DEPARTMENT OF PEDIATRICS AND

COMMUNICABLE DISEASES

15th Annual Pediatric Research Symposium September 30 – October 1, 2004

Towsley Center

DEPARTMENT OF PEDIATRICS AND COMMUNICABLE DISEASES C.S. MOTT CHILDREN’S HOSPITAL UNIVERSITY OF MICHIGAN 1500 East Medical Center Drive, D3202 MPB Ann Arbor, MI 48109-0718 (734) 647-9928

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PROGRAM-AT-A-GLANCE September 30 – October 1, 2004

TIME

ACTIVITY AND LOCATION

THURSDAY, SEPTEMBER 30

4:00 pm – 6:00 pm Reception and Combined Poster Session

Towsley Lobby

FRIDAY, OCTOBER 1

7:00 am – 8:00 am Registration, Continental Breakfast, and Poster Viewing Towsley Lobby

8:05 am – 11:15 am Plenary Session Dow Auditorium

11:15 am – 1:00 pm Breakout Sessions

Basic Science: Molecular Mechanisms Towsley Dining Room

Basic Science: Pathophysiologic Pathways

Sheldon Auditorium

Clinical Investigation G2315 Towsley

Health Services Research

G2314 Towsley 1:00 pm – 2:30 pm Lunch

Pick up boxed lunch at the Registration Desk

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Welcome This year’s program includes keynote lectures from Dr. Phyllis A. Dennery, Division of Neonatology, Children’s Hospital of Philadelphia; and Dr. Ron G. Rosenfeld, Senior Vice President for Medical Affairs for the Lucile Packard Foundation for Children’s Health. From the 70 abstracts submitted by UM Pediatrics faculty, fellows, residents, and students, the following awardees have been chosen to provide oral presentations of their work on Friday, October 1. Faculty awardee presentations will be during the morning Plenary Session; Fellow and Student awards will be presented during the Breakout Sessions: Maide O. Raeker, Best Abstract Catherine E. Keegan, Basic Science Research Faculty Award Michael D. Cabana, Clinical Investigation/Health Services Research Faculty Award Sung Won J. Choi, Basic Science Research Fellow Award Jeffrey G. Gossett, Clinical Investigation/Health Services Research Fellow Award Molly B. Pendergast, Medical Student Award Yu-Hwai Tsai, Undergraduate Award This meeting has been made possible in part through the generosity of our supporters and the research efforts of faculty, fellows, residents, medical and graduate students. We are very proud of the extent and breadth of our research programs and activities, and we trust you will enjoy the day’s activities. We would like to thank the abstract reviewers for their time and effort in the review process in this important endeavor.

Department of Pediatrics and Communicable Diseases Valerie P. Castle, M.D. John J. LiPuma, M.D. Professor and Chair Associate Chair for Research

Daniel S. Wechsler, M.D., Ph.D. Symposium Program Chair

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Research Advisory Committee Members

John D. Barks, MD Associate Professor,

Neonatal/Perinatal Medicine

Richard F. Ransom, PhD Research Investigator, Nephrology

John R. Charpie, MD, PhD Clinical Associate Professor,

Cardiology

Mark W. Russell, MD Assistant Professor, Cardiology

Sarah J. Clark, MPH Research Investigator and General

Pediatrics Assoc Director for Research

William E. Smoyer, MD Associate Professor and Director,

Nephrology

Alex R. Kemper, MD Assistant Professor, General

Pediatrics

Delia M. Vázquez, MD Associate Professor, Endocrinology

John J. LiPuma, MD (Chair)

Professor, Infectious Diseases Daniel S. Wechsler, MD, PhD

Associate Professor, Hematology/Oncology

Frank W. Moler, MD Associate Professor, Critical Care

Medicine

Acknowledgements

The University of Michigan Department of Pediatrics would like to thank the following for their generous support:

Amgen Eli Lilly

Mead Johnson Nutritionals Nabi Pharmaceuticals

Ross Products Division, Abbott Laboratories TAP Pharmaceuticals

The University of Michigan Medical School is accredited by the Accreditation Council for Continuing Medical Education (ACCME) to provide continuing medical education for physicians. The University of Michigan Medical School takes responsibility for the content, quality, and scientific integrity of this CME activity. The University of Michigan Medical School designates this educational activity for a maximum of 4.75 category 1 credits towards the AMA Physician's Recognition Award. Each physician should claim only those credits that he/she actually spent in the activity.

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Keynote Speakers

Dr. Phyllis A. Dennery is the former Associate Chair for the Division of Pediatric Neonatal and Perinatal Medicine and Director of Pediatric Neonatal Research at Stanford University School of Medicine. She is currently Associate Professor of Pediatrics/Neonatology at the University of Pennsylvania School of Medicine and Chief of Neonatology at the Children’s Hospital of Philadelphia. Dr. Dennery earned her B.Sc. in Bio-Genetics from McGill University, Quebec, Canada in 1980 and her M.D. from Howard University, Washington, D.C. in 1984. Her research involves the role and regulation of heme oxygenase (HO) during lung development and the description of lung HO expression throughout development. She has authored numerous papers and received several awards, including the Ross Young Investigator Award and a Andrew W. Mellon Fellowship. She is an active member of many scientific organizations including, The American Thoracic Society, the Society for Pediatric Research (SPR)/Pediatric Academic Societies (PAS) and the American Pediatric Society. Dr. Ron G. Rosenfeld is the former Chair of Pediatrics at the Oregon Health and Sciences University in Portland, Oregon. He is currently the Senior Vice President for Medical Affairs for the Lucile Packard Foundation for Children’s Health, Palo Alto, California. Dr. Rosenfeld earned a BA, with highest honors, from Columbia University in 1968, and an MD with honors from Stanford University in 1973. He continued postgraduate work at Stanford, ultimately joining its faculty and becoming a Professor of Pediatrics in 1989. Dr. Rosenfeld is an internationally renowned authority on the endocrine basis of growth and development. He has been at the forefront of understanding the biology of growth hormones and growth factors for more than 25 years. His research has focused on the study of insulin-like growth factors during fetal and postnatal growth, as well the role of these factors in cancer. Dr. Rosenfeld has authored more than 500 publications and 8 books and received numerous awards, including a Mellon Foundation Fellowship, the Basil O’Connor Award from the March of Dimes, and a NIH Career Development Award.

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University of Michigan Medical School Department of Pediatrics

15th ANNUAL RESEARCH SYMPOSIUM

Thursday, September 30 & Friday, October 1, 2004 Towsley Center

THURSDAY, SEPTEMBER 30, 2004 4:00 – 6:00 pm Reception and Combined Poster Session

Towsley Center Lobby FRIDAY, OCTOBER 1, 2004 7:00 am Registration, Continental Breakfast, and Poster Viewing Towsley Center Lobby 8:05 Welcome – Dow Auditorium

Dr. John LiPuma, Professor and Associate Chair for Research Dr. Valerie Castle, Professor and Chair, Department of Pediatrics Dr. Daniel Wechsler, Professor and Symposium Program Chair

Introudction of Keynote Speaker – Daniel Wechsler

8:15 Keynote Speaker HEME OXYGENASE AS A SIGNALING MOLECULE.

Phyllis A. Dennery, Children’s Hospital of Philadelphia 9:15 OBSCURIN IS REQUIRED FOR STRIATED MUSCLE

DEVELOPMENT AND SOMITOGENESIS IN ZEBRAFISH. MO Raeker, F Su, SB Sutter, S Lyons, and MW Russell, Ann Arbor,

MI. (Best Abstract Awardee) Abstract 1 9:30 UROGENITAL AND CAUDAL DYSGENESIS IN

ADRENOCORTICAL DYSPLASIA (ACD) MICE IS CAUSED BY A SPLICING MUTATION IN A NOVEL TELOMERIC REGULATOR. CE Keegan, JE Hutz, T Else, M Adamska, SP Shah, AE Kent, JM Howes, WG Beamer, and GD Hammer, Ann Arbor, MI and Bar Harbor, ME. (Basic Science Faculty Awardee)

Abstract 2

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9:45 RANDOMIZED CONTROLLED TRIAL OF PHYSICIAN EDUCATION TO IMPROVE PATIENT OUTCOMES FOR ASTHMA. MD Cabana, KK Slish, D Evans, R Mellins, RW Brown,

X Lin, B Nan, and NM Clark, Ann Arbor, MI and New York, NY. (Clinical Investigation/Health Services Research Faculty Awardee) Abstract 3

10:00 – 10:15 Break

Introudction of Keynote Speaker – Daniel Wechsler 10:15 Keynote Speaker

WHY DO WE GROW (OR NOT GROW): THE EXPANDING DEFINITION OF IGF DEFICIENCY" Ron G. Rosenfeld, Oregon Health and Sciences University

11:15 Introduction – Daniel Wechsler

Basic Science – Pathophysiologic Pathways Sheldon Auditorium

Patrick D. Boyer, Presiding 11:30 TEMPORAL AND REGION SPECIFIC ABNORMALITIES IN

ENTERIC NERVOUS SYSTEM STEM CELL DEVELOPMENT IN THE HIRSCHSPRUNG RAT. Y Tsai and CE Gariepy, Ann Arbor, MI. (Undergraduate Awardee) Abstract 4

11:45 CAMPYLOBACTER JEJUNI GENES INVOLVED IN INVASION

INTO GUT EPITHELIAL CELLS. GC Fogg and VJ DiRita, Ann Arbor, MI. Abstract 5

12:00 ROLE OF SELECTINS AND SELECTIN LIGAND RECEPTORS

IN THE DEVELOPMENT OF IDIOPATHIC PNEUMONIA SYNDROME AFTER ALLOGENIC STEM CELL TRANSPLANTATION. SW Choi, K Olkiewicz, C Liu, JB Lowe, G Hildebrandt, L Corrion, and KR Cooke, Ann Arbor, MI. (Basic Science Fellow Awardee) Abstract 6

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12:15 ABROGATION OF CXCR3 RECEPTOR:LIGAND INTERACTIONS REDUCES LEUKOCYTE RECRUITMENT TO THE LUNG AND THE SEVERITY OF EXPERIMENTAL IDIOPATHIC PNEUMONIA SYNDROME. KR Cooke, LA Corrion, KM Olkiewicz, S Choi, C Liu, and GC Hildebrandt, Ann Arbor, MI and Gainesville, FL. Abstract 7

12:30 DIFFERENTIAL SUSCEPTABILITY OF NORMAL AND

CYSTIC FIBROSIS AIRWAY EPITHELIAL CELL CULTURES TO BURKHOLDERIA CEPACIA COMPLEX. U Sajjan, M Hershenson, J Forstner, S Keshavjee, and JJ LiPuma, Ann Arbor, MI and Toronto, Ontario, Canada. Abstract 8

12:45 OXIDANT STRESS AND MYOCARDIAL DYSFUNCTION

AFTER ISCHEMIA-REPERFUSION INJURY IN NEONATAL RABBITS. CL Buller and JR Charpie, Ann Arbor, MI.

Abstract 9

Basic Science – Molecular Mechanisms Towsley Dining Room

Donna M. Martin, Presiding 11:30 MUTATIONS IN A NOVEL GENE, NPHP5, CAUSE

NEPHRONOPHTHISIS WITH EARLY-ONSET RETINITIS PIGMENTOSA. EA Otto, B Loeys, S Fan, H Khanna, U Muerb, JF O'Toole, M Attanasio, B Utsch, A Kispert, M Tsuda, DS Williams, B Margolis, A Swaroop, and F Hildebrandt, Ann Arbor, MI, Baltimore, MD, Hannover, Germany, Hyogo, Japan, and San Diego, CA.

Abstract 10 11:45 INCREASED HSP27 EXPRESSION MIMICS THE

PROTECTION FROM PODOCYTE INJURY AND ACTIN FILAMENT DISRUPTION CONFERRED BY DEXAMETHA-SONE. RF Ransom, V Vega-Warner, J Klein, and WE Smoyer, Ann Arbor, MI and Louisville, KY. Abstract 11

12:00 HOMO-OLIGOMERIZATION IN API2-MALT1 FUSION

PROTEIN-MEDIATED ONCOGENESIS. LM McAllister-Lucas, P Kuffa, P Lucas, N Inohara, and G Nunez, Ann Arbor, MI.

Abstract 12

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12:15 PERTURBED ENDOCYTOSIS: A NOVEL LEUKEMOGENIC MECHANISM IN AML. M Pendergast, AC Walker, MM Chao, and DS Wechsler, Ann Arbor, MI. (Medical Student Awardee)

Abstract 13 12:30 TARGETED ADAMTS13 DEFICIENCY IN MICE DOES NOT

RESULT IN CONGENITAL THROMBOTIC THROMBOCYTO-PENIC PURPURA. DG Motto, W Zhang, G Zhu, D Ginsburg, W Zhang, G Zhu, J Homeister, and D Ginsburg, Ann Arbor, MI.

Abstract 14 12:45 ROLE OF THE TANDEM MYOSIN LIGHT CHAIN KINASE

FAMILY MEMBERS OBSCURIN-MLCK AND SPEG IN MYOCYTE DIFFERENTIATION. SB Sutter, MO Raeker, AB Borisov, and MW Russell, Ann Arbor, MI. Abstract 15

Clinical Research G2315 Towsley

John E. Levine, Presiding 11:30 ETANERCEPT FOR THE TREATMENT OF IDIOPATHIC

PNEUMONITIS POST ALLOGENEIC STEM CELL TRANSPLANTATION. GA Yanik, JE Levine, JP Uberti, RJ Hutchinson, JL Ferrara, and KC Cooke, Ann Arbor, MI.

Abstract 16 11:45 IMPLEMENTING INSULIN PUMP THERAPY WITH

PRESCHOOLERS WITH TYPE 1 DIABETES MELLITUS: A PILOT INVESTIGATION OF MEDICAL AND QUALITY OF LIFE OUTCOMES. L Opipari-Arrigan, EM Fredericks, N Burkhart, L Dale, M Hodge, JC Kichler, P Olton, and C Foster, Ann Arbor, MI. Abstract 17

12:00 CATHETER BASED DECOMPRESSION OF THE LEFT

ATRIUM IN PATIENTS WITH HYPOPLASTIC LEFT HEART SYNDROME AND RESTRICTIVE ATRIAL SETPUM IS SAFE AND EFFECTIVE. JG Gossett, AP Rocchini, TR Lloyd, RG Ohye, EL Bove, and JN Graziano, Ann Arbor, MI. (Clinical Investigation/Health Services Research Fellow Awardee)

Abstract 18

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12:15 TRANSCATHETER CRYOTHERAPY FOR TREATMENT OF SUPRAVENTRICULAR TACHYCARDIA IN YOUNG PATIENTS. PS Fischbach, EV Saarel, and M Dick II, Ann Arbor, MI and Cleveland, OH. Abstract 19

12:30 COMPARISON OF HIGH RESOLUTION COMPUTERIZED

TOMOGRAPHY (HRCT) OF THE CHEST AND PULMONARY FUNCTION TESTING IN EVALUATING THE EFFECT OF TOBRAMYCIN SOLUTION FOR INHALATION (TSI) IN CYSTIC FIBROSIS (CF) SUBJECTS WITH MILD LUNG DISEASE. SZ Nasr, PJ Strause, and P Eckhardt, Ann Arbor, MI. Abstract 20

12:45 BARRIERS TO SCREENING INFANTS FOR RETINOPATHY

OF PREMATURITY AFTER DISCHARGE OR TRANSFER FROM A NEONATAL INTENSIVE CARE UNIT. MA Attar, M Gates, A Iatrow, S Lang, and S Bratton, Ann Arbor, MI.

Abstract 21

Health Services Research G2314 Towsley

Sarah J. Clark, Presiding 11:30 USE OF ANTI-INFLAMMATORY MEDICATIONS AND

EMERGENCY DEPARTMENT RELIANCE AMONG CHILDREN WITH ASTHMA. KJ Dombkowski, DR Gill, and SJ Clark, Ann Arbor, MI. Abstract 22

11:45 BLOOD LEAD TESTING AMONG MEDICAID-ENROLLED

CHILDREN. AR Kemper, LM Cohn, KE Fant, and KJ Dombkowski, Ann Arbor, MI. Abstract 23

12:00 PARENTS' TRUST IN THEIR CHILD'S PHYSICIAN. KL Moseley, SJ Clark, A Gebremarium, MJ Sternthal, and AR Kemper,

Ann Arbor, MI. Abstract 24 12:15 VARIATION IN RESUSCITATION MEDICATION DOSES

LISTED ON CODE CARDS OF PEDIATRIC TRAINING INSTITUTIONS. EY Yoon and MM Davis, Ann Arbor, MI.

Abstract 25

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12:30 DISPARITIES IN HEALTH CARE UTILIZATION AND EXPENDITURES FOR LOW-INCOME VS HIGHER INCOME OVERWEIGHT YOUTH. MM Davis, A Gebremariam, and IR Nabry, Ann Arbor, MI. Abstract 26

1:00 Lunch and Poster Session 1 FETAL PROGRAMMING: TESTOSTERONE TREATMENT

DURING PREGNANCY ADVANCES PLACENTOME DIFFERENTIATION. OI Astapova, TL Steckler, LM Jackson, PS MohanKumar, SP Ford, DL Foster, and V Padmanabhan, Ann Arbor, MI, East Lansing, MI, and Laramie, MI. Abstract 27

2 A NEW GENE LOCUS FOR NEPHRONOPHTHISIS MAPS TO

CHROMOSOME 19P. M Attanasio, B Utsch, JL Weber, HC Hennies, J Hoefele, E Otto, and F Hildebrandt, Ann Arbor, MI, Marshfield, WI, and Berlin, Germany. Abstract 28

3 BILATERAL ALTERATIONS OF HIPPOCAMPAL

DENDRITIC MORPHOLOGY AFTER UNILATERAL CEREBRAL HYPOXIA-ISCHEMIA IN THE IMMATURE RAT. JD Barks, BT Felt, YQ Liu, J Shao, S Choi, T Schallert, YQ Liu, and B Kolb, Ann Arbor, MI, Austin, TX, and Lethbridge, Alberta, Canada. Abstract 29

4 DIFFERENTIAL REGULATION OF OBSCURIN AND

OBSCURIN-MLCK EXPRESSION DURING CARDIAC AND SKELETAL MUSCLE GROWTH. AB Borisov, MO Raeker, A Kontrogianni, RJ Bloch, and MW Russell, Ann Arbor, MI and Baltimore, MD. Abstract 30

5 ESSENTIAL ROLE OF OBSCURIN IN

MYOFIBRILLOGENESIS AND MYOFIBRILLAR ALIGNMENT: FAILURE TO INCORPORATE MYOSIN INTO THE SARCOMERE IN CARDIAC MYOCYTES DEPLETED OF OBSCURIN USING RNAI-MEDIATED INHIBITION.

AB Borisov, SB Sutter, A Kontrogianni, RJ Bloch, and MW Russell, Ann Arbor, MI and Baltimore, MD. Abstract 31

6 INTEGRATING A SOCIOCULTURAL MEDICINE

CURRICULUM IN PEDIATRICS. M Bozynski, T Tang, H Haftel, R Anderson, J Mitchell, and V Press, Ann Arbor, MI. Abstract 32

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7 DEVELOPMENT OF A TRANSGENIC MOUSE MODEL IN UNDERSTANDING THE MECHANISM OF HUMAN BETA GLOBIN SWITCHING. AD Campbell, Q Lui, O Tanabe, and JD Engel, Ann Arbor, MI. Abstract 33

8 DEVELOPMENTAL IRON DEFICIENCY IMPAIRS

WATERMAZE PERFORMANCE FOR RATS. BT Felt, H Tian, J Shao, JD Barks, and T Schallert, Ann Arbor, MI and Austin, TX. Abstract 34

9 MECHANISMS OF LEUKEMIC TRANSFORMATION BY MLL-

CALM: IDENTIFICATION OF A CALM-DERIVED TRANSCRIPTIONAL REGULATORY DOMAIN. EJ Fox, MM Chao, and DS Wechsler, Ann Arbor, MI. Abstract 35

10 ASSESSMENT OF RIGHT VENTRICULAR FUNCTION IN

THE FETUS WITH HYPOPLASTIC LEFT HEART SYNDROME: USE OF THE MYOCARDIAL PERFORMANCE INDEX. SK Gelehrter and CA Gomez, Ann Arbor, MI.

Abstract 36 11 DEVELOPMENT OF A 3-DIMENSIONAL PHYSIOLOGICAL

MODEL OF THE INTERNAL ANAL SPHINCTER BIOENGINEERED IN-VITRO FROM ISOLATED SMOOTH MUSCLE CELLS. L Hecker, K Baar, and KN Bitar, Ann Arbor, MI.

Abstract 37 12 NEUROBLASTOMA CELL TYPE HETEROGENEITY

DEFINED BY GENOMIC AND PROTEOMIC METHODS. JA Jarzembowski, R Kuick, AW Opipari, and VP Castle, Ann Arbor,

MI. Abstract 38 13 GENETIC ASSOCIATIONS IN NONTYPEABLE

HAEMOPHILUS INFLUENZAE STRAINS CONTRIBUTING TO OTITIS MEDIA. PC Juliao, J Xie, CF Marrs, and JR Gilsdorf, Ann Arbor, MI. Abstract 39

14 BOWMAN'S CAPSULE EXPRESSION OF A PODOCALYXIN

TRANSGENE: A NOVEL METHOD TO MARK THE PARIETAL EPITHELIUM. DB Kershaw, S Pauly, and Y Li, Ann Arbor, MI. Abstract 40

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15 THE YEAST ASPARTYL PROTEASE YAPSIN 1 IS A

COMPONENT OF THE TRANSCRIPTIONAL RESPONSE TO CELL WALL STRESS AND REQUIRED FOR CELL WALL INTEGRITY. DJ Krysan and RS Fuller, Ann Arbor, MI.

Abstract 41 16 DETECTION OF PODOCYTE ABNORMALITIES IN

NEPHROLOGIC DISORDERS, DIABETES AND SYSTEMIC LUPUS ERYTHEMATOSUS. DM Kurnit K Yang, H Yang, Y Tang, and A Khafagi, Ann Arbor, MI. Abstract 42

17 FETAL PROGRAMMING: DEVELOPMENTAL

PROGRESSION OF ESTRADIOL POSITIVE FEEDBACK DEFECTS IN PRENATALLY TESTOSTERONE-TREATED SHEEP. JS Lee, M Manikkam, HN Sarma, C Herkmer, DL Foster, and V Padmanabhan, Ann Arbor, MI. Abstract 43

18 VITAMIN D DEFICIENCY: AN UNRECOGNIZED PROBLEM

IN INFANTS AND CHILDREN IN BOSTON. JM Lee, JR Smith, BL Philipp, and MF Holick, Ann Arbor, MI and Boston, MA.

Abstract 44 19 SECRETION AND POST-TRANSLATIONAL PROCESSING OF

A NOVEL TESTIS INSULIN-LIKE PROTEIN. C Lu, S Singh, RB Gibbs, WH Walker, RK Menon, and S Singh, Ann Arbor, MI and Pittsburgh, PA. Abstract 45

20 FETAL PROGRAMMING: PROGESTERONE TREATMENT

DURING THE POSTPUBERTAL ANESTROUS SEASON PARTIALLY OVERCOMES FOLLICULAR DEFICIT BUT FAILS TO RESTORE NORMAL LUTEAL FUNCTION. M Manikkam, TL Steckler, EK Inskeep, and V Padmanabhan, Ann Arbor, MI. Abstract 46

21 MUTATIONS IN NPHS1 AND NPHS2 IN PATIENTS WITH

STEROID-RESISTANT NEPHROTIC SYNDROME. BE Mucha, F Hildebrandt, RG Ruf, and M Schultheiss, Ann Arbor, MI and Berlin, Germany. Abstract 47

22 BIOCHEMICAL SIGNALING MECHANISMS REGULATING

RHINOVIRUS-INDUCED AIRWAY EPITHELIAL CELL RESPONSES. D Newcomb, Y Jia, S Nanua, Y Ads, L Zhou, U Sajjan, and M Hershenson, Ann Arbor, MI. Abstract 48

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23 RETINITIS PIGMENTOSA AND RENAL FAILURE IN A PATIENT WITH MUTATIONS IN INVERSIN. JF O'Toole, E Otto, Y Frishberg, and F Hildebrandt, Ann Arbor, MI and Jerusalem, Israel. Abstract 49

24 DISTRIBUTION OF PEDIATRIC CRITICAL CARE

RESOURCES IN THE UNITED STATES. FO Odetola, SJ Clark, GL Freed, SL Bratton, and MM Davis, Ann Arbor, MI.

Abstract 50 25 THE K.I.D.S. PROJECT: MEDICAL AND PSYCHOSOCIAL

OUTCOMES OF AN INTERVENTION TO KICK IN DIABETES SUPPORT FOR ECONOMICALLY DISADVANTAGED ADOLESCENTS WITH TYPE 1 DIABETES MELLITUS. L Opipari-Arrigan, JC Kichler, EM Fredericks, N Burkhart, L Dale, S Eder, A Silverman, S Bratton, and C Foster, Ann Arbor, MI and Milwaukee, WI. Abstract 51

26 DIRECT ASSOCIATION OF RHOA WITH SPECIFIC

DOMAINS OF PKCα. H Pang, S Somara, SB Patil, and KN Bitar, Ann Arbor, MI. Abstract 52

27 TACROLIMUS IN STEROID RESISTANT NEPHROTIC

SYNDROME. KE Papez, M Mills, SC White, PD Brophy, and DB Kershaw, Ann Arbor, MI. Abstract 53

28 HEMODIALYSIS AND HEMOFILTRATION IN PEDIATRICS:

A NEW APPROACH TO INTOXICATION. KE Papez, LS Allred, TA Mottes, TL Kudelka, TR Kelly, BJ Adams, RS Nievaard, JJ Lin, DB Kershaw, and PD Brophy, Ann Arbor, MI. Abstract 54

29 MORTALITY AND PROGRESSION OF CHRONIC KIDNEY

DISEASE (CKD) AMONG VETERANS WITH DIABETES. UD Patel, AO Ojo, EW Young, and RA Hayward, Ann Arbor, MI. Abstract 55 30 RECEIPT OF CARDIOVASCULAR (CV) RISK-REDUCTIVE

MEDICATIONS AMONG VETERANS WITH DIABETES AND CHRONIC KIDNEY DISEASE (CKD). UD Patel, AO Ojo, EW Young, and RA Hayward, Ann Arbor, MI. Abstract 56

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31 ASSOCIATION OF HSP27 WITH MYPT, ROCK II AND PHOSPHO-RHOA IN THE PARTICULATE FRACTION DURING SUSTAINED CONTRACTION OF SMOOTH MUSCLE OF THE COLON. SB Patil and KN Bitar, Ann Arbor, MI. Abstract 57

32 NESTIN-CRE FATE MAPPING IN THE DEVELOPING MOUSE

EMBRYO. H Ramaprakash, JM Skidmore, AM Sclafani, and DM Martin, Ann Arbor, MI. Abstract 58

33 PITX2 AND LMX1B INTERACTIONS IN THE DEVELOPING

MAMMALIAN SUBTHALAMIC NUCLEUS. JM Skidmore and DM Martin, Ann Arbor, MI. Abstract 59

34 CALCIUM-DEPENDENT PKC-MEDIATED ACETYL-

CHOLINE-INDUCED PHOSPHORYLATION OF TROPOMYOSIN IN COLONIC SMOOTH MUSCLE CELLS.

S Somara and KN Bitar, Ann Arbor, MI. Abstract 60 35 DEFINING THE DISTRIBUTION OF RALSTONIA SPECIES IN

CYSTIC FIBROSIS BY PCR ANALYSIS. T Spilker, R Reik, T Coenye, P Vandamme, and JJ LiPuma, Ann Arbor, MI and Gent, Belgium. Abstract 61

36 FETAL PROGRAMMING: PRENATAL EXPOSURE TO

BISPHENOL-A AND METHOXYCHLOR DISRUPTS OVARIAN FOLLICULAR DYNAMICS. TL Steckler, M Manikkam, EK Inskeep, and V Padmanabhan, Ann Arbor, MI and Morgantown, WV. Abstract 62

37 HISTONE DEACETYLASE INHIBITORS AS AN APPROACH

TO TREAT NEUROBLASTOMA. C Subramanian, AW Opipari, R Kwok, and VP Castle, Ann Arbor, MI. Abstract 63

38 CHRONIC GRAFT VERSUS HOST DISEASE AFTER

ALLOGENEIC PERIPHERAL BLOOD STEM CELL TRANSPLANTATION IN CHILDREN. CD Thornburg, GA Yanik,

RJ Hutchinson, KR Cooke, JL Ferrara, and JE Levine, Ann Arbor, MI. Abstract 64 39 MODULATION OF HOST CHEMOKINE RESPONSES BY

LATENT GAMMAHERPESVIRUS INFECTION. JB Weinberg, GS Stempfle, and KR Spindler, Ann Arbor, MI. Abstract 65

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40 INFLAMMATORY RESPONSES IN THE LUNG AND BRAIN INDUCED BY ACUTE RESPIRATORY INFECTION WITH MOUSE ADENOVIRUS TYPE 1. JB Weinberg, GS Stempfle, and KR Spindler, Ann Arbor, MI. Abstract 66

41 THE C/EBPα MYELOID TRANSCRIPTION FACTOR

REGULATES EXPRESSION OF MXI0. BF Wolf and DS Wechsler, Ann Arbor, MI. Abstract 67

42 SUBVENTRICULAR ZONE PROLIFERATION AFTER

αAMINO-3-HYDROXY-5-METHYL-4-ISOXAZOLEPROPIONIC ACID (AMPA) RECEPTOR-MEDIATED NEONATAL BRAIN INJURY. G Xu, FS Silverstein, J Ong, and JD Barks, Ann Arbor, MI. Abstract 68

43 DIFFERENTIAL EXPRESSION OF MXI0 AND MXI1 IN CELL

DIFFERENTIATION. D Zheng and DS Wechsler, Ann Arbor, MI. Abstract 69

44 REGULATION OF CELL SIZE AND CONTRACTILE

PROTEIN EXPRESSION IN AIRWAY SMOOTH MUSCLE CELLS. L Zhou, A Goldsmith, and MB Hershenson, Ann Arbor, MI. Abstract 70

45 UTILIZATION OF INTRACRANIAL PRESSURE MONITORS

IN CRITICALLY ILL CHILDREN WITH MENINGITIS. FO Odetola, JM Tilford, and MM Davis, Ann Arbor, MI and Little

Rock, AR. Abstract 71

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Plenary Session

1 OBSCURIN IS REQUIRED FOR STRIATED MUSCLE DEVELOP-MENT AND SOMITOGENESIS IN ZEBRAFISH. M Raeker, F Su, S Sutter, S Lyons, M Russell, University of Michigan, Ann Arbor, MI Division of Pediatric Cardiology Obscurin is a giant sarcomere-associated protein whose interactions with titin and ankyrin suggest that it has an important role in myofibril assembly and structural support. Obscurin and its invertebrate orthologue Unc-89 are expressed in striated muscle across a range of species, from Drosophila to human, suggesting its functions are evolutionarily conserved. Hypothesis: If obscurin is required for vertebrate myofibrillogenesis, it should be expressed in a tissue- and stage-specific manner and inhibition of its expression should cause defects of cardiac and skeletal muscle development. Methods/Results: RNA and antibody in situ hybridization localized obscurin expression in developing zebrafish embryos to the somites and central nervous system by 48 hours post-fertilization (hpf) and to the heart by 72 hpf. Obscurin morpholino antisense oligonucleotides targeting the translation initiation site (MO1) and 5’UTR (MO2) or a control morpholino (5 base pair mismatch to MO1 or MO2) were injected into zebrafish embryos (1-4 cell stage). Both MO1 and MO2 caused similar developmental defects, including diminished numbers and marked disarray of skeletal myofibrils, somite segmentation defects, and abnormalities of cardiac structure and function. No abnormalities were observed in the control-injected embryos. Decreased amount and organization of striated muscle and increased severity of cardiac defects was noted with increased morpholino doses. Cardiac abnormalities of obscurin-depleted embryos ranged from mild cardiac dilatation and dysfunction to an unlooped tube-like heart with marked pericardial edema. Conclusions: This is the first demonstration that obscurin is required for vertebrate cardiac and skeletal muscle development. The diminished capacity to generate new myofibrils in response to obscurin depletion suggests that it may have a vital role in the causation of or adaptation to cardiac and skeletal myopathies. (Best Abstract Awardee)

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2 UROGENITAL AND CAUDAL DYSGENESIS IN ADRENOCORTICAL DYSPLASIA (acd) MICE IS CAUSED BY A SPLICING MUTATION IN A NOVEL TELOMERIC REGULATOR. CE Keegan, JE Hutz, T Else, M Adamska, SP Shah, AE Kent, JM Howes, WG Beamer, and GD Hammer, University of Michigan, Ann Arbor, MI.

Division of Genetics Adrenocortical dysplasia (acd) is a spontaneous autosomal recessive mouse mutant with developmental defects in organs derived from the urogenital ridge. In surviving adult mutants, adrenocortical dysplasia and hypofunction are predominant features. Adults are infertile due to lack of mature germ cells, and fifty percent develop hydronephrosis due to ureteral hyperplasia. In addition, mutants exhibit growth retardation from birth, and many have decreased fur and flaky skin. We report the identification of a splice donor mutation in a novel gene, which is the mouse ortholog of a newly discovered telomeric regulator. This gene (Acd) has recently been characterized by others as a novel component of the protein complex that controls telomere elongation by telomerase. Characterization of Acd transcripts in mutant animals reveals two abnormal transcripts, consistent with a splicing defect. Expression of a wild-type Acd transgene in acd mutants rescues the observed phenotype. Most mutants die within 1-2 days of life on the original genetic background. Analysis of these mutant embryos reveals variable, yet striking defects in caudal specification, limb patterning and axial skeleton formation. In the tail bud, reduced expression of Wnt3a and Dll1 correlates with phenotypic severity of caudal regression. In the limbs, expression of Fgf8 is expanded in the dorsal-ventral axis of the apical ectodermal ridge and shortened in the anterior-posterior axis, consistent with the observed loss of anterior digits in older embryos. The axial skeleton of mutant embryos shows abnormal vertebral fusions in the cervical, lumbar, and caudal regions. The growth retardation, skin, and germ cell abnormalities observed in adult acd mutants are reminiscent of premature aging syndromes caused by genomic instability in humans. However, this is the first report to show that a telomeric regulator is required for proper urogenital ridge differentiation, axial skeleton specification, and limb patterning in the mouse. Further studies are in progress to evaluate for evidence of genomic instability in acd mutant animals. (Basic Science Faculty Awardee)

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3 RANDOMIZED CONTROLLED TRIAL OF PHYSICIAN EDUCATION TO IMPROVE PATIENT OUTCOMES FOR ASTHMA. MD Cabana, KK Slish, D Evans, R Mellins, RW Brown, X Lin, B Nan, NM Clark, University of Michigan School of Public Health and School of Medicine, Ann Arbor, MI. We conducted a group-randomized controlled trial to evaluate an asthma education seminar in improving the care delivered by primary care physicians. The goal of the seminar is to improve physician communication skills as well as patient outcomes for asthma. A sample of pediatric practices in 10 regions in the United States were recruited and then randomly assigned to receive the seminar versus no seminar. The seminar included 2 interactive sessions (2.5 hrs. each) that reviewed national guidelines, communication skills (e.g., interactive conversation, goal-setting), and key asthma educational messages (e.g., use of controller medications). The format included short lectures, case discussions and a video modeling communication techniques. We interviewed a random sample of each physician’s panel of asthma patients (2-12 yrs. of age with no other pulmonary diseases) at baseline and one-year after the seminar. We collected information on parent perceptions of physician communication, child’s frequency of asthma symptoms, emergency department (ED) and inpatient hospitalizations for asthma, severity of illness, medication use, and demographic information (patient age, gender, insurance status and passive tobacco exposure). We used multivariate regression models to determine differences in physician communication behavior and patient asthma outcomes, controlling for demographic characteristics and intervention exposure. We used general estimating equations to control for clustering. One-hundred-four pediatricians and a random sample of 870 of their asthma patients participated. One year after the intervention, we completed follow-up interviews with the parents of 731 of the 870 (84%) patients. Patient had a mean age of 7 yrs; 65% were male; 13% had Medicaid; 38% had persistent asthma. Compared to controls, physicians that attended the seminar were more likely to inquire about concerns about asthma; encourage patients to be active; and set goals for successful treatment (p<0.05). Patients of physicians that attended the educational seminar had decreased days limited by asthma symptoms, as well as decreased ED visits for asthma (p<0.05), but no difference in inpatient hospitalizations. This innovative educational seminar was effective in improving physician communication skills as well as patient asthma outcomes. Skills-based interactive physician education can be an effective method to improve the quality of physician communication, asthma care and patient outcomes. (Clinical Investigation/Health Services Research Faculty Awardee)

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Basic Science – Pathophysiologic Pathways 4 TEMPORAL AND REGION SPECIFIC ABNORMALITIES IN ENTERIC NERVOUS SYSTEM STEM CELL DEVELOPMENT IN THE HIRSCHSPRUNG RAT Y Tsai and C Gariepy, University of Michigan, Ann Arbor, MI

Division of Pediatric Gastroenterology Loss of signaling through the endothelin-B receptor (ETB) causes congenital distal intestinal aganglionosis (Hirschsprung disease) in humans and other mammalian species. Several different roles for ETB in enteric nervous system (ENS) development are reported including prevention of progenitor differentiation, promotion progenitor proliferation and modulation of progenitor migration within the developing gut. While a defect in the colonic ENS is obvious postnatally in these mutant animals, recent studies in mice demonstrate a role for ETB in small bowel ENS development and several lines of evidence suggest that critical ETB signaling occurs as ENS precursors colonize the cecal region of the developing gut. We determined the time-course of ENS development in wildtype and ETB-null rats via whole mount immunohistochemistry for p75 (the neurotrophin receptor). At embryonic day (E) 14.5, p75 staining in the proximal gut was indistinguishable between wildtype and ETB-null rats, though failure of cecal ENS development in ETB-null rats was already clear. Over the following 3 days, p75 staining progressed caudally in wildtype rats with staining present in the distal gut by E17.5. We then isolated and cultured ENS progenitors from the embryonic small bowel, cecal region and colon of wildtype and ETB-null rats at key time points during ENS development. We found a significantly reduced number of multipotent ENS progenitors behind the progenitor colonization wavefront in ETB-null compared to wildtype rats that was not accompanied by abnormalities in cell-cycle distribution. We also observed that failure of ENS progenitor colonization of the cecum is associated with an abnormality in cellular proliferation. Conclusions: Lack of ETB signaling leads to a reduction in multipotent ENS precursors in the small bowel of ETB-null rats just as these cells would normally be colonizing the cecum. This reduction occurs in the absence of changes in cell-cycle distribution, suggesting that the precursor cells become restricted in their developmental potential. Cecal ENS development is particularly associated with significant cellular proliferation. Regional differences in ENS progenitor development will likely prove important in strategies involving their transplantation or pharmacologic manipulation to correct ENS deficits. (Undergraduate Awardee)

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5 CAMPYLOBACTER JEJUNI GENES INVOLVED IN INVASION INTO GUT EPITHELIAL CELLS. GC Fogg and VJ DiRita, University of Michigan, Ann Arbor, MI.

Division of Pediatric Infectious Diseases Purpose: To determine specific genes in the C. jejuni genome that mediate invasion and intracellular survival in cultured intestinal epithelial cells. Background: C. jejuni is a widespread human enteric pathogen that is a leading cause of bacterial gastroenteritis worldwide. Histopathology on infected tissues and several studies with cultured intestinal epithelial cell lines have demonstrated that C. jejuni is capable of invading and persisting in the gut epithelium. Methods: Using a library of 1,100 signature tagged transposon mutants, we screened individual mutants in a gentamicin protection assay to identify mutants incapable of invading and surviving within cultured gut epithelial cells. The genes responsible for the observed invasion defect were determined by direct chromosomal sequencing. We hypothesize that these genes are involved in various mechanisms critical to bacterial invasion and intracellular survival. Results: We identified 31 mutants that had at least a 10-fold decrease in invasion as compared to wild type C. jejuni. These mutants represented disruption of 13 unique genetic loci throughout the bacterial genome. Several of these mutants have insertions in genes involved in flagellar biosynthesis, regulation of the flagellar transcriptional cascade or the motor proteins necessary for rotation of the flagellar apparatus. We have also identified four genes that do not have a previously known function and have designated them cigA-E (Campylobacter invasion genes). Since motility seems to play a central role in the invasion process, these cig-mutants were tested for their motility phenotype. Two of these mutants are motile, and two are non-motile. Comparative sequence analysis of the predicted protein products of these genes suggests that all of these genes are potentially found in the outer membrane or periplasmic space. This suggests that these proteins may play a role at the interface between the bacterium and the host cell. Mutational analysis of these four genes is currently in progress. Conclusions: Our genetic screen has illustrated the importance that motility and the flagellar apparatus plays in invasion by C. jejuni. We have also identified four novel genes that are also important for efficient invasion into host cells. Two of the cig-genes seem to mediate motility, while the other two cig-genes participate in an invasion pathway independent of motility.

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6 ROLE OF SELECTINS IN THE DEVELOPMENT OF IDIOPATHIC PNEUMONIA SYNDROME (IPS) FOLLOWING ALLOGENEIC (ALLO) STEM CELL TRANSPLANTATION (SCT). SW Choi, K Olkiewicz, C Liu, JB Lowe, G Hildebrandt, L Corrion, and KR Cooke, University of Michigan. IPS is a fatal complication following allo-SCT. The pathophysiology of IPS involves soluble cytokines and cellular effectors, but the mechanisms by which donor cells traffic to the lung remain largely unknown. Selectins are expressed on the vascular endothelium, interact with receptor ligands on activated leukocytes, and mediate the tethering of leukocytes to the endothelium during inflammation. We hypothesized that if the development of IPS is dependent upon the expression of selectins, abrogation of selectin receptor:ligand interactions will reduce leukocyte recruitment to the lung after allo-SCT. We used an established murine IPS model to test this hypothesis. Lethally irradiated B6 mice received SCT from either syngeneic (B6) or allogeneic, MHC matched (LP) donors. Pulmonary E selectin mRNA expression significantly increased in allo-SCT recipients at weeks 4 (>44%) and 6 (>24%) compared to syn controls. To investigate the role of selectin receptor:ligand interactions in the progression of IPS, we used mice deficient in both P- and E-selectin (P/E sel dko) as recipients. Evaluation of lung injury 4 weeks following SCT revealed that compared to B6 wt controls, B6 P/E sel dko mice receiving allo-SCT had significantly decreased broncho-alveolar lavage (BAL) fluid cellularity (0.63±0.07 vs. 1.52±0.41) and complete abrogation of lung pathology (0.3±0.3 vs. 3.8±0.6). We next used B6 mice lacking fucosyltransferase IV- and VII (FucT dko) as SCT donors in a second irradiated (P>F1) SCT system. FucT dko mice are deficient in L-, E-, and P-selectin ligands and demonstrate significant defects in T-cell trafficking to inflamed tissues. Allo (B6>F1) and syn (F1>F1) controls were again included. By week six after SCT, recipients of allo FucT dko T cells developed less severe IPS as measured by histopathology (3.4±0.4 vs. 6.0±0.8) along with the total number of cells (1.2±0.1 vs. 1.5±0.2) and the number of CD8+ cells (0.09±0.02 vs. 0.28±0.07) present in the BAL fluid compared to mice receiving allo-SCT from wild type donors. Paradoxically, abrogation of selectin receptor:ligand interactions resulted in enhanced systemic GVHD as measured by mortality and clinical score (changes in weight, skin integrity, fur texture, posture and activity). Our data uncover a significant role for selectins in the recruitment of donor cells to the lung after allo-SCT. The increase in systemic GVHD seen in our experiments suggests that these interactions may be target organ specific. Studies are ongoing to further evaluate this possibility and to discern whether strategies can be developed to exploit this mechanistic insight in order to successfully treat or prevent the development of IPS after allo-SCT. (Basic Science Fellow Awardee)

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7 ABROGATION OF CXCR3 RECEPTOR:LIGAND INTERACTIONS REDUCES LEUKOCYTE RECRUITMENT TO THE LUNG AND THE SEVERITY OF EXPERIMENTAL IDIOPATHIC PNEUMONIA SYNDROME. KR Cooke, LA Corrion, KM. Olkiewicz, S Choi, C Liu and GC Hildebrandt, The University of Michigan, Ann Arbor, MI. Idiopathic pneumonia syndrome (IPS) is a frequently fatal complication after allogeneic stem cell transplantation (allo-SCT) The pathophysiology of IPS involves the secretion of inflammatory cytokines including IFNγ and TNFα along with the recruitment of donor T cells to the lung. CXCR3 is a chemokine receptor that is expressed on activated Th1/Tc1 T cell subsets and the expression of its ligands CXCL9 (Mig) and CXCL10 (IP-10) can be induced in a variety of cell types by IFNγ alone or in combination with TNFα. We hypothesized, that recruitment of donor T cells to the lung is dependent, at least in part, upon interactions between the chemokines MIG and IP-10 and their receptor CXCR3. We tested this hypothesis using an established murine SCT model where in lethally irradiated bm1 mice receive SCT from either syngeneic (bm1) or allogeneic, MHC class I disparate (B6) donors. MIG and IP-10 BAL levels were significantly elevated in allo-SCT recipients compared to syn controls at weeks 1 (MIG: 162.8 ± 37.6 vs. 0; IP-10: 41.1 ± 4.2 vs. 0 pg/ml) and 4 (MIG: 153.5 ± 41.7 vs. 21.6 ± 9.0; IP-10: 202.0 ± 61.1 vs. 3.8 ± 0.9 pg/ml) after SCT and correlated with the infiltration of donor CXCR3+ T cells into the lung. To examine the effect of these chemokines on T cell recruitment, we treated allo-SCT recipients with polyclonal antibodies (Abs) against MIG and IP-10 or with control Ab from day 0 until day 28. Compared to allo-SCT controls, lung pathology at day 28 was significantly decreased in the Ab-treated animals (5.3 ± 0.4 vs. 3.5 ± 0.4) and was associated with significantly decreased numbers of total cells, CD8+T cells and TNFα levels in the BAL fluid. MG/IP-10 neutralization also reduced damage to the GI tract at week 1 after SCT and resulted in less severe systemic GVHD as measured by survival and clinical score. In a final set of experiments, we used B6 CXCR3-/- mice as allo SCT donors and found that mice receiving CXCR3 -/- cells had decreased pulmonary injury at week 4 as measured by lung pathology (4.8 ± 0.5 vs. 9.1 ± 1.0), BAL cellularity (1.2 ± 0.1 vs. 2.5 ± 0.4 x106), and CD8+T cells (0.06 ± 0.01 vs. 0.17 ± 0.03 x106) compared to recipients of allo-SCT from wild type controls. Our data demonstrate that chemokine interactions between CXCR3, MIG and IP-10 are important for donor T cell recruitment into the lung and the development of IPS. Approaches focusing on the abrogation of these interactions may prove successful in preventing or treating this serious complication after allogeneic SCT.

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8 DIFFERENTIAL SUSCEPTABILITY OF NORMAL AND CYSTIC FIBROSIS AIRWAY EPITHELIAL CELL CULTURES TO BURKHOLDERIA CEPACIA COMPLEX U Sajjan,1 M Hershenson,1 J Forstner,2 S Keshavjee,2 and JJ LiPuma,1

1University of Michigan, Ann Arbor, MI; 2The Hospital for Sick Children, Toronto, Canada

Divisions of Infectious Diseases and Pulmonology The Burkholderia cepacia complex (Bcc) is comprised of at least ten distinct bacterial species that are important respiratory pathogens in persons with cystic fibrosis (CF). Patients infected with Bcc exhibit an unpredictable clinical outcome ranging from prolonged carriage to rapidly fatal necrotizing pneumonia and sepsis. A major cause of this mortality is an exaggerated inflammatory response to infection. Bcc have been shown previously to stimulate a pro-inflammatory response from human monocytes and undifferentiated lung epithelial cells that is significantly greater than that seen with other CF pathogens. In this study, we compared the capacity of clinical and environmental Bcc to persist and stimulate IL-8 from well-differentiated CF and non-CF airway epithelial cell cultures. We also examined the location of bacteria in the infected cultures. Airway epithelial cells from CF and non-CF patients were grown in an air/liquid interface model to promote differentiation to a pseudostratified mucociliary-epithelia. The CF cell cultures exhibited goblet cell hyperplasia, increased secretion of mucus and higher basal levels of IL-8 compared to similarly cultured non-CF cells. Unlike the non-CF cells, the CF cells did not express the CF transmembrane conductance regulator (CFTR) on their apical surfaces. 24 hours after infection of the apical surface of cell cultures with low inocula (103 -104 CFU) of Bcc, the CF cultures had more cell-associated bacteria and secreted greater IL-8 than the non-CF cultures. In general, clinical isolates stimulated higher IL-8 secretion than environmental isolates. By immunofluorescent and transmission electron microscopy (TEM), bacteria were detected in the apical mucus layer in non-CF cultures, whereas in CF cultures bacteria were present on the cell surface and deeper in the cell layer. TEM demonstrated bacteria within the cytoplasm of CF cells. In summary, Bcc stimulate a greater pro-inflammatory response from CF airway epithelial cells than from normal cells. Distinct Bcc strains differ significantly in their capacity to persist and activate a pro-inflammatory response from epithelial cells. Some Bcc strains are also able to invade CF cells in vitro. These results suggest that CF respiratory epithelia are more susceptible to infection by Bcc than non-CF epithelia, and that the intense pro-inflammatory stimulation of human cells by Bcc contributes to the pathogenesis of infection due to these species in CF.

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9 OXIDANT STRESS AND MYOCARDIAL DYSFUNCTION AFTER ISCHEMIA-REPERFUSION INJURY IN NEONATAL RABBITS. CL Buller, JR Charpie, University of Michigan, Ann Arbor, MI

Division of Cardiology Congenital heart disease surgery in neonates often requires cardiopulmonary bypass and circulatory arrest. Myocardial ischemia-reperfusion (IR) injury contributes to postoperative ventricular dysfunction and increased morbidity and mortality. In adults with coronary artery disease, oxygen-derived free radicals (OFR) contribute to myocardial reperfusion injury. We hypothesized that OFR contribute to ventricular dysfunction after myocardial IR in neonates. Neonatal (7- to 14-day old) rabbit hearts were perfused with oxygenated Krebs-Henseleit buffer at 38°C in a Langendorff preparation. Left ventricular developed pressure (LVDP) was measured continuously via a balloon-tipped catheter in the LV apex. Control kit hearts were continuously perfused with buffer at 38oC throughout the experiments. Global ischemic injury was simulated by stopping coronary perfusion for 15, 30, or 60 minutes, followed by reperfusion with warmed buffer for 1 hour. Western blot analysis was performed on LV tissue after completion of the Langendorff protocol with antibodies for gp91phox (an NADPH oxidase subunit) and caspase-3. Additional LV sections were isolated for fluorescence microscopy with dihydrorhodamine (DHR), specific for peroxynitrite production. Fifteen minutes of warm ischemia had no adverse effect on LV function compared to pre-ischemia. In contrast, percent LVDP recovered (mean±SEM) was reduced after both 30 (64.2±14%) and 60 minutes (7.4±3%, p= 0.008) of ischemia followed by reperfusion. Densitometric analysis revealed greater production of gp91phox and a significant increase in caspase-3 activation (IR 0.9±0.04 vs. control 0.4±0.005, p=0.003) in hearts that experienced IR injury. DHR staining qualitatively confirmed an increase in endomyocardial oxidant stress that positively correlated with the length of ischemia. Administration of N-acetylcysteine, a potent antioxidant, prior to ischemia completely attenuated LV dysfunction after IR injury (132.7±19.4%). These results suggest that increased oxidant stress through NADPH oxidase may be associated with caspase-3 activation in immature myocardium following IR injury, and that antioxidant administration can prevent ventricular dysfunction.

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Basic Science – Molecular Mechanisms

10 MUTATIONS IN A NOVEL GENE, NPHP5, CAUSE NEPHRONOPHTHISIS WITH EARLY-ONSET RETINITIS PIGMENTOSA. EA Otto1, B Loeys2, S Fan3, H Khanna4, U Muerb1, JF O’Toole1, M Attanasio1, B Utsch1, A Kispert5, M Tsuda 6, DS Williams7, B Margolis3, A Swaroop4 and F Hildebrandt*1. 1Departments of Pediatrics and of *Human Genetics, University of Michigan, Ann Arbor, MI, United States; 2Department of Human Genetics, Johns Hopkins University, Baltimore, MD, United States; 3Department of Internal Medicine, University of Michigan, Ann Arbor, MI, United States; 4Department of Ophthalmology, University of Michigan, Ann Arbor, MI, United States; 5Institute of Molecular Biology, Medizinische Hochschule Hannover, Hannover, Germany, 6Life Science, Himeji Institute of Technology, Hyogo, Japan and 7Department of Pharmacology, University of California at San Diego, San Diego, CA, United States .

Departments of Pediatrics and Human Genetics Nephronophthisis (NPHP) is an autosomal recessive cystic renal disease. NPHP is the most common inherited cause of renal failure in childhood. Four causative genes (NPHP1-4) have been identified, three of which encode novel proteins (nephrocystin-1, -3, -4). Recently, we identified mutations in inversin as causing NPHP2 and localized inversin and nephrocystins to primary cilia of renal epithelial cells, thus linking NPHP to primary cilia, polycystic kidney disease, and to left-right axis development (Nature Genet 34:p413; p455-, p355, 2003). 10% of patients with mutations in either NPHP gene have retinitis pigmentosa (RP), termed Senior-Loken syndrome (SLSN). We here performed a genome wide search for linkage and identified a new locus (NPHP5) for NPHP on chromosome 3q within a critical region of 7.8 cM. Maximum 2-point LOD score was Zmax=3.5 (2=0). By directly sequencing cDNAs of 10 candidate genes we identified 8 distinct recessive mutations in a novel gene (NPHP5) in 15 patients with SLSN. All mutations were truncating. RP was present in 100% of cases. The NPHP5 gene product, nephrocystin-5 is highly conserved in evolution. It occurs in a complex with RPGR, which is mutated in the most frequent form of X-linked RP. Nephrocystin-5 localizes to primary cilia of renal epithelial cells and to the retina, thus explaining the renal-retinal phenotype. We thus identified mutations in a novel gene as the cause of NPHP and SLSN.

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11 INCREASED HSP27 EXPRESSION MIMICS THE PROTECTION FROM PODOCYTE INJURY AND ACTIN FILAMENT DISRUPTION CONFERRED BY DEXAMETHASONE. RF Ransom*, V Vega-Warner*, J Klein**, and WE Smoyer* *Department of Pediatrics, University of Michigan, Ann Arbor, MI and **Departments of Biochemistry and Molecular Biology, University of Louisville, Louisville, KY.

Department of Pediatrics Glucocorticoids, the primary therapy for nephrotic syndrome, have historically been hypothesized to act on circulating lymphocytes, although the glomerular podocyte is the cell type most directly affected in this disease. However, our studies have shown that glucocorticoids can act directly on cultured murine podocytes to protect from and ameliorate injury from puromycin aminonucleoside and to decrease direct actin filament disruption by the monomer sequestering agent latrunculin B. A subsequent proteomic analysis of the alterations in protein expression in podocytes induced by treatment with the glucocorticoid dexamethasone revealed, among other changes, an increase in the expression of the small heat shock protein αB-crystallin. The increase in αB-crystallin expression induced by dexamethasone was confirmed by quantitative Western blotting, and blots probed for the related small heat shock protein hsp27 revealed that the expression of this protein in podocytes was also increased by dexamethasone. The amount of αB-crystallin in dexamethasone-treated podocytes increased significantly by 1 d after treatment to 301% of the amount in time-matched controls, and increased further to 463% of controls by 7 d. The maximum increase in hsp27 was observed earlier at 1 d after treatment (425% of controls) and hsp27 remained comparably elevated for at least 7 d. We previously reported that increased hsp27 expression in podocytes protected from, while decreased hsp27 expression exacerbated, injury from puromycin aminonucleoside. We have now found that increased hsp27 expression in podocytes provided protection from direct microfilament disruption by latrunculin. These results, in conjunction with our prior findings demonstrating that the expression of both αB-crystallin and hsp27 are increased in cultured podocytes by dexamethasone, suggest that our observation of dexamethasone-induced protection of cultured podocytes from injury and microfilament disruption is a result of increases in the expression of these small heat shock proteins.

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12 HOMO-OLIGOMERIZATION IN API2-MALT1 FUSION PROTEIN-MEDIATED ONCOGENESIS. L McAllister-Lucas, P. Kuffa, P Lucas, N Inohara, G Nunez, University of Michigan, Ann Arbor, MI.

Division of Hematology/Oncology Background: Mucosa associated Lymphoid Tissue (MALT) lymphoma is the most common form of extra-nodal lymphoma worldwide. The t(11;18) chromosomal translocation occurs in 50% of cases of MALT lymphoma and is associated with treatment resistance. This translocation results in fusion of the coding regions of two genes; inhibitor of apoptosis 2, (API2), and a previously uncharacterized gene designated MALT1. This fusion leads to expression of a chimeric protein composed of N-terminal sequence of API2 fused to C-terminal sequence of MALT1. We have demonstrated that the API2-MALT1 fusion oncoprotein constitutively activates the pro-survival transcription factor NF-κB. Genetic and biochemical studies have established that wild-type MALT1 is an essential mediator of NF-κB activation that is required for normal lymphocyte proliferation in response to antigen. The BCL10 adaptor protein regulates MALT1 activity by binding to and mediating the oligomerization of MALT1, thereby rendering MALT1 capable of activating NF-κB. The mechanism by which API2-MALT1 perturbs normal BCL10/MALT1 signaling and contributes to lymphomagenesis is not known. We hypothesize that the API2 moiety of API2-MALT1 mediates homo-oligomerization, thereby leading to constitutive oligomerization of the MALT1 moiety and unregulated induction of the NF-κB prosurvival signal. Results: To test our hypothesis, we have generated expression constructs encoding FLAG epitope-tagged API2-MALT1 and Myc epitope-tagged API2-MALT1, as well as FLAG and Myc-tagged wild-type MALT1. Using a co-immunoprecipitation assay, we have demonstrated that the API2-MALT1 proteins homodimerize in the absence of BCL10, whereas wild-type MALT1 proteins do not. We have also generated expression plasmids encoding API2-MALT1 fusion proteins that contain point mutations predicted to disrupt API2-mediated homo-oligomerization. If these mutants are unable to oligomerize, we will then test whether loss of oligomerization correlates with loss of NF-kB activation and/or loss of transforming capability. Conclusion: We present the first evidence that, unlike wild-type MALT1, the API2-MALT1 fusion oncoprotein can form homo-oligomers in the absence of interaction with BCL10. These results support the notion that the API2 moiety of API2-MALT1 mediates constitutive / unregulated MALT1 oligomerization, and therefore, constitutive activation of NF−κB. These observations advance our understanding of MALT lymphoma pathogenesis, and could yield novel targets for the rational design of pharmaceutical agents, such as blockers of API2-mediated oligomerization or inhibitors of MALT1, for the treatment of MALT lymphoma.

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13 PERTURBED ENDOCYTOSIS: A NOVEL LEUKEMOGENIC MECHANISM IN AML. M Pendergast, AC Walker, MM Chao, DS Wechsler, University of Michigan, Ann Arbor, MI

Division of Pediatric Hematology-Oncology Background: Chromosomal translocations involving the MLL gene are found in a significant fraction of poor prognosis childhood leukemias. We recently identified a novel fusion protein, MLL-CALM, in an infant with fatal AML. The native CALM protein plays a key role in clathrin-dependent endocytosis (CDE) and intracellular vesicle transport. Interestingly, CALM was first described as a partner for the AF10 gene – itself an MLL partner – in aggressive leukemias and lymphomas. Recently, it was shown that mutations in calm are responsible for hematopoietic abnormalities in inbred fit1 mice. Taken together, these observations suggest an important role for CALM in both normal and malignant hematopoiesis. We hypothesize that disrupted CDE resulting from the presence of CALM-containing fusion proteins interferes with downregulation of growth factor signaling, thereby contributing to leukemogenesis. Objective: Determine whether expression of MLL-CALM or CALM-AF10 interferes with transferrin (TF) CDE, and whether MLL-CALM expression reduces the IL-3 growth factor dependence of FL5.12 cells. Design/Methods: The relative ability of leukemia cell lines that harbor MLL and CALM translocations to endocytose AlexaFluor633-conjugated TF was assessed by flow cytometry. The degree of Texas Red-TF endocytosis in COS7 cells expressing GFP-tagged MLL-CALM and CALM-AF10 was quantitated using densitometry. Finally, IL-3-dependent FL5.12 cells engineered to stably express MLL-CALM were grown in decreasing amounts of IL-3-containing WEHI conditioned medium media and survival was determined. Results: U937 cells (that harbor the CALM-AF10 translocation) showed reduced endocytosis of AF633-TF, but cells with other MLL translocations showed robust AF633-TF uptake. COS-7 cells expressing MLL-CALM or CALM-AF10 showed 40-50% less endocytosis of TR-TF compared with untransfected cells or cells transfected with empty vector. IL-3-dependent FL5.12 cells transfected with MLL-CALM expression vector grew better in low concentrations of IL-3-containing WEHI conditioned media in comparison with FL5.12 transfected with empty vector. Conclusions: Expression of MLL-CALM or CALM-AF10 interferes with endocytosis and renders FL5.12 cells less dependent on exogenous IL-3. The ability of MLL-CALM and CALM-AF10 to interfere with CDE may result in sustained growth factor signaling and contribute to the development of aggressive hematopoietic malignancies. (Medical Student Awardee)

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14 TARGETED ADAMTS13 DEFICIENCY IN MICE DOES NOT RESULT IN CONGENITAL THROMBOTIC THROMBOCYTOPENIC PURPURA. D. Motto, W. Zhang, G. Zhu, J. Homeister, and D. Ginsburg, University of Michigan, Ann Arbor, MI.

Division of Hematology/Oncology Thrombotic Thrombocytopenic Purpura (TTP) is a life threatening systemic illness characterized by the formation of blood clots in the microcirculation and the clinical pentad of fever, hemolytic anemia, thrombocytopenia, neurological symptoms, and renal dysfunction. Pathogenesis of TTP involves the blood clotting protein von Willebrand Factor (VWF) and the newly-described ADAMTS13 metalloprotease. VWF is synthesized by endothelial cells and stored in the Weibel-Palade bodies in the form of high molecular weight multimers (UL-VWF). UL-VWF is the most adhesive and reactive form of VWF and may lead to spontaneous platelet aggregation if not further processed by ADAMTS13. The currently favored hypothesis regarding TTP pathogenesis states that loss of ADAMTS13 function results in the accumulation of UL-VWF in the plasma, in turn leading to the development of micro-thrombi in the vasculature and the subsequent pathology observed in TTP. To investigate the function of ADAMTS13 in vivo, we disrupted the murine ADAMTS13 gene and analyzed the resulting ADAMTS13 deficient mice for development of pathology consistent with TTP. Interestingly, we found that ADAMTS13 -/- mice resulting from heterozygous intercross matings were born close to the expected Mendelian frequency of 25% +/+, 50% +/-, and 25% -/-, indicating that murine ADAMTS13 deficiency is not embryonic or perinatal lethal. The ADAMTS13 -/- mice were physically indistinguishable from their +/+ and +/- littermates, and did not die at an increased rate. Laboratory analysis demonstrated no differences in baseline RBC and platelet counts, PT, PTT, or bleeding time. Furthermore, no evidence of TTP was observed in peripheral blood smear analysis or tissue histopathological survey. In conclusion, unlike most humans with familial ADAMTS13 deficiency, mice with targeted disruption of the ADAMTS13 gene do not develop TTP, either in the perinatal period or as they age. These results demonstrate that ADAMTS13 deficiency in mice is not sufficient for development of TTP, and may indicate the existence of additional environmental triggers (e.g. infection) or genetic modifiers (e.g. VWF level). The ADAMTS13 -/- mice are the focus of ongoing studies designed to elucidate these potential genetic and environmental factors that may contribute to the pathogenesis of TTP.

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15 ROLE OF THE TANDEM MYOSIN LIGHT CHAIN KINASE FAMILY MEMBERS OBSCURIN-MLCK AND SPEG IN MYOCYTE DIFFERENTIATION. SB Sutter, MO Raeker, AB Borisov, MW Russell, University of Michigan, Ann Arbor, MI

Division of Pediatric Cardiology During development, cardiac myocytes terminally differentiate and withdraw from the cell cycle such that, by shortly after birth, the population of myocytes capable of cell division is negligible. The factors that maintain this state of terminal differentiation are poorly understood but appear to involve both cell cycle regulation and intracellular signaling. Recently two novel muscle-specific kinases with a unique tandem arrangement of myosin light chain kinase (MLCK)-like domains, obscurin-MLCK and SPEG, were noted to be expressed late in myocyte differentiation. Hypothesis: The tandem MLCKs, SPEG and obscurin-MLCK, promote and maintain terminal differentiation of cardiac myocytes. Methods/Results: Adult rat cardiac myocytes were grown in primary culture in the presence of 10% serum, allowing them to de- and then re-differentiate over a two week period. Both obscurin-MLCK and SPEG localized to the Z band in mature, fully differentiated adult cardiac myocytes. As the cells de-differentiated, the tandem kinases became diffusely localized in the cytoplasm where they remained until late in the re-differentiation process. Overexpression of the single kinase isoform of obscurin-MLCK delayed myocyte de-differentiation with many cells remaining either rod-like or rounded even after 10 days in culture, by which time control cells had flattened and were well into the re-differentiation process. Conversely, inhibition of obscurin-MLCK or SPEG expression, using adenoviral delivery of small interfering RNA (RNAi) constructs, was associated with normal or accelerated de-differentiation but incomplete re-differentiation as indicated by a paucity of mature sarcomeric structures. While control cells demonstrated vigorous rhythmic contractions, only rare beating cells noted after obscurin-MLCK or SPEG RNAi treatment. Furthermore, SPEG RNAi-treated cells demonstrated a marked increase in the number of nuclei per cell, suggesting that some of the myocytes had re-entered the cell cycle and undergone karyokinesis. Conclusions: Both tandem MLCKs, obscurin-MLCK and SPEG, appear to be required for normal myocyte differentiation and for maintenance of the terminally differentiated state. Therefore, either alone or in combination with cell cycle regulation, it may be possible to manipulate the activity of these kinases to encourage terminally differentiated cardiac myocytes to de-differentiate, re-enter the cell cycle and proliferate, thereby enabling the heart to regenerate or repair damaged tissue.

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Clinical Research 16 SOLUBLE TUMOR NECROSIS FACTOR RECEPTOR: ENBREL (ETANERCEPT) FOR THE TREATMENT OF IDIOPATHIC PNEUMONIA SYNDROME FOLLOWING ALLOGENEIC STEM CELL TRANSPLANTATION. GA Yanik, JE Levine, JP Uberti, JLM Ferrara, R Hutchinson, and KR Cooke. University of Michigan Medical Center, Ann Arbor, MI. Idiopathic Pneumonia Syndrome (IPS), a diffuse, non-infectious process that occurs in 10% of allogeneic marrow transplants, is associated with a mortality > 70%. Etanercept is a soluble tumor necrosis factor (TNF) receptor fused to the Fc portion of a human IgG1. Fifteen patients (median 18 yrs, 1 - 60 yrs), each meeting the diagnostic criteria for IPS, were enrolled in a trial using etanercept to treat IPS post transplant. Broncho-alveolar lavage (BAL) specimen were obtained pre and post therapy, undergoing analysis for both infectious pathogens and for inflammatory cytokine markers (TNF, TNFR1, sCD-14, LBP, and MCP-1). Patients in whom the pre-therapy BAL was positive for a potential pathogen (by specific stain or culture) were ineligible for therapy. Etanercept was administered subcutaneously at a dose of 0.4 mg/kg twice weekly, for a maximum of 8 doses. All patients required supplemental oxygen at therapy onset, with seven patients requiring mechanical ventilation. Etanercept therapy was initiated a median of 17 days (range 11-87 days) post transplant. RESULTS: Nine of 15 patients had a complete response, defined as the ability to withdraw completely from supplemental oxygen support. In responders, the median time to complete response was 6 days (range 3-18 days) and median time to normalization of radiographic findings was 6 days (range 2-17 days). Three patients died while on therapy, from progressive organ dysfunction. Post therapy BAL fluid analysis noted significant reductions in all inflammatory cytokines tested, including TNFa, TNFR1, sCD14, LBP and MCP-1 (Table below). CONCLUSION: Etanercept therapy was associated with favorable responses in patients with IPS. In addition, the decline in BAL fluid cytokine levels noted post therapy support a mechanistic role for TNFa in the pathogenesis of this disorder.

BAL Fluid Analysis, Pre and Post Etanercept Therapy n TNF TNFR1 sCD-14 LBP MCP-1 Controls - A 3 5.2 (0 -15.6) 21 (18.2-23.5) 0 (0) 0 (0) 43.4 (15-72) Controls - B 10 5.4 (0 -22.8) 112 (19-373) 1895(0-8821) 0 (0) 282 (24-975) Subjects, Pre 7 92.6 (0 -500) 861(82-1500) 82,610 (0-250K) 331 (0-1000) 20,152 Subjects, Post 5 15 (10 -22.9) 175 (90-364) 3041 (0-6200) 0 (0) 67 (60-80.8)

Controls were a group of healthy volunteers (A) or transplant patients without IPS (B). Values in pg/ml.

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17 IMPLEMENTING INSULIN PUMP THERAPY IN PRESCHOOLERS WITH TYPE 1 DIABETES MELLITUS: A PILOT INVESTIGATION OF MEDICAL AND QUALITY OF LIFE OUTCOMES. L. Opipari-Arrigan, E.M. Fredericks, N. Burkhart, L. Dale, M. Hodge, J. Kichler, P. Olton, C. Foster, Department of Pediatrics, University of Michigan Health System, Ann Arbor, MI.

Department of Pediatrics BACKGROUND: Recent studies suggest that continuous subcutaneous insulin infusion therapy (CSII) may be at least as effective as multiple insulin injections (INJ) in achieving glycemic control in preschoolers with Type 1 diabetes mellitus (IDDM). Yet, there is little empirical data regarding the impact of CSII on quality of life for preschool age children and their families. OBJECTIVE: To assess whether CSII is comparable to INJ with respect to glycemic control and adverse events, and to determine if CSII improves quality of life in families of preschoolers with IDDM. DESIGN/METHODS: 16 children with at least a one year history of IDDM were randomly assigned to receive either CSII or INJ. Mean age at baseline was 4.4 0.7 years. Families attended monthly study visits for 6 months. Hemoglobin A1C (HbA1C) was used to measure glycemic control. Glucose variability was measured at baseline and 6-months follow-up using continuous blood glucose sensing. Adverse events were reviewed monthly. Measures of quality of life were collected at baseline and 6-months follow-up. RESULTS: Mean HbA1C levels from baseline (CSII 8.3 1.4%, INJ 8.0 0.8%) to 6-months (CSII 8.4 0.8%, INJ 8.2 0.4%) remained stable and group differences were not significant. No significant group differences were found in duration of hypo- or hyperglycemic events or frequency of adverse events. The CSII group reported a significant decrease in diabetes-related worry (p=0.01), while the INJ group reported increased frequency of stress associated with their child's medical care (p<0.05), and a trend toward increased overall parenting stress (p<0.1). Following the study, 100% of the CSII group remained on pump therapy and 50% of the INJ group initiated CSII, suggesting overall parental satisfaction with CSII. CONCLUSIONS: HbA1c levels remained stable with the initiation of CSII and there were no significant group differences with respect to adverse events or glycemic control. CSII led to decreased diabetes-related worry for parents and was associated with high parental satisfaction. This pilot investigation suggests that CSII therapy is comparable to intensive insulin injection therapy, but may be associated with higher treatment satisfaction and improved quality of life.

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18 CATHETER BASED DECOMPRESSION OF THE LEFT ATRIUM IN PATIENTS WITH HYPOPLASTIC LEFT HEART SYNDROME AND RESTRICTIVE ATRIAL SEPTUM IS SAFE AND EFFECTIVE. JG Gossett, AP Rocchini, TR Lloyd, RG Ohye, EL Bove, JN Graziano, University of Michigan, Ann Arbor, MI INTRODUCTION: Infants with Hypoplastic Left Heart Syndrome (HLHS) and restrictive or intact atrial septum (rAS) present with severe cyanosis, pulmonary edema and are critically ill. A previous report from our institution on emergent Norwood (NWD) for HLHS with rAS showed 10% survival. HYPOTHESIS: Trans-catheter left atrial (LA) decompression in HLHS with rAS safely and effectively relieves the LA hypertension, improves oxygenation, and improves NWD survival. METHODS: Patients (pts) with HLHS and rAS requiring emergent pre-NWD intervention between 1996 and 2004 were identified. Hospital charts, catheterization records, and echocardiograms were reviewed. RESULTS: Of 33 pts identified, 30 had catheter procedures and 3 had surgical septectomies. Of the 30 catheterizations, 28 had atrial septostomies: 23 static balloon dilations (3 with bioptome), 4 Rashkind septostomies and 1 intra-atrial stent. Trans-septal puncture was required in 16/28 (57%). Two procedures were aborted due to perforation (1) or inability to enter the LA (1). An additional 6 pts required a subsequent surgical septectomy, for a failure rate of 27% (8/30). There were no catheter-related mortalities. Major complications occurred in 3 pts (10%) who had atrial perforations requiring intervention. Minor complications included transient arrhythmias in 12 (40%), and asymptomatic needle perforations in 5 (17%). For pts with data available pre- and post-procedure, the mean ASD gradient by catheterization fell from 16.7+/-4.9 to 6.3+/-3.4 mmHg (p<0.001; n=18). Mean LA pressure dropped from 21.8 +/-5.5 to 13.1+/-6.5 mmHg (p<0.001; n=16). Mean PaO2 rose from 29.5+/-9.1 to 36.5+/-5.1 torr (P<0.001; n=23). The size of the LA did not predict treatment failure. Of the 30 pts, 16/29 (55%) survived to discharge with 1 still hospitalized. Of the 22 pts successfully decompressed, 15/21 (71%) were discharged with 1 still hospitalized. Twelve have undergone hemi-Fontan, and 9 Fontan. CONCLUSIONS: Trans-catheter decompression of the LA for patients with HLHS and rAS can be performed safely, reduces the trans-atrial gradient and improves systemic oxygenation. Mortality for these patients remains high, but early catheter intervention improves survival compared to emergent NWD. (Clinical Investigation/Health Services Research Fellow Awardee)

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19 TRANSCATHETER CRYOTHERAPY FOR TREATMENT OF SUPRAVENTRICULAR ARRHYTHMIAS IN YOUNG PATIENTS. PS Fischbach, EV Saarel, M Dick, II. University of Michigan, Ann Arbor, MI Introduction: There are few reports of transcatheter cryotherapy (CRYO) for treatment of supraventricular tachycardia (SVT) in young patients. Purpose: To report the initial experience with CRYO ablation at our institution. Methods and Results: Thirty consecutive subjects (mean age 13.6+2, range 2.4-33 yrs, median 14 yrs; 19 female) underwent transcatheter CRYO at the University of Michigan between 8/1/03 and 4/30/04. All patients had documented SVT prior to electrophysiologic study (EPS). Twenty-two subjects had AV nodal re-entry tachycardia (AVNRT), 6 atrioventricular re-entry tachycardia (AVRT) (3 with 2 accessory pathways (APs) including 2 right free wall, 3 right anteroseptal, 2 midseptal, 1 right posterolateral, and 1 left posteroseptal), 1 ectopic junctional tachycardia (JET), and 1 junctional ectopic tachycardia (JET). Success (see table) = loss of pre-excitation, slow pathway ablation (AVNRT), and non-inducible SVT. N in Table = # procedures. Five minor complications occurred: Transient (T)

Substrate N CRYO mapsmean (range)

CRYO ablationsmean (range)

Success CRYO

SuccessRFA

AVNRT 22 12 (0-49) 6 (1-22) 20/22 2/2 APs/AVRT 9 8 (6-11) 8 (0-29) 3/9 4/6

AET 2 1 (0-4) 31 (6-56) 1/2 0/1 JET 1 7 19 1/1

complete heart block (CHB) in both patients with anteroseptal APs during RFA, TCHB in 1 patient with an anteroseptal AP during CRYO mapping, T 2nd degree HB in the patient with JET during CRYO mapping, and transient 1st degree HB in a single patient with AVNRT during CRYO mapping. There were no major complications. Conclusions: Transcatheter CRYO proved safe for use in young patients. CRYO was more effective for treatment of AVNRT and JET than AVRT in this early experience.

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20 COMPARISON OF HIGH RESOLUTION COMPUTERIZED TOMOGRAPHY (HRCT) OF THE CHEST AND PULMONARY FUNCTION TESTING IN EVALUATING THE EFFECT OF TOBRAMYCIN SOLUTION FOR INHALATION (TSI) IN CYSTIC FIBROSIS (CF) SUBJECTS WITH MILD LUNG DISEASE. SZ Nasr, P Eckhardt, PJ Strause, University of Michigan., Ann Arbor, MI. Objective: To evaluate the usefulness of HRCT of the chest compared to spirometry measures (PFTs) in evaluating the effects of tobramycin solution for inhalation (TSI) in CF patients six years of age and older. Design: 32 subjects with CF and mostly mild lung disease were enrolled in a randomized, double-blind, placebo-controlled study. Duration was 28 days; 31 subjects completed the study. HRCT and PFTs were evaluated at baseline and at the end of the study. 2 pediatric radiologists scored all films using a validated system. Results: HRCT scores decreased - 4.06 +/- 3.20 (mean +/- se) for TSI subjects and increased 1.75 +/- 1.78 for placebo subjects (p= 0.13), suggesting a trend of improvement in pulmonary status in the TSI arm relative to the placebo arm. Mean FEF25-75% predicted increased 6.08 +/- 4.86 for TSI subjects and decreased - 0.60 +/- 2.34 for placebo subjects (p=0.23). FEV1% predicted increased slightly for both TSI and placebo subjects (1.29 +/- 3.33 for TSI and 1.17 +/- 1.4 for placebo p=0.97). Two HRCT subscores (atelectasis and inhomogeneity) were observed to be highly discordant, in that observed changes in these scores were contradictory to observed pulmonary function changes and other HRCT subscores. A modified global score was calculated by dropping atelectasis and inhomogeneity from the global score, which resulted in a larger observed HRCT treatment effect in the TSI group. The modified HRCT global scores decreased –6.68 +/-3.09 (mean +/- se) for TSI subjects and increased 0.02 +/- 2.0 (mean +/- se) for the placebo subjects (p=0.07). Assuming that observed differences in this study represent true treatment effects, we calculated sample sizes that would be required to show statistical significance by difference in modified HRCT score, global HRCT score, FEF25-75 % predicted or FEV1 % predicted. A total of 60-70,100, 200, and over 800 patients would be necessary to demonstrate a significant TSI treatment effect in this demographic population using the respective methods. Conclusion: HRCT can be a robust measure of change in CF mild pulmonary disease, requiring a smaller sample size than that required to demonstrate a significant treatment effect by pulmonary function testing alone. Our study also suggests that two HRCT subscores, atelectasis and inhomogeneity, may not be useful measures in CF mild lung disease. This study was funded by Chiron Corp, Emeryville CA.

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21 BARRIERS TO SCREENING INFANTS FOR RETINOPATHY OF PREMATURITY AFTER DISCHARGE OR TRANSFER FROM A NEONATAL INTENSIVE CARE UNIT. M Attar, M Gates, A Iatrow, S Lang, S Bratton, Department of Pediatrics, University of Michigan, Ann Arbor, Michigan.

Department of Pediatrics Neonatal intensive care unit (NICU) practices may influence the delivery of ophthalmology care for infants eligible for back transfer to a referring community hospital. We conducted this study to assess barriers to screening for Retinopathy of Prematurity (ROP) after discharge or transfer from a NICU. Study Design: Retrospective study of 78 infants who needed ophthalmology examinations at the time of their discharge or transfer from the NICU. These infants either needed screening for ROP or had retinal examinations in the NICU and needed further follow-up. Results: 64% of infants received appropriate eye care. Infants who did not receive the follow-up care had greater mean gestational age (mean SD; 30.7±2.3 vs. 29.4±2.6 weeks, p=0.02) and birth weights (mean SD; 1581±366 vs. 1329±504 gm, p=0.007) compared to infants who received the recommended care. There were no statistical differences between the two groups when comparing their race, maternal age, maternal marital status, insurance coverage, or site of birth. Forty-three (55.1%) infants were transferred back, 26 (33.3%) infants were discharged home from the NICU, while nine (11.6%) infants were discharged home from the regional center after their transfer to the general pediatrics service. Infants discharged from the NICU were significantly more likely to receive recommended eye care compared to infants transported back to the community hospital or transferred to the pediatric service at the regional center (Relative Risk Ratio 1.5, 95% confidence interval 1.1-2.1, p=0.01). Infants transferred or discharged from the NICU not screened for ROP (n=49) had lower completion rates compared to infants who had their first retinal examination in the NICU and needed follow-up (n=29) (89% vs. 49%, p<0.0001). Infants whose ophthalmology follow up was recommended in the discharge summary and had their appointments arranged prior to discharging them from the community hospital or the regional center were ten time more likely to get the appropriate eye care (Relative Risk Ratio 10.7, 95% Confidence Interval 2.8-40.3, p<0.0001) compared to infants who did not have arrangements and recommendations. Parents were compliant (n=56, 89%) with pre-arranged appointments. Conclusions: Infants transferred back or discharged from the NICU before having a retinal examination represent a high-risk group for not receiving eye screening. Scheduling appointments for these infants when they leave the NICU may improve their ROP screening and care.

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Health Services Research

22 USE OF ANTI-INFLAMMATORY MEDICATIONS AND EMERGENCY DEPARTMENT RELIANCE AMONG CHILDREN WITH ASTHMA. KJ Dombkowski, DR Gill and SJ Clark, Child Health Evaluation and Research Unit, University of Michigan, Ann Arbor, MI.

Division of General Pediatrics Background: National guidelines recommend anti-inflammatory (AI) medications for long-term control of persistent asthma. A high degree of emergency department reliance (EDR), defined as the proportion of ambulatory asthma visits occurring in the ED, is one indicator of inadequate control. Objective: To examine AI medication use and associated changes in annual EDR among children with persistent asthma. Design/Methods: Retrospective analysis of 2001-2002 administrative claims for 5,409 continuously enrolled Michigan Medicaid children 5-21 years old with persistent asthma. Subjects were determined to have persistent asthma in both study years using National Committee for Quality Assurance (NCQA) HEDIS criteria. AI medication use in 2001 and 2002 was determined from pharmacy claims. The outcome measured was mean annual asthma EDR. Comparisons of EDR were conducted using chi square tests of association and multivariate analysis was conducted using logistic regression. Results: In 2001, 47.5% of children had no asthma ED visits and at least one outpatient visit; 17.3% had at least one asthma ED visit, but no outpatient visit; 20.2% had both type of visits, and 15.0 % had neither. Both outpatient and ED utilization was significantly lower in 2002 (p< .001). Overall EDR was 30.8% in 2001 and 26.9% in 2002 (p<.001). EDR was lowest in both years among children using AI medications throughout the study period. EDR varied significantly by demographic characteristics and AI use (p<001). Multivariate models confirmed the association between AI use in both years and lower EDR, controlling for age, gender, race, urban location, disability status, and year (OR=0.30, p< .001). Conclusions: Interventions to ensure appropriate medication use may be an effective mechanism to reduce EDR by children with persistent asthma.

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23 BLOOD LEAD TESTING AMONG MEDICAID-ENROLLED CHILDREN. AR Kemper, LM Cohn, KE Fant, KJ Dombkowski, Child Health Evaluation and Research Unit, Division of General Pediatrics, University of Michigan, Ann Arbor, MI.

Child Health Evaluation and Research Unit Background: Federal regulations mandate that Medicaid-enrolled children be tested for lead poisoning at 1 and 2 years, or between 3 and 5 years if not previously tested. Objective: To measure the rate of lead testing among Medicaid-enrolled children in Michigan and the subsequent proportion of children with elevated lead levels; and, to determine factors associated with testing and elevated lead levels. Methods: We performed a retrospective analysis of children ≤ 5 years of age continuously enrolled in Medicaid during 2002. Results: There were 216,578 children included in the analysis. The overall rate of lead testing was 19.6% (95%CI:19.4%-19.8%) of which 8.3% (95%CI:8.0%-8.5%) had a level ≥ 10 µg/dL. Across all ages, 4-year-old children had the greatest odds of being tested. Hispanic or non-white children, or those living in high-risk areas for lead exposure were more likely to be tested and more likely to have an elevated blood lead level. However, 1.2% of tested children without these risk factors had a level ≥ 10 µg/dL. Enrollment in Medicaid managed care was associated with an increased likelihood of testing. After adjusting for other factors, those in managed care for >75% of their enrollment in 2002 had 1.24 (95%CI 1.23-1.25) greater odds of being tested than those in fee-for-service for >75% of their enrollment. Conclusions: The rate of testing is low. Patterns suggest testing is targeted to those at highest risk, potentially leading some children with elevated lead levels to be missed. Managed care programs may offer insight into how to increase testing.

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24 PARENTS’ TRUST IN THEIR CHILD’S PHYSICIAN. KL Moseley, SJ Clark, A Gebremarium, MJ Sternthal, and AR Kemper, University of Michigan Background: In previous studies of adult patients, higher levels of trust as measured by the Trust in Physician scale (TiPS) and similar instruments predict continuity, satisfaction with care, and adherence to treatment. Objective: To field-test a modified TiPS for parents of pediatric patients to explore race-based differences. Design/Methods: A cross-sectional study of parents accompanying pediatric patients at university-affiliated outpatient clinics. Verbal consent was obtained and parents completed a self-administered anonymous survey containing basic demographic information and the TiPS. The TiPS is an 11-item inventory with a scoring range of 11-55, measuring patient trust in a specific physician in the domains of competence, confidentiality, and concern for patient best interest . Answers are on a 5-point Likert scale. Analysis: Total TiPS

scores were analyzed by demographic variables using STATA8. A non-parametric one-way ANOVA (Kruskal-Wallis test) was used to compare mean total TiPS scores. Results: 562 of 711 parents completed surveys (79% response rate). Parental race was African American (AA), 22%, White, 67%, and Other, 11%. Mean total TiPS score for AA parents was 44.7 (SD 6.3), White, 45.7 (SD 6), and Other, 44.4 (SD 5.2, p=.16), consistent with previous studies of adult patients. Scores for parents with a college degree (45.2, SD 5.8) were similar to those of parents without one (45.6, SD 6.2, p=.48). Those with any public insurance had a significantly higher TiPS score (46.7, SD 5.4) than those with private insurance (44.8, SD 6.2, p=0.01), indicating higher physician trust.Conclusions: These results suggest that parental trust in their child s physician does not vary by race and is comparable to levels of trust found adults’ assessments of their own physicians. While levels of trust did not vary by educational status, there was a significant difference in TiPS scores by insurance type. Unlike prior studies of adult patients, parents who were enrolled in public insurance plans had significantly higher scores than parents with private insurance, indicating greater trust in their child’s physician. Further study is needed to determine how differential levels of physician trust may affect continuity, satisfaction and adherence in pediatric populations.

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25 VARIATION IN RESUSCITATION MEDICATION DOSES LISTED ON CODE CARDS OF PEDIATRIC TRAINING INSTITUTIONS. EY Yoon1, MM Davis 1,2, University of Michigan, Ann Arbor, MI 1Division of General Pediatrics and 2Division of General Internal Medicine

Division of General Pediatrics Background: Prior work suggests that medication errors are common in pediatric inpatient settings and that the majority of medication errors are dosing errors. Many pediatric training institutions provide “code cards” to house staff that list medications and recommended doses for emergent resuscitation. Objective: To describe similarities and differences in resuscitation medication doses listed on code cards of pediatric training institutions in the U.S. Design/Methods: We contacted all pediatric training institutions in the United States to request their code cards. For each medication measure (e.g., low end of dosing range for a given medication) listed on code cards, we identified the predominant dose across all cards received. For each medication measure, we calculated the difference from the predominant dose and expressed the difference in percent. The percent difference of each medication measure was then expressed as a median value across code cards. Results: We evaluated 132 code cards provided by 112 pediatric training institutions (response rate=61%); 16% of the institutions used multiple cards. Approximately half of the institutions used either the Pediatric Advanced Life Support (PALS) card (41%) or the PALS Survival card (12%). 57% of institutions used their own code cards. The majority (80%) of non-PALS/Survival cards listed more than half of the resuscitative medications that appeared on PALS/Survival cards. The most frequently listed resuscitative medications on the non-PALS/Survival cards were atropine (97%), epinephrine (97%), sodium bicarbonate (93%), adenosine (90%) and lidocaine bolus (90%). The majority (96%) of predominant doses on non-PALS/Survival cards were consistent with doses listed on PALS/Survival cards. However, the median percent difference from the predominant dose of each medication measure across non-PALS/Survival cards ranged from 0%-200% and more than a quarter (26%) of medication measures had a median percent difference of ≥100%. In addition, 80% of the most frequently listed resuscitative medications on the non-PALS/Survival cards had median percent difference of ≥100%. Conclusions: Although the resuscitation medication dosing recommendations listed on PALS/Survival cards were predominant across code cards of different pediatric training institutions, there was large variation in dosing recommendations of the most frequently listed resuscitative medications. Future studies should explore the extent to which this variation is associated with clinical outcomes.

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26 DISPARITIES IN HEALTH CARE UTILIZATION AND EXPENDITURES FOR LOW-INCOME VS HIGHER-INCOME OVERWEIGHT YOUTH. MM Davis, A Gebremariam, IR Mabry, University of Michigan, Ann Arbor, MI Background: The rise in prevalence of childhood overweight (body mass index [BMI] ≥95th percentile for age and sex) has occurred disproportionately among children of minority backgrounds and has also been associated with living in poverty. We compared patterns of health care utilization and expenditures among low-income (<200% federal poverty level) versus higher-income overweight youth in order to identify potential disparities in opportunities to address youth overweight in the clinical setting. Methods: We used data from the 2001 Medical Expenditure Panel Survey (MEPS), an annual household survey representative of the US non-institutionalized civilian population. The MEPS data include parent-reported child heights and weights that allow calculation of BMI; given expected biases in parent-reported data, we standardized the distribution of overweight youth by age and sex to the concurrent prevalence of youth overweight from the National Health and Nutrition Examination Survey. The MEPS data also include household income that permitted us to compare low-income to higher-income overweight children regarding MEPS measures of health care utilization (office-based, emergency department, inpatient) and expenditures (out-of-pocket, pharmacy, total). All comparisons were adjusted for child age and sex, self-reported health status, race/ethnicity, family structure, census region, and insurance status. Results: Of 1153 overweight youth in the sample, 26.3% (weighted) lived in low-income households. Low-income overweight youth had similar inpatient and emergency department utilization to higher-income overweight youth, but significantly lower office-based utilization (1.78 vs 3.21 visits per year; p<.05). Low-income overweight youth also exhibited significantly lower annual out-of-pocket ($171 vs $245), pharmacy ($79 vs $143), and total expenditures ($680 vs $1461; all p<.05) than higher-income overweight youth. These differences were of comparable or greater magnitude to differences measured among normal-weight youth in the same sample. Discussion: This is the first study to describe lower health care utilization and lower expenditures for low-income versus higher-income overweight youth, while adjusting for factors such as race/ethnicity and health status known to be associated with overweight status. Such differences suggest that overweight youth in low-income households have fewer opportunities for medical interventions regarding their overweight status, potentially worsening racial disparities in overweight prevalence.

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Poster Session 27 FETAL PROGRAMMING: TESTOSTERONE TREATMENT DURING PREGNANCY ADVANCES PLACENTOME DIFFERENTIATION. O Astapova1,3, TL Steckler1,3, LM Jackson2,3, PS MohanKumar4, SP Ford5, DL Foster2,3, V Padmanabhan1,2,3. 1Departments of Pediatrics, 2Obstetrics & Gynecology and 3the Reproductive Sciences Program, University of Michigan, Ann Arbor, Michigan; 4College of Veterinary Medicine, Michigan State University, East Lansing, MI; 5Department of Animal Science, University of Wyoming, Laramie, WY.

Departments of Pediatrics and the Reproductive Sciences Program

During pregnancy in sheep, placentomes facilitate nutrient exchange between mother and fetus. As pregnancy advances, progressive morphological differentiation of Type A placentomes into Types B, C and D increases the proportion of fetal surface area and the efficiency of nutrient exchange. In intrauterine growth retardation models, this differentiation occurs earlier to compensate for maternal undernutrition. Recently, we determined that prenatal testosterone (T) treatment produces growth retardation in sheep (Endocrinology 145:790, 2004). In the present study, we tested the hypothesis that prenatal T exposure accelerates placentome differentiation. Fetal number and weight, as well as placentome number, weight (g) and type were determined in control (C) and T-treated (100 mg, im, twice weekly, Days 30 to 90 of gestation) sheep on Days 65 and 90 of gestation (Day 65: n=10 C, n=10 T; Day 90: n=10 C, n=12 T). T-treated females had on average 14% fewer placentomes than controls at both times (P=0.059). There was a gestational day by treatment interaction on placentome weight (P<0.01), which was evident in T-treated females as a decrease on Day 65 and an increase on Day 90. There were fewer Type A placentomes in T-treated sheep at both times (P<0.05). There were no treatment differences in Type B or D placentomes. The decrease in Type A was compensated by an increase (P<0.0001) in number and weight of Type C placentomes at both Day 65 and 90. The advanced differentiation to Type C increased placentome efficiency (fetal weight/placentome weight) on Day 65 of gestation (C: 0.23±0.03 vs. T: 0.35±0.02; P<0.01). Despite the reduced number, placentome efficiency of T-treated sheep was similar to controls on Day 90 of gestation (C: 1.41±0.08 vs. T: 1.23±0.07). Upon visual inspection, placentomes of T-treated sheep appeared to be more vascular suggesting that the increased efficiency of T-treated sheep may be a function of increased blood flow. In undernutrition models as placentomes advance from Type A through D, their vascularity increases (SF Ford, personal observation). Our findings suggest that prenatal T advances placentome differentiation and that this may serve as a compensatory mechanism to increase nutrient delivery between mother and fetus to reduce growth-retardation. Supported by NIH HD- 41098.

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28 A NEW GENE LOCUS FOR NEPHRONOPHTHISIS MAPS TO CHROMOSOME 19P. M Attanasio1, B Utsch1, JL Weber2, HC Hennies3, J Hoefele1, E Otto1, F Hildebrandt1, 1Department of Pediatrics, University of Michigan, Ann Arbor MI, USA, 2Center for Medical Genetics, Marshfield Clinic Research Founation, Marshfield, Wisconsin, USA, 3Gene Mapping Centre, Max Delbrück-Centrum, Berlin, Germany

Department of Pediatrics Nephronophtisis (NPH) is a rare autosomal recessive disease and represents the most frequent genetic cause of chronic renal insufficiency in the first two decades of life. (1) Senior-Löken Syndrome (SLS) is the association of NPH with (retinitis pigmentosa). (2) Four genes (NPHP 1, 2, 3 and 4) have been identified by positional cloning as causative for NPHP and SLS. Their products are expressed in primary cilia of renal epithelial cells, therby linking the pathogenesis of NPHP to polycystic kidney disease and ciliary function. (3, 4). Since mutations have only been detected in 30% of all patients with NPHP/SLS, we performed a new total genome scan on 23 families with NPH and 9 with SLS. Pedigrees were both multiplex and non multiplex. Six of the NPH and 4 of the SLS families are known to be consanguineous. Four hundred and eleven microsatellite markers were genotyped at an average distance of 10 cM. Two-point LOD scores were calculated seeparately for the two phenotypic groups plotted across the genome using MAKESCAN program.(5) Patients were grouped in two cohorts depending of the presence or the abscence of retinitis pigmentosa. Diagnosis were made by experienced Pediatric Nephrologists and Ophtalmologists. Most of the patients received renal biopsy that confirmed the clinical diagnosis. In the cohort of NPHP patients the highest LOD Score suggestive for linkage was found on chromosome 19p for marker D19S591 at relative position 9.84 cM (Zmax =3.8 at θ=0.0). In the cohort of SLS patients the highest LOD Score suggestive for linkage was found on chromosoe 19q at D19589 at 87.66 cM (Zmax =2.16 at θ=0.05, 1.82 at theta 0.0). We conclude that both loci represent likely new gene loci for NPHP and SLS, respectively. Further studies aim at refinement by haplotype analysis and at identification of new genes causing NPH and SLS. (1) Hildebrandt F et al. Clin Investig 70:802-808, 1992 (2) Senior et al. Am J Ophtalmol 52:625-633, 1961 (3) Otto et al. Nature Genet 34(4):413-20, 2003 (4) Olbrich et al. Nature Genet 34(4):455-9, 2003 (5) Hildebrandt et al, Biomed Res 26: 592-599, 1993

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29 BILATERAL ALTERATIONS OF HIPPOCAMPAL DENDRITIC MORPHOLOGY AFTER UNILATERAL CEREBRAL HYPOXIA-ISCHEMIA IN THE IMMATURE RAT. J Barks, YQ Liu, B Kolb, Pediatrics, University of Michigan, Ann Arbor, MI and Psychology, University of Lethbridge, Lethbridge, Alberta, Canada.

Department of Pediatrics Background: Lasting cognitive deficits can be a consequence of hypoxic-ischemic (HI) injury to the immature brain. In neonatal rats, these deficits include impaired spatial learning, which is a hippocampal function; deficits could be due to neuronal loss and/or altered synaptogenesis. Little is known about the effect of neonatal HI brain injury on synaptic indices, either in injured or unaffected brain tissue. Quantification of dendritic morphology, e.g. dendritic length, branching and spine density in Golgi-stained tissue, is frequently used to evaluate the effects of brain injury on synaptic organization. We hypothesized that unilateral neonatal cerebral HI elicits bilateral dendritic abnormalities. Design/Methods: We evaluated hippocampal dendritic trees in seven-day-old (P7) male rats (n=12) that underwent either unilateral cerebral HI (n=6, right carotid ligation followed by 1.5h in 8% O2) or a sham procedure (neck incision followed by 1.5h in 8% O2). These conditions elicit mild to moderate neuronal loss in ipsilateral striatum, cortex and hippocampus, with no visible contralateral abnormalities. Rats were weaned at P21 and were housed in standard cages without behavioral testing until P60. Brains were perfused with saline followed by 1% paraformaldehyde, placed in Golgi-Cox solution for 2 wk, then sectioned at 200 µm. Spine density was calculated for 10 CA1 pyramidal neurons/side/rat by tracing a length of a third order terminal basilar dendrite (at least 70 µm long) at 2000X. The exact length of the dendritic segment was calculated, and the number of spines along the entire length counted and expressed as spines/10 µm. Total dendritic branch points/neuron were counted and dendritic length/neuron was estimated by counting dendritic intersections with a series of concentric spheres at 20 µm intervals centered on the soma. Results: In the basilar dendritic tree, hippocampal pyramidal neuron spine density was reduced both ipsilaterally (spines/10µm, mean±SD: Sham 10.6±0.6 vs. HI 8.4±0.3, p<0.001, t-test) and contralaterally (Sham 10.7±0.4 vs. HI 9.1±0.7, p<0.001, t-test). Basilar dendritic length was reduced bilaterally and dendritic branching was reduced ipsilaterally. Conclusions: These findings indicate that abnormalities of synaptic organization persist into adulthood after neonatal HI brain injury, and these abnormalities affect regions of the brain distant from the site of hypoxic-ischemic neuronal injury. These dendritic changes might be amenable to reversal by therapeutic interventions such as behavioral enrichment or trophic factor treatment.

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30 DIFFERENTIAL REGULATION OF OBSCURIN AND OBSCURIN-MLCK EXPRESSION DURING CARDIAC AND SKELETAL MUSCLE GROWTH. AB Borisov1, MO Raeker1, A Kontrogianni-Konstantopoulos2, RJ Bloch2, MW Russell1, 1University of Michigan, Ann Arbor; 2University of Maryland, Baltimore

Division of Pediatric Cardiology Obscurin is a novel giant multidomain protein recently identified in cardiac and skeletal muscle. We found two domains with high similarity to myosin light chain kinase (MLCK) that are encoded by alternative transcripts of the obscurin gene. Little is known concerning the functions and the developmental expression patterns of obscurin and obscurin-associated MLCK during cardiac and skeletal myogenesis. We hypothesized that both obscurin and the kinase would be progressively upregulated and co-localized as muscle cells differentiate. Our in vivo and in vitro studies demonstrated that obscurin and obscurin-MLCK were expressed autonomously during muscle growth and hypertrophy and had distinct cellular localization in differentiating cardiac and skeletal myocytes. Both obscurin and obscurin-MLCK were expressed in the mouse heart by day 10 post conception. We detected obscurin in the heart earlier than in the somatic musculature, and its expression was progressively upregulated during muscle differentiation in rats and mice. Developmental upregulation of obscurin in differentiating cardiac and skeletal myocytes was associated with assembly and accumulation of mature myofibrils, where it was localized in the areas of M-lines and Z-bands. Unlike obscurin, obscurin-MLCK was expressed mainly in the nuclei with some weak to moderate immunopositivity in the Z-bands at advanced stages of myogenesis. Of special interest is the fact that, unlike obscurin, this protein was found in subpopulations of mononucleated myoblasts before their fusion into myotubes suggesting its differential regulation in cardiac and skeletal muscle. Thus our results demonstrate the differential spatial expression, distinct functional roles and different developmental control of these proteins. At the same time, early upregulation and the overlapping expression patterns in embryonic and adult tissues suggest that obscurin and obscurin-MLCK are synergistically involved in the progression of cardiac and somatic myogenesis.

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31 ESSENTIAL ROLE OF OBSCURIN IN MYOFIBRILLOGENESIS AND MYOFIBRILLAR ALIGNMENT: FAILURE TO INCORPORATE MYOSIN INTO THE SARCOMERE IN CARDIAC MYOCYTES DEPLETED OF OBSCURIN USING RNAi-MEDIATED INHIBITION. AB Borisov, S Sutter, A Kontrogianni-Konstantopoulos, RJ Bloch, MV Westfall, MW Russell, University of Michigan, Ann Arbor, MI. Division of Pediatric Cardiology Myofibrillogenesis involves the assembly of new contractile units (sarcomeres) and their alignment and integration into higher order structures (myofibrils). It occurs during development as myocytes differentiate and is re-initiated in adult skeletal and cardiac muscle in response to exercise or injury. The giant protein titin is required for normal myofibril assembly, potentially serving as a ruler for the appropriate spacing of the contractile apparatus. Recently a second giant protein, obscurin, was identified and determined to interact with titin during this process. In this study, adenoviral delivery of siRNA contructs was used to limit obscurin expression in remodeling rat cardiac myocytes in vitro. Gradual depletion of obscurin from cardiac myofibrils resulted in their unbundling and progressive malalignment. Even mild depletion was associated with irregularities of alignment and the separation of adjacent myofibrils. Complete depletion was associated with the absence of distinct striations as determined by alpha-actinin immunolabeling and new myofibril assembly was unable to proceed past its earliest stages. Premyofibrillar structures such as linear arrays of Z dots formed but were unable to coalesce into mature Z bands, and sarcomeric myosin could not be incorporated into the nascent myofibril. This is the first demonstration of the requirement of obscurin for mature myofibril assembly and for the structural support of existing myofibrils. As such it may have important roles in adaptive processes requiring myocyte remodeling and new myofibril assembly such as during myocardial hypertrophy and in response to cardiac failure. Further elucidation of the functions and interactions of this novel sarcomeric protein may have important implications for the treatment of congestive heart failure, through its capacity to structurally reinforce the myofibrillar apparatus and enable the generation of new contractile structures.

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32 INTEGRATING A SOCIOCULTURAL MEDICINE CURRICULUM IN PEDIATRICS. M Bozynski, T Tang, H Haftel, R Anderson, J Mitchell, and V Press

Department of Pediatric Education The growing diversity of the US population demands that physicians be well grounded in Sociocultural Medicine. This curriculum targets residents (including Pediatric, Family Medicine and Emergency Medicine) and medical students. The curriculum seeks to integrate a longitudinal and comprehensive sociocultural medicine curriculum within Pediatric Education. This curriculum involves a three-pronged approach targeting the multi-systemic levels of the individual physician, educational infrastructure, and the evolving culture of medical training. Individual Physician. Promote cultural humility and lifelong learning. Cultural humility refers to the attitude in which physicians acknowledge their own biases affecting health care delivery and engage in continual reflection and self-directed learning. Also implicit in this attitude is the recognition that the patient/family is the teacher of their own unique culture and the acceptance to challenge traditional power differentials in the patient-physician relationship. Educational Infrastructure. Imbed socio-cultural awareness and competency into mainstream education activities. Learning experiences will be integrated within all traditional educational structures including Grand Rounds, noon conferences, clinical rotations, morning reports, didactic forums, and other learning venues. Evolving culture of medical training . Launch an innovative teaching method to meet the changing culture of resident training. Given the recent policy to reduce resident work hours (80-hour week) and the geographic dispersal of residents across multiple training sites, we propose to implement a interactive web-based bulletin board to conduct faculty-proctored case-based discussions focused on socio-cultural topics and challenges. A series of 4 faculty-developed video cases will be utilized to develop residents’ recognition, response, and reflection to socio-cultural challenges in health care.

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33 DEVELOPMENT OF A TRANSGENIC MOUSE MODEL IN UNDERSTANDING THE MECHANISM OF HUMAN BETA GLOBIN SWITCHING. A Campbell, Q Liu, O Tanabe, and JD Engel, University of Michigan, Ann Arbor, MI, Department of Cell and Developmental Biology and Department of Pediatrics. Introduction: There are five human beta-type globin genes (ε, Gγ Αγ, δ, α & β) that are under control of the Locus Control Region (LCR). The molecular basis of how the LCR directs the transcription of the beta globin genes at considerable distances (up to 50-60kb upstream) still remains controversial. . Two main models proposed that attempt to explain how the LCR directs the globin gene are 1) Looping model-individual globin gene promoters compete (gene competition) for LCR activity throughout development. 2),Linking model, depends on unknown chromatin structural determinants that lie between the LCR and the globin genes directing transcription. Our lab has exploited the use of transgenic mice harboring the human beta globin gene within a 150kb yeast artificial chromosome (YAC).. Purpose: To develop a mouse model harboring the human beta globin YAC to address the following questions: 1) Does distance from the LCR play a major, a minor, or no role at all in globin gene transcriptional activation? 2) Does looping/competition or linking most accurately describe the basis for differential β type globin gene transcriptional activity? Methods/DataThe standard 150 kb YAC was modified into a new mutant 200kb YAC. First, a linearized targeting construct corresponding to the 5’ end of the adult βglobin gene was incorporated into the YAC, selected normally by URA3. The targeting plasmid included 1 kbp of the ligated cohesive ends of λ DNA, flanked by loxP511 and loxP514 sites. Following (-)selection on FOA, the targeting plasmid and selection marker was excised leaving behind the λDNA flanked by the specifically arranged loxP recombination sites. In a second round of targeting and selection, a URA3-bearing (50kb) full length λ DNA was electorporated into yeast cells bearing the modified β globin promoter, followed by selection for homologously recombined YACs. This final product resulted in a 200 kbp human β globin YAC bearing an insertion of λ DNA between the LCR and the β globin gene. PCR, and southern blot analysis of the modified 200 kb YAC was performed to verify the correct structure of this transgene. This mutant 200kb YAC was isolated and then microinjected into fertilized eggs to establish transgenic lines. Summary: 1) A modified 200kb Human Beta Globin YAC was developed and has the correct structure and size. 2) Transgenic Mice harboring this modified 200kb is currently being generated.

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34 DEVELOPMENTAL IRON DEFICIENCY IMPAIRS RAT WATERMAZE PERFORMANCE. BT Felt1, H. Tian1, J Shao1, J. Barks1, T Schallert2, 1Center for Human Growth and Development, University of Michigan, Ann Arbor, MI, 2Department of Psychology, University of Texas, Austin, TX.

Center for Human Growth and Development Background: Iron deficiency (ID), a common nutritional disorder during development, has been associated with cognitive and behavioral impairments. Previous studies suggest developmental ID in rats affects hippocampal dendritic morphology and alters performance in the Morris Water Maze (MWM). Objective: To explore the nature of MWM deficits in rats that had ID during development. Design/Methods: 8-week old dams were randomized to iron sufficient (IS) or iron deficient (ID) groups. IS or ID diets were given during gestation and lactation. All pups received the IS diet after postnatal day (P)20. At P35, 8 to 9 IS and ID group males began one of two MWM assessments. 1st: Place learning (standard MWM) and memory (platform removed at 24 hours – probe) were assessed. Rats continued with trials to enhance learning to search the target quadrant then had a second probe trial. 2nd: IS and ID rats had thigmotaxis training before standard MWM assessment and probe. Data (days to criteria, latency, quadrant preference and thigmotaxis) were analyzed using one-way and repeated measures ANOVA (RPM). Results: 1st: ID group rats had longer latencies on place learning (RPM p<0.001). Two ID rats reached criteria (ID+) but six did not (ID-). ID- rats had significantly more thigmotaxis in place learning trials than IS rats (RPM p<0.002) but ID+ and IS rats did not differ. In the probe, IS rats had greater quadrant preference (30.94 ± 9.03) than ID- rats (-19.62 ± 8.84) and less thigmotaxis. After additional trials, latency was similar for IS and all ID rats. However, IS vs ID- remained different on probe quadrant preference (25.49 ± 5.83 vs -20.46 ± 10.32, p<0.05). 2nd: After thigmotaxis training, IS rats still had shorter latencies than ID rats in the standard MWM (RPM, p<0.001) and greater quadrant preference than ID- rats on the probe (10.29 ± 11.31 vs -33.30 ± 0.01). Conclusions: Previously ID rats demonstrate poorer MWM performance that appears related to persistent thigmotaxis behavior. Although learning improved for ID rats with additional training, memory for platform location did not. Thigmotaxis training did not impair learning measures for IS rats. The results suggest that developmental ID is associated with poorer ability to leave thigmotaxis and switch-strategies in spatial learning and memory tasks.

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35 MECHANISMS OF LEUKEMIC TRANSFORMATION BY MLL-CALM: IDENTIFICATION OF A CALM-DERIVED TRANSCRIPTIONAL REGULATORY DOMAIN. EJ Fox, MM Chao, DS Wechsler, University of Michigan, Ann Arbor, MI Division of Pediatric Hematology-Oncology Background: MLL translocations are common in infant leukemias, and >50 distinct translocation partners have been described. We recently identified the CALM gene as a novel MLL partner in an infant with aggressive AML. Interestingly, CALM was first discovered as a translocation partner for AF10, which is also an MLL fusion partner in aggressive leukemias and lymphomas. The native CALM protein exhibits predominantly cytoplasmic localization, and participates in clathrin-dependent endocytosis and intracellular vesicle transport. We have previously shown that expression of MLL-CALM immortalizes murine hematopoietic progenitor and that the fusion of the carboxy terminus of CALM to MLL alters MLL transcriptional activity. We hypothesize that CALM possesses a specific transcriptional activation domain (TAD) which modulates MLL transcriptional activity, thereby contributing to leukemogenesis. Objective: To delineate the specific CALM domains which constitute the CALM TAD, and to determine the specificity of CALM-dependent transcriptional activation. Methods: We prepared a set of expression vectors in which various portions of CALM are fused to the GAL4 DNA-binding domain. These vectors were co-transfected into COS-7 cells with a GAL4-luciferase reporter plasmid, and luciferase activity was measured 48 hours after transient transfection. Similar luciferase activity assays were performed with CALM expression constructs lacking the GAL4 DNA binding domain, and with luciferase reporter plasmids that did not contain GAL4 DNA binding sites. Results: Significant luciferase activity was only observed in constructs containing distal CALM carboxy amino acids (aa 436-660). Mutation of an NR (Nuclear Receptor) Box motif did not affect transcription. We found that two endocytosis-related NPF domains play opposite roles: deletion of NPF#1 dramatically reduces, while mutation of NPF#2 increases transcriptional activity. Expression constructs lacking GAL4 DNA binding domains have no activity, and GAL4 binding sites are required for luciferase activity. Conclusions: We have begun to identify specific crucial residues in the CALM TAD. The presence of the TAD suggests that altered transcriptional regulation of MLL-dependent genes may play an important role in MLL-CALM (and possibly CALM-AF10) dependent transformation. Our observations suggest that other MLL partners with native cytoplasmic localization may possess unrecognized transcriptional activity.

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36 ASSESSMENT OF RIGHT VENTRICULAR FUNCTION IN THE FETUS WITH HYPOPLASTIC LEFT HEART SYNDROME: USE OF THE MYOCARDIAL PERFORMANCE INDEX. S Gelehrter, C Gomez, University of Michigan, Ann Arbor, MI

Division of Pediatric Cardiology Background: Right ventricular (RV) systolic and diastolic dysfunction are risk factors for poor outcome in infants with hypoplastic left heart syndrome (HLHS), but have been difficult to quantitate in the fetus. The myocardial performance index (MPI), or Tei index, is a Doppler derived measure of ventricular function that reflects both systolic and diastolic function, is independent of ventricular geometry and heart rate, and is easily obtainable and reproducible. Our goal was to assess RV function in fetuses with HLHS using the MPI as well as other measures of ventricular function. Methods: Echocardiograms from fetuses with HLHS at our institution from August, 2002 to March, 2004 were retrospectively reviewed. The MPI was calculated from spectral Doppler measurement of RV ejection time and the interval between cessation and onset of tricuspid inflow. Peak E and A waves were measured from the tricuspid inflow Doppler for calculation of E/A ratio. Subjective assessment of RV systolic function was made on each study. RV shortening fraction (SF) was measured where possible. Results: Seventy echocardiograms were reviewed in 46 patients. The RV MPI could be measured in 50 echocardiograms (37 patients). Gestational age ranged from 19 to 39 weeks. All patients had subjectively normal RV contractility. Where a RV SF could be measured, the mean was 0.37 +/- 0.11. The mean RV MPI in all patients was 0.44 +/- 0.15 (range: 0.21 to 0.79) and increased with gestational age. In second trimester fetuses the mean RV MPI was 0.34 +/- 0.10, and in third trimester fetuses it increased to 0.46 +/- 0.16 (p=0.05). The mean E/A ratio in all patients was 0.68 +/- 0.11 (range: 0.44 to 0.93). There was no change with gestational age. Conclusion: Measurement of the RV MPI is feasible in fetuses with HLHS. The mean RV MPI is higher than published normals, and there was a trend towards increasing MPI with gestational age. The tricuspid E/A ratio was constant throughout gestation, differing from the normal increase with gestational age. As systolic function in all the fetuses remained subjectively normal, the rising MPI and constant tricuspid E/A wave ratio are suggestive of developing diastolic dysfunction. Further study is needed to evaluate the clinical implications of RV diastolic dysfunction in fetuses with HLHS.

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37 DEVELOPMENT OF A 3-DIMENSIONAL PHYSIOLOGICAL MODEL OF THE INTERNAL ANAL SPHINCTER BIOENGINEERED IN-VITRO FROM ISOLATED SMOOTH MUSCLE CELLS. L Hecker1, K Baar2, and KN Bitar1, 1Division of Pediatric Gastroenterology and 2Division of Mechanical Engineering, University of Michigan, Ann Arbor, MI.

Division of Pediatric Gastroenterology Background: Fecal incontinence affects people of all ages and social backgrounds and can have devastating psychological and economic consequences. This disorder is associated with reduced anal closure pressure and mechanical stress maintaining tissue coaptation and closure of the anal canal, which is largely attributed to decreased mechanical efficiency of the smooth muscle in the internal anal sphincter (IAS). However, little is known about the pathophysiological mechanisms responsible for the malfunction of sphincteric smooth muscle at the cellular level. Objective: To develop a 3-Dimensional Physiological Model of the Internal Anal Sphincter Bioengineered in-vitro from Isolated Smooth Muscle Cells. Methods: Smooth muscle cells isolated from the IAS of rabbits were seeded on top of a loose fibrin gel. As the cells proliferated and became confluent, they contracted the gel and self-organized into a 3-D cylindrical ring of sphincteric tissue around a 5mm diameter SYLGARD mold. Results: 1) The bioengineered IAS constructs generated a calcium-dependent basal tone. 2) Upon stimulation with 10-7M acetylcholine, bioengineered IAS constructs showed a peak contraction of 9.8±3.8µN at 30 seconds, which was sustained for 4 minutes. 3) bioengineered IAS constructs showed a dose-dependent force generation in response to acetylcholine. 4) Stimulation with 10-6M cAMP resulted in a calcium-independent sustained relaxation of -16.3±2.0µN from the basal tone. Summary: IAS constructs are highly reproducible, can be maintained in culture under physiological conditions, and are functional for up to 40 days. Conclusions: The is the first model of a bioengineered ring of Gastrointestinal smooth muscle sphincter, which resulted in a new model of the IAS bioengineered from sphincteric smooth muscle cells in vitro. This bioengineered IAS ring responds functionally and may be used in the elucidation of the mechanisms causing sphincter malfunction.

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38 NEUROBLASTOMA CELL TYPE HETEROGENEITY DEFINED BY GENOMIC AND PROTEOMIC METHODS. JA Jarzembowski, R Kuick, AW Opipari, and VP Castle, University of Michigan, Ann Arbor, MI.

Departments of Pediatrics and Pathology Neuroblastoma is a neural crest-derived malignancy responsible for 15% of childhood cancer deaths, and often resistant to treatment. It is composed of at least two cell populations: an aggressive neuroblastic component resembling primitive neural cells, and a benign-acting stromal component resembling mature neural connective tissue and Schwannian stroma. Chemotherapy can cure some neuroblastomas, but it also causes a subset of tumors to progress and/or recur. In this context, tumors are predominantly composed of neuroblastic cells. In vitro, cell lines derived from primary tumors can spontaneously convert between both cell types (N-type or S-type), providing a model system for studying their differences. We probed a Affymetrix microarray chip with RNA isolated from cultured cell lines in order to identify genes that are differentially expressed in N- and S-type cells. Candidate genes revealed by this method were verified by immunohistochemical staining of tissue microarrays constructed from over 100 primary neuroblastoma tumors, cultured cell lines, and other neural and non-neural tumors. This methodology was used to simultaneously characterize expression in terms of overall level, subcellular localization, and neuroblastic versus stromal localization. Principal component analysis of the Affymetrix expression data allowed N- and S-type cell lines to be easily distinguished. Tumor expression data analyzed according to this approach showed that tumor specimens segregated according to neuroblastic and stromal content. Hierarchical clustering supported the histological distinction between N- and S-type cell lines. Analysis of gene expression showed that 75 genes were ≥5-fold upregulated in N-type cells and 365 genes were ≥5-fold upregulated in S-type cells (p<0.01). Differential expression of 25 genes of particular interest and novelty was confirmed by immunohistochemistry. These exciting results provide insight into new molecular pathways that may be involved in neuroblastoma tumorigenesis and the control of tumor cell differentiation and stem cell-like behavior. Knowledge of the genetic signature of subpopulations of neuroblastoma cells will pave the way to a better understanding of in vivo behavior, particularly with respect to predicting chemotherapeutic response and clinical outcome.

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39 GENETIC ASSOCIATIONS IN NONTYPEABLE HAEMOPHILUS INFLUENZAE STRAINS CONTRIBUTING TO OTITIS MEDIA. PC Juliao, J Xie, CF Marrs and JR. Gilsdorf, University of Michigan, Ann Arbor, MI.

Division of Infectious Disease Nontypeable Haemophilus influenzae (NTHi) is an important cause of acute otitis media (OM). Although the pathogenic mechanisms are not well understood, NTHi may contain special virulence features that facilitate development of OM. Eight genetic regions, previously identified by genomic subtraction, are suggested to play a role in virulence. Six of the genetic regions, associated with seven genes (Hi0002, Hi0003, Hi0004, fdnG, fdoH, fdoI, and hmwA), are more prevalent in NTHi isolates from OM cases compared to throat isolates from healthy children. In contrast, a genetic region associated with fdhD was more prevalent in healthy throat isolates than OM isolates, suggesting a protective role in otitis media. In this study, we examine the co-occurrence of these genetic regions in NTHi strains. A sample of 136 isolates (90 throat and 46 middle ear) previously characterized for presence of the genetic regions was tested for co-occurrence of these regions by the kappa test (k) using SAS (v8). DNA sequence analysis was performed to determine allelic variation. Pairwise comparisons showed that the presence of five genetic regions (associated with genes Hi0002, Hi0003, Hi0004, fdnG, fdoH, fdoI) are significantly correlated (k range 0.4 to 0.9, p<0.0001), which is explained by their physical location in Hi strain Rd. The presence of the genetic region spanning fdoH and fdoI is also significantly correlated with the presence of a genetic region (hmwA) located elsewhere within the genome, suggesting a functional linkage (k=0.6, p <0.0001). The fdhD genetic region was negatively correlated in its presence with hmwA genetic region (k= –0.2, p <0.0001) as well as the genetic region spanning fdoH and fdoI (k= –0.3, p <0.0001). Further analyses of genetic regions associated with fdoH and fdoI demonstrated allelic differences compared to strain Rd. Preliminary analyses suggest that these genetic regions may contribute synergistically to the pathogenesis of NTHi otitis media. Hence, exploration into virulence of NTHi should not be limited to individual genes, but rather viewed as an interplay of many genes possibly influenced by allelic variation.

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40 BOWMAN’S CAPSULE EXPRESSION OF A PODOCALYXIN TRANSGENE: A NOVEL METHOD TO MARK THE PARIETAL EPITHELIUM. S Pauly, Y Li, DB Kershaw, University of Michigan, Ann Arbor Pediatric Nephrology Podocalyxin (Podxl) is a transmembrane sialomucin that is widely expressed on the surface of podocytes (glomerular viserical epithelial cells), vascular endothelium, and bone marrow. Mice lacking Podxl die in the first day of life with severe kidney defects. In order to study the role of Podxl in the glomerular filter we made transgenic mice with a 3007 bp human Podxl promoter and the full length rabbit Podxl transgene. Results: We have obtained litters from 16 founders. Podxl expression was assessed by IHC using specific anti-rabbit Podxl antibodies and the Vector lab’s mouse on mouse kit to minimize background. Analysis of the expression of these founder lines are showed that 5 lines expressed strongly in the kidney, spleen and thymus. No signal above background was seen in the 11 other organs examined .An additional 5 lines showed weaker kidney expression and no/minimal vascular expression in the heart and moderate expression in the thymus. 6 lines produced minimal / no expression in the kidney and thymus. Localization of the expression of the transgene in the kidney showed strong signal of the parietal epithelium of Bowman’s capsule and a lack of signal from the visceral epithelium (podocytes). In addition in some founder lines additional expression was present in the interstitial peritubular region of the medulla. Podxl has been shown to modify cell-cell junctions however on transmission electron microscopy of the parietal epithelium no differences we seen between wild-type and transgenic mice. Discussion: In many diseases of the kidney the normal relationship of the parietal and visceral glomerular epithelium is altered. The parietal epithelium of Bowman’s capsule appears to be important in the cell proliferation and migration that occurs in the process of crescentic glomerulonephritis. No parietal epithelial specific promoter constructs (kidney) have been described to study this cell type. We have inadvertently created transgenic construct that expresses strongly on the parietal epithelium with no expression elsewhere in the kidney that may be useful in studying the role of the parietal epithelium in glomerular disease and tracing cell lineage in glomerular inflammatory disease.

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41 THE YEAST ASPARTYL PROTEASE YAPSIN 1 IS A COMPONENT OF THE TRANSCRIPTIONAL RESPONSE TO CELL WALL STRESS AND REQUIRED FOR CELL WALL INTEGRITY. DJ Krysan and RS Fuller, University of Michigan

Division of Pediatric Infectious Disease Background: The yapsins (YPS1,2,3,6,&7) are a family of five GPI-linked aspartyl proteases in S. cerevisiae with close homologues in the important human pathogen, Candida albicans. Based primarily on experiments with strains containing deletions of multiple yapsins, we demonstrated previously that the yapsins are involved in cell wall glucan homeostasis and are required for maintenance of cell wall integrity. In this work, we show that YPS1 plays a unique role in cell wall processes and that it is part of the transcriptional response to cell wall stress. Results: Of the five yapsin single null mutants, yps1∆ is uniquely hypersensitive to the specific 1,3-β-glucan synthase inhibitor, caspofungin, and to the general cell wall perturbant, caffeine. Overexpression of YPS1 partially suppresses both caspofungin and congo red (a dye that disrupts glucan fibrils) toxicity in wild type cells, indicating that Yps1p may process proteins that stabilize cell wall glucan. A number of genome-wide expression studies of cell wall stress identified YPS1 as a gene induced under such conditions, suggesting that differential expression may account for the unique phenotypes associated with yps1∆. Using a lacZ reporter construct containing the promoter region of YPS1, we have shown that YPS1 is induced during periods of cell wall stress and remodeling and that this expression is dually regulated by the PKC1-MPK1, mitogen-activated protein kinase cascade as well as the calcineurin pathway. This pattern of expression and regulation closely parallels that of the stress-induced subunit of glucan synthase, FKS2, and implies that YPS1 functions in conjunction with FKS2 to remodel cell wall β-glucan during cell wall stress. Conclusions: Yps1p is part of the transcriptional response to cell wall stress and is required for maintenance of cell wall integrity under severe stress. Yps1p appears to stabilize cell wall glucan and, when expressed at high levels, leads to partial resistance to the clinically important antifungal, caspofungin. Since close homologues of YPS1 exist in pathogenic fungi, these results suggest that yapsin homologues represent potential drug targets as well as possible mediators of antifungal resistance.

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42 DETECTION OF PODOCYTE ABNORMALITIES IN NEPHROLOGIC DISORDERS, DIABETES AND SYSTEMIC LUPUS ERYTHEMATOSUS. DM Kurnit, K Yang, H Yang, Y Tang, A Khafagi, University of Michigan, Ann Arbor, MI.

Genetics Division The glomerular podocytes are commonly affected in nephrologic disorders,

diabetes and lupus, with a substantial plurality of subjects showing abnormalities. We elaborated a highly sensitive and specific assay that detected nephrin in urine sediment, the urine mRNA for nephrin (UMN) assay. This assay required detection down to several molecules of nephrin at the attomolar (10-18 M level). To accomplish this, we used the Mass Array system of Sequenom that monitors PCR by mass spectrometry. The absence of background signal in normal subjects demonstrated its specificity. In contrast, a plurality of subjects with nephrologic disorders, diabetes and SLE showed abnormalities.

In diabetes, we observed that UMN abnormalities preceded microalbuminuria in 30% of subjects with diabetes, demonstrating that this may be an early test to detect kidney problems. This was true in both adults and pediatric populations. Alternatively, later in the course of disease when microalbuminuria was fixedly abnormal but ACE inhibitors or ARBs were used, the UMN could be zero (normal) in some subjects whereas other subjects (at least 30%) continued to manifest abnormal UMN values. These presumably constitute the subjects at risk to develop chronic kidney disease. In SLE, we observed that both pediatric and adult subjects with renal involvement could intermittently show UMN anomalies, again indicating that UMN could be used to guide clinical treatments for this disorder.

We detected UMN anomalies in a variety of nephrologic disorders associated with podocyte pathology, including membranous progressive glomerulonephritis, acute glomerulonephritidies and nephrotic syndrome, Henoch-Schonlein purpura and focal segmental glomerulosclerosis. In multiple cases, the UMN test paralleled the clinical findings, demonstrating the utility of the quantitative aspects of this test. In several cases, there were sharp spikes of UMN that disappeared upon subsequent visits. These unanticipated spikes of UMN may explain ‘chronic’ renal disorders where kidney function is impaired without ‘acute’ episodes being appreciated. In sum, the UMN test has the potential to reveal the current status of the renal podocyte by a non-invasive analysis of urine.

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43 FETAL PROGRAMMING: DEVELOPMENTAL PROGRESSION OF ESTRADIOL POSITIVE FEEDBACK DEFECTS IN PRENATALLY TESTOSTERONE-TREATED SHEEP. JS Lee, M Manikkam, HN Sarma, C Herkimer, DL Foster, and V Padmanabhan Department of Pediatrics, Obstetrics & Gynecology

Ecology & Evolutionary Biology and the Reproductive Sciences Program

Female lambs exposed prenatally to testosterone (T) have a progressive loss of ovarian cyclicity culminating in reproductive failure (Endocrinology 144:1426, 2003). Perhaps this is a function of compromised estradiol (E) positive feedback. We tested the hypothesis that prenatal T treatment (100 mg T propionate twice weekly, days 30 to 90 of gestation to the mother) leads to progressive loss of E feedback. Competency of the surge system to respond to positive feedback effects of E was tested in control (n=7) and prenatally T-treated females (n=7) at the following weeks of age: 12 (pre-pubertal); 23 (puberty was 26.8±0.9 weeks in control; 30.6±1.8 weeks in T-treated, mean±SE); 54 (during their first postpubertal anestrus season). Before testing, follicular waves were synchronized by administering Nal-Glu, a GnRH antagonist (50µg/kg sc every 12h) for 72h followed by sc insertion of a 3-mm E implant to maintain early follicular levels of E for the next 72h. For testing, follicular phase E rise was produced by insertion of 4, 30-mm E implants. Blood samples were collected for LH measurements every 2h for 72h. All control animals at all ages produced LH surges defined as an increase in LH of twice the baseline value persisting for at least 8h. This was not the case for prenatally T-treated females where 3 of 7 produced LH surges at 12 weeks, 2 of 7 at 23 weeks and 5 of 6 at 54 weeks. The latent period between insertion of the E implants and the LH surge was increased (p<0.05) by prenatal T (control vs prenatally T-treated: 12 weeks, 8.7±0.5 vs 16.3±2.7 days; 23 weeks, 15.5±3.9 vs 34.0±1.0 days; 54 weeks, 13.7±0.9 vs 25.6±5 days) at all ages. Magnitude of LH surge was reduced at 12 weeks (48.4±10.1 ng/mL vs 12.6±5.3, p<0.05) and 54 weeks (52.0±14.4 vs 31.2±18.3, p=0.07) of age in prenatally T-treated animals. There was no age/treatment difference in the duration of the LH surge. These findings fail to support our hypothesis because the compromised positive feedback system in the developing female improves after the pubertal breeding season. The amelioration of estradiol positive feedback mechanism post-pubertally raises the possibility that repetitive cycles during the pubertal breeding season are beneficial. This may be transient because reproductive cycles deteriorate over time. NIH HD41098

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44 VITAMIN D DEFICIENCY: AN UNRECOGNIZED PROBLEM IN INFANTS AND MOTHERS IN BOSTON. JM Lee1, JR Smith2, BL Philipp3 and MF Holick4 1Department of Pediatric Endocrinology, University of Michigan, Ann Arbor, MI, United States; 2Department of Pediatric Endocrinology, Children's Hospital, Boston, Boston, MA, United States; 3Department of Pediatrics, Boston Medical Center, Boston, MA, United States and 4Department of Endocrinology, Diabetes and Nutrition, Boston Medical Center, Boston, MA, United States.

Department of Pediatric Endocrinology Background: Vitamin D deficiency places a child at risk for nutritional rickets and may also increase their risk of type 1 diabetes mellitus, autoimmune diseases, and common cancers. Little data exist about the prevalence of Vitamin D deficiency in mothers and their newborns in the US. Objective: To determine the prevalence of Vitamin D deficiency in healthy mothers and their newborns in Boston, MA (42 degrees North) in the winter months. Design/Methods: A convenience sample of 49 mother/infant pairs were enrolled from October 2002 to February 2003 at an inner-city hospital in Boston. Venous blood samples to measure 25(OH)D levels were obtained from all mothers and 39 infants. 5 infants had cord blood samples for 25(OH)D analyzed. All infants were healthy and >37 weeks and no mothers had a history of parathyroid or renal disease. A questionnaire was administered to all mothers, asking about dietary history, medications, and sun exposure. The serum assay was performed using the method of Chen et al. The limit of detection was 12.5 nmol/l. Values below the limit of detection were assigned 10 nmol/l. The assay has an intra-assay coefficient of variation of 8% and an inter-assay coefficient of 12%. Results: A majority of mothers (n=36, 73.5%) and a majority of the infants (n=36, 81.8%) were found to be vitamin D deficient, defined as a 25-hydroxyvitamin D level <50 nmol/l. The table shows the demographic characteristics of the mothers. 65% of mothers were black and 24% were caucasian. 20% identified as Hispanic. The average age was 28 6 years. The mean levels of 25(OH)D for mothers was 40.2 36.1 nmol/l, and for infants was 28.6 25.8 nmol/l. The correlation between 25(OH)D levels in infants and mothers was statistically significant(p<0.001). Consumption of milk, fish, or multivitamins, or the number of hours of sun exposure did not demonstrate a statistically significant correlation with 25(OH)D levels. Conclusions: Vitamin D deficiency was a common finding among mostly black and hispanic women and their babies screened at birth in Boston MA in winter months.

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45 SECRETION AND POST-TRANSLATIONAL PROCESSING OF A NOVEL TESTIS INSULIN-LIKE PROTEIN. C Lu1*, S Singh1, RB Gibbs2, WH Walker3 and RK Menon1, 1Pediatrics, University of Michigan, Ann Arbor, MI; 2Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA, and 3 Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA. Department of Pediatrics Insulin-like peptide 6 (Insl6) is recently identified protein of the insulin family whose biological function is yet to be determined. Our previous studies demonstrated that this protein is most abundant in testis and that the germ cell is the principal site of expression of Insl6. Immunofluorescence studies of both ectopically expressed Insl6 and testicular tissue demonstrated that Insl6 is localized to the endoplasmic reticulum. In this study, we present evidence to demonstrate that 1) Insl6 is a secreted protein. Secreted Insl6 could be detected from supernatant of CHO cells which stably express Insl6 protein. 2) The Insl6 protein is linked by disulfide bonds. This result was obtained by observing a conformational change in the Insl6 protein subjected to non-reducing and reducing conditions. 3) Insl6 is synthesized as a precursor and is a substrate for the prohromone convertase-4 (PC-4). In the absence of PC-4, there were two secreted forms of Insl6, the precursor form (26Kd) and a smaller form (6Kd). Ectopic expression of PC-4 resulted in the disappearance of the precursor (26kD) form of Insl6 with retention of the smaller (6Kd) form. Prior reports have demonstrated that the PC-4 null mouse is infertile; however a putative target(s) for PC-4 in the testis that could ex plain the phenotype of the PC-4 null mouse model was not known. Our results suggest that the Inls6 protein could be an endogenous target for PC-4 action in the male germ cell. In conclusion, Insl6 is a secreted protein which is linked by disulfide bonds and processed by PC4.

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46 FETAL PROGRAMMING: PROGESTERONE TREATMENT DURING THE POSTPUBERTAL ANESTROUS SEASON PARTIALLY OVERCOMES FOLLICULAR DEFICIT BUT FAILS TO RESTORE NORMAL LUTEAL FUNCTION. M Manikkam, TL Steckler, EK Inskeep, and V Padmanabhan, University of Michigan, Ann Arbor, MI

Division of Pediatrics Endocrinology Prenatal testosterone (T) treatment leads to progressive deterioration of the reproductive axis [Endocrinology 144:1426, 2003]. Recently we found ovarian follicular disruption in prenatally T-treated sheep during the postpubertal period, manifested as a reduction in number of 2-mm follicles, an increase in the size of the two largest follicles and their prolonged persistence [Biol Reprod 68(Suppl 1):329, 2003]. Because progesterone (P) treatment restores cyclicity in cows with cystic follicles, we hypothesized that cyclic P treatment during postpubertal anestrus would prevent further follicular disruption and rescue the prenatally T-treated sheep from becoming acyclic. A subset of postpubertal control (n=7) and prenatally T-treated Suffolk (n=6) sheep were implanted with 2 controlled internal drug releasing devices (CIDRs) sc for 13-14 days every 17 days throughout the postpubertal anestrous season (Control: CP; Prenatally T-treated: TP). A group of control (C; n=6) and prenatally T-treated (T; n=7) sheep served as corresponding controls. We expected the T but not the TP animals to become anovulatory in the following breeding season. Daily ovarian ultrasonography was performed for 21 days following administration of PGF2α twice, 11 days apart. Daily blood samples were taken during this period to monitor changes in P and track CL, if present on the ovary. Results revealed that 100% of C and CP ewes studied had CL (5 C and 3 CP). Only 4/7 T sheep and 3/5 TP-sheep (1 died) developed CL. Prenatal T-treatment reduced total number of 2-mm follicles (T: 68.2 ± 11.3 vs. C: 96.1 ± 3.2; P < 0.05) and follicles emerging to 3-mm (T: 29.1 ± 3.5 vs. C: 45.3 ± 2.3; P < 0.05), increased size of the largest follicle (T: 13.6 ± 1.5 vs. C: 7.5 ± 0.2 mm; P < 0.05) and duration it was present on the ovary (T: 10.7 ± 2.2 vs. C: 5.5 ± 1.2 days; P < 0.05). P treatment restored the number of 2-mm follicles and follicles emerging to 3-mm to control values, but failed to improve CL and large follicle dynamics and prevent cycle deterioration. The failure of cyclic P treatment to completely overcome follicular deficits indicates that progressive loss of cycles is prenatally programmed or alternatively the low concentration of P achieved from sc P implants (24% of cycling levels) may have been insufficient to overcome the deficit.

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47 MUTATIONS IN NPHS1 AND NPHS2 IN PATIENTS WITH STEROID-RESISTANT NEPHROTIC SYNDROME. BE Mucha, M Schultheiss, RG Ruf, F Hildebrandt, University of Michigan, Ann Arbor, MI; Berlin, Germany

Division of Pediatric Nephrology More than 25% of pediatric patients with primary steroid-resistant nephrotic syndrome (NS) have autosomal recessive mutations in the NPHS2 gene (Podocin) (Ruf et al. 2004; Karle et al. 2002). The congenital NS (CNS) of the Finnish type is caused by recessive mutations in the NPHS1 gene (Nephrin). In a few patients mutations in both genes were described (Koziell et al. 2002). Therefore we tested the hypothesis that the presence of a NPHS1 mutation in NPHS2 patients would modify the age of onset of disease. We performed mutational analysis of all exons of NPHS1 in 63 unrelated patients with steroid-resistant nephrotic syndrome (congenital and late onset) who were previously shown to have NPHS2 mutations and 12 CNS patients without NPHS2 mutations. In 49 patients with two recessive mutations in NPHS2 only one patient (F1268) had an additional novel heterozygous mutation in NPHS1; the age of onset (AO) was 1.6 years. In 14 patients with only one mutation in NPHS2, four patients with two mutations in NPHS1 were identified. All four patients had CNS and were heterozygous carriers of the frequent NPHS2 mutation, G686A/R229Q. 11/12 patients with congenital NS who did not carry any mutations in NPHS2 had two recessive mutations in NPHS1 (classic nephrotic syndrome of the Finnish type). None of the described mutations were found in 90 healthy controls. Patient F1268 has a homozygous mutation in the NPHS2 gene leading to a truncated protein (insT460-467/V165X). The amino acid of her novel heterozygous NPHS1 mutation (G1271C/V991L) is not conserved in evolution and could be a rare polymorphism. Her age of onset was 1.6 years compared to the median AO of 2.4 years of all NPHS2 patients, no biospy result was available. Four patients with one NPHS2 mutation had recessive mutations of the NPHS1 gene which alone are sufficient to cause a congenital age of onset. However, given the limited scope of this investigation we are unable to draw any genotype/phenotype correlations.

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48 BIOCHEMICAL SIGNALING MECHANISMS REGULATING RHINOVIRUS-INDUCED AIRWAY EPITHELIAL CELL RESPONSES. D Newcomb, Y Jia, S Nanua, Y Ads, L Zhou, U Sajjan, M Hershenson. University of Michigan, Ann Arbor, MI

Division of Pediatric Pulmonology Rhinovirus infection is an important cause of asthma exacerbations, yet the mechanisms underlying this process are poorly understood. We examined the signaling mechanisms elicited by rhinovirus (RV)-39 infection of 16HBE14o- human bronchial epithelial cells. RV39 infection induced expression of interleukin (IL)-8, granulocyte-macrophage colony stimulating factor and intercellular adhesion molecule (ICAM)-1, the receptor for RV39 and other major subgroup RVs. RV39 infection also induced the phosphorylation and/or activation of a number of signaling intermediates including, Jun N-terminal kinase (JNK), phosphatidylinositol (PI) 3-kinase and Akt, as well as the DNA binding and transactivation of AP-1 and NF-kB. Inhibition of JNK attenuated RV39-induced transcription from the IL-8 promoter via the AP-1 site, which serves as a basal level enhancer in this context. On the other hand, inhibition of PI 3-kinase and Akt attenuated both NF-kB transactivation and IL-8 expression. Inhibition of PI 3-kinase did not attenuate viral endocytosis, suggesting that PI 3-kinase functions as an upstream activator of NF-kB during RV39 infection. We hypothesized that RV-induced respiratory exacerbations might result from synergistic or additive effects of RV and pro-asthmatic cytokines on airway epithelial cell pro-inflammatory responses. 16HBE14o- cells were simultaneously infected with RV39 and treated with either IL-13 or tumor necrosis factor (TNF)-alpha. The combination of RV and IL13 led to synergistic increases in both IL-8 expression and AP-1 transactivation, whereas the combination of RV and TNF alpha had additive effects on IL-8 expression and NF-kB transactivation. Both IL-13 and TNF alpha caused modest increases in ICAM-1 expression, but the RV titer of IL-13 and TNF alpha-treated cells was not increased, suggesting that increased ICAM-1 expression is not responsible for the observed additive or synergistic responses. We conclude that RV and pro-asthmatic cytokines have synergistic or additive effects on airway epithelial cell IL-8 expression, AP-1 transactivation (in the case of IL-13) and NF-kB transactivation (TNF alpha).

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49 RETINITIS PIGMENTOSA AND RENAL FAILURE IN A PATIENT WITH MUTATIONS IN INVERSIN JF O Toole 1, E Otto 2, Y Frishberg 3 and F Hildebrandt 2, University of Michigan, Ann Arbor, MI, 1 Department of Internal Medicine, Division of Nephrology, and 2 Department of Pediatrics and Communicable Disease, Division of Nephrology, 3 Shaare Zedek Medical Center, Jerusalem, Israel

Division of Pediatric Nephrology Nephronophthisis (NPHP) is an autosomal recessive kidney disease characterized by corticomedullary cyst development, interstitial fibrosis and end-stage renal disease within the first two decades of life. Three genes have been reported to cause NPHP with a recessive inheritance pattern: NPHP1, NPHP3 and NPHP4. Mutations in NPHP1, NPHP3 and NPHP4 have been associated with a 10% incidence of retinitis pigmentosa. We recently reported mutations in the gene for inversin, INVS, as a cause of infantile NPHP in seven families (Nat Genet. 34:413, 2003). Here we present the first report of retinitis pigmentosa and renal failure in a patient with homozygous mutations INVS. The patient was evaluated for mutational analysis of INVS using direct sequencing due to early onset (2 years of age) of renal failure requiring dialysis. Retinitis pigmentosa was documented by ophthalmologic examination. The parents of the patient are first-degree cousins. Results of mutational analysis of all INVS exons revealed a homozygous mutation in exon 13, C2719T, with a predicted coding sequence change of R907X. The truncation of the protein is predicted to occur before the second “destruction box” and the second IQ domain. This mutation segregated from the mother, DNA was unavailable from the father. This mutation was previously published with the initial report of INVS mutations in NPHP type 2, but the family in which they occurred had no evidence of retinal abnormalities. These findings demonstrate that INVS mutations are associated with retinitis pigmentosa, as are the other genes, which cause NPHP. While mutations in both copies of INVS are sufficient to cause renal failure, the incidence of retinitis pigmentosa is low. This suggests a role for modifier genes in the development of retinal abnormalities in the setting of NPHP.

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50 DISTRIBUTION OF PEDIATRIC CRITICAL CARE RESOURCES IN THE UNITED STATES. FO Odetola, SJ Clark, GL Freed, SL Bratton, MM Davis, University of Michigan, Ann Arbor. Department of Pediatrics and Communicable Diseases Background: Prior studies describe growth in pediatric intensive care units (PICUs) in number and bed size over time. Concerns exist regarding equitable distribution of these units and access to pediatric critical care resources. Objective: To characterize the distribution of pediatric critical care resources in the United States in 2004. Methods: In January through May 2004, we conducted a cross-sectional survey of medical directors of intensive care facilities for children other than preterm neonates. Results: Pediatric critical care medical directors from 252 of 337 eligible hospitals responded (response rate = 75%). The median number of beds was 12 (interquartile range [IQR]: 8-17), with a median of 58 admissions per PICU bed (IQR: 44-70) in 2003. The median number of admissions per PICU bed was not statistically different among PICUs of different sizes. Approximately 6% of hospitals shared PICU space with critically ill adults. 94% of the PICUs had a pediatric intensivist. The median ratio of pediatric intensivists to PICU beds was 1:3, and pediatric intensivists provided 24-hour coverage in 68% of the PICUs. While 100% of the PICUs had capacity for mechanical ventilation and vascular pressure monitoring, less than 80% had facilities for renal replacement or nitric oxide therapies. Advanced patient care technology, particularly renal replacement and inhaled nitric oxide therapies, was significantly more likely to be available in PICUs of larger size than in smaller facilities; for example, 96% of PICUs with >19 beds had hemodialysis capabilities compared to 43% of PICUs with <7 beds (p<0.01). Conclusion: Facilities for critically ill children are broadly distributed across the U.S. Pediatric intensivist staffing is similar across PICUs after adjustment for bed size. Larger PICUs have significantly greater resource-capacity for high-intensity renal and respiratory therapy. The impact of PICU resource availability on referral patterns and outcome of pediatric critical illness warrants further study.

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51 THE K.I.D.S. PROJECT: MEDICAL AND PSYCHOSOCIAL OUTCOMES OF AN INTERVENTION TO KICK IN DIABETES SUPPORT FOR ECONOMICALLY DISADVANTAGED ADOLESCENTS WITH TYPE 1 DIABETES MELLITUS. L Opipari-Arrigan, J Kichler, E Fredericks, N Burkhart, L Dale, S Eder, A Silverman, S Bratton, C Foster., University of Michigan

Department of Pediatrics BACKGROUND: Adolescents with Type 1 diabetes mellitus (IDDM) have difficulty maintaining glycemic control and general health status. Economically disadvantaged adolescents are at an even greater risk for worse outcomes with IDDM, and utilize a disproportionately higher number of health care resources. There is limited empirical data on how interventions targeting low income youth could improve their functioning. OBJECTIVE: Pilot a cost-effective, self-management protocol for economically disadvantaged adolescents with IDDM and their families. Evaluate the efficacy of self-management protocol in health status, self-care behavior, quality of life, family involvement, emotional well-being, and glycemic control. METHODS: 29 adolescents, age 10 to 14 years, with IDDM (>1 year duration) who received Medicaid or other state insurance assistance and their parents completed a 6-session, family centered, self-management intervention consisting of communication training, problem solving, goal setting and diabetes education. Participants completed measures of general and diabetes quality of life, regimen adherence, diabetes responsibility, and diabetes support at baseline and post-treatment. Hemoglobin A1c (HbA1c) levels were obtained at these points. The data were analyzed using paired t-tests and linear regressions. RESULTS: HbA1c levels remained stable from baseline (mean = 9.3, SD =1.43) to post-treatment (mean = 9.2, SD = 1.24). Adolescents reported working together more collaboratively with their parents on their health maintenance for diabetes at post-treatment (p<.01) and experiencing less non-supportive family behaviors related to diabetes (p<.05). Parents reported improvements in their child s diabetes symptoms (p<.05), fewer barriers to diabetes treatment (p <.05), and better communication about diabetes (p <.05). Adolescents reported a decrease in their general depressive symptomatology (p<.05). Parental report suggested that adolescents had increased emotional functioning. The pre-to post-change in the adolescents’ emotional functioning was found to account for 30.2% of the variance in pre-to post-improvements of HbA1c levels at post-treatment (p<.01). CONCLUSIONS: A family-based self-management intervention targeted at economically disadvantaged adolescents can improve short term diabetes related health and behavioral functioning.

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52 DIRECT ASSOCIATION OF RHOA WITH SPECIFIC DOMAINS OF PKCα. H Pang, S Somara, SB Patil, KN Bitar, University of Michigan, Ann Arbor, MI,

Division of Pediatric Gastroenterology PKCα plays important roles in smooth muscle contraction. It is characterized by an NH2-terminal regulatory domain (C1 and C2) containing binding sites for calcium, phosphatidylserine, and diacylglycerol, which mediate membrane association and activation; a small central hinge region and a COOH-terminal catalytic domain (C3 and C4) containing the active site. Rho is a small guanosine triphosphatase (GTPase) involved in smooth muscle contraction. Previous studies in our laboratory showed that agonist induced contraction of smooth muscle from the rabbit colon is associated with translocation of PKCα and of RhoA to the membrane. The goal of this study is to assess whether PKCα and RhoA participate in a direct protein-protein interaction and determine the domains of PKCα involved in direct interaction with RhoA. Methods: an assay system using purified recombinant fusion proteins was employed. His-tagged individual domains and different combinations of domains of PKCα were constructed to carry out this study. Results: 1) GST-RhoA directly bound to full length PKCα (82.57 ± 15.26% increase of binding above control). 2) GST-RhoA bound to His-PKCα (C1) (70.48 ± 20.78 % increase), His-PKCα (C2) (72.26 ± 29.96 % increase) and His-PKCα (C4) (90.58 ± 26.79 % increase). 3) GST-RhoA did not bind to His-PKCα (C3) domain (0.64 ± 5.18 % above control). 4) GST-RhoA bound to the different combinations of PKCα domains, His-PKCα (C2C3C4) (94.09 ± 12.13 % above control), His-PKCα (C1C2) (60.78 ± 13.78 % above control), His-PKCα (C3C4) (85.10 ± 16.16 % above control), 5) GST-RhoA did not bind to His-PKCα (C1C2C3) (0.47 ± 1.26 % above control), nor to His-PKCα (C2C3) (7.45 ± 10.76 % above control). Conclusions: Our results provide evidence for the existence of an inter-domain interaction among C1, C2 and C3 domains independently of C4 domain, which retains the PKCα molecular in an inactive conformation. When C1 domain was removed, His-PKCα (C2C3C4) and His-PKCα (C3C4) bound to RhoA well. These fragments of PKCα mimic the active form of PKCα, removing the pseudosubstrate (at C1 domain) which occupies the active site of C4 domain, thus allows substrate binding. It suggests that RhoA may be the substrate of PKCα.

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53 TACROLIMUS IN STEROID RESISTANT NEPHROTIC SYNDROME. KE Papez, M Mills, SC White, PD Brophy, DB Kershaw, University of Michigan, Ann Arbor, MI Division of Pediatric Nephrology To date there have been limited descriptions of the use of tacrolimus (TAC) for treatment of childhood steroid resistant nephrotic syndrome (SRNS). We present our experience with TAC in patients (Pts) with SRNS. Eleven pediatric Pts with SRNS were treated with TAC. Patients presented with nephrotic syndrome (NS) at a mean age of 5.1 (19 mos-16 yrs) and TAC was initiated after an average of 5.7 years of disease (5 mos-17 years). Previous treatment included prednisone (11), oral cyclophosphamide (CYP) (4), IV pulse CYP (1), mycophenolate mofetil (MMF) (3) and cyclosporine A(CSA) (10). CSA was used per our published experience. Supportive therapies (ACE inhibitors, ARB, and statins) were used as indicated by Pts conditions. Pts had biopsy findings of focal segmental glomerulosclerosis (4), IgM Nephropathy (6), and thin basement membrane disease with mild mesangial proliferation (1). TAC was initiated in 10 Pts due to failure of treatment with multiple regimens; 6 Pts on CSA and prednisone; 2 Pts on CSA, prednisone, and MMF; 1 Pt on prednisone and MMF; 1 Pt on oral CYP and prednisone. All but 1 Pt had tried CSA. Five Pts were started on TAC due to medication toxicities; 3 Pts failing CSA and prednisone above had CSA toxicity on biopsy, 1 Pt failing CSA and prednisone had behavioral steroid toxicity, and 1 Pt with controlled nephrosis on CSA developed CSA-associated hypertensive encephalopathy and seizures. All but one obese patient was started on 0.1 - 0.23 mg/kg/day of tacrolimus BID and initial target levels were 8-10 ng/mL. Overall, 4 of 11 Pts had no response to TAC therapy. The remaining 7 developed remission 3-20 weeks after initiation, although 3 subsequently had relapses. Three pts had TAC withdrawn due to GI distress, diabetes mellitus (DM), or interstitial fibrosis after 1, 19,and 30 months of therapy, respectively. Of the 3 pts with CSA toxicity on biopsy and multiple relapses, 2 are relapse free at 4 and 6 months, on TAC and TAC/MMF, respectively. The third patient had TAC withdrawn due to DM, and has progressed to ESRD. The Pt who had hypertensive encephalopathy associated with CSA tolerated TAC with 1 relapse in 12 months. The Pt treated previously with only CYP and prednisone responded 4 weeks after TAC initiation and is 19 months relapse free, and is weaning off TAC. In our limited experience many Pts with difficult SRNS respond well to TAC and non-response to CSA does not predict a non-response to TAC.

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54 HEMODIALYSIS AND HEMOFILTRATION IN PEDIATRICS: A NEW APPROACH TO INTOXICATION. KE Papez, LS Allred, TA Mottes, TL Kudelka, TR Kelly, BJ Adams, RS Nievaard, JJ Lin, DB Kershaw, and PD Brophy, University of Michigan, Ann Arbor, MI Division of Pediatric Nephrology Pediatric patients represent a significant portion of poisoning morbidity. In general, toxins amenable to removal by hemodialysis have been approached with serial intermittent hemodialytic (IHD) therapies. Presently at our institution, an approach similar to the treatment of inborn errors of metabolism has become preferred. This approach entails the use of an initial IHD session followed by the use of continuous hemofiltration (HF). Unlike repetitive IHD, this combination therapy aids in blunting the rebound phenomena common with intoxications. We have recently utilized this approach in two pediatric patients. The first patient was a 14-year-old girl who presented to the emergency department with altered mental status of one day’s duration. The laboratory analyses demonstrated a metabolic acidosis with acute renal insufficiency. A volatile acid screen demonstrated ethylene glycol intoxication. The initial ethylene glycol level was noted to be 24.4mg/dL. The second patient was an 11-year-old girl who had been on chronic lithium. She was noted to have developed significant episodes of ataxia, athetosis, and dysarthria. On evaluation, she showed EKG changes with first degree heart block with prolonged PR and QT intervals. Her initial lithium level was 7.34 mEq/L at presentation. A creatinine of 1.2 and a slightly compressed anion gap were consistent with lithium intoxication. Both patients had triple lumen catheters placed and had initial IHD runs of 3hrs with a BFR of 250 mL/min and dialysate rate of 700 mL/min. Subsequently, both were placed on HF (24 and 48 hours, respectively) using the PRISMA®, BFR of 180 mL/min and a standard phosphorus and potassium-containing D/FRF solution at a combined rate of 3.5 L/hr, while using citrate anticoagulation. Both patients clinically and chemically improved, tolerated the successive therapies well, and had an attenuation of rebound toxin levels with this method. Both were discharged from the PICU. The successful approach to both of these patients provides a rationale for the use of HF following IHD for the treatment of acute ingestions. During these therapies attention to the dialysate bath and replacement fluid must address the patients’ metabolic derangements. We feel that this approach provides the patient with a more sustained clearance of toxin than serial IHD.

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55 MORTALITY AND PROGRESSION OF CHRONIC KIDNEY DISEASE (CKD) AMONG VETERANS WITH DIABETES. UD Patel,1,2, AO Ojo, 1, EW Young,1,2, and RA Hayward,2, 1 Div of Nephrology, University of Michigan, and 2 HSR&D, AAVA Healthcare System; All Ann Arbor, MI, United States

Division of Nephrology Background: Despite high risk of cardiovascular (CV) mortality even among patients with early CKD, CV risk-reductive therapies remain underutilized. We evaluated current medical care and examined the influence of CV risk-reduction strategies on early progression of CKD and death among diabetic veterans. Methods: 14,020 patients within a 7 hospital service network in 1998-9 were identified by computerized records. CKD stages were identified using standard National Kidney Foundation definitions. Results: Mean age was 66+11 years and mean estimated GFR was 74+25 mL/min/1.73m2. Most patients had mild to moderate CKD (Stages 1-3: 6%, 46%, 26% respectively). At baseline, medication prescribing rates for ACE-inhibitors/angiotensin receptor-blockers (AA), beta-blockers (BB), and statins (ST) were much lower than recommended in practice guidelines (58%, 25%, 41% respectively). During a 3-year period, the annual death rate in this cohort was 6% while the annual CKD progression rate (defined as a change from Stage 2 to 3) was 3%. Multivariable logistic regression adjusting for casemix (age, race, proteinuria, Hgb A1c, comorbidities, outpatient visits, and use of other CV medications) showed that each of the three medications was associated with lower mortality [Odds ratio (OR) (95%CI)]: AA 0.89 (0.78, 1.03), BB 0.86 (0.80, 0.93), and ST 0.77 (0.67, 0.93). Increased mortality was predicted by baseline CKD stage (OR 1.25 per stage), proteinuria (OR 1.90), and age>65 (OR 2.65). After adjusting for casemix, use of AA, BB and ST were not associated with significantly lower rates of CKD progression. However, CKD progression was strongly predicted by baseline CKD stage (OR 7.29 per stage), as well as proteinuria (OR 1.74), and age>65 (OR 1.67). Conclusions: In a large cohort of diabetic veterans with early CKD, use of CV risk-reductive medications is associated with decreased mortality, but not with prevention of CKD progression. However, even in this very high-risk, high-benefit population, these potentially life-saving medications are dramatically underutilized. These results highlight that even for those with early CKD that the dominate risk is CV mortality, rather than progression to dialysis. Further work directed at delineating and overcoming barriers to delivery of these CV medications in those with CDK is underway.

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56 RECEIPT OF CARDIOVASCULAR (CV) RISK-REDUCTIVE MEDICATIONS AMONG VETERANS WITH DIABETES AND CHRONIC KIDNEY DISEASE (CKD). UD Patel,1,2, AO Ojo,1 , EW Young,1,2, RA Hayward,2. 1 Division of Nephrology, University of Michigan, and 2 HSR&D, AAVA Healthcare System; All Ann Arbor, MI Division of Nephrology Background: Despite high risk of CV mortality among patients with CKD, CV risk-reductive therapies remain underutilized; we evaluated CV prescribing patterns and predictors among diabetic veterans. Methods and Results: 14,020 patients within a 7 hospital service network in 1998-9 were identified by computerized records. Mean age was 66+11 years and mean estimated GFR was 74+25 mL/min/1.73m2. Most patients had mild-moderate CKD (Stages 1-3: 6%, 46%, 26% respectively). CV medication prescribing was suboptimal but increased significantly during the 3-year study period: increasing for ACE-inhibitors (AA) from 55% to 66% of patients, angiotensin receptor-blockers (ARB) from 3% to 8%, beta-blockers (BB) from 25% to 41%, and statins (ST) 41% to 55% (p<0.001 for all; Fig.). Unlike prior studies, these rates did not decline significantly with progressive stages of CKD. While AA and ST remained stable among CKD stages, ARB and BB increased with progressive stages at 3 years (Fig 1B). After multivariable logistic regression to adjust for casemix (age, race, proteinuria, Hgb A1c, comorbidities, outpatient visits), use of each medicine significantly increased the likelihood of receiving the other medications [e.g. receiving ST was associated with, Odds ratio(OR) for AA 2.18, ARB 1.99,and BB 2.42]. While baseline CKD stage predicted use of AA, BB, & ST (OR:1.09,1.07,1.09 respectively), proteinuria only predicted use of AA & BB (OR:1.24,1.10 respectively).

Conclusions: In a cohort of diabetic veterans with CKD, use of CV risk-reductive medications increased over a 3-year period, but still remains highly underutilized. The strong associations with use of other concurrent medications suggest that there may be a significant provider-level effect. Further studies examining reasons for failure to prescribe these medications are necessary to determine specific provider- and patient-level barriers.

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57 ASSOCIATION OF HSP27 WITH MYPT, ROCK II AND PHOSPHO-RHOA IN THE PARTICULATE FRACTION DURING SUSTAINED CONTRACTION OF SMOOTH MUSCLE OF THE COLON. SB Patil, KN Bitar, University of Michigan, Ann Arbor, MI,

Division of Pediatric Gastroenterology Myosin light chain Phosphorylation (MLC) by myosin light chain kinase (MLCK) is the predominant process in the initiation of smooth muscle contraction, whereas MLC de-phosphorylation results in muscle relaxation. Agonist induced activation of RhoA/Rho kinase pathway results in inhibition of myosin phosphatase and maintenance of MLC20 phosphorylation. In addition, phosphorylation of HSP27 is critical for the maintenance of smooth muscle contraction. We hypothesize that HSP27 mediated-redistribution of MYPT and ROCKII and, association of ROCKII with RhoA is important during agonist induced smooth muscle contraction. Methods: Isolated smooth muscle from rabbit colon were stimulated with acetylcholine for 30s or 4 minutes in the presence or absence of Y27632 (a ROCKII inhibitor) or calphostinC (a PKC inhibitor). MLC phosphorylation was measured by IEF of cell lysates. MYPT phosphorylation was measured by western blot using anti-Phospho MYPT antibody. Serine phosphorylation of RhoA, PKCα were measured by immuno-precipitating the samples with anti-phospho-serine antibody followed by immuno-blot with anti-RhoA or anti-PKCα antibodies. Results: Acetylcholine induced: 1) sustained contraction and phosphorylation of MLC concomitant with phosphorylation of MYPT (113.43 and 132.72 % at 30s and 4 min) which was inhibited by Y27632 (102.85 and 105.97 % at 30s and min respectively, 2) sustained translocation of ROCKII to the particulate fraction (135.18.1 %), 3) increased association of ROCK II with RhoA (216.324.1 %) and with HSP27 (155.610.7%) which was inhibited by Y27632, 4) sustained serine phosphorylation of PKCα and of RhoA. Y27632 inhibited both RhoA and PKCα phosphorylation, 5) sustained association of MYPT with HSP27 (160.44 and 215.95 % at 30s and 4 min) which was inhibited by Y27632 (55.47 and 53.28 % at 30s and 4 min), 6) a decrease in the total phosphatase activity (63.09 and 61.80 % at 30s and 4 minutes) which was inhibited by Y27632 (99.86 and 101.82 % at 30s and 4 min). Conclusions: 1) During sustained contraction of smooth muscle cells, acetylcholine mediates independent phosphorylation and translocation of RhoA and of PKCα. 2) During acetylcholine-induced contraction, MYPT is phosphorylated and associates with translocated HSP27 in the particulate fraction. 3) HSP27-mediated translocation of the complex, MYPT-HSP27-RhoA/ROCKII, to the particulate fraction is essential for sustained contraction.

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58 NESTIN-CRE FATE MAPPING IN THE DEVELOPING MOUSE EMBRYO. H Ramaprakash, A. Sclafani, J.M. Skidmore, D.M. Martin, The University of Michigan, Ann Arbor, Michigan.

Departments of Pediatrics and Human Genetics Cre-lox recombination strategies are commonly used to target genetic rearrangements in a spatially and temporally restricted manner, and can produce isolated gene deletions when complete loss of function produces an undesirable effect such as embryonic lethality. Nestin, an intermediate filament expressed in neural progenitor cells in the brain, has been used to drive expression of Cre recombinase in neural-specific Cre mouse deleter strains. In this report we crossed Nestin-Cre mice with the Rosa26R reporter strain and analyzed β-galactosidase activity at e10.5-e14.5. We found moderate Cre activity in the e10.5 midbrain, tectum, tegmentum, dorsal and ventral hindbrain regions, and spinal cord and branchial arches. Between e10.5 and e12.5 there was increased Cre activity in the spinal cord and in the retina and oral ectoderm. By e14.5 there was prominent Cre activity throughout the central nervous system neuroepithelium, both in the ventricular and differentiating zones. In the pituitary, there was minimal Cre activity at e10.5, with increased activity at e12.5 and e14.5. Additional sites of less prominent Cre activity included thyroid, thymus, heart, lung, kidney, adrenal, and mesenchyme of the lung and gut. These results indicate developmental and regional specificity of Nestin-Cre activity, not only in the developing brain but also in the pituitary, spinal cord, developing craniofacial tissues and other organs. These data highlight the utility of Nestin-Cre mice for targeting genes in the developing brain and face and for lineage tracing in multiple tissues.

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59 PITX2 AND LMX1B INTERACTIONS IN THE DEVELOPING MAMMALIAN SUBTHALAMIC NUCLEUS. JM Skidmore, DM Martin, University of Michigan, Ann Arbor, MI. Regional specialization of the developing neuroepithelium is a central issue in developmental neurobiology. In the mouse forebrain and hindbrain, transcription factors including Dlx, Pax, and Engrailed genes function to specify distinct neuronal identities with specific neurotransmitter and anatomical phenotypes. Pitx2, a paired-like homeodomain transcription factor, is likely to have a role in regional specialization given its requirement for normal development of neurons in the murine subthalamic nucleus (STN), a key component of basal ganglia and a target for deep brain stimulation in Parkinson’s disease. The mechanisms regulating Pitx2-mediated STN development are not known. Here we report an analysis of STN gene expression in midgestation wildtype and Pitx2 mutant mouse embryos, to identify potential downstream targets of Pitx2 in this brain region. Pitx2 co-localizes at the cellular level with Lmx1b, a LIM-homeodomain transcription factor that specifies mesencephalic dopaminergic neurons in the developing mouse substantia nigra. We detected loss of LMX1B protein in the developing STN of Pitx2 mutant embryos. LMX1B expression is preserved in other brain regions of Pitx2 mutant mouse embryos, suggesting regionally specific interactions of Pitx2 and Lmx1b during brain development. Ongoing studies using gene targeting and expression analysis will help clarify the precise genetic pathways by which Pitx2 and Lmx1b confer STN neuronal differentiation and lineage specification.

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60 CALCIUM-DEPENDENT PKC-MEDIATED ACETYLCHOLINE-INDUCED PHOSPHORYLATION OF TROPOMYOSIN IN COLONIC SMOOTH MUSCLE CELLS. S Somara, KN Bitar, University of Michigan, Ann Arbor, MI,

Division of Pediatric Gastroenterology Background: Mg2+/ATPase activity of the actomyosin complex is crucial for muscle contraction. Phosphorylation of αα−tropomyosin has been reported to be associated with increased myosin Mg2+/ATPase activity in vitro in rabbit striated muscle. The role of phosphorylated tropomyosin in smooth muscle is unclear. Objective: To investigate the phosphorylation of smooth muscle tropomyosin and the role of calcium as well as PKC in this phosphorylation. Methods: Smooth muscle cells from rabbit colon were isolated in buffer with and without calcium. Whole cell lysates of freshly isolated cells were preincubated with/without calphostin C, a PKC inhibitor and stimulated with acetylcholine (0.1 µM) for 30 sec or 4 min. The samples were immunoprecipitated with phosphoserine antibody followed by immunoblotting with tropomyosin antibody. Results: 1) Stimulation of colonic smooth muscle with acetylcholine resulted in a significant and sustained increase in serine phosphorylation of tropomyosin (408 ± 12 and 389 ± 28 percent increase; p≤0.001, n=3 at 30 sec and 4 min respectively). 2) Preincubation with calphostin-C inhibited acetylcholine-induced increase in tropomyosin phosphorylation (112 + 14 and 135 ± 20 percent decrease at 30 s and 4 min respectively P≤ 0.06, n=3). 3) Acetylcholine-induced serine phosphorylation was greatly reduced in the absence of calcium (144 + 5 at 30 sec and 133 + 6 percent at 30 sec and 4 min respectively; P≤0.01; n=3). 4) Sequence analysis of smooth muscle tropomyosin for PKC consensus sequences revealed that tropomyosin protein contains five potential PKC serine phosphorylation sites. Summary: 1-In colonic smooth muscle cells, acetylcholine induced a sustained phosphorylation of tropomyosin. 2-Acetylcholine-induced phosphorylation of tropomyosin in colonic smooth muscle was PKC-mediated 3-The PKC-mediated phosphorylation of tropomyosin is also calcium-dependent. 4-The five potential PKC phosphorylatable sites on smooth muscle tropomyosin are S63, S123, S158, S215, and S262. The PKC phosphorylatable sites on smooth muscle tropomyosin are novel and different from the ser283 site, a known phosphorylatable site expressed only in striated muscle. Conclusion: In colonic smooth muscle where troponin is absent, agonist-induced, calcium-dependent, PKC-mediated phosphorylation of tropomyosin could have a role in activation of actomyosin Mg2+/ATPase.

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61 DEFINING THE DISTRIBUTION OF RALSTONIA SPECIES IN CYSTIC FIBROSIS BY PCR ANALYSIS. T Spilker,1 R Reik,1 T Coenye,2 P Vandamme, 2 JJ LiPuma1 1University of Michigan, Ann Arbor, MI; 2Universiteit Gent, Gent, Belgium

Division of Infectious Diseases Ralstonia pickettii has long been recognized as an opportunistic bacterial pathogen in persons with CF. Recently, we described several novel Ralstonia species that also have been recovered from CF sputum culture. We sought to define the distribution of Ralstonia species recovered from respiratory specimens from persons with CF. We developed a novel PCR assay targeting 16S rDNA sequences specific for all species within the genus Ralstonia. A second 16S rDNA-directed assay was designed to be specific for the recently defined species R. respiraculi. In an assessment of 187 CF sputum bacterial isolates that had been previously identified by PCR assay or 16S rDNA sequence analysis as representing 13 Ralstonia species and 24 other CF relevant bacterial species, we found the Ralstonia genus-level PCR to have a sensitivity of 98% and a specificity of 100%. To assess the performance of the R. respiraculi-specific PCR assay, we examined 60 CF sputum isolates. This group included 16 isolates identified as R. respiraculi by 16S rDNA sequence analysis and 44 isolates representing 12 additional Ralstonia species. We found the R. respiraculi PCR assay to have a sensitivity of 100% and a specificity of 95% (with two R. metallidurans isolates giving a positive result). To investigate the distribution of Ralstonia species among infected CF patients, we used these novel assays, 16S rDNA sequence analysis, and three previously reported PCR assays (specific for R. pickettii, R. mannitolilytica or R. insidiosa) to assess 113 Ralstonia isolates recovered from 113 CF patients in the US. R. mannitolilytica accounted for 42.5% of isolates, while R. pickettii and R respiraculi each accounted for 16.7%. Other Ralstonia species were infrequently recovered, with R. gilardii and R. insidiosa accounting for 6.7% and 2.5% of isolates, respectively. R. eutropha, R. paucula and R. taiwanensis were each represented by a single isolate (or 0.8% of the total). Eight isolates (6.7%) were clearly identified as Ralstonia species by 16S rDNA sequence analysis, but could not be classified as belonging to a previously defined species. These data (i) demonstrate the utility of the novel PCR assays for identification of Ralstonia spp and R. respiraculi, (ii) reveal the unexpected dominance of R. mannitolilytica among Ralstonia-infected CF patients, and (iii) indicate that additional as yet undefined Ralstonia species are likely involved in pulmonary infection in CF.

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62 FETAL PROGRAMMING: PRENATAL EXPOSURE TO BISPHENOL-A AND METHOXYCHLOR DISRUPTS OVARIAN FOLLICULAR DYNAMICS. TL Steckler, M Manikkam, EK Inskeep, and V Padmanabhan University of Michigan, Department of Pediatrics and the Reproductive Sciences Program, Ann Arbor, MI

Department of Pediatrics and the Reproductive Sciences Program Exposure of sheep fetuses to testosterone (T) and not dihydrotestosterone disrupts estradiol (E) positive feedback (Rev Reprod 3:130, 1998) and leads to multifollicular ovaries (Mol Cell Endocrinol 185:51, 2001) suggesting that such disruptions are regulated by conversion of T to E. These findings raise concerns as to the threat that unintended exposure to exogenous steroids poses to reproductive health. Recently, we found that in utero exposure to bisphenol-A and methoxychlor (BPA, MXC; estrogenic endocrine disrupting chemicals (EDCs)) had differential effects on the generation of the preovulatory LH surge following induction of luteolysis with PGF2a; BPA sheep had reduced latency and/or reduced LH surge amplitude while MXC sheep had increased latency (Biol Rep 68(Suppl 1):293, 2003). Differences in latency of LH surge in BPA/MXC sheep suggest disruptions in follicular dynamics and/or compromised E positive feedback. In this study, we tested the hypothesis that exposure of sheep fetuses to BPA or MXC impairs follicular selection dynamics. Suffolk ewes were treated with MXC (n=6), BPA (n=8) (5 mg/kg/day in cotton seed oil) or vehicle (C; n=8) s.c. from days 30 to 90 of gestation. At ~19 months of age, transrectal ovarian ultrasonography was performed daily for 21 days. Daily changes in appearance and disappearance of ovarian follicles, the follicular sizes and presence of corpora lutea (CL) were monitored in both ovaries. The total number of follicles present was greater (C: 93.7±2.8; MXC: 112.4±7.2; P<0.02; by analysis of variance), an increased size of the second largest follicle (C: 5.0±0.7; MXC: 7.0 ± 0.5; P<0.05), and a tendency for an increased mean duration of presence of the second largest follicle (C: 5.5 ± 0.63 d; MXC: 7.2 ± 0.5 d; P<0.08) was observed in the MXC group. An increase in the diameter (mm) of the second largest follicle (C: 5.0±0.7; BPA: 7.1±0.4; P<0.02) characterized BPA group. No differences in CL dynamics were evident. The differential effects of BPA/MXC on ovarian follicular dynamics observed suggest that prenatal exposure to MXC has a greater impact on follicle selection dynamics. Our findings substantiate the concerns regarding the deleterious effects that unintended exposure to EDCs during pregnancy might have on fertility. NIH HD 41098 & Office of the Vice President for Research, U of M.

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63 HISTONE DEACETYLASE INHIBITORS AS AN APPROACH TO TREAT NEUROBLASTOMA. C Subramanian,* AW Opipari,#; R Kwok,# and VP Castle,* *Department of Pediatrics, # Department of OBGYN, University of Michigan, Ann Arbor, MI.

Department of Pediatrics Neuroblastoma, a cancer that develops in infants and young children, is poorly responsive to available drugs and for this reason is usually incurable. Histone deacetylase inhibitors (HDACIs) are the new class of chemotherapeutic drugs and are proving to be useful against several types of cancer. Low toxicity of these drugs as well as the novelty of these drugs towards NB pathogenesis prompted us to investigate the role of HDACI as potential therapeutics for treating NB. Although, clinical development of this class of drugs is advanced, the molecular mechanism of action of this class of drugs is almost entirely undefined. We have found that the most aggressive type of NB cell lines, represented by neuroblastic cell lines (N-type) in vitro are more sensitive to killing by HDACIs like TSA than less aggressive Stromal (S-type) cell lines that are also derived from NB tumors. Moreover, When NB cells are interconverted from undifferentiated intermediate type to N-type cell lines in vitro they become highly sensitive to TSA treatment. We have identified the mechanism by which HDACIs exert their cytotoxic response on NB cell lines. Our results indicate that it is independent of signals including caspases, p53 and protein synthesis. Interestingly, our results showed that HDACI cytotoxic response is dependent on translocation of Bax into mitochondria and release of Bax from KU70. Even though, key roles of KU70, a best known autoantigen, in multiple nuclear processes like DNA repair, chromosome maintenance, transcriptional regulation, and V(D)J recombination are well known, only recently Bax has been identified as its cytoplasmic binding partner. Studies carried out by Cohen et al. (Molecular Cell, 13, 627-638, 2004) identified that acetylation of KU70 at lysine residues 539 and 542 controls Bax mediated apoptosis. Future studies are underway for defining how HDAC activity in NB translates in to Bax translocation and the role of KU70 in this process using KU70 lysine mutant (K539, 542R) that cannot be acetylated. The results from the study will test our hypothesis that acetylation of KU70 is essential for TSA induced killing of NB cells which will shed further light on the mechanism by which HDACIs, particularly TSA, acts on neuroblastoma.

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64 CHRONIC GRAFT VERSUS HOST DISEASE AFTER ALLOGENEIC PERIPHERAL BLOOD STEM CELL TRANSPLANTATION IN CHILDREN. CD Thornburg, GA Yanik, RJ Hutchinson, KR Cooke, JL Ferrara, JE Levine, University of Michigan, Ann Arbor, MI. Peripheral blood stem cell transplantation (PSCT) has been explored as an alternative to bone marrow transplantation. Compared to bone marrow, peripheral blood stem cells result in more rapid engraftment. Some studies have suggested a survival advantage for PSCT, but other studies reveal increased chronic graft-versus-host disease (CGVHD). The incidence, severity, and duration of CGHVD following PSCT in children are poorly understood. In order to characterize the incidence and course of chronic graft-versus-host disease in a pediatric population, we retrospectively reviewed the results of 30, consecutive, related allogeneic peripheral blood stem cell transplants in subjects (less than 21 years) with malignant and non-malignant conditions. CGVHD developed in 23/29 (79%) of evaluable subjects. The most common sites of CGHVD were the skin, liver, mouth and eyes. Three patients died from CGVHD, and the majority of severe CGVHD associated complications occurred in these patients. The mean duration of follow-up after day 100 was 1031 days, and the mean duration of immunosuppressive therapy for CGHVD was 751 days. Intensive therapy with high-dose steroids or greater than two immunosuppressive agents accounted for twenty-four percent of the treatment time. Treatment related complications included hyperglycemia [3/29], hypertension [5/29], cataracts [2/29] and avascular necrosis [2/29]. Despite the high incidence of CGVHD and prolonged treatment, all survivors attended grade school, high school or college at an age appropriate level. This study improves the understanding of the clinical burden of chronic graft-versus-host disease in children undergoing peripheral blood stem cell transplant.

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65 MODULATION OF HOST CHEMOKINE RESPONSES BY LATENT GAMMAHERPESVIRUS INFECTION. J Weinberg, G Stempfle, K Spindler, Departments of Pediatrics and Microbiology and Immunology, University of Michigan, Ann Arbor, MI.

Division of Pediatric Infectious Diseases The gammaherpesviruses have evolved multiple immunomodulatory mechanisms to escape immune control, including genes that affect complement deposition, MHC class I expression and chemokine responses. To test the hypothesis that gHV-mediated immunomodulation is capable of affecting host responses to other pathogens, we evaluated chemokine responses to acute mouse adenovirus 1 (MAV-1) infection in mice latently infected with murine gammaherpesvirus 68 (MHV-68). BALB/c mice were initially infected intranasally with MHV-68. After recovering for a minimum of 30 days, the mice were infected intraperitoneally with MAV-1. MHV-68-naïve mice subsequently infected with MAV-1 served as controls. In both groups of mice, MAV-1 infection upregulated the expression of the chemokines CXCL10/IP-10, CCL2/MCP-1 and TCA-3 between 3 and 7 dpi. Prior to MAV-1 infection, baseline expression of the chemokines CCL5/RANTES, CCL3/MIP-1α, CCL4/MIP-1β and MIP-2 was elevated in mice previously infected with MHV-68 compared to levels measured in MHV-68-naïve mice. MAV-1 infection did not substantially upregulate the expression of these chemokines in either group of mice, but rather decreased their expression to levels seen in MHV-68-naïve mice infected with MAV-1. No significant differences were detected between groups in the upregulation of other cytokines such as IL-1α, IL-1β, IL-6, IL-10, IL-12, IL-18 and IFN-γ. MAV-1 gene expression was not affected by latent gammaherpesvirus, as expression of the MAV-1 hexon gene in the spleen and brain at multiple time points did not differ significantly between groups. No elevations in MHV-68 viral load were measured following MAV-1 coinfection, suggesting that coinfection did not induce reactivation of latent MHV-68. Taken together, these data suggest that latent MHV-68 infection does not modulate susceptibility to subsequent infection with MAV-1. Low-level persistent or latent MHV-68 infection may continue to stimulate chemokine responses in the spleen at late time points post-infection, and acute MAV-1 infection is capable of downregulating this persistent response.

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66 INFLAMMATORY RESPONSES IN THE LUNG AND BRAIN INDUCED BY ACUTE RESPIRATORY INFECTION WITH MOUSE ADENOVIRUS TYPE 1. J Weinberg, G Stempfle, K Spindler, Departments of Pediatrics and Microbiology and Immunology, University of Michigan, Ann Arbor, MI

Division of Pediatric Infectious Diseases Adenovirus infection is a common cause of upper and lower respiratory tract infections in humans. Inflammatory responses to adenovirus infection contribute to clinical disease and are a substantial hindrance to the use of adenoviral vectors for gene therapy. Studies of adenovirus pathogenesis are limited by the strict species-specificity of the adenoviruses. We used mouse adenovirus 1 (MAV-1) to study host inflammatory responses to acute lung infection in C57BL/6 mice. Mice were infected intranasally with virus or were mock-infected with conditioned media. Organs were harvested at baseline and at multiple time points post-infection. MAV-1 replication, assessed using ribonuclease protection assay (RPA) to measure MAV-1 hexon gene expression, was detected in the lungs between 4 and 7 dpi and became undetectable in the lungs at 14 dpi. MAV-1 gene expression was detected only at 14 dpi in the brain. Chemokine gene expression was also measured using RPA. In the lungs, peak levels of most chemokine transcripts occurred at 7 dpi, while RANTES expression continued to increase at 14 dpi. No differences in viral gene expression, chemokine response or cellular infiltrate were noted in the lungs of mice infected with a virus deleted for the E1A gene, an adenovirus gene that has been implicated as a potential immunomodulatory gene. Chemokine expression was absent in the brains of mock-infected mice and in MAV-infected mice at 7 dpi, while multiple chemokines were upregulated in the brains of infected mice at 14 dpi. Histopathology revealed a mild, predominantly mononuclear cellular infiltrate in lungs at 7 dpi. No infiltrate was seen in lungs harvested at 14 dpi or in mock-infected lungs. A marked perivascular cellular infiltrate was present in brains infected with wildtype virus at 14dpi. Together, these data demonstrate systemic viral dissemination and inflammatory responses following acute respiratory infection with MAV-1. MAV-1 infection of mice has the potential to serve as a model for inflammatory changes seen in human adenovirus disease.

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67 THE C/EBPα MYELOID TRANSCRIPTION FACTOR REGULATES EXPRESSION OF MXI0. BF Wolf and DS Wechsler. The University of Michigan, Ann Arbor, MI Section of Pediatric Hematology-Oncology Background: The MYC family plays a key role in the control of proliferation and differentiation in normal and malignant cells. c-Myc is a transcription factor that activates transcription of genes related to cell growth, division, and apoptosis. Mxi1, a homologous yet distinct relative of c-Myc, represses transcription of c-Myc-regulated genes. We have previously shown that Mxi1 expression antagonizes c-Myc in brain and prostate tumor cell lines, resulting in suppression of cell proliferation. We recently identified Mxi0, an alternatively transcribed Mxi1 isoform that lacks growth suppressive activity. While Mxi1 and Mxi0 are co-expressed in many cell lines and tissues, the relative levels of Mxi0 are consistently higher in malignant cells. This discrepancy suggests that expression of Mxi0 and Mxi1 may be differentially regulated. Objective: To identify transcription factor(s) critical to MXI0 transcription. Methods/Results: We have identified a 17 base pair region 260 bp upstream of the MXI0 ATG, deletion of which dramatically reduces transcription through the MXI0 promoter. Sequence analysis indicates the presence of overlapping putative AP2 (previously shown to regulate MXI1) and C/EBPα transcription factor binding sites in this area. We have prepared MXI0 promoter luciferase reporter constructs in which the AP2 and C/EBPα sites have been mutated. These mutants show reduced activity in comparison with the wild type promoter in K562 cells. When co-transfected with increasing amounts of AP2 expression plasmid into AP2-deficient HEPG2 cells, the presence of AP2 appeared to have no specific effect on transcription. However, similar studies in C/EBPα-deficient 293T cells suggested that C/EBPα specifically activates MXI0 at low concentrations, but is repressive at high levels. To confirm this interaction, we performed gel shift assays using 32P-labeled oligonucleotides and nuclear extracts from C/EBPα transfected 293T cells. These studies showed reduced C/EBPα binding to mutant oligonucleotides in comparison with wild type. In the presence of C/EBPα-specific antiserum, supershifted DNA-protein assemblies were detected. Conclusions: AP2 co-transfection experiments indicate that AP2 is not critical for MXI0 transcription. However, our studies suggest that C/EBPα participates in regulation of MXI0 transcription. Since C/EBPα plays a crucial role in myeloid differentiation, it is of interest that we have recently found that MXI0 levels first increase then decrease as myeloid U937 cells are induced to differentiate (DSW, unpublished observations). This raises the possibility that MXI0 is a downstream mediator of C/EBPα activity.

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68 SUBVENTRICULAR ZONE PROLIFERATION AFTER α-AMINO-3-HYDROXY-5-METHYL-4-ISOXAZOLEPROPIONIC ACID (AMPA) RECEPTOR-MEDIATED NEONATAL BRAIN INJURY. G Xu, J Ong, FS Silverstein, J Barks, University of Michigan, Ann Arbor, MI

Departments of Pediatrics and Neurology Background: The forebrain subventricular zone (SVZ) contains stem cells capable of proliferating to generate new mature neurons and oligodendrocytes (OL). Although some studies suggest acute loss of SVZ OL precursors after unilateral hypoxic-ischemic (HI, unilateral carotid ligation followed by timed hypoxia exposure) neonatal brain injury, recovery of myelin basic protein (MBP) immunostaining in the white matter 2 weeks after HI raises the possibility that stem cells in the SVZ might proliferate to repopulate the injured white matter. As an alternative to the HI model, we developed a model of neonatal brain injury that focuses on a unique mechanism of increased vulnerability of immature OLs, AMPA-receptor mediated excitotoxicity. We previously demonstrated that intracerebroventricular (icv) injection of the glutamate analog AMPA in seven-day-old (P7) rats caused a reduction in expression of two OL-specific genes, proteolipid protein and DM-20 24-48h later. We hypothesized that the immature SVZ would respond to AMPA-induced injury with cellular proliferation. Methods: P7 rat pups received either left or right (n=12) stereotaxic icv injections of S-AMPA, 2.5 nmol in 5 µl water (pH 7.2). Controls included right icv injections of PBS 5 µl (pH 7.2, n=5) and non-injected littermates (n=7). On P8 or P14, brains were perfused with 4% paraformaldehyde, cryoprotected in graded sucrose and sectioned serially at 60 µm. A series of every fourth section was processed for immunohistochemistry using an antibody to the cell proliferation marker Ki-67. Bilateral SVZ was outlined and Ki-67+ cells were counted stereologically using the Optical Disector method (Stereologer, SPA, Alexandria VA). Results: Bilateral enlargement of the lateral ventricles (greater on the side of injection) was common in the AMPA-injected group. At 24h after icv AMPA injection, there was a bilateral increase in the number of Ki-67+ cells/SVZ, compared to normals, with no difference between the ipsilateral and contralateral SVZ (p<0.05, Kruskal-Wallis with Dunn’s post-test). By 7days the median number of Ki-67+ cells/SVZ was still greater than in normal controls (p<0.05), but was lower than at 24h post-injection (p<0.05). Conclusion: This preliminary study using the novel marker Ki-67 suggests that there is a proliferative response in the immature SVZ in response to an AMPA receptor-mediated excitotoxic stimulus. However the phenotype and ultimate fate of the proliferating cells remain to be characterized.

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69 DIFFERENTIAL EXPRESSION OF MXI0 AND MXI1 IN CELL DIFFERENTIATION. D Zheng and DS Wechsler. The University of Michigan, Ann Arbor, MI Division of Pediatric Hematology-Oncology Background: The c-Myc transcription factor regulates expression of genes related to cell metabolism, growth and proliferation. Mxi1, a Myc family member, antagonizes the growth-promoting effects of c-Myc by repressing transcription – its overexpression inhibits cell proliferation and is associated with cellular differentiation. We recently identified Mxi0, an alternatively transcribed Mxi1 isoform that fails to repress transcription. While Mxi1 has a punctate nuclear distribution, Mxi0 is diffusely cytoplasmic in all cell lines examined. Relative levels of Mxi0 and Mxi1 vary, with the Mxi0/1 ratio being higher in less differentiated tissues and cells. While MXI1 mRNA levels increase when myeloid U937 cells are induced to differentiate, changes in MXI0 mRNA, and Mxi0 and Mxi1 protein levels in differentiation have not been addressed. Objective: To study expression and localization patterns of Mxi0 and Mxi1 in cell differentiation. Methods/Results: Differentiation was induced by 18 nM TPA in U937 cells, and by 10 µM 13-cis retinoic acid in SH-SY5Y neuroblastoma cells. MXI0 and MXI1 mRNA expression levels (by RT-PCR) and Mxi0 and Mxi1 protein (by Western blot and immunofluorescence) were measured at various time points (t=3h to 10d). In U937 cells, MXI0 mRNA expression increased dramatically 6h after TPA induction, followed by a decrease back to baseline by 12h. In contrast, MXI1 mRNA levels increased modestly, but only after 24h. Similar changes were seen at the protein level for both Mxi0 and Mxi1. RA-induced differentiation of SH-SY5Y cells resulted in similar MXI0 and MXI1 mRNA and protein expression patterns. Localization was analyzed by immunofluorescence for GFP fusions and endogenous proteins. Mxi0-GFP exhibits three distinct patterns – cytoplasmic, perinuclear and nuclear; Mxi1-GFP was only identified in the nucleus. Immunofluorescence studies showed that endogenous Mxi0 is cytoplasmic, while Mxi1 is nuclear. During differentiation, Mxi0 shuttles between the cytoplasm and nucleus, while Mxi1 remains nuclear. Conclusions: The dramatic increase in Mxi0 expression within 6h of differentiation induction in both hematopoietic and neuroblastoma cells indicates that Mxi0 plays an important role early in differentiation. In the face of modest Mxi1 changes, these observations suggest that perturbation of the Mxi0/Mxi1 ratio may modulate the effects of c-Myc, acting as a switch that commits cells to differentiation pathways. Our observation that Mxi0 shuttles from the cytoplasm to the nucleus during differentiation suggests that altered subcellular localization may affect the activity of Mxi0 in differentiation.

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70 REGULATION OF CELL SIZE AND CONTRACTILE PROTEIN EXPRESSION IN AIRWAY SMOOTH MUSCLE CELLS. L Zhou, A Goldsmith, M Hershenson. University of Michigan, Ann Arbor, MI

Division of Pediatrics Pulmonology Airway smooth muscle hypertrophy is a feature of severe asthma. However, the molecular mechanisms underlying this process are poorly understood. We established a conditionally-immortalized human airway smooth muscle cell line with a temperature-sensitive SV40 large T antigen. At the non-permissive temperature, cells arrest in mid-G1 of the cell cycle and demonstrate features of hypertrophy seen in asthma including increased cell size by flow cytometry and increased expression of contractile proteins such as alpha-smooth muscle actin and myosin light chain kinase (MLCK). Hypertrophic cells did not show increased contractile protein mRNA, suggesting that protein expression is under translational control. To further characterize this process, we pre-treated cells with a battery of chemical inhibitors consisting of rapamycin (an inhibitor of mTOR), LY294002 (an inhibitor of phosphatidylinositol 3-kinase), SB202190 (p38 mitogen-activated protein kinase) and PD98059 (extracellular signal regulated kinase). Rapamycin, LY294002 and, to a lesser extent, SB202190, each decreased cell size. LY294002 also decreased contractile protein expression but not steady-state actin mRNA expression, consistent with the notion that contractile protein is controlled at the level of mRNA translation. However, rapamycin had no effect on actin or MLCK protein expression, demonstrating that while cellular hypertrophy and contractile protein expression may occur concurrently, these processes are regulated independently. Since cell size was decreased by rapamycin, we examined the phosphorylation or expression of mTOR and potential downstream effectors including ribosomal S6 kinase and proteins of the eukaryotic initiation factor (eIF)-4F complex. Hypertrophy was associated with phosphorylation of mTOR, S6 kinase, eIF4E-binding protein (4E-BP) and eIF4E, as well as increased expression of the RNA helicase eiF4AII. We conclude that increased airway smooth muscle cell size, but not contractile protein expression, is dependent on mTOR. Future studies will determine the requirement and sufficiency of mTOR downstream effectors in this process.

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71 UTILIZATION OF INTRACRANIAL PRESSURE MONITORS IN CRITICALLY ILL CHILDREN WITH MENINGITIS. FO Odetola*, JM Tilford**, MM Davis*, *Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor; MI, **Department of Pediatrics. University of Arkansas for the Medical Sciences, Little Rock, AR.

Department of Pediatrics and Communicable Diseases Background: Intracranial pressure monitoring (ICPM) may reduce mortality and morbidity associated with childhood meningitis. However, indications for ICPM are unclear, and patterns of ICPM use in children with meningitis are unknown. Objectives: To describe patient- and hospital-level factors associated with the use of ICPM in critically ill children with meningitis. Design/ Methods: A retrospective cohort study of children 0-18 years old hospitalized with meningitis requiring mechanical ventilation was conducted using the Kids’ Inpatient Database (KID). The KID includes over 2 million pediatric discharge records obtained from 22 states in 1997 and 27 states in 2000. Records were weighted to obtain national estimates of meningitis hospitalizations and ICPM use. ICPM cases were identified using ICD-9 procedure codes. Meningitis was defined using ICD-9 codes for a primary or secondary diagnosis of meningitis. Hospitalizations with ICD-9 codes for traumatic brain injury or ventriculo-peritoneal shunts were excluded, as were pre-transfer hospitalizations. Using data pooled from 1997 and 2000, bivariate comparisons of factors related to ICPM use were performed, and multivariate logistic regression models were fit to determine which factors were independently associated with ICPM use. Results: There were 1067 and 1170 hospitalizations for childhood meningitis requiring mechanical ventilation in 1997 and 2000, respectively. Most (79%) of the hospitalizations involved infants (<1 year old). In bivariate analyses, ICPM use differed by age (4% in infants, 14% in children 1-4 years old, 21% in children 5-18 years old; p<0.01) and by census region. There was no difference in ICPM use by gender, race, etiologic organism, median household income, primary payer, hospital characteristics (rural/urban, teaching status, bed size), or year. In multivariate analyses, increasing age in 1-year increments (OR 1.14, 95%CI 1.09-1.20) and living in the West (OR 2.59, 1.38-4.87) compared to other census regions were independently associated with ICPM use, while controlling for other factors. Conclusion: The findings indicate heretofore unrecognized variation in the use of ICPM for childhood meningitis by child age and by census region. Further study is warranted to examine the reasons for this variation and effects on clinical outcomes related to ICPM use.