Lymphocyte Responses to an Aqueous Extract from Cow Hair and Dander

The proliferative responsiveness to cow dander antigens of lymphocytes from 23 asthmatic patients with allergy to cow hair and dander, from 10 patients with allergic asthma to other antigens and from 31 non-atopic subjects was investigated. Lymphocytes from patients with allergic asthma due to cow epithelium antigens showed a statistically significant decreased responsiveness to an extract from cow dander, compared to that seen with lymphocytes from newborn infants, lymphocytes from normal controls and from asthmatic patients without allergy to cow. The response to the cow dander extract seemed to be an accessory cell dependent T lymphocyte response.     

Allergens in Cow Hair and Dander

Quantitative immunoelectrophoretic methods were used for the analysis of the allergens in cow hair and dander and for comparison with related preparations. The immunoelectro-phoretic precipitation pattern of an extract from cow hair and dander showed 17 precipitates. Crossed radioimmunoelectrophoresis showed four of these to be major allergens of the extract, and these allergens are common major allergens in four investigated cow breeds. The allergens are associated predominantly with hair and dander, but they were also demonstrated in cow saliva, urine, whey, amniotic fluid and beef, as well as in cow-hide products. Allergens displaying partial immunological identity with the major allergens of cow hair and dander were found in extracts from goat and sheep pelts, in products made of these materials such as carpet and knitting wool, and in carpets made of animal hair.     

Quantitative Immunoelectrophoretic Analysis of Extract from Cow Hair and Dander

Quantitative immunoelectrophoresis used for the analysis of a diabased, centrifuged and freeze-dried extract from cow hair and dander revealed 17 antigens. Five of these were identified as serum proteins. Partial identity to antigens of serum and extract from hair and dander of goat, sheep, swine, horse, dog, cat and guinea pig, and to antigens of house dust was demonstrated. Sera from 36 patients with manifest allergy to cow hair and dander selected on the basis of case history, RAST, skin and provocation test, were examined in crossed radioimmunoelectrophoresis (CRIE); sera from five persons with high serum IgE, but without allergy to cow hair and dander, and sera from five normal individuals were controls 31/36 of the sera contained IgE with specific affinity for two of the antigens of the extract. Further, two major and six minor allergens were identified. The control sera showed no specific IgE binding. A significant positive correlation was found between RAST and CRIE for the first group of patients. The approximate molecular weights of the four major allergens obtained by means of gel chromarography were: 2.4 × 10⁴, 2 × 10⁴, 2 × 10³ dalton, respectively. Using Con-A and Con-A Sepharose in crossed immunoaffinoelectrophoresis, eight of the antigens were revealed to contain groups with affinity for Con-A.     

Human Serum IgE against Two Major Allergens from Cow Hair and Dander: Determination of Affinity and Quantity of Antigen-Specific IgE

The quantity and quality of antigen-specific IgE against purified major allergens from cow hair and dander was measured by a double-antibody radioimmunoassay. In serum from patients with allergic asthma to cow the affinity constant varied from 1 to 150 X 10(9) l/mol and the number of antibody combining sites from 0.01 to 0.4 X 10-9 mol/l serum for each antigen.     

Standardization of Allergen Extracts with Appropriate Methods

The paper presents recommendations for standardization of allergen extract by the combined use of skin prick tests and radioallergosorbent tests. These tests provide appropriate means for quantitation of the total allergenic activity of an extract. Advantages and limitations are discussed with reference to the literature.     

Humoral and Cell-Mediated Immune Responses to Dog Dander and Hair in Asthmatic Children

Dog dander and hair (DDH) specific IgA, IgG and IgE antibodies from serum samples Of 202 asthmatic children, and in nasal secretion from 4 to 15 years were measured by enzyme-linked immunosorbent assay. The results were compared with clinical history, and with allergy test (skin prick test, provocation test and RAST) using the same DDH extract. A blood sample for the in vitro lymphocyte stimulation test was obtained from 40 children, and a nasal secretion sample for analysis of the local DDH-specific IgA, IgG and IgE antibody levels was collected form 35 of them. In children of dog-keeping families, higher serum levels of DDH-specific antibodies, especially IgE antibodies, were observed if the dog had been in the home already during the first years of the child's life. The serum levels of DDH-specific antibodies, however, did not correlate with the degree of the present exposure to dogs. The serum levels of DDH-specific or total IgE, or with the results of skin prick or provocation test. The serum levels of DDH-specific IgA were highest in children who were subjectively most sensitive to dogs. Nasal levels of DDH-specific IgE correlated positively with serum specific IgE levels. The correlation was weaker between nasal and serum titers of specific IgG, and not significant between nasal and serum IgA antibody levels, Specific IgE antibody levels were higher, while specific IgA and IgG antibody levels were lower, in nasal secretion form subjects with nasal symptoms on contact with dogs, when compared with subjects with other complaints (asthma, conjunctival or skin reactions). DDH-specific IgG levels correlated negatively with specific IgE level in the nasal secretion from subjects with a positive provocation test result, while the correlation was positive in subjects with a negative provocation test. The in vitro lymphocyte response to DDH did not correlate with the results of allergy test, or with the levels of DDH-specific antibodies in serum or in nasal secretion.     

HEP versus PNU Standardization of Allergen Extracts in Skin Prick Testing

During 1 year, all patients referred to an allergy outpatient clinic for adults were skin prick tested with a panel of standard allergen extracts from two manufacturers using different methods of standardization. One company referred to the histamine equivalent prick (HEP) and the other used the more traditional protein nitrogen units (PNU). Standard extracts and five-fold dilution were tested. The results indicate that the ratio of concentration between two extracts of the same allergen should be measured by the absolute difference of the wheal diameters. We found significant differences between corresponding extracts from the two manufacturers.     

Current Status of Standardization of Inhalant Allergen Extracts in Korea

Allergy diagnosis and immunotherapy in Korea rely mostly on imported allergen extracts. However, some allergens that are not important in Western countries are not commercially available, and even the same species of allergen source often displays differences in allergenicity due to amino acid sequence polymorphisms. Therefore, it is essential to prepare allergen extracts that reflect regional characteristics. Allergen standardization has been performed since 2009 with the support of the Korea Center for Disease Control and Prevention. Here, we summarize the current status of allergen standardization, focusing on the house dust mite and cockroach. Pollen allergens that are under investigation are also briefly described.     

Molecular and immunological characterization of Can f 4: a dog dander allergen cross‐reactive with a 23 kDa odorant‐binding protein in cow dander

Background Dog dander is an important cause of respiratory allergy but its content of allergenic components is still incompletely known. While Can f 1, 2, 3 and 5 have been studied in detail, only fragmentary information is available on the lipocalin Can f 4. Objective To purify, clone and characterize dog dander allergen Can f 4. Methods Can f 4 was purified from dog dander extract by size exclusion, ion exchange and reverse phase chromatography. A cDNA encoding Can f 4 was cloned and used to produce recombinant Can f 4 in Escherichia coli. A 23 kDa protein from cow dander, displaying cross‐reactivity with Can f 4, was purified and identified by amino acid sequencing and mass spectrometry. IgE antibody binding to dog and cow dander extract and to individual dog allergens among 37 dog allergic subjects and 44 pollen allergic controls was studied using ImmunoCAP. Results A dog genome segment containing the Can f 4 gene was bioinformatically identified and enabled the cloning of Can f 4 cDNA. Recombinant Can f 4 displayed close immunological and biochemical similarity to purified natural Can f 4 and bound IgE antibodies from 13/37 (35%) sera of dog allergic subjects. Can f 4 reactive sera showed IgE binding to a 23 kDa protein present in cow dander extract, related to a family of odorant‐binding proteins. The dog and cow proteins shared 37% sequence identity and their cross‐reactivity was demonstrated by IgE inhibition experiments. Conclusion Recombinant Can f 4 brings the panel of available dog allergens closer to completion and will be important in component‐resolved diagnostics in allergy to animal epithelial allergens. Cite this as: L. Mattsson, T. Lundgren, P. Olsson, M. Sundberg and J. Lidholm, Clinical & Experimental Allergy, 2010 (40) 1276–1287.     

Allergen extract-induced interleukin-10 in human memory B cells inhibits immunoglobulin E production

Background Elevated specific IgE antibody levels are common in atopic individuals, caused by T-helper type 2-dominated B cell activation. The induction of antigen-specific IL-10 secreting T cells is discussed as an important mechanism during specific immunotherapy. By contrast the presence and function of B cell-derived IL-10 is not well defined yet.
Objective We investigated whether type-I allergen extracts induce IL-10 expression in human B cells and analysed its functional role on IgE production.
Methods Human peripheral B cells were stimulated with grass pollen, house dust mite (HDM) ( Dermatophagoides pteronyssimus ; Der p) and dog allergen extract. Expression of IL-10 by activated human B cells was determined by flow cytometric analysis and ELISA. Functional analysis considering immunoglobulin production was assayed by ELISA.
Results The allergen extracts studied induced IL-10 expression in B cells. However, the ability to induce IL-10 differed between the allergen extracts. The most potent allergen extract was dog (169±28 pg/mL), followed by grass pollen (141±10 pg/mL) and HDM allergen (125±11 pg/mL). Upon allergen extract stimulation only CD27 expressing memory B cells produced IL-10 and co-expressed the very early activation antigen CD69. The addition of allergen extracts to B cells activated by anti-CD40 and IL-4 selectively inhibited IgE which was dependent on allergen extract-induced IL-10. By contrast the other immunoglobulin subclasses like IgA, IgG or IgM were not altered upon allergen extract challenge.
Conclusion Our data indicate that allergen-activated memory B cells can modulate IgE production through secretion of IL-10.     

The Use of a Reference Allergenic Extract in the Evaluation of Allergen Products

Two commercially available mugwort (Artemisia vulgaris (Av] pollen extracts Av-ALK and Av-PS were compared with a dialysed reference mugwort pollen extract (Av-REF) by means of crossed immunoelectrophoresis (CIE), crossed radio immunoelectrophoresis (CRIE), RAST-inhibition, RAST-screening and skin prick test. Av-ALK and Av-PS both contained approximately half the number of precipitation arcs detectable in Av-REF, and Av-PS seemed to lack two intermediate allergens and two minor allergens, while Av-ALK seemed to lack one minor allergen. The RAST-inhibition curve obtained for Av-PS did not parallel the curves obtained for Av-ALK and Av-REF, indicating that the allergen composition of Av-PS differs from that of the others. Further, RAST-screening experiments support the CIE observation that Av-PS seemed to lack two of the intermediate allergens. The HEP values determined for Av-ALK and Av-PS diverge considerably from those stated by the manufacturers. The results obtained demonstrate that the outcome of a comparison between a reference allergenic extract and allergenic products depends very much on the method employed.     

Safety and Utility of Rush Immunotherapy with Aqueous Allergen Extracts for Treatment of Respiratory Allergies

BackgroundGenerally, allergen immunotherapy must be administered for three to five years. Meanwhile, rush immunotherapy (RIT) shortens the required duration for the build-up phase, thereby improving the therapy''s convenience compared with conventional immunotherapy (CIT). However, RIT is often performed with modified allergens. Therefore, this study aimed to investigate the safety and utility of RIT with aqueous allergens.MethodsMedical records of 98 patients sensitized with at least one inhalant allergen who had received subcutaneous immunotherapy for allergic rhinitis with or without asthma were retrospectively reviewed. All patients were classified into three groups: depot-RIT (n = 25), receiving RIT with depot allergen; aqueous-RIT (n = 48), receiving RIT with aqueous allergen; and aqueous-CIT (n = 25), receiving CIT with aqueous allergen. Patients who had received immunotherapy targeting only house dust mites were excluded.ResultsThe proportions of patients presenting with a systemic reaction to depot-RIT, aqueous-RIT, or aqueous-CIT were 80.0%, 85.4%, and 48.0%, respectively (P = 0.002). The proportions of patients experiencing severe systemic reaction were 4.0%, 16.7%, and 8.0% in depot-RIT, aqueous-RIT and aqueous-CIT, respectively (P = 0.223). The proportions of depot-RIT and aqueous-RIT patients presenting with systemic reaction or severe systemic reaction did not differ significantly (P = 0.553 and P = 0.118, respectively). Significantly fewer depot-RIT (1.0 ± 0.2) and aqueous-RIT patients (2.0 ± 1.3) required outpatient clinical visits during the build-up phase, compared to those administered aqueous-CIT (13.6 ± 1.9; P < 0.001). Moreover, the build-up phase decreased to 17.4 ± 1.8 days in depot-RIT and 23.7 ± 10.9 days in aqueous-RIT, compared to 92.0 ± 12.5 days in aqueous-CIT (P < 0.001).ConclusionRIT with aqueous allergen reduced the build-up phase duration and frequency of hospital visits, with acceptable safety levels. RIT with aqueous allergen may, therefore, be suitable for broad application to patients with respiratory allergies.     

Varying Allergen Composition and Content Affects the in vivo Allergenic Activity of Commercial Dermatophagoides pteronyssinus Extracts

Background: Diagnosis and immunotherapy of house-dust mite (HDM) allergy is still based on natural allergen extracts. The aim of this study was to analyze commercially available Dermatophagoides pteronyssinus extracts from different manufacturers regarding allergen composition and content and whether variations may affect their allergenic activity. Methods: Antibodies specific for several D. pteronyssinus allergens (Der p 1, 2, 5, 7, 10 and 21) were used to analyze extracts from 10 different manufacturers by immunoblotting. Sandwich ELISAs were used to quantify Der p 1 and Der p 2 in the extracts. Mite-allergic patients (n = 45) were skin-tested with the extracts and tested for immunoglobulin E (IgE) reactivity to a panel of 10 mite allergens (Der p 1, 2, 4, 5, 7, 8, 10, 14, 20 and 21) by dot blot. Results: Only Der p 1 and Der p 2 were detected in all extracts but their concentrations and ratios showed high variability (Der p 1: 6.0-40.8 μg ml(-1); Der p 2: 1.7-45.0 μg ml(-1)). At least 1 out of 4 allergens (i.e. Der p 5, 7, 10 and 21) was not detected in 8 of the studied extracts. Mite-allergic subjects showed different IgE reactivity profiles to the individual mite allergens, the extracts showed different allergenic activity in skin-prick tests and false-negative results. Conclusions: Commercially available D. pteronyssinus extracts lack important allergens, show great variability regarding allergen composition and content and some gave false-negative diagnostic test results in certain patients.     

王棕花粉变应原的分析与鉴定

目的对我国南方常见的棕榈科植物王棕花粉（Roystonea regia pollen）变应原蛋白进行分离、分析与鉴定,为标准化变应原疫苗的研制提供基础。方法取常规方法制备的王棕花粉浸出液,采用SDS．PAGE分离王棕花粉蛋白质组分,测定其相对分子量,同时用10例对王棕花粉过敏的患者血清作Western-blot鉴定其变应原及主要变应原成分。结果SDS．PAGE显示王棕花粉有10条可辨蛋白带,其中主要条带有8条,分别为100000、66000、38000、36000、29000、30000、24000、16000和14000Mr,Western—blot结果表明,10例王棕花粉过敏患者血清全部呈阳性反应,有66000、24000、16000和14000Mr共4条致敏条带,其中分子量在16000和14000Mr的蛋白为主要变应原。结论王棕花粉变应原的分析与鉴定为临床王棕花粉变态反应疾病的诊断和治疗奠定了基础。     

208例哮喘患儿吸入性过敏原的调查分析

本文报道了208例哮喘患儿吸入性过敏原的调查结果. 采用变应原浸液皮肤点刺方法. 皮试阳性者169例, 占81.25%.变应原浸液内含18种常见吸入性过敏原. 尘螨的阳性率最高达78.85%, 其次是室尘的阳性率为35.58%, 烟的阳性率为32.69%.无一例对其它变应原呈阳性反应, 过敏原皮试阳性率无性别差异, 与有无个人及家庭过敏史无关. 哮喘婴幼儿皮试阳性率低于哮喘患儿, 哮喘皮试阳性率在年龄组之间有显著差异(P＜0.01), 有随着年龄加大而渐增的趋势. 结果提示: 尘螨、空气污染和被动吸烟是诸多哮喘触发因素中具高度危险性的因素, 加强这方面的对策研究对儿童哮喘的防治具有意义.     

Allergen sensitization and bronchial hyper-responsiveness to adenosine monophosphate in asthmatic patients

BACKGROUND: Indirect bronchoprovocation using adenosine monophosphate (AMP) is related to atopic phenotype expression. OBJECTIVE: To evaluate the putative relationship between skin prick allergen sensitization and bronchial hyper-responsiveness to AMP in a retrospective cross-sectional database analysis. METHODS: We retrospectively evaluated two groups of non-smoking asthmatics (forced expiratory volume in 1 s (FEV)1>/=60% predicted) who were reactive (responders) or unreactive (controls) to inhaled AMP. The main outcome measure was the difference in sensitization to individual allergens and the total atopic load according to the presence or absence of bronchial hyper-responsiveness. RESULTS: We initially identified 180 (44%) non-smoking asthmatics with PC20/=1600 mg/mL. For those who had a skin prick test, the responders (n=151) and controls (n=151) were found to be matched for age, sex, inhaled corticosteroid dose and number of patients using inhaled corticosteroids. There were significant differences in the number of responders vs. controls in terms of sensitization to house dust mite (77% vs. 62%, P=0.004), aspergillus (19% vs. 9%, P=0.014), cat (61% vs. 48%, P=0.028), total atopic load (493 vs. 380 positive tests, P<0.001) and forced mid-expiratory flow (60% vs. 68% predicted, P<0.001). CONCLUSION: Sensitization to common aeroallergens increased the likelihood of bronchial inflammation as reflected by bronchial hyper-responsiveness to inhaled AMP, independently of both FEV1 or inhaled corticosteroid use. This in turn suggests an association between allergen exposure and AMP responsiveness in asthmatics. Further prospective long-term evaluation is indicated to assess whether allergen avoidance strategies can modify the airway response.     

Extraction and Degradation of Timothy Pollen Allergen during Simulated in vivo Conditions

Extraction and degradation of timothy pollen in saline has been compared with extraction in nasal secretion and gastric fluid. By measuring the absolute amount of one important allergenic substance by quantitative immunoelectrophoresis and the total allergenic activity by RAST as a function of time it was shown that the allergens were extracted extremely fast and that both the total allergenic activity and the concentration of one allergen readied maximum after about 20 min. Compared with saline, extraction under the simulated in vivo conditions gave a lower maximum level and a faster degradation of allergens.