Child Health Research Center Annual Retreat

Co-hosted by

The Department of Pediatrics Yale University

and The Department of Medicine Children’s Hospital Boston

Hotel Viking Newport, Rhode Island November 1–3, 2007

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TIME ACTIVITY AND LOCATION

THURSDAY, NOVEMBER 1, 2007

12:00 pm – 5:30 pm Registration The Bellevue Ballroom Foyer

4:00 pm - 5:30 pm

Poster Presenters Set-up Posters The Bellevue Ballroom

5:30 pm – 6:00 pm

Reception The Bellevue Ballroom

6:00 pm – 7:00 pm Dinner The Bellevue Ballroom

7:30 pm – 8:30 pm Keynote Speaker: Mr. Frank Brady Chairman, Medical Missions for Children "Lessons Learned While Building a Global

Telemedicine Network" The Bellevue Ballroom

8:30 pm – 10:00 pm Poster Session (posters on display the entire meeting)

The Bellevue Ballroom

FRIDAY, NOVEMBER 2, 2007

7:30 am – 8:00 am Registration and Breakfast The Bellevue Ballroom

8:00 am – 12:00 pm Oral Presentations The Bellevue Ballroom

12:00 pm – 1:00 pm Program Director’s Business Meeting Salons ABC

12:00 pm – 1:20 pm Lunch Salon AB

1:20 pm – 5:00 pm Oral Presentations The Bellevue Ballroom

6:00 pm

Dinner The Bellevue Ballroom

8:00 pm Ghosts of Newport Tour Washington Square

(outdoor tour)

SATURDAY, NOVEMBER 3, 2007

8:00 am – 11:00 am Breakfast/Informal Discussion Salon AB

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Author Abstract and Biosketch Index

Program Page # Bio and Abstract Pg# Juan Pablo Abonia 9 81 Suneet Agarwal 7 55 Shahab Asgharzadeh 8 59 Anne Blaschke 10 87 Josh Bonkowsky 4 13 Sara DiVall 8 65 Patricia Dubin 10 85 Anthony French 10 91 Rasheed Gbadegesin 4 15 Edward Gilmore 4 17 Oscar Gomez-Duarte 9 93 Julie Goodwin 10 77 Ruchi Gupta 7 49 Paula Hertel 5 19 Rachel Katzenellenbogen 5 21 Paul Kingma 5 23 Carrie Kitko 5 25 Mark Levin 5 27 Corinne Linardic 8 63 Carey Lumeng 10 95 Veronica Mas Casullo 7 51 Sean McGhee 9 73 Valerie McLin 7 53 Steven Mittelman 5 29 Kenan Onel 8 69 Tamara Pozos 10 89 Sridhar Rao 5 31 Michelle Rheault 9 79 Lawrence Rhein 6 33 Amy Roberts 7 57 Robert Roghair 6 35 Mary Beth Ross 6 37 Shannon Ross 9 83 Matthew Ryan 9 71 Brian Sims 6 39 Christopher Spurney 8 61 Maria L. Sulis 6 41 Kimara Targoff 9 75 Trent Tipple 8 67 Adeline Vanderver 6 43 Rachel Wolfson 6 45 David Ziring 7 47

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CHILD HEALTH RESEARCH CENTER ANNUAL RETREAT

November 1-3, 2007 Hotel Viking, Newport, Rhode Island

THURSDAY, NOVEMBER 1, 2007 12:00 pm – 5:30 pm Registration – The Bellevue Ballroom, Foyer 4:00 pm Poster Set-up – The Bellevue Ballroom 5:30 pm Reception – The Bellevue Ballroom 6:00 pm Dinner – The Bellevue Ballroom 7:30 pm Keynote Speaker – The Bellevue Ballroom

Mr. Frank Brady Chairman, Medical Missions for Children

Lessons Learned While Building a Global Telemedicine Network

8:30 pm – 10:00 pm Poster Session – The Bellevue Ballroom

POSTER SESSION – THE BELLEVUE BALLROOM 1 TEMPORAL-SPATIAL MAPPING OF BASAL GANGLIA

PATHFINDING IN ZEBRAFISH. JL Bonkowsky and CB Chien, Salt Lake City, UT. University of Utah School of Medicine Page 13

2 MUTATIONS IN PLCE1/NPHS3 ARE A MAJOR CAUSE

OF ISOLATED DIFFUSE MESANGIAL SCLEROSIS (IDMS). RA Gbadegesin, M Zenker, R Wiggins, S Heeringa, J Liu, C Vlangos, B Hoskins, B Hinkes, and F Hildebrandt, Durham, NC, and Ann Arbor, MI. Duke University Page 15

3 A NOVEL GENETIC DISEASE MICROCEPHALY WITH

SEIZURES ASSOCIATED MUTATIONS IN A DNA-REPAIR GENE. EC Gilmore, J Shen, and CA Walsh, Boston, MA. Children's Hospital Boston Page 17

4 MATERNAL IMMUNIZATION USING ROTAVIRUS VIRUS-LIKE PARTICLES CAN PREVENT ROTAVIRUS-

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ASSOCIATED BILIARY ATRESIA IN NEONATAL MOUSE PUPS. PM Hertel, SE Crawford, MJ Finegold, and MK Estes, Houston, TX. Baylor College of Medicine Page 19 5 NFX1-123 AND CYTOPLASMIC POLY(A) BINDING

PROTEINS AUGMENT ACTIVATION OF TELOMERASE BY HPV 16E6. RA Katzenellenbogen, P Vliet-Gregg, and DA Galloway, Seattle, WA. University of Washington Page 21

6 SYSTEMIC SURFACTANT PROTEIN D INHIBITS

INFLAMMATION INDUCED BY INTRAVENOUS LIPOPOLYSACCHARIDE. PS Kingma, SK Mierke, and JA Whitsett, Cincinnati, OH. Cincinnati Children's Hospital Medical Center Page 23

7 CHRONIC GVHD (cGVHD): ANALYSIS OF INCIDENCE

AND POTENTIAL FOR DISCOVERY OF BIOMARKERS. CL Kitko, S Paczesny, J Richardson, S Choi, G Yanik, J Ferrara, and JE Levine, Ann Arbor, MI. University of Michigan

Page 25 8 NOVEL POPULATION OF MELANOCYTE-LIKE CELLS

IN THE HEART TRIGGER ATRIAL ARRHYTHMOGENESIS. MD Levin, MM Lu, NB Petrenko, D Lang, JT Jacobson, CF Spurney, CB Brown, L Huang, F Beerman, JA Epstein, and VV Patel, Philadelphia, PA, and Washington, DC and Epalinges, Switzerland. Children’s Hospital of Philadelphia Page 27

9 EXPLORING THE EFFECTS OF OBESITY TO

ACCELERATE LEUKEMIA RELAPSE USING A MOUSE MODEL. SD Mittelman, JW Behan, JP Yun, and N Heisterkamp, Los Angeles, CA. Children’s Hospital Los Angeles Page 29

10 THE ROLE OF SALL4 IN ES CELL PLURIPOTENCY. S Rao, and S Orkin, Boston, MA. Children's Hospital Boston Page 31

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11 FcγRIII IS PROTECTIVE AGAINST PSEUDOMONAS AERUGINOSA PNEUMONIA. LM Rhein, M Perkins, NP Gerard, and C Gerard, Boston, MA. Children's Hospital Boston

Page 33 12 MATERNAL LOW PROTEIN DIET AND FETAL

GLUCOCORTICOID EXPOSURE PROGRAM ADULT MOUSE CARDIOVASCULAR AND ENDOCRINE STATUS. RD Roghair, JL Segar, KA Volk, G Aldape, CM Kiroff, TD Scholz, and FS Lamb, Iowa City, IA. University of Iowa Page 35

13 IDENTIFICATION OF POTENTIAL TARGET GENES

OF THE CHIMERIC TRANSCRIPTION FACTOR, E2A-PBX1 BY siRNA KNOCKDOWN. ME Ross, C Chen, BH Connell, K Foley, and J Townsend, Columbus, OH. Ohio State University Page 37

14 THE ACTIVITY OF THE CYSTINE GLUTAMATE

EXCHANGER IS REQUIRED FOR OLIGODENDROCYTE SURVIVAL. B Sims and H Sontheimer, Birmingham, AL. University of Alabama at Birmingham Page 39

15 NEW ONCOGENIC EVENTS IN T-CELL

LEUKEMOGENESIS. M Sulis, O Williams, S Pallikuppam, T Palomero, and A Ferrando, New York, NY. Columbia University Page 41

16 ALTERED PROTEIN TRAFFICKING IN EIF2B

RELATED DISORDERS IN RESPONSE TO ENDOPLASMIC RETICULUM STRESS. AL Vanderver, M Mintz, R Schiffmann, and Y Hathout, Washington, DC and Bethesda, MD. Children's National Medical Center

Page 43 17 HMGB1 CAUSES HUMAN LUNG ENDOTHELIAL CELL

(EC) BARRIER DISRUPTION. RK Wolfson, ET Chiang, and JGN Garcia, and, Chicago, IL. University of Chicago

Page 45

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18 ENDOCANNABINOID-MEDIATED IMMUNOREGU-LATION OF EXPERIMENTAL COLITIS. DA Ziring and J Braun, Los Angeles, CA. UCLA David Geffen School of Medicine Page 47

19 GEOGRAPHIC VARIABILITY IN CHILDHOOD ASTHMA PREVALENCE IN CHICAGO. R Gupta, X Zhang, L Sharp, J Shannon, and K Weiss, Chicago IL and Washington DC. Northwestern Feinberg School of Medicine Page 49

20 HERPES SIMPLEX VIRUS IMPEDES DENDRITIC CELL FUNCTION POSSIBLE MECHANISM OF IMMUNE EVASION. V Mas Casullo, V Shende, and BC Herold, New York, NY. Mount Sinai Medical Center Page 51

FRIDAY, NOVEMBER 2, 2007 7:30 Breakfast – The Bellevue Ballroom

ORAL PRESENTATIONS The Bellevue Room

Session A

Development, Genetics and Cancer

Jordan Kriedberg, Presiding 8:00 THE ROLE OF WNT SIGNALING IN THE

DEVELOPMENT OF THE VISCERAL MESODERM. VA McLin, RD Shah, CH Hu, and M Jamrich, Houston, TX.

Baylor College of Medicine Page 53 8:20 ROLE OF THE TRANSCRIPTION FACTOR NANOG IN

EMBRYONIC DEVELOPMENT AND NUCLEAR REPROGRAMMING. S Agarwal, A Yabuuchi, A Takeuchi, and GQ Daley, Boston, MA. Children's Hospital Boston

Page 55 8:40 SARCOMERE GENE MUTATIONS AND NOONAN

SYNDROME CONGENITAL HYPERTROPHIC

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CARDIOMYOPATHY. AE Roberts, VA Joshi, and RS Kucherlapati, Boston, MA. Children’s Hospital Boston

Page 57 9:00 PROGNOSTIC SIGNIFICANCE OF GENOMIC

ALTERATIONS OF METASTATIC NEUROBLASTOMAS. S Asgharzadeh, R Pique-Regi, Y Tu, R Sposto, H Shimada, A Ortega, and RC Seeger, Los Angeles, CA. Children’s Hospital Los Angeles Page 59

9:20 CARDIOMYOPATHY IN THE DYSTROPHIN

DEFICIENT MOUSE HEART: CHARACTERIZATION USING ECHOCARDIOGRAPHY AND GENE EXPRESSION PROFILING. CF Spurney, S Knoblach, EE Pistilli, K Nagaraju, and EP Hoffman, Washington, DC. Children’s National Medical Center Page 61

9:40 BREAK – Poster Viewing and Discussion

Session B Development, Genetics and Cancer

Scott A. Rivkees, Presiding 10:00 GENETIC MODELING OF ALVEOLAR

RHABDOMYOSARCOMA. CM Linardic, K Etheridge, S Naini, S Adam, R Bentley, S Qualman, and C Counter, Durham, NC and Columbus, OH. Duke University Page 63

10:20 INSULIN STIMULATES GONADOTROPIN-RELEASING

HORMONE (GNRH) PROMOTER ACTIVITY IN VIVO. SA DiVall, L Su, A Wolfe, and S Radovick, Baltimore, MD. Johns Hopkins University Page 65

10:40 EFFECTS OF SUBLETHAL HYPEROXIC EXPOSURE

ON THE THIOREDOXIN SYSTEM IN NEWBORN MICE. TE Tipple, SE Welty, LD Nelin, and LK Rogers, Columbus, OH. The Ohio State University College of Medicine Page 67

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11:00 THE GENETIC DETERMINANTS OF APOPTOSIS. K Onel, O Yildiz, B Yilmaz, J Badner, A Kowalska, R

Diederich, and J Badner, Chicago, IL. University of Chicago Page 69 11:20 MARKED BILE DUCT PROLIFERATION IN MICE

HETEROZYGOUS FOR JAG1 AND FRINGE ALLELES. MJ Ryan, K Kusumi, N Spinner, L Underkoffler, A Nelson, M Segalov, P Russo, and KM Loomes, Philadelphia, PA and Phoenix, AZ. The Children's Hospital of Philadelphia

Page 71 11:40 COPY NUMBER VARIATIONS IN DEL22Q11.2

SYNDROME. SA McGhee, M Suchard, and ERB McCabe, Los Angeles, CA. David Geffen School of Medicine at UCLA

Page 73 12:00 LUNCH – SALON AB

Session C Development, Infection and Immunity

Margaret K. Hostetter, Presiding 1:20 NKX GENES REGULATE HEART TUBE EXTENSION IN

ZEBRAFISH. KL Targoff, T Schell, and D Yelon, New York, NY. Columbia University Page 75

1:40 A CRITICAL ROLE OF THE VASCULAR ENDOTHELIUM IN THE DEVELOPMENT OF GLUCOCORTICOID MEDIATED HYPERTENSION. JE Goodwin, J Zhang, and D Geller, New Haven, CT. Yale

University School of Medicine Page 77 2:00 FUNCTIONAL SIMILARITIES BETWEEN

SYNAPTOPODIN AND TROPOMYOSIN IN STRESS FIBER FORMATION. M Rheault and P Mundel, New York, NY. Mount Sinai School of Medicine Page 79

2:20 THE INVOLVEMENT OF MAST CELLS IN

EOSINOPHILIC ESOPHAGITIS. JP Abonia, C Blanchard,

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and ME Rothenberg, Cincinnati, OH. Cincinnati Children's Hospital Medical Center Page 81

2:40 BLOOD VIRAL LOAD AND HEARING LOSS IN YOUNG

CHILDREN WITH CONGENITAL CYTOMEGALOVIRUS INFECTION. SA Ross, Z Novak, WJ Britt, KB Fowler, RF Pass, and SB Boppana, Birmingham, AL. The University of Alabama at Birmingham Page 83

3:00 BREAK –Poster Viewing and Discussion

Session D

Infection and Immunity

Gary Fleisher, Presiding 3:20 IL-17-INDEPENDENT ACTIONS OF IL-23 IN

PSEUDOMONAS AERUGINOSA PULMONARY INFECTION. PJ Dubin and JK Kolls, Pittsburgh, PA. Children’s Hospital of Pittsburgh Page 85

3:40 RAPID MOLECULAR DIAGNOSTICS FOR SERIOUS

BACTERIAL INFECTION. AJ Blaschke, L Meyers, K Kitanovic, AT Pavia, CL Byington, and MA Poritz, Salt Lake City, UT. University of Utah Page 87

4:00 MMP-9 IS REQUIRED FOR TUBERCULOUS

GRANULOMA FORMATION IN VIVO. TC Pozos, H Volkman, J Zheng, JF Rawls, and L Ramakrishnan, Seattle, WA and Chapel Hill, NC. University of Washington Page 89

4:20 RAPID EMERGENCE OF ESCAPE MUTANTS

FOLLOWING INFECTION WITH MURINE CYTOMEGALOVIRUS IN IMMUNODEFICIENT MICE. AR French, T Geurs, J Pingel, and W Yokoyama, St Louis, MO. Washington University School of Medicine Page 91

4:40 GENETIC AND EVOLUTIONARY ANALYSIS OF

LONGUS PILUS OF ENTEROTOXIGENIC ESCHERICHIA COLI. OG Gomez-Duarte, J Bai, S

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Chattopadhyay, SJ Weissman, and EV Sokurenko, Iowa City, IA and Seattle, WA. University of Iowa Children's Hospital

Page 93 5:00 OBESITY ALTERS THE ACTIVATION STATE OF

ADIPOSE TISSUE MACROPHAGES. CN Lumeng, JL Bodzin, and AR Saltiel, Ann Arbor, MI. University of Michigan

Page 95 6:00 DINNER – The Bellevue Ballroom 8:00 GHOSTS OF NEWPORT TOUR – Washington Square

(outdoor tour)

SATURDAY, NOVEMBER 3, 2007

8:00 am – 11:00 am Breakfast/Informal Discussion – Salon AB

(Posters need to be taken down)

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Biography and Abstracts of Presenters

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Josh Bonkowsky, MD, PhD Instructor University of Utah Dr. Josh Bonkowsky is an instructor in the Division of Pediatric Neurology at the University of Utah. He received his undergraduate degree in Biochemistry from Harvard University, and then did his MD/PhD training at the University of California, San Diego. Subsequently, he completed pediatric and child neurology residencies at the University of Utah and Primary Children’s Medical Center, Salt Lake City. Dr. Bonkowsky’s research is on neurogenetics and disorders of neurodevelopment. His laboratory focus is characterization of CNS development in the forebrain and basal ganglia, and its genetic regulation in normal and disease states. Using zebrafish, he has developed transgenic lines expressing fluorescent markers in live embryos to visualize neuron pathfinding. His clinical research is on the causes and biomarkers of inherited leukodystrophies.

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Fig. 1: Different enhancer:gfp constructs in zebrafish CNS. Lateral views (except D), anterior left, dorsal top. Arrow: telencephalic

TEMPORAL-SPATIAL MAPPING OF BASAL GANGLIA PATHFINDING IN ZEBRAFISH J.L. Bonkowsky, C.B. Chien, Department of Pediatrics, University of Utah, Salt Lake City, UT Background: The basal ganglia brain nuclei are integral to motor function, cognitive development, and emotional regulation. Despite basal ganglia vulnerability and involvement in cerebral palsy and other neurodevelopmental conditions, development and formation of their connections remain poorly understood. Objectives: My goal is to characterize the development and genetic control of basal ganglia connectivity, using zebrafish (Danio rerio). Zebrafish offers key advantages: rapid CNS development; direct visualization of neurons and axons; and economic large-scale genetic screens. Methods: We sought to identify zebrafish basal ganglia neurons. To show that these neurons are equivalent to their mammalian homologs, we examined expression of conserved genes, and their loss-of-function phenotype. To visualize axon pathfinding, we generated constructs and transgenic fish that express GFP in subsets of basal ganglia neurons. To investigate the genetic control of connectivity, we are examining pathfinding genes including the robos, slits, and ephrins. Results: We identified mesodiencephalic dopaminergic (mesDA) basal ganglia neurons in zebrafish. There was conserved expression of mesDA genes to their mammalian homologs, and we found loss of dopa-minergic-specific markers using a loss-of-function approach. We generated enhancer:gfp constructs with specific expression in basal ganglia neurons and their axons (Fig.1), and are examining the role of the robos in mesDA pathfinding. Conclusions: We identified mesDA neurons in the zebrafish and showed they are likely homologous to mammalian mesDA neurons. Our enhancer:gfp constructs and transgenic fish allow us to analyze mesDA neuron pathfinding. The ability to characterize the spatial and temporal roles of genes in basal ganglia development enables us to develop molecular models of basal ganglia function and connectivity.

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Rasheed A. Gbadegesin, MD Jr. Faculty Duke University Rasheed Gbadegesin M.D. received his medical degree from the College of Medicine University of Ibadan Nigeria. He did his residency training at the University College Hospital Ibadan Nigeria. He was awarded the prestigious Fellowship of the International Society of Nephrology and was a Fellow at the University of Manchester United Kingdom. He later immigrated to the USA and was a resident in pediatrics in New York and later a Pediatric Nephrology Fellow at the University of Michigan Ann Arbor. He spent the last 2 years of his Fellowship in the Laboratory of Friedhelm Hildebrandt. He worked under the direction of Dr. Hildebrandt at this time and he was part of the team that successfully identified mutations in PLCE1 as a cause of early onset nephrotic syndrome. This finding has been the subject of high profile publication in Nature Genetics. He presented results from this work at the Pediatric Academy Society meeting in May 2007 and he won the ASPN Fellows’ Basic Science award for this presentation. Dr. Gbadegesin is currently a junior Faculty at the Duke University Durham, NC and he is currently continuing his work on the molecular genetics of glomerular diseases in the laboratory of Dr Michelle Winn.

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MUTATIONS IN PLCE1/NPHS3 ARE A MAJOR CAUSE OF ISOLATED DIFFUSE MESANGIAL SCLEROSIS (IDMS) RA Gbadegesin, B Hinkes, B Hoskins, C Vlangos, J Liu, S Heeringa, R Wiggins, M Zenker, and F Hildebrandt, Department of Pediatrics, University of Michigan, Ann Arbor and Duke University, Durham, NC. Background: Diffuse mesangial sclerosis (DMS) is a histologically distinct variant of nephrotic syndrome (NS) characterized by early onset and by progression to end stage kidney disease. We recently identified by positional cloning recessive mutations in the gene PLCE1/NPHS3 as a cause of NS that manifest as DMS or rarely as FSGS (1). Objectives: In order to ascertain the frequency of PLCE1 mutations in isolated DMS (IDMS), we performed mutational analysis in children with IDMS for mutations in PLCE1 as well as WT1, and LAMB2, two other genes that cause DMS. Methods: We identified 41 children with IDMS from a worldwide cohort of 1,368 children with NS. The 41 children from 36 different families were analyzed for mutations in PLCE1 by multiplex capillary heteroduplex analysis and direct sequencing, by direct sequencing of exons 8 and 9 of WT1, and of all the exons of LAMB2. Results: The median (range) age at onset of NS was 11 (1-72) months. We detected homozygous or compound heterozygous mutations in PLCE1 in 11/36 (30.5%) families and WT1 mutations in 3/36 (8.3%). We found no disease causing mutation in LAMB2. All the PLCE1 mutations are truncating or splice site mutations. All mutations were absent from 150 healthy control chromosomes. One child with truncating PLCE1 mutations had responded to treatment with cyclosporine and has been disease free for more than 13 years (1). Conclusions: PLCE1 mutations are a major cause IDMS in this worldwide cohort and account for almost 1/3 of cases with IDMS. One child with truncating mutations was found to be therapy responsive. If this observation is confirmed in a larger study, mutation in PLCE1 may serve as a biomarker for selecting patients with IDMS who may benefit from treatment. (1) Hinkes B. et al. Nat Genet 38: 1397-1405; 2006

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Ed Gilmore, MD, PhD Children’s Hospital Boston I grew up in rural Northeastern Ohio and did my undergraduate work at Ohio State University with a B.S. in Molecular Genetics. Next, I attended Medical and Graduate school at Case Western Reserve University as a Medical Scientist Training Program fellow. I chose neuroscience as a field and the laboratory of Karl Herrup, Ph.D. as a Ph.D. advisor because I wanted training in mouse genetics. I thought mice were a powerful system because of their ability to be used for models of human genetic diseases. My Ph.D. thesis involved studying brain abnormalities in Cyclin-dependent kinase 5 deficient mice. My work showed there were cell autonomous neuronal migration abnormalities in both the cerebral cortex and cerebellum. After graduating from medical school, I did my pediatrics training at Children’s Hospital of Michigan (Wayne State University, Detroit, MI) and my adult and pediatric neurology training at Massachusetts General Hospital (Harvard University, Boston, MA). After completing my clinical training, I knew that I wanted to re-start my research career with the eventual goal of developing an independent research laboratory. I choose the laboratory of Christopher Walsh, M.D., Ph.D. because of his lab’s ability identify novel human genetic changes leading to brain abnormalities and also to understand the mechanisms of disease through mouse models. Through my current project of studying a novel human genetic neurological disease, microcephaly with seizures that is caused by a mutation in a DNA repair gene, I hope to understand the mechanism of this disease. Through this and other work, I hope to understand the role of DNA repair in the developing brain with the eventual goal of improving the lives of children with various abnormalities.

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A NOVEL GENETIC DISEASE MICROCEPHALY WITH SEIZURES ASSOCIATED MUTATIONS IN A DNA-REPAIR GENE. Edward C. Gilmore1, Jun Shen2, Christopher A. Walsh1,2 1 Division of Genetics ,Children’s Hospital Boston, Harvard Medical School, Boston, MA, 2 Howard Hughes Medical Institute, Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA. Microcephaly is a condition defined by head size less than 2 standard deviations below the mean. When severe, microcephaly is frequently associated with mental retardation. The laboratory of Christopher Walsh at Children’s Hospital Boston is interested in identifying genetic conditions leading to abnormal brain development to understand the causes of mental retardation and other conditions. The Walsh lab in association with international collaborators has identified a new condition with severe microcephaly associated and infantile onset of intractable epilepsy we have called microcephaly with seizures. The patients have severe developmental delay and intractable epilepsy. They have very small brains on MRI, but no obvious structural abnormalities that often lead to epilepsy such as neuronal migration defects. Through genetic mapping and candidate gene sequencing in consanguineous families of Middle-Eastern origin, along with Europeans as well, the genetic abnormality was demonstrated to be a DNA repair gene. We hope to develop a greater understanding of mechanism of disease leading both microcephaly and seizures in these patients.

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Paula M. Hertel, MD Jr. Faculty Baylor College of Medicine I am originally from Minnesota and went to college and medical school there, but am currently based in Houston, where I have done my residency in pediatrics, my fellowship in pediatrics GI/nutrition/hepatology, and am now junior faculty at Baylor College of Medicine and Texas Children’s Hospital. Beginning this academic year, my clinical focus will be pediatric hepatology, and I will spend the bulk of my time continuing my studies on a mouse model of rotavirus-associated biliary atresia under the guidance of my wonderful mentor, virologist Mary Estes, Ph.D. Biliary atresia is a devastating disease of infants and children that results in suffering for so many of my patients and their families. This is highly motivating when it comes to the search for mechanisms and potential treatments for this disease!

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MATERNAL IMMUNIZATION USING ROTAVIRUS VIRUS-LIKE PARTICLES CAN PREVENT ROTAVIRUS-ASSOCIATED BILIARY ATRESIA IN NEONATAL MOUSE PUPS. PM Hertel, SE Crawford, MJ Finegold, MK Estes, Baylor College of Medicine, Houston, TX. Background: Biliary atresia (BA) is a progressive, irreversible obliterating disease of bile ducts that develops during infancy and is believed to be caused by rotavirus in some cases. Objectives: To test the hypothesis that maternal immunization using recombinant VP 2/6/7 rotavirus virus-like particles (VLPs), which contain immunogenic rotavirus capsid proteins but are noninfectious, will prevent rotavirus-associated biliary atresia in mouse pups. Design/Method: Serum rotavirus antibody titers were determined in 18 Balb/c mouse dams with varying rotavirus exposure histories. Dams were then given intramuscular injections with either 20 µg rotavirus VLPs plus adjuvant or adjuvant alone before mating. When litters were born, maternal serum antibody titers were repeated. Pups were inoculated with 107 pfu live RRV intraperitoneally on the 2nd day of life, and assessed daily from 5-11 days post-inoculation (dpi) for signs of cholestasis. BA was defined as observation of at least one acholic (pale) stool using a colorimetric stool rating scale or at least one observation of 3+ bilirubinuria using a rapid test strip as well as gross abnormalities in bile duct morphology (strictures and/or dilatations) at 11 dpi. All mice were euthanized at 11 dpi, and bile ducts and livers were resected and examined grossly and microscopically. Results: Eighteen dams inoculated with VLPs + adjuvant had significant augmentation of serum rotavirus antibody titers versus dams inoculated with adjuvant alone (p<0.01). Twenty-four of 31 pups (77.4%) born to 10 dams with low (≤1:800) peripartum serum rotavirus titers and 1 of 31 pups (3.2%) born to 8 dams with low (≤1:1600) titers developed BA (p<0.01). Pups with BA had significantly higher serum rotavirus antigen levels (p<0.01) and lower antibody titers (p<0.01) than healthy pups. Conclusions Reached: Immunization using rotavirus virus-like particles significantly augments serum immunity against rotavirus in mouse dams. Maternal rotavirus antibody titers of ≥1:800 result in significant protection of offspring against BA following neonatal rotavirus infection.

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Rachel A. Katzenellenbogen, MD Acting Assistant Professor University of Washington Rachel A. Katzenellenbogen is an Acting Assistant Professor in Pediatrics in the division of general pediatrics and adolescent medicine at the University of Washington. After growing up in the Midwest, she received her undergraduate degree in Chemistry at Harvard University in 1994. She then entered The Johns Hopkins School of Medicine in Baltimore, MD where she received her medical degree in 1999. She received her pediatric residency training, as well as her fellowship in adolescent medicine and STD research, at the University of Washington in Seattle, WA. During her fellowship she began to pursue her research in high-risk human papillomavirus (HPV) infections under the mentorship of Dr. Denise A. Galloway at the Fred Hutchinson Cancer Research Center. She is continuing her research on high-risk HPV and how the infection leads to cancer.

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NFX1-123 AND CYTOPLASMIC POLY(A) BINDING PROTEINS AUGMENT ACTIVATION OF TELOMERASE BY HPV 16E6. RA Katzenellenbogen1,2, P Vliet-Gregg1, DA Galloway2, Dept. of Pediatrics, 1University of Washington, Seattle, WA, 2Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA. Background: Human papillomavirus (HPV) is the most common sexually transmitted infection, and HPV types are categorized as high risk or low risk based on their association with anogenital cancer. The high risk HPV E6 protein binds to E6 Associated Protein (E6AP) and promotes degradation of p53 and activation of hTERT, the catalytic subunit of telomerase. These are key steps in HPV-positive cancer progression. E6 also binds to two isoforms of the cellular protein NFX1 (Nuclear Factor binds to the X1 box), NFX1-91 and NFX1-123, which have opposing affects on hTERT transcription. Objective: To determine how NFX1-123 affects the activation of hTERT in HPV 16E6-expressing cells. Design/Methods: FLAG-tagged NFX1-123, a short hairpin RNA for NFX1-123, or vector controls were introduced into HPV 16E6 expressing primary keratinocytes (HFKs), and Real Time PCR and telomeric repeat amplification protocol (TRAP) were used to study telomerase activity. Protein purification and mass spectrometry with NFX1-123 were used to identify protein partners. GST pulldown assays and co-immunoprecipitation assays confirmed and delineated regions necessary for binding. HPV 16E6 expressing HFKs were transfected with an hTERT promoter-reporter construct, NFX1-123 WT and mutant constructs, and cytoplasmic poly(A) binding proteins (PABPCs) to study hTERT promoter-driven expression. Results: Overexpression of NFX1-123 increased telomerase activity and hTERT mRNA levels, and knock down of NFX1-123 decreased telomerase activity and hTERT mRNA levels, in HPV 16E6 expressing HFKs. NFX1-123 interacted with HPV 16E6 and also interacted with PABPCs via its N-terminal PAM2 motif. When the PAM2 motif of NFX1-123 was mutated or deleted, the ability of NFX1-123 to augment expression from the hTERT promoter by HPV 16E6 was reduced. Co-expression of NFX1-123 and PABPCs synergistically increased E6 activation of the hTERT promoter. Conclusions: NFX1-123 is necessary for the full activation of hTERT expression by HPV 16E6, and overexpression of NFX1-123 augments the activation of hTERT. The interaction of NFX1-123 and PABPCs via the PAM2 motif is important for augmentation of activation. These results indicate E6 regulates hTERT at both the transcriptional and post-transcriptional level.

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Paul Kingma, MD, PhD Assistant Professor Cincinnati Children’s Hospital Medical Center I completed my undergraduate studies at Calvin College in Grand Rapids, Michigan and began my formal research training with enrollment in the Medical Scientist Training Program at Vanderbilt University Medical School. At Vanderbilt I joined the laboratory of Dr. Neil Osheroff in the Department of Biochemistry and received my M.D., as well as a Ph.D. in 2000. Following medical school, I trained as a pediatric resident at Cincinnati Children’s Hospital Medical Center (CCHMC), Cincinnati, Ohio. During my residency training I enrolled in the American Board of Pediatrics Integrated Research Pathway. This pathway provided protected research time under the mentorship of Dr. Jeffrey Whitsett, M.D. in the Section of Neonatology, Perinatal and Pulmonary Biology. I remained at CCHMC after my residency training and entered fellowship training in neonatology. During my fellowship, I continued my work with Dr. Whitsett on the role of surfactant protein D structural domains in alveolar macrophage regulation. I joined the faculty at CCHMC as an Assistant Professor in July 2005. My research interests now focus on determining the role of surfactant protein D in acute respiratory distress syndrome and systemic sepsis.

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SYSTEMIC SURFACTANT PROTEIN D INHIBITS INFLAMMATION INDUCED BY INTRAVENOUS LIPOPOLYSACCHARIDE. PS Kingma, SK Mierke, and JA Whitsett, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio. Background: Surfactant protein D (SP-D) is a member of the collectin family of innate host defense proteins that has critical roles in both clearing infectious pathogens from the lung and regulating pulmonary host defense cells. In addition to the respiratory tree, SP-D is also detected in lower concentrations in plasma and many other non-pulmonary tissues. Plasma levels of SP-D increase during infection and other proinflammatory states in a manner similar to intrapulmonary SP-D; however the source and functions of extrapulmonary SP-D are largely unknown. Objective: We hypothesized that systemic SP-D may clear infectious pathogens and regulate host defense cells in extrapulmonary systems. Method: To determine if SP-D inhibited inflammation induced by systemic lipopolysaccharide (LPS), E.coli 0111:B4 LPS was administered to mice via tail vein injection with and without SP-D and the inflammatory response was measured. Results: Whereas systemic SP-D did not increase the clearance of LPS from plasma, SP-D significantly decreased levels of IL-6 and TNFα in a concentration dependent manner with a maximum reduction of 40% and 50% in IL-6 and TNFα levels, respectively. Inhibition of LPS-induced inflammation by SP-D correlated with SP-D LPS binding suggesting SP-D mediated inhibition of LPS requires direct SP-D LPS interactions. Analysis of tissue specific IL-6 gene expression suggests that SP-D inhibits LPS-induced systemic inflammation in vascular cells. Conclusions Reached: Taken together, the above results suggest that circulating SP-D inhibits inflammation induced by systemic LPS by direct interactions with vascular cells and raises the possibility that a physiological purpose of increasing systemic SP-D levels during infection is to scavenge systemic infectious pathogens and limit inflammation induced tissue injury.

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Carrie L. Kitko, MD Lecturer University of Michigan Carrie L. Kitko is currently a Lecturer in Pediatrics and Internal Medicine in the Blood and Marrow Transplant Program at the University of Michigan. Her previous research focused on the analysis of protein levels at day 7 following allogeneic hematopoietic stem cell transplantation (HSCT). In this work, it was demonstrated that day 7 elevations of tumor necrosis factor receptor 1 (TNFR1) were associated with greater severity of acute graft versus host disease (GVHD), increased transplant related mortality and decreased one year overall survival. Dr. Kitko is currently working on applying these same techniques to patients at risk for developing chronic GVHD. She has currently has identified 327 patients transplant at the University of Michigan between January 2002 and December 2006 that are at risk for chronic GVHD. She is currently analyzing stored patient serum samples from various time points post HSCT in hopes of identifying a protein fingerprint for chronic GVHD.

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CHRONIC GVHD (cGVHD): ANALYSIS OF INCIDENCE AND POTENTIAL FOR DISCOVERY OF BIOMARKERS. CL Kitko, S Paczesny, J Richardson, S Choi, G Yanik, J Ferrara, JE Levine, University of Michigan, Ann Arbor MI. Background: Chronic GVHD occurs in up to 80% of allogeneic bone marrow transplant (BMT) patients and is a major cause of morbidity and mortality. Known risk factors for developing cGVHD include acute GVHD and unrelated donor source. Manifestations of this disease resemble autoimmune disorders, including scleroderma, sicca syndrome and auto-antibody formation. A new staging system proposed by NIH categorizes cGVHD by type of onset [de novo, acute followed by chronic, and progressive (chronic developing before acute resolves)] and stage (mild, moderate, and severe). At onset, platelet counts >100,000/mm3 are associated with superior survival. Biomarkers other than platelet count, as have been developed for acute GVHD, are highly desirable. Objective: To establish a cGVHD clinical outcomes database for BMT patients using the NIH staging system and to compare the incidence and stage of cGVHD to prior reports for the design of future biomarker studies. Design/Methods: Between January 2005 and December 2006 blood samples were obtained at day 100 from 150 BMT patients at the University of Michigan who were evaluable for cGVHD. 18 month overall survival was calculated for these patients based on type of cGVHD at onset, stage, and platelet count. Results: The median day of onset for cGVHD was 150d. The 18 month OS for no cGVHD (n=39) was 53%, for de novo cGVHD (n=42) was 85%, for acute followed by chronic (n=45) was 70% and for progressive cGVHD (n=24) was 40% (p<0.001 by log rank analysis). The 18 month OS for mild cGVHD (n=22) was 90%, for moderate cGVHD (n=57) was 72%, and for severe (n=31) was 55% (p=0.01 for the log rank test of trend). Platelet count > 100,000/mm3 was the most significant discriminator of 18 month OS (78%, n=81 vs 29%, n=29; p<0.001). Conclusions Reached: The cGVHD outcomes in our patients according to type of onset, stage, and platelet count are similar to those reported in the literature. We therefore plan to screen previously collected day 100 samples for biomarkers in order to develop diagnostic, predictive and prognostic tests for cGVHD.

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Mark Levin, MD Attending Cardiologist Children’s Hospital of Philadelphia Dr. Levin is an attending cardiologist at the Children’s Hospital of Philadelphia. He obtained his M.D. from Boston University in 1998 and subsequently completed a residency in pediatrics at Babies’ and Children’s Hospital in New York City. He completed his general cardiology fellowship at the Children’s Hospital of Philadelphia in 2004. Since that time, Dr. Levin has been performing post-doctoral research on a project in the Molecular Cardiology Research Center under both Dr. Jonathan Epstein, whose laboratory focuses on cardiac development and Dr. Vickas Patel, whose lab is interested in mechanisms of cardiac arrhythmias.

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NOVEL POPULATION OF MELANOCYTE-LIKE CELLS IN THE HEART TRIGGER ATRIAL ARRHYTHMOGENESIS. MD Levin, 1, 2, MM Lu2, NB Petrenko2, D Lang2, JT Jacobson2, CF Spurney3, CB Brown2, L Huang2, F Beermann4, JA Epstein2, VV Patel2. 1 Dept of Pediatrics, Div. of Cardiology, Children’s Hospital of Philadelphia, Philadelphia, PA 2Cardiovascular Institute, The U of Pennsylvania School of Medicine, Philadelphia, PA 3Children’s National Medical Center, Washington, D.C, 4Swiss Institute for Experimental Cancer Research, Epalinges, Switzerland. Background: Atrial fibrillation (AF) is the most common cardiac arrhythmia requiring medical intervention. It is often initiated by ectopic beats arising from the pulmonary veins, but the source of these beats remains unknown. A number of cell populations migrate to and invade the developing heart, including neural crest cells, proepicardial cells and secondary heart field cells. We report here a novel population of melanocyte-like cells characterized by Dopachrome Tautomerase (DCT) expression that appear to be a source of triggers for atrial arrhythmogenesis. Studies implicate increased reactive species in human AF, and DCT is thought to scavenge reactive species in dermal melanocytes. Objectives: We investigated the contribution of these cardiac melanocyte-like cells (CMLC) to AF in a mouse model with targeted deletion of the DCT gene. Methods: We employed cellular fate mapping, mRNA and protein expression techniques to differentiate CMLCs from myocytes, fibroblasts, neurons and neural crest cells. Invasive electrophysiology studies were performed in DCT null mice, and current clamp analysis was performed on primary cultures of murine cardiac myocytes and CMLCs. Results: Like melanocytes, CMLCs express tyrosinase (TYR), TYR related protein 1, and Microphthalmia Associated Transcription Factor (MITF); these cells are distinct from neural crest cells in that they do not fate map to WNT1 expressing cells and do not express smooth muscle actin. CMLCs fail to co-express myocyte (MF20), neurofilament (2H3), or fibroblast (fibronectin) markers. They populate the atrioventricular canal, atria and pulmonary veins in the embryonic and mature heart. Atrial burst pacing induced 27 episodes of AF (781.3±417ms) in DCT null mice (9/11), while the same protocol induced 2 episodes (172 and 1540 ms) in control mice (1/12) (p=.0004). CMLCs are excitable and exhibit prolonged action potentials compared to myocytes. Moreover, CMLCs lacking DCT have prolonged action potential duration compared to the same cells with intact DCT, suggesting this property may trigger atrial arrhythmias. Conclusions: We suggest that this novel population of melanocyte-like cells in the heart contributes to atrial arrhythmogenesis, likely by increasing triggered activity secondary to reactive species modification of ion channels.

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Steven Mittelman, MD, PhD Attending Physician Children’s Hospital Los Angeles Dr. Steven Mittelman is an attending physician in the Division of Endocrinology, an investigator in the Saban Research Institute at Children’s Hospital Los Angeles, and an Assistant Professor of Pediatrics and Physiology at the University of Southern California Keck School of Medicine. He received his MD and PhD degrees in the University of Southern California Keck School of Medicine. His PhD work was done in the laboratory of Dr. Richard Bergman, in the Department of Physiology and Biophysics. His PhD thesis, entitled “The Indirect Effect of Insulin to Control Liver Glucose Production: Testing the Single Gateway Hypothesis”, elucidated the physiological mechanisms by which insulin controls liver glucose production in mammals. Further post-doctoral work in the same laboratory examined beta-cell compensation for obesity-induced insulin resistance. At Childrens Hospital Los Angeles, Steven completed his residency in pediatrics and fellowship in pediatric endocrinology. During his subspecialty training, Steven researched the roles of appetite-regulating hormones in obesity under the mentorship of Dr. Tom Buchanan, and began his work exploring the relationship between obesity and leukemia relapse with Dr. Nora Heisterkamp. His laboratory is currently investigating why obese children have increased risk of leukemia relapse, using both mouse models and tissue culture. In addition to the K12 from the CHRC, Steven is supported by funds from the Division of Endocrinology, the Saban Research Institute, and a pilot grant from the USC Center for Transdisciplinary Research on Energetics and Cancer.

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EXPLORING THE EFFECT OF OBESITY TO ACCELERATE LEUKEMIA RELAPSE USING A MOUSE MODEL. SD Mittelman, JW Behan, JP Yun, N Heisterkamp, Childrens Hospital Los Angeles, Los Angeles, CA. Background: Obesity contributes to an increased risk of cancer incidence and mortality. A recent retrospective study demonstrated that children who are overweight at the time of diagnosis of high-risk acute lymphoblastic leukemia (ALL) have a ~50% increased rate of relapse after treatment. Objective: We investigated whether obesity would accelerate ALL in mice using three different approaches. Design/Method: In our first experiment, BCR/ABL transgenic mice, which develop spontaneous leukemia, were randomized at weaning to laboratory chow (n=20) or a high-fat diet (60% of calories from fat, n=18), and observed for leukemia onset. In our second experiment, wild-type mice raised on either a high-fat diet (n=10) or a control diet (10% of calories from fat, n=14) were transplanted with leukemia cells from a BCR/ABL transgenic mouse, and time to leukemia onset was observed. In our third experiment, 6 lean and 6 obese mice were transplanted with leukemia cells and then, after a 4-day engraftment period, treated with vincristine (0.5 mg/kg/week IP for 4 weeks). Mice were monitored until onset of leukemia. Results: There was a tendency for the obese transgenic mice to develop spontaneous ALL earlier than control mice, though this did not reach statistical significance (p=0.17). In post-hoc analysis, obese mice that lived beyond the median age of survival (120 days) developed ALL significantly earlier than the lean controls (p<0.05), implying a possible time-dependent effect of obesity to accelerate leukemia onset. In transplanted mice, there was no difference in the time to onset of leukemia in obese vs. control mice (31±4 vs. 29±4 days post-transplant, p=n.s.), implying that obesity likely does not significantly accelerate leukemia cell proliferation in vivo. When mice were transplanted and then treated with vincristine, only 2 of 6 mice in each dietary group developed leukemia. The obese mice developed leukemia at 35.5±4.9 days post-transplant, which was significantly sooner than the lean mice in which leukemia developed 77.5±6.4 days post-transplant (p<0.05). Conclusions Reached: These results indicate that diet-induced obesity may accelerate the progression of spontaneous leukemia in a transgenic leukemia model, but not in a model of leukemia transplantation. However, obesity may accelerate leukemia relapse after chemotherapy. Whether this latter effect is due to altered pharmacokinetics associated with obesity or a direct effect of obesity on leukemia cells remains to be determined. Further studies are needed to more clearly define the role of human obesity in leukemia treatment and outcome.

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Sidhar Rao, MD, PhD Fellow Children’s Hospital Boston I was born in Minnesota, and did my undergraduate work at the University of Minnesota. Next I went to the University of Chicago for my M.D., Ph.D. with my thesis in the laboratory of Dr. M. Celeste Simon focusing on the role of Ets transcription factors in B-lymphocyte development. After completing my MD/PhD I did a residency in Pediatrics at Children’s Hospital, Boston followed by a Pediatric Hematology/Oncology fellowship at Dana Farber Cancer Institute and Children’s Hospital, Boston. I am currently a post-doctoral fellow in the laboratory of Dr. Stuart H. Orkin, with my work focusing on the transcriptional network controlling Embryonic Stem Cell (ESC) pluripotency.

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THE ROLE OF SALL4 IN ES CELL PLURIPOTENCY. S Rao, S Orkin, Dana Farber Cancer Institute and Children’s Hospital Boston, Boston, MA. Background: The pluripotency of murine Embryonic Stem Cells (mESCs) is tightly regulated by a number of transcription factors including Sall4, the causative gene of Okihiro syndrome. The exact mechanism by which Sall4 regulates the transcriptional and/or epigenetic state of mESCs remains to be elucidated. Objectives: To determine the mechanism by which Sall4 regulates mESC pluripotency. Design/Method: To use a combination of affinity purification/mass spectroscopy, genome-wide location analysis, and expression profiling of mESCs to integrate the target genes and regulatory network controlled by Sall4. Results: Initial proteomics work reveals that full-length Sall4 interacts with other Sall-proteins in mESCs and components of the nucleosomal remodeling and histone deacetylase complex (NuRD). Interestingly, gel filtration shows that the two predominant iosoforms of Sall4 (Sall4a and Sall4b) form distinct complexes of different molecular weight. Genome-wide location analysis and expression profiling is ongoing and will reveal the genes critically regulated by both isoforms of Sall4. Conclusions reached: Preliminary data seems to indicate that the two isoforms of Sall4 form distinct protein complexes within mESCs, and thereby likely play different roles in transcriptional regulation.

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Lawrence Rhein, MD Instructor Children’s Hospital Boston Lawrence Rhein graduated from Harvard University cum laude, majoring in Biology. He received his MD degree from the University of Pennsylvania and completed his residency and Chief Residency at Boston Children’s Hospital. He then completed dual fellowships in Pediatric Pulmonology and Neonatology at Boston Children’s Hospital. He is an Instructor in Pediatrics at Harvard Medical School, and directs the Center for Healthy Infant Lung Development. His research focuses on the role of chemokines and their receptors in models of infection.

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FcγRIII IS PROTECTIVE AGAINST PSEUDOMONAS AERUGINOSA PNEUMONIA. L Rhein, M Perkins, NP Gerard, and C Gerard, Children’s Hospital, Department of Medicine, Harvard Medical School, Boston, MA. Background: Fcγ receptors, selective for the Fc region of IgG, play a critical role in inflammatory responses in immune-complex diseases of the lung. Regulation of Fcγ receptor expression is accomplished at least in part by the complement anaphylatoxin, C5a, and interaction with its primary receptor, the C5aR. We previously showed that activation of the C5aR is critical for mucosal defense against Pseudomonas aeruginosa (PSA) lung infections. The relationship of the change in C5aR expression to the FcγRII/III balance is not established, nor is a distinct role for FcγRIII in defense against bacterial pathogens. Objectives: To analyze the complex relationships among the Fcγ receptors and the complement anaphylatoxin C5a signaling pathways in pulmonary infection by PSA. Design/Method: We infected wild type (WT) and FcγRIII-deficient mice with PSA and compared survival, bacterial clearance, cytokine expression. We also analyzed RNA expression of FcγRII and FcγRIII in C5aR-deficient mice after PSA infection. Results: All of the FcγRIII-/- mice died within 72 hours of infection, whereas only 50% of WT animals were dead after 8 days of observation (n=9-14 per group, P=0.006). A significant increase in colony-forming units (CFU) was recovered from the lungs of FcγRIII -/- mice compared to WT animals 24 hours following infection (1.04 ±0.41x105 CFU/gram lung tissue for FcγRIII-/- vs 1.96±0.61x104

CFU/gram lung tissue for WT mice, n=10-11 mice per group, P=0.022). Compared with PSA-infected WT mice, FcγRIII-/- mice had ~40% more IL-6 (460±52 pg/ml for FcγRIII-/- vs 270±74 for WT mice, P=0.025), and ~60% more TNF-α (276±86 pg/ml for FcγRIII-/- vs 108±46 pg/ml for WT mice, P=0.048, n=9-11 mice per group). WT mice revealed a significant decrease in the lung FcγRII/FcγRIII mRNA ratio, but no change was observed in C5aR-/- animals (P=0.04 for PSA infected vs uninfected WT mice, n=4 per group. The change in FcγRII/FcγRIII ratio was similar following intratracheal administration of recombinant C5a (P=0.009 for WT mice with or without C5a administration, n=4 per group). Conclusions Reached: FcγRIII mediates protection against mortality from PSA pneumonia and contributes to the pulmonary clearance of PSA. PSA infection alters the ratio of activating FcγRIII and inhibitory FcγRII via a C5a/C5aR-dependent mechanism.

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Robert Roghair, MD Assistant Professor University of Iowa I joined the faculty of the University of Iowa College of Medicine as an Associate in Pediatrics June 2005. I was promoted to Assistant Professor of Pediatrics the following year. I have benefited from NIH financial support ranging from Pediatric Research Loan Repayment Program (LRP) awards to Neonatology fellowship stipends (NRSA T32 HL07413 and HD041922) and ongoing mentored Career Development Awards (K08 HD050359 and my department’s CHRCDA grant). Along with my primary career mentors, Drs. Jeffrey Segar, Thomas Scholz and Fred Lamb, I have been investigating the fetal origins of adult cardiovascular disease for the past 6 years. While we continue to utilize an early gestation dexamethasone model to explore the origins of ovine coronary artery dysfunction, we have recently extended our observations into complementary systems, including a rat model of gestational onset diabetes mellitus and a mouse model of naturally occurring growth restriction. The present abstract focuses on our newest investigations seeking to define the mechanisms responsible for programmed hypertension. These studies have increasing evaluated the role of inciting genetic and epigenetic alterations, as well as potentially palliative or preventative neonatal nutritional or pharmacologic intervention.

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MATERNAL LOW PROTEIN DIET AND FETAL GLUCOCORTICOID EXPOSURE PROGRAM ADULT MOUSE CARDIOVASCULAR AND ENDOCRINE STATUS RD Roghair, JL Segar, KA Volk, G Aldape, CM Kiroff, TD Scholz, FS Lamb; Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, IA Background: Low birth weight is a risk factor for hypertension and diabetes. In male rats, maternal low protein diet inhibits placental 11-β-hydroxysteroid dehydrogenase (11βHSD), increases fetal glucocorticoid exposure, and programs cardiovascular and endocrine dysfunction in adulthood. Female rats are partially protected from programmed hypertension by enhanced estrogen-stimulated nitric oxide synthase activity. Development of an analogous murine model would facilitate etiologic definition. Methods: Iso-caloric low protein (LP, 9% Pro) or normal protein (NP, 18% Pro) diets were provided to dams from E0 to E19. Dams on NP received either saline or the 11βHSD inhibitor carbenoxolone (CX, 12.5 mg/kg sc from E12 to E19). All dams received NP while nursing. Following weaning at 21d, offspring were randomized to either ongoing NP (18% Pro, 6% Fat) or conversion to high fat (HF; 16% Pro, 41% Fat). At 6 months, blood pressures were recorded by radiotelemetry while mice received baseline diet (0.25% NaCl), high salt diet (9% NaCl), or high salt diet with the nitric oxide synthase inhibitor LNAME (1mg/ml drinking water). N = 5-7 mice of each sex per prenatal/postnatal diet. Results: Although dam caloric intake was not altered by LP or CX, both interventions reduced pup birth weight (LP: 1.7 +/- 0.1g; CX: 1.9 +/- 0.1g; NP: 2.2 +/- 0.2g). Compared to adult NP controls, female LP and CX offspring had decreased fat pad weight and decreased blood pressures on either baseline or high salt diet (all P<0.05). This programmed decrease in female blood pressure was blocked by LNAME. In contrast, male LP and CX offspring had increased fat pad weight and increased baseline blood pressures (P <0.05). This programmed male hypertension was exacerbated by both dietary salt and LNAME. Postnatal HF selectively increased control male blood pressure and fat pad weight, thereby matching the phenotypes seen following prenatal LP or CX exposure. Conclusions: Maternal LP or CX impairs fetal growth and programs hypertension in male mice. This programmed hypertension is partially masked by compensatory nitric oxide synthase activation and appears to be related to excessive postnatal fat deposition. Female mice are protected from programmed hypertension with a combination of decreased adiposity and enhanced nitric oxide synthase activation. In a species amenable to genetic manipulation, maternal LP diet or fetal exposure to maternally-derived glucocorticoids programs adult cardiovascular and endocrine status.

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Mary Beth Ross, MD, PhD Assistant Professor Columbus Children's Research Institute Mary Beth is an Assistant Professor in the Research Institute at the Nationwide Children’s Hospital in Columbus, Ohio. She received her MD and PhD in the Medical Scientist Training Program at the State University of New York at Buffalo. Her PhD dissertation was in the field of immunology titled “The Regulation of Natural Killer Cell Cytokine Production” under the mentorship of Michael A. Caligiuri, MD. Subsequently, she completed General Pediatrics Residency at the St. Louis Children’s Hospital which is affiliated with Washington University. Subspecialty training in Pediatric Hematology-Oncology was completed at St. Jude Children’s Research Hospital where she worked with Dr. James Downing delineating gene expression profiles of pediatric acute lymphoblastic leukemia (ALL). Dr. Ross’ clinical and research interest is in pediatric leukemia. A variety of underlying genetic lesions have prognostic value in pediatric ALL. Indeed, some translocations are currently in use for assignment of risk directed therapy. One such example is t(1;19) which results in the formation of a chimeric transcription factor, E2A-PBX1. We are trying to utilize high throughput techniques to identify what genes are potentially altered by the presence of the E2a-PBX1 transcription factor. Understanding how these unique protein products alter the cell on a whole genome basis may give us insight into the leukemogenic process or new targets of therapy.

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IDENTIFICATION OF POTENTIAL TARGET GENES OF THE CHIMERIC TRANSCRIPTION FACTOR, E2A-PBX1 BY siRNA KNOCKDOWN. C. Chen, B.H. Connell, K. Foley, J. Townsend, M.E. Ross. Center for Childhood Cancer, The Research Institute at Nationwide Children’s Hospital; Columbus, OH Background: Nonrandom chromosomal translocations are frequently seen in pediatric acute lymphoblastic leukemia. E2A-PBX1 results from the fusion of the regulatory and 5’ regions of E2A with the 3’ regions of PBX1 in the t(1;19)(q23;p13) translocation. In vitro, E2A-PBX1 has been shown to have transcriptional activity, though little is known about which genes have altered expression in the presence of E2A-PBX1. Objectives: To identify potential target genes of E2A-PBX1. Design/Method: An E2A-PBX1 containing cell line (697) was transfected with E2A-PBX1 specific siRNA utilizing an Amaxa nucleofector2. Biologic replicates were performed by transfection of separate cultures on separate days. E2A-PBX1 mRNA was knocked down to 25% as demonstrated by quantitative RT-PCR. Message was calculated by the ΔΔCt method normalizing to eEF2. Knockdown of E2A-PBX1 was also demonstrated at the protein level by western analysis. E2A-PBX1 was detected utilizing the G289-1 antibody purchased from Becton Dickinson and the ECF secondary antibody-chemfluorescence system from Amersham. Once knockdown of E2A-PBX1 was demonstrated at both the message and protein level, genome wide gene expression was assessed by Affymetrix U133 2.0 Plus arrays. RNA quality was assessed utilizing the HP Bioanalyzer 2100. Hybridizations were prepared and performed according to current Affymetrix protocols in the Functional Genomics Core at the Research Institute. Microarray data was normalized with RMA. Differentially expressed genes were selected by Significance Analysis of microarrays (SAM). Results: In E2A-PBX1 siRNA knocked down cells, 78 probe sets had differential expression relative to cells undergoing mock transfection and cells transfected with nontargeting siRNA. The 78 probe sets represent 49 known genes and 8 ESTs. The false discovery rate was less than 5%. Genes of specific interest include: WNT-16, ANKS1B(EB-1), FAT, and RORB, which other laboratories have previously demonstrated to have an association with E2A-PBX1. 22% of the genes whose expression was altered by E2A-PBX1 siRNA are also a part of the previously published E2A-PBX1 gene expression list. [Ross, Blood 2003] Conclusions Reached: We have utilized siRNA to E2A-PBX1 to identify potential target genes of E2A-PBX1. These results will need to be confirmed by additional methods.

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Brian Sims MD, PhD Assistant Professor University of Alabama at Birmingham Brian Sims became Assistant Professor of Pediatrics and Cell Biology July 1st, 2006 in the Division of Neonatology. He finished University of Alabama School of Medicine Class of 2000. His PhD is in Cell Biology with a concentration in Neurobiology. His training includes a residency at St. Louis Children's Hospital in Pediatrics and Fellowship in Neonatology at UAB. While at St. Louis Children's Hospital he began investigating mouse embryonic stem cells as a model system to study premature oligodendrocytes. These cells have been implicated in white matter injury of premature neonatal brains ultimately leading to cerebral palsy. His research interest is to better characterize these cells and investigate whether these premature oligodendrocytes can be protected from injury. The ultimate goal is to decrease the white matter injury seen in neonates by understanding the biology of these premature oligodendrocytes

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THE ACTIVITY OF THE CYSTINE GLUTAMATE EXCHANGER IS REQUIRED FOR OLIGODENDROCYTE SURVIVAL. B Sims1,2, H Sontheimer2, Departments of Pediatrics1 and Neurobiology2, University of Alabama School of Medicine, Birmingham, AL. Background: White Matter Injury (WMI) is a devastating injury in the preterm neonate. Periventricular Leukomalacia is found in a majority of premature neonates with WMI. Both excitotoxic and oxidative mechanisms have been linked to oligodendrocyte precursor cell (OPC) damage which ultimately leads to PVL. The cystine/glutamate exchanger (System Xc-) is expressed in OPCs and may be protective against oxidative injury. In most cells, System Xc- responsible for producing intracellular glutathione to protect cells from oxidative injury. Objectives: The purpose of this study is to determine the role of System Xc- activity in OPC vulnerability. Design/Methods: Mouse embryonic stem cells were differentiated into oligoedendrocyte precursors using the standard 4-/4+ Embryoid Body Protocol. Cells were plated on polyornithine coated coverslips/plates at a concentration of 3*106 cells/plate. Cells were grown to in Dulbecco’s Modified Essential medium and Modified Sato medium 1:1 for 3 then prepared for various experiments. Immunocytochemistry was used to detect OPCs- preoligodendrocytes (PreOLs) and immature oligodendrocytes (OLs). O4, NG2 and O1 antibodies were used to define OPCs. RT-PCR was used to determine if System Xc- mRNA was present after 24-48 hours in the presence of inhibitors of System Xc- (S4-CPG and sulfasalazine). Mouse brain total RNA was used as a positive control for System Xc-. Negative controls for RT-PCR were samples without RNA template and the internal standard was β-actin. A glutathione detection kit (ApoDetect) was used for glutathione determination. Results: S4-CPG and Sulfasalazine at 100uM caused a significant decrease in O4+ cells (74+ 8.3%), p < 0.01 and O1+ cells (37 + 5.4%), p< 0.01 compared to controls. All RT-PCR experiments were positive for the cystine/glutamate exchanger mRNA. Glutathione levels were not detectable in both O4+ and O1+ cells after addition of inhibitor. Conclusions: System Xc- activity has a significant role in oligodendrocyte survival. Glutathione levels were decreased after blockage of System Xc- suggesting that glutathione depletion may have a pivotal role in the vulnerability of OPCs.

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Maria L. Sulis, MD Assistant Professor Columbia University I joined the faculty of the Department of Pediatrics, Division of Pediatric Oncology at the Children’s Hospital off New York, Columbia University in 2003 as an assistant professor of clinical pediatrics. I did the pediatric training at ST. Vincent’s Hospital in New York and completed the fellowship in pediatric hematology and oncology at the Children’s Hospital of New York. I joined the laboratory of Dr. Ramon Parsons during the last 18 months of the fellowship and worked on the regulation of the PTEN tumor suppressor gene. Since two years ago I have been working in the laboratory of Dr. Adolfo Ferrando. The main focus of my research is T-cell leukemias and in particular the NOTCH1 transcription factor which is mutated in more than half of T-cell leukemias. We have found a yet unknown mutation of NOTCH1 in the exon 28 that expands the extracellular portion of the receptor resulting in a marked increase in the activity of NOTCH1. I am currently exploring the biochemical mechanism of ligand independent activation related to this new mutant. In parallel I am studying alternative mechanisms of activation of NOTCH1 pathway in the absence of NOTCH1 mutations such as altered mechanisms of protein degradation. The long term objective is to identify new therapeutic targets specific for T-cell leukemias.

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NEW ONCOGENIC EVENTS IN T-CELL LEUKEMOGENESIS. ML Sulis, O Williams, S Pallikuppam, T Palomero, A Ferrando. Children Hospital of New York, Columbia University, New York, NY. Background: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy that accounts for 15% of childhood leukemias. 50% of human T-ALL carry activating mutations in NOTCH1, which lead to ligand independent activation or stabilization of the active form of NOTCH1. The mechanisms of transformation of the T-ALLs that do not carry the prototypical NOTCH1 mutations remain unclear. Objectives: Based on the central role of NOTCH1 in the development of T-cell precursors and in T-cell leukemogenesis, we postulate that aberrant activation of NOTCH1 signaling may be a universal mechanism of T-cell transformation. Our objective is to identify and characterize novel mechanisms of aberrant NOTCH1 activation in T-ALL. Methods: We analyzed the levels of intracellular NOTCH1 (ICN1) protein in a broad panel of T-ALL lines by western blot and found that Jurkat, TAIL7 and HSB2 cells express high levels of ICN1 despite lacking the prototypical mutations in exons 26, 27 and 34. Direct sequencing analysis of RT-PCR products encompassing the complete coding sequence of NOTCH1 was carried out. Results: We found that the Jurkat cell line carries an in-frame insertion of CAGG followed by an internal tandem duplication (ITD) of 47 bp in exon 28. This mutation introduces 17 aminoacids in the extracellular juxtamembrane region of the receptor. PCR amplification of exon 28 from genomic DNA of 200 primary T-ALL lymphoblast samples identified 7 patients with ITD of similar length. We compared the activity of wild type NOTCH1, the Jurkat mutation and a prototypical mutant allele of NOTCH1, L1601P by luciferase reporter assay and found that the new Jurkat mutant allele induced over 200-fold activation of the NOTCH1 reporter compared to controls. To investigate whether the activation of NOTCH1 generated in the exon 28 mutants is dependent on the length of the insertion or on the specific sequence, we introduced artificial insertions of random peptides of different length (5,8,11,14 aa) and found induction of the NOTCH1 reporter in all of the artificial mutants, up to 70-fold in the 14aa insertion. Conclusion: We have identified a novel mechanism of ligand independent activation of NOTCH1 mediated by an ITD in exon 28. The expansion of the extracellular juxtamembrane region of the receptor caused by these mutant alleles result in high levels of activity. We also demonstrated that the activity is dependent on the number of residues introduced and not on the specific sequence. Further investigation on the biochemical mechanism of activation is ongoing.

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Adeline Vanderver, MD Director of the Myelin Disorders Clinic and Staff Clinician Children’s National Medical Center Dr. Adeline Vanderer, MD is presently Director of the Myelin Disorders Clinic and a staff clinician in the Neurology Department at Children’s National Medical Center, as well as a research scientist in the Children’s Research Institute. Dr. Vanderver completed her fellowship training in Neurology at Children’s National Medical Center, and her pediatrics residency at the AI DuPont Hospital for Children/ Thomas Jefferson University. She is currently completing a fellowship in Biochemical Genetics at the National Human Genome Research Institute at the NIH. Dr. Vanderver is an Assistant Professor in Neurology and Pediatrics at The George Washington University. Throughout her career, Dr. Vanderver has been involved in clinical and applied basic science research. Her clinical research includes diagnosis of unclassified leukodystrophies, phenotypic characterization of known leukodystrophies, as well as the approach to neurogenetic disorders. Her basic science research focuses on biomarker discovery, mutation analysis, and the mechanisms of disease in leukodystrophies. Recently, Dr Vanderver has identified a novel biomarker that shortens the time of diagnosis of Vanishing White Matter disease (a type of leukodystrophy) from months to 48 hours, as well as working on possible therapeutic targets. Dr. Vanderver is the recipient of the prestigious American Academy of Neurology Foundation Clinical Research Training Fellowship. Dr Vanderver’s career goals are increasing understanding of the causes of leukodystrophies and developing diagnostic tools as well as treatment options in these devastating disorders.

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ALTERED PROTEIN TRAFFICKING IN EIF2B RELATED DISORDERS IN RESPONSE TO ENDOPLASMIC RETICULUM STRESS. A Vanderver, M Mintz, R Schiffmann*, Y Hathout, Children’s National Medical Center, Washington, DC, *National Institutes of Health, Bethesda, MD BACKGROUND: EIF2B related disorders, caused by mutations in ubiquitously expressed eukaryotic translation initiation factor 2B (EIF2B), have predominantly glial cell findings. Recent studies have revealed an altered endoplasmic reticulum (ER) stress response in eIF2B mutated cells, however how this results in altered myelin maintenance is still unexplained. OBJECTIVE: Explain how an altered ER stress response in eIF2B related disorders (Vanishing White Matter Disease) results in loss of myelin homeostasis. METHODS: Primary fibroblast cultures from 2 EIF2B mutated patients (R195H and R113H) were compared to 2 fibroblast controls using stable isotope labeling of amino acids in culture (SILAC). Control cells were grown in 13C6,15N2-Lys and 13C6-Arg labeled medium, while primary EIF2B mutated patient cells were grown in standard media. All cells were submitted to ER stress inducing agents (thapsigargin). Fully labeled control fibroblasts and unlabeled primary EIF2B mutated fibroblasts were mixed at 1:1 ratio, then processed for protein identification using ER fractionation, SDS-PAGE gel electrophoresis, and analysis by LC-MS/MS. Peptide ratios from labeled and unlabeled peptides were obtained. Proteins were identified using Mascot and Sequest search engines against indexed databases. Labeled and unlabeled peptides ratios were determined with ZoomQuant. A similar paradigm was applied to rodent oligodendrocyte lines with stable siRNA of the eiF2B5 subunit, and reintroduction of the R195H mutation. RESULTS: Comparative proteomic approaches revealed an elevation in the ER fraction of EIF2B mutated versus normal cells of proteins known to be either secreted or membrane-associated, suggesting abnormal retention of these proteins in the ER lumen of EIF2B mutated cells. Additional results include a marked decrease in the levels of proteins involved in lysosomal biogenesis as well as proteins involved in intracellular trafficking and endosome biogenesis. CONCLUSIONS REACHED: Mutations in EIF2B result in abnormal protein trafficking in response to cellular stress. This may explain why mutations of this ubiquitously expressed protein result in myelin destruction, since oligodendrocytes are uniquely sensitive to disturbances in protein stoichiometry. Our recent description of the association between EIF2B mutations and decreased asialotransferrin in the CSF also supports the hypothesis that this disease is the result of altered protein translation and modifications. This novel insight offers the potential for therapeutic strategies targeting the ER stress response pathway and improves our understanding of myelin homeostasis.

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Rachel K. Wolfson, MD Instructor University of Chicago Dr. Rachel Wolfson is an Instructor in the section of Pediatric Critical Care Medicine at the University of Chicago. She received her BS in Biology from the University of Michigan (Ann Arbor, MI) and her MD from the University of Chicago Pritzker School of Medicine (Chicago, IL). Dr. Wolfson completed both her residency in Pediatrics and her fellowship in Pediatric Critical Care Medicine at the University of Chicago. Her research focuses on the role of HMGB1, a late mediator of sepsis and the systemic inflammatory response, in mediating endothelial dysfunction in sepsis.

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HMGB1 CAUSES HUMAN LUNG ENDOTHELIAL CELL (EC) BARRIER DISRUPTION. RK Wolfson, ET Chiang, JGN Garcia. University of Chicago, Chicago, IL. Background: High-mobility group 1 (HMGB1), initially described as a nuclear transcription factor, functions as a late mediator of sepsis and the systemic inflammatory response syndrome (SIRS), significant causes of morbidity and mortality in pediatric patients. Acute lung injury (ALI) is intimately associated with disruption of pulmonary endothelial integrity, a defining feature of sepsis and SIRS. Limited reports have associated HMGB1 with development of ALI, though none have directly measured the effect of HMGB1 on endothelial barrier function, and the mechanisms of HMGB1-induced effects on vascular function are unknown. Objectives: We evaluated the in vitro effect of HMGB1 on human lung endothelial cell (EC) barrier function assessed by transendothelial cell resistance (TER) and by the development of paracellular gaps. Design/Methods: Human pulmonary artery EC were grown on gold electrodes to measure TER using electrical cell impedance sensing or grown on glass coverslips for immunofluorescence assessment (VE-cadherin and F-actin) with measurements of paracellular gaps. Human EC were pretreated with either low-dose lipopolysaccharide (LPS) (16 hours) or with 18% cyclic stretch (48 hours), strategies which mimic sepsis and high tidal volume mechanical ventilation respectively. Results: HMGB1 causes rapid (<1hr), dose-dependent EC barrier disruption measured by both TER and paracellular gap area. LPS pretreatment sensitizes EC to subsequent HMGB1 treatment, increasing paracellular gap formation. Pretreatment with 18% cyclic stretch sensitizes EC to subsequent HMGB1 treatment as measured by TER. Conclusions Reached: HMGB1 causes rapid endothelial barrier disruption in a dose-dependent manner which is exaggerated by prior treatment with LPS and 18% cyclic stretch, which sensitize human EC to subsequent HMGB1 treatment. These results increase our understanding of the role of HMGB1 in acute lung injury.

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David Ziring, MD Assistant Professor David Geffen School of Medicine at UCLA Dr. Ziring is a pediatric gastroenterologist and basic immunologist who has served on the UCLA School of Medicine faculty since he joined the Department of Pediatrics in 2005. He graduated from UCLA Department of Pediatrics completing his fellowship in pediatric gastroenterology in 2005. Dr. Ziring earned his B.S. in Zoology from UC Davis and his M.S. degrees in Applied Physiology and in Clinical Immunology as well as his MD from the Chicago Medical School. He is completing his work in the National Institutes of Health (NIH) sponsored K30 program in translational research. Dr. Ziring serves on the Medical Advisory Committee for the Southern California chapter of the Crohns and Colitis Foundation of America (CCFA) and is a member of the board of the IBD Support Foundation. He has research grants sponsored by the CCFA and the NIH. His laboratory research centers on the immunoregulation of the intestine and the modeling of human Inflammatory Bowel Disease (IBD). Dr. Ziring is presently testing promising therapeutic compounds in mice for the treatment of human IBD, research that is also helping to understand key signals that immune cells use to control inflammation in the gut.

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ENDOCANNABINOID-MEDIATED IMMUNOREGULATION OF EXPERIMENTAL COLITIS. D Ziring, J Braun, David Geffen School of Medicine at UCLA, Los Angeles, CA. Background: Inflammatory bowel disease results from the dysregulation of immune homeostasis and an inappropriate response to commensal bacteria. The cells that regulate intestinal inflammation and the local small molecules that control their actions are not well understood. Recent work indicates that cannabinoid agonists play important biologic and potential therapeutic roles on cells involved in inflammation. Peripheral cannabinoid (CB2) receptors are found predominantly on cells of the immune system. The capacity of cannabinoids to alter immune function is well established, particularly on the production and responsiveness to certain cytokines by macrophages and dendritic cells. We have shown the CB2 receptor is required on immune cells for the development of immunoregulatory cell subsets. Objectives:We were prompted to determine if CB2 receptor agonists could augment protection against murine modeled colitis. We thus modeled both acute and immune colitis via DSS and Galphai2 -/- T cell transfer, respectively. We then performed in vitro assays of CB2 agonist on the responsiveness of innate immune cell types such as macrophages and dendritic cells to TLR4 agonist. Methods: We administered DSS to C3H/HeJ mice with concurrent administration of CB2 agonist, AM1241, or vehicle control (DMSO) intraperitoneally (IP). We monitored the mice for weight loss and hematochezia. We withdrew the DSS after 5 days and sacrificed the mice on day 8 to collect tissue for histologic colitis scoring. In a separate experiment, we isolated splenic T cells from sick Galphai2 -/- mice and administered them IP to Rag 1 -/- mice and similarly administered AM1241 or DMSO. Finally, bone marrow derived macrophages and dendritic cells were cultured with LPS or vehicle control in the presence or absence of AM1241. Intracellular TNFα expression was measured by flow cytometry. Results: Surprisingly, we found that mice injected with AM1241 had more severe colitis as modeled by DSS. Conversely, AM1241 was protective against the development of immune Galphai2 -/- mediated T cell colitis as measured by both weight loss and histology. AM1241 dampened the responsiveness of both macrophages and dendritic cells to TLR4 agonist as measured by their production of TNFα. Conclusion: AM1241, a CB2 receptor-specific agonist, protects against immune but not acute colitis. DSS injury models acute, T cell-independent colitis, likely mimicking the initiating events in chronic intestinal inflammation. CB2 specific agonists appear to augment the activity of cells engaged in the immunoregulation of gut inflammation, rather than by attenuating effector function.

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Ruchi Gupta, MD, MPH Assistant Professor Children’s Memorial Hospital Dr. Gupta received her BS in Biology from the University of Louisville in 1994 and obtained her degree in Medicine from the University of Louisville School of Medicine in 1998. She completed her residency in pediatrics at Children’s Hospital and Regional Medical Center in Seattle, Washington in 2001, and worked for a year at Children’s Hospital Boston. Dr. Gupta completed a fellowship in Pediatric Health Services Research at Children’s Hospital Boston and received a Masters in Public Health from the Harvard School of Public Health in 2004. Dr. Gupta currently works at Children’s Memorial Hospital and the Institute for Healthcare Studies at Northwestern Feinberg School of Medicine in the areas of childhood asthma with mentor Dr. Kevin Weiss and food allergy with mentor Dr. Xiaobin Wang.

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GEOGRAPHIC VARIABILITY IN CHILDHOOD ASTHMA PREVALENCE IN CHICAGO. R Gupta1,2, X Zhang3, L Sharp4, J Shannon5, K Weiss1,6, 1Institute for Healthcare Studies, Northwestern Feinberg School of Medicine, Chicago IL. 2Children’s Memorial Research Center, Children’s Memorial Hospital, Chicago IL. 3 The Robert Graham Center: Policy Studies in Family Medicine and Primary Care, American Academy of Family Physicians, Washington D.C. 4 Department of Medicine, Section on Health Promotion, University of Illinois at Chicago, Chicago, IL5 Division of Pulmonary and Critical Care Medicine, Department of Medicine, Cook County Hospital, Chicago, IL6 Division of General Internal Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL. Background: Childhood asthma prevalence has been shown to be higher in urban communities overall without an understanding of differences by neighborhood. Objectives: To characterize the geographic variability of childhood asthma prevalence among neighborhoods in Chicago. Design/Methods: Asthma screening was conducted among children attending 105 Chicago schools as part of the Chicago Initiative to Raise Asthma Health Equity (CHIRAH). Additional child information included age, gender, race/ethnicity, and household members with asthma. Surveys were geocoded and linked with neighborhoods. Neighborhood information on race, education and income were based on 2000 census data. Bivariate and multilevel analyses were performed. Results: Of the 48, 917 surveys, 41,255 (84.3%) were geocoded into 287 neighborhoods. Asthma prevalence among all children was 12.9%. Asthma rates varied among neighborhoods from 0% to 44% (IRQ: 8-24%) Asthma prevalence (mean, standard deviation, range) in predominantly Black neighborhoods (19.9, +/- 7, 4-44%) was higher than in predominantly White neighborhoods (11.4, +/- 4.7, 2-30%) and predominantly Hispanic neighborhoods (12.1, +/- 6.8, 0-29%). Although gender, age, household members with asthma and neighborhood income significantly affected asthma prevalence, they did not explain the differences seen between neighborhoods. Race explained some (about 80%) but not all of this variation. Conclusions Reached: Childhood asthma prevalence varies widely by neighborhood within this urban environment. Adjacent areas in Chicago were identified with significantly different asthma prevalence. A better understanding of the impact of neighborhood characteristics may lend insight into potential interventions to reduce childhood asthma.

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Veronica Mas Casullo, MD Instructor Mount Sinai School of Medicine Dr. Mas Casullo completed a fellowship in Pediatrics Infectious Diseases at Mount Sinai School of Medicine. During her training, her research focused on studying the mechanism of action of topical microbicides to prevent HSV-2 infection. She joined the division of Pediatric Infectious Diseases at Mount Sinai Medical Center in 2004. She is currently working on the characterization of the interaction between Human Monocyte derived Dendritic Cells and Herpes Simplex Virus type 2. Her research focuses on HIV-HSV co-infection, trying to elucidate the molecular bases to support the epidemiological link between HSV and HIV.

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HERPES SIMPLEX VIRUS IMPEDES DENDRITIC CELL FUNCTION POSSIBLE MECHANISM of IMMUNE EVASION. V Mas Casullo, V Shende, and BC Herold, Mount Sinai Medical Center, New York, NY Background: These studies were designed to explore the impact of HSV-2 on immature myeloid derived dendritic cells (mDCs) and to test the hypothesis that HSV modifies mDNCs to escape its own immune surveillance. Design/Methods: Monocytes were isolated form PBMC through positive selection on magnetic beads and the cells were cultured in medium supplemented with recombinant human GM-CSF and IL-4. The response to HSV-2 was assessed by exposing the cells to HSV-2 or, as a control, the TLR agonist, LPS. Phenotypic changes were assessed by FACS, cytokine and chemokine responses by Bioluminex, and functional responses by assessing ability of the DCs to present antigen (Ag) to T cells in a mixed lymphocyte reaction (MLR) and ability to migrate in response to CCL19. Activation of NFkB pathways was evaluated by electrophoretic mobility shift assays (EMSA). Results: Exposure of mDCS to HSV failed to trigger DC maturation. While LPS upregulates CCR7, CD83, CD86 and HLADR and downregulates CCR5 and DCSIGN, HSV failed to downregulate CCR5 and DCSIGN and had little or no effect on any of the other cell surface markers. Subsequent exposure to LPS failed to trigger any response suggesting that the HSV-exposed cells were anergic. Consistent with this observation, HSV- exposed DCs failed to migrate in response to CCL19 or present Ag to T cells in a MLR reaction. Despite the inappropriate maturational response, HSV-exposed mDCS released 100-1000 fold more pro-inflammatory cytokines (IL-6, IL-1α and IL-1β) and chemokines (RANTES, MIP1α and MIP1β) than mock-exposed cells (p < 0.001). HSV also activated nuclear transport of NFkB, which may contribute to the observed cytokine and chemokine response. Conclusions: These findings demonstrate that HSV-2 induces paradoxical changes in immature MDCs characterized by functional anergy, but release of inflammatory cytokines and chemokines. These responses could provide an immune escape mechanism to augment HSV’s own infectivity and facilitate HIV co-infection. The pro-inflammatory response may recruit or activate HIV target cells and enhance HIV replication. The inability of HSV to downregulate CCR5 or DCSIGN could also enhance HIV infection.

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Valerie A McLin, MD Assistant Professor Baylor College of Medicine Texas Children's Liver Center I attended medical school at the University of Geneva, Switzerland, and completed a residency combining pediatrics and pediatric surgery at Geneva Children's Hospital. Early in residency, I spent one year working with liver transplant patients, and developed an interest in pediatric gastroenterology and hepatology. In 2001 I moved to Cincinnati, Ohio, for a fellowship in pediatric gastroenterology, hepatology and nutrition, and extended the fellowship by one year to further develop my skills in developmental biology of the liver under the mentorship of Dr. Aaron Zorn. At the same time, I participated in a tertiary hepatology referral clinic under the leadership of Dr. William Balistreri. I moved to Houston in the fall of 2005, where I joined the Texas Children's Liver Center as an assistant professor of Pediatrics. I am currently working under the mentorship of Dr. Milan Jamrich, and am happy to say that I recently secured a K08 grant from the NIH. The work presented at this meeting overlaps both the CHRCDA K12 and K08.

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THE ROLE OF WNT SIGNALING IN THE DEVELOPMENT OF THE VISCERAL MESODERM. VA McLin, RD Shah, CH Hu, M Jamrich. Baylor College of Medicine, Houston, TX. Background: The epithelium of the vertebrate gastrointestinal tract is derived from the endoderm and the muscular layers and mesenchyme are derived from the visceral mesoderm. The epithelium has been the focus of most studies to date because of the clinical implications of epithelial biology. However, very little is known about the development of the visceral mesoderm or how it contributes to patterning of the gastrointestinal tract. Across species, the transcription factor FoxF1 has been shown to be essential for the formation of the visceral mesoderm, but very little is known about its molecular regulation. In vertebrates, members of the biologically important Wnt/βcatenin signaling cascade are expressed in the developing visceral mesoderm, at the right time and place to regulate mesoderm patterning possibly through FoxF1. Hypothesis: Selective inhibition of Wnt signaling in the developing visceral mesoderm will cause abnormal gut development through FoxF1 downregulation. Design: We performed targeted microinjections and transgenesis to inhibit conditionally Wnt signaling in the developing visceral mesoderm of Xenopus laevis. We used GSK3β mRNA microinjection and the FoxF1 regulatory sequence to drive a dominant negative Wnt receptor in the developing visceral mesoderm (FoxF1-Nxfz8). Embryos were assayed by morphology, histology, and gene expression. Results: Embryos in which βcatenin was downregulated through GSK3β overexpression in the visceral mesoderm displayed focal downregulation of FoxF1 expression in the targeted cells. The effect of non-cell autonomous Wnt signaling on the visceral mesoderm was examined in the FoxF1-Nxfz8 transgenic embryos. 18/25 embryos displayed impaired gut coiling, abnormal organ positioning and incomplete lumen formation. These changes were most noticeable in the cranial and caudal most parts of the embryonic gut. Conclusions: Non-cell autonomous inhibition of Wnt signaling in the visceral mesoderm leads to abnormal gut coiling, organ placement, and lumen formation, affecting predominantly the proximal and distal portions of the GI tract. Cell autonomous inhibition of Wnt signaling in the lateral plate mesoderm downregulates FoxF1 expression, suggesting that in part, FoxF1 expression requires Wnt/βcatenin signaling.

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Suneet Agarwal, MD, PhD Instructor Children’s Hospital, Boston I was born in Toronto, Canada and grew up in Akron, Ohio. I attended Brown University and completed my Sc.B. in Biochemistry and Molecular Biology in 1993. I received my M.D. and Ph.D. degrees from Harvard Medical School in 2001. The subject of my thesis work with Dr. Anjana Rao was the regulation of endogenous cytokine gene loci during T helper cell differentiation. I completed residency and fellowship training in Pediatric Hematology/Oncology at Children’s Hospital Boston in 2006, and I am currently Instructor in Pediatrics at Harvard Medical School. My current post-doctoral work with Dr. George Daley at Children’s Hospital Boston is on the mechanisms of nuclear reprogramming, which continues to fuel my long-term interests in epigenetics and cell-fate specification.

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ROLE OF THE TRANSCRIPTION FACTOR NANOG IN EMBRYONIC DEVELOPMENT AND NUCLEAR REPROGRAMMING. S Agarwal, A Yabuuchi, A Takeuchi, GQ Daley, Children’s Hospital Boston, Boston, MA. Background: Adult cells can be reprogrammed to a pluripotent state by nuclear transfer (NT), raising the prospect of creating patient-specific stem cells for use in regenerative medicine. However, nuclear reprogramming is an exceedingly inefficient process, and the mechanisms are largely unknown. The identification of factors involved in reprogramming may help overcome technical and ethical barriers to stem cell therapy. The transcription factor Nanog has been shown to be important in the maintenance of pluripotency. Based on its embryonic expression pattern, we hypothesize that Nanog is a “master regulator” which activates pluripotency gene programs and specifies cell fate during embryogenesis. We further hypothesize that Nanog will be able to improve nuclear reprogramming efficiency. Objectives: We aim to determine whether: (1) Nanog specifies pluripotent cell fate in the developing embryo, and (2) ectopic Nanog expression can improve the efficiency of nuclear reprogramming in an NT assay. Design/Method: We engineered an embryonic stem (ES) cell line with doxycycline-inducible expression of Nanog, allowing us to control the ectopic expression of Nanog. These cells were cultured in the presence or absence of doxycycline, and used as donor cells for NT. Treatment with or without doxycycline was continued during the in vitro culture of the NT embryos, and developmental progression through cleavage and compaction stages was tracked. The efficiency of ES cell line derivation from NT embryos reaching the blastocyst stage was quantified. Results: Inducible Nanog NT embryos cultured with or without doxycycline displayed no significant differences in morphology during development. Overall, the rate of ES lines produced from fused NT embryos was almost 2-fold higher with in the presence versus absence of ectopic Nanog induction (15% vs 8%, respectively). The rate of progression to blastocyst stage was approximately 40% higher in the presence versus absence of overexpressed Nanog (37% versus 26%, respectively). However, the rate of ES line derivation from NT blastocysts developed in the presence or absence of doxycycline was less markedly different (39% versus 31%, respectively). Conclusion Reached: Our results suggest that overexpressing Nanog in NT donor cells increases the ability to generate ES lines. Thus Nanog may enhance nuclear reprogramming. However, Nanog may primarily enhance development of NT blastocysts, more so than increasing ES line derivation from blastocysts (which is a more precise measure of reprogramming efficiency). Further experiments will aim to distinguish these possibilities.

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Amy E. Roberts, M.D. Faculty Children’s Hospital Boston Amy Roberts, M.D. received her undergraduate degree in Biology and Women’s Studies from Swarthmore College and her medical degree from Dartmouth Medical School. During her pediatrics residency at the University of Massachusetts Medical School she became interested in clinical genetics and worked with Laurie Demmer, M.D. studying primary care physicians’ knowledge of the ethics of genetic testing. Following her residency, Dr. Roberts was accepted into the Harvard Medical School Genetics Training Program. In her second year of training she began to work with her research mentor, Raju Kucherlapati, Ph.D., on a genotype-phenotype correlation study for children and adults with a cardiovascular disorder, Noonan syndrome. A multi-center research protocol was established to recruit patients from across the country and around the world. Upon completion of her genetics training, Dr. Roberts joined the Cardiovascular Genetics Program in the Department of Cardiology at Children’s Hospital Boston and continues her Noonan syndrome research. She is board certified in both pediatrics and medical genetics. Her clinical practice includes children with heart disease and a broad range of genetic diagnoses including cardiomyopathy and Williams, Noonan, Cardiofaciocutaneous, Velocardiofacial, Ehlers Danlos, Marfan, Alagille, and Turner syndromes. Dr. Roberts also serves as a clinical geneticist at the Harvard-Partners Center for Genetics and Genomics and as associate physician and instructor in medicine at Brigham and Women’s Hospital. Dr. Roberts received the 2006 John M. Opitz Young Investigator Award for a research paper investigating the clinical presentation of children with subtelomeric chromosomal deletions and duplications. Dr. Roberts was the lead author of a paper published in 2007 that describes that mutations in the SOS1 gene are responsible for a significant proportion of cases of Noonan syndrome.

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SARCOMERE GENE MUTATIONS AND NOONAN SYNDROME CONGENITAL HYPERTROPHIC CARDIOMYOPATHY. AE Roberts1,3, VA Joshi, RS Kucherlapati1 1 Harvard Medical School/Partners Healthcare Center for Genetics and Genomics, 2Department of Pathology Massachusetts General Hospital, 3 Children’s Hospital Boston Hypertrophic cardiomyopathy (HCM), an autosomal dominant disorder of variable penetrance, has an estimated prevalence of 1 in 500 making this the most common heritable cardiovascular disease. Mutations in sarcomere genes have been shown to cause HCM. Noonan syndrome (NS) is an autosomal dominant disorder characterized by dysmorphic facial features, short stature, skeletal abnormalities, learning disorders, and congenital heart disorders. HCM is estimated to affect approximately 20% of children with Noonan syndrome with cardiac features presenting at birth, infancy, or childhood. Approximately 50% of those with a diagnosis of NS will have a PTPN11 mutation. We present a fifteen-month-old girl diagnosed with congenital HCM and pulmonary valve stenosis that was unresolved after balloon dilation.. Exam at 15 months was notable for facial and skeletal features of NS. The family history was noncontributory. Two DNA sequence variants were detected: 1493G>T (R498L) in the NS associated PTPN11 gene (de novo) and (P955fs) in the MYBPC3 sarcomere gene (maternally derived). The R498L variant has been reported in one individual with LEOPARD syndrome. P955fs has been reported as pathogenic and is known to segregate with the clinical features of isolated HCM in two families. MYBPC3 mutations are thought to cause HCM of adult onset, although milder disease with later onset, less hypertrophy, lower penetrance, and a better prognosis has been observed. Individuals with LEOPARD Syndrome mutations present with HCM more frequently than individuals with NS, and the HCM observed can be severe and present prenatally. It is possible that the PTPN11 mutation alone can account for the cardiac features observed in this individual, but it could be postulated that there is a synergistic effect with the MYBPC3 mutation. It has been shown that PTPN11 mutations are not causative of isolated nonsyndromic HCM but the co-incidence of sarcomere gene mutations in those with HCM and NS associated with a PTPN11 mutation is not known. It can be predicted, based on the incidence in the general population, that roughly 1:1,000,000 individuals would have both a PTPN11 and sarcomere variant. In cases of NS with severe or early onset HCM, other factors, especially sarcomere dysfunction, could contribute to the cardiac features observed.

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Shahab Asgharzadeh, MD Assistant Professor Children’s Hospital Los Angeles Dr. Asgharzadeh studies genetic features of neuroblastomas that predict their clinical behavior. His goal is to develop accurate prognostic classifiers using microarray data that can be translated clinically for treatment planning. Dr. Asgharzadeh received his baccalaureate degree in Biomedical Engineering from Northwestern University. He then received his M.D. degree from University of Illinois followed by training in Pediatrics at the University of Chicago. He completed a fellowship in clinical medical ethics at University of Chicago followed by a fellowship in pediatric hematology-oncology at Childrens Hospital Los Angeles, an affiliate of University of Southern California. Dr. Asgharzadeh joined the faculty of the University of Southern California in 2005 and practices as a pediatric neuro-oncologist at Children’s Hospital Los Angeles.

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PROGNOSTIC SIGNIFICANCE OF GENOMIC ALTERATIONS OF METASTATIC NEUROBLASTOMAS. S Asgharzadeh1,2, R Pique-Regi3, Y Tu1, R Sposto1,2, H Shimada1,2, A Ortega3, RC Seeger1,2. 1Childrens Hospital Los Angeles, 2Keck School of Medicine and 3Viterbi School of Engineering, University of Southern California, Los Angeles, CA Background: Patients with metastatic neuroblastomas have quite different outcomes depending upon their age at diagnosis and MYCN amplification status. Accurate classification of patients, using genetic features of their tumors identified through microarray studies, would improve prediction of outcome and facilitate more appropriate selection of therapy. Objectives: 1) Develop algorithms for analysis of high-density single nucleotide polymorphism (SNP) arrays that would lead to improved identification of copy number alterations (CNA) in neuroblastoma tumors; 2) Identify subgroups of stage 4 neuroblastoma patients based on genomic features (amplifications or deletions) obtained from SNP arrays; and 3) Develop a genomic classifier that would improve prediction of outcome. Method: 43 patients with metastatic neuroblastoma (18 MYCN amplified, 28 non-amplified) tumors were analyzed using Affymetrix 50K Xba SNP arrays. A novel algorithm was developed using Sparse Bayesian Learning (SBL) to identify CNA. The accuracy of the algorithm was determined using simulated genomic data and four neuroblastoma cell lines’ data generated on Affymetrix 50K Xba, 250K Sty, 250K Nsp, and Illumina-550K platforms. Principal component analysis (PCA) was used to define regions with DNA copy number abnormalities and diagonal linear discriminant analysis (DLDA) was used to build and assess error rates of classifiers using combination of these regions. Loss of heterozygosity data were also used as features to build prognostic models. Results: The SBL algorithm had greater accuracy identifying CNAs as compared to other algorithms (e.g. CBS, HMM, GLAD and with 100-fold increase in computational speed as compared to CBS. Of the 106 major breakpoints identified in the 4 neuroblastoma cell lines tested, 67% were represented on all three microarray platforms, and these CNAs included all the major known neuroblastoma CNAs. In a proof of principle experiment, the PCAs for chromosomes 2p, 1p, and 19q were the top features identified in a DLDA analysis distinguishing tumors based on their MYCN status with an error rate of 9%. Analysis of loss of heterozygosity data for the 28 patients whose tumors lacked MYCN amplification, identified low and high risk subgroup with PFS of 64% and 32%, respectively. Conclusions: Accurate identification of genomic features of metastatic neuroblastomas from SNP arrays could be used to identify patients who have distinct outcomes even though their tumors are clinically indistinguishable.

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Christopher F. Spurney, MD Assistant Professor Children’s National Heart Institute Children’s National Medical Center I am currently a pediatric cardiologist at Children’s National Medical Center in Washington, DC. I joined the faculty in July 2004 as an Assistant Professor of Pediatrics and obtained the Child Health Research Career Development Award. The goal of my training is to become an independent researcher in the field of cardiomyopathy secondary to muscular dystrophies. I attended the College of Arts and Sciences at Cornell University in Ithaca, NY (1987 -1991). After college, I spent two years as a lab technician in the Clinical Brain Disorders Branch of the National Institute of Mental Health in Bethesda. I attended New York Medical College from 1994-1998. I completed a general pediatric residency at the Babies and Children’s Hospital of the Columbia University College of Physicians and Surgeons in New York City in 2001. In 2004, I completed my pediatric cardiology fellowship at Children’s National Medical Center.

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CARDIOMYOPATHY IN THE DYSTROPHIN DEFICIENT MOUSE HEART: CHARACTERIZATION USING ECHOCARDIOGRAPHY AND GENE EXPRESSION PROFILING. C Spurney, S Knoblach, EE Pistilli, K Nagaraju, EP Hoffman, Center for Genetic Medicine Research, Children’s Research Institute, Children’s National Medical Center, Washington, DC Background: Duchenne muscular dystrophy (DMD) causes dilated cardiomyopathy in the second decade of life in affected males and later in life in female carriers. As treatment of the muscular and respiratory systems improves, cardiac failure is an increasing cause of death in DMD patients. Objectives: Using the dystrophin deficient mdx mouse model, the genetic homologue to human dystrophin deficiency, we studied the cardiac structure, function and gene expression at 9-10 months of age. Design/ Methods: Mdx (n=14) and wild type (n=10) mice were anesthetized using inhaled isoflorane and echocardiography was performed using a 35 MHz transducer. B-mode, M-mode and spectral Doppler images were obtained and function was measured. Total RNA was extracted from the left ventricles of mdx and wild type mice and used to generate gene expression profiles using the Affymetrix GeneChip® Mouse Expression Set 430 2.0. Altered genes were validated from independent samples using TaqMan® real time PCR quantification. Results: Echocardiography showed a significant decrease in percent shortening fraction of mdx mice compared to wild type. Histology of the left ventricles of mdx mice also showed significantly increased patchy fibrosis compared to wild type. Focusing on profibrotic pathways, gene expression profiling and real time PCR of the left ventricles of mdx mice showed increased expression of NADPH oxidase 4 (Nox4) and lysyl oxidase (Lox). Comparing these results with real time-PCR of dystrophin deficient skeletal muscle, Lox was similarly increased, but Nox4 mRNA showed no changes in mdx mice. Conclusions: This is the first report of gene expression in the left ventricles of dystrophin deficient mice showing echocardiographic evidence of cardiomyopathy. Up-regulation of Nox4 is unique to cardiac tissue and has recently been shown to be involved in other models of cardiac fibrosis. The pathways regulated by this gene may be putative targets for therapeutic interventions to treat the cardiomyopathy seen in DMD.

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Corinne Linardic, MD, PhD Assistant Professor Duke University Corinne Linardic was born in Oradell, New Jersey and attended Duke University in Durham, NC for her undergraduate education, where she was graduated with a B.S. in Zoology and completed requirements for a B.A. in English literature. She continued at Duke, receiving both her M.D. and Ph.D. degrees, working in the Department of Cell Biology with Yusuf Hannun on the nascent field of ceramide signaling and its role in apoptosis and protein trafficking. She was a pediatrics intern and resident at the Children’s Hospital of Philadelphia. Her Pediatric Hematology-Oncology Fellowship training began at Children’s Hospital of Philadelphia, and was completed at Duke University Medical Center, where she is now an assistant professor in the Division of Pediatric Hematology-Oncology. Dr. Linardic’s clinical and research interests are in pediatric and adolescent musculoskeletal tumors. Recently, in collaboration with her mentor Chris Counter, she has created a genetically defined, human cell-based model for the pediatric malignancy rhabdomyosarcoma, and is using this approach to dissect molecular pathways dysregulated in this cancer.

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GENETIC MODELING OF ALVEOLAR RHABDOMYOSARCOMA. K Etheridge*, S Naini*, S Adam*, R Bentley*, S Qualman§, C Counter*, and C Linardic*, *Duke University Medical Center, Durham, NC, and §Columbus Children’s Research Institute, Columbus, OH. Background: Rhabdomysarcoma (RMS), a cancer with features of skeletal muscle, is the most common soft tissue sarcoma of childhood. Despite therapeutic advances, children with the alveolar variant of RMS (aRMS) have a 5-yr survival of <30%. Up to 70% of aRMS tumors express the PAX3-FKHR (PF) gene mutation, suggesting that its specific early expression underlies aRMS. We have developed genetically defined models of RMS, based on the step-wise conversion of primary human skeletal muscle myoblasts (HSMMs) to their tumorigenic counterpart. Although successful in generating models for less aggressive RMS variants, a model for aRMS eluded us. Objectives: To develop a new model for aRMS that incorporated the PF gene, and take advantage of our prior observations in which expression of PF in HSMMs, in cooperation with loss of the p16 tumor suppressor, was found to enable bypass of the senescence checkpoint, providing an initiating “oncogenic hit.” Design/Methods: HSMMs cultured in vitro were stably transduced with amphotrophic retrovirus encoding PF cDNA, and allowed to proliferate past the senescence checkpoint. Post-senescent HSMMs were then additionally transduced with hTERT, MycN, and RAS cDNAs to corrupt the telomerase, Myc and Ras pathways, which from our prior studies are known to be critical components of tumorigenesis in human cells. Importantly, these pathways are also corrupt in naturally-occurring human aRMS. Immunoblotting and PCR-based assays were used to verify transgene expression. Resulting cell lines were evaluated for transforming ability in vitro using soft agar assays, and for tumorigenic ability in vivo using xenograft growth in SCID-beige mice. Assays were done in triplicate so that average and SD values could be obtained. H&E and immunohistochemistry were used to assess resemblance of xenografts to human aRMS tumors. Results: Combined stable expression of PF, hTERT, and MycN in HSMM cells was necessary and sufficient to generate cells that when assayed as xenografts, generated tumors precisely mimicking aRMS. Expression of oncogenic RAS, while it hastened tumor formation, was not necessary, suggesting that PF assumes the role of RAS in aRMS tumorigenesis. Conclusions: Combined expression of PF, hTERT, and MycN is necessary and sufficient to convert HSMMs to tumorigenic cells that form aRMS in vivo. This model is the first to define the combination of genetic lesions required to form human aRMS, and may provide a platform upon which to rationally design new therapeutic approaches.

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Sara DiVall, MD Instructor Johns Hopkins University Dr. DiVall received her BS in Biochemistry and Molecular Biology from University of Wisconsin-Madison in 1995 and obtained her degree in Medicine from the University of Wisconsin School of Medicine in 1999. She completed her residency in pediatrics at Albany Medical Center in 2002, and began her fellowship in Pediatric Endocrinology at the University of Chicago in 2003, working in the laboratory of Dr. Sally Radovick and Dr. Andrew Wolfe. She followed her research mentors to Johns Hopkins University in 2005 and completed her fellowship in 2006. She continues to work with Drs. Radovick and Wolfe studying the neuroendocrine regulation of reproduction. Specifically, Dr. DiVall studies the effects on insulin and IGF-1 on gonadotropin-releasing hormone neuron function.

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INSULIN STIMULATES GONADOTROPIN-RELEASING HORMONE (GNRH) PROMOTER ACTIVITY IN VIVO S DiVall, L Su, A Wolfe, and S Radovick, Johns Hopkins University School of Medicine, Baltimore, Maryland. Background: The molecular mechanism directing the integration of nutrition status with reproductive function at the level of the hypothalamus is not fully elucidated. Previously our laboratory and others demonstrated that insulin increased gonadotropin-releasing hormone (GnRH) transcription in immortalized GnRH-producing neuronal cell lines. Objective: To investigate if insulin affects GnRH promoter activity in vivo and to map the insulin responsive region on the GnRH promoter. Design: In vivo: Transgenic mice expressing the GnRH promoter fused to a luciferase reporter gene were fasted for 18 hours then administered insulin and glucose. After 60 minutes, hypothalami were harvested and a luciferase assay performed. In vitro: Immortalized GnRH secreting cells were transfected with constructs containing serial deletions of the GnRH promoter fused to a luciferase reporter gene. Luciferase assays were then performed after one hour of insulin treatment. Results: Mice with the -3446 bp fragment of the promoter fused to luciferase (3446LUC) treated with insulin+glucose had luciferase activity nearly 3-fold greater then untreated 3446LUC mice. Similarly, 2078LUC mice treated with insulin+glucose had luciferase activity 2-fold greater than untreated 2078LUC mice. In contrast, mice with the 356LUC transgene treated with insulin+glucose did not have a significant difference in luciferase activity from untreated mice. Blood glucose levels were not significantly different between treated and untreated mice. In vitro, cells transfected with a construct containing -1005 bp of the GnRH promoter (1005LUC) had a nearly 2-fold greater luciferase activity compared to untreated cells. In contrast, cells transfected with the 803LUC construct did not have a significant difference in luciferase activity from untreated cells. Conclusions: 1) insulin stimulates GnRH promoter activity measured as luciferase expression in vivo and in vitro; 2) the region of the GnRH promoter between -2078 and -356 contains an in vivo insulin response element; and 3) an insulin response element is present in vitro between -1005 and -803 bp of the GnRH promoter. Collectively, these findings suggest that insulin may be a metabolic signal regulating the reproductive axis by increasing GnRH gene expression. Further studies to define the in vivo role of insulin signaling within the GnRH neuron are underway.

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Trent Tipple, MD Assistant Professor The Ohio State University College of Medicine Trent E. Tipple is an investigator in the Center for Perinatal Research at Columbus Children’s Research Institute and an Assistant Professor of Pediatrics at The Ohio State University College of Medicine in Columbus, Ohio. He received his medical degree from Indiana University School of Medicine and completed his pediatric residency and neonatal-perinatal medicine fellowship at Columbus Children’s Hospital. Dr. Tipple’s clinical interest in the pathogenesis of bronchopulmonary dysplasia (BPD) has led him to investigate redox regulation of lung growth and development. Utilizing a murine model of BPD, his research is focused on understanding the effects of hyperoxic exposure on thioredoxin, a redox active protein that is dynamically altered by changes in oxygen tension and is linked to both proliferative and apoptotic pathways. Additionally, Dr. Tipple participates in clinical care and teaching at both Columbus Children’s Hospital and The Ohio State University Medical Center.

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EFFECTS OF SUBLETHAL HYPEROXIC EXPOSURE ON THE THIOREDOXIN SYSTEM IN NEWBORN MICE. TE Tipple, SE Welty, LD Nelin, and LK Rogers, The Ohio State University College of Medicine, Columbus, OH Background: Prolonged exposure of neonatal mice to sublethal hyperoxia causes functional and pathological changes similar to those seen in infants with bronchopulmonary dysplasia (BPD), these lung changes are characterized by abnormal lung development. Thioredoxin-1 (Trx1) is important in antioxidant defense, regulation of cellular proliferation, and regulation of gene expression. Trx1 expression is increased in the lung by hyperoxic exposure in a baboon model of BPD. Objective: The objective of the present studies was to test the hypothesis that neonatal mice exposed to sublethal hyperoxia will exhibit alterations in normal developmental pulmonary and hepatic levels of Trx1. Design: Within 24 h of birth, C3H/HeN mice were exposed to sublethal hyperoxia (FiO2 = 0.85) or were kept in room air (FiO2 = 0.21). The dams nursing the pups were rotated between hyperoxia and room air every 24 hours. At 1, 3, 7 and 14 d, mice were euthanized, lungs and livers removed, and Trx1 levels determined by western blotting. Band intensities were quantified and normalized to ß-actin. Data, expressed as means ± SEM, and were analyzed by 2-way ANOVA with Student-Neuman-Keuls tests post-hoc. Results: In newborn mice exposed to sublethal hyperoxia for up to 14 days lung development is altered characterized by fewer and larger alveoli and decreased secondary crest formation. In room air controls, hepatic Trx1 levels nearly doubled by day 3 (1.0±0.2 vs 0.6±0.1, day 1, p<0.05) and were 4 times higher by day 7 (2.6±0.1, p<0.05 compared to day 1). No time-dependent effects on Trx1 levels in the lungs of room air controls were observed. Exposure to sublethal hyperoxia increased hepatic Trx1 levels on day 1 (1.4±0.1, p<0.05). Hepatic Trx1 levels were not different than room air controls on days 3, 7 or 14. Pulmonary Trx1 levels were not different than room air controls at any of the time points. Conclusions: Hepatic levels of Trx1 increase, while pulmonary Trx1 levels remain constant through the first 2 weeks of life. Hepatic but not pulmonary levels of Trx1 increase with exposure to sub-lethal hyperoxia. We speculate that alterations of hepatic Trx1 caused by hyperoxic exposure may contribute to hyperoxia-induced alterations in lung development in this rodent model of BPD.

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Kenan Onel, MD, PhD Assistant Professor University of Chicago Dr. Onel studies host factors that predispose an individual towards cancer. His goal is to identify genetic variants that alter the function of the pathways that are the barriers against cancer, with the hope that these can be translated clinically into biomarkers of cancer susceptibility. Dr. Onel received his baccalaureate and master’s degree in post-World War I European history from Yale University. He then received his M.D. and Ph.D. degrees from Cornell University Medical College. His Ph.D. was in Molecular Biology, working on the structure and function of the REC1 gene of Ustilago maydis under the tutelage of Bill Holloman, Ph.D. Dr. Onel’s pediatrics training was at Babies and Children’s Hospital of New York, Columbia-Presbyterian Medical Center. Following that, he completed a fellowship in pediatric hematology/oncology at the Memorial Sloan-Kettering Cancer Center. Simultaneously, he undertook a post-doctoral fellowship at the Rockefeller University and Memorial Sloan-Kettering, working on a functional analysis of the p53 pathway under the joint mentorship of Arnie Levine, Ph.D., and Carlos Cordon-Cardo, M.D., Ph.D. Dr. Onel joined the faculty of the University of Chicago in 2004. Coincidentally, the White Sox won the World Series the following year.

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THE GENETIC DETERMINANTS OF APOPTOSIS. O. Yildiz, B. Yilmaz, A. Kowalska, R. Diederich, J. Badner, and K. Onel, The University of Chicago, Chicago, IL. Background: When exposed to potentially oncogenic stresses, cellular response pathways are activated. Together, the coordinated response of these pathways determines the ultimate fate of a stressed cell. A prediction is that genetic variation that alters any component of these pathways would also alter cancer susceptibility or chemoresponsiveness. Apoptosis is one phenotype commonly deranged in cancer. Indeed, in murine models, it is the cellular function selected against in the development of hematopoietic malignancies. Hence, the identification of genetic determinants of apoptosis in lymphocytes is likely to provide significant insight into genes and pathways deregulated in oncogenesis. Objectives: In peripheral blood mononuclear cells (PBMCs) freshly isolated from healthy volunteers, we have shown that there is unexpected heterogeneity in the elicited apoptotic response as measured by TUNEL (range: 20 – 70%). Despite this tremendous heterogeneity among individuals, damage-induced apoptosis is highly reproducible within an individual. This suggests that damage-induced apoptosis is a genetically determined response program. The purpose of this project is first, to test the hypothesis that damage-induced apoptosis is a heritable trait, and second, if so, to map these determinants using an in vitro tractable genetic system. Design/Methods: The Centre d’Etude du Polymorphisme Human (CEPH) pedigrees are extensively genotyped multigenerational lineages of primarily Caucasian descent collected specifically for genetic research. Lymphoblastoid cell lines (LCLs) have been made from each member of these families. Each line is exposed to increasing doses of UV-irradiation to induce DNA damage (0 – 250J), and apoptosis is quantified by annexin V positivity. By contrasting the variation between families to that within a family, heritability is calculated for each dose of UV. Linkage analysis is then performed to map genomic response loci that may contain candidate genetic determinants of apoptosis for the most heritable phenotypes. Results: We have thus far completely phenotyped six families (=90 individuals) for five different UV doses. While damage-induced apoptosis is not heritable at low doses of UV (50J). It is, however, highly heritable at 100J (26% due to genetic factors, p = 0.014), and 250J (42% genetic, p = 0.0001). Conclusions Reached: Taken together, these data suggest that damage-induced apoptosis is a genetically determined trait resulting from inherited polymorphic variation in stress response pathways. Our results indicate that these loci can be successfully mapped using our familial linkage strategy. If so, our objectives are, first, to identify these genetic determinants, and second, to translate these findings from the laboratory to the clinic as biomarkers of cancer susceptibility, and as candidate molecular targets for novel therapeutic interventions.

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Matthew J. Ryan, MD Attending Physician The Children's Hospital of Philadelphia Matthew J. Ryan obtained his MD degree from The New Jersey Medical School, University of Medicine and Dentistry of New Jersey, and subsequently trained at The University of Chicago Children’s Hospital for his pediatric residency. Dr. Ryan completed his training in Pediatric Gastroenterology at The Children's Hospital of Philadelphia where he is currently an Attending Physician in the Division of Gastroenterology and Nutrition. Dr. Ryan works in the laboratory of Kathleen Loomes, MD in The Abramson Research Center, performing research on signaling pathways that govern bile duct development and growth in murine models. His clinical interests include Alagille Syndrome and cholestatic liver disease.

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MARKED BILE DUCT PROLIFERATION IN MICE HETEROZYGOUS FOR JAG1 AND FRINGE ALLELES. M Ryan1,2, K Kusumi3, N Spinner2, L Underkoffler1, A Nelson1, M Segalov1, P Russo1,2, K Loomes1,2. 1The Children's Hospital of Philadelphia, Philadelphia PA; 2The University of Pennsylvania School of Medicine, Philadelphia PA; 3Arizona State University, Phoenix AZ Background: The Notch signaling pathway is involved in cell fate determination in many organ systems. JAG1 encodes a Notch ligand, and mutations in this gene can cause Alagille Syndrome (AGS), a multisystem disorder characterized by bile duct paucity and other abnormalities. There is a tremendous variability in phenotypes even among individuals with the same JAG1 mutations, suggesting other modifier genes play a role. In mammals, three Fringe genes (Lunatic (Lfng), Radical (Rfng) and Manic (Mfng)) encode glycosyltransferases that modify Notch ligand-receptor interactions. Objectives: We have examined mice heterozygous for mutations in Jag1 and each of the Fringe genes to determine if they affect Notch signaling in liver growth and development. Methods: Genetic crosses were performed to generate mice with the Jag1+/- Lfng +/-, Jag1+/- Rfng +/- and Jag1+/- Mfng +/- genotypes, along with littermate controls. Liver morphology was evaluated at various time points by histology and immunohistochemistry (e.g., cytokeratins, PCNA). To determine whether epithelial to mesenchymal cell transition (EMT) was occurring, double labeling was done with CK19 antibody and either FSP-1 or HSP47 (EMT markers). Results: Adult Jag1+/- Lfng +/- and Jag1+/- Rfng +/- mice had a significant increase in the number of bile ducts surrounding portal tracts when compared to control littermates, whereas Jag1+/- Mfng +/- livers were normal. In all of the crosses, hepatocytes appeared normal with no signs of cholestasis. The extrahepatic biliary tree was patent and appeared normal. In newborn mice, bile duct morphology was normal. Despite the obvious increase in the number of mature bile ducts, there was no significant increase in the number of actively proliferating cells between the double heterozygous mutants and their littermate controls. Additionally, when evaluating for EMT, antibody markers did not co-localize to the bile duct cells, suggesting EMT was not integral to the development of the phenotype. Conclusions: Mice heterozygous for mutations in Jag1 and Lfng or Rfng have a striking proliferation of bile ducts in the adult liver, whereas Jag1+/- Mfng +/- mice have no hepatic phenotype. Further studies are needed to determine the mechanism whereby decreased expression of Jag1 and Lfng or Rfng leads to bile duct proliferation. These data suggest LFNG and RFNG may be potential candidates to be modifiers of the AGS hepatic phenotype in children.

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Sean McGhee, MD Clinical Instructor David Geffen School of Medicine at UCLA Sean McGhee is a clinical instructor in pediatric immunology at the David Geffen School of Medicine at UCLA and the Mattel Children's Hospital at UCLA. He attended medical school at Stanford University, and did his residency, chief residency and fellowship training at UCLA. His research interests are in the epidemiology of primary immune deficiencies.

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COPY NUMBER VARIATIONS IN DEL22Q11.2 SYNDROME. S McGhee, M Suchard, ERB McCabe, David Geffen School of Medicine at UCLA, Los Angeles, CA. Background: Genetic polymorphisms, including single nucleotide polymorphisms, small insertions, and deletions, contribute to phenotypic changes and may be associated with disease. Large-scale copy number variations (CNVs) are a part of this variability and can be determined genome-wide using representational oligonucletoide microarray analysis (ROMA). Such CNVs are found in normal patients and new CNVs frequently arise in individual patients. Objectives: We investigated the hypothesis that patients with del22q11.2 syndrome might have a broader susceptibility to larger or more frequent CNVs. CNVs and the del22q11.2 deletion both may arise from unequal crossing-over events, and we hypothesized that del22q11.2 patients may have an abnormal mechanism resulting in a predisposition to larger CNVs. Design/Method: 12 patients with del22q11.2 syndrome and 8 healthy controls were assessed for genome-wide copy number changes using ROMA. Results: There was an average of 16 CNVs per genome and this average did not differ between del22q11.2 syndrome patients and controls (Wilcoxon p=0.516). The median CNV size also did not differ between patients and controls (157,555 vs. 153,157 respectively). We also determined whether patients with del22q11.2 syndrome were more likely to have larger CNVs. Here also, the frequency of CNVs greater than 500 Kb was not significantly different than controls (Fisher p=0.21, OR of 1.74, 95% CI 0.74-4.08). Conclusions: We conclude that patients with del22q11.2 syndrome do not differ from normal controls with respect to the frequency or size of CNVs, suggesting that the unequal crossing over event that produces both CNVs and the del22q11.2 deletion result from similar mechanisms in patients and controls.

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Kimara L. Targoff, MD Instructor Children’s Hospital of New York – Presbyterian Hospital of Columbia University Dr. Kimara L. Targoff is an Instructor in Pediatrics in the Division of Cardiology at the Morgan Stanley Children’s Hospital of New York – Presbyterian Hospital of Columbia University. Dr. Targoff’s current research is performed in the laboratory of Dr. Deborah Yelon in the Developmental Genetics Program and Department of Cell Biology at the Skirball Institute of Biomolecular Medicine at New York University School of Medicine. Dr. Targoff is focusing on the role of nkx genes during cardiac morphogenesis in zebrafish. Specifically, she is working to enhance our understanding of the molecular and cellular functions of nkx genes through detailed analysis of ventricular and atrial cell number, cell shape and size, and cell movement in nkx-deficient embryos. In addition, Dr. Targoff plans to examine the influence of nkx genes on the subcellular processes, such as the establishment of apicobasal polarity, that are essential for cardiomyocyte organization within the cardiac chambers.

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NKX GENES REGULATE HEART TUBE EXTENSION IN ZEBRAFISH. KL Targoff, T Schell, and D Yelon, Department of Pediatrics, Division of Pediatric Cardiology, Columbia University, New York, NY. Developmental Genetics Program and Department of Cell Biology, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY. Background: The cardiac homeodomain transcription factor Nkx2-5 is a key causative gene associated with congenital heart disease in humans. Previous studies in mouse have shown that Nkx2-5 is essential for cardiac chamber morphogenesis and myocardial maturation. Although Nkx2-5 clearly plays a fundamental role in cardiac development, we do not understand how it regulates the underlying cellular mechanisms that guide cardiac chamber formation. Objectives: With the benefit of a transparent zebrafish embryo, we can combine embryologic and genetic approaches to analyze cardiac cell behavior during morphogenesis. Design/Methods: The zebrafish genome contains two Nkx2-5 homologs that are expressed in cardiomyocytes, nkx2.5 and nkx2.7. In order to establish the roles of nkx genes during cardiac chamber formation in zebrafish, we set out to create a loss-of-function model using antisense morpholino oligonucleotides. Results: Here we show that nkx2.5 and nkx2.7 play a previously unappreciated, crucial role in the process of heart tube extension. Injection of anti-nkx2.5 and anti-nkx2.7 morpholinos disrupts cardiac morphogenesis, leading to the formation of a small, misshapen ventricular chamber and an enlarged, bulbous atrial chamber. Morphogenetic defects are first evident during the earliest stages of heart tube assembly. In wild type embryos, a critical transition occurs as bilateral cell populations fuse and extend into a long and narrow heart tube. In contrast, nkx-deficient embryos have an abnormally short and wide ventricular portion of the heart tube and a disorganized, sprawling atrial component. As a first step toward assessing the cellular mechanisms underlying this phenotype, we determined that chamber specific precursor cell populations are established normally in nkx-deficient embryos. However, as morphogenesis proceeds, the loss of nkx gene function leads to a mild reduction in the number of ventricular cardiomyocytes and a surplus of atrial cardiomyocytes. Conclusions Reached: Therefore, we conclude that nkx genes play multiple roles in guiding heart tube extension through differential effects on ventricular and atrial cell number.

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Julie Goodwin, MD Associate Research Scientist Yale University Julie attended Dartmouth College where she graduated cum laude with a major in Engineering Sciences in 1996. It was through coursework in biomedical engineering that she became interested in pursuing a career in medicine. She attended medical school at Boston University School of Medicine and received her M.D. degree in 2001. From there Julie moved to Washington D.C. to do her internship and residency in pediatrics at Georgetown University Hospital which she completed in 2004. She decided to sub-specialize in Pediatric Nephrology and recently completed her fellowship at Yale University School of Medicine in June 2007. She is currently a junior faculty member at Yale in the position of Associate Research Scientist. While at Yale, her research interest has been trying to determine the mechanism of glucocorticoid-induced hypertension using mouse models that allow tissue-specific knock-out of the glucocorticoid receptor. She is the recipient of a National Kidney Foundation Research Fellowship Award and has presented her data at recent ASN and PAS meetings.

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A CRITICAL ROLE OF THE VASCULAR ENDOTHELIUM IN THE DEVELOPMENT OF GLUCOCORTICOID MEDIATED HYPERTENSION. J Goodwin, J Zhang, D Geller, Yale University School of Medicine, New Haven, CT. Background: Acute hypertensive events are a common clinical occurrence in patients with kidney disease. A substantial body of evidence implicates glucocorticoids (GCs), a common treatment for may renal conditions, as an important contributor to hypertension (HTN). While activation of the mineralocorticoid receptor (MR) by GCs with resulting increased renal sodium reabsorption has traditionally been thought to the mechanism of this HTN, recent work indicates that the glucocorticoid receptor (GR) may also play an important role. Objective: To determine if GR in the vascular endothelium (VE) contributes to the development of GC-mediated HTN. Design/Methods: We used a mouse model permitting tissue-specific excision of wild-type (WT) GR from VE via Cre-lox recombination and Tie-1 Cre. Effective VE GR knockout was confirmed by immunofluorescent staining of mouse blood vessels. Gender-matched, littermate Cre- controls and VE GR knock-out mice were given dexamethasone (DEX) in their drinking water at a dose of 15 mg/L for 1 week. Blood pressure (BP) in unrestrained animals was measured continuously during this period by an implantable catheter. Results: Control and VE GR KO mice developed normally and had similar body weight. However, KO mice (n=7) had a small but significant increase in mean BP at baseline compared to WT controls (n=5) (112.2 mm Hg vs. 104.6 mm Hg, p=0.040). When treated with DEX, control mice exhibited a significant rise in mean BP of 9.74 mm Hg within 48 hours while KO mice had a mean BP rise of only 3.69 mg Hg in the first 48 hrs of DEX therapy (p=0.004). After 1 week of treatment with DEX mean BP in the controls increased by 13.14 mm Hg while that in the KO animals increased by 2.73 mm Hg (p=0.008). Conclusions reached: These data suggest that:

(a) acute GC-induced HTN is likely mediated via GR in vascular tissues and is likely not mediated through MR,

(b) VE GR may play a role in blood pressure homeostasis (c) VE GR may mediate both the acute and chronic BP response to GCs in

this model

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Michelle Rheault, MD Fellow Mt. Sinai School of Medicine Michelle Rheault completed her Pediatric residency and Pediatric Nephrology fellowship at the University of Minnesota in Minneapolis. In 2005, she joined the lab of Dr. Peter Mundel at Mount Sinai School of Medicine in New York as a postdoctoral fellow. She later joined the Division of Pediatric Nephrology as an Instructor in 2006. Dr. Rheault’s research is focused on elucidating the mechanism of actin cytoskeletal rearrangements in podocytes during initiation and recovery from nephrotic syndrome. Specifically, she is exploring the functional similarities between synaptopodin, an actin-associated protein found in highly dynamic cell compartments such as the podocyte foot process and dendritic spine apparatus, and tropomyosin, a ubiquitously expressed actin binding protein, in the formation of actin stress fibers.

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FUNCTIONAL SIMILARITIES BETWEEN SYNAPTOPODIN AND TROPOMYOSIN IN STRESS FIBER FORMATION. M. Rheault and P. Mundel, Mount Sinai School of Medicine, New York, NY. Background: Synaptopodin is the founding member of a unique class of proline-rich actin-associated proteins highly expressed in dynamic cell compartments such as the podocyte foot process in the kidney and the dendritic spine apparatus in the brain. Tropomyosin is a ubiquitously expressed protein that also binds actin and plays an essential role in regulating muscle contraction. Both synaptopodin and tropomyosin are essential for the regulation of actin cytoskeletal dynamics and in the development of actin stress fibers. Synaptopodin induces stress fibers by competitive blocking of Smurf-1 mediated ubiquitination of RhoA, thereby preventing the targeting of RhoA for proteasomal degradation (Asanuma et. al., Nat Cell Biol, 2006). We hypothesize that tropomyosin induces stress fibers in a similar manner. Objectives: To determine the similarities and differences in the molecular mechanisms by which synaptopodin and tropomyosin induce stress fibers. Methods: NIH3T3 cells, which have stress fibers at baseline, were utilized in this study. Transient knockdown of tropomyosin was performed by transfection (FuGENE, Roche) with a pSuper plasmid (Oligoengine) containing a verified tropomyosin shRNA sequence or a control sequence with GFP expressed as a marker of transfection. Immunofluorescence microscopy, Western blotting, and in vitro co-immunoprecipitation assays were performed via standard techniques. Tm311 (Sigma) and NT (proprietary) primary antibodies were utilized to visualize tropomyosin and synaptopodin, respectively. Results: Tropomyosin is present in NIH3T3 cells and localizes along stress fibers. Gene silencing of tropomyosin abrogates stress fibers. Co-transfection of synaptopodin into tropomyosin gene-silenced NIH3T3 cells leads to restoration of stress fibers. Thus, synaptopodin can replace the function of tropomyosin in these cells, suggesting a functional similarity in their mechanism of action. In co-immunoprecipitation assays in HEK cells, we demonstrate that tropomyosin specifically binds RhoA and Smurf-1, similar to synaptopodin. Conclusions: Synaptopodin and tropomyosin share functional similarities in the regulation of actin stress fibers. Tropomyosin may induce stress fibers through RhoA pathways. Further studies are ongoing to determine the molecular mechanisms by which tropomyosin induces stress fibers.

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Juan Pablo Abonia, MD Assistant Professor Cincinnati Children’s Hospital Medical Center Dr. J. Pablo Abonia is currently an Assistant Professor at Cincinnati Children’s Hospital Medical Center in the Division of Allergy and Immunology, affiliated with the University of Cincinnati College of Medicine. His current research work is focused upon two primary areas related to mast cell biology. The first research area deals with mast cells in research subjects suffering from eosinophilic esophagitis. In these patients, along with the eosinophilic infiltration of the esophagus, there is a concomitant esophageal mastocytosis. Dr. Abonia’s research attempts to identify what role mast cells play in epithelial proliferation and eosinophilic recruitment. The second research area deals with the recruitment of mast cell progenitors during inflammatory processes. This work has recently demonstrated the requirement for the α4β1 & α4β7 integrins along with the VCAM-1 addressin in recruiting mast cell progenitors to the mouse lung during allergic inflammation. Further, the presence of CXCR2 chemokine receptor within the lung tissue is necessary for the up-regulation of VCAM-1 and MCp recruitment during this acute inflammatory process.

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THE INVOLVEMENT OF MAST CELLS IN EOSINOPHILIC ESOPHAGITIS. JP Abonia, C Blanchard and ME Rothenberg, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH. Background: Eosinophilic esophagitis (EE) is emerging disorder with poorly understood pathogenesis. Whereas prior studies have primarily focused on the role of eosinophils in disease diagnosis and pathogenesis, we now focus our attention on the involvement of mast cells. Objectives: We sought to define mast cell levels, phenotypes and genes that in EE patients. Design/Method: Fifty-four esophageal biopsies were obtained from patients undergoing evaluation for EE and were assessed by H&E staining along with tryptase immunohistochemistry (IHC). Esophageal RNA microarray hybridization was performed using Affymetrix U133 Plus 2.0 GeneChip (54,681 genes) and analyzed using GeneSpring GX. Results: Along with gross changes in esophageal appearance, eosinophilic infiltration, and basal cell hyperplasia, there was a concomitant esophageal mastocytosis; levels of mast cells were 12.3/HPF and 5.9/HPF in EE and normal esophageal biopsies respectively (p<0.01). The level of esophageal mast cells correlated with the degree of epithelial hyperplasia (p<0.0001) and their degranulation index were increased as assessed by immunohistochemical detection of extracellular mast cell granule proteases. Esophageal mastocytosis was independent of patient age, gender, atopic status, and therapy with proton pump inhibitors. Global expression profile analysis revealed that mast cell levels correlated with the dysregulation of 0.4% (223 genes) of the genome, and overlapped with 15% (63 genes) of the eosinophil-associated transcriptome (434 genes). The expression of the MC proteases tryptase (TPSB2) and carboxypeptidase A (CPA3), but not chymase (CMA1), strongly correlated with mast cells and distinguished EE from non-eosinophilic chronic esophagitis indicating the predominant involvement of a mast cell with a T cell-dependent mucosal phenotype. Mast cell levels correlated with stem cell factor (p<0.001) and CXCL6 (p<0.001), and eotaxin-3 (p<0.01) levels to a lesser extent. Finally, treatment of EE patients with swallowed fluticasone propionate normalized levels of mast cells and several mast cell associated genes including both CPA3 and TPSB2 mRNA in responder patients. Conclusions: Taken together, we propose involvement of stem cell factor and CXCL6-mediated esophageal mastocytosis and mast cell activation in the pathogenesis of EE and that this process is reversible with fluticasone therapy and distinguishes EE from chronic esophagitis.

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Shannon Ross, MD, MSPH Assistant Professor University of Alabama at Birmingham Shannon Ross, MD, MSPH received her MD from the University of Alabama at Birmingham and went on to do her Pediatric residency and Pediatric Infectious Diseases Fellowship at UAB. She received her MSPH from UAB in 2006. She is currently Asst. Professor of Pediatrics at the University of Alabama at Birmingham. Her research interests include the pathogenesis of hearing loss in congenital cytomegalovirus infection. In addition, she is also interested in maternal CMV infection and the relationship between STD’s and CMV.

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BLOOD VIRAL LOAD AND HEARING LOSS IN YOUNG CHILDREN WITH CONGENITAL CYTOMEGALOVIRUS INFECTION. S Ross, Z Novak, W Britt, K Fowler, R Pass, S Boppana, The Department of Pediatrics, The University of Alabama at Birmingham, Birmingham, AL. Background: Congenital cytomegalovirus (CMV) infection is a leading cause of hearing loss in children but the pathogenesis of CMV related sensorineural hearing loss (SNHL) is poorly understood. Higher peripheral blood CMV viral load in early infancy has been associated with SNHL. However, the relationship between virus burden and hearing outcome in older infants and young children with congenital cytomegalovirus infection has not been explored. Objectives: To determine if increased viral load beyond early infancy is associated with hearing loss and to explore the dynamics of blood CMV viremia in young children. Methods: Blood samples were obtained from 138 children with congenital CMV infection and CMV DNA was measured with a real-time quantitative PCR assay. Viral load measurements were analyzed in five different age groups: <2 months, 2-6 months, 6-12 months, 12-24 months and >24 months by non-parametric methods as well as X2 for trend analysis. Results: Among 57 patients in whom more than one blood sample was available, 60% (34/57) had detectable CMV DNA for the entire follow-up period (range 0.5-42 months). Among the 23 children that cleared their blood of CMV, the average age of clearance was 14.7 ± 7.8 months. Approximately half (22/48, 46%) of children with normal hearing cleared their blood of CMV DNA whereas only 11% (1/9) of children with SNHL achieved an undetectable viral load (p=0.07). Symptomatic infants less than two months of age had higher amounts of CMV DNA than asymptomatic infants (p=0.007). Additionally, higher viral load in the first six months of life was associated with hearing loss. The negative predictive value for hearing loss of a viral load less than <20,000ge/ml among children less than 2 months of age was 93% and among children 2-6 months of age was 95%. No association between virus burden and hearing loss was found in older infants and children. Conclusions: CMV DNA can persist in the blood for 2 years or more in children with congenital CMV infection. Viral load <20,000ge/ml is associated with a low risk of hearing loss in children 6 months of age and younger.

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Patricia J. Dubin, MD Assistant Professor Children’s Hospital of Pittsburgh Patricia J. Dubin, MD is an Assistant Professor of Pediatrics at the Children’s Hospital of Pittsburgh at the University of Pittsburgh Medical Center. She completed her pediatric residency at Yale-New Haven Children’s Hospital and pediatric pulmonary training at Children’s Hospital of Pittsburgh. Her clinical interest in cystic fibrosis has led her to study the immune regulation of P. aeruginosa pulmonary infection which is the most significant cause of morbidity and mortality in this group of patients. Utilizing acute and chronic murine models of pulmonary infection, she has focused her studies on the roles of IL-23 and IL-17 in regulating neutrophil recruitment during P. aeruginosa pulmonary infection. This work has been supported by the American Lung Association, the American Thoracic Society and the Cystic Fibrosis Foundation. In addition to her research, Dr. Dubin is actively engaged in clinical care and teaching at Children’s Hospital of Pittsburgh and the University of Pittsburgh School of Medicine.

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IL-17-INDEPENDENT ACTIONS OF IL-23 IN PSEUDOMONAS AERUGINOSA PULMONARY INFECTION. PJ Dubin, JK Kolls, Children’s Hospital of Pittsburgh, Pittsburgh, PA. Background: Chronic P. aeruginosa (PA) pulmonary infection is the most significant cause of morbidity and mortality in individuals with cystic fibrosis (CF). Because PA is a highly adaptable pathogen, the CF host immune response is unable to eradicate it and causes significant collateral airway damage through recruitment and activation of neutrophils. IL-23 and IL-17 constitute a pro-inflammatory axis that is critical to neutrophil recruitment in PA infection. Objectives: Define the IL-17-independent role of IL-23 in the acute host inflammatory response to PA pulmonary infection. Determine if IL-23 acts independently or additively/synergistically with IL-1β in effecting this response. Design/Method: Experiment 1: WT and IL-23-deficient mice were infected with PA by intratracheal (IT) instillation. BAL inflammatory cell counts and cytokines/chemokines were measured at 3 hours. Experiment 2: Recombinant murine IL-23, IL-1β and IL-23 + IL-1β were instilled into WT and IL-23-deficient mice via IT. BAL inflammatory cell counts and cytokines/chemokines were measured at 3 hours. Results: Experiment 1: IL-23 deficient mice had significantly lower percent neutrophils (p<0.02) and lower MIP1a, KC, and IL-6 (p<0.001) compared to WT mice. At this early timepoint, IL-17 was undetectable in both groups and bacterial load was not significantly different between groups to explain these differences. Experiment 2: In in vivo studies, the IT administration of IL-23 + IL-1β elicited neutrophil recruitment and cytokine/chemokine induction in excess of that of IL-23 alone and IL-1β alone (p<0.01). These results suggest that IL-23 and IL-1β act synergistically. Statistical analysis for all studies was completed with t test or ANOVA as appropriate. Conclusion: In addition to its role in the IL-23/IL-17 proinflammatory axis, IL-23 is critically important to neutrophil recruitment in PA infection through its synergistic actions with IL-1β, acting in an IL-17-independent manner. These findings have not been reported before and highlight the critical role that IL-23 plays as a master regulator in both the innate and adaptive immune response to PA pulmonary infection.

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Anne Blaschke, MD, PhD Instructor University of Utah Dr. Anne Blaschke is an instructor in the Division of Pediatric Infectious Diseases at the University of Utah. Dr. Blaschke grew up in California, and received her undergraduate degree in Biology from Brown University. She received her M.D. and Ph.D degrees at the University of California, San Diego and completed her residency in Pediatrics and fellowship in Pediatric Infectious Diseases at the University of Utah and Primary Children's Medical Center, Salt Lake City. Dr. Blaschke’s research focuses on development of PCR-based rapid molecular testing for infectious disease. She is currently working in collaboration with Salt Lake City-based Idaho Technology, Inc., specialists in innovative PCR technology. Dr. Blaschke’s career goal is to become an expert in the development and implementation of rapid, point-of-care molecular diagnostics. She envisions developing novel testing to guide immediate and appropriate care for children, as well as rapid tests valuable in the identification and management of epidemic illness. Clinically, Dr. Blaschke studies rates and risks factors associated with antibiotic-resistant bacterial infection in children.

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Specific Amplification of E.coli and S.agalactiae using nmPCR

Included in both reactions: E.coli, S.agalactiae, S.pneumoniae,S.aureus N.meningitidis, L.monocytogenes , and negative control

A. E.coli rpoB inner primers

E.coli

B. S.agalactiae rpoB inner primers

S.agalactiae

RAPID MOLECULAR DIAGNOSTICS FOR SERIOUS BACTERIAL INFECTION. A.J. Blaschke1, L. Meyers2, K. Kitanovic2, A.T. Pavia1 C.L. Byington1, M.A. Poritz2 1University of Utah, Salt Lake City, UT, 2Idaho Technology, Inc (ITI), Salt Lake City, UT Background: Ten percent of infants presenting with fever have a serious bacterial infection (SBI). Expert consensus guidelines recommend a burdensome and expensive management strategy pending bacterial culture results. Rapid and accurate diagnostics would allow prompt identification of infants with SBI, and prevent unnecessary hospitalization and antibiotic treatment of low-risk infants. Objectives: Develop a nested multiplex PCR (nmPCR) assay, the Febrile Infant Risk Stratification Tool (“FIRST Assay”), to rapidly and accurately identify common bacterial pathogens of febrile infants. Design: Nested PCR is a sensitive and specific 2-stage PCR strategy. In the first stage, “outer primers” generate a long amplicon that is the target for the second stage reaction. “Inner primers” generate a shorter product that is detected. In nmPCR, the first-stage reaction contains multiple primer sets. ITI has developed a portable real-time PCR platform that uses nmPCR to analyze up to 120 nucleic acid targets in an unprocessed clinical sample, with a time-to-result of less than 90 minutes. Primers were designed based on alignment of bacterial DNA sequences from GenBank, and clinical bacterial isolates were used in assay development. Results: The FIRST Assay targets conserved housekeeping genes, including the RNA polymerase beta subunit (rpoB) and the DNA gyrase subunit b (gyrB). Degenerate outer primers are broad-range, and amplify targets in diverse bacterial species; inner primers target pathogenic species of interest. Identification of 14 bacterial pathogens, including E. coli and Group B Streptococcus was tested using this system. With only 4 outer primer pairs, appropriate targets from both rpoB and gyrB were amplified in all bacteria tested. Inner primers were specific for their target species (see Figure). All 14 species were accurately identified as confirmed by sequencing of both genes. Conclusions: We have shown proof-of-principal for the use of nmPCR targeting multiple conserved housekeeping genes for the accurate identification of bacteria causing infection in febrile infants. Broad-range outer primers provide the potential to easily modify the inner targets for different patient populations. When fully developed, the FIRST Assay will be integral to the rapid, accurate evaluation for SBI in infants with fever.

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Tamara Pozos, MD, PhD Acting Instructor University of Washington/Children’s Hospital Dr. Tamara Pozos is an Acting Instructor in Pediatrics, Infectious Diseases, at the University of Washington/Children’s Hospital in Seattle, WA. She received her MD/PhD from Stanford University in 1998, followed by pediatric residency training at the University of California from 1998-2002. She moved to Seattle for Pediatric Infectious Diseases fellowship training and joined the faculty in 2006.

Her overall research interest is the immunologic basis of the heightened susceptibility of infants and children to severe M. tuberculosis infection compared to adults. The zebrafish embryo-M. marinum system allows the in vivo study of TB pathogenesis in a genetically tractable system. Using this model she has shown that MMPs, especially MMP-9, are required for the earliest steps of granuloma formation and growth. Follow-up studies will investigate the role of MMPs in mammalian macrophages and mouse models of TB. Her ultimate goal is to characterize the earliest steps of tuberculosis pathogenesis to identify better diagnostic and therapeutic options for infants and children infected with TB.

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MMP-9 IS REQUIRED FOR TUBERCULOUS GRANULOMA FORMATION IN VIVO. TC Pozos1, H Volkman2, J Zheng1, J Rawls3, L Ramakrishnan2. 1Departments of Pediatrics (Infectious Diseases), 2.Microbiology, University of Washington, Seattle Washington and 3Cell and Molecular Physiology; University of North Carolina, Chapel Hill, NC. Background: Tuberculous granuloma formation begins with macrophage recruitment to sites of infection, followed by subsequent migration and aggregation of infected macrophages. The Mycobacterium RD1 locus, a region missing in the attenuated vaccine strain BCG, encodes a secretion system that promotes mycobacterial virulence. RD1-deficient (ΔRD1) bacteria fail to elicit efficient granuloma formation in vivo despite their ability to grow in infected macrophages. Objectives: To identify and characterize host innate immune determinants required for macrophage migration and aggregation into granulomas. Methods: Zebrafish are a natural host of Mycobacterium marinum (Mm), a close genetic relative of M. tuberculosis. By 5 days after infection of zebrafish embryos with wild-type (WT) Mm, infected and uninfected macrophages have aggregated into granulomas. We therefore analyzed gene expression in WT or ΔRD1 Mm infection at 5 days post-infection using the zebrafish Affymetrix array and confirmed findings with quantitative real-time PCR. We then localized gene expression over the course of infection using in situ hybridization and indirect immunofluorescence. The impact on infection of gene deletion was assayed with morpholino antisense technology. Results: 20 immune or inflammation-related genes are differentially regulated by Mm infection in whole animals by 5 days post-infection. We have focused on matrix metalloproteinase -9 (MMP-9) given its known role in tuberculosis (TB) in mammals. MMP-9 expression is 2.1 fold higher in WT versus ΔRD1 infection even when ending bacterial burden is normalized. MMP-9 protein localizes to uninfected and not infected macrophages at 1 day post-infection. Reduction of MMP-9 expression by morpholino treatment results in attenuated bacterial growth and deficient granuloma formation, reproducing the phenotype of the mycobacterial RD1 mutant. Conclusions: These data show that mycobacteria induce granuloma formation through RD1-dependent regulation of MMP-9. Selective MMP-9 expression on uninfected macrophages, and potentially early downregulation of MMP-9 in the infected macrophages themselves, may provide a mechanism for enlargement of an infection focus at an optimal tissue site. Characterizing the roles of MMPs in early infection will lead to new insights into innate immune responses to early mycobacterial infection and may offer new therapeutic modalities for TB.

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Anthony French, MD, PhD Assistant Professor Washington University School of Medicine Anthony French, M.D., Ph.D. is Assistant Professor of Pediatrics at Washington University School of Medicine. He received his M.D. and Ph.D. degrees from the University of Illinois at Urbana-Champaign where his graduate work centered on mechanisms of receptor-mediated endocytosis and intracellular sorting. Dr. French completed his residency in Pediatrics at the Mayo Clinic in Rochester, MN and his fellowship in Pediatric Rheumatology at Washington University School of Medicine in St. Louis. He joined the faculty in the Division of Rheumatology and Immunology in 2003 at Washington University upon completion of his fellowship, where his clinical interests include juvenile rheumatoid arthritis, juvenile dermatomyositis, vasculitis, and pediatric lupus. Dr. French’s postdoctoral and ongoing research is focused on the role of natural killer (NK) cells in the early innate immune response. Natural killer cells play a crucial role in anti-pathogen host defense and are particularly important in mediating resistance to viral infections. Abnormalities in either NK cell numbers and/or function have also been consistently identified in a number of autoimmune disorders such as rheumatoid arthritis. Current research is motivated by the hypothesis that a clearer understanding of in vivo NK cell responses and homeostasis may lead to novel therapeutic interventions in autoimmune diseases. For this work, Dr. French has been the recipient of several awards including an institutional Howard Hughes Medical Institute Faculty Development Award, March of Dimes Basil O’Connor Starter Scholar Award, and Clinical Investigator Development Award from the NIH.

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RAPID EMERGENCE OF ESCAPE MUTANTS FOLLOWING INFECTION WITH MURINE CYTOMEGALOVIRUS IN IMMUNODEFICIENT MICE. A. French, T. Geurs, J. Pingel, W. Yokoyama, Washington University School of Medicine, St. Louis MO. Background: Natural killer (NK) cells play a crucial role in the initial host defense against pathogens such as murine cytomegalovirus (MCMV). They respond rapidly and effectively control pathogen replication while the adaptive immune system is being activated. Objective: We hypothesized that in the absence of an adaptive immune system, an effective initial NK cell response would exert enough selective pressure to permit the specific outgrowth of MCMV escape mutants. Design/Methods: MCMV-resistant immunodeficient mice (C57BL/6) lacking T and B cells were infected with low doses of MCMV. Survival and viral titers were monitored. MCMV isolates collected late during in infection in immunodeficient mice were sequenced and characterized. Results: An effective NK cell response was not sufficient for long-term pathogen control as demonstrated by the late recrudescence of disease and mortality in immunodeficient mice infected with MCMV. MCMV isolates collected late during infection in these mice had nearly uniformly mutated a viral open reading frame (m157) recognized by the specific NK cell receptor responsible for resistance to MCMV. We demonstrated that these mutant viruses were no longer effectively controlled by NK cells. Conclusions: In the absence of an adaptive immune response, NK cells suppress the initial infection but exert enough selective pressure to drive the outgrowth of MCMV mutants that escape recognition by NK cells. These findings suggest that late recrudescence of viral infections in certain clinical settings may also be due to viral escape from NK cells or other components of innate immunity.

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Oscar G. Gomez-Duarte, MD, PhD Assistant Professor University of Iowa Children’s Hospital I obtained my MD at the National University of Colombia, School of Medicine in Colombia and PhD degree from the University of Maryland School of Medicine. I did an Infectious Disease Fellowship at Children's Hospital & Regional Medical Center at the University of Washington in Seattle and am currently a Tenure track Assistant Professor in the Department of Pediatrics, Division of Infectious Diseases at the University of Iowa Children’s Hospital of Iowa in Iowa City. My Research Interests are:

• Development of molecular epidemiology tools for rapid and affordable diagnosis of diarrheogenic E. coli strains from stool samples of children with diarrhea in developing countries.

• Characterization of fimbrial adherence factors of enterotoxigenic E. coli (ETEC).

• Identification of chromosomally-encoded factors involved in the pathogenesis of ETEC diarrheal disease.

• Development of attenuated-bacterial live vaccines and protein-based vaccines against ETEC traveler's and infant diarrhea.

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GENETIC AND EVOLUTIONARY ANALYSIS OF LONGUS PILUS OF ENTEROTOXIGENIC ESCHERICHIA COLI. O. G. Gomez-Duarte1, J. Bai1, S. Chattopadhyay2, S. J. Weissman2, and E. V. Sokurenko2, 1University of Iowa Children’s Hospital, Iowa City, IA. 2University of Washington, Seattle, WA Background. Enterotoxigenic Escherichia coli (ETEC) is an important cause of watery diarrhea in children living in the developing world, as well as in travelers to these areas. A significant number of human ETEC strains produce a type IV pilus named Longus. Objectives. The purpose of this work is to identify the genetic structure encoding Longus, the genetic variation of the structural subunit, and the associated genetic components that may contribute to the evolution and pathogenesis of ETEC. Materials and Methods. Longus genes were cloned and sequenced from the virulence plasmid of a prototype ETEC wild type strain. The structural and non-structural Longus gene sequences from a collection of longus-positive ETEC strains was analyzed by the ClustalX 1.83 and PAUP* 4.0b software systems. Results. We identified and characterized the genes involved in the biosynthesis of Longus. Sequence analysis identified a region of 14 kilobases containing 16 open reading frames, fourteen of them with 57 to 95% identity at the protein level to CFA/III, another type IV pilus of ETEC. Furthermore, sequence analysis of the Longus structural subunit gene, lngA, from different ETEC isolates demonstrated that lngA alleles segregated into three structurally distinct groups, while corresponding biogenesis genes were nearly identical. Within the lngA groups, strong positive selection for single amino acid replacements was identified, with the variability being driven by either immune escape or adaptive functional changes. DNA sequencing downstream the Longus cluster identified an insertion sequence and the heat stable enterotoxin gene. The close association of these two virulence-associated regions with Longus, are suggestive of gene co-regulation and co-evolution as described for the pilus and toxin genes of V. cholerae. Conclusions. Molecular evolutionary analysis indicates frequent horizontal transfer of lngA between Longus gene clusters of different E. coli. Based on the occurrence of conserved genes, Longus fimbriae are potentially expressed by 38% of ETEC, making it the most common ETEC-specific colonization factor identified to date. Our findings provide the basis for understanding the evolution, diversity and biogenesis machinery of Longus and other type IV pili in ETEC.

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Carey N. Lumeng MD, PhD Clinical Lecturer University of Michigan Dr. Carey Lumeng received his undergraduate education at Princeton University where he majored in Molecular Biology and was mentored by Dr. Thomas Silhavy. He continued his studies in the Medical Scientists Training Program (MSTP) at the University of Michigan Medical School and the Department of Human Genetics. In the laboratory of Dr. Jeffrey Chamberlain, his PhD dissertation centered around the pathogenesis of Duchenne muscular dystrophy. He identified a new family of serine kinases that associate with the dystrophin glycoprotein complex at post-synaptic structures in skeletal muscle and received a Dean’s Award for Research Excellence from the Michigan Medical School in 2000. After completing his MSTP training, he continued his clinical training in Pediatrics in the Boston Combined Residency Program at Boston University and Boston Children’s Hospital. During residency, he completed a research project with Dr. Alan Fine of Boston University on pulmonary stem cells that received an award from the Society of Pediatric Research in 2003. He next pursued subspecialty training in Pediatric Pulmonology at the University of Michigan and completed his fellowship in 2006. During his fellowship, he pursued research on the links between inflammation and obesity in the laboratory of Dr. Alan Saltiel. His work characterizing the biology of adipose tissue macrophages in obesity was featured on the cover of The Journal of Clinical Investigation in January 2007. He is currently a Clinical Lecturer in the Pediatric Pulmonary Division in the Department of Pediatrics at the University of Michigan and was supported on the University of Michigan CHRC grant from 2006-2007.

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OBESITY ALTERS THE ACTIVATION STATE OF ADIPOSE TISSUE MACROPHAGES. CN Lumeng, JL Bodzin, and AR Saltiel. University of Michigan, Ann Arbor, MI. Background: Obesity induces a state of chronic inflammation that has been hypothesized to mediate the pathogenesis of obesity-associated health conditions. Adipose tissue secretes inflammatory cytokines such as tumor necrosis factor α (TNFα) in obese states and adipose tissue macrophages (ATMs) have been identified as the primary source of these factors. Importantly, ATMs have been shown to be required for the development of insulin resistance in animal models of type 2 diabetes suggesting that ATMs link inflammation and metabolic changes in obesity. Objectives: To characterize the properties of ATMs in lean and obese mice and to assess the contribution of ATMs to the systemic inflammatory changes seen in obesity. Methods: Male C57Bl/6, C-C chemokine receptor 2 (CCR2) knockout, and HIV-LTR-Luciferase (HLL) reporter mice were fed a chow diet (5% kcal from fat) or a high-fat diet (HFD) (45% kcal from fat) for 20 weeks. ATMs were isolated from epididymal fat pads after collagenase digestion, centrifugation, and flow cytometry. Gene expression was analyzed by quantitative RT-PCR. Bioluminescence of chow and HFD fed HLL mice were analyzed with a CCD camera (IVIS) after luciferin injection. Results: ATMs from chow-fed mice expressed genes characteristic of alternatively activated or M2-polarized macrophages (Ym1, ArgI, Il10). In contrast, ATMs from HFD-fed mice downregulated these M2 genes and increased expression of genes seen in M1 classical macrophage activation (Nos2, Tnfa). Confocal microscopy of fat pads confirmed these findings and suggested that the transition between M2 to M1 activation states occurs around dying adipocytes. ATMs from obese CCR2 knockout mice retained an M2 gene expression profile consistent with their attenuated adipose tissue inflammation. IL-10 blocked insulin-resistance in adipocytes caused by TNFα in vitro, suggesting that M2 macrophages preserve normal adipocyte function in the face of inflammatory challenges. While ATMs were M1 activated with obesity, similar changes were not observed in pulmonary or peritoneal macrophages. Analysis of live NFκB-luciferase transgenic reporter mice demonstrated a 5.8-fold activation of NFκB specifically in adipose tissue with obesity. Conclusions: Obesity alters the inflammatory capacity of ATMs in a way that can directly influences adipocyte metabolic capacity. Future studies will examine the mechanism of IL-10 action on adipocytes and if modulation of ATM activation state can attenuate inflammation and improve insulin sensitivity in vivo.

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Registrants Name and Email Addresses

Juan Pablo Abonia pablo.abonia@cchmc.org Suneet Agarwal suneet.agarwal@childrens.harvard.edu Page Anderson ander005@mc.duke.edu Michael Artman michael-artman@uiowa.edu Shahab Asgharzadeh shahab@chla.usc.edu James Bale james.bale@hsc.utah.edu John Barnard barnardj@ccri.net Anne Blaschke anne.blaschke@hsc.utah.edu Joshua Bonkowsky jbonkowsky@gmail.com Laurance Boxer laboxer@umich.edu William Britt rdawson@peds.uab.edu Garrett Brodeur brodeur@email.chop.edu Valerie Castle vcastle@umich.edu Edward Clark ed.clark@hsc.utah.edu Alan Cohen cohen@email.chop.edu Sherin Devaskar Sdevaskar@mednet.ucla.edu Sara DiVall sdivall1@jhmi.edu Patricia Dubin dubipj@chp.edu Gary Fleisher Gary.Fleisher@childrens.harvard.edu Anthony French french_a@kids.wustl.edu Rasheed Gbadegesin rasheed.gbadegesin@duke.edu Bruce Gelb bruce.gelb@mssm.edu Charles Gomer cgomer@chla.usc.edu Oscar Gomez oscar-gomez@uiowa.edu Julie Goodwin julie.goodwin@yale.edu Thomas Green tgreen@northwestern.edu Ruchi Gupta r-gupta@northwestern.edu Morey Haymond mhaymond@bcm.edu Paula Hertel phertel@bcm.tmc.edu Friedhelm Hildebrandt fhilde@umich.edu Margaret Hostetter anna.iuliano@yale.edu Rachel Katzenellenbogen rkatzen@u.washington.edu Paul Kingma paul.kingma@cchmc.org Carrie Kitko ckitko@umich.edu Jay Kolls Joy.capo@chp.edu Jordan Kreidberg Jordan.Kreidberg@childrens.harvard.edu Stephan Ladisch sladisch@cnmc.org Mark Levin levinm@email.chop.edu Corinne Linardic linar001@mc.duke.edu John LiPuma jlipuma@umich.edu Carey N. Lumeng clumeng@umich.edu Veronica Mas Casullo veronica.mascasullo@mssm.edu Sean McGhee smcghee@mednet.ucla.edu

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Valerie McLin mclin@bcm.edu Steven Mittelman smittelman@chla.usc.edu Louis Muglia muglia_l@kids.wustl.edu Kenan Onel konel@uchicago.edu David Perlmutter david.perlmutter@chp.edu Richard Polin rap32@columbia.edu Tamara Pozos tamz@u.washington.edu Sridhar Rao sridhar.rao@childrens.harvard.edu Michelle Rheault michelle.rheault@mssm.edu Lawrence Rhein Lawrence.Rhein@childrens.harvard.edu Scott Rivkees scott.rivkees@yale.edu Amy Roberts aeroberts@partners.org Robert Roghair robert-roghair@uiowa.edu Mary Beth Ross rossm@ccri.net Shannon Ross sross@peds.uab.edu Marc Rothenberg marc.rothenberg@cchmc.org Matthew Ryan ryanma@email.chop.edu Alan Schwartz schwartz@kids.wustl.edu Brian Sims bsims@peds.uab.edu Christopher Spurney cspurney@cnmc.org Joseph St. Geme j.stgeme@duke.edu Fielding Stapleton bruder.stapleton@seattlechildrens.org Maria Luisa Sulis mls95@columbia.edu Kimara Targoff kl284@columbia.edu Trent Tipple tipplet@pediatrics.ohio-state.edu Roberta Williams rwilliams@chla.usc.edu Karen Winer winerk@mail.nih.gov Rachel Wolfson rwolfson@peds.bsd.uchicago.edu David Ziring dziring@mednet.ucla.edu