The Pediatric Infectious Disease Journal • Volume 33, Number 6, June 2014
12. Jungbluth GL, Welshman IR, Hopkins NK. Linezolid pharmacokinetics in pediatric patients: an overview. Pediatr Infect Dis J. 2003;22(9 suppl): S153–S157. 13. Park IN, Hong SB, Oh YM, et al. Efficacy and tolerability of daily-half dose linezolid in patients with intractable multidrug-resistant tuberculosis. J Antimicrob Chemother. 2006;58:701–704. 14. Koh WJ, Kwon OJ, Gwak H, et al. Daily 300 mg dose of linezolid for the treatment of intractable multidrug-resistant and extensively drug-resistant tuberculosis. J Antimicrob Chemother. 2009;64:388–391. 15. Khaliq Y, Zhanel GG. Fluoroquinolone-associated tendinopathy: a critical review of the literature. Clin Infect Dis. 2003;36:1404–1410. 16. Consolidated action plan to prevent and combat multidrug and extensively drug-resistant Tuberculosis in the WHO European Region 2011–2015. Available at: http://www.euro.who.int/__data/assets/pdf_file/0007/147832/ wd15E_TB_ActionPlan_111388.pdf. Accessed September 1, 2012. 17. Direção Geral de Saúde in Programa Nacional de luta contra a tuberculose. Tuberculose Multirresistente, Sinopse para Seleção dos Regimes Terapêuticos 2011. Available at: http://www.dgs.pt/?cr=20229. Accessed September 1, 2012.
PERIOSTIN IS UPREGULATED IN CORONARY ARTERIOPATHY IN KAWASAKI DISEASE AND IS A POTENTIAL DIAGNOSTIC BIOMARKER Rebecca Reindel, MD,* Kwang-Youn A. Kim, PhD,† Susan C. Baker, PhD,‡ Stanford T. Shulman, MD,* Elizabeth J. Perlman, MD,§ Mark W. Lingen, DDS, PhD,¶ Cynthia Trevenen, MD,‖ and Anne H. Rowley, MD* Abstract: Periostin was upregulated 11-fold in acute and chronic Kawasaki Disease coronary arteries compared with controls (P = 0.003). Kawasaki Disease patients had significantly elevated serum periostin values compared with febrile controls (P = 0.0086). There was no relationship between serum periostin values and age, gender or acute phase reactants; there was a relationship between serum periostin and maximal coronary artery Z scores that did not reach significance (P = 0.08). Periostin may prove to be useful as a component of a future diagnostic biomarker panel for Kawasaki Disease. Key Words: Kawasaki disease, biomarker, periostin Accepted for publication December 4, 2013. From the *Department of Pediatrics, Northwestern University Feinberg School of Medicine, Ann & Robert H. Lurie Children’s Hospital of Chicago; †Department of Preventive Medicine, Northwestern University Feinberg School of Medicine; ‡Department of Microbiology/Immunology, Loyola University Stritch School of Medicine, Maywood; §Department of Pathology, Northwestern University Feinberg School of Medicine, Ann and Robert H. Lurie Children’s Hospital of Chicago; ¶Department of Pathology, University of Chicago Pritzker School of Medicine; and ‖Department of Pathology and Laboratory Medicine, Alberta Children’s Hospital, Calgary, Alberta, Canada. This work was supported by the National Institutes of Health HL 63771 and HL109955 to AHR, the American Heart Association of Metropolitan Chicago, the Max Goldenberg Foundation and the Center for Kawasaki Disease at the Ann & Robert H. Lurie Children’s Hospital of Chicago. The authors have no other funding conflicts of interest to disclose. Address for correspondence: Anne H. Rowley, MD, 310 E Superior, Morton 4-685B, Chicago, IL 60611.E-mail: [email protected]. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (www.pidj.com). Copyright © 2014 by Lippincott Williams & Wilkins DOI: 10.1097/INF.0000000000000233
K
awasaki Disease (KD) is a medium-sized muscular arterial disease of early childhood with a predilection for the coronary arteries, resulting in potentially life-threatening dilatation, aneurysms, thrombosis and stenosis. The etiologic agent remains unknown, and the diagnosis can be difficult because clinical features overlap with other childhood illnesses.1 Light and transmission electron microscopic studies of KD coronary arteries reveal © 2014 Lippincott Williams & Wilkins
Periostin in Kawasaki Disease
3 distinct linked vasculopathic processes (1) acute self-limited neutrophilic necrotizing arteritis, (2) subacute/chronic vasculitis and (3) luminal myofibroblastic proliferation, a progressive stenosing intraluminal proliferative lesion composed of smooth muscle cell-derived myofibroblasts and their extracellular matrix (ECM) products.2 Serum markers indicating coronary artery damage from the 3 processes, if identified, could prove useful in KD diagnosis. We previously reported dysregulation of several ECM molecules in KD coronary arteries by real-time RT-PCR (reverse transcriptase polymerase chain reaction) ECM gene array analysis.3 Periostin, a matricellular protein that plays a role in vascular and cardiac responses to injury and repair,4 was not included on the commercially available ECM array used in our previous study but was of interest to us as a potential biomarker of KD because it is a secreted protein. In this study, we determined whether periostin was upregulated in KD coronary arteries and whether serum levels of periostin were elevated in children with acute KD.
METHODS Patients/Tissues/Sera This study was approved by the Ann & Robert H. Lurie hildren’s Hospital of Chicago Institutional Review Board. Clinical C and demographic information on KD patients and febrile controls are shown in Tables (Supplemental Digital Content 1–4, , http:// links.lww.com/INF/B785, http://links.lww.com/INF/B786, http:// links.lww.com/INF/B787 and http://links.lww.com/INF/B788). The mean age of the 15 afebrile controls was 3 years. Coronary arteries from KD patients were individually embedded, while control epicardial coronary arteries with normal pathologic findings were microdissected from myocardial tissue blocks. All KD patients had significant coronary arteriopathy; See Table, Supplemental Digital Content 1, http://links.lww.com/INF/B785, for case numbers from our previously published study reporting the pathologic features of these cases.2 KD sera were obtained before treatment. Coronary artery dilation was defined as a Z score of the right or left anterior descending coronary arteries of >2.
RNA Extraction From Formalin-fixed, ParaffinEmbedded Tissues RNA was extracted from formalin-fixed, paraffin-embedded coronary artery tissue sections (7–10 microns) using the Qiagen RNeasy FFPE kit (Qiagen/SA Biosciences, Valencia, CA) as per manufacturer instructions, except that proteinase K lysis was performed for 1 hour at 56°C.
cDNA Synthesis and cDNA Quality Assessment Single strand cDNA was synthesized from 300 ng of extracted RNA using the Qiagen/SA Biosciences RT2 preAMP cDNA Synthesis Kit. The quality of each cDNA sample was assessed in triplicate by real-time PCR using SYBR Green and primers for the RNA housekeeping gene RPL13A and for human genomic DNA contamination (HGDC; Qiagen/SA Biosciences). Samples were considered to be of good quality if the C(t) values for HGDC were at least >3 (8 fold-change) for RPL13A and the RPL13A C(t) was
12. Jungbluth GL, Welshman IR, Hopkins NK. Linezolid pharmacokinetics in pediatric patients: an overview. Pediatr Infect Dis J. 2003;22(9 suppl): S153–S157. 13. Park IN, Hong SB, Oh YM, et al. Efficacy and tolerability of daily-half dose linezolid in patients with intractable multidrug-resistant tuberculosis. J Antimicrob Chemother. 2006;58:701–704. 14. Koh WJ, Kwon OJ, Gwak H, et al. Daily 300 mg dose of linezolid for the treatment of intractable multidrug-resistant and extensively drug-resistant tuberculosis. J Antimicrob Chemother. 2009;64:388–391. 15. Khaliq Y, Zhanel GG. Fluoroquinolone-associated tendinopathy: a critical review of the literature. Clin Infect Dis. 2003;36:1404–1410. 16. Consolidated action plan to prevent and combat multidrug and extensively drug-resistant Tuberculosis in the WHO European Region 2011–2015. Available at: http://www.euro.who.int/__data/assets/pdf_file/0007/147832/ wd15E_TB_ActionPlan_111388.pdf. Accessed September 1, 2012. 17. Direção Geral de Saúde in Programa Nacional de luta contra a tuberculose. Tuberculose Multirresistente, Sinopse para Seleção dos Regimes Terapêuticos 2011. Available at: http://www.dgs.pt/?cr=20229. Accessed September 1, 2012.
PERIOSTIN IS UPREGULATED IN CORONARY ARTERIOPATHY IN KAWASAKI DISEASE AND IS A POTENTIAL DIAGNOSTIC BIOMARKER Rebecca Reindel, MD,* Kwang-Youn A. Kim, PhD,† Susan C. Baker, PhD,‡ Stanford T. Shulman, MD,* Elizabeth J. Perlman, MD,§ Mark W. Lingen, DDS, PhD,¶ Cynthia Trevenen, MD,‖ and Anne H. Rowley, MD* Abstract: Periostin was upregulated 11-fold in acute and chronic Kawasaki Disease coronary arteries compared with controls (P = 0.003). Kawasaki Disease patients had significantly elevated serum periostin values compared with febrile controls (P = 0.0086). There was no relationship between serum periostin values and age, gender or acute phase reactants; there was a relationship between serum periostin and maximal coronary artery Z scores that did not reach significance (P = 0.08). Periostin may prove to be useful as a component of a future diagnostic biomarker panel for Kawasaki Disease. Key Words: Kawasaki disease, biomarker, periostin Accepted for publication December 4, 2013. From the *Department of Pediatrics, Northwestern University Feinberg School of Medicine, Ann & Robert H. Lurie Children’s Hospital of Chicago; †Department of Preventive Medicine, Northwestern University Feinberg School of Medicine; ‡Department of Microbiology/Immunology, Loyola University Stritch School of Medicine, Maywood; §Department of Pathology, Northwestern University Feinberg School of Medicine, Ann and Robert H. Lurie Children’s Hospital of Chicago; ¶Department of Pathology, University of Chicago Pritzker School of Medicine; and ‖Department of Pathology and Laboratory Medicine, Alberta Children’s Hospital, Calgary, Alberta, Canada. This work was supported by the National Institutes of Health HL 63771 and HL109955 to AHR, the American Heart Association of Metropolitan Chicago, the Max Goldenberg Foundation and the Center for Kawasaki Disease at the Ann & Robert H. Lurie Children’s Hospital of Chicago. The authors have no other funding conflicts of interest to disclose. Address for correspondence: Anne H. Rowley, MD, 310 E Superior, Morton 4-685B, Chicago, IL 60611.E-mail: [email protected]. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website (www.pidj.com). Copyright © 2014 by Lippincott Williams & Wilkins DOI: 10.1097/INF.0000000000000233
K
awasaki Disease (KD) is a medium-sized muscular arterial disease of early childhood with a predilection for the coronary arteries, resulting in potentially life-threatening dilatation, aneurysms, thrombosis and stenosis. The etiologic agent remains unknown, and the diagnosis can be difficult because clinical features overlap with other childhood illnesses.1 Light and transmission electron microscopic studies of KD coronary arteries reveal © 2014 Lippincott Williams & Wilkins
Periostin in Kawasaki Disease
3 distinct linked vasculopathic processes (1) acute self-limited neutrophilic necrotizing arteritis, (2) subacute/chronic vasculitis and (3) luminal myofibroblastic proliferation, a progressive stenosing intraluminal proliferative lesion composed of smooth muscle cell-derived myofibroblasts and their extracellular matrix (ECM) products.2 Serum markers indicating coronary artery damage from the 3 processes, if identified, could prove useful in KD diagnosis. We previously reported dysregulation of several ECM molecules in KD coronary arteries by real-time RT-PCR (reverse transcriptase polymerase chain reaction) ECM gene array analysis.3 Periostin, a matricellular protein that plays a role in vascular and cardiac responses to injury and repair,4 was not included on the commercially available ECM array used in our previous study but was of interest to us as a potential biomarker of KD because it is a secreted protein. In this study, we determined whether periostin was upregulated in KD coronary arteries and whether serum levels of periostin were elevated in children with acute KD.
METHODS Patients/Tissues/Sera This study was approved by the Ann & Robert H. Lurie hildren’s Hospital of Chicago Institutional Review Board. Clinical C and demographic information on KD patients and febrile controls are shown in Tables (Supplemental Digital Content 1–4, , http:// links.lww.com/INF/B785, http://links.lww.com/INF/B786, http:// links.lww.com/INF/B787 and http://links.lww.com/INF/B788). The mean age of the 15 afebrile controls was 3 years. Coronary arteries from KD patients were individually embedded, while control epicardial coronary arteries with normal pathologic findings were microdissected from myocardial tissue blocks. All KD patients had significant coronary arteriopathy; See Table, Supplemental Digital Content 1, http://links.lww.com/INF/B785, for case numbers from our previously published study reporting the pathologic features of these cases.2 KD sera were obtained before treatment. Coronary artery dilation was defined as a Z score of the right or left anterior descending coronary arteries of >2.
RNA Extraction From Formalin-fixed, ParaffinEmbedded Tissues RNA was extracted from formalin-fixed, paraffin-embedded coronary artery tissue sections (7–10 microns) using the Qiagen RNeasy FFPE kit (Qiagen/SA Biosciences, Valencia, CA) as per manufacturer instructions, except that proteinase K lysis was performed for 1 hour at 56°C.
cDNA Synthesis and cDNA Quality Assessment Single strand cDNA was synthesized from 300 ng of extracted RNA using the Qiagen/SA Biosciences RT2 preAMP cDNA Synthesis Kit. The quality of each cDNA sample was assessed in triplicate by real-time PCR using SYBR Green and primers for the RNA housekeeping gene RPL13A and for human genomic DNA contamination (HGDC; Qiagen/SA Biosciences). Samples were considered to be of good quality if the C(t) values for HGDC were at least >3 (8 fold-change) for RPL13A and the RPL13A C(t) was
