Damp building moulds: Assessment of sensitization in patients and studies into mechanisms of airway inflammation using experimental models

Exposure to water-damaged buildings and the associated health problems have evoked concern and created confusion during the past 20 years. Individuals exposed to moisture problem buildings report adverse health effects such as non-specific respiratory symptoms. Microbes, especially fungi, growing on the damp material have been considered as potential sources of the health problems encountered in these buildings. Fungi and their airborne fungal spores contain allergens and secondary metabolites which may trigger allergic as well as inflammatory types of responses in the eyes and airways. Although epidemiological studies have revealed an association between damp buildings and health problems, no direct cause-and-effect relationship has been established. Further knowledge is needed about the epidemiology and the mechanisms leading to the symptoms associated with exposure to fungi. Two different approaches have been used in this thesis in order to investigate the diverse health effects associated with exposure to moulds. In the first part, sensitization to moulds was evaluated and potential cross-reactivity studied in patients attending a hospital for suspected allergy. In the second part, one typical mould known to be found in water-damaged buildings and to produce toxic secondary metabolites was used to study the airway responses in an experimental model. Exposure studies were performed on both naive and allergen sensitized mice. The first part of the study showed that mould allergy is rare and highly dependent on the atopic status of the examined individual. The prevalence of sensitization was 2.7% to Cladosporium herbarum and 2.8% to Alternaria alternata in patients, the majority of whom were atopic subjects. Some of the patients sensitized to mould suffered from atopic eczema. Frequently the patients were observed to possess specific serum IgE antibodies to a yeast present in the normal skin flora, Pityrosporum ovale. In some of these patients, the IgE binding was partly found to be due to binding to shared glycoproteins in the mould and yeast allergen extracts. The second part of the study revealed that exposure to Stachybotrys chartarum spores induced an airway inflammation in the lungs of mice. The inflammation was characterized by an influx of inflammatory cells, mainly neutrophils and lymphocytes, into the lungs but with almost no differences in airway responses seen between the satratoxin producing and non-satratoxin producing strain. On the other hand, when mice were exposed to S. chartarum and sensitized/challenged with ovalbumin the extent of the inflammation was markedly enhanced. A synergistic increase in the numbers of inflammatory cells was seen in BAL and severe inflammation was observed in the histological lung sections. In conclusion, the results in this thesis imply that exposure to moulds in water damaged buildings may trigger health effects in susceptible individuals. The symptoms can rarely be explained by IgE mediated allergy to moulds. Other non-allergic mechanisms seem to be involved. Stachybotrys chartarum is one of the moulds potentially responsible for health problems. In this thesis, new reaction models for the airway inflammation induced by S. chartarum have been found using experimental approaches. The immunological status played an important role in the airway inflammation, enhancing the effects of mould exposure. The results imply that sensitized individuals may be more susceptible to exposure to moulds than non-sensitized individuals.

Airway Inflammation and Bronchial Hyperresponsiveness in Elite Cross-Country Skiers and in Patients with Newly Diagnosed Asthma: A Bronchial Biopsy Study

The objective of these studies was to evaluate possible airway inflammation and remodeling at the bronchial level in cross-country skiers without a prior diagnosis of asthma, and relate the findings to patients with mild chronic asthma and patients with newly diagnosed asthma. We also studied the association of airway inflammatory changes and bronchial hyperresponsivess (BHR), and treatment effects in cross-country skiers and in patients with newly diagnosed asthma. Bronchial biopsies were obtained from the subjects by flexible bronchoscopy, and the inflammatory cells (eosinophils, mast cells, T-lymphocytes, macrophages, and neutrophils) were identified by immunohistochemistry. Tenascin (Tn) immunoreactivity in the bronchial basement membrane (BM) was identified by immunofluorescence staining. Lung function was measured with spirometry, and BHR was assessed by methacholine (skiers) or histamine (asthmatics) challenges. Skiers with BHR and asthma-like symptoms were recruited to a drug-intervention study. Skiers were given treatment (22 weeks) with placebo or budesonide (400 µg bid). Patients with newly diagnosed asthma were given treatment for 16 weeks with placebo, salmeterol (SLM) (50 µg bid), fluticasone propionate (FP) (250 µg bid), or disodium cromoglicate (DSCG) (5 mg qid). Bronchial biopsies were obtained at baseline and at the end of the treatment period. In the skiers a distinct airway inflammation was evident. In their bronchial biopsy specimens, T-lymphocyte, macrophage, and eosinophil counts were, respectively greater by 43-fold (P<0.001), 26-fold (P<0.001, and 2-fold (P<0.001) in skiers, and by 70-fold (p>0.001), 63-fold (P<0.001), and 8-fold (P<0.001) in asthmatic subjects than in controls. In skiers, neutrophil counts were more than 2-fold greater than in asthmatic subjects (P<0.05). Tn expression was higher in skiers than in controls and lower in skiers than in mild asthmatics. No significant changes were seen between skiers with or without BHR in the inflammatory cell counts or Tn expression. Treatment with inhaled budesonide did not attenuate asthma-like symptoms, the inflammatory cell infiltration, or BM Tn expression in the skiers. In newly diagnosed asthmatic patients, SLM, FP, and DSCG reduced asthma symptoms, and need for rescue medication (P<0.04). BHR was reduced by doubling doses 2.78, 5.22, and 1.35 respectively (all P<0.05). SLM and placebo had no effect on cell counts or Tn expression. FP and DSCG reduced eosinophil counts in the bronchial biopsy specimens (P<0.02 and <0.048, respectively). No significant change in tenascin expression appeared in any treatment group. Regarding to atopy, no significant differences existed in the inflammatory cell counts in the bronchial mucosa of subjects with newly diagnosed asthma or in elite cross country skiers. Tn expression in the BM was significantly higher in atopic asthma than in those with nonatopic asthma. Airway inflammation occurred in elite cross-country skiers with and without respiratory symptoms or BHR. Their inflammatory cell pattern differed from that in asthma. Infiltration with eosinophils, macrophages, and mast cells was milder, but lymphocyte counts did not differ from counts in asthmatic airways. Neutrophilic infiltration was more extensive in skiers than in asthmatics. Remodeling took place in the skiers’ airways, as reflected by increased expression of BM tenascin These inflammatory changes and Tn expression may be caused by prolonged exposure of the lower airways to inadequately humidified cold air. In skiers inflammatory changes and remodeling were not reversed with anti-inflammatory treatment. In contrast, in patients with newly diagnosed asthma, anti-inflammatory treatment did attenuate eosinophilic inflammation in the bronchial mucosa. In skiers, anti-inflammatory treatment did not attenuate BHR as it did in asthmatic patients. The BHR in skiers was attenuated spontaneously during placebo treatment, with no difference from budesonide treatment. Lower training intensity during the treatment period may explain this spontaneous decrease in BHR. The origin of BHR probably differs in skiers and in asthmatics. No significant association between BHR and inflammatory cell counts or between BHR and Tn expression was evident in cross-country skiers or asthmatic subjects. Airway remodeling differed between atopic and nonatopic asthma. As opposed to nonatopic asthma, Tn expression was higher in atopic asthma and is related to inflammatory cell densities.

Epithelial sodium channel in airway epithelium of the newborn infant : Implications for postnatal pulmonary adaptation

The aim of the present thesis was to study the role of the epithelial sodium channel (ENaC) in clearance of fetal lung fluid in the newborn infant by measurement of airway epithelial expression of ENaC, of nasal transepithelial potential difference (N-PD), and of lung compliance (LC). In addition, the effect of postnatal dexamethasone on airway epithelial ENaC expression was measured in preterm infants with bronchopulmonary dysplasia (BPD). The patient population was formed of selected term newborn infants born in the Department of Obstetrics (Studies II-IV) and selected preterm newborn infants treated in the neonatal intensive care unit of the Hospital for Children and Adolescents (Studies I and IV) of the Helsinki University Central Hospital in Finland. A small population of preterm infants suffering from BPD was included in Study I. Studies I, III, and IV included airway epithelial measurement of ENaC and in Studies II and III, measurement of N-PD and LC. In Study I, ENaC expression analyses were performed in the Research Institute of the Hospital for Sick Children in Toronto, Ontario, Canada. In the following studies, analyses were performed in the Scientific Laboratory of the Hospital for Children and Adolescents. N-PD and LC measurements were performed at bedside in these hospitals. In term newborn infants, the percentage of amiloride-sensitive N-PD, a surrogate for ENaC activity, measured during the first 4 postnatal hours correlates positively with LC measured 1 to 2 days postnatally. Preterm infants with BPD had, after a therapeutic dose of dexamethasone, higher airway epithelial ENaC expression than before treatment. These patients were subsequently weaned from mechanical ventilation, probably as a result of the clearance of extra fluid from the alveolar spaces. In addition, we found that in preterm infants ENaC expression increases with gestational age (GA). In preterm infants, ENaC expression in the airway epithelium was lower than in term newborn infants. During the early postnatal period in those born both preterm and term airway epithelial βENaC expression decreased significantly. Term newborn infants delivered vaginally had a significantly smaller airway epithelial expression of αENaC after the first postnatal day than did those delivered by cesarean section. The functional studies showed no difference in N-PD between infants delivered vaginally and by cesarean section. We therefore conclude that the low airway epithelial expression of ENaC in the preterm infant and the correlation of N-PD with LC in the term infant indicate a role for ENaC in the pathogenesis of perinatal pulmonary adaptation and neonatal respiratory distress. Because dexamethasone raised ENaC expression in preterm infants with BPD, and infants were subsequently weaned from ventilator therapy, we suggest that studies on the treatment of respiratory distress in the preterm infant should include the induction of ENaC activity.

Cardiac function before and after treatment for various types of loading conditions and in myocardial restriction : A prospective study on pediatric patients with two- and three-dimensional echocardiography and measurement of serum natriuretic peptides

Background: The incidence of all forms of congenital heart defects is 0.75%. For patients with congenital heart defects, life-expectancy has improved with new treatment modalities. Structural heart defects may require surgical or catheter treatment which may be corrective or palliative. Even those with corrective therapy need regular follow-up due to residual lesions, late sequelae, and possible complications after interventions. Aims: The aim of this thesis was to evaluate cardiac function before and after treatment for volume overload of the right ventricle (RV) caused by atrial septal defect (ASD), volume overload of the left ventricle (LV) caused by patent ductus arteriosus (PDA), and pressure overload of the LV caused by coarctation of the aorta (CoA), and to evaluate cardiac function in patients with Mulibrey nanism. Methods: In Study I, of the 24 children with ASD, 7 underwent surgical correction and 17 percutaneous occlusion of ASD. Study II had 33 patients with PDA undergoing percutaneous occlusion. In Study III, 28 patients with CoA underwent either surgical correction or percutaneous balloon dilatation of CoA. Study IV comprised 26 children with Mulibrey nanism. A total of 76 healthy voluntary children were examined as a control group. In each study, controls were matched to patients. All patients and controls underwent clinical cardiovascular examinations, two-dimensional (2D) and three-dimensional (3D) echocardiographic examinations, and blood sampling for measurement of natriuretic peptides prior to the intervention and twice or three times thereafter. Control children were examined once by 2D and 3D echocardiography. M-mode echocardiography was performed from the parasternal long axis view directed by 2D echocardiography. The left atrium-to-aorta (LA/Ao) ratio was calculated as an index of LA size. The end-diastolic and end-systolic dimensions of LV as well as the end-diastolic thicknesses of the interventricular septum and LV posterior wall were measured. LV volumes, and the fractional shortening (FS) and ejection fraction (EF) as indices of contractility were then calculated, and the z scores of LV dimensions determined. Diastolic function of LV was estimated from the mitral inflow signal obtained by Doppler echocardiography. In three-dimensional echocardiography, time-volume curves were used to determine end-diastolic and end-systolic volumes, stroke volume, and EF. Diastolic and systolic function of LV was estimated from the calculated first derivatives of these curves. Results: (I): In all children with ASD, during the one-year follow-up, the z score of the RV end-diastolic diameter decreased and that of LV increased. However, dilatation of RV did not resolve entirely during the follow-up in either treatment group. In addition, the size of LV increased more slowly in the surgical subgroup but reached control levels in both groups. Concentrations of natriuretic peptides in patients treated percutaneously increased during the first month after ASD closure and normalized thereafter, but in patients treated surgically, they remained higher than in controls. (II): In the PDA group, at baseline, the end-diastolic diameter of LV measured over 2SD in 5 of 33 patients. The median N-terminal pro-brain natriuretic peptide (proBNP) concentration before closure measured 72 ng/l in the control group and 141 ng/l in the PDA group (P = 0.001) and 6 months after closure measured 78.5 ng/l (P = NS). Patients differed from control subjects in indices of LV diastolic and systolic function at baseline, but by the end of follow-up, all these differences had disappeared. Even in the subgroup of patients with normal-sized LV at baseline, the LV end-diastolic volume decreased significantly during follow-up. (III): Before repair, the size and wall thickness of LV were higher in patients with CoA than in controls. Systolic blood pressure measured a median 123 mm Hg in patients before repair (P < 0.001) and 103 mm Hg one year thereafter, and 101 mm Hg in controls. The diameter of the coarctation segment measured a median 3.0 mm at baseline, and 7.9 at the 12-month (P = 0.006) follow-up. Thicknesses of the interventricular septum and posterior wall of the LV decreased after repair but increased to the initial level one year thereafter. The velocity time integrals of mitral inflow increased, but no changes were evident in LV dimensions or contractility. During follow-up, serum levels of natriuretic peptides decreased correlating with diastolic and systolic indices of LV function in 2D and 3D echocardiography. (IV): In 2D echocardiography, the interventricular septum and LV posterior wall were thicker, and velocity time integrals of mitral inflow shorter in patients with Mulibrey nanism than in controls. In 3D echocardiography, LV end-diastolic volume measured a median 51.9 (range 33.3 to 73.4) ml/m² in patients and 59.7 (range 37.6 to 87.6) ml/m² in controls (P = 0.040), and serum levels of ANPN and proBNP a median 0.54 (range 0.04 to 4.7) nmol/l and 289 (range 18 to 9170) ng/l, in patients and 0.28 (range 0.09 to 0.72) nmol/l (P < 0.001) and 54 (range 26 to 139) ng/l (P < 0.001) in controls. They correlated with several indices of diastolic LV function. Conclusions (I): During the one-year follow-up after the ASD closure, RV size decreased but did not normalize in all patients. The size of the LV normalized after ASD closure but the increase in LV size was slower in patients treated surgically than in those treated with the percutaneous technique. Serum levels of ANPN and proBNP were elevated prior to ASD closure but decreased thereafter to control levels in patients treated with the percutaneous technique but not in those treated surgically. (II): Changes in LV volume and function caused by PDA disappeared by 6 months after percutaneous closure. Even the children with normal-sized LV benefited from the procedure. (III): After repair of CoA, the RV size and the velocity time integrals of mitral inflow increased, and serum levels of natriuretic peptides decreased. Patients need close follow-up, despite cessation of LV pressure overload, since LV hypertrophy persisted even in normotensive patients with normal growth of the coarctation segment. (IV): In children with Mulibrey nanism, the LV wall was hypertrophied, with myocardial restriction and impairment of LV function. Significant correlations appeared between indices of LV function, size of the left atrium, and levels of natriuretic peptides, indicating that measurement of serum levels of natriuretic peptides can be used in the clinical follow-up of this patient group despite its dependence on loading conditions.

Pharmacokinetics, efficacy, and safety of pravastatin in children : Studies in children with heterozygous familial hypercholesterolemia and in pediatric cardiac transplant recipients

Background. Hyperlipidemia is a common concern in patients with heterozygous familial hypercholesterolemia (HeFH) and in cardiac transplant recipients. In both groups, an elevated serum LDL cholesterol level accelerates the development of atherosclerotic vascular disease and increases the rates of cardiovascular morbidity and mortality. The purpose of this study is to assess the pharmacokinetics, efficacy, and safety of cholesterol-lowering pravastatin in children with HeFH and in pediatric cardiac transplant recipients receiving immunosuppressive medication. Patients and Methods. The pharmacokinetics of pravastatin was studied in 20 HeFH children and in 19 pediatric cardiac transplant recipients receiving triple immunosuppression. The patients ingested a single 10-mg dose of pravastatin, and plasma pravastatin concentrations were measured up to 10/24 hours. The efficacy and safety of pravastatin (maximum dose 10 to 60 mg/day and 10 mg/day) up to one to two years were studied in 30 patients with HeFH and in 19 cardiac transplant recipients, respectively. In a subgroup of 16 HeFH children, serum non-cholesterol sterol ratios (102 x mmol/mol of cholesterol), surrogate estimates of cholesterol absorption (cholestanol, campesterol, sitosterol), and synthesis (desmosterol and lathosterol) were studied at study baseline (on plant stanol esters) and during combination with pravastatin and plant stanol esters. In the transplant recipients, the lipoprotein levels and their mass compositions were analyzed before and after one year of pravastatin use, and then compared to values measured from 21 healthy pediatric controls. The transplant recipients were grouped into patients with transplant coronary artery disease (TxCAD) and patients without TxCAD, based on annual angiography evaluations before pravastatin. Results. In the cardiac transplant recipients, the mean area under the plasma concentration-time curve of pravastatin [AUC(0-10)], 264.1 * 192.4 ng.h/mL, was nearly ten-fold higher than in the HeFH children (26.6 * 17.0 ng.h/mL). By 2, 4, 6, 12 and 24 months of treatment, the LDL cholesterol levels in the HeFH children had respectively decreased by 25%, 26%, 29%, 33%, and 32%. In the HeFH group, pravastatin treatment increased the markers of cholesterol absorption and decreased those of synthesis. High ratios of cholestanol to cholesterol were associated with the poor cholesterol-lowering efficacy of pravastatin. In cardiac transplant recipients, pravastatin 10 mg/day lowered the LDL cholesterol by approximately 19%. Compared with the patients without TxCAD, patients with TxCAD had significantly lower HDL cholesterol concentrations and higher apoB-100/apoA-I ratios at baseline (1.0 ± 0.3 mmol/L vs. 1.4 ± 0.3 mmol/L, P = 0.031; and 0.7 ± 0.2 vs. 0.5 ± 0.1, P = 0.034) and after one year of pravastatin use (1.0 ± 0.3 mmol/L vs. 1.4 ± 0.3 mmol/L, P = 0.013; and 0.6 ± 0.2 vs. 0.4 ± 0.1, P = 0.005). Compared with healthy controls, the transplant recipients exhibited elevated serum triglycerides at baseline (median 1.3 [range 0.6-3.2] mmol/L vs. 0.7 [0.3-2.4] mmol/L, P=0.0002), which negatively correlated with their HDL cholesterol concentration (r = -0.523, P = 0.022). Recipients also exhibited higher apoB-100/apoA1 ratios (0.6 ± 0.2 vs. 0.4 ± 0.1, P = 0.005). In addition, elevated triglyceride levels were still observed after one year of pravastatin use (1.3 [0.5-3.5] mmol/L vs. 0.7 [0.3-2.4] mmol/L, P = 0.0004). Clinically significant elevations in alanine aminotransferase, creatine kinase, or creatinine ocurred in neither group. Conclusions. Immunosuppressive medication considerably increased the plasma pravastatin concentrations. In both patient groups, pravastatin treatment was moderately effective, safe, and well tolerated. In the HeFH group, high baseline cholesterol absorption seemed to predispose patients to insufficient cholesterol-lowering efficacy of pravastatin. In the cardiac transplant recipients, low HDL cholesterol and a high apoB-100/apoA-I ratio were associated with development of TxCAD. Even though pravastatin in the transplant recipients effectively lowered serum total and LDL cholesterol concentrations, it failed to normalize their elevated triglyceride levels and, in some patients, to prevent the progression of TxCAD.

Cyclosporine population pharmacokinetics in pediatric renal transplant recipients

Cyclosporine is an immunosuppressant drug with a narrow therapeutic index and large variability in pharmacokinetics. To improve cyclosporine dose individualization in children, we used population pharmacokinetic modeling to study the effects of developmental, clinical, and genetic factors on cyclosporine pharmacokinetics in altogether 176 subjects (age range: 0.36–20.2 years) before and up to 16 years after renal transplantation. Pre-transplantation test doses of cyclosporine were given intravenously (3 mg/kg) and orally (10 mg/kg), on separate occasions, followed by blood sampling for 24 hours (n=175). After transplantation, in a total of 137 patients, cyclosporine concentration was quantified at trough, two hours post-dose, or with dose-interval curves. One-hundred-four of the studied patients were genotyped for 17 putatively functionally significant sequence variations in the ABCB1, SLCO1B1, ABCC2, CYP3A4, CYP3A5, and NR1I2 genes. Pharmacokinetic modeling was performed with the nonlinear mixed effects modeling computer program, NONMEM. A 3-compartment population pharmacokinetic model with first order absorption without lag-time was used to describe the data. The most important covariate affecting systemic clearance and distribution volume was allometrically scaled body weight i.e. body weight**3/4 for clearance and absolute body weight for volume of distribution. The clearance adjusted by absolute body weight declined with age and pre-pubertal children (< 8 years) had an approximately 25% higher clearance/body weight (L/h/kg) than did older children. Adjustment of clearance for allometric body weight removed its relationship to age after the first year of life. This finding is consistent with a gradual reduction in relative liver size towards adult values, and a relatively constant CYP3A content in the liver from about 6–12 months of age to adulthood. The other significant covariates affecting cyclosporine clearance and volume of distribution were hematocrit, plasma cholesterol, and serum creatinine, explaining up to 20%–30% of inter-individual differences before transplantation. After transplantation, their predictive role was smaller, as the variations in hematocrit, plasma cholesterol, and serum creatinine were also smaller. Before transplantation, no clinical or demographic covariates were found to affect oral bioavailability, and no systematic age-related changes in oral bioavailability were observed. After transplantation, older children receiving cyclosporine twice daily as the gelatine capsule microemulsion formulation had an about 1.25–1.3 times higher bioavailability than did the younger children receiving the liquid microemulsion formulation thrice daily. Moreover, cyclosporine oral bioavailability increased over 1.5-fold in the first month after transplantation, returning thereafter gradually to its initial value in 1–1.5 years. The largest cyclosporine doses were administered in the first 3–6 months after transplantation, and thereafter the single doses of cyclosporine were often smaller than 3 mg/kg. Thus, the results suggest that cyclosporine displays dose-dependent, saturable pre-systemic metabolism even at low single doses, whereas complete saturation of CYP3A4 and MDR1 (P-glycoprotein) renders cyclosporine pharmacokinetics dose-linear at higher doses. No significant associations were found between genetic polymorphisms and cyclosporine pharmacokinetics before transplantation in the whole population for which genetic data was available (n=104). However, in children older than eight years (n=22), heterozygous and homozygous carriers of the ABCB1 c.2677T or c.1236T alleles had an about 1.3 times or 1.6 times higher oral bioavailability, respectively, than did non-carriers. After transplantation, none of the ABCB1 SNPs or any other SNPs were found to be associated with cyclosporine clearance or oral bioavailability in the whole population, in the patients older than eight years, or in the patients younger than eight years. In the whole population, in those patients carrying the NR1I2 g.-25385C–g.-24381A–g.-205_-200GAGAAG–g.7635G–g.8055C haplotype, however, the bioavailability of cyclosporine was about one tenth lower, per allele, than in non-carriers. This effect was significant also in a subgroup of patients older than eight years. Furthermore, in patients carrying the NR1I2 g.-25385C–g.-24381A–g.-205_-200GAGAAG–g.7635G–g.8055T haplotype, the bioavailability was almost one fifth higher, per allele, than in non-carriers. It may be possible to improve individualization of cyclosporine dosing in children by accounting for the effects of developmental factors (body weight, liver size), time after transplantation, and cyclosporine dosing frequency/formulation. Further studies are required on the predictive value of genotyping for individualization of cyclosporine dosing in children.

The role of inflammation and matrix proteins in airway remodelling

Asthma is a chronic inflammatory disorder of the airways. Remodelling in asthma is defined as the structural changes seen in the airways of asthmatics in comparison to healthy controls. Progressive loss of lung function also seen in asthma might be caused by remodelling. The research aims of this thesis were to investigate inflammation and remodelling in the airways of different types of asthmatics and smokers. The association between inflammation and remodelling was also examined in a mouse model of allergic airway inflammation. Healthy smokers showed increased numbers of macrophages in the BAL with no changes in the inflammatory cells in biopsies. Macrophages seemed to be quite quiescent, since mRNA expression for a wide variety of inflammatory mediators, especially chemokines CCL3, CCL4, CCL5 and CCL20, secreted by macrophages was significantly lower than in healthy non-smokers. Attenuated macrophage activity in the airway lumen may render smokers more susceptible to airway infections and have an impact on the development of other airway pathology. Patients with diisocyanate-induced asthma (DIA) on inhaled corticosteroids (ICS) who still had non-specific bronchial hyperreactivity (NSBHR) at the end of the follow-up showed increased expression of TNF-α, IL-6 and IL-15 mRNA in BAL cells compared to those without NSBHR. In addition to being markers for poor prognosis and possible slight glucocorticoid resistance, these cytokines might aid in guiding the treatment of DIA. The increase in the thickness of tenascin-C layer in the bronchial basement membrane (BM) was much less than usually seen in other types of asthma, which might not make tenascin-C a good marker for DIA. OVA-induced tenascin-C expression in the lung was attenuated in STAT4-/- mice with impaired Th1-type immunity compared to WT mice. Interestingly, STAT6-/- mice with impaired Th2-type immunity showed tenascin-C expression levels similar to those of WT mice. The clearest difference between these two knockout strains in response to OVA was that STAT4-/- mice exhibited no upregulation of IFN-γ and TNF-α mRNA expression. Thus, tenascin-C expression was unexpectedly more related to Th1 type reactions. In vitro studies confirmed the results. Human fibroblasts stimulated by TNF-α and IFN-γ showed increased expression of tenascin-C. Patients with newly diagnosed asthma showed increased expression of laminin α2 in the bronchial BM in comparison to patients with asthma symptoms only and healthy controls. Both patients with asthma and those with only asthma symptoms showed increased expression of the laminin β2 chain in comparison to controls. Thus, laminin α2 expression differentiated patients with clinical asthma from patients with symptoms only. Furthermore, the expression of laminin α2 and β2 was associated with NSBHR, linking very specific remodelling events to clinical findings.

Snoring and sleep apnea in children

Snoring is a primary and major clinical symptom of upper airway obstruction during sleep. Sleep-disordered breathing ranges from primary snoring to significant partial upper airway obstruction, and obstructive sleep apnea. Adult snoring and obstructive sleep apnea have been extensively studied, whereas less is known about these disorders in children. Snoring and more severe obstructive sleep apnea have been shown to have a harmful effect on the neurobehavioral development of children, but the mechanisms of this effect remains unknown. Furthermore, the correlation of this effect to objective sleep study parameters remains poor. This study evaluated the prevalence of snoring in preschool-aged children in Finland. Host and environmental risk factors, and neurobehavioral and neurocognitive symptoms of children suffering from snoring or obstructive sleep apnea were also investigated. The feasibility of acoustic rhinometry in young children was assessed. The prevalence and risk factors of snoring (I) were evaluated by a questionnaire. The random sample included 2100 children aged 1-6 years living in Helsinki. All 3- to 6-year-old children whose parents reported their child to snore always, often, or sometimes were categorized as snorers, and invited to participate to the clinical study (II-IV). Non-snoring children whose parents were willing to participate in the clinical study were invited to serve as controls. Children underwent a clinical ear-nose-throat examination. Emotional, behavioral, and cognitive performances were evaluated by Child Behavioral Checklist (CBCL), Wechsler Preschool and Primary Scale of Intelligence (WPPSI-R) and NEPSY-A Developmental Neuropsychological Assessment (NEPSY). Nasal volume was measured by acoustic rhinometry, and nasal resistance by rhinomanometry. Lateral and posteroanterior cephalometry were performed. A standard overnight ambulatory polysomnography was performed in the home environment. Twenty-six healthy children were tested in order to assess the feasibility of acoustic rhinometry in young children (V). Snoring was common in children; 6.3% of children snored always or often, whereas 81.3% snored never or occasionally. No differences were apparent between snorers and non-snorers regarding age, or gender. Pediatric snoring was associated with recurrent upper respiratory infections, otitis media, and allergic rhinitis. Exposure to parental tobacco smoke, especially maternal smoking, was more common among snorers. Rhinitis was more common among children who exposured to tobacco smoke. Overnight polysomnography (PSG) was performed on 87 children; 74% showed no signs of significant upper airway obstruction during sleep. Three children had obstructive apnea/hypopnea index (OAHI) greater than 5/h. Age, gender, or a previous adenoidectomy or tonsillectomy did not correlate with OAHI, whereas tonsillar size did correlate with OAHI. Relative body weight and obesity correlated with none of the PSG parameters. In cephalometry, no clear differences or correlations were found in PSG parameters or between snorers and non-snorers. No correlations were observed between acoustic rhinometry, rhinomanometry, and PSG parameters. Psychiatric symptoms were more frequent in the snoring group than in the nonsnoring group. In particular, anxious and depressed symptoms were more prevalent in the snoring group. Snoring children frequently scored lower in language functions. However, PSG parameters correlated poorly with neurocognitive test results in these children. This study and previous studies indicate that snoring without episodes of obstructive apnea or SpO2 desaturations may cause impairment in behavioral and neurocognitive functions. The mechanism of action remains unknown. Exposure to parental tobacco smoke is more common among snorers than non-snorers, emphasizing the importance of a smoke-free environment. Children tolerated acoustic rhinometry measurements well.