Biomarkers in Inflammatory Childhood Diseases

Mediators of Inflammation

Guest Editors: Wilco de Jager, Dirk Holzinger, and Ger T. Rijkers Biomarkers in Inflammatory Childhood Diseases Mediators of Inflammation Biomarkers in Inflammatory Childhood Diseases

This is a special issue published in “Mediators of Inflammation.” All articles are open access articles distributed under the Creative Com- mons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Editorial Board

Anshu Agrawal, USA Nina Ivanovska, Bulgaria Vera L. Petricevich, Mexico Muzamil Ahmad, India Yong Jiang , China Peter Plomgaard, Denmark Simi Ali, UK Yona Keisari, Israel Marc Pouliot, Canada Philip Bufler, Germany Alex Kleinjan, The Netherlands Michal Amit Rahat, Israel Hidde Bult, Belgium Magdalena Klink, Poland Jean-Marie Reimund, France Elisabetta Buommino, Italy Elzbieta Kolaczkowska, Poland Alexander Riad, Germany Dianne Cooper, UK Dmitri V. Krysko, Belgium Huub Savelkoul, The Netherlands Guanglin Cui, Norway Philipp M. Lepper, Germany Natalie J. Serkova, USA Fulvio D’Acquisto, UK Changlin Li, USA Sunit Kumar Singh, India Pham My-Chan Dang, France E. Lopez-Collazo,´ Spain Dirk E. Smith, USA Beatriz De las Heras, Spain Antonio Maccio,` Italy Helen Steel, South Africa Chiara De Luca, Italy A. Malamitsi-Puchner, Greece Dennis D. Taub, USA Yves Denizot, France Sunil Kumar Manna, India Kathy Triantafilou, UK Clara Di Filippo, Italy Francesco Marotta, Italy Fumio Tsuji, Japan Bruno L. Diaz, Brazil Donna-Marie McCafferty, Canada Peter Uciechowski, Germany Maziar Divangahi, Canada Celine´ Mehats,´ France Giuseppe Valacchi, Italy Amos Douvdevani, Israel Barbro Melgert, The Netherlands Luc Vallieres,` Canada Stefanie B. Flohe,´ Germany Vinod K. Mishra, USA Jan van Amsterdam, The Netherlands Taniaˆ S. Frode,¨ Brazil Eeva Moilanen, Finland Elena Voronov, Israel Julio Galvez, Spain Eric F. Morand, Australia Jyoti J. Watters, USA Christoph Garlichs, Germany Jonas Mudter, Germany Soh Yamazaki, Japan Ronald Gladue, USA Marja Ojaniemi, Finland Satoru Yui, Japan Hermann Gram, Switzerland Sandra Oliveira, Brazil Teresa Zelante, Singapore Oreste Gualillo, Spain Andrew Parker, Switzerland Dezheng Zhao, USA Elaine Hatanaka, Brazil Jonathan Peake, Austria Freek Zijlstra, The Netherlands Contents

Biomarkers in Inflammatory Childhood Diseases, Wilco de Jager, Dirk Holzinger, and Ger T. Rijkers Volume 2013, Article ID 745493, 2 pages

IL-1𝛽 and IL-6 Upregulation in Children with H1N1 Influenza Virus Infection,AntonioChiaretti, Silvia Pulitano,` Giovanni Barone, Pietro Ferrara, Valerio Romano, Domenico Capozzi, and Riccardo Riccardi Volume 2013, Article ID 495848, 8 pages

Evaluation of Clinical and Immunological Responses: A 2-Year Follow-Up Study in Children with Allergic Rhinitis due to House Dust Mite, Heleen Moed, Roy Gerth van Wijk, Rudi W. Hendriks, and J. C. van der Wouden Volume 2013, Article ID 345217, 8 pages

Evaluation of Adipokines: Apelin, Visfatin, and Resistin in Children with Atopic Dermatitis, Edyta Machura, Maria Szczepanska, Katarzyna Ziora, Dariusz Ziora, Elzbieta Swietochowska, Malgorzata Barc-Czarnecka, and Alicja Kasperska-Zajac Volume 2013, Article ID 760691, 8 pages

Circulating Cytokines and Growth Factors in Pediatric Pulmonary Hypertension,MarkDuncan, BrandieD.Wagner,KeriMurray,JennaAllen,KelleyColvin,FrankJ.Accurso,andD.DunbarIvy Volume 2012, Article ID 143428, 7 pages

Symptoms, but Not a Biomarker Response to Inhaled Corticosteroids, Predict Asthma in Preschool Children with Recurrent Wheeze,E.M.M.Klaassen,K.D.G.vandeKant,Q.Jobsis,¨ S. T. P. Høvig, C. P. van Schayck, G. T. Rijkers, and E. Dompeling Volume 2012, Article ID 162571, 7 pages

Update of Faecal Markers of Inflammation in Children with Cystic Fibrosis,JungM.Lee, Steven T. Leach, Tamarah Katz, Andrew S. Day, Adam Jaffe, and Chee Y. Ooi Volume 2012, Article ID 948367, 6 pages Hindawi Publishing Corporation Mediators of Inflammation Volume 2013, Article ID 745493, 2 pages http://dx.doi.org/10.1155/2013/745493

Editorial Biomarkers in Inflammatory Childhood Diseases

Wilco de Jager,1 Dirk Holzinger,2,3 and Ger T. Rijkers4,5

1 Centre of Molecular and Cellular Intervention, Department of Paediatric Immunology, University Medical Centre Utrecht, Lundlaan 6, 3584 EA Utrecht, The Netherlands 2 Department of Paediatric Rheumatology and Immunology, University Children’s Hospital Muenster, Muenster, Germany 3 Institute of Immunology, University Hospital Muenster, 48147 Muenster, Germany 4 Department of Science, University College Roosevelt, Utrecht University Lange Noordstraat 1, 4330 AB Middelburg, The Netherlands 5 LaboratoryofMedicalMicrobiologyandImmunology,St.AntoniusHospital,P.O.Box2500,3430EMNieuwegein,TheNetherlands

Correspondence should be addressed to Wilco de Jager; [email protected]

Received 14 May 2013; Accepted 14 May 2013

Copyright © 2013 Wilco de Jager et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Biomarkers are of crucial importance in modern medicine [2]. Therefore, insight and progression of complex paediatric for risk-assessment, disease prevention, early diagnosis, drug diseases, in particular inflammatory diseases, can benefit target identification, drug response and monitoring of dis- from establishment and validation of a comprehensive set of ease activity. Biomarkers can be defined as characteristics biomarkers. which are measured and evaluated as indicators of a normal In this special Issue on Biomarkers in Inflammatory biologic,orpathologicprocess,orpharmacologicresponses Childhood Diseases, various paediatric diseases are discussed towards a therapeutic intervention [1]. Although the term which are either chronic such as cystic fibrosis and allergic biomarker is relatively new, biomarkers have been used for rhinitis or asthma, or acute disease with life threatening considerable time, in fact as long as clinical medicine. For complications such as paediatric pulmonary hypertension example, body temperature is a well-known biomarker for and H1N1 infection. Of interest to note is that there are fever; serum levels of C-reactive protein (the most performed several contributions which use or discuss non invasive assay in hospital laboratories) is a marker for inflammation sample to acquire biological material of a diseased organ, andglucoselevelsareusedfordiagnosisaswellastherapeutic such as exhaled breath condensate from asthmatic children, readout for controlling diabetes. and faeces of cystic fibrosis patients with potential intestinal ForthisspecialissueonBiomarkersinInflammatory inflammation. Furthermore, for optimal data mining several Childhood Diseases we took particular interest in studies researchers also implemented state of the art new multiplex dealing with biomarkers for immune mediated childhood technologies which can handle micro volumes of biological diseases. Besides the fact that biomarkers for childhood samples, and produce more insight information in compari- diseases are, due to ethical restrictions, small population son with classical methodologies. sizes, and limited sample volumes less available, in most Duncan and colleagues from Colorado, USA, report cases the normal reference range is extrapolated from adult promising new data on circulating cytokines, in particular populations or animal models and therefore not always EGF, IL-6 and VEGF, which could predict adverse events in applicable for paediatric patients. As the pediatric population children with pulmonary hypertension. In a small cohort of is in the process of development, both mentally and physically children with H1N1 infection, Chiaretti et al. from Rome, they are at risk for other hazards in comparison with adults. Italy, show that elevated IL-1𝛽 and IL-6 are associated with Furthermore, when studying the various parameters of the disease severity, but not with ultimate outcome. Moed et al. human immune system changes are observed during aging have investigated the value of a set of both cellular as well as which have to be taken in account in paediatric patients cytokine biomarkers in children with allergic rhinitis. While 2 Mediators of Inflammation the biomarker profile was of use in establishing the severity of the allergic rhinitis, long term follow-up demonstrated reduction of allergic symptoms with persistence of the in vitro cellular abnormalities. Also in predicting which children with wheezing symptoms would progress to asthma, as was studied by Klaassen et al. in Maastricht, clinical symptoms are better predictors than biomarkers in exhaled breath condensate.Incysticfibrosis,pulmonaryinflammationisamajor clinical expression of the disease and in the majority of cases respiratory failure is the cause of mortality. Accumulating data now show that intestinal inflammation contributes to the disease and Lee et al. from Sidney, Australia review this evidence, in particular the value of faecal inflammatory markers such as calprotectin and S100A12. The final paper deals with childhood obesity. Adipose tissue is a major source for adipokines, which include hormones such as leptin and adiponectin, cytokines, as well as proteins like apelin and resistin. Machura et al. from Zabrze in Poland have evaluated these adipokines in children with atopic dermatitis and find apelin and visfatin excellent biomarkers for atopic dermatitis. This special issue on Biomarkers in Inflammatory Child- hood Diseases thus reflects the diversity of research in this field and shows the potential of biomarker research in paediatrics. Taken together, the strength of combining noninvasive sampling of biological samples such as saliva, urine, faeces and exhaled breath condensates, as well as multiplex analysis methodologies which can handle small sized sample volumes holds the future for biomarker research in paediatric populations. Wilco de Jager Dirk Holzinger GerT.Rijkers

References

[1] A. J. Atkinson Jr., W. A. Colburn, V. G. DeGruttola et al., “Biomarkers and surrogate endpoints, preferred definitions and conceptual framework,” Clinical Pharmacology & Therapeutics, vol. 69, pp. 89–95, 2001. [2]P.D.Rymkiewicz,Y.X.Heng,A.Vasudev,andA.Larbi,“The immune system in the aging human,” Immunologic Research, vol.53,no.1–3,pp.235–250,2012. Hindawi Publishing Corporation Mediators of Inflammation Volume 2013, Article ID 495848, 8 pages http://dx.doi.org/10.1155/2013/495848

Research Article IL-1𝛽 and IL-6 Upregulation in Children with H1N1 Influenza Virus Infection

Antonio Chiaretti,1 Silvia Pulitanò,2 Giovanni Barone,1 Pietro Ferrara,1 Valerio Romano,1 Domenico Capozzi,1 and Riccardo Riccardi1

1 Department of Pediatrics, Catholic University of the Sacred Heart, A. Gemelli Hospital, Policlinico Gemelli, Largo Gemelli, 1-00168 Rome, Italy 2 Pediatric Intensive Care Unit, Catholic University of the Sacred Heart, A. Gemelli Hospital, 00168 Rome, Italy

Correspondence should be addressed to Antonio Chiaretti; [email protected]

Received 22 June 2012; Accepted 31 March 2013

Academic Editor: Ger Rijkers

Copyright © 2013 Antonio Chiaretti et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The role of cytokines in relation to clinical manifestations, disease severity, and outcome of children with H1N1 virus infection remains thus far unclear. The aim of this study was to evaluate interleukin IL-1𝛽 and IL-6 plasma expressions and their association with clinical findings, disease severity, and outcome of children with H1N1 infection. We prospectively evaluated 15 children with H1N1 virus infection and 15 controls with lower respiratory tract infections (LRTI). Interleukin plasma levels were measured using immunoenzymatic assays. Significantly higher levels of IL-1𝛽 and IL-6 were detected in all patients with H1N1 virus infection compared to controls. It is noteworthy to mention that in H1N1 patients with more severe clinical manifestations of disease IL-1𝛽 and IL-6 expressions were significantly upregulated compared to H1N1 patients with mild clinical manifestations. In particular, IL-6 was significantly correlated with specific clinical findings, such as severity of respiratory compromise and fever. No correlation wasfound between interleukin expression and final outcome. In conclusion, H1N1 virus infection induces an early and significant upregulation of both interleukins IL1𝛽 and IL-6 plasma expressions. The upregulation of these cytokines is likely to play a proinflammatory role in H1N1 virus infection and may contribute to airway inflammation and bronchial hyperreactivity in these patients.

1. Introduction syndrome (ARDS) [4, 5], requiring admission in Pediatric Intensive Care unit (PICU) and mechanical ventilation [6]. In the last years the world has been facing a new pandemia Several hypotheses to explain this particular virulence of caused by an H1N1 influenza virus, the so-called H1N1/09 H1N1 in children were advocated, including downregulation virus, which contains a unique combination of gene segments of type 1 interferon expression, apoptosis, and hyperin- that has never been identified in humans or animals [1]. duction of proinflammatory cytokines7 [ ]. Upregulation of This new pandemic strain is of particular concern because inflammatory cytokines, such as the TNF-a, IL-1𝛽,IL-6,and of its efficient person-to-person transmission responsible for IL-10, and a cytokine-mediated inflammatory response have increased virulence and morbidity in humans [2, 3]. also been documented as responsible of severity of viral lung The novel influenza H1N1 virus was identified as acause infections [8]. Different viruses, such as respiratory syncytial of febrile respiratory infections ranging from self-limited virus (RSV) and adenovirus, enhanced the production of IL- to severe illness both in adults and children. Recent data 6 by human macrophages influencing the susceptibility and reported that most cases of H1N1 infection with high rate of severity of respiratory infections [9]. In addition, pulmonary hospitalizations occurred in children who aged 5–14 years. and systemic inflammatory stimuli, such as hypoxia and A small percentage of these patients can develop more fever, induce the biosynthesis of interleukins (ILs) in most cell complicated and severe symptoms, such as elevated fever, types, including respiratory endothelium and mast cells [10, violent dry cough, pneumonia, and acute respiratory distress 11], thus determining the increase of vascular permeability 2 Mediators of Inflammation and leukocyte accumulation in lung tissue [12, 13]. In the Oral Oseltamivir (60 mg twice daily for 5 days) was literature the inflammatory role of IL-6 and IL-1𝛽 in both administered to all 15 patients with the diagnosis of influenza systemic and respiratory disorders such as meningitis, head H1N1 virus infection, and supportive therapy for ARDS was injury, and ARDS has also been reported [14, 15]. Moreover, started based on the severity of respiratory failure (Table 1). recent studies demonstrated that influenza virus A elicits an Fever was treated aggressively with paracetamol, while dry acute inflammatory response characterized by the production cough with aerosol therapy. Chest X-ray was performed of pro-inflammatory cytokines, such as IL-33 and IL-6, in within the first 6 hours of hospital admission. Eventual chest infected lungs, suggesting a key role for these interleukins CT scan was performed in all children with H1N1 infection in the pathogenesis of respiratory epithelial cell damage with particular severity of respiratory impairment or with and lung inflammation [16, 17].However,theroleofmost specific findings at standard chest radiography (i.e., PNX, cytokines in relation to clinical findings, disease severity, and pneumopericardium, or pneumomediastinum). All patients outcome of children with H1N1 virus infection remains thus were isolated at the moment of the admission based on far unclear. Attempting to elucidate the immune mechanisms their clinical symptoms suspected for H1N1 infection or of inflammation and to clarify the role of interleukins IL- other acute respiratory illness. The throat/nose swabs and 1𝛽 andIL-6inchildrenwithH1N1virusinfection,we blood samples for both laboratory studies and cytokines evaluated the plasma levels of these cytokines in 15 children determination were taken at the moment of the admission. with H1N1 infection and 15 controls with lower respiratory All the throat/nose swabs were sent to the microbiology for tract infections (LRTI), to determine whether a correlation influenza virus detection and were analyzed for influenza with the expression of these molecular markers and clinical A, B, subtypes of A by influenza real-time RT-PCR test, findings of these patients exists. and RSV infection. Tables 1 and 2 reported the clinical and demographic characteristics of both patients and controls 2. Patients and Methods studied. The outcome of patients was assessed upon discharge 2.1. Study Population. Weconductedaprospectiveobserva- from the hospital using the Glasgow Outcome Score (GOS), tional clinical study among children admitted from October which assigns a score of 1 to children who died, 2 to persistent 2009 to December 2010 with the diagnosis of influenza H1N1 vegetative state, 3 to severe neurologic deficits, 4 to mild virus infection and LRTI to the Pediatric Intensive Care Unit neurologic deficits, and 5 to completely healthy children [18, (PICU) and Pediatric Infectious Disease Unit (PIDU) of the 19]. “Agostino Gemelli” Hospital, Catholic University Medical School, Rome, Italy. Patients with H1N1 influenza virus infec- 2.2. Plasma Sample Collection. In H1N1 patients we collected tion were grouped according to age, etiology of virus infec- blood samples using indwelling radial artery catheters in tion, findings of chest radiograph, clinical and laboratory children admitted to the PICU or arterial puncture in characteristics, respiratory care, and final outcome (Table 1). children admitted to the PIDU after local painful treatment. We also decided to differentiate the patients with H1N1 virus All samples were obtained in the acute phase of the illness, infection in two groups (severe and mild manifestations atthemomentoftheadmissionofthepatients,andbefore of H1N1 infection) based on the severity of the symptoms starting any treatment. The plasma samples were submitted andontheadmissiontothePICU.Weconsideredsevere for microbiological and biochemical analysis (leukocyte and manifestations of H1N1 influenza infection, the presence platelet counts, serum C-reactive protein concentration, pro- of hypoxia at admission (SpO2 less than 82% in room calcitonin, glucose-protein concentration, electrolytes, acid- air), ARDS requiring mechanical ventilation or noninvasive base study, BUN, etc.). ventilation (NIV) by Helmet, oxygen supplementation by To measure interleukin levels all blood samples were Ventimask or CPAP by face mask, severity of fever (more than centrifuged for 10 min at 5,000 rpm, and the supernatants ∘ ∘ 39 C at the moment of admission), presence and duration were immediately stored at −70 Cuntilanalysis. of cough, presence of specific radiological findings, such Ascontrols,weusedbloodradialarterysamplescollected as pneumothorax (PNX), pneumopericardium, and pneu- from children with the diagnosis of LRTI who had undergone momediastinum, and other specific clinical manifestations, bloodsampleanalysisatthemomentoftheiradmissiontothe such as neurological involvement. Based on these admission PICU or PIDU. parameters, nine patients with severe manifestations of H1N1 The study was approved by the Institutional Review influenza virus infection were admitted to the PICU, while Board, and the parents of participating children were in- the other 6 patients with mild symptoms of H1N1 infection formed about study and provided written informed consent. were admitted to the PIDU. Regarding the control group, 8 infants with severe RSV bronchiolitis were admitted to the 2.3. Interleukin Assays. IL-1𝛽 and IL-6 were measured from PICU, while the other 7 children with LRTI to the PIDU. blood samples using commercial immunoenzymatic kits Six infants with RSV bronchiolitis admitted to the PICU (Human Quantikine by R&D Systems) following the instruc- underwent oxygen supplementation and NIV by Helmet, tions of the manufacturer. The sensitivity of the assay was while the other 2 patients required mechanical ventilation. typically 0.70 pg/mL for IL-6 and 1 pg/mL for IL-1𝛽;nocross- The other 7 infants belonging to the control group required reactivity or interference with other related interleukins was only oxygen supplementation and symptomatic treatment observed. Results were represented in pg/mL, and all assays (Table 2). were performed in duplicate. Mediators of Inflammation 3 (GOS) Outcome nts. (days) in hospital Length of stay None 7 5 None 27 5 None 11 5 None 18 5 NoneNone 9 9 5 5 None 10 5 Complications Pneumorrachis 19 5 Pneumopericardium 18 5 Pneumomediastinum 11 5 and and and therapy Oseltamivir Oseltamivir Oseltamivir Oseltamivir Oseltamivir Oseltamivir Oseltamivir Oseltamivir Oseltamivir Oseltamivir Ceftriaxone, Ceftriaxone, Ceftriaxone, Ceftriaxone, Ceftriaxone, Ceftriaxone, Ceftriaxone, Ceftriaxone, Ceftriaxone, Antimicrobial Klaritromicin, Clarithromycin, Clarithromycin, Clarithromycin, by Ventimask 2 EI/MV (4 days) (4 days) Ventimask Ventimask Ventimask Ventimask Ventimask supplementation supplementation Oseltamivirsupplementation Oseltamivir None None 4 6 5 5 Respiratory care 2 2 2 supplementation by supplementation by supplementation by supplementation by supplementation by CPAP by Helmet O O 2 2 2 2 2 NIV by full-face mask O O O O O (1 day) + O (2 days ), CPAP by Helmet (11 days), CPAP by Helmet NIV by full-face mask, EI/MV lung X-ray Chest bilateral Bilateral Bilateral Bilateral infiltrates infiltrates Interstitial Interstitial pneumonia pulmonary pulmonary pulmonary pulmonary Pneumonia Pneumonia Pneumonia pneumonia, Hyperinflated consolidation consolidation Pneumothorax pleural effusion Pneumothorax, at 2 % % % % % % % % % SpO admission in room air 55 94 95 Normal Normal None None Oseltamivir Oseltamivir None None 3 3 5 5 790 691 6 93 Normal None Oseltamivir None 3 5 492NormalO Duration of cough (days) 58 78 46 78 68 70 66 81 77 82 47 50 66 76 35 82 49 73 ...... 0 0 0 0 0 0 0 0 0 ± ± ± ± ± ± ± ± ± 6 7 1 3 4 2 7 2 5 ...... day of Table 1: Clinical and radiological findings, respiratory assessment, complicationsand of infectedH1N1 children. In bold the more severe H1N1 patie 38.5 ± 0.5 38.7 ± 0.2 38.2 ± 0.4 38.3 ± 0.7 38.4 ± 0.6 38.1 ± 0.4 admission Fever at first Patients 139 239 339 439 539 639 739 839 939 10 11 12 13 14 15 CPAP: continuous positive airway pressure; EI: endotracheal intubation; MV: mechanical ventilation. 4 Mediators of Inflammation (GOS) Outcome hospital (days) Length of stay in None 4 5 None 5 5 Complications therapy Ceftriazone Amoxicillin None 3 5 Amoxicillin None 4AmoxicillinAmoxicillin None 5 Amoxicillin None 3 None 2Amoxicillin 5 3 None 5 5 4 5 Amoxicillin Amoxicillin, Antimicrobial Claritromicina 2 2 2 2 2 2 2 and O and O and O and O and O and O and O face mask supplementation Amoxicillinsupplementationsupplementation Claritromicina Nonesupplementation Amoxicillin Nonesupplementation Amoxicillinsupplementation None Amoxicillin 5 Amoxicillin None 5 None 4 None 5 3 5 3 3 5 5 5 5 Respiratory care 2 2 2 2 2 2 supplementation by supplementation supplementation supplementation supplementation supplementation supplementation supplementation CPAP by Helmet CPAP by Helmet CPAP by Helmet CPAP by Helmet CPAP by Helmet O O O 2 O Mechanical ventilation Mechanical ventilation lung lung lung lung lung lung X-ray Chest atelectasia Interstitial Segmental atelectasia atelectasia atelectasia Segmental Segmental Segmental Segmental pulmonary pneumonia pulmonary pulmonary pulmonary pulmonary Hyperinflated hyperinflated Hyperinflated Hyperinflated Hyperinflated Hyperinflated atelectasia and % % % % % % % % at admission 2 in room air SpO 391% 3391%NormalO 386% 93% Normal None Amoxicillin None 2 5 493% 4490%NormalO 88% Normal O Duration of cough (days) 34 84 25 82 15 85 23 81 44 82 63 81 64 80 35 82 ...... 0 0 0 0 0 0 0 0 ± ± ± ± ± ± ± ± Table 2: Clinical and radiological findings, respiratory assessment, complicationsand LRTI of children. In bold thesevere more patients. LRTI 4 5 1 3 4 2 5 1 ...... 37.5 ± 0.2 37.5 ± 0.3 37.7 ± 0.5 37.5 ± 0.3 38.1 ± 0.4 38.3 ± 0.6 37.8 ± 0.5 of admission Fever at first day Patients 137 237 338 438 537 637 738 838 9 10 11 12 13 14 15 CPAP: continuous positive airway pressure; EI: endotracheal intubation; MV: mechanical ventilation. Mediators of Inflammation 5

2.4. Statistical Analysis. The nonparametric Mann-Whitney 175 𝑃 < 0.0001 test and t-test were used to perform statistical comparisons 150 between children with H1N1 virus infection and LRTI control group for continuous variables. Analysis of variance was per- 125 𝛽 formed using Tukey-Kramer test to compare levels of IL-1 100 and IL-6 in the studied population. Linear regression analysis was used to evaluate the correlation between interleukin 75 expression and clinical manifestations in H1N1 patients. 50 2 Coefficient of determination (𝑅 ) was taken as a measure 25 of the goodness of fit of the model. A P value < 0.05 was considered significant. Statistical and database software used (pg/mL) concentrations Interleukin 0 1𝛽 included GraphPad version 5.0 (GraphPad Software, San IL- IL-6 Diego, CA, USA) and Microsoft Office Excel 2007 (Microsoft Figure 1: Levels of IL-1𝛽 andIL-6inH1N1patientsareshownin Corporation, Redmond, WA, USA), respectively. the figure. Box plot representation was used. The level of IL-6 was significantly higher than the level of IL-1𝛽 (𝑃 < 0.0001). 3. Results

3.1. Clinical and Laboratory Differences between H1N1 Patients 80 and Controls. We include in this study 15 patients with H1N1 𝑃 < 0.0001 virus infection and 15 children with LRTI. Patients with H1N1 infection aged 2.8 years to 17.3 years, with a mean 60 age of 7.9 years, while children with LRTI aged 1.1 years to 6.3 years, with a mean age of 3.7 years. Nine children 40 with severe H1N1 virus infection were admitted to our PICU due to the severity of their respiratory compromise, while the other 6 patients to the PIDU. Among children with 20 LRTI,8outof15wereadmittedtothePICUwiththe diagnosis of severe RSV bronchiolitis, while the other 7 Interleukin concentrations (pg/mL) concentrations Interleukin were admitted to the PIDU (4 with diagnosis of non-RSV 0 1𝛽 bronchiolitis and 3 with diagnosis of influenza A (H2N3) IL- IL-6 virus infection). Regarding clinical differences between the Figure 2: Levels of IL-1𝛽 andIL-6inLRTIpatientsareshowninthe two groups, H1N1 patients experienced higher median fever ∘ ∘ figure. Box plot representation was used. IL-6 level was significantly (39.2 C) compared to controls (37.7 C) (𝑃 < 0.0001). Cough higher compared to IL-1𝛽 (𝑃 < 0.0001). was a common symptom in both groups. However, H1N1 patients more frequently suffered from a dry and longer cough compared to LRTI patients (median 6 days versus interleukins. In these patients we found significantly𝑃< ( 4days)(𝑃 < 0.0001). The most frequent pulmonary 0.0001)higherlevelsofIL-6(108.1 ± 22.8 pg/mL) compared abnormalities at chest X-ray were represented by pneumonia to IL-1𝛽 (17.2 ± 7.9 pg/mL) (Figure 1). Also in LRTI patients and pulmonary consolidation in the H1N1 patients, while in themeanplasmalevelsofIL-6weresignificantlyhigher LRTI children we detected atypical findings, such as hyper- compared to the levels of IL-1𝛽 (49.0 ± 10.3 pg/mL versus inflatedlungsandsegmentalpulmonaryatelectasias.Two 8.1 ± 2.2 pg/mL) (𝑃 < 0.0001)(Figure 2). patients with H1N1 infection showed PNX, while another three children showed severe respiratory complications, such as pneumopericardium, pneumomediastinum, and pneu- 3.3. Plasma Level Differences of Interleukin Expression between morrhachis at chest CT scan (Table 1). No pulmonary or H1N1 Patients and Controls. Significantly higher levels of systemic complications were referred to LRTI group. No interleukin IL-6 and IL-1𝛽 were demonstrated in all patients differences in clinical manifestations, such as gastrointestinal with H1N1 infection compared to controls. Compared and neurological symptoms, have been reported between the with LRTI patients, H1N1 patients displayed significantly groups. Regarding laboratory tests (blood cells and platelet increased plasma levels of IL-6 (108.1 ± 22.8 pg/mL versus count, serum C-reactive protein, procalcitonin, GOT, GPT, 49.0 ± 10.3 pg/mL; 𝑃 < 0.0001)andIL-1𝛽 (17.2 ± 7.9 pg/mL CTN, and urea) no significant differences were detected versus 8.1 ± 2.2 pg/mL; 𝑃 < 0.0002)(Figure 3). between H1N1 patients and LRTI controls. All children, both patients and controls, had a good outcome without any 3.4. Correlation between Interleukin Expression with Disease significant complications (GOS 5), but H1N1 patients hada Severity and Clinical Manifestations in H1N1 Patients. To significantly longer time of hospitalization compared to the elucidate the association between interleukin expression and 𝑃 = 0.0013 control group (9 days versus 3 days: ). disease severity, we analyzed their plasma levels both in patients with severe (9 patients) and mild symptoms (6 3.2. Interleukin Expression in H1N1 Patients and Controls. patients) of H1N1 influenza virus infection. Compared to In H1N1 patients we detected different plasma levels of the mild patients, severe H1N1 patients produced significant 6 Mediators of Inflammation

150 40 𝑃 < 0.0001 39.8 125 39.6 39.4 100 39.2 39

75 Fever 38.8 38.6 50 38.4 38.2 25 𝑃 = 0.0002 38 60 70 80 90 100 110 120 130 140 150

Interleukin concentrations (pg/mL) concentrations Interleukin 0 IL-1𝛽 H1N1 IL-1𝛽 controls IL-6 H1N1 IL-6 controls IL-6 (pg/mL)

Figure 3: Box plot representation was used. H1N1 patients had Figure 5: A scatter plot shows the relationship between fever and significantly higher levels of IL-1𝛽 (𝑃 = 0.0002) and IL-6 (𝑃< IL-6 plasma levels. The line represents the regression line (linear × 𝑅2 = 0.64 0.0001)whencomparedtocontrols. regression equation: fever = 0.02 IL-6 + 36.8 ).

150 95 𝑃 < 0.0001 90 125 85 80 100 75 70

75 admission at 65 2 60

50 SpO 55 𝑃 < 0.0001 50 25 45 60 70 80 90 100 110 120 130 140 150

Interleukin concentrations (pg/mL) concentrations Interleukin 0 IL-6 (pg/mL) IL-1𝛽 severe IL-1𝛽 mild IL-6 severe IL-6 mild Figure 6: A scatter plot show the relationship between SpO2 at Figure 4: Box plot representation was used. H1N1 patients with admission in room air and IL-6 plasma level. The line represents the 𝛽 𝑃 < 0.0001 severe disease had significantly higher levels of IL-1 ( ) regression line (linear regression equation: SpO2 = −0.38 × IL-6+ 2 and IL-6 (𝑃 < 0.0001)whencomparedtopatientswithmild 122.9 𝑅 = 0.53). symptoms.

and significant up-regulation of interleukin IL-1𝛽 and IL-6 𝛽 22.6 ± 4.7 9.1 ± higher levels of IL-1 ( pg/mL versus plasma levels suggesting that these cytokines are responsible 2.8 𝑃 < 0.0001 124.1 ± 11.8 pg/mL; )andIL-6( pg/mL versus for different molecular reactions leading to airway inflamma- 84.0 ± 8.6 𝑃 < 0.0001 pg/mL; )(Figure 4). tion and disease severity. Compared to LRTI controls, H1N1 Moreover, to verify whether there was a correlation infected children showed a strongly higher production of between interleukin up-regulation and clinical manifesta- both IL-1𝛽 and IL-6 soon after virus lung infection, and this tions in H1N1 patients, we compared the plasma levels of overexpression seems to correlate with the severity of clinical these cytokines with some clinical symptoms referred to the compromise assessed upon admission. We also observed that patients. In particular, we detected a positive correlation in H1N1 patients with more severe clinical manifestations of between plasma level of IL-6 and fever with a coefficient disease, plasma levels of IL-1𝛽 and IL-6 were significantly 𝑃 = 0.0004 of determination of 0.64 ( )(Figure 5). Finally upregulatedcomparedtoH1N1mildpatientsandthatthis we found a negative correlation between IL-6 plasma level over expression was correlated with some specific clinical and SpO2 at admission in room air with a coefficient of manifestations and a longer time of hospitalisation. More 𝑃 = 0020 determination of 0.53 ( )(Figure 6). No significant in particular, IL-6 up-regulation was significantly correlated correlations were reported between interleukin expression with the severity of respiratory compromise, testified by and other clinical and laboratoristic parameters, such as bio- alowerSpO2 at admission and higher fever observed in chemical markers of inflammation (C-reactive protein and this subset of children, as previously reported in patients procalcitonin), respiratory care, systemic complications, and, with H1N1 virus infection [7]. No differences were reported finally, outcome of all children, with H1N1 virus infection. between plasma expression of these factors and final outcome of patients and controls. 4. Discussion To date it is difficult to explain the exact role of ILs in the mechanisms of virus host response, because both Our study, despite the limited patient sample so far evaluated, pro-inflammatory and immunoprotective actions have been provides evidence that H1N1 virus infection induces an early reported in previous researches. H1N1 virus infection causes Mediators of Inflammation 7 the activation of the host macrophages and lymphocytes release may contribute to the development of airway inflam- determining the release of pro-inflammatory cytokines. The mation and bronchial hyperreactivity [34]. In our study, increased expression of pro-inflammatory cytokines into the IL-1𝛽 and IL-6 up-regulation, observed early after H1N1 lung tissue may lead to higher blood vessel permeability, virus infection, was consistent with the timing of cytokine phagocytic cell recruitment, apoptosis of lung epithelial expression in experimental models of virus infected human cells, and release of neutrophil-derived enzymes, such as alveolar macrophages, suggesting that this over expression myeloperoxidase and elastase, responsible of severity of acute plays a key role in the mechanisms of inflammatory lung lung injury [20].Ourresultsareinagreementwiththese response [35]. The significant correlation between ILs upreg- studies, as we showed a significant correlation between ILs ulation and severity of H1N1 virus infection observed in up-regulation and severity of respiratory compromise in our patients might reflect an endogenous attempt against children with H1N1 virus infection. There are some possible molecular mechanisms activated in the epithelium cells explanations for this relationship. Up-regulation of IL-1𝛽 of infected lung suggesting that ILs up-regulation acts in and IL-6 may affect lung functioning because hypoxia is in different fashion to amplify and propagate inflammation in turn responsible for the endogenous cytokine production the airways. However, given that the statistical power to find after H1N1 lung infections [10]. Cytokine up-regulation may a statistically significant association in a model of 15 patients cause epithelial cell damage through different mechanisms. is very low, we need to be very cautious on interpreting these ILs have a direct toxic effect by increasing the production data, because only limited information is available on ILs of nitric oxide synthase, cyclooxygenase, and free radicals expressioninchildrenwithvirallunginfections. and by favouring the release of the excitatory amino acid In conclusion, our observations provide new evidence in experimental model of neurotoxicity and also in patients that an immune response is activated at the early stage of with severe sleep apnea [21, 22],thus determining impaired pandemic H1N1 influenza virus infection with up-regulated pulmonary function [23]. Previous studies, in fact, reported production of plasma interleukins IL-1𝛽 and IL-6. These find- the correlation between IL-1𝛽 and IL-6 up-regulation and ings are consistent with previous experimental and clinical some clinical and radiological findings, such as pneumonia studiesconfirmingakeyroleforbothoftheseinterleukins and ARDS, both in experimental animal models and in in the pathogenesis of airway inflammation and bronchial children with naturally acquired seasonal influenza A [24– hyperreactivity during virus lung infections. The increased 27]. More in particular, IL-1𝛽 and IL-6 have been identified expression of these cytokines may together be the underlying as specific markers of the severity of acute lung injury during cause of the observed clinical symptoms in severe H1N1 H1N1 influenza virus infection [8] and it has also been patients, and defining the relationships between ILs expres- reported that IL-1𝛽 is an early and useful biomarker of the sion and the pathophysiology and clinical manifestations of severity and progression of lung inflammation in patients H1N1 may help to shed light on the molecular pathogenesis undergoing mechanical ventilation and unresponsive to anti- of H1N1 influenza and other human viral lung infections. Fur- microbiological treatment [28]. 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Research Article Evaluation of Clinical and Immunological Responses: A 2-Year Follow-Up Study in Children with Allergic Rhinitis due to House Dust Mite

Heleen Moed,1 Roy Gerth van Wijk,2 Rudi W. Hendriks,3 and J. C. van der Wouden4

1 Department of General Practice, Erasmus MC-University Medical Center Rotterdam, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands 2 Department of Allergology, Erasmus MC-University Medical Center Rotterdam, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands 3 Department of Pulmonary Medicine, Erasmus MC-University Medical Center Rotterdam, P.O. Box 2040, 3000 CA Rotterdam, The Netherlands 4 Department of General Practice, VU University Medical Center, 1081 HZ, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands

Correspondence should be addressed to Heleen Moed; [email protected]

Received 6 June 2012; Revised 3 April 2013; Accepted 4 April 2013

Academic Editor: Ger Rijkers

Copyright © 2013 Heleen Moed et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background. Allergic rhinitis is a disease with polarization towards Th2 and a defect of regulatory T cells. Immunological changes have been reported after immunotherapy treatment. However, there is not much known about the natural course of allergic rhinitis with respect to clinical manifestation and the relation with immunological responses. Objective. To evaluate clinical symptoms of allergic rhinitis, in relation to in vivo allergen-specific skin responses and in vitro allergen-specific effector and regulatory T cells determined at baseline and after two years. Methods. From a large trial, 59 children were randomly selected. The following variables were compared: clinical symptoms, allergen skin tests, specific IgE, T-cell proliferation, IL-5, IL-13, IFN-gamma, IL-10, TGF-beta, + CD4 CD25hi cells, and Foxp3 expression. Results. Allergic symptoms had decreased after two years. Whereas skin test reactions correlated between years 0 and 2, there was no change in the size of the reaction. Also, proinflammatory reactions did not change after two years, with a positive correlation between years 0 and 2. No relevant changes were observed with respect to regulatory cells. Conclusion. Whereas, comparable to immunotherapy, allergic complaints decrease, the immunological changes of specific T-cell activity (both effector cells and regulator cells) which are observed after immunotherapy, do not change.

1. Introduction congestion as the predominant symptom. During this phase, inflammatory cells such as eosinophils and T cells infiltrate Allergic rhinitis (AR) is a common chronic illness affecting the mucosa. The Th2-polarized immune response especially, 10% to 20% of the children worldwide [1], and its prevalence with secretion of cytokines such as IL-4, IL-5 and IL-13 and a has significantly increased among children over the last two defect of Th1 cells producing IFN-gamma, play a central role decades. AR is clinically defined as a symptomatic disorder in the late phase allergic response [2–4]. ofthenoseinducedbyanIgE-mediatedinflammationafter It has been observed that the immune response to aller- exposure to an allergen. In the immediate response to an gensistheresultofafinebalancebetweenallergen-specific antigen (the early-phase allergic reaction), histamine and Th2 cells and T regulatory cells (Tregs) [5]. Tregs include + other inflammatory mediators are released from mast cells a broad spectrum of CD4 T-cell subpopulations such as in the nasal mucosa [2]. This causes the characteristic nasal natural thymic-derived Treg cells (nTreg) characterized by + + symptoms which include sneezing, itching, rhinorrhoea, their CD4 CD25 phenotype expressing high levels of Foxp3 and nasal congestion. A late-phase allergic reaction occurs [6]andinducibleTr1 cells (iTreg) which suppress immune approximately 4 to 12 hours after antigen exposure, with nasal function by the secretion of IL10 and TGF-beta [7–9]. 2 Mediators of Inflammation

From the recent literature it becomes clear that it may below). Before scoring symptoms, nasal corticosteroids were be possible to interfere with the natural course of AR withheld for 4 weeks before the study period. During the total by specific immunotherapy (SIT)9 [ –13]. SIT may induce study period patients were allowed to use rescue medication changes that skew Th2-toTh1-type responses [14]andby (provided by us, i.e., levocetirizine tablets, xylometazoline means of tolerance induction. One of the key mechanisms nasal spray, and levocabastine eyedrops) or another allergy behind this tolerance induction includes induction of Treg- or asthma medication as long as they wrote it down on their type cytokines (IL-10 and TGF-beta) [9, 10]andincreased diary cards (see below). The presence of asthma was assessed + + frequencies of CD4 CD25 cells [11]. using the International Study of Asthma and Allergies in There is an unexplained variability in the clinical course Childhood (ISAAC) core questionnaire [17]. of allergic rhinitis—with persistence in some individuals and either progression or remission in others. There is a lack of 2.3. Measurement of Nasal, Eye, or Asthma Symptoms. All longitudinal investigations examining the natural history of participants or their parents scored their nasal, eye, and AR.Inthepresentstudy,weinvestigatethenaturalcourse asthma (lung) symptoms on diary cards at baseline (1 month of AR regarding clinical manifestations of the disorder and inOctoberorinNovember)andaftertwoyears(3monthsin immunological responses in a followup of 2 years in children September–December). Nasal symptoms (sneezing, itching withhousedustmiteallergy.Weevaluateclinicalsymptoms, nose, watery running nose, and nasal blockage), eye symp- in vivo skin responses due to allergen and in vitro allergen- toms (itching, tearing, and redness), and asthma symptoms specific effector T cells and Treg cells during the natural (wheeze/breathless and dry cough during night) were scored course of the disease. on a 0–3 scale (0 = none, 1 = mild, 2 = moderate, and 3 = severe). In total, a maximal daily cumulative nasal symptom 2. Methods score of 12, eye symptom score of 9, and lung symptom score of 6 could thus be obtained. 2.1. Study Design. Thisstudyisapartoftherandomized A mean symptom score was determined by calculating placebo-controlled trial ISRCTN91141483 which evaluates themeandailyscoreovertheentirediaryperiod(i.e.,one sublingual immunotherapy with low-dose house dust mite month at baseline and three months after 2 years). Only allergen (2.03 mcg/mL or 700 BU/mL Der p1) in children with diaries with at least 50% of the filled-out pages were included AR [15, 16]. Patients entered the study either in September– in the analyses. In case patients used additional medication December 2005 or in September–December 2006 for a period fortheirallergyorasthma,theywereaskedtodocumenttheir of approximately two years. Written informed consent was use in the patient diary throughout the 2-year period. obtained.ThestudywasapprovedbytheEthicalReview Board of Erasmus MC-University Medical Center Rotterdam. 2.4. Skin Testing. Allergy skin testing was performed at As described elsewhere, [16] neither this low-dose form baseline and after 2 years by intracutaneous injection of of SLIT was effective with regard to the reduction of allergy 0.02 mL Dermatophagoides pteronyssinus in the forearm complaints, asthma complaints, intake of rescue medication (concentration 30 SQ U/mL, manufactured by ALK-Abello,´ and disease-specific quality of life, nor was there any differ- Nieuwegein, The Netherlands). We chose to perform an ence in reported side effects (both local and general) between intracutaneous skin test rather than the usual skin prick test SLIT or placebo treatment, indicating that the dosis of the because intracutaneous injection of the allergen is the most investigated product was a low to mediate reaction. Analysis feasible and convenient way to induce a late-phase response of placebo (𝑛=30)andverum(𝑛=29)groupsseparatelyin after the early-phase skin response [18]. As a positive control, children participating in the present study revealed neither histamine (concentration of 0.01 mg/mL) was injected, and differences between verum and placebo regarding clinical the negative control was dilution buffer. Reactions were read symptoms (𝑃 valuevariedbetween0.06and0.86)norin after 15 min (early response) and after 6 h (late response). The vivo skin responses (𝑃 value between 0.17 and 0.80) or in 2 areaoftheskinresponseinmm was measured by a specially vitro measurements of pro-inflammatory markers𝑃 ( value developed scanning programme. The early-phase response between 0.16 and 0.85) or regulatory T cells (𝑃 value between was expressed as a histamine equivalent intra-cutaneous 0.10 and 0.89). Therefore, we decided to merge the two groups index or HEIC index. The late-phase response was expressed and consider them as one group which received placebo 2 during two years. This merged group forms the basis for the as the area of the skin response in mm . Children were not present study. allowed to take antihistamines within 24 h before skin testing.

2.5. Detection of House Dust Mite-Specific IgE. Serum IgE 2.2. Patient Selection. From the main trial, fifty-nine children antibodies to D. pteronyssinus were determined using the were randomly selected and invited to participate in this elab- CAP-RAST system (Pharmacia, Uppsala, Sweden), according orate study. As in the main study, children (aged 6–18 years) to the manufacturers instructions. with AR and established HDM allergy were selected from the electronic medical records in general practice. The inclusion criteria were presence of specific IgE antibodies to HDM in 2.6. House-Dust-Mite-Specific T-Cell Proliferation. Blood was serum (≥0.7 kU/L), a history of allergic rhinitis during at least drawn at baseline and after 2 years before skin testing. 1 year and a nasal symptom score of at least 4 out of 12 (see Peripheral blood mononuclear cells (PBMCs) were isolated Mediators of Inflammation 3 from heparinized blood by density centrifugation on Ficoll- Table 1: Baseline characteristics of the study population. Paque Plus (GE Healthcare, Uppsala, Sweden). PBMCs were 𝑁=59 used in a lymphocyte proliferation test (LPT). Cells were resuspended in complete medium (RPMI + HEPES + gluta- Age, years (mean, SD) 11.6 (3.0) max supplemented with gentamicin (Gibco, Gibco BRL, Life Gender, male (𝑛, %) 28 (47.5%) Technologies, Rockville, MD, USA) and 5% heat-inactivated Wheeze/dyspnea last year (𝑛, %) 37 (62.7%) human serum (Sanquin, Rotterdam, The Netherlands) and Multisensitization (𝑛, %) 49 (83.1%) stimulated by culturing in the presence or absence of 2 IR/mL D. pteronyssinus (Stallergenes,` France). All cell cultures were performed in quadruplicate in a final volume of 200 mL with 6 a cell concentration of 2 × 10 cells/mL in 96-well round- (Applied Biosystems), preformulated primers (hFoxP3 and bottomed microtitre plates. To determine HDM-specific ∘ housekeeping gene hHPRT) and probe mixes (“Assay on proliferative responses, cells were cultured for 5 days at 37 C, Demand”, Applied Biosystems). PCR conditions were 2 min 5% CO2,and95%humidity.Duringthelast16h,cellswere ∘ ∘ 3 at 50 C, 10 min at 95 C, followed by 40 cycles of 15 s at pulsed with 0.5 𝜇Ci/well H thymidine (Pharmacia, UK). ∘ ∘ 𝛽 95 Cand60C for 1 min using an ABI PRISM 7300 (Applied Radioactivity was measured with a -plate reader, and the Biosystems). PCR amplification of the housekeeping gene proliferative capacity was assessed by the stimulation index 3 was performed during each run for each sample to allow (SI), calculated as the ratio of mean Hthymidineuptake normalization between samples. in stimulated to that in non-stimulated cultures. The SI was considered positive when it exceeded 2.0. 2.10. Statistical Analysis. Values are presented as mean (with standard deviation, SD), median (with interquartile range, 2.7. House-Dust-Mite-Specific Cytokine Production by ELISA. IQR), and median estimated difference between baseline To determine HDM-specific cytokine production, 1 mL of × 6 (year 0) and year 2. Comparisons were performed by non- PBMCs with a concentration of 2 10 cells/mL was cultured parametric analysis, using Wilcoxon signed rank test for in duplicate in a 24-well plate with or without 2 IR/mL D. comparisons between baseline data and data after 2 years. The pteronyssinus (Stallergenes` France). After 5 days of culture, ∘ Spearman rank test (𝑅𝑠) was used to assess the correlations supernatants were harvested and stored at −20 Cuntiltest- 𝑃 𝛾 betweenyear0andyear2.A value of 0.05 was considered ing. IL-5, IL-13, IFN- , IL10, and TGF-beta cytokine produc- statistically significant. All analyses were performed using tion was measured following the manufacturer’s instructions statistical software SPSS (version 18). (eBioscience, San Diego, CA, USA or R&D Systems, Abing- don, UK). HDM-induced cytokine production was assessed by subtracting the cytokine concentration of nonstimulated 3. Results from that of stimulated culture supernatants. The baseline characteristics of the 59 children included in the study are described in an earlier study [19]. Of the 59 + 25hi 2.8. Detection of CD4 CD Cells by FACS. For pheno- children, 42 (71.8%) completed the 2-year study period. The typical analysis, cells were washed with FACS buffer (0.05% most important values are summarized in Table 1.Mean NaN3, 2% BSA in PBS) and stained for 30 min on ice and median values (with SD or IQR) of clinical symptoms, + hi protected from light. For the detection of CD4 CD25 in vivo allergen-induced skin tests, and in vitro specific T cells the following anti-human antibodies were used: pro-inflammatory and regulatory responses are displayed in FITC-conjugated anti-hCD3 (UCHT1, eBioscience), PE- Table 2. conjugated anti-hCD25 (M-A251, BD) APC-conjugated anti- Clinical symptoms of the nose, eye and lung all decreased hCD4 (RPA-T4, eBioscience). Aspecific binding was pre- significantly two years after baseline measurement. Table 3 vented by the use of 2% Heat Inactivated (HI) human AB shows a median paired estimated difference of −1.34 (𝑃< serum during staining. Afterwards the cells were washed 0.0001)fornosesymptoms,−0.29 (𝑃 = 0.02)foreye three times with FACS buffer and measured. Data acquisi- symptoms and −0.33 (𝑃 < 0.0001)forlungsymptoms.There tion was performed by flow cytometry (FACSCalibur; BD was a significant positive correlation between values of year Biosciences) and data analysis was performed using FlowJo 0andyear2forallclinicalsymptoms(𝑅𝑠 between 0.56 and software (Treestar, Coata Mesa, CA)). 0.61; 𝑃 < 0.0001). Reactions of in vivo HDM specific skin tests (both early as 2.9.MeasurementofFoxp3LevelsbyMeansofReal-Time well as late skin reactions) did not significantly change when Quantitative PCR. Quantitative RT-PCR for hFoxP3 was measured two years after the baseline measurement (Table 3, performed on RNA isolated from HDM-stimulated PBMC’s 𝑃 = 0.09 or 𝑃 = 0.07,resp.,Table 3). Whereas there were no (as described above). Total RNA was isolated with RNeasy significant changes in time, skin responses showed a positive Mini Kit (Qiagen) and treated with DNAseI, according to correlation when skin responses of year 0 and year 2 were manufacturer’s protocol. 100 ng RNA was used as a template compared (𝑅𝑠 = 0.44; 𝑃 = 0.005 for the early skin response for cDNA synthesis, using Superscript II reverse transcriptase or 𝑅𝑠 = 0.5; 𝑃 = 0.001 for the late skin response). (Invitrogen) and random hexamer primers. Quantitative Relevant proinflammatory variables for AR (i.e., HDM PCR was performed with Taqman Universal PCR Mastermix specific IgE, T-cell proliferation, IL-5, IL-13, and IFN-gamma) 4 Mediators of Inflammation

Table 2: Mean (SD), median (IQR), and number of different variables for year 0 and year 2.

Year 0 Year 2 Mean SD Median IQR 𝑁 Mean SD Median IQR 𝑁 Clinical symptoms Nose (score from 0–12) 3.8 2.2 3.62 3.46 59 2.3 1.95 1.84 2.06 52 Eye (score from 0–9) 1.15 1.32 0.67 1.39 59 0.65 1.1 0.21 0.79 52 Lung (score from 0–6) 0.87 1.13 0.37 1.33 59 0.36 0.9 0.028 0.15 52 HDM-specific skin test Early skin test (HEIC) 1.72 0.61 1.69 0.76 52 1.96 0.78 1.7 0.87 42 Late skin test (mm2) 871.4 771.6 759.2 1389.8 59 1112.4 904.6 1044.5 1418.28 42 HDM-specific proinflammatory response Specific IgE (kU/L) 43.23 34.78 38.1 62.45 59 47.58 34.81 45.6 62.3 53 Proliferation (SI) 6.84 6.18 4.78 5.52 59 5.59 6.73 3.29 5.22 41 IL-5 (pg/mL) 892.93 1963.66 329.66 793.15 59 384.4 549.35 194.82 515.00 41 IL-13 (pg/mL) 848.54 1243.31 313.05 1206.3 59 875.31 783.25 1183.44 1494.64 41 IFN-gamma (pg/mL) 97.81 123.61 55.67 139.94 59 58.39 101.85 26.93 72.73 41 HDM-specific regulatory response CD4+CD25hi (% of CD3+) 0.44 0.26 0.39 0.32 59 0.88 0.46 0.73 0.44 41 FoxP3 (relative expression) 3.22 2.41 2.39 2.47 59 2.8 1.75 2.4 2.65 41 IL-10 (pg/mL) 81.75 157.33 21.32 95.02 59 21.81 39.0 9.24 24.7 41 TGF-beta (pg/mL) 105.13 258.26 0.00 27.27 59 221.35 383.86 58.72 305.75 41 SD: standard deviation. IQR: Inter Quartile Range. were comparable between year 0 and year 2 (Table 3, 𝑃 > 0.05 4. Discussion except for IFN-gamma which showed a significant decrease). Whereasvaluesdidnotsignificantlyincreaseordecrease, Results of the present study show clinical symptoms rated by there was a significant correlation between data from year 0 allergic patients at baseline and two years later in combination with immunological parameters measured in vivo or in vitro. andyear2forallthesemarkers(𝑅𝑠 between 0.35 and 0.86), In a time period of two years, clinical symptoms of the nose, except for IL-5 (𝑅𝑠 = 0.27). eye, and lung significantly decrease in children with house- + 25hi The frequency of regulatory CD4 CD cells had dust-mite-induced AR. Although children rate their allergy increased two years after baseline measurement. However, complaints as being less than at the beginning of the study, other markers characteristic for regulatory cells (Foxp3, IL- this decrease is not reflected in a decrease of one of the 10, TGF-beta) did not increase in time (in fact for IL-10 a immunological parameters. decrease could be detected, Table 3). Correlations between Apossibleexplanationforthereductionofsymptoms year 0 and year 2 for these variables were nonsignificant is that allergic rhinitis decreases in time. Whereas there (except for IL-10). are many studies determining symptoms during followup of Figure 1 summarizes the above-described findings with SLIT treatment, there is little information on the possibility a decrease of clinical symptoms after 2 years in combi- of spontaneous remission of allergic rhinitis [12, 20]. Some nation with a positive correlation between data of year 0 studies have reported the relationship between age and and year 2 (represented by nasal symptoms in Figure 1(a)), improvement or decline of rhinitis over the years [21–23]. comparable skin-test values for year 0 and year 2, with During an 8-year follow-up of patients with allergic rhinitis, a positive correlation between the two years (early skin Nihlen´ et al. [21] found a 20% remission rate, which was test is presented in Figure 1(b)), no significant change in higher in older patients. Broder et al. [22] described that proinflammatory reactions (in Figure 1(c) represented as remission with allergic rhinitis was more likely when the spec IgE) with a high correlation between measurements duration of the disease was shorter than 5 years. + 25hi Another plausible explanation for the reduction of symp- during the two years, and an increase of CD4 CD toms might be that, due to participation in a study, patients Treg cells without a correlation between the two years of are more aware of their allergy and take more care of measurement (Figure 1(d)). As can be seen in this figure, their complaints, which may result in a reduction of their there is no difference in any of the values between patients symptoms. In this study, patients took (placebo) medication receiving placebo treatment or verum-SLIT treatment (which during a period of two years. The effect of placebo treatment they received during a period of two years in the original is substantial in patients participating in a study evaluating trial). SLIT. Mediators of Inflammation 5

Table 3: Paired estimated difference between year 2, and 0 (median, CIs and 𝑃-value) and Spearman correlation.

Paired estimated difference Correlation ∗ # † Median 95% CI Sign. 𝑅𝑠 Sign. Clinical symptoms Nose (score from 0–12) −1.34 −1.86 −0.85 <0.0001 0.56 <0.0001 Eye (score from 0–9) −0.29 −0.62 −0.05 0.02 0.56 <0.0001 Lung (score from 0–6) −0.33 −0.58 −0.13 <0.0001 0.61 <0.0001 HDM-specific skin test Early skin test (HEIC) 0.16 −0.03 0.35 0.09 0.44 0.005 Late skin test (mm2)199.85−12.55 458.2 0.07 0.5 0.001 HDM-specific pro-inflammatory response Specific IgE (kU/L) 1.95 −0.79 6.45 0.17 0.86 <0.0001 Proliferation (SI) −0.67 −2.02 0.89 0.39 0.38 0.015 IL-5 (pg/mL) −132.1 −450.62 69.51 0.19 0.27 0.087 IL-13 (pg/mL) 133.92 −210.18 538.48 0.46 0.36 0.022 IFN-gamma (pg/mL) −44.84 −82.33 −4.00 0.01 0.35 0.027 HDM-specific regulatory response CD4+CD25hi (% of CD3+) 0.44 0.3 0.56 <0.0001 0.18 0.27 FoxP3 (relative expression) −0.12 −0.67 0.41 0.64 0.24 0.13 IL-10 (pg/mL) −39.34 −67.66 −10.56 0.002 0.357 0.024 TGF-beta (pg/mL) 41.43 0.00 144.01 0.18 0.032 0.84 ∗ CI: confidence interval Hodges Lehman. #𝑃-value, Wilcoxon. † 𝑅𝑠 : Spearman rank correlation between year 0 and year 2.

Whereasfromthisstudyitbecomesclearthatcomplaints The literature shows that SIT is able to reduce allergic of patients with AR decrease over time, it is demonstrated in a complaints in combination with a reduction of allergen- birth cohort study that the prevalence of patients developing induced T-cell proliferation [14]andashiftintheTh2/Th1 AR increases over the time [24]. Therefore, when children cytokine balance, favouring type-1 T cells [29]. This shift get older the change of developing AR is increasing, but for could be a result of either decreased IL-4 production after the patients with complaints of AR the change of having allergen stimulation [14]orenhancedIFN-𝛾 expression [30]. comparable complaints two years later is decreasing. Some studies have found a reduction of allergen-specific IgE Skin responses (measured directly after intradermal aller- in combination with an increase of specific IgG levels due gen injection and a late response with induction of inflam- to SIT [30–32]. In the large randomized study [16], of which matory cells) remain constant over time. Whereas these this present study is a component, we determined IgG1 and responses are comparable at baseline and after two years, IgG4levelsin163childrenatyear0andyear2.Theseresults these data show a positive correlation, which means that show that IgG levels remain constant over the time with a values which are high at baseline are also high after two very high correlation between year 0 and year 2 (𝑅 is 0.74 years, and low values remain low during the two years. It for IgG and 0.83 for IgG4), which emphasises that the dose of therefore appears that sensitization persists even in the cases allergen administrated might have been insufficient to induce with remission of symptoms. This is underlined by a study tolerance. of Bodtger and Linneberg [25] who describe that in adult Different studies demonstrate the effect of immunother- patientswithhousedustmiteallergy,only5.5%ofthepatients apy on regulatory mechanisms [9–12]. They describe a sig- show a remission of specific IgE after eight years, whereas nificant increase of IL-10, and TGF-𝛽 in patients undergoing remissionofsymptomsoccurredin32.4%ofthepatients. SIT compared to untreated atopic patients (or patients receiv- + + Also, a recent study of Kong et al. [26] describes that five years ing placebo). Moreover, the number of CD4 CD25 cells after skin testing 96% remain skin test positive. expressing the transcription factor Foxp3 was increased after AR is characterized by high levels of allergen-specific allergen stimulation in the immunotherapy group [13]. In IgE and secretion of the Th2 cytokines IL-4, IL-5 and IL- our study, results related to regulatory T cells are conflicting. + + 13 [3, 27, 28]. In this study, we do not see a decrease of Whereas we see an increase of CD4 CD25 cells, there is these inflammatory markers two years after the baseline no correlation between data of year 0 and data of year 2. measurement, whereas patients rate their symptoms as better Moreover, other markers regarding regulation of the immune thanbefore.Incontrasttoourfindings,inpatientswhodonot response (i.e., Foxp3, IL-10 and TGF-beta) do not increase. receive immunotherapy, allergen-specific T-cell responses In fact, IL-10 decreases in time. From this, it is unlikely that canbereducedbymeansofspecificimmunotherapy(SIT). regulatory cells play a role in this present study; self-rated 6 Mediators of Inflammation

12 𝑅𝑠 = 0.56 𝑃 < 0.0001 4 𝑅𝑠 = 0.44 𝑃 = 0.005

10 3 8

6 2

Nose—year 0 Nose—year 4

Early skin test—year 0 skin test—year Early 1 2

0 0

0 2 4 6 8 10 12 0 1 2 3 4 Nose—year 2 Early skin test—year 2 (a) (b)

100 𝑅 = 0.86 𝑃 < 0.0001 𝑠 2 𝑅𝑠 = 0.18 𝑃 = 0.27

80 1.5

60 —year 0 —year hi 1

40 CD25 + Specific IgE—year 0 IgE—year Specific

CD4 0.5 20

0 0

0 20 40 60 80 100 0 0.5 1 1.5 2 + Specific IgE—year 2 CD4 CD25hi —year 2 (c) (d)

Figure 1: Correlation between clinical and immunological values determined at baseline and two years later. (a) patient rated nose symptoms + (𝑛=52), (b) response of early skin test (𝑛=42), (c) specific IgE (𝑛=53), and (d) regulatory CD4 CD25hi Tcells(𝑛=41). Open bullets: verum SLIT-treatment during two years, closed bullets: placebo treatment during two years (see Section 2), dashed lines: median values, and 𝑅𝑠: Spearman Rank test.

allergysymptomsdodecrease,butthisisnottheresultof and regulator cells) which are observed after immunotherapy an increase of regulatory cells. However, we have to keep in do not change. This decrease in clinical symptoms might be mindthatthenumberofpatientsusedtoanswerthisquestion explained by the placebo effect, since patients participated issmall,andweshouldbeconservativewithrespecttothis in a study and took (placebo) medication during a period statement. of two years. It may be concluded that SIT plays an active Alimitationofthepresentstudyisthatonly42outof59 role in tolerance induction in the patients immune system; children (71%) wanted to participate two years later. Some of whereas during the two years of this study, allergic complaints these children were lost to followup (a total of seven children), reduce without any difference in immunological parameters and others still participated in the main SLIT study but did as determined in this study. notwanttodoadditionalskintestingorvenapuncture(atotal of ten children). However, the total number of 42 children is Acknowledgments still an acceptable number to study possible immunological mechanisms in relation to symptoms in a period of two years. The authors thank all patients and their parents for partic- From this study, it can be concluded that whereas, compa- ipation in the study and Margaretha Lambers (Department rabletoSIT,allergiccomplaintsdecrease,theimmunological of Lung Diseases, Erasmus MC) for performing the labora- changes of HDM-specific T-cell activity (both effector cells tory tests. This study was sponsored by ARTU-Biologicals, Mediators of Inflammation 7

Lelystad, The Netherlands. The authors declare that they have [15]C.M.A.deBot,H.Moed,M.Y.Bergeretal.,“Ran- noconflictofinterests. domized double-blind placebo-controlled trial of sublingual immunotherapy in children with house dust mite allergy in primary care: study design and recruitment,” BMC Family References Practice,vol.9,article59,2008. [1] J. L. Brozek,˙ J. Bousquet, C. E. Baena-Cagnani et al., “Allergic [16]C.M.deBot,H.Moed,M.Y.Bergeretal.,“Sublingual Rhinitis and its Impact on Asthma (ARIA) guidelines: 2010 immunotherapy not effective in house dust mite allergic chil- Revision,” JournalofAllergyandClinicalImmunology,vol.126, dren in primary care,” Pediatric Allergy and Immunology,vol. no. 3, pp. 466–476, 2010. 23,no.2,pp.151–159,2012. [2] I. Hansen, L. Klimek, R. Mosges,¨ and K. Hormann,¨ “Mediators [17] M. I. Asher, U. Keil, H. R. 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[30] S. R. Durham and S. J. Till, “Immunologic changes associated with allergen immunotherapy,” JournalofAllergyandClinical Immunology,vol.102,no.2,pp.157–164,1998. [31] M. G. Tari, M. Mancino, and G. Monti, “Efficacy of sublingual immunotherapy in patients with rhinitis and asthma due to house dust mite. A double-blind study,” Allergologia et Immunopathologia,vol.18,no.5,pp.277–284,1990. [32] K. Gehlhar, M. Schlaak, W.M. Becker, and A. Bufe, “Monitoring allergen immunotherapy of pollen-allergic patients: the ratio of allergen-specific IgG4 to IgG1 correlates with clinical outcome,” Clinical and Experimental Allergy,vol.29,no.4,pp.497–506, 1999. Hindawi Publishing Corporation Mediators of Inflammation Volume 2013, Article ID 760691, 8 pages http://dx.doi.org/10.1155/2013/760691

Clinical Study Evaluation of Adipokines: Apelin, Visfatin, and Resistin in Children with Atopic Dermatitis

Edyta Machura,1 Maria Szczepanska,1 Katarzyna Ziora,1 Dariusz Ziora,2 Elzbieta Swietochowska,3 MaBgorzata Barc-Czarnecka,1 and Alicja Kasperska-Zajac4

1 DepartmentofPediatrics,MedicalUniversityofSilesia,Ulica3-goMaja13-15,41-800Zabrze,Poland 2 Department of Pneumonology and Tuberculosis, Medical University of Silesia, Ulica Koziołka 1, 41-803 Zabrze, Poland 3 Department of Biochemistry, Medical University of Silesia, Ulica Jordana 19, 41-808 Zabrze, Poland 4 Department of Internal Diseases, Allergology and Clinical Immunology, Medical University of Silesia, Ulica Ceglana 35, 40-952 Katowice, Poland

Correspondence should be addressed to Edyta Machura; [email protected]

Received 9 July 2012; Revised 27 December 2012; Accepted 28 December 2012

Academic Editor: Wilco de Jager

Copyright © 2013 Edyta Machura et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Very little is known about the role of adipokines in atopic dermatitis (AD) in children. This study aimed at analyzing the serumlevelsofresistin,apelin,andvisfatininchildrenwithADinrelationtobodyweight,ADseverity,andgender.Serum concentration of adipokines was measured in 27 children with AD and in 46 healthy subjects. Selected biochemical parameters were evaluated and skin prick test was performed. Serum levels of resistin and apelin were significantly higher, whereas serum visfatin concentration was significantly lower in children with AD versus healthy controls, although an increase in resistin levels was exclusively demonstrated in boys. In AD group, a significant increase in apelin levels in girls was documented. There was no relationship between adipokines levels and the degree of allergic sensitization. Receiver operating characteristic curve analysis demonstrated that the serum apelin cutoff value differentiating children with AD from those without was >137.8 pg/mL. Resistin and visfatin cutoff values were >3.8 ng/mL and ≤ 2.13 ng/mL, respectively. Apelin and visfatin can serve as excellent indicators to distinguish children with AD from those without disease.

1. Introduction compounds was observed in obesity and obesity-related disorders [5]. In addition, several studies suggest that some Increasing literature evidence indicates that obesity is a of adipokines are involved in the pathogenesis of other risk factor for the development of asthma [1, 2]. Exact inflammatory disorders including asthma [7], inflammatory mechanisms for the links between obesity and asthma are not bowel disease [8], rheumatoid arthritis [9], and psoriasis well understood, although a possible role for adipokines has [10]. Several reports pointed at the proinflammatory role of been indicated [3, 4]. leptin and anti-inflammatory role of adiponectin in asthma, Recent studies showed that adipose tissue is far more but the data are currently equivocal [4, 7]. It seems that than a site for energy storage and it is in fact an active another adipokine, resistin, may be also involved in asthma endocrine, paracrine, and also immune organ secreting mul- pathogenesis and severity [11]. Our recent studies revealed tiple bioactive mediators, called adipokines. These adipokines altered apelin and visfatin levels in childhood atopic asthma include hormones (leptin, adiponectin), cytokines (TNF- [12, 13]. 𝛼, IL-6, IL-10, and visfatin), and other proteins (apelin, In contrast to asthma, studies in children that investigate resistin), which participate in numerous physiological and the relationship between obesity and AD are few but with pathological processes [5, 6]. The deregulated synthesis controversial results [2, 14]. Also data related to the role and and/or secretion of adipokines towards the proinflammatory potential significance of adipokines in AD are limited7 [ ]. 2 Mediators of Inflammation

Since AD is characterized as a chronic inflammatory dis- 6000, Roche Diagnostic, Switzerland). Low-density lipopro- order, we hypothesized that abnormalities in adipokine tein cholesterol (LDL-C) was calculated by the Friedewald serum levels might occur. Therefore in the present study, formula (LDL-C = TC − HDL-C − TG/5). we estimated apelin, visfatin, and resistin serum levels in children with AD. The adipokine concentrations were related 2.1.3. Apelin, Visfatin, and Resistin Measurements. Serum to body weight and AD severity. Additionally, possible dif- visfatin C-terminal and apelin-12 concentrations were mea- ferences in adipokine levels between boys and girls were also sured by commercial enzyme immunoassay kit according to explored. the manufacturer’s protocol (human recombinant visfatin C- terminal, human recombinant apelin-12 (Phoenix Pharma- 2. Materials and Methods ceuticals, Inc., Burlingame, CA, USA)). The concentrations of apelin-12 and visfatin C-terminal were determined on Twenty-seven children (mean age 9.9 ± 0.77 range 4.3–17.5y) the basis of standard curve carried out for serial dilution suffering from mild to severe AD were enrolled into the available in kit standards. Absorbance was measured using 𝜇 study. AD was diagnosed according to the criteria described spectrophotometer ( Quant, Microplate Reader, Bio-Tek, by Hanifin and Rajka15 [ ]. The severity of dermatitis was Winooski, VT, USA) at 450 nm wavelengths. Acquired data estimated according to the SCORAD index (ranging from 0 were analyzed using KC Junior Software (v.1.31.5, Bio-Tek to 103 points) [16].Basedonthetotalscore,6childrenwere Instruments, Winooski, VT, USA). The sensitivity values classified as mild (<25 points), 12 children as moderate (25– for visfatin and apelin-12 were 2.17 ng/mL and 0.07 ng/mL, 50 points), and 9 children as severe (>50 points) AD. All the respectively. The intra- and interassay errors for visfatin were < < < children had positive skin prick tests (SPTs) to ≥1allergens.A 10% and 15% and 8.2% and 15% for apelin-12, respectively. positive SPT was defined as a mean diameter of at least 3mm Serum resistin concentration was measured by com- inthepresenceofnegativediluentsandpositivehistamine mercially available enzyme-linked immunosorbent assay kit controls. The degree of allergic sensitization was measured by (Mediagnost, Reutlingen, Germany). The concentration of the whole size of SPTs. Children with concomitant asthma resistin was determined on the basis of standard curve carried andrhinitiswereexcludedfromthisstudy.Thepatients out for serial dilution available in kit standards (human were not treated with any antihistamines, topical steroids, or recombinant resistin). Immunocomplex detection was deter- calcineurin inhibitors for at least 1 week before enrolment mined on the basis of reaction with rabbit polyclonal anti- into the study (only emollients were applied). They were free body antihuman immunoglobulin (IgG) conjugated with of any systemic steroids during the preceding 8 weeks. The horseradish peroxides and then with TMB substrate solution control group consisted of 46 healthy children (mean age (TMB substrate, slow kinetic, Sigma-Aldrich, St. Louis, MO, 11.04 ± 0.62,range4−17 y) with a negative history of allergic USA). Absorbance was measured using spectrophotometer 𝜇 disease, the normal level of total serum IgE, and negative ( Quant, Microplate Reader, Bio-Tek, Winooski, VT, USA) results of skin prick test to a panel of aeroallergens (dust at 450nm wavelengths. Acquired data were analyzed using mite, mixed grass or tree pollen, cat and dog; Allergopharma, KCJuniorSoftware(v.1.31.5,Bio-TekInstruments,Winooski, Reinbek, Germany). Children included into the control group VT, USA). The sensitivity for resistin was 0.012 ng/mL, and attended the outpatient pediatric clinic for nonimmunolog- the intra- and interassay errors were 4.66% and 4.79%, ical, noninflammatory health problems and needed venous respectively. puncture. The present study was approved by the Ethics Committee 2.2. Statistical Analysis. The statistical analysis was per- of the Medical University of Silesia in Katowice. Written formed using Statistica 6.0 software (StatSoft, Inc., Tulsa, informed consent was obtained from children’s parents. OK, USA) and data presented as mean values ± SE. Mann- Whitney 𝑈 test was used for comparisons between groups. Correlations were calculated using the Spearman rank test. 2.1. Specific Measurements Accuracy of the diagnostic adipokines measurement test was assessed using the receiver operating characteristic (ROC) 2.1.1. Anthropometric Measurements. Body mass index (BMI) −2 curve analysis. ROC analysis was performed using MedCalc (body weight (kg)/height squared (m ), and standard devi- software(v11.3.5.0).Foralltests,valuesof𝑃 < 0.05 were ation (SD) score for BMI (BMI-SDS) were calculated accord- considered statistically significant. ing to the current Polish population normal range [17]. 3. Results 2.1.2. Biochemical Measurements. The fasting blood samples werecollectedinthemorningbetween7:00AMand9:30AM Characteristics of the 27 children with AD and the 46 healthy following an overnight fasting. The serum was separated by control subjects are presented in Table 1.Bothgroupswere ∘ centrifugation at 1300 g for 10 min at +4 C and immediately similar in age (𝑃=NS). In AD group, 25.79% (𝑛=6)of ∘ stored at −70 C until analyzed. Biochemical parameters, children were obese as well as 13.04% (𝑛=6)inhealthy including C-reactive protein (CRP), triglycerides (TGs), total children. Despite this, the mean values of BMI and BMI- cholesterol (TC), and high-density lipoprotein cholesterol SDS of AD children were similar as compared to those of (HDL-C), were measured, using chemistry analyzer (Cobas the healthy group. Normal weight was defined as BMI-SDS Mediators of Inflammation 3

Table 1: Demographic and clinical characteristics of children with Table 2: Mean values of adipokine serum levels in children with atopic dermatitis and healthy children. atopic dermatitis and healthy children.

Healthy Mean adipokine Children with AD Healthy children Children with AD 𝑛=27 𝑛=46 Characteristics 𝑛=27 children serum levels ( ;M/F:10/17) ( ;M/F:19/27) ( ) 𝑛=46 ( ) Apelin, pg/mL ± 9.9 ± 0.99 11.7 ± 3.7 ∗ Age, years, mean SE All subjects 157.52 ± 2.76 130.18 ± 0.89 𝑛 ∗ Sex, M: 10; F: 17 M: 19; F: 27 Boys 149.10 ± 4.42 124.69 ± 0.76 Duration of AD, years, ‡ ∗∧ 6.24 ± 0.37 — Girls 162.47 ± 3.01 134.05 ± 0.84 mean ± SE ± 47.67 ± 67 Visfatin, ng/mL SCORAD, mean SE ∗ All subjects 1.84 ± 0.04 2.54 ± 0.03 Serum total IgE, IU/mL, 768.23 ± 174.3# 50.11 ± 0.93 1.78 ± 0.03 2.37 ± 0.03∗ mean ± SE Boys ‡ 1.88 ± 0.05 2.66 ± 0.03∗∧ Eosinophils, % (𝑛, 8.5 ± 1.38 2.21 ± 0.4 Girls 𝜇 ± † cells/ L), mean SE (488.4 ± 80.61) (155.05 ± 13.42) Resistin, ng/L 4.07 ± 0.03 3.82 ± 0.05† Positive SPT, 𝑛 (%) 100 (100) 0 All subjects ∗ Total SPT wheal size, mm, Boys 4.04 ± 0.05 3.46 ± 0.04 22.9 ± 2.43 — ∧ mean ± SE Girls 4.09 ± 0.03 4.07 ± 0.04 BMI 18.1 ± 1.44 18.76 ± 0.56 Data as shown as mean ± standard error. 𝑃 values from Mann-Whitney’s 𝑈- ∗ test. 𝑃 < 0.001 AD versus control. BMI-SDS 0.83 ± 0.41 0.18 ± 0.26 † 𝑃 < 0.003 AD versus control. 2.68 ± 1.9 1.01 ± 0.2 ‡ CRP (mg/L) 𝑃 < 0.01 AD girls versus AD boys. ∧ TG (mmol/L) 1.09 ± 0.13 0.97 ± 0.04 𝑃 < 0.001 healthy girls versus healthy boys. TC (mmol/L) 4.34 ± 0.015 3.99 ± 0.03 LDL-C (mmol/L) 2.29 ± 0.015 1.99 ± 0.06 Table 3: The adipokine serum levels/BMI ratio in AD children and healthy controls. HDL-C (mmol/L) 1.86 ± 0.01 1.5 ± 0.02 BMI: body mass index; BMI-SDS: body mass index-standard deviation Adipokine serum Children with AD Healthy children 𝑛=27 𝑛=46 scores; CRP: C-reactive protein; FEV1, HDL-C: high-density lipoprotein levels/BMI ( ;M/F:10/17) ( ;M/F:19/27) cholesterol; IgE: immunoglobulin E; IU: international unit; LDL-C: low- Apelin pg/mL/BMI densitylipoproteincholesterol;SE:standarderror;SPT:skinpricktest;TC: (kg/m2) total cholesterol; TG: triglyceride. ∗ ‡ † # 9.06 ± 0.20 7.18 ± 0.19 All 𝑃 values from Mann-Whitney’s 𝑈-test. 𝑃 < 0.05, 𝑃 < 0.01,and 𝑃< All subjects 0.0002. Boys 8.54 ± 0.28 7.3 ± 0.66 ∗ Girls 9.37 ± 0.42 7.09 ± 0.36 between −2.0 and +2.0. Obesity was defined as BMI-SDS > Visfatin ng/mL 2 2.0. Underweight was defined as BMI-SDS <−2. Children BMI/(kg/m ) ∗ with AD had higher IgE levels and higher percentage and All subjects 0.11 ± 0.00 0.14 ± 0.00 ∗ blood eosinophil numbers. Boys 0.10 ± 0.01 0.14 ± 0.01 ∗ Girls 0.11 ± 0.01 0.14 ± 0.01 3.1. Comparison of Serum Adipokine Levels between Children Resistin ng/mL/BMI with AD and Control Group. Mean values of serum levels (kg/m2) of apelin, visfatin, and resistin in all children are shown in All subjects 0.23 ± 0.01 0.21 ± 0.01 Table 2. Serum levels of apelin and resistin were significantly Boys 0.23 ± 0.02 0.20 ± 0.01 higher in the total group of AD children than those of control 0.24 ± 0.01 0.21 ± 0.01 group (𝑃 < 0.001, 𝑃 < ).0.003 However, the increase in the Girls Data as shown as mean ± standard error. P values from Mann-Whitney’s 𝑈- resistin level was seen only in boys. In contrast, serum visfatin ∗ 𝑃 < 0.001 level was significantly lower in AD children as compared to test. AD versus control . that of healthy subjects (𝑃 < 0.001). In AD children, after stratifying by gender, there was a significant increase in apelin adipokine levels are dependent on the amount of adipose 𝑃 < 0.01 levels in girls as compared to those of boys ( ). tissue, adipokine levels were adjusted for BMI by dividing the On the other hand, no differences in any measured clinical measured concentration by BMI. Mean values of adipokine and laboratory parameters, including age, BMI, BMI-SDS, or serum levels/BMI ratios are shown in Table 3.Wefound severity of disease between boys and girls, were observed. that apelin levels/BMI ratio was significantly higher and In the control group, serum levels of all adipokines were visfatin level/BMI ratio was lower in children with AD when 𝑃 < 0.001 significantly higher in girls than in boys ( ). compared with healthy children (𝑃 < 0.001). However, a significant increase in apelin level/BMI ratio was seen only in 3.2. Comparison of Serum Adipokine Levels/BMI Ratio girls. The apelin/BMI ratio showed no difference in compar- between Children with AD and Control Group. As serum ison to the control group (𝑃 = 0.08). No difference between 4 Mediators of Inflammation

BMI adjusted serum concentrations of resistin between AD Table 4: Significant correlations between adipokine serum levels children and healthy subjects was found. After stratifying by and other parameters in children with atopic dermatitis and healthy gender, there was no difference in adipokines/BMI ratio in children. theparticulargroupsbothinADandinhealthychildren. Examined significant correlation 𝑅𝑃 Among the 73 children enrolled in this study, there were Atopic dermatitis 18 normal-weight children with AD and 38 healthy normal Visfatin and triglycerides 0.97 0.02 weights. No differences in serum resistin, visfatin, and apelin levels between normal weight and obese AD children were Visfatin and apelin 0.49 0.008 − observed as well (data not shown). Resistin and age 0.41 0.03 SCORAD and IgE 0.65 0.0002 Healthy controls 3.3. Correlations between Adipokines and Clinical and Labo- Visfatin and TG 0.97 0.03 ratory Parameters. We have documented only few significant Resistin and TG 0.99 0.001 correlations between adipokines levels and other parameters (Table 4). Apelin and TG 0.99 0.0003 In children with AD, a strong positive correlation was Resistin and apelin 0.98 0.019 found between visfatin concentration and TG levels. In Resistin and apelin 0.9 0.004 addition, serum apelin levels correlated positively with serum Apelin and visfatin 0.96 0.03 visfatin levels. Serum resistin levels inversely correlated with Spearman’s rank test, 𝑃 < 0.05; TG: triglyceride. age of AD children. Adipokines showed no correlation with BMI and BMI-SDS, SCORADindex, duration of AD, and ratio was seen only in girls. Moreover, the difference in all with degree of allergic sensitization. SCORADindex values adipokine levels between AD and healthy children was found, correlated exclusively with serum IgE levels. No correlation despite similar body weight. This finding suggests that these was found between BMI and severity of AD. adipokines may be implicated in the immunopathogenesis of No correlation between age, BMI and BMI-SDS, IgE, and AD, independently of their confirmed role in obesity. serum adipokines was found in examined healthy children, DataaboutthebehaviourofadipokinesinADarelimited. but strong positive correlation between serum TG levels and Only three studies evaluating leptin or adiponectin in AD resistin, visfatin, and apelin was observed. In addition in were conducted. Nagel et al. showed a negative association control group, serum levels of resistin correlated significantly between serum adiponectin levels and atopic dermatitis, and with both serum visfatin and apelin levels; also serum apelin ever diagnosed eczema, and found no association between level correlated with serum visfatin level. circulating leptin concentration and AD symptoms in chil- In order to evaluate the diagnostic value of serum dren [7]. They also did not confirm the significant difference adipokine levels for differentiation children with AD from in serum leptin between AD children and control group. all examined children, we performed receiver operating It was in accordance with the study of Bostanci et al. This characteristic analysis (ROC). When the cutoff value for manuscript has documented additionally that local steroid apelin was set to 137.8 pg/mL, the sensitivity and specificity application did not influence circulating leptin levels [18]. for AD were 96.3% (95% CI: 81.0–99.9) and 86.96% (95% In contrast to this data, Kimata documented elevated serum CI: 73.7–95.1), respectively. For resistin, the cut-off value leptin in AD children [19]. The role of adipokines such as was ≥3.8 ng/mL, the sensitivity and specificity for AD were apelin, visfatin, and resistin in patients with AD has not been 96.3% (95% CI: 81.0−99.0) and 52.17 (95% CI: 36.9–67.1), investigated so far. and for visfatin the cut-off value was ≤2.13, the sensitivity Apelin is a more recently identified adipokine, which and specificity were 92.59% (95% CI: 75.7–99.1%) and 97.83 exists in different isoforms, and acts thorough the binding to (95% CI: 88.5–99.9%) (Figure 1). The area under the ROC a specific G-protein-coupled receptor named API, present on for apelin was 0.981, and for visfatin was 0.988 indicating endothelial cells, vascular smooth myocytes, and cardiomy- that serum levels of both adipokines are the best biomarkers ocytes [20, 21]. Apelin synthesis in adipocytes is strongly differentiating subjects with atopic dermatitis from healthy upregulated by insulin, and plasma apelin level markedly children (Figure 2). increases in obese and hyperinsulinemic mice and humans [22] its excretion is induced by hypoxia [23]. In addition, 4. Discussion TNF-alpha may act as a key player in the upregulation ofapelinexpressioninadipocytesbothinobeseandlean The current study was designed to examine the circulating human [24]. It has been reported that apelin has a regulatory apelin, resistin, and visfatin levels in children with AD, effect on cardiovascular system, appetite, drinking behav- who had no current additional allergic symptoms such as iors, neoangiogenesis, lymphangiogenesis, fibrogenesis, and asthma and allergic rhinitis. To eliminate the possible effect tumor growth [25, 26].However,thedataonapelinare of obesity on adipokine levels, BMI adjusted serum concen- controversial. Lately, Reinehr et al. in their longitudinal study tration of adipokines was also assessed. We demonstrated revealed the lack of association between apelin and obesity in that in children with AD elevated apelin concentrations and children [27]. apelin/BMI ratio are accompanied by decreased visfatin levels The possible role of apelin in allergic inflammation has andvisfatin/BMIratio.However,anincreaseinapelin/BMI not been widely studied in both human and animal models, Mediators of Inflammation 5

190 3

180 2.8 2.6 170 2.4 160 (ng/mL) 2.2 ≤2.13 150 Sensitivity: 2 92.6 Apelin (pg/mL) Apelin 140 > Visfatin Specificity: 137.8 1.8 Sensitivity: 97.8 130 96.3 Specificity: 1.6 87 120 1.4 Control AD Control AD (a) (b) 4.6

4.4

4.2

(ng/mL) 3.8 >3.8 Sensitivity: 3.6 96.3 Specificity: Resistin 3.4 52.2

3.2

3 Control AD (c)

Figure 1: Interactive dot diagram comparing levels of (a) apelin, (b) visfatin, and (c) resistin levels between children with atopic dermatitis and healthy children. yet. However, in our previous papers we confirmed high increases the surface expression of costimulatory molecules, apelin concentration in children with asthma [12]. for example, CD54, CD40, and CD80 in monocytes [8]. The origin and the role of increased apelin levels in serum Moreover, visfatin enhances production of chemokines such among examined children with AD remain unclear. Since as CXCL8, CXCL10, and CCL20 in human keratinocytes neoangiogenesis is a feature of AD skin lesion [28], it seems [30]. Increased visfatin expression and/or visfatin plasma that apelin may be involved also in this chronic inflammatory concentrations have been identified in a variety of chronic disorder. inflammatory diseases, including rheumatoid arthritis9 [ ], Surprisingly, among children with AD we observed a inflammatory bowel disease [8], and psoriasis [10]. There are decreased serum level of visfatin compared with healthy chil- conflicting reports about the relationship between obesity dren, and this finding suggests that visfatin may have a protec- and circulating levels of visfatin. Some researchers reported tive effect against AD in children. We have also documented increased visfatin plasma levels in obese adult women [31] in children with atopic asthma similar reduced concentration or obese children [32], whereas others observed no apparent of visfatin [13]. In this study, we excluded children presenting relationship between overweight and/or obesity and serum simultaneously with both AD and asthma symptoms to visfatin concentrations [33], or even noted a negative corre- link the adipokines changes exclusively to AD. Visfatin lation [34]. (PBEF-pre-B cell, colony-enhancing factor) is a growth factor We observed significantly higher levels of serum resistin for early B-lymphocytes, and it is highly expressed and in AD than in controls, but this feature was seen only in boys. secreted by human visceral fat (hence the term “visfatin”) However, BMI adjusted serum concentration of resistin was [29]. Plasma concentration of visfatin strongly correlates similar in both groups. Our results suggest that the global with the amount of fat. Visfatin upregulates the production increase in resistin levels in our AD boys might correlate of the pro- and anti-inflammatory cytokines, for example, with the amount of adipose tissue. Further studies should interleukin-1𝛽,(IL-1𝛽), TNF-𝛼, IL-1Ra, IL-6, and IL-10 and explore if resistin may be one of the factors explaining 6 Mediators of Inflammation

100 The lack of relationship between adipokine levels and BMI or BMI-SDS may be explained by differences in fat distribution. Unfortunately in this study, the measures of central 80 obesity were not estimated. Since visceral fat is supposed to be more metabolically active than subcutaneous fat, the correlation of adipokine levels and central obesity requires further evaluation. We have not found difference in serum 60 adipokine levels in different degrees of both AD severity and allergic sensitization and total IgE. Additionally, the degree of AD severity showed no correlation with obesity. Recently, Sensitivity 40 Silverberg et al. in a retrospective case-control study found an association between prolonged obesity (>2,5 years) in early childhood and increased odds ratio of AD and severity of 20 AD. Nevertheless, they performed their examination without the application of a widely established AD scoring systems (SCORAD, Eczema Area and Severity Index) [2]. We demonstrated a significant difference in serum apelin 0 levels between boys and girls including children with AD. 02040 60 80 100 In the control group, sex differences in apelin, resistin, and 100-specificity visfatin levels were observed. It should be emphasized, that Apelin (pg/mL) other studies in children and adolescents have shown girls to Visfatin (ng/mL) have greater resistin concentration [39], but gender-related Resistin (ng/mL) differences in visfatin levels were not observed31 [ ]. The most interesting finding, which is the strength of the current study, is that apelin and visfatin serve as good markers Adipokine serum Area under the Standard error 95% confidence differentiating AD children from healthy ones. However, it is levels ROC curve interval Apelin 0.981 0.019 0.918–0.999 difficult to establish if altered adipokine levels play a causative role in AD or if they have changed secondary to chronic Resistin 0.683 0.067 0.563–0.787 allergic inflammation. Visfatin 0.988 0.012 0.928–1.000 The study has some limitations. Firstly, the sample size was rather small to represent all the children with atopic Figure 2: Comparison of receiver operating characteristic (ROC) dermatitis. This was because the children were selected curves for apelin, resistin, and visfatin between children with atopic according to specific criteria—high total and specific IgE or dermatitis and healthy children. positive SPT, without rhinitis or asthma. It is very difficult to collect an extensive number of AD children without the relation between obesity and AD. There are only few concomitant disorder which may affect adipokine concentra- publications on resistin in human asthma. One study of chil- tions. Secondly, the wide range of age in examined children dren showed a protective association between serum resistin with AD, although similar to control, may be considered as a concentration and risk for asthma [35]. On the contrary, drawback of this study. Thirdly, a cross-sectional analysis of LaRochelle et al. documented that plasma resistin levels this study precludes any temporal or cause-effect conclusions. were significantly higher in adult patients with moderate- In conclusion, this preliminary study provides the evi- to-severe persistent asthma than in control and increased dence of significantly higher apelin and decreased level of with disease severity [11].However,Arshietal.didnot visfatin in atopic dermatitis. Additional prospective studies find any difference in resistin levels between children with with a larger number of patients and application of other asthma and healthy children [36]. Resistin is expressed in adipokines are needed to clarify the precise mechanism brown and white adipose tissues and is a member of the and their significance in the immunopathogenesis of AD in family of cysteine-rich proteins called resistin-like molecules children. [37]. Circulating monocytes and macrophages seem to be responsible for resistin production in human [5]. Resistin has proinflammatory properties and upregulates the expression Funding of monocyte chemoattractant protein 1 (MCP-1) as well as This work was financially supported by Grants KNW-1-103/10 vascular cell adhesion molecule 1 (VCAM-1) and intercellular from the Medical University of Silesia in Katowice, Poland. adhesion molecule 1 (ICAM-1) in endothelial cells, which have been known to contribute in the pathogenesis of allergic inflammation5 [ ]. Macrophage exposure to resistin has been References showntoinduceexpressionoftumornecrosisfactor-alpha (TNF-alpha) and IL-12 and IL-6 [38]. [1]M.E.Jensen,C.E.Collins,P.G.Gibson,andL.G.Wood, In the current study, also obese AD children had sim- “The obesity phenotype in children with asthma,” Paediatric ilar levels of all adipokines as normal-weight AD children. Respiratory Reviews, vol. 12, no. 3, pp. 152–159, 2011. Mediators of Inflammation 7

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Research Article Circulating Cytokines and Growth Factors in Pediatric Pulmonary Hypertension

Mark Duncan,1, 2, 3, 4 Brandie D. Wagner,5 Keri Murray,1 Jenna Allen,1 Kelley Colvin,6 Frank J. Accurso,1 and D. Dunbar Ivy6

1 Section of Pediatric Pulmonology, School of Medicine, University of Colorado and Children’s Hospital Colorado, Aurora, CO 80045, USA 2 Obesity Research Center, King Saud University, Riyadh, Saudi Arabia 3 Biodesix Inc., Boulder, CO 80045, USA 4 Division of Endocrinology, Metabolism and Diabetes, School of Medicine, University of Colorado Denver, MS 8106, 12801 E. 17th Avenue, RC 1 South, Room 7103, Aurora, CO 80045, USA 5 Department of Biostatistics and Informatics, Colorado School of Public Health, University of Colorado Denver, Aurora, CO 80045, USA 6 Section of Pediatric Cardiology, School of Medicine, University of Colorado and Children’s Hospital Colorado, Aurora, Colorado 80045, USA

Correspondence should be addressed to Mark Duncan, [email protected]

Received 23 June 2012; Revised 25 October 2012; Accepted 15 November 2012

Academic Editor: Dirk Holzinger

Copyright © 2012 Mark Duncan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background. Management of pediatric pulmonary hypertension (PH) remains challenging. We have assessed a panel of circulating proteins in children with PH to investigate their value as predictive and/or prognostic biomarkers. From these determinations, we aim to develop a practical, noninvasive tool to aid in the management of pediatric PH. Methods. Twelve cytokines and growth factors putatively associated with lung or vascular disease were examined in plasma specimens from 70 children with PH using multiplex protein array technology. Associations between hemodynamics, adverse events, and protein markers were evaluated. Results. Epidermal growth factor (EGF) and IL-6 were associated with important hemodynamics. Of the twelve proteins, VEGF and IL-6 were significantly, univariately associated with the occurrence of an adverse event, with odds ratios (95% confidence intervals) of 0.56 (0.33–0.98) and 1.69 (1.03–2.77), respectively. When hemodynamic predictors were combined with protein markers, the ability to predict adverse outcomes within the following year significantly increased. Conclusions. Specific circulating proteins are associated with hemodynamic variables in pediatric PH. If confirmed in additional cohorts, measurement of these proteins could aid patient care and design of clinical trials by identifying patients at risk for adverse events. These findings also further support a role for inflammation in pediatric PH.

1. Introduction objective measures that can aid in managing these patients [2]. Specifically, there is a need for better tools for prognosis Pulmonary hypertension (PH) encompasses a diverse group and assessment of the progression of the disease [1, 3, 4]. of disorders, all characterized by an elevation in pulmonary Outcomes of most interest to clinicians and PH patients arterial pressure and pulmonary vascular resistance (PVR) include death, hospitalization for PH-related events, right [1]. Pediatric PH is associated with significant morbidity ventricular failure, initiation of intravenous prostanoid and mortality, and differs from PH as manifest in adults in therapy, and lung transplantation. Although definitive, these several important ways, such as the development of PH in endpointsareoftendifficult to study in pediatric PH a growing lung [1]. Medical treatment of pediatric PH has due to their infrequent occurrences and the requirement been challenging due to a lack of accessible, noninvasive, for long-term followup. Currently, disease progression is 2 Mediators of Inflammation most commonly assessed through a 6-minute walk dis- Table 1: Patient demographics and clinical measurements. tance and hemodynamic assessments, but both of these = suffer from practical drawbacks and neither is thoroughly Patient demographics (no of subjects 70) validated in the pediatric population. These limitations in current outcomes and prognostic variables have spurred our Female—no. (%) 37 (52.9%) search for reliable, noninvasive, objective, and cost-effective APAH—no. (%) 45 (64.3%) alternatives that are easily measured in children [1, 4]. Congential heart disease 31 (44.3%) Circulating protein biomarkers have the potential to meet Chronic lung disease 11 (15.7%) these objectives [5]. Clotting disorders 10 (14.3%) The role of inflammation in the pathobiology of PH Age (years)—median (IQR) 8.0 (4.4–13.0) has recently been emphasized [6–9]. Cytokine- and growth factor-dependent mechanisms lead to inflammatory cell Therapies recruitment and are prominent in PH [9–11]. Of these Mono therapy 23 (32.9%) factors, interleukin (IL)-6 is possibly the most studied [7, 12– Dual therapy 23 (32.9%) 15]. For example, IL-6 administration in rats induces PH, Triple therapy 10 (14.3%) and IL-6 augments hypoxia-induced PH. In addition, it has Calcium channel blockers 22 (31.4%) been reported recently that transgenic mice overexpressing PDE-5 inhibitors 29 (41.4%) IL-6 display PH, vascular remodeling, and an exaggerated Endothelin receptor blockers 25 (35.7%) response to hypoxia [16]. Further, circulating levels of cytokines are reportedly important in the pathogenesis of Prostacyclin 23 (32.9%) a wide range of other conditions including chronic heart Epoprostenol 12 (17.1%) failure [17], acute renal failure [18], and sepsis [19]. IV Treprostinil 8 (11.4%) Recent developments in protein array technology allow Inh Iloprost 3 (4.3%) high throughput, multiplex analysis with excellent sen- CATH variables—median (IQR) (n = 59) sitivity, precision, and specificity. The approach is also Pulmonary artery pressure, mm Hg 34 (23–56) cost effective and uses minimal sample because all the Pulmonary capillary wedge pressure, analytes are measured on a single chip simultaneously. The 8 (6–10) mm Hg requirement for low sample volumes is of special interest in the management of pediatric patients. We therefore Right atrial pressure, mm Hg 5 (3–7) applied a protein array approach that provides accurate and Cardiac index, L/min × m2 3.5 (3.0–4.3) Pulmonary vascular resistance index, precise quantification of twelve proteins with known lung 5.6 (4.1–13.9) or vascular effects in a cohort of pediatric patients with PH. wood units × m2 Our objective was to determine whether there is a clinically PVR/SVR 0.48 (0.27–0.76) relevant association between any of the panel constituents, Vasoreactivity (% change in mPAP −21.1 (−29.5 to −12.8) individually or combined, with hemodynamic parameters, w/NO) disease prognosis, and relevant adverse outcomes. Our Follow-up time (months)—median 36 (12–89) hypothesis was that measurement of these factors could be (min–max) prognostic in pediatric PH. Adverse outcomes 16 (22.9%) Within 12 months 9 (12.9%) 2. Materials and Methods First observed outcome 2.1. Patient Population. Prior approval for these studies was Expired 10 obtained from the Colorado Multiple Institutional Review Transplantation 0 Board (COMIRB, approval #05-0551). Written informed consent was obtained from the parents or guardians of Initiation of IV prostanoids 8 all children who served as subjects of the investigation, and when appropriate, from the subjects themselves. These studies comply with the Health Insurance Portability & in the cardiac catheterization laboratory during a routine Accountability Act of 1996. Plasma samples were collected pulmonary hypertension visit. The data were obtained with from 70 pediatric patients with PH ranging in age from the patient breathing room air, prior to testing pulmonary newborn to 21.3 years old. Samples were obtained between reactivity with the use of vasodilator therapy. For right- November 2001 and May 2008. All patients were being heart catheterization, we used a Swan-Ganz catheter in the treated with appropriate therapies to manage their PH femoral vein or internal jugular vein to determine mean during sample collection (Table 1)[20]. Patients were clas- right atrial pressure (RAP), mean pulmonary artery pres- sified as idiopathic pediatric pulmonary arterial hyperten- sure (PAP), pulmonary capillary wedge pressure (PCWP), sion (IPAH) or associated pulmonary arterial hypertension cardiac index (CI), and pulmonary vascular resistance index (APAH) according to published criteria [2]. (PVRi). Invasive arterial monitoring was used to measure the systemic vascular resistance index (SVRi). We used Fick 2.2. Data Variables. In this study, disease prognosis/severity with assumed oxygen consumption in those patients with was determined using hemodynamic parameters measured shunts and thermodilution in all others to measure cardiac Mediators of Inflammation 3 output and then calculate cardiac index. Most subjects predictive ability of the logistic regressions to an independent were ventilated because they were children. In addition, the set of observations, c-indices were calculated using the reactivity to inhaled nitric oxide was calculated by taking leave-out-one cross-validation approach. All analyses were the difference in mean PAP between room air and with performed using SAS version 9.2 software (SAS Institute Inc., nitric oxide. The adverse outcome used in the predictive Cary, NC, USA, 2008). models was defined as initiation of intravenous prostanoids (epoprostenol or treprostinil), transplantation, and/or death occurring within 12 months from the collection of the blood 3. Results sample. Admission for right heart failure is a rare event in children and was not included in the adverse outcome The study population consisted of 70 pediatric patients definition. with PH: 36% IPAH and 64% with APAH. Of these, 80% of patients were treated with at least one therapy (Table 1). The median PAP was elevated at 34 mm Hg (the 2.3. Circulating Protein Measurements. Blood (5 mL) was ◦ interquartile range (IQR) was 23–56 mm Hg) with a median drawn, plasma isolated, and this was frozen at −70 C RAP value of 5 mm Hg (IQR, 3–7 mm Hg). Nine (13%) until analysis. Protein determinations were performed on an patients experienced an adverse event within 12 months of Evidence Analyzer (Randox Laboratories, Northern Ireland) sample collection. These patients had worse mPAP levels according to the manufacturer’s protocol for the simulta- ranging from 30 to 76 with a median of 65.5 mm Hg as well neous quantification of the following analytes: interleukin- as a median PVRI of 15.5 Wood Units × m2 ranging from 1alpha (IL-1α), interleukin-1beta (IL-1β), interleukin-2 (IL- 5.1–20.4. Only 39 of the 48 patients with vasoreactivity tests 2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 had complete data in order to assess the responder status (IL-8), interleukin-10 (IL-10), tumor necrosis factor alpha based on the pediatric definition [23]. Based on these data (TNFα), gamma interferon cytokine (IFNγ), monocyte 2 (5%) were classified as responders and neither of these chemotactic protein-1 (MCP-1), endothelial growth factor patients experienced an adverse event. Individual protein (EGF), and vascular endothelial growth factor (VEGF). measurements are displayed separately by diagnostic group Values for individual proteins measured by this multiplexed (Figure 1). There were no significant differences between the protein array technology have been shown to correlate with IPAH and APAH groups for any of the protein levels. single ELISA measurements. Further, intra- and interassay Cardiac catheterization was performed for all patients coefficients of variation of less than 10% are typical for in the initial evaluation for PH. Thereafter, only 59 (84%) most protein measured by this multiplexed approach with of the patients had a catheterization performed on the this system [21]. Determinations were made in duplicate same day as blood collection for the correlation analysis, and all calculations were based on the average of the two and of these, 48 individuals also had a vasoreactivity test measurements for each sample. Values below the limit of in response to inhaled nitric oxide (NO). The following detection were set to a value of 0. pairs of hemodynamic and circulating protein markers had correlation coefficients (r) significantly different from zero: 2.4. Statistical Analysis. Descriptive statistics were calculated IL-1α and CI (r =−0.31, CL = −0.53 to −0.04); EGF and using mean ± standard deviations or medians and the mPAP (r =−0.45, CL = −0.63 to −0.21); EGF and PVRi interquartile range (IQR) for continuous variables and (r =−0.37, CL = −0.57 to −0.12); EGF and PVR/SVR percentages for categorical variables. Protein measurements (r =−0.42, CL = −0.62 to −0.17); IL-6 and mPAP (r = 0.27, were transformed (log base 2) and anchored at 1. Compar- CL = 0.01 to 0.49); IL-6 and PVRi (r = 0.32, CL = 0.06 to isonsacrossgroupsweremadebyWilcoxonranksumor 0.53). None of the biomarkers correlated with the change in chi-squared tests, as appropriate. Spearman rank correlation mean PAP in response to inhaled NO. coefficients were used to estimate the association between To assess the potential additive predictive ability of the hemodynamic variables and protein markers. Logistic the proteins we measured, a series of logistic regression regressions were fit to investigate the association between the models predicting adverse outcome within 1 year were dichotomous adverse outcome variable (outcome observed estimated. The first of these were univariate models that within 12 months) and the protein measurements. Mean were estimated to determine the association of each protein standardization and principal component analysis (PCA) individually with the adverse outcome variable. VEGF and were performed for dimension reduction across the protein IL-6 were significantly associated with the adverse outcome: measurements, resulting in a single value representing a (OR (95% CI) were as follows: 0.56 (0.33–0.98) and 1.69 weighted combination of all twelve markers. A score statistic (1.03–2.77), respectively. In addition to VEGF, IL-1β,IL- was used to select the principal component (PC) most 2, IL-4, MCP1, and EGF also had negative associations, associated with the outcome and the best subset of two PCs indicating that lower values of these proteins were associated and clinical variables in a multivariate logistic regression. with the adverse event outcome. PCA was performed on The predictive ability of the model was investigated using a the protein marker data to condense the 12 measurements c-index (area under the ROC curve). The improvement in to a few orthogonal components. The 3rd component was risk prediction associated with the addition of the protein significantly associated with the outcome, with higher values biomarker PC was assessed by calculation of a net reclassi- corresponding to an increased risk of an adverse event. fication improvement (NRI) measure [22]. To estimate the The risk associated with this protein index was weighted 4 Mediators of Inflammation

1024 APAH improve our ability to predict outcomes in children with PH. 512 IPAH n = (n = 25) ( 45) Speciﬁcally, the addition of a protein index, weighted mostly 256 by IL-6, IL-10, and MCP-1 concentrations, signiﬁcantly 128 increased the probabilities for those patients with an adverse 64 event compared to hemodynamic measurements alone. 32 When investigating the plasma proteins univariately, VEGF

(pg/mL) 16 8 and IL-6 were associated with occurrence of an adverse event 4 andEGFandIL-6werecorrelatedwithmeanPAPand 2 PVRi. In addition, we identified a combination of all twelve 1 proteins which associated well with adverse events and had IL1a IL1b IL2 IL4 IL6 IL8 IL10 VEGF IFNGTNFα MCP1 EGF similar predictive ability compared to the top hemodynamic Figure 1: Biomarker characteristics: the distribution of the 12 predictor. cytokine and growth factor measurements based on disease clas- Interest in the role of cytokines and growth factors in the sification. The y-axis is displayed on the log (base 2) scale. Filled development of PH has grown. Recent evidence is consistent areas indicate the interquartile range (distance between 25th and with the view that circulating factors and inflammatory 75th percentiles), the middle line corresponds to the median, and cells contribute to remodeling in chronic PH [11, 24, 25]. the whiskers contain data within 1.5 interquartile ranges. None of ff Circulating and lung levels of the proinflammatory cytokine the di erences was statistically significant. IL-6 are increased in patients with idiopathic PH and PH associated with inflammatory diseases [12–15]. IL-6 may also be a mediator of disease in PH because overexpression of IL- by higher values of IL-1α and IL-6 and lower VEGF values 6 in a mouse model exacerbated PH by both proproliferative (Figure 2). This finding corresponds well with the results and antiapoptotic mechanisms [16]. Other studies confirm from the univariate analyses and gave a c-index of 0.81. an important role for IL-6 in PH. For example, IL-6 treated Score statistics were used to identify the top predictive mice are prone to develop hypoxia-induced pulmonary clinical markers and determine the added value of a protein hypertension [26] which may be mediated in part by marker index over the top clinical predictor. The resulting an inflammatory process [27]. In our study, we found clinical model identified PAP as the top predictor. Using that increased levels of circulating IL-6 were significantly this model as a base model, the PCs of the protein associated with an increased risk of an adverse event but measurements were added as predictors and the selection when considered multivariately with PAP, CI, and VEGF, process was repeated. After mPAP, the 4th PC was the top it was replaced with an anti-inflammatory cytokine, IL-10. predictor indicating it contains information orthogonal to Interestingly, IL-6 and IL-10 levels were positively associated the hemodynamic variable. The risk associated with the 4th with each other. PC was weighted by higher values of MCP1 and IL-6 and Recently there has also been increased interest in the role lower IL-10 values. To evaluate the added predictive value of of VEGF in pulmonary arterial hypertension. Studies indi- the protein measurements in addition to mPAP, ROC curves, cate VEGF may be an important mediator of lung growth. corresponding c-indices, and NRI measures were utilized VEGF is responsible for angiogenesis and vasculogenesis, (Figure 3). The inclusion of these two proteins increased the and animal models indicate that when VEGF signaling is c-index from 0.81 to 0.90. The predicted probabilities from impaired, this contributes to the pathogenesis of PH [28, both models were also compared. Significant improvement 29]. Alternatively, growth factors may be important for the was seen with the addition of the protein measurements (NRI maintenance of continued lung growth in patients with P value = 0.01) indicating that their inclusion enhanced the pulmonary arterial hypertension, and a favorable phenotype models ability to correctly classify patients. This result was may be associated with increased VEGF and EGF. Farkas et verified with a resampling approach used to estimate the al. [30] found a similar inverse relationship between VEGF models predictive ability when applied to an independent set and PAP in rats and Lassus et al. [31] reported decreased of observations. The estimated c-indices for the model with circulating plasma levels of VEGF in human infants with mPAP alone, and with mPAP and the protein measurements, PH. were 0.69 and 0.82, respectively. The search for biomarkers that are useful in the management of pediatric PH is in its infancy, but the ideal 4. Discussion marker would be obtained noninvasively, easily measured, and would offer high sensitivity and specificity. Currently, the Our data (Table 1) indicate a surprisingly high morbidity and brain natriuretic peptide system and neurohumoral markers mortality, even in children with mild pulmonary hyperten- have been evaluated in children with PH [32–34]. sion, and they underscore the fact that this is a very high-risk Our study demonstrates that quantification of a panel group. This observation stimulated our efforts to develop of cytokines and growth factors has potential applications a practical and noninvasive approach to the management in clinical trial design by identifying patients at risk for of pediatric pulmonary hypertension that can be used in a an adverse event. In addition, patients at higher risk for routine setting. Of interest and clinical relevance is the fact an adverse event may need to be treated more aggressively that we have shown that by simply adding the quantification with continuous intravenous therapy or listed for lung of a set of plasma proteins, it is possible to markedly transplantation. Mediators of Inflammation 5

4 0.6 0.5 3 0.4 0.3 2 0.2 1 0.1 0

3rd PC 3rd 0 −0.1 PC loadings −0.2 −1 −0.3 − . −2 0 4 −0.5 −3 −0.6 β α Non-event Event<1 year α IL-2 IL-4 IL-6 IL-8 EGF IL-10 IL-1 IL-1 IFNG TNF VEGF MCP-1 3rd PC (11.4%) 4th PC (10.5%) (a) (b)

Figure 2: Dimension reduction using principal component analysis: the third PC was found to be the most associated with outcome. (a) The distribution of the 3rd PC is displayed separately by outcome. Filled areas indicate the interquartile range (distance between 25th and 75th percentiles), the middle line corresponds to the median, and the whiskers contain data within 1.5 interquartile ranges. (b) The PC loadings for the 3rd and 4th PCs are displayed. The 3rd PC is heavily loaded by positive IL-1α and IL-6 and negative VEGF values, whereas the 4th PC is heavily weighted by positive IL-6 and MCP-1 and negative IL-10 values.

1 1

0.8 0.8

0.6 0.6

Sensitivity 0.4 0.4

0.2 0.2

0 + base from Probabilities model protein 0 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1 1-speciﬁcity Probabilities from base model PAP (c = 0.81) Non-event PAP and Pr PC (c = 0.9) Event <1 year (a) (b)

Figure 3: Added value of proteins toward outcome prediction. (a) Comparison of the ROC curves indicate an improvement in discriminative ability with the addition of the 4th PC calculated from the protein measurements. c-indices for each model are also displayed. (b) The reclassification of estimated probabilities for the logistic regression model which included protein markers as a predictor versus the model based on PAP alone. The dots represent those observations associated with an event and the open circles with a nonevent. Higher percentages of events and nonevents are desired above and below the reference line, respectively. The corresponding reclassification index was significant (P = 0.01) indicating a significant improvement in prediction with the addition of the proteins.

5. Conclusions prediction and prognostic evaluation of pediatric patients with PH. It is envisioned that future use of biomarkers This study adds to the growing body of literature indicating will guide appropriate treatment selection. The potential for that inflammation is important in pediatric PH and that growth factors, EGF and VEGF, to aid in the management of circulating (blood) biomarkers can be important tools in PH is important and novel. Serial sampling and assessment 6 Mediators of Inflammation of longitudinal changes might provide important additional American Journal of Respiratory and Critical Care Medicine, insights, but these findings indicate that even a single point vol. 182, no. 12, pp. 1554–1562, 2010. in time determination can predict the future clinical course. [4]I.M.Robbins,T.M.Moore,C.J.Blaisdell,andS.H.Abman, We intentionally examined a heterogeneous group of patients “Improving outcomes for pulmonary vascular disease,” Amer- in this study in order to explore patterns of biomarker ican Journal of Respiratory and Critical Care Medicine, vol. 185, expression. It is clear that the next step is to validate these no. 9, pp. 1015–1020, 2012. findings in a larger scale study including several groups of [5] E. Soon, A. M. Holmes, C. M. Treacy et al., “Elevated levels well-characterized subjects. We suggest that, based on our of inflammatory cytokines predict survival in idiopathic and familial pulmonary arterial hypertension,” Circulation, vol. findings, additional evaluation of cytokines and growth fac- 122, no. 9, pp. 920–927, 2010. tors is warranted because there is growing evidence that these [6] R. T. Schermuly, H. A. Ghofrani, M. R. Wilkins, and F. determinations may be relevant for disease management. Grimminger, “Mechanisms of disease: pulmonary arterial hypertension,” Nature Reviews Cardiology,vol.8,no.8,pp. Abbreviation 443–455, 2011. [7] R. Mathew, “Inflammation and pulmonary hypertension,” APAH: Associated pulmonary arterial hypertension Cardiology in Review, vol. 18, no. 2, pp. 67–72, 2010. CI: Cardiac index [8] L. C. Price, S. J. Wort, F. Perros et al., “Inflammation in EGF: Endothelial growth factor pulmonary arterial hypertension,” Chest, vol. 141, no. 1, pp. IFNγ: Gamma interferon cytokine 210–221, 2012. IL: Interleukin [9]S.Hall,P.Brogan,S.G.Haworth,andN.Klein,“Contribution IPAH: Idiopathic pulmonary arterial hypertension of inflammation to the pathology of idiopathic pulmonary IQR: Interquartile range arterial hypertension in children,” Thorax,vol.64,no.9,pp. NO: Nitric oxide 778–783, 2009. MCP-1: Monocyte chemoattractant protein-1 [10] P. M. Hassoun, L. Mouthon, J. A. Barbera` et al., “Inflamma- tion, growth factors, and pulmonary vascular remodeling,” NRI: Net reclassification improvement Journal of the American College of Cardiology, vol. 54, no. 1, PAP: Pulmonary artery pressure supplement 1, pp. S10–S19, 2009. PCWP: Pulmonary capillary wedge pressure [11] R. M. Tuder, J. C. Marecki, A. Richter, I. Fijalkowska, and PH: Pulmonary hypertension S. Flores, “Pathology of pulmonary hypertension,” Clinics in PVR: Pulmonary vascular resistance Chest Medicine, vol. 28, no. 1, pp. 23–42, 2007. RAP: Right arterial pressure [12] M. Humbert, G. Monti, F. Brenot et al., “Increased SVR: Systemic vascular resistance interleukin-1 and interleukin-6 serum concentrations in TNFα: Tumor necrosis factor alpha severe primary pulmonary hypertension,” American Journal VEGF: Vascular endothelial growth factor. of Respiratory and Critical Care Medicine, vol. 151, no. 5, pp. 1628–1631, 1995. [13] K. Hashimoto, K. Nakamura, H. Fujio et al., “Epoprostenol Authors’ Contribution therapy decreases elevated circulating levels of monocyte chemoattractant protein-1 in patients with primary pul- M. Duncan and B. D. Wagner contributed equally to this monary hypertension,” Circulation Journal,vol.68,no.3,pp. work. 227–231, 2004. [14] R. M. Tuder, M. Chacon, L. Alger et al., “Expression of Acknowledgments angiogenesis-related molecules in plexiform lesions in severe pulmonary hypertension: evidence for a process of disordered This study was supported in part by UO1-HL081335, Clini- angiogenesis,” Journal of Pathology, vol. 195, no. 3, pp. 367– cal Proteomics Center in Lung Disease, NIH, Colorado CTSA 374, 2001. Grant 1 UL1 RR025780 from NCRR/NIH, P50 HL084923, [15] T. M. Yu, Y. H. Chen, J. Y. 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Research Article Symptoms, but Not a Biomarker Response to Inhaled Corticosteroids, Predict Asthma in Preschool Children with Recurrent Wheeze

E. M. M. Klaassen,1 K. D. G. van de Kant,1 Q. Jobsis,¨ 1 S. T. P. Høvig,1 C. P. van Schayck,2 G. T. Rijkers,3, 4 and E. Dompeling1

1 Department of Paediatric Pulmonology, School for Public Health and Primary Care (CAPHRI), Maastricht University Medical Centre (MUMC), P.O. Box 5800, 6202 AZ Maastricht, The Netherlands 2 Department of General Practice, CAPHRI, MUMC, P.O. Box 5800, 6202 AZ Maastricht, The Netherlands 3 Department of Medical Microbiology and Immunology, Sint Antonius Hospital, P.O. Box 2500, 3430 EM Nieuwegein, The Netherlands 4 Department of Sciences, Roosevelt Academy, P.O. Box 94, 4330 AB Middelburg, The Netherlands

Correspondence should be addressed to E. M. M. Klaassen, [email protected]

Received 10 July 2012; Revised 14 November 2012; Accepted 15 November 2012

Academic Editor: Dirk Holzinger

Copyright © 2012 E. M. M. Klaassen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background. A reliable asthma diagnosis is challenging in preschool wheezing children. As inhaled corticosteroids (ICS) are more eﬀective in asthmatics than in children with transient wheeze, an ICS response might be helpful in early asthma diagnosis. Methods. 175 children (aged two–four years) with recurrent wheeze received 200 μg Beclomethasone extra-ﬁne daily for eight weeks. Changes in Exhaled Breath Condensate (EBC) biomarkers (pH, interleukin (IL)-1α, IL-2, IL-4, IL-5, IL-10, IFN-γ, sICAM, and CCL-11), Fractional exhaled Nitric Oxide (FeNO), airway resistance, and symptoms were assessed. At six years of age a child was diagnosed as transient wheezer or asthmatic. Adjusted logistic regression analysis was performed with multiple testing correction. Results. 106 transient wheezers and 64 asthmatics were analysed at six years of age. Neither changes in EBC biomarkers, nor FeNO, airway resistance, or symptoms during ICS trial at preschool age were related to asthma diagnosis at six years of age. However, asthmatics had more airway symptoms before the start of the ICS trial than transient wheezers (P<0.01). Discussion. Although symptom score in preschool wheezing children at baseline was associated with asthma at six years of age, EBC biomarkers, airway resistance, or symptom response to ICS at preschool age could not predict asthma diagnosis at six years of age.

1. Introduction of airway inflammation require invasive procedures such as bronchoalveolar lavage, bronchial biopsy, and induced spu- Asthma is one of the most commonly occurring chronic tum and therefore are not routinely used. Effective asthma diseases in children. It mostly develops in early childhood treatment is available in the form of inhaled corticosteroids and is characterized by recurrent episodes of wheezing, ff breathlessness, and coughing due to airway inflammation (ICS). ICS are less e ective in children with airway symptoms ffi and airway hyperresponsiveness [1]. However, in the major- not due to asthma [1, 3]. As a result of di culties in ity of children symptoms are virally induced and transient proper asthma diagnosis, true asthmatics are often “under- (so-called transient wheezers) [2]. So, the associated airway treated,” whereas children with transient symptoms are symptoms are subjective and not specific for asthma. More- often “overtreated” [4]. Although international guidelines over, young children are not able to perform lung function advocate a trial treatment with ICS for a likely diagnosis of tests. Consequently, a diagnosis of asthma at young age asthma in young children, so far studies addressing response hasproventobedifficult [1, 2]. Objective measurements to ICS as a diagnostic tool in children have been scarce [1]. 2 Mediators of Inflammation

Exhaled Breath Condensate (EBC) is a noninvasive tool Children were followed-up until six years of age. At this age to assess biomarkers of airway inflammation [5]. Based on a definite asthma diagnoses was made (see further). the ICS response of inflammation biomarkers in EBC at The study protocol was approved by the Dutch Central preschool age, a prediction of asthma at a later age might be Committee on Research Involving Human Subjects (CCMO: possible. Besides, ICS-induced changes in Fractional exhaled NL17407.000.07/2007-001817-40, The Hague, The Nether- Nitric Oxide (FeNO), airway resistance, and symptoms lands). All parents gave written informed consent. might contribute to an early asthma diagnosis. Therefore, our study aim was to assess whether an ICS response based 2.3. Asthma Diagnosis. At the age of six years, a definite on changes in biomarkers in EBC, FeNO, airway resistance, diagnosis (transient wheezer or true asthmatic) was made and symptoms in preschool-aged wheezing children has a by two paediatric pulmonologists. An asthma diagnosis was predictive value for an asthma diagnosis at age six years. based on symptoms, lung function (reversibility to a β2- agonist and bronchial hyperresponsiveness), and medication use. Next to this clinical assessment a diagnosis was assessed 2. Methods by a computer calculated algorithm. In this algorithm, asthma was diagnosed in case two out of the following three 2.1. Study Population. Participants from the ADEM study features were present: (1) positive symptoms (coughing ≥1 (Asthma DEtection and Monitoring study registered at night(s) a week, ≥2 episodes of wheezing during the last clinicaltrial.gov: NCT 00422747) were included. The ADEM twelve months, some days wheeze during colds or exercise study is a long-term case-control study that started in 2006 or night time, two out of the following three items; some in The Netherlands and aimed to develop a noninvasive days wheeze during colds or during exercise in combination instrument for early asthma diagnosis by using biomarkers with night time coughing; every day, most days or several of airway inflammation in exhaled breath (condensate), and days wheeze during colds, during exercise or during the night early lung function measurements (airway resistance). The time; cough during the night time) [7, 8], (2) presence of study protocol has been described previously [6]. In total, lung function abnormalities (a 20% fall in Forced expiratory 252 children aged two to four years were included: 202 volume in one second (FEV ) induced by a provocative children with recurrent wheezing symptoms (≥2 wheezing 1 concentration of histamine <2 mg/mL, or bronchodilator episodes until inclusion according to the International Study reversibility ≥9% (see below)) [9], and (3) ICS use. In of Asthma and Allergies in Childhood (ISAAC) question- case of disagreement between the clinical diagnosis and the naire) and 50 healthy controls without wheezing episodes computer calculated diagnosis, children were reassessed by until inclusion [7]. For the present analysis only the children the two paediatric pulmonologists who decided on the final with recurrent wheezing symptoms were included. diagnosis. Bronchial hyperresponsiveness and reversibility measurements were performed according to the ERS guideline 2.2. Study Design. The main study objective was to assess [10]. FEV was assessed in each child by means of the whether an ICS response in biomarkers in EBC at preschool 1 MasterScreen Pneumo (Jaeger, Wuerzburg, Germany). The age was able to predict an asthma diagnosis at six years of highest FEV of three technically satisfactory curves was used age. Besides, the diagnostic value of the ICS response in 1 for analysis. A challenge test was performed using histamine FeNO, airway resistance, and symptoms for the prediction dissolved in physiologic saline in doubling concentrations of asthma at age six years was assessed. For this purpose, from 0.032 to 16 mg/mL. Concentrations were increased an ICS trial was started in preschool children with recurrent until a fall of ≥20% (PC )inFEV was measured or the final wheeze. Airway medication (such as ICS and β -agonists) 20 1 2 dilution was reached. Thereafter, all children directly received was temporarily stopped prior to measurement. Children 400 μg salbutamol, and FEV was reevaluated after fifteen who were not able to discontinue ICS from four weeks 1 minutes. The change in FEV was expressed as a percentage before the trial were excluded from analyses. In case of 1 of the predicted value. clear symptoms of an airway infection, the measurement was postponed for four weeks. During the initial visit EBC was collected, FeNO and airway resistance were assessed, 2.4. Biomarkers of Inflammation in Exhaled Breath Conden- and questionnaires were completed (for details see below). sate. EBC was collected during ten minutes of tidal breathing Subsequently, recurrent wheezing children received 100 μg into a mask with a two-way nonrebreathing valve connected ICS (Beclomethasone) extra-fine two times a day via the to a closed glass condenser with recirculation unit developed AeroChamber (Trudell Medical International, ON, Canada) at our institute [5]. Directly after EBC collection and pH for eight weeks. Symptom relieve by using salbutamol measurement (Radiometer, Zoetermeer, The Netherlands), (Airomir, Teva Pharma NL, Haarlem, The Netherlands) samples were stored at −80◦C until further analysis. A distributed by an AeroChamber was allowed during the selection of proinflammatory cytokines ((interleukin (IL)- trial. At the end of the ICS trial, all measurements were 1α), T-helper 1 cytokines (interferon-γ (IFN-γ)andIL-2), reassessed. Compliance to ICS was assessed by weighting T-helper 2 cytokines (IL-4 and IL-5)), an antiinflammatory ICS canisters before and after the trial. Children who used cytokine (IL-10), a chemokine (Eotaxin (CCL-11)), and an a minimum of 80% of the prescribed medication were adhesion molecule (soluble Intercellular Adhesion Molecule considered compliant (used for the per protocol subanalysis). 1 (sICAM1)) were used for analysis. These biomarkers were Mediators of Inflammation 3 analysed by using the multiplex immunoassay (Luminex children with asthma and healthy controls aged 6–16 years, Corporation, Austin, TX, USA) as described previously differences in concentrations of IL-1α, IL-2, IL-4, IFN-γ, [5, 11–13]. Values under the detection limit received CCL-11, and sICAM were found of, respectively, 4.5, 13.2, a randomly generated value between zero and the detection 2.9, 11.3, 0.9, and 80.0 pg/mL [17]. If we assume comparable limit [12]. differences in for example CCL-11 in the present population between asthmatics and transient wheezers with a power of 2.5. Fractional Exhaled Nitric Oxide. As previously 90% and an alpha of 0.05, 39 asthmatics and 78 transient described, FeNO was collected offline in a 500 mL inert wheezers are needed. balloon during tidal breathing and measured by using a nitric oxide monitoring system (NIOX, Aerocrine, Solna, 3. Results Sweden) [6]. 3.1. Baseline Characteristics. Of the 202 recurrent wheezers 2.6. Airway Resistance. Airway resistance was measured included in the ADEM study, 175 parents gave informed during tidal breathing by means of the MicroRint (Micro consent to participate in the ICS trial. One participant was Medical, Rochester Ltd, UK) as described before [6, 14]. excluded from analysis as an asthma diagnosis could not The median of at least 5 technically acceptable airway be assessed due to missing data. Four children (all true interruptions during an expiratory peak flow was used for asthmatics) were unable to stop their regular ICS medication analysis. Measurements were performed before and fifteen and were therefore excluded from further analysis. In total, minutes after inhalation of 300 μg of salbutamol via a spacer 170 (65 asthmatics and 105 transient wheezers) children (AeroChamber). were included in the intention to treat analysis and 103 (42 asthmatics and 61 transient wheezers) in the per protocol 2.7. Symptom Score. Parents completed a questionnaire analysis (as they used a minimum of 80% prescribed ICS). on airway symptoms [8]. Based on this questionnaire a Figure 1 presents the study flow chart. Agreement between symptom score was computed by adding six items (cough clinical and computed diagnosis was reached in 83% before during daytime, cough at night, wheeze during daytime, and 89% after clinical reassessment. wheeze at night, dyspnoea during daytime, and dyspnoea at Baseline characteristics are displayed in Table 1.Pre- night) on a Likert scale (every day = 1, most days = 2, some school children in the asthmatic group used significantly β days = 3, a few days = 4, and not at all = 5). The minimal score more ICS and short-acting 2-agonists in the preceding year was six, and the maximal score was 30 (no day and night time compared to the transient wheeze group. Besides, atopy symptoms). at preschool age was significantly more frequent in true asthmatics. At six years of age, ICS and short-acting β2- agonists were used in 40/43% of the asthmatics and 2/6% 2.8. Statistical Analysis. Data were analysed using SPSS 18 of the transient wheezers, respectively. Atopy was present in (SPSS Inc., Chicago, IL, USA). Due to the skewed distri- 45% of the asthmatics and 31% of the transient wheezers at butions, biomarkers in EBC were log transformed which six years of age. successfully imparted a normal distribution. Differences in baseline characteristics between transient wheezers and true asthmatics were evaluated by using the chi-square test for 3.2. Analysis at Baseline. EBC and FeNO were successfully categorical variables and the independent t-test for con- collected in all children (100%). With the exception of tinuous parametric variables. The effect of each individual Eotaxin (89%), all EBC markers were above the detection potential predictor (both before the ICS trial and changes limit in more than 98% of the samples. Prebronchodilator during ICS) on asthma diagnosis at six years of age was and postbronchodilator airway resistance were successfully tested by separate logistic regression models. Models were assessed in 95% and 97% of the children, respectively. The adjusted for possible confounders (gender, atopy, eczema, symptom score was present in all children (100%). No statis- previous ICS use, first degree relative with asthma, smoking tically significant differences in biomarkers of inflammation exposure, and season of measurement). Data were presented in EBC, FeNO, or airway resistance were observed between for intention to treat analysis unless otherwise specified. P both groups at preschool age (Table 2). However, children values were corrected for multiple testing by False Discovery with an asthma diagnosis at six years of age had significantly Rate (FDR) for each hypothesis [15]. Differences were more symptoms (lower symptom score) at preschool age considered statistically significant when the FDR corrected compared to transient wheezers (odds ratio = 0.85; 95% P value was <0.05. confidence interval = 0.77–0.93; P<0.01 FDR corrected).

2.9. Power Analysis. The primary outcome measure is the 3.3. Changes Induced by Inhaled Corticosteroids. No statis- definite asthma diagnosis at six years of age. In a population tically significant changes between transient wheezers and of young children with recurrent airway symptoms, the asthmatics in biomarkers in EBC, FeNO, airway resistance or prevalence of asthma at six years of age is 30% [16]. In the symptom score were induced by the ICS trial (Table 3). FeNO current study, including 202 children with recurrent wheeze, levels in the total group neither showed significant changes this will result in at least 50 children with asthma taken into (median ppb (interquartile range) before the ICS trial: 8.4 account a drop out rate of 10%. Based on an earlier study in (4.2–14.3), after the ICS trial: 9.6 (4.5–15.4); P = 0.45). 4 Mediators of Inflammation

ADEM study recurrent wheeze N = 202

Informed consent for the ICS study N = 175

Incomplete data N = 1

Included N = 174

Not able to stop ICS N = 4

Intention to treat N = 170

Not compliant N = 67

Per protocol N = 103

ADEM: Asthma detection and monitoring; ICS: inhaled corticosteroids. Per protocol: only compliant children (at least 80% of the prescribed study medication was used). Figure 1: Study ﬂow chart.

Table 1: Characteristics at baseline for transient wheezers and true treatment of biomarkers of inflammation in EBC, FeNO, asthmatics. airway resistance, and symptoms at preschool age could Transient predict an asthma diagnosis at six years of age. Changes in Asthma Total wheeze biomarkers in EBC, FeNO, airway resistance, or symptoms N = 65 N = 170 N = 105 induced by an eight-week ICS treatment at preschool age Mean age (SD) 3.3 (0.6) 3.3 (0.6) 3.3 (0.6) were not related to an asthma diagnosis at six years of age. However, it was demonstrated that children with asthma at n Gender: male/female ( ) 55/50 37/28 92/78 six years of age had more respiratory symptoms at preschool † ∗ Atopy ,% 20 35 26 age compared to transient wheezers. Also, medication use Eczema, % 36 47 40 and atopy at baseline were significantly higher in asthmatics Use of ICS, %∗ 12 26 18 compared to transient wheezers. Collection of EBC is a noninvasive method in which Use of short-acting ∗ 31 54 40 biomarkers expected to reflect airway inflammation can be β2-agonists, % assessed, and most influence of the systemic metabolism is Asthma first degree 36 44 39 avoided. Previously our research group has shown differences relatives, % of biomarkers of inflammation between asthmatics and Smoking exposure, % 30 31 31 healthy children [17]. Moreover, a higher concentration for ∗P<0.05 between transient wheezers and true asthmatics; †atopy is defined multiple biomarkers was noted in children with more severe as a positive (≥0.35 kU/L); Phadiatop Infant test (Phadiatop Infant, Phadia, wheeze compared to children without wheeze in the present Uppsala, Sweden); SD: standard deviation. population [13]. In adult populations with asthma, it was demonstrated that several biomarkers in EBC significantly changed after ICS treatment [18, 19]. Differences of ICS 3.4. Subanalysis per Protocol. Results of the study did not response based on changes in biomarkers in children have change when only compliant children were included in the not yet been investigated. In the present study, none of the analysis (per protocol analysis). selected biomarkers in EBC before or changes during the ICS treatment were associated with an asthma diagnosis at 4. Discussion six years of age. Besides, FeNO assessed before or a change during ICS treatment at preschool age was not associated Presently, no adequate diagnostic tool is available for an with an asthma diagnosis at six years of age. FeNO levels early asthma diagnosis in preschool wheezing children. in the total group neither showed significant changes. Data This study addressed the question whether response to ICS on the effectiveness of predicting an ICS response by FeNO Mediators of Inflammation 5

Table 2: Baseline analysis at preschool age.

Median (IQR) transient wheeze Median (IQR) asthma OR 95% CI IL-1α, in pg/mL 34.0 (15.6–83.7) 44.2 (18.3–97.6) 1.10 0.84–1.44 IL-2, in pg/mL 55.5 (35.7–76.8) 57.0 (43.1–81.2) 1.32 0.80–2.16 IL-4, in pg/mL 8.8 (4.8–13.9) 9.6 (6.0–14.5) 1.15 0.70–1.88 IL-5, in pg/mL 29.8 (17.8–58.7) 37.2 (19.4–80.6) 1.21 0.85–1.73 IL-10, in pg/mL 4.5 (2.8–6.9) 5.2 (3.2–8.4) 1.09 0.72–1.65 IFN-γ, in pg/mL 28.1 (17.5–44.6) 27.1 (16.0–44.2) 0.88 0.67–1.16 sICAM, in pg/mL 190.4 (94.8–416.2) 265.5 (104.1–516.4) 1.08 0.79–1.48 CCL-11, in pg/mL 8.3 (5.2–12.5)) 8.0 (5.3–14.6) 0.90 0.71–1.15 pH 6.0 (5.7–6.3) 5.8 (5.6–6.2) 0.66 0.34–1.28 FeNO, in ppb 6.7 (3.7–13.3) 8.9 (5.4–18.1) 1.02 0.98–1.05 Airway resistance before medication§ 1.4 (0.3) 1.5 (0.4) 2.15 0.83–5.56 Airway resistance after medication§ 1.2 (0.4) 1.3 (0.3) 2.06 0.70–6.01 Bronchodilator response§ 0.1 (0.3) 0.0 (0.5) 1.44 0.45–4.55 Total symptom score∗ 27 (25–29) 25 (20–28) 0.85 0.77–0.93 ∗ False Discovery Rate (FDR) corrected P<0.01; §mean (standard deviation). IL: interleukin; IFN-γ: interferon-γ; sICAM: soluble Intercellular Adhesion Molecule; CCL-11: Eotaxin; FeNO: Fractional exhaled Nitric Oxide; OR: odds ratio; 95% CI: 95% conﬁdence interval. Analysis adjusted for gender atopy, eczema, previous inhaled corticosteroids use, asthma ﬁrst relative, smoking exposure, and season of measurement. The transient wheeze group is the reference group.

Table 3: Changes induced by inhaled corticosteroids related to an antieosinophilic inflammation effect [1, 3, 24]. Therefore, asthma diagnosis. ICS are expected to be more effective in asthmatics compared to transient wheezers. However, increasing evidence points OR 95% CI towards overlap between eosinophilic and neutrophilic IL-1α 0.96 0.78–1.19 inflammation in both asthma and transient wheeze [26, 27]. IL-2 0.99 0.71–1.38 Due to this overlap, changes in inflammation biomarkers IL-4 0.90 0.64–1.28 induced by ICS treatment might be less pronounced, and IL-5 0.97 0.76–1.24 these changes may not be able to distinguish between transient wheezers and true asthmatics, as was observed IL-10 0.87 0.65–1.16 in our study. It would be interesting to study differences IFN-γ 0.93 0.73–1.20 with healthy children as well. Previously we did demonstrate sICAM 0.97 0.77–1.22 differences between baseline measurements of biomarkers in CCL-11 0.94 0.75–1.17 EBC in healthy children and children with recurrent wheeze pH 0.99 0.61–1.60 in the same cohort [12]. However, due to ethical constraints the healthy children have not been included in the trial. FeNO 1.01 0.99–1.04 Airway resistance in preschool children has been found Airway resistance before medication 0.80 0.33–1.93 to be predictive for asthma symptoms at six years of age Airway resistance after medication 1.51 0.55–4.16 [28]. Besides, bronchodilator reversibility was able to predict Total symptom score decreased Ref. effectiveness of anti-inflammatory therapy in young children Total symptom score equal 0.74 0.27–2.04 with asthma [20]. No ICS response for airway resistance or Total symptom score increased 0.65 0.30–1.39 reversibility was demonstrated in the present study. These results should be interpreted with caution as the children IL: interleukin; IFN-γ: interferon-γ; sICAM: soluble Intercellular Adhesion Molecule; CCL-11: Eotaxin; FeNO: Fractional exhaled Nitric Oxide; OR: participating in our study in general had mild symptoms odds ratio; 95% CI: 95% confidence interval; Ref.: reference category. before and during ICS treatment, thus leaving little room for Analysis adjusted for gender atopy, eczema, previous inhaled corticosteroids improvement. use, asthma first relative, smoking exposure, and season of measurement. Even though most children had only mild symptoms, The transient wheeze group is the reference group. Due to violation of symptoms at preschool age were positively associated with linearity assumption in log transformation, results for total symptom score are split up into three equal categories. an asthma diagnosis at six years of age. This association remained highly significant after multiple testing corrections. However, changes in symptoms during ICS treatment were in children are conflicting [20–22]. Asthma is expected to not related to the asthma diagnosis at six years of age. This be predominated by an eosinophilic airway inflammation result can be interpreted twofold. First, as symptoms were while transient wheeze is characterised by a neutrophilic mild there was not enough room for improvement during inflammation [23–25]. ICS are believed to mainly have an ICS treatment. Second, improvement in symptoms was 6 Mediators of Inflammation equal for asthmatics and for transient wheezers. Either way, hampered. Although no predictive value of biomarkers in differentiation of true asthmatics from transient wheezers EBC could be demonstrated in the present study, EBC basedonsymptomreductionduetoICStreatmentwasnot biomarkers might still be of additional value for an asthma possible in our study. It will be interesting to address this diagnosis. In the future, we will determine whether profiles issue in a population with more severe symptoms. of EBC biomarkers in combination with other parameters The major strength of this study is the longitudinal such as clinical features, exhaled volatile organic compounds, design that enabled the assessment of an ICS response of and gene expression can be of value in predicting an asthma several biomarkers at preschool age and an asthma diagnosis diagnosis. at six years of age. A second strength of this study is the high follow-up rate. Information to assess an asthma diagnosis was available in all but one child at six years of age. Thirdly, Conflict of Interests measurements were feasible and noninvasive which made The authors state that they have no conflict of interests to them suitable for young children. Feasibility of assessing declare. biomarkers in EBC was increased by the use of a double- jacketed condenser with heated breath recirculation unit as was developed at our institute [5].Duetothisefficient Acknowledgments condenser system EBC could be collected in a relatively short collection time. Besides, multiple biomarkers could be mea- The authors thank medical students of the Maastricht sured in a small sample by the highly sensitive multiplexed University Kiki Vangangelt, Nedim Dzino, Imke Duijf, liquid bead arrays with good to excellent reproducibility Brenda Thonissen,¨ Esther Kalicharan and Emily Cohen [11, 17]. Finally, we chose to administer Beclomethasone for their outstanding assistance during the measurements; extra-fine. Previous research demonstrated that the use of Frank Nijpels and other workers from Instrument Devel- small particles in young children are effective and safe opment Engineering and Evaluation (IDEE), the Maastricht and are expected to result in increased and more uniform University for their contribution to the development of deposition in the lower respiratory tracts. This minimizes the condenser; TEVA Pharma NL for providing the study the necessary dosage for administration [29, 30]. However, medication and the AeroChambers; the parents and children some limitations need to be addressed. Firstly, measurement for their participation. of biomarkers of inflammation in EBC is a technique that still needs more validation and standardisation. Despite these References restrictions, our research group has extensive experience on the use of EBC as a noninvasive technique to assess [1] S. E. Pedersen, S. S. Hurd, R. F. Lemanske et al., “Global strat- biomarkers in airway inflammation [5, 6, 17]. Secondly, as egy for the diagnosis and management of asthma in children asthma is heterogeneous by nature, difficulties might arise 5 years and younger,” Pediatric Pulmonology,vol.46,no.1,pp. when assessing an asthma diagnosis. In the present study, 1–17, 2011. both a clinical and a computer algorithm were used for an [2] O. E. Savenije, R. Granell, D. Caudri et al., “Comparison of asthma diagnosis. The degree of accordance between the childhood wheezing phenotypes in 2 birth cohorts: ALSPAC and PIAMA,” Journal of Allergy and Clinical Immunology, vol. clinical and algorithm diagnosis was high, and mismatches 127, no. 6, pp. 1505–1512.e14, 2011. were reevaluated in order to prevent an incorrect diagnoses. [3] S. Schokker, E. M. W. Kooi, T. W. de Vries et al., “Inhaled cor- Finally, it can be argued whether a period of eight weeks ffi ticosteroids for recurrent respiratory symptoms in preschool ICSissu cient to assess changes in biomarkers in EBC, children in general practice: randomized controlled trial,” FeNO, airway resistance, and symptoms at preschool age to Pulmonary Pharmacology and Therapeutics,vol.21,no.1,pp. predict an asthma diagnosis at six years of age. According 88–97, 2008. to international guidelines a treatment trial with ICS of at [4] D. Caudri, A. H. Wijga, H. A. Smit et al., “Asthma symptoms least eight weeks in preschool children can give guidance to and medication in the PIAMA birth cohort: evidence for the presence of asthma [1]. Therefore, changes in biomarkers under and overtreatment,” Pediatric Allergy and Immunology, in EBC, airway resistance, FeNO, and symptoms are to be vol. 22, no. 7, pp. 652–659, 2011. expected after eight weeks. [5] P. P. Rosias, C. M. Robroeks, K. D. van de Kant et al., “Fea- sibility of a new method to collect exhaled breath condensate in pre-school children,” Pediatric Allergy and Immunology, vol. 21, no. 1, pp. e235–e244, 2010. 5. Conclusions [6]K.D.VanDeKant,E.M.Klaassen,Q.Jobsis,¨ A. J. Nijhuis, O. In conclusion, in this study we demonstrated that, although C. Van Schayck, and E. Dompeling, “Early diagnosis of asthma in young children by using non-invasive biomarkers of airway symptoms at preschool age were predictive for an asthma inflammation and early lung function measurements: study diagnosis at age six years, changes in biomarkers of inflam- protocol of a case-control study,” BMC Public Health, vol. 9, mation in EBC, FeNO, airway resistance, and symptoms article 210, 2009. induced by ICS at preschool age could not predict an asthma [7] R. Beasley, U. Keil, E. Von Mutius, and N. Pearce, “Worldwide diagnosis at six years of age. 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[8]C.V.E.Powell,P.McNamara,A.Solis,andN.J.Shaw,“A [23] D. Snijders, S. Agostini, F. Bertuola et al., “Markers of parent completed questionnaire to describe the patterns of eosinophilic and neutrophilic inflammation in bronchoalve- wheezing and other respiratory symptoms in infants and olar lavage of asthmatic and atopic children,” Allergy, vol. 65, preschool children,” Archives of Disease in Childhood, vol. 87, no. 8, pp. 978–985, 2010. no. 5, pp. 376–379, 2002. [24] D. C. Cowan, J. O. Cowan, R. Palmay, A. Williamson, and [9] R. Pellegrino, G. Viegi, V.Brusasco et al., “Interpretative strate- D. R. Taylor, “Effects of steroid therapy on inflammatory cell gies for lung function tests,” European Respiratory Journal, vol. subtypes in asthma,” Thorax, vol. 65, no. 5, pp. 384–390, 2010. 26, no. 5, pp. 948–968, 2005. [25]P.M.C.Pitrez,S.Brennan,andP.D.Sly,“Inflammatory [10] M. R. Miller, J. Hankinson, V. 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Review Article Update of Faecal Markers of Inﬂammation in Children with Cystic Fibrosis

Jung M. Lee,1 Steven T. Leach,1 Tamarah Katz, 2 Andrew S. Day,3 Adam Jaffe,1, 2 and Chee Y. Ooi1, 4

1 School of Women’s and Children’s Health, Faculty of Medicine, University of New South Wales, High Street, Sydney, NSW, Australia 2 Department of Respiratory Medicine, Sydney Children’s Hospital Randwick, Sydney, NSW, Australia 3 Department of Paediatrics, University of Otago (Christchurch), Christchurch, New Zealand 4 Department of Gastroenterology, Sydney Children’s Hospital Randwick, Sydney, NSW, Australia

Correspondence should be addressed to Chee Y. Ooi, [email protected]

Received 22 June 2012; Accepted 1 August 2012

Academic Editor: Wilco de Jager

Copyright © 2012 Jung M. Lee et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

There is evidence of intestinal inflammation in patients with CF. Intestinal inflammation may negatively impact the nutritional status of patient with CF, which adversely affects pulmonary function and survival. This paper provides an up-to-date review of intestinal inflammation in CF and an evaluation of utility of two specific faecal inflammatory markers (S100A12 and calprotectin).

1. Introduction primary cause of malabsorption is due to maldigestion from pancreatic exocrine insufficiency. However, children with Cystic fibrosis (CF) is the most common, life-shortening, CF can continue to have malabsorption despite pancreatic recessive disease in Caucasians with an average life enzyme replacement therapy (PERT) administration. It has expectancy of 40 years [1]. In the majority of cases, been previously suggested that the presence of an acidic mortality in CF is due to respiratory failure. CF is caused intestinal milieu, that impairs enzyme activity, contributes by mutations in the gene that encodes for the cystic fibrosis to the failure of PERT in nutrient assimilation in CF. More transmembrane conductance regulator (CFTR) protein [2, recently, intestinal inflammation has been hypothesized as 3]. The CFTR protein functions on the apical surface of another contributing factor. epithelial cells as a cyclic AMP-dependent chloride and a bicarbonate channel [4, 5]. Absent or defective CFTR leads to viscous luminal secretions in affected organs particularly 2. Intestinal Inflammation in Cystic Fibrosis in the lungs, intestines, and pancreas. The nutritional status of CF patients is a major deter- There is increasing evidence of intestinal inflammation in minant of pulmonary and survival outcomes. Longitudinal CF. Although the exact underlying mechanism is unknown, cohort studies in CF report a distinct survival advantage there have been several pathogenic mechanisms proposed among patients with better nutritional status [6–8]. More (Figure 1). As a primary consequence of a defective CFTR, specifically, poor nutritional status is not only strongly intestinal mucus secretions are viscid and inspissated due linked to poorer lung function but was also an independent to dehydration, are in an acidic milieu, and have altered risk factor for early death in children with CF. Several glycosylation of mucins [10, 11]. Such environmental factors contribute to impaired nutritional status in CF. These changes may predispose to alterations in the balance and/or include malabsorption, recurrent sinopulmonary infections, composition of the gut flora (dysbiosis), resulting in mucosal increased energy expenditure, and suboptimal intake [9]. inflammation. Indeed, evidence to support the presence The malabsorptive state in CF is likely multifactorial. The of alterations in gut microbiota (or dysbiosis) has been 2 Mediators of Inflammation

Repeated and/or chronic Low pH due to exocrine antibiotic exposure pancreatic insufﬁciency Detectable in stool

Intestinal dysmotility

S100A12 S100A9

Lumen Bacterial S100A8 Thick mucus overgrowth and altered Mucus bacterial layer composition CFTR

Intestinal epithelial cells Mucosa Neutrophils

Inﬂammatory Increased stimuli inﬂammatory (e.g., IL-1, LPS ) response

Figure 1: Proposed pathogenesis of intestinal inflammation in cystic fibrosis. reported in murine models of CF. Quantitative PCR of intercellular adhesion molecule (ICAM)-1, IL-2 receptor bacterial 16S RNA in CF mice revealed a >40-fold increase alpha (CD25), IL-2, and interferon-γ was observed in in bacterial load in the small intestine alone [11]. In CF the lamina propria of duodenal mucosal specimens from mice with proven dysbiosis, expression of inflammation- CF children [14]. Interestingly, 2 CF patients who had related genes decreased when these mice were treated with commenced PERT prior to the endoscopy had evidence antibiotics, hence supporting the link between intestinal of intestinal inflammation. Furthermore, small intestinal dysbiosis and inflammation in CF. Furthermore reduction inflammation was not observed in subjects with non-CF PI of intestinal bacteria significantly improved the growth of chronic pancreatitis, suggesting that PI itself is unlikely the CF mice but had no effect on growth of wild-type mice cause of intestinal inflammation. [10]. Intestinal dysmotility was also observed in CF mice, Bruzzese and colleagues [15] were the first investigators and attributed to the further development of dysbiosis to evaluate noninvasive methods for measuring intestinal and bacterial overgrowth [11]. Secondary factors such as inflammation using faecal calprotectin and rectal nitric intestinal resection (e.g., due to meconium ileus) or early oxide production. Mean (SD) faecal calprotectin levels were and/or repeated antibiotic exposure to treat sinopulmonary significantly higher in 30 children with PI CF compared to infection(s) may also influence gut microbial ecology. It has 30 healthy controls (219 (94) μg/g versus 46 (31) μg/g), but also been suggested that inflammation is induced by an significantly lower than 15 children with active inflammatory increased and abnormal antigenic load, which is a result bowel disease (IBD) (309 (86) μg/g). However the incidence of the inadequate digestive enzymes present in the small of intestinal inflammation was similar with 29 of the 30 CF intestines [12]. and all 15 of the IBD children having abnormal levels of Smyth et al. [13] previously reported measurements calprotectin. Twenty of these children with CF, 20 controls, of intestinal inflammatory proteins from whole gut lavage and 15 children with active IBD went on to have rectal of 21 children with pancreatic insufficient (PI) CF and nitric oxide production measured by the rectal dialysis bag 12 controls. This study demonstrated increased produc- technique. Similarly, nitric oxide levels were significantly tion of inflammatory biomarkers including albumin, IgG, higher in children with CF than controls but did not differ IgM, interleukin- (IL-) 1β, IL-8, neutrophil, elastase, and to those with IBD. Bruzzese and colleagues then went on eosinophilic cationic protein in children with CF. In a to give 10 CF children a probiotic, Lactobacillus casei strain separate study that compared duodenal mucosal specimens GG (LGG) once a day for 4 weeks and remeasured faecal (obtained endoscopically) from 14 PI CF patients, 20 healthy calprotectin and rectal nitric oxide levels. Notably, both controls, and 4 non-CF patients with PI chronic pancreatitis, biomarkers of calprotectin and nitric oxide were significantly an increased mononuclear cell infiltrate which expressed reduced in 8 and 5 of the 10 children, respectively, suggesting Mediators of Inflammation 3 an association between intestinal inflammation and modifi- biomarkers include S100A12, calprotectin (S100A8/S100A9), cations in intestinal microflora. IL-8, albumin, lactoferrin, IL-1, IgM, IgG, neutrophil elas- In a more recent study, Werlin et al. [16]conductedwire- tase, tumour necrosis factor-α, and eosinophil cationic less capsule endoscopy in 41 CF patients including 13 who protein (ECP) [13, 15, 16]. The S100 proteins, calprotectin were pancreatic sufficient (PS), thereby permitting direct and S100A12, will be discussed in detail. inspection of the small intestinal mucosa for endoscopic Both of these S100 biomarkers have been validated abnormalities. Signs of small intestinal inflammation include for use in another chronic inflammatory condition of the edema, erythema, mucosal breaks, and frank ulceration in intestines, namely, IBD. Furthermore, both calprotectin and 26 of the CF patients (of which 6 were PS). Those with S100A12 have been used as markers of airway and/or PI generally had more pronounced mucosal changes than systemic inflammation in CF. On the other hand, the other those with PS. When faecal calprotectin was obtained from a biomarkers listed have been poorly studied in both CF and subset of these CF patients, it was normal in all 9 PS patients intestinal inflammation. (23.2 ug/g) and elevated in those with PI (257.7 ug/g). Bruzzese et al. evaluated whether abnormal intestinal 3.1. S100 Proteins. The human calcium-binding S100 pro- microflora may result in an abnormal immune/ teins have been proposed to be sensitive markers of inflam- inflammatory response elsewhere, such as in the lungs mation. S100 proteins comprise a family of more than [17]. Using a randomized, placebo-controlled, crossover 20 calcium-binding proteins with various intracellular and study they looked at whether or not the administration extracellular roles including calcium sensing, regulation of of LGG is associated with a reduction in pulmonary cell growth, cell migration, and protein phosphorylation exacerbations in CF. Thirty-eight children and adults with PI [19–22]. A number of S100 proteins, in particular S100A8 CF were randomized to 6 months of LGG supplementation (MRP8, calgranulin A), S100A9 (MRP14, calgranulin B) and or placebo and then after a 4-week, wash-out period crossed S100A12 (calgranulin C) have innate immune functions, over to the placebo and LGG group respectively. The are expressed in phagocytes [21] and are associated with incidence of pulmonary exacerbations and the number of inflammation. hospital admissions were significantly higher in the placebo group than the LGG group. Outcomes were consistently better when looking at groups in parallel or longitudinally. 3.2. Calprotectin (S100A8/S100A9). The proteins S100A8 A similar study was conducted by Weiss et al. [18]. and S100A9 are found in granulocytes, monocytes, and early This study involved 10 patients with PI CF who received differentiation stages of macrophages and each has individual probiotics (Lactobacillus acidophilus, Lactobacillus bulgaricus, intracellular and extracellular functions [22]. Furthermore, Bifidobacterium bifidum, and Streptococcus thermophiles) S100A9 associates with S100A8 in a complex referred every day for 6 months. A significant reduction in pulmonary to as calprotectin, which is a heterogeneous arrangement exacerbation was observed during the 6 months intervention of S100A8 and S100A9. Importantly, calprotectin is also period. Although Bruzzese et al. [17] and Weiss et al. [18] induced under inflammatory conditions. Thus, calprotectin showed a reduction in pulmonary exacerbations, concurrent is not a disease-specific marker for CF. Nevertheless, over two measurement of biomarkers of intestinal inflammation was decades ago the protein complex, calprotectin, was identified not performed. in the serum of individuals with CF and was termed the CF- In addition, a possible trend between intestinal inflam- associated antigen (CFA). This complex is a heterodimer that mation (as measured by faecal calprotectin) and nutritional is secreted by a novel pathway and is present systemically or status was reported by Werlin et al. [16]. The interpretations in particular fluids in a variety of inflammatory conditions arising from this study, however, were limited by the [23]. S100A8 and S100A9 are found in various cell types, reporting of mean BMI values despite having a mixed including neutrophils, monocytes, macrophages, dendritic paediatric and adult population. Based on the studies cells, epithelial cells, microvascular endothelial cells, and conducted so far, there is convincing evidence that intestinal fibroblasts, generally as a result of activation [24]. The inflammation is present in CF, particularly those with PI. S100A8/S100A9 complex comprises 60% of the cytoso- However the potential clinical implication of this has not lic protein in neutrophils. Inflammatory triggers such as been fully explored. Gastrointestinal inflammation is a mod- cytokines (e.g., IL-1) or bacterial products (e.g. LPS) activate ifiable factor, which may impair nutrient absorption and, monocytes and intestinal epithelial cells [25]. This stimulates therefore, negatively impact on growth. Not all patients with specific expression and secretion of calprotectin by intestinal intestinal inflammation had overt gastrointestinal symptoms epithelial cells resulting in the elevated fecal calprotectin and endoscopic evaluation is invasive, costly, and time levels in faeces. Calprotectin also activates macrophages with consuming. The ability to identify and monitor intestinal an interaction with toll-like receptor 4 (TLR4) inducing inflammation noninvasively is therefore advantageous. a positive feedback. Calprotectin activates endothelial cells inducing expression of endothelial adhesion molecules. This activation stimulates leucocyte adherence promoting 3. Faecal Inflammatory Markers recruitment of leucocytes to inflamed intestinal tissue. TLR4 is activated on neutrophils amplifying inflammation by As discussed, several biomarkers have been previously used releasing cytokines (IL-1, TNF-α) and reactive oxygen species to detect intestinal inflammation in patients with CF. These (ROS). Calprotectin is released by neutrophils from this 4 Mediators of Inflammation activation [26]. Furthermore, calprotectin may also alter the TNF, and ROS production. This positive feedback triggers an gut microbiota by chelating zinc, an essential metabolite for additional release of S100A12 by mucosal and intraluminal bacteria survival [27]. granulocytes. S100A12 is therefore closely related to granu- Studies reporting the use of faecal calprotectin in patients locyte activation. with CF have been already been discussed. Faecal calprotectin The role of S100A12 in intestinal inflammation in CF has has been more extensively evaluated as a biomarker of not been fully evaluated [37]. Similar to calprotectin, the role intestinal inflammation in IBD. Faecal calprotectin is able to of S100A12 as a biomarker of intestinal inflammation has discriminate adults with active IBD from those with irritable been better defined in IBD, where faecal S00A12 is shown bowel syndrome with 100% sensitivity and 97% specificity to be a valid and reliable noninvasive biomarker of intestinal using a cutoff of 30 mg/g [28, 29]. Similar distinctions inflammation. S100A12 was elevated in stools collected from have also been made for paediatric patients with active children shown to have extensive bowel inflammation (n = IBD [29–31]. von Roon et al. evaluated the diagnostic 20; 142.4 ± 125 mg/kg) compared to controls with no precision of fecal calprotectin for IBD using prospective bowel condition (n = 24; 2.8 ± 4.8 mg/kg) (P<0.0001) studies comparing fecal calprotectin against the histological [38]. Furthermore, receiver operator curve analysis indicated diagnosis and calculated a sensitivity of 95% and a specificity that faecal S100A12 distinguished gut inflammation with of 91% for the diagnosis of IBD (versus non-IBD diagnoses) 96% sensitivity and 92% specificity with cutoff of 10 mg/kg [32]. Furthermore, faecal calprotectin levels were associated and area under the curve of 99.2%. In addition, S100A12 with the degree of IBD activity evaluated with clinical, levels were stable in stool even when stool samples were endoscopic, and histological parameters [28–30, 33]. stored at room temperature for up to 7 days. A further In addition, calprotectin has also been measured in the study expanded upon these findings for both S100A12 (and serum and respiratory tract secretions in CF patients. Golden calprotectin) [31]. Children with intestinal symptoms were et al. [34] measured serum calprotectin levels in children enrolled prospectively with faecal S100A12 and calprotectin with CF, many of who had infective pulmonary exacerba- measured. Half of these children were diagnosed with tions at the time of measurement. Serum calprotectin levels active IBD upon review of mucosal biopsies. Both S100A12 were higher in the CF group than in healthy controls. It (median 55.2 mg/kg) and calprotectin (1265 mg/kg) were was concluded that serum calprotectin provided a better elevated in the children with active IBD (n = 31) in assessment of acute airway inflammation than traditional contrast to the children without any inflammatory changes serum biomarkers of inflammation (such as white-cell count present (n = 30) (S100A12 median 1.1 mg/kg, P<0.0001; and C-reactive protein). calprotectin median 30.5 mg/kg; P<0.0001). Furthermore, Recent data suggests that there may be interactions S100A12 (using a cutoff of 10 mg/kg) gave a sensitivity between the production of S100 proteins and the underlying and specificity of 97%, respectively, for the detection of genetic abnormalities in CF. Renaud et al. [2]reported inflammation whereas calprotectin (cutoff 50 mg/kg) gave a potential regulation of the expression of the S100A8 and sensitivity of 100% and specificity of 67%. S100A9 genes by mutations in CFTR. This association InCF,Foelletal.[21] ascertained that S100A12 levels supports the possibility that CF may have a primary were raised in the serum and sputum of affected patients. inflammatory component, due to altered epithelial barrier The population included individuals with end-stage lung function and decreased innate immunity at this level. This disease and others having an infective exacerbation (rather may occur at the respiratory and gastrointestinal epithelial than young children). Mucosal expression of S100A12 was borders. Thomas et al. [35] also suggested that inflammation increased in lung biopsy samples (immunohistochemistry) is a primary component of CF related to an underlying and levels of S100A12 were increased in serum and spu- abnormal immune response. In this study, CF mice (carrying tum samples. Furthermore, the levels of this protein were the G551D mutation) had an altered immune response to increased at the time of an infective exacerbation and fell bacterial products (e.g., LPS). after successful treatment, potentially playing the role of a surrogate serum marker for infectious exacerbations.

3.3. S100A12. In contrast to the broad expression pattern of 4. Areas for Future Research calprotectin, S100A12 is more restricted primarily to granulocytes [21, 22, 24]. S100A12 acts independently without Future work is required to better understand the underlying S100A8 or S100A9 during calcium dependent signalling. mechanisms in the development of intestinal inflammation. S100A12 is exclusively released after inflammatory activation As alterations in the gut microbiota have been implicated, of granulocytes. S100A12 exerts inflammatory effects on a the use of next generation high throughput sequencing variety of cell types through binding to the receptor of technology to investigate the gut microbiota in CF should advanced glycation end products (RAGE). RAGE ligation be considered. The direct effects of intestinal inflammation activates Nuclear-Factor-(NF-) κB and related intracellular on nutritional and pulmonary status have not yet been fully signalling pathway with the consequent production of pro- investigated. Further work is also required to determine the inflammatory mediators [36]. The interaction with RAGE most reliable biomarker(s) of intestinal inflammation in CF. and receptors on endothelial cells also induces leucocyte In addition, the roles of targeted anti-inflammatory therapies adhesion and transmigration. Stimulation of granulocytes in for the gut as well as use of pro- and prebiotics require further an autocrine fashion results in release of cytokines like IL-1, evaluation, with promising clinical implications. Mediators of Inflammation 5

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Maiuri, G. de Ritis et al., “Evidence of chronic markers. Faecal biomarkers of inflammation, especially inflammation in morphologically normal small intestine of calprotectin and S100A12, have promising roles in the cystic fibrosis patients,” Pediatric Research,vol.47,no.3,pp. identification of these patients. Greater insights into the 344–350, 2000. intestinal milieu in CF and its systemic implications are [15] E. Bruzzese, V. Raia, G. Gaudiello et al., “Intestinal inflam- much needed. mation is a frequent feature of cystic fibrosis and is reduced by probiotic administration,” Alimentary Pharmacology and Acknowledgments Therapeutics, vol. 20, no. 7, pp. 813–819, 2004. [16] S. L. Werlin, I. Benuri-Silbiger, E. 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