CLINICS January 2014

Volume 69Number 1 - January/2014

CLINICSEditors

Wagner Farid Gattaz, Edmund Chada BaracatFaculdade de Medicina da Universidade de Sao Paulo

Sao Paulo, SP, Brazil

Editorial Board

Abhijit ChandraKing George’s Medical CollegeLucknow, India

Adamastor Humberto PereiraUniversidade Federal do Rio Grandedo SulPorto Alegre, RS, Brazil

Adauto CasteloUniversidade Federal de Sao PauloSao Paulo, SP, Brazil

Ademar LopesFundacao Antonio Prudente, Hospitaldo CancerSao Paulo, SP, Brazil

Alberto Azoubel AntunesFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Alexandre Roberto PreciosoFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Andrea SchmittUniversity of GoettingenGoettingen, Germany

Arnaldo Valdir Zumiotti

Faculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Artur Brum-FernandesUniversite de SherbrookeQuebec, Canada

Carmita Helena Najjar AbdoFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Cesar Gomes VictoraFaculdade de Medicina da UniversidadeFederal de PelotasPelotas, RS, Brasil

Daniel Romero MunozFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Edmund NeugebauerWitten/Herdecke UniversityWitten, North Rhine - Westphalia,Germany

Egberto Gaspar de Moura Jr.Universidade do Estado do Rio deJaneiroRio de Janeiro, RJ, Brazil

Ernest Eugene MooreUniversity of Colorado DenverDenver, CO, USA

Euclides Ayres CastilhoFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Fabio Biscegli JateneFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Francisco LaurindoFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Hiroyuki HirasawaChiba University School of MedicineChiba, Japan

Irismar Reis de OliveiraFaculdade de Medicina da UniversidadeFederal da BahiaSalvador, BA, Brasil

Irshad ChaudryUniversity of AlabamaBirmingham, AL, USA

Ivan Cecconello

Faculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Ke-Seng ZhaoSouthern Medical UniversityGuangzhou, China

Laura Cunha RodriguesLondon School of Hygiene andTropical Medicine - Universityof LondonLondon, UK

Marcelo ZugaibFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Marco Martins AmatuzziFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Maria Aparecida Shikanai YasudaFaculdade de Medicina da Universidadede Sao PauloSao Paulo, SP, Brazil

Mauro PerrettiWilliam Harvey Research InstituteLondon, UK

Area Editors

Ana Maria de Ulhoa EscobarFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Anna Sara Shafferman LevinFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Antonio Egidio NardiUniversidade Federal do Rio de JaneiroRio de Janeiro, RJ, Brazil

Anuar Ibrahim MitreFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Berenice Bilharinho MendoncaFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Bruno ZilbersteinFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Carlos SerranoFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Carmen Silvia Valente BarbasFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Claudia Regina Furquim de AndradeFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Emilia Inoue SatoUniversidade Federal de Sao PauloSao Paulo, SP, Brazil

Fulvio Alexandre ScorzaUniversidade Federal de Sao PauloSao Paulo, SP, Brazil

Geraldo BusattoFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Gustavo Franco CarvalhalFaculdade de Medicina da Pontifıcia UniversidadeCatolica do Rio Grande do SulPorto Alegre, Rio Grande do Sul, Brazil

Heitor Franco de Andrade Jr.Faculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Jesus Paula CarvalhoFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Joaquim Prado Moraes-FilhoFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Jose Luiz Gomes do AmaralUniversidade Federal de Sao PauloSao Paulo, SP, Brazil

Ludhmila Abrahao HajjarFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Luız Eugenio Garcez-LemeFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Luiz Fernando OnuchicFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Lydia Masako FerreiraUniversidade Federal de Sao PauloSao Paulo, SP, Brazil

Marcos IntagliettaUniversity of California, San DiegoSan Diego, CA, USA

Maria Cecılia SolimeneFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Mauricio EtchebehereUniversidade Estadual de CampinasCampinas, SP, Brazil

Michele CorrealeUniversity of FoggiaFoggia, Italy

Naomi Kondo NakagawaFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Nelson WoloskerFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Newton Kara-JuniorFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Olavo Pires de CamargoFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Oswaldo Keith OkamotoUniversidade de Sao PauloSao Paulo, SP, Brazil

Patricia Rieken Macedo RoccoUniversidade Federal do Rio de JaneiroRio de Janeiro, RJ, Brazil

Paulo HoffFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Raul CoimbraUniversity of California, San DiegoLa Jolla, CA, USA

Renato Delascio LopesDuke University Medical CenterDurham, NC, USA

Ricardo Bassil LasmarUniversidade Federal FluminenseNiteroi, RJ, Brazil

Rosa Maria Rodrigues PereiraFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Rubens Belfort Jr.Universidade Federal de Sao PauloSao Paulo, SP, Brazil

Ruth GuinsburgUniversidade Federal de Sao PauloSao Paulo, SP, Brazil

Ruy Jorge Cruz JuniorUniversity of PittsburghPittsburgh, PA, USA

Sandro EstevesANDROFERT - Andrology & HumanReproduction ClinicCampinas, SP, Brazil

Sergio Paulo BydlowskiFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Simone AppenzellerUniversidade Estadual de CampinasCampinas, SP, Brazil

Sophie Francoise Mauricette DerchainFaculdade de Ciencias Medicas, UniversidadeEstadual de CampinasCampinas, SP, Brazil

Suely Kazue Nagahashi MarieFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Valeria AokiFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Michael Gregory SarrMayo ClinicRochester, MN, USA

Milton de Arruda MartinsFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Mitchell C. PosnerThe University of Chicago Medical CenterChicago, IL, USA

Moyses SzkloJohns Hopkins Bloomberg School of Public HealthBaltimore, USA

Navantino AlvesFaculdade de Ciencias Medicas de Minas GeraisBelo Horizonte, MG, Brazil

Noedir Antonio Groppo StolfFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Pedro Puech-LeaoFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Peter LibbyBrigham and Women’s HospitalBoston, Boston, MA, USA

Philip CohenUniversity of Houston Health CenterHouston, Texas, USA

Rafael Andrade-AlegreSanto Tomas HospitalRepublic of Panama, Panama

Ricardo Antonio RefinettiFaculdade de Medicina da Universidade Federal doRio de JaneiroRio de Janeiro, RJ, Brazil

Roberto ChiesaSan Raffaele Hospital

Milan, Italy

Ronald A. AshersonNetcare Rosebank HospitalRosebank, Johannesburg, South Africa

Samir RasslanFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Tarcisio Eloy Pessoa de BarrosFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Valentim GentilFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

Wagner Farid GattazFaculdade de Medicina da Universidade de Sao PauloSao Paulo, SP, Brazil

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Alberto Jose da Silva DuarteAluisio Augusto Cotrim SeguradoAna Claudia Latronico XavierBerenice Bilharinho de MendoncaCarlos Roberto Ribeiro de CarvalhoClarice TanakaClaudia Regina Furquim de AndradeCyro FestaDalton de Alencar Fischer ChamoneDaniel Romero MunozEdmund Chada BaracatEduardo MassadEloisa Silva Dutra de Oliveira BonfaEuripedes Constantino MiguelFabio Biscegli JateneFlair Jose CarrilhoGerson ChadiGilberto Luis CamanhoGiovanni Guido CerriIrene de Lourdes NoronhaIrineu Tadeu VelascoIvan Cecconello

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Clinics. Sao Paulo: Scientific Journal of Hospital das Clınicas da Faculdade de Medicina da Universidade de Sao Paulo, 2005-

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CLINICS

CONTENTS

Clinics 2014 69(1):1–74

CLINICAL SCIENCES

Treatment of AO/OTA 31-A3 intertrochanteric femoral fractures with a percutaneous compression

plateFei Luo, Jie Shen, Jianzhong Xu, Shiwu Dong, Qiang Huang, Zhao Xie . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1

Clinical dysphagia risk predictors after prolonged orotracheal intubationGisele Chagas de Medeiros, Fernanda Chiarion Sassi, Laura Davison Mangilli, Bruno Zilberstein,

Claudia Regina Furquim de Andrade. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8

PITX2C loss-of-function mutations responsible for idiopathic atrial fibrillationXing-Biao Qiu, Ying-Jia Xu, Ruo-Gu Li, Lei Xu, Xu Liu, Wei-Yi Fang, Yi-Qing Yang, Xin-Kai Qu . . . . . . . . . . . . . 15

Comparison of the TruView infant EVO2 PCDTM and C-MAC video laryngoscopes with direct

Macintosh laryngoscopy for routine tracheal intubation in infants with normal airwaysHaitham Mutlak, Udo Rolle, Willi Rosskopf, Richard Schalk, Kai Zacharowski, Dirk Meininger, Christian Byhahn . . . .23

The thickness of posterior cortical areas is related to executive dysfunction in Alzheimer’s diseaseLuciano de Gois Vasconcelos, Andrea Parolin Jackowski, Maira Okada de Oliveira, Yona Mayara Ribeiro Flor,

Altay Alves Lino Souza, Orlando Francisco Amodeo Bueno, Sonia Maria Dozzi Brucki . . . . . . . . . . . . . . . . . . . . . . 28

Sex differences in 24-hour ultra-marathon performance - A retrospective data analysis from

1977 to 2012Laura Peter, Christoph Alexander Rust, Beat Knechtle, Thomas Rosemann, Romuald Lepers . . . . . . . . . . . . . . . . . . 38

Angiotensin II type 1 and 2 receptors and lymphatic vessels modulate lung remodeling and fibrosis in

systemic sclerosis and idiopathic pulmonary fibrosisEdwin Roger Parra, Aline Domingos Pinto Ruppert, Vera Luiza Capelozzi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Risk factors associated with death in Brazilian children with severe dengue: a case-control studyMaria dos Remedios Freitas Carvalho Branco, Expedito Jose de Albuquerque Luna, Leonidas Lopes Braga Junior,

Ricardo Villar Barbosa de Oliveira, Lıvia Teresa Moreira Rios, Maria do Socorro da Silva, Maria Nilza Lima Medeiros,

Gilnara Fontinelle Silva, Fernanda Campos Amaral Figueiredo Nina, Taliane Jardim Lima, Jayron Alves Brito,

Avessandra Costa Cardoso de Oliveira, Claudio Sergio Pannuti . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

BASIC RESEARCH

Mibefradil reduces blood glucose concentration in db/db miceYujie Lu, Min Long, Shiwen Zhou, Zihui Xu, Fuquan Hu, Ming Li . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

ISSN-1807-5932

RAPID COMMUNICATION

Upregulation of SOCS-1 by Nutlin-3 in acute myeloid leukemia cells but not in primary normal cellsVeronica Tisato, Alessia Norcio, Claudio Celeghini, Daniela Milani, Arianna Gonelli, Paola Secchiero . . . . . . . . . . . . 68

Treatment of AO/OTA 31-A3 intertrochanteric femoralfractures with a percutaneous compression plateFei Luo,I# Jie Shen,I# Jianzhong Xu,I Shiwu Dong,II Qiang Huang,I Zhao XieI*I The Third Military Medical University, Southwest Hospital, Department of Orthopaedics, Chongqing, China. II The Third Military Medical University, Key

Laboratory of Biomechanics and Tissue Engineering under the National Education, Department of Anatomy, Chongqing, China.

OBJECTIVE: AO/OTA 31-A3 intertrochanteric femoral fractures have completely different fracture line directionsand biomechanical characteristics compared with other types of intertrochanteric fractures. The choice of thefixation method has been a focus of dispute among orthopedic trauma surgeons. The purpose of this study wasto review the outcomes of these fractures treated with a percutaneous compression plate at our institute.

METHOD: Seventeen patients with AO/OTA 31-A3 intertrochanteric femoral fractures were treated with apercutaneous compression plate at our institute from January 2010 to December 2011. The clinical data andimaging results were retrospectively analyzed.

RESULTS: The medical complication of popliteal vein thrombosis occurred in one patient. Sixteen patients werefollowed up for 12 to 21 months. Two patients had malunion and mild pain. Fracture collapse occurred in twopatients, with one having head penetration. These two patients had moderate pain. There were no occurrencesof nonunion or reoperation. The mean Harris hip score obtained during the last follow-up was 84.1 (61-97).Patients with a poor quality of reduction were more likely to have pain results (p = 0.001). A trend existedtoward the presence of a poor quality of reduction (p = 0.05) in patients with a collapse of fracture. Patientswith poor preoperative mobility were more likely to have a lower Harris hip score (p = 0.000).

CONCLUSION: The percutaneous compression plate is an alternative device for the treatment of AO/OTA 31-A3intertrochanteric femoral fractures. Good fracture reduction and an ideal placement position of the neck screware important in the success of the device.

KEYWORDS: AO/OTA 31-A3 Intertrochanteric Femoral Fractures; Percutaneous Compression Plate; Sliding HipScrew; Intramedullary Nail.

Luo F, Shen J, Xu J, Dong S, Huang Q, Xie Z. Treatment of AO/OTA 31-A3 intertrochanteric femoral fractures with a percutaneous compressionplate. Clinics. 2014;69(1):1-7.

Received for publication on February 6, 2013; First review completed on March 6, 2013; Accepted for publication on April 12, 2013

E-mail: [email protected]

*corresponding author

#contributed equally to this work

Tel.: 86 023 68765785

& INTRODUCTION

AO/OTA 31-A3 intertrochanteric femoral fractures aredifferent from A1 and A2 intertrochanteric fractures becauseof their unique anatomical and biomechanical characteris-tics. According to the report by Haidukewych et al. (1), A3fractures accounted for 2.2% of hip fractures and 5.3% ofintertrochanteric fractures. Currently, there are two maintypes of implants available for these fractures, namely,extramedullary and intramedullary implants. The treatmentof these fractures with a sliding hip screw (SHS) has the

disadvantages of massive trauma, poor stability, and a highincidence of fixation failures. Haidukewych et al. (1), Rokitoet al. (2), and Henry et al. (3) all believed that this device wasnot suitable for AO/OTA 31-A3 intertrochanteric femoralfractures. Treating these fractures with the intramedullarynail has an advantage with respect to mechanics andefficacy in clinical applications (4-6). However, it is notminimally invasive. Some unstable proximal femoral frac-tures, such as intertrochanteric fractures with extension intothe piriform fossa (7), fractures in patients with shortskeletons and narrow femoral canals (8), and fractures inwhich a closed reduction can only be performed in theabduction position, are assessed as difficult to treat withnails. Although intramedullary nails may have advantagesover extramedullary fixation for AO type A3 fractures, thereis currently insufficient evidence to confirm a significantsuperiority of these nails over extramedullary devices (9).Thus, there is considerable controversy regarding the choiceof treatment.

Copyright � 2014 CLINICS – This is an Open Access article distributed underthe terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided theoriginal work is properly cited.

No potential conflict of interest was reported.

DOI: 10.6061/clinics/2014(01)01

CLINICAL SCIENCE

1

In recent years, minimally invasive surgical techniques havebeen popularized. Clinical studies on the percutaneous compres-sion plate (PCCP) treatment of intertrochanteric fractures haveshown that it has the advantages of being minimally invasive (10-12) and stable (10,13); thus, the PCCP may become the implant ofchoice for intertrochanteric fractures in elderly patients withmultiple comorbidities (14-16). Current studies have been mostlyrestricted to PCCP treatment for A1 and A2 fractures (10-13,17,18). AO/OTA 31-A3 intertrochanteric femoral fractureswere considered to be an absolute contraindication for the PCCPby Peyser et al. (19). However, Simecek et al. (20) reported thatthe PCCP is suitable for all types of intertrochanteric fractures,including A3 fractures. No clinical study specifically on thePCCP treatment of AO/OTA 31-A3 intertrochanteric femoralfractures has been reported. In this retrospective study, wepresent the results of PCCP implants used to treat these fractures.

& MATERIALS AND METHODS

Seventeen patients with AO/OTA 31-A3 intertrochantericfemoral fractures were treated with the PCCP (Orthofix,Italy) at our institute from January 2010 to December 2011.Based on the AO/OTA classification (21), the fractures wereclassified as 1 case of type 31-A3.1, 5 cases of type 31-A3.2,and 11 cases of type 31-A3.3. The mean age of the patientswas 77.8 (37-90) years, with 5 males and 12 females (Table 1).

Attempts were made to stabilize preexisting conditionsbefore surgery. Local nerve blockade anesthesia was applied.All operations were performed and completed by the sameassociate professor using C-arm fluoroscopy. The fractureswere reduced using an orthopedic traction bed. Traction androtation, when needed, were used with the posterior reductiondevice to achieve and maintain reduction during the surgicalprocedure, as observed in the anteroposterior and lateral views.

EthicsThis observational and retrospective study was approved

by the ethics committee of the Southwest Hospital, theThird Military Medical University, 30 Gao-tan-yan Street,Shapingba District, Chongqing, China.

Surgical techniqueAfter basic fracture reduction, the PCCP was inserted

with a 2-cm skin incision. After adjusting the plate position,the first neck screw was inserted into the appropriateposition in the femoral neck, with the entrance pointavoiding the fracture line (to avoid splitting and re-displacement in the proximal fracture when drilling)(Figures 1 and 2). Then, three shaft screws were fixedthrough the second incision. Finally, the second neck screwwas placed in the same way as the first screw. The woundwas irrigated and closed without the use of a suction drain.

Postoperative protocolAntibiotics were applied for 3-5 days postoperatively, and

anticoagulant therapy was not administered before or aftersurgery. After an X-ray examination was performed on thesecond postoperative day, the patients were encouragedto perform functional exercises with the affected limb.Touchdown weight-bearing with the help of crutches or aframe began two weeks after the surgery. One month afterthe surgery, progressive weight bearing was encouraged astolerated. Full weight bearing was encouraged 3 months aftersurgery, based on evidence of callus formation on radio-graphs. The patients were examined clinically and radio-graphically with a minimum follow-up period of 12 months.

Clinical assessmentThe operation time, perioperative blood loss, occurrence of

medical complications, length of stay in the hospital, andpreoperative functional activity status were recorded.Complications such as pain, particularly in the thigh, protrusionor cutouts of the neck screw, collapse of the fracture, headpenetration, malunion and nonunion were recorded. The func-tional assessment of the affected limb was based on the Harris hipscore (22), with possible ratings of excellent (90–100 points), good(80–89 points), fair (70–79 points), or poor (,70 points).

Radiographic assessmentPostoperative radiographs were assessed to determine

fracture reduction and the position of the neck screw. The

Table 1 - Evaluation of demographic characteristics, radiological results, mobility, and complications.

Case Gender Age (years)

AO Fracture

Types Reduction

Position of

Neck Screw Mobility Complications

Pre-op Post-op

1 Male 58 A3.3 Good Good Independent Independent

2 Female 88 A3.3 Good Good Independent One aid

3 Female 90 A3.2 Good Good One aid One aid

4 Male 88 A3.3 Bad Bad Independent One aid Mild pain

5 Female 87 A3.3 Bad Bad Independent Independent Mild pain

6 Male 37 A3.3 Good Good Independent Independent Loosening of sleeve of neck screw

7 Female 70 A3.2 Acceptable Good Independent Independent Loosening of sleeve of neck screw

8 Female 82 A3.3 Bad Good Independent Wheelchair Collapse of fracture, moderate pain

9 Female 84 A3.3 Good Good Independent Independent Protrusion of neck screw

10 Female 72 A3.3 Bad Good Independent Wheelchair Collapse of fracture, moderate pain, head

penetration

11 Female 71 A3.2 Acceptable Good Independent Independent

12 Female 82 A3.1 Good Good Independent One aid

13 Female 86 A3.3 Good Good One aid Frame Protrusion of neck screw

14 Male 81 A3.3 Good Bad One aid Deceased

15 Male 80 A3.2 Acceptable Good Independent One aid Protrusion of neck screw, popliteal vein

thrombosis

16 Female 82 A3.2 Acceptable Bad Independent Independent

17 Female 85 A3.3 Good Bad One aid Frame

AO/OTA 31-A3 fractures treated with a PCCPLuo F and Shen J et al.

CLINICS 2014;69(1):1-7

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reduction was regarded as acceptable if there was nosignificant varus malalignment of the proximal fragment orif there was less than 10 mm of translation between themain fragments (23). The optimal position of the neck screw(17,24) was in the center or the distal third of the femoralneck on the anteroposterior view and in the middle third ofthe femoral neck on the lateral view. Fractures were judgedto be healed radiographically if bridging callus was evidenton three of four cortices, as observed on two anteroposteriorand lateral views (1). A deviation of the neck-shaft angle ofmore than 15˚ was defined as malunion.

Statistical analysisThe data were analyzed using the SPSS 13.0 statistical

software package (SPSS Inc., Chicago, IL, USA). Age,operation time, blood loss, and Harris hip scores werecompared between the groups by independent t-tests.Patient gender and quality of the reduction were comparedusing Fisher’s exact tests. A two-tailed value of p,0.05 wasconsidered statistically significant.

& RESULTS

No deaths occurred during the perioperative period. Themean operating time was 41.5 (26-73) min, the meanintraoperative blood loss was 41.3 (10-80) ml, and the meanhospital stay was 8.5 (5-14) days. Based on the preoperativeX-rays, one patient was diagnosed with a type 31-A2fracture, which was confirmed as type 31-A3 after theintraoperative reduction. The medical complication ofpopliteal vein thrombosis occurred in one patient, and noother medical complications occurred in the remaining 16cases.

Clinical outcomesOne patient died of a lung infection after 25 postoperative

days. Sixteen patients attended the follow-up examinationsfor a minimum of 12 months. The mean follow-up periodwas 14 (12–21) months. There were no occurrences ofnonunion or reoperation. Pain was absent in 12 patients,mild in 2 patients, and moderate in 2 patients (Table 1).According to the Harris hip score, 6 patients had excellentresults, 6 had good results, 2 had fair results, and 2 had poorresults. The mean Harris hip score of the last follow-up was84.1 (61-97). Additionally, the mobility of 8 patients basicallyrecovered to the preoperative level, and that of theremaining 8 patients decreased to different extents. Twopatients, who suffered collapse of the fracture, depended ona wheelchair. As expected in a group of elderly patients,mobility was reduced after the surgery (Figure 3).

Radiographic outcomesAccording to the definition of the quality of the reduction,

9 patients showed good reduction, 4 had an acceptablereduction, and 4 had a poor reduction, with a bad positionof the neck screw found in 5 patients (Table 1). Imagingfollow-ups were conducted in 16 cases, with completeimaging data (including preoperative, postoperative, andfinal follow-up X-rays). All patients achieved fractureunions without additional procedures. Two patients had amalunion, with femoral neck-shaft angles with varusdeformities being greater than those of the contralateralsides by 15 . Two patients had a loosening of the sleeve ofthe neck screw, and three patients had protrusion of theneck screw. Collapse of the fracture occurred in twopatients, with one having head penetration (Figure 4). Theinitial and follow-up radiographs of a sample case arepresented in Figure 5.

Statistical outcomesWe also attempted to identify the factors predictive of

pain or fracture collapse following minimally invasiveosteosynthesis with the PCCP. Factors such as age, gender,operation time, blood loss, and quality of the reduction wereanalyzed. Although the factors were compared betweenpatients with and without pain (Table 2), those patients with

Figure 1 - Intraoperative photograph of one patient. The PCCPwas inserted in a submuscular manner and temporarily fixedwith a butterfly pin and percutaneous bone hook to the femurvia the proximal and distal incisions, respectively. The mainsleeve was brought through one of the oblique holes in theproximal plate.

Figure 2 - Intraoperative radiograph of one patient. Theentrance point avoided the fracture line during the placementof the first neck screw.

CLINICS 2014;69(1):1-7 AO/OTA 31-A3 fractures treated with a PCCPLuo F and Shen J et al.

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a poor quality of the reduction were more likely toexperience pain (p = 0.001). No significant difference existedin the presence of a poor quality of the reduction betweenthe groups with and without fracture collapse, whereas atrend existed toward the presence of a poor quality ofreduction (p = 0.05) in the fracture collapse group (Table 3).Furthermore, we found that patients with poor preoperativemobility were more likely to have a lower Harris hip score(p = 0.000) (Table 4).

& DISCUSSION

AO/OTA 31-A3 intertrochanteric femoral fractures haveunique anatomical and biomechanical characteristics, with

the major fracture line running from distal-lateral toproximal-medial. When an SHS is used for fixation, thefracture line is almost parallel to the orientation of the neckscrew, and the effect of sliding compression can contrarilycause a lateral displacement of the proximal fracture,leading to protrusion of the neck screw. Meanwhile, aproximal fracture can easily split during drilling because theintegrity of the lateral wall of the femur is destroyed and theentrance point of the neck screw is located just at thefracture line. Haidukewych et al. (1) reported that the failurerate was as high as 56%.

The internal fixation treatment with an intramedullarynail for these fractures involves central fixation andtheoretically provides better biomechanical stability (25).

Figure 3 - Graph showing the level of patient mobility before and after the fracture.

Figure 4 - A 72-year-old female patient with a type 31-A3.3 fracture. A) Initial radiograph. B) Lateral radiograph of the proximal femurat the 6th postoperative week. C) Lateral radiograph of the proximal femur at the 12th postoperative month showing the headpenetration of the proximal neck screw.


CLINICS 2014;69(1):1-7

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Kokoroghiannis et al. (26) considered intramedullary de-vices to be more appropriate for the very unstable AO31-A3fractures. Although such devices have mechanical advan-tages in theory, the reported failure rates of intramedullarynails when used to treat these fractures vary greatly due tothe different numbers of clinical cases (4,5,23,27-29). Someunstable proximal femoral fractures (7,8) are difficult to fixwith an intramedullary nail. Additionally, intramedullaryfixation is associated with a large amount of hidden bloodloss (30), with no significant difference in the soft tissuedamage compared with extramedullary fixation (31). Thus,the use of intramedullary nails is not considered to be agenuine minimally invasive treatment. Although the tech-nology of the intramedullary nail has been continuouslyimproved, the systematic review by Norris et al. (32)reported that the incidence of a second fracture afterinternal fixation with an intramedullary nail was approxi-mately 1.7%.

Hip fractures involve a complex coupling of fragilepatients with fragile bones, which an ideal treatmentmethod should be able to address. Gotfried (17) believedthat the application of the PCCP could meet these require-ments. The PCCP consists of one steel plate, two neckscrews, and three cortical bone screws. Closed reduction isapplied during surgery. The proximal fracture is fixed bytwo neck screws with rotational stability; meanwhile, thesescrews are placed in a parallel direction to restrict the lateraldisplacement of the fragments in the proximal fracture,yielding more biomechanical advantages than the SHS (33).The diameters of the two neck screws (7.2 mm) in the PCCPare smaller than those of the SHS. The small diameter andgradual drilling can protect the lateral wall and reduce theoccurrence of an intraoperative split or postoperativefracture collapse (10,13).

In our study, the mean operating time was 41.5 (26-73)min, and the mean perioperative blood loss was 41.3 (10-80)ml. These outcomes demonstrated that the PCCP has theadvantage of being minimally invasive in the treatment oftype A3 fractures. The short operation time and lowperioperative blood loss associated with this method areextremely important in elderly patients in whom otherdiseases may take precedence, thereby requiring rapidfracture fixation. All patients achieved fracture unionswithout the use of additional procedures. The meanHarris hip score obtained during the last follow-up was84.1 (61-97). Although the results were satisfactory, therewere some orthopedic complications in our study. Thesleeve of the neck screw was loose in two cases. Thiscomplication may have been due to a technical problemwhere the screw was not tightened between the sleeve andplate in some patients, causing the gradual loosening of thesleeve from the plate. Protrusion of the neck screw occurredin three cases. The cause of this protrusion is most likely theshearing force caused by the tendency for a lateraldisplacement of the proximal end and a medial displace-ment of the distal end in type A3 fractures. However, thesecomplications did not affect the fracture healing, demon-strating that dual axial fixation of the PCCP can effectivelycontrol the lateral displacement of the fragments inproximal fractures. Because of poor intraoperative reduc-tion, four patients had pain (p = 0.001). Two patients hadmalunion, with a varus malalignment of the proximalfragment. However, the X-rays obtained during follow-upshowed fracture healing. These two patients had thecapability to perform normal activities in daily life.Collapse of the fracture occurred in two patients and ledto head penetration in one patient. This complication mayhave been due to poor reduction of the fracture (p = 0.05).Furthermore, both of these patients were older than 60 yearsold and had severe osteoporosis. Two patients depended ona walker frame because they all also had sequelae ofcerebrovascular disease and depended on a walking aidbefore the surgery (p = 0.000).

According to existing studies, for AO/OTA 31-A3intertrochanteric femoral fractures, regardless of intrame-dullary or extramedullary fixation, fracture reduction andthe implant placement position are the two key factorsaffecting the efficacy of the surgery(1,4-6,23,34). A poorreduction of the fracture and a bad position of the neckscrew are important causes of fixation failure. However,based on a retrospective study of 101 cases with reverseobliquity and transverse fractures of the trochanteric region

Figure 5 - The initial and follow-up radiographs of a sample case. A) Initial radiograph. B) Radiograph 1 day postoperatively. C)Anteroposterior radiograph at 10 months postoperatively demonstrating the fracture healing. D, E) Anteroposterior and lateralradiographs at 21 months postoperatively showing the fracture healing with no obvious changes in the proximal femur.

Table 2 - Factors that may be predictive of a ‘‘pain’’ result.

Pain-absent

(N = 12) Pain (N = 4) p-value

Age (years) 76.1¡15.3 82.3¡7.3 0.458a

Gender (female) 9 (75%) 3 (75%) 1.000b

Operation time (min) 41.3¡11.0 41.7¡5.9 0.933a

Blood loss (ml) 37.9¡17.2 50.0¡21.6 0.271a

Quality of reduction

(poor)

0 (0%) 4 (100%) 0.001b

aAnalyzed using the independent t-test.bAnalyzed using Fisher’s test.


5

of the femur, Brammar et al. (35) found that the incidence ofscrew cut-out was independent of the degree of fracturereduction and the position of the neck screw in cases ofeither intramedullary or extramedullary fixation. Theyattributed this result to the small number of cases withcut-out failure. Based on the results of our clinicalobservation and follow-up, the reduction, reduction main-tenance, and placement of the neck screw during thesurgical procedure are the key steps for a successful surgeryin the PCCP treatment of AO/OTA 31-A3 intertrochantericfemoral fractures. The dual axial fixation of the device,which contributes to rotational stability and thus providescontrolled fracture impaction, has a strong anti-cut-outability. The occurrence of the lateral displacement of thefragments in the proximal fracture and coxa vara waseffectively controlled if the fracture achieved good reduc-tion. In this study, varus malunion and collapse of thefracture occurred in four patients because of poor reduction.The two neck screws were placed in an orderly manner,with the goal of avoiding the fracture line as much aspossible. Progressive drilling with a small-diameter drill bitranging from 7.0-9.3 mm was performed. This protocol isimportant to prevent the splitting and re-displacement ofthe proximal fragment during the drilling process. Theoptimal position of the neck screw was in the center or thedistal third of the femoral neck on the anteroposterior viewand in the middle third of the head on the lateral view. Inthis study, the position of the neck screw was not good infive cases, which may be attributed to poor reduction in twocases and the entrance point avoiding the fracture lineduring the placement of the first neck screw in the otherthree cases. For these patients with poor reduction and/or abad neck screw position, functional exercise in the earlypostoperative period and weight bearing in the later periodis an effective method to avoid complications.

Undoubtedly, this study also had some limitations. First,because of the absence of a control group, the results ofthe retrospective study remain to be further confirmedby prospective randomized controlled trials. Second, thenumber of cases was small because of the low morbidity ofthese fractures, making a significant conclusion between theoccurrence of fracture collapse and the quality of thereduction difficult. Third, the follow-up period was short,

and the long-term outcomes need to be evaluated throughobservations during a further follow-up.

According to the results of this study, the PCCP is analternative device treatment for AO/OTA 31-A3 intertro-chanteric femoral fractures. Good fracture reduction and anideal placement position of the neck screw are important inthe success of the device. Furthermore, the preoperativemobility of patients is also an important factor in post-operative functional recovery. Although fracture collapseoccurred in two patients, this outcome could be improvedby anatomic reduction and a more accurate placement of theneck screw in the femoral head.

& AUTHOR CONTRIBUTIONS

Xie Z and Xu JZ conceived and designed the study. Shen J and Luo F

wrote the manuscript. Luo F and Huang Q participated in data

management and performed the statistical analysis. Xie Z performed the

operations. Dong SW critically commented on the manuscript. All authors

read and approved the final version of the manuscript.

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Table 3 - Factors that may be predictive of a ‘‘collapse of fracture’’ result.

No collapse of fracture (N = 14) Collapse of fracture (N = 2) p-value

Age (years) 77.7¡14.7 77.0¡7.1 0.948a

Gender (female) 10(71.4%) 2(100%) 1.000b

Operation time (min) 40.9¡10.2 45.0¡7.1 0.594a

Blood loss (ml) 38.9¡16.2 55.0¡35.4 0.263a

Quality of reduction (poor) 2 (14.3%) 2 (100%) 0.05b

aAnalyzed using the independent t-test.bAnalyzed using Fisher’s test.

Table 4 - Preoperative mobility is predictive of the Harriship score.

Independent

(N = 13) One aid (N = 3) p-value

Harris hip score 87.0¡9.8 78.1¡7.9 0.000a

aAnalyzed using the independent t-test.


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33. Krischak G, Augat P, Beck A, Arand M, Baier B, Blakytny R, et al.Biomechanical comparison of two side plate fixation techniques in anunstable intertrochanteric osteotomy model: sliding hip screw andpercutaneous compression plate. Clin Biomech. 2007;22(10):1112-8,http://dx.doi.org/10.1016/j.clinbiomech.2007.07.016.

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35. Brammar TJ, Kendrew J, Khan RJK, Parker MJ. Reverse obliquity andtransverse fractures of the trochanteric region of the femur; a review of101 cases. Injury. 2005;36(7):851-7, http://dx.doi.org/10.1016/j.injury.2005.02.004.


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Clinical dysphagia risk predictors after prolongedorotracheal intubationGisele Chagas de Medeiros,I Fernanda Chiarion Sassi,I Laura Davison Mangilli,I Bruno Zilberstein,II Claudia

Regina Furquim de AndradeI

I Faculdade de Medicina da Universidade de Sao Paulo, Physiotherapy, Speech-Language and Hearing Sciences, and Occupational Therapy, Sao Paulo/SP,

Brazil. II Faculdade de Medicina da Universidade de Sao Paulo, Department of Gastroenterology, Sao Paulo/SP, Brazil.

OBJECTIVES: To elucidate independent risk factors for dysphagia after prolonged orotracheal intubation.

METHODS: The participants were 148 consecutive patients who underwent clinical bedside swallowingassessments from September 2009 to September 2011. All patients had received prolonged orotrachealintubations and were admitted to one of several intensive care units of a large Brazilian school hospital. Thecorrelations between the conducted water swallow test results and dysphagia risk levels were analyzed forstatistical significance.

RESULTS: Of the 148 patients included in the study, 91 were male and 57 were female (mean age, 53.64 years).The univariate analysis results indicated that specific variables, including extraoral loss, multiple swallows, cervicalauscultation, vocal quality, cough, choking, and other signs, were possible significant high-risk indicators ofdysphagia onset. The multivariate analysis results indicated that cervical auscultation and coughing wereindependent predictive variables for high dysphagia risk.

CONCLUSIONS: Patients displaying extraoral loss, multiple swallows, cervical auscultation, vocal quality, cough,choking and other signs should benefit from early swallowing evaluations. Additionally, early post-extubationdysfunction recognition is paramount in reducing the morbidity rate in this high-risk population.

KEYWORDS: Deglutition; Deglutition Disorders; Orotracheal Intubation; Clinical/Bedside Assessment.

Medeiros GC, Sassi FC, Mangilli LD, Zilberstein B, Andrade CR. Clinical dysphagia risk predictors after prolonged orotracheal intubation. Clinics.2014;69(1):8-14.

Received for publication on June 11, 2013; First review completed on June 25, 2013; Accepted for publication on July 7, 2013


Tel.: 55 11 3091-8406

& INTRODUCTION

Swallowing is a complex process that requires the precisetiming and coordination of more than 25 muscles (1),including multiple oral-facial, pharyngeal, laryngeal, res-piratory, and esophageal muscles (2), as well as 6 cranialnerves and frontal lobes (3). Alterations in this process, ordysphagia, can result in profound morbidity and canincrease the probability of aspiration and delay proper oralnutrition administration (1). To prevent aspiration, a bolusof food or fluid reaching the posterior oral cavity stimulatesneuroreceptors that trigger respiratory muscles to haltrespiration, usually during exhalation (2-4).

It is no surprise that orotracheal tubes can disturb theseintricately choreographed events and cause post-extubationdysphagia (2). Prolonged intubation, typically defined as an

intubation lasting longer than 48 hours (3,5,6), is thought tocontribute to swallowing dysfunction. The development ofpost-extubation swallowing dysfunction is well documentedin the literature and occurs with a high prevalence, with 44 to87% of these patients developing the condition (5,7). Factorsthat lead to post-extubation swallowing dysfunction aremultifactorial and include oropharyngeal muscle inactivity,glottis injury, mucosal inflammation leading to the loss oftissue architecture, and vocal cord ulcerations. Additionally,the lingering effects of narcotics and anxiolytics can bluntprotective airway reflexes (6,8). The clinical significance ofpost-extubation dysfunction is profound, as it can result inincreased morbidity and mortality. Specific risk factors forthese outcomes, however, have not been described forintensive care unit (ICU) patients who have receivedprolonged orotracheal intubation.

Various techniques have been developed to assessswallowing functions, including manometry, manofluoro-graphy, scintigraphy, electromyography, pH monitoring,and ultrasound analyses (5). Traditionally, videofluoro-scopy has been considered the gold standard for swallow-ing evaluations (5,9,10). The clinical utility of this test iscompromised, however, by the need to transport moder-ately ill patients to the radiology department, as well as the



DOI: 10.6061/clinics/2014(01)02

CLINICAL SCIENCE

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requirement of specialized equipment and personnel thatare not readily available in many hospitals (11). Thus,screening protocols that are designed to identify patients athigh risk for developing dysphagia are needed. Theseclinical screening procedures should be effective, based onthe presence of specific symptoms, in determining whichpatients should undergo a more specific form of assessment.

Speech-language pathologists are trained to evaluate andtreat oral motor function disorders objectively, managefacial and cervical muscle rehabilitation, and advisephysicians regarding tube changes and the reintroductionof oral food intake (12,13). The aim of the participation ofthese professionals in multidisciplinary teams is to preventand reduce complications resulting from oral motor func-tion alterations (12,14,15), thereby reducing the length ofhospital stay and readmission rates due to complications(16). Previous studies have already addressed the effective-ness of clinical swallowing assessment protocols (17). Theclinical assessment sensitivity for predicting aspirationsis still limited, however, because it remains difficult todetect all silent aspirations; therefore, speech pathologistsmust have reliable instruments when first evaluating post-orotracheal extubation patients (11).

The objective of this study was to elucidate the indepen-dent factors that predict dysphagia risk after prolongedorotracheal intubation (OTI) in ICU patients. Our hypoth-esis, based on the existing literature, was that clinicaldysphagia predictors would include multiple swallows perbolus, limited laryngeal elevation during swallowing, andalterations in vocal quality after swallowing.


Using the medical records from the Hospital das Clinicasda Faculdade de Medicina da Universidade de Sao Paulo,Brazil, we conducted a retrospective, observational cohortstudy on extubated ICU patients who had undergone a

bedside swallow evaluation (BSE) by a speech pathologist.The study was approved by the Scientific and EthicsCommittee of the Institution (CAPPEsq HCFMUSP 0224/10). Additionally, this study was approved as a retro-spective document review; therefore, patient consent wasnot required.

Patient PopulationPatients were eligible if they met all of the following

criteria: (a) the patient was admitted to an ICU (InstitutoCentral do Hospital das Clınicas da Faculdade de Medicinada Universidade de Sao Paulo) between September 2009and September 2011; (b) the patient received prolongedintubation (.48 hours); (c) BSE was administered by aspeech pathologist 24 to 48 hours following extubation; and(d) the patient was older than 18 years of age, (e) had clinicaland respiratory stability, and (f) had a Glasgow Coma Scalescore that was .14 points. The decision to consult a speechpathologist was left to the primary treating physician’sdiscretion. Patients were excluded if they (a) were using atracheostomy tube, (b) presented with neurological diseases,(c) presented with esophageal dysphagia, or (d) hadundergone head or neck surgical procedures.

Of the 1,080 ICU patients who received a BSE, 456 hadbeen subjected to an OTI; however, only 85% (388) hadrecords of a prolonged OTI. Of the remaining patients, 148met the inclusion criteria (Figure 1).

In our hospital, the weaning and discontinuation ofventilatory support protocol are based on the AmericanAssociation for Respiratory Care and the American Collegeof Critical Care Medicine guidelines (18). These criteria areas follows: (a) evidence for some reversal of the underlyingcause of respiratory failure; (b) adequate oxygenation(PaO2/Fio2 ratio .150 to 200, requiring positive end-expiratory pressure [PEEP] #5 to 8 cm H2O; Fio2 #0.4 to0.5) and a pH $7.25; (c) hemodynamic stability, as defined

Figure 1 - The patient population.

CLINICS 2014;69(1):8-14 Clinical dysphagia predictorsMedeiros GC et al.

9

by the absence of active myocardial ischemia and clinicallysignificant hypotension (i.e., a condition requiring novasopressor therapy or a low-dose vasopressor therapy,such as ,5 m/kg/min of dopamine or dobutamine); and (d)the capability to initiate an inspiratory effort.

Measurements: Clinical Swallowing AssessmentThe BSE included the application of the Dysphagia Risk

Evaluation Protocol (DREP) (19), followed by the classifica-tion of the functional swallowing level according to theAmerican Speech-Language-Hearing Association NationalOutcome Measurement System (ASHA NOMS) (20).

DREP (19) is a Brazilian bedside assessment protocoldesigned for early dysphagia risk detection. In our hospital,this is the standard protocol used to assess swallowingdysfunction in patients. DREP has already been validated(21) and includes items previously described as beingeffective in identifying high-risk dysphagia patients(13,22,23). It includes the controlled administration of waterand puree/solid volumes. DREP determines whether apatient should receive larger volumes and different texturesof food and liquids, as well as the amount of monitoringnecessary for safe feeding. The protocol is divided into 2sections, a water swallow test and a puree/solid swallowtest, and the results are marked as either pass or fail for eachof the observed items. As determined by the authors of theprotocol, patient swallowing was assessed during theadministration of 5 ml of water (via a syringe); 3, 5, and10 ml of fruit puree (from a spoon); and half a piece ofbread. The tests were repeated, if necessary, up to 3 times toconfirm the results. The assessment procedures consisted of11 items for the water swallow test and 12 items for thepuree/solid swallow test. Patients were placed in theupright position so that their sitting position would notinterfere with the research results (24). The assessed itemsand the criteria used to interpret the results are describedbelow.

A. The Water Swallow Test (5 ml of water administeredfrom a syringe)

a. Extraoral loss: Pass - Water does not escape fromthe patient’s lips, and the patient manages the bolusadequately. Fail - The patient has difficulty mana-ging the bolus, which causes drooling/spillagefrom the mouth.

b. Oral transit time: Pass - The patient swallows thebolus within 4 seconds. Fail - Patient takes longerthan 4 seconds to swallow the bolus or does notswallow it.

c. Nasal reflux: Pass - Water does not escape from thepatient’s nasal cavities. Fail - Water comes out ofthe patient’s nasal cavities.

d. Multiple swallows per bolus: Pass - The patientonly needs 1 swallow per bolus. Fail - The patientneeds more than 1 swallow per bolus, which causesdrooling/spillage from the mouth. Additionally,the patient needs cues to complete the task.

e. Laryngeal elevation (monitored by positioning theindex and middle fingers over the hyoid bone andthe thyroid cartilage): Pass - The patient reaches anaverage elevation of 2 fingers. Fail - The patient

does not present laryngeal elevation or presents anaverage elevation of less than 2 fingers.

f. Cervical auscultation (a stethoscope is placed at thelateral aspects above the cricoid cartilage and infront of the sternocleidomastoid muscle and largevessels): Pass - The patient presents the 3 char-acteristic sounds (two clicks followed by anexpiratory sound), indicating that the bolus hasgone through the pharynx. Fail - The patient doesnot present any sound or presents sounds otherthan those described above.

g. Oxygen saturation (baseline oxygen saturation isregistered prior to the swallow test using a monitoror pulse oximetry): Pass - The patient does notpresent oxygen saturation changes of more than 4units. Fail - The patient presents oxygen saturationchanges of more than 4 units.

h. Voice quality: Pass - The patient does not presentany alterations within the first minute after swal-lowing. Fail - The patient’s voice becomes gurgly(‘‘wet’’) within the first minute after swallowing.

i. Cough: Pass - The patient does not cough withinthe first minute after swallowing. Fail – The patientcoughs (voluntary or not) with or without throatclearing within the first minute after swallowing.

j. Choking: Pass - The patient does not choke afterswallowing. Fail - The patient chokes during and/or after swallowing.

k. Other signs (cardiac and respiratory frequencies):Pass - The patient does not present significantlyincreased cardiac (60-100 beats per minute) andrespiratory frequency (12-20 breaths per minute)changes. Fail - The patient presents signs ofcyanosis, bronchospasm, and significant vital signalterations.

B. The puree/solid swallow test (3, 5, and 10 ml of fruitpuree offered from a spoon along with a half a piece ofbread)

a. Extraoral loss: Pass - Water does not escape fromthe patient’s lips, and the patient manages the bolusadequately. Fail - The patient has difficulty mana-ging the bolus, which causes drooling/spillagefrom the mouth.

b. Oral transit time: Pass - The patient swallows thebolus within 20 seconds. Fail - Patient takes longerthan 20 seconds to swallow bolus or does notswallow it.

c. Nasal reflux: The same as above.

d. Oral residue: Pass - An absence or up to 25% of thebolus residue is observed in the patient’s oralcavity. Fail - More than 25% of the bolus residue isobserved in the patient’s oral cavity.

e. Multiple swallows per bolus: Pass - The patientrequires 1-3 swallows per bolus. Fail - The patientrequires more than 3 swallows per bolus, presentswith drooling/spillage from the mouth, and needscues to complete the task.

f. Laryngeal elevation: The same as above.

g. Cervical auscultation: The same as above.

h. Oxygen saturation: The same as above.

Clinical dysphagia predictorsMedeiros GC et al.

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i. Voice quality: The same as above.

j. Cough: The same as above.

k. Choking: The same as above.

l. Other signs: The same as above.

The ASHA NOMS swallowing level scale is a multi-dimensional tool designed to measure both the supervisionand diet levels required by assigning a single numberbetween 1 and 7 (Table 1). For this study, the patients’specific diets and supervision levels were used to calculateeach patient’s ASHA NOMS swallowing scale score. Thespeech-language pathologist was certified in assigning theASHA NOMS swallowing levels. The ASHA NOMS levelwas determined based on the DREP results.

Statistical AnalysisOnly the results obtained from the water swallow test

were used for the analyses. The statistical analysis includedassessment of the correlation between the water swallowtest and the dysphagia risk level (i.e., ASHA NOMS). Thepurpose of this analysis was to identify which items(including extraoral loss, oral transit time, nasal reflux,multiple swallows per bolus, laryngeal elevation, cervicalauscultation, oxygen saturation, coughing, choking, andother signs) were the most significant predictors of highdysphagia risk in the investigated population.

The variables were descriptively presented in contingencytables containing the absolute (n) and relative (%) frequen-cies. A logistic regression model was used to examine the

relationships between the independent dysphagia riskvariables. All variables were analyzed using a univariatemodel to determine statistical significance (p#0.10). Allsignificant variables and the interactions between them wereused to obtain selections for a multivariate model (p#0.05),according to the ‘‘enter’’ procedure. The variables thatremained in the model were independent predictor variables.

& RESULTS

The selected sample consisted of 91 males and 57 females,with a mean age of 53.64 years (range: 18-90 years). Patientspresented an average of 1.08 OTIs (range: 1-2 OTIs) and anaverage of 182.4 hours of intubation (range: 48-720 hours).The average overall time required to perform the swallow-ing assessment following extubation was 36.0 hours (range:24-48 hours).

Tables 2 and 3 display the water swallow test results andthe distribution of patients according to their ASHA NOMSlevels, respectively. In the present study, the oxygensaturation and vital sign monitoring were recategorized as‘‘other signs’’. Additionally, for statistical purposes, theASHA NOMS values were also recategorized, whereby L1represented Levels 1 to 4 and L2 represented Levels 5 to 7.

Table 4 presents the logistic regression model (univariateanalysis) results, which were based on the independentdysphagia risk variables. For this analysis, ASHA NOMSLevel 1 patients were considered a high dysphagia riskpopulation; however, ASHA NOMS Level 2 patients wereconsidered a low dysphagia risk population. The univariateanalysis results indicated that the extraoral loss, multipleswallows, cervical auscultation, vocal quality, cough, andchoking variables were possible significant high-risk indi-cators of dysphagia. Nasal reflux was not considered in thisanalysis because none of the patients failed the nasal refluxtest. The ‘‘other signs’’ variable was also not consideredbecause only 1 patient failed this item.

Table 2 - The water swallow test results.

Variables Pass Fail

n % n %

Extraoral loss 130 88 18 12

Oral transit time 135 91 13 9

Nasal reflux 148 100 0 0

Multiple swallows 61 41 87 59

Laryngeal elevation 83 56 65 44

Cervical auscultation 99 67 49 33

Voice quality 127 86 21 14

Cough 84 57 64 43

Choking 128 87 20 13

Other signs 147 99 1 1

n – number of patients, % of patients.

Table 3 - The ASHA NOMS results.

L n %

1 72 49

2 76 51

L – level, n – number of patients, % of patients.

Table 1 - ASHA NOMS swallowing level scale.

Level 1 The individual is not able to swallow safely with their mouth. All nutrition and hydration is received through non-oral means

(i.e., a nasogastric tube).

Level 2 The individual is not able to swallow safely for nutritional and hydration purposes but may achieve some consistency with

consistent maximal cues during therapy sessions only. An alternative feeding method is required.

Level 3 An alternative feeding method is required, as the individual receives less than 50% of his/her nutrition and hydration by mouth,

swallowing is safe with the consistent use of moderate cues to utilize compensatory strategies, and/or the patient requires

maximum diet restriction.

Level 4 Swallowing is safe but usually requires moderate cues to use compensatory strategies, the individual has moderate diet

restrictions, and/or the patient still requires tube feeding and/or oral supplements.

Level 5 Swallowing is safe with minimal diet restrictions and/or the patient occasionally requires minimal cues to use compensatory

strategies. The patient may occasionally self-cue. All nutrition and hydration needs are met by mouth at mealtime.

Level 6 Swallowing is safe, the individual eats and drinks independently, and the individual may rarely require minimal cueing. The

individual usually self-cues when difficulty occurs and may need to avoid or requires additional time (due to dysphagia) to

consume specific food items (e.g., popcorn and nuts).

Level 7 The individual’s ability to eat independently is not limited by altered swallowing functions. Swallowing is safe and efficient for

all food consistencies. Compensatory strategies are effectively used when needed.


11

Table 5 presents the logistic regression model (multi-variate analysis) results of the independent variablesassociated with dysphagia risk. According to these results,cervical auscultation and cough were independent predic-tive variables of high dysphagia risk.

& DISCUSSION

This study represents the largest prolonged orotrachealintubation Brazilian patient group that has been screenedfor possible signs of dysphagia. To our knowledge, this isone of the few studies that have investigated possibledysphagia risk predictors based on clinical symptoms inICU patients. Early post-extubation dysfunction recognitionis paramount in reducing the rate of morbidity in this high-risk population.

Extended intubation durations have been correlated withdysphagia (5,25-27) and have also been reported to beindependent predictors of dysphagia severity (1,28). Post-extubation, a higher dysphagia risk was reported in patientswith Glasgow Coma Scale scores #14 (6) and in patientsaged $55 years (1,6). In contrast, another study found thatneither age nor intubation duration was correlated withincreased swallowing dysfunction in post-orotracheal intu-bation patients (29). Prolonged intubation swallowingdisorders extend the time before oral myofunctional/swallowing assessments can begin and the time to returnto normal oral feeding while also delaying subsequenthospital discharges (27,28,30).

Screening procedures are generally designed to be quick(,15 minutes), to be relatively non-invasive, and to providelittle risk to the patient while identifying the dysphagiasymptoms that may require an in-depth diagnostic assess-ment (23). In developing countries, the prolonged intensivemedical and nursing care that is required by many patientsplaces additional demands on already stretched healthcare

budgets (31). Moreover, routine post-extubation swallowingstudies can lead to additional, and possibly unnecessary,imaging, increasing healthcare resource use. Our resultsindicated that altered cervical auscultation and coughingduring water swallow tests increased the likelihood ofdysphagia in patients who underwent prolonged orotra-cheal intubation (11).

It is critical that health professionals distinguish betweenscreenings and diagnoses. A screening does not define thenature of a patient’s problem; it simply identifies the patientas being at risk for a significant problem/disorder (inthis case, dysphagia) (23). The variables that were strongpredictors of high dysphagia risk in our study are alsoconsidered to be variables associated with the possiblepresence of an aspiration (23,32,33). The reasons foraspirations have been discussed frequently in the literature,in which multiple causes have been postulated. It is knownthat alterations in the chemo- and/or mechanoreceptors(located in the pharyngeal and laryngeal mucosae) that areinvolved in the swallowing reflex are altered by thepresence of an orotracheal tube (1). Inhibition of the sensorylarynx abilities led to the absence of coughing or any otherbehavioral aspiration signs in patients following liquidbolus ingestion. Furthermore, this effect was observedimmediately and 4 hours following extubation, and thedetrimental effects were significantly reduced within8 hours post-extubation (34). Additionally, most mucosallesions caused by orotracheal tubes are healed 3 daysfollowing extubation (35).

Swallowing dysfunctions may persist, however, despitethe removal of orotracheal tubes and the necessaryspontaneous recovery periods. The swallowing dysfunctionmechanisms following an extubation are thought to be acombination of muscle ‘‘freezing’’ (which may be attribu-table to the lack of swallowing while intubated) and the lossof proprioception (which may be attributable to mucosallesions) (6).

Cervical auscultation is increasingly being used tosupplement clinical swallowing assessments. The soundsassociated with swallowing have been investigated usingaccelerometers and microphones to identify acoustic char-acteristics (36). Additionally, these sounds may also predictaspiration onset (37). The use of cervical auscultation varieswith respect to its reported reliability (38) and validitycompared with videofluoroscopic swallow studies (VFSS)(39,40). VFSS itself, however, has poor intra- and inter-raterreliability (41-43). According to Lazareck and Moussavi (37),swallowing sound assessments have great potential toreduce the need for VFSS and to assist in overall clinicalswallowing assessments. It is clear from the literature that,despite the methods used to assess swallowing, providingthe necessary training is indispensable.

A study by Bordon et al. (1) analyzed swallowingdysfunction risk factors after prolonged intubation intrauma patients. The authors also used a simple bedsidespeech pathology assessment (specifically, their swallowingfailure indicators included coughing when drinking liquidsand the presence of multiple swallows). Patients with andwithout post-extubation dysphagia were then compared byunivariate analysis with respect to their age, the number ofventilator and ICU days, the presence or absence ofpneumonia, and other variables. The authors suggestedthat older patients (above 55 years of age) with extendedICU stays and ventilator requirements may benefit from

Table 4 - Logistic regression (univariate analysis) resultsbased on independent dysphagia risk variables.

Variables Odds ratio CI (95%) p-value

Extraoral loss 7.758 1.174–35.104 0.008*

Oral transit time 11.143 1.409–88.106 0.022*

Nasal reflux - - -

Multiple swallows 2.164 1.111–4.218 0.023*

Laryngeal elevation 1.750 .902–3.394 0.098

Cervical auscultation 26.833 7.784–92.504 ,0.001*

Vocal quality 20.968 2.731–160.978 0.003*

Cough 42.273 13.752–129.939 ,0.001*

Choking 19.603 2.548–150.839 0.004*

Other signs - - -

CI – confidence interval, *significant results.

Table 5 - A logistic regression (multivariate analysis) ofthe independent variables associated with dysphagia risk.

Variables Odds ratio CI (95%) p-value

Extra oral loss 1.837 0.192–17.574 0.598

Multiple swallows 2.056 0.698–6.059 0.191

Cervical auscultation 12.709 2.940–54.931 0.001*

Vocal quality 9.115 0.935–88.853 0.057

Cough 14.817 3.444–63.740 ,0.001*

Choking 2.489 0.194–31.958 0.484

CI – confidence interval, *significant results.


CLINICS 2014;69(1):8-14

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early swallowing evaluations. Similarly, Leder et al. (25)investigated aspiration incidence following extubation incritically ill trauma patients using a bedside transnasalfiberoptic endoscopic swallowing evaluation. The authorsnoted that aspiration was identified in 45% of the subjects,44% of whom were silent aspirators. The authors arguedthat aspiration identification may reduce the likelihood ofpulmonary complications following an extubation.

Although there are a few potential limitations of ourstudy (e.g., it was a single-institution investigation and thusmay only reflect local patient characteristics, and the studydid not include any confirmatory fluoroscopic imaging todocument silent or subclinical aspirations), our findingsdemonstrated that the overall swallowing deficit rates inpatients subjected to prolonged orotracheal intubations (i.e.,ASHA NOMS Level 1) were comparable to those that havebeen previously published. Studies performed with a directlaryngoscope indicated that approximately 56% of theobserved critically ill patients displayed evidence ofswallowing dysfunction (5,8). Similarly, studies performedwith fiberoptic endoscopic evaluations (FEES) demon-strated that swallowing dysfunction occurred in approxi-mately 52% of patients after prolonged intubation (29).

In the literature, there is no clear definition of whichpatients are at risk for dysphagia. The results of our studyindicate that if patients who are subjected to prolongedorotracheal intubation present with altered cervical auscul-tation and coughing during a water swallow test, thesepatients should be given an early and more detailedswallowing assessment. Moreover, these assessmentsshould possibly include imaging examinations beforerestarting oral nutrition. A similar study, performed withtrauma patients subjected to mechanical ventilation (3),investigated whether a BSE could identify swallowingdysfunction in this patient group. One of the main findingsof this study was that the patients who failed the BSErequired longer mechanical ventilation than those whopassed the BSE. Additionally, 78% of the patients intubatedfor more than 72 hours failed the BSE. All patients whopassed the BSE, however, were discharged from the hospitalwithout a clinical aspiration event. The authors alsoidentified independent risk factors for BSE failures, whichincluded tracheostomy, older age, prolonged mechanicalventilation, delirium tremens, traumatic brain injury, andspine fracture.

Finally, we would like to state that we did not study thelong-term consequences of post-extubation dysphagia inour cohort because our end point was an evaluationperformed within 48 hours of the observed extubations.Future studies at our institution will attempt to answer thisand other questions.


Medeiros GC was responsible for the data collection and analysis,

interpretation of the results, and manuscript writing. Sassi FC organized

and conducted the statistical analyses, interpreted the results, and wrote a

major portion of the manuscript. Mangilli LD participated in the data

collection and analyses and organized and conducted the statistical

analyses. Zilberstein B was responsible for the medical criteria adopted

in the experimental design and contributed to the data analysis and

manuscript preparation. Andrade CR was responsible for the research and

experimental design and contributed to the data analysis and manuscript

preparation.

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17. Nishiwaki K, Tsuji T, Liu M, Hase K, Tanaka N, Fujiwara T.Identification of a simple screening tool for dysphagia in patients withstroke using factor analysis of multiple dysphagia variables. J RehabilMed. 2005;37(4):247-251, http://dx.doi.org/10.1080/16501970510026999.

18. Maclntyre NR. Evidence-based guidelines for weaning and discontinu-ing ventilatory support: a collective task force facilitated by a theAmerican College of Chest Phsysicians; the American Association forRespiratory Care; and the American College of Critical Care Medicine.Chest. 2001;120(6 Suppl):375-96, http://dx.doi.org/10.1378/chest.120.6_suppl.375S.

19. Padovani AR, Moraes DP, Mangilli LD, Andrade CRF. Protocolofonoaudiologico de avaliacao do risco para disfagia (PARD). Rev SocBras Fonoaudiol. 2007;12(3):199-205, http://dx.doi.org/10.1590/S1516-80342007000300007.

20. Rockville MD. American Speech-Language-Hearing AssociationNational Outcome Measurement System (NOMS). Adult Speech-Language Pathology training manual, ASHA, 1998.

21. Padovani AR, Moraes DP, Mangilli LD, Andrade CRF. Protocolo deavaliacao fonoaudiologica de risco para disfagia (PARD). In: AndradeCRF, Limongi SCO (Eds). Disfagia – pratica baseada em evidencias, 1 ed.Sao Paulo: Sarvier, 2012. p.62-73.

22. Smith Hammond CA, Goldstein LB. Cough and aspiration of food andliquids due to oral-pharyngeal dysphagia: ACCP evidence-based clinicalpractice guidelines. Chest. 2006;129(1 Suppl):154S-168S, http://dx.doi.org/10.1378/chest.129.1_suppl.154S.

23. Logemann JA, Veis S, Colangelo L. A screening procedure fororopharyngeal dysphagia. Dysphagia. 1999;14(1):44-51, http://dx.doi.org/10.1007/PL00009583.

24. Goldsmith T. Evaluation and treatment of swallowing disordersfollowing endotracheal intubation and tracheostomy. Int Anesthesiol


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25. Leder SB, Cohn SM, Moller BA. Fiberoptic endoscopic documentation ofthe high incidence of aspiration following extubation in critically illtrauma patients. Dysphagia. 1998;13(4):208-12, http://dx.doi.org/10.1007/PL00009573.

26. Postma G, McGuirt F, Butle SG, Ress CJ, Crandall HL, Tansavatdi K.Abnormalities in hospitalized patients with dysphagia. Laryngoscope.2007;117(10):1720-2, http://dx.doi.org/10.1097/MLG.0b013e31811ff906.

27. Rumbach AF, Ward EC, Cornwell PL, Bassett LV, Muller JM. Clinicalprogression and outcome of dysphagia following thermal burn injury: aprospective cohort study. J Burn Care Res. 2012;33(3):336-46, http://dx.doi.org/10.1097/BCR.0b013e3182356143.

28. Barker J, Martino R, Reichart B, Hickey EJ, Ralph-Edwards A. Incidenceand impact of dysphagia in patients receiving prolonged endotrachealintubation after cardiac surgery. Can J Surg. 2009;52(2):119-24.

29. El Solh A, Okada M, Bhat A, Pietrantoni C. Swallowing disorders postorotracheal intubation in the elderly. Intensive Care Med. 2003;29(9):1451-5, http://dx.doi.org/10.1007/s00134-003-1870-4.

30. Ward EC, Green K, Morton AL. Patterns and predictors of swallowingresolution following adult traumatic brain injury. J Head Trauma Rehabil.2007;22(3):184-91, http://dx.doi.org/10.1097/01.HTR.0000271119.96780.f5.

31. Thwaites CL, Farrar JJ. Preventing and treating tetanus. BMJ. 2003;326(7381):117-8, http://dx.doi.org/10.1136/bmj.326.7381.117.

32. Smith-Hammond CA, Goldstein LB, Horner RD, Ying J, Gray L, Gonzales-Rothi L, Bolser DC. Prediciting aspiration in patients with ischemic stroke:comparison of clinical signs and aerodynamic measures of voluntarycough. Chest. 2009;135(3):769-77, http://dx.doi.org/10.1378/chest.08-1122.

33. Camargo FP, Ono J, Park M, Caruso, Carvalho CRR. An evaluation ofrespiration and swallowing interaction after orotracheal intubation. Clinics.2010;65(9):919-22, http://dx.doi.org/10.1590/S1807-59322010000900015.

34. Burgess GE, Cooper JR, Marino RJ, Peuler MJ, Warriner RA. Laryngealcompetence after tracheal extubation. Anesthesiology. 1979;51(1):73-7,http://dx.doi.org/10.1097/00000542-197907000-00016.

35. Stauffer JL, Olson DE, Petty TL. Complications and consequences ofendotracheal intubation and tracheotomy. Am J Med. 1981;70(1):65-76.

36. Cichero JAY, Murdoch BE. Acoustic signature of the normal swallow:characterization by age, gender, and bolus volume. Ann Otol RhinolLaryngol. 2002;111(7 Pt 1):623-32.

37. Lazareck LJ, Moussavi ZMK. Classification of normal and dysphagicswallows by acoustical means. IEEE Trans Biomed Eng. 2004;51(12):2103-112, http://dx.doi.org/10.1109/TBME.2004.836504.

38. Borr C, Hielscher-Fastabend M, Phil D, Lucking A. Reliability andvalidity of cervical auscultation. Dysphagia. 2007;22(3):225-34, http://dx.doi.org/10.1007/s00455-007-9078-3.

39. Mann G, Hankey GJ. Initial clinical and demographic predictors ofswallowing impairment following acute stroke. Dysphagia. 2001;16(3):208-5, http://dx.doi.org/10.1007/s00455-001-0069-5.

40. Mari F, Matei M, Ceravolo MG, Pisani A, Montesi A, Provinciali L.Predictive value of clinical indices in detecting aspiration in patientswith neurological disorders. J Neurol Neurosurg Psychiatry. 1997;63(4):456-60, http://dx.doi.org/10.1136/jnnp.63.4.456.

41. Kuhlemeier KV, Yates P, Palmer JB. Intra- and interrater variation in theevaluation of videofluorographic swallowing studies. Dysphagia. 1998;13(3):142-7, http://dx.doi.org/10.1007/PL00009564.

42. McCullough GH, Wertz RT, Rosenbek JC, Millis RH, Ebb WG, Ross KB.Inter- and intrajudge reliability for videofluoroscopic swallowingevaluation measures. Dysphagia. 2001;16(2):110-1, http://dx.doi.org/10.1007/s004550010004.

43. Leslie P, Drinnan MJ, Finn P, Ford GA, Wilson JA. Reliability andvalidity of cervical auscultation: a controlled comparison using video-fluoroscopy. Dysphagia. 2004;19(4):231-40.


CLINICS 2014;69(1):8-14

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PITX2C loss-of-function mutations responsible foridiopathic atrial fibrillationXing-Biao Qiu, Ying-Jia Xu, Ruo-Gu Li, Lei Xu, Xu Liu, Wei-Yi Fang, Yi-Qing Yang, Xin-Kai Qu

Shanghai Jiao Tong University, Shanghai Chest Hospital, Department of Cardiology and Cardiovascular Research, Shanghai, China.

OBJECTIVE: This study aimed to identify novel PITX2c mutations responsible for idiopathic atrial fibrillation.

METHODS: A cohort of 210 unrelated patients with idiopathic atrial fibrillation and 200 unrelated, ethnicallymatched healthy individuals used as controls were recruited. The whole coding exons and splice junctions ofthe PITX2c gene, which encodes a paired-like homeobox transcription factor required for normal cardiovascularmorphogenesis, were sequenced in 210 patients and 200 control subjects. The causative potentials of theidentified mutations were automatically predicted by MutationTaster and PolyPhen-2. The functionalcharacteristics of the PITX2c mutations were explored using a dual-luciferase reporter assay system.

RESULTS: Two novel heterozygous PITX2c mutations (p.Q105L and p.R122C) were identified in 2 of the 210unrelated patients with idiopathic atrial fibrillation. These missense mutations were absent in the 400 controlchromosomes and were both predicted to be pathogenic. Multiple alignments of PITX2c protein sequencesacross various species showed that the altered amino acids were highly evolutionarily conserved. A functionalanalysis demonstrated that the mutant PITX2c proteins were both associated with significantly reducedtranscriptional activity compared with their wild-type counterparts.

CONCLUSION: The findings of this study associate PITX2c loss-of-function mutations with atrial fibrillation,supporting the hypothesis that dysfunctional PITX2c confers enhanced susceptibility to atrial fibrillation andsuggesting potential implications for early prophylaxis and allele-specific therapy for this common arrhythmia.

KEYWORDS: Atrial Fibrillation; Transcriptional Factor; PITX2c; Genetics; Reporter Gene.

Qiu XB, Xu YJ, Li RG, Xu L, Liu X, Fang WY, et al. PITX2C loss-of-function mutations responsible for idiopathic atrial fibrillation. Clinics.2014;69(1):15-22.

Received for publication on May 19, 2013; First review completed on June 10, 2013; Accepted for publication on July 10, 2013


Tel.: 86 21 22200000

& INTRODUCTION

Atrial fibrillation (AF), a supraventricular tachycardiacharacterized by rapid and chaotic atrial electrical activitywith subsequent deterioration of atrial mechanical function,is the most prevalent form of sustained cardiac arrhythmiain humans worldwide and is responsible for approximatelyone-third of all hospital admissions with miscellaneousheart rhythm disturbances (1). The prevalence of AF isapproximately 1% in the general population and increasesstrikingly with advanced age, increasing from ,1% inindividuals younger than 60 years to approximately 10%in subjects older than 80 years (1). According to theFramingham Heart Study, the lifetime risk for developingAF is approximately 25% for people older than 40 years (2).The prevalence of AF in the United States is currently

estimated at 2.3 million and is projected to exceed 10 millionby 2050 (3). This common arrhythmia significantly con-tributes to a degraded quality of life, reduced exercisecapacity, cognitive impairment or dementia, tachycardio-myopathy, thromboembolism, congestive heart failure,and even death (1,4,5). The mortality of patients with AFis approximately twice that of age- and gender-matchedpatients with normal sinus rhythms, independent of pre-existing cardiovascular conditions (6). AF patients have a2- to 7-fold increased risk for ischemic stroke compared withindividuals without AF, and 15%–20% of all strokes areascribed to AF (7). Note that the risk of thromboembolismattributable to AF also increases markedly with increasedage, ranging from 1.5% among patients aged 50–59 years to23.5% among patients aged 80–89 years (7). Consequently,AF imposes a substantial economic burden on health caresystems because of its increased morbidity- and mortality-associated therapeutic interventions. This socioeconomicburden is anticipated to continually increase in the future aslife expectancies increase (8). Despite its high prevalenceand important clinical significance, the molecular basis ofAF remains poorly understood.

Frequently, AF is associated with diverse structuralheart diseases or systemic disorders, such as ischemic heart



DOI: 10.6061/clinics/2014(01)03

CLINICAL SICENCE

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disease, valvular heart disease, congenital heart disease,pulmonary heart disease, cardiomyopathy, cardiothoracicsurgery, congestive heart failure, essential hypertension,and hyperthyroidism (1,9). Other risk factors for AF includeaging, obesity, sleep apnea, cigarette smoking, excessivealcohol consumption, and exposure to drugs or toxicants(1,10). However, in 12%–30% of all AF patients and 20%–45% of younger AF patients, AF occurs alone in the absenceof the above-mentioned underlying diseases or precipitatingfactors; this condition is defined as idiopathic or lone AFand termed familial AF, as at least 15% of patients havepositive family histories of AF (1,11). A growing numberof epidemiological studies have demonstrated the familialaggregation of AF and the enhanced susceptibility to AF inthe close relatives of AF patients, indicating that genetic riskfactors play key roles in the pathogenesis of AF in a subsetof patients (12). A whole-genome scan with polymorphicmicrosatellite markers and linkage analysis mapped theAF susceptibility loci on human chromosomes 10q22, 6q14-16, 11p15.5, 5p15, 10p11-q21, and 5p13, for which the AF-causing mutations in 2 genes, KCNQ1 on chromosome11p15.5 and NUP155 on chromosome 5p13, were identifiedand functionally characterized (13). Analyses of the candi-date genes revealed a long list of AF-associated genes,including KCNE2, KCNH2, KCNJ2, KCNA5, SCN5A,ANP, GJA5, GATA4, GATA5, GATA6, and NKX2-5 (13-21). Nevertheless, AF is genetically heterogeneous, and thegenetic defects underlying AF in an overwhelming majorityof patients remain to be identified.

Recently, a genome-wide association study identified 2sequence variants (rs2200733 and rs10033464) on chromo-some 4q25 that were strongly associated with an enhancedvulnerability for AF (22). This association was subsequentlyreplicated in a study of 4 large populations with ambulatoryAF (23) and was also reported for post-cardiac surgery AFin a setting thought to be related to inflammation (24). More-over, the 2 sequence variants were observed to increase therisk for both the early and late recurrences of AF aftercatheter ablation (25) and acted as genetic modifiers of rareion channel mutations associated with familial AF (26). ThePITX2 gene is closest to these sequence variants; it a memberof the pituitary homeobox (PITX) family of transcriptionfactors, which play a pivotal role in embryonic morphogen-esis. PITX2c is the predominant isoform that is expressedin embryonic and adult hearts (27). Emerging evidenceunderlines the essential role of PITX2c in the embryonicdevelopment of the left atrium, cardiac conduction system,and pulmonary venous myocardium, a major source ofectopic activity that is implicated in initiating and main-taining AF (28). The abnormal expression of PITX2c hasbeen associated with an increased predisposition to AF (29-31). These findings justify screening for PITX2c as a primecandidate gene for idiopathic AF.


Ethics statementThis study was performed in compliance with the ethical

principles of the revised Declaration of Helsinki (SomersetWest, Republic of South Africa, 1996). The research protocolwas reviewed and approved by the local institutional ethicscommittee, and written informed consent was obtainedfrom all participants prior to the study.

Study subjectsA cohort of 210 unrelated patients with idiopathic AF was

recruited from the Han Chinese population. Patients withenlarged left atriums ($40 mm in left atrial diameter) wereexcluded. A total of 200 ethnically matched, unrelatedhealthy individuals were enrolled as the controls. Peripheralvenous blood samples were prepared, and clinical data,including medical records, electrocardiogram, and echo-cardiography reports, were collected. The study subjectswere clinically classified using a consistently applied set ofdefinitions (11,18). Briefly, the AF diagnosis was made usinga standard 12-lead electrocardiogram demonstrating no Pwaves and irregular R-R intervals, regardless of the clinicalsymptoms. Idiopathic AF was defined as AF occurring inindividuals without other cardiac or systemic diseases usinga physical examination, electrocardiogram, transthoracicechocardiogram, or extensive laboratory tests. Subjects wereclassified as ‘healthy’ if they were asymptomatic and hadnormal electrocardiograms. In addition, paroxysmal AF wasdefined as AF lasting more than 30 seconds that terminatedspontaneously. Persistent AF was defined as AF lastingmore than 7 days and requiring either pharmacologictherapy or electrical cardioversion for termination. AF thatwas refractory to cardioversion or that was allowed tocontinue was classified as long-lasting persistent AF.

Genetic scanGenomic DNA was extracted from the peripheral venous

blood lymphocytes of all participants with a WizardGenomic DNA Purification Kit (Promega, Madison, WI,USA). The whole coding region and splice junction sitesof PITX2c was sequenced in 210 unrelated patients withidiopathic AF, and the genotyping PITX2c in the 200 controlindividuals was performed subsequently to identify thepresence of mutations in the patients. The referentialgenomic DNA sequence of PITX2c was derived fromthe GenBank (accession No. NC_000004), which is at theNational Center for Biotechnical Information (NCBI, http://www.ncbi.nlm.nih.gov/). The primer pairs used to amplifythe coding exons and exon-intron boundaries of PITX2cby polymerase chain reaction (PCR) were designed withthe help of the online Primer 3 program (http://frodo.wi.mit.edu), as shown in Table 1. PCR was performed usingHotStar Taq DNA Polymerase (Qiagen, Hilden, Germany)on a Veriti Thermal Cycler (Applied Biosystems, FosterCity, CA, USA) under standard conditions and concentra-tions of reagents. Amplified products were analyzed on 1%agarose gels stained with ethidium bromide and purifiedwith QIAquick Gel Extraction Kit (Qiagen). Both strands ofeach amplicon were sequenced with a BigDyeH Terminatorv3.1 Cycle Sequencing Kit (Applied Biosystems) under anABI PRISM 3130 XL DNA Analyzer (Applied Biosystems).The sequencing primers were the same as those pre-viously designed for specific region amplification. TheDNA sequences were analyzed with the DNA SequencingAnalysis Software v5.1 (Applied Biosystems). The variantwas validated by re-sequencing an independent PCR-generated amplicon from the subject and meeting thequality control thresholds with a call rate .99%. In addition,for an identified sequence variant, the Exome Variant Server(EVS; http://evs.gs.washington.edu/EVS) and NCBI’s singlenucleotide polymorphism (SNP; http://www.ncbi.nlm.nih.gov/SNP) online databases were searched to confirm itsnovelty.

PITX2c mutations in AFQiu XB et al.

CLINICS 2014;69(1):15-22

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Alignment of multiple PITX2c protein sequencesMultiple PITX2c protein sequences across various species

were aligned using the online program MUSCLE, version3.6 (http://www.ncbi.nlm.nih.gov/).

Prediction of the disease-causing potential of aPITX2c sequence variation

The disease-causing potential of a PITX2c sequencevariation was predicted using MutationTaster (an onlineprogram at http://www.mutationtaster.org), which auto-matically provides a probability for the variation to be eithera pathogenic mutation or a benign polymorphism. Notethat the p-value used here is the probability of the correctprediction rather than the probability of error, as used int-test statistics (i.e., a value close to 1 indicates a highaccuracy of the prediction). Another online program,PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2), wasalso used to evaluate the mutational pathogenicity.

Expression plasmids and site-directed mutagenesisThe recombinant expression plasmid PITX2c-pcDNA4,

which was constructed by M. Hermina Strungaru et al. (32),was a gift from Prof. Georges Christe, Physiopathologie desTroubles du Rythme Cardiaque, Faculte de Pharmacie deLyon, Universite Lyon 1, Lyon, France. The atrial natriureticfactor (ANF)-luciferase reporter plasmid, which contains the2600-bp 5’-flanking region of the ANF gene, namely ANF(-2600)-Luc, was kindly provided by Dr. Ichiro Shiojima,from the Department of Cardiovascular Science andMedicine, Chiba University Graduate School of Medicine,Chuo-ku, Chiba, Japan. Each of the identified mutations wasintroduced into the wild-type PITX2c using a QuickChangeII XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla,CA, USA) with a complementary pair of primers. Themutants were sequenced to confirm the desired mutationsand to exclude any other sequence variations.

Luciferase reporter gene assayChinese hamster ovary (CHO) cells were cultured in

Dulbecco’s modified Eagle’s medium supplemented with10% fetal calf serum, 100 units/ml penicillin, and 100 mg/mlstreptomycin. CHO cells were grown 24 h prior to thetransfection. The ANF(-2600)-Luc reporter construct andan internal control reporter plasmid pGL4.75 (hRluc/CMV,Promega) were used in transient transfection assays toexplore the transactivational activity of the PITX2c mutants.The CHO cells were transfected with 2 mg of wild-typePITX2c–pcDNA4, mutant PITX2c–pcDNA4 (Q105L orR122C), or empty vector pcDNA4, 2.0 mg of ANF(-2600)-Luc reporter construct, and 0.04 mg of pGL4.75 controlreporter vector using Lipofectamine 2000 TransfectionReagent (Invitrogen, Carlsbad, CA, USA). For the co-transfection experiments, 1 mg of wild-type PITX2c–pcDNA4, 1 mg of mutant PITX2c–pcDNA4 (Q105L orR122C), 2.0 mg of ANF(-2600)-Luc, and 0.04 mg of pGL4.75were used. The transfected cells were incubated for 24 h, and

then they were lysed and assayed for the reporter activities.Firefly luciferase and Renilla luciferase activities were mea-sured with the Dual-Glo luciferase assay system (Promega).The activity of the ANF promoter was presented as thefold activation of Firefly luciferase relative to the Renillaluciferase. A minimum of three independent experimentswere performed for wild-type or mutant PITX2c.

Statistical analysisThe data are expressed as the means¡SD. Continuous

variables were tested for normality of distribution, andStudent9s unpaired t-test was used to compare the numericvariables between 2 groups. The categorical variables werecompared between 2 groups using Pearson’s chi-squaredtest or Fisher9s exact test, as appropriate. A 2-sided p-value,0.05 was considered statistically significant.

& RESULTS

Clinical characteristics of the study populationA cohort of 210 unrelated patients with idiopathic AF

were enlisted, clinically evaluated, and compared with 200ethnically matched, unrelated healthy individuals. None ofthe participants had the traditional risk factors for AF. Therewere no significant differences between the patient andcontrol groups in the baseline characteristics, including age,gender, body mass index, blood pressure, fasting bloodglucose, serum lipid, left atrial dimension, left ventricularejection fraction, heart rate at rest, and lifestyle (data notshown). The baseline clinical characteristics of the 210patients with idiopathic AF are summarized in Table 2.

PITX2c mutationsTwo heterozygous missense mutations in PITX2c were

identified in 2 of the 210 unrelated patients with idiopathicAF. The total population prevalence of PITX2c mutationsbased on the patient cohort was approximately 0.95%.Specifically, a substitution of thymine (T) for adenine (A)in the second nucleotide of codon 105 (c.314A.T), whichpredicted the transition of glutamine (Q) into leucine (L) atamino acid 105 (p.Q105L), was identified in a 52-year-oldfemale patient who was initially diagnosed with AF at theage of 35 years. A change of cytosine (C) into thymine (T) inthe first nucleotide of codon 122 (c.364C.T), correspondingto the transversion of arginine (R) into cysteine (C) at aminoacid 122 (p.R122C), was found in a 48-year-old male patientwho was first diagnosed with AF at the age of 21 years. The2 mutation carriers had no apparent malformations in theeyes, teeth, umbilicus, or heart and had no positive familyhistory. No relatives from these 2 mutation carriers wereavailable for PITX2c genotyping. The sequence chromato-grams showing the detected heterozygous PITX2c muta-tions of c.314A.T and c.364C.T in contrast to their controlsequences are shown in Figures 1A and 1B, respectively.A schematic diagram of PITX2c showing the structuraldomains and the locations of the identified mutations is

Table 1 - Primers to amplify the coding exons and exon-intron boundaries of PITX2c.

Exon Forward primer (59 to 39) Reverse primer (59 to 39) Size (bp)

1 CAG,CTT,GGC,TTG,AGA,ACT,CG TGA,CTT,CCT,TGG,GGC,GAG,AG 442

2 CAG,CTC,TTC,CAC,GGC,TTC,TG GCT,GCC,TTC,CAC,ATT,CTC,TC 387

3 AAT,CTG,CAC,TGT,GGC,ATC,TG AGT,CTT,TCA,AGG,GCG,GAG,TT 677

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presented in Figure 2 (33,34). These missense mutationswere neither observed in the control population norreported in the EVS and NCBI SNP databases.

Alignments of multiple PITX2c protein sequencesacross species

The alignment of PITX2c protein sequences across 11species ranging from humans to mammals and insectsshowed that the altered amino acid residues were com-pletely (for p.R122) or highly (for p.Q105) evolutionarilyconserved among PITX2c orthologs, which suggestedthat these particular arginine and glutamine residues inthe homeodomain of PITX2c are functionally important(Figure 3).

Disease-causing potential of the PITX2c variationsThe PITX2c sequence variations of c.314A.T and

c.364C.T were both automatically predicted by Mutation-Taster to be disease-causing mutations with the samep-value of 1.0. No SNP in the altered regions was found inthe MutationTaster database. The 2 PITX2c sequencevariations were also predicted by PolyPhen-2 to most likelybe damaging, with scores of 0.999 for c.314A.T (sensitivity0.14; specificity 0.99) and 1.000 for c.364C.T (sensitivity0.00, specificity 1.00).

Transcriptional activity of the PITX2c mutantsThe same amounts of wild-type (2 mg), Q105L-mutant

(2 mg), and R122C-mutant PITX2c (2 mg) activated theANF promoter by ,9-fold, ,3-fold, and ,2-fold increases,respectively, compared with the empty plasmid. When thesame amount of wild-type PITX2c (1 mg) was cotransfectedwith Q105L-mutant PITX2c (1 mg) or R122C-mutant PITX2c(1 mg), the induced activation of the ANF promoter was a

,5-fold increase, compared with the empty plasmid(Figure 4).

& DISCUSSION

In the present study, 2 novel heterozygous PITX2cmutations, p.Q105L and p.R122C, were identified in 2unrelated patients with idiopathic AF; these mutationswere absent in the 400 reference chromosomes and wereboth predicted to be pathogenic by MutationTaster andPolyPhen-2. A cross-species alignment of PITX2c proteinsequences showed that the altered amino acids are highlyevolutionarily conserved. The functional analysis demon-strated that the mutant PITX2c proteins are associated withsignificantly decreased transcriptional activity. Therefore, itis likely that having functionally impaired PITX2c predis-poses a patient to AF.

PITX2 is expressed as several protein isoforms, which aregenerated by differential mRNA splicing and alternativetranslation initiation sites. In the human and the mouse,the transcriptionally functional isoforms are PITX2-a, -b,and -c, which share an identical homeodomain and carboxylterminus but differ in their amino termini. Humans possessan additional isoform, the D isoform, which lacks theamino-terminal domain and most of the homeodomain (31).PITX2c is the major isoform that is expressed asymme-trically in the developing and adult heart and plays a crucialrole in normal cardiovascular genesis and maturation(30,31). The human PITX2c gene maps to chromosome4q25, which consists of 3 exons coding for a protein of 324amino acids (35). One of the most important functionaldomains of PITX2c is the homeodomain that recognizesand binds to the specific consensus DNA sequence 59-TAATCC-39. This homeodomain is essential for DNAbinding, nuclear translocation, and interaction with other

Table 2 - Baseline clinical characteristics of the study subjects.*

Clinical characteristics Patient group (n = 210) Control group (n = 200)

Male (%) 96 (46) 92 (46)

Age at the initial AF diagnosis (years) 53.2¡8.7 NA

Age at the time of the study (years) 56.7¡10.1 58.3¡9.5

Type of AF at presentation

Paroxysmal AF (%) 147 (70) 0 (0)

Persistent AF (%) 38 (18) 0 (0)

Long-lasting persistent AF (%) 25 (12) 0 (0)

Positive family history of AF (%) 32 (15) 0 (0)

History of cardioversion (%) 178 (85) 0 (0)

Implanted cardiac pacemaker (%) 6 (3) 0 (0)

Resting heart rate (beats per minute) 76.5¡11.8 77.2¡10.5

Systolic blood pressure (mmHg) 130.4¡12.6 131.0¡13.3

Diastolic blood pressure (mmHg) 85.8¡7.3 86.2¡8.1

Body mass index (kg/m2) 22.7¡2.0 23.0¡2.4

Left atrial dimension (mm) 38.2¡3.6 37.5¡3.9

Left ventricular ejection fraction (%) 62.8¡7.2 63.4¡6.7

Fasting blood glucose (mmol/L) 4.5¡0.4 4.6¡0.5

Total cholesterol (mmol/L) 4.2¡0.3 4.1¡0.4

Triglycerides (mmol/L) 1.6¡0.2 1.5¡0.3

Medications

Amiodarone (%) 160 (76) 0 (0)

Warfarin (%) 151 (72) 0 (0)

Digoxin (%) 44 (21) 0 (0)

Beta-blocker (%) 17 (8) 0 (0)

Calcium channel blocker (%) 10 (5) 0 (0)

*There were no significant differences in the baseline characteristics between the patient and control groups.

NA indicates not applicable or not available.


CLINICS 2014;69(1):15-22

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transcription factors (35). The PITX2c mutations of p.Q105Land p.R122C, which are identified in the present study,are both located in this homeodomain; thus, they may beexpected to influence the transcriptional activity of PITX2cby interfering with its DNA-binding ability.

Previous studies have corroborated that PITX2c isan upstream regulator of multiple target genes that are

expressed in the heart during embryogenesis, includingthe gene that encodes ANF (36). Therefore, the functionalrole of a PITX2c mutation can be characterized by an assayof the transcriptional activity of the ANF promoter in thecells expressing PITX2c mutants, in contrast to the wild-typecounterpart. In this study, the functional characteristics ofthe 2 novel PITX2c mutations identified in the AF patients

Figure 1 - Sequence electropherograms showing the PITX2c mutations in contrast with their corresponding controls. The arrowsindicate the heterozygous nucleotides of A/T (Figure 1A, mutant) or C/T (Figure 1B, mutant) or the homozygous nucleotides of A/A(Figure 1A, wild-type) or C/C (Figure 1B, wild-type). The rectangle designates the nucleotides comprising a codon of PITX2c.

Figure 2 - A schematic representation of the PITX2c protein structure with the atrial fibrillation-related mutations indicated. Themutations identified in patients with atrial fibrillation are shown above the structural domains. NH2 means amino-terminus; TAD1,transcriptional activation domain 1 (amino acids 1–91); HD, homeodomain (amino acids 92–151); NLS, nuclear localization signal(amino acids 145–161); TID1, transcriptional inhibitory domain 1 (amino acids 162–212); TAD2, transcriptional activation domain 2(amino acids 213–285); TID2, transcriptional inhibitory domain 2 (amino acids 286–324); and COOH, carboxyl-terminus.


19

were delineated by a transcriptional activity analysis, theresults of which showed that both mutations were asso-ciated with a significantly reduced transcriptional activityon a downstream gene. This result suggests that the dys-functional PITX2c resulting from mutations is potentially analternative pathological mechanism in AF.

The finding that functionally impaired PITX2c contributesto AF may be partially attributed to the abnormal deve-lopment of cardiovascular system, especially pulmonaryvenous myocardium (37,38). PITX2c is abundantly ex-pressed in the atria and pulmonary myocardium, down-regulating the sinoatrial nodal gene program, for example,Shox2, HCN4 and Cav3.1, and upregulating a gene programcharacteristic of a working myocardium phenotype, forexample, Nkx2.5, Cx40, Cx43, ANP, and Kir2.1 (27,29,31,37).Therefore, PITX2c loss-of-function mutations presumablypredispose a patient to AF by inducing the identity switchof the atrial and pulmonary myocardium to a sinoatrialnode–like phenotype, thereby forming an electrophysiolo-gical substrate that favors AF. Another equally compellingexplanation is that dysfunctional PITX2c may change theexpression profile of ion channels in the atrium, includingan increased expression of the potassium-channel geneKCNQ1, which alters atrial repolarization, as suggested bythe gene expression analysis and functional studies inPITX2-deficient mice (29,31,39).

It has been shown that some downstream genes aretransactivated by PITX2c (27), and mutations in multipletarget genes, including Nkx2.5, Cx40, Cx43, and ANP, have

been causally implicated in AF (14,21,40-42). This resultimplies that mutated PITX2c may confer vulnerability to AFby downregulating the expressions of these target genes.Therefore, additional experiments investigating whetherPITX2c mutations can modify the expression levels of some

Figure 3 - Alignment of the multiple PITX2c protein sequences across species. The altered amino acids of p.Q105 and p.R122 are highlyevolutionarily conserved among the various species.

Figure 4 – The functional defects associated with the PITX2cmutations. The activation of an atrial natriuretic factor (ANF)promoter-driven luciferase reporter in the CHO cells by PITX2cwild-type (WT), Q105L-mutant, or R122C-mutant, alone or incombination, demonstrated a significantly decreased transacti-vational activity by the mutant proteins. The experiments wereperformed in triplicate, and the means and standard deviationsare shown. ** indicates p,0.001 and * denotes p,0.01, whencompared with the same amount (2 mg) of wild-type PITX2c.


CLINICS 2014;69(1):15-22

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of the genes involved in the AF pathogenesis are stillrequired.

Notably, PITX2 mutations were previously implicated intype 1 Axenfeld-Rieger syndrome, type 2 iridogoniodysgen-esis, Peters’ anomaly, ring dermoid of cornea, and con-genital cardiac malformation (33,43-45). In this study, themutations identified in the AF patients were in the home-odomain shared by PITX2a, PITX2b, and PITX2c, whichimplies that mutated PITX2 may also be responsible for AF.

In addition, Yang et al. (46) previously screened 152 indexpatients with familial AF who were enlisted from the HanChinese population. They identified 2 novel heterozygousPITX2c mutations, including c.110C.G, corresponding top.S37W, and c.840 T.A, which resulted in p.Y280X in 2of the 152 AF probands, with a mutational prevalence ofapproximately 1.32%. The authors’ analysis of the pedigreesshowed that each mutation that co-segregated with AFwas transmitted in an autosomal dominant manner in thefamily, with complete penetrance. However, the functionalcharacteristics of the PITX2 mutations associated withfamilial AF remain to be addressed.

In conclusion, this study links PITX2c loss-of-functionmutations to AF for the first time, provides evidencethat functionally impaired PITX2c is associated with anincreased vulnerability to AF, and identifies the potentialimplications for early prophylaxis and allele-specific thera-pies for this common arrhythmia.

& ACKNOWLEDGMENTS

We thank the study participants for their devotion to the study. This work

was partially supported by grants from the National Natural Science Fund

of China (81070153, 81270161 and 30570768), the National Basic

Research Program of China (2010CB912604), and the Personnel

Development Foundation of Shanghai, China (2010019).


Qiu XB contributed to the experimental design, the clinical and

experimental research, the data analysis and interpretation, and the

manuscript writing. Xu YJ, Li RG, Xu L, Liu X, and Fang WY

contributed to the clinical research and the data analysis and interpreta-

tion. Yang YQ and Qu XK contributed to the study design, the

experimental research, the data analysis and interpretation, and the initial

drafting and revision of the manuscript.

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Comparison of the TruView infant EVO2 PCDTM andC-MAC video laryngoscopes with direct Macintoshlaryngoscopy for routine tracheal intubation ininfants with normal airwaysHaitham Mutlak,I Udo Rolle,II Willi Rosskopf,I Richard Schalk,I Kai Zacharowski,I Dirk Meininger,I

Christian ByhahnIII

I Johann Wolfgang Goethe-University Hospital, Clinic of Anaesthesiology, Intensive Care Medicine and Pain Therapy, Frankfurt, Germany. II Johann

Wolfgang Goethe-University Hospital, Department of Pediatric Surgery, Frankfurt, Germany. III Protestant Hospital Oldenburg, European Medical School

Oldenburg-Groningen, Department of Anesthesiology and Intensive Care Medicine, Oldenburg, Germany.

OBJECTIVE: Videolaryngoscopy has mainly been developed to facilitate difficult airway intubation. However,there is a lack of studies demonstrating this method’s efficacy in pediatric patients. The aim of the present studywas to compare the TruView infant EVO2 and the C-MAC videolaryngoscope with conventional directMacintosh laryngoscopy in children with a bodyweight #10 kg in terms of intubation conditions and the timeto intubation.

METHODS: In total, 65 children with a bodyweight #10 kg (0-22 months) who had undergone elective surgeryrequiring endotracheal intubation were retrospectively analyzed. Our database was screened for intubationswith the TruView infant EVO2, the C-MAC videolaryngoscope, and conventional direct Macintosh laryngo-scopy. The intubation conditions, the time to intubation, and the oxygen saturation before and afterintubation were monitored, and demographic data were recorded. Only children with a bodyweight #10 kgwere included in the analysis.

RESULTS: A total of 23 children were intubated using the C-MAC videolaryngoscope, and 22 children wereintubated using the TruView EVO2. Additionally, 20 children were intubated using a standard Macintosh blade.The time required for tracheal intubation was significantly longer using the TruView EVO2 (52 sec vs. 28 sec forC-MAC vs. 26 sec for direct LG). However, no significant difference in oxygen saturation was found afterintubation.

CONCLUSION: All devices allowed excellent visualization of the vocal cords, but the time to intubation wasprolonged when the TruView EVO2 was used. The absence of a decline in oxygen saturation may be due toapneic oxygenation via the TruView scope and may provide a margin of safety. In sum, the use of the TruViewby a well-trained anesthetist may be an alternative for difficult airway management in pediatric patients.

KEYWORDS: Videolaryngoscopy; Tracheal Intubation; Infants.

Mutlak H, Rolle U, Rosskopf W, Schalk R, Zacharowski K, Meininger D, et al. Comparison of the TruView infant EVO2 PCDTM and C-MAC videolaryngoscopes with direct Macintosh laryngoscopy for routine tracheal intubation in infants with normal airways. Clinics. 2014;69(1):23-27.

Received for publication on June 11, 2013; First review completed on July 7, 2013; Accepted for publication on July 12, 2013


Tel.: +49 69 6301 7532

& INTRODUCTION

Direct laryngoscopy can be challenging in small children.Failure to successfully intubate the trachea and to secure apatient’s airway remains a leading cause of morbidity and

mortality in the operative setting (1). In children, adverserespiratory effects are responsible for the largest proportionof perioperative critical events (2).

The airway of infants is special, differing significantlyfrom that of older children. Anatomic differences include alarge head that tends to flex the short neck and obstruct theairway, a large tongue, a short jaw, a long palate, a longepiglottis, a more cephalad-located larynx, and a soft airwaythat may lead to airway obstruction (3).

Despite the development of numerous pediatric airwaymanagement tools and techniques, ‘‘classic’’ endotrachealintubation remains the gold standard in securing the airway(4). However, in the last several years, there has been



DOI: 10.6061/clinics/2014(01)04

CLINICAL SCIENCE

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intensive development of tools for the management ofdifficult pediatric airways. Several studies have shown thatvideolaryngoscopy provides a better laryngeal view thandoes direct laryngoscopy in adult patients or mannequinswith either apparently normal or potentially difficultairways (5-10). Most of the published data related tovideolaryngoscopy have been obtained from adults, as theimplementation of videolaryngoscopy in pediatric airwayshas only been investigated in a few recent publications(11-14). Therefore, videolaryngoscopy in children is adeveloping area of research, and several different tools forindirect laryngoscopy are available today.

The C-MAC videolaryngoscope system (Karl Storz GmbH& Co. KG, Tuttlingen, Germany; Figure 1) is a novel devicethat can be used with special Macintosh laryngoscopeblades of different sizes (2,3,4) and a difficult-airway blade(D-Blade) (15,16). A camera providing an 80˚ angle of viewand a light source are recessed from the tip of the blade. Theelectronic unit sits in a handle attached to the laryngoscopeblade and is connected by a wire to a portable TFT videomonitor. The system allows for the Macintosh laryngoscopeblade to be used for direct and indirect laryngoscopy andfor the D-Blade to be used for only indirect laryngoscopy.The low profile of the original British Macintosh blades mayprove advantageous, especially when the mouth opening islimited.

In 2006, the Truphatek TruView EVO2 system (TruphatekInternational Limited, Netanya, Israel) was introduced foradult airway management (17), and in 2009, the device

began to be used in pediatric anesthesia (18). The TruViewsystem is a device with an integrated optical lens system and aunique blade tip angulation that provides an optimal line ofsight, allowing a view of the glottis via the prismatic lenswithout having to align the oral, pharyngeal, and tracheal axes(Figure 2). The blade of the laryngoscope has a magnified opticside port that provides a wide, magnified laryngeal view at a 46˚anterior refracted angle. The tool has a narrower blade tip thandoes the Macintosh blade and an integrated oxygen jet-cleaningsystem (flow 2-5 l/min-1) to prevent fogging and provideapneic oxygenation. In its commercial version, a portable TFTmonitor can be attached. An infant blade is also available andis recommended for use in children with a bodyweight of1-10 kg. To provide the correct angulation and rigidity in theendotracheal tube, a special stylet (the OptiShapeTM) comeswith the TruView.

This study was designed to compare intubation conditionsusing the TruView EVO2, the smallest available C-MACvideolaryngoscope (blade size 2), and standard direct laryngo-scopy with a Macintosh blade in children with a bodyweight#10 kg. In contrast to the wide use of the Miller blade in thissubset of patients, at our institution, the Macintosh blade is thestandard blade for primary intubation, even in infants. Here, thestudy endpoints were the time to intubation (TTI) andvisualization of the glottic structures.


Approval for this retrospective observational study wasobtained from the local ethics committee of the MedicalFaculty of Goethe University Frankfurt. The C-MAC and theTruView EVO2 devices are integral parts of the airwaymanagement process at our institution and are regularlyused for airway management in children. For quality-control reasons, we collect data on intubation conditionsand the handling of each device after introduction into anew environment. In this study, children were consideredappropriate for recruitment if they were from 1-10 kg inweight and had undergone elective surgery with plannedendotracheal intubation. The exclusion criteria included thepresence of a high risk of pulmonary aspiration, thepresence of any pathology of the head or neck, hemody-namic instability, and emergency surgery. The included

Figure 1 - The C-MAC video laryngoscope system (Karl StorzGmbH & Co. KG, Tuttlingen, Germany.

Figure 2 - Truphatek TruView EVO2 system with attached videomonitor (Truphatek International Limited, Netanya, Israel).

Videolaryngoscopy for intubation in infantsMutlak H et al.

CLINICS 2014;69(1):23-27

24

children were retrospectively analyzed over a period of 4months. The laryngoscopists were board certified andexperienced in standard direct laryngoscopy and indirectlaryngoscopy with the C-MAC device. Prior to clinicalutilization of the TruView EVO2 device, the physiciansperformed 10 mannequin intubations and three humanintubations with the device, before any data collection wasstarted.

In the operating theater, all children were monitoredusing electrocardiograms, noninvasive arterial blood pres-sure measurements, pulse oximetry, capnography, andinspired oxygen concentration measurements. Additionally,all children underwent inhalational induction with sevo-flurane. Prior to laryngoscopy, the lungs were ventilatedwith 4-8% sevoflurane in oxygen. A neuromuscularrelaxant (mivacurium 0.2 mg kg-1) was administeredroutinely. When the anesthesia depth and neuromuscularblockade were deemed suitable for intubation, laryngo-scopy was performed. All intubations were performed witha styletted endotracheal tube without a cuff. In the TruViewEVO2 group, a special stylet, the OptiShapeTM, was used.Based on prior experience in the use of the C-MACvideolaryngoscope, angulation of the ETT in this groupwas 60-70˚at the tip to achieve a better guidance toward theglottis.

The primary outcome parameter in this study was theTTI, defined as the time from the end of preoxygenation(mask taken from the face) to the first detection of endtidalCO2. Timing was determined by a member of the researchteam using a stopwatch (Finanzplatzuhr, Sinn, Frankfurt amMain, Germany). The laryngeal view was also graded accordingto the Cormack-Lehane (CL) score (19). Furthermore, the lowestperipheral oxygen saturation (LpO2) during intubation wasnoted. After intubation, the ease of intubation with the respectivedevice was recorded using a 10 cm visual analog scale (VAS),with 0 being extremely difficult and 10 being easy and comfortable.

StatisticsAn analysis of the data confirmed that a Gaussian

distribution was not present. All data were summarizedusing the median and range. The statistical analysis wasperformed using a software package (GraphPad InStatVersion 3.06; GraphPad Software Inc., San Diego, CA).The data were analyzed with the nonparametric Kruskal-Wallis one-way analysis of variance (ANOVA) test, and apost-hoc analysis was performed using Dunn’s multiplecomparison test. Statistical significance was assumed with aprobability of type I error of less than 5% (p,0.05).

& RESULTS

A total of 65 children with normal airways wereretrospectively included over a period of 4 months: 23children in the C-MAC group (C-MAC), 22 children in theTruView EVO2 group (TruView), and 20 children in theMacintosh blade group (Macintosh). The children in allgroups were similar in terms of age, but there were

statistically significant differences between the groups inthe mean weights and heights. The patient characteristicsare summarized in Table 1.

The time required for tracheal intubation was signifi-cantly longer in the TruView group (52 sec [20-102 sec])than in the C-MAC (28 sec [8-93 sec]) and Macintosh (26 sec[18-95 sec]) groups (Table 2). In most children, the devicesproduced excellent visualization of the vocal cords. Onlyone child in the C-MAC group and another in the Macintoshgroup were graded as CL III. The lowest peripheral oxygensaturation values during the intubation attempt were 94%in the C-MAC group, 86% in the TruView group, and 93% inthe Macintosh group. Additionally, the ease of use,as analyzed by a VAS, was 9 (8-10) in the C-MAC group,8 (6-10) in the TruView group, and 10 (0-10) in theMacintosh group.

No complications directly related to the intubationattempt, no dental injuries, and no airway injuries wereobserved in any of the groups.

& DISCUSSION

We aimed to compare the effectiveness of two differentvideolaryngoscopes, the C-MAC videolaryngoscope and theTruView EVO2 laryngoscope, with that of direct laryngo-scopy using a standard Macintosh blade in pediatricpatients with a weight #10 kg. This weight class wasselected because both video-assisted devices are certified foruse in this patient population, according to the manufac-turer’s instructions.

The presented data were limited by a lack of randomiza-tion that led to significant differences in the demographicdata of each group. However, on average, the time requiredfor tracheal intubation with the TruView EVO2 laryngo-scope was more than 20 sec longer than for the C-MAC ordirect laryngoscopy. This finding was both statistically andclinically significant. This difference could have resultedfrom the more extensive experience of the participatinganesthesiologists with the Macintosh blade of the C-MACvideolaryngoscope. Although the anesthesiologists whoparticipated in this study had practiced with the TruViewdevice several times before, their experience with this newtechnique was less extensive than their experience with theC-MAC videolaryngoscope, which is frequently used in ourdepartment in adult patients with difficult airways. Inaddition to this limited experience in the use of the TruViewprior to the start of the study, its use was furthercomplicated by the requirement to perform intubation inan indirect manner. The tube also needs to be advancedblindly until its tip enters the visual field of the TruViewand is displayed on the attached monitor. Finally, due to themagnified laryngeal view at a 46˚ anterior refracted angle,tube insertion through the vocal cords remains difficult. Thestylet (OptiShapeTM), with its preformed angulation, is notfixed with the tube, so posterior slipping of the tube into theesophagus is a concern when using the TruView EVO2laryngoscope. Overall, this tool’s use requires good hand-eye

Table 1 - Patient characteristics. The data are presented as the median and range.

C-MAC (n = 23) TruView (n = 22) Macintosh (n = 20) p-value

Age (months) 9.0 (1-22)* 4.0 (0-21) 7.5 (0-20) *p,0.05 vs. TruView EVO2

Weight (kg) 8.6 (2.7-10)* 5.2 (2.7-10) 7.5 (2.5-10) *p,0.05 vs. TruView EVO2

Height (cm) 70 (50-85)* 60 (50-80) 66.5 (50-95) *p,0.05 vs. TruView EVO2

CLINICS 2014;69(1):23-27 Videolaryngoscopy for intubation in infantsMutlak H et al.

25

coordination. Indirect laryngoscopy was also performedusing the C-MAC device, but this device is subjectivelyeasier to use due to the similarity in shape with the Macintoshblade.

Malik et al. found that the duration of tracheal intubationwith the TruView EVO2 was longer than with otherlaryngoscopes (20). However, the authors used the devicesin adults with immobilized cervical spines. Although ourresults are in agreement with those obtained by Malik andcolleagues, the direct comparability is limited. In contrast toour data, Malik et al. concluded that using the TruView withits optical entrance attached to the top of the blade and foggingon the distal lens were the primary reasons for the prolongedduration of tracheal intubation. In our study, fogging of theTruView lens was reduced by insufflation of oxygen directlyto the top of the blade through a special port. Beyond theantifogging effects, the supplemental oxygen insufflationallowed for apneic oxygenation during laryngoscopy andtube placement. The TTI with the TruView was 56 sec onaverage, and the good tolerance of this long-lasting intubationprocedure might have been due to the supplemental oxygeninsufflation. Only one child experienced a short period ofoxygen desaturation (SpO2 86%). In this case, the TTI was52 sec, and the oxygen saturation was 100% before and 96%after preoxygenation, perhaps due to a leaky mask and anagitated child. In contrast, in the C-MAC group, the TTI wassignificantly shorter. Accordingly, the lowest peripheraloxygen saturation was higher (94% in one child), even thoughno supplemental oxygen was administered in this group.Under standard direct intubation with a Macintosh blade, thelowest peripheral oxygen saturation was 93% in our study.

Although a good view of the vocal cords is occasionallydifficult to obtain by conventional direct laryngoscopy,tracheal tube placement is generally easier than whenindirect techniques are used because the anatomical andoptical axes are aligned. In contrast, indirect techniques mayallow for good visualization of the vocal cords in mostpatients, but endotracheal tube placement is occasionallydifficult and even impossible because of the divergentanatomical and optical axes (6,15).

Kim et al. compared the GlideScope with a Macintoshblade in 203 children with and without backward, upward,and rightward pressure (BURP) and found that use of theGlideScope was associated with a better laryngoscopic viewbut required a longer TTI (11). Li et al. and Barak et al. alsodemonstrated a longer duration of intubation when theTruView was used compared with the Macintosh blade.Despite the longer duration of the intubation, the laryngo-scopic view was rated as good (21,22).

The main limitations of the study were its retrospectivecharacter and the variable experience of the participating

anesthesiologists with the different devices, leading tosignificantly faster intubation using the Macintosh blade ofthe C-MAC videolaryngoscope. Additionally, the fact thatno power analysis preceded the study may have addition-ally limited its informative content. However, a post-hocpower analysis revealed a power of 0.98, which issignificant. Calculations were performed using the TTI asthe main outcome variable, along with the number of cases,the mean and standard deviation of the statisticallysignificant groups (TruView and Macintosh blade), and atwo-tailed alpha value of 0.05. It should be noted that thesignificant differences in the weights and heights of thesubjects did not influence the main results.

In our study, we found that in small children weighing#10 kg, tracheal intubation using the TruView EVO2 tooknearly twice as long as using the smallest available C-MACvideolaryngoscope blade or conventional direct laryngo-scopy. Visualization of the vocal cords was excellent, but theintroduction of the tube with the preformed stylet waschallenging in certain cases. Nevertheless, no significantdifference in SpO2 was found after intubation. This phenom-enon may be due to apneic oxygenation via the TruViewscope and gives a margin of safety in this special subset ofpatients. Furthermore, in infants with difficult airways, theuse of the TruView scope by an anesthetist with sufficientexpertise in the TruView system may represent an alternativefor the management of difficult airways. However, withoutfurther investigation in a clinical trial, this hypothesisremains speculative. The previously described visualizationlimitation of fogging when using the TruView device was notobserved in our study. In particular, the application ofoxygen through a special port on this device reduced foggingand blew secretions away from the optical lens.

& ACKNOWLEDGMENTS

This work has not been funded by any sources. No ethical, financial, or

other conflict of interest exists regarding the subject matter or materials. No

person involved in this study has any financial relationship or competing

interests with the manufacturers of either the TruView EVO2 or the C-

MAC laryngoscopes. The C-MAC video laryngoscope (Karl Storz GmbH

& Co. KG, Tuttlingen, Germany) and the TruView infant EVO2

laryngoscope (Truphatek International LtdH, Netanya, Israel) are regularly

used and are part of the Clinic of Anaesthesiology equipment inventory.

There was no source of external funding.


Mutlak H, Byhahn C, Meininger D, Zacharowski K, Schalk R, and Rolle

U wrote the paper and prepared the manuscript. Mutlak H, Meininger D,

and Byhahn C conceived the study and performed the statistical analysis.

Rosskopf W, Meininger D, and Mutlak H performed most of the tracheal

intubations.

Table 2 - Detailed outcome data for all devices. The data are presented as the median and range.

C-MAC (n = 23) TruView (n = 22) Macintosh (n = 20) p-value

CL I 19 21 17 n.s.

CL II 3 1 2 n.s.

CL III 1 - 1 n.s.

CL IV - - - n.s.

TTI (sec) 28 (8-93) 52* (20-102) 26 (18-95) *p,0.05 vs. C-MAC and Macintosh

LpO2 (%) 99 (94-100) 100 (86-100) 100 (93-100) n.s.

VAS (cm) 9 (8-10) 8* (6-10) 9 (0-10) *p,0.05 vs. C-MAC and Macintosh

CL = Cormack & Lehane grade; TTI = Time to intubation; LpO2 = Lowest peripheral oxygen saturation; VAS = Visual analog scale.

Videolaryngoscopy for intubation in infantsMutlak H et al.

CLINICS 2014;69(1):23-27

26

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2. Gregory GA, Riazi J. Classification and assessment of the difficultpediatric airway. Anesthesiol Clin North America. 1998;16(4):729-41,http://dx.doi.org/10.1016/S0889-8537(05)70058-4.

3. Holm-Knudsen RJ, Rasmussen LS. Paediatric airway management: basicaspects. Acta Anaesthesiol Scand. 2009;53(1):1-9, http://dx.doi.org/10.1111/j.1399-6576.2008.01794.x.

4. Goldmann K. Recent developments in airway management of thepaediatric patient. Curr Opin Anaesthesiol. 2006;19(3):278-84, http://dx.doi.org/10.1097/01.aco.0000192786.93386.d5.

5. Aziz M, Dillman D, Kirsch JR, Brambrink A. Video laryngoscopy withthe macintosh video laryngoscope in simulated prehospital scenarios byparamedic students. Prehosp Emerg Care. 2009;13(2):251-5, http://dx.doi.org/10.1080/10903120802706070.

6. Byhahn C, Iber T, Zacharowski K, Weber CF, Ruesseler M, Schalk R, et al.Tracheal intubation using the mobile C-MAC video laryngoscope ordirect laryngoscopy for patients with a simulated difficult airway.Minerva Anestesiol. 2010;76(8):577-83.

7. Jungbauer A, Schumann M, Brunkhorst V, Borgers A, Groeben H.Expected difficult tracheal intubation: a prospective comparison of directlaryngoscopy and video laryngoscopy in 200 patients. Br J Anaesth.2009;102(4):546-50, http://dx.doi.org/10.1093/bja/aep013.

8. Low D, Healy D, Rasburn N. The use of the BERCI DCI VideoLaryngoscope for teaching novices direct laryngoscopy and trachealintubation. Anaesthesia. 2008;63(2):195-201, http://dx.doi.org/10.1111/j.1365-2044.2007.05323.x.

9. McElwain J, Malik MA, Harte BH, Flynn NM, Laffey JG. Comparison ofthe C-MAC videolaryngoscope with the Macintosh, Glidescope, andAirtraq laryngoscopes in easy and difficult laryngoscopy scenarios inmanikins. Anaesthesia. 2010;65(5):483-9, http://dx.doi.org/10.1111/j.1365-2044.2010.06307.x.

10. Ng I, Sim XL, Williams D, Segal R. A randomised controlled trialcomparing the McGrath(H) videolaryngoscope with the straight bladelaryngoscope when used in adult patients with potential difficultairways. Anaesthesia. 2011;66(8):709-14, http://dx.doi.org/10.1111/j.1365-2044.2011.06767.x.

11. Kim JT, Na HS, Bae JY, Kim DW, Kim HS, Kim CS, et al. GlideScopevideo laryngoscope: a randomized clinical trial in 203 paediatric patients.Br J Anaesth. 2008;101(4):531-4, http://dx.doi.org/10.1093/bja/aen234.

12. Riveros R, Sung W, Sessler DI, Sanchez IP, Mendoza ML, Mascha EJ, et al.Comparison of the Truview PCDTM and the GlideScope(H) videolaryngoscopes with direct laryngoscopy in pediatric patients: a rando-mized trial. Can J Anaesth. 2013;60(5):450-7.

13. Trevisanuto D, Fornaro E, Verghese C. The GlideScope video laryngo-scope: initial experience in five neonates. Can J Anaesth. 2006;53(4):423-4.

14. Wald SH, Keyes M, Brown A. Pediatric video laryngoscope rescue for adifficult neonatal intubation. Paediatr Anaesth. 2008;18(8):790-2, http://dx.doi.org/10.1111/j.1460-9592.2008.02542.x.

15. Cavus E, Kieckhaefer J, Doerges V, Moeller T, Thee C, Wagner K. TheC-MAC videolaryngoscope: first experiences with a new device forvideolaryngoscopy-guided intubation. Anesth Analg. 2010;110(2):473-7,http://dx.doi.org/10.1213/ANE.0b013e3181c5bce5.

16. Cavus E, Neumann T, Doerges V, Moeller T, Scharf E, Wagner K, et al.First clinical evaluation of the C-MAC D-Blade videolaryngoscopeduring routine and difficult intubation. Anesth Analg. 2011;112(2):382-5,http://dx.doi.org/10.1213/ANE.0b013e31820553fb.

17. Matsumoto S, Asai T, Shingu K. Truview video laryngoscope in patientswith difficult airways. Anesth Analg. 2006;103(2):492-3, http://dx.doi.org/10.1213/01.ANE.0000227064.72260.19.

18. Singh R, Singh P, Vajifdar H. A comparison of Truview infant EVO2laryngoscope with the Miller blade in neonates and infants. PaediatrAnaesth. 2009;19(4):338-42, http://dx.doi.org/10.1111/j.1460-9592.2009.02929.x.

19. Cormack RS, Lehane J. Difficult tracheal intubation in obstetrics.Anaesthesia. 1984;39(11):1105-11, http://dx.doi.org/10.1111/j.1365-2044.1984.tb08932.x.

20. Malik MA, Maharaj CH, Harte BH, Laffey JG. Comparison of Macintosh,Truview EVO2, Glidescope, and Airwayscope laryngoscope use inpatients with cervical spine immobilization. Br J Anaesth. 2008;101(5):723-30, http://dx.doi.org/10.1093/bja/aen231.

21. Barak M, Philipchuck P, Abecassis P, Katz Y. A comparison of theTruview blade with the Macintosh blade in adult patients. Anaesthesia.2007;62(8):827-31, http://dx.doi.org/10.1111/j.1365-2044.2007.05143.x.

22. Li JB, Xiong YC, Wang XL, Fan XH, Li Y, Xu H, et al. An evaluation of theTruView EVO2 laryngoscope. Anaesthesia. 2007;62(9):940-3, http://dx.doi.org/10.1111/j.1365-2044.2007.05182.x.

CLINICS 2014;69(1):23-27 Videolaryngoscopy for intubation in infantsMutlak H et al.

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The thickness of posterior cortical areas is related toexecutive dysfunction in Alzheimer’s diseaseLuciano de Gois Vasconcelos,I Andrea Parolin Jackowski,II Maira Okada de Oliveira,III Yona Mayara Ribeiro

Flor,II Altay Alves Lino Souza,I Orlando Francisco Amodeo Bueno,I Sonia Maria Dozzi BruckiIII

I Universidade Federal de Sao Paulo, Psychobiology Department, Sao Paulo/SP, Brazil. II Universidade Federal de Sao Paulo, Psychiatry Department,

Laboratorio Interdisciplinar de Neurociencias Clınicas (LiNC), Sao Paulo/SP, Brazil. III Faculdade de Medicina da Universidade de Sao Paulo, Hospital das

Clınicas, Cognitive Neurology and Behavior Group, Sao Paulo/SP, Brazil.

OBJECTIVE: To establish whether alterations of brain structures in Alzheimer’s disease are associated withexecutive dysfunction.

METHODS: Nineteen patients with Alzheimer’s disease and 22 older control subjects underwent acomprehensive evaluation. The clock drawing test, digit span test, executive motor function test, BehavioralAssessment of the Dysexecutive Syndrome battery (Rule Shift Cards test), and Stroop test were used to evaluateexecutive dysfunction. A multiparametric approach using the FreeSurfer image analysis suite provided adescription of volumetric and geometric features of the gray matter structures.

RESULTS: The cortical thickness maps showed a negative correlation between the Behavioral Assessment of theDysexecutive Syndrome battery (Rule Shift Cards test) and the right middle frontal gyrus; a positive correlationbetween the executive motor function test and the left superior parietal gyrus, left middle temporal gyrus,bilateral supramarginal gyri, right middle frontal gyrus, and right precuneus; a negative correlation betweenthe Stroop test (part III) and the right superior parietal gyrus; and a negative correlation between the Strooptest (part III) and the right middle temporal gyrus.

CONCLUSION: Executive dysfunction in Alzheimer’s disease is correlated with alterations not only in the frontalareas but also within many temporal and parietal regions.

KEYWORDS: Executive Functions; Alzheimer’s Disease; Magnetic Resonance Imaging.

Vasconcelos LG, Jackowski AP, Oliveira MO, Ribeiro Flor YM, Souza AA, Bueno OF, et al. The thickness of posterior cortical areas is related toexecutive dysfunction in Alzheimer’s disease. Clinics. 2014;69(1):28-37.



Tel.: 55 11 5084 0505

& INTRODUCTION

Although the prevailing concept of Alzheimer’s disease(AD) as an episodic memory disorder is well supported,there are many examples of clinical heterogeneity (1).Several non-amnestic presentations of the pathophysiologi-cal process of AD exist, and probable AD is diagnosed evenif executive function is the main cognitive deficit (2).

Executive function is a multidimensional cognitivedomain that includes attention, sequencing, goal formation,planning, execution of goal-directed plans, effective perfor-mance, insight, will, abstraction, and judgment (3).Executive dysfunctions have heterogeneous manifestations,and they occur almost universally in all stages of dementia

(4). Furthermore, these dysfunctions are associated withgreater risk for the development of AD (5). Executivedysfunction is also associated with greater dementiaseverity, rapid disease progression, disability, behavioraldisorders, and higher mortality (6–9).

Approaches that focus on the localization of executiveabilities within the frontal lobe have often been criticized;critics have favored a perspective that emphasizes theconnectivity between the frontal regions and the moreposterior and subcortical brain areas (3). The prefrontalcortex receives inputs from higher-order association corticalareas such as the posterior parietal lobe, superior temporallobe, and paralimbic regions (10).

Many studies have explored the neural basis of executivedysfunction in AD. Although most of these studiescorrelated changes in the frontal structures with executiveperformance impairment, many others correlated executivedysfunction with posterior cortical areas (11–15).

Automated magnetic resonance imaging (MRI) thicknessmeasures of individual brain regions can identify mildcognitive impairment and AD with great accuracy,specificity, consistency, and reproducibility across multiple



DOI: 10.6061/clinics/2014(01)05

CLINICAL SCIENCE

28

independent cohorts. These measures correlate strongly withclinical measures of cognitive decline as well as cellularbiomarkers (16–18). Using software tools, a single volumetricT1-weighted MRI scan can be completely processed withlittle to no manual intervention in a relatively short amount oftime. Evidence from the literature suggests that corticalthickness can predict the risk of conversion from mildcognitive impairment to AD with a higher degree of accuracythan clinical and neuropsychological assessments (19,20).Therefore, this automated measure provides a cost-effectiveand efficient method for the early diagnosis of AD and mildcognitive impairment. Furthermore, these measurementsmay serve as a quantitative and biologically meaningfulendpoint in therapeutic trials.

The questionable description of executive functions ashigher-level cognitive functions mediated primarily by thefrontal lobes and the lack of a definitive role for specific brainstructures in certain executive tasks should be better clarified.Moreover, the lack of a clear correlation between corticalthickness and executive function performance in healthysubjects and the limited number of studies assessing thecorrelation between the posterior associative cortical thick-ness and executive functions should be further evaluated.

The aim of this study was to establish whether alterationsin gray matter volume and cortical thickness of brainstructures are associated with executive dysfunction inpatients with mild AD and healthy controls.

& METHODS

SubjectsNineteen patients with mild AD and 22 older control

subjects were recruited from a multidisciplinary memoryclinic. The control subjects did not have any cognitivecomplaints or functional impairment, and all of theparticipants in the patient group fulfilled the NationalInstitute of Neurological and Communicative Disorders andStroke and the AD and Related Disorders Associationcriteria for probable AD (2). Patients had FunctionalAssessment Staging (21) scores of 3 or 4 and had beenreceiving a stable dose of a cholinesterase inhibitor for atleast 2 months. Controls had Functional Assessment Stagingscores of 1 or 2.

Exclusion criteria included significant symptoms ofdepression (15-item Geriatric Depression Scale score $6)(22); significant radiological evidence of ischemic braindisease; a Modified Hachinski Ischemic score .4 (23); aprevious cerebrovascular event, a Mini-Mental StateExamination score ,20 (24) or evidence of other degen-erative or secondary dementias; end-stage chronic diseaseor an unstable medical condition; a psychiatric history;antipsychotic or psychoactive medication adjustments in the2 months prior to study enrollment; significant visual orhearing impairment; age ,60 years; schooling of less than 2years; and any other condition that could prevent thepatient from undergoing an MRI examination or cognitiveassessment.

Patients with high levels of depressive symptoms wereexcluded to avoid bias in the cognitive evaluation becausesuch symptoms strongly influence performance on cogni-tive assessments (25). Both groups were also pairedaccording to educational level to reduce the effect of thisvariable on the cortical thickness results. A flow diagram ofsubject inclusion and exclusion is shown in Figure 1.

ProceduresEach subject and the caregivers of the patients with AD

underwent a complete interview with a consultant geria-trician. The physician collected demographic and medicalinformation, including history of hypertension or diabetesmellitus, body mass index, and waist-to-hip ratio.

A functional status assessment was performed using theFunctional Activities Questionnaire (23) and the Disabi-lity Assessment for Dementia (only in patients) (27).Neuropsychiatric symptoms were evaluated using theNeuropsychiatric Inventory (28). The comprehensive cogni-tive evaluation included executive tests, such as the clock

Figure 1 - Flow diagram of the subject selection procedure. For thecontrol group, 66 elderly individuals were invited to participate inthe study. Of these individuals, 21 missed the clinical assessment orwere not included due to their meeting clinical exclusion criteria.Of the 45 remaining subjects, 17 were excluded because theymissed the neuropsychological assessment. Of the 28 remainingsubjects, four missed the magnetic resonance imaging (MRI)assessment. The images of three elderly controls were notincluded in the study because they were considered to be of lowquality. Regarding the patient group, 46 AD patients were invitedto participate in the study. Of these patients, 16 missed the clinicalassessment or were not included due to their meeting clinicalexclusion criteria. Of the 30 remaining subjects, seven wereexcluded because they missed the neuropsychological assessment.Of the 23 remaining subjects, two missed the MRI assessment. Theimages of two AD patients were not included in the study becausethey were considered to be of low quality.

CLINICS 2014;69(1):28-37 Cortical thinning and executive dysfunction in ADVasconcelos LG et al.

29

drawing test (29), the digit span test, an executive motorfunction test (30), the Behavioral Assessment of theDysexecutive Syndrome (BADS) (Rule Shift Cards subtest)(31), and the Stroop test. Each participant also underwentMRI.

To evaluate executive motor function, a modified versionof the Neuropsi battery subtest was used (30). Each subjectwas asked to pay attention to a sequence of three handpositions, which was performed three times by theexaminer. The subject was asked to reproduce the sequencein the correct order three times. No verbal cues were given,but the examiner did indicate whether the reproduction wascorrect or incorrect. To perform this task, the subject had toplace his or her dominant hand in three different positionssequentially: a fist resting horizontally, a palm restingvertically, and a palm resting horizontally. If the subject wasunable to reproduce the sequence after three attempts, thescore was 1. If the subject was able to reproduce thesequence after two attempts, the score was 2. If the subjectwas able to reproduce the sequence in the first attempt, thescore was 3.

MRI data acquisition, analysis, and post-processingMRI of the brain was obtained in all subjects using a 1.5-T

scanner [Magnetom Sonata (Maestro Class) Siemens AG,Medical Solutions, Erlangen, Germany] with an eight-channel head coil. To minimize variation, a single investi-gator positioned all of the subjects using the orbitomeatalline as a landmark. Two conventional sequences wereperformed to exclude structural lesions: a) axial T2-weighted FLAIR (fluid-attenuated inversion recovery) in aplane parallel to the anterior commissure-posterior com-missure (AC-PC) line [TR (repetition time) = 8500 ms, TE(echo time) = 107 ms, IT (inversion time) = 2500 ms, slicethickness = 5.0 mm, slice interval = 1.5 mm, FOV (field ofview) = 240 mm, matrix size = 2566256, NEX = 1] and b)sagittal T1-gradient echo volumetric acquisition for multi-planar reconstruction (TR = 2000 ms, TE = 3.42 ms, flipangle = 15 degrees, FOV = 256 mm, 1.0-mm slice thickness

with no gaps, total of 160 slices per slab, matrixsize = 2566256, NEX = 1).

The quality of the structural MRI data was rated by twoexperienced neuroimaging researchers according to a three-point rating scale: 0 = no motion artifacts, excellent quality;1 = a few motion artifacts, fair quality; and 2 = moderate/severe motion artifacts, poor quality. Only datasets withscores of 0 were considered to be of sufficient quality forresearch purposes. The criteria used to define quality were(a) signal-to-noise ratio; (b) tissue contrast; and (c) artifacts,including c1) motion artifacts (ghosting and smearing), c2)edge artifacts (ghosting, chemical shifts, and ringing), c3)distortions, and c4) aliasing (wrap-around) artifacts. All theMRI exams were performed between 1 (minimum) and 8(maximum) weeks after the neuropsychological evaluation.The interval was not different between the AD and controlsubjects.

T1-weighted images were processed using the recon-allpipeline of the FreeSurfer package, which is documentedand freely available for download online (32,33). Asummary of the options used in the recon-all pipeline anda detailed description of this methodology are included inthe supplementary material.

Statistical analysisDemographic, clinical, cognitive, functional, and beha-

vioral data were analyzed with SPSS 18 (SPSS, Chicago, IL,USA). Prior to conducting the analyses, the measurementswere tested for normality using the Shapiro-Wilk test.

Demographic, clinical, and neuropsychological data, aswell as data on brain structure volumes (Table 1, supple-mentary material), are presented as the mean ¡ standarddeviation. Student’s t-tests (at a significance level of p,0.05)were used to compare the data of AD patients and controls.

To evaluate whether there were correlations betweenexecutive functions and brain structures, the volumetricmeasures were first transformed to Z scores using theformula [(value - mean)/SD], and a stepwise back-ward linear regression was performed. Type I errors in the

Table 1 - Demographic, medical, and cognitive data description.

Variable

Controls (n = 22, 12 females):

Mean (SD); range

Alzheimer’s disease (n = 19,

10 females): Mean (SD); range

Differences between

groups (t; p-value)

Age (years) 70.14 (5.67); 60–80. 75.42 (4.81); 66–86. 23.187; 0.003*

Education (years) 9.14 (5.26); 2–18. 7.68 (4.42); 3–16. 0.947; 0.349

Diabetes (%) 22 21 0.126; 0.900

Hypertension (%) 64 58 0.367; 0.715

Waist-to-hip ratio 0.94 (0.81); 0.7–1.1. 0.92 (0.71); 0.8–1.1. 21.037; 0.306

Body mass index 27.21 (3.71); 19– 32 26.10 (3.62); 21–36. 0.965; 0.340

Modified Hachinski scale 0.95 (0.84); 0–3. 0.68 (0.58); 0–2. 1.175; 0.247

Duration of cholinesterase inhibitor use

(months)

NA 42.63 (27.35); 4–106. NA

Mini Mental State Examination 28.82 (0.90); 27–30. 24.00 (2.62); 20–29. 8.083; 0.000*

Neuropsychiatric Inventory NA 20.36 (19.25); 0–77 NA

Stroop test part III (time - seconds) 48.77 (19.96); 25–103. 67.63 (28.50); 35–155. 22.480; 0.018*

Stroop test part III (errors) 1.59 (2.30); 0–9. 4.95 (4.50); 0–18. 23.067; 0.004*

Digit Span Backwards 3.86 (1.32); 0–6. 3.00 (1.29); 0–4. 2.110; 0.041*

Executive motor function test 2.45 (0.67); 1–3. 1.26 (1.14); 0–3. 4.127; 0.000*

Behavioral Assessment of Dysexecutive

Syndrome: Rule Shift Cards test - rule 2 (time -

seconds)

37.00 (8.25); 25–60. 41.21 (12.70); 26–76. 21.275; 0.210

Behavioral Assessment of Dysexecutive

Syndrome: Rule Shift Cards test - rule 2 (errors)

3.32 (3.92); 0–10. 7.05 (3.45); 0–11. 23.211; 0.003*

Clock drawing test 7.95 (2.36); 4–10. 6.11 (2.74); 2–10. 2.319; 0.026*

NA: not available; * statistically significant difference.

Cortical thinning and executive dysfunction in ADVasconcelos LG et al.

CLINICS 2014;69(1):28-37

30

follow-up multiple comparisons were controlled via Bonferroniadjustment (at a significance level of 0.015). The scores on theexecutive function tests represented the independent variablesused to predict alterations in brain structures.

The stepwise backward linear regression included thevariables of both groups (n = 41) and a ‘dummy’ variable(elderly controls vs. AD subjects). All correlations werecontrolled for age, gender, and intracranial volume.

Ethics statementThis study was approved by the Joint Ethics Committee of

the Universidade Federal de Sao Paulo, and participants (orthe guardian or caregiver of the patients with AD) providedwritten informed consent in accordance with the De-claration of Helsinki.

& RESULTS

Demographic, clinical, cognitive, functional, andbehavioral data

Table 1 shows the baseline characteristics of the studypopulation. The mean age of the total sample was 72.5 years(SD 5.8, range 60–86 years). The mean ages of the elderlycontrols (n = 22, 12 females) and the AD patients (n = 19, 10females) were 70.14 years (SD 5.67, range 60–80 years) and75.42 years (SD 4.81, range 66–86 years), respectively. TheAD subjects were significantly older than the elderlycontrols (t 23.187; p = 0.003). The mean educational levels(years) of the elderly controls and AD patients were 9.14years (SD 5.26, range 2–18 years) and 7.68 years (SD 4.42,range 3–16 years), respectively. No significant differenceswith respect to educational level were observed between thegroups (t = 0.947; p = 0.349).

One patient scored 29 on the MMSE. This subject hadbeen followed over the previous 2 years because of mildexecutive cognitive impairment. During the follow-upperiod, a progressive cognitive and functional decline wasobserved through neuropsychological and clinical evalua-tions. The patient developed dementia and was thereforeincluded in the study.

The mean scores on the geriatric depression scale forpatients and controls were 2 (range 0–5) and 1.3 (range 0–5),respectively. No significant differences with respect toprevalence of depressive symptoms were observed betweenthe groups. The mean score of patients on the FunctionalActivities Questionnaire was 9.8 (SD 4.7, range 2–22). Thecontrol group did not show any functional impairment.The Disability Assessment for Dementia and theNeuropsychiatric Inventory were also administered topatients with AD to complete the functional and behavioralassessment. The mean scores were 87% (SD 11, range 60–100%) and 22 (SD 19, range 0–77), respectively.

Volumetric assessmentCompared with controls, patients with AD exhibited

significantly smaller volumes of the bilateral caudal middlefrontal gyri, isthmus of cingulate, left pars opercularis, rightpars orbitalis, left pars triangularis, rostral middle frontalgyri bilaterally, superior frontal gyri bilaterally, frontal polebilaterally, middle temporal gyri bilaterally, precuneusbilaterally, superior parietal gyri bilaterally, inferior parietalgyri bilaterally, supramarginal gyri bilaterally, and leftfusiform gyrus. A detailed description of the volumetric

neuroimaging data of the participants is provided in thesupplementary material (Table 1, Supplementary Material).

The volume of the right superior parietal gyrus correlatednegatively with results on the Stroop test part III (errors)(beta = 20.093, t = 20.359, p = 0.012) and differentiated theAD group from the healthy controls (beta = 20.986,t = 23.071, p = 0.005).

Cortical thickness mapsThe cortical thickness maps of the patients and control

subjects showed a negative correlation between the BADSscore (Rule Shift Cards test, rule 2, errors) and the thicknessof the right rostral middle frontal gyrus; see Figure 2,images 1A and 2A. A positive correlation between theexecutive motor function test and the left superior parietalgyrus, left middle temporal gyrus, bilateral supramarginalgyri, right caudal middle frontal gyrus, and right precuneusthickness was noted (see Figure 2, images 1B, 2B, 1C, and2C). There was a negative correlation between the results ofthe Stroop test part III (errors) and the right superiorparietal gyrus (see Figure 2, image 1D). There was anegative correlation between the results of the Stroop testpart III (time) and the right middle temporal gyrus (seeFigure 2, image 2D). Table 2 provides the parameters of thelesion extension and the location of the findings shown inFigure 2. Scatterplot graphs of the correlations between theexecutive motor function test scores and the left middletemporal gyrus and bilateral supramarginal gyri corticalthickness are provided (Figure 2).

Analysis by group of the cortical thickness maps showedthat the main differences between elderly controls and ADpatients were in structures of the frontal, parietal, andtemporal lobes; the fusiform bilaterally; and a few areas ofthe occipital lobe. A detailed description of the related brainstructures, parameters of lesion extension, and location ofcortical thickness differences is provided in the supplemen-tary material (Table 2).

& DISCUSSION

Our results showed that executive dysfunctions in mildAD may be correlated with the thinning of the parietal andtemporal cortices.

A correlation between the volume and cortical thicknessof the right superior parietal gyrus and scores on executivefunction tests was observed. The volumetric correlationcould be used to differentiate AD patients from controls.

The cortical thickness of the left superior parietal gyrus,bilateral supramarginal gyri, right precuneus, and leftmiddle temporal gyrus correlated positively with perfor-mance on the executive motor function test. The executivefunctions assessed by this cognitive test, such as workingmemory, planning, and praxis, did not correlate with thestructures mentioned in previous studies.

The right superior parietal gyrus and the right middletemporal gyrus correlated negatively with the scores of theStroop test part III, supporting the role of these structures ininhibitory control. Similar results are not found in theliterature, although one study correlated response inhibitionwith the right parietal cortices in bipolar disorder type 1patients (34).

The anatomical correlations of the Stroop test and theexecutive motor function test occurred predominantly andwith higher intensity in the right hemisphere, confirming


31

previous studies (34,35). These findings highlight thecapacity of the practical cognitive tests (mentioned above)to detect executive dysfunction in patients with mild AD.

Many other studies using different neuroimaging meth-ods have correlated parietal and temporal structures withexecutive functions. Few of these studies used corticalthickness as a variable (11,15). Voxel-based morphometrystudies revealed that patients with AD without the epsilon 4allele of apolipoprotein had poorer executive task perfor-mance and greater frontoparietal atrophy (11) and that grey

matter reduction of the bilateral insula and left lateraltemporal lobe was a predictor of clinical progression ofdysexecutive mild cognitive impairment (36). Radionuclidestudies have revealed correlations between executive func-tions and the parietal and temporal regions (12,37).Functional MRI studies have correlated the right frontalregions and the associative parietotemporal areas withexecutive deficits in patients with AD (13,38).

The relationship between cortical thickness and perfor-mance on cognitive tests has not been fully elucidated and

Figure 2 - Cortical thickness maps of associations between brain regions and executive functions. Red, orange, and yellow colorsrepresent positive correlations, and blue represents negative correlations. 1A and 2A illustrate the negative correlation between theBADS score (Rule Shift Cards test, rule 2, errors) and the thickness of the right rostral middle frontal gyrus; 1B, 2B, 1C, and 2C illustratethe positive correlation between the executive motor function test and the left superior parietal gyrus, left middle temporal gyrus,bilateral supramarginal gyri, right caudal middle frontal gyrus, and right precuneus thickness; 1D illustrates the negative correlationbetween the results of the Stroop test part III (errors) and the right superior parietal gyrus; and 2D illustrates the negative correlationbetween the results of the Stroop test part III (time) and the right middle temporal gyrus. The scale indicates z-scores. Scatterplots ofthe correlations between the executive motor function test scores and the left middle temporal gyrus and bilateral supramarginal gyricortical thickness are provided. Table 2 provides the parameters of the lesion extension and the location of the findings shown in thisfigure.


CLINICS 2014;69(1):28-37

32

warrants further investigation. We found positive correla-tions between cortical thickness and cognitive test perfor-mance, both in controls and patients. However, previousstudies have yielded different results that showed aninverse relationship between cortical thickness and perfor-mance on executive function tests in control subjects (39,40).

The differential aspects of this study should be men-tioned. The selective pathological involvement of someneocortical areas and temporal lobe structures, which iscommon in AD (41), was also observed in our neuroimagingfindings and correlated with executive dysfunction.Previous studies based on analysis of the cortical surfacehave also shown that AD patients present cortical thinningin the various areas of the frontal, parietal, and temporallobes (42,43). Cardiovascular risk, an important factor incognitive decline and executive impairment and a possibleconfounder in AD studies, was considered in this investiga-tion (44,45).

The populations in developing countries are exposed tovarious adverse conditions. A combined disadvantage ineducation, income, wealth, and occupation was associatedwith poor cognitive function in late life (46). Education hasbeen found to be the most consistent socioeconomic factorassociated with cognitive dysfunction (47). Our sample hada mean education level of 8 years, higher than the averageyears of schooling of the adult Brazilian population, whichis estimated at 7.4 years (48). Most of the studies in this fieldwere performed in developed countries with more highlyeducated individuals than our sample (11,15,18,36).Although our study population had a higher educationlevel than most of the country, our sample is morerepresentative of the local population than those used inother studies, and our results could be used as a referencefor future studies evaluating the cortical thickness of ADpatients with a low education level.

The results of this study should be interpreted withcaution because our study presents a few limitations. Themain limitations include the small sample size and the agedifference between patients and control subjects. Otherpossible sources of bias could be 1) the use of age as acovariate and the omission of education level as a covariatein the stepwise backward linear regression; 2) the MRIcontraindications; 3) spurious correlations due to the largeamount of data used in the neuroimaging analysis; 4)restriction to subjects with a high burden of cerebrovasculardisease; and 5) use of the FreeSurfer package template,which is based on MRI scans of young, healthy subjects.

The poor ecological validity of some executive tests and thecomplex interdependence of the executive functions in othercognitive domains should be mentioned as a potential problemin the assessment of the executive functions of AD patients (3).

Cholinesterase inhibitors have been shown to decreasehippocampal and cortical atrophy (49,50) and improvecognitive performance in AD patients (51). Although thetreatment time varied between the patients (4–107 months),the patients had similar clinical staging (FunctionalAssessment Staging score of 3 or 4). To the best of ourknowledge, no studies have assessed the effects of choli-nesterase inhibitors on cortical thickness.

In the early phases of AD, before the intense period ofneuronal loss, synaptic reorganization changes to compen-sate for the degenerative effects of brain damage are usuallyobserved (52). The differences between groups observed inthis study could also be a result of this process and not onlya direct effect of AD neuropathology.

This study has implications for our understanding of howfunctional deficits in patients are associated with their under-lying structural basis. Neuroimaging techniques have demon-strated that executive abilities are not confined to the frontalarea of the brain but instead consist of complex interactionsamong different brain regions (53). Our results are consistentwith those of other AD studies, which have suggested thatexecutive function may not depend entirely on the prefrontalcortex but on other posterior cortical areas as well.

The association between modern neuroimaging methodsand practical tests, such as the Stroop test and the executivemotor function test, could be very useful for identifyingexecutive dysfunction in patients with AD. Future neuroi-maging studies addressing the connection between theseposterior cortical areas and the relationships betweencortical thickness and education level would add to theunderstanding of the neural basis of AD.

Executive dysfunction in mild AD is associated withabnormalities not only with the frontal areas but also withmany temporal and parietal regions. The pathophysiologyof executive dysfunction is complex and includes abnorm-alities in multiple brain regions and, most likely, theconnections between them.

& ACKNOWLEDGMENTS

This study was supported by the Associacao Fundo de Incentivo a Pesquisa

(AFIP), Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP

#2008/11282-9), and Conselho Nacional de Desenvolvimento Cientıfico e

Tecnologico (CNPq).

Table 2 - Correlations between executive function tests and cortical thickness of brain structures: parameters of lesionextension and location of the findings shown in Figure 2.

Executive function tests Brain structure Size (mm2) Talairach coordinates

BADS score (Rule Shift Cards test, rule 2, errors) Right rostral middle frontal gyrus 1658.60 12.31 107.15–21.49

Executive motor function test Left superior parietal gyrus 42.99 226.70 253.4 40.6

Left middle temporal gyrus 35.31 257.8 258.6 0.2

Left supramarginal gyrus 925.10 31.82 211.13 3.09

Right supramarginal gyrus 823.56 61.6 239.5 27.0

Right caudal middle frontal gyrus 8783.39 28.7 18.7 43.6

Right precuneus 1616.14 27.73 252.84 20.06

Stroop test part III (errors) Right superior parietal gyrus 530.65 30.6 245.3 61.5

Stroop test part III (time) Right middle temporal gyrus 4467.34 39.93 226.11 241.86


33


Jackowski AP, Brucki SM, and Bueno OF designed the study, supervised

the data collection, analyzed the data, and reviewed the paper.

Vasconcelos LG collected and analyzed the data and wrote the paper.

Oliveira MO and Flor YM collected and analyzed the data. Souza AA

analyzed the data and wrote part of the results section.

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& APPENDIX - SUPPLEMENTARY MATERIAL

Cortical thickness surface-based analysis: FreeSurfersoftware

The FreeSurfer package is software used for the assess-ment and visualization of structural and functional brainimaging data. It is fully automated structural imagingsoftware for processing neuroimaging data.

The FreeSurfer package is documented and freely avail-able for download online (https://surfer.nmr.mgh.harvard.edu/fswiki/FreeSurferWiki).

The software employs a method that is reproducible,consistent, and precise.

The main steps of this approach are gray/white mattersegmentation, pial and white matter surface modeling,transformation of the cortical surface to spherical coordi-nates, nonlinear surface registration based on curvature(gyrus and sulcus), analysis of multiple subjects, andautomated parcellation of cortical areas. A set of fivemorphometric parameters per vertex is used as an inputto the multimodal classifier: average convexity or concavity,mean radial curvature, metric distortion, cortical thickness,

and surface area. The average convexity or concavity is usedto quantify the primary folding pattern of a surface. Thisparameter can capture large-scale geometric features,indicating the depth-height above the template surface ofthe FreeSurfer and the sulcal depth or gyral height. Themean radial curvature is used to assess folding of thecortical surface. Metric distortion is calculated as the degreeof displacement of the cortical surface when registered tothe FreeSurfer template.

The FreeSurfer uses as a template the MNI 305 atlas. It is atemplate based on an average of 305 T1-weighted MRI scansof young, healthy subjects, linearly transformed to Talairachspace. All the images assessed in the study were fitted tothis template to enable comparison between them.

Cortical thickness and surface area were used to quantifyvolumetric differences. Significant difference maps wereconstructed using a general linear model, assuming asignificance level of 5%, corrected for multiple comparisonsusing the false discovery rate.

Summary of the Recon-all of the FreeSurfersoftware

N Step 1:

N Motion correction

N Intensity normalization

N Talairach transformation: Transformation from theoriginal volume to the MNI305 atlas

N Removal of the skull

N Step 2

N Topological normalization

N Topological correction

N Gaussian atlas classification

N Subcortical segmentation

N White matter segmentation

N Surface smoothing

N Surface inflation

N Cortical parcellation

N Step 3

N Spherical registration

N Cortical spherical parcellation

N Parcellation statistics: Summary of cortical parcella-tion statistics for each structure, including: 1. structurename; 2. number of vertices; 3. total surface area(mm2); 4. total gray matter volume (mm3); 5. averagecortical thickness (mm); 6. standard error of corticalthickness (mm); 7. integrated rectified mean curvature;8. integrated rectified Gaussian curvature; 9. foldingindex; and 10. intrinsic curvature index.


35

Supplementary Table 1 - Volumes [mm3] of different brain structures measured with an automated volumetric method(FreeSurfer).

Brain structure

volume (mm3)

Left hemisphere:

mean (SD)

Differences between

groups (t; p-value)

Right hemisphere:

mean (SD)

Differences between

groups (t; p-value)

Caudal anterior cingulate Control 1685.23 (434.84) 20.087; 0.931 2056.09 (506.72) 1.513; 0.138

Patient 1697.74 (483.08) 1832.26 (428.73)

Caudal middle frontal Control 5849.32 (980.91) 2.902; 0.006* 5346.50 (1132.18) 2.207; 0.033*

Patient 4940.79 (1021.33) 4701.26 (626.98)

Isthmus cingulate Control 2509.59 (484.52) 2.428; 0.020* 2379.59 (377.58) 2.631; 0.012*

Patient 2181.74 (358.88) 2083.11 (337.96)

Lateral orbitofrontal Control 7392.73 (883.47) 1.797; 0.080 7416.23 (855.54) 0.832; 0.411

Patient 6934.47 (724.62) 7192.89 (859.13)

Medial orbitofrontal Control 5382.05 (898.86) 1.527; 0.135 4976.59 (596.86) 1.472; 0.149

Patient 4983.89 (747.20) 4669.37 (739.16)

Paracentral Control 2938.86 (593.83) 1.941; 0.059 3392.91 (579.42) 1.523; 0.136

Patient 2609.79 (472.53) 3139.37 (469.71)

Pars opercularis Control 4435.59 (851.00) 2.521; 0.016* 3542.05 (717.96) 1.844; 0.073

Patient 3853.21 (577.81) 3196.21 (419.41)

Pars orbitalis Control 2007.95 (373.62) 1.983; 0.054 2612.77 (373.91) 2.893; 0.006*

Patient 1822.16 (175.96) 2300.37 (307.44)

Pars triangularis Control 3307.91 (581.73) 2.088; 0.043* 3917.32 (641.66) 1.701; 0.097

Patient 2966.84 (441.38) 3592.79 (568.64)

Rostral anterior cingulate Control 2720.32 (470.85) 1.432; 0.160 2222.45 (465.68) 1.498; 0.142

Patient 2513.68 (448.79) 2042.42 (257.33)

Rostral middle frontal Control 14221.68 (1798.14) 2.834; 0.007* 15501.50 (2029.12) 2.794; 0.008*

Patient 12770.32 (1422.34) 13800.11 (1840.23)

Superior frontal Control 19761.64 (2997.74) 2.791; 0.008* 19168.27 (2624.50) 3.779; 0.001*

Patient 17521.11 (1936.71) 16500.89 (1722.51)

Frontal pole Control 713.41 (181.72) 3.527; 0.001* 952.59 (178.94) 5.105; 0.000*

Patient 542.89 (114.51) 686.16 (151.06)

Superior temporal Control 11017.32 (1678.8) 1.874; 0.68 10467.68 (1396.09) 1.244; 0.221

Patient 10161.68 (1146.63) 9937.89 (1326.80)

Middle temporal Control 9677.50 (1394.84) 2.031; 049* 10797.59 (1296.25) 4.130; 0.000*

Patient 8686.00 (1729.73) 9025.37 (1451.89)

Inferior temporal Control 10044.91 (1718.88) 1.843; 0.73 10144.77 (1779.39) 1.631; 0.111

Patient 9104.84 (1515.960 9217.16 (1846.91)

Para- hippocampal Control 1984.68 (278.07) 2.041; 0.48 1886.32 (351.04) 1.441; 0.261

Patient 1804.53 (285.11) 1763.37 (335.98)

Postcentral Control 8457.32 (1209.36) 0.479; 0.634 8230.00 (1060.68) 1.294; 0.203

Patient 8246.05 (1608.57) 7789.89 (1115.48)

Precuneus Control 8381.86 (872.34) 3.710; 0.001* 8882.95 (1015.76) 3.900; 0.000*

Patient 7256.16 (1070.60) 7612.00 (1068.80)

Superior parietal Control 11734.91 (1091.82) 3.382; 0.002* 11404.82 (1242.52) 3.439; 0.001*

Patient 10278.68 (1644.83) 9844.47 (1657.50)

Inferior parietal Control 11814.50 (2018.33) 2.267; 0.029* 137299.55 (20629.7) 2.607; 0.013*

Patient 10474.37 (1722.94) 121180.53 (18645.8)

Supramarginal Control 9614.05 (1476.46) 2.575; 0.014* 9000.36 (1232.09) 2.507; 0.016*

Patient 8496.47 (1272.31) 8084.63 (1084.58)

Fusiform Control 9403.27 (1293.04) 3.404; 0.002* 87853.64 (13171.89) 1.750; 0.088

Patient 8161.11 (995.04) 81200.00 (10807.03)

Intracranial Control 1520000.31 (167.94) 0.354; 0.726

Patient 1501000.57 (170.88)

*Statistically significant difference.


CLINICS 2014;69(1):28-37

36

Supplementary Table 2 - Differences in brain structures,lesion extension parameters, and locations of corticalthickness between elderly control subjects andAlzheimer’s disease patients.

Brain structure Size (mm2) Talairach Coordinates

Left hemisphere

Caudal middle frontal 65.72 238.1 19.9 29.7


Superior frontal 1076.06 29.6 20.1 59.6

Pars opercularis 96.65 248.4 22.1 18.2

Pars triangularis 491.21 246.9 25.9 5.7

Precentral 11.08 247.7 0.2 8.8

Isthmus cingulate 232.86 215.8 249.0 0.9

Posterior cingulate 164.68 24.1 212.1 37.7

Lateral orbitofrontal 77.61 233.6 24.3 219.0

Precuneus 794.88 24.8 258.3 13.6

Precuneus 364.24 29.2 250.6 65.3

Superior parietal 42.99 226.7 253.4 40.6

Inferior parietal 499.03 236.6 270.8 45.8

Superior temporal 941.20 247.6 210.6 211.5


Middle temporal 35.31 257.8 258.6 0.2

Lateral occipital 58.40 234.1 282.0 8.4

Cuneus 114.62 24.5 283.2 17.4

Fusiform 2617.10 228.9 245.6 219.0

Insula 13.64 230.9 229.4 15.3

Right hemisphere


Parsopercularis 300.05 45.7 14.4 21.0

Precentral 541.95 15.3 226.8 59.1

Medial orbitofrontal 20.53 7.0 19.4 211.9

Precentral 494.04 45.5 28.5 37.7

Precentral 16.44 30.5 214.5 59.2






Postcentral 112.83 49.8 221.5 54.3

Supramarginal 823.56 61.6 239.5 27.0



Entorhinal 4813.42 23.7 27.0 232.7



Lingual 538.99 20.2 273.9 27.0

Fusiform 21.84 41.2 247.3 218.3


37

Sex differences in 24-hour ultra-marathon perfor-mance - A retrospective data analysis from 1977 to2012Laura Peter,I Christoph Alexander Rust,I Beat Knechtle,I,II Thomas Rosemann,I Romuald LepersIII

I University of Zurich, Institute of General Practice and Health Services Research, Zurich, Switzerland. II Gesundheitszentrum St. Gallen, St. Gallen,

Switzerland. III University of Burgundy, Faculty of Sport Sciences, Dijon, France.

OBJECTIVES: This study examined the changes in running performance and the sex differences between womenand men in 24-hour ultra-marathons held worldwide from 1977 to 2012.

METHOD: Changes in running speed and ages of the fastest 24-hour ultra-marathoners were determined usingsingle- and multi-level regression analyses.

RESULTS: From 1977 to 2012, the sex differences in 24-hour ultra-marathon performance were 4.6¡0.5% for allwomen and men, 13.3% for the annual fastest finishers, 12.9¡0.8% for the top 10 and 12.2¡0.4% for the top100 finishers. Over time, the sex differences decreased for the annual fastest finishers to 17%, for the annual 10fastest finishers to 11.3¡2.2% and for the annual 100 fastest finishers to 14.2¡1.8%. For the annual fastestmen, the age of peak running speed increased from 23 years (1977) to 53 years (2012). For the annual 10 and100 fastest men, the ages of peak running speed were unchanged at 40.9¡2.5 and 44.4¡1.1 years, respectively.For women, the ages of the annual fastest, the annual 10 fastest and the annual 100 fastest remainedunchanged at 43.0¡6.1, 43.2¡2.6 and 43.8¡0.8 years, respectively.

CONCLUSION: The gap between the annual top, annual top 10 and annual top 100 female and male 24-hourultra-marathoners decreased over the last 35 years; however, it seems unlikely that women will outrun men in24-hour ultra-marathons in the near future. The fastest 24-hour ultra-marathoners worldwide achieved theirpeak performance at the age of master athletes (.35 years).

KEYWORDS: Ultra-endurance; Running; Athlete.

Peter L, Rust CA, Knechtle B, Rosemann T, Lepers R. Sex differences in 24-hour ultra-marathon performance - A retrospective data analysis from1977 to 2012. Clinics. 2014;69(1):38-46.

Received for publication on April 19, 2013; First review completed on June 23, 2013; Accepted for publication on July 16, 2013


Tel.: 41 0 71 226 82 82

& INTRODUCTION

Over the last several decades, the sex difference inrunning performance has been a matter of particular interest(1-5). When women’s running performance improvedduring the 1980s (6), the discussion arose whether it wouldbe possible for women to outrun men (1,3-5,7-9). In 1992, acontroversial paper published in ‘‘Nature’’ reported that themean running speed for women and men would intersect inthe future, first at the marathon distance in approximately1998 (6). However, marathon-running performance reacheda plateau for women since the mid 1980s, with a sexdifference of approximately 12% (10).

Several studies addressed the question whether the sexdifference in running performance would decrease withincreasing race distance (1,2,4,5,9,11). It was assumed thatwomen might be able to achieve a performance equivalentto men in ultra-marathon running (1,5,9). However, analysisof the world’s best running times at distances from 100meters (m) to 200 kilometers (km) demonstrated a 7-19% sexdifference in running performance, with an increase in sexdifference for distances longer than the marathon (4).Similar findings have been reported for long-distancetriathlons. Knechtle et al. (12) investigated the world’sfastest performances in ultra-triathlons held between 1985and 2009 and demonstrated that the sex difference inperformance increased with increasing race distance. Menwere approximately 19% faster than women in Double (i.e.,7.6 km swim, 360 km cycle and 84.4 km run) and TripleIronman ultra-triathlons (i.e., 11.4 km swim, 540 km cycleand 126.6 km run) and approximately 30% faster in DecaIron ultra-triathlons (i.e., 38 km swim, 1,800 km cycle and422 km run). In Ironman triathlons, the sex difference inswimming was stable over the last 20 years; however, the



DOI: 10.6061/clinics/2014(01)06

CLINICAL SCIENCE

38

difference increased slightly in cycling and decreased inrunning (13). The sex difference for swimming performancewas smaller than for running and cycling. Thus, the sexdifference in the Ironman triathlon differed between thedisciplines (14).

Sex differences in endurance performances appear to bebiological in origin (2). The major reasons for the observedperformance differences include the larger body size ofmen (15) and the lower body fat percentage compared towomen (15,16,17). Men have a greater maximal oxygenuptake (VO2 max) compared to women (10,15,16), whichleads to a relevant advantage in aerobic performance(2,16,18). Men also have higher hemoglobin and hemato-crit counts relative to women (15). Another advantage formen is their greater training motivation (19). Women alsolack experience in long-distance running competitions(2,4), which might be a disadvantage relative to men.However, there are also reasons why women couldoutperform men at longer distances. Women have greaterfat stores (15-17) and tend to oxidize fat at a higher ratethan men (20,21). Another assumption is that women seemto have less damage to skeletal muscle cells relative to menduring and/or following prolonged exercises (22), whichmay also be an advantage at ultra-endurance distances.Women have lower body mass (23) and likely need lessenergy for shifting their body mass from one place toanother.

The sex difference in ultra-endurance running (eventslasting.6 hours) (24) has been investigated in 100-mileultra-marathons held in North America (1,5,8,9,11) andfor the world’s best running times for distances up to200 km (4). Recent studies investigating the performancein ultra-marathon running have mainly focused on asingle race or a series of races rather than on a series ofraces held worldwide. The aim of this study was toinvestigate the changes in sex differences in ultra-endurance distances on the basis of the complete, world-wide 24-hour ultra-marathons held between 1977 and2012. A 24-hour ultra-marathon is a time-limited race.Competitors have to cover the largest possible distancewithin 24 hours with performances from approximately146 km (25) to 300 km. The world record for this typeof marathon is 303.5 km (www.ultra-marathon.org). Wehypothesized that the sex difference at the 24-hour ultra-marathon distance has been decreasing over the pastseveral decades.


All procedures used in this cross-sectional data analysismet the ethical standards of the Swiss Academy of MedicalSciences (www.samw.ch/en/Ethics/Guidelines/Currently-valid-guidelines.html) and were approved by the Institu-tional Review Board of Kanton St. Gallen, Switzerland(decision letter of June 1, 2010) with a waiver of therequirement for informed consent of the participants giventhe fact that the study involved the analysis of publiclyavailable data.

Data sampling and data analysisAll competitors who ever participated in a 24-hour ultra-

marathon between 1977 and 2012 were analyzed regardingthe associations between age, sex and race performance(Table 1). The data set for this study was obtained from the

Table 1 - Number of total finishers for each country,sorted by the percentage of female finishers.

Country Women Men Overall

Percentage of

women

New Zealand 31 26 57 54.4

Romania 2 2 4 50

Ukraine 112 273 385 29.1

United States 788 1984 2772 28.4

Canada 57 151 208 27.4

Latvia 21 58 79 26.6

Great Britain 164 455 619 26.5

Croatia 5 14 19 26.3

Japan 190 543 733 25.9

Hungary 76 223 299 25.4

Serbia 41 121 162 25.3

Turkey 2 6 8 25

Macedonia 1 3 4 25

Suisse 158 513 671 23.6

Estonia 9 30 39 23.1

Slovenia 5 17 22 22.7

Denmark 55 191 246 22.4

Finland 58 207 265 21.9

South Korea 13 47 60 21.7

Germany 1563 5722 7285 21.5

Luxembourg 3 11 14 21.4

Uruguay 10 37 47 21.3

Taiwan 59 223 282 20.9

Italy 200 767 967 20.7

Sweden 79 309 388 20.4

Kazakhstan 4 16 20 20

Mexico 1 4 5 20

Russia 448 1829 2277 19.7

Brazil 178 739 917 19.4

Czech Republic 111 461 572 19.4

Australia 80 378 458 17.5

Argentina 30 145 175 17.1

Poland 56 310 366 15.3

France 1403 8099 9502 14.8

Austria 293 1709 2002 14.6

Spain 44 316 360 12.2

Belarus 13 96 109 11.9

Netherlands 55 413 468 11.8

Ireland 5 54 59 8.5

South Africa 3 38 41 7.3

Belgium 21 404 425 4.9

Greece 9 246 255 3.5

Slovakia 5 152 157 3.2

Portugal 1 30 31 3.2

Norway 1 78 79 1.3

Lithuania 27 27 0

Bulgaria 21 21 0

Algeria 20 20 0

India 12 12 0

Cyprus 8 8 0

Mali 8 8 0

Moldova 7 7 0

Montenegro 7 7 0

Monaco 5 5 0

Iraq 3 3 0

Morocco 3 3 0

Somalia 3 3 0

Uzbekistan 3 3 0

Bosnia and Herzegovina 1 1 0

Chile 1 1 0

Egypt 1 1 0

Iran 1 1 0

Iceland 1 1 0

Malaysia 1 1 0

Senegal 1 1 0

Singapore 1 1 0

Tunisia 1 1 0

6,463 27,586 34,049 19

CLINICS 2014;69(1):38-46 Sex differences in 24-hour ultra-marathon runningPeter L et al.

39

race website ‘‘Deutsche Ultramarathon-Vereinigung’’ (www.ultra-marathon.org). To facilitate reading and to increasethe comparability of similar analyses of races of variousdistances, all race distances were transformed to runningspeed (km N h-1) before analysis. Running speed wascalculated using the equation [running speed in km N h-1]= [race distance achieved in km]/24 hours (h). Todetermine changes in peak performance in running andthe age of peak running speed, running speed and the ageof the annual top, annual top 10 and annual top 100women and men were analyzed. The sex difference inrunning performance was calculated using the equation([km N h-1 in women] - [km N h-1 in men])/[km N h-1 inmen] 6 100, where the sex difference was calculated forevery pairing of equally placed competitors (e.g., betweenmale and female 1st place, between male and female 2nd

place, etc.) before calculating the mean values andstandard deviations of all the pairings. To facilitatereading, all sex differences were transformed to absolutevalues prior to analysis. The performance density for bothwomen and men was calculated using the equation([running speed of the 10th place or of the 100th placefinisher] - [running speed of the 1st place finisher])/[running speed of the 1st place finisher] 6 100. Theperformance density expresses the differences in runningtime between the 1st place and the 10th place finishers andbetween the 1st place and the 100th place finishers aspercentages of the winner’s times, which indicates thedensities of both the 10 and 100 fastest competitors in therace.

Statistical analysesThe data in the text and figures are reported as

means¡standard deviations (SD). To increase the relia-bility of the data analyses, each set of data was tested fornormal distribution and homogeneity of variances priorto statistical analyses. Normal distribution was testedusing a D’Agostino and Pearson omnibus normality test,and the homogeneity of variances was tested using aLevene’s test. Trends in participation were analyzedusing regression analysis with the ‘‘straight line’’ and‘‘exponential growth equation’’ models, whereas for eachset of data (e.g., each age group), both models werecompared using Akaike’s Information Criteria (AIC) todecide which model resulted in the highest probability ofcorrectness.

Single- and multi-level regression analyses were used toinvestigate changes in performance and the ages of thefinishers. A hierarchical regression model was used to avoidthe impact of a cluster effect on the results of the analysis ofoverall performance and sex difference if one competitorfinished more than once in the annual top, annual top 10or annual top 100. Furthermore, performance regressionanalyses were corrected for competitors’ ages to preventmisinterpretation of the ‘‘age effect’’ as a ‘‘time effect.’’ Toanalyze differences between two groups, the Student’s t-testwas used for cases of normally distributed data, and theMann-Whitney test was used for non-normally distributeddata. Statistical analyses were performed using IBM SPSSStatistics software (Version 21, IBM SPSS, Chicago, IL, USA)and GraphPad Prism (Version 6.01, GraphPad Software, LaJolla, CA, USA). Significance was accepted at p,0.05 (two-sided for t-tests).

& RESULTS

Complete data containing both sex and age were availablefor a total of 34,049 racers, of which 6,463 were women(19%) and 27,586 were men (81%).

Participation trendsThe number of races increased exponentially from one race

in 1977 to 81 races in 2012. The numbers of annual overallfinishers and both annual female and male finishers alsoincreased exponentially over the time period studied. In 1977,one man finished a 24-hour ultra-marathon. In 2012, 6,463women and 27,586 men finished 24-hour ultra-marathons.Women accounted for 19% of the overall field. Table 1displays the number of male and female finishers by countryof origin. New Zealand was the only country where theproportion of female finishers was greater than male finishers.Apart from South Africa, there were no female finishers fromAfrican countries. The proportion of women trended higherin countries with larger numbers of total finishers.

Sex difference in running speedFigure 1A presents the running speeds for the fastest

women and men ever finishing 24-hour ultra-marathons.The fastest woman ever ran at 10.64 km/h, and the fastestman ever ran at 12.27 km/h, which is a sex difference of13.3%. Considering the 10 fastest women and the men ever,the women ran at 10.25¡0.21 km/h and the men at11.77¡0.26 km/h, with a running speed sex difference of12.9¡0.8%. For the 100 fastest finishers ever, the women ranat 9.68¡0.27 km/h and the men at 11.04¡0.32 km/h, witha sex difference of 12.2¡0.4%. When all female andmale finishers were considered, the women ran at 5.59¡1.92 km/h and the men at 5.86¡1.92 km/h, with a sexdifference of 4.6¡0.5%. The sex difference between thefastest 100 women and men was less than the sex differencebetween the fastest 10 women and men (Figure 1B).Running speed for the annual fastest men increased from8.71 km/h (1977) to 12.27 km/h (2012); for women, theannual fastest speed increased from 9.03 km/h (1982) to10.18 km/h (2012) (Figure 2A). This increase remained truewhen controlling for multiple finishes (Table 2). The sexdifference in running speed decreased over time to 17% in2012 (Figure 2A) and was also true when controlling formultiple finishes (Table 2). For the annual 10 fastestfinishers (Figure 2B), the men’s running speed increasedfrom 7.86¡0.81 km/h (1982) to 11.07¡0.51 km/h (2012),and the women’s running speed increased from6.23¡2.34 km/h (1986) to 9.81¡0.25 km/h (2012); theseresults remained true when controlling for multiple finishes(Table 2). The sex difference in running speed decreasedfrom 33.2¡21.8% (1986) to 11.3¡2.2% (2012) and remainedtrue when controlling for multiple finishes (Table 2).Considering the annual 100 fastest finishers (Figure 2C),the men’s running speed increased from 7.25¡1.69 km/h(1989) to 9.95¡0.57 km/h (2012), and the women’s runningspeed increased from 6.24¡1.65 km/h (1999) to8.54¡0.64 km/h (2012); these results were also true whencontrolling for multiple finishes (Table 2). The sex differencedecreased from 30.7¡12.8% (1999) to 14.2¡1.8% (2012), allresults remained true when controlling for multiple finishes(Table 2). The performance densities from the 10th place tothe fastest competitor increased for men from -27% (1982) to-13.3% (2012) and increased for women from -78.5% (1986)

Sex differences in 24-hour ultra-marathon runningPeter L et al.

CLINICS 2014;69(1):38-46

40

to -6.4% (2012) (Figure 3A). The performance density fromthe 100th finisher to the fastest finishers increased for menfrom -67% (1989) to -24.4% (2012) and increased for womenfrom -76.2% (1999) to -24.5% (2012) (Figure 3B).

The age of peak running speedThe highest number of finishes for both men and women

were from competitors aged 45-49. The age of the annual

fastest men increased from 23 years (1977) to 53 years (2012)(Figure 4A), including when controlling for repeated finishes(Table 2). The age of the annual fastest women remainedunchanged at 43.0¡6.1 years (Figure 4A), including whencontrolling for repeated finishes (Table 2). For the annual 10fastest finishers, the mean ages remained unchanged at43.2¡2.6 years for women and 40.9¡2.5 years for men(Figure 4B), including when controlling for repeated finishes

Figure 1 - Running speeds (Panel A) and sex differences (Panel B) for the fastest competitors ever.


41

(Table 2). When considering the annual 100 fastest finishers,the mean ages remained unchanged at 43.8¡0.8 years forwomen and 44.4¡1.1 years for men (Figure 4C), includingwhen controlling for repeated finishes (Table 2).

& DISCUSSION

The main findings of this study were first, that there wereincreases in the numbers of both female and male 24-hour

ultra-marathoners from 1977-2012. Second, a decrease in thesex difference over this time was observed for the annualfastest, the annual top 10 and the annual top 100 24-hourultra-marathoners. Third, the fastest 24-hour ultra-marath-oners worldwide were master athletes (.35 years).

Increasing numbers of 24-hour running finishersA main finding regarding participation was that the

number of finishers increased exponentially for bothwomen and men over the studied time period. NewZealand was the only country where the proportion offemale finishers was higher than the proportion of malefinishers. Apart from South Africa, there were no femalefinishers from African countries. For all countries, thereappeared to be a trend toward a higher proportion of femalefinishers in countries with larger numbers of total finishers.These results may suggest that men were forerunners in theperformance of new emerging sports, while women beganlater. Women started participating later in sporting compe-titions such as the marathon (15). In 1972, sex discriminationwas prohibited in the USA (US Department of Labor, 1972),and in 1984, the first Olympic marathon for women tookplace (15). Today, the possibility for women to pursue theirinterests in sports is higher than in the past. Nowadays, it ispossible for female ultra-marathoners to look after theirchildren, work a full time job and train for ultra-marathons(26).

Based on the analysis of single ultra-marathons within asingle country, the number of female ultra-marathonersincreased during the same period (1998 to 2011), whereasthe number of male ultra-marathoners remained unchangedin a mountain ultra-marathon such as the 78-km ‘‘SwissAlpine’’ held in Davos, Switzerland (27). The number ofmale participants was also reported to be stable in the 100-mile ultra-marathons in the USA (7,11). Hoffman andWegelin (7), Hoffman (11) and Eichenberger et al. (27)performed their analyses in Western countries; therefore,nationality may also have an influence on the number ofcompetitors.

Sex difference in performance for 24-hourultra-marathoners

The running speeds of the annual fastest, the annual top10 and the annual top 100 women increased over thestudied time period; as hypothesized, the sex differencecorrespondingly decreased during this same timeframe.Women lack experience in participating in enduranceperformance competitions (4). Over the last several decades,women have had more time to improve their experience inultra-endurance running competitions; therefore, they haveenhanced their ultra-marathon performances. Thus, theincrease in the number of female competitors could be apossible explanation for the decrease in the sex differenceamong 24-hour ultra-marathoners.

We found differences of approximately 13% between boththe annual winners and the annual top 10 finishers, and 12%for the annual top 100 finishers. This sex difference wassimilar to the sex difference observed in terms of marathondistance, which has remained stable since the mid 1980s(10). However, this sex difference was lower than the 20%sex difference Hoffman (8) reported for the winners of100-mile ultra-marathons in North America. One explana-tion for this difference could be that the best ultra-marathoners opted to participate in the more famous

Figure 2 - Changes in running speed and sex differences inrunning speed for the annual fastest (Panel A), the annual 10fastest (Panel B) and the annual 100 fastest (Panel C) competitorsover time.


CLINICS 2014;69(1):38-46

42

100-mile runs than in the 24-hour ultra-marathon, or it couldbe because Hoffman (5,8) analyzed races only in NorthAmerica. Coast et al. (4) described a 14% difference for the100-mile distance and a 19.1% difference for the 200-kmdistance. In a 24-hour ultra-marathon, the mean distance isapproximately 146 km (25); however, this distance might beconsiderably longer for the fastest women and men. Thus, thesex difference in the present 24-hour ultra-marathoners wasalso less than the difference suggested by Coast et al. (4).

The sex difference between the 100 fastest women andmen was less than the sex difference between the fastest 10women and men. The sex difference of all female and malefinishers was only 4.6%, whereas the sex differences of theannual top 10 and the annual top 100 finishers were bothgreater than 12%. Indeed, there was a higher sex differenceamong elite 24-hour ultra-marathon finishers compared tothe average finishers. These results correspond to the trendreported by Hoffman (8) for 100-mile runs. He reported sexdifferences of 20% for the fastest finish time and only 3% forthe average finish time (8).

The average time differences between the winner and the10th place athlete decreased for both women and men. In

2012, this time difference was smaller for women than men.The density for the top 10 finishers was higher for womenthan for men. The densities for the top 100 men (24.4%) andtop 100 women (24.5%) were approximately the same in2012. Lepers et al. reported the same trend for Ironmantriathletes: the performance densities for elite women andmen seemed to become similar (13).

The age of peak running performance in 24-hour ultra-marathons. An interesting finding regarding the age of the24-hour ultra-marathoners was that the mean ages wereunchanged over the entire study period - approximately 41years for the annual top 10 men and approximately 44 yearsfor the annual top 100 men. For women, mean ages ofthe top 10 and top 100 finishers remained unchanged atapproximately 44 years.

The ages of the fastest women and men increased in singleultra-marathons held within a single country, such the‘‘Swiss Alpine’’ from 1998 to 2011 in Switzerland (27) andthe 100-mile runs from 1978 to 2008 in the USA (8). In the 100-mile runs in North America, the fastest times were achievedby competitors in the 30-39 age group for men and the 40-49age group for women (8). The fastest running times in the

Table 2 - Multi-level regression analyses for changes in running speed over time, sex differences in running and ages offinishers (Model 1), with corrections for multiple finishes (Model 2) and ages of athletes with multiple finishes (Model3).

Model ß SE (ß) Stand. ß T p-value

Running speed in men

Annual fastest 1 0.072 0.012 0.717 5.911 ,0.0001

2 0.072 0.012 0.717 5.911 ,0.0001

3 0.075 0.013 0.747 5.891 ,0.0001

Annual 10 fastest 1 0.066 0.005 0.630 14.245 ,0.0001

2 0.066 0.005 0.630 14.245 ,0.0001

3 0.067 0.005 0.633 14.344 ,0.0001

Annual 100 fastest 1 0.065 0.002 0.476 26.510 ,0.0001

2 0.065 0.002 0.476 26.510 ,0.0001

3 0.066 0.002 0.483 27.009 ,0.0001

Running speed in women

Annual fastest 1 0.099 0.026 0.579 3.826 0.001

2 0.099 0.026 0.579 3.826 0.001

3 0.099 0.027 0.578 3.739 0.001

Annual 10 fastest 1 0.102 0.007 0.678 15.101 ,0.0001

2 0.102 0.007 0.678 15.101 ,0.0001

3 0.103 0.007 0.685 15.225 ,0.0001

Annual 100 fastest 1 0.150 0.007 0.516 22.524 ,0.0001

2 0.150 0.007 0.516 22.524 ,0.0001

3 0.150 0.007 0.516 22.497 ,0.0001

Sex difference in running speed

Annual fastest 1 -0.600 0.256 -0.399 -2.343 0.026

2 -0.600 0.256 -0.399 -2.343 0.026

Annual 10 fastest 1 -0.759 0.056 -0.639 -13.612 ,0.0001

2 -0.759 0.056 -0.639 -13.612 ,0.0001

Annual 100 fastest 1 -1.139 0.040 -0.602 -28.174 ,0.0001

2 -1.139 0.040 -0.602 -28.174 ,0.0001

Age in men

Annual fastest 1 0.071 0.023 0.063 3.077 0.002

2 0.071 0.023 0.063 3.077 0.002

Annual 10 fastest 1 0.023 0.045 0.030 0.518 0.605

2 0.023 0.045 0.030 0.518 0.605

Annual 100 fastest 1 0.193 0.118 0.275 1.642 0.110

2 0.193 0.118 0.275 1.642 0.110

Age in women

Annual fastest 1 -0.058 0.124 -0.086 -0.465 0.645

2 -0.058 0.124 -0.086 -0.465 0.645

Annual 10 fastest 1 -0.087 0.052 -0.103 -1.693 0.092

2 -0.087 0.052 -0.103 -1.693 0.092

Annual 100 fastest 1 0.048 0.050 0.026 0.959 0.338

2 0.048 0.050 0.026 0.959 0.338


43

‘‘Swiss Alpine’’ were achieved by competitors in the 30-34age group for women and the 40-44 age group for men (27).

Reaburn and Dascombe (28) described a curvilineardecline in endurance performances beginning at the age of35 years, and they defined endurance athletes older than

35 years as being ‘‘master athletes.’’ Master athletes aresystematically trained for special forms of sports designedfor older adults. However, in the 24-hour ultra-marathons,the male and female world elite athletes are older than 40years. Additionally, in the ‘‘Swiss Alpine’’ ultra-marathon

Figure 3 - Differences in running speed between the 1st and the 10th place finishers (Panel A) and between the 1st and the 100th placefinishers (Panel B) expressed as percentages of the winning times for both women and men.


CLINICS 2014;69(1):38-46

44

(27) and in the 100-mile runs (8), the fastest finishers weremaster athletes, according to Raeburn’s definition. Ourresults are in agreement with Reaburn’s definition of masterathletes. In 24-hour ultra-marathons, the male world eliteathletes were, on average, 41 years old; consequently,master athletes have to be older. Therefore, our results

suggest a change for the age definition of master athletes interms of ultra-endurance running.

Limitations and implications for future researchThe strength of this study is that the results are based on

an extensive dataset with a large sample size. This datasetprovides a good overview of the sex differences among 24-hour ultra-marathons worldwide. However, the national-ities of the participants are unknown, and we do nothave data concerning athlete physiology, psychology, bodycomposition, training, previous experience, weather, raceroute, etc. A recent study identified that one’s longesttraining run and the best marathon time were importantpredictor variables for successful 24-hour ultra-marathonperformance. Anthropometric characteristics, such as bodyfat or skinfold thickness, did not influence performance (25).For the 100-mile ultra-marathons, Hoffman and Fogard (29)investigated factors related to successful finishing, such asbody mass index, training volume, ambient temperatures atthe events, medication use, etc. Krouse et al. (26) investi-gated the influence of motivation, goal orientation andtraining for female ultra-runners and reported that generalhealth orientation and psychological coping were thestrongest motivational factors for female ultra-marathoners.

To summarize, there were constant increases in thenumbers of female and male 24-hour ultra-marathonersduring the 1977-2012 time period. The running speeds of theannual fastest women and men increased, and the sexdifferences in running speed decreased over these years.The ages of peak running speed were unchanged for theannual top 10 and annual top 100 women and men.Although women’s running speeds improved, the sexdifferences in performance decreased over time. Therefore,it seems unlikely that women will be able to outrun men in24-hour ultra-marathons in the near future. The fastest24-hour ultra-marathoners worldwide achieved their peakperformance at the age of master athletes (.35 years).Future studies are needed to investigate longer distance-and time-limited races. Most likely, the age of peak runningspeed increases in proportion to the length of the race.


Peter L collected the data and drafted the manuscript. Rust CA performed

the statistical analyses. Knechtle B designed the study and helped drafting

the manuscript. Rosemann T critically revised the manuscript for

important intellectual content. Lepers R assisted in the statistical analyses

and helped drafting the manuscript.

& REFERENCES

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2. Cheuvront SN, Carter R, Deruisseau KC, Moffatt RJ. Running per-formance differences between men and women: an update. SportsMed. 2005;35(12):1017-24, http://dx.doi.org/10.2165/00007256-200535120-00002.

3. Dyer KF. Catching up the men: Women in sport. Junction Books; 1982.4. Coast JR, Blevins JS, Wilson BA. Do gender differences in running

performance disappear with distance? Can J Appl Physiol. 2004;29(2):139-45.

5. Hoffman MD. Ultramarathon trail running comparison of performance-matched men and women. Med Sci Sports Exerc. 2008;40(9):1681-6,http://dx.doi.org/10.1249/MSS.0b013e318177eb63.

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7. Hoffman MD, Wegelin JA. The Western States 100-Mile Endurance Run:participation and performance trends. Med Sci Sports Exerc.2009;41(12):2191-8, http://dx.doi.org/10.1249/MSS.0b013e3181a8d553.

Figure 4 - Changes in the ages of peak running speed for theannual fastest (Panel A), the annual 10 fastest (Panel B) and theannual 100 fastest (Panel C) competitors over time.


45

8. Hoffman MD. Performance trends in 161-km ultramarathons. Int J SportsMed. 2010;31(1):31-7.

9. Speechly DP, Taylor SR, Rogers GG. Differences in ultraenduranceexercise in performance-matched male and female runners. Med SciSports Exerc. 1996;28(3):359-65.

10. Sparling PB, O’Donnell EM, Snow TK. The gender difference in distancerunning performance has plateaued: an analysis of world rankings from1980 to 1996. Med Sci Sports Exerc. 1998;30(12):1725-9, http://dx.doi.org/10.1097/00005768-199812000-00011.

11. Hoffman MD, Ong JC, Wang G. Historical analysis of participation in161 km ultramarathons in North America. Int J Hist Sport. 2010;27(11):1877-91.

12. Knechtle B, Knechtle P, Lepers R. Participation and performance trendsin ultra-triathlons from 1985 to 2009. Scand J Med Sci Sports. 2011;21(6):e82-90, http://dx.doi.org/10.1111/j.1600-0838.2010.01160.x.

13. Lepers R. Analysis of Hawaii ironman performances in elite triathletesfrom 1981 to 2007. Med Sci Sports Exerc. 2008;40(10):1828-34, http://dx.doi.org/10.1249/MSS.0b013e31817e91a4.

14. Lepers R, Maffiuletti A. Age and gender interactions in ultraenduranceperformance: insight from the triathlon. Med Sci Sports Exerc. 2011;43(1):134-9, http://dx.doi.org/10.1249/MSS.0b013e3181e57997.

15. Lynch SL, Hoch AZ. The female runner: gender specifics. Clin SportsMed. 2010;29(3):477-98, http://dx.doi.org/10.1016/j.csm.2010.03.003.

16. Drinkwater BL. Women and exercise: physiological aspects. Exerc Sport SciRev. 1984;12:21-51, http://dx.doi.org/10.1249/00003677-198401000-00004.

17. Durnin JV, Womersley J. Body fat assessed from total body densityand its estimation from skinfold thickness: measurements on 481men and women aged from 16 to 72 years. Br J Nutr. 1974;32(1):77-97.

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20. Hellstrom L, Blaak E, Hagstrom-Toft E. Gender differences in adrenergicregulation of lipid metabolism during exercise. Int J Sports Med. 1996;17(6):439-47.

21. Tarnopolsky MA. Gender differences in substrate metabolism duringendurance exercise. Can J Appl Physiol. 2000;25(4):312-27.

22. Tiidus PM. Estrogen and gender effects on muscle damage, inflamma-tion and oxidation stress. Can J Appl Physiol. 2000;25(4):274-87.

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24. Zaryski C, Smith DJ. Training principles and issues for ultra-enduranceathletes. Curr Sports Med Rep. 2005;4(3):165-70, http://dx.doi.org/10.1097/01.CSMR.0000306201.49315.73.

25. Knechtle B, Knechtle P, Rosemann T, Lepers R. Personal best marathontime and longest training run, not anthropometry, predict performancein recreational 24-hour ultrarunners. J Strength Cond Res. 2011;25(8):2212-8, http://dx.doi.org/10.1519/JSC.0b013e3181f6b0c7.

26. Krouse RZ, Ransdell LB, Lucas SM, Pritchard ME. Motivation, goalorientation, coaching, and training habits of women ultrarunners.J Strength Cond Res. 2011;25(10):2835-42, http://dx.doi.org/10.1519/JSC.0b013e318204caa0.

27. Eichenberger E, Knechtle B, Rust CA, Rosemann T, Lepers R. Age andsex interactions in mountain ultramarathon running- the Swiss AlpineMarathon. Open Access J Sports Med. 2012;3:73-80.

28. Reaburn P, Dascombe B. Endurance performance in master athletes. EurRev Aging Phys Act. 2008;5(1):31-42, http://dx.doi.org/10.1007/s11556-008-0029-2.

29. Hoffman MD, Fogard K. Factors related to successful completion ofa 161-km ultramarathon. Int J Sports Physiol Perform. 2011; 6(1):25-37.


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Angiotensin II type 1 and 2 receptors and lymphaticvessels modulate lung remodeling and fibrosis insystemic sclerosis and idiopathic pulmonary fibrosisEdwin Roger Parra, Aline Domingos Pinto Ruppert, Vera Luiza Capelozzi

Faculdade de Medicina da Universidade de Sao Paulo, Department of Pathology, Laboratory of Histomorphometry and Pulmonary Genetics,

Sao Paulo/SP, Brazil.

OBJECTIVE: To validate the importance of the angiotensin II receptor isotypes and the lymphatic vessels insystemic sclerosis and idiopathic pulmonary fibrosis.

METHODS: We examined angiotensin II type 1 and 2 receptors and lymphatic vessels in the pulmonary tissuesobtained from open lung biopsies of 30 patients with systemic sclerosis and 28 patients with idiopathicpulmonary fibrosis. Their histologic patterns included cellular and fibrotic non-specific interstitial pneumoniafor systemic sclerosis and usual interstitial pneumonia for idiopathic pulmonary fibrosis. We usedimmunohistochemistry and histomorphometry to evaluate the number of cells in the alveolar septae and thevessels stained by these markers. Survival curves were also used.

RESULTS: We found a significantly increased percentage of septal and vessel cells immunostained for the angiotensintype 1 and 2 receptors in the systemic sclerosis and idiopathic pulmonary fibrosis patients compared with the controls.A similar percentage of angiotensin 2 receptor positive vessel cells was observed in fibrotic non-specific interstitialpneumonia and usual interstitial pneumonia. A significantly increased percentage of lymphatic vessels was present inthe usual interstitial pneumonia group compared with the non-specific interstitial pneumonia and control groups. ACox regression analysis showed a high risk of death for the patients with usual interstitial pneumonia and a highpercentage of vessel cells immunostained for the angiotensin 2 receptor in the lymphatic vessels.

CONCLUSION: We concluded that angiotensin II receptor expression in the lung parenchyma can potentially controlorgan remodeling and fibrosis, which suggests that strategies aimed at preventing high angiotensin 2 receptorexpression may be used as potential therapeutic target in patients with pulmonary systemic sclerosis and idiopathicpulmonary fibrosis.

KEYWORDS: Systemic Sclerosis; Idiopathic Pulmonary Fibrosis; Immunohistochemistry; Angiotensin; Survival.

Parra ER, Ruppert AD, Capelozzi VL. Angiotensin II type 1 and 2 receptors and lymphatic vessels modulate lung remodeling and fibrosis in systemicsclerosis and idiopathic pulmonary fibrosis. Clinics. 2014;69(1):47-54.



Tel.: 55 11 3066 7427

& INTRODUCTION

Diffuse parenchymal lung diseases (DPLD), also knownas interstitial lung diseases (ILD), encompass a heteroge-neous group of disorders, including idiopathic pulmonaryfibrosis (IPF) and systemic sclerosis (SSc). These disordersresult from the remodeling of the lung parenchyma byvarying inflammation and fibrosis patterns that frequentlyaffect the alveolar septae and vessels and their respectiveepithelial, endothelial and muscular linings (1). However,

patients with interstitial pneumonia associated with SSchave a better prognosis than patients with idiopathicinterstitial pneumonia (2-6) because of their increasedresponse to immunosuppressive therapies and the preva-lence of septal cellular thickening, which is associated withthe pattern of nonspecific interstitial pneumonia (NSIP)(7-12). The prognosis is not as good for IPF patients with apattern of usual interstitial pneumonia (UIP) (12). Thus,there is great interest in developing methods that canidentify the mechanisms of parenchymal and vascularremodeling. We must identify these pathogenic mechanismsto avoid destroying the lungs and to provide effectivetreatment.

Many studies have investigated the parenchymal andvascular remodeling markers in DPLD to identify themechanisms behind the disease progression and shortenedsurvival rates (13-17). Because epithelial and endothelialcells and fibroblasts represent an abundant and functionally



DOI: 10.6061/clinics/2014(01)07

CLINICAL SCIENCE

47

important group of cells in lung fibrosis (18), a group ofangiotensins that exert biological effects on these cell typeshave been described (19) and, therefore, may be targeted aspotentially useful modulators of organ remodeling andfibrosis (20-22). Of these angiotensins, angiotensin II (ANGII) has shown promise. ANG II is generated by the cleavageof angiotensin I by the angiotensin-converting enzyme(ACE). ANG II binds to and acts via 2 high-affinity receptorisotypes, the angiotensin II type-1 (AGTR-1) and type-2(AGTR-2) receptors, both of which belong to the7-transmembrane G protein–coupled receptor family(23,24). In some studies, AGTR-2 has been detected indifferent adult human cells and in fetal tissue, in which itseems to play a major role during embryonic development(25-27). Other studies have shown that AGTR-2 inhibits orcounteracts the AGTR-1 mediated effects by directlyinteracting with AGTR-1 (28), but there has been uncertaintyabout the signaling pathway activated by AGTR-2 (29-31).These studies have indicated that the ACE-ANGII-AGTRaxis contributes to the fibrotic response in the lung;however, the role that the AGTRs play in developing lungfibrosis in IPF and SSc remains unclear. To validate theimportance of the ACE-ANGII-AGTR axis, to explore thequantitative relationship between this factor and patientoutcomes and to examine the relationship between AGTRsand other parenchymal and vascular factors, we studiedthese markers in 30 SSc and 26 IPF patients.

& PATIENTS AND METHODS

Between January 2002 and July 2004, 30 consecutivepatients with SSc and ILD (identified using high-resolutioncomputed tomography [HRCT]) and 26 IPF patients under-went open lung biopsies at the Clinical Hospital, Universityof Sao Paulo Medical School (1,32). The median patient agewas 64 years (range, 57-71 years) for IPF (16 men, 10women) and 45 years (range, 35-53 years) for SSc (allwomen). All patients fulfilled the diagnostic and subtypecriteria for SSc (33,34) and IPF (35,36). Open lung biopsywas performed using formal thoracotomy, thereby avoidinghoneycombing areas. The 56 patients provided writteninformed consent (protocol number 0960/08), and the studywas approved by the local ethics committee. The patients’clinical records were analyzed. The HRCT and pulmonaryfunction test (PFT) were performed up to 3 months beforethe biopsies. The pulmonary function tests included theforced expiratory volume in 1 s (FEV1), forced vital capacity(FVC), FEV1/FVC ratio 100, total lung capacity (TLC) andresidual volume and carbon monoxide transfer factor(DLCO). The TLC, residual volume and residual volume/TLC percentages were measured using the helium dilutionmethod with a MasterScreen apparatus (Erich JaegerGmbH, Wurzburg, Bavaria, Germany), and the DLCO andDLCO/alveolar volume were measured using the singlebreath-holding helium dilution method (37). The lungfunction measurements are reported as percentages of thepredicted values. The cardiac parameters of these patientswere normal. The HRCTs were performed using 1.0- or 1.5-mm-thick sections with patients in the supine position andwith full inspiration at 10-mm intervals. A specialized chestradiologist and a pneumologist analyzed the images at 3pre-established levels (i.e., the trachea, carina and pulmon-ary veins) for the presence of any signs of ILD, such as

ground glass, consolidation, reticular opacities, honeycomb-ing and bronchiectasis.

Histological analysisThe pulmonary tissue was fixed in 10% neutral buffered

formalin and embedded in paraffin. Thin sections werestained with hematoxylin and eosin. Additional subserialsections from the paraffin blocks were used for theimmunohistochemistry. Two pathologists who specializein lung diseases and were blinded to the clinical aspects ofthe patients classified the lung specimens according to thenew ILD classification consensus (1). The pathologists madetheir final diagnoses by consensus. UIP, the histologicpattern of IPF, was characterized by a patchy subpleuraland paraseptal distribution of the parenchymal injury.Temporal heterogeneity was observed at low magnifica-tion, with areas of normal lung parenchyma alternatingwith alveolar collapse, interstitial mononuclear infiltrates,septal fibromyxoid tissue (fibroblastic foci) and honeycomblung. All SSc patients had histologic patterns that wereconsistent with non-specific interstitial pneumonia, asdefined by temporally homogeneous septal inflammatorythickening and interstitial fibrosis. Normal lung tissue,which was obtained from 10 individuals (3 males and 7females; median age 47 years, range 31 to 60 years) whohad died suddenly from non-pulmonary causes, served asthe control.

ImmunohistochemistryA standard peroxidase technique was used with Harris’s

hematoxylin as the counterstain to identify AGTR-1 andAGTR-2 expression in the alveolar septae and vascular wallcells. D2-40 was used to identify lymphatic vessels in thecontrol, IPF-UIP pattern (normal, collapsed and fibroblasticfoci areas) and SSc-NSIP pattern lungs. Biotinylated goatpolyclonal antibodies were used. Anti-AGTR-1, anti-AGTR-2 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA)and anti-D2-40 (Signet Laboratory Inc., USA) antibodieswere incubated with the tissue sections at 1:50, 1:50 and1:100 dilutions, respectively. A Max Polymer Novolinkamplification kit (Leica, Newcastle Inc., UK) was used forsignal amplification, and 3.39-diaminobenzidine tetrachlor-ide (0.25 mg dissolved in 1 mL 0.02% hydrogen peroxide)was used as a precipitating substrate for signal detection.The specificity of the primary antibodies was confirmed bythe appropriate reagent controls (omitting the primaryantibody or substituting non-immune serum for theprimary antibody in the staining protocol), which revealedno staining. The positive expression of AGTR-1, AGTR-2and D2-40 was indicated by brown cytoplasmic staining ofthe cells.

HistomorphometryThe area fraction occupied by AGTR-1/AGTR-2+ cells in

the alveolar septae and vascular walls and lymphatic vesselsalong the peripheral and periaxial interstitium was deter-mined with a point-counting procedure (38). We used a 100-point grid with a known area (187.500 mm2 at 4006magnification) attached to the microscope eyepiece (38).Ten fields were chosen from across multiple sites, account-ing/adjusting for the cellularity or number of positivelyimmunostained cells per connective tissue area (38). Thus,the septal area in each field was determined according to thenumber of points falling within the positive cells in the field

Angiotensin receptors and pulmonary remodelingParra ER et al.

CLINICS 2014;69(1):47-54

48

of view (as a proportion of the total grid area). Afterwards,the number of positive cells within the septal area wascounted. The immunostaining cellularity was determined asthe number of positive cells in each field divided by theseptal area. The final results were expressed as themean¡standard deviation (SD) of the lung specimens foreach patient in 10 random, non-coincident microscopicfields.

To control for variation in the scoring between our 2histologists (ERP and VLC), 20% of the stained slides wereindependently scored by both observers. The coefficient ofvariance between the cell counts for the 2 observers was ,5%.

Statistical analysisThe data are reported as the means¡SD. The statistical

analyses were performed using an analysis of variance(ANOVA) followed by the appropriate post hoc tests,including Bonferroni correction and Student’s t-test forcomparisons between 2 groups. The survival curves weredetermined using the Kaplan-Meier method, and the risk ofdeath was estimated using a Cox regression analysis. Thestatistical program SPSS, Inc. (USA) was used, and p,0.05was considered to be significant.

& RESULTS

Clinical featuresThe clinical features of the 56 patients included in this

study are shown in Table 1. All studied patients showed arestrictive lung function pattern that was characterized by adecrease in TLC (the mean values were 81% of thatpredicted for SSc-NSIP and 78% of that predicted for IPF-UIP) and an increased FEV 1/FVC ratio/100 (the meanvalue was 107% of that predicted for SSc-NSIP vs. 90% ofthat predicted for IPF-UIP). The mean predicted DLCOvalues were significantly decreased in the IPF-UIP patients(55%) compared with the SSc-NSIP (66%) patients, and theywere significantly greater when comparing the DLCO/VAin the IPF-UIP (47%) vs. the SSc-NSIP (77%) patients. Nodifference was found in the FEV1, FCV, TLC and RV valuesbetween the SSc-NSIP and IPF-UIP groups (Table 1).

Angiotensin II type 1 and 2 receptorsAlveolar septae and vessels from the normal control lungs,

NSIP and UIP histologic patterns are shown in Figure 1(immunostained with the AGTR-1 and AGTR-2 antibodies).Different immunostaining intensity was observed in theepithelial, endothelial, myofibroblast and smooth musclecells from the alveolar septae and vessels in the SSc-NSIP(cellular SSc-NSIP and fibrotic SSc-NSIP) and IPF-UIP groupscompared to the normal control lungs. Table 2 summarizesthe morphometric results. A significantly higher percentageof the septal and vessel cells immunostained for AGTR-1/AGTR-2 in the cellular SSc-NSIP, fibrotic SSc-NSIP and IPF-UIP lungs was observed compared to the controls. In the SSc-NSIP and IPF-UIP lungs, a similar proportion of the septalcells immunostained for AGTR-1 was observed, except forthe IPF-UIP lungs, in which the AGTR-2 immunoreactivityrepresented a significantly reduced proportion of the septalcells compared to the SSc-NSIP lungs. Of equal significancewas the increased percentage of the vessel cells that wereimmunostained for AGTR-1 in the cellular SSc-NSIP and IPF-UIP lungs. Concerning AGTR-2, a similar percentage of thevessel cells was present in the fibrotic SSc-NSIP and IPF-UIPlungs. When we compared the total cellular expression ofAGTR-1 in the SSc-NSIP and IPF-UIP groups, a similarproportion was observed; however, both were increasedcompared to the control group (Figure Q), except for the IPF-UIP lungs, in which the total cellular AGTR-2 expression hada significantly smaller proportion than that of the SSc-NSIPpattern lungs (Figure R).

Distribution of lymphatic vesselsFigure 2 shows the peripheral, interlobular and peri-

bronchovascular lymphatic vessels from the normal controllungs and the SSc-NSIP and IPF-UIP histologic patterns,immunostained for D2-40. A different proportion of thelymphatic vessels in the peripheral, interlobular andperibronchovascular interstitium was visualized in theSSc-NSIP (cellular SSc-NSIP and fibrotic SSc-NSIP) andIPF-UIP lungs compared to the control lungs. Table 2summarizes the morphometric results. In the peripheral,interlobular and peribronchovascular interstitium of theIPF-UIP group, the lymphatic vessels showed a significantlyincreased area compared to the SSc-NSIP lungs (Figure G).A significantly higher percentage of lymphatic vessels waspresent in the peripheral, interlobular and periaxial inter-stitium of the IPF-UIP pattern compared to the SSc-NSIPhistologic pattern (Figure H).

Pulmonary function testsA significant inverse association was found between the

vascular AGTR-2 and DCLO/VA (R = -0.74, p = 0.001) in theSSc-NSIP and (R = -0.78, p = 0.001) UIP groups. Equallysignificant was the negative association between thelymphatic vessel area in SSc-NSIP and the predicted valuesof DLCO (R = -0.53, p = 0.04).

Survival analysisNineteen patients died during the 180-month follow-up

period (5 SSc-NSIP and 14 IPF-UIP). A preliminaryexamination of the Kaplan-Meier survival curves showedthat in this study, the patients with the SSc-NSIP patternwith percentages of vessel cells immunostained for AGTR-1that were ,2.49% and AGTR-2 percentages ,7.46% had

Table 1 - Clinical data of the patients with systemicsclerosis and idiopathic pulmonary fibrosis.

SSc-NSIP

(n = 30) IPF-UIP (n = 26) p-value

Age, years 45¡8 64¡7 ,0.001

Sex (female/male) 30 10/16 1.2

Spirometry

FEV1 (% predicted) 70¡13.8 76¡20.01 0.23

FVC (% predicted) 65¡13.8 69¡16.8 0.35

FEV1/FVC 107¡8.69 90¡18.7 ,0.001

TLC (% predicted) 81¡11.5 78¡21.9 0.64

RV (% predicted) 117¡35.5 101¡62.04 0.36

DLCO (% predicted) 66¡21.6 55¡19.1 0.01

DLCO/VA (% predicted) 77¡35.2 47¡22.74 0.008

The data are expressed as the means¡SD or as counts (percentages);

p,0.05 was significant.

Abbreviations: NSIP, non-specific interstitial pneumonia; UIP, usual

interstitial pneumonia; FEV1, forced expiratory volume in 1 s; FVC, forced

vital capacity; TLC, total lung capacity; RV, residual volume; DLCO,

diffusing capacity of the lung for carbon monoxide; VA, alveolar volume.

CLINICS 2014;69(1):47-54 Angiotensin receptors and pulmonary remodelingParra ER et al.

49

higher survival rates than the patients with percentages ofvessel cells immunostained for AGTR-1 that were .2.49%and AGTR-2 percentages .7.46. The multivariate analysesby the Cox regression model showed statistical significance

(log-likelihood = 59.39, p,0.0001) with a high risk of deathfor the patients with the IPF-UIP pattern (p = 0.01) and ahigh percentage of vessel cells immunostained for theAGTR-2 and lymphatic vessels (Table 3).

Figure 1 - Cellular expression of the angiotensin II type 1 receptor (AGTR-1) and AGTR-2 divided in septal areas and intrapulmonaryvessels from normal control lungs, systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF). In (A) and (B), we observe a diffuseand minimal AGTR-1 (arrows) expression in the alveolar septal areas and in the intrapulmonary vessels of the controls. The AGTR-1expression shows greater increases in the thickened alveolar septal region from the cellular SSc-NSIP (arrows, E) than in the fibrotic SSc-NSIP (arrows, I). In the intrapulmonary vessels, the expression of AGTR-1 (arrows) is minimal and similar in both groups (cellular SSc-NSIP, F and fibrotic SSc-NSIP, J). Increased expression of AGTR-1 (arrows) is observed in the septal areas (M) and intrapulmonary vessels(N) of IPF-UIP. In (C) and (D), we observed AGRT-2 (arrows) expression in the septal areas and intrapulmonary vessels from the controls.In (G) and (K), we observed a similar expression of AGTR-2 (arrows) in the septal areas of cellular and fibrotic SSc-NSIP. Increasedexpression of AGTR-2 (arrows) is observed in the intrapulmonary vessels of fibrotic SSc-NSIP (L) compared to cellular SSc-NSIP (H). Theexpression of AGTR-2 (arrows) is more evident in the septal areas of IPF-UIP (O) than in the intrapulmonary vessels (P) of this histologicpattern. Q and R show the total values of AGTR-1 and AGTR-2 and the results analyzed by a one-way ANOVA with the Bonferronimultiple comparisons test for the control, SSc-NSIP and IPF-UIP groups. (*) Significantly higher AGTR-1 expression was observed in theSSc-NSIP and IPF-UIP lungs compared with the control group (p = 0.001 and p = 0.001, respectively). A similar situation was observedwith AGTR-2 expression from the SSc-NSIP and IPF-UIP patterns compared to the control group (p,0.001 and p,0.001, respectively). ({)Significantly higher expression of AGTR-2 in the SSc-NSIP group was observed when compared with the IPF-UIP group (p = 0.04) byStudent’s t-test.

Table 2 - Summary of the morphometric resultsa.

Variables Control Cellular SSc-NSIP Fibrotic SSc-NSIP SSc-NSIP UIP

Septal AGTR-1 1.45¡1.02 10.72¡6.20 12.18¡8.00 11.41¡6.97 10.10¡22.23

Vascular AGTR-1 1.06¡1.03 4.36¡5.35 0.51¡1.15 2.53¡4.33 2.13¡1.64

Septal AGTR-2 0.03¡0.02 12.75¡8.05 12.78¡6.96 12.77¡7.36 7.78¡4.31

Vascular AGTR-2 0.01¡0.04 1.26¡1.35 6.91¡4.64 3.83¡4.30 6.06¡4.66

Total AGTR-1 1.25¡0.84 7.54¡4.49 6.34¡3.96 6.97¡4.18 6.26¡10.93

Total AGTR-2 0.02¡0.02 6.51¡3.88 9.85¡5.09 8.18¡4.73 6.38¡3.63

Lymphatic density 1.68¡0.38 2.72¡0.62 2.92¡0.69 2.80¡0.65 3.73¡1.19

Lymphatic area 2.81¡0.90 4.28¡1.32 4.33¡0.64 4.25¡1.08 5.52¡2.24

aThe units of ‘‘% of points’’ indicate the number of points overlying the phenomena of interest divided by the total number of points overlying the septae

and vessels. In morphometry, this process is called a point fraction and is often symbolized as Pp. The Pp has been shown to approximate the volume

fraction or Vv.


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Figure 2 - D2-40 cell expression in the lymphatic vessels of the normal control lungs, systemic sclerosis (SSc) and idiopathic pulmonaryfibrosis (IPF). The lymphatic vessels (arrows) were observed in the sub pleural (A) and interlobular septal interstitium (B) in the controllungs. In cellular SSc-NSIP, the lymphatic vessels (arrows) were observed in the thickened alveolar septal region (C) and were infrequentcompared to the fibrotic SSc-NSIP pattern (arrows, D). In the UIP pattern, we observed a higher proportion of lymphatic vessels (arrows)in the interlobular and subpleural spaces (E and F). G and H represent the total values of the lymphatic dilatation and density, and theresults analyzed by the one-way ANOVA followed by the Bonferroni multiple comparisons test for the control, SSc-NSIP and IPF-UIPgroups. (*) A significantly increased lymphatic dilatation (G) was observed in the IPF-UIP group compared to the SSc-NSIP (p = 0.01) andcontrol (p = 0.01) groups. Similarly, a significantly higher lymphatic density (H) was observed in the IPF-UIP group compared to the SSc-NSIP (0.02) and control (0.01) groups.


51

& DISCUSSION

Unlike SSc-NSIP, the likely reason that IPF-UIP patientsdo not respond to immunosuppressive therapies and theirclinical course is marked by inexorable deterioration is theimbalance between parenchymal and vascular remodeling.Therefore, it is important to determine whether additionalhistological details can help us understand the clinicaldifferences between these diseases. After pulmonary injury,parenchymal and vascular remodeling contribute toincreased pulmonary epithelial and endothelial permeabil-ity. This early phase is followed by a subacute fibroproli-ferative phase, which may allow repair of the injured lungor may result in progressive obliteration of the interstitial,alveolar and vascular compartments of the lung through afibroproliferative process that may be established by 24 hafter the injury (39).

Recent studies have shown that ANG II contributes topulmonary fibrosis progression, which is evidenced notonly by its potent vasoconstrictor activity but also by itsinfluence on organ remodeling and fibrosis (20,21,22). TheAGTR-1 and AGTR-2 binding of ANG II is facilitated by thecleavage of ANG I by the ACE. Both receptors belong to the7-transmembrane G protein–coupled receptor family(23,24). Whereas most of the ANG II-mediated effects, suchas cell growth, inflammation or extracellular matrix synth-esis, have been shown to be mediated via AGTR-1, thefunction of AGTR-2 has been less clearly investigated(40,41). During the development of fibrosis, AGTR expres-sion undergoes an expression shift in favor of higher AGTR-2 levels, thereby tilting the cellular response to ANG II(42-44). Thus, for all these reasons, we should not besurprised to learn that immunohistochemical staining forAGTRs provides important information about the organremodeling process in diffuse parenchymal lung diseases,and our results now confirm the pathogenetic importance ofAGTRs in IPF and SSc.

We found a significantly greater percentage of septal andvessel cells immunostained for AGTR-1 in SSc-NSIP andIPF-UIP compared with the normal lung. In addition, SSc-NSIP and IPF-UIP had a similar proportion of septal cellsimmunostained for AGTR-1. We also found an increasedpercentage of vessel cells immunostained for AGTR-1 incellular SSc-NSIP and IPF-UIP. Previous reports in theliterature have shown the potential role of ANG II inexperimental lung fibrosis and in IPF (42-44), while thepotency of AGTR-1 in fostering lung fibrosis has beenpreviously demonstrated in multiple in vitro cell cultureconditions as well as in vivo animal models (45,42). Inexperimental animal models of lung fibrosis, using bleo-mycin exposure or radiation, the fibrotic response was

attenuated by the ACE inhibitors and by specific AGTR-1antagonism (45,46). Studies of the inhibition of the ANG IIpathway have demonstrated opposing results. Some studieshave shown beneficial results and blocked the fibrosis,principally in bleomycin-induced pulmonary fibrosis (47);others have failed to document a beneficial effect of theANG II blockade in experimental models of lung fibrosis,indicating that the fibrotic disease process is dependent notonly on AGTR-1 activity (48). We found that a smallpercentage of vessel cells in SSc-NSIP are AGTR-1+. Incontrast to the data available for AGTR-1, the function ofAGTR-2 is much less well explored. In our study, there wasan increased percentage of vessel cells immunostained forAGTR-2 in SSc-NSIP and IPF-UIP, suggesting an importantrole in the pathogenesis of these disorders. In heart tissue,the AGTR-2 is considered to be a neutralizer, modulatingthe actions of AGTR-1 (28,29), but many signaling mechan-isms and receptor functions are unclear, particularly the roleof AGTR-2 in pulmonary fibrosis.

We also found a significantly higher percentage oflymphatic vessels in the peripheral, interlobular andperiaxial interstitium of IPF-UIP compared with the SSc-NSIP histologic pattern. In IPF-UIP, the lymphatic vesselsshowed a significantly increased area compared with SSc-NSIP. The lymphatic vessels contribute to fibrosis matura-tion and scar formation through the drainage of excessiveproteins and fluid during fibrosis (49). In the present study,D2-40+ lymphatics were abundant in the peripheral,interlobular and peribronchovascular interstitium. Pre-vious studies performed by our group have shown thatthe lymphatic vessels play an important role in earlyremodeling in the development of pulmonary fibrosis (50).The current study clearly demonstrated that the lymphaticvessels were poorly detected in the affected areas of earlyremodeling, such as cellular SSc-NSIP, compared to thefibrotic SSc-NSIP and IPF-UIP patterns.

Our study has clinical and functional importance. Asignificant inverse association was found between vascularAGTR-2 and DCLO/VA in the SSc-NSIP and IPF-UIPgroups. Of equal significance was the negative associationbetween the lymphatic vessel area in SSc-NSIP and thepredicted values of DLCO. To establish the relevance ofthese findings to disease progression in the patients, theAGTRs were evaluated as a function of survival (controlledfor age) in the IPF-UIP and SSc-NSIP histologic patterns.The multivariate analyses using the Cox regression modelshowed a high risk of death for the patients with the IPF-UIP pattern presenting a high percentage of vessel cellsimmunostained for AGTR-2 in the lymphatic vessels.

We concluded that the AGTRs and lymphatic vessels inthe lung parenchyma offer the potential to control the organ

Table 3 - Cox proportional hazards regression to ascertain the individual contribution of the histological patterns (NSIPand UIP) and the morphological factors associated with survival and to compare the adjusted survival between thegroups (-2 log-likelihood = 59.93; Chi-squared = 40.10; p,0.001).

b SE Wald test p-value Exp (b) 95.0% CI for Exp (b)

Lower Upper

NSIP pattern -12.70 182.18 5.97 0.94 0.00 0.00 0.60E149

UIP pattern -1.60 0.65 5.97 0.01 0.20 0.56 0.78

AGTR2 vascular 0.26 0.89 9.23 0.002 1.30 1.10 1.55

Lymphatic density -1.20 0.25 6.92 0.008 0.297 0.122 0.73

b= beta coefficient; SE = standard error; Exp (b) = exponential beta; CI = Confidence interval.


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remodeling and fibrosis involved in SSc-NSIP and IPF-UIPprogression, which suggests that strategies aimed atpreventing high AGTR synthesis and low lymphangiogen-esis may have a greater impact on SSc and IPF. Furtherstudy using a randomized and prospective trial is necessaryto finalize this conclusion.

& ACKNOWLEDGMENTS

We are grateful to Sandra de Morais Fernezlian, a biologist from the

immunohistochemistry laboratory, for the helpful immunohistochemistry

staining results and to Prof. Carlos Roberto Ribeiro de Carvalho and

Ronaldo Adib Cairalla, from the Department of Pneumology, for their

patient databases. This study was supported by the National Council for

Scientific and Technological Development (CNPq) and the Foundation for

the Support of Research of the State of Sao Paulo (2008/53022-3 and

2011/06312-9).


All authors designed the study, analyzed and interpreted the data and

wrote the manuscript.

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Risk factors associated with death in Brazilianchildren with severe dengue: a case-control studyMaria dos Remedios Freitas Carvalho Branco,I,II Expedito Jose de Albuquerque Luna,II Leonidas Lopes

Braga Junior,III Ricardo Villar Barbosa de Oliveira,III Lıvia Teresa Moreira Rios,III Maria do Socorro da Silva,IV

Maria Nilza Lima Medeiros,IV Gilnara Fontinelle Silva,I Fernanda Campos Amaral Figueiredo Nina,I Taliane

Jardim Lima,I Jayron Alves Brito,I Avessandra Costa Cardoso de Oliveira,I Claudio Sergio PannutiII

I Universidade Federal do Maranhao, Departamento de Patologia, Sao Luıs/MA, Brazil. II Universidade de Sao Paulo, Instituto de Medicina Tropical de Sao

Paulo, Departamento de Molestias Infecciosas e Parasitarias (LIMHC), Sao Paulo/SP, Brazil. III Hospital da Universidade Federal do Maranhao, Sao Luıs/MA,

Brazil. IV Vigilancia Epidemiologica Municipal de Sao Luıs, Sao Luıs/MA, Brazil.

OBJECTIVE: The purpose of this case-control study was to evaluate risk factors associated with death in childrenwith severe dengue.

METHODS: The clinical condition of hospitalized patients with severe dengue who died (cases, n = 18) wascompared with that of hospitalized patients with severe dengue who survived (controls, n = 77). The inclusioncriteria for this study were age under 13 years; hospital admission in Sao Luis, northeastern Brazil; andlaboratory-confirmed diagnosis of dengue.

RESULTS: Severe bleeding (hemoptysis), a defining criterion for dengue severity, was the factor most stronglyassociated with death in our study. We also found that epistaxis and persistent vomiting, both included aswarning signs in the World Health Organization (WHO) classification of dengue, were strongly associated withdeath. No significant association was observed between any of the laboratory findings and death.

CONCLUSIONS: The finding that epistaxis and persistent vomiting were also associated with death in childrenwith severe dengue was unexpected and deserves to be explored in future studies. Because intensive care unitsare often limited in resource-poor settings, any information that can help to distinguish patients with severedengue with a higher risk to progress to death may be crucial.

KEYWORDS: Dengue; Child; Risk Factors; Death; Brazil; Case-Control Studies.

Branco MR, Luna EJ, Braga Jr LL, Oliveira RV, Rios LT, Silva MS, et al. Risk factors associated with death in Brazilian children with severe dengue:a case-control study. Clinics. 2014;69(1):55-60.

Received for publication on May 3, 2013; First review completed on May 28, 2013; Accepted for publication on July 19, 2013


Tel.: 55 98 8803-2488

& INTRODUCTION

The incidence of dengue virus infection is rising inendemic areas in tropical and subtropical regions world-wide. In the Americas, the cumulative number of denguecases in the last 30 years exceeded 5 million. In this timeperiod, Brazil had the largest number of reported denguecases (54.5% of those reported in the Americas) and the sixthlargest number of dengue hemorrhagic fever (DHF) cases(1). In Brazil, the first dengue outbreak occurred in 1981-1982, in Boa Vista, Roraima State, with isolation of theDENV-1 and DENV-4 serotypes (2). This outbreak was

contained by local vector control measures, and no dengueactivity was reported for the next 4 years (3). In 1986, theDENV-1 serotype was introduced into Rio de Janeiro. Sincethe re-emergence of dengue in Brazil in 1986, the countryhas had several epidemics and has reported the highestnumber of cases in the world on multiple occasions (4). Inthe Americas, the dengue case-fatality rate has increasedover the past decade (1). The incidence of severe cases ofdengue in Brazil has increased since 2001 (3,5), with adramatic increase in severe cases and dengue-related deathsin patients younger than 15 years of age since 2007,particularly in the northeastern region of the country (6-7).

Three serotypes of dengue virus (DENV-1, DENV-2, andDENV-3) have been endemic in Brazil since 2000 (4,8).Endemic circulation of the fourth serotype (DENV-4) hasrecently also been confirmed in Brazil (9-10). In thenortheastern state of Maranhao, high household infestationrates of Aedes aegypti have been observed since 1995,particularly on Sao Luıs Island (11). The first few cases ofdengue caused by DENV-1 in Maranhao were reported in



DOI: 10.6061/clinics/2014(01)08

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55

1994, followed by outbreaks in 1995-96 and 1997-98 (12). Aseroepidemiologic survey conducted in 1996 revealed a41.5% prevalence of dengue antibodies among residents ofSao Luıs Island (13). After the introduction of DENV-2 intoMaranhao in 2001, the incidence of dengue in the stateincreased (12); however, the first deaths from DHF onlyoccurred after the introduction of DENV-3 (5,12). Anincrease in the incidence of DHF and an increase in case-fatality rates in children under age 15 during a 2006-2007epidemic were predominantly associated with infectionwith the DENV-2 serotype. The risk factors for DHF and fordengue shock syndrome (DSS) in children have beenaddressed in previous studies (14-20). However, mostclinical and laboratory findings associated with death inchildren have been compiled through descriptive studies. Inthis case-control study, we report the risk factors associatedwith death in dengue patients younger than 13 years.


Study design and patientsThis study was performed in Sao Luıs, the capital of the

northeastern Brazilian state of Maranhao. The inclusioncriteria for this study were age under 13 years and hospitaladmission in Sao Luis with laboratory-confirmed acutedengue infection. The patients who died (n = 18) wereselected among patients admitted to any hospital in SaoLuıs from April 2006 (the beginning of the epidemic)through December 2007. The controls (n = 77) were allpatients with severe dengue admitted to the Hospital of theUniversidade Federal do Maranhao (HUUFMA) during thesame period who survived. The controls were selected onlyin the HUUFMA because it is a state referral hospital for thetreatment of severe and complicated dengue cases wherereliable medical records were available. It is a publichospital that is part of the national public health system.Additionally, we conducted a subset analysis, consideringonly the cases admitted to the HUUFMA.

Laboratory diagnosis of acute dengue infectionThe diagnosis of acute dengue infection was confirmed by

detection of dengue-specific IgM antibodies using animmunoglobulin M antibody-capture enzyme-linked immu-nosorbent assay (MAC-ELISA) or by DENV detection inserum, blood, or viscera by reverse transcription-polymer-ase chain reaction (RT-PCR). These tests were conducted ina Central Public Health Reference Laboratory.

Dengue case classificationThe 2009 World Health Organization (WHO) ‘‘Dengue

guidelines for diagnosis, treatment, prevention, and con-trol’’ document was used for dengue case classification andfor determining levels of severity. In this classification,severe dengue is defined by the presence of severe plasmaleakage leading to shock or fluid accumulation withrespiratory distress; and/or severe bleeding, as evaluatedby the clinician; and/or severe organ involvement (liver,central nervous system [CNS], heart and other organs) (21).

Clinical and laboratory dataDemographics, medical history, clinical findings, results

of laboratory tests and imaging as well as informationabout treatment and patient outcomes were obtained frommedical records and investigation forms of the National

Mandatory Reporting System (SINAN) for dengue cases.Patients who died were identified through data availablein three different systems: the SINAN, the MortalityInformation System (SIM) and the Hospital AdmissionInformation System of the National Public Health System(SIH-SUS). Additional clinical and pathological informationregarding fatal cases was obtained from death certificates,necropsy reports and interviews with family memberscompiled by the Municipal Dengue Control Program ofSao Luıs.

The plasma leakage criteria were cavity effusion (foundon imaging or on necropsy), hemoconcentration (an increasein hematocrit of $20% of baseline or a decrease inhematocrit following volume-replacement treatment of$20% of baseline), hypoalbuminemia (serum albumin,3.5 g/dL) or hypoproteinemia (serum protein ,6.0 g/dL).

Data analysisClinical and laboratory data were entered into a database

using Epi Info 3.5 software (Centers for Disease Control andPrevention, Atlanta, GA, USA). STATA 10.0 software(StataCorp LP, College Station, TX, USA) was used forstatistical analysis. For quantitative variables, measures ofcentral tendency and dispersion were calculated.Qualitative variables are presented as frequencies andproportions. A logistic regression model was used tocalculate the unadjusted associations between the outcome(death) and various independent variables. Odds ratios and95% confidence intervals were calculated. The number ofcases was too small to allow for adjustment in a multiplelogistic regression model.

EthicsThe study protocol was approved by the Institutional

Review Board of the HUUFMA. Sources included informa-tion obtained from medical records, death certificates,necropsy reports and SINAN investigation forms.Confidentiality and subject anonymity was ensuredthroughout the investigation. Written informed consentwas not obtained because the study primarily relied onsecondary data. In interviews with the family members ofpatients who died of dengue, oral consent was obtained anddocumented.

& RESULTS

During the study period, 33 patients under 13 years ofage died of suspected dengue. Ten of these 33 patientswere admitted to the ICU of the HUUFMA, and 23 wereadmitted to one of the seven other hospitals in Sao Luıs. Ofthe 33 patients, 18 had laboratory-confirmed dengueinfection and were included in the study as cases. Thir-teen of the remaining 15 patients who died could bethoroughly investigated, and 4 had signs of severe plasmaleakage, suggesting severe dengue. However, because alaboratory diagnosis of dengue could not be confirmed,these 4 patients were not included in the study. Of the 18cases included in the study, 5 were admitted to the ICU ofthe HUUFMA.

Of 396 patients younger than 13 years with suspecteddengue who were admitted to the HUUFMA during thestudy period, 77 had laboratory-confirmed dengue in itsmost serious presentation (shock) and were included inthe study as controls. Demographic information, DENV

Risk factors for death in children with dengueBranco MR et al.

CLINICS 2014;69(1):55-60

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serotypes, ICU admission and the durations of fever andhospital stay are shown in Table 1. All cases and controlshad fever. The durations of fever and hospital stay wereshorter among cases than controls (Table 1).

Simple logistic regression analysis showed that epistaxis,hemoptysis and persistent vomiting were clinical signssignificantly associated with death (Table 2). Table 3 showsthe laboratory and imaging results as well as the analysis ofthe plasma leakage criteria (cavity effusion, hemoconcentra-tion, hypoalbuminemia and hypoproteinemia). No signifi-cant association was observed between any of the laboratoryfindings and death.

To control for possible selection bias, we performed asubset analysis including only the cases admitted to theHUUFMA (Table 4). In this subset, most of the findingswere maintained. The association with ‘‘persistent vomit-ing’’ was no longer significant; however, epistaxis remaineda significant factor associated with death.

& DISCUSSION

In our study, no significant association was observedbetween any of the laboratory findings and death. Thisfinding supports the 2009 WHO dengue criteria for severedengue, which emphasize clinical signs over laboratoryfindings (21). Severe plasma leakage leading to shock didnot appear to be a significant risk factor because all controlshad severe dengue with shock. The factor most stronglyassociated with death in our study was severe bleeding(hemoptysis). Hemoptysis is considered a defining criterionfor dengue severity, according to the revised WHOclassification (21) that was recently assessed in a multi-center study (22). However, we found that epistaxis andpersistent vomiting, which are considered warning signs inthe revised WHO dengue case classification, were alsostrongly associated with death. Epistaxis remained signifi-cantly associated with death in the subset analysis includingonly the cases admitted to the HUUFMA. From a clinicalpoint of view, epistaxis and persistent vomiting are notintrinsically severe, unlike hemoptysis and shock, which cancause a rapid progression of the patient to death. However,epistaxis and persistent vomiting can be surrogate markersfor severe dengue, even if we do not currently have a logicalexplanation for this finding.

Signs and symptoms associated with death in childrenhave been addressed in previous descriptive studies. In Thaichildren, bleeding was one of the risk factors for DSS (17). InColombian children with DHF and atypical manifestationsof dengue, all those who had hemoptysis died (23). InMalaysian children with severe dengue infections, asignificant association was found between major bleedingand death (p = 0.001) (24). In an Indonesian study of 30children with dengue who died, 16.7% had epistaxis (25). Inthe 1981 Cuban epidemic, of the 13 children with DHF/DSSwho died, 12 had vomiting, and 3 had epistaxis (26).

In a descriptive study of 15 Colombian children youngerthan 13 with DHF who died, the cause of death wasmyocarditis in 9, acute hepatitis in 3 and disseminatedintravascular coagulation in the remaining 3, indicating thatmortality due to DHF was not caused only by hypovolemicshock (27). Some information from the cited descriptive

Table 1 - Demographic and clinical features of childrenwith severe dengue.

Demographic or clinical feature Cases Controls

No. of patients 18 77

Time of occurrence

2006 3 44

2007 15 33

Boys, no. (%) 6 (33.3) 34 (44.2)

Admission to ICU, n (%) 11 (61.1) 12 (15.6)

Serotype (n) DENV-2 (5) (0)

Necropsy 6 0

Comorbidities 1 (5.5) 0

Suspected dengue on admission, n (%) 9/13 (69.2) 76/77 (98.7)

Age, mean years ¡ SD (median) 4.17¡3.29 (4) 4.04¡2.72 (4)

Age range in years 0-10 0-12

Age, minimum in months 6 4

Fever duration, mean days ¡ SD

(median)

3.77¡1.89 (3) 5.30¡2.53 (5)

Duration of fever, range in days 1-8 1-13

Hospital stay, mean days ¡ SD

(median)

2.20¡2.01 (2) 7.26¡3.63 (6)

Hospital stay, range in days 0-6 3-24

Table 2 - Association between clinical characteristics and death in children with severe dengue.

Signs and symptoms Cases N (%) Controls N (%) Odds ratio 95% CI p-value

Cold extremities 8/17 (47.1) 14/77 (18.2) 4.00 1.31-12.19 0.015

Cyanosis 10/17 (58.8) 17/77 (22.1) 5.04 1.67-15.24 0.004

Dehydration 14/15 (93.3) 53/77 (68.8) 6.34 0.79-51.01 0.083

Dyspnea 11/17 (64.7) 36/77 (46.7) 2.09 0.70-6.21 0.186

Edema 8/17 (47.1) 47/77 (61.0) 0.57 0.20-1.63 0.293

Lethargy 13/17 (76.5) 36/77 (46.7) 3.70 1.11-12.37 0.034

Persistent vomiting 7/17 (41.2) 11/77 (14.3) 4.20 1.32-13.37 0.015

Prostration 11/17 (64.7) 9/77 (11.7) 13.85 4.12-46.62 ,0.001

Restlessness 6/17 (35.3) 13/77 (16.9) 2.68 0.84-8.56 0.095

Shock 18/18 (100.0) 77/77 (100.0) NCa NC NC

Bleeding (any kind) 15/18 (83.3) 77/77 (100.0) NC NC NC

Ecchymoses 6/18 (33.3) 13/77 (16.9) 2.46 0.78-7.75 0.124

Epistaxis 5/17 (29.4) 7/77 (9.1) 4.17 1.13-15.3 0.032

Gastrointestinal bleeding 14/18 (77.8) 41/77 (53.3) 3.07 0.93-10.18 0.066

Gum bleeding 1/17 (5.9) 10/77 (13.0) 0.42 0.05-3.51 0.422

Hemoptysis 4/17 (23.5) 1/77 (1.3) 23.38 2.42-226.1 0.006

Petechiae 6/18 (33.3) 54/77 (70.1) 0.21 0.07-0.64 0.006

Positive tourniquet test 1/3 (33.3) 15/31 (48.4) 0.53 0.04-6.51 0.622

aNC = not calculated.

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studies on death in children with dengue is summarized inTable 5.

The DENV-2 serotype predominated in Maranhao in 2006and 2007. Unfortunately, genotyping was not performed forall patients in the outbreak in Sao Luıs; the DENV serotypewas determined in only 5 cases, and all 5 were DENV-2.

In our study, different factors may have contributed to thepoor prognosis of the cases. The pediatricians had noexperience in the diagnosis and management of patientswith dengue, and health teams were not prepared toprovide emergency care to patients with severe dengueduring the early phase of the epidemic. Only 69.2% of caseswere diagnosed as dengue at the time of hospital admission,compared to 98.7% of controls. This finding suggests that adelay in dengue diagnosis may have worsened the prog-nosis. Additionally, 5 patients had no access to an ICU bed,and 2 arrived at the hospital with advanced disease. Similarfindings were observed in a descriptive study on 14 deathsdue to dengue in 2 municipalities in northeastern Brazil (30).

The durations of fever and hospital stay were shorter incases than in controls, suggesting that the clinical conditionof the cases on admission was worse than that of thecontrols, which may have contributed to the worse out-comes observed for the cases. It is important to consider thatthe earlier a risk factor for death can be identified, thegreater the possibility of introducing appropriate therapeu-tic interventions to prevent death. If high-risk factors hadbeen promptly recognized and the children had beenproperly treated, it is possible that progression to profoundshock and death could have been prevented.

The selection of the cases at 7 different hospitals, whichcould have health care resources of varying quality, mayhave contributed to the deaths of the case patients.

The small number of cases in this study resulted in verywide confidence intervals, including the variables stronglyassociated (odds ratio$3) with death. This limitation alsoprevented us from adjusting the data in a multivariatelogistic regression model. Our unadjusted analysis showed

Table 3 - Laboratory and imaging findings of children with severe dengue.

Variable Cases N (%) Controls N (%) Odds ratio 95% CI p-value

ALT $1000 U/L 1/12 (8.3) 1/70 (1.4) 6.27 0.36-107.78 0.206

AST $1000 U/L 2/12 (16.7) 3/73 (4.1) 4.67 0.69-31.45 0.114

Hematocrit .45% 3/12 (25.0) 21/77 (27.3) 0.89 0.22-3.60 0.869

Leukocyte count .10 000/mm3 5/12 (41.7) 13/77 (16.9) 3.52 0.96-12.82 0.057

Platelet count

,150 000/mm3 11/14 (78.6) 73/77 (94.8) 0.20 0.04-1.02 0.053

,100 000/mm3 11/14 (78.6) 61/77 (79.2) 0.96 0.24-3.86 0.956

,50 000/mm3 6/14 (42.9) 35/77 (45.5) 0.90 0.28-2.84 0.857

Findings on imaging exams

Ascites 7/8 (87.5) 36/54 (66.7) 3.50 0.40-30.66 0.258

Pleural effusion

on ultrasonography 5/8 (62.5) 39/54 (72.2) 0.64 0.14-3.02 0.574

on radiography 5/9 (55.6) 56/71 (78.9) 0.33 0.08-1.40 0.135

Thicker gallbladder wall 3/8 (37.5) 29/54 (53.7) 0.52 0.11-2.38 0.398

Plasma leakage criteria

Cavity effusion 17/18 (94.4) 69/74 (93.2) 1.23 0.13-11.25 0.853

Hemoconcentration 5/7 (71.4) 49/76 (64.5) 1.38 0.25-7.58 0.713

Serum albumin ,3.5 g/dL 7/7 (100.0) 64/70 (91.4) NCa NC NC

Serum protein ,6.0 g/dL 5/5 (100.0) 43/51 (84.3) NC NC NC


Table 4 - Association between clinical characteristics and death in children admitted to the Hospital of the UniversidadeFederal do Maranhao with severe dengue.

Signs and symptoms Cases N (%) Controls N (%) Odds ratio 95% CI p-value

Cold extremities 3/5 (60.0) 14/77 (18.2) 6.75 1.03-44.26 0.047

Cyanosis 5/5 (100.0) 17/77 (22.1) NCa NC NC

Dehydration 4/5 (80.0) 53/77 (68.8) 1.81 0.19-17.08 0.604

Dyspnea 5/5 (100.0) 36/77 (46.7) NC NC NC

Edema 5/5 (100.0) 47/77 (61.0) NC NC NC

Lethargy 4/5 (80.0) 36/77 (46.7) 4.56 0.49-42.64 0.184

Persistent vomiting 0/5 (0.0) 11/77 (14.3) NC NC NC

Prostration 3/5 (60.0) 9/77 (11.7) 11.33 1.66-77.27 0.013

Restlessness 2/5 (40.0) 13/77 (16.9) 3.28 0.50-21.64 0.217

Shock 5/5 (100.0) 77/77 (100.0) NC NC NC

Bleeding (any kind) 5/5 (100.0) 77/77 (100.0) NC NC NC

Ecchymoses 2/5 (40.0) 13/77 (16.9) 3.28 0.50-21.24 0.217

Epistaxis 4/5 (80.0) 7/77 (9.1) 40.0 3.91-409.05 0.002

Gastrointestinal bleeding 5/5 (100.0) 41/77 (53.3) NC NC NC

Gum bleeding 1/5 (20.0) 10/77 (13.0) 1.67 0.17-16.54 0.659

Hemoptysis 2/5 (40.0) 1/77 (1.3) 50.67 3.53-726.74 0.004

Petechiae 4/5 (80.0) 54/77 (70.1) 1.70 0.18-16.08 0.642

Positive tourniquet test 1/2 (50.0) 15/31 (48.4) 1.07 0.06-18.62 0.965



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that severe bleeding manifested by hemoptysis was stronglyassociated with death, providing additional support for therevised WHO dengue case classification (21). However, thefinding that epistaxis and persistent vomiting were alsoassociated with death in children with severe dengue wasunexpected and deserves to be explored in future studies.Because intensive care units are often limited in resource-poor settings, any information that can help to distinguishpatients with severe dengue with a higher risk to progress todeath may be crucial.

& ACKNOWLEDGMENTS

We would like to thank Dr. Wellington da Silva Mendes, who participated

in this study and died in 2009. His premature death at the age of 48 was a

severe loss to the Universidade Federal do Maranhao (UFMA), and we are

honored to have worked with him.We are grateful to the staff of the

Hospital of the Universidade Federal do Maranhao (HUUFMA),

particularly to the teams working in the Infectious Diseases ward,

Laboratory, Ultrasonography and ICU. We are also grateful to the

technicians and managers of Epidemiological Surveillance in the state of

Maranhao and Sao Luıs County. This research was funded by grants BIC-

UFMA 00366/07, BIC-UFMA 00377/07 and BD-00266/09 from

Fundacao de Amparo a Pesquisa e ao Desenvolvimento Cientıfico e

Tecnologico do Maranhao (FAPEMA), Sao Luıs, Maranhao, Brazil. GFS

was the recipient of a junior research fellowship in 2007-2008 from the

Universidade Federal do Maranhao, Sao Luıs, Maranhao, Brazil. The

funders had no role in the study design, data collection and analysis,

decision to publish or preparation of the manuscript.


Branco MR, Luna EJ and Pannuti CS conceived and designed the study,

performed the final data analysis and prepared the first draft. Branco MR,

Braga Jr LL, Oliveira RV, Rios LT, Silva MS, Medeiros MN, Silva GF,

Nina FC, Lima TJ, Brito JA and Oliveira AC collected the data. All

authors contributed to the revision of the manuscript and read and

approved its final version.

& REFERENCES

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10. Brazil. Ministerio da Saude. Secretaria de Vigilancia em Saude. Isolamentodo sorotipo DENV 4 em Manaus/AM. Available from: http://portal.saude.gov.br/portal/arquivos/pdf/isolamento_denv4_manaus.pdf. Acce-ssed 2011 Jan 8.

11. Rebelo JM, Costa JM, Silva FS, Pereira YN, da Silva JM. [Distributionof Aedes aegypti and dengue in the State of Maranhao, Brazil].Cad Saude Publica. 1999;15(3):477-86, http://dx.doi.org/10.1590/S0102-311X1999000300004.

12. Goncalves Neto VS, Rebelo JM. [Epidemiological characteristics ofdengue in the Municipality of Sao Luıs, Maranhao, Brazil, 1997-2002].Cad Saude Publica. 2004;20(5):1424-31.

13. Vasconcelos PF, Lima JW, Raposo ML, Rodrigues SG, da Rosa JF,Amorim SM, et al. [A seroepidemiological survey on the island of SaoLuis during a dengue epidemic in Maranhao]. Rev Soc Bras Med Trop.1999;32(2):171-9, http://dx.doi.org/10.1590/S0037-86821999000200009.

14. Loke H, Bethell DB, Phuong CX, Dung M, Schneider J, White NJ, et al.Strong HLA class I-restricted T cell responses in dengue hemorrhagicfever: a double-edged sword? J Infect Dis. 2001;184(11):1369-73, http://dx.doi.org/10.1086/324320.

15. Loke H, Bethell D, Phuong CXT, Day N, White N, Farrar J, et al.Susceptibility to dengue hemorrhagic fever in Vietnam: evidence of anassociation with variation in the vitamin D receptor and Fc gammareceptor Ila genes. Am J Trop Med Hyg. 2002;67(1):102-6.

16. Pichainarong N, Mongkalangoon N, Kalayanarooj S, Chaveepo-jnkamjorn W. Relationship between body size and severity of denguehemorrhagic fever among children aged 0-14 years. SoutheastAsian J Trop Med Public Health. 2006;37(2):283-8.

17. Tantracheewathorn T, Tantracheewathorn S. Risk factors of dengueshock syndrome in children. J Med Assoc Thai. 2007;90(2):272-7.

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19. Nguyen TP, Kikuchi M, Vu TQ, Do QH, Tran TT, Vo DT, et al. Protectiveand enhancing HLA alleles, HLA-DRB1*0901 and HLA-A*24, for severeforms of dengue virus infection, dengue hemorrhagic fever and dengueshock syndrome. PloS Negl Trop Dis. 2008;2(10):e304.

20. Libraty DH, Acosta LP, Tallo V, Segubre-Mercado E, Bautista A, Potts JA,et al. A prospective nested case-control study of dengue in infants:rethinking and refining the antibody-dependent enhancement denguehemorrhagic fever model. PloS Med. 2009;6(10):e1000171, http://dx.doi.org/10.1371/journal.pmed.1000171.

21. World Health Organization. Dengue: guidelines for diagnosis, treatment,prevention and control. Available from: http://whqlibdoc.who.int/publications/2009/9789241547871_eng.pdf. Accessed 2009 Nov 29.

22. Barniol J, Gaczkowski R, Barbato EV, Cunha RV, Salgado D, Martınez E,et al. Usefulness and applicability of the revised dengue case classification

Table 5 - Summary of descriptive studies on dengue deaths in children.

Author(s), year Forms of disease Age Number of patients

Number of

deaths

Symptoms associated with death or

cause of death, n (%)

Mendez, Gonzalez, 2006 (23) DHF/DSS 0 m-12 y 168 10 Hepatitis 6/10 (60.0), Neurological

alterations 6/10 (60.0)

Sumarmo et al., 1983 (25) DF/DHF/DSS 0 m-14 y 30 30 GI bleeding 24/30 (80.0), Hepatomegaly

16/30 (53.3), Petechiae 16/30 (53.3)

Guzman et al., 1984 (26) DHF/DSS 2 m-12 y 13 13 Vomiting 12/13 (92.3), Hematemesis 12/

13 (92.3), Ascites 9/13 (69.2)

Salgado et al., 2008 (27) DHF/DSS 0 y-12 y 13 13 Myocarditis 9/13 (69.2), Hepatitis 3/13

(23.1), DIC 3/13 (23.1)

Kamath, Ranjit, 2006 (28) DHF/DSS Children 109 9 Shock/DIC/ARDS 4/9 (44.4), Shock/DIC 2/9

(22.2), Neurological alterations 2/9 (22.2)

Kouri et al., 1989 (29) DHF/DSS Children 124 57 GI bleeding

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by disease: multi-centre study in 18 countries. BMC Infect Dis. 2011;11:106,http://dx.doi.org/10.1186/1471-2334-11-106.

23. Mendez A, Gonzalez G. [Abnormal clinical manifestations of denguehemorrhagic fever in children]. Biomedica. 2006;26(1):61-70.

24. Lum LC, Goh AY, Chan PW, El-Amin AL, Lam SK. Risk factors forhemorrhage in severe dengue infections. J Pediatr. 2002;140(5):629-31,http://dx.doi.org/10.1067/mpd.2002.123665.

25. Sumarmo H, Wulur E, Jahja E, Gubler DJ, Suharyono W, Sorensen K.Clinical observations on virologically confirmed fatal dengue infec-tions in Jakarta, Indonesia. Bull World Health Organ. 1983;61(4):693-701.

26. Guzman MG, Kourı G, Morier L, Soler M, Fernandez A. Casos mortalesde dengue hemorragico en Cuba, 1981. Bol Of Sanit Panam. 1984;97(2):111-7.

27. Salgado DM, Panqueba CA, Vega MR, Garzon M, Castro D, RodriguezJA. [Dengue hemorrhagic fever mortality in children: beyond shock].Infect. 2008;12(1):21-27.

28. Kamath SR, Ranjit S. Clinical features, complications and atypicalmanifestations of children with severe forms of dengue hemorrhagicfever in South India. Indian J Pediatr. 2006;73(10):889-95.

29. Kouri GP, Guzman MG, Bravo JR, Triana C. Dengue haemorrhagicfever/dengue shock syndrome: lessons from the Cuban epidemic, 1981.Bull World Health Organ. 1989;67(4):375-80.

30. Figueiro AC, Hartz ZM, Brito CA, Samico I, Siqueira Filha NT, CazarinG, et al. [Death from dengue fever as a sentinel event for evaluation ofquality of healthcare: a case study in two municipalities in NortheastBrazil, 2008]. Cad Saude Publica. 2011 Dec;27(12):2373-85, http://dx.doi.org/10.1590/S0102-311X2011001200009.


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Mibefradil reduces blood glucose concentration indb/db miceYujie Lu,I Min Long,I Shiwen Zhou,II Zihui Xu,I* Fuquan Hu,III Ming LiIV

I Third Military Medical University, Xinqiao Hospital, Department of Endocrinology, Chongqing, China. II Third Military Medical University, Xinqiao

Hospital, Clinical Pharmacology Institution, Chongqing, China. III Third Military Medical University, Xinqiao Hospital, Department of Microbiology

Chongqing, China. IV Tulane University, Department of Physiology, New Orleans/LA, United States.

OBJECTIVE: Numerous recent studies suggest that abnormal intracellular calcium concentration ([Ca2+]i) is acommon defect in diabetic animal models and patients. Abnormal calcium handling is an important mechanismin the defective pancreatic b-cell function in type 2 diabetes. T-type Ca2+ channel antagonists lower bloodglucose in type 2 diabetes, but the mechanism remains unknown.

METHODS: We examined the effect of the Ca2+ channel antagonist mibefradil on blood glucose in male db/dbmice and phenotypically normal heterozygous mice by intraperitoneal injection.

RESULTS: Mibefradil (15 mg/kg, i.p., b.i.d.) caused a profound reduction of fasting blood glucose from430.92¡20.46 mg/dl to 285.20¡5.74 mg/dl in three days. The hypoglycemic effect of mibefradil wasreproduced by NNC 55-0396, a compound structurally similar to mibefradil but more selective for T-type Ca2+

channels, but not by the specific L-type Ca2+ channel blocker nicardipine. Mibefradil did not show suchhypoglycemic effects in heterozygous animals. In addition, triglycerides, basal insulin and food intake weresignificantly decreased by mibefradil treatment in the db/db mice but not in the controls. Western blot analysis,immunohistochemistry and immunofluorescence staining showed a significantly increased expression of T-typeCa2+ channel a-subunits Cav3.1 and Cav3.2 in liver and brain tissues from db/db mice compared to those fromheterozygous animals.

CONCLUSIONS: Collectively, these results suggest that T-type Ca2+ channels are potential therapeutic targets forantidiabetic drugs.

KEYWORDS: Diabetes Mellitus; Hypoglycemic Effect; Insulin; Food Intake; T-type Ca2+ Channel Antagonist.

Lu Y, Long M, Zhou S, Xu Z, Hu F, Li M. Mibefradil reduces blood glucose concentration in db/db mice. Clinics. 2014;69(1):61-67.

Received for publication on April 28, 2013; First review completed on May 15, 2013; Accepted for publication on May 23, 2013


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*corresponding author

& INTRODUCTION

The hallmark of type 2 diabetes mellitus is chronichyperglycemia under both fasting and postprandial condi-tions. Numerous recent studies in diabetic animal modelsand patients suggest that abnormal intracellular calciumconcentration ([Ca2+]i) is a common defect in both insulin-dependent (type 1) and insulin-independent (type 2)diabetes (1). Abnormal calcium handling is an importantmechanism in the defective pancreatic b-cell function intype 2 diabetes (2). Dysregulation of [Ca2+]i may represent acommon factor underlying metabolic, cardiovascular, ocularand neural complications of diabetes mellitus (3). Clinical

administration of an L-type Ca2+ antagonist has producedno detrimental or beneficial effects on glucose tolerance(4,5). Cerebrocrast, an L- and T-type calcium channelinhibitor, decreases blood glucose and food intake andincreases glucose uptake by the brain (6,7). However, thespecific effects of T-type Ca2+ channel antagonists on bloodglucose regulation remain unknown.

T-type Ca2+ channels are different from other types of Ca2+

channels with regard to their kinetics, pharmacologicalproperties and activation/inactivation voltage range (8,9). T-type Ca2+ currents have been described in human pancreaticislet cells (10,11). Studies on human islets have shown that adesensitization of glucose-induced insulin secretion is asso-ciated with [Ca2+]i elevation (12). T-type Ca2+ current densityand the channels’ mRNA levels increase markedly in ratpancreatic islets treated with high glucose (13).

Mibefradil blocks both L-type and T-type Ca2+ channels(14). Clinically, mibefradil has significant therapeutic advan-tages in reducing blood pressure (15,16), preventing bloodpressure-related arterial hypertrophy (17), lowering heartrate (18) and preventing and reducing hyperinsulinemia (19).



DOI: 10.6061/clinics/2014(01)09

BASIC RESEARCH

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An analog of mibefradil, (1S,2S)-2-(2-(N-[(3-benzoimidazol-2-yl)propyl]-N-methylamino) ethyl)-6-fluoro 1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride[NNC 55-0396], is more selective for T-type Ca2+ channels (20).The molecular structure of NNC55-0396 is more similar to thatof mibefradil than to that of the L-type Ca2+ channel blockernicardipine (Figure 1). The clinical effect of NNC 55-0396 isunclear.


Experimental animalsExperimental (male BKS Cg-Dock 7 m+/+Lepr/J, db/db,

36–40 g) and control (male Dock7m+/+Leprdb, db/+, hereafter‘‘wild-type’’, 23–26 g) eight-week-old mice purchased fromJackson Laboratory (Bar Harbor, ME) were randomly dividedinto two main groups. The mice were further randomlyseparated into two sub-groups, for a total of four groups(n = 6 mice per group): the control placebo group, controldrug intervention group, db/db placebo group and db/dbdrug intervention group. Mice from both drug intervention

groups were injected with mibefradil (i.p., 15 mg/kg perinjection, b.i.d, for all experiments; Sigma-Aldrich, St. Louis,MS) in 100 ml normal saline with 0.05% DMSO. Normal salinewith 0.05% DMSO was given to both placebo groups in anidentical manner. During the drug intervention, fasting bloodglucose (food deprivation for eight hours), blood pressure,body weight, food intake and insulin tolerance (by insulintolerance test (ITT)) were continually measured. At the end oftreatments, blood, liver and brain samples were collected.

The animal experimental protocol was approved by theInstitutional Animal Care and Use Committee of the ThirdMilitary Medical University. The organs of the animals wereremoved for histochemical analysis after euthanasia.

Immunoassay for insulin, glycosylated hemoglobinA1c and total cholesterol measurements

Mouse cardiac blood (0.8 ml) was stored at 4 C for three tofour hours. After coagulation and clot retraction, sampleswere centrifuged at X1,000g for ten minutes, and serum wasstored at 220 C for later use. Serum basal insulin wasmeasured using an insulin ELISA kit from Merodia (Uppsala,Sweden); HbA1c and total cholesterol were measured byusing ELISA kits from R&D Systems (Minneapolis, MN).

Western blot analysisThe tissue samples (50 mg) were homogenized in lysis

buffer (Thermo Fisher Scientific Inc, Waltham, MA) contain-ing a protease inhibitor cocktail (Roche, Basel, Sweden) andthen centrifuged at X9,000g rpm for 15 minutes at 4 C. Thesupernatants were denatured and loaded for electrophoresisat a volume containing 90 mg total protein. After separation,the proteins were transferred to polyvinylidene fluoride(PVDF) membranes. Blotted membranes were incubatedovernight with primary antibodies against ß-tubulin (SantaCruz Biotechnology Inc, Santa Cruz, CA, 1:200), a1G (Bioss,China, 1:200) and a1H (Santa Cruz Biotechnology Inc,1:200). The membranes were then incubated with appro-priate secondary antibodies at room temperature for1.5 hours before detection with a chemiluminescence kit(Beyotime, BeyoECL Plus, China).

Immunohistochemical analysisThe expression levels of Cav3.1 and Cav3.2 were

evaluated using standard immunohistochemistry methods.

Figure 1 - Chemical structure of calcium channel blockers.

Figure 2 - The hypoglycemic and hypolipidemic effects of Ca2+ channel antagonists in db/db mice. A) Time-dependent changes in bloodglucose concentration during drug treatment. B) Serum HbA1c in mice with and without mibefradil injection. C) Serum totalcholesterol in mice with and without mibefradil injection. Abbreviations: WT, nondiabetic wild-type mice; db/db, db/db mice; NS,normal saline; Mib, mibefradil; NNC, NNC 55-0396; Nic, nicardipine. * p,0.05, n = 6, compared with WT NS group; ** p,0.05, n = 6,compared with NS group of the same type of mice at the same time. Statistical analyses were performed with Student’s t-test (A) orANOVA (B and C) according to the specific application.

Function of T-type Ca2+ channels in hyperglycemiaLu Y et al.

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Fresh tissue samples were washed with PBS, followed byparaformaldehyde fixation (4%) and paraffin embedding. Thesamples were sliced into 4 mm serial sections for xylenedewaxing and alcohol dehydration. After blocking nonspecificantigens with goat serum, the samples were incubated over-night at 4 C with diluted primary antibodies (50 ml). Then,biotin-labeled secondary antibodies were added for incubationat room temperature for one hour in the dark. The samples werethen stained with DAPI for five minutes in the dark, dried andsealed with anti-fluorescence quenching mounting mediumand kept at 4 C in the dark. Samples without primaryantibodies were employed as negative controls.

Statistical analysisAll data are expressed as the mean ¡ SEM. Statistical

analyses were performed with Student’s t-test or ANOVAaccording to the specific application. P-values #0.05 wereconsidered significant. Data were organized with AdobeIllustrator CS3 and analyzed with SPSS 17.0 (StatisticalProduct and Service Solutions, IBM, Chicago, USA). Statisticalsignificance for Western blot experiments was determined byanalyzing gray-level values using Quantity One software.

& RESULTS

The effects of Ca2+ channel antagonists on blood glucosewere examined in db/db mice. As shown in Figure 2A,

mibefradil (15 mg/kg, b.i.d) intraperitoneal injection sig-nificantly decreased blood glucose concentration from430.92¡20.46 mg/dl to 285.20¡5.74 mg/dl (n = 6, p,0.05)by day 3. Fasting blood glucose decreased to a level similarto that of wild-type controls at day 5. The injection of normalsaline (with 0.05% DMSO vehicle) had no effect on bloodglucose in db/db mice. The application of NNC 55-0396, anantagonist that is more specific to the T-type Ca2+ channel(21) at the same dose (15 mg/kg, b.i.d), also effectivelyreduced blood glucose. In contrast, the L-type Ca2+ channelblocker nicardipine (12.5 mg/kg, b.i.d) had no significanteffect on blood glucose when administered at a concentra-tion commonly used for in vivo study (22). These resultsindicate that Ca2+ channel antagonists have a profoundblood glucose-lowering effect in db/db mice, and this effectis likely attributed to the inhibition of T-type Ca2+ channelsrather than any effects on L-type Ca2+ channels. Theapplication of mibefradil, NNC 55-0396 or nicardipine forone week had no significant effect on fasting blood glucosein wild-type mice (Figure 2A), indicating that the effects ofT-type Ca2+ channel blockers are specific to the db/db mice.

The effects of T-type Ca2+ channel blockers on bloodglucose were also evaluated by measuring hemoglobinglycosylation in db/db and wild-type mice. Similar to theabove results, mibefradil effectively reduced HbA1c indb/db mice from 301.5¡9.50 to 236.9¡9.83 (p,0.05, n = 6;Figure 2B). Interestingly, mibefradil slightly reduced HbA1c

Figure 3 - Effects of mibefradil on insulin sensitivity and basal insulin release in diabetic and nondiabetic mice. A and B) Glucosedisposal measured by insulin tolerance test (ITT) before (A) and after (B) mibefradil treatment for seven days. C) Difference in the areaunder the curve before and after drug injection. D) Effect of mibefradil on basal insulin in diabetic and nondiabetic mice. * p,0.05,n = 6, compared with WT NS group; ** p,0.05, n = 6, compared with NS group of the same type of mice at the same time. Statisticalanalyses were performed with Student’s t-test (D) or ANOVA (C), according to the specific application.

CLINICS 2014;69(1):61-67 Function of T-type Ca2+ channels in hyperglycemiaLu Y et al.

63

in wild-type animals (from 250.0¡8.28 to 221.7¡4.50 nmol/L, p,0.05, n = 6), which indicates a physiological role for T-type Ca2+ channels in glucose regulation in normal mice.The effect of mibefradil on blood triglycerides wasevaluated in db/db mice. Figure 2C shows that after oneweek of mibefradil treatment, plasma cholesterol wassignificantly reduced (p,0.05, n = 6) in a manner similar tothe reduction of blood glucose. In contrast, there was nosignificant change in cholesterol level between the wild-typemice with and without the treatment with mibefradil.Cholesterol was significantly different between db/db andwild-type control groups (p,0.05, n = 6).

To delineate the mechanism underlying the effects of T-type Ca2+ channel antagonists on blood glucose in db/dbmice, an ITT was performed after six hours of fasting. Micewere injected intraperitoneally with porcine insulin (XinbaiPharmaceutical, Nanking, China) at 0.75 unit/kg of bodyweight. Blood glucose was measured from tail bleeds takenat the indicated times. Figures 3A and 3B show the results ofITTs before and after three days of mibefradil injection inwild-type and db/db mice. The ITT results show nosignificant difference in the glucose tolerance in responseto insulin stimulation between the mibefradil-treated andsaline-treated groups (Figures 3A, 3B, 3C). This similar

response may be attributed to the existing high level ofinsulin in the db/db mice, which may have attenuated theeffects of further insulin application. In contrast, basalinsulin (after food deprivation for eight hours) in db/db micewas significantly reduced by seven days of treatment withmibefradil, from 4.80¡0.35 to 3.21¡0.12 ng/ml (p,0.05,n = 6) (Figure 3D). This finding indicates that T-type Ca2+

channel antagonists suppress insulin release from pancrea-tic b-cells of db/db mice. Therefore, T-type Ca2+ channelsmay play a role in the pathogenesis of hyperinsulinemia inthis type 2 diabetic model.

The maintenance of the basal glucose level is regulated bythe release of glucose from the liver and the uptake ofglucose into muscle, adipose and brain tissue. The profoundblood glucose-lowering effect of T-type Ca2+ channel

Figure 4 - Western blot analysis of T-type Ca2+ channel expressionin liver and brain tissues of diabetic and nondiabetic mice.Increased expression of a1G (Cav3.1) and a1H (Cav3.2) subunits

of T-type Ca2+ channels (n = 3). The molecular weights of Cav3.1and Cav3.2 are ,262 kDa. * p,0.05, n = 6, compared with WT NSgroup. Statistical analyses were performed with ANOVA.

Figure 5 - Immunohistochemical and immunofluorescence stain-ing showing an increased expression of a1G and a1H subunits ofT-type Ca2+ channels in liver and brain slide preparations. A andC) Immunohistochemical staining for T-type Ca2+ channel a1G (A)and a1H (C) subunits in liver slide preparations from non-diabeticmice (scale bar = 20 mm). B and D) Immunohistochemical stainingof db/db animal liver preparations (scale bar = 20 mm). E and G)Immunofluorescence staining showing the expression of a1G (E)

and a1H (G) subunits of T-type Ca2+ channels in brain slidepreparations from nondiabetic mice (scale bar = 25 mm). F and H)Immunofluorescence staining of brain slide preparations fromdb/db mice (scale bar = 25 mm).


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antagonists under high insulin concentrations suggests thatthe target organs of mibefradil may include the liver and thebrain, which is the upper-level control center for hepaticglucose production. We examined the protein levels of Cav3.1(a1G) and Cav3.2 (a1H) a1 subunits of T-type Ca2+ channelsin the liver and brain of db/db mice and control wild-typemice. As shown in Figure 4, both Cav3.1 and Cav3.2 in theliver and brain were significantly higher in db/db mice. Thesedata may explain the difference in the blood glucose-loweringeffects of mibefradil and NNC 55-0396 observed in db/db and

wild-type animals. These findings also suggest that thetargets of mibefradil and NNC 55-0396 are in the liver andthe brain in addition to pancreatic b-cells. Immunohisto-chemistry confirmed the results of Western blot analysis.Figure 5 shows that the expression of a1G and a1H washigher in db/db liver (Figure 5B and 5D) and brain (Figure 5Fand 5H) preparations compared to those in the wild-typeanimals.

Because db/db mice are characterized by continualfeeding as a result of a mutation in the leptin receptor, we

Figure 6 - Effects of mibefradil and/or nicardipine on the food consumption, mean blood pressure and heart rate of db/db andnondiabetic mice. A) Effect of mibefradil injection on the food intake of diabetic and nondiabetic mice. B) Effects of mibefradil andnicardipine on the mean blood pressure of diabetic and nondiabetic mice. C) Effects of mibefradil and nicardipine on the heart rate ofdiabetic and nondiabetic mice. * p,0.05, n = 6, compared with WT NS group; ** p,0.05, n = 6, compared with NS group of the sametype of mice at the same time. Statistical analyses were performed with Student’s t-test (A) or ANOVA (B and C) according to thespecific application.


65

also set up experiments to evaluate the effect of mibefradiland NNC 55-0396 on the feeding behavior of these animals.Mibefradil significantly reduced the food consumption ofdb/db mice but had no significant effect on the controlanimals (Figure 6A). This result also suggests mibefradil hasa central nervous system (CNS) target that may affect thefeeding behavior of the diabetic animals (15,16).

Mibefradil also has antihypertensive effects; thus, weexamined the effects of mibefradil and NNC 55-0396 on thecardiovascular functions of db/db mice. The mean bloodpressure (MBP) and heart rate after drug injection in the db/db and wild-type mice showed no significant difference inMBP (Figure 6B) or heart rate (Figure 6C) among mibefradil-,NNC 55-0396- and vehicle-treated groups. The physiologicalfunctions of T-type Ca2+ channels in cardiovascular regula-tion in db/db mice remain unclear.

& DISCUSSION

The db/db mouse is a well-established diabetic rodentmodel for studying the mechanisms of hyperglycemia,hyperinsulinemia, hyperlipidemia and related metabolicabnormalities. The present study is the first attempt toutilize this model for characterizing the role of T-type Ca2+

channels in hyperglycemia in vivo. The hallmark of diabetesmellitus is chronically high fasting blood glucose, resultingfrom impaired insulin sensitivity in the peripheral tissuesand defective insulin production by pancreatic b-cells.Using the db/db model, we showed that T-type Ca2+

channels might be novel therapeutic targets for hyperglyce-mia and that antagonists of T-type Ca2+ channels mightlower basal glucose by reducing liver glucose output anddecreasing basal insulin release or synthesis by pancreaticb-cells (Figure 7).

Basal insulin release from pancreatic b-cells may becontrolled by a different mechanism from glucose-stimu-lated insulin release. Hyperinsulinemia is commonlyobserved in pre-diabetic and type 2 diabetic patients.Chronic exposure to elevated insulin may result in thedevelopment of insulin resistance (23) and, in our opinion,may also lead to the attenuation of first-phase insulinrelease observed in type 2 diabetic patients. Therefore, thefact that T-type Ca2+ channel antagonists can reduce thebasal insulin is certainly an encouraging result because itmay not have a significant impact on glucose-stimulatedinsulin release, which is mostly mediated by the activationof L-type Ca2+ channels.

We have shown that T-type Ca2+ channel antagonistssignificantly reduced basal insulin release in wild-typeanimals compared to untreated db/db diabetic animals,indicating that the basal release of insulin from pancreatic b-cells might involve a T-type Ca2+ channel-mediatedmechanism. However, residual basal insulin is still elevatedafter T-type Ca2+ channel antagonist treatment, suggestingthat T-type Ca2+ channel antagonists may also suppressglucose output from the liver during fasting.

As shown in Figure 3, the results of ITT indicated thattreatment with T-type Ca2+ channel antagonists failed toimprove glucose disposal in db/db mice compared tountreated animals. We are currently unable to draw adefinitive conclusion about the effects of these drugs onimproving insulin sensitivity in muscle, adipose or braintissue. It is possible that the db/db mouse is not a suitablemodel for studying the role of T-type Ca2+ channels in

insulin sensitivity because the hyperglycemia of the db/dbmice cannot be reversed by an injection of insulin (TheJackson Laboratory, http://jaxmice.jax.org/list/ra66.html).

The overexpression of T-type Ca2+ channel a1G and a1Hsubunits in db/db mice compared with the wild-type animalssuggests that either a genetic predisposition favors the T-typeCa2+ channels or a secondary pathological regulation of theseproteins occurs in these mice. Nevertheless, the fact that T-type Ca2+ channel antagonists have an effect on glucoseregulation in wild-type animals (Figure 2B) suggests that T-type Ca2+ channels may also have physiological functions innon-diabetic animals at low expression levels.

Further investigation is required to confirm the effects ofT-type Ca2+ channel antagonists on metabolic regulation inthe CNS. The results of the present study show an increasedexpression of T-type Ca2+ channel a1G and a1H subunits inthe brain of db/db mice, suggesting that these channels mayplay critical roles in the mechanisms of hyperglycemia andhyperlipidemia in these animals. We also show that T-typeCa2+ channel antagonists caused a decrease in appetite inthe later phases of drug treatment, as well as a decrease inbody weight (data not shown), in db/db mice, indicating thatT-type Ca2+ channels indeed play an important role inmetabolic regulation in db/db mice; nevertheless, furtherinvestigation of this subject is necessary.

& ACKNOWLEDGMENTS

This work was supported by The Natural Science Foundation of the

Chongqing Science & Technology Commission (No. 2008B119) and a

Clinical Research Award [2008C169] from Third Military Medical

University. The authors thank Dr. Zhiming Zhu (High Blood Pressure

Endocrine Division, Daping Hospital, Third Military Medical University)

for kindly providing advice on mouse blood pressure and heart rate

monitoring instruments and Dr. Guansong Wang (Institute of Respiratory

Diseases, Xinqiao Hospital, Third Military Medical University) for

generously providing the experimental conditions. The authors also thank

Dr. Jonathan Pottle and Dr. Suresh C. Sikka for assistance in the

preparation of this manuscript.


Lu Y, Li M and Xu Z participated in the research design. Lu Y, Long M

and Xu Z conducted the experiments. Lu Y, Long M, Li M, Zhou S, Hu F

Figure 7 - A diagram showing the possible targeting sites ofmibefradil on the system regulating basal glucose. (+), secretionof hormones or nerve signals increased by the activity of T-typeCa2+ channels.


CLINICS 2014;69(1):61-67

66

and Xu Z contributed to the new reagents or analytic tools. Lu Y, Zhou S,

Hu F, Li M and Xu Z performed the data analysis. Li M, Lu Y and Xu Z

wrote the manuscript.

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Upregulation of SOCS-1 by Nutlin-3 in acute myeloidleukemia cells but not in primary normal cellsVeronica Tisato,I Alessia Norcio,II Claudio Celeghini,III Daniela Milani,I Arianna Gonelli,I Paola SecchieroI

I University of Ferrara, Department of Morphology, Surgery and Experimental Medicine and LTTA Centre, Ferrara, Italy. II Institute for Maternal and Child

Health, IRCCS ‘‘Burlo Garofolo’’, Trieste, Italy. III University of Trieste, Department of Life Sciences, Trieste, Italy.

OBJECTIVE: It has been shown that SOCS-1 plays an important role in the proper control of cytokine/growthfactor responses and acts as a tumor suppressor in acute myeloid leukemias. Therefore, the objective of thepresent study was to evaluate the in vitro effect of treatment with Nutlin-3, a small molecule inhibitor of theMDM2/p53 interaction, on the expression of the suppressor of cytokine signaling 1 in primary acute myeloidleukemia cells and in myeloid cell lines with differential p53 status.

METHOD: The expression of the suppressor of cytokine signaling 1 was quantitatively analyzed by real-time PCR inmyeloid p53wild-type (OCI and MOLM) and p53null HL-60, leukemic cell lines, in patient-derived acute myeloidleukemia blasts, and in primary normal cell types, such as macrophages, endothelial cells, and bone marrowmesenchymal stem cells. The p53-dependence of the suppressor of cytokine signaling 1 upregulation that is inducedby Nutlin-3 was analyzed in experiments performed using siRNA for p53, while the functional upregulation of thesuppressor of cytokine signaling 1 was analyzed by assessing the levels of phosphorylated STAT-3.

RESULTS: Nutlin-3 significantly upregulated the transcription of the suppressor of cytokine signaling 1 in p53wild-type

OCI and MOLM but not in p53deleted p53null HL60, myeloid leukemic cell lines, as well as in primary acute myeloidleukemia blasts. Conversely, and somewhat unexpectedly, Nutlin-3 did not modulate the suppressor of cytokinesignaling 1 expression in primary normal macrophages, endothelial cells, and bone marrow mesenchymal stem cells.The p53-dependent upregulation of the suppressor of cytokine signaling 1 by Nutlin-3 was associated with thedownregulation of phosphorylated STAT-3, a major molecular target of the suppressor of cytokine signaling 1.

CONCLUSION: Overall, our data suggest a potential role for the suppressor of cytokine signaling 1 as a therapeutictarget of Nutlin-3 in p53 wild-type acute myeloid leukemias.

KEYWORDS: Nutlin-3; SOCS-1; AML.

Tisato V, Norcio A, Celeghini C, Milani D, Gonelli A, Secchiero P. Upregulation of SOCS-1 by Nutlin-3 in acute myeloid leukemia cells but not inprimary normal cells. Clinics. 2014;69(1):68-74.



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& INTRODUCTION

The selective small molecule inhibitor Nutlin-3 bindsMDM2 in the p53 binding pocket with high selectivity andcan release p53 from negative control, leading to an effectivestabilization of p53 and activation of the p53 pathway (1). Anumber of studies have demonstrated that Nutlin-3 induces exvivo cytotoxic cell death of p53wild-type acute myeloid leuke-mias (AMLs) (2–7), and Nutlin-3 was recently shown toupregulate the suppressor of cytokine signaling 1 (SOCS-1) inprimary B leukemic cells through the mirR-155 pathway (8,9).Since the cloning of SOCS-1, it has become evident that the

SOCS proteins are important for the proper control of cytokineand growth factor responses, and the absence of SOCSproteins leads to excessive cytokine signaling (10). In addition,SOCS-1 also acts as a tumor suppressor, as documented by thefact that SOCS-1 silencing by DNA hypermethylation at thegene promoter region has been found in both solid tumors,such as hepatocarcinomas (11), and AMLs (12–17).

Based on these findings, in the present study, weevaluated the effect of Nutlin-3 treatment on SOCS-1expression in primary AML cells, as well as in myeloid celllines with differential p53 status. Additionally, the effect ofNutlin-3 exposure on SOCS-1 expression was evaluated inprimary normal cells characteristic of the bone marrowmicroenvironment, such as primary macrophages, endothe-lial cells, and multipotent stromal cells (MSCs).


Cell cultureThe myeloid p53wild-type (OCI and MOLM) and p53null

(HL-60) leukemic cell lines were purchased from the ATCC



DOI: 10.6061/clinics/2014(01)10

RAPID COMMUNICATION

68

(American Type Culture Collection, Manassas, VA). MOLMand HL-60 leukemic cell lines were cultured in RPMI-1640containing 10% FBS (both from Gibco BRL, Grand Island,NY), while OCI cells were cultured in alpha-MEM (LONZA,Basel, Switzerland) containing 10% FBS, as previouslydescribed (18).

Primary peripheral blood samples were collected in heparin-coated tubes from five AML patients and six healthy blooddonors after informed consent was obtained in accordance withthe Declaration of Helsinki and in agreement with institutionalguidelines. Peripheral blood mononuclear cells (PBMC) fromAML patients and healthy donors were isolated by gradientcentrifugation with lymphocyte cell separation medium(Cedarlane Laboratories, Hornby, ON). The percentage ofblasts among leukemic PBMC ranged from 60–85% for allpatients, as assessed by light microscopy and verified bystandard flow cytometry analysis. AML patient cells wereseeded at a density of 16106 cells/ml in RPMI containing 10%FBS (both from Gibco BRL). To obtain primary normal adherentmacrophages, blood donor PBMCs were seeded at a density of56106 cells/ml, and non-adherent cells were removed after 18hours. Adherent cells were cultivated in fresh RPMI mediumcontaining 10% FBS, as previously described (19).

Human umbilical vein endothelial cells (HUVECs) werepurchased from BioWhittaker (Walkersville, MD) andgrown on 0.2% gelatin-coated tissue culture plates inM199 endothelial growth medium supplemented with 20%FBS, heparin, and 50 mg/ml ECGF (all from BioWhittaker),as previously described (20). In all experiments, cells wereused between the 3rd and 5th passage in vitro. Bone marrow-derived MSCs were purchased from LONZA and grown inMSC Growth Medium (MSC-GM, LONZA), as previouslydescribed (21,22).

Culture treatments and evaluation of cellcytotoxicity

Cells were treated by adding Nutlin-3 (10 mM; obtainedfrom Cayman Chemical, Ann Arbor, MI) to the culturemedium. After treatment, cell viability was monitored up to48 hours by Trypan blue dye exclusion, as previouslydescribed (23). In parallel, the degree of apoptosis wasquantified by Annexin V-FITC/propidium iodide (PI)staining (Immunotech, Marseille, France) followed by flowcytometry analysis, as previously described (24,25). Toanalyze the cell cycle profile, cells were incubated with50 mM 5-bromodeoxyuridine (BrdU; Sigma Aldrich) at 37 Cfor 1 hour, the anti-BrdU antibody (BD BiosciencesPharmingen, Franklin Lakes, NJ) was bound to BrdU, andthe complex was detected by an FITC-conjugated secondaryantibody (Beckman-Coulter, Marseille, France) (26,27). Afteradditional staining with 50 mg/ml PI (Sigma-Aldrich), thecell samples were analyzed by flow cytometry.

RNA analysisAliquots of untreated and Nutlin-3-treated cells were

harvested for RNA extraction 24–48 hours post-treatment.Total RNA was extracted using the Qiagen RNeasy Plusmini kit (Qiagen, Hilden, Germany) according to thesupplier’s instructions. The integrity of the total RNApreparation was assessed using an Agilent 2100Bioanalyzer. RNA was transcribed into cDNA using theGEArray AmpoLabeling-LPR Kit (Superarray BioscienceCorporation, Frederick, MD). Investigations of SOCS-1 andmiR-155 gene expression were both carried out in RNA

samples with the real-time thermal analyzer Rotor-GeneTM

6000 (Corbett, Cambridge, UK) using SYBR Green-basedtechnology and the RT-PCR primer set for human SOCS-1cDNA or miR-155 (SABioscience, Frederick, MD). Geneexpression of the target sequences was normalized withrespect to the expression of endogenous controls. Eachsample was tested in triplicate.

Western blot analysesCells were lysed in ice-cold RIPA buffer (50 mM Tris pH

7.5, 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.25%sodium desoxycholate) supplemented with protease inhibi-tors (Complete, Roche; Germany) on ice for 1 hour (28).Before gel migration, samples were added to loading buffer(250 mM Tris pH 6.8, 2% SDS, 40% glycerin, 20% beta-mercaptoethanol) and boiled for 2 minutes. Equal amountsof protein for each sample were migrated in acrylamide gelsand blotted onto nitrocellulose filters, as previouslydescribed (29), before incubation with the following mono-clonal antibodies: anti-p53, anti-SOCS-1, anti-STAT-3, andanti-phospho-STAT-3 (all from Santa Cruz Biotechnology,Santa Cruz, CA), as well as anti-tubulin (Sigma-Aldrich).After incubation with peroxidase-conjugated anti-mouseIgG, specific reactions were revealed with the ECL detectionkit (Amersham Pharmacia Biotech).

Multiplex immunoassayThe MILLIPLEX MAP Human Multi-Pathway 9-plex

Magnetic Bead Signaling kit phosphoprotein (MerckMillipore, Billerica, MA, USA) was used to detect changesin phosphorylated ERK/MAP kinase 1/2, Akt, STAT-3,JNK, p70 S6 kinase, NF-kB, STAT-5A/B, CREB, and p38 incell lysates using the Luminex system, according to themanufacturer’s instructions. Briefly, cells were seeded at adensity of 16106/ml and treated with Nutlin-3 (10 mM). Atdifferent time points, cells were harvested in MILLIPLEXMAP lysis buffer (Merck Millipore) in the presence of theProtease Inhibitor Cocktail Set III (Calbiochem, San Diego,CA). Each lysate was diluted in the MILLIPLEX MAP AssayBuffer 2 (Merck Millipore), incubated at 4 C overnight, andanalyzed according to the assay protocol. MedianFluorescence Intensity (MFI) was measured with theLuminex System and normalized for mg of protein.

Transfection experimentsOCI cells (1.256106) were resuspended in 0.1 ml of

NucleofectorTM solution V from the human Nucleofectorkit V (Amaxa, Cologne, Germany). Two mg of plasmid DNA(GFP-construct) or 1 mg of siRNA was mixed with the 0.1 mlof cell suspension, transferred into a 2.0-mm electroporationcuvette, and nucleofected using an Amaxa Nucleofector IIapparatus, following the manufacturer’s guidelines and aspreviously described (30). After transfection, cells wereimmediately transferred into complete medium and cul-tured in six-well plates at 37 C. Transfection efficiency wasestimated in each experiment by scoring the number ofGFP-positive cells by flow cytometry analysis. siRNAs weredesigned and manufactured by Ambion Inc. (Austin, TX)according to the current guidelines for effective gene knock-down by this method and were validated in preliminaryexperiments. Negative control siRNA, comprised of a 19-bpscrambled sequence with 39 dT overhangs (Ambion’sSilencer negative control siRNA), was used to demonstratethat transfection did not induce non-specific effects on gene

CLINICS 2014;69(1):68-74 SOCS-1 upregulation by Nutlin-3 in AMLTisato V et al.

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expression. In miR-155 functional studies, cells weretransfected with the hsa-miR-155 anti-miR oligo to inhibitthe activity of endogenous miR-155. In parallel, cells weretransfected with the FAM-labeled miR negative controloligo as a non-targeting negative control and to monitortransfection efficiency. All oligos were obtained fromAmbion.

Statistical analysisDescriptive statistical analyses were conducted. For each

set of experiments, values are reported as the mean¡SD.Data were analyzed with Student’s t test, and statisticalsignificance was defined as p,0.05.

& RESULTS

Upregulation of SOCS-1 expression in p53wild-type

AML cells by Nutlin-3In the first group of experiments, we investigated whether

Nutlin-3 affected the expression of SOCS-1 mRNA in AMLcells. For this purpose, we assessed both patient (n = 5) AMLblasts and myeloid cell lines, characterized by either p53wild-type

(OCI and MOLM) or p53null (HL-60) status. As reported inTable 1, Nutlin-3 induced significant cytotoxicity in all patientAML blast cultures and p53wild-type myeloid cell lines but not inp53null HL-60 cells, which is consistent with the ability ofNutlin-3 to induce apoptosis and cell cycle arrest (2,4).Moreover, as shown in Figure 1A, treatment with Nutlin-3induced a significant (p,0.05) increase in SOCS-1 mRNA levelsin all primary AML blasts and in p53wild-type OCI and MOLMcells but not in p53null HL-60 cells. In an attempt to also evaluateSOCS-1 protein, we analyzed cell lysates by the Western blotassay. However, under our experimental conditions, thecommercially available antibodies did not allow for clear andreproducible protein detection (data not shown). The datashown in Figure 1A suggest but do not prove that the Nutlin-3-mediated upregulation of SOCS-1 in leukemic cells requires afunctional p53 pathway. Therefore, to ascertain the role of p53in Nutlin-3-induced upregulation of SOCS-1, we used pre-determined optimal experimental conditions to transfect siRNAagainst p53 in OCI cells to specifically knock down p53 geneexpression. As shown in Figure 1B, p53 knock down counter-acted the ability of Nutlin-3 to increase the level of p53 proteinin OCI cells and significantly (p,0.05) counteracted the abilityof Nutlin-3 to upregulate SOCS-1 expression. These datademonstrate that the ability of Nutlin-3 to transcriptionally

upregulate SOCS-1 expression in myeloid leukemic cells isdependent on p53.

The recent data demonstrating that Nutlin-3 downregu-lates miR-155 in primary B-CLL (8), along with a key role formiR-155 in regulating SOCS-1 expression (9,31,32), togethersupported the investigation of miR-155 knock down onSOCS-1 expression in response to Nutlin-3 in AML cellmodels. For this purpose, OCI cells were transfected eitherwith the hsa-miR-155 anti-miR oligo to inhibit the activity ofendogenous miR-155 or with the miR negative control oligobefore exposure to Nutlin-3. Treatment with Nutlin-3increased the levels of SOCS-1 mRNA in control (miRnegative control oligo) transfected cells, and this effect wasenhanced in cells transfected with anti-miR-155 (Figure 1C).Although these experiments did not address a potentialdirect link between Nutlin-3 and miR-155, which also acts asoncomiR in AML (33,34), they confirm an important role formiR-155 in SOCS-1 transcriptional modulation and showthat anti-miR-155 enhances the ability of Nutlin-3 toupregulate SOCS-1 mRNA expression in AML cells.

Because SOCS-1 is known to affect the intracellularsignaling pathways downstream of several cytokines andone of its major targets is represented by the inhibition ofthe JAK/STAT-3 pathway (10,11), we analyzed the func-tionality of Nutlin-3-mediated induction of SOCS-1 byassessing the levels of phosphorylated STAT-3. For thispurpose, leukemic cells were exposed to Nutlin-3 (10 mM),and at different time points, the phosphorylation pattern ofthe main intracellular pathways was evaluated by a multi-plex assay. Among the activated pathways (Figure 2),phosphorylation levels of both ERK1/2 and p38, althoughcharacterized by different baseline kinetics, were unaffectedby Nutlin-3 exposure (Figure 2A). Of interest, STAT-3phosphorylation, characterized by a progressive increaseover time in untreated cultures, was significantly inhibitedafter 24 hours of Nutlin-3 treatment, as further confirmed byWestern blot analysis (Figure 2B). Overall, these results areconsistent with those of a previous study showing thatERK1/2 is not affected by SOCS-1 (35), and they validate theability of SOCS-1 to downregulate the JAK/STAT-3 path-way upon Nutlin-3 treatment of leukemic cells.

Lack of SOCS-1 modulation by Nutlin-3 in primarynormal macrophages, endothelial cells, and MSC

Because SOCS-1 plays a role in different cell types (10), weanalyzed whether Nutlin-3 was able to also modulate SOCS-1 in primary normal cells, such as normal macrophages,endothelial cells, and bone marrow MSCs, which representthe relevant cytotypes present in the bone marrow micro-environment. As shown in Figure 3, Nutlin-3 did notsignificantly modulate SOCS-1 expression in any of theprimary cell types investigated, suggesting that the ability ofNutlin-3 to upregulate SOCS-1 was confined to p53wild-type

malignant AML cells (Figure 3).

& DISCUSSION

Previous findings indicated that SOCS-1 may function as atumor suppressor and that it is frequently found to besilenced in hematopoietic malignancies. In this context, wehave demonstrated that a novel non-genotoxic activator ofthe p53 pathway, Nutlin-3, is able to significantly upregulateSOCS-1 expression in primary AML blasts as well as inp53wild-type myeloid OCI and MOLM cell lines, but not in

Table 1 - Effect of Nutlin-3 treatment on myeloidleukemic cell viability.

Myeloid

leukemic cells

Cell viability of Nutlin-3 treated

cultures (% of untreated cultures)a

OCI 30¡7

MOLM 6¡3

HL60 88¡10

Pt. 1 23¡6

Pt. 2 29¡8

Pt. 3 36¡9

Pt. 4 22¡5

Pt. 5 32¡6

aLeukemic cells were exposed to Nutlin-3 (10 mM) and cell viability was

analysed at 48 hours of treatment. Data are reported as means¡SD of at

least three independent experiments.

SOCS-1 upregulation by Nutlin-3 in AMLTisato V et al.

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70

p53null HL-60 cells. In addition, we have demonstrated thatthe upregulation of SOCS-1 was functional because thisupregulation resulted in a significant downregulation of

STAT-3 phosphorylation levels. It is important to note thatthe activation of STAT-3 has been frequently reported in bothprimary human acute leukemia and leukemic cell lines (36),

Figure 1 - Transcriptional upregulation of SOCS-1 by Nutlin-3 in AML cells. In A, AML cell lines and primary AML patient (Pt.) cells were exposedto Nutlin-3 (10 mM). Levels of SOCS-1 mRNA were analyzed by quantitative RT-PCR. The results are expressed as the fold of increase in SOCS-1modulation by Nutlin-3 after 24 hours of treatment with respect to the control untreated cultures (set to 1) (hatched line). Data are reported asthe mean¡SD of the results from at least three experiments, each performed in triplicate. In B, OCI cells were transfected with controlscrambled (scr.) siRNA or p53 siRNA before treatment with Nutlin-3. p53 protein levels were analyzed by Western blot, and tubulin staining isshown as the loading control. Representative examples of Western blot results, from three independent experiments are shown. In parallel,levels of SOCS-1 mRNA were expressed as the fold increase with respect to the scrambled control transfected cultures. In C, OCI cells transfectedwith either hsa-miR-155 anti-miR oligo or miR negative control oligo were exposed to Nutlin-3 and SOCS-1 mRNA was expressed as the foldincrease in modulation. Asterisks, p,0.05 with respect to the untreated cultures or to the scrambled control transfected cultures.


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as well as that STAT-3 represents a major molecular target ofSOCS-1 (10,11). However, no modulation of SOCS-1 wasobserved in primary normal macrophages, endothelial cells,or bone marrow MSC, suggesting that Nutlin-3 selectivelyupregulated SOCS-1 in malignant cells but not in normal cellsof the bone marrow microenvironment. To date, we do nothave an explanation for this differential activity of Nutlin-3 inmyeloid leukemia cells and primary normal cells. We cannotexclude the possibility that Nutlin-3-mediated upregulationof SOCS-1 is also dependent on its ability to arrest cell cycleprogression in highly cycling malignant cells, as someexperimental observations (generated by blocking the cellcycle with different pharmacological agents) potentiallysupport this hypothesis. However, these data are still toopreliminary to draw any major conclusions. Additionally,a cycle-related effect of Nutlin-3 in upregulating SOCS-1was also suggested by the more robust SOCS-1 induction

in leukemic cell lines (3- to 8-fold) compared withprimary AML (2- to 4-fold), a result also previouslyreported for primary B-CLL cells (2- to 4-fold; 9). Thus,further investigation is clearly needed to elucidate themolecular mechanisms underlining the differential beha-vior of Nutlin-3 with respect to SOCS-1 induction innormal cells compared with malignant cells.

A second interesting finding of our study was theidentification that the Nutlin-3-mediated upregulation ofSOCS-1 was potentiated by a concomitant downregulationof miR-155, which is known to act as an oncomiR in AML(33,34). Thus, SOCS-1 represents a potential common targetbetween Nutlin-3 and antago-miR-155.

In conclusion, our data demonstrate that Nutlin-3, a smallmolecule with great therapeutic potential in hematologicalmalignancies (37,38), upregulates the SOCS-1 pathway inleukemic cells but apparently not in normal quiescent cells.

Figure 2 - Phosphorylation patterns of Nutlin-3-treated AML cells. Leukemic cells were exposed to Nutlin-3 (10 mM), andphosphoprotein levels were analyzed in cell lysates by the Multiplex assay at the indicated time points. Phosphoprotein levels areexpressed as the median fluorescence intensity (MFI). Data are reported as the mean¡SD. In A, phosphorylation levels of ERK1/2 andp38 in untreated and Nutlin-3-treated cultures are shown. In B, phosphorylation levels of STAT-3 in untreated and Nutlin-3-treatedcultures, analyzed with the Multiplex assay and validated by Western blot analysis, are shown. Asterisks, p,0.05 with respect to theuntreated cultures.


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Due to the role of SOCS-1 as an oncosuppressor, therapeuticcombinations based on Nutlin-3 warrant further investiga-tions with regard to the treatment of AML.

& ACKNOWLEDGMENTS

This work was supported by the Regione Friuli Venezia Giulia - AITT Project.


Tisato V, Norcio A, Celeghini C, Gonelli A, and Milani D performed the

experiments. Tisato V and Norcio A analyzed the data. Secchiero P

designed the study and wrote the manuscript. All authors have read and

approved the manuscript.

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Figure 3 - Lack of transcriptional modulation of SOCS-1 byNutlin-3 in normal cells. Normal adherent PBMC, endothelialcells, and MSC were exposed to Nutlin-3 (10 mM). Levels of SOCS-1 mRNA were analyzed by quantitative RT-PCR. The results areexpressed as the fold increase in SOCS-1 modulation by Nutlin-3after 24 hours of treatment with respect to the control untreatedcultures (set to 1) (hatched line). Data are reported as themean¡SD of the results from at least three experiments, eachperformed in triplicate.


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Av. - São Paulo - - Tel.: 11 3898-7850 / Fax: 11 3898-7878Rebouças, 600 05402-000 - Brasil 55 55 - [email protected]

www.convencoesreboucas.com.br

No Rebouças, pequenos eventos

tornam-se grandes.

Seja pequeno, médio ou grande, o Rebouças tem o espaço idealpara todo o tipo de evento. A melhor logística e infraestrutura deapoio maximizam os recursos operacionais e potencializam osencontros científicos, promocionais, institucionais ou culturais.

O é isso: um dos mais bemplanejados e tradicionais espaços receptivos do País,dimensionado para sediar congressos, convenções, exposições,seminários, simpósios, cursos, lançamento de produtos, entreoutros, bem no coração da cidade de São Paulo.

Centro de Convenções Rebouças

Atualmente possui 8 ambientes climatizados e modernizados queatendem até 1.200 participantes, além de uma equipe preparadae empenhada em acompanhar cerca de 280 eventos por ano.

EM BREVE, estará ampliando suas instalações, que permitirá odobro da capacidade de público.

CLINICS January 2014

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