Nordic Congress of Allergology
A glimpse into ongoing research in allergy and immunology in Iceland
In this short compendium Icelandic investigators were asked to give a brief description of their ongoing research in the field of allergy and immunology. The topics covered are lectures that will be presented at the meeting.
R 1 - Inflammatory Responses in Respiratory Syncytial Virus Infection in Infants; Cytokines, Chemokines
Kristjánsson S
Respiratory Syncytial Virus (RSV) is the leading cause of respiratory tract infection during infancy. Early childhood RSV infection has been considered to be a risk factor for developing bronchial asthma. Cytotoxic T cells are required for virus clearance, but Th2 cells have been implicated in enhanced pathology following RSV infection. Lung eosinphilia has been associated with a local reduction in IFN-g production and increased IL-4 production.
Recently we finished the following pilot study. Seventeen children < 18 months of age with a RSV infection (mean age 3,3 months) were enrolled. RSV infection was confirmed by culture and immunofluorescence of nasal secretion. Urine was sampled before 12.00 am. The children were seen 3 weeks, 3 months and 2.5 years after the RSV infection. Skin Prick Test (SPT) for 10 different allergens was performed
Compared to the initial values U-EPX was slightly elevated (p=0.07) in the RSV group at the 3 month control (131 vs. 192 mg/mmol creatinine). At the 3 month visit U-EPX in the RSV group was significantly increased (p=007) compared to the age matched controls (192 vs. 101 mg/mmol creatinine).
The increased U-EPX values at 3 months after the RSV infection compared to the controls and also the slightly increased U-EPX values at that time indicate that the RSV infection causes activation of eosinophils and probably leads to a Th2 type inflammation in the majority of the children. However these effects were not seen at the visit 2.5 years after the RSV infection and wheezing was not correlated to U-EPX.
The following project has been ongoing the last 2.5 years. Children with diagnosed RSV infection are being followed until they are at least 7 years of age. The first results are presented here.
We measured the levels of these cytokines in relation to eotaxin and eosinophil production of the Eosinophil Cationic Protein in nasal secretion (N-ECP). ECP was also measured in serum (S-ECP). Thirty-nine infants < 7 months of age with a RSV infection (mean age 3 months) were enrolled in the study. RSV infection was confirmed by culture and immunofluorescence of nasal secretion. Fifty healthy infants in the same age group with no prior infection (outside the RSV season) were included as controls.
IL-4, IFN-g and eotaxin in nasal secretions were measured with sandwich ELISA (R&D Systems, Oxon, UK) and ECP with UniCap (Pharmacia &Upjohn, Uppsala, Sweden).
There was a significant statistical difference between the RSV and control groups in levels of IL-4 (0.79 vs. 0.31 pg/mL), (p=0,008) and IFN-g (0.55 vs. 0.18 pg/mL), (p=0,021). N-ECP levels were increased in the RSV group (p<0.001) compared to the control group (378,6 vs. 54,3 µg/L), but S-ECP levels were comparable (3.9 vs. 5.0 µg/L). In the RSV group there was a significant correlation between IL-4 and N-ECP (R=0.60, p<0.001) and a negative correlation between IFN-g and N-ECP (R=-0.39, p=0.015). There was a disparity in the levels of IL-4 and IFN-g in the RSV group (R=-0.22, p=0.182) as well as in the control group (R=-0.076, p=0.598). The
IL-4/IFN-g ratios were similar in the RSV group and control group (1.4 and 1.7). A negative correlation was found between IL-4 and eotaxin (R=-0.48, p=0.002).
In the RSV group eotaxin was significantly higher in infants > 3 months of age compared to infants < 3 months of age (37.8 vs. 13.5 pg/mL, p=0.003). The reverse was true for IL-4 which was primarily found in infants < 3 months of age (1.1 vs. 0.3 pg/ml, p=0,049).
Increased IL-4 in in nasal secretion in the RSV group compared to healthy controls as well as a good correlation between IL-4 and N-ECP levels in RSV infected infants strongly indicates a relative increase of local production of Th2 cytokines in the RSV group.
R 2 - Genetic factors separating different asthma phenotypes
Björnsdóttir US
In collaboration with DeCode Genetics, we have been using gene array technology to separate different asthma phenotypes.
1. Atopic vs nonatopic asthma
Atopy and asthma often coincide, however immunopathology, cellular, molecular and genetic mechanisms may be different.
We used gene array technology to examine whether different gene expression profiles can separate atopy and asthma. Asthma phenotype and severity was assessed by medical history, physical examination, methacholine challenge tests and PFT values. Atopy was verified by skin prick testing. Patients were classified into three groups in addition to controls: atopics (allergic rhinitis) without asthma or with only mild exercise induced asthma, allergic rhinitis with moderate asthma and nonatopics with moderate asthma. Total RNA was extracted from peripheral blood mononuclear cells treated with the inflammatory cytokines IL-1b and TNFa. RNA expression was examined using Affymetrix Hu95A chips, each recognising over 12600 genes. Expression profiles were analysed by comparing the mean change of signal intensity in cytokine stimulated vs not stimulated peripheral blood mononuclear cells in the 4 different phenotypes.
Certain proinflammatory genes discriminated atopic from nonatopic subjects, irrespective of asthma (p < 0.05). These included IL-10, STAT-1 and several IFN related cytokines. Another group of genes separated asthmatic subjects from those with only allergic rhinitis p < 0.05). These included IL-2R, IL-8, IL-6, TIMP and NFkb.
We have thus found that multiple genes known to play an important role in the regulation and action of inflammatory responses were differentially expressed between the three study groups: subjects with allergic rhinitis without asthma or with only exercise induced symptoms, allergic rhinitis with moderate asthma and nonatopic subjects with moderate asthma.
2. Glucocorticoid sensitive vs resistant asthmatics
Inhaled glucocorticoids (GCs) are widely used to control moderate to severe asthma. Asthma prevalence in Iceland is 5-6% and over 40% of patients use inhaled GCs on a daily basis. The effective dose for each patient can vary as much as 10 fold, however little is known about the molecular mechanisms that underlie these differences in GC sensitivity. In this set of experiments, we used affymetrix technology; performed on RNA samples from PBMCs collected from 50 asthmatic patients. Patients were classified as GC resistant (n=25) if dependent on a GC inhalation dose of 1600 mg per day or higher and GC sensitive (n=25) if dependent on a daily GC dose between 200-800 mg. The two groups were adjusted by age and sex. Asthma disease severity was assessed by PFTs and Mch challenge tests and prevalence of atopy was confirmed by skin prick test and total serum IgE values. The PBMCs were treated in an in vitro setting with IL-1b/TNFa in the absence (woGC) and presence (wGC) of pre-treatment with GC. Total RNA was extracted from the treated PBCM and gene expression was examined by Affy-hu95A chips which each has 12607 genes. RNA expression was analysed by comparing the mean difference change of signal intensity of wGC to woGC in each group. Asthma and atopy scores for the resistant and sensitive asthmatics were were comparable.
We found forty genes to be significantly differently expressed between the GCSAs and GCRAs. These genes included various cytokines/chemokines, their receptors, transcription factors and regulators of signal transduction and apoptosis. Examples of such genes are IL-6, IL-8, CD44, NF-kappa-B and jun-B which are all more significantly downregulated by GC in the GCSAs compared to GCRAs.
A group of genes involved in interferon pathways, transcription factors and IL-10 that separated atopics from nonatopics - irrespective of asthma status. Also a group of proinflammatory genes, cytokines and chemokines - in addition to factors associated with airway remodelling that separarted asthmatics from nonasthmatics - irrespective of atopic status.
Where will the future take us? Global gene profiling may give us the possibility to confirm current hypothesis on differences in gene expression and inflammatory pathways already known in asthma. This could in turn improve our understanding of yet unknown disease mechanisms and factors and the possibility of target directed therapy.
R 3 - Does dietary fish-oil improve health?
Haraldsson Á
In many countries, dietary fish-oil is considered healthy and is claimed to diminish infections, decrease autoimmune reactions and improve health in general. The reason for this remains unclarified.
In recent years, we have conducted several animal studies in order to investigate the possible effect of dietary fish-oil on the immune system.
The effect of dietary fish-oil supplementation on survival after infection was first investigated. The study revealed that the survival of NMRI mice after Klebsiella Pneumoniae intramuscular infection was significantly better in the group fed fish-oil enriched diet as compared to control groups (P=0.0034).
These results raised the question whether the dietary fish-oil might have any direct effect on bacterial growth in vivo. We therefore conducted a study where bacteria were counted in blood and in the infected muscle at various time intervals after infection with Klebsiella Pneumoniae. The bacterial count was not significantly different between the groups. Our conclusion therefore was that the fish-oil did not affect the bacterial growth in vivo.
Subsequently, we studied the effect of dietary fish-oil on TNF-a and IL-1 production and were not able to detect significant difference in these cytokine productions between the animals fed fish-oil enriched diet or not.
If the beneficial effect of dietary fish-oil is due to influence on the immune response it should be more or less independent of the infection site. Consequently, we studied the survival of mice after pulmonary infections with Klebsiella Pneumoniae. Again, the survival of the mice fed the fish-oil enriched diet and infected in the lungs with Klebsiella pneumoniae was significantly better compared to the survival of mice fed the corn-oil enriched diet (p=0,0001 and p=0,0013). We therefore concluded that the beneficial effect of dietary fish-oil on infection was independent of the site of infection. These results are in accordance with the hypothesis that dietary fish-oil influences the immune response.
Most of the infection studies have been done using the Gram negative bacteria Klebsiella pneumoniae as an infective agent. However, Gram positive bacteria, such as Streptococcus pneumoniae, are a very common microbes, in particular in children. The immune response against Gram positive bacteria is somewhat different to the response to Gram negative agents. Subsequently, we evaluated the effect of dietary fish-oil on infections due to Gram positive or Gram negative bacteria, by studying the survival of mice infected in the lungs with either Klebsiella pneumoniae or Streptococcus pneumoniae. The data for the groups infected with Klebsiella pneumoniae confirmed our earlier results (p=0.0001 and 0.0013) but, interestingly, there was no difference in the survival of mice infected with Streptococcus pneumoniae serotype 3, receiving either fish-oil or corn-oil enriched diets (p=0.74 and p=0.15). These results indicate that dietary fish-oil has beneficial effect on survival of mice after experimental infection with Klebsiella pneumoniae but not after infection with Streptococcus pneumoniae serotype 3. It indicates a more specific effect on the immune system than anticipated.
It has been postulated that fish-oil,in particular the omega-3 fatty acids, alter the leukotrien metabolism, shifting the production from the more active substances, LTB4, PGE2, and TXA2, to the less active, LTB5, PGE3, and TXA3. Consequently, we studied the effect of dietary fish-oil on the leukotriene metabolism by feeding the mice fish-oil or corn-oil with or without a leukotrien inhibitor (5-LO inhibitor, Zileuton® Abott). Interestingly, the beneficial effect of fish-oil was prevented by the leukotriene inhibitor. Our results thus indicates that the beneficial effects of fish oil are, at least in part, due to altered leukotriene metabolism.
Taken together, our studies indicate that fish-oil in fact alters the immune response and increases survival after some infections in experimental animals. Our hypothesis is that the fish-oil is slightly immunosuppressive, possibly due to the altered leukotriene metabolism, shifting towards less active substances. This immunosuppression would diminish the overwhelming septic shock syndrome in infected animals and enabling survival due to slower immune response. This immunosuppression could also account for the effect on autoimmune reactions. The effect on different infectious agents and the possible pathophysiological effect on the leukotriene metabolism needs still further investigation.
R 4 - Linkage of asthma to chromosome 14q24: "asthma gene one"
Hákonarson H, Björnsdóttir US, Stefánsson K
Asthma is a complex genetic disorder with a heterogeneous phenotype, largely attributed to the interactions among many genes and between these genes and the environment. Numerous loci and candidate genes have been reported to show linkage and association to asthma and atopy. While some studies reporting these observations are compelling, no asthma gene conferring high risk has been mapped such that it meets stringent criteria for genome-wide significance. We have performed a genome-wide scan with 976 microsatellite markers using 175 extended Icelandic families with 596 asthma patients. The families were identified by cross-matching a list of asthma patients from the Allergy/ Pulmonary Divisions of the National University Hospital of Iceland with a genealogy database of the entire Icelandic nation. We detected linkage of asthma to chromosome 14q24 with an allele-sharing lod score of 2.66. After increasing the marker density within the locus to a microsatellite every 0.2 cM on the average, the lod score rose to 4.00. We designate this locus, asthma locus one (AS1). Taken together, these results provide evidence for a novel susceptibility gene for asthma on chromosome 14q24.
R 5 - Asthma - Gastroesophageal Reflux, Sleep
Gíslason Þ
Based on recent epidemiological data from the European Community Respiratory Health Survey (ECRHS) newly developed asthma is often associated with female gender and obesity. Similar results have been reported from Canada (1) and decreased physical activity does not explain the association of new asthma to weight gain (2).
Both gastroesophageal reflux (GER) and obstructive sleep apnea syndrome (OSAS) increase in prevalence with increased body weight. GER has been associated with respiratory symptoms and many studies have shown a high prevalence of GER among patients with asthma (3,4). In a recent report even asthmatics without reflux symptoms had a high prevalence (62%) of abnormal 24-h esophageal tests (5). The relationship between GER and respiratory symptoms has primarily been assessed in highly selected populations at secondary or tertiary referral hospitals. There are few epidemiological studies in the general population and little is known about a possible association between sleep, GER and respiratory symptoms in an unselected random population.
Between 1990 and 1993 the ECRHS was conducted in different centers throughout the world (6). In four of the participating centers - Reykjavik Iceland, Uppsala and Göteborg, Sweden, and Antwerp, Belgium - a questionnaire relating to sleep disturbances was added to the second part of the ECRHS study (7-9). Altogether 2,202 subjects (20-48 years) were randomly selected from the general population in addition to 459 with reported asthma (10). The investigation also included a structured interview, spirometry, methacholine challenge, peak flow diary and skin prick tests. In the random population sample, 101 (4.6%) subjects reported GER defined as heartburn or belching after retiring to bed at least once per week. Subjects with GER were more often overweight and had more frequent symptoms of sleep disordered breathing than participants not reporting GER. Participants with GER were more likely to report wheezing (adjusted odds ratio [OR] = 2.5), breathlessness at rest (OR = 2.8), nocturnal breathlessness (OR = 2.9) and had increased peak flow variability compared to the subjects without GER. Physician diagnosed current asthma was reported by 9% of subjects with GER compared to 4% of those not reporting GER (p<0.05). Subjects with the combination of asthma and GER had a higher prevalence of sleep-related symptoms like snoring, daytime sleepiness etc than subjects with asthma alone (10).
Based on available data, it is possible that episodes of upper airway obstruction during sleep are associated with large intrathoracic/esophageal negative pressures swings (11), which result in an increased transdiaphragmatic pressure gradient, and may, at least in theory, lead to regurgitation of gastric acid into the esophagus. Such sleep-related GER could potentially cause respiratory dysfunction, bronchoconstriction, coughing, wheezing, laryngospasm and sleep disturbance. If the strong association we find between OSAS and GER is confirmed by others, traditional evaluation and treatment of asthmatic/OSAS patients may change. Therapeutic trials with continuous positive airway pressure (CPAP) may be indicated. This is especially true in complex cases with combined OSAS, GER and resistant respiratory symptoms. Recent studies reporting that CPAP reduces GER in OSAS (12), that swallowing reflex is improved after CPAP (13) and that CPAP even improves the control of asthma (14) support this approach. A therapeutic trial with a potent proton inhibitor should be considered in patients with resistant asthma, even in subjectsnot having GER or OSAS symptoms.
R 6 - Compliance in childhood asthma
Jónasson G
Most physicians who treat asthma in children are undoubtedly well aware of the fact that drug compliance in asthma therapy is generally poor, but some may think that even if this is the case, it surely does not apply for the vast majority of their own patients. However, physicians do not (unfortunately!) have any special ability to assess drug adherence of their patients. This statement is partly based on the fact ( as some studies have shown) that patients with poor compliance have no special observable characteristics that help to identify them.
It is therefore likely that clinicians overestimate patient compliance, and at times fail to realize that non-compliance is the most common reason for inadequate treatment response in children receiving therapy with inhaled corticosteroids. This in turn sometimes resulting in hospital admission due to acute asthma. Non-compliance is also considered to be an important cause of asthma mortality.
In clinical trials intended to assess the efficacy of a certain intervention such as drug treatment, patients are selected by means of specific inclusion criteria. Subjects should be interested in participating and willing to comply with the rules of the trial. In return the patient receives regular follow-up, attention and motivation, which again can result in increased compliance during the trial. When conclusions are to be drawn from such controlled trials it is important to monitor patient compliance in an objective way. This can be important regarding both the assessment of a possible dose-response effect (including side effects) of a given treatment, as well as general conclusions to be drawn from the trial.
163 children (7-16 years, 56 girls/107 boys) with mild asthma were included in a double blind, randomised study. After a two-week run-in period, the children received inhaled budesonide 100 mg or 200 mg daily, and/or placebo for 12 weeks. All patients used daily diary cards throughout the study. Compliance was also assessed by counting the number of remaining doses in the inhalers returned at the end of the study. Results from 160 patients were analysed. Mean compliance according to the diary was 93%, whereas estimated mean compliance when counting remaining doses in the inhaler was 77%. This discrepancy increased considerably as the measured compliance decreased.
122 children from the same study group were included in a follow-up study for 24 months and their drug adherence assessed by counting the number of remaining doses in the inhaler when the study medication was returned at six months intervals. A statistically significant decrease in measured drug adherence was found from three to nine months and further to 24 months reaching mean values of 40.6% and 46.9% for inhaled morning and evening medication respectively. Drug adherence declined more rapidly in the placebo group (compared to active treatment) and this difference became significant after two years of treatment.
Measured drug adherence diminishes significantly when treating children with mild asthma in a long term trial. This emphasises the importance of monitoring compliance in clinical trials.
R 7 - T Lymphocyte-Mediated Changes in Airway Smooth Muscle Responsivenes Are Attributed to Induced Autocrine Release and Actions of Interleukin (IL)-5
Hákonarson H
Bi-directional stimulatory cross-talk was recently found to exist between activated T cells and ASM cells, a process that involves co-ligation of specific cellular adhesion/co-stimulatory molecules that results in the induction of pro-asthmatic-like changes in ASM responsiveness.
In this study we examined whether the cooperative intercellular signaling between activated T cells and ASM cells is coupled to the induced expression and actions of IL-5 and IL-1ß
Agonist-induced constrictor and relaxant responses were examined in ASM segments exposed to resting and anti-CD3-activated T cells, in the absence and presence of either an IL-5mAb or the rhIL-1ra. In addition, mRNA and protein expression of IL-5 and IL-1ß were assayed under control and anti-CD3-stimulated conditions.
We found that relative to inactive T cells, incubation of ASM tissues with anti-CD3-activated T cells induced pro-asthmatic-like changes in agonist-mediated ASM responsiveness. This T cell-induced perturbation in ASM responsiveness was ablated by pretreating the tissues with either IL-5r mAb or IL-1ra. Moreover, exposure of ASM cells to anti-CD3-activated T cells elicited an initial increased mRNA expression and release of IL-5, followed by an enhanced expression and release of IL-1ß, and the induced release of these cytokines was prevented in ASM cells that were pretreated with IL-5r mAb.
Collectively, these observations provide new evidence demonstrating that exposure of naïve ASM cells to activated T cells induces the sequential release of IL-5 and IL-1ß from the ASM cells, and that the latter cytokines act in an autocrine manner to elicit the pro-asthmatic phenotype of altered ASM responsiveness.
R 8 - A protective immune response - experience from the field of pneumococcal vaccines
Sigurðardóttir SÞ, Jónsdóttir I
Streptococcus pneumoniae (pneumococcus) is an important cause of morbidity and mortality among infants and young children. Protection against pneumococcal infection is by opsonization of pneumococci with serotype-specific polysaccharide antibodies and complement, leading to phagocytosis and killing by polymorphonuclear leukocytes. Children less than two years of age are unable to produce protective antibodies to most of these polysaccharides which are T-cell independent antigens. By conjugating pneumococcal polysaccharides to protein, they have been rendered immunogenic in this age group. The pneumococcal conjugate vaccines (Pnc) have been shown to be safe and induce protective antibody response in infants at an age when they are unable to respond to pneumococcal capsular polysaccharides. Vaccine trials in Iceland have shown that the IgG response to the Pnc is carrier and serotype specific. The vaccine induced IgG antibodies were functional as demonstrated with opsonophagocytosis and a good IgG booster response with avitity maturation indicated memory induction. On the other hand, naturally aquired immunity may reflect antibodies that are nonspecific and nonfunctional. Most purified PPS used for antibody measurements are contaminated with cell wall polysaccharide (CWPS), thus antibodies to CWPS which has failed to demonstrate a protective role in humans may give false positive results in ELISA. This may be overcome to some extend by neutalization with CWPS.
To investigate if the carrier is limitting for the antibody response, two 11-valent Pnc vaccines, F3 (type 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F conjugated to tetanus or diphtheria toxoids) and F3bis (same serotypes but both carrier proteins for 6B, 9V, 18C and 23F) (Aventis Pasteur) were compared in 146 Icelandic infants. They were randomized to receive either vaccine at 3, 4, 6 and 13 months. Blood samples were obtained at 3, 7, 13, 14 and 24 months. Both vaccines were equally safe and induced significant primary and booster IgG responses. Mixing two conjugates of the less immunogenic serotypes containing two protein carriers did not improve the primary immune response. At 24 months of age the vaccinated children had higher antibody levels to all 11 serotypes than unvaccinated age-matched controls.
Along with antibody levels, immunological memory is improtant for long term protection against infection. Avidity maturation and a rapid IgG response has been used as an indirect indicator of memory induction. When toddlers were vaccinated with one dose of F3bis at 17 months of age, 95% produced antibodies against 9 of 11 pneumococcal types that were above the level of 0.15 mg/ml. When they 10 months later, were boosted with PPS vaccine, one log rise in IgG to 11 serotypes was observed in one week with significant increase in avidity, indicating immunological memory at an age when children usually show marginal response to PPS vaccine.
Other investigators have demaonstrated 97.5% efficacy of Pnc vaccins against invasive disease and 57% decrease in otitis media caused by the vaccine serotypes. Prevention of nasopharyngeal carriage is important for induction of heard immunity and reduction of transmission but where the vaccines have been used in large scale, heard immunity has also been observed. A reduction has also been observed in antibiotic resistant strains as most of those belong to serotypes that are included in the vaccines.
R 9 - Prevalence of Atopy and Atopic diseases
Clausen M
The prevalence of adult atopic diseases in Iceland is lower than in Western Europe but there are no data concerning children. As part of the phase-2 of the International Study of Asthma and Allergy in Children (ISAAC) we investigated the prevalence of atopic diseases and sensitivity to common allergens, in 10-11 year old schoolchildren. 946 children and their parents answered questionnaire about atopic diseases. Skin prick tests (SPT) with 6 allergens were performed on 774 children and signs of atopic eczema registered. The prevalence of allergic rhinitis was 11% and asthma 9%. The reported prevalence of eczema was 26.9%, however, only 10.3% had signs when inspected. A positive SPT was recorded in 24.9% of the children, i.e. 18.7% to mixed grasses, 12.9% to cat, 3.6% to mixed trees, 2.9% to D. pter, 1.4% to D. farinae and 0.5% to alternaria. The high prevalence of atopic diseases in children at 10-11 years is surprising as the prevalence in adults is very low in Iceland. The findings resemble those in developing countries. Other changes in environmental factors would be operative, however, as Iceland has had an affluent lifestyle for a considerable time. The high prevalence of sensitivity to grass pollen may be explained by the long grass pollen season.
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R 1 - Inflammatory Responses in Respiratory Syncytial Virus Infection in Infants; Cytokines, Chemokines
and Eosinophil Cationic Protein
Kristjánsson SRespiratory Syncytial Virus (RSV) is the leading cause of respiratory tract infection during infancy. Early childhood RSV infection has been considered to be a risk factor for developing bronchial asthma. Cytotoxic T cells are required for virus clearance, but Th2 cells have been implicated in enhanced pathology following RSV infection. Lung eosinphilia has been associated with a local reduction in IFN-g production and increased IL-4 production.
Recently we finished the following pilot study. Seventeen children < 18 months of age with a RSV infection (mean age 3,3 months) were enrolled. RSV infection was confirmed by culture and immunofluorescence of nasal secretion. Urine was sampled before 12.00 am. The children were seen 3 weeks, 3 months and 2.5 years after the RSV infection. Skin Prick Test (SPT) for 10 different allergens was performed
Compared to the initial values U-EPX was slightly elevated (p=0.07) in the RSV group at the 3 month control (131 vs. 192 mg/mmol creatinine). At the 3 month visit U-EPX in the RSV group was significantly increased (p=007) compared to the age matched controls (192 vs. 101 mg/mmol creatinine).
The increased U-EPX values at 3 months after the RSV infection compared to the controls and also the slightly increased U-EPX values at that time indicate that the RSV infection causes activation of eosinophils and probably leads to a Th2 type inflammation in the majority of the children. However these effects were not seen at the visit 2.5 years after the RSV infection and wheezing was not correlated to U-EPX.
The following project has been ongoing the last 2.5 years. Children with diagnosed RSV infection are being followed until they are at least 7 years of age. The first results are presented here.
We measured the levels of these cytokines in relation to eotaxin and eosinophil production of the Eosinophil Cationic Protein in nasal secretion (N-ECP). ECP was also measured in serum (S-ECP). Thirty-nine infants < 7 months of age with a RSV infection (mean age 3 months) were enrolled in the study. RSV infection was confirmed by culture and immunofluorescence of nasal secretion. Fifty healthy infants in the same age group with no prior infection (outside the RSV season) were included as controls.
IL-4, IFN-g and eotaxin in nasal secretions were measured with sandwich ELISA (R&D Systems, Oxon, UK) and ECP with UniCap (Pharmacia &Upjohn, Uppsala, Sweden).
There was a significant statistical difference between the RSV and control groups in levels of IL-4 (0.79 vs. 0.31 pg/mL), (p=0,008) and IFN-g (0.55 vs. 0.18 pg/mL), (p=0,021). N-ECP levels were increased in the RSV group (p<0.001) compared to the control group (378,6 vs. 54,3 µg/L), but S-ECP levels were comparable (3.9 vs. 5.0 µg/L). In the RSV group there was a significant correlation between IL-4 and N-ECP (R=0.60, p<0.001) and a negative correlation between IFN-g and N-ECP (R=-0.39, p=0.015). There was a disparity in the levels of IL-4 and IFN-g in the RSV group (R=-0.22, p=0.182) as well as in the control group (R=-0.076, p=0.598). The
IL-4/IFN-g ratios were similar in the RSV group and control group (1.4 and 1.7). A negative correlation was found between IL-4 and eotaxin (R=-0.48, p=0.002).
In the RSV group eotaxin was significantly higher in infants > 3 months of age compared to infants < 3 months of age (37.8 vs. 13.5 pg/mL, p=0.003). The reverse was true for IL-4 which was primarily found in infants < 3 months of age (1.1 vs. 0.3 pg/ml, p=0,049).
Increased IL-4 in in nasal secretion in the RSV group compared to healthy controls as well as a good correlation between IL-4 and N-ECP levels in RSV infected infants strongly indicates a relative increase of local production of Th2 cytokines in the RSV group.
R 2 - Genetic factors separating different asthma phenotypes
Björnsdóttir US
In collaboration with DeCode Genetics, we have been using gene array technology to separate different asthma phenotypes.
1. Atopic vs nonatopic asthma
Atopy and asthma often coincide, however immunopathology, cellular, molecular and genetic mechanisms may be different.
We used gene array technology to examine whether different gene expression profiles can separate atopy and asthma. Asthma phenotype and severity was assessed by medical history, physical examination, methacholine challenge tests and PFT values. Atopy was verified by skin prick testing. Patients were classified into three groups in addition to controls: atopics (allergic rhinitis) without asthma or with only mild exercise induced asthma, allergic rhinitis with moderate asthma and nonatopics with moderate asthma. Total RNA was extracted from peripheral blood mononuclear cells treated with the inflammatory cytokines IL-1b and TNFa. RNA expression was examined using Affymetrix Hu95A chips, each recognising over 12600 genes. Expression profiles were analysed by comparing the mean change of signal intensity in cytokine stimulated vs not stimulated peripheral blood mononuclear cells in the 4 different phenotypes.
Certain proinflammatory genes discriminated atopic from nonatopic subjects, irrespective of asthma (p < 0.05). These included IL-10, STAT-1 and several IFN related cytokines. Another group of genes separated asthmatic subjects from those with only allergic rhinitis p < 0.05). These included IL-2R, IL-8, IL-6, TIMP and NFkb.
We have thus found that multiple genes known to play an important role in the regulation and action of inflammatory responses were differentially expressed between the three study groups: subjects with allergic rhinitis without asthma or with only exercise induced symptoms, allergic rhinitis with moderate asthma and nonatopic subjects with moderate asthma.
2. Glucocorticoid sensitive vs resistant asthmatics
Inhaled glucocorticoids (GCs) are widely used to control moderate to severe asthma. Asthma prevalence in Iceland is 5-6% and over 40% of patients use inhaled GCs on a daily basis. The effective dose for each patient can vary as much as 10 fold, however little is known about the molecular mechanisms that underlie these differences in GC sensitivity. In this set of experiments, we used affymetrix technology; performed on RNA samples from PBMCs collected from 50 asthmatic patients. Patients were classified as GC resistant (n=25) if dependent on a GC inhalation dose of 1600 mg per day or higher and GC sensitive (n=25) if dependent on a daily GC dose between 200-800 mg. The two groups were adjusted by age and sex. Asthma disease severity was assessed by PFTs and Mch challenge tests and prevalence of atopy was confirmed by skin prick test and total serum IgE values. The PBMCs were treated in an in vitro setting with IL-1b/TNFa in the absence (woGC) and presence (wGC) of pre-treatment with GC. Total RNA was extracted from the treated PBCM and gene expression was examined by Affy-hu95A chips which each has 12607 genes. RNA expression was analysed by comparing the mean difference change of signal intensity of wGC to woGC in each group. Asthma and atopy scores for the resistant and sensitive asthmatics were were comparable.
We found forty genes to be significantly differently expressed between the GCSAs and GCRAs. These genes included various cytokines/chemokines, their receptors, transcription factors and regulators of signal transduction and apoptosis. Examples of such genes are IL-6, IL-8, CD44, NF-kappa-B and jun-B which are all more significantly downregulated by GC in the GCSAs compared to GCRAs.
A group of genes involved in interferon pathways, transcription factors and IL-10 that separated atopics from nonatopics - irrespective of asthma status. Also a group of proinflammatory genes, cytokines and chemokines - in addition to factors associated with airway remodelling that separarted asthmatics from nonasthmatics - irrespective of atopic status.
Where will the future take us? Global gene profiling may give us the possibility to confirm current hypothesis on differences in gene expression and inflammatory pathways already known in asthma. This could in turn improve our understanding of yet unknown disease mechanisms and factors and the possibility of target directed therapy.
R 3 - Does dietary fish-oil improve health?
Haraldsson Á
In many countries, dietary fish-oil is considered healthy and is claimed to diminish infections, decrease autoimmune reactions and improve health in general. The reason for this remains unclarified.
In recent years, we have conducted several animal studies in order to investigate the possible effect of dietary fish-oil on the immune system.
The effect of dietary fish-oil supplementation on survival after infection was first investigated. The study revealed that the survival of NMRI mice after Klebsiella Pneumoniae intramuscular infection was significantly better in the group fed fish-oil enriched diet as compared to control groups (P=0.0034).
These results raised the question whether the dietary fish-oil might have any direct effect on bacterial growth in vivo. We therefore conducted a study where bacteria were counted in blood and in the infected muscle at various time intervals after infection with Klebsiella Pneumoniae. The bacterial count was not significantly different between the groups. Our conclusion therefore was that the fish-oil did not affect the bacterial growth in vivo.
Subsequently, we studied the effect of dietary fish-oil on TNF-a and IL-1 production and were not able to detect significant difference in these cytokine productions between the animals fed fish-oil enriched diet or not.
If the beneficial effect of dietary fish-oil is due to influence on the immune response it should be more or less independent of the infection site. Consequently, we studied the survival of mice after pulmonary infections with Klebsiella Pneumoniae. Again, the survival of the mice fed the fish-oil enriched diet and infected in the lungs with Klebsiella pneumoniae was significantly better compared to the survival of mice fed the corn-oil enriched diet (p=0,0001 and p=0,0013). We therefore concluded that the beneficial effect of dietary fish-oil on infection was independent of the site of infection. These results are in accordance with the hypothesis that dietary fish-oil influences the immune response.
Most of the infection studies have been done using the Gram negative bacteria Klebsiella pneumoniae as an infective agent. However, Gram positive bacteria, such as Streptococcus pneumoniae, are a very common microbes, in particular in children. The immune response against Gram positive bacteria is somewhat different to the response to Gram negative agents. Subsequently, we evaluated the effect of dietary fish-oil on infections due to Gram positive or Gram negative bacteria, by studying the survival of mice infected in the lungs with either Klebsiella pneumoniae or Streptococcus pneumoniae. The data for the groups infected with Klebsiella pneumoniae confirmed our earlier results (p=0.0001 and 0.0013) but, interestingly, there was no difference in the survival of mice infected with Streptococcus pneumoniae serotype 3, receiving either fish-oil or corn-oil enriched diets (p=0.74 and p=0.15). These results indicate that dietary fish-oil has beneficial effect on survival of mice after experimental infection with Klebsiella pneumoniae but not after infection with Streptococcus pneumoniae serotype 3. It indicates a more specific effect on the immune system than anticipated.
It has been postulated that fish-oil,in particular the omega-3 fatty acids, alter the leukotrien metabolism, shifting the production from the more active substances, LTB4, PGE2, and TXA2, to the less active, LTB5, PGE3, and TXA3. Consequently, we studied the effect of dietary fish-oil on the leukotriene metabolism by feeding the mice fish-oil or corn-oil with or without a leukotrien inhibitor (5-LO inhibitor, Zileuton® Abott). Interestingly, the beneficial effect of fish-oil was prevented by the leukotriene inhibitor. Our results thus indicates that the beneficial effects of fish oil are, at least in part, due to altered leukotriene metabolism.
Taken together, our studies indicate that fish-oil in fact alters the immune response and increases survival after some infections in experimental animals. Our hypothesis is that the fish-oil is slightly immunosuppressive, possibly due to the altered leukotriene metabolism, shifting towards less active substances. This immunosuppression would diminish the overwhelming septic shock syndrome in infected animals and enabling survival due to slower immune response. This immunosuppression could also account for the effect on autoimmune reactions. The effect on different infectious agents and the possible pathophysiological effect on the leukotriene metabolism needs still further investigation.
R 4 - Linkage of asthma to chromosome 14q24: "asthma gene one"
Hákonarson H, Björnsdóttir US, Stefánsson K
Asthma is a complex genetic disorder with a heterogeneous phenotype, largely attributed to the interactions among many genes and between these genes and the environment. Numerous loci and candidate genes have been reported to show linkage and association to asthma and atopy. While some studies reporting these observations are compelling, no asthma gene conferring high risk has been mapped such that it meets stringent criteria for genome-wide significance. We have performed a genome-wide scan with 976 microsatellite markers using 175 extended Icelandic families with 596 asthma patients. The families were identified by cross-matching a list of asthma patients from the Allergy/ Pulmonary Divisions of the National University Hospital of Iceland with a genealogy database of the entire Icelandic nation. We detected linkage of asthma to chromosome 14q24 with an allele-sharing lod score of 2.66. After increasing the marker density within the locus to a microsatellite every 0.2 cM on the average, the lod score rose to 4.00. We designate this locus, asthma locus one (AS1). Taken together, these results provide evidence for a novel susceptibility gene for asthma on chromosome 14q24.
R 5 - Asthma - Gastroesophageal Reflux, Sleep
and Obesity
Gíslason Þ
Based on recent epidemiological data from the European Community Respiratory Health Survey (ECRHS) newly developed asthma is often associated with female gender and obesity. Similar results have been reported from Canada (1) and decreased physical activity does not explain the association of new asthma to weight gain (2).
Both gastroesophageal reflux (GER) and obstructive sleep apnea syndrome (OSAS) increase in prevalence with increased body weight. GER has been associated with respiratory symptoms and many studies have shown a high prevalence of GER among patients with asthma (3,4). In a recent report even asthmatics without reflux symptoms had a high prevalence (62%) of abnormal 24-h esophageal tests (5). The relationship between GER and respiratory symptoms has primarily been assessed in highly selected populations at secondary or tertiary referral hospitals. There are few epidemiological studies in the general population and little is known about a possible association between sleep, GER and respiratory symptoms in an unselected random population.
Between 1990 and 1993 the ECRHS was conducted in different centers throughout the world (6). In four of the participating centers - Reykjavik Iceland, Uppsala and Göteborg, Sweden, and Antwerp, Belgium - a questionnaire relating to sleep disturbances was added to the second part of the ECRHS study (7-9). Altogether 2,202 subjects (20-48 years) were randomly selected from the general population in addition to 459 with reported asthma (10). The investigation also included a structured interview, spirometry, methacholine challenge, peak flow diary and skin prick tests. In the random population sample, 101 (4.6%) subjects reported GER defined as heartburn or belching after retiring to bed at least once per week. Subjects with GER were more often overweight and had more frequent symptoms of sleep disordered breathing than participants not reporting GER. Participants with GER were more likely to report wheezing (adjusted odds ratio [OR] = 2.5), breathlessness at rest (OR = 2.8), nocturnal breathlessness (OR = 2.9) and had increased peak flow variability compared to the subjects without GER. Physician diagnosed current asthma was reported by 9% of subjects with GER compared to 4% of those not reporting GER (p<0.05). Subjects with the combination of asthma and GER had a higher prevalence of sleep-related symptoms like snoring, daytime sleepiness etc than subjects with asthma alone (10).
Based on available data, it is possible that episodes of upper airway obstruction during sleep are associated with large intrathoracic/esophageal negative pressures swings (11), which result in an increased transdiaphragmatic pressure gradient, and may, at least in theory, lead to regurgitation of gastric acid into the esophagus. Such sleep-related GER could potentially cause respiratory dysfunction, bronchoconstriction, coughing, wheezing, laryngospasm and sleep disturbance. If the strong association we find between OSAS and GER is confirmed by others, traditional evaluation and treatment of asthmatic/OSAS patients may change. Therapeutic trials with continuous positive airway pressure (CPAP) may be indicated. This is especially true in complex cases with combined OSAS, GER and resistant respiratory symptoms. Recent studies reporting that CPAP reduces GER in OSAS (12), that swallowing reflex is improved after CPAP (13) and that CPAP even improves the control of asthma (14) support this approach. A therapeutic trial with a potent proton inhibitor should be considered in patients with resistant asthma, even in subjectsnot having GER or OSAS symptoms.
R 6 - Compliance in childhood asthma
Jónasson G
Most physicians who treat asthma in children are undoubtedly well aware of the fact that drug compliance in asthma therapy is generally poor, but some may think that even if this is the case, it surely does not apply for the vast majority of their own patients. However, physicians do not (unfortunately!) have any special ability to assess drug adherence of their patients. This statement is partly based on the fact ( as some studies have shown) that patients with poor compliance have no special observable characteristics that help to identify them.
It is therefore likely that clinicians overestimate patient compliance, and at times fail to realize that non-compliance is the most common reason for inadequate treatment response in children receiving therapy with inhaled corticosteroids. This in turn sometimes resulting in hospital admission due to acute asthma. Non-compliance is also considered to be an important cause of asthma mortality.
In clinical trials intended to assess the efficacy of a certain intervention such as drug treatment, patients are selected by means of specific inclusion criteria. Subjects should be interested in participating and willing to comply with the rules of the trial. In return the patient receives regular follow-up, attention and motivation, which again can result in increased compliance during the trial. When conclusions are to be drawn from such controlled trials it is important to monitor patient compliance in an objective way. This can be important regarding both the assessment of a possible dose-response effect (including side effects) of a given treatment, as well as general conclusions to be drawn from the trial.
163 children (7-16 years, 56 girls/107 boys) with mild asthma were included in a double blind, randomised study. After a two-week run-in period, the children received inhaled budesonide 100 mg or 200 mg daily, and/or placebo for 12 weeks. All patients used daily diary cards throughout the study. Compliance was also assessed by counting the number of remaining doses in the inhalers returned at the end of the study. Results from 160 patients were analysed. Mean compliance according to the diary was 93%, whereas estimated mean compliance when counting remaining doses in the inhaler was 77%. This discrepancy increased considerably as the measured compliance decreased.
122 children from the same study group were included in a follow-up study for 24 months and their drug adherence assessed by counting the number of remaining doses in the inhaler when the study medication was returned at six months intervals. A statistically significant decrease in measured drug adherence was found from three to nine months and further to 24 months reaching mean values of 40.6% and 46.9% for inhaled morning and evening medication respectively. Drug adherence declined more rapidly in the placebo group (compared to active treatment) and this difference became significant after two years of treatment.
Measured drug adherence diminishes significantly when treating children with mild asthma in a long term trial. This emphasises the importance of monitoring compliance in clinical trials.
R 7 - T Lymphocyte-Mediated Changes in Airway Smooth Muscle Responsivenes Are Attributed to Induced Autocrine Release and Actions of Interleukin (IL)-5
and IL-1ß
Hákonarson H
Bi-directional stimulatory cross-talk was recently found to exist between activated T cells and ASM cells, a process that involves co-ligation of specific cellular adhesion/co-stimulatory molecules that results in the induction of pro-asthmatic-like changes in ASM responsiveness.
In this study we examined whether the cooperative intercellular signaling between activated T cells and ASM cells is coupled to the induced expression and actions of IL-5 and IL-1ß
Agonist-induced constrictor and relaxant responses were examined in ASM segments exposed to resting and anti-CD3-activated T cells, in the absence and presence of either an IL-5mAb or the rhIL-1ra. In addition, mRNA and protein expression of IL-5 and IL-1ß were assayed under control and anti-CD3-stimulated conditions.
We found that relative to inactive T cells, incubation of ASM tissues with anti-CD3-activated T cells induced pro-asthmatic-like changes in agonist-mediated ASM responsiveness. This T cell-induced perturbation in ASM responsiveness was ablated by pretreating the tissues with either IL-5r mAb or IL-1ra. Moreover, exposure of ASM cells to anti-CD3-activated T cells elicited an initial increased mRNA expression and release of IL-5, followed by an enhanced expression and release of IL-1ß, and the induced release of these cytokines was prevented in ASM cells that were pretreated with IL-5r mAb.
Collectively, these observations provide new evidence demonstrating that exposure of naïve ASM cells to activated T cells induces the sequential release of IL-5 and IL-1ß from the ASM cells, and that the latter cytokines act in an autocrine manner to elicit the pro-asthmatic phenotype of altered ASM responsiveness.
R 8 - A protective immune response - experience from the field of pneumococcal vaccines
Sigurðardóttir SÞ, Jónsdóttir I
Streptococcus pneumoniae (pneumococcus) is an important cause of morbidity and mortality among infants and young children. Protection against pneumococcal infection is by opsonization of pneumococci with serotype-specific polysaccharide antibodies and complement, leading to phagocytosis and killing by polymorphonuclear leukocytes. Children less than two years of age are unable to produce protective antibodies to most of these polysaccharides which are T-cell independent antigens. By conjugating pneumococcal polysaccharides to protein, they have been rendered immunogenic in this age group. The pneumococcal conjugate vaccines (Pnc) have been shown to be safe and induce protective antibody response in infants at an age when they are unable to respond to pneumococcal capsular polysaccharides. Vaccine trials in Iceland have shown that the IgG response to the Pnc is carrier and serotype specific. The vaccine induced IgG antibodies were functional as demonstrated with opsonophagocytosis and a good IgG booster response with avitity maturation indicated memory induction. On the other hand, naturally aquired immunity may reflect antibodies that are nonspecific and nonfunctional. Most purified PPS used for antibody measurements are contaminated with cell wall polysaccharide (CWPS), thus antibodies to CWPS which has failed to demonstrate a protective role in humans may give false positive results in ELISA. This may be overcome to some extend by neutalization with CWPS.
To investigate if the carrier is limitting for the antibody response, two 11-valent Pnc vaccines, F3 (type 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F and 23F conjugated to tetanus or diphtheria toxoids) and F3bis (same serotypes but both carrier proteins for 6B, 9V, 18C and 23F) (Aventis Pasteur) were compared in 146 Icelandic infants. They were randomized to receive either vaccine at 3, 4, 6 and 13 months. Blood samples were obtained at 3, 7, 13, 14 and 24 months. Both vaccines were equally safe and induced significant primary and booster IgG responses. Mixing two conjugates of the less immunogenic serotypes containing two protein carriers did not improve the primary immune response. At 24 months of age the vaccinated children had higher antibody levels to all 11 serotypes than unvaccinated age-matched controls.
Along with antibody levels, immunological memory is improtant for long term protection against infection. Avidity maturation and a rapid IgG response has been used as an indirect indicator of memory induction. When toddlers were vaccinated with one dose of F3bis at 17 months of age, 95% produced antibodies against 9 of 11 pneumococcal types that were above the level of 0.15 mg/ml. When they 10 months later, were boosted with PPS vaccine, one log rise in IgG to 11 serotypes was observed in one week with significant increase in avidity, indicating immunological memory at an age when children usually show marginal response to PPS vaccine.
Other investigators have demaonstrated 97.5% efficacy of Pnc vaccins against invasive disease and 57% decrease in otitis media caused by the vaccine serotypes. Prevention of nasopharyngeal carriage is important for induction of heard immunity and reduction of transmission but where the vaccines have been used in large scale, heard immunity has also been observed. A reduction has also been observed in antibiotic resistant strains as most of those belong to serotypes that are included in the vaccines.
R 9 - Prevalence of Atopy and Atopic diseases
in Iceland
Clausen M
The prevalence of adult atopic diseases in Iceland is lower than in Western Europe but there are no data concerning children. As part of the phase-2 of the International Study of Asthma and Allergy in Children (ISAAC) we investigated the prevalence of atopic diseases and sensitivity to common allergens, in 10-11 year old schoolchildren. 946 children and their parents answered questionnaire about atopic diseases. Skin prick tests (SPT) with 6 allergens were performed on 774 children and signs of atopic eczema registered. The prevalence of allergic rhinitis was 11% and asthma 9%. The reported prevalence of eczema was 26.9%, however, only 10.3% had signs when inspected. A positive SPT was recorded in 24.9% of the children, i.e. 18.7% to mixed grasses, 12.9% to cat, 3.6% to mixed trees, 2.9% to D. pter, 1.4% to D. farinae and 0.5% to alternaria. The high prevalence of atopic diseases in children at 10-11 years is surprising as the prevalence in adults is very low in Iceland. The findings resemble those in developing countries. Other changes in environmental factors would be operative, however, as Iceland has had an affluent lifestyle for a considerable time. The high prevalence of sensitivity to grass pollen may be explained by the long grass pollen season.
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