PCSK2 expression in neuroendocrine tumors points to a midgut, pulmonary, or pheochromocytoma-paraganglioma origin.

Neuroendocrine tumors (NETs) are often diagnosed from the metastases of an unknown primary tumor. Specific immunohistochemical (IHC) markers indicating the location of a primary tumor are needed. The proprotein convertase subtilisin/kexin type 2 (PCSK2) is found in normal neural and neuroendocrine cells, and known to express in NETs. We investigated the tissue microarray (TMA) of 86 primary tumors from 13 different organs and 9 metastatic NETs, including primary tumor-metastasis pairs, for PCSK2 expression with polymer-based IHC. PCSK2 was strongly positive in all small intestine and appendiceal NETs, the so-called midgut NETs, in most pheochromocytomas and paragangliomas, and in some of the typical and atypical pulmonary carcinoid tumors. NETs showing strong positivity were re-evaluated in larger tumor cohorts confirming the primary observation. In the metastases, the expression of PCSK2 mirrored that of the corresponding primary tumors. We found negative or weak staining in NETs from the thymus, gastric mucosa, pancreas, rectum, thyroid, and parathyroid. PCSK2 expression did not correlate with Ki-67 in well-differentiated NETs. Our data suggest that PCSK2 positivity can indicate the location of the primary tumor. Thus, PCSK2 could function in the IHC panel determined from screening metastatic NET biopsies of unknown primary origins.

Sputum Vitamin D Binding Protein (VDBP) GC1S/1S Genotype Predicts Airway Obstruction: A Prospective Study in Smokers with COPD.

Introduction: The vitamin D binding protein (VDBP, also known as GC-globulin) and vitamin D deficiency have been associated with chronic obstructive pulmonary disease (COPD). rs7041 and rs4588 are two single nucleotide polymorphisms of the VDBP gene, including three common allelic variants (GC1S, GC1F and GC2). Previous studies primarily assessed the serum levels of vitamin D and VDBP in COPD. However, less is known regarding the impact of the local release of VDBP on COPD lung function. Thus, we examined the association of sputum and plasma VDBP with lung function at baseline and at four years, and examined potential genetic polymorphism interactions. Methods: The baseline levels of sputum VDBP, plasma VDBP and plasma 25-OH vitamin D, as well as the GC rs4588 and rs7041 genotypes, were assessed in a 4-year Finnish follow-up cohort (n = 233) of non-smokers, and smokers with and without COPD. The associations between the VDBP levels and the longitudinal decline of lung function were further analysed. Results: High frequencies of the haplotypes in rs7041/rs4588 were homozygous GC1S/1S (42.5%). Higher sputum VDBP levels in stage I and stage II COPD were observed only in carriers with GC1S/1S genotype when compared with non-smokers (p = 0.034 and p = 0.002, respectively). Genotype multivariate regression analysis indicated that the baseline sputum VDBP and FEV1/FVC ratio at baseline independently predicted FEV1% at follow-up. Discussion and Conclusion: The baseline sputum VDBP expression was elevated in smokers with COPD among individuals with the GC1S/1S genotype, and predicted follow-up airway obstruction. Our results suggest that the GC polymorphism should be considered when exploring the potential of VDBP as a biomarker for COPD.

Nocturnal asthma is affected by genetic interactions between RORA and NPSR1.

BACKGROUND: Neuropeptide S Receptor 1 ( NPSR1) and Retinoid Acid Receptor-Related Orphan Receptor Alpha (RORA ) interact biologically, are both known candidate genes for asthma, and are involved in controlling circadian rhythm. Thus, we assessed (1) whether interactions between RORA and NPSR1 specifically affect the nocturnal asthma phenotype and (2) how this may differ from other asthma phenotypes. METHODS: Interaction effects between 24 single-nucleotide polymorphisms (SNPs) in RORA and 35 SNPs in NPSR1 on asthma and nocturnal asthma symptoms were determined in 1432 subjects (763 asthmatics [192 with nocturnal asthma symptoms]; 669 controls) from the Multicenter Asthma Genetic in Childhood/International Study of Asthma and Allergies in Childhood studies. The results were validated and extended in children from the Manchester Asthma and Allergy Study (N = 723) and the Children Allergy Milieu Stockholm and Epidemiological cohort (N = 1646). RESULTS: RORA* NPSR1 interactions seemed to affect both asthma and nocturnal asthma. In stratified analyses, however, interactions mainly affected nocturnal asthma and less so asthma without nocturnal symptoms or asthma severity. Results were replicated in two independent cohorts and seemed to remain constant over time throughout youth. CONCLUSION: RORA* NPSR1 interactions appear to be involved in mechanisms specific for nocturnal asthma. In contrast to previous studies focusing on the role of beta 2 receptor polymorphisms in nocturnal asthma as a feature of asthma control or severity in general, our data suggest that changes in circadian rhythm control are associated with nighttime asthma symptoms.

Neuropeptide S (NPS) variants modify the signaling and risk effects of NPS Receptor 1 (NPSR1) variants in asthma.

Single nucleotide polymorphisms (SNPs) close to the gain-of-function substitution, Asn(107)Ile (rs324981, A>T), in Neuropeptide S Receptor 1 (NPSR1) have been associated with asthma. Furthermore, a functional SNP (rs4751440, G>C) in Neuropeptide S (NPS) encodes a Val(6)Leu substitution on the mature peptide that results in reduced bioactivity. We sought to examine the effects of different combinations of these NPS and NPSR1 variants on downstream signaling and genetic risk of asthma. In transfected cells, the magnitude of NPSR1-induced activation of cAMP/PKA signal transduction pathways and downstream gene expression was dependent on the combination of the NPS and NPSR1 variants with NPS-Val(6)/NPSR1-Ile(107) resulting in strongest and NPS-Leu(6)/NPSR1-Asn(107) in weakest effects, respectively. One or two copies of the NPS-Leu(6) (rs4751440) were associated with physician-diagnosed childhood asthma (OR: 0.67, 95%CI 0.49-0.92, p = 0.01) and together with two other linked NPS variants (rs1931704 and rs10830123) formed a protective haplotype (p = 0.008) in the Swedish birth cohort BAMSE (2033 children). NPS rs10830123 showed epistasis with NPSR1 rs324981 encoding Asn(107)Ile (p = 0.009) in BAMSE and with the linked NPSR1 rs17199659 (p = 0.005) in the German MAGIC/ISAAC II cohort (1454 children). In conclusion, NPS variants modify asthma risk and should be considered in genetic association studies of NPSR1 with asthma and other complex diseases.

Characterization of sputum biomarkers for asthma-COPD overlap syndrome.

Asthma-COPD overlap syndrome (ACOS) is a commonly encountered chronic airway disease. However, ACOS is still a consensus-based clinical phenotype and the underlying inflammatory mechanisms are inadequately characterized. To clarify the inflammatory mediatypical for ACOS, five biomarkers, namely interleukin (IL)-13, myeloperoxidase (MPO), neutrophil gelatinase-associated lipocalin (NGAL), chitinase-like protein (YKL-40), and IL-6, were selected. This study hypothesized that sputum biomarkers relevant for airway inflammation in asthma (IL-13), COPD (MPO, NGAL), or in both asthma and COPD (YKL-40, IL-6) could be used to differentiate ACOS from COPD and asthma. The aim of this study was to characterize the inflammatory profile and improve the recognition of ACOS. Induced sputum levels of IL-13, MPO, NGAL, YKL-40, and IL-6 were measured by enzyme-linked immunosorbent assay/Luminex assay in a Finnish discovery cohort (n=90) of nonsmokers, smokers, and patients with asthma, COPD, and ACOS and validated in a Japanese cohort (n=135). The classification accuracy of potential biomarkers was compared with area under the receiver operating characteristic curves. Only sputum NGAL levels could differentiate ACOS from asthma (P<0.001 and P<0.001) and COPD (P<0.05 and P=0.002) in the discovery and replication cohorts, respectively. Sputum NGAL levels were independently correlated with the percentage of pre-bronchodilator forced expiratory volume in 1 second predicted in multivariate analysis in the discovery and replication cohorts (P=0.001 and P=0.002, respectively). In conclusion, sputum biomarkers reflecting both airway inflammation and remodeling of the tissue show potential in differentiation between asthma, COPD, and ACOS.

Gremlin-1 Overexpression in Mouse Lung Reduces Silica-Induced Lymphocyte Recruitment - A Link to Idiopathic Pulmonary Fibrosis through Negative Correlation with CXCL10 Chemokine.

Idiopathic pulmonary fibrosis (IPF) is characterized by activation and injury of epithelial cells, the accumulation of connective tissue and changes in the inflammatory microenvironment. The bone morphogenetic protein (BMP) inhibitor protein gremlin-1 is associated with the progression of fibrosis both in human and mouse lung. We generated a transgenic mouse model expressing gremlin-1 in type II lung epithelial cells using the surfactant protein C (SPC) promoter and the Cre-LoxP system. Gremlin-1 protein expression was detected specifically in the lung after birth and did not result in any signs of respiratory insufficiency. Exposure to silicon dioxide resulted in reduced amounts of lymphocyte aggregates in transgenic lungs while no alteration in the fibrotic response was observed. Microarray gene expression profiling and analyses of bronchoalveolar lavage fluid cytokines indicated a reduced lymphocytic response and a downregulation of interferon-induced gene program. Consistent with reduced Th1 response, there was a downregulation of the mRNA and protein expression of the anti-fibrotic chemokine CXCL10, which has been linked to IPF. In human IPF patient samples we also established a strong negative correlation in the mRNA expression levels of gremlin-1 and CXCL10. Our results suggest that in addition to regulation of epithelial-mesenchymal crosstalk during tissue injury, gremlin-1 modulates inflammatory cell recruitment and anti-fibrotic chemokine production in the lung.

Lung tissue proteomics identifies elevated transglutaminase 2 levels in stable chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease characterized by irreversible airflow limitation. Cigarette smoking represents the main risk factor, but the specific mechanisms of COPD are not completely understood. Our aim was to identify COPD-specific proteomic changes involved in disease onset and severity. A comparative proteomic analysis of 51 lung tissues from nonsmokers, smokers, smokers with mild to moderate (stage I-II) COPD, severe to very severe COPD (stage III-IV), and patients with α-1-antitrypsin deficiency (AATD) and idiopathic pulmonary fibrosis (IPF) was performed by cysteine-specific two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Selected COPD-specific changes were validated by immunoblotting and further by ELISA in 120 induced sputum and plasma samples from nonsmokers, smokers, and patients with COPD (stage I-III). Altogether 82 altered proteins were identified comprising COPD-, AATD-, and IPF-specific, overlapping, and unspecific changes. Cathepsin D (CTSD), dihydropyrimidinase-related protein 2 (DPYSL2), transglutaminase 2 (TGM2), and tripeptidyl-peptidase 1 (TPP1) were validated as COPD-specific. TGM2 was not associated with smoking and correlated with COPD severity in lung tissue. TGM2 levels in sputum and plasma were elevated in patients with COPD (stage II-III) and correlated with lung function. In conclusion, new proteins related to COPD onset and severity could be identified with TGM2 being a novel potential diagnostic and therapeutic target for COPD. Further studies in carefully characterized cohorts are required to validate the identified changes.

Inhalation of rod-like carbon nanotubes causes unconventional allergic airway inflammation.

BACKGROUND: Carbon nanotubes (CNT) represent a great promise for technological and industrial development but serious concerns on their health effects have also emerged. Rod-shaped CNT are, in fact, able to induce asbestos-like pathogenicity in mice including granuloma formation in abdominal cavity and sub-pleural fibrosis. Exposure to CNT, especially in the occupational context, happens mainly by inhalation. However, little is known about the possible effects of CNT on pulmonary allergic diseases, such as asthma. METHODS: We exposed mice by inhalation to two types of multi-walled CNT, rigid rod-like and flexible tangled CNT, for four hours a day once or on four consecutive days. Early events were monitored immediately and 24 hours after the single inhalation exposure and the four day exposure mimicked an occupational work week. Mast cell deficient mice were used to evaluate the role of mast cells in the occurring inflammation. RESULTS: Here we show that even a short-term inhalation of the rod-like CNT induces novel innate immunity-mediated allergic-like airway inflammation in healthy mice. Marked eosinophilia was accompanied by mucus hypersecretion, AHR and the expression of Th2-type cytokines. Exploration of the early events by transcriptomics analysis reveals that a single 4-h exposure to rod-shaped CNT, but not to tangled CNT, causes a radical up-regulation of genes involved in innate immunity and cytokine/chemokine pathways. Mast cells were found to partially regulate the inflammation caused by rod-like CNT, but also alveaolar macrophages play an important role in the early stages. CONCLUSIONS: These observations emphasize the diverse abilities of CNT to impact the immune system, and they should be taken into account for hazard assessment.

Soluble receptor for advanced glycation end-products and progression of airway disease.

BACKGROUND: The receptor for advanced glycation end-products (RAGE) is highly expressed in the lung, where it is believed to have a homeostatic role. Reduced plasma levels of soluble RAGE (sRAGE) have been reported in patients with chronic obstructive pulmonary disease (COPD). The aim of the present study was to evaluate the association of plasma sRAGE levels with a longitudinal decline of lung function. We have also measured plasma levels of high mobility group box 1 (HMGB1), a RAGE ligand which has been associated with chronic inflammatory diseases including COPD. METHODS: Baseline plasma concentrations of sRAGE and HMGB1 were measured in non-smokers (n = 32), smokers without COPD (n = 212), and smokers with COPD (n = 51), and the associations of the plasma sRAGE and HMGB1 levels with longitudinal declines of lung function during a 4-year follow-up period were analysed. RESULTS: The plasma levels of sRAGE were significantly lower in smokers without COPD and in smokers with COPD, as compared to those of non-smokers. Plasma sRAGE levels positively correlated with FVC and FEV1 and inversely correlated with BMI and pack-years. Lower sRAGE levels were associated with greater declines of FEV1/FVC over 4 years in all participants. Moreover, multivariate regression analysis indicated that the baseline plasma sRAGE concentration was an independent predictor of FEV1/FVC decline in all groups. A subgroup analysis showed that decreased sRAGE levels are significantly associated with a more rapid decline of FEV1/FVC in smokers with COPD. There was no significant correlation between plasma HMGB1 levels and longitudinal decline of lung function. CONCLUSIONS: Lower plasma concentrations of sRAGE were associated with greater progression of airflow limitations over time, especially in smokers with COPD, suggesting that RAGE might have a protective role in the lung.

Enhanced expression of neuropeptide S (NPS) receptor in eosinophils from severe asthmatics and subjects with total IgE above 100IU/ml.

Eosinophils are inflammatory cells of particular relevance to asthma exacerbations. Neuropeptide S (NPS) receptor was identified in a search for asthma susceptibility genes, where the risk haplotypes of the NPS receptor gene associated with total serum IgE above 100IU/ml and asthma. The aim of the present study was to investigate and compare expression of NPS receptor in human peripheral blood eosinophils derived from subjects with total serum IgE above and below 100IU/ml and patients with different phenotypes of asthma. Additionally, we aimed to study the function of NPS receptor in human eosinophils. We found higher NPS receptor protein expression in eosinophils derived from subjects with high IgE when compared to those from subjects with low IgE and the level of NPS receptor positively correlated with serum IgE. NPS receptor expression was also higher in eosinophils from patients with severe asthma than in cells from mild asthmatics or healthy controls. The receptor agonist NPS was a chemotactic agent for eosinophils. NPS also increased N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated CD11b integrin levels in eosinophils from subjects with high IgE. Furthermore, eosinophils from those subjects exhibited Ca(2+) mobilization but not cAMP rise in response to NPS. Altogether, NPS receptor may have a pathological role in individuals with severe asthma and/or elevated serum IgE levels as eosinophils from these patients express higher levels of NPS receptor protein and respond to NPS by enhanced migration and adhesion molecule expression.

Rule-based models of the interplay between genetic and environmental factors in childhood allergy.

Both genetic and environmental factors are important for the development of allergic diseases. However, a detailed understanding of how such factors act together is lacking. To elucidate the interplay between genetic and environmental factors in allergic diseases, we used a novel bioinformatics approach that combines feature selection and machine learning. In two materials, PARSIFAL (a European cross-sectional study of 3113 children) and BAMSE (a Swedish birth-cohort including 2033 children), genetic variants as well as environmental and lifestyle factors were evaluated for their contribution to allergic phenotypes. Monte Carlo feature selection and rule based models were used to identify and rank rules describing how combinations of genetic and environmental factors affect the risk of allergic diseases. Novel interactions between genes were suggested and replicated, such as between ORMDL3 and RORA, where certain genotype combinations gave odds ratios for current asthma of 2.1 (95% CI 1.2-3.6) and 3.2 (95% CI 2.0-5.0) in the BAMSE and PARSIFAL children, respectively. Several combinations of environmental factors appeared to be important for the development of allergic disease in children. For example, use of baby formula and antibiotics early in life was associated with an odds ratio of 7.4 (95% CI 4.5-12.0) of developing asthma. Furthermore, genetic variants together with environmental factors seemed to play a role for allergic diseases, such as the use of antibiotics early in life and COL29A1 variants for asthma, and farm living and NPSR1 variants for allergic eczema. Overall, combinations of environmental and life style factors appeared more frequently in the models than combinations solely involving genes. In conclusion, a new bioinformatics approach is described for analyzing complex data, including extensive genetic and environmental information. Interactions identified with this approach could provide useful hints for further in-depth studies of etiological mechanisms and may also strengthen the basis for risk assessment and prevention.

Interaction between retinoid acid receptor-related orphan receptor alpha (RORA) and neuropeptide S receptor 1 (NPSR1) in asthma.

Retinoid acid receptor-related Orphan Receptor Alpha (RORA) was recently identified as a susceptibility gene for asthma in a genome-wide association study. To investigate the impact of RORA on asthma susceptibility, we performed a genetic association study between RORA single nucleotide polymorphisms (SNPs) in the vicinity of the asthma-associated SNP (rs11071559) and asthma-related traits. Because the regulatory region of a previously implicated asthma susceptibility gene, Neuropeptide S receptor 1 (NPSR1), has predicted elements for RORA binding, we hypothesized that RORA may interact biologically and genetically with NPSR1. 37 RORA SNPs and eight NPSR1 SNPs were genotyped in the Swedish birth cohort BAMSE (2033 children) and the European cross-sectional PARSIFAL study (1120 children). Seven RORA SNPs confined into a 49 kb region were significantly associated with physician-diagnosed childhood asthma. The most significant association with rs7164773 (T/C) was driven by the CC genotype in asthma cases (OR = 2.0, 95%CI 1.36-2.93, p = 0.0003 in BAMSE; and 1.61, 1.18-2.19, p = 0.002 in the combined BAMSE-PARSIFAL datasets, respectively), and strikingly, the risk effect was dependent on the Gln344Arg mutation in NPSR1. In cell models, stimulation of NPSR1 activated a pathway including RORA and other circadian clock genes. Over-expression of RORA decreased NPSR1 promoter activity further suggesting a regulatory loop between these genes. In addition, Rora mRNA expression was lower in the lung tissue of Npsr1 deficient mice compared to wildtype littermates during the early hours of the light period. We conclude that RORA SNPs are associated with childhood asthma and show epistasis with NPSR1, and the interaction between RORA and NPSR1 may be of biological relevance. Combinations of common susceptibility alleles and less common functional polymorphisms may modify the joint risk effects on asthma susceptibility.

Transcriptome analysis reveals upregulation of bitter taste receptors in severe asthmatics.

The causes of severe childhood asthma are poorly understood. Our aim was to define global patterns of gene expression in children with severe therapy-resistant and controlled asthma. White blood cells were isolated and the global transcriptome profile was characterised using the Affymetrix Human Gene ST 1.0 chip in children with severe, therapy-resistant asthma (n=17), controlled asthma (n=19) and healthy controls (n=18). Receptor expression was studied in separated leukocyte fractions from asthmatic adults (n=12). Overall, 1378 genes were differentially expressed between children with severe/controlled asthma and controls. Three significantly enriched Kyoto Encyclopedia of Genes and Genomes pathways were represented: natural killer cell-mediated cytotoxicity (upregulated in controlled asthma); N-glycan biosynthesis (downregulated in severe asthma); and bitter taste transduction receptors (TAS2Rs) (upregulated in severe asthma). Quantitative PCR experiments confirmed upregulation of TAS2Rs in severe asthmatics. TAS2R expression was replicated in leukocytes from adult asthmatics, in which TAS2R agonists also inhibited LPS-induced cytokine release. Significant correlations between expression of TAS2Rs and clinical markers of asthma severity were found in both adults and children. In conclusion, specific gene expression patterns were observed in children with severe, therapy-resistant asthma. The increased expression of bronchodilatory TAS2Rs suggests a new target for the treatment of asthma.

The bitter taste receptor (TAS2R) agonists denatonium and chloroquine display distinct patterns of relaxation of the guinea pig trachea.

Activation of taste receptors (TAS2Rs) by bitter taste agonists has been reported to cause bronchodilation. The aim of this study was to extend the information on the effects of bitter taste agonists on responses induced by different contractile mediators in a standard airway physiology preparation. Isometric responses were assessed in guinea pig trachea (GPT). TAS2R agonists were administered either to segments precontracted with different agonists for contraction or given before challenge with the different contractile stimuli, including antigen in tissues from ovalbumin-sensitized animals. TAS2R mRNA expression on GPT epithelium and smooth muscle was measured with real-time PCR. Denatonium, chloroquine, thiamine, and noscapine induced concentration-dependent relaxations (R(max): 98.3 ± 1.6, 100.0 ± 0.0, 100.0 ± 0.0, and 52.3 ± 1.1% of maximum, respectively, in the presence of indomethacin) in segments precontracted with carbachol. The receptors for denatonium (TAS2R4, TAS2R10) and chloroquine (TAS2R3, TAS2R10) were expressed in GPT. Whereas denatonium selectively inhibited contractions induced by carbachol, chloroquine uniformly inhibited contractions evoked by prostaglandin E(2), the thromboxane receptor agonist U-46619, leukotriene D(4), histamine, and antigen. The effects of denatonium, but not those of chloroquine, were partly inhibited by blockers of the large Ca(2+)-activated K(+) channels and decreased by an increase of the level of precontraction. In conclusion, TAS2R agonists mediated strong relaxations and substantial inhibition of contractions in GPT. Chloroquine and denatonium had distinct patterns of activity, indicating different signaling mechanisms. The findings reinforce the hypothesis that TAS2Rs are potential targets for the development of a new class of more efficacious agonists for bronchodilation.

A novel screening method detects herpesviral DNA in the idiopathic pulmonary fibrosis lung.

BACKGROUND: Herpesviruses could contribute to the lung epithelial injury that initiates profibrotic responses in idiopathic pulmonary fibrosis (IPF). METHODS: We identified herpesviral DNA from IPF and control lung tissue using a multiplex PCR-and microarray-based method. Active herpesviral infection was detected by standard methods, and inflammatory cell subtypes were identified with specific antibodies. Patients that underwent lung transplantation were monitored for signs of herpesviral infection. RESULTS: A total of 11/12 IPF samples were positive for Epstein-Barr virus (EBV) and 10/12 for human herpesvirus 6B (HHV-6B) DNA. Control lung samples (n = 10) were negative for EBV DNA, whereas three samples were positive for HHV-6B. EBV-encoded RNA (EBER) was identified in nine IPF samples and localized mainly to lymphocytic aggregates. HHV-6B antigens were detected in mononuclear cells in IPF lung tissue. CD20+ B lymphocytic aggregates that were surrounded by CD3+ T cells were abundant in IPF lungs. CD23+ cells (activated B cells, EBV-transformed lymphoblasts, and dendritic cells) were observed in the aggregates. IPF patients had no signs of increased herpesviral activation after lung transplantation. CONCLUSIONS: Inflammatory cells are the main source of herpesviral DNA in the human IPF lung. Diagnostic tools should be actively used to elucidate whether herpesviral infection affects the pathogenesis, progression, and/or exacerbation of IPF.

The asthma candidate gene NPSR1 mediates isoform specific downstream signalling.

BACKGROUND: Neuropeptide S Receptor 1 (NPSR1, GPRA, GPR154) was first identified as an asthma candidate gene through positional cloning and has since been replicated as an asthma and allergy susceptibility gene in several independent association studies. In humans, NPSR1 encodes two G protein-coupled receptor variants, NPSR1-A and NPSR1-B, with unique intracellular C-termini. Both isoforms show distinct expression pattern in asthmatic airways. Although NPSR1-A has been extensively studied, functional differences and properties of NPSR1-B have not yet been clearly examined. Our objective was to investigate downstream signalling properties of NPSR1-B and functional differences between NPSR1-A and NPSR1-B. METHODS: HEK-293 cells transiently overexpressing NPSR1-A or NPSR1-B were stimulated with the ligand neuropeptide S (NPS) and downstream signalling effects were monitored by genome-scale affymetrix expression-arrays. The results were verified by NPS concentration-response and time series analysis using qRT-PCR, cAMP and Ca²âº assays, and cAMP/PKA, MAPK/JNK and MAPK/ERK pathway specific reporter assays. RESULTS: NPSR1-B signalled through the same pathways and regulated the same genes as NPSR1-A, but NPSR1-B yielded lower induction on effector genes than NPSR1-A, with one notable exception, CD69, a marker of regulatory T cells. CONCLUSIONS: We conclude that NPSR1-B is regulating essentially identical set of genes as NPSR1-A, with few, but possibly important exceptions, and that NPSR1-A induces stronger signalling effects than NPSR1-B. Our findings suggest an isoform-specific link to pathogenetic processes in asthma and allergy.

Transcription factor GATA-6 is expressed in quiescent myofibroblasts in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) (histopathology of usual interstitial pneumonia [UIP]) is a progressive disease with poor prognosis. Characteristic features of IPF/UIP include fibroblastic foci, which are patchy lesions of focal, disarranged myofibroblasts. GATA-6 is a transcription factor linked with cell differentiation. Its role in the development of IPF has not previously been investigated. We hypothesized that GATA-6 participates in the differentiation of fibroblasts into myofibroblasts in IPF/UIP lungs. The expression patterns of GATA-6, the mesenchymal marker alpha-smooth muscle actin (alpha-SMA), and markers for proliferation (Ki67) and apoptosis (caspase-3) were analyzed in human IPF/UIP tissue samples. The effects of GATA-6 overexpression and silencing were studied in cell cultures. The results show that the alpha-SMA-positive fibroblastic foci in IPF/UIP lungs are positive for GATA-6, but negative for Ki67 and caspase-3. Cultured human IPF/UIP fibroblasts expressed GATA-6 mRNA, whereas cells from the normal adult lung did not. In cultured A549 lung epithelial cells, the induction of GATA-6 by transforming growth factor-beta1 resulted in simultaneous expression of alpha-SMA and decrease of E-cadherin. The inhibition of GATA-6 expression in fibroblasts showed that GATA-6 mediates the alpha-SMA-inducing signal of transforming growth factor-beta1. In conclusion, the hallmark of IPF/UIP histopathology, the fibroblast focus, consists of differentiated, quiescent cells that prominently express GATA-6.

ELMOD2, a candidate gene for idiopathic pulmonary fibrosis, regulates antiviral responses.

Viral infections and abnormal host response are thought to cause epithelial injury in idiopathic pulmonary fibrosis (IPF). To understand IPF pathogenesis, we have used overexpression cell models and expression microarrays to discover genes networked with ELMO domain containing 2 (ELMOD2) gene genetically implicated in IPF. The identified pathways were confirmed in vitro, and ELMOD2 protein expression was characterized in tissue samples. Here 303 genes were significantly altered after ELMOD2 transfection of human alveolar epithelial A549 cell line. The enriched pathways were interferon induction, viral response, antigen processing and presentation, and I-/nuclear factor-kappaB signaling. ELMOD2 showed immunoreactivity in macrophages and type II alveolar epithelial cells in normal human lung. In A549 cells, forced expression of ELMOD2 increased type I and type III interferon mRNA expression, and ELMOD2-specific siRNA molecules inhibited expression of these antiviral cytokines in response to Toll-like receptor three (TLR3) activation. In human macrophages silencing of ELMOD2 inhibited TLR3-dependent expression of type I and type III interferon genes. Influenza A virus infection decreased ELMOD2 mRNA expression in A549 cells and macrophages suggesting negative regulation in viral infections. In summary, our results show that TLR3 pathway is dependent on ELMOD2.-Pulkkinen, V., Bruce, S., Rintahaka, J., Hodgson, U., Laitinen, T., Alenius, H., Kinnula, V. L., Myllärniemi, M., Matikainen, S., Kere, J. ELMOD2, a candidate gene for idiopathic pulmonary fibrosis, regulates antiviral responses.

Neuropeptide s receptor 1 gene polymorphism is associated with susceptibility to inflammatory bowel disease.

BACKGROUND & AIMS: The neuropeptide S receptor (NPSR1) gene has been associated recently with asthma and maps in a region of chromosome 7 previously linked also to inflammatory bowel disease (IBD). NPSR1 is expressed on the epithelia of several organs including the intestine, and appears to be up-regulated in inflammation. We tested NPSR1 gene polymorphism for association with IBD and verified whether the expression of its 2 major isoforms (NPSR1-A and NPSR1-B) is altered in the intestine of IBD patients. METHODS: Eight NPSR1 polymorphisms were genotyped in 2490 subjects from 3 cohorts of IBD patients and controls from Italy, Sweden, and Finland. Real-time polymerase chain reaction and immunohistochemistry were used to quantify NPSR1 messenger RNA (mRNA) and protein expression in intestinal biopsy specimens from IBD patients and controls. RESULTS: Global analysis of the whole dataset identified strong association of a NPSR1 haplotype block with IBD (P = .0018) and its 2 major forms: Crohn's disease (CD) (P = .026) and ulcerative colitis (UC) (P = .003). Genetic effects caused by individual haplotypes were identified mainly for the predisposing haplotype H2 in CD (P = .0005) and the protective haplotype H8 in UC (P = .003). NPSR1 mRNA and protein levels were increased in IBD patients compared with controls, and the risk haplotype H2 correlated with higher expression of both NPSR1-A (P = .024) and NPSR1-B (P = .047) mRNAs. CONCLUSIONS: NPSR1 polymorphism is associated with IBD susceptibility. Specific NPSR1 alleles might act as genetic risk factors for chronic inflammatory diseases of the epithelial barrier organs.

G protein-coupled receptor for asthma susceptibility associates with respiratory distress syndrome.

BACKGROUND: Respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD) have some common features with asthma. AIM: To study whether G protein-coupled receptor for asthma susceptibility (GPRA) contributes to RDS or BPD. METHODS: A haplotype association study was performed in a case-control setting of 521 Finnish infants (including 176 preterm neonates with RDS and 37 with BPD). Immunoreactivity of GPRA isoforms A and B was determined in pulmonary samples of fetuses, term infants and preterm infants with RDS or BPD. GPRA mRNA expression was determined by quantitative real-time polymerase chain reaction (PCR) in samples from nasal respiratory epithelium of adults, term infants and preterm infants. RESULTS: In infants with RDS born at 32-35 weeks of gestation, GPRA haplotype H1 was significantly underrepresented in RDS, whereas haplotype H4/H5 was associated with an increased risk. As in asthma, GPRA B isoform was induced in bronchial smooth muscle cells in RDS and BPD. In nasal respiratory epithelium, relative GPRA mRNA expression was strong in adults, weak in preterm and slightly higher in term samples. CONCLUSIONS: The results suggest that near-term RDS and asthma share the same susceptibility and protective GPRA haplotypes. Altered GPRA expression may play a role in the pathogenesis of RDS and BPD in preterm infants.