Biomarkers of chronic airflow limitation and COPD identified by mass spectrometry.

Rationale: COPD affects 300 million people worldwide and is the third leading cause of death according to World Health Organization global health estimates. Early symptoms are subtle, and so COPD is often diagnosed at an advanced stage. Thus, there is an unmet need for biomarkers that can identify individuals at early stages of the disease before clinical symptoms have manifested. To date, few biomarkers are available for clinical diagnostic use in COPD. Methods: We evaluated a panel of serum biomarkers related to inflammation and infection for their ability to discriminate between 77 subjects with chronic airflow limitation (CAL) and 142 subjects with COPD, versus 150 healthy subjects (divided into two control groups that were matched with regards to age, gender and smoking to CAL and COPD). Healthy subjects and CAL were from Burden of Obstructive Lung Disease (BOLD), a population-based study. CAL was defined by post-bronchodilatory forced expiratory volume in 1 s/forced vital capacity ratio <0.7 in the BOLD population. COPD subjects were from Tools for Identifying Exacerbations (TIE), a COPD patient cohort. Quantification of 100 biomarker candidates was done by liquid chromatography-tandem mass spectrometry. Results: Several protein-derived peptides were upregulated in CAL, compared to controls; most notably peptides representing histidine-rich glycoprotein (HRG), α1-acid glycoprotein (AGP1), α1-antitrypsin (α1AT) and fibronectin. Out of these, HRG-, AGP1- and α1AT-specific peptides were also elevated in the COPD cohort. Conclusion: HRG, AGP1 and α1AT biomarkers distinguish subjects with CAL and COPD from healthy controls. HRG and AGP1 represent novel findings.

Rhinovirus-induced wheeze was associated with asthma development in predisposed children.

AIM: This study explored whether early-life factors, such as rhinovirus-induced wheeze and allergic sensitisation, were related to asthma at 11 years of age. METHODS: We focused on 107 children aged 6-48 months, who attended the paediatric emergency department at Astrid Lindgren's Children's Hospital in Stockholm, Sweden, with acute wheeze in 2008-2012. They also attended follow-up visits at 11 years of age and were compared with 46 age-matched healthy controls. Odds ratios (OR) with 95% confidence intervals (CI) were calculated with logistic regression. RESULTS: We found that 62.6% of the acute wheeze cases had asthma at 11 years of age. Rhinoviruses at inclusion were the only common airway viruses associated with an increased asthma risk (OR 2.4, 95% CI 1.02-5.6). Other increased risks were parental heredity for asthma and/or allergies (adjusted OR 3.4, 95% CI 1.1-9.9) and allergic sensitisation at 2 years of age (adjusted OR 3.0, 95% CI 1.02-8.7). The highest prevalence of asthma was when children had both rhinovirus-induced wheeze at inclusion and allergic sensitisation at 7 years of age. CONCLUSION: Our findings highlight the importance of hereditary factors and allergic sensitisation on the development of asthma and suggest that rhinoviruses are associated with asthma development in predisposed children.

Inflammatory related plasma proteins involved in acute preschool wheeze.

BACKGROUND: Preschool wheeze is a risk factor for asthma development. However, the molecular mechanism behind a wheezing episode is not well understood. OBJECTIVE: Our aims were to assess the association of plasma proteins with acute preschool wheeze and to study the proteins with differential expression at the acute phase at revisit after 3 months. Additionally, to investigate the relationship between protein expression and clinical parameters. METHOD: We measured 92 inflammatory proteins in plasma and clinical parameters from 145 children during an episode of preschool wheeze (PW) and at the revisit after 3 months (PW-R, n = 113/145) and 101 healthy controls (HC) aged 6-48 months in the GEWAC cohort using the antibody-mediated proximity extension-based assay (Olink Proteomics, Uppsala). RESULTS: Of the 74 analysed proteins, 52 were differentially expressed between PW and HC. The expression profiles of the top 10 proteins, Oncostatin M (OSM), IL-10, IL-6, Fibroblast growth factor 21 (FGF21), AXIN1, CXCL10, SIRT2, TNFSF11, Tumour necrosis factor ß (TNF-ß) and CASP8, could almost entirely separate PW from HC. Five out of 10 proteins were associated with intake of oral corticosteroids (OCS) 24 h preceding blood sampling (OSM, CASP8, IL-10, TNF-ß and CXCL10). No differences in protein expression were seen between PWs with or without OCS in comparison to HC. At the revisit after 3 months, differential protein expressions were still seen between PW-R and HC for three (IL-10, SIRT2 and FGF21) of the 10 proteins. CONCLUSION: Our results contribute to unravelling potential immunopathological pathways shared between preschool wheeze and asthma.

Serum Eosinophilic Cationic Protein Is a Reliable Biomarker for Childhood Asthma.

BACKGROUND: Eosinophilic cationic protein (ECP) is associated with airway inflammation and asthma. However, the clinical value of measuring ECP in childhood asthma is not fully known. We aimed to study the diagnostic performance of serum ECP and other common asthma biomarkers, individually and in combinations. METHODS: In a cross-sectional study, 5-16-year-old children with current asthma (CA) (n = 37), transient asthma (TA) (n = 43), (previous history of wheezing/asthma), and healthy children (HC) (n = 86) were investigated for ECP, blood eosinophil count (B-Eos), fractional exhaled nitric oxide (FeNO), and lung function, i.e., spirometry (forced expiratory volume during the first second [FEV1]/forced vital capacity [FVC] ratio). RESULTS: Both ECP and B-Eos were higher in CA compared to TA (p < 0.01) and HC (p < 0.0001). ECP and B-Eos were also higher in TA compared to HC (p < 0.05 and p < 0.001, respectively). FeNO was higher in CA (p < 0.0001) and TA (p < 0.01) compared to HC but similar between the asthma groups. The FEV1/FVC ratio was lower in CA compared to TA and HC (both p < 0.01) but similar between TA and HC. The best diagnostic performance regarding CA was found for ECP and B-Eos with receiver operating characteristics area under curve (AUC) of 0.801 and 0.810, respectively. The optimal cutoff for ECP (29 µg/L) yielded a sensitivity and specificity of 70.3% and 81.4%. The corresponding AUCs for FeNO and FEV1/FVC were 0.732 and 0.670, respectively. ECP and B-Eos showed the highest AUCs (0.669 and 0.673) for differentiation between CA and TA. Combining ECP with FeNO and FEV1/FVC increased the odds ratio (OR) for having CA from OR 3.97-10.3 for the single biomarkers to OR 20.2 (95% confidence interval: 5.76-68.6). CONCLUSION: Our results show that serum ECP is a reliable biomarker in the diagnosis of childhood asthma, with additional value in combination with FeNO and FEV1/FVC, and that ECP can be an alternative to B-Eos.

Development of Sensitization to Multiple Allergen Molecules from Preschool to School Age Is Related to Asthma.

INTRODUCTION: Allergic sensitization in early life has been identified as a strong risk factor for subsequent asthma in childhood. It is still unclear why only a part of sensitized children develop asthma, and the role of specific allergen molecules in asthma pathogenesis is ambiguous [Pharmacol Ther. 2009 Feb;121(2):174-84]. We assessed the sensitization to multiple allergen molecules longitudinally and explored its relation to persistent asthma at 7 years. METHODS: Seventy-two children included during an acute wheezing episode (cases) were followed prospectively from early preschool age (EPA) to age 7, and compared to 43 healthy controls at EPA. Allergen molecules were analyzed at EPA and age 7 using ImmunoCAP Solid-phase Allergen Chip (ISAC). Asthma diagnosis at 7 years was based on symptoms, medication, and spirometry. RESULTS: At EPA, cases compared to controls showed a tendency toward having a higher prevalence of allergic sensitization (23.6% vs. 9.3%, p = 0.055). The prevalence of sensitization increased in cases from EPA to 7 years (23.6% vs. 38.9%; p = 0.048) as well as the median number (range) of immunoglobulin E (IgE)-reactive molecules 3 (3-14) versus 6.5 (1-21); p = 0.024. Sensitization to each additional molecule from EPA to the age of 7 was significantly related to asthma at 7 (OR = 1.25, 95% confidence interval [1.01, 1.54]). CONCLUSION: Polysensitization, assessed by allergen molecules, had a significant impact on persistent asthma at school age. The extent of sensitization, illustrated by molecular spreading from preschool to school age, was related to asthma diagnosis at 7 years in children with a history of wheezing at early life.

Importance of type and degree of IgE sensitisation for defining fractional exhaled nitric oxide reference values.

BACKGROUND: Fractional exhaled nitric oxide (FENO) is a marker of type 2 airway inflammation used in clinical practice in asthma. However, reference values are needed to broaden the clinical use of FENO and this is within the scope of a newly started Global Lung Function Initiative task force. We aim to study FENO levels with special emphasis on the upper limit of normal (ULN) in relation to the type and degree of IgE sensitisation. METHODS: FENO was measured in 1855 non-smoking, respiratory healthy subjects from the Swedish CArdioPulmonary bioImage Study (SCAPIS). Atopic subjects (n = 424), defined as being IgE-sensitised to aeroallergens (ImmunoCAP Phadiatop™, ≥0.35 PAU/l) were compared to non-atopic subjects (<0.35 PAU/l, n = 1431). Atopic subjects were further characterised according to their grade of IgE sensitisation (IgE antibody tertiles: (T1<1.16, T2 1.16-3.72 and T3 >3.72 PAU/l) and sensitisation to perennial (cat or mite) or seasonal (birch) allergens. RESULTS: Subjects IgE-sensitised to cat or mite had higher FENO compared to non-atopic subjects (FENO (ppb): median 20.0 vs. 15.0, and ULN 50.4 vs. 33.0, p < 0.001). This was seen to a lesser extent for subjects IgE-sensitised to birch only (median 18.0 vs. 15.0, and ULN 38.0 vs. 33.0, p = 0.048). Atopic subjects with a high degree of IgE sensitisation (Phadiatop: >3.72 PAU/l) had the highest FENO compared to non-atopic subjects (median 20.0 vs. 15.0, and ULN 56.0 vs. 33.0, p < 0.001). CONCLUSIONS: The type and degree of IgE sensitisation should be considered in generating FENO reference values.

Development of an automated ImmunoCAP research assay for eosinophil derived neurotoxin and its use in asthma diagnosis in children.

BACKGROUND: Eosinophil Derived Neurotoxin (EDN) is an eosinophil granule protein that is released during eosinophil activation. EDN has proven to be a promising marker for eosinophilic inflammation both in asthma and in wheezing symptoms in children. OBJECTIVES: Here we present a novel ImmunoCAPTM automated immunofluorescence research assay for measurement of EDN and its potential use in diagnosis of asthmatic children. METHODS: We report the analytical performance of the assay in serum, heparin- and EDTA-plasma in terms of precision, linearity, sensitivity, interfering substances and specimen handling. We also compared the EDN research assay with established methods for asthma diagnosis: fraction of exhaled nitric oxide (FeNO) and blood eosinophil fraction (EOS%) to demonstrate the diagnostic value of EDN in childhood asthma. RESULTS: The total precision (measured using percentage CV) was ≤5.8% for both serum and plasma. The dilution analysis yielded linear results across the dynamic range of the assay for both serum and plasma. No notable interferences of endogenous substances were observed.The median concentration of EDN was significantly higher in the asthma group compared to the healthy controls and the EDN correlates well with EOS%. CONCLUSIONS: we have shown that EDN can be measured reliably and robustly with the ImmunoCAP platform in both serum and plasma samples. EDN can be used to distinguish asthmatic from healthy children and correlates well with EOS% and could be a valuable complement to both EOS% and FeNO.

Population genomics of Mesolithic Scandinavia: Investigating early postglacial migration routes and high-latitude adaptation.

Scandinavia was one of the last geographic areas in Europe to become habitable for humans after the Last Glacial Maximum (LGM). However, the routes and genetic composition of these postglacial migrants remain unclear. We sequenced the genomes, up to 57× coverage, of seven hunter-gatherers excavated across Scandinavia and dated from 9,500-6,000 years before present (BP). Surprisingly, among the Scandinavian Mesolithic individuals, the genetic data display an east-west genetic gradient that opposes the pattern seen in other parts of Mesolithic Europe. Our results suggest two different early postglacial migrations into Scandinavia: initially from the south, and later, from the northeast. The latter followed the ice-free Norwegian north Atlantic coast, along which novel and advanced pressure-blade stone-tool techniques may have spread. These two groups met and mixed in Scandinavia, creating a genetically diverse population, which shows patterns of genetic adaptation to high latitude environments. These potential adaptations include high frequencies of low pigmentation variants and a gene region associated with physical performance, which shows strong continuity into modern-day northern Europeans.

Fecal Calprotectin and Eosinophil-derived Neurotoxin in Healthy Children Between 0 and 12 Years.

OBJECTIVES: In young children, the use of fecal calprotectin (fCP) as a biomarker is limited because reference values have not been widely accepted up to now. Moreover, reference values for fecal eosinophil-derived neurotoxin (fEDN) in children have not been established. The aim of the present study was to investigate fCP and fEDN levels in young healthy children to establish reference values. METHODS: Stool samples were obtained from healthy children ages 0 to 12 years. fCP and fEDN levels were analyzed using the EliA Calprotectin 2 assay (Phadia AB) and a novel research assay (on the ImmunoCAP platform), respectively. RESULTS: In the 174 included children (87 boys), 95th Percentile values ranged from 1519 mg/kg at 0 months to 54.4 mg/kg at 144 months for fCP and from 9.9 mg/kg at 0 months to 0.2 mg/kg at 144 months for fEDN. There was a statistically significant association between age and fCP concentrations (P < 0.001) and age and fEDN concentrations (P < 0.001). We also found a statistically significant association between fEDN and fCP concentrations (rho = 0.52, P < 0.001). According to our results, we provide a nomogram and we suggest 3 different age groups for evaluation of fCP and fEDN concentrations, the 95th percentile being respectively 910.3 and 7.4 mg/kg for 0-12 months, 285.9 and 2.9 mg/kg for >1 to 4 years, and 54.4 and 0.2 mg/kg for >4 to 12 years. DISCUSSION: By using an improved analytical method, we have confirmed that young healthy children have higher fCP concentrations than healthy adults. We, for the first time, report reference values for fEDN concentrations in a pediatric population. The proposed nomograms and reference values for fCP and fEDN are aimed at facilitating the applicability of biomarkers for both neutrophil- and eosinophil-mediated intestinal inflammation in children in clinical practice.

NY-ESO-1 protein formulated in ISCOMATRIX adjuvant is a potent anticancer vaccine inducing both humoral and CD8+ t-cell-mediated immunity and protection against NY-ESO-1+ tumors.

NY-ESO-1 is a 180 amino-acid human tumor antigen expressed by many different tumor types and belongs to the family of "cancer-testis" antigens. In humans, NY-ESO-1 is one of the most immunogenic tumor antigens and NY-ESO-1 peptides have been shown to induce NY-ESO-1-specific CD8(+) CTLs capable of altering the natural course of NY-ESO-1-expressing tumors in cancer patients. Here we describe the preclinical immunogenicity and efficacy of NY-ESO-1 protein formulated with the ISCOMATRIX adjuvant (NY-ESO-1 vaccine). In vitro, the NY-ESO-1 vaccine was readily taken up by human monocyte-derived dendritic cells, and on maturation, these human monocyte-derived dendritic cells efficiently cross-presented HLA-A2-restricted epitopes to NY-ESO-1-specific CD8(+) T cells. In addition, epitopes of NY-ESO-1 protein were also presented on MHC class II molecules to NY-ESO-1-specific CD4(+) T cells. The NY-ESO-1 vaccine induced strong NY-ESO-1-specific IFN-gamma and IgG2a responses in C57BL/6 mice. Furthermore, the NY-ESO-1 vaccine induced NY-ESO-1-specific CD8(+) CTLs in HLA-A2 transgenic mice that were capable of lysing human HLA-A2(+) NY-ESO-1(+) tumor cells. Finally, C57BL/6 mice, immunized with the NY-ESO-1 vaccine, were protected against challenge with a B16 melanoma cell line expressing NY-ESO-1. These data illustrate that the NY-ESO-1 vaccine represents a potent therapeutic anticancer vaccine.