<p>In this thesis the oxidative metabolism by blood eosinophils from birch pollen allergic subjects was studied and compared to that by eosinophils from healthy controls, during and out of the pollen season. The effects and mechanisms of <i>in vitro</i> IL-5 priming on blood eosinophils were investigated and compared to the effects of <i>in vivo</i> priming during pollen exposure.</p><p>The main findings of this work were that the oxidative metabolism by blood eosinophils taken from pollen allergic subjects is reduced during the pollen season. The eosinophils taken from asymptomatic allergics have a reduced capacity to produce oxygen free radicals as compared to non-allergic controls. The oxidative metabolism by blood eosinophils from allergic subjects is primed <i>in vivo</i> during the pollen season, as compared to the healthy controls and as compared to out of season. IL-5 primed the oxidative metabolism by eosinophils from allergic subjects in a similar way as eosinophils from healthy controls, both during and out of pollen exposure. The total and tyrosine phosphorylation patterns obtained were identical in eosinophils from allergic subjects and non-allergic controls during the pollen season. Spontaneous phosphorylation was the same in both groups and different from that after IL-5 priming. The oxidative metabolism of blood eosinophils is composed of different stages. The initial stage, measured as the t_½rises of the CL curves, is an indication of the state of priming of the cell, while the end stage, measured as the peaks of the CL curves, is an estimate of the total radical production by the cells. IL-5 priming affected these two stages differently and the two stages are regulated by different signal transduction pathways and IL-5 priming causes a by-passing of MEK.</p><p>In conclusion, in this thesis it is shown that blood eosinophils from allergic subjects are primed <i>in vivo</i> during exposure to their allergen. This <i>in vivo</i> priming leads on one hand to a reduced oxidative metabolism during the pollen season, but also to a faster onset of radical production as a response to certain stimuli. Our data do not provide any evidence of IL-5 involvement in the <i>in vivo</i> priming of blood eosinophils from allergic patients during pollen exposure.</p>

<p>The accumulation of eosinophil granulocytes (EOS) at sites of inflammation is a common feature of astma, allergic rhinitis and inflammatory bowel disease. The aim of the present investigation was to study the mechanisms involved in this accumulation.</p><p>Bronchoalveolar lavage (BAL) fluid obtained from patients with birch-pollen allergy lavaged during season exhibited increased eosinophil chemotactic activity compared with pre-season BAL fluid from the same patients. We identified IL-5, IL-8 and RANTES as the main eosinophil chemotactic agents in the BAL fluid. Only EOS from allergic donors responded to IL-8. IL-2 inhibited albumin-stimulated eosinophil migration towards buffer or chemoattractants. EOS from allergic subjects were less sensitive to this inhibition than EOS from normal subjects, and in vitro priming of the EOS with IL-5 prevented the inhibitory effect of IL-2. We therefore hypothesise that IL-2 acts as an autocrine regulator of EOS migration, and that this inhibitory effect may be down-regulated in allergy, resulting in increased migration of EOS towards chemotactic factors. The stimulation of eosinophil migration by albumin is mediated by PI3 kinase. Decreased expression of CD49d and CD49f caused by albumin may decrease the adhesiveness of the EOS, which in turn may facilitate migration. We found a higher chemotactic activity in perfusion fluids from patients with ulcerative colitis than from control patients. The chemotactic activity correlated with the concentrations of eosinophil granule proteins in the perfusion fluids. IL-5 and TNF-α were identified as two of the chemotactic agents in the perfusion fluid that were inhibited by steroid treatment. Agents with steroid-insensitive chemotactic activity remain to be identified.</p>

<p>Otitis media with effusion (OME) is the major form of chronic relapsing inflammatory disease of the middle ear, constitutes the most common diagnosis for children under 15 years old and is the major cause of auditory dysfunction in pre-school children. OME is a disease more commonly found in allergic children. These studies sought to investigate the inflammatory response in the middle ear of patients and test the hypothesis that an allergic-like response might occur in the ear. Atopy was diagnosed by standard in vitro tests. Immunochemical techniques used to study classic allergic rhinitis and asthma were extrapolated to the evaluation of OME children whose effusion persisted beyond 2 months. Not only eosinophil cationic protein (ECP), tryptase, CD3-positive and IL-5 producing cells, but also myeloperoxidase (MPO) was found in middle ear fluid and/or mucosa in the majority of patients with OME and atopy. </p><p>Initially, levels of ECP, MPO, and tryptase were measured in effusions from 97 random OME patients whose atopic status was determined by in vitro testing to 12 inhalants and 5 foods. The response of eosinophils, neutrophils and mast cells in the middle ear was distinctly different between atopic and non-atopic patients (p<0.001) with higher levels of the cell markers in the atopic group of patients. This suggested that 1) perhaps OME was predominantly a disease of atopics and that 2) they differed in their response from non-atopics.</p><p>Tryptase was measured in middle ear effusions from 38 patients with OME, 94.7% of whom were atopic by in vitro testing. Tryptase was elevated only in the effusion of atopic patients as compared to 5 controls (p<0.01). Biopsies stained histochemically for tryptase showed evidence of mast cells in the mucosa and submucosa from 6 of 8 OME ears but absent in 4 normals.</p><p>Middle ear biopsies, embedded in a plastic resin to improve the structural preservation, from 5 patients with OME and 5 normals were evaluated for the presence of eosinophils and neutrophils with monoclonal antibodies against 4 specific granule proteins. Eosinophils and neutrophils were present in the mucosa and mucus in significantly higher numbers than in the control group.</p><p>In an effort to determine whether the middle ear itself might be involved in allergic disease, evidence that some of the cells, mediators and cytokines associated specifically with a Th-2 response were sought for in the middle ear mucosa of these children. Middle ear biopsies from 7 atopic patients with OME and 4 controls demonstrated the presence of activated eosinophils, CD-3+ T cells and IL-5 mRNA cells only in the mucosa from atopic OME children. </p><p>Conclusion: Effusion and mucosal biopsies containing ECP, tryptase, and/or IL-5 mRNA cells, CD3+ T cells, eosinophils, and mast cells indicate that many of the mediators and cells essential to the production of a Th-2 immune mediated response are present in ears with chronic effusion. The increased levels of MPO in atopic patients further suggest that the general inflammatory response to putative inciting agents such as bacterial and viral products may be altered in atopy. These studies support the hypothesis that the exaggerated inflammation within the middle ear associated with most cases of OME is possibly the result of an atopic response within the middle ear itself.</p>

<p>This thesis studied environmental factors in relation to asthma and respiratory symptoms among schoolchildren in two countries. In Sweden, 1014 pupils (5-14 year) in 8 schools participated. Wheeze was reported by 7.8%, current asthma by 5.9%, doctor-diagnosed asthma by 7.7%, cat allergy by 6.8% and dog allergy by 4.8%. Current asthma was less common among those consuming more fresh milk and fish. Doctor-diagnosed asthma was less common among those consuming olive oil. Cat, dog and horse allergens were common in settled dust and related to respiratory symptoms. Pupils consuming butter and fresh milk had less respiratory symptoms in relation to allergen exposure. In schools with increased levels of microbial volatile organic compounds and selected plasticizers (Texanol and TXIB) asthma and respiratory symptoms were more common.</p><p>In Korea, 2365 pupils (9-11 year) in 12 schools participated (96%). In total, wheeze was reported by 8.0%, current asthma by 5.7%, doctor-diagnosed asthma by 5.4%, cat allergy by 2.6% and dog allergy by 4.9%. Contamination of dog and mite (<i>Dermatophagoides farinae</i>) allergen was common while cat allergen was uncommon. Remodelling, changing floor and building dampness at home were positively associated with asthma and respiratory symptoms. The strongest associations were found for floor dampness. Indoor/outdoor concentration of NO₂, formaldehyde and ultrafine particles (UFP) at schools were positively associated with asthma and respiratory symptoms. </p><p>When comparing Sweden and Korea, Korean pupils had more breathlessness and asthma but reported less cat and pollen allergy. Swedish schools had CO₂-levels below 1000 ppm, while most Korean schools exceeded this standard. Since both home and school environment may affect pupil’s asthma and respiratory symptoms, air quality should be an important health issue. Moreover, changes in dietary habits may be beneficial to decrease asthma and allergies. Furthermore, interaction between diet and environment needs to be further investigated.</p>

In this thesis the oxidative metabolism by blood eosinophils from birch pollen allergic subjects was studied and compared to that by eosinophils from healthy controls, during and out of the pollen season. The effects and mechanisms of in vitro IL-5 priming on blood eosinophils were investigated and compared to the effects of in vivo priming during pollen exposure. The main findings of this work were that the oxidative metabolism by blood eosinophils taken from pollen allergic subjects is reduced during the pollen season. The eosinophils taken from asymptomatic allergics have a reduced capacity to produce oxygen free radicals as compared to non-allergic controls. The oxidative metabolism by blood eosinophils from allergic subjects is primed in vivo during the pollen season, as compared to the healthy controls and as compared to out of season. IL-5 primed the oxidative metabolism by eosinophils from allergic subjects in a similar way as eosinophils from healthy controls, both during and out of pollen exposure. The total and tyrosine phosphorylation patterns obtained were identical in eosinophils from allergic subjects and non-allergic controls during the pollen season. Spontaneous phosphorylation was the same in both groups and different from that after IL-5 priming. The oxidative metabolism of blood eosinophils is composed of different stages. The initial stage, measured as the t½rises of the CL curves, is an indication of the state of priming of the cell, while the end stage, measured as the peaks of the CL curves, is an estimate of the total radical production by the cells. IL-5 priming affected these two stages differently and the two stages are regulated by different signal transduction pathways and IL-5 priming causes a by-passing of MEK. In conclusion, in this thesis it is shown that blood eosinophils from allergic subjects are primed in vivo during exposure to their allergen. This in vivo priming leads on one hand to a reduced oxidative metabolism during the pollen season, but also to a faster onset of radical production as a response to certain stimuli. Our data do not provide any evidence of IL-5 involvement in the in vivo priming of blood eosinophils from allergic patients during pollen exposure.

The accumulation of eosinophil granulocytes (EOS) at sites of inflammation is a common feature of astma, allergic rhinitis and inflammatory bowel disease. The aim of the present investigation was to study the mechanisms involved in this accumulation. Bronchoalveolar lavage (BAL) fluid obtained from patients with birch-pollen allergy lavaged during season exhibited increased eosinophil chemotactic activity compared with pre-season BAL fluid from the same patients. We identified IL-5, IL-8 and RANTES as the main eosinophil chemotactic agents in the BAL fluid. Only EOS from allergic donors responded to IL-8. IL-2 inhibited albumin-stimulated eosinophil migration towards buffer or chemoattractants. EOS from allergic subjects were less sensitive to this inhibition than EOS from normal subjects, and in vitro priming of the EOS with IL-5 prevented the inhibitory effect of IL-2. We therefore hypothesise that IL-2 acts as an autocrine regulator of EOS migration, and that this inhibitory effect may be down-regulated in allergy, resulting in increased migration of EOS towards chemotactic factors. The stimulation of eosinophil migration by albumin is mediated by PI3 kinase. Decreased expression of CD49d and CD49f caused by albumin may decrease the adhesiveness of the EOS, which in turn may facilitate migration. We found a higher chemotactic activity in perfusion fluids from patients with ulcerative colitis than from control patients. The chemotactic activity correlated with the concentrations of eosinophil granule proteins in the perfusion fluids. IL-5 and TNF-α were identified as two of the chemotactic agents in the perfusion fluid that were inhibited by steroid treatment. Agents with steroid-insensitive chemotactic activity remain to be identified.

Otitis media with effusion (OME) is the major form of chronic relapsing inflammatory disease of the middle ear, constitutes the most common diagnosis for children under 15 years old and is the major cause of auditory dysfunction in pre-school children. OME is a disease more commonly found in allergic children. These studies sought to investigate the inflammatory response in the middle ear of patients and test the hypothesis that an allergic-like response might occur in the ear. Atopy was diagnosed by standard in vitro tests. Immunochemical techniques used to study classic allergic rhinitis and asthma were extrapolated to the evaluation of OME children whose effusion persisted beyond 2 months. Not only eosinophil cationic protein (ECP), tryptase, CD3-positive and IL-5 producing cells, but also myeloperoxidase (MPO) was found in middle ear fluid and/or mucosa in the majority of patients with OME and atopy. Initially, levels of ECP, MPO, and tryptase were measured in effusions from 97 random OME patients whose atopic status was determined by in vitro testing to 12 inhalants and 5 foods. The response of eosinophils, neutrophils and mast cells in the middle ear was distinctly different between atopic and non-atopic patients (p&lt;0.001) with higher levels of the cell markers in the atopic group of patients. This suggested that 1) perhaps OME was predominantly a disease of atopics and that 2) they differed in their response from non-atopics. Tryptase was measured in middle ear effusions from 38 patients with OME, 94.7% of whom were atopic by in vitro testing. Tryptase was elevated only in the effusion of atopic patients as compared to 5 controls (p&lt;0.01). Biopsies stained histochemically for tryptase showed evidence of mast cells in the mucosa and submucosa from 6 of 8 OME ears but absent in 4 normals. Middle ear biopsies, embedded in a plastic resin to improve the structural preservation, from 5 patients with OME and 5 normals were evaluated for the presence of eosinophils and neutrophils with monoclonal antibodies against 4 specific granule proteins. Eosinophils and neutrophils were present in the mucosa and mucus in significantly higher numbers than in the control group. In an effort to determine whether the middle ear itself might be involved in allergic disease, evidence that some of the cells, mediators and cytokines associated specifically with a Th-2 response were sought for in the middle ear mucosa of these children. Middle ear biopsies from 7 atopic patients with OME and 4 controls demonstrated the presence of activated eosinophils, CD-3+ T cells and IL-5 mRNA cells only in the mucosa from atopic OME children. Conclusion: Effusion and mucosal biopsies containing ECP, tryptase, and/or IL-5 mRNA cells, CD3+ T cells, eosinophils, and mast cells indicate that many of the mediators and cells essential to the production of a Th-2 immune mediated response are present in ears with chronic effusion. The increased levels of MPO in atopic patients further suggest that the general inflammatory response to putative inciting agents such as bacterial and viral products may be altered in atopy. These studies support the hypothesis that the exaggerated inflammation within the middle ear associated with most cases of OME is possibly the result of an atopic response within the middle ear itself.

The eosinophil cationic protein (ECP) is usually associated with the eosinophil granulocyte. In this thesis the presence and production of this protein has been studied in two other cells. The circulating monocyte was found to contain ECP mRNA and small amounts of ECP, one thousand times less than that found in the eosinophil. The production decreased by differentiation of the myelomonoblastic cell line U937 into a macrophage phenotype. Submucosal lung macrophages did not stain for ECP and alveolar macrophages did not contain ECP mRNA. The circulating neutrophil contains ECP at a level hundred fold less than the eosinophil. We found that the protein is located to the primary granules of the neutrophil but could detect no ECP mRNA in the cell. It was shown in vitro that the protein was taken up by the cell and partly transported to the primary granules. The uptake did not seem to be receptor mediated. Upon stimulation of the neutrophils, ECP previously taken up, was re-secreted. The ECP protein is heterogeneous both to molecular characteristics and to function. To evaluate if a genetic component is involved, the ECP gene was analysed in 70 individuals. Three single nucleotide polymorphisms (SNP´s) were found, denoted 277(C&gt;T), 434(G&gt;C) and 562(G&gt;C). The two first were located to the mature peptide-coding region and would change the amino acids, arg45cys and arg97thr. The prevalence of the most common SNP, 434, was evaluated in two eosinophil-related diseases, allergy/asthma and Hodgkin Lymphoma (HL). Forty-three HL patients were evaluated and it was found that the 434GG was significantly more prevalent in patients having nodular sclerosis (NS) as compared to other histologies (p=0.03). Erythrocyte sedimentation rate was also related to the 434GG genotype (p=0.009). In 209 medical students 434GG was more common (p=0.002) in those who indicated allergy. The genotype was unrelated to the production of IgE antibodies to allergens. In analysis of 76 subjects with asthma it was found that the 434GG genotype was significantly more common among allergic asthmatics (p=0.04). Asthma and HL-NS are characterized by fibrosis and eosinophils and ECP has been suggested in fibrosis development.

Chlamydia pneumoniae is a human respiratory tract pathogen. Seroepidemiological studies indicate that C. pneumoniae infection is most common in school-aged children and infrequently detected in younger children. The aims of this study were to further elucidate the prevalence of C. pneumoniae in paediatric populations and to describe the clinical implications of these infections. The study population consisted of 367 children with respiratory tract diseases, 453 presumed healthy children at day-care, 69 children undergoing adenoidectomy and 1585 children from a population based cohort. Family members to infected day-care children were investigated. The laboratory methods used were polymerase chain reaction (PCR) on specimen from upper respiratory tract, serology by microimmunofluorescence (MIF), and immunohistochemistry (IHC) on adenoid tissue specimen. Personal data and medical history were obtained by the means of questionnaires and by the study of patient records. In children younger than five years, the prevalence of C. pneumoniae was 17% as detected by PCR. This prevalence started to increase with increasing age from two years of age. The corresponding increase in serology as detected by MIF started at the age of four years. The prevalence at day-care centres varied from 4 to 39%. Both PCR and MIF underestimated the prevalence of C. pneumoniae detected by IHC. Families to infected children were investigated: mothers were more often infected than fathers were. Most C. pneumoniae infections in small children were confined to the upper respiratory tract. These infections were usually mild or asymptomatic. Symptomatic disease may be of prolonged nature. No subsequent illness after C. pneumoniae infection was detected at follow-up after four years. In general, no association between C. pneumoniae and asthma was found, but C. pneumoniae may be of importance for asthma in some susceptible individuals. Previous C. pneumoniae infection reduced the risk for later atopy. In conclusion, C. pneumoniae is a common finding in small children and most often causes relatively mild disease. If the acquisition of this infection early in life will have any implications for future health remains to be investigated.

Immunoglobulin E (IgE) is an important mediator in atopic allergies. This thesis describes the development of a therapeutic vaccine against IgE and its effects in rats and dogs. The development of an assay to determine IgE levels in dogs, and the finding of a chromosome region in rats that affects IgE levels are also reported. The vaccine is a chimeric molecule consisting of the constant domains Cε2, Cε3 and Cε4 from IgE. The target domain of the vaccine is the Cε3 domain in the recipient species, which is the domain directly involved in receptor binding, while the flanking regions, Cε2 and Cε4, are from a distantly related mammal. In rats, the vaccine induced an immune response against circulating IgE, which decreased IgE levels by 90% and substantially reduced their allergic symptoms. Further, the effects of adjuvants in rats and dogs were evaluated, and when co-administered with the vaccine certain adjuvants were shown to increase the immune response against IgE. Mineral-oils were the most potent adjuvants in inducing a response against IgE, but metabolizable oils spiked with immunostimulatory substances were also efficient. It was also shown that the therapeutic vaccine could induce a decrease in IgE levels in adult dogs, even though their initial levels were exceptionally high compared with humans. The IgE levels in 76 dogs ranged between 1 and 41 μg/ml while humans normally have around 150 ng/ml. However, the high IgE levels did not correlate to any specific breed, nor did they distinguish between dogs that were diagnosed as healthy and those suffering from atopic eczema, autoimmunity or skin parasites. Regulation of total IgE levels probably involves many genes. In the final phase of the study, one candidate locus known to be involved in arthritis susceptibility in rats was investigated, and was found also to affect IgE levels.