共查询到20条相似文献,搜索用时 31 毫秒
1.
Emara M Royer PJ Abbas Z Sewell HF Mohamed GG Singh S Peel S Fox J Shakib F Martinez-Pomares L Ghaemmaghami AM 《The Journal of biological chemistry》2011,286(15):13033-13040
Dendritic cells are professional antigen-presenting cells that are specialized in antigen uptake and presentation. Allergy to cat has increased substantially in recent years and has been shown to be positively associated with asthma. We have recently shown that the mannose receptor (MR), a C-type lectin expressed by dendritic cells, recognizes various glycoallergens from diverse sources and is involved in promoting allergic responses to a major house dust mite allergen in vitro. Here we investigated the potential role of MR in allergic responses to Fel d 1, a major cat allergen. Fel d 1 binding to MR was confirmed by ELISA. Using blocking, gene silencing (siRNA) experiments, and MR knock-out (MR(-/-)) cells, we have demonstrated that MR plays a major role in internalization of Fel d 1 by human and mouse antigen-presenting cells. Intriguingly, unlike other glycoallergens, recognition of Fel d 1 by MR is mediated by the cysteine-rich domain, which correlates with the presence of sulfated carbohydrates in natural Fel d 1. WT and MR(-/-) mice were used to study the role of MR in allergic sensitization to Fel d 1 in vivo. MR(-/-) mice sensitized with cat dander extract and Fel d 1 produced significantly lower levels of total IgE, Fel d 1-specific-IgE and IgG1, the hallmarks of allergic response, compared with WT mice. Our data show for the first time that Fel d 1 is a novel ligand of the cysteine-rich domain of MR and that MR is likely to play a pivotal role in allergic sensitization to airborne allergens in vivo. 相似文献
2.
Grönlund H Bergman T Sandström K Alvelius G Reininger R Verdino P Hauswirth A Liderot K Valent P Spitzauer S Keller W Valenta R van Hage-Hamsten M 《The Journal of biological chemistry》2003,278(41):40144-40151
Dander from the domestic cat (Felis domesticus) is one of the most common causes of IgE-mediated allergy. Attempts to produce tetrameric folded major allergen Fel d 1 by recombinant methods with structural features similar to the natural allergen have been only partially successful. In this study, a recombinant folded Fel d 1 with molecular and biological properties similar to the natural counterpart was produced. A synthetic gene coding for direct fusion of the Fel d 1 chain 2 N-terminally to chain 1 was constructed by overlapping oligonucleotides in PCR. Escherichia coli expression resulted in a non-covalently associated homodimer with an apparent molecular mass of 30 kDa defined by size exclusion chromatography. Furthermore, each 19,177-Da subunit displayed a disulfide pattern identical to that found in the natural Fel d 1, i.e. Cys3(1) Cys73(2), Cys44(1)-Cys48(2), Cys70(1)-Cys7(2), as determined by electrospray mass spectrometry after tryptic digestion. Circular dichroism analysis showed identical folds of natural and recombinant Fel d 1. Furthermore, recombinant Fel d l reacted specifically with serum IgE, inducing expression of CD203c on basophils and lymphoproliferative responses in cat-allergic patients. The results show that the overall fold and immunological properties of the recombinant Fel d 1 are very similar to those of natural Fel d 1. Moreover, the recombinant Fel d 1 construct provides a tool for defining the three-dimensional structure of Fel d 1 and represents a reagent for diagnosis and allergen-specific immunotherapy of cat allergy. 相似文献
3.
Pets are an important source of indoor allergens. The aim of the study was to compare cat and dog allergen levels in cars, schools and homes. The study was carried out in 17 cars, 14 classrooms and 19 dwellings located in the highly industrialized and urbanized region of Poland. Dust and air samples were analyzed for Fel d 1 and Can f 1 using a double monoclonal ELISA assay. The highest amounts of cat and dog allergens (Fel d 1: 1169 μg/g; Can f 1: 277 μg/g) were found in dwellings with pets. Allergen concentrations were correlated with the number of animals kept at home. Although concentrations on automobile seats were lower, Fel d 1 levels exceeded 8 μg/g in 23.5 % of cars and high levels of Can f 1 (>10 μg/g) were found in 17.6 % of cars. The study revealed that cars of pet owners may be reservoirs of cat and dog allergens even when animals are not transported in them. In schools, concentrations of pet allergens did not reach high levels, but the moderate levels of Fel d 1 (≥1–8 μg/g) and Can f 1 (≥2–10 μg/g) were detected in 42.9 and 7.1 % of the investigated classrooms. Concentrations of cat and dog allergen in schools were higher than in homes without pets. While airborne Fel d 1 and Can f 1 levels were found low, residential allergen concentrations in settled dust and air were correlated. The study results suggest that classrooms and cars of pet owners may be important sites of exposure to cat and dog allergens, though the highest concentrations of Fel d 1 and Can f 1 are found in homes of pet owners. 相似文献
4.
Kaiser L Velickovic TC Badia-Martinez D Adedoyin J Thunberg S Hallén D Berndt K Grönlund H Gafvelin G van Hage M Achour A 《Journal of molecular biology》2007,370(4):714-727
Felis domesticus allergen 1(Fel d 1) is a 35 kDa tetrameric glycoprotein formed by two heterodimers which elicits IgE responses in 95% of patients with allergy to cat. We have previously established in vitro conditions for the appropriate folding of recombinant Fel d 1 using a direct linkage of chain 1 to chain 2 (construct Fel d 1 (1+2)) and chain 2 to chain 1 (construct Fel d 1 (2+1)). Although the crystal structure of Fel d 1 (2+1) revealed a striking structural similarity to that of uteroglobin, a steroid-inducible cytokine-like molecule with anti-inflammatory and immunomodulatory properties, no functional tetrameric form of Fel d 1 could be identified. Here we present the crystal structure of the Fel d 1 (1+2) tetramer at 1.6 A resolution. Interestingly, the crystal structure of tetrameric Fel d 1 reveals two different calcium-binding sites. Symmetrically positioned on each side of the Fel d 1 tetramer, the external Ca(2+)-binding sites correspond to a putative Ca(2+)-binding site previously suggested for uteroglobin. The second Ca(2+)-binding site lies within the dimerization interface, stabilizing the formation of the Fel d 1 tetramer, and inducing important local conformational changes that directly govern the shape of two water-filled cavities. The crystal structure suggests a potential portal for an unknown ligand. Alternatively, the two cavities could be used by the allergen as a conditional inner space allowing for the spatial rearrangement of centrally localized side-chains, such as Asp130, without altering the overall fold of the molecule. The striking structural similarity of the major cat allergen to uteroglobin, coupled to the identification in the present study of a common Ca(2+)-binding site, let us speculate that Fel d 1 could provoke an allergic response through the modulation of phospholipase A2, by sequestering Ca ions in a similar manner as previously suggested for uteroglobin. 相似文献
5.
Kaiser L Grönlund H Sandalova T Ljunggren HG van Hage-Hamsten M Achour A Schneider G 《The Journal of biological chemistry》2003,278(39):37730-37735
The domestic cat (Felis domesticus) is one of the most important causes of allergic asthma worldwide. The dominating cat allergen, Fel d 1, is composed of two heterodimers. Recently, it has been shown that recombinant Fel d 1, consisting of chain 2 and chain 1 fused together without additional linker, has immunological properties indistinguishable from the natural heterodimeric protein. Herein, we report the crystal structure of recombinant monomeric Fel d 1 at 1.85-A resolution, determined by multi-wavelength anomalous diffraction using selenomethionine substituted protein. Fel d 1 is an all-helical protein and consists of eight helices. The two halves of the recombinant Fel d 1 molecule, corresponding to the wild-type Fel d 1 chains, are very similar in three-dimensional structure, despite the lack of significant sequence identity. The structure of the Fel d 1 presents a striking similarity to that of uteroglobin, a steroid-inducible cytokine-like molecule with anti-inflammatory and immunomodulatory properties. An internal, asymmetric cavity is formed in the Fel d 1 that could bind an endogenous ligand. The distribution of residues lining this cavity suggests that such a ligand must be amphipathic. The structure of Fel d 1 displays the localization of three previously defined Fel d 1 IgE epitopes on the surface of the protein. The three-dimensional structure provides a framework for rational design of hypoallergenic mutants aimed for treatment of cat allergy. 相似文献
6.
Seppälä U Hägglund P Wurtzen PA Ipsen H Thorsted P Lenhard T Roepstorff P Spangfort MD 《The Journal of biological chemistry》2005,280(5):3208-3216
Fel d 1 is a major cat allergen inducing allergic rhinitis and asthma in sensitized individuals. It has a more complex structure when compared with other allergens and therefore expression of recombinant Fel d 1 has been considered a challenge. The present study shows for the first time that a Baculovirus expression system is able to produce an intact rFel d 1 molecule that is glycosylated and structurally equivalent to the natural cat allergen, nFel d 1. Enzymatic digestion of rFel d 1 and further analysis by use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) resulted in a complete coverage of the amino acid sequence of rFel d 1. In addition, the three disulfide bridges at the positions alpha70-beta7, alpha44-beta48, and alpha3-beta73 were verified. The N-glycan structure of rFel d 1 was investigated by a combination of MALDI-TOF MS and monosaccharide analysis by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAC). The N-glycosylation analyses of rFel d 1 refer to a pattern of glycoforms including core alpha1.3-fucosylation that is different from nFel d 1. Further characterization by use of human serum IgE, histamine release, and lymphocyte proliferation assays demonstrated that the immunological characteristics of rFel d 1 are similar to those of nFel d 1. Detailed characterization of both natural and recombinant allergens provides tools to explore immunological mechanisms associated with allergen sensitization and desensitization. 相似文献
7.
Natt Tasaniyananda Urai Chaisri Anchalee Tungtrongchitr Wanpen Chaicumpa Nitat Sookrung 《PloS one》2016,11(3)
Cats (Felis domesticus) are rich source of airborne allergens that prevailed in the environment and sensitized a number of people to allergy. In this study, a mouse model of allergic rhinitis caused by the cat allergens was developed for the first time and the model was used for testing therapeutic efficacy of a novel intranasal liposome-entrapped vaccines made of native Fel d 1 (major cat allergen) in comparison with the vaccine made of crude cat hair extract (cCE). BALB/c mice were sensitized with cCE mixed with alum intraperitoneally and intranasally. The allergic mice were treated with eight doses of either liposome (L)-entrapped native Fel d 1 (L-nFD1), L-cCE), or placebo on every alternate day. Vaccine efficacy evaluation was performed one day after provoking the treated mice with aerosolic cCE. All allergenized mice developed histological features of allergic rhinitis with rises of serum specific-IgE and Th2 cytokine gene expression. Serum IgE and intranasal mucus production of allergic mice reduced significantly after vaccination in comparison with the placebo mice. The vaccines also caused a shift of the Th2 response (reduction of Th2 cytokine expressions) towards the non-pathogenic responses: Th1 (down-regulation of the Th1 suppressive cytokine gene, IL-35) and Treg (up-regulation of IL-10 and TGF-β). In conclusions, a mouse model of allergic rhinitis to cat allergens was successfully developed. The intranasal, liposome-adjuvanted vaccines, especially the refined single allergen formulation, assuaged the allergic manifestations in the modeled mice. The prototype vaccine is worthwhile testing further for clinical use in the pet allergic patients. 相似文献
8.
Chaithanya Madhurantakam Ola B. Nilsson Hannes Uchtenhagen Tiiu Saarne Tatyana Sandalova Adnane Achour 《Journal of molecular biology》2010,401(1):68-83
The dog lipocalin allergen Can f 2 is an important cause of allergic sensitization in humans worldwide. Here, the first crystal structure of recombinant rCan f 2 at 1.45 Å resolution displays a classical lipocalin fold with a conserved Gly-Xaa-Trp motif, in which Trp19 stabilizes the overall topology of the monomeric rCan f 2. Phe38 and Tyr84 localized on the L1 and L5 loops, respectively, control access to the highly hydrophobic calyx. Although the rCan f 2 calyx is nearly identical with the aero-allergens MUP1, Equ c 1 and A2U from mouse, horse and rat, respectively, no IgE cross-reactivity was found using sera from five mono-sensitized subjects. However, clear IgE cross-reactivity was demonstrated between Can f 2 and the cat allergen Fel d 4, although they share less than 22% sequence identity. This suggests a role for these allergens in co-sensitization between cat- and dog-allergic patients. 相似文献
9.
Epitope mapping of two major inhalant allergens, Der p I and Der f I, from mites of the genus Dermatophagoides 总被引:2,自引:0,他引:2
M D Chapman P W Heymann T A Platts-Mills 《Journal of immunology (Baltimore, Md. : 1950)》1987,139(5):1479-1484
The repertoire of antigenic sites on two major dust mite allergens, Der p I of Dermatophagoides pteronyssinus and Der f I of D. farinae, was studied using murine (BALB/c) monoclonal antibodies (Mab), polyclonal rabbit IgG antibodies, and human IgE antibodies. Fifty-three IgG Mab were analyzed from six different fusions (five vs Der p I, one vs Der f I). By antigen binding radioimmunoassay (RIA), most Mab were either Der p I or Der f I specific, and only 2/53 bound to both allergens. Epitope mapping studies using cold Mab to inhibit the binding of six 125I labeled Mab to solid phase allergen defined four nonrepeated, nonoverlapping epitopes on Der p I, a single species-specific epitope on Der f I and a cross-reacting epitope present on each allergen. All but one of the 53 Mab bound to one of these six epitopes. Seventy percent (25/35) of anti-Der p I Mab were directed to the same epitope, suggesting that this epitope is immunodominant for BALB/c mice. Similarly, 88% (16/18) of anti-Der f I Mab bound to the same epitope on Der f I. Parallel cross-inhibition curves were obtained using the species-specific Mab, 10B9, and the cross-reacting Mab, 4C1, to compete for binding to Der p I, suggesting that the epitopes defined by these two Mab on Der p I are adjacent to one another. Both murine Mab and polyclonal rabbit IgG antibodies to cross-reacting sites on both allergens were used to inhibit binding of human IgE antibodies to Der p I by using 19 sera from mite allergic patients. Cross-reacting rabbit IgG antibodies strongly inhibited all sera tested (mean 79.5% +/- 7.7) and two Mab, 10B9 and 4C1, partially inhibited (38% +/- 12). However, the four Mab directed against separate species-specific epitopes (including murine immunodominant sites) showed little or no inhibition (less than or equal to 20%). Our results suggest that most of the epitopes defined by Mab are not the same as, or close to, those defined by human IgE antibody. The striking differences in the repertoires of murine IgG and human IgE antibody responses to Der p I and Der f I could be explained by genetic differences or by altered antigen processing and presentation occurring as a result of different modes of immunization in mice and in mite allergic humans. 相似文献
10.
Summary Quantification of specific allergens in household dust samples may provide important information for selecting appropriate allergen control methods, and monitoring efficacy and compliance. The purpose of this study was to investigate the source of variation in mite and cat allergen measurements associated with dust sample collection. Discrete and composite dust samples were collected on a filter using a special vacuum sampling device. Aqueous extracts of the dust samples were prepared thenDer p I,Der f I, andFel d I were quantitated by enzyme immunoassays (EIA). Mite and cat allergens were frequently detected in dust samples from human dwellings, and the amounts of these allergens varied significantly (p<0.01) among dwellings. The differences of allergen measurements among duplicate samples taken immediately and up to three weeks later appear to be much smaller than differences among houses and between rooms. Variation among dust samples taken from living rooms and bedrooms of the same dwelling suggest differences in allergen reservoirs. Composite samples formed by sampling specific objects within a room may provide a reliable estimate of allergen exposure in that room. Dust samples from discrete objects are useful to find and monitor specific reservoirs of mite and cat allergens. 相似文献
11.
The relative binding site location and species specificity of 19 mouse hybridoma antibodies, produced in four laboratories, to Dermatophagoides pteronyssinus major fecal allergen, Der p I, was studied by using immobilized mAb and inhibitions of radiolabeled Ag binding. Four mAb groups were defined, within which 4, 6, 8, and 5 mAb, respectively, cross-inhibited each other. Five mAb were members of both group 2 and 3, demonstrating a considerable overlap of epitopes between the corresponding antibody-binding regions. The degree of mAb species specificity, as assessed by inhibition with cold Der p I and Ag Der m I and Der f I from the related species, Dermatophagoides microceras and Dermatophagoides farinae, was highly variable even for mAb binding to the same region on the Ag. Five cases of cross-reactivity between Der p I and Der m I and one case of cross-reactivity between Der p I and Der f I were found. The N-terminal 30 amino acids of the three species showed 7 substitutions between Der p I and Der m I/Der f I and 2 between Der f I and Der p I/Der m I. Single mAb inhibited up to 65% of labeled Der p I binding to immobilized human IgE from allergic patients' sera and up to 24% of labeled Der p I binding to immobilized rabbit antibodies. The spectrum of species specificities in human IgE sera, as assessed by inhibitions with cold Ag, was similar to that of the mAb. No evidence for the presence of strictly sequential epitopes, reactive with either mAb or human IgE was found, as judged from the weak inhibitory activity of acid-denatured Der p I. 相似文献
12.
M D Chapman W M Sutherland T A Platts-Mills 《Journal of immunology (Baltimore, Md. : 1950)》1984,133(5):2488-2495
Monoclonal antibodies were raised against Antigen P1, the major allergen of the house dust mite (Dermatophagoides pteronyssinus). The majority were Antigen P1 specific, isotype IgG1, and did not react with a comparable D. farinae allergen. These antibodies bound 38 to 50% of 125I Antigen P1 in antigen-binding assays (titer greater than or equal to 1/1,000,000), and the quantities of IgG antibody in ascites were 2 to 4 logs greater than those in polyclonal mouse antiserum or in serum from a mite-allergic patient. Two IgM antibodies showed weak binding to Antigen P1 but reacted strongly with D. pteronyssinus in enzyme immunoassay (titer greater than or equal to 1/100,000). Assessments of the specificity of the IgG antibodies by using two inhibition radioimmunoassays suggested that they were directed against two different epitopes. Antibodies 10B9 F6 and 5H8 C12 were purified by preparative isoelectric focusing (isoelectric points of pI 6.25 and 7.4, respectively) and radiolabeled with 125I. Cross-inhibition experiments, using ascites dilutions to inhibit binding of each radiolabeled antibody to Antigen P1, confirmed that these antibodies recognized two distinct epitopes. Analysis of antibodies from 39 clones/hybrids showed that the majority were directed against the same epitopes as either 10B9 F6 or 5H8 C12 (3 out of 39 [8%] and 29 out of 39 [74%], respectively). None of the monoclonal antibodies significantly inhibited (greater than 10%) human IgE binding to Antigen P1 in the radioallergosorbent test. However, 12 of 14 sera from mite allergic patients inhibited binding by the monoclonal antibodies. One serum from a mite-allergic patient inhibited binding of both 10B9 F6 and 5H8 C12 by greater than 85% and showed parallel inhibition curves. The results suggest that these monoclonal antibodies could be used to assay Antigen P1 in both D. pteronyssinus and house dust extracts. It should also be possible to use monoclonal antibodies in inhibition assays to define the antigenic/allergenic determinants recognized by human IgG and IgE antibodies on this mite allergen. 相似文献
13.
M Barel A Fiandino A X Delcayre F Lyamani R Frade 《Journal of immunology (Baltimore, Md. : 1950)》1988,141(5):1590-1595
Glycoprotein (gp) 140, the EBV/C3dR of B lymphocytes, is a membrane site involved in human cell regulation. To analyze the specificities of the binding sites for EBV and for C3d on the gp 140 molecule, two distinct approaches were used. First, anti-EBV/C3dR mAb were prepared against highly purified EBV/C3dR. Nine anti-EBV/C3dR mAb were obtained. Four of these anti-EBV/C3dR mAb inhibited C3d binding but not EBV binding on gp 140, whereas four others exerted an inverse effect. These differences could not be due to differences in isotype, antibody concentration, affinity constant, and number of molecules bound on cell surface, as these parameters were identical for the nine used mAb. Second, polyclonal anti-idiotypic antibodies (Ab2) were prepared against F(ab)'2 fragments of polyclonal anti-EBV/C3dR (Ab1). Ab2 recognized the variable portion of Ab1 as controlled by immunoblotting experiments. Ab2, which did not react with the cell surface, inhibited Ab1 binding on Raji cells. Ab2 mimicked the EBV/C3dR by its properties to bind to particle-bound C3d and EBV, preventing their binding on Raji cell surface. C3d binding specificities contained in Ab2 were isolated by affinity chromatography on C3b/C3bi-Sepharose. These specificities, being the internal image of C3d binding site of EBV/C3dR, reacted with Ab1 and inhibited particle-bound C3d binding on Raji cells but did not react with EBV. Taken together, these data support strongly that gp 140, the EBV/C3dR, carried two distinct binding sites, one for EBV and one for C3d. 相似文献
14.
M Akagawa T Mori T Ando H Okudaira 《Bioscience, biotechnology, and biochemistry》1992,56(11):1725-1727
An enzyme linked immunosorbent assay system using a monoclonal antibody, 15E11, specific for a major allergen Der f II in house dust mite, was developed. This system detected only Der f II in the presence of Der p II and other allergens. The Der f II contents in several house dust samples significantly correlated with the numbers of the mites in the same house dust samples (n = 14, r = 0.88, p < 0.001). These data showed that this system was useful for specific measurement of Der f II in house dusts. 相似文献
15.
Fiftyfour allergic patients with bronchial asthma or obstructive lung disease were investigated at admission and discharge after a 3 months hospitalization period in the alpine valley Davos. An intracutaneous skin test procedure was carried out with 16 common inhalant allergens. Histamine 0.01 mg/ml was used as a positive control and a phosphate buffered saline with 0.03% HSA and 0.5% phenol were used as negative controls. A liophylized control for house dust mite was used for every patient on admission and discharge, in order to control loss of allergen potency of the vial. Furthermore, an additional freshly prepared vial of house dust mite allergen was analyzed after it was opened and stored at 4°C after 3 months. The wheal and flare reactions were calculated separately for the early reaction (at 15 minutes) and the late reaction (at 6 hours). The sum of wheal/histamine index and fare/histamine index were calculated for the early phase reactions and analyzed according two-tailed paired student's t tests. Using RAST inhibition, the liophylized house dust mite showed the same allergen concentration as in the fluid form. The 3 months old vial was analyzed and showed the same allergen concentration as was expected. Results for histamine reactions on admission and discharge showed no significant difference. Patients showed no reactions on the negative controls. Differences were found between skin test reactions on admission and discharge for the sum of early wheal reactions (p = 0.0001), and to certain allergen wheal such as house dust mite and some other common allergens. We conclude that during a 12 weeks stay in the alpine climate Davos, intracutaneous early wheal reactions to certain allergens are decreasing possibly reflecting a decreased exposure. The late reaction showed no significant change. 相似文献
16.
Bermuda grass pollen (BGP) contains a very complex mixture of allergens, but only a few have been characterized. One of the allergens, with an apparent molecular mass of 21 kDa, has been shown to bind serum IgE from 29% of patients with BGP allergy. A combination of chromatographic techniques (ion exchange and reverse phase HPLC) was used to purify the 21 kDa allergen. Immunoblotting was performed to investigate its IgE binding and lectin-binding activities, and the Lysyl-C endopeptidase digested peptides were determined by N-terminal sequencing. The cDNA sequence was analyzed by RACE PCR-based cloning. The protein mass and the putative glycan structure were further elucidated using MALDI-TOF mass spectrometry. The purified 21 kDa allergen was designated Cyn d 24 according to the protocol of International Union of Immunological Societies (IUIS). It has a molecular mass of 18,411 Da by MALDI-TOF analysis and a pI of 5.9. The cDNA encoding Cyn d 24 was predicted to produce a 153 amino acid mature protein containing tow conserved sequences seen in the pathogen-related protein family. Carbohydrate analysis showed that the most abundant N-linked glycan is a alpha(3)-fucosylated pauci-mannose (Man3GlcNAc2) structure, without a Xyl beta-(1,2)-linked to the branching beta-Man. Thus, Cyn d 24 is a glycoprotein and the results of the sequence alignment indicate that this novel allergen is a pathogenesis-related protein 1. To the best of our knowledge, this is the first study to identify any grass pollen allergen as a pathogenesis-related protein 1. 相似文献
17.
A direct on sampling filter in solution (DOSIS) method for quantification of airborne cat allergens has been developed. In this method, the allergens firmly adsorbed to a porous polytetrafluoroethylene filter are reacted with specific antibodies conjugated to alkaline phosphatase, generating a matrix-bound allergen-antibody-phosphatase complex. The treated filter is subsequently floated on a commercially available chemiluminescent phosphatase substrate solution. Aliquots of this solution are removed and analyzed luminometrically. The light intensity of the product is linearly related to the amount of allergen over a large mass range, 0-100 SQ units (1 SQ unit is about 146 pg of the allergenic protein Fel d 1). DOSIS demonstrated intra- and interassay precisions of 9% and 8% and 14% and 21% for the levels 4 and 20 SQ units per filter, respectively. The limit of quantification was estimated to 0.4 SQ units (58 pg Fel d 1) of cat allergen per filter. Application of DOSIS to analysis of cat allergen concentrations of indoor air in homes with and without cats revealed, on average, a six times higher concentration in the former (142 SQ units/m(3)) as compared to the latter (24 SQ units/m(3)). The recorded concentrations for airborne cat allergen in homes with cats are in accordance with previously reported figures. Allergen-specifically stained sampling filters revealed the particulate nature of airborne cat allergen which seemed predominantly to be carried by numerous large dust particles. 相似文献
18.
Fel d I is the major cat allergen that induces asthma and allergic rhinitis in humans. To investigate the mechanism of allergic responses to this allergen, a mouse model was developed. Mice sensitized to chain 1 of Fel d I exhibited T cell responses, B cell responses, and mast cell responses when challenged with the protein. Subcutaneous injections of peptides containing the dominant T cell epitopes of the allergen induced T cell tolerance in presensitized mice. When challenged with the allergen intratracheally, these tolerized mice produced a decreased amount of histamine in vivo. The decrease in histamine release was not solely dependent on the reduction of allergen-specific IgE. These data show that mast cell activity in mice with an ongoing sensitivity to allergen can be regulated through peptide-induced T cell tolerance. 相似文献
19.
Analysis of 6-keto PGF1 alpha, 5-HETE, and LTC4 in rat lung: comparison of GM/MS, RIA, and EIA 总被引:4,自引:0,他引:4
J Y Westcott S Chang M Balazy D O Stene P Pradelles J Maclouf N F Voelkel R C Murphy 《Prostaglandins》1986,32(6):857-873
The recent availability of fast and sensitive radioimmunoassay (RIA) and enzyme immunoassay (EIA) procedures to measure icosanoids has led to utilization of these techniques by many investigators. A major concern has been that techniques based on immunoreactivity may lack specificity, in particular if complex biologic fluids or tissue extracts are evaluated. The purpose of this investigation was the comparison of icosanoid measurements obtained either with EIA or RIA with those obtained by gas chromatography/mass spectrometry (GC/MS). Rats were injected with Salmonella enteritidis endotoxin, killed at various times after the injection and the lung extract assayed for 6-keto-PGF1 alpha, 5-HETE and LTC4. By EIA lung tissue was found to contain large quantities of 6-keto-PGF1 alpha after endotoxin stimulation. Comparisons made between EIA and GC/MS analysis showed good correlation between 6-keto-PGF1 alpha amounts in lung as determined by each technique. It was also determined that little purification of lung extract was needed to obtain reliable quantitation of 6-keto-PGF1 alpha, probably due to the specificity of the antibody and the large quantity of this prostaglandin produced. Crudely purified (Sep-Pak) lung extracts gave 5-HETE levels by RIA which were highly correlated with GC/MS values, but RIA values were 70% higher than those obtained by GC/MS. The presence of other components in lung extract which cross react with this 5-HETE antibody was probably responsible for the higher values obtained by RIA. LTC4 was measured by immunoassay in crude lung extracts, as well as after Sep-Pak purification and HPLC purification. LTC4 levels were identical in unpurified lung extract and after Sep-Pak purification, but decreased substantially after HPLC purification. Thus, by validating the icosanoid immunoassays, we have found that they can give accurate and reproducible results in lung tissue, although LTC4 and 5-HETE must be purified prior to analysis. 相似文献
20.
J J Tsai O Cromwell P Maestrelli R E O'Hehir R Moqbel A B Kay 《Journal of immunology (Baltimore, Md. : 1950)》1989,142(5):1661-1668
Human PBMC cultured with PHA produced a factor which enhanced neutrophil LTB4 production. After progressive purification by gel filtration and chromatofocusing, leukotriene release enhancing factor (LREF) gave a single band on SDS-PAGE corresponding to a Mr of 60 kDa and a isoelectric point of 5.0 to 5.2. Human T lymphocytes were identified as a source of LREF because a long term human CD4+ T cell line, specific for house dust mite (Dermatophagoides farinae) generated leukotriene release enhancing activity (LREA) when stimulated with the inducing Ag together with irradiated accessory cells. Cultures of accessory cell and Ag alone did not induce neutrophil activation. Similarly, control cultures using an irrelevant allergen (grass pollen) failed to produce this activity. Stimulation of PBMC with anti-CD3 mAb also generated LREA. LREA had other neutrophil-associated biologic activities. These included the enhancement of neutrophil adherence to nylon wool, increased cytotoxicity of opsonised schistosomula of Schistosoma mansoni and upregulation of C receptors (CR1 and CR3). LREA had minimal neutrophil chemotactic activity. Therefore, LREF has potential as a mediator of inflammation and provides a link between lipid mediators, neutrophils, and cell-mediated immunity in the context of allergic diseases. 相似文献