allergic patients.

Optimizing diagnostic tests for persulphate-induced respiratory diseases.

BACKGROUND: Persulphates from hair bleaching products are considered the major cause of occupational-rhinitis and asthma in hairdressers. The specific inhalation challenge (SIC) is considered 'reference standard' for diagnosing persulphate-induced asthma and rhinitis; however, the currently validated method of performing SIC with persulphate powder is time consuming with a duration of up to 4 days. The value of skin prick tests (SPTs) and histamine release tests (HRTs) with persulphates is unknown. The aim of this study was to establish a novel rapid SIC with persulphate powder to test for both rhinitis and asthma simultaneously in 1 day. In addition, we assessed the suitability of SPTs and HRTs for detecting persulphate-induced respiratory diseases. METHODS: The study population included 19 hairdressers with a history of work-related rhinitis and/or asthma symptoms, 12 symptomatic controls (10 with concurrent allergic asthma and rhinitis and two with non-allergic asthma), and 40 healthy controls. A previous severe asthmatic reaction and/or anaphylactic reaction to persulphates was considered an exclusion criterion for hairdressers. The 19 hairdressers and 12 symptomatic controls had SIC performed with 3 × 5 min exposures to potassium persulphate powder in a provocation chamber. All participants, including the 40 healthy controls, were subjected also to SPTs and HRTs with three persulphate salts at concentrations of 2-20 % and 0.03-1 %, respectively. RESULTS: None of the symptomatic controls had a nasal or bronchial response to SIC with potassium persulphate. Six hairdressers presented a nasal and two a bronchial response. No severe reactions occurred. No positive SPTs were recorded, neither among hairdressers, symptomatic controls, nor healthy controls. All three groups showed nonspecific non-IgE mediated histamine release to persulphates in HRT. CONCLUSIONS: The proposed method for performing SIC showed a high specificity for detecting persulphate-induced asthma and rhinitis. The rapid SIC was able to produce positive nasal and bronchial responses in symptomatic hairdressers without any severe reactions occurring. SPTs and HRTs cannot predict asthma or rhinitis caused by persulphates.

Improved vaccination response during ranitidine treatment, and increased plasma histamine concentrations, in patients with B cell chronic lymphocytic leukemia.

Patients with B cell chronic lymphocytic leukemia (B-CLL) have decreased capacity to mount relevant antibody responses upon immunization, and development of hypogammaglobulinemia is part of the natural history of the disease. We investigated the influence of histamine type-2 (H2) receptor blockade by ranitidine on the in vivo antibody production in B-CLL patients following vaccination. Anti-polysaccharide antibodies in B-CLL patients, vaccinated with a tetanus-toxoid conjugated vaccine against Haemophilus influenzae type-B (Hib), reached long-term protective levels in more than 90% of B-CLL patients randomized to ranitidine treatment, as compared to 43% of the untreated patients (P = 0.024). No difference in the response to vaccination against influenza virus types A and B protein could be detected between the two groups. Plasma histamine levels were 2-fold to 20-fold higher in 23 out of 31 B-CLL patients, compared to normal controls, and these levels showed a significant positive correlation to disease duration. These findings indicate the possibility of improving in vivo antibody production against a highly relevant pathogen in B-CLL patients by histamine type-2 receptor blockade, and the combined finding of an immune-stimulatory effect of ranitidine and increased plasma histamine levels, strongly suggests the involvement of histamine in the pathogenesis of B-CLL immunodeficiency.

Comparison of four variants of a major allergen in hazelnut (Corylus avellana) Cor a 1.04 with the major hazel pollen allergen Cor a 1.01.

The aim of this study was to produce the Bet v 1-related major hazelnut allergen Cor a 1.0401 and variants thereof as recombinant allergens, and to compare their immuno-reactivity with the major hazel pollen allergen using sera of patients whose hazelnut allergy recently was confirmed by double-blind placebo-controlled food challenges (DBPCFC) in a multicenter study. Total RNA was isolated from immature hazelnuts and transcribed into cDNA. Full length coding DNA obtained by PCR-strategy was subcloned into pTYB11 vector and expressed in E. coli ER2566 cells. Native non-fusion target proteins were purified by DTT-induced self-cleavage of the intein-tagged N-terminal fusion proteins. IgE reactivity of the recombinant allergens was tested by enzyme allergosorbent test (EAST), EAST-inhibition, immunoblot-inhibition and histamine release assays. Four recombinant allergens were produced showing deduced amino acid sequence identities among each other of 97-99%, and were considered as variants Cor a 1.0401 (GenBank Accession no.: AF136945), Cor a 1.0402 (AF323973), Cor a 1.0403 (AF323974) and Cor a 1.0404 (AF323975). Cor a 1.0402 and 03 only differed in a C4S exchange. Cor a 1.0404 had a unique proline residue in position 99. Surprisingly, only 63% identity was revealed with hazel pollen Cor a 1. EAST with 43 sera of patients with positive DBPCFC to hazelnut indicated IgE reactivity to Cor a 1.0401 in 95% of the sera, to Cor a 1.0402 in 93%, to Cor a 1.0403 in 91%, and in only 74% of the sera to the proline variant Cor a 1.0404. The allergenic activity of the four variants was confirmed by histamine release assays in 15 hazelnut-allergic patients stimulated with the four variants and controls. Eleven sera were positive with extract from native hazelnut, 13 with rCor a 1.0401, 12 with rCor a 1.0402, 11 with rCor a 1.0403, and only two with rCor a 1.0404 containing the proline exchange. The high IgE binding variant Cor a 1.0401 showed only partial IgE cross-reactivity with pollen Cor a 1. IgE-binding and histamine release capacity led to a concordant ranking of the allergenic activity of the recombinant variants: Cor a 1.0401>Cor a 1.0402 and 03>Cor a 1.0404 (the proline variant). Similar results for Cor a 1.0402 and 03 suggest a minor influence in IgE binding of cysteine in position 4, whereas proline in position 99 appears to be responsible for the decrease in IgE reactivity in Cor a 1.0404. It appears that the epitopes of hazelnut Cor a 1.04 are less related to pollen Cor a 1 than to Bet v 1 from birch pollen. Low IgE binding variants or mutants of Cor a 1.04 are candidate compounds for developing a novel and safe approach of specific immunotherapy of hazelnut allergy.

Acute effects of substance P and calcitonin gene-related peptide in human skin--a microdialysis study.

Upon activation nociceptors release neuropeptides in the skin provoking vasodilation and plasma protein extravasation in rodents, but only vasodilation in humans. Pivotal peptides in the induction of neurogenic inflammation comprise calcitonin gene-related peptide and substance P, the latter being suggested to act partly via degranulation of mast cells. In this study substance P and calcitonin gene-related peptide-induced vasodilation, protein extravasation, histamine release, and sensory effects were investigated simultaneously in human skin by dermal microdialysis. The vasodilatory prostaglandin E(2) and the mast cell activator codeine served as positive controls. Substance P and calcitonin gene-related peptide applied intradermally via large cut-off plasmapheresis capillaries induced dose-dependent local vasodilation, but only SP provoked protein extravasation in concentrations greater than 10(-9) M. Substance P-induced (10(-8)-10(-6) M) protein extravasation was not accompanied by histamine release and was unaffected by cetirizine (histamine H1 blocker, 200 microg per ml). Only the highest concentration of substance P (10(-5) M) induced significant histamine release. Neither neuropeptide caused any axon reflex erythema or any itch or pain sensation, whereas mast cell degranulation by codeine dose dependently provoked itch, flare, protein extravasation, and histamine release. In human skin calcitonin gene-related peptide and substance P induce vasodilation by a mechanism not involving histamine. No evidence for neuropeptide-induced activation of nociceptors was obtained. Our results suggest that endogenous calcitonin gene-related peptide and substance P have no acute sensory function in human skin. The lack of neurogenic protein extravasation in humans can most probably be attributed to low local concentrations of this neuropeptide still sufficient to exert trophic and immunomodulatory effects (10(-11) M), but too low to induce protein extravasation (10(-8) M) or even mast cell degranulation (10(-5) M). J Invest Dermatol 115:1015-1020 2000

A method for production and determination of histamine releasing activity from human peripheral blood mononuclear cells.

Histamine releasing factors, i.e. cytokines capable of inducing histamine release from basophils or mast cells, have been suggested to be involved in the pathogenesis of, for example, allergic late-phase reactions. Here we describe a controlled method for production and determination of histamine releasing activity (HRA) from human peripheral blood mononuclear cells (MNC). MNC were incubated with concanavalin A (Con A) for 2 h and cultured for another 40 h in fresh serum free medium. The culture supernatants were concentrated 19-25 fold by ultrafiltration (molecular weight cut-off: 3000 Da). The preparations of HRA induced dose- and Ca2+-dependent histamine release from leukocytes. Supernatants of parallel cultures of unstimulated MNC did not induce histamine release. The HRA was neither due to exogenous histamine releasing compounds (e.g. Con A) nor to residual histamine in the preparations of HRA. The kinetics of HRA induced histamine release (half-maximal release after > 40 min) were slower and more protracted than those of anti-IgE induced histamine release. However, based on a comparison between HRA induced histamine release from leukocytes and purified (97%) basophils, this did not appear to be due to an indirect effect on the basophils. Finally, neither the production of nor the response to HRA was dependent on the allergic status of the donor.

Histamine immunocytochemistry: a new method for detection of basophils in peripheral blood.

We report that basophils in peripheral blood can be stained using histamine immunocytochemistry. The staining is based on the fixation of leucocytes with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (CDI) and the subsequent incubation of these cells with antisera raised against histamine conjugated to different carrier proteins using CDI. The staining appears to be specific for basophils and stained cells can be examined using both fluorescence microscopy and flow cytometry. In addition, histamine immunocytochemistry can be combined with conventional immunocytochemistry by incubating leucocytes with antibodies to cell surface antigens prior to or following fixation of the cells with CDI. Thus, histamine immunocytochemistry may be a valuable tool in future studies of human basophils.

Type I reactions directed against Pneumocystis carinii in AIDS patients.

Type I allergy directed against Pneumocystis carinii (PC) has been investigated in 14 patients with AIDS. The Pneumocystis carinii pneumonia often shows a rapid and severe course, and type I allergy against the parasite might be a pathogenic co-factor in the interstitial lung inflammation. In twelve of the AIDS patients the clinical symptoms and course of illness indicated a PC pneumonia. The basophil histamine release test was used as a sensitive test to detect type I allergy against PC. Eight of the patients showed significant histamine release when stimulated with PC. In contrast, only two patients in the group of 12 HIV antibody-positive homosexual men and none in the control group of 13 heterosexual men released histamine. The histamine release was mediated by an immunological reaction, since the release was abolished and regained by removal from and refixation to the cell surface of the cell-bound immunoglobulins before the antigen challenge. The results suggest an involvement of type I allergy as a pathogenic co-factor in Pneumocystis carinii pneumonia.