Dander Extract Research Articles

The determinants of bronchial hyperresponsiveness in patients with allergic rhinitis

Patients with allergic rhinitis and bronchial hyperresponsiveness (BHR) may be at higher risk of developing asthma. To investigate whether reactivity to aeroallergens in skin prick testing (SPT) and serum eosinophil cationic protein levels can be used to predict BHR in allergic rhinitis patients. Fifty-nine consecutive patients with allergic rhinitis underwent SPTs using grass, tree, weed, parietaria, Alternaria, Aspergillus, mites, and cat and dog dander extracts. Methacholine challenge tests were performed using spirometry. Methacholine-induced BHR was detected in 23 patients (39%). Of 59 patients, 14 had 1 positive SPT response, 35 had 2 to 4 positive responses, and 10 had more than 4 positive responses. There was a significant inverse correlation between methacholine provocation concentration that caused a decrease in forced expiratory volume in 1 second of 20% (PC20) and the number of positive SPT responses (r = -0.28; P = .03). The BHR-positive patients had a mean of 4 positive SPT responses, whereas BHR-negative patients had a mean of 2.6 (P = .04). Nine BHR-positive patients (39%) and only 1 BHR-negative patient (3%) had more than 4 positive SPT responses (P < .001). There was no correlation between serum eosinophil cationic protein levels and methacholine PC20 doses. There was a strong association between hyperresponsiveness to methacholine and both cat and dog dander sensitivity (P < .001 and P = .001, respectively). Allergic rhinitis patients with SPT responses to a higher number of allergens are more likely to have BHR. Whether the number of positive SPT responses correlates with the risk of developing asthma in allergic rhinitis patients remains to be determined.

Immunological response to administration of standardized cat allergen extract at differing doses

Rationale Allergen immunotherapy is an effective method of treatment for allergic rhinitis and asthma. However, the therapeutic maintenance doses, timing, and mechanisms of immunotherapy are unknown. We conducted a study to assess regulatory T cells and their cytokine response to four different doses of cat allergen extract. Methods Twenty eight cat-allergic patients, randomized in a double-blinded study, were assigned to one of four treatment arms: placebo and cat dander extract containing 0.6 μg Fel d1, 3 μg Fel d1, and 15 μg Fel d1. Flow cytometry was used to assess relative amounts of IL-4 and IFN-G produced by CD4 cells and TGF-B and IL-10 produced by CD4/CD25 immunoregulatory cells. ANOVA analysis was performed prior to the initiation of the buildup phase of immunotherapy and at the beginning of maintenance immunotherapy (two months later), and will be performed after one year of therapy. Results This is an interim analysis comparing baseline with two months of immunotherapy. The high dose immunotherapy group (15 μg Fel d1) exhibited a statistically significant increase in IFN-G (0.05 vs 2.15; p=0.008) and a decrease in the relative amount of CD4/CD25 cells (2.51 vs 1.01; p=0.046). The low dose group (0.6 μg Fel d1) exhibited a statistically significant decrease in TGF-B (102.92 vs 38.16; p=0.002), an increase in IL-10 (7.12 vs 9.02; p=0.019), and an increase in IL-4 (0.23 vs 10.67; p=0.013). Conclusions During the buildup phase of low dose cat immunotherapy, regulatory cell production of TGF-B decreases, and IL-4 and IL-10 increase. In the high dose buildup phase, IFN-G increases.

Determinants of bronchial hyperresponsiveness (BHR) in patients with allergic rhinitis (AR)

Rationale Patients with AR and BHR have a higher risk of developing asthma. In this study, we investigated whether reactivity to aeroallergens in skin prick test (SPT) and serum eosinophil cationic protein (sECP) levels could be used to predict BHR in AR. Methods Fifty-nine consecutive patients diagnosed with AR between February 2001 and February 2002 were studied. SPTs were performed using grass, tree, weed, parietaria, Alternaria, Aspergillus, mites and cat and dog dander extracts. Methacholine challenge test was performed using spirometry. Results Methacholine induced BHR was detected in 23 patients (39%). Of the 59 patients, 14 had one positive SPT response, 35 had 2-4 and 10 had >4 positive responses. There was a significant inverse correlation between methacholine PC 20 doses and the number of positive SPTs (p=0.031, r=−0.28). BHR(+) patients had a mean of 4 positive SPTs while BHR(-) patients had a mean of 2.6 (p=0.04). Of the 23 BHR(+) patients, 39% had >4 positive SPTs. In contrast, only 2.7% of the BHR(-) patients had >4 positive SPTs (p=0.0001). The mean methacholine PC 20 dose was 5.05 ± 9.72mg/ml in patients with >4 positive SPTs, 23.4 ± 14mg/ml in patients with 2-4 and 23.6 ± 13.4mg/ml in patients with one positive response (p=0.001 for >4 vs. 2–4, P=0.002 for >4 vs. 1). There was no correlation between sECP levels and methacholine PC 20 doses. Conclusions AR patients with SPT responses to a higher number of allergens are more likely to have BHR. Whether the number of positive SPT responses correlates with the risk of developing asthma in AR remains to be determined.

Identificación de los taxones de pólenes alergénicos en pacientes polínicos para conocer la temporada de riesgo

BackgroundDetermining the risk season for the presence of pollen in the atmosphere aids primary care physicians in the diagnosis and treatment of allergic diseases. Our objective was to identify the taxa of pollen that cause allergic rhinoconjunctivitis in a sample of patients from a health center who presented seasonal symptoms MethodsWe designed an observational, crosssectional, non-randomized study to be carried out in the Cazoña Health Center in Santander, Spain. We selected 30 volunteers of both sexes, aged between 13 and 69 year old, who suffered seasonal rhinoconjunctivitis symptoms and who had always lived in Santander. Patients underwent skin-prick tests with the 25 pollen extracts routinely used in Spain, house dust mite (HDM), cat dander and Alternaria extracts by means of the Prick-Film? system. The test result was expressed as the percentage of the papule area caused by histamine. Measurement was performed by scanning the copied papule area with the Prick-Scan? program for PC ResultsAll patients were positive to grass pollen, 26 % were sensitized to grass pollen only and 23% were also sensitized to other non-pollen allergens. Twenty-seven percent tested positive to Plantago, 20 % to Quercus and 13 % to Morus; the remaining pollen extracts were positive in less than 10 %. Fifty-six percent of the patients were positive to HDM and 6% to cat dander ConclusionsA warning period for pollinosis patients in the city of Santander can be defined. In our case, the important period is the grass pollen season, since the remaining pollen taxa sensitized few patients. Most of our patients were also sensitized to HDM

A double-blind, placebo-controlled immunotherapy dose-response study with standardized cat extract

Background: Allergen immunotherapy with doses of cat extract containing approximately 15 μg of the major allergen, Fel d 1, have been proved clinically effective in several double-blind, placebo-controlled studies. However, the maintenance doses used in allergy practice in the United States are often considerably less than this proven dose. Objective: The purpose of this investigation was to determine whether maintenance immunotherapy with cat dander extract containing 0.6 μg or 3.0 μg of Fel d 1 was more effective than placebo and similar in efficacy to treatment with extracts containing 15.0 μg Fel d 1, immunologic parameters being used as the outcome. Methods: Twenty-eight cat-allergic patients were randomly entered, 7 in each group, into a double-blind, placebo-controlled comparison of the immunologic response to treatment with placebo or cat dander extract containing 0.6 μg, 3.0 μg, or 15.0 μg of Fel d 1. Maintenance doses were achieved in 8 visits over a period of 4 weeks through use of a cluster regimen; each subject then received 1 weekly maintenance injection before posttreatment measurements were made. The response to immunotherapy was assessed before immunotherapy and after the first weekly maintenance injection. Studies included responses to titrated skin prick tests to cat extract and an unrelated allergen and serum allergen-specific IgE and IgG4. Titrated nasal challenges were performed with cat extract; measurement of mRNA and secreted cytokines (IL-4, IL-5, and IFN-γ) was done at 6 hours. Serum cytokines (IL-4, IL-5, IFN-γ) were measured, and flow cytometric analysis of intracellular cytokines (IL-4, IL-5, IFN-γ) was performed. Cat allergen–stimulated lymphocyte proliferation was performed with measurement of cytokines in the supernatant (IL-4, IL-5, IFN-γ). Results: All 28 subjects completed the study. Significant and dose-dependent differences were encountered in the titrated skin prick tests (P = .008), the cat-specific IgG4 (P = .01), and the reduction in CD4+/IL-4+ PBMCs on flow cytometry (P = .03). There were no significant differences between placebo and cat dander extract containing Fel d 1 0.6 μg. Both extracts containing 3.0 μg and 15.0 μg produced significant decreases in skin prick test sensitivity (P = .02 and P = .002, respectively). The extracts containing 3.0 μg and 15.0 μg produced significant increases in cat-specific IgG4 (P = .01 and P = .006, respectively). Only the 15.0-μg-per-dose extract produced a significant reduction in the percent of CD4+/IL-4+ PBMCs (P = .003). Conclusion: In this double-blind, placebo-controlled study, a maintenance dose of cat dander extract containing 15.0 μg Fel d 1 produced the most consistent immunologic response. A dose of 3.0 μg reduced skin prick test sensitivity and increased cat-specific IgG4 but did not reduce the circulating CD4+/IL-4+ PBMCs, a change that is likely related to the clinically significant response to allergen immunotherapy. These findings suggest that a maintenance dose of 15.0 μg of Fel d 1 is most apt to be clinically effective for allergen immunotherapy. (J Allergy Clin Immunol 2003;111:155-61.)

Inhibition of human T-cell responses by allergen peptides.

Of the many significant advances in the field of immunology that have occurred in the last 30 years arguably the most important, at least from a T-cell biologists point of view, has been the definition of major histocompatibility complex (MHC) restriction1 and the crystal structure of human leucocyte antigen (HLA) molecules.2–4 These demonstrations have enabled detailed study of the way in which T cells recognize antigen and also how this process differs from that in B cells. With a few notable exceptions, production of specific immunoglobulin by B cells is T-cell dependent. Furthermore, recruitment of non-lymphoid effector cells into areas of inflammation is a process largely marshalled by the T lymphocyte. It follows therefore that inhibition of T-cell function may have beneficial outcomes in situations in which detrimental immune responses occur and give rise to pathology. Suppression of T-cell function has been achieved with the use of pharmacological agents, such as glucocorticosteroids and cyclosporin, and more recently with biological agents, such as monoclonal antibodies directed against CD3 and CD4. Whilst achieving the desired aim of immunosuppression, these approaches suffer from serious weaknesses, the most obvious of which is a lack of specificity for the antigen(s) which drive the inflammatory process. The same may be said to be true of other therapeutic approaches currently being developed which aim to neutralize the effects of individual mediators in the inflammatory cascade. Here, a notable success has been the use of monoclonal antibodies to tumour necrosis factor (TNF) to treat patients with rheumatoid arthritis.5 However, the long-term consequences of removing individual mediators remain to be seen. Of concern is the recent report of subjects receiving anti-TNF therapy developing a lupus-like syndrome thought to be a direct result of the therapy itself.6 The challenge of introducing specificity into strategies aimed at modulating T-cell responses is one that has been met with considerable success in animal models by the use of peptide epitopes from the antigen in question. Peptide-induced tolerance has been demonstrated in models of experimental autoimmune encephalomyelitis (EAE),7,8 collagen-induced arthritis9 and, more recently, in models of allergic disease. Briner and colleagues sensitized mice to the cat allergen Fel d I and subsequently demonstrated the ability of allergen-derived peptides to inhibit T-cell cytokine and antibody production.10 Hoyne and colleagues demonstrated the ability of peptides from the house dust mite allergen Der p II to down-regulate T-cell and antibody responses to challenge with intact protein and also to prevent sensitization by prior administration.11,12 Bauer administered a dominant T-cell epitope of Bet v I to CBA/J mice either prior to or following sensitization with the whole allergen. No change in antibody isotype was observed but T-cell proliferative responses were inhibited by both treatments.13 More recently, mice sensitized to bee venom allergen phospholipase A2 (PLA2), were treated by intranasal administration of a mixture of three long peptides spanning the entire molecule. A marked reduction in specific immunoglobulin E (IgE) was observed coupled with a decreased interleukin-4 (IL-4) to interferon-γ (IFN-γ) ratio. Pre-administration of peptide resulted in a failure to develop IgE sensitization to subsequent immunization with the whole molecule.14 In addition to animal models, the ability of high-dose peptides to modulate human T-cell responses has also been demonstrated. In vitro studies with human T cells were performed by Lamb and colleagues, who demonstrated the induction of antigen-specific non-responsiveness following the incubation of T-cell clones with supraoptimal doses of specific ligand in the absence of professional antigen-presenting cells.15 Collectively, these studies provided a logical basis for the translation of a peptide-based therapeutic approach in man.

Anaphylaxis to deer dander in a child: a case report

Hypersensitivity to deer dander is rarely reported, with only 26 cases in the literature. Ours is the youngest reported case and the first reported case of anaphylaxis on exposure to a live deer. Evaluation of a case of anaphylaxis in a young boy upon exposure to a deer. A 4-year-old boy experienced hives, swelling, and shortness of breath requiring epinephrine following a deer exposure. He had one mild reaction 5 days prior to his anaphylaxis with an indirect exposure. A deer dander extract was made from fur supplied by the patient's mother. IgE-mediated reactivity was positive for deer and cattle by both selective skin prick method and RAST results. Hypersensitivity to wild animals can lead to life threatening anaphylaxis, even in children. Passive transfer of antigen may occur, but needs further investigation.

Autoallergy: A pathogenetic factor in atopic dermatitis?

Long before the discovery of IgE it was reported that human dander extract can elicit immediate-type skin reactions in patients with severe atopy and that this skin sensitivity can be passively transferred with serum. Several recent findings have rekindled the interest in this phenomenon and led to the concept that IgE autoreactivity may play a pathogenetic role in severe and chronic forms of atopy. The elucidation of the nature of several environmental allergens has revealed striking structural and immunologic similarities with human proteins. It was also reported that patients predominantly with severe and chronic manifestations of atopy (eg, atopic dermatitis) contain IgE autoantibodies against a wide variety of proteins expressed in histogenetically unrelated human cell types and tissue specimens. Last, complementary DNAs coding for autoallergens were isolated from human expression complementary DNA libraries and recombinant autoallergens were produced. The autoallergens characterized to date represent mainly intracellular proteins, but some of them could be detected as IgE immune complexes in sera of sensitized patients. We suggest that at least two pathomechanisms could play a role in autoallergy. First, autoallergens may cross-link effector cell-bound IgE autoantibodies and, by release of inflammatory mediators, lead to immediate-type symptoms. Second, IgE-mediated presentation of autoallergens may activate autoreactive T cells to release proinflammatory cytokines, contributing to the magnitude of the allergic tissue reaction. (J Allergy Clin Immunol 2000;105:432-7.)

Purified natural and recombinant Fel d 1 and cat albumin in in vitro diagnostics for cat allergy

Background: Current diagnostics and therapeutics for cat allergy are based on cat epithelial extracts originating from highly variable source materials. This gives rise to several problems: variability of allergen composition, contamination with house dust mite allergens, and potential transfer of pathogenic agents. Objective: The aim of this study was to investigate the feasibility of replacing cat epithelial extracts with purified natural or recombinant allergens. Methods: Sera (n = 509) were selected on the basis of a positive cat RAST result and tested in a RAST for IgE reactivity to purified Fel d 1, cat albumin (CA), or both. The analysis was performed with both natural and recombinant allergens. In addition, some sera were further analyzed by means of immunoblotting. A serum pool was used for cat RAST inhibition with purified natural and recombinant allergens as inhibitors. Results: Natural and recombinant Fel d 1 caused very similar results: 94.1% and 96.1% positive test results, respectively. In general, the negative sera were low responders to cat extract. The addition of CA (16.7% positive sera) resulted in a decrease in the number of discrepencies between purified allergens and whole extract to 2.8%. Only for 2% of all sera, sensitization to cat was largely explained by IgE reactivity to CA. IgE reactivity to Fel d 1 accounts for 88% of the total IgE response to cat allergens, as was demonstrated by RAST, with Fel d 1 concentrations nearing saturation. Recombinant Fel d 1 performed equally well in the RAST analysis. Recombinant CA was succesfully expressed in the yeast Pichia pastoris , and its immune reactivity closely resembled that of its natural counterpart. Conclusion: Natural and recombinant Fel d 1 and CA are good candidates for replacing ill-defined cat dander extracts in diagnostics for cat allergy. Although CA is not essential for the vast majority of cat-sensitized patients, some subjects are selectively sensitized to this serum protein. (J Allergy Clin Immunol 1999;104:1223-30.)

Evaluating the relevance of aeroallergen sensitization in atopic eczema with the atopy patch test: A randomized, double-blind multicenter study

Background: IgE-mediated sensitization of questionable clinical relevance is routinely demonstrated in patients with atopic eczema (AE) by skin prick test or radioallergosorbent test (RAST). We report the results of a randomized, double-blind multicenter study in patients with AE on the relevance and technical aspects of aeroallergen patch testing (atopy patch test, APT). Methods: Two hundred fifty-three adult patients with AE in remission were tested in 7 study centers on clinically uninvolved, unabraded back skin with 3000 to 10,000 PNU/g of Dermatophagoides pteronyssinus, cat dander, grass, birch, and mugwort pollen allergen extracts in petrolatum. After skin prick and RAST and a detailed history on aeroallergen-induced eczema flares was obtained, reading was done at 48 to 72 hours. Results: Previous eczema flares after contact with specific allergens were reported by 10% (mugwort pollen) to 52% (D pteronyssinus) of patients. The percentage of clear-cut positive APT reactions ranged from 44% with D pteronyssinus to 5% with mugwort. Positive skin prick (36% to 65%) and RAST (49% to 75%) results were more frequent. APT results showed significant concordances with history, skin prick, and RAST for D pteronyssinus, cat dander, and grass pollen ( P < .001). Optimal test concentrations were 5000 (grass pollen) and 7000 PNU/g ( D pteronyssinus , cat dander). The APT had a higher specificity (69% to 92% depending on allergen) with regard to clinical relevance of an allergen compared with skin prick test (44% to 53%) and RAST (42% to 64%). Conclusion: Aeroallergens are able to elicit eczematous skin reactions after epidermal application. The relevance of aeroallergens for AE flares may be evaluated by APT. (J Am Acad Dermatol 1999;40:187-93.)

Washing the clothes of cat owners is a simple method to prevent cat allergen dispersal

Washing the clothes of cat owners is a simple method to prevent cat allergen dispersal

Separation of horse dander allergen proteins by two-dimensional electrophoresis--molecular characterisation and identification of Equ c 2.0101 and Equ c 2.0102 as lipocalin proteins.

The aim of this work was to identify which proteins in horse dander extracts are allergens and to characterise them. Two-dimensional PAGE showed that horse dander preparations are composed of up to 50 proteins, all having acidic isoelectric points in the pH range 3-4.5. Immunoblots of two-dimensional PAGE were used to compare the reactivity of the proteins with IgE from 23 allergic patients. Patient sera were divided into two main groups recognising either allergens of 18.5 kDa or proteins of 27-29 and 31 kDa. The proteins of 27-29 kDa and 31 kDa were all N-glycosylated and their glycan chains seem to play a role in the binding of IgE from allergic patients. The sugar composition of their carbohydrate moiety was determined and lectin-binding experiments indicated presence of terminal sialic acid linked alpha-(2-->6) to galactose, galactose linked beta-(1-->4) to N-acetylglucosamine, and possibly presence of sialic acid linked alpha-(2-->3) to galactose. The 27-29-kDa glycoproteins had heterogeneous isoelectric points, most probably due to different degrees of sialylation in their oligosaccharide chains. The two 18.5-kDa allergens exhibited slightly different isoelectric points and their N-terminal sequences were identical, showing that they most likely were isoforms of the same protein. Sequence analyses revealed that their N-terminal sequences are similar to proteins belonging to the lipocalin family. We named the two 18.5-kDa proteins Equ c 2.0101 and Equ c 2.0102, according to International Allergen Nomenclature recommendations [King, T. P., Hoffman, D., Lowenstein, H., Marsh, D. G., Platts-Mills, T. A. E. & Thomas, W. (1995) J. Allergy Clin. Immunol. 96, 5-14]. The N-terminal of the allergens of 27-29 kDa were blocked and their sequences were not determined. Their amino acid compositions were determined and comparison with acidic mammalian proteins in the Swiss-Prot database revealed high scores with lipocalin proteins. This suggests that the glycosylated horse dander allergens belong to the lipocalin family, like Equ c 2.0101 and Equ c 2.0102.

The major dog allergens, Can f 1 and Can f 2, are salivary lipocalin proteins: cloning and immunological characterization of the recombinant forms.

Canis familiaris allergen 1 (Can f 1) and Canis familiaris allergen 2 (Can f 2) are the two major allergens present in dog dander extracts. We now report the isolation of cDNAs encoding both proteins and present their nucleotide and deduced amino acid sequences. Can f 1, produced by tongue epithelial tissue, has homology with the von Ebner's gland (VEG) protein, a salivary protein not previously thought to have allergenic properties. Can f 2, produced by tongue and parotid gland, has homology with mouse urinary protein (MUP), a known allergen. Both VEG protein and MUP are members of the lipocalin family of small ligand-binding proteins. Recombinant forms of Can f 1 and Can f 2 were produced and tested for immunoglobulin E (IgE) reactivity. Among dog-allergic subjects, 45% had IgE directed exclusively to rCan f 1, and 25% had IgE to both rCan f 1 and rCan f 2. In addition, both recombinant proteins were able to cross-link IgE and elicit histamine release from peripheral blood leucocytes in vitro. These findings confirm that Can f 1 and Can f 2 are major and minor dog allergens, respectively, and demonstrate that recombinant forms of dog allergens retain at least some IgE-binding epitopes.

Clinical efficacy of specific immunotherapy to cat dander: a double‐blind placebo‐controlled trial

To assess the efficacy of specific immunotherapy with standardized cat dander extract, using objective endpoints and simulated 'natural' exposure to cats. Double-blind, randomized, placebo-controlled study carried out at a UK Allergy research clinic. Twenty-eight patients with moderate to severe allergic rhinoconjunctivitis with asthma due to cat allergy. Subjects were stratified for cat sensitivity, cat ownership and asthma, and the groups were well matched for all relevant parameters. Symptom scores and peak flow rate during and after exposure to cats in a cat-room. Skin tests and conjunctival provocation thresholds. The actively treated group showed a marked reduction in symptoms during the cat exposure (mean score 61.6-17.1; P < 0.001) with no change in the placebo group (64.7 vs 62.1). The active group also showed a reduced peak flow response to cat exposure (mean fall of 85 L/min pretreatment, 29 L/min after treatment, P < 0.005) as well as reductions in conjunctival provocation sensitivity, skin sensitivity to cat extract and skin sensitivity to house dust mite (D.pteronyssinus). Skin reactivity to histamine and codeine were unaltered. No significant adverse reactions were encountered. Specific immunotherapy seems to be an effective treatment for cat allergy. Allergy to cats is common and often poorly controlled on conventional pharmacotherapy. Although cat allergy has not traditionally been considered as a valid indication for immunotherapy in the UK, it should now be considered as a legitimate treatment, especially for those who are unable to avoid exposure.

Rush immunotherapy results in allergen-specific alterations in lymphocyte function and interferon-γ production in CD4+ T cells

Background: Allergen immunotherapy results in a number of changes in clinical, inflammatory, and immunologic parameters. However, the basis for the specificity of this form of therapy is unknown, especially in the context of changes in T- and B-lymphocyte function after desensitization to specific allergens. Objective: This study was designed to determine the immunologic consequences of rush immunotherapy. Methods: We studied 10 patients who had positive skin test responses to the house dust mite Dermatophagoides pteronyssinus (Dpt) and cat dander extract. Each received rush immunotherapy to mite, but not cat dander, over a 2- to 4-week period until maintenance was achieved. Patients were evaluated before and when maintenance was achieved for skin test and nasal reactivity to mite and cat dander; antibody levels to the allergen were monitored, as were lymphocyte proliferative responses and cytokine production. Results: Rush immunotherapy to house dust mite resulted in a significant reduction in skin and nasal reactivity to mite allergen, but not to cat allergen, in 10 of 10 patients. This was accompanied by a rise in serum anti-Dpt IgE, whereas anti-cat IgE was not altered (7 of 7 patients). In seven of seven patients there was an increase in anti-Dpt IgG 4 levels. T-cell proliferative responses to mite antigen were suppressed, and numbers of CD8 T cells increased in frequency. There was a marked increase in interferon-γ production, particularly by CD4 T cells in 10 of 10 patients. The correlation between the increases in interferon-γ production and the changes in cutaneous reactivity was highly significant. Conclusion: We show that rush immunotherapy is immunologically specific in eliciting changes in T- and B-cell responses to the desensitization antigen. The specificity and potential benefit of immunotherapy may be linked to the increase in interferon-γ production by allergen-activated CD4+ T lymphocytes. (J Allergy Clin Immunol 1997;99:530-8.)

Biologic allergen assay for in vivo test allergens with an in vitro model of the murine type I reaction

Background: The determination of the biologic activity of allergenic extracts in human beings is limited for ethical and practical reasons. The establishment of a simplified in vitro model, which mimics a main event of the type I reaction, should provide true benefit for manufacturers, researchers, and clinicians. Objective: This study was designed to develop and evaluate a mediator release assay based on rat basophil leukemia cells for the purpose of investigating allergenic extracts. Methods: Rat basophil leukemia cells were passively sensitized with murine IgE raised against allergens and stimulated by serial dilutions of allergenic extracts in a dose-related manner. The allergen-specific degranulation was monitored by measuring the release of β-hexosaminidase. Results: The investigation of standardized commercial allergen products for in vivo diagnostics (birch pollen, cat dander, and bee venom) allowed a quantitative description of differences in biologic activity in accordance with the declared activity units. The Fel d 1 content was determined in 17 cat dander extracts and correlated well with the results of a two-site binding ELISA ( r= 0.93, log/log). Extremely low allergen amounts in the range of 10 to 100 pg/ml could be easily detected. Moreover, the cross-reactivity pattern of patients allergic to birch pollen could be reproduced with extracts of hazel, alder, apple, and celery. Conclusion: The assay is suitable for supplementing quality control of allergenic extracts and for the determination of biologic activity of final allergen products. As a research tool, it allows the study of IgE cross-linking properties of modified and recombinant allergens at an early stage before they are tested in human beings. (J Allergy Clin Immunol 1997;99:227-32.)

False-positive skin prick test responses to commercially available dog dander extracts caused by contamination with house dust mite (Dermatophagoides pteronyssinus)allergens

BACKGROUND: In an outpatient population, a high frequency of positive skin prick test responses to dog dander was found in the absence of detectable IgE to dog dander in the RAST. The majority of these patients were sensitized to house dust mites (Dermatophagoides pteronyssinus) and had no obvious dog-related allergic symptoms. These findings prompted us to investigate whether dog dander skin test preparations are contaminated with house dust mite allergens in amounts sufficient to cause false-positive skin prick test responses in patients sensitized to house dust mites. METHODS: Antigen detection assays with monoclonal and polyclonal antibodies were used to determine concentrations of the major allergen Can f 1 from dog dander and the major allergens Der p 1 and Der p 2 from house dust mites in five commercially available dog dander skin prick test preparations (A to E). RESULTS: Can f 1 concentrations varied for the different extracts (A: 170 μg/ml, B: 11.1 μg/ml, C: 13.3 μg/ml, D: 3.8 μg/ml, and E: 59.4 μg/ml). Der p 1 was detectable in all extracts (A: 33.4 ng/ml, B: 5.1 ng/ml, C: 29.6 ng/ml, D: 0.4 ng/ml, and E: 1.9 ng/ml), and Der p 2 was detectable in some of the commercially available dog dander skin prick test preparations tested (A: 31.3 ng/ml, B: 3.0 ng/ml, and C: 7.5 ng/ml). The median house dust mite threshold in the skin prick test was found to be 5.8 ng/ml of Der p 1 (range, 3.5 to 20.8 ng/ml) in nine patients tested. CONCLUSION: Contamination of commercially available dog dander skin prick test preparations with the major allergens (Der p 1 and Der p 2) of the house dust mite (D. pteronyssinus) was demonstrated. These contaminations cause false-positive responses to skin prick tests with dog dander in patients sensitized to house dust mite. (J Allergy Clin Immunol 1996;98:1028-34.)

Anaphylactic reaction after bites by Glossina morsitans(tsetse fly) in a laboratory worker

Anaphylactic reaction after bites by Glossina morsitans(tsetse fly) in a laboratory worker

Determination of the stability of diluted allergen extracts using a concentration step prior to EAST inhibition

Generally the stability of diluted allergen extracts, as used for skin testing, provocation testing and immunotherapy can not be measured using a normal enzyme allergosorbent test (EAST) inhibition method. The aim of this study was to determine the stability of diluted allergen extracts using an ultrafiltration step prior to the standard EAST inhibition procedure, in which the allergen extract was concentrated 100-fold. This concentration procedure was validated for Dermatophagoides pteronyssinus, timothy pollen, birch pollen and cat dander extracts and used in a stability study in which three batches were stored for 1 year at 6 degrees C and 25 degrees C. There was no difference in relative potency before and after concentration of birch and timothy pollen extracts. D. pteronyssinus and cat dander extracts showed a significant decrease of 25% and 35% of the relative potency after concentration. The mean coefficient of variation of 12 determinations of the stability study was 11.8%. For all allergens the 30 BU/mL or approximately 0.00025 mg/mL solution was stable for 12 months at both temperatures, except for D. pteronyssinus which declined rapidly at 25 degrees C.

Molecular analysis of allergenic proteins in bovine dander

An analytic procedure was established to characterize bovine dander proteins with allergenic properties. The proteins from dander extract were separated by size-exclusion gel filtration, and the fractions were studied with SDS-PAGE followed by immunoblotting. An 11-kDa allergen was found in the same gel filtration fractions as 20- and 22-kDa allergens, and this suggests that the 11-kDa allergen is a dimer in its native form. Our method also detected two separate 22-kDa allergens. The primary structure of the major bovine dander allergen (BDA20) was also studied. A protein sequencer was used to determine the amino acid sequences of enzymatically cleaved peptides. The homology searches revealed that BDA20 is not a previously known bovine protein.