bluuebunny/crossref_metadata_2025 huggingface dataset:
The Importance of Serine Proteinases as Aeroallergens Associated with Asthma
Authors: Shen Horng-Der , Tam Ming F. , Chou Hong , Han Shou-Hwa
Published: April 2003

Penicillium and Aspergillus species have been identified as prevalent indoor airborne fungi that are associated with extrinsic bronchial asthma. We have recently analyzed the IgE–binding components in 8 prevalent Penicillium and Aspergillus species (P. citrinum, P. notatum, P. oxalicum, P. brevicompactum, A. fumigatus, A. flavus, A. oryzae and A. niger) by immunoblotting and N–terminal amino acid sequence analysis. Our results show that the alkaline and/or vacuolar serine proteinases are the major allergens in these prevalent fungal species. IgE cross–reactivity among these major allergens was also detected. Results obtained provide an important basis for clinical allergy. In addition, monoclonal antibodies against alkaline and/or vacuolar serine proteinase allergens have been generated. These antibodies can be applied for the standardization of allergenic extracts.
Identification and Characterisation of Two Allergens from the Dust Mite Acarus siro, Homologous with Fatty Acid–Binding Proteins
Authors: Eriksson Tove L.J. , Whitley Paul , Johansson Eva , van Hage-Hamsten Marianne , Gafvelin Guro
Published: April 2003

Background: Dust mites are a major cause of allergic disease worldwide. The dust mite Acarus siro is an inducer of occupational allergy among farmers, but sensitisation has also been found in non–farming populations. Methods: A degenerate primer was designed to the N–terminal amino acid sequence of a 15–kD IgE–binding protein in A. siro extract. The cDNA sequence was obtained by using reverse transcriptase polymerase chain reaction, standard cloning and sequencing techniques. The protein was expressed in Escherichia coli with a 6–histidine tag at its C–terminus. Immunoblotting of the recombinant protein and whole extract was performed using patient sera. Results and conclusion: 15 and 17–kD allergens were identified in a fraction of A. siro extract. The cDNA of the 15–kD allergen was isolated, cloned and sequenced and the allergen was expressed as a recombinant protein. The calculated molecular weight of the cDNA–encoded protein is 14.2 kD. The predicted amino acid sequence has one potential N–glycosylation site at position 4–6 and a cytosolic fatty acid–binding protein signature at position 5–22. The protein has 64% sequence identity with Blo t 13, an allergen from the dust mite Blomia tropicalis, as well as homology with several other fatty acid–binding proteins (FABPs) from different organisms. The allergen was named Aca s 13 and was recognised strongly by 3 of 13 (23%) of the subjects investigated. The amino acid sequence of the 17–kD protein was partly determined and it also showed high sequence homology with Blo t 13 and FABPs.
Alkaline Serine Proteinase Is a Major Allergen of Aspergillus flavus, a Prevalent Airborne Aspergillus Species in the Taipei Area
Authors: Chou Hong , Lin Win-Ling , Tam Ming F. , Wang Soo-Ray , Han Shou-Hwa , Shen Horng-Der
Published: April 2003

Background: Aspergillus species are prevalent indoor airborne fungi and have been identified to be a causative agent of human allergic disorders. In the present study, we identified, purified and characterized the allergen(s) from Aspergillus flavus, a predominant airborne Aspergillus species in the Taipei area. Methods: The IgE–binding components of A. flavus were identified by SDS–PAGE immunoblotting with sera from asthmatic patients. The N–terminal amino acid sequences of the major allergens were determined by Edman degradation. The allergenic cross–reactivity among allergens from different fungi was analyzed by immunoblot inhibition using sera from asthmatic patients. The detected major allergen was purified from the culture medium. It was further characterized in terms of its N–terminal amino acid sequence, its IgE–binding activity and its enzymatic activity. Results:  The results of the immunoblot analysis indicate that a 34–kD component that has high IgE–binding (63%) frequency is a major allergen of A. flavus. The N–terminus of this 34–kD major allergen (GLTTQKSAP) has high sequence identity with that of the 34–kD alkaline serine proteinase major allergen of A. oryzae. Results from immunoblot inhibition studies indicate that IgE cross–reactivity occurs among the 34–kD major allergens of A. flavus, A. fumigatus and Penicillium citrinum. The 34–kD major allergen of A. flavus was purified from the culture medium by ammonium sulfate precipitation and DEAE ion exchange chromatography. The N–terminal amino acid sequence of the purified allergen (Asp fl 13) is identical to that determined previously for the 34–kD major allergen in the crude extract of A. flavus. The IgE immunoblot reactivity to the 34–kD major allergen in the crude extract can be dose–dependently inhibited by the purified Asp fl 13. The degree of IgE binding to the 34–kD major allergen in the crude extract correlates with that of the purified Asp fl 13 in sera of 8 asthmatic patients. The purified Asp fl 13 has proteolytic activity with casein as substrate at pH 8.0. This enzymatic activity can be inhibited by either phenylmethylsulfonyl fluoride or diethylpyrocarbonate. Conclusion: Our results suggest that the 34–kD alkaline serine proteinase is a major allergen of A. flavus. There was IgE cross–reactivity among allergens of A. flavus, A. fumigatus and P. citrinum.
Anaphylactic Response to Parasite Antigens: IgE and IgG1 Independently Induce Death in Trichinella–Infected Mice
Authors: Bruschi Fabrizio , Pozio Edoardo , Watanabe Naohiro , Gomez-Morales M. Angeles , Ito Mamoru , Huang Yong , Binaghi Ruben
Published: April 2003

The response of animals infected with different Trichinella species (T. spiralis, T. britovi, T. pseudospiralis) to antigens of different Trichinella species was evaluated in outbred mice (CD1) and inbred mice (BALB/c, C3H, SJL, C57BL/6). In mice deficient for IgE or IgG1, T. spiralis only was used. In homologous conditions (i.e., same Trichinella species for antigens and infectious agent) mortality was very high (up to 100% in SJL and C57BL/6). The anaphylactic response in heterologous conditions was much lower and death was observed only in C57BL/6 and SJL mice. Experiments with deficient mice have shown that both specific IgE and IgG1 independently induce a fatal anaphylactic response.
A Repeat Polymorphism in Interleukin–4 Gene Is Highly Associated with Specific Clinical Phenotypes of Asthma
Authors: Chouchane L. , Sfar I. , Bousaffara R. , El Kamel A. , Sfar M.T. , Ismaïl A.
Published: April 2003

Background: IL–4 is a determining factor in immunologic mechanisms to allergy and inflammation. The authors designed a case–controlled study to investigate the potential association of a repeat polymorphism in IL–4 gene with specific clinical phenotypes of asthma. Methods: The authors used the polymerase chain reaction to characterize the variation of the IL–4 intron 2 region in 145 unrelated Tunisian patients with asthma and 160 healthy control subjects. In order to strengthen the case–controlled study, analysis of IL–4 polymorphism was performed in families of several asthmatic patients. Asthma scores were determined and correlated with this polymorphism. Results: Analysis of IL–4 polymorphism in patients with allergic asthma and in control subjects demonstrated a significant association between the IL–4 A1 allele and asthma. Further evidence of the strong association found between IL–4 intron 2 polymorphism and asthma was provided by the finding that asthma is transmitted in association with the inheritance of the IL–4 A1 marker. When patients were stratified into two groups according to the degree of the severity of asthma, the IL–4 A1 allele was specifically not associated with mild asthma, but highly associated with the moderate and severe forms of the disease. The relative risk (RR) of severe asthma is especially high in patients carrying the A1/A3 genotype (RR = 3.94, p = 0.0001). Conversely, a major decrease in the frequency of the IL–4 A3/A3 genotype was observed in patients with severe asthma, resulting in a significantly negative RR of this clinical phenotype of asthma (RR = 0.165, p = 0.0001). Conclusions: Tunisian persons carrying the IL–4 A1/A3 genotype may have an increased risk of severe asthma.
Fragrance and Contact Allergens in vitro Modulate the HLA–DR and E–Cadherin Expression on Human Epidermal Langerhans Cells
Authors: Verrier Anne Claude , Schmitt Daniel , Staquet Marie Jeanne
Published: April 2003

Background: Epidermal Langerhans cells (LCs) play a critical role in the induction of contact hypersensitivity. The LCs leave the skin, move to the regional lymph nodes and present the allergens embedded in the HLA–DR molecule to naive T–lymphocytes. To allow LC emigration from the epidermis, E–cadherin must be downregulated. In this study, we have examined the early events that occur in the human epidermis after exposure to three strong contact sensitizers and two commonly used fragrances by examining alterations of E–cadherin and HLA–DR expression. Methods: To determine whether E–cadherin and HLA–DR levels were modulated by allergens, flow cytometry was utilized to evaluate E–cadherin and HLA–DR expression on human epidermal LCs exposed to the different chemicals for 4 h at 37°C. Results: In vitro stimulation with the contact sensitizers isoeugenol, cinnamaldehyde, 2,4,6–trinitrobenzenesulfonic acid, Bandrowski’sbase, or p–phenylene diamine resulted in a dose–dependent decrease of HLA–DR expression on the surface of LCs without affecting the number of positive cells. These contact allergens induced a downregulation of E–cadherin expression as well as a significant decrease of the percentage of E–cadherin–positive cells. Incubation with an irritant, sodium lauryl sulfate, did not significantly change HLA–DR and E–cadherin expression. Conclusions: Based on the alteration of E–cadherin and HLA–DR expression of human LCs under short–term exposure conditions, there was a clear difference between contact sensitizers and a well–characterized irritant. For the first time, the ability of fragrance allergens in dipropylene glycol, a widely used vehicle in fragrance and cosmetic industries, was demonstrated to induce human LC phenotypic alterations. In combination with a series of in vitro tests, this rapid and simple method should help to detect the sensitizing potential of a substance to be applied onto the human skin as an alternative to animal testing.
A Novel Assay for Serum Complement Activity: C42 Generation Assay
Authors: Kobayashi Emi , Kitano Etsuko , Kitamura Hajime
Published: April 2003

Background: In most clinics, laboratory tests for serum complement are limited to immunochemical determinations of C3 and C4 and are occasionally extended to the hemolytic titration of total complement functional activity (CH50). However, these tests are often not sufficient for the analysis of low CH50 serum. Methods: A novel assay for serum complement activity, the C42 generation assay, has been developed. The principle of this assay is based on the hemolysis of sensitized sheep erythrocytes (EA) by complement components in two sera: C42 (the classical pathway C3 convertase) is generated on EA by C1, C4 and C2 in the first serum, followed by a second reaction leading to hemolysis by C3–C9 supplied by the addition of the second serum in the presence of EDTA. Results: This methodology permits the evaluation of two distinct serum complement activities of a test serum. The combined activity of C1, C4 and C2, as well as the combined activity of C3–C9, can be estimated from the observed degrees of hemolysis. Information obtained from this assay is helpful for the analysis of test serum determined to have decreased CH50. Several clinical cases are presented in which this assay was utilized. Conclusions: The C42 generation assay is another functional assay of serum complement which can provide information beyond that obtained from the typical serum CH50 assay. Since intermediate cells or isolated complement components are not necessary, this assay can be employed rapidly and economically in a clinical setting.
Characterization of IgE–Reactive Autoantigens in Atopic Dermatitis 1. Subcellular Distribution and Tissue–Specific Expression
Authors: Seiberler Susanne , Bugajska-Schretter Agnes , Hufnagl Peter , Binder Bernd R. , Stöckl Johannes , Spitzauer Susanne , Valent Peter , Valenta Rudolf
Published: April 2003

We have previously reported that patients suffering from atopic dermatitis (AD) frequently display IgE autoantibody reactivity to human proteins expressed in cell lines of different histogenetic origins. Molecular cloning and in situ staining experiments revealed that certain IgE–reactive autoantigens were expressed preferentially in target organs of atopy while others could be detected in various tissues and cell types. Here we use serum IgE from AD patients to investigate the distribution of autoantigens in subcellular fractions of the human epithelial cell line A431 and in human tissue specimens. Results obtained showed that IgE–reactive autoantigens can be detected in the nuclear > microsomal > mitochondrial > cytoplasmic fraction of A431 cells as well as in a variety of human tissue specimens (brain, bone, intestine, liver, lung, muscle, skin, uterus) and effector cells of atopy (basophils, mast cells, T cells). If IgE autoreactivity plays a pathogenetic role in severe forms of atopy, organ–specific manifestations (e.g. AD) may result from the transport to and deposition of IgE–reactive autoantigens in certain target organs (skin) rather than from a preferential expression of the autoantigens in the affected tissues.
Reactivity of T Cells with Grass Pollen Allergen Extract and Allergoid
Authors: Kahler H. , Stüwe H.-Th. , Cromwell O. , Fiebig H.
Published: April 2003

Background: Successful allergen–specific immunotherapy is achieved with progressively increasing doses of allergen or allergoid. In order to gain further insight into the mechanism of action of allergoids several in vitro investigations were conducted. Methods: Peripheral blood mononuclear cells (PBMC) from grass pollen allergic and nonallergic subjects were stimulated with either grass pollen extract or allergoid and the proliferation and cytokine production (IL–5, IFN–γ) were measured. Similar investigations were performed with Phl p 5–specific T cell lines (TCL) and clones (TCC). Dendritic cells and PBMC were compared in terms of their relative efficacies as antigen–presenting cells. Results: Both allergen and allergoid induced proliferation and Th2 and Th1 cytokine synthesis by PBMC of allergic subjects, whereas PBMC of nonallergic subjects did not produce IL–5. The maximum level of IL–5 was obtained with a lower concentration than was necessary for maximal IFN–γ production. Higher stimulation doses of allergen and allergoid shifted the cytokine profiles towards a Th1 phenotype. TCL and TCC clearly showed reactivity with both allergen and allergoid when using autologous PBMC for antigen presentation, but compared with the native allergen the reactivity of the allergoid was reduced with most of the TCC. Using dendritic cells for antigen presentation a pronounced increase of stimulation of the TCC especially for the allergoids becomes obvious. Conclusion: In common with grass pollen allergen the corresponding allergoids possess a strong allergen–specific T cell–stimulating capacity. However, the degree of T cell stimulation by the allergoid seems to be dependent on the type of the antigen–presenting cell. Both, allergen and allergoid, can modulate T cell responses in a dose–dependent manner.
Repeated Inhalation of Low Doses of Cat Allergen That Do Not Induce Clinical Symptoms Increases Bronchial Hyperresponsiveness and Eosinophil Cationic Protein Levels
Authors: de Blay Frédéric , Krieger Pascale , Spirlet François , Moreau Lionel , Duvernelle Catherine , Kassel Olivier , Kopferschmitt Marie Christine , Gasser Bernard , Demangeat Christine , Pauli Gabrielle , Frossard Nelly
Published: April 2003

The aim of this study was to investigate whether repeated exposure to subclinical doses of cat allergens, not inducing asthma symptoms, could affect eosinophil cationic protein (ECP) levels in bronchoalveolar lavage (BAL) or in peripheral blood, without the appearance of clinical symptoms. Twelve patients with mild asthma, all sensitized to cats and not exposed to cat allergen at home, underwent a series of inhalations of cat allergen or placebo for 8 days over 2 weeks. A methacholine challenge was performed before and after the allergen and saline exposures, and BAL and blood were sampled for ECP measurements and eosinophil counts. No patients experienced asthma symptoms. However, PD20 methacholine (geometric mean) decreased significantly from 263 μg before to 126 μg after inhalation of allergen. Inhalation of saline did not induce any significant change in PD20. The change in log PD20 before and after cat allergen exposure was statistically different from the change in log PD20 before and after saline. Median ECP levels in BAL and serum increased significantly after allergen exposure, from 0.8 to 3.1 μg/l (p<0.02) and from 15.9 to 31.4 μg/l (p<0.05), respectively. No change was observed after saline inhalations. The change in BAL and serum ECP levels was statistically significant compared to that in the control group. The number of eosinophils did not change, however, nor did IL–5 and RANTES levels in BAL and serum. In conclusion, our results show that (1) exposure of asthma patients to repeated low doses of allergen, which did not provoke any clinical symptoms, is capable of inducing a local eosinophil activation associated with an increase in nonspecific bronchial hyperresponsiveness and (2) the increase in serum ECP levels due to eosinophil activation precedes the occurrence of asthma symptoms and may thus be a marker of allergen exposure in allergic asthma.
Nuclear Factor Kappa B Mediates Interleukin–8 Production in Eosinophils
Authors: Yamashita Naomi , Koizumi Hirotaka , Yamashita Naohide , Murata Machiko , Mano Kenji , Ohta Ken
Published: April 2003

Background: Recent reports indicate that in response to various stimuli, eosinophils produce a variety of cytokines (e.g. IL–8) which play pivotal roles in allergic inflammation. In that regard, the transcription factor, nuclear factor, Kappa B (NF–κB), is an important activator of tumor–necrosis–factor–alpha (TNF–α)–induced IL–8 gene expression in monocytes, lymphocytes and neutrophils. We therefore investigated the role played by NF–κB in cytokine production induced by stimulation of eosinophils with the proinflammatory cytokines, granulocyte–monocyte colony–stimulating factor (GM–CSF) and TNF–α. Methods: Peripheral blood samples were obtained from human subjects with slight to moderate eosinophilia. NF–κB activation elicited by exposing cells to GM–CSF and/or TNF–α was investigated using immunohistochemistry and gel shift assays. To functionally assess the effects of NF–κB translocation, IL–8 production was also examined using an enzyme–linked immunosorbent assay. Results: Stimulation of eosinophils with GM–CSF + TNF–α induced significant increases in the synthesis and secretion of IL–8 which were associated with translocation of NF–κB p50 into the nucleus. The binding of NF–κB to the DNA was verified by the gel shift assays. IL–8 production was significantly inhibited by N–acetyl–L–cysteine, FK506 and MG–132, inhibitors of NF–κB activation and translocation. Conclusion: On the basis of our findings, we conclude that activation and translocation of NF–κB plays a crucial role in the signal–transduction pathway leading to the synthesis and release of IL–8 by eosinophils.
Expression of Mast Cell Proteases in Rat Lung during Helminth Infection: Mast Cells Express both Rat Mast Cell Protease II and Tryptase in Helminth Infected Lung
Authors: Tomita Masaki , Itoh Hiroshi , Kobayashi Takahiko , Onitsuka Toshio , Nawa Yukifumi
Published: April 2003

Background: The phenotype of proliferated mast cells in Nippostrongylus brasiliensis–infected rat lung has been identified as mucosal mast cells (MMC) but not connective tissue mast cells (CTMC). However, a previous study of ours showed that the expression of rat mast cell tryptase (RMCT) mRNA, which has been reported to be confined to CTMC, significantly increased in rat lung 14 days after infection. Methods: The expression of four mast cell proteases in rat lung during the course of infection with N. brasiliensis was examined by RNA blot analysis. Immunohistochemical analysis of RMCT and rat mast cell protease (RMCP) II, which has been reported to be confined to MMC was also performed. Results: The number of lung mast cells did not change until 7 days after infection, then gradually increased until 21 days after infection. The expression of the RMCP II gene had increased 14 and 21 days after infection. In addition, the expression of the RMCP I and RMCT genes had also increased at the same time points, but RMCP III had not. By immunohistochemistry, most of the mast cells in infected lung were identified as RMCP II+/RMCT– (MMC), but both RMCP II+ and RMCT+ mast cells were also observed. Conclusions: The present results suggest that mast cell phenotype alteration or a distinct mast cell subset might be present in N. brasiliensis–infected rat lung, and therefore N. brasiliensis–infected rat lung may be a useful tool for studying the differentiation mechanism of mast cells.
Neurokinin–1 Receptor Antagonist Inhibits Short–Term Sulfuric–Acid–Induced Airway Hyperresponsiveness in Sensitized Guinea Pigs
Authors: Teramoto Shin , Tanaka Hiroshi , Kaneko Satoshi , Abe Shosaku
Published: April 2003

Background: Tachykinins are involved in the development of bronchial inflammation and airway hyperresponsiveness (AHR); however, the role of the neurokinin–1 (NK1) receptor in acid–aerosol–induced bronchial impairment in asthmatic patients remains controversial. Methods: To investigate the effects on the NK1 receptor antagonist FK888 the neurokinin–2 (NK2) receptor antagonist SR48968 on sulfuric–acid (H2SO4)–induced AHR in guinea pigs, specific airways resistance (sRaw) and airways responsiveness to methacholine (MCh) were measured before and after 6 h of exposure to H2SO4 aerosol (pH 1.7, 82 mg/m3) in ovalbumin–sensitized guinea pigs. Results: Airway responsiveness to MCh significantly increased (p<0.05) after the exposure, however sRaw did not. Treatment with FK888 significantly inhibited (p<0.05) H2SO4–induced AHR in a dose–dependent manner, as did SR48968. Conclusions: These results suggest that not only NK2 but also NK1 receptors might have important roles in the development of acid–aerosol–induced AHR.
IL-4 Induces Eotaxin Production in Corneal Keratocytes but Not in Epithelial Cells
Authors: Fukagawa Kazumi , Nakajima Toshiharu , Saito Hirohisa , Tsubota Kazuo , Shimmura Shigeto , Natori Michiya , Hirai Koichi
Published: April 2003

Background: In severe allergic eye diseases, the breakdown of epithelial barrier function can lead to severe corneal damage such as erosions or ulcers which often resist treatment. Although eosinophils are thought to play a crucial role in corneal tissue damage in severe ocular allergy, the mechanisms of eosinophil recruitment to the cornea has not been fully clarified. Eotaxin has been found in tears of severe allergic patients with corneal ulcer. In this study, we investigated whether the Th2 cytokine interleukin-4 (IL-4) induces eotaxin production in human corneal epithelial cells and keratocytes. Methods: Primary cultures of human corneal epithelial cells and keratocytes were incubated with IL-4 and/or TNF-α for 48 h. Released eotaxin was measured by ELISA, and the eotaxin proteins were visualized by immunocytochemistry. Eotaxin mRNA expression in cultured cells was analyzed by RT-PCR. Results: IL-4 induced eotaxin production in keratocytes in a dose- and time-dependent manner which was enhanced by TNF-α. There was no detectable eotaxin produced by corneal epithelial cells (<5 pg/ml). The cytoplasm of keratocytes incubated with IL-4 stained positively against anti-eotaxin antibodies, while eotaxin mRNA was detected in keratocytes incubated with IL-4. Conclusions: Human corneal keratocytes, but not epithelial cells, are capable of producing eotaxin by stimulation with IL-4. Our results suggest that eotaxin production in keratocytes induced by IL-4 may play an important role in eosinophil recruitment to corneal ulcers in allergic ocular disease. Eotaxin production by keratocytes may explain the severity of allergic disease involving the corneal stroma.
Purification of Group 2 Dermatophagoides pteronyssinus Allergen and Prevalence of Its Specific IgE in Asthmatics
Authors: Tsai Jaw-Ji , Shen Horng-Der , Chua Kaw-Yan
Published: April 2003

Group 2 allergens are a major cause of sensitization in patients allergic to house dust mites. This study was performed to determine the prevalence of hypersensitivity to group 2 allergens (Der p 2) of Dermatophagoides pteronyssinus (Dp) in asthmatic patients in Taiwan. To facilitate the analysis of Der p 2-specific IgE, we raised a panel of monoclonal antibodies (MoAbs) to Der p 2 antigens. Purified Der p 2 was obtained after MoAb affinity column purification. There were 82 asthmatic patients (41 adults and 41 children) with hypersensitivity to Dp who were analyzed for hypersensitivity to Der p 2. All of them were both skin test- and serology test-reactive to Dp. Using purified Der p 2, 87.8% (72/82) of patients had a skin-test-positive reaction. Six adults (6/41) and 4 children (4/41) had negative skin tests for Der p 2. Ten families (both parents and children were asthmatics) of the 82 patients were selected for Der p 2 skin testing and Der p 2-specific IgE determination using immunoblot analysis. Results showed that 90% (18/20) of patients’ skin reactions to Der p 2 and serum contained specific IgE to Der p 2. Because 87.8% (85.4% of adults and 90.2% of children) of the asthmatic patients with Dp hypersensitivity were allergic to Der p 2, its role in the pathogenesis of asthma in Taiwan appears to be important. Purified Der p 2 allergens can be further used for allergen skin testing and immunotherapy.
Safety of Sublingual-Swallow Immunotherapy in Children and Adults
Authors: André C. , Vatrinet C. , Galvain S. , Carat F. , Sicard H.
Published: April 2003

Background: Immunotherapy is an established treatment of allergic diseases. The safety of this treatment, particularly when administered without direct medical surveillance, as in the case of the sublingual-swallow route needs to be established. The aim of this paper is to review the safety of the sublingual-swallow immunotherapy as reported in eight double-blind, placebo-controlled trials carried out in France, Italy and Greece. Methods: Six hundred and ninety subjects, 472 adults and 218 children, took part in trials of specific immunotherapy (SIT) for the treatment of rhinoconjunctivitis and/or asthma. Three hundred and forty-seven patients received SIT and 343 patients received placebo. Treatment with specific immunotherapy with allergen extracts or placebo was administered using the sublingual-swallow technique. The allergens administered were grass, ambrosia, parietaria and olive pollens, and mites. The daily dose taken during maintenance therapy ranged from 100 to 300 IR (index of reactivity) and cumulative doses ranged from 4,500 to 104,000 IR. Treatment duration ranged from 4 months to 2 years. Adverse events reported either spontaneously by the patient or on direct questioning by the investigator were analysed. Results: One hundred and forty-five unusual events were reported in the subjects receiving active SIT and 79 in those receiving placebo (p < 0.001). Of these 85 were children aged 15 years or less (50 received active SIT, 35 placebo) and 139 were adults (95 received SIT, 44 placebo). Unusual events involving the buccal cavity (61 SIT, 13 placebo) and the gastro-intestinal tract (47 SIT, 15 placebo) were significantly more frequent in the SIT-treated patients (p < 0.001). Wheezing (9 SIT, 21 placebo) was more frequent in the placebo-treated patients (p < 0.05). There were no differences in the frequency of unusual events between adults and children and in the frequency of events involving other body systems. No event was reported as serious. Two events reported as laryngeal oedema were not considered to be accurate descriptions. Conclusions: No serious adverse event was reported in the studies monitored, confirming the good safety profile of the sublingual-swallow method both in children and adults with rhinitis or moderate asthma.
Athymic Nude Rats Develop Severe Pulmonary Hypertension following Monocrotaline Administration
Authors: Miyata Masayuki , Sakuma Fumitaka , Ito Masayuki , Ohira Hiromasa , Sato Yukio , Kasukawa Reiji
Published: April 2003

Objective: To investigate the significance of the presence or absence of the thymus in the development of pulmonary hypertension (PH) following monocrotaline (MCT) administration, the degree of MCT-induced PH (MCT-PH) in athymic nude (F344/N Jcl-rnu) rats was compared with that in their euthymic (rnu/+) littermates. Methods: Histopathological studies of the lung in terms of interstitial edema, congestion, thickening of the alveolar wall, inflammatory cell infiltration and degeneration of arteries were performed by staining with hematoxylin-eosin (HE), elastin van Gieson and Masson’s trichrome. The medial wall thickness of the small pulmonary arteries and the weight ratio of the right ventricular free wall (RV) to that of the left ventricle plus septum [RV/(LV + S) weight ratio] were used as indices of the degree of PH. Toluidine blue staining was performed to estimate the number of the mast cells in the lung interstitium. Results: Interstitial edema was significantly severer in MCT- injected euthymic rats than in MCT-injected athymic nude rats (p < 0.01); in contrast, the thickening of the alveolar wall was severer in MCT-injected athymic nude rats than in MCT-injected euthymic rats (p < 0.05). The degree of MCT-PH, as determined by the medial wall thickness of the small pulmonary arteries and RV/(LV + S) weight ratio in MCT-injected athymic nude rats, was significantly severer than in MCT-injected euthymic rats (p < 0.05 and p < 0.01, respectively). The number of mast cells was significantly greater in MCT-injected athymic nude rats than in MCT-injected euthymic rats (p < 0.01). The degree of the medial wall thickness of the small pulmonary arteries was significantly correlated with RV/(LV + S) weight ratio (p < 0.05) as well as with the number of mast cells in MCT-injected rats. Conclusions: Athymic nude rats developed severer PH than euthymic rats along with a greater number of mast cells and severer histopathological changes, such as thickening of the alveolar wall. Mast cell proliferation was considered to play a pivotal role in the development of PH in MCT-PH.
Lipid Transfer Protein: A Pan-Allergen in Plant-Derived Foods That Is Highly Resistant to Pepsin Digestion
Authors: Asero Riccardo , Mistrello Gianni , Roncarolo Daniela , de Vries Sacco C. , Gautier Marie-Françoise , Ciurana Caroline L.F. , Verbeek Eddy , Mohammadi Tamimount , Knul-Brettlova Vlasta , Akkerdaas Jaap H. , Bulder Ingrid , Aalberse Rob C. , van Ree Ronald
Published: April 2003

Background: Lipid transfer proteins (LTPs) are small molecules of approximately 10 kD that demonstrate high stability. They have recently been identified as allergens in the Rosaceae subfamilies of the Prunoideae (peach, apricot, plum) and of the Pomoideae (apple). They belong to a family of structurally highly conserved proteins that are also present in non-Rosaceae vegetable foods. Objective: The aim of this study was to investigate the cross-reactivity to non-Rosaceae LTPs, and to study the role of protein stability in allergenicity. Methods: Thirty-eight patients with a positive SPT to Rosaceae fruit extracts enriched for LTP were characterized by interview and SPT. To investigate IgE cross-reactivity between Rosaceae and non-Rosaceae LTPs, RAST and RAST inhibition as well as ELISA and ELISA inhibition were performed, using whole food extracts and purified LTPs. Both purified natural LTPs (peach, carrot and broccoli) and Pichia pastoris recombinant LTPs (carrot and wheat) were included. Pepsin digestion was used to address the role of stability in the allergenicity of LTPs. Results: IgE antibodies to Rosaceae LTPs reacted to a broad range of vegetable foods, including Gramineae (cereals), Leguminosae (peanut), Juglandaceae (walnut), Anacardiaceae (pistachio), Brassicaceae (broccoli), Umbelliferae (carrot, celery), Solanaceae (tomato), Cucurbitaceae (melon), and Actinidiaceae (kiwi). Binding and inhibition studies with purified natural and recombinant LTPs confirmed their role in this cross-reactivity. Many of these cross-reactivities were accompanied by clinical food allergy, frequently including systemic reactions. Antibody binding to LTP was shown to be resistant to pepsin treatment of whole extract or purified LTP. Conclusion: LTP is a pan-allergen with a degree of cross-reactivity comparable to profilin. Due to its extreme resistance to pepsin digestion, LTP is a potentially severe food allergen.
An Amplified ELISA Inhibition Method for the Measurement of Airborne Soybean Allergens
Authors: Cruz Maria-Jesús , Rodrigo Maria-José , Antó Josep-Maria , Morell Ferran
Published: April 2003

Background: Measurement of the soybean aeroallergen in Barcelona and other cities where soybean is unloaded is of increasing importance in controlling population exposure and evaluating the influence of such exposure on the persistence of asthma symtpoms. Objective: The aims of the study were: (1) to standardize an amplified ELISA inhibition method for the quantification of soybean aeroallergen and (2) to compare this method to a previously described RAST inhibition method. Methods and Results: An amplified competitive ELISA inhibition method with a biotin-streptavidin system was carried out using a pool of sera from soybean-sensitized patients. The results were expressed as U/ml using a low-molecular-mass soybean allergen as reference standard. Reproducibility was calculated by statistically comparing the slope of the regression lines of the standard curve of 4 consecutive assays and by determining the coefficient of variation (CV) of the percent inhibition data for each point of several independent standard curves, each from the same assay (intra-assay) and also from a separate assay (inter-assay). No significant differences in the slopes were obtained by analysis of covariance (ANCOVA) F = 1.04. The CV between assays varied between 4 and 22% (for the assay range used in the reference standard) and was greater than the CV within assays (5–10%). Only values with a CV(%) smaller than 20% were considered acceptable. 78.5% of the samples satisfied this criterion. The RAST inhibition and ELISA inhibition methods were compared by difference plots from the values of 338 air filter eluates. The intraclass correlation coefficient was 0.456 (p < 0.001). After the results of both methods were classified as lower and higher than 165 U/m3, the kappa index was 0.46 (p < 0.001). Conclusions: The data obtained in the present study are comparable to those reported from other similar immunoassays. Moreover, despite the difficulty in comparing air-sampling values from different laboratories, the kappa index may be taken to represent fairly good agreement beyond chance between both methods. All these data demonstrate that the present immunoassay is useful for measuring airborne soybean aeroallergens and can also be applied to evaluate the relationship between exposure and the development of asthma symptoms.
Development of a Novel Enzyme-Linked Immunosorbent Assay for Blood and Urinary Eosinophil-Derived Neurotoxin: A Preliminary Study in Patients with Bronchial Asthma
Authors: Morioka Junichiro , Kurosawa Motohiro , Inamura Hiroaki , Nakagami Rieko , Mizushima Yutaka , Chihara Junichi , Yokoseki Tatsuki , Kitamura Sinobu , Omura Yoshiaki , Shibata Masao
Published: April 2003

Background: Eosinophil-derived neurotoxin (EDN), also called eosinophil protein X (EPX), has been suggested to be a useful marker of eosinophilic inflammation. However, no commercial enzyme-linked immunosorbent assay (ELISA) kit for EDN is available yet. Methods: EDN was purified from pooled urine from healthy male volunteers. Polyclonal and monoclonal anti-EDN antibodies were subsequently raised, and a sandwich ELISA for EDN was established. EDN levels in serum, plasma and urine from asymptomatic patients with bronchial asthma were measured by the ELISA method. Some of the blood samples were also measured by a commercial radioimmunoassay (RIA) kit. Results: The ELISA method detected human EDN with a minimum detection limit of less than 0.62 ng/ml and did not cross-react with other eosinophil granule cationic proteins including eosinophil cationic protein. The intra- and interassay coefficients of variation of the ELISA method ranged from 2.6 to 3.6% and from 6.5 to 9.4%, respectively. Good linearity was observed with serially diluted different samples, and the recoveries of the purified EDN added to serum samples ranged from 85 to 110%. Median EDN concentrations in serum (36.9 vs. 19.1 ng/ml), plasma (23.0 vs. 14.5 ng/ml) and urine (118.2 vs. 72.1 μg/mmol Cr) were significantly raised in asymptomatic asthmatic patients compared with healthy control subjects. EDN levels in serum, plasma and urine from the patients significantly correlated with the number of peripheral blood eosinophils, but not total serum IgE levels. A significant relationship between EDN values measured by the EPX-RIA kit and the EDN-ELISA method was observed. Conclusions: We have developed a novel efficient ELISA method to specifically measure blood and urinary EDN, which may be useful to study the role of eosinophils in allergic diseases including bronchial asthma.
Dexamethasone and Cyclosporin A Affect the Maturation of Monocyte-Derived Dendritic Cells Differently
Authors: Manome Hideaki , Aiba Setsuya , Singh Sanjay , Yoshino Yumiko , Tagami Hachiro
Published: April 2003

In contrast to the confirmed effects of glucocorticoids (GCs) and cyclosporin A (CyA) on T cells, the effects of both agents on antigen-presenting cells (APCs), especially on dendritic cells (DCs), are still poorly understood. In this study, we cultured monocyte-derived DCs (MoDCs) under a variety of stimulations in the presence or absence of these immunosuppressants and compared their effects on the activation of MoDCs by these stimulations. The stimulations used were the following: three bacterial toxins, including lipopolysaccharide (LPS), staphylococcal enterotoxin A (SEA) and streptococcal pyrogenic exotoxin A (SPEA), the combination of IL-1β and TNF-α, and an agonistic anti-CD40 antibody. All of these stimulations increased the expression of CD54, CD83, CD86, and HLA-DR antigen, and the production of TNF-α in MoDCs. When MoDCs were treated with dexamethasone (Dex) during the stimulation, Dex significantly suppressed the augmentation of CD86 expression and TNF-α production induced by all of these stimulations. In contrast, when MoDCs were treated with CyA, it inhibited only the effects induced by the superantigens, SEA and SPEA, but not that induced by LPS, the combination of cytokines, or anti-CD40 antibody. The augmentation of CD54 or HLA-DR antigen expression was not significantly suppressed by either Dex or by CyA. When we used MoDCs pretreated with each of these stimulations + Dex or + CyA as APCs, however, significant suppression of T cell proliferation was observed only in the case of the pretreatment with IL-1β/TNF-α + Dex. The allogeneic T cell stimulation by MoDCs pretreated with the other combinations did not significantly differ from that treated with the stimulation alone. Our present study succeeded in demonstrating a clear difference between Dex and CyA in the activation of MoDCs. These differences may induce a significant difference in their final immunological responses.
Immunological Activity of Recombinant Ole e 1 in Patients with Olea europaea Pollinosis
Authors: Quiralte J. , González E. , Arias de Saavedra J.M. , Villalba M. , Florido J.F. , Sáenz de San Pedro B. , Rodríguez R.
Published: April 2003

Background: Recombinant allergens have potential advantages over conventional allergenic extracts. However, these recombinant allergens should be evaluated for their antigenic activity and compared with their natural counterparts before being used for clinical purposes. Methods: We studied 33 patients with seasonal rhinitis and/or bronchial asthma and a positive skin prick test to Olea europaea pollen extract, 10 atopic patients with no history of pollinosis and a negative skin prick test to O. europaea extract and 10 healthy controls. Skin prick tests and determination by ELISA of specific IgE to natural Ole e 1 (nOle e 1) and recombinant Ole e 1 (rOle e 1) expressed in Pichia pastoris were performed in all patients and controls. Inhibition assays were performed between nOle e 1 and rOle e 1 by ELISA. Results: All patients with O. europaea pollinosis had positive skin test responses to both commercial O. europaea extract and nOle e 1 allergen, and all reacted to rOle e 1 on the skin prick test. The nonatopic and atopic control subjects with negative olive pollen skin test results did not react to rOle e 1 on the skin prick test, even at the highest concentrations, confirming the specificity of this test. We found a weak correlation between the wheal surface area produced by the prick test with nOle e 1 and the wheal surface area produced by rOle e 1 at 10 μg/ml (r = 0.42, p < 0.05). Comparison of specific IgE against both nOle e 1 and rOle e 1 in the patients did not reveal any significant difference. There was a strong correlation between the amount of specific IgE against nOle e 1 and rOle e 1 (r = 0.99, p < 0.01). The two proteins displayed the same extent of binding inhibition to IgE antibodies in ELISA inhibition experiments. Conclusions: These results confirm the immunological activity of rOle e 1 expressed in P. pastoris and indicate that Ole e 1 is one of the major allergens in O. europaea pollinosis as evaluated by skin prick test and serological methods. The correlation between rOle e 1 and nOle e 1 in skin test results and serologic data indicates the potential of recombinant allergens for clinical applications and diagnosis of O. europaea pollen allergy.
Production of Enzymatically and Immunologically Active Der f 1 in Escherichia coli
Authors: Takahashi Kyoko , Takai Toshiro , Yasuhara Takaomi , Yuuki Toshifumi , Ohtake Yasuyuki , Yokota Toyokazu , Okumura Yasushi
Published: April 2003

Der f 1 is a major house dust mite allergen belonging to the cysteine protease family. Because of the great demand for clinical and research use of this allergen, much effort to establish an efficient method of preparing purified Der f 1 has been made. We constructed an isopropyl-β-D-thiogalactopyranoside-inducible expression plasmid to produce the pro-form of Der f 1 in Escherichia coli. The recombinant product was accumulated as insoluble inclusion bodies in cells. The solubilized inclusion bodies were found to be successfully renatured by two-step gel filtration chromatography. About 70 mg of pro Der f 1 were properly refolded by this method from 1 liter of culture. Acid treatment of the renatured pro Der f 1 resulted in the autocatalytic removal of the pro-sequence. The obtained mature form of Der f 1 bound IgE in patient sera and induced the release of histamine from peripheral blood leukocytes equally to native Der f 1. Furthermore, mature Der f 1 obtained by this method had identical protease activity with native Der f 1. We also discussed the contribution of the pro-sequence and the sugar chain of Der f 1 to its antigenic and enzymatic activity. This is the first report to produce an active mature form of recombinant Der f 1 in E. coli.
Binding of Glucocorticoids to Human Nasal Tissue in vitro
Authors: Esmailpour Nasser , Högger Petra , Rohdewald Peter
Published: April 2003

Background: Intranasal application of glucocorticoids is an efficacious treatment of allergic rhinitis and some cases of nonallergic rhinitis. However, no data on binding of glucocorticoids to nasal tissue are available. Pronounced binding of the compound to the target tissue is favorable as it might serve as a local deposit delivering the glucocorticoid to specific receptors and it slows down the efflux of the compound into systemic circulation. Methods: Human nasal tissue was incubated with fluticasone propionate, budesonide, flunisolide and beclomethasone-17-monopropionate. Kinetics of binding and redistribution of the tissue-bound fraction into human plasma was monitored. Results: Binding of glucocorticoids to human nasal tissue was fast and highest for the lipophilic fluticasone propionate, followed by beclomethasone-17-monopropionate. Also, highest concentrations of these lipophilic glucocorticoids remained in nasal tissue after equilibration of drug-saturated tissue with plasma. Conclusions: Lipophilic compounds exhibit a high tissue binding and retention which is an important property of topically applied glucocorticoids. It is the basis for prolonged action and low concentration of the compound in systemic circulation.
Resting B Cells Are Not Antigen-Presenting Cells in the Induction of Oral Tolerance of Specific Th2 Immune Responses in Mice
Authors: Peng Ho-Jen , Chang Zo-Nan , Kuo Shu-Wen , Lee Chin-Cheng , Tzau Yi-Yiu
Published: April 2003

Background: It has been shown that antigen presentation by resting B cells can induce tolerance to intravenously administered protein antigens, but the role of resting B cells in the induction of oral tolerance is unclear. Methods: Mice continuously treated since birth with rabbit anti-mouse IgM serum for 5 weeks were depleted of B cells. When 4 weeks old, B cell-depleted mice drank 10% chicken egg white (EW) for 5 days. Ten weeks later, they were immunized with 10 μg of ovalbumin in alum and their T helper 2 (Th2) immune responses were tested. Results:  Th2 cell-mediated IgE and IgG1 antibody responses and spleen cell production of IL-4 and IL-5 were suppressed by prior EW feeding during anti-IgM treatment. When anti-IgM-treated spleen cells collected 1 week after EW ingestion were transferred to naïve recipients, active suppression of Th2 immune responses was also demonstrated. Conclusions:  Although resting small B cells aggregate in the mantle zone of follicles of intestinal Peyer’s patches, the present data suggest that they are not antigen-presenting cells in the induction of oral tolerance of Th2 immune responses to oral antigens.