bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin‐10 generation in atopic children following oral Lactobacillus rhamnosus GG
Authors: Pessi T. , Sütas Y. , Hurme M. , Isolauri E.
Published: March 2003

Oral Lactobacillus rhamnosus GG ingestion for 5 days to 4 weeks has been shown to alleviate clinical symptoms of gastrointestinal inflammation and atopic dermatitis.

To determine whether oral Lactobacillus rhamnosus GG may act by generating immunosuppressive mediator in atopic children.

Lactobacillus rhamnosus GG (ATCC 53103) at a daily dose of 2 × 1010 cfu was added for 4 weeks to the diets of nine children (mean age, 21 months) with atopic dermatitis. Blood and faecal samples were collected before supplementation and at early (2 weeks) and late stage (4 and 8 weeks from the beginning). The concentrations of interleukin‐6 (IL‐6), IL‐10, IL‐12, tumour necrosis factor‐α (TNFα) and interferon‐γ (IFNγ) in sera, as well as the production of IL‐2, IL‐4, IL‐10 and IFNγ in mitogen‐induced peripheral blood mononuclear cells, were assessed. Secretory IgA and TNFα were also determined in faeces.

The serum IL‐10 concentration differed significantly between before, early and late samples (P < 0.001) due to the elevation of serum IL‐10 in the later phase of oral Lactobacillus rhamnosus GG ingestion. The enhancement of IL‐10 production in mitogen‐induced cultures preceded the rise in serum IL‐10.

The enhanced IL‐10 generation in vivo substantiates the anti‐inflammatory properties of specific probiotic bacteria strains, and provides an additional reason for considering such treatments for patients with intestinal inflammation.
Studies on linkage and association of atopy with the chromosomal region 12q13–24
Authors: Heinzmann A. , Grotherr P. , Jerkic S.‐P. , Lichtenberg A. , Braun S. , Kruse S. , Forster J. , Kuehr J. , Deichmann K. A.
Published: March 2003

Background

Several studies have shown linkage of bronchial asthma, allergic rhinitis and total serum IgE concentration to the chromosomal region 12q13–24 in ethnical diverse populations. This region harbours a number of candidate genes for asthma and atopy, including stem cell factor (SCF), leukotriene A4 hydrolase (LTA4H), thyroid receptor 2 (TR2), and signal transducer and activator of transcription 6 (STAT6). However, the same region was shown as well to be linked to other diseases with inflammatory character. So far no variants in any of these genes have been published which would allow association studies and confirm the pathogenicity of any of these genes.

Objective

We wanted to test for linkage of the chromosomal region 12q13–24 with the atopic phenotype without regard to clinical manifestations. Furthermore we screened for common nucleotide polymorphisms in candidate genes to enable association studies.

Methods

We employed sib‐pair linkage analysis and transmission disequilibrium testing with regard to four highly polymorphic microsatellite markers in 12q13–24 in atopic nuclear families. In addition, we looked for polymorphisms in the genes coding for SCF, LTA4H, TR2 and STAT6 performing SSCP‐analysis and direct genomic sequencing.

Results

We found no evidence for linkage of the genomic region 12q13–24 to elevated total serum IgE levels, specific sensitization to common inhalant allergens or atopy. Furthermore we identified three nucleotide polymorphisms including one common variant in the gene coding for SCF. No association of this polymorphism and any of the atopic phenotypes was seen.

Conclusion

We conclude from our data that genes in the chromosomal region 12q13–24 and in particular SCF are unlikely to exert a major effect on the induction of the atopic phenotype in our Caucasian population. However, we did not focus on the asthmatic and thereby inflammatory aspect of atopy which might explain these results in contradiction to previous studies.
Mast cells and their secretory granules are angiogenic in the chick embryo chorioallantoic membrane
Authors: Ribatti D. , Crivellato E. , Candussio L. , Nico B. , Vacca A. , Roncali L. , Dammacco F.
Published: March 2003

Background Many data suggest that the density of mast cells is highly correlated with the extent of both normal and pathological angiogenesis.

Objective In this study we have compared in an in vivo assay, the chick embryo chorioallantoic membrane, the angiogenic potential of mast cell suspensions isolated from rats, degranulated mast cells and their secretory granules.

Methods Gelatin sponges adsorbed with cell suspensions of rat mast cells, degranulated mast cells and their secretory granules were implanted on the top of the chorioallantoic membrane at day 8 of incubation. At day 12 the angiogenic response was evaluated macroscopically, microscopically and by a morphometric method of ‘point counting’.

Results Isolated mast cells and their secretory granules, but not degranulated mast cells, induced an angiogenic response in the chorioallantoic membrane. The addition of antifibroblast growth factor‐2 or antivascular endothelial growth factor antibodies reduced the angiogenic response of both mast cells and their secretory granules by 50% and 30%, respectively.

Conclusion These data support the evidence that the angiogenic properties of mast cells depend on the angiogenic molecules contained in their secretory granules and indicate that fibroblast growth factor‐2 and vascular endothelial growth factor are the angiogenic cytokines primarily and perhaps synergistically responsible for this vasoproliferative activity.
The development of an antibody to trimellitic anhydride
Authors: Griffin P. , Allan L. , Beckett P. , Elms J. , Curran A. D.
Published: March 2003

Acid anhydrides are a group of highly reactive chemicals used widely in the formulation of paints and plastics. Exposure to acid anhydrides causes several occupational lung diseases such as pneumonititis and asthma. Whilst anhydrides, specifically trimellitic anhydride (TMA), have been shown to bind to lung tissue in an animal model, further investigation has been hampered by the lack of a reagent which would enable the identification of primary target proteins for the binding of TMA.

Our objective was to develop an antibody to TMA which would enable in vitro studies of TMA interactions with lung epithelial proteins.

We developed a monoclonal antibody which binds soley to TMA. We have demonstrated that the antibody can be used to detect TMA bound to different human proteins with little non‐specific binding to unconjugated proteins. We then exposed cells of the A549 lung epithelial cell line to TMA in vitro and have shown by western blotting that binding occurs in the 20‐35 Kd weight range.

We have developed a specific and sensitive reagent to detect TMA bound to proteins. We have used this to show that when TMA is incubated with a lung epithelial cell line, that the TMA binds to proteins with a restricted molecular weight range. These results suggest that the current paradigm for the detection of IgE to small molecular weight reactive chemicals, which presupposes that the chemical binds to serum albumin, may need further investigation.
Rye γ‐70 and γ‐35 secalins and barley γ‐3 hordein cross‐react with ω‐5 gliadin, a major allergen in wheat‐dependent, exercise‐induced anaphylaxis
Authors: Palosuo K. , Alenius H. , Varjonen E. , Kalkkinen N. , Reunala T.
Published: March 2003

Patients with wheat‐dependent, exercise‐induced anaphylaxis experience severe allergic reactions when exercising after ingestion of wheat. The major wheat allergen associated with these reactions is a ω‐5 gliadin, and patients following a gluten‐free diet have remained free of symptoms.

The aim of this study was to examine whether allergens cross‐reacting with wheat ω‐5 gliadin are present in rye, barley and oats.

Sera from 23 adult patients with wheat‐dependent, exercise‐induced anaphylaxis were examined. Cereal allergens cross‐reacting with wheat ω‐5 gliadin were identified by immunoblot inhibition. The cross‐reactive allergens were purified by gel filtration and reversed‐phase chromatography and submitted to amino acid sequencing. Cross‐reactivity was further studied by IgE ELISA and ELISA inhibition, and in vivo reactivity by skin prick testing.

In immunoblotting rabbit anti‐ω‐5 gliadin antibodies bound to 70 kDa and 32 kDa proteins in rye and a 34‐kDa protein in barley, but not to proteins in oats. N‐terminal sequencing identified these proteins as rye γ‐70 secalin, rye γ‐ 35 secalin and barley γ‐3 hordein, correspondingly. In ELISA 21/23 (91%) patients with wheat‐dependent, exercise‐induced anaphylaxis showed IgE antibodies to purified γ‐70 secalin, 19/23 (83%) to γ‐35 secalin and 21/23 (91%) to γ‐3 hordein. In ELISA inhibition ω‐5 gliadin inhibited over 90% of the IgE binding of pooled patient sera to solid‐phase γ‐secalins and γ‐3 hordein. Skin prick testing gave positive reactions to γ‐70 secalin in 10/15 (67%) patients, to γ‐35 secalin in 3/15 (20%) patients and to γ‐3 hordein in 7/15 (47%) patients.

The results of this study show that γ‐70 and γ‐35 secalins in rye and γ‐3 hordein in barley cross‐react with ω‐5 gliadin, a major allergen in wheat‐dependent, exercise‐induced anaphylaxis. These findings suggest that also rye and barley may elicit symptoms in patients with wheat‐dependent, exercise‐induced anaphylaxis.
Distribution of HLA‐A, B alleles and polymorphisms of TAP and LMP genes in Korean patients with atopic dermatitis
Authors: Lee H. J. , Ha S. J. , Han H. , Kim J. W.
Published: March 2003

Background Atopic dermatitis has been seen to result from multifactorial inheritance, with interaction between genetic and environmental factors. The genetic association may differ according to the ethnic backgrounds.

Objective The purpose of this study was to investigate the genetic factors in Korean atopic dermatitis patients by studying the human leucocyte antigen (HLA) class I association and polymorphisms of transporters associated with antigen presentation (TAP) and low‐molecular‐weight polypeptide (LMP) genes.

Methods HLA‐A and B genotyping was performed in 53 atopic dermatitis patients and 184 healthy controls using the standard microlymphocytotoxicity technique. TAP1, TAP2, LMP2, and LMP7 gene polymorphisms were anaylzed using the polymerase chain reaction (PCR)‐single strand conformation polymorphism (SSCP), PCR‐amplification refractory mutation system (ARMS), and PCR‐restriction fragment length polymorphism (RFLP).

Results Allele frequency of HLA‐A24 was significantly increased in patients with atopic dermatitis compared to controls (P < 0.05). HLA‐B alleles showed no differences in distribution between patients and controls. Genotype, phenotype, and allele frequencies of TAP1 gene also revealed no differences in distribution between patients and controls. Analysis of TAP2 gene polymorphisms showed increased frequencies of the TAP2*C allele and TAP2*A/TAP2*C genotype in atopic dermatitis patients compared to controls (P < 0.05). Distribution of LMP2 and LMP7 gene polymorphisms was similar for patients and controls.

Conclusion This study demonstrates an association of atopic dermatitis with HLA‐A24 and TAP2*C alleles in Korean patients. Discrepancy with the previous reports might be related to different patient characteristics and ethnic variations.
cDNA cloning and molecular identification of the major oyster allergen from the Pacific oyster Crassostrea gigas
Authors: Leung P. S. C. , Chu K. H.
Published: March 2003

Background Shellfish is one of the most common food allergens. Despite the recent cloning and molecular identification of the major heat stable crustacean allergens in shrimp, lobster and crab, there have been no similar studies on molluscs to which a significant portion of populations allergic to shellfish are also hypersensitive. Recent biochemical evidence suggests that tropomyosin is also an allergen in molluscs, but data on the molecular cloning, nucleotide sequencing, expression and IgE binding to mollusc tropomyosin are lacking.

Objective This study was undertaken to clone, identify and determine the primary structure of a major IgE‐reactive mollusc allergen in oyster at the DNA and protein level.

Methods We constructed an expression cDNA library from the Pacific oyster Crassostrea gigas. This library was screened for IgE binding clones using sera from 15 subjects with a well‐documented history of type I hypersensitivity reactions to oysters. An IgE reactive clone was selected and sub‐cloned into plasmids for nucleotide sequence determination and expression in E. coli.

Results We identified a 1.3‐kb cDNA designated as Cra g 1.03. Expression of Cra g 1.03 in plasmid vector pGEX produced a 59‐kDa recombinant fusion protein reactive to the IgE antibodies from patients with oyster allergies but not non‐allergic controls. Cra g 1.03 has an open reading frame of 233 amino acids and demonstrates marked similarity in amino acid composition and peptide sequence with mollusc and crustacean tropomyosins. Absorption of oyster allergic sera with Cra g 1.03 totally removed IgE reactivity to oyster extract. Moreover, absorption of allergic sera with recombinant shrimp tropomyosin (Met e 1), lobster tropomyosin (Pan s 1) and crab tropomyosin (Cha f 1) removed most of the IgE reactivity to Cra g 1.03.

Conclusion Cra g 1.03 is the first oyster allergen identified at the molecular level. Nucleotide and amino acid comparison shows that this protein is the oyster tropomyosin.
The involvement of matrix metalloproteinase‐9 in airway inflammation of patients with acute asthma
Authors: Lee Y. C. , Lee H. B. , Rhee Y. K. , Song C. H.
Published: March 2003

Background Bronchial asthma is an inflammatory disease of the airway characterized by airway remodelling, and is due at least in part to an excess of extracellular matrix (ECM) deposition in the airway wall, which leads to subepithelial collagen deposition. Matrix metalloproteinase‐9 (MMP‐9) is the major proteolytic enzyme that induces bronchial remodelling in asthma. MMP‐9 is also important in the migration of inflammatory cells through basement membrane components.

Objectives We evaluated whether airway inflammatory cells correlated with levels of MMP‐9 in acute asthma and we examined the time course of sputum levels of MMP‐9 activity in patients with spontaneous asthma exacerbation.

Methods We performed zymographic analysis and checked levels of MMP‐9 by means of enzyme immunoassay. MMP‐9 levels were also evaluated during a spontaneous attack of asthma.

Results Pro‐MMP‐9 activities and concentrations of MMP‐9 in asthmatic patients significantly exceeded those of control subjects (P < 0.01). The activities of pro‐MMP‐9 were significantly higher in acute asthmatic patients than in stable asthmatic patients (P < 0.01). The elevated MMP‐9 activities significantly decreased after 7 and 28 days of therapy. In acute asthmatic patients, the levels of sputum MMP‐9 significantly correlated with the total macrophage + neutrophil + eosinophil cell numbers.

Conclusion These data suggest that airway inflammation after asthma exacerbation correlates with the overproduction of MMP‐9, which then leads to airway remodelling.
Removal of bronchoalveolar cells augments the late eosinophilic response to segmental allergen challenge
Authors: Hunt L. W. , Gleich G. J. , Kita H. , Weiler D. A. , Schroeder D. R. , Vuk‐Pavlovic Z. , Sur S.
Published: March 2003

Background In patients with quiescent asthma, macrophages are the most prevalent cells recovered by bronchoalveolar lavage (BAL). Through activation via their FcεRII receptors or by acting as antigen‐presenting cells, macrophages could, in theory, promote the late airway response to allergen.

Objective

In order to investigate the importance of macrophages and other airway luminal cells in inducing the late airway response, a novel washout experiment was designed.

Methods Five patients with ragweed‐allergic asthma underwent bronchoscopy and segmental bronchial challenge with either normal saline or short ragweed extract in two segments of one lung. In a third segment of the opposite lung, 12 successive BALs (25 mL each) were performed, followed by challenge with an identical dose of short ragweed (washed‐challenged segment). After 24 h, all three challenged segments underwent BAL.

Results Initially, in the washed‐challenged segment, over 80% (mean 80.4%, range 68–88%) of the recoverable airway dwelling cells were removed. Unexpectedly, 24 h later these same washed‐challenged segments contained more eosinophils in the BAL than the challenged segments from the opposite lung (P = 0.033).

Conclusions Removing the majority of airway luminal cells followed by allergen bronchoprovocation increased the number of eosinophils recovered 24 h after challenge. Our results suggest that in quiescent allergic asthma, the airway luminal cells are protective and attenuate the late eosinophilic response to allergen challenge.
Stressful life events promote the manifestation of asthma and atopic diseases
Authors: Kilpeläinen M. , Koskenvuo M. , Helenius H. , Terho E. O.
Published: March 2003

Background Psychosocial stress is known to aggravate asthma. Less is known about the impact of stressful life events on the expression of asthma and atopic disorders.

Objective To determine whether the onset of asthma, allergic rhinitis or conjunctivitis, and atopic dermatitis, are associated with stressful life events.

Methods A postal survey on risk factors for asthma and atopic diseases was carried out among 10 667 Finnish first‐year university students aged 18–25 years. Stressful life events, (i) severe disease or death of a family member, and (ii) parental or personal conflicts, were retrospectively recorded during the preceding year, 1–5 years, 6–10 years, and more than 10 years prior to the survey response. In a case‐control setting, conditional multiple logistic regression analysis was used to assess the temporal association between major stressful life events occurring during a period either preceding, concomitant or subsequent with subject's diagnoses.

Results Concomitant parental and personal conflicts increased the risk of asthma (OR 1.72, 95% CI 1.10–2.69) when adjusted by parental asthma, education and passive smoking at early age. Concomitant severe disease or death of mother, father or spouse (OR 1.52, 95% CI 1.09–2.16) and precedent parental and personal conflicts (OR 1.75, 95% CI 1.15–2.77) increased the risk of manifestation of allergic rhinoconjunctivitis when adjusted for parental atopic disease, education and passive smoking. Subjects' asthma and atopic dermatitis, but not allergic rhinoconjunctivitis, were related to excess of subsequent stressful life events.

Conclusion An association between stressful life events and subjects' asthma, allergic rhinoconjunctivitis and atopic dermatitis is suggested.
Influence of respiratory syncytial virus infection on cytokine and inflammatory responses in allergic mice
Authors: Barends M. , Boelen A. , De Rond L. , Kwakkel J. , Bestebroer T. , Dormans J. , Neijens H. , Kimman T.
Published: March 2003

Background Th2 lymphocyte responses are associated with inflammation and disease during allergic responses. Exposure to particular environmental factors during the expression of allergy could result in more pronounced Th2‐like immune responses and more severe disease. One factor might be a respiratory virus infection.

Objective The aim of our study was to investigate the influence of respiratory syncytial virus (RSV) infection on the expression of ovalbumin (OVA)‐induced allergy in BALB/c mice.

Methods We determined OVA‐specific IgE in serum, cytokine profiles and histopathological lesions in lungs of OVA‐allergic mice after RSV infection.

Results OVA sensitization and challenge induced OVA‐specific IgE in serum, Th2 cytokine mRNA expression, and mononuclear and eosinophilic inflammation in the lungs. RSV inoculation during the challenge period enhanced OVA‐induced IL‐4 and IL‐5 mRNA expression in lung tissue. RSV further enhanced the OVA‐induced hypertrophy of mucous cells and eosinophilic infiltration in lung tissue. Surprisingly, RSV infection decreased Th2 cytokine secretion and eosinophilic influx in bronchoalveolar lavage of OVA‐allergic mice. Because inactivated RSV did not influence these responses, replication of RSV appeared essential for the modification of OVA‐induced Th2 cytokine expression. RSV did not change OVA‐specific IgE levels in serum. Furthermore, the RSV‐induced IL‐12 mRNA expression in lung tissue of OVA‐allergic mice was diminished, but IFN‐γ mRNA expression was not affected.

Conclusion RSV infection enhanced particular OVA‐induced Th2 cytokine mRNA responses and pulmonary lesions in allergic mice and thus aggravated allergic respiratory disease.
Expression of Th1, Th2 and immunosuppressive cytokine gene transcripts in canine atopic dermatitis
Authors: Nuttall T. J. , Knight P. A. , McAleese S. M. , Lamb J. R. , Hill P. B.
Published: March 2003

Background Atopic dermatitis is a common inflammatory skin disease of humans and dogs. Human atopic dermatitis is associated with Th2‐type responses, although Th1 cytokines can be identified in chronic lesions. In contrast, tolerance to environmental allergens in healthy individuals is mediated by regulatory T cells.

Objective This study examined the expression of the immunosuppressive cytokines TGF‐β and IL‐10, the Th2‐type cytokines IL‐4 and IL‐6, and the Th1‐type cytokines IFN‐γ, TNF‐α, IL‐2, IL‐12p35 and IL‐12p40, in canine atopic dermatitis.

Materials and methods RNA was isolated from lesional atopic, non‐lesional atopic and healthy canine skin samples. Semi‐quantitative reverse transcriptase polymerase chain reactions (RT‐PCRs) were carried out using specific primers and one‐way analyses of variance used to compare cytokine expression in each group.

Results Canine atopic dermatitis was associated with over‐expression of IL‐4 mRNA and reduced transcription of TGF‐β compared with healthy skin (P < 0.05). Higher levels of IFN‐γ, TNF‐α and IL‐2 mRNA were seen in lesional compared with non‐lesional and healthy skin (P < 0.05). There were no significant differences in IL‐10, IL‐6, IL‐12p35 or IL‐12p40 transcription between the three groups.

Conclusions This is the first report to demonstrate that canine atopic dermatitis is associated with over‐production of IL‐4. Clinical tolerance in healthy individuals appears to be associated with TGF‐β, although it is unclear if this reflects an active mechanism or simply non‐responsiveness of the immune system. Th1 cytokines may be induced by subsequent self‐trauma and secondary infections in atopic skin. We believe that these results better characterize spontaneously occurring canine atopic dermatitis. We further propose that this should be investigated as a possible animal model of human atopic dermatitis.
Mouldy houses influence symptoms of asthma among atopic individuals
Authors: Dharmage S. , Bailey M. , Raven J. , Abeyawickrama K. , Cao D. , Guest D. , Rolland J. , Forbes A. , Thien F. , Abramson M. , Walters E. H.
Published: March 2003

Background The influence of current levels of indoor fungi on asthma is a controversial issue that needs to be resolved in order to advise patients appropriately.

Objective To assess the seasonal variation in indoor fungal levels and the impact of these levels on asthma among mould‐sensitized individuals.

Methods Thirty‐five young adults with current asthma and sensitization to fungi were visited four times over 1 year. At each home visit a questionnaire was administered and samples of dust and air were collected. Participants also recorded information on symptoms, peak expiratory flows (PEF) and medication use. Dust samples were analysed for house dust mite allergen (Der p 1) and total fungal biomass (ergosterol). Total and genus‐specific fungal propagules were identified in air samples. Seasonal variation in allergen levels and significant independent effects of fungal levels on peak flow variability (PFV) were identified by repeated measures analysis of variance.

Results Significant seasonal variations were observed in viable airborne fungi, ergosterol levels in the floor dust and PFV. PFV correlated significantly with symptom scores and the dose of reliever medication. PFV was also significantly associated with smoking and visible mould. The association between visible mould and PFV was independent of season, smoking and the dose of reliever medication. However, there was no association between total fungi, specific fungi or ergosterol and PFV. Der p 1 levels had no significant influence on asthma, even in HDM‐sensitized individuals.

Conclusions Mouldy homes adversely influence asthma in asthmatics sensitized to fungi.
Prevalence of wheeze, bronchial hyper‐responsiveness and asthma in the elderly Chinese
Authors: Choy D. K. L. , Hui D. S. C. , Li S. T. , Ko F. W. S. , Ho S. , Woo J. , Lai C. K. W.
Published: March 2003

Background Asthma is a common health problem affecting patients of all ages. Because of the ease of sampling, epidemiological studies have concentrated mainly on the paediatric and general population.

Objective This study aimed to determine the prevalence of wheeze, bronchial hyper‐responsiveness and asthma amongst our elderly population and deduce any clinical and laboratory risk factors that might identify elderly asthmatics at an earlier stage.

Methods Two thousand and thirty‐two elderly Chinese aged ≥ 70 years, randomly selected from a registered list of all recipients of Old Age and Disability Allowances in Hong Kong, were administered a questionnaire on lung health. Two hundred and fifty subjects were invited to attend our laboratory for skin tests and pulmonary function tests and 179 agreed. Of these, 173 (96.6%) and 176 (98.3%) had eosinophil count and serum IgE levels measured, respectively. Two definitions of asthma were used: (1) bronchial hyper‐responsiveness (BHR) plus current wheeze, and (2) history of wheezing without previous diagnostic labels of emphysema or chronic bronchitis.

Results Fifteen patients (out of 179: 8.4%) reported wheezing over the past 1 year. Fifty‐one patients (28.5%) demonstrated BHR on spirometry or histamine challenge tests. Seven patients had both symptoms of wheezing and evidence of BHR. The prevalence of asthma using this definition is therefore 3.9% (95% CI 1.6–7.9%). Nine patients had symptoms of wheezing without previous diagnostic labels of chronic bronchitis or emphysema and, using this definition, the prevalence is 5.0% (95% CI 2.3–9.3%). Using multiple logistic regression studies, sex, social class, age, smoking habits, serum IgE levels and eosinophil counts did not predict a diagnosis of asthma using either definition. We found no association between a positive skin test and any respiratory symptoms or illnesses including asthma.

Conclusion Wheeze, bronchial hyper‐responsiveness and asthma are prevalent amongst our elderly population. However, there were no identifiable demographic and laboratory risk factors in this study that may help us predict a diagnosis of asthma.
Role of interleukin‐13 in eosinophil accumulation and airway remodelling in a mouse model of chronic asthma
Authors: Kumar R. K. , Herbert C. , Yang M. , Koskinen A. M. L. , McKenzie A. N. J. , Foster P. S.
Published: March 2003

Background Interleukin‐13 is believed to be important in asthmatic inflammation and airway hyper‐reactivity.

Objective To investigate the role of IL‐13 in chronic asthma, using an improved experimental model of asthma that reproduces most of the morphological features of the human disease.

Methods BALB/c mice or gene‐targeted mice deficient in their ability to produce IL‐13 or the IL‐4 receptor α‐chain (IL‐4Rα) were sensitized to ovalbumin and exposed to aerosolized antigen for 30 min/day on 3 days/week for 6 weeks. Intraepithelial eosinophils, accumulation of chronic inflammatory cells in the airway wall, subepithelial fibrosis, epithelial hypertrophy and numbers of mucous cells were quantified histomorphometrically. Airway hyper‐reactivity (AHR) to a cholinergic agonist was assessed by barometric plethysmography.

Results Compared with wild‐type animals, IL‐13 ‐/‐ mice exhibited diminished accumulation of eosinophils and chronic inflammatory cells, as well as reduced subepithelial fibrosis, epithelial hypertrophy and mucous cell hyperplasia (P < 0.01 for all comparisons). In contrast, AHR was still demonstrable in IL ‐13 ‐/‐ mice. In IL‐4Rα ‐/‐ mice the inflammatory response, subepithelial fibrosis and AHR were similar to wild‐type mice, although the receptor‐deficient mice had significantly less epithelial hypertrophy and mucous cell hyperplasia.

Conclusion These results imply a critical role for IL‐13 in accumulation of intraepithelial eosinophils in chronic asthma, as well as in epithelial and subepithelial remodelling. In addition, they suggest that in chronic asthma, IL‐13 may be capable of signalling via a pathway that does not involve IL‐4Rα.
Studies on the carbohydrate moiety of Pla l 1 allergen. Identification of a major N‐glycan and significance for the immunoglobulin E‐binding activity
Authors: Calabozo B. , Barber D. , Polo F.
Published: March 2003

Background Pla l 1, the major allergen of Plantago lanceolata pollen, is a glycoprotein that contains an N‐glycosylation site. Carbohydrate moieties of many allergenic glycoproteins have been reported to be IgE‐binding determinants responsible for cross‐reactivity among different species.

Objective To identify the kind of linkages and the type of glycans present in Pla l 1 and to investigate their contribution to the allergic response to this allergen.

Methods Pla l 1 was deglycosylated by N‐glycosidase A and the IgE‐binding ability of the unglycosylated protein was evaluated by dot‐blot. Identification of β1 → 2 xylose and/or α1 → 3 fucose residues in Pla l 1 N‐glycan was carried out by incubation with specific antibodies from rabbit antiserum against HRP (anti‐HRP). The contribution of this N‐glycan to total IgE reactivity was analysed quantitatively by pre‐incubation of Pla l 1 with anti‐HRP prior to incubation with sera. The role of the carbohydrate moiety of Pla l 1 in cross‐reactivity was studied by RAST using unrelated glycoproteins with known sugar composition and structure.

Results The effectiveness of N‐glycosidase A to deglycosylate Pla l 1 and the ineffectiveness of the treatment with PNGase F indicate that Pla l 1 carries a complex N‐glycan with an α1 → 3 fucose residue in its structure. Furthermore, the presence of β1 → 2 xylose and/or α1 → 3 fucose residues was identified in this N‐glycan by means of an ELISA. Pre‐incubation of Pla l 1 with an anti‐HRP antibody caused a weak but significant reduction in IgE reactivity. Some sera from P. lanceolata‐allergic patients reacted positively with four glycoproteins that bear N‐glycans of complex type but not with fetuine.

Conclusions Pla l 1 is a glycoprotein that carries at least a complex, major N‐linked glycan, with a α1 → 3 fucose residue in its structure and probably also a β1 → 2 xylose. This glycan moiety does not seem to constitute a relevant allergenic epitope of Pla l 1.
Uncoupled regulation of leukotriene C4 synthase in platelets from aspirin‐intolerant asthmatics and healthy volunteers after aspirin treatment
Authors: Tornhamre S. , Ehnhage A. , Kölbeck K. G. , Edenius C. , Lindgren J. Å.
Published: March 2003

Background We have reported that thromboxane A2 induces suppression of leukotriene (LT) C4 synthase activity in human platelets.

Aim In the present study, we describe a mechanism whereby aspirin treatment can lead to increased formation of LTC4, which is a potent bronchoconstrictor and inflammatory mediator. This mechanism is also demonstrated to be present in platelets from aspirin‐intolerant asthmatics (AIA).

Methods The effect of arachidonic acid or platelet agonists on LTC4 synthase activity was investigated in platelets obtained from healthy volunteers, aspirin‐intolerant asthmatics or aspirin‐tolerant asthmatics after in vivo treatment or in vitro pre‐incubation with aspirin.

Results Incubation of normal platelets with arachidonic acid or collagen provoked approximately 50% reduction of platelet LTC4 synthase activity, as determined by the conversion of LTA4 to LTC4. However, the inhibitory effect of arachidonic acid or collagen was not observed after oral administration of aspirin prior to collection of the platelets. Arachidonic acid‐induced inhibition of LTC4 synthase activity was totally abolished in platelets collected from peripheral blood already 30 min after aspirin ingestion but was fully restored in platelets collected 3 to 7 days after the administration of aspirin. Treatment of platelet suspensions with aspirin in vitro dose‐dependently counteracted the suppressive effect of arachidonic acid on LTC4 formation, with total reversal at approximately 40 µm. In contrast, the major aspirin metabolite, salicylic acid did not alter arachidonic acid‐induced reduction of LTC4 synthase activity. Similarly, LTC4 synthase activity in platelets from AIA and aspirin‐tolerant asthmatics (ATA) was reduced by approximately 50% after pre‐treatment with arachidonic acid in vitro. Again the inhibitory effect was abolished when platelets were pre‐incubated in the presence of aspirin.

Conclusion The results indicate that oral aspirin administration can lead to uncoupling of thromboxane A2‐dependent negative feedback mechanisms, which may normally restrict the production of cysteinyl leukotrienes. This mechanism can be of potential interest in aspirin‐induced asthma.
Zafirlukast has no beneficial effects in the treatment of chronic urticaria
Authors: Reimers A. , Pichler C. , Helbling A. , J. Pichler W. , Yawalkar N.
Published: March 2003

Background Leukotriene receptor antagonists have shown some efficacy in the treatment of asthma. Injection of LTC4, LTD4 and LTE4 into the skin leads to a weal‐and‐flare reaction, suggesting an involvement of leukotrienes in the pathogenesis of urticaria. Indeed, various reports have indicated a beneficial effect for leukotriene receptor antagonists in patients with chronic urticaria.

Objective To determine the therapeutic effect of the leukotriene receptor antagonist zafirlukast in patients with chronic urticaria.

Methods The study was a double‐blind, placebo‐controlled, cross‐over study lasting for 12 weeks. Fifty‐two patients with chronic urticaria were investigated at a university hospital. The patients were randomized to receive 20 mg zafirlukast b.i.d. or placebo and cross‐over was scheduled after 6 weeks. The efficacy of the treatment was evaluated by a daily symptom score, six physical examinations, the requirement of rescue antihistamines (acrivastine) and an overall assessment by the patient and the investigating physician.

Results Forty‐six patients completed the study: zafirlukast was well tolerated without alteration of the investigated laboratory parameters. In comparison with placebo, treatment with zafirlukast resulted in no significant positive effect for any of the efficacy measures. Moreover, we were unable to identify any subgroup of patients with chronic urticaria responding with a therapeutic benefit.

Conclusions The leukotriene receptor antagonist zafirlukast does not provide a significant therapeutic benefit at a dose of 20 mg b.i.d. in patients with chronic urticaria.
Tumour necrosis factor‐α‐induced expression of intercellular adhesion molecule‐1 on human eosinophilic leukaemia EoL‐1 cells is mediated by the activation of nuclear factor‐κB pathway
Authors: Ip W. K. , Wong C. K. , Lam C. W. K.
Published: March 2003

Background Intercellular adhesion molecule‐1 (ICAM‐1) has been shown to mediate the adhesion and migration of eosinophils to the site of allergic inflammation. However, molecular mechanisms regulating the expression of ICAM‐1 in eosinophils are still being elucidated. We investigated the effect of tumour necrosis factor‐alpha (TNF‐α) on ICAM‐1 expression of eosinophils.

Methods The surface expression of ICAM‐1 on a human eosinophilic leukaemic cell line, EoL‐1, was assessed by immunocytochemical staining. The phosphorylation of inhibitor kappa B‐alpha (IκB‐α) and p38 mitogen‐activated protein kinase (MAPK) was detected by Western blot. Nuclear factor kappa‐B (NF‐κB) pathway‐related genes were evaluated by the cDNA expression array system, whereas the activity of NF‐κB was measured by electrophoretic mobility shift assay (EMSA).

Results TNF‐α was found to induce the cell surface expression of ICAM‐1. A specific proteasome inhibitor N‐cbz‐Leu‐Leu‐leucinal (MG‐132), but not a p38 MAPK inhibitor (SB 203580), was found to suppress the TNF‐α‐induced expression of ICAM‐1 on EoL‐1 cells. The gene expressions of ICAM‐1, NF‐κB and IκBα were up‐regulated after the stimulation with TNF‐α. Further, TNF‐α was shown to induce IκB‐α phosphorylation and degradation, thereby indicating the activation of NF‐κB. In EMSA, there was a shifted NF‐κB band on TNF‐α‐treated cells with or without SB 203580, but no shifted band was observed on MG‐132‐treated cells.

Conclusion In vitro studies of EoL‐1 cells, an eosinophilic leukaemic cell line, confirmed that NF‐κB plays an important role in the expression of ICAM‐1 and recruitment of eosinophils in allergic inflammation.
Indirect demonstration of the lifetime function of human thymus
Authors: Marušić M , Turkalj-Kljajić M , Petrovečki M , Užarević B , Rudolf M , Batinić D , Ugljen R , Anić D , Ćavar Z , Jelić I , Malenica B
Published: March 2003

The aim of this study was to test the hypothesis that human thymus maintains its function as the site of early T cell development throughout life, but to a progressively diminishing extent. Mononuclear cell suspensions prepared from the samples of 39 human thymuses were analysed for the total number of cells per gram of thymus tissue, percentage of single marker-positive CD2, CD4 and CD8 cells, percentages of double-positive CD4 CD8 and CD2 CD8 cells, double-negative CD4 CD8 cells, absolute numbers of these cells per gram of tissue, and extent of the in vitro proliferation upon stimulation with concanavalin A (Con A), phytohaemagglutinin (PHA) and pokeweed mitogen (PWM) mitogens. The main outcome measures were flow cytometric data on thymus lymphoid cell composition (according to CD classification), expressed as percentages and numbers of cells per gram of thymus tissue. The total number of mononuclear cells expressed per gram of thymus tissue exponentially decreased with age. The slope of none of the analysed cell subpopulations differed from the slope of the line constructed for age-related decline of the total number of mononuclear cells (−0.024 on a semilogarithmic scale). The thymuses of all ages contained all analysed cell subpopulations in approximately the same proportions: percentages of these cell subpopulations did not change with age, except for all CD4+ (P = 0.017) and double-positive CD4+ CD8+ (P = 0.016) cells, which tended to decrease with age. The extent of proliferation of thymus cells upon stimulation with T and B cell mitogens was unrelated to age. We conclude that the thymus retains its function as the site of differentiation of T lymphocytes throughout life. With respect to the number of involved lymphoid cells, the function exponentially decreases with age.
β2-Glycoprotein I-dependent and -independent anticardiolipin antibody in non-obese diabetic (NOD) mice
Authors: Anzai K , Nagafuchi S , Niho Y , Kikuchi M , Ono J
Published: March 2003

In the present study we investigate whether or not anticardiolipin antibody (aCL) is produced in NOD mice, which is a representative animal model of insulin-dependent diabetes mellitus (IDDM). We found that IgG class aCL appeared in 6.9% of non-diabetic NOD mice at weeks 5–15. The rates increased with age to 31.6% at weeks 16–25 and 71.9% at weeks 26–35. In addition, the titre and incidence of aCL were higher in diabetic mice than in non-diabetic mice. It was also found that aCL in NOD mice involved β2-glycoprotein I (β2-GPI)-dependent and -independent aCL, when β2-GPI was added to the aCL assay system. The IgG subclass of both β2-GPI-dependent and -independent aCL belonged exclusively to IgG2a. In addition, immunohistochemical studies revealed the predominant accumulation of IgG2a- or IgG3-positive B lymphocytes within insulitis. These results suggest that the autoimmunity in NOD mice may thus be associated with Th1 predominant immunological response. In conclusion, aCL with multiple antigenic specificity were produced in NOD mice along with the development of insulitis and diabetes. NOD mice should thus be added to the list of animal models possessing antiphospholipid antibody.
Effects of adjuvants on the immune response to allergens in a murine model of allergen inhalation: cholera toxin induces a Th1-like response to Bet v 1, the major birch pollen allergen
Authors: Wiedermann U , Jahn-Schmid B , Fritsch R , Bauer L , Renz H , Kraft D , Ebner C
Published: March 2003

Based on the fact that type I allergies are frequently elicited by inhalant allergens, we have established a model of aerosol inhalation leading to allergic sensitization in BALB/c mice. Using this model we studied the effects of aluminium hydroxide (Al(OH)3), known to enhance IgE antibody responses, compared with cholera toxin (CT), a potent mucosal adjuvant, on the immune response to birch pollen (BP) and its major allergen Bet v 1. Two groups of BALB/c mice were either systemically immunized with recombinant Bet v 1 in Al(OH)3 and subsequently aerosol exposed to BP allergen, or aerosolized with BP and CT. IgE-mediated skin reactions were only elicited in the mice which had received Bet v1/Al(OH)3. Allergen-specific serum IgE and IgG1 antibodies dominated in the Al(OH)3 group, IgG2a antibody levels to BP and rBet v 1 were markedly higher in the sera of mice exposed to CT with the allergen. IgA antibodies were only detected in the bronchial lavage of the CT-treated group. Moreover, the latter group displayed consistently higher T cell proliferative responses to BP and interferon-gamma production in vitro. Thus, the systemic immunization with rBet v 1 in Al(OH)3 before inhalation of the BP extract promoted a Th2-like immune response, while CT mixed with the aerosolized BP extract rather induced a Th1-like immune response. In an attempt to reverse these ongoing immune responses we could achieve a shift towards a Th0 response. Immunization with BP extract without adjuvant treatment led to undetectable antibody or cellular immune responses. We conclude from the present study that the induction of an immune response to BP allergen after aerosol inhalation can be directed towards a Th1- or a Th2-like response. Once established, the immune response can be modulated.
Spontaneous immunoglobulin-producing capacity of cultures from lupus patients and normal donors following depletion of cells expressing CD19 or CD38
Authors: Bourne T , Zukowska-Cooper M , Salaman M R , Seifert M H , Isenberg D A
Published: March 2003

Cells spontaneously secreting IgG or IgM (ISC) are present at a high level in the blood of patients with systemic lupus erythematosus (SLE). By use of magnetic-bead techniques, mononuclear cells from such patients and healthy donors were fractionated according to expression of CD19 or CD38 and the cell fractions were then cultured in the absence of added mitogen/antigen for 5/6 days. Supernatant IgG and IgM were determined and, in addition, in the CD38 experiments ISC were enumerated both before and after culture. Much of the immunoglobulin-producing capacity of unfractionated cells (UFC) from both donor groups was recovered in the CD19− fraction, and no immunoglobulin was produced by CD19+ cells suggesting, unexpectedly, that ISC were not expressing CD19. By contrast, CD38 fractionation resulted in nearly all ISC passing to the CD38+ fraction which produced levels of immunoglobulin approaching 50% that of UFC. On culture of CD38− cells there was a build up in the number of IgG and IgM ISC, this being particularly striking in the controls with numbers well in excess of those in UFC. Not all these new ISC became CD38+, but the maturation process was more efficient in the SLE patients. The possibility is discussed that the spontaneous response in the CD38− populations is due to removal of CD38+ natural killer (NK) cells. Removal of ISC that are present preculture is a helpful initial step in studying ISC generation in the disease.
1,25-Dihydroxyvitamin D3 restores sensitivity to cyclophosphamide-induced apoptosis in non-obese diabetic (NOD) mice and protects against diabetes
Authors: Casteels K , Waer M , Bouillon R , Depovere J , Valckx D , Laureys J , Mathieu C
Published: March 2003

The activated form of vitamin D, 1,25(OH)2D3, and its analogues can prevent type I diabetes in NOD mice. Protection is achieved without signs of systemic immunosuppression and is associated with a restoration of the defective immune regulator system of the NOD mice. The aim of the present study was to investigate whether this restoration of regulator cell function is the only mechanism in the prevention of diabetes by 1,25(OH)2D3. We tested therefore if 1,25(OH)2D3 could prevent cyclophosphamide-induced diabetes, since diabetes occurring after cyclophosphamide injection is believed to be due to an elimination of suppresser cells. NOD mice treated with 1,25(OH)2D3 (5 μg/kg every 2 days) from the time of weaning were clearly protected against diabetes induced by cyclophosphamide (200 mg/kg body wt at 70 days old) (2/12 (17%) versus 36/53 (68%) in control mice, P < 0.005). By co-transfer experiments it was demonstrated that cyclophosphamide had indeed eliminated the suppresser cells present in 1,25(OH)2D3-treated mice. Since cyclophosphamide injection did not break the protection offered by 1,25(OH)2D3, it was clear that diabetogenic effector cells were affected by 1,25(OH)2D3 treatment as well. This was confirmed by the finding that splenocytes from 1,25(OH)2D3-treated mice were less capable of transferring diabetes in young, irradiated NOD mice, and by the demonstration of lower Th1 cytokine levels in the pancreases of 1,25(OH)2D3-treated, cyclophosphamide-injected mice. This better elimination of effector cells in 1,25(OH)2D3-treated mice could be explained by a restoration of the sensitivity to cyclophosphamide-induced apoptosis in both thymocytes and splenocytes, in normally apoptosis-resistant NOD mice. Altogether, these data indicate that the protection against diabetes offered by 1,25(OH)2D3 may be independent of the presence of suppresser cells, and may involve increased apoptosis of Th1 autoimmune effector cells.
Cloning and characterization of a latex allergen (Hev b7): homology to patatin, a plant PLA2
Authors: Kostyal D A , Hickey V L , Noti J D , Sussman G L , Beezhold D H
Published: March 2003

We previously identified a 46-kD protein allergen in latex as having amino acid sequence homology to the patatin gene family. The objective of this study was to characterize this protein by molecular techniques. RNA was isolated from the latex or leaf material from Hevea brasiliensis and from potato tubers. Specific polymerase chain reaction (PCR) primers were designed from the amino acid sequence and reverse transcriptase (RT)-PCR amplified a specific product from latex RNA that was subsequently cloned and sequenced. This product was 1493 bp in length with an 1167 bp open reading frame. The deduced amino acid sequence encodes for a 389 aa protein, pI 4.82 with 43% homology to tobacco patatin. Northern analysis of potato, Hevea leaf, and latex RNA demonstrated the message to be most abundant in latex, weakly present in Hevea leaf, but no hybridization occurred with potato RNA. Patatin has lipid acyl-transferase and PLA2-like activity, suggesting it plays a role as a defence-related protein. Other defence-related proteins in latex such as hevein, glucanase, and hevamine are also allergens. Increased production of defence-related proteins as a result of increased tapping of the rubber trees to meet the demand for latex may explain the increased allergenicity of latex.