The 2nd Annual - DOC by o262mlx9


									              The 2nd Annual
Johns Hopkins Bayview Research Symposium

                  Sponsored by
       The Bayview Scientific Advisory Board

             Friday, December 7, 20007
                  9:30AM – 3:30PM
     The Johns Hopkins Asthma & Allergy Center

                       TABLE OF CONTENTS
Tsadik T. Abraham, Ross H. Lowe, Stephane O. Pirnay, William D. Darwin, and Marilyn A. Huestis.
Simultaneous GC/EI-MS Determination of ∆9-Tetrahydrocannabinol, 11-Hydroxy-∆9-tetrahydrocannabinol,
and 11-Nor-9-carboxy-∆9-tetrahydrocannabinol in Human Urine Following Tandem Enzyme – Alkaline
Hydrolysis. (NIH/NIDA)

Balanle Asiyanbola, MD. Pattern of recurrence following pancreaticoduodenectomy for pancreatic
adenocarcinoma is associated with number of positive nodes and lymph node ratioHepatic . (SURGERY)

Lori L. Beason-Held, Michael A. Kraut, Luigi Ferrucci, Wendy Elkins, Alan B. Zonderman, and Susan M.
Resnick. Long-term Statin Users Show Changes in Brain Function Over Time. (NIH/NIDA and JOHNS

Wendy L. Bennett, Pamela Ouyang, Albert W. Wu, Kerry J. Stewart. Fatness and fitness: how do they
affect health-related quality of life in people with diabetes? (MEDICINE - GENERAL INTERNAL MEDICINE)

Edward S. Bessman, M.D. Geriatric Altered Mental Status in the Emergency Department: Assessing and
Mitigating Providers’ Knowledge Deficit. (EMERGENCY MEDICINE)

Satyanarayan Bhat, Paul Savage, and Stephen Milner. Antimicrobial peptides in burns and sepsis.

Satyanarayan Bhat and Stephen Milner. Ceragenin CSA-13 prevents endotoxin shock in neonatal rats and
mice. (SURGERY)

Clifton O Bingham III, Jon T Giles, Patricia E Jones, Joan M Bathon, Susan A Bartlett. High Prevalence
of Moderate to Severe Periodontal Disease in Rheumatoid Arthritis Patients. (MEDICINE –

Julius Birnbaum, Douglas Kerr, Michelle Petri. Transverse Versus Longitudinal Myelitis in SLE.

J. M. Bossert, G. C. Poles, K. A. Wihbey, E. Koya, Y. Shaham. Role of D1-family dopamine receptors in the
medial and lateral accumbens shell in context-induced relapse to heroin seeking. (NIH/NIDA)

C. M. Boyd, J. Wolff, C. Cigolle, C. Blaum. Denominators for Pay for Performance Standards Among

Leff B, Burton J, Burton L, Guido S, Steinwachs D, Mader S, Naughton B, Burl J, Clark B, Greenough
WB. Stress Experienced by Family Members of Patients Treated in Hospital at Home Compared with
Traditional Acute Hospital Care. (MEDICINE – GERIATRICS)

Junran Cao, Satoshi Ikemoto. Rewarding Effects of Neuropeptides Injected into the Brain. (NIH/NIDA)

E.S. Chen, M.D., Y. Zhang, M.D., Ph.D., and D.R. Moller, M.D. Pulmonary Granulomatous Inflammation in
Response to Mycobacterial Catalase-Peroxidase Protein: An Experimental Model of Sarcoidosis.
(MEDICINE – Pulmonary)

Aiwu Cheng, Ph.D., Naomi Kamimura, Ph.D., Mark P. Mattson, Ph.D. Function of mitochondrial
biogenesis and fission/fusion machineries in neural development and neurodegeneration. (NIH/NIDA)
M.S. Chisolm, M. Tuten, E.C. Strain, and H.E. Jones Screening for co-occurring mood disorder among
pregnant substance-dependent patients: Characteristics of the Addiction Severity Index and the Beck
Depression Inventory. (PSYCHIATRY)

Grant V. Chow, David D. Spragg, Glenn A. Hirsch, Alan Cheng, Joseph E. Marine. Prognostic
significance of cardiac troponin I levels in patients presenting with supraventricular tachycardia.

TE Taylor-Clark and BJ. Undem. 4-hydroxynonenal: a potential endogenous activator of Transient
Receptor Potential A1 (TRPA1) channels on sensory neurons. (MEDICINE – ALLERGY AND CLINICAL

Anthony DeFulio, Mick Needham, Wendy D. Donlin, Conrad J. Wong, and Kenneth Silverman.
Employment-Based Abstinence Reinforcement as a Maintenance Intervention for the Treatment of
Persistent Cocaine Use in Methadone Patients. (PSYCHIATRY)

Alice Dobi, Elyssa B Margolis, and Marisela Morales. Glutamatergic neurons of the ventral tegmental
area establish synaptic contacts on local dopaminergic and non-dopaminergic neurons. (NIH/NIDA)

Kenneth W. Fisbein, Yehezkiel A. Gluzband, Masura Kaku, Hasina Ambia-Sobhan, Sue A. Shapses,
Mitsuo Yamauchi, and Richard G. Spencer. Effects of Formalin Fixation and Collagen Cross-Linking in T2
and Magnetization Transfer in Bovine Nasal Cartilage. (NIH/NIA)

Franckowiak SC, Ling G, Greenough WB III, Wagner DA, Andersen RE. Prediction of Hydration Status
using a Handheld, Portable Impedance Analyzer. (MEDICINE – GERIATRICS)

Criag J. Galban and Richard G. Spencer. Measurement of Spin-Lattice Relaxation Times and Chemical
Exchange Rate in Multiple-Site Systems Using Progressive Saturation. (NIH/NIA)

L. Gao, N. M. Rafaels, N. Hansel, D. N. Grigoryev, C. Cheadle, D. D. Sin, R. Wise, L. Hummers, F. Wigley,
R. A. Mathias, T. H. Beaty, P. Hassoun, K. C. Barnes. Common Variants in Vascular Endothelial Growth
Factor (VEGF) are Associated with Distinct Pulmonary Phenotypes, Scleroderma-related Pulmonary
Arterial Hypertension (SSc-PAH) and Chronic Obstructive Pulmonary Disease (COPD). (MEDICINE –

Udi E. Ghitza, Sunila G. Nair, Sam A. Golden, Sarah M. Gray, Jamie L. Uejima, Jennifer M. Bossert and
Yavin Shaham. Peptide YY3-36 decreases reinstatement of high-fat food seeking during dieting in a rat
relapse model. (NIH/NIDA)

B. Robert Gibson, Panagis Galiatsatos, Lisa Eaton, Rania Abu-Hamdah, Stephen M. Milner, Dariush
Elahi, Dana K. Andersen. Intensive Insulin Therapy is associated with an Equivalent Survival Benefit in the
Burn Intensive Care Unit (BICU) as in the Surgical Intensive Care Unit (SICU). (Surgery)

M. Giunta, S. Camandola, S.G.Cella, M. P. Mattson. Decreased Expression of CD36 Precedes Amyloid
Accumulation in the 3xTgAD Mouse Model of Alzheimer’s Disease. (NIH/NIA)

M. Gleichmann, L. Collis, P. Smith, M.P. Mattson. Single Neuron Oxygen Consumption Responses to
Glutamate. (NIH/NIA)

Amit Golding, Felipe Andrade. The Environment Induced by Active Lupus Plasma Suppresses the
Expansion of FoxP3 High-Expressing Regulatory T Cells. (MEDICINE – RHEUMATOLOGY)
Myriam Gorospe, PhD. A predominantly nuclear RNA-binding protein …(NIH/NIA)

Rebecca F. Gottesman, MD PhD, Jonathan T. Kleinman, BS, Cameron Davis, BA, Jennifer Heidler-Gary,
MA, Melissa Newhart, BS & Argye E. Hillis, MD. Estimating Diffusion-Perfusion Mismatch Using the
Teresa R. Gray, Diaa M. Shakleya, Marilyn A. Huestis. A novel method for detection and quantification
of nicotine and metabolites in human meconium by LC-MS/MS using atmospheric pressure chemical

Guerrero-Cazares, Hugo; Achanta, Pragathi; Frazier, James; Attenello, Frank; Zamora-Berridi, Grettel;
Chaichana, Kaisorn; Choi, Alyssa; Niranjan, Ashwini; Quiñones-Hinojosa, Alfredo. In Vitro
Characterization of Human Glioblastoma Multiforme and Subventricular Zone Derived Astrocytes in
Response to EGF, PDGF and Cerebrospinal Fluid. (NEUROSURGERY)

S. Gulyani; B.C. Jones; R.P. Allen; M. P. Mattson; C.J. Earley. Adenosine A2A Receptors are Up-Regulated
in Inbred Mice with Low Levels of Iron in the Striatum. (NEUROLOGY)

Zhihong Guo, Haiyang Jiang, Xiangru Xu, Wenzhen Duan, and Mark P. Mattson. Leptin-Mediated Cell
Survival Signaling in Hippocampal Neurons Mediated by JAK /STAT3 and Mitochondrial Stabilization.

Katrina Hein and Marisela Morales. The alpha7 subunit of the nicotinic receptor is expressed in a subset
of hippocampal interneurons containing cholecystokinin. (NIH/NIDA)

GG Hesketh, M Shah, F Akar, Y Guo, GF Tomaselli, JE Van Eyk. Phosphorylation Dependent Targeting of
Connexin 43 to Lipid Rafts is Increased in Heart Failure. (MEDICINE – Cardiology)

Suzanne Jan De Beur. FGF-23 Controls NaPiIIa Trafficking Via Crosstalk Between the PI-3 kinase and

Sojkova Jitka, Kraut Michael A, Beason-Held Lori, Wu Xiaoying, Davatzikos Christos, Resnick Susan M.
Effect of Regional Volume Changes on PET Activation Patterns in the Aging Brain. (NIH/NIA)

Josiah Johnston, Wendy Iser, Catherine Wolkow and Ilya Goldberg. Quantitative image pattern analysis
of tissues in C. elegans identifies distinct morphological states during aging. (NIH/NIA)

Kane, LA1, Youngman, MJ2, Jensen, RE2, and Van Eyk, JE1,3. Analysis of the structure and function ATP
Synthase complex from site specific yeast mutants of the β Subunit mimicking known phosphorylations.

Jean Kim, Lowella F Heinecke, David Proud, Robert P Schleimer. In vitro and in vivo induction of B7-H1
and B7-DC expression by human rhinovirus infection of airway epithelial cells. (OTOLARYNGOLOGY –

Amy M. Knight, MD, Steven J. Kravet, MD, G. Michael Harper, MD, Dmitry Kiyatkin, MD, Bruce Leff,
MD. The Good News about CPOE and Medical Student Ordering Ability. (MEDICINE- GENERAL INTERNAL

Amy M. Knight, MD, Joseph A. Carrese, MD, Scott M. Wright, MD. Qualitative Assessment of the Long-
term Impact of a Faculty Development Program in Teaching Skills. (MEDICINE – GENERAL INTERNAL
Eisuke Koya, Jamie L. Uejima, Jennifer M. Bossert, Yavin Shaham, Bruce Hope. Medial prefrontal
cortex activity and its role in incubation of cocaine craving. (NIH/NIDA)

Edward Kraus, Diane Lepley, Dorry Segev, Mary Leffell, Donna Lucas, Mark Haas, Lorraine Racusen,
Serena Bagnascu, Vanessa Collins, Chris Simpkens, Dan Warren, Robert Montgomery, Andrea
Zachary. Donor Specific Antibody (DSA) and C4d + in Surveillance Biopsies Following Positive Cross Match
Live Donor Kidney Transplantation. (MEDICINE – CARDIOLOGY)

Leng SX, Finucane T, Boult L, Zheng L, Greenough WB. Inadvertent self-healing in desperate times.
Lancet. 2007;370:1458. (MEDICINE – GERIATRICS)

Lixin Liu PhD Guy Marti MD Xianjie Zhang MD PhD Stephen Milner MD Gregg Semenza MD PhD,
John W Harmon MD. Non-Viral Plasmid Transfection with Hypoxia Inducible Factor- 1 Alpha Improves
Diabetic Wound Healing. (SURGERY)

Mahmoud Malas, MD; Rafael Llinas, MD; James Black, MD; Umair Qazi , MD; Bruce Perler, MD. CREST,
Carotid Revascularization Endarterectomy vs. Stenting Trial, Unexpected Findings.

M McNabney, J Wolff, L Semanick, J Kasper, C Boult. Long-stay nursing home residents who may not
require institutional care. (MEDICINE – GERIATRICS)

Stephen Milner MD. Antimicrobial Peptides and Burn Wounds. (SURGERY)

Eric T. Moolchan, MD, Craig S. Parzynski, BA, Debra L. Zimmerman, RN, BSN, Maria Jaszyna-Gasior,

Sunila G. Nair, Sam A. Golden and Yavin Shaham. Differential effects of the orexin 1 receptor antagonist
SB 334867 on high-fat food self-administration and reinstatement of food seeking in rats. (NIH/NIDA)

Pamela Ouyang, Dhananjay Vaidya, Sherita H. Golden, Moyses Szklo, Adrian Dobs, Kiang Liu, Pamela
J. Schreiner, Susan M. Gapstur. The association of endogenous sex hormones and the metabolic
syndrome in men and women: The Multi-Ethnic Study of Atherosclerosis (MESA).

Park E, Christmas C, Schmaltz HN, Durso SC. The perceived change of diverse clinical-educators through
an intensive course on teaching geriatrics. (MEDICINE – GERIATRICS)

Park E, Durso SC, Confessore GJ. A case study of educational practices for the aging global population.

Park E. Learner autonomy and global competitiveness. (MEDICINE – GERIATRICS)

Park E. Learner autonomy and learner orientation. (MEDICINE – GERIATRICS)

Jessica M. Peirce, Christopher K. Burke, Kenneth B. Stoller, Karin J. Neufeld, Van L. King, Robert K.
Brooner. Longitudinal Pattern of Traumatic Event Exposure and PTSD in Opioid Dependence Treatment.

Vani Rao, MD; Jennifer Spiro, MS; Cynthia Munro PhD; Dzung Pham, PhD; Mahaveer
Degoankar Alena Horska, PhD; David M. Yousem, MD, and Peter B. Barker, D.Phil. Neuroanatomical
Correlates of Post-TBI Depression. (PSYCHIATRY)
Heather L. Rogers, Roy C. Ziegelstein, James A. Fauerbach, Gina M. Magyar-Russell, and Marlene S.
Williams Depressive Symptoms and Platelet Aggregation after Acute Coronary Syndrome. (PSYCHIATRY)

John T. Schroeder, Kristin L. Chichester, Anja P. Bieneman Toll-like Receptor-9 Suppression in
Plasmacytoid Dendritic Cells Following IgE-dependent Activation is Mediated by Autocrine TNF-a.
(MEDICINE - Allergy and Clinical Immunology)

Philip Seo, Peter A. Merkel, Ulrich Specks, Gary S. Hoffman, Carol A. Langford, Robert Spiera, Cees G.
Kallenberg, Anthony M. Turkiewicz, E. William St. Clair, John H. Stone. Evidence for the ANCA-
Associated Vasculitis Index of Damage (AVID). (MEDICINE – RHEUMATOLOGY)

S. H. Shippey, R. E. Gutman, L. H. Quiroz, V. L. Handa. Contemporary Approaches to Cystocele Repair: A
Survey of AUGS Members. (OB/GYN)

Shultes-von-Schlageter M, Park E, Tucker PM. Inter-Site Consistency at a Multi-Site. Psychiatry

Frederick E. Sieber, M.D., Khwaja Zakriya, M.D., Simon Mears, M.D., Ph.D., Alan Gottshalk, M.D.,
Ph.D., Constantine Lyketsos, M.D., Ph.D. The Role of Anesthetic Induced Depth of Hypnosis (AIDH) in

D. Siluk, R.V. Oliveira, M.E. Rodriguez-Rosas, S. Ling, A. Bos, L. Ferrucci, I.W. Wainer. Application of an
HPLC Method to Determine Vitamin A and Vitamin E Isomers Concentrations in Human Plasma. (NIH/NIA)

Danuta Siluk, Donald Mager, Naomi Gronich, Darrell Abernethy, Irving W.Wainer.
Development and Validation of an Enantioselective LC-MS Method for the Simultaneous Determination of
R,S-Propranolol and R,S-Hyoscyamine in Human Plasma. (NIH/NIA)

T. G. Son, S. Camandola, R. Telljohann, D. Hyun, D. Jo, Q. Yu, N. H. Greig and M. P. Mattson.
In Search of Neuroprotective Hormetic Phytochemicals. (NIH/NIA)

David A. Spector, Qing Yang, Leonid Klopouh, Edward J. Weinman, Deborah A. Steplock, Rajatsubhra
Biswas, Jie Liu, James B. Wade. The ROMK (Kir1.1) Potassium Channel Is Present in Mammalian Urinary
Tract Epithelia and Muscle. (MEDICINE – RENAL)

David A. Spector, Qing Yang, and James B. Wade. High urea and creatinine concentrations and urea
transporter B in mammalian urinary tract tissues. (MEDICINE – RENAL)

KE Steele, T Magnuson, MA Schweitzer, A Kumar, H Kuwabawa, J Galecki, J Brasic, M Alexander, W
Ye, DF Wong. Alterations in Central Dopamine Receptors Before and After Gastric Bypass Surgery.

Kerry J. Stewart, EdD; Brian H. George, MS; Kristina Potrekus, MS; Anita C. Bacher; MSN, Harry A.
Silber, MD, PhD, Nae-Yuh Wang; PhD, Edward P. Shapiro, MD, Pamela Ouyang, MD.
Increased Fatness and Reduced Fitness are Associated with Aortic Stiffness in Persons with Type 2
Diabetes and Hypertension. (Medicine - Clinical and Research Exercise Physiology)

Sun-Young Oh, Ph.D., Tao Zheng, M.D., John Schroeder, Ph.D., Becky Vonakis, Ph.D., Oksoon Choi,
Ph.D., Zhou Zhu, M.D., Ph.D. Role of Mast Cells in the Development of Pulmonary Allergic Inflammation
of SHP-1 Deficient Mice. (MEDICINE - Allergy and Clinical Immunology)
Patricia Tagliaferro, Hui-Ling Wang and Marisela Morales. Corticotropin releasing factor receptor 1
(CRF-R1) in the ventral tegmental area is preferentially encoded and translated by dopaminergic neurons.

Dhananjay Vaidya, Moyses Szklo, Mary Cushman, Paul Holvoet, Hossein Bahrami, Nancy S. Jenny,
Pamela Ouyang. The Metabolic Syndrome is associated with Circulating Markers of Endothelial
Dysfunction in the Multi-Ethnic Study of Atherosclerosis (MESA). (MEDICINE – CARDIOLOGY)

Ryan Vandrey, George Bigelow, and Maxine Stitzer. Brief cocaine abstinence induced by voucher and
cash-based incentives. (PSYCHIATRY)

R. Wan, M. Brown, A. Cheng, T. Brown, D. Mager†, and M. Mattson. Brain Derived Neurotrophic Factor
Regulates Energy Metabolism and Autonomic Activity. (NIH/NIA)

Dongqing Wang, Xianmin, Xia, Alexis Oetting, Paul Yen. Histone Acetylation and the Role of
Corepressor in Negative-Regulation of TSHα-Subunit Gene by thyroid hormone. (MEDICINE –

Hui-Ling Wang, Patricia Tagliaferro and Marisela Morales. The laterodorsal tegmental nucleus have
three distinct sub-populations of neurons: cholinergic, glutamatergic and GABAergic. (NIH/NIDA)

Michael R. Weed, Ph.D. , Terry Clayton, and James M. Cook, Ph.D. RY-023, a selective inverse agonist at
the benzodiazepine binding site on GABA-A ÿ5 receptor, improves performance in a delayed-match-to-
sample task in rhesus monkeys. (PSYCHIATRY)

Xianmin Xia, Guihua Wang, Aiwu Cheng, Xuelai Luo, Jianping Gong, Junbo Hu, Paul M. Yen. A peptide
inhibitor targeting the regulatory subunit of PI 3-kinase, p55PIK, is effective in human thyroid cancer

Xiangru Xu, Ming Zhan, Wenzhen Duan, Vinayakumar Prabhu , Randall Brenneman, William Wood,
Jeff Firman, Huai Li, Peisu Zhang, Carol Ibe, Alan B. Zonderman, Dan L. Longo, Suresh Poosala, Kevin
G. Becker, and Mark P. Mattson. Gene expression atlas of the mouse central nervous system: impact and
interactions of age, energy intake and gender. (NIH/NIA)
Simultaneous GC/EI-MS Determination of ∆9-Tetrahydrocannabinol, 11-Hydroxy-∆9-
tetrahydrocannabinol, and 11-Nor-9-carboxy-∆9-tetrahydrocannabinol in Human Urine
Following Tandem Enzyme – Alkaline Hydrolysis

Tsadik T. Abraham*, Ross H. Lowe, Stephane O. Pirnay, William D. Darwin, and Marilyn A. Huestis

Chemistry and Drug Metabolism, IRP, National Institute on Drug Abuse, National Institutes of
Health, Baltimore, MD, 21224, USA

A sensitive and specific GC/EI-MS method was modified and validated for the simultaneous
extraction and quantification of ∆9-tetrahydrocannabinol (THC), 11-hydroxy-∆9-
tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-∆9-tetrahydrocannabinol
(THCCOOH) in human urine. THC and its hydroxylated and carboxylated metabolites are
conjugated prior to urinary excretion. Gas chromatography/mass spectrometry (GC/MS)
methods require hydrolysis of urine conjugates prior to extraction and analysis to capture
total analyte concentration.

To ensure complete hydrolysis of conjugates and capture of total analyte content, urine
samples were hydrolyzed by two methods in series. Initial hydrolysis was with Escherichia coli
-glucuronidase (Type IX–A). Two mL urine fortified with THC-d3, 11-OH-THC-d3 and
THCCOOH-d3 was hydrolyzed with 0.5 mL of 20,000 units/mL of E. coli -glucuronidase (5000
u/mL) for 16 h at 37oC in a shaking water bath followed by a second hydrolysis utilizing 80μL
of 10N NaOH at 60oC for 20 min. Specimens were adjusted to pH 5-6.5 with 50 µL
concentrated glacial acetic acid. Two mL of acetonitrile were added to precipitate protein
followed by 2 mL 2N sodium acetate buffer (pH 4.0). Specimens were centrifuged and
supernatants applied to conditioned solid phase extraction (SPE) columns. SPE columns
were washed with 3 mL deionized water, 2 mL 0.1N hydrochloric acid /acetonitrile (70:30 v/v),
and dried by full vacuum for 10 min. After priming the sorbent bed with 0.2 mL hexane,
analytes were eluted with 5 mL elution solvent (hexane:ethyl acetate 80:20 v/v) into tubes
containing 0.5 mL ethanol. Extracts were reconstituted with 25 µL acetonitrile, transferred to
autosampler vials, and 20 µL BSTFA was added. Vials were capped and derivatized at 85°C for
30 min. Extracted analytes were simultaneously quantified by gas chromatography–mass
spectrometry with electron impact ionization (GC/EI-MS).

THC and 11-OH-THC were recovered by E.coli -glucuronidase hydrolysis but not by alkaline
hydrolysis or when no hydrolysis was performed. Alkaline hydrolysis with 10 N NaOH
provided more effective recovery of THCCOOH than enzyme, and the tandem enzyme-
alkaline hydrolysis procedure demonstrated the most complete recovery of analytes.
Combining enzyme and alkaline hydrolysis in sequence enabled efficient recovery of THC, 11-
OH-THC, and THCCOOH conjugates. The three analytes were quantified simultaneously with
dynamic ranges of 2.5 to 300 ng/mL. Extraction efficiencies were 57.0 - 59.3% for THC, 68.3 -
75.5% for 11-OH-THC, and 71.5 - 79.7% for THCCOOH. Intra and inter-assay precision across the
linear range of the assay ranged from 0.1 - 4.3% and 2.6 - 7.4%, respectively. Accuracy was
within 15% of target concentrations. This method was applied to the analysis of urine
specimens collected from individuals participating in controlled and withdrawal cannabis
administration studies, and may be a useful analytical procedure for determining recency of
cannabis use in forensic toxicology applications.
Keywords: Tetrahydrocannabinol, GC/MS, Urine and Hydrolysis
Long-term Statin Users Show Changes in Brain Function Over Time

Lori L. Beason-Held1, Michael A. Kraut2, Luigi Ferrucci1,
Wendy Elkins1, Alan B. Zonderman1, and Susan M. Resnick1

    Intramural Research Program, National Institute on Aging, NIH
    Dept. of Radiology, Johns Hopkins University School of Medicine

Statins were developed to treat hyperlipidemia by lowering low density lipoproteins (LDL)
levels. Pleiotropic effects such as stabilization of atherosclerotic plaque formation,
enhancement of endothelial function, decreased blood clot formation, and anti-inflammatory
and antioxidant actions have also led to their use in treatment of cardiovascular disease,
diabetes, organ transplants and in individuals at risk for stroke and myocardial infarction in
the absence of hyperlipidemia. The effect of statin use on brain function, however, remains
unclear. To address this issue, we examined PET regional cerebral blood flow (rCBF) data
from older participants in the Baltimore Longitudinal Study of Aging (BLSA). Differences in
resting-state rCBF were investigated in statin users (n=16; mean age Year 1 = 66.0) across 1-5
years of drug treatment relative to non-users (n=16; mean age Year 1 = 66.1). There were no
significant differences between groups at baseline (pre-medication). However, statin users
showed greater rCBF decreases over time in putamen, premotor, primary auditory and
somatosensory regions and greater relative increases over time in the cerebellum, visual
cortex and thalamus compared to non-users. rCBF changes in the putamen occurred in early
years, changes in premotor, somatosensory, visual and thalamic regions occurred after
longer treatment in later years, and changes in auditory and cerebellar regions occurred
throughout the 5 years of statin use. Together, these results show that statin use affects
brain function in older individuals. The results further suggest that the regional pattern of
change is related to duration of treatment.
Fatness and fitness: how do they affect health-related quality of life in people with

Wendy L. Bennett1, Pamela Ouyang2, Albert W. Wu1, Kerry J. Stewart2
Divisions of 1General Internal Medicine and 2Cardiology, Department of Medicine,
Johns Hopkins School of Medicine, Baltimore, MD

Disease complications reduce health-related quality of life (HRQOL) in people with type 2
diabetes mellitus. Less is known about the association of modifiable risk factors such as
obesity and fitness with HRQOL.

We hypothesized that decreased total body fatness and increased levels of fitness would be
associated with higher HRQOL and attenuate the adverse effect of diabetes.

Cross-sectional study using baseline data from subjects recruited for two exercise training

One study enrolled subjects with and the other without diabetes. All subjects had mild
hypertension, a sedentary lifestyle and were without other medical conditions that
precluded exercise.

Aerobic fitness was assessed as maximal oxygen uptake (VO2max) during treadmill testing,
total body fatness by dual-energy x-ray absorptiometry (percent body fat) and HRQOL by the
Medical Outcomes Study SF-36. We used multivariate linear regression analyses to test the
association of HRQOL outcomes with diabetes, fitness and fatness.

There were 121 non-diabetics and 71 diabetics. Diabetics were younger (p<0.01), more likely
to be men (p<0.01), non-white (p<0.01) and meet criteria for metabolic syndrome (p=0.03).
Diabetics had significantly lower mean scores for general health perceptions (GHP) and
physical component score (PCS). In a multivariate regression analysis, diabetes (p<0.01) and
lower VO2max (p=0.02) but not percent body fat, were associated with reduced GHP. The
effect size was greatest for diabetes (effect size= - 3) even after accounting for fitness,
fatness and other clinical variables. In a second multivariate analysis higher percent body fat
(p=0.02) and having diabetes (p=0.04), but not VO2max, were associated with reduced PCS.
The largest effect size was for percent body fat (effect size= - 2.4).

Lower levels of fatness and improved fitness reduced the negative association of diabetes
with HRQOL. These data suggest that patients with diabetes that make lifestyle changes
focused on physical activity and weight reduction may be able to improve their HRQOL.
Geriatric Altered Mental Status in the Emergency Department: Assessing and Mitigating
Providers’ Knowledge Deficit

Edward S. Bessman, M.D.
Department of Emergency Medicine
Johns Hopkins Bayview Medical Center

Introduction: Emergency Medicine is one of the specialties partnering with the American
Geriatrics Society in the “Geriatrics for Specialists” initiative designed to foster increased
education in geriatrics for non-geriatricians.

Objective: This study was undertaken to 1) evaluate emergency department (ED) providers’
fund of knowledge of basic principles for the assessment of geriatric patients who present
with altered mental status and 2) compare a standard case-based lecture format to a novel
templated assessment instrument as a means to mitigate any knowledge deficits.

Methods: A tool was developed to measure learners’ knowledge of certain fundamental
aspects of assessment of geriatric patients with altered mental status (figure 1). This tool was
administered in two settings: 1) immediately before and after a case-based lecture and 2) one
week before and three weeks after the introduction of a templated chart for geriatric
assessment in the ED (figures 2A and2B). Posters (figure 3) announcing the introduction of
the new chart were placed in two locations in the ED but no further educational efforts were
made. Study design eliminated crossover between the two settings.

Settings: The ED at Johns Hopkins Bayview Medical Center and one of the weekly
educational conferences of the Johns Hopkins Emergency Medicine residency program.

Results: Out of a possible score of 9 points, the average pre-intervention score on the
assessment tool for the ED group was 3.79 (SD 1.07) and for the lecture group was 4.00 (SD
1.70), p=0.83. After the intervention, the respective scores had increased significantly in both
settings: 6.69 (SD 2.21) for the ED (p<0.01) and 8.12 (SD 1.54) for the lecture setting (p<0.01).
The greater improvement in the score for the lecture group compared to the ED group was
small but significant (p<0.05).

Conclusion: ED providers’ knowledge of basic principles for the assessment of geriatric
patients who present with altered mental status is deficient. As expected, this deficit can be
mitigated by a structured lecture format. Interestingly, the introduction of a templated
charting tool without additional educational efforts works almost as well as a means for
improving knowledge. Further study should be undertaken to investigate which technique
results in the more enduring improvement.
Antimicrobial peptides in burns and sepsis
Satyanarayan Bhat1, Paul Savage2, and Stephen Milner1

Johns Hopkins Burn Center/ Michael D. Hendrix Burn Research Center, Johns Hopkins
University, Baltimore, MD; 2Department of Chemistry and Biochemistry, Brigham Young
University, C100 BNSN, Provo, UT 84602, USA

Burns are associated with immunosuppression and sepsis for which no effective treatment is
currently available and accounts for over 54% of deaths in the United States. Defensins and
cathelicidins are families of cationic antimicrobial peptides (AMP) which play a significant role
in innate and adaptive immunity and are widely expressed in burn affected organs such as
skin, lung, kidney and intestine as well as in leukocytes. These natural antimicrobial agents
directly kill bacteria, fungi and enveloped viruses and chemoattract immature dendritic cells,
neutrophils, macrophages and certain T and B-lymphocytes. In a series of experiments we
have demonstrated that following a thermal injury there is a deficicency in the protective
barrier. Levels of Human β-defensin-1-3 (HBD1-3) and the cathelicidin LL-37 were significantly
decreased in burned skin epithelia. In addition, HBD-2 was absent in burn blister fluid and
poorly expressed in lung fluid after inhalation injury. Surviving skin adnexae, however, in the
deeper dermis and subcutaneous tissues retained expression of AMPs. In addition,
heatshock treatment to keratinocytes also decreases HBD-2 expression and increases cell
size. LL-37 and also its synthetic anlogue CSA-13 binds to LPS and prevents endotoxin shock.
Therefore, the combination of antibacterial action and LPS neutralization may render CSA-13
a novel drug for prevention and treatment of septic shock.
Title: High Prevalence of Moderate to Severe Periodontal Disease in Rheumatoid Arthritis

Clifton O. Bingham III, Jon T. Giles, Patricia E. Jones, Joan M. Bathon, Susan J. Bartlett. Johns
Hopkins University, Baltimore, MD

PURPOSE: Periodontal disease (PD) and rheumatoid arthritis (RA) are inflammatory diseases
with similar cytokines, proteases, inflammatory markers, bone and collagen degradation, and
even HLA associations. Emerging evidence suggests PD may be common in RA patients, but
few studies have evaluated periodontal disease in patients with both early and established

METHODS: Data are drawn from initial subjects enrolled in a prospective observational study
of RA patients with both newly diagnosed and established disease. Eligibility includes RA
diagnosis and having more than 20 teeth in the functional dentition. Comprehensive oral
health evaluations are performed by a single trained dental research hygienist including
plaque index, gingivitis, bleeding, pocket depth, and attachment loss at six sites per tooth.
Patients also complete an oral health questionnaire to determine the prevalence of oral
health conditions including periodontal disease and xerostomia. RA subjects are classified as
having PD when there are at least 4 sites with pocket depth ³ 3mm and at least 4 sites of
attachment loss ³3 mm.

RESULTS: Of an initial 21 subjects with RA who were screened, 28% were ineligible to
participate because of having fewer than 20 functional teeth. Among the 15 subjects thus far
RA enrolled, 7 have disease duration of less than 1 yr (mean 7.8 mo). Average disease
duration among those with established RA is 8.5 yrs. Early RA subjects had more active
disease based on 28 joint counts than patients with greater than one year of disease (swollen
joints 7.4 versus 5.3, tender joints 7.4 versus 2.6, respectively). Based on comprehensive
periodontal examinations, more than half (56 %) of all RA subjects fulfilled the definition of
moderate to severe PD with a greater proportion of early (67%) versus established (50%)

CONCLUSIONS: These initial data suggest that in the community, having adequate teeth for
functional dentition may be common in many RA patients. Among those RA patients with
more than 20 teeth, we found that more than half have moderate to severe periodontal
disease, a value higher than previously described in RA. PD was detected in patients with
both early and established RA, suggesting that PD may precede rather than result from RA
and may be involved in RA disease development. These data indicate the importance of
routine dental evaluation and periodontal care for patients with RA.

Funding: NIH/NIDCR R03-DE018094, Maryland Chapter Arthritis Foundation Institutional
Grant, and Arthritis Foundation Arthritis Investigator Award

Disclosures: C.O. Bingham III, None; J.T. Giles, None; P.E. Jones, None; J.M. Bathon,
None; S.J. Bartlett, None.
Title: Transverse Versus Longitudinal Myeltis in SLE

Author(s): Julius Birnbaum, Douglas Kerr, Michelle Petri. Johns Hopkins University,
Baltimore, MD

Purpose:. Prior reports of lupus myelitis have not explored whether regional versus
longitudinal patterns of inflammation have discriminating features. Whereas transverse
myelitis (TM) refers to regional inflammation limited to less than 3 vertebral segments,
longitudinal myelitis (LM) is defined by inflammation spanning 3 or more vertebral segments.
This distinction has important clinical, mechanistic and prognostic implications. Relapsing-
remitting MS is associated with TM, with sensory more than motor symptoms. In contrast,
Devic’s syndrome is associated with longitudinal myelitis, with significant motor deficits at
clinical nadir.

Methods: TM versus LM was diagnosed on the basis of criteria described above. The
Kurtzke’s Expanded Disability Status Scale (EDSS), a scale of disability used to define
morbidity in patients with MS and Devic’s syndrome, was calculated. Relevant details of
symptoms and neurological exams were abstracted from review of records, with the EDSS
score calculated at date of last visit. A score of six or greater indicates dependence on
ambulatory assist devices for even minimal distances.

Results: This study represents the largest case-series ever reported of lupus patients with
myelitis. Ten patients presented with a longitudinal pattern of inflammation, and 4 presented
with a transverse pattern of inflammation. In contrast to idiopathic TM, (relapsing disease
seen in only 15% of patients), lupus myelitis was associated with relapses in both TM (2/4
cases) and LM (7/14 cases). A total of 11 flares were observed in the 50% of cases with
relapsing TM, and a total of 31 flares were observed in the 50% of cases with relapsing LM.
Optic neuritis (RR=7.0, 95% CI [1.1, 43.0], p=0.02) and anti-Ro antibodies (RR=4.2, 95%CI[0.7,
24.9], p=0.08) were associated with relapsing disease. TM demonstrated a statistically
significant association with anticardiolipin antibodies (RR=10.0, 95%CI[1.6,64.1],p=0.01). Both
TM and LM were associated with rapid deterioration of gait dysfunction over a similar period
of follow-up; 50% of TM patients (median FU of 8.6 years), and 80% of LM patients (median
FU of 7.3 years) developed EDSS scores of six or greater.

Conclusions: (1) Anticardiolipin antibodies were associated with TM rather than LM. This
suggests that antiphospholipid antibodies may cause regionally limited spinal cord disease by
an ischemic mechanism, and suggests that further studies are warranted to see if
antithrombotic therapy can modify progression of neurological morbidity in lupus TM
patients. (2) Lupus myelitis is associated with a polyphasic course in half of LM and TM cases.
Optic neuritis and anti-Ro antibodies were associated with a relapsing course, and may serve
as important prognostic indicators of future myelitis flares. (3) After 20 years of onset, only
25% of patients with relapsing-remitting MS have EDSS scores of 3 or greater; in contrast, this
study demonstrates that lupus myelitis carries the more grave prognosis of graduating to an
EDSS score of 6 or greater, in a median follow-up of 8 years.

Disclosures: J. Birnbaum, None; D. Kerr, None; M. Petri, None.
C. M. Boyd1, J. Wolff1, C. Cigolle2, C. Blaum2
1. Medicine, Health Policy and Management, Johns Hopkins University, Baltimore, MD, USA.
2. University of Michigan Medical School, Ann Arbor, MI, USA.

Medicare pay-for-performance may rely on single-disease performance standards developed
in younger, employed populations. How these standards apply in an older, multimorbid
population is unknown.

Using the 2004 5% Standard Analytic File of all Medicare-covered service utilization, we
examined the comorbidity, utilization, expenditure, and provider use patterns of Medicare
beneficiaries eligible/ineligible for the age-based denominators of diabetes quality standards
used in a Medicare Demonstration Project. Using a standard approach to identifying chronic
conditions, all ICD codes within claims data were mapped to 41 mutually exclusive chronic

Overall, 21%(n=340,925) of the sample had diabetes. Of those with diabetes, 38.6%(n=131,476)
were ineligible for diabetes quality standards due to age>75 years. 15.5%(n=52,924) were <65
years. Each group had significant comorbidity: the <65 year age group(mean number of
chronic conditions, 4.3[SD 3.2] [out of possible 41]); 65-75 year age group(mean 4.2[SD 2.9]);
>75 year age group (mean 5.0[SD 3.2]). Most common comorbidities among the eligible 65-75
year group were hypertension(73%), lipid disorders(63%), coronary heart disease(30%), eye
disorders(27%), arthritis(24%), chronic lung disease(21%), heart failure (20%), conduction
disorders(16%), thyroid disorders(15%), and cancer(13%). The estimated yearly costs for the
>75 group and the 65-75 group were $33 and $31 billion, respectively, with higher mean costs
in the >75 group ($12650 vs. $9917 per person) resulting from observed greater use of
hospital, skilled nursing facility, home health, and Part B services. Among all beneficiaries
with diabetes, the mean number of unique outpatient physicians visited for evaluation and
management was 3.3; the mean number of outpatient physician visits was 9.1. 41% of
beneficiaries with diabetes did not see a geriatrician, family practitioner, internist, or general
practitioner for the majority of their outpatient visits.

Denominators used in current pay-for-performance endeavors may exclude the most
complex, multimorbid, and high-utilization Medicare beneficiaries. Eligible Medicare
beneficiaries have a substantial multimorbidity burden, making clinical management more
complex. The large number of physicians seen may make identification of a single
responsible physician an important challenge to pay for performance programs.

Financial Disclosure (List all funders who provided support for this research) : Johns
Hopkins Bayview Center for Innovative Medicine
John A. Hartford Center of Excellence

Junran Cao*, Satoshi Ikemoto.

National Institute on Drug Abuse, National Institutes of Health, Department of Health and
Human Services, Baltimore, Maryland 21224, USA.

Neuropeptide S (NPS) is a recently identified neuropeptide. NPS-containing cell bodies are
localized in the lower brainstem just adjacent to the locus coeruleus and its receptors are
expressed in the brain areas that are implicated in motivation and reward. Indeed, NPS
appears to play an important role in arousal. We examined possible rewarding effects of NPS
injected into the lateral ventricle, using intracranial self-administration and conditioned place
preference procedures. Each male Wistar rat was implanted with a permanent unilateral
guide cannula ended just above the lateral ventricle. For intra-ventricular self-administration,
rats received NPS at the dose of 3.39 pmol per infusion in sessions 2-4 and vehicle in sessions
1 and 5 followed by three sessions with 0.339, 3.39 and 33.9 pmol doses. Each session lasted
for 90 min, and sessions were separated by 24 hours. Rats learned quickly to self-administer
the 3.39 pmol dose into the lateral ventricle. It is unclear whether rats self-administer NPS at
a lower dose. Effects of two doses of NPS were evaluated for conditioned place preference.
Each rat was confined in a compartment for 20 min immediately after intraventricular
injections of NPS (100 or 1,000 pmol) and confined in another compartment for 20min after
vehicle injections. Injections were separated by 24 hours. Pairings of NPS and vehicle
injections with these compartments were alternated over 8 days. Place preference of each
rat was examined in the first session, prior to conditioning, and the last session, after
conditioning, by allowing them to have free access to both compartments for 15 min without
injections. The high dose (1,000 pmol) of NPS induced conditioned place preference,
whereas the low dose (100 pmol) induced conditioned place avoidance. These data suggest
that NPS injections into the lateral ventricle are rewarding, although NPS injections at some
doses may also be aversive. These findings and the previous finding that intra-ventricular
injections of NPS elicit motor stimulant effects raise the possibility that the NPS system
interacts with the mesolimbic dopamine system.

Supported by the Intramural Research Program of NIDA, NIH and the NIH Grant DA021880.
Pulmonary Granulomatous Inflammation in Response to Mycobacterial Catalase-Peroxidase
Protein: An Experimental Model of Sarcoidosis

E.S. Chen, M.D., Y. Zhang, M.D., Ph.D. and D.R. Moller, M.D.
Johns Hopkins Univ., Baltimore, MD, United States.

Our recent studies identified catalase-peroxidase (mKatG) protein and DNA in a subset of
sarcoidosis tissues and provided evidence that mKatG is a target of the adaptive immune
response in sarcoidosis, suggesting that mKatG is a pathogenic antigen (J Exp Med 2005;
201:755-67). To examine the mechanisms of mKatG-induced granuloma formation, we tested
recombinant mKatG in a rodent model of granulomatous inflammation. Lewis rats were
sensitized to mKatG protein in Complete Freunds Adjuvant and exposed 2w later to mKatG-
linked sepharose beads or sham-reacted control beads via IV injection as previously
described by other investigators. Granuloma size (mean radius of inflammation around bead)
and cellularity were quantified by immunohistochemistry. A robust monocyte / macrophage
dominant cellular infiltrate with giant cells formed around mKatG beads at 4d in contrast to

granulomas though o
mKatG is a comparatively potent antigen in this model. Consistent with this premise, mKatG-

4d). Cultured splenocytes isolated from rats exposed to mKatG coated beads produced
significantly higher amounts of interferon-
compared to splenocytes isolated from rats exposed to uncoated control beads (uncoated vs
mKatG, 122 17pg/mL vs 286 51pg/mL, p<0.05). These results demonstrate that mKatG protein
has the ability to induce a vigorous antigen-specific Th1 granulomatous response in an
experimental rodent model, supporting a role for mKatG as a pathobiologically relevant
antigen in systemic sarcoidosis.

Aiwu Cheng, Ph.D.1, Naomi Kamimura1, Ph.D.1,2, Mark P. Mattson, Ph.D.1, 1Laboratory of
Neurosciences, National Institute on Aging, Intramural Research Program, Baltimore, MD
21224 and 2Department of Biochemistry and Cell Biology, Institute of Development and Aging
Sciences, Nippon Medical School, Japan.


Mitochondria are the primary sources of energy and oxyradicals in cells, and participate in
intermediary metabolism, calcium signaling and apoptosis. Mitochondria undergo
biogenesis, as well as frequent fission and fusion that regulate their morphology, number
and function. The fission and fusion processes are regulated by several GTPase proteins:
mitofusin (mfn1, mfn2) and OPA1 are essential for mitochondrial fusion whereas dynamin
related protein (Drp1) and fission1 (fis1) regulate mitochondrial fissions.

We provide evidences that excessive mitochondrial fission is an early and pivotal event in
neuronal apoptosis induced by an array of insults including A-beta 1-42, DNA damage inducer
Etoposide and the mitochondrial uncoupler FCCP. Moreover, A-beta 42 inhibits the
mitochondrial biogenesis and interferes with the tanslocation of mitochondria into neuronal
processes in young immature neurons. In physiological conditions, mitochondrial mass
(biogenesis) and mitochondria fission/fusion proteins (Drp1, Mfn2 and OPA1) are significantly
up-regulated during neuronal differentiation and maturation. Therefore neural development
events such as neurite outgrowth and synaptogenesis might also be modulated by
mitochondrial biogenesis and dynamics. Lastly, we found that neurotrophic factor BDNF can
increase mitochondrial biogenesis through up-regulation of PGC1alpha, a tissue-specific
transcriptional coactivator that has been implicated in mitochondrial biogenesis in heart and
skeletal muscle. In contrast, BDNF does not change the expression of mitochondrial fission
and fusion proteins. By using adeno-PGC1alpha, adeno-drp1 and its mutant, we will further
elucidate the functions of mitochondrial biogenesis and dynamics (fission and fusion) in
neural development and disease.

Our studies will provide critical evidence and insight in elucidating function of mitochondria
in the neural development and neurological disorders.
Screening for co-occurring mood disorder among pregnant substance-dependent patients:
Characteristics of the Addiction Severity Index and the Beck Depression Inventory

*M.S. Chisolm
M. Tuten
E.C. Strain
H.E. Jones
Department of Psychiatry and Behavioral Sciences
The Johns Hopkins University School of Medicine
Baltimore, MD 21224 USA
The Center for Addiction and Pregnancy
Johns Hopkins Bayview Medical Center
Baltimore, MD 21224 USA

Objective: This study compares the test characteristics of the Addiction Severity Index (ASI)
and the Beck Depression Inventory (BDI) when used as screening tests for current mood
disorder in pregnant substance-dependent women.

Methods: One hundred eighty seven patients completed the ASI, BDI and the Structured
Clinical Interview for DSM-IV (SCID-I). Receiver operating characteristic (ROC) curves were
constructed for the ASI and the BDI as predictors of current mood disorder. Sensitivities,
specificities, predictive values, and likelihood ratios were calculated for various cutoff values.

Results: Of patients diagnosed with a current mood disorder (n=51), the mean ASI psychiatric
Interviewer Severity Rating (ISR) was 4.4 and the mean BDI score was 17.1. Those without a
current mood disorder (n=136) had a mean ASI psychiatric ISR of 2.7 and a mean BDI score of
14.0. Areas under ROC curves were 0.73 for the ASI and 0.59 for the BDI. Using a cutoff
range of 4 or above, the sensitivity of the ASI ISR to detect current mood disorder was 0.82,
the specificity was 0.49, the positive predictive value was 0.38 and the negative predictive
value was 0.88. Using a cutoff range of 17 or above, the sensitivity of the BDI score to detect
current mood disorder was 0.49, the specificity was 0.62, the positive predictive value was
0.33 and the negative predictive value was 0.76.

Conclusion: The ASI psychiatric ISR was able to predict the presence of a mood disorder with
better sensitivity and specificity compared to the BDI.

Supported by NIDA R01-DA12403, K02-DA00332.
The authors have no financial relationships that are related to the topic of this presentation.
A version of this abstract was presented at the 2007 meeting of the College on Problems of
Drug Dependence.
Prognostic significance of cardiac troponin I levels in patients presenting with
supraventricular tachycardia

Author Block: Grant V. Chow, David D. Spragg, Glenn A. Hirsch, Alan Cheng, Joseph E.
Marine, Johns Hopkins Medical Institutions, Baltimore, MD

While cardiac troponin I (cTnI) elevation in patients with supraventricular tachycardia
(SVT) has been reported, the incidence, predictors, and prognostic significance of cTnI
elevation associated with SVT presentation are not known.

We screened records of all patients discharged from our institution between 1/1/02 and
12/31/06 for diagnosis of SVT (ICD-9 code 427.0). Patients with a diagnosis of SVT at
presentation (confirmed by 12-lead ECG) who had at least one cTnI level measured were
reviewed for baseline information and clinical follow-up. Patients with atrial fibrillation
and atrial flutter were excluded. Elevated cTnI was defined as > 0.1 ng/mL. Univariate and
stepwise multivariable logistic and Cox proportional-hazards regressions were used to
assess predictors of cTnI elevation and future events, respectively. The primary endpoint
was combination of death, myocardial infarction, and cardiovascular rehospitalization.

81 patients met criteria for the study (mean age 61.9 +/-15.6 yrs., 55.6 % female). 26
patients (32.1%) had elevated cTnI (mean 0.81+/- 1.6 ng/mL). Mean follow-up period was
2.2 +/-1.7 yrs., during which 30 patients (37%) reached the primary endpoint. Univariate
predictors of elevated cTnI included history of coronary artery disease (OR 3.7, 95%CI
1.04-13.0, p=0.04), LV ejection fraction < 50% (OR 5.1, 95%CI 1.57-16.4, p<0.01), renal
dysfunction (OR 3.8, 95%CI 1.4-10.2, p<0.01), and either ST segment depression (OR 4.9,
95%CI 1.3-17.8, p=0.02 ) or LBBB (OR 17.5, 95%CI 1.9-162.4, p= 0.01) during SVT. Predictors
of elevated cTnI after multivariate analysis included LV dysfunction (OR 7.5, 95%CI 1.8-
31.6, p<0.01), ST depression (OR 6.9, 95%CI 1.2-35.2, p=0.02), and LBBB (OR 13.5, 95%CI 1.2-
152.2, p=0.04). After multivariable adjustment, the presence of elevated cTnI with SVT
was associated with increased risk of the primary endpoint (HR 2.6, 95%CI 1.1-6.1, p=0.02).

Mild elevation of cTnI is common in patients presenting to hospital with SVT and confers
increased risk of future cardiovascular events. These data suggest that patients with SVT
and elevated cTnI warrant further evaluation for cardiovascular comorbidities.
4-hydroxynonenal: a potential endogenous activator of Transient Receptor Potential A1
(TRPA1) channels on sensory neurons

TE Taylor-Clark and BJ. Undem
 Division of Allergy and Clinical Immunology
Johns Hopkins School of Medicine, Baltimore, MD

Stimulation of TRPA1 receptors leads to activation of subtypes of capsaicin-sensitive sensory
nerves (Nature, 427, 260-5; 2004). Stimulants of TRPA1 include allyl isothiocyannate (the
active ingredient in horseradish and mustard oil), cinnamaldehyde and acrolein.

4-hydroxynonenal (4-HNE) is a peroxidation metabolite of linoleic and arachidonic acids. It is
produced in Inflammatory diseases including asthma and COPD. Based on its similarity to
acrolein, we hypothesized that it may be an endogenous activator of TRPA1. We found that
4-HNE mimics mustard oil in its ability to increase intraneuronal calcium (as determined using
a Fura 2AM assay) in neurons isolated from mouse trigeminal ganglia. Approximately 45% of
all mouse trigeminal neurons were stimulated by 4-HNE. Although most of these neurons
were also capsaicin sensitive, the 4-HNE response was not inhibited by the TRPV1-selective
antagonist I-RTX (1uM). The 4-HNE response was, however, virtually abolished by ruthenium
red (30uM), a nonselective antagonist of TRP channels, including TRPA1. Mustard oil (100uM)
activated approximately 55% of mouse trigeminal neurons. Pretreatment with 4-HNE reduced
the number of mustard oil-sensitive neurons to 17% without affecting the proportion of
capsaicin-sensitive neurons (50% without 4-HNE, 60% after 4-HNE), suggesting that 4-HNE
selectively desensitized mustard oil responses. We also evaluated trigeminal neurons that
project sensory fibers specifically to the upper airways (using retrograde tracing techniques)
and found that both 4-HNE and mustard oil activate approximately 60% of these nerves. We
next showed that 4-HNE stimulates (increases calcium) in HEK cells transfected with TRPA1,
but not in wild-type HEK cells. In conclusion, these data support the hypothesis that 4-HNE is
an endogenous activator of TRPA1 receptors and that these receptors, once activated, can
stimulate trigeminal sensory neurons, including upper airway-specific neurons.

Funded by NIH, and by a grant from GlaxoSmitghKline.
Glutamatergic neurons of the ventral tegmental area establish synaptic contacts on local
dopaminergic and non-dopaminergic neurons
Alice Dobi, Elyssa B Margolis* and Marisela Morales
National Institute on Drug Abuse, Intramural Research Program, Cellular Neurophysiology.
5500 Nathan Shock Drive, Baltimore, MD. 21224. *Ernest Gallo Clinic and Research Center,
University of California, San Francisco, Emeryville, CA 94608.

The ventral tegmental area (VTA) plays a role in reward and motivation. In addition to
                                        -amino butyric acid (GABA), glutamatergic neurons
have recently been found in the VTA. VTA glutamatergic neurons express transcripts
encoding the vesicular glutamate transporter type 2 (VGluT2). To investigate whether VTA
glutamatergic neurons establish local synapses, we marked axon terminals of VTA neurons
by local injection of the anterograde axonal tracer Phaseolus vulgaris-leucoagglutinin (PHAL)
and used double immunolabeling and electron microcopic analysis to determine if PHAL-
positive terminals within the VTA contained VGluT2. PHAL-positive terminals established
either asymmetric or symmetric synapses, predominantly on dendrites, more than half of all
PHAL-positive terminals that made recognizable synapses contained VGluT2. Double labeled
PHAL/VGluT2 terminals established predominantly asymmetric synapses while most PHAL-
positive terminals lacking VGluT2 formed symmetric synapses. To determine whether
DAergic neurons receive glutamatergic inputs from local neurons, we used triple
fluorescence immunolabeling with antibodies against PHAL, VGluT2 and tyrosine hydroxylase
(TH, a marker for DAergic neurons). By confocal fluorescence microscopy we observed
double labeled PHAL/ VGluT2 varicosities contacting TH-positive dendrites. By electron
microscopy, we further determined that double labeled PHAL/VGluT2 axon terminals formed
synaptic contacts on dendrites of both TH-immunoreactive and TH-negative cells. In whole
cell recordings of VTA neurons we observed that blocking local action potential activity
significantly decreased the frequency of synaptic glutamatergic events in DAergic and non-
DAergic neurons. In summary, we provide anatomical and electrophysiological evidence that
local glutamatergic neurons establish intrinsic synapses within the VTA.
Common Variants in Vascular Endothelial Growth Factor (VEGF) are Associated with Distinct
Pulmonary Phenotypes, Scleroderma-related Pulmonary Arterial Hypertension (SSc-PAH)
and Chronic Obstructive Pulmonary Disease (COPD)

L. Gao1, N. M. Rafaels1, N. Hansel2, D. N. Grigoryev1, C. Cheadle1, D. D. Sin5, R. Wise2, L.
Hummers3, F. Wigley3, R. A. Mathias6, T. H. Beaty4, P. Hassoun2, K. C. Barnes1
Div. Allergy & Clinical Immunology, 2Div. Pulmonary & Critical Care Medicine, 3Div.
Rheumatology and 4Bloomberg School of Public Health, JHU, Baltimore, MD, 5Univ. of British
Columbia, Vancouver, Canada, 6NHGRI, Baltimore, MD.

Rationale: Pulmonary arterial hypertension (PAH) is the leading cause of mortality in patients
with scleroderma-related diseases. Gene expression profiling studies suggested that VEGF is a
candidate gene for SSc-PAH and serum levels of VEGF were elevated in scleroderma patients.
Increased serum and expression levels of VEGF also have previously been observed in
patients with COPD. We compared patients with SSc-PAH and COPD to check for differences
in effects of VEGF.

Methods: Genotyping was performed on the ABI 7900HT Sequence Detection System on 16
tagging single nucleotide polymorphisms (SNPs) in VEGF in 100 European American SSc-PAH
cases and 107 healthy controls. A subset of 7 of these SNPs was also genotyped on 449
European American COPD patients. Decline of lung function and serum levels of
inflammatory biomarkers (IL-6, IL-1RA, C-reactive protein) were also examined. Linear
regression models were used to test for association while adjusting for potential
confounding factors.

Results: Significant associations were observed for markers rs1547651 (in the VEGF gene
promoter) and an intronic SNP (rs833068) with the SSc-PAH phenotype (P=0.02 and 0.007,
respectively). SNPs (rs833067 and rs998584) in VEGF were also significantly associated with
serum levels of all 3 biomarkers among COPD patients (P=0.012-0.0015). Lung function was
marginally associated with one of these variants.

Conclusions: These data support a pleiotropic role for VEGF in the pulmonary pathogenesis of
SSc-PAH and COPD. Validation of these findings is ongoing in other complex lung traits,
including asthma and acute lung injury.
       Peptide YY3-36 decreases reinstatement of high-fat food seeking during dieting in a rat
       relapse model

       Udi E. Ghitza, Sunila G. Nair, Sam A. Golden, Sarah M. Gray, Jamie L. Uejima,
       Jennifer M. Bossert and Yavin Shaham

       Behavioral Neuroscience Branch, NIDA/IRP/NIH/DHHS, Baltimore, MD

       A major problem in treating obesity is high rates of relapse to maladaptive food-taking habits
       during dieting. This relapse is often provoked by acute re-exposure to palatable food, food-
       associated cues, or stress. We used a reinstatement model, commonly used to study relapse
       to abused drugs, to explore the effect of peptide YY3-36 (PYY3-36) on reinstatement of high-
       fat (35%, 45-mg-pellets) food-seeking induced by acute exposure to the pellets (pellet-
       priming), a cue previously associated with pellet delivery (pellet-cue), or yohimbine (2 mg/kg,
       a pharmacological stressor). Rats were placed on a restricted diet (16 g chow/d), and lever-
       pressed for the pellets for 9-12 sessions (6-h/d, every 48-h); pellet delivery was paired with a
       tone-light cue. They were then given 10-20 extinction sessions wherein lever-presses were
       not reinforced with the pellets, and subsequently tested for reinstatement of food-seeking.
       Systemic PYY3-36 injections (100-200 µg/kg) decreased pellet-priming- and pellet-cue-induced
       reinstatement of food-seeking, but not yohimbine-induced reinstatement. Arcuate nucleus
       (Arc) injections of PYY3-36 (0.4 μg/side) decreased pellet-priming-induced reinstatement.
       The attenuation of pellet-priming-induced reinstatement by systemic PYY3-36 was reversed
       by systemic (2 mg/kg) but not Arc (0.5 μg/side) injections of the Y2 receptor antagonist
       BIIE0246. Arc PYY3-36 injections did not decrease pellet-cue-induced reinstatement. Finally,
       systemic PYY3-36 injections had minimal effects on ongoing food self-administration, or
       heroin-priming- or heroin-cue-induced reinstatement of heroin-seeking. These data identify
       an effect of systemic PYY3-36 on relapse to food-seeking that is independent of Y2-receptor
       activation in Arc, and suggest that PYY3-36 should be considered for the treatment of relapse
       to maladaptive food-taking-habits during dieting.

This work was supported by the Intramural Research Program of the National Institutes of Health, National
Institute on Drug Abuse.

 B. Robert Gibson, Panagis Galiatsatos, Lisa Eaton, Rania Abu-Hamdah, Stephen M. Milner, Dariush Elahi, Dana
 K. Andersen

 Department of Surgery, Johns Hopkins University School of Medicine, Johns Hopkins Bayview Medical
 Center, Baltimore, MD

 INTRODUCTION: Intensive insulin therapy in the surgical ICU has been shown to reduce sepsis and mortality,
 but the benefit of intensive insulin therapy in the burn ICU remains unclear.

 METHODS: We studied the normalization of blood glucose level with intensive insulin therapy in patients
 admitted to the surgical ICU and burn ICU, whose demographics, clinical data, and outcomes were entered
 into a prospectively collected database. From October 2006 to July 2007, 667 patients were admitted to our
 mixed-service surgical ICU and 303 patients were admitted to our regional burn ICU, of which 79 patients
 met criteria (plasma glucose > 150 mg/dl) for initiation of intensive insulin therapy. Thirty-seven patients
 required intensive insulin therapy for at least 72 hours, and we analyzed these patients to determine if tight
 glycemic control (glucose ≤ 150 mg/dl by day 3) is associated with reduced morbidity and mortality. We also
 assessed the degree of success and adverse events in the maintenance of tight glycemic control in the burn
 ICU compared to the surgical ICU, with an intensive insulin therapy protocol that required glucose
 determinations at least every 2 – 4 hours. Hypoglycemia was defined as <60 mg/dl.

 RESULTS: Data are shown as means ± SEM:

   Day 3 avg          N      Gender M/F       Age           BMI          Mortality      % Sepsis Hypoglycemic Events
Glucose (mg/dL)                             (years)                                                 (<60 mg/dL)
   SICU ≤150         10          2/8       64 ± 5.6       38.9 ± 5.8         0           1 (10%)          0
   SICU >150         12          6/6        71 ± 2.8      37.2 ± 3.0      7 (58%)       7 (58%)           4
   BICU ≤150          5          2/3       48 ± 12.5      26.7 ± 1.2      1 (20%)       3 (60%)           2
   BICU >150         10          9/1       54 ± 4.2       34.1 ± 2.7      5 (50%)       7 (70%)           1

 Admission glucose levels and average levels for day 1, 2 and 3 for the groups below or equal to 150 mg/dL
 were 206, 144, 137 and 125 mg/dL in the burn ICU; the corresponding levels in the surgical ICU were 275, 151,
 115 and 110 mg/dL. The levels for the groups above 150 mg/dL were 222, 204, 179 and 228 mg/dL in the burn
 ICU and 205, 165, 158 and 193 mg/dL in the surgical ICU (p=NS for comparisons between groups). Percent
 burn surface area in the burn ICU patients was 10 and 45 in the ≤150 and >150 mg/dL groups, respectively.
 Daily amounts of insulin were ≈ 2 fold higher in the SICU (P<0.05). Overall only 41% of surgical ICU/burn ICU
 patients achieved glycemic goal of ≤150 mg/dL by day 3 (45% in the surgical ICU and 33% in the burn ICU,
 p=NS), and hypoglycemic events (<60 mg/dL) were seen in 19% of patients (none with medical sequellae).
 Poor glycemic control was strongly associated with mortality in both burn ICU and surgical ICU pts (55%), and
 correlated highly with septic events.

 CONCLUSIONS: Mortality is associated with poor glycemic control in both SICU and BICU patients. Sepsis is a
 causative factor in 10/13 (77%) of deaths. In addition, BICU mortality is associated with % total body surface
 area burn, and elevated BMI. We conclude that both surgical ICU and burn ICU patients benefit equally from
 tight glycemic control obtained by intensive insulin therapy.
Single Neuron Oxygen Consumption Responses to Glutamate

M. Gleichmann1, L. Collis2, P. Smith2, M.P. Mattson1.
  Laboratory of Neurosciences, National Institute on Aging Intramural Research Program,
Baltimore, MD.
  Biocurrents Research Center, MBL, Woods Hole

Glutamate is the major excitatory neurotransmitter in the brain and plays fundamental roles
in essentially all behaviors. Excessive activation of glutamate receptors is believed to
contribute to neuronal cell death in a number of neurological disorders including stroke,
epilepsy and traumatic brain injury. The process of excitotoxicity involves opening of ligand-
activated glutamate receptor channels (NMDA-, AMPA-, Kainate-receptors) and subsequent
influx of calcium and sodium. However, the consequences of calcium influx that damage
neurons are less well understood. In this project we tested the hypothesis that glutamate
alters cellular energy metabolism in neurons resulting in energy failure under excitotoxic
conditions. We employed a novel self-referencing oxygen-sensing electrode to measure the
oxygen consumption rate of single neurons and small neuronal clusters in primary rat cortical
cell cultures. Our results show that glutamate, at concentrations ranging from 10 M to 33
M, causes oxygen consumption to increase to maximal levels (200-250% of baseline
consumption) within 5 to 10 min. This rapid increase in oxygen consumption is followed by a
steady decline to levels close to zero. This pattern of oxygen consumption was virtually
identical to that seen in neurons treated with the mitochondrial membrane uncoupler FCCP.
Single neuron oxygen consumption, together with simultaneous assessment of cytosolic
calcium concentrations and mitochondrial membrane potential by confocal microscopy,
showed that a sudden increase in cytosolic calcium was followed by an increase in oxygen
consumption. Once oxygen consumption declined, a reduction in mitochondrial membrane
potential occurred. The NMDA receptor antagonist MK 801 prevented neuronal death only
when given before calcium levels and oxygen consumption peaked. ATP levels in cortical
neuron cultures declined to 47% of control levels within 15 min of exposure to 33 M
glutamate. After 15 min of glutamate exposure the ATP/ADP ratio declined from 2.92 to 0.67
(i.e. a decline to 22.8% of control levels) and cell viability was reduced to around 50% of
control 24 hours after exposure. This constellation of maximal oxygen consumption with
simultaneous or subsequent decline in ATP levels leads us to conclude that ATP depletion is
an early and crucial event contributing to glutamate toxicity.
Title: The Environment Induced by Active Lupus Plasma Suppresses the Expansion of FoxP3
High-Expressing Regulatory T Cells

Authors: Amit Golding, Felipe Andrade. Johns Hopkins Medical Institutions, Baltimore, MD.

Purpose: CD4+CD25+FoxP3+ regulatory T cells (Tregs) have an important role in preventing
autoimmune disease. In systemic lupus erythematosis (SLE), Tregs have been shown to be
diminished in number and function in patients with active disease. It is not clear whether the
primary defect exists in the Tregs themselves, or rather in the pro-inflammatory “lupus
environment.” Our aim was to explore how Tregs from normal donors would behave when
exposed to an active lupus environment. We therefore determined the effect of active
lupus plasma on the expansion of normal CD4+CD25+FoxP3+ regulatory T cells in vitro.
Methods: Treg assays were performed as previously described (Gavin et al PNAS April, 2006).
Briefly, peripheral mononuclear cells (PBMCs) from normal donors were incubated with
soluble anti-CD3 resulting in polyclonal activation of T cells. Expansion of Tregs was
determined at days 0, 3, 7 and 10 by detection of CD4+CD25+FoxP3+IFN-
cytometry (IFN-                                               effectors that are IFN-   and also express
FoxP3, albeit at lower levels). All assays were performed in 5% heat-inactivated human
serum, with or without normal plasma, and with or without increasing amounts of active SLE
plasma. To ensure that the FoxP3-high population represented expansion of pre-existing
“natural” Tregs rather than de novo expression of FoxP3 in non-Tregs, the specificity of this
population was confirmed by CD25-depletion of fresh PBMCs using magnetic beads.
Results: In the presence of normal plasma, maximal expansion of CD25+ FoxP3+-high IFN-
   Tregs occurred at day 3, similar to prior studies. In contrast, when cells were incubated
with active lupus plasma, we observed a dramatic reduction in FoxP3+-high Tregs (see
Figure). Interestingly, this effect could be seen even at a level as low as 0.2% of SLE plasma
added to the culture.
Conclusions: This study demonstrates that the abnormal number and decreased function of
Tregs in patients with SLE might result from the inadequate expansion of natural Tregs
during immune activation due to some factor in the active lupus environment. The
observation that a Treg defect can be imposed upon Tregs from normal donors suggests that
the failing of Tregs in SLE is likely not a cell-intrinsic defect. We have found that addition of a
small amount of active plasma from patients with active SLE profoundly inhibits the ability of
normal Tregs to expand and upregulate FoxP3, considered the master regulator of Treg
development. Our current objectives are to determine what factors in the SLE plasma are
responsible for suppressing the expansion of FoxP3-high Tregs and to determine which
targets on Tregs are acted upon by the lupus environment.
Myriam Gorospe, PhD
Senior Investigator, RNA Regulation Section
Laboratory of Cellular and Molecular Biology
National Institute on Aging, National Institutes of Health
5600 Nathan Shock Drive
Baltimore, MD 21224, USA

A predominantly nuclear RNA-binding protein, HuR translocates to the cytoplasm in
response to stress and proliferative signals, whereupon it stabilizes target mRNAs or
modulates their translation. Here, we present evidence that HuR phosphorylation on
Ser-202 by the G2 phase kinase Cdk1 influences its subcellular distribution: i) HuR was
specifically phosphorylated in synchronous G2-phase cultures, ii) interventions to
reduce Cdk1 activity promoted HuR's cytoplasmic localization, and iii) interventions to
activate Cdk1 lowered HuR's cytoplasmic abundance. In keeping with the prominently
cytoplasmic location of the nonphosphorylatable point mutant HuR(S202A), phospho-
HuR(S202) was shown to be predominantly nuclear using a novel anti-phospho-
HuR(S202) antibody. The enhanced cytoplasmic presence of unphosphorylated HuR
was linked to its decreased association with 14-3-3 and to its heightened binding to
target mRNAs. Our findings suggest that Cdk1 phosphorylates HuR during G2, thereby
retaining it in the nucleus in association with 14-3-3 and hindering its
posttranscriptional function and antiapoptotic influence.
Estimating Diffusion-Perfusion Mismatch Using the NIHSS-Plus

Rebecca F. Gottesman, MD PhD, Jonathan T. Kleinman, BS, Cameron Davis, BA, Jennifer
Heidler-Gary, MA, Melissa Newhart, BS & Argye E. Hillis, MD. Neurology, Johns Hopkins
University School of Medicine, Baltimore, MD.

Diffusion-perfusion (DWI/PWI) mismatch is assessed in acute stroke patients to determine
the difference between infarcted brain tissue and surrounding dysfunctional area at risk for
infarction. Presence of a mismatch may lead to further stroke intervention, including tPA or
endovascular treatment. However, this technology is neither available at all hospitals nor
feasible in all patients. We hypothesized that we could estimate DWI/PWI mismatch by
combining actual DWI volume with estimated perfusion abnormality, using clinical tests. We
studied 87 consecutive acute nondominant hemispheric stroke patients with DWI/PWI and
cognitive testing. We have previously described the NIHSS-plus, with additional points for
errors on line cancellation and visual extinction tasks. Using this scale, we calculated
predicted PWI (pPWI) volume with a linear spline regression model. Compared to DWI
volume, we calculated percent radiographic mismatch (PWI-DWI/DWI *100) and clinical
mismatch (pPWI-DWI/DWI *100), and coded these each as presence (>20%) or absence of
mismatch. Having an estimated mismatch had 90% sensitivity in prediction of actual
mismatch. Specificity was 36%, and the area-under-the-curve (for ROC analysis) was 0.63. The
high sensitivity means most patients with an actual mismatch will be identified clinically,
indicating potential benefit from intervention to restore blood flow.
A novel method for detection and quantification of nicotine and metabolites in human
meconium by LC-MS/MS using atmospheric pressure chemical ionization

Teresa R. Gray*, Diaa M. Shakleya, Marilyn A. Huestis
Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug
Abuse, National Institutes of Health, Baltimore, MD 21224, USA

Nicotine exposure in utero is associated with decreased fetal growth, alterations to lung and
nervous system development, behavioral disorders and increased risk of nicotine addiction
later in life. Meconium is often used to detect in utero drug exposure because of its easy and
non-invasive collection and long window of detection.

Objective: A liquid chromatography/atmospheric pressure chemical ionization tandem mass
spectrometry (LC-APCI-MS/MS) method for the determination of nicotine, cotinine, trans-3’-
hydroxycotinine, nornicotine and norcotinine in meconium was developed and validated.
 Method: Meconium specimens spiked with analytes and deuterated internal standards were
sonicated in acidic methanol. After centrifugation and supernatant evaporation, enzymatic
hydrolysis with β-glucuronidase was performed overnight to cleave glucuronide conjugates
of nicotine, cotinine and trans-3’-hydroxycotinine. Analytes were isolated using mixed mode
solid phase extraction and analyzed by LC-APCI-MS/MS operating in multiple reaction
monitoring (MRM) mode. The analytical method was validated over four days for limit of
quantification, accuracy, precision, recovery, matrix effects, hydrolysis efficiency, carryover
and interference.

Results: The limits quantification were 1.25 ng/g for cotinine, trans-3’-hydroxycotinine, and
norcotinine, and 5 ng/g for nornicotine and nicotine; the upper limit of quantification for all
analytes was 500 ng/g. Correlation coefficients for each calibration curve were >0.99. Intra-
and inter-day precision ranged from 3 to 10 % relative standard deviation (RSD) and 5 to 20 %
RSD, respectively. Intra- and inter-day accuracy was greater than 76%. Recoveries were all
above 61%. Validation parameters were tested at 8, 80 and 400 ng/g. Ion suppression due to
matrix effect ranged from 20-70%, depending on the analyte. Hydrolysis efficiencies for
glucuronide conjugates of nicotine, cotinine and trans-3’-hydroxycotinine were 90, 80, 15%,
respectively; the hydrolysis procedure did not affect unbound analytes. Analyte conversion
to nornicotine was detected following stronger basic and acidic hydrolysis conditions.
Analyte stability was assessed under the following conditions: 24 h at room temperature, 72
h at 4°C, three -20°C freeze-thaw cycles, and 24 h in the autosampler; losses of less than 30%
were observed under each condition. No analyte carryover was observed at two times the
upper limit of quantification. No interference by illicit and therapeutic drugs was observed.

Conclusion: The first LC-APCI-MS/MS method for the identification and quantification of
nicotine, cotinine, trans-3’-hydroxycotinine, nornicotine and norcotinine in meconium is
described. The method will be used in studies to correlate meconium concentrations to
neonatal outcome measurements.

Key Words: LC-APCI-MS/MS, Nicotine, Meconium

Guerrero-Cazares, Hugo; Achanta, Pragathi; Frazier, James; Attenello, Frank; Zamora-Berridi,
Grettel; Chaichana, Kaisorn; Choi, Alyssa; Niranjan, Ashwini; Quiñones-Hinojosa, Alfredo.
Dep. of Neurosurgery, Johns Hopkins University.

Neural stem cells (NSC) are characterized by their ability to self renew, proliferate, and
differentiate into the three main CNS cell lineages. A specific population of astrocytes in the
Subventricular Zone (SVZ) has proven to be the bona fide neural stem cells. Some brain
tumors, such as glioblastoma multiforme (GBM) contain a cell subpopulation called Brain
Tumor Stem Cells (BTSCs) that have been found to share characteristics with the NSCs.
Evidence in rodents suggests that NSCs can give rise to BTSCs. The evidence includes the
anatomical relation between the SVZ and some GBMs’ location and the tumorigenic
phenotype that NSCs present in response to growth factors such as PDGF or EGF. To test this
hypothesis, we collected intraoperative tissue and cerebrospinal fluid (CSF) from patients
diagnosed with GBM, as well as SVZ and Cortex from patients without brain cancer diagnosis
(SVZ and NC-Cx respectively). We purified the astrocytes and characterized the GBM gene
expression profile depending on their anatomical contact with the SVZ, based on T1 MRI
scanning. We performed a neurosphere assay to test the stem cell properties of the different
samples. We also evaluated the migration and proliferation of the cells in vitro, in response to
the addition of EGF, PDGF or CSF. Our preliminary results show that the gene expression
profiles differ between samples from GBMs that are touching the ventricle and those that
are not. Our results also show that both GBM and SVZ but not NC-Cx derived cells are able to
form neurospheres and differentiate into Tuj-1 (neuroblasts) and GFAP (astrocytes) positive
cells. The addition of EGF or PDGF increased the migration of GBM and SVZ derived cells,
while it did not have a significant effect on NC-Cx cells. CSF had the same effect in a dose
dependant manner, even though there is no significant difference between the one derived
from GBM patients and the one from non-cancer patients. Our results confirm the similarity
between SVZ and GBM derived cells and begin to shed light into the hypothesis that some
GBMs may arise from normal NSCs residing in the human SVZ. This malignant transformation
could be stimulated by growth factors such as EGF or PDGF as well as other signals contained
in the CSF.
Adenosine A2A Receptors are Up-Regulated in Inbred Mice with Low Levels of Iron in the

S. Gulyani1; B.C. Jones2; R.P. Allen1; M. P. Mattson3; C.J. Earley1

1. Neurology, Johns Hopkins Univ., Baltimore, MD, USA, 2. Department of Nutritional Sciences,
The Pennsylvania State University, University Park, Pennsylvania, USA. 3. Laboratory of
Neurosciences, National Institute on Aging Intramural Research Program, Baltimore, MD, USA

Restless legs syndrome (RLS) is a neurological disorder associated with low brain iron levels
whose symptoms are responsive to treatment with dopaminergic agents and intravenous
iron therapy. Iron deprived rats with brain iron deficiency have been found to have
decreased dopamine D2 receptors. Because adenosine can influence dopaminergic
neurotransmissions in the basal ganglia via postsynaptic interactions between adenosine A2a
and D2 receptors, we tested the hypothesis that iron deficiency alters A2a receptor (A2aR)
levels in the striatum. We measured levels A2aR in the striatum of female mice from two BXD
recombinant inbred strains showing different concentrations of striatal iron, viz., 22 vs 12 µg
iron/g tissue. Densitometric immunoblot analysis showed that levels of A2AR levels were
significantly greater in striatal samples from the low-iron strain compared to the high-iron
strain 1 (p <0.01). These results are consistent with our earlier finding that iron deficiency up
regulates A2aR in cell culture models and striatum of iron-deficient mice. At the biochemical
level, it was previously shown that, in crude membrane preparations from rat striatum,
stimulation of A2aR produces a decrease in the affinity of D2 receptors for agonists. Thus, the
observed up regulation of A2aR with decreased iron might be one factor contributing to the
down regulation of D2 receptor protein observed in the iron deficiency model of RLS.
Leptin-Mediated Cell Survival Signaling in Hippocampal Neurons Mediated by JAK /STAT3
and Mitochondrial Stabilization
Zhihong Guo1, Haiyang Jiang1, Xiangru Xu 1, Wenzhen Duan1 and Mark P. Mattson 1, 2. 1
Laboratory of Neurosciences and National Institute on Aging Intramural Research Program,
5600 Nathan Shock Drive, Baltimore, MD 21224. 2 Department of Neuroscience, Johns
Hopkins University School of Medicine, Baltimore, MD 21205.

Leptin plays a pivotal role in the regulation of energy homeostasis and metabolism, primarily
by acting on neurons in the hypothalamus that control food intake. However, leptin
receptors are more widely expressed in the brain suggesting additional, as yet unknown,
functions of leptin. Here we show that both embryonic and adult hippocampal neurons
express leptin receptors coupled to activation of STAT3 and PI3 kinase- Akt signaling
pathways. Leptin protects hippocampal neurons against cell death induced by neurotrophic
factor withdrawal, and excitotoxic and oxidative insults. The neuroprotective effect of leptin
is antagonized by the JAK2-STAT3 inhibitor AG-490, STAT3 decoy DNA and PI3-kinase/Akt
inhibitors, but not by an inhibitor of mitogen-activated protein kinases. Leptin induces the
production of manganese superoxide dismutase and the anti-apoptotic protein Bcl-xL,
stabilizes mitochondrial membrane potential and lessens mitochondrial oxidative stress.
Leptin receptor deficient mice (db/db mice) are more vulnerable to seizure-induced
hippocampal damage, and intraventricular administration of leptin protects neurons against
seizures. Our findings suggest that, by enhancing mitochondrial resistance to apoptosis and
excitotoxicity, leptin signaling serves a neurotrophic function in the developing and adult
The alpha7 subunit of the nicotinic receptor is expressed in a subset of hippocampal
interneurons containing cholecystokinin

Katrina Hein and Marisela Morales

National Institute on Drug Abuse, Intramural Research Program, Cellular Neurophysiology.
5500 Nathan Shock Drive, Baltimore, MD. 21224.

The hippocampus has multiple kinds of nicotinic receptors made of different subunits.
                                                  -             -nAch) subunit is the only one
that makes functional homomeric ion channel receptors, and within the hippocampus is
highly expressed in pyramidal (glutamatergic) neurons. However, we detected high levels of
  -nAch mRNA not only in pyramidal neurons, but also in interneurons in the hippocampus
and the dentate gyrus. We applied a double in situ hybridization procedure to investigate the
phenotype of hippocampal neurons expres
mRNA was detected with radioactive riboprobes, and glutamic acid decarboxilase (GAD;
                           -aminobutyric acid GABA) mRNA was detected with non-
radioactive riboprobes. We found that the vast ma                                         -nAch
mRNA co-                                                                                        -
nAch mRNA resembles that of cells containing cholecystokinin (CCK), we investigated by a
combination of in situ hybridization and immunohistochemistry whether hippocampal
                            -                                                               -nAch
/CCK double labeled cells in the hippocampus, but the proportion of these neurons varied
across the hippocampus (about 70% o
radiatum and s. oriens, 50% in the s. lucidum and 20% in the s. oriens).

subunit are expressed not only in glutamatergic neurons, but also in a subset of GABAergic
                                                                     -nAch receptor is
involved in GABAergic and glutamatergic neurotransmission.

GG Hesketh, M Shah, F Akar, Y Guo, GF Tomaselli, JE Van Eyk, Dept. of Medicine, Division of
Cardiology, Johns Hopkins University, Baltimore, MD, USA

Heart failure (HF) is associated with decreased ventricular conduction velocity (CV)
and an increase in fatal ventricular arrhythmias, due in part to decreased gap junctional
communication. HF is associated with decreased expression and altered subcellular
localization of connexin 43 (Cx43), the major gap junction protein of the ventricle. We
observe that hyperphosphorylated Cx43 targets to lipid raft (LR) domains in the heart,
and this targeting is increased in a canine model of pacing induced HF. In light of
emerging evidence that LRs may underlie a unique endocytic mechanism, we
hypothesize that phosphorylation dependent targeting of Cx43 to LRs may precede
internalization and degradation, and contribute to Cx43 changes in HF. To begin to test
our hypothesis we first attempted to characterize the phosphorylation state of LR
targeted Cx43. We identified 8 phosphorylated residues in immunoprecipitated Cx43
using mass spectrometry. The LR targeted form of Cx43 from both normal and HF dog
hearts contained a cluster of 4 phosphorylation sites (S325, T326, S328, S330). In HF,
T326 was also found to be phosphorylated in non-lipid raft forms of Cx43. T326 is a
novel phosphorylation site. We conclude that 1) Hyperphosphorylated Cx43 targets to
LR 2) LR targeted Cx43 increases in HF 3) Specific phosphorylation sites associate with
LR targeted Cx43, and may be altered in HF. These studies may begin to illuminate
mechanisms of altered Cx43 expression and decreased CV in HF.
FGF-23 Controls NaPiIIa Trafficking Via Crosstalk Between the PI-3 kinase and
MAPkinase Pathways

Suzanne Jan de Beur, M.D.
Department of Medicine - Endocrinology

Fibroblast Growth Factor 23 (FGF-23) is an important physiological regulator of
phosphate homeostasis and its excess or deficiency has been implicated in several human
disorders of phosphate homeostasis. FGF-23 exerts its phosphaturic action by
promoting internalization of the type IIa and type IIc sodium-phosphate co-transporters
(NaPiIIa, NaPiIIc) from the renal brush border membrane and preventing the
compensatory increase in 1,25(OH)2D3 by reducing renal 1α hydroxylase expression.
FGF-23 can bind several known FGF receptors (FGFR) and activate MAP kinase
signaling. However, it is not known which of FGFR signaling pathways mediate the
diverse actions of FGF-23. We sought to distinguish which FGFR signaling pathway
regulates FGF-23-mediated NaPiIIa internalization. Previously, we reported that FGF-23
stimulates ERK1/2 (ERK) phosphorylation and that inhibiting ERK signaling with
U0126 abolished FGF-23-mediated NaPiIIa internalization in opossum kidney cells
(OK/e). To interrogate other FGFR signaling pathways such as the phosphatidylinositol-
3 kinase (PI-3 kinase) pathway, we measured phosphorlation of Akt by immunoblot but
observed no increase in pAkt upon stimulation with FGF-23 (0.1 ng/ml-100ng/ml). Yet,
surprisingly, inhibition of PI-3 kinase with LY294002 partially blocked FGF-23-
mediated NaPiIIa internalization. In tyrosine kinase receptor signaling in general and
FGFR signaling specifically, the MAP kinase pathway may be activated by a number of
upstream molecules including Ras, phospholipase C (PLC via protein kinase C
(PKC), and PI-3 kinase via PDK-1. In order, to further dissect the inputs to the MAP
kinase pathway, we measured pERK by immunoblot in FGF-23 stimulated OK/e cells
treated with the farnesyl transferase inhibitor (FTI-277) that induces accumulation of
non-farnesylated cytoplasmic Ras to form inactive Ras/Raf complexes. OK/e cells were
preincubated with 2 m FTI-277 for 1-24 hrs and then stimulated with FGF-23 (10
ng/ml) or PTH (1-84, 10-7M). MAP kinase activation was measured by immunoblot of
whole cell lysate with a pERK antibody. In OK/e cells treated with FGF-23, FTI-277 did
not appreciably reduce pERK compared to untreated FGF-23-stimulated cells. In
contrast, PTH-stimulated ERK phosphorylation was completely abolished with FTI-277
treatment. U0126 blocked both FGF-23 and PTH-stimulated ERK phosphorylation.
Taken together, these data suggest that the PI3 kinase pathway regulates FGF-23-
mediated NaPiIIa internalization through crosstalk with the MAP kinase pathway and
that this is a distinct signaling pathway from PTH-mediated NaPiIIa trafficking.
Effect of Regional Volume Changes on PET Activation Patterns in the Aging Brain

Sojkova Jitka 1,2, Kraut Michael A2, Beason-Held Lori1,2, Wu Xiaoying3, Davatzikos Christos3,
Resnick Susan M1

 Lab of Cognition and Personality, Intramural Research Program, National Institute on Aging,
Baltimore, MD

Russell H. Morgan Department of Radiology and Radiological Sciences,          Johns Hopkins
University School of Medicine, Baltimore, MD

 Section on Biomedical Image Analysis, Department of Radiology, University of Pennsylvania,
Philadelphia, PA

Background: While longitudinal decreases in brain volume have been documented to occur
in nondemented elderly, the relationship between these volumetric changes and longitudinal
changes in PET activation patterns remains unclear.

Methods: 99 nondemented elderly participants (68.99 +/- 7.33 years old at baseline) in the
Baltimore Longitudinal Study of Aging underwent up to 9 years of structural MRI and
[15O]H2O PET imaging during a resting condition and during performance of verbal and figural
delayed recognition memory tasks. Effects of brain volume changes on linear changes in the
task specific PET activation patterns (task recognition – rest) from years 1 to 9 were assessed
using voxel-based analysis, adjusting for baseline age and health status at each visit.

Results: Initial evaluation of rCBF changes specific to performance of the verbal memory
task revealed longitudinal increases in left cuneus (BA18) and right orbitofrontal (BA11) areas,
whereas longitudinal decreases were noted in right superior temporal (BA22) and right
inferior frontal (BA47) regions. Changes in rCBF specific to the figural task consisted of
longitudinal increases in left cuneus (BA18), left precuneus (BA 7), and anterior cingulate (BA
24) cortex whereas decreases were noted in right superior temporal (BA22) and inferior
frontal (BA45) cortex. When volume changes in these regions were taken into account, no
significant differences in the patterns of rCBF increase or decrease were observed for either
verbal or figural conditions.

Conclusion: In the two recognition tasks tested, longitudinal changes in brain tissue volume
had no significant effect on the spatial extent and location of brain activation changes that
occurred over the 8 years of follow-up. This finding suggests that localized longitudinal
volume changes do not substantially influence longitudinal functional changes in this sample
of nondemented older individuals.

This research was supported by the Intramural Research Program of the NIH, National
Institute on Aging.
Quantitative image pattern analysis of tissues in C. elegans identifies distinct
morphological states during aging

Josiah Johnston, Wendy Iser, Catherine Wolkow and Ilya Goldberg
National Institute on Aging, Intramural Research Program, NIH,
Baltimore, MD USA

Organ development and aging can be assessed by changes in tissue
morphology and function. Quantification of morphological change
usually consists of assessing individual markers at discrete
intervals. An alternative approach is to consider changes in overall
structure rather than specific markers. Here, we describe a whole-
image analysis strategy based on pattern recognition that can
quantitatively track progression through global morphological
change. We have used this approach to study age-associated changes
in the morphology of the C. elegans pharynx, a neuromuscular organ
involved in feeding. Pattern analysis of pharynx images obtained by
differential interference contrast microscopy identified distinct
structural states representing progression from early adulthood to
mid-life and older ages. The patterns of morphology were altered in
mutants affected in pharynx function. A similar analysis using
longitudinal data identified a relationship between mid-life pharynx
morphology and pharynx function at later ages. In summary, pattern
analysis of an image series offers a novel and generally accessible
approach for quantifying morphological changes and identifying
structural biomarkers.
Analysis of the structure and function ATP Synthase complex from site specific yeast
mutants of the β Subunit mimicking known phosphorylations.

Kane, LA1, Youngman, MJ2, Jensen, RE2, and Van Eyk, JE1,3. Johns Hopkins University,
Departments of 1Biological Chemistry, 2Cell Biology and 3Medicine.

Introduction: Recently, we discovered that the β subunit of the mitochondrial ATP synthase
undergoes modification upon a 60 minute treatment of myocytes with adenosine and
identified 5 novel phosphorylation sites on the protein. Two sites are buried within the ATP
synthase complex and the others are located on the external face. The functional
consequences of phosphorylation and complex assembly are assessed. Methods: A model
system, S. cerevisiae, was chosen for high sequence homology of the β subunits and for ease
of cloning protocols. Non-phosphorylatable (S/T to A) and pseudophosphorylated (T to E or S
to D) analogs of 4 sites were created, T91, S246, T295 and T351. Isolation of intact F1/Fo
complex was performed using a sucrose centrifugation for all strains and equal protein
amounts were used for future assays. Strains were compared to WT and a deletion strain for
ATPase activity of isolated complex (measuring release of Pi from ATP), and complex
assembly (whole mitochondrial Blue Native (BN)-PAGE). Results: On non-fermentable media
all strains had WT growth, except the T295E strain, which has decrease growth. ATPase
assays (n=6) on T295E strain showed a significant reduction in activity compared to WT (0.01
± 0.004 and 0.1 ± 0.01 respectively, p<0.0001) and was equivalent to a deletion strain. Both
internal strains T351A and T351E, had significantly decreased function (0.041 ± 0.006 and
0.035 ± 0.007, p<0.0001). One external site strain T91E had significantly decreased function as
compared to both the T91A and WT. BN-PAGE gels revealed a complex assembly defect in the
T295E, T351E mutants which lack the free F1 component, normally found in abundance. Other
strains had small changes in assembly. Conclusions: This data suggests that ATP synthase can
be modulated by phosphorylation (both activity and assembly) and may have implications to
preconditioning where the phosphorylations were first identified.

This work supported by: This work is supported by an American Heart Association Pre-
doctoral Fellowship Award (Lesley Kane #0715247U) and by the NIH contract #P01HL081427
(Jennifer Van Eyk).
In vitro and in vivo induction of B7-H1 and B7-DC
expression by human rhinovirus fnfection of airway
epithelial cells

Jean Kim1, Lowella F Heinecke2, David Proud3, Robert P
Schleimer4. 1Otolaryngology and Medicine, Johns Hopkins
University School of Medicine, 5501 Hopkins Bayview
Circle, Rm 3B65A, Baltimore, MD, 21224, 2Medicine,
Johns Hopkins University School of Medicine, 5501
Hopkins Bayview Circle, Baltimore, MD, 21224,
3Department of Physiology & Biophysics, University of
Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N
4N1, Canada, 4Allergy-Immunology, Northwestern
Feinberg School of Medicine, 240 E. Huron St., Room M-
318, Chicago, IL, 60611

We have found that human airway epithelial cells express
costimulatory molecules B7-H1, B7-H2, B7-H3 and B7-
DC mRNA and cell-surface protein. IFN! and TNF"
selectively induce B7-H1 and B7-DC and glucocorticoid
fluticasone (FP) inhibits this induction. We now report that
human rhinovirus infection (HRV-16), a key trigger of
exacerbations of chronic rhinosinusitis and asthma, results
in selective induction of B7-H1 and B7-DC expression in
airway epithelial cells both in vitro and in vivo. In vitro
exposure of human primary bronchial epithelial cells
(PBEC) to HRV-16 (TCID50 5 X 103.1) resulted in
induction of cell surface expression of B7-H1 as measured
by flow cytometry (from 42±9 to 56±8 MFI, p<0.05) and
B7-DC (from 5±1 to 9±2 MFI, p<0.01) (n=6).
Pretreatment with FP (10-7 M) inhibited the induction of
B7-H1 by 64% (p<0.05) and B7-DC by 95% (p<0.01).
Additionally, in vitro exposure of PBEC to TLR3 agonist
dsRNA (25 ug/ml), a surrogate for HRV16 infection,
mirrored the effect of HRV-16 infection. Nasal scrapings
taken at the time of peak symptom scores 4 days after
infection of 6 human subjects with HRV-16 showed
selective increases in levels of mRNA for B7-H1 (8.5±2.5
fold, p<0.03) and B7-DC (3.2±1.1 fold, p<0.07). These
data show that exposure to TLR3 agonist dsRNA or HRV-
16 infection induces B7-H1 and B7-DC on epithelial cells
and this may influence the development of adaptive
immune responses in the airways.
The Good News about CPOE and Medical Student Ordering Ability

Amy M. Knight, MD1, Steven J. Kravet, MD1, G. Michael Harper, MD2, Dmitry Kiyatkin, MD1,
Bruce Leff, MD1
    Johns Hopkins Bayview Medical Center, Baltimore, MD
    University of California, San Francisco, CA

When order quality was compared for medical students who trained at hospitals using CPOE to those who
trained using handwritten orders, no significant differences were found.
Introduction: Some educators believe CPOE may enhance medical students’ learning experiences by
exposing them to recommended standards of care and decision support. However, others have
expressed concern that medical education is hindered by CPOE, by potentially decreasing time for
interaction between students and housestaff, and by automating the placement of certain orders for
particular situations.
Methods: Subjects included 136 Johns Hopkins University School of Medicine students who began the
two-month Basic Medicine clerkship between March 2003 and April 2004. Demographic information
was collected at the beginning of the clerkship. Students spent the first half of the clerkship either at
a hospital using a home-grown CPOE system (69 students), at a hospital using only paper orders (22
students), or at a hospital that switched midway through the study period from paper orders (22
students) to a commercial CPOE system (23 students). After the first month of the clerkship students
were asked to write mock admission orders for a hypothetical elderly patient with pneumonia,
hypoxia, uncontrolled diabetes, mild renal failure, and evidence of dehydration. A group of internal
medicine faculty and fellows identified orders that were at least moderately important should such a
patient actually be admitted to the hospital. The recommended orders were grouped according to
whether they were Basic Orders (ADC VAA DIML), Lifesaving Orders (acceptable oxygen therapy,
acceptable IVF type and rate, appropriate antibiotic chosen, and acceptable dose, route and frequency
for the antibiotic), and Other Higher Level Orders.
Using a tool with high inter-observer reliability, student orders were rated for the presence or absence
of each of the recommended orders, and combined scores calculated for each student. Score
distributions were dichotomized at the median, and Chi Square tests were used to compare students
who trained using paper orders (n=44) to those who trained using CPOE (n=92).
Results: 59% of the students were male, 56% white, and 87% in their 3 rd year of medical school. 120
students (88%) attempted to write orders for the mock patient. 63% of students included 9 or more of
the 10 Basic Orders in their mock admission orders. 62% included at least one of the five Lifesaving
Orders. 61% included 7 or more of the 14 Other Higher Level Orders. 57% included 17 or more of all 29
orders recommended by the faculty and fellows for this patient. Students who trained at hospitals
using CPOE were just as likely as those who trained using paper orders to attempt to write orders
(p=0.50) and to just as likely to include Basic Orders (p=0.28), Lifesaving Orders (p=0.85), Other Higher
Level Orders (p=0.75), and all recommended orders (p=0.93). When students who trained at the
hospital that switched from paper orders to CPOE were compared to each other, no significant
differences were found.
Conclusion: Fears concerning the effects of CPOE on medical student learning experience and abilities
do not appear to be justified. Multi-institution studies are warranted, however, as CPOE use increases
at teaching hospitals across the country.
Qualitative Assessment of the Long-term Impact of a Faculty Development Program in
Teaching Skills

Amy M. Knight, MD, Joseph A. Carrese, MD, Scott M. Wright, MD

Introduction: The long-term impact of faculty development programs is poorly understood,
and most assessments of them have been quantitative in nature.

Methods: A survey was done in July 2002 of the 242 faculty members and fellows who had
participated in a nine-month faculty development program in teaching skills from 1987
through 2000. The survey included two quantitative questions and an open-ended
qualitative question about the impact of the program on their professional and personal

Results: Two hundred past participants (83%) responded to the survey. Participants from
early and recent cohorts were similarly represented. Eighty-two percent of respondents said
program participation had “a moderate” or “a lot” of impact on their professional life, and
49% said their personal life had been affected to this degree. Four major domains, each
containing three or more subcategories, emerged from qualitative analysis. The domain
intrapersonal development included changes participants reported in themselves and in their
approach to self-management. Interpersonal development contained subcategories relating
to how participants interact with others. Subcategories in the domain development as a
teacher related to increased teaching ability and enjoyment. The domain career
development included professional growth and career opportunities attributed to program

Conclusions: Longitudinal faculty development programs can have broad and sustained
positive effects on the professional and personal lives of participants. Qualitative evaluation
methods may result in a richer and deeper understanding of the impact of these programs.

Recently published as: Knight AM, Carrese JA, Wright SM. Qualitative Assessment of the
Long-term Impact of a Faculty Development Program in Teaching Skills. Medical Education
2007; 41:592-600.
Medial prefrontal cortex activity and its role in incubation of
cocaine craving

Eisuke Koya, Jamie L. Uejima, Jennifer M. Bossert, Yavin Shaham*,
Bruce Hope

Behavioral Neuroscience Branch, NIDA/IRP/DHHS, National Institutes of
Health, Baltimore, MD 21224

Re-exposure to cocaine-associated cues induces craving in human
addicts and cocaine seeking in laboratory animals. Cue-induced
cocaine seeking progressively increases with cocaine withdrawal
duration, a phenomenon we termed “incubation of cocaine craving”. We
previously found that exposure to cocaine cues in extinction tests
increases ERK phosphorylation (a marker of neuronal activity and
plasticity) in the dorsal and ventral medial prefrontal cortex after
30 but not 1 d of withdrawal from cocaine self-administration (Koya
et al. SFN, 2006). Here, we assessed whether increased ERK activity
in the medial prefrontal cortex mediates enhanced cue-induced cocaine
seeking after 30 withdrawal days by using U0126, an inhibitor of ERK
phosphorylation. We trained rats to press a lever for cocaine for 10
days (0.75 mg/kg/infusions, 6 h/d); cocaine infusions were paired
with a tone-light cue. We then assessed cue-induced cocaine seeking
in 30 min extinction tests after 30 days of withdrawal from self-
administered cocaine. Different groups of rats were injected with
vehicle or U0126 (1.0 microgram/side) into the dorsal or ventral
medial prefrontal cortex. We found that U0126 decreased p-ERK
immunoreactivity in the dorsal and ventral medial prefrontal cortex,
but had no effect on cue-induced cocaine seeking in the extinction
tests. Together with our previous findings, our results suggest that
cue-induced ERK activation in the prefrontal cortex does not mediate
the expression of incubation of cocaine craving. We currently use a
mixture of baclofen and muscimol to explore whether generalized
inhibition of medial prefrontal cortex activity attenuates enhanced
cocaine seeking after 30 withdrawal days.
Donor Specific Antibody (DSA) and C4d + in Surveillance Biopsies
Following Positive Cross Match Live Donor Kidney Transplantation.

Edward Kraus,1 Diane Lepley,2 Dorry Segev,2 Mary Leffell,1 Donna Lucas,1
Mark Haas,3 Lorraine Racusen,3 Serena Bagnascu,3 Vanessa Collins,2 Chris
Simpkins,2 Dan Warren,2 Robert Montgomery,2 Andrea Zachary.1 1Medicine,
Johns Hopkins, Baltimore, MD; 2Surgery, Johns Hopkins, Baltimore, MD;
3Pathology, Johns Hopkins, Baltimore, MD.

Background: The incidence and determinants of subclinical Antibody Mediated
Rejection (AMR) following kidney transplantation in high risk positive cross match
(+XM) recipients is unknown. If recognized early, injury due to AMR might be
modifi able if not preventable. Methods: We reviewed 82 protocol Bx obtained 1, 3,
6, and 12 months post transplant from 90 +XM live donor kidney transplant recipients
(10/1/99 - 11/8/05) to determine the incidence of C4d + as a marker of potential
subclinical AMR. In all cases, the presence/absence of donor-specifi c antibody (DSA)
was known. Bx were excluded for clinical cause, ie. rising serum creatinine, failure of
serum creatinine to fall to expected level, follow-up of Rx of rejection, and proteinuria.
All patients underwent desensitization with plasmapheresis, CMV Ig, and in some
cases anti-CD20 antibody and/or splenectomy. Maintenance immunosuppression was
prednisone, tacrolimus or sirolimus and MMF. Results: There was no association
between the presence of DSA and subclinical cellular rejection (Banff > 1a). 30% of
the Bx were C4d + (1m 29%, 3m 32%, 6m 22%, 12m 39%). Although DSA was present
with all C4d+ Bx , 51% of DSA + Bx (1m 65%, 3m 46%, 6m 60%, 12 m 22%) were
C4d-. Comparing DSA+ C4d+ Bx (17 pts) with DSA+ C4d- Bx (16 pts), there were no
differences in donor and rec age, gender, race, relationship, cause ESRD, # transplants,
CMV status, HLA mm. For C4d+ Bx vs C4d - Bx strength of DSA, as determined by
reactivity in a cytotoxicity XM, was higher pretreatment (p<0.025) but not at the times
of transplantation or Bx. DSA to DRw51-53 pretreatment was present for 47% C4d+ but
only 19% C4d- recipients.(p=NS). Conclusions: There is good concordance between
the presence of DSA and C4d+ staining on protocol biopsies. For +XM recipients, the
strength of DSA prior to desensitization and possibly the specifi city of DSA are risk
factors for development of C4d+. Potentially, with further defi nition of these factors,
monitoring protocols could be refi ned to prevent or ameliorate antibody mediated
processes leading to CAN and transplant GN.

Presented American Transplant Congress San Francisco CA, May 4-9, 2007
Inadvertent self-healing in desperate times
Sean X Leng, Thomas Finucane, Lisa Boult, Larry Zheng, William B Greenough III
Lancet 2007; 370: 1458

Johns Hopkins University School of Medicine, Baltimore, MD, USA (S X Leng MD,
Prof T Finucane MD, L Boult MD, L Zheng MD, Prof W B Greenough III MD)
Correspondence to:
Dr Sean X Leng, Johns Hopkins
University School of Medicine,
5505 Hopkins Bayview Circle,
Baltimore, MD 21224, USA

In February, 2007, a 47-year-old woman was admitted to our hospital, with stage IV pressure
ulcers. She had become paraplegic in 1999, after a bullet damaged the spine at the level of
the 9th thoracic vertebra. Pressure ulcers developed in 2006. In December, 2006, the patient
devel oped a high fever and was found to have worsening pressure ulcers and meticillin-
resistant Staphylococcus aureus bacteraemia. Her condition improved with intravenous
vancomycin and wound debridement. Because she was doubly incontinent, a Foley catheter
was inserted, and a colostomy was done to reduce wound contamination. The patient was
transferred to a nursing home, but developed intermittent fever, anorexia, and severe left
hip pain. After several weeks, she was transferred to our hospital.

On admission, she was cachectic, at 48·2 kg, and had five large stage IV pressure ulcers on
the lower back and hips (fi gure). Her white-blood-cell count, and serum concentration of C-
reactive protein (CRP) were 14·2×109 per L, and 167 mg/L, respectively. Her serum
concentrations of haemoglobin and albumin were only 87 g/L and 19 g/L, respectively. We
started to treat her with vancomycin. For wound care, we used an antimicrobial gel. CT
showed erosion of the femoral head and neck, inferior pubic ramus, and acetabulum, with fl
uid and air in the left hip joint. CT-guided aspiration yielded clear liquid, cultures of which
were negative. Despite treatment, the patient’s condition worsened. The hip pain and fever
continued; the white-blood-cell count and serum concentration of CRP rose as high as
18·5×109 per L and 257 mg/L, respectively; the serum albumin concen tration fell to 13 g/L, and
the patient’s weight to 43·6 kg. 6 weeks after admission, radiography showed that the
femoral head and neck were separated from the rest of the femur, and medially displaced.
Hours later, a bone fragment fell out of a pressure sore, while the dressing was being
changed. The patient’s condition then started to improve. The fever and pain ceased
immediately, and the pressure ulcers began to heal. 8 weeks after the fragment fell out, the
patient’s weight had increased to 58·2 kg; the serum
concentrations of haemoglobin and albumin were 128 g/L and 32 g/L, respectively; the white-
blood-cell count and serum concentration of CRP were 8·25×109 per L and
22 mg/L, respectively. At the end of August, 2007, the patient remained in hospital; she had
no fever, her weight was stable at 59 kg, and her wounds continued to heal.

Pressure ulcers are common in elderly people, and people with spinal-cord injuries.1,2 For
several years after being shot, our patient had been cared for by relatives and had been able
to live at home. When a close relative died, the standard of care provided by her family
decreased substantially, and a fragmented health system did not fully meet her needs. We
think that the osteomyelitis was caused by infection spreading from an ulcer, either directly
or through the bloodstream.3 The infected proximal femur developed avascular necrosis.
Because the blood supply to the proximal femur was so poor,
intravenous antibiotics were largely ineffective. Ordinarily, we would have done an excision
arthroplasty, known as the Girdlestone procedure,4 to remove the infected bone.
However, the patient was so ill that surgery was judged to be unaccept ably risky.
Fortunately, the necrotic bonedropped off , eerily mimicking the Girdlestone procedure.

1 Byrne DW, Salzberg CA. Major risk factors for pressure ulcers in the
spinal cord disabled: a literature review. Spinal Cord 1996; 34: 255–63.
2 Bates-Jensen BM. Quality indicators for prevention and
management of pressure ulcers in vulnerable elders.
Ann Intern Med 2001; 135: 744–51.
3 Lew DP, Waldvogel FA. Osteomyelitis. Lancet 2004; 364: 369–79.
4 Horan FT. Robert Jones, Gathorne Girdlestone and excision
arthroplasty of the hip. J Bone Joint Surg Br 2005; 87: 104–06.

Lixin Liu, Guy P Marti, Xianjie Zhang, Xiaofei Wei, Huafeng Zhang, Ye V Liu, Manuel Nastai,
Stephen Milner, Gregg L Semenza, John W Harmon

Introduction: We explored the possibility that age induced impairment in cutaneous wound
healing in diabetic mice would correlate with reduced production of the transcription factor
Hypoxia Inducible Factor-1 Alpha (HIF-1α) and its downstream targets.

Methods and Results: Real time reverse transcriptase PCR assessment was carried out at
intervals in wound tissue from young (6-8 week) and older (18-21 week) mice. HIF-1α mRNA
levels after wounding were greater in younger as compared to older diabetic animals;
Unwounded 26±7 vs 1± 0, day 3 after wound 41± 3 vs 15± 9, and day 5 after wound 168± 55 vs
13± 4 (ANOVA and Tukey p< .05). The mRNA levels for HIF-1-regulated gene products,
including VEGF, Angiopoietin 1, Angiopoietin 2, PDGFB and PLGF, were also diminished in the
older animals, suggesting that inadequate production of HIF-1α may explain the impairment
in wound healing in older diabetic mice. We then assessed if electroporation assisted HIF-1α
plasmid transfection could improve wound healing in diabetic mice. We injected gWIZ-CA5, a
constitutively active form of HIF-1α, intradermally followed by electroporation. The gWIZ-CA5
induced a 700-fold increase in HIF-1α mRNA at the injection site at day 3. To assess the effect
of HIF-1 α on wound healing, four 5mm diameter wounds were created on the dorsum of
diabetic mice. The wound area in the gWIZ-CA5 treated group was statistically smaller than
that of the empty vector control group at days 5, 7 and 10. Over 60% of wounds were 95%
healed in the presence of HIF-1 α versus 12% in the empty vector treated group by day 10.

Conclusion: Taken together these findings suggest that HIF-1 α CA5 has potential to improve
wound healing that has been impaired by diabetes.

Acknowledgements: Support by American Diabetes Association grant 1-05-RA-50 and the
Hendrix Burn Fund
The Carotid Revascularization Endarterectomy vs. Stenting Trial (CREST)

Hopkins Principal Investigator: Mahmoud B. Malas M.D. Assistant Professor, Chief of
Endovascular Surgery, Johns Hopkins Bayview Medical Center.
Contact: Tel: 410-550-5332 email:

Hopkins Co-Investigators: Co-Investigators: Eric Aldrich M.D, James Black M.D., Heitham
Hassoun M.D., Bruce Perler M.D., Rafael Llinas M.D.

Hopkins Study Coordinator: Umair Qazi M.D., M.P.H.

BACKGROUND: Carotid endarterectomy (CEA) and best medical therapy have been proven
superior to best medical therapy alone for symptomatic (>50%) and asymptomatic (>60%)
stenosis. Carotid angioplasty with stent (CAS) offers a less invasive alternative. However
establishing safety, efficacy, and durability of CAS requires rigorous comparison with CEA.

OBJECTIVE: The Carotid Revascularization Endarterectomy vs. Stenting Trial (CREST), is
a prospective, randomized, parallel, two-arm, multi-center clinical trial funded by NINDS
comparing the efficacy of carotid artery stenting (CAS) to carotid endarterectomy (CEA)
in preventing stroke, myocardial infarction and death in patients with symptomatic
carotid stenosis >50% or asymptomatic carotid stenosis >60%

INTERVENTION(S): Carotid Endarterectomy and Carotid Artery Stenting.
The devices approved for use in the trial are the RX ACCULINK™ and ACCULINK™ Carotid
Stent Systems and the RX ACCUNET™ and ACCUNET™ Embolic Protection Devices.

POPULATION STUDIED: Symptomatic patients with TIA, amaurosis fugax, or non-disabling
stroke within 180 days of randomization and ipsilateral carotid stenosis ≥50% and
asymptomatic patients without recent (within 180 days) transient ischemic attack or non-
disabling stroke with ipsilateral carotid stenosis of ≥60% are eligible for randomization.
About 2500 subjects would be randomized.

OUTCOME MEASURE(S): Primary endpoints are a composite of stroke, myocardial
infarction or death within 30 days, and stroke ipsilateral to the study artery after 30 days.
Follow-up includes alternating clinic visits or telephone contact every three months.

TRIAL STATUS: CREST received a favorable review by NINDS in late 2006 and is slated for
renewal for 2007 through 2011. Enrollment in the lead-in phase is now complete and the
preliminary results of the lead-in phase are also now available. Current enrollment is about
2000 patient with a target of 2500. CREST is the largest randomizing trial to address the
efficacy of CAS versus CEA with 2000 randomized and 1301 participants in the lead-in

Trial Web Site:
Long-stay nursing home residents who may not require institutional care.

M McNabney, J Wolff, L Semanick, J Kasper, C Boult. Johns Hopkins University, Baltimore,

Purpose: To estimate the percentage of older long-stay nursing home (NH) residents who
have minimal functional dependency and to describe their characteristics.

Methods: Analysis of cross-sectional data from the 1999 National Nursing Home Survey. We
classified long-stay (>100 days) older NH residents as either high-functioning (0-2 ADL
dependencies) or low-functioning (3-5 ADL dependencies). To further explore the high-
functioning residents’ need for NH care, we then determined whether each had recently
received specified rehabilitative and skilled nursing services (PT, OT, ST, mental health
services, severe vision or hearing impairment, catheter or ostomy use) or had a mobility
impairment or a mental disorder (including dementia).

Results: Within this nationally representative sample of older long-stay NH residents, 19.8%
were dependent in 0-2 ADL. These high-functioning residents had a mean age of 83.8 years
and a mean length of NH stay of 961 days; 72% were female, 90% were white, and 60% were
Medicaid recipients. Among these high-functioning residents, 13.1% (2.6% of the total sample)
had not received any of the specified rehabilitative or skilled nursing services in the past 30
days and did not have a mobility impairment or mental disorder/dementia. Among the
remaining 86.9% of high-functioning residents, 38% had received rehabilitative/skilled nursing
services recently, 39% had impaired mobility and 62% had a mental disorder/dementia.

Conclusions: One-fifth of the nation’s 1.2 million long-stay NH residents have minimal
functional dependency, suggesting that many could be served well in community-based,
rather than institutional, settings. Identifying and focusing attention on newly admitted NH
residents who have significant potential for discharge may increase their rate of return to the
community – and decrease the nation’s population of (and financial burden for) long-stay NH

Eric T. Moolchan, MD, Craig S. Parzynski, BA, Debra L. Zimmerman, RN, BSN, Maria Jaszyna-
Gasior, MD, PhD

Adult slow nicotine metabolizers smoke fewer cigarettes daily and have lower smoke
exposure, carbon monoxide and plasma nicotine levels compared with normal and fast
metabolizers. Linked to this, evidence is emerging that nicotine metabolism might also
influence smoking topography (mean and total puff volume), a quantifiable measure of
smoking behavior. The present study investigated the association of adolescent nicotine
metabolism with smoking topography in a sample of tobacco cessation treatment-seeking
adolescent smokers.. The ratio of 3-hydroxycotinine to cotinine (3OHCOT/COT) served to
quantify nicotine metabolism. Smoking topography measures obtained prior to treatment
included mean puff volume, total puff volume, inter-puff interval, puff duration, and puff
velocity. Participants were 77 adolescent smokers (66% female, 66% European American,
mean age 15.2 ± 1.2, mean Cigarettes Per Day (CPD) 19.0 ± 8.6, mean Fagerström Test for
Nicotine Dependance (FTND) 7.0 ± 1.2) who took part in a three-arm randomized double-
blind, double-dummy, placebo-controlled nicotine replacement therapy trial. Partial Pearson
correlation coefficient analysis controlling for CPD, ethnicity and gender indicated that the
relationship between 3OHCOT/COT and puff volume (r = .249), 3OHCOT/COT and total puff
volume (r = .218) approached significance (p = .053, p = .081 respectively) suggesting that as
nicotine metabolism increases, puff volume and possibly smoke exposure also increase. No
significant relationship between 3OHCOT/COT and puff duration, inter-puff interval or puff
velocity was found. These findings suggest that, as among adult smokers, the rate of
metabolism may alter select parameters of smoking behavior among dependent adolescent
smokers. A broader sample of adolescent smokers is necessary to conclude on the
relationship between rate of nicotine metabolism and smoking behavior.

Keywords: adolescent, metabolism, nicotine, smoking

Corresponding author: Eric T. Moolchan, Teen Tobacco Addiction Treatment Research Clinic.
5500 Nathan Shock Dr., Baltimore, MD, 21224

Differential effects of the orexin 1 receptor antagonist SB 334867 on high-fat food self-
administration and reinstatement of food seeking in rats

Sunila G. Nair, Sam A. Golden and Yavin Shaham

Behavioral Neuroscience Branch, NIDA/IRP/NIH/DHHS, Baltimore, MD

Background and purpose: Many studies have demonstrated a role of orexin 1 (hypocretin 1)
receptors in home-cage food consumption in rodents. However, the role of these receptors
in operant food self-administration or relapse to food seeking in animal models is unknown.

Experimental approach: In Experiment 1, we trained food-restricted rats (16-20 g/day) to
lever press for high-fat (35%) pellets (3-6 h/day, every other day). We then tested the effect
of the orexin 1 receptor antagonist SB 334867 (10, 20 mg/kg, i.p) on pellet self-administration.
In Experiment 2, we trained rats to self-administer pellets, and following extinction of the
food-reinforced responding, we tested the effect of orexin A (3, 6 μg, i.c.v) on reinstatement
of food-seeking and the effect of SB 334867 on this reinstatement. In Experiment 3, we
tested the effect of SB 334867 on reinstatement induced by non-contingent pellet exposure
(pellet-priming) or the pharmacological stressor yohimbine (2 mg/kg, i.p).

Key results: SB 334867 attenuated high-fat pellet self-administration. In contrast, SB 334867
had no effect on reinstatement of lever presses induced by orexin A, pellet-priming or

Conclusions and implications: These data indicate that during dieting, orexin 1 receptors
contribute to operant high-fat pellet self-administration, but not in reinstatement of food
seeking induced by acute re-exposure to the food itself or by the induction of a stress-like
state by yohimbine.
The association of endogenous sex hormones and the metabolic syndrome in men and women: The
Multi-Ethnic Study of Atherosclerosis (MESA)

Pamela Ouyang1, Dhananjay Vaidya1, Sherita H. Golden1, Moyses Szklo1, Adrian Dobs1, Kiang Liu2, Pamela
J. Schreiner3, Susan M. Gapstur2. 1Johns Hopkins University, Baltimore, MD, 2Northwestern University,
Chicago, IL, 3University of Minnesota, Minneapolis, MN.

Background: There are conflicting reports regarding the role of endogenous sex hormones and
metabolic risk factors. We investigated the cross-sectional association of the Metabolic Syndrome
(MetSyn) with endogenous sex hormones (total testosterone, T; estradiol, E2;
dehydroepiandrostenedione, DHEA; sex hormone binding globulin, SHBG).

Methods: We studied postmenopausal women who were not on hormone therapy (n=2041) and men
(n=3149) at the baseline visit of MESA, a NIH multicenter cohort study of individuals without clinical
cardiovascular disease. Association of MetSyn presence (NCEP-ATP-III) with all log-transformed sex
hormone levels was estimated using logistic regression adjusted for age, ethnicity (White, Chinese,
Black, Hispanic), current smoking, and lipid lowering medication use. Interactions by sex were formally
tested and sex-stratified analyses are presented. Similar analyses were performed for each component
of MetSyn, adjusting for all other components.

Results: The figure shows that E2 was directly and SHBG was inversely associated with the odds of
MetSyn in both men and women, DHEA and T were inversely associated with MetSyn in men only (sex
interaction significant for T). This qualitative interaction of T association with better metabolic status in
men was seen for the components of abdominal obesity (p<0.001), impaired fasting glucose (p=0.004),
and low HDL-cholesterol (p=0.050), independent of the other metabolic components. The T associations
did not differ by race/ethnicity.

Conclusion: Lower E2 and higher SHBG are associated with better metabolic status in both
men and women. Higher androgenicity is independently associated with lower metabolic
component presence and MetSyn in men, but not in women.

Regression s (% difference in odds of MetSyn per 1% higher hormone level)
  2%      T            E2         DHEA       SHBG

              ***            ***
                                   ***                          *               Men

   0%                                                                           Women
                   *<0.05, **<0.01, ***<0.001
   -2%                                                              ***
The perceived change of diverse clinical-educators through an intensive course on teaching

Park E, Christmas C, Schmaltz HN, Durso SC.International Journal of Self-Directed Learning.

The short-term intensive course has been adopted as an efficient model in continuing
education for busy professionals. Literature reports learner perception is an important
aspect to determine the success of education through the model. The purposes of this study
were to describe how diverse clinician-educators experience the perceived change through
such model, the Geriatrics Mini-Fellowship (GMF), and to explore emerging patterns linking
learner autonomy to selected learning outcomes through longitudinal observations. The
purposeful sample for this qualitative study consisted of ten clinician-educators from
different disciplines, who reported their perceived changes during GMF. A hermeneutic
phenomenological study approach was used for analyses. In essence, five core themes
characterized the experience of change through GMF. For the group, learner autonomy
appeared to associate with some immediate program outcomes but not with the longer
outcomes of the target teaching behaviors.
A case study of educational practices for the aging global population.

Park E, Durso SC, Confessore GJ.Lifelong Education and Libraries 2006;6:67-79.

Over the past half century, the population of older adults has increased substantially. The
United Nations projects continued rapid worldwide growth of this group. Adult educators
need to understand the educational needs of such a rapidly growing population. Although
many leaders in the adult education sector have discussed this issue, there is little
comprehensive information about the global spectrum of current educational needs and
practices for this population. In order to understand these contemporary global practices, a
representative global organization was studied regarding their activities on this issue. A
purposeful qualitative sample of 163 cases addressing the contemporary multi-sector
educational needs of the elderly was identified from the 3159 presentation abstracts
published in the proceedings of The 2001 17th International Association of Gerontology
World Congress (now International Association of Gerontology and Geriatrics). The authors
concentrated on assessing abstracts of presentations by authors from the East Asia Forum
on Adult Education (EAFAE) membership countries and special administrative regions (SAR)
versus the remaining regions of the world. EAFAE's membership includes China, Hong Kong
S.A.R., Japan, Korea, Macau S.A.R., Singapore, and Taiwan.
Learner autonomy and global competitiveness.

Park E. Korean Federation of Lifelong Learning, Seoul, Korea 2006 December.

Global competitiveness depends of the capacity of the workforce to engage in effective and
efficient lifelong learning. The literature indicates that enhance learner autonomy is a
necessary condition to achieve optimal learning productivity including areas of employment
stability and performance over the lifespan. This featured presentation introduced a model
that clarifies the relationship of learner autonomy, the principle of double-loop learning, and
productive lifelong learning outcomes. Simply stated, individuals who hold high levels of
autonomy can maximize the effect of learning ,as they engage in double-loop thinking. This
simultaneously provides learning about the content and learning about the way they learn.
The author assessed learner autonomy among 600 male and female professional-degree
holders in East Asia and the United States. The data indicated that American adult learners
showed relatively higher degrees of learner autonomy compared to East Asians. In the East
Asian sample, men were found to exhibit higher degrees of learner autonomy than do
women. It was suggested that the greatest relative gains in global competitiveness would be
realized by enhancing the relative learner autonomy of women as a matter of East Asian
workforce development.
Learner autonomy and learner orientation.

Park E. East Asia Forum for Adult Education (EAFAE) 7th General Assembly, Seoul, Korea 2006

The concept of learner orientation as a mechanism for understanding what brings the
individual to a particular learning event grew out of Cyril O. Houle’s qualitative research
conducted in the late 1950s and early 1960s. Houle described three general categories of
learner orientation: Goal- , Activity-, and Learning-Orientation. His typology has provided a
helpful way of answering the question, “Why do people bother to learn?” Park provided a
new dimension for analyzing why individuals engage in the learning episodes: “Required
versus Not-required.” Her data revealed the traditional Activity-Orientation type was actually
a mix of Social, Required, and Goal Orientation. The empirical evidence suggested that
educators need to assess the new dimensions of “Required” when they attempt to
understand the primary orientation of individual learners through assessing the orientation
of Goal, Required, Social, and Learning. She concluded that the relative learner autonomy of
an individual associates to his or her learner orientation with respect to the learning episode.
Longitudinal Pattern of Traumatic Event Exposure and PTSD in Opioid Dependence

Jessica M. Peirce, Christopher K. Burke, Kenneth B. Stoller, Karin J. Neufeld, Van L. King,
Robert K. Brooner
Johns Hopkins University School of Medicine, Baltimore, MD

Research has found little direct influence of traumatic event exposure or PTSD on substance
abuse outcomes (e.g., Mills et al., 2007). However, most studies assess traumatic events and
PTSD at only one timepoint. The present study is a prospective evaluation of traumatic event
exposure and PTSD in substance abuse treatment patients. Participants were admitted to an
opioid dependence treatment program and were also enrolled in a parent study of
psychiatric evaluation and treatment. Traumatic events were assessed monthly with the
Traumatic Life Events Questionnaire (TLEQ; Kubany et al., 2000). PTSD was diagnosed at
baseline with the SCID and assessed at 4 months with the Modified PTSD Symptom Scale
(Falsetti et al., 1993). To date, 92 participants (57 women; 35 men) have completed six
months of the ongoing 12-month longitudinal study. Average age is 40 years; sample racial
composition is 52% Caucasian; 35% African American; 13% multiracial or other. Every
participant had a traumatic event history; the average number of events was 23 (SD=12;
range 3-67). There were no gender differences in traumatic event exposure history. Lifetime
history of PTSD was diagnosed in 30% of participants; 57% of that group (n=16) met current
PTSD criteria. Women were more likely to receive a lifetime PTSD diagnosis than men (40% vs.
14%; p<.05). During treatment, 20% of patients endorsed a new traumatic event at each of six
monthly follow-ups. Women were overall less likely to report traumatic event re-exposure
than men, and also appeared to have lower rates of re-exposure over time. Two-thirds of the
sample were re-exposed to at least one traumatic event during the six months of the study.
The most common event re-exposure was unexpected death of or injury to a loved one,
followed by witnessing, being threatened with, or experiencing a physical assault. PTSD at
intake was not associated with PTSD at the 4-month follow-up [RR=.98 (95% CI: .21-4.57)],
indicating considerable fluctuation in diagnostic status. Traumatic event re-exposure during
treatment was associated with increased risk of PTSD at Month 4 for men [RR=1.67 (95% CI:
1.17-2.38)], but not for women [RR=.59 (95% CI: .23-1.5)]. PTSD at intake was associated with
lower six-month retention. These data suggest that substance dependent patients continue
to experience significant re-exposure to high-risk traumatic events while in treatment,
although women are somewhat less likely to report re-exposure than men. In addition,
patients’ PTSD symptoms fluctuate considerably during treatment, suggesting that
assessment at only one timepoint would be inadequate to accurately assess risk for poor
treatment response.
Neuroanatomical Correlates of Post-TBI Depression

Vani Rao, MD1; Jennifer Spiro, MS1; Cynthia Munro PhD2 ; Dzung Pham3, PhD;Mahaveer
Degoankar3 Alena Horska3, PhD; David M. Yousem3, MD, and Peter B. Barker2, D.Phil.

1. Division of Geriatric Psychiatry & Neuropsychiatry, Department of Psychiatry, Johns
Hopkins School of Medicine. Baltimore, MD 21205
2. . Division of Medical Psychology, Deparment of Psychiatry, Johns Hopkins School of
Medicine. Baltimore, MD 212 87
3. Division of Neuroradiology, Russell H Morgan Department of Radiology and Radiological
Science, Johns Hopkins School of Medicine. Baltimore, MD 21287

Background: Depression is a common psychiatric sequela of traumatic brain injury (TBI). To
our knowledge, there are only two prior studies on the neuroanatomical correlates of TBI-

Objective: To determine the neuroanatomical correlates of post-TBI depression

Methods: Individuals with no past history of major depression (MD), who had sustained
closed traumatic brain injury within 3 months to 5 years before presentation were recruited.
Individuals who had developed MD for the first time after TBI were recruited as Cases and
those who had not developed MD were recruited as Controls. Cases and Controls were
compared on demographic variables, neuropsychological tests , brain metabolic ratios of N-
acetyl aspartate/Creatine (NAA/Cr) and regional brain volumes.

Results: Cases (N=10) when compared to Controls (N=7) were significantly older (52.4 versus
27.4 yrs, P<.001). All Controls had moderate/severe TBI compared to only 60% of cases
(p=0.03). On tests of cognitive functioning, the cases and controls differed only on test of
frontal functioning (Trails B) and trended towards significance on tests of temporal
functioning. On assessment of brain metabolic concentraions, Cases had significantly
reduced NAA/Cr ratio in the right basal ganglia. On volumetric analyses Cases had
significantly reduced grey matter volume in the right frontal lobe, and trended towards
significance in bilateral temporal volumes.

Conclusion: The results suggest a possible role of fronto-temporal lobe and basal ganglia
pathology in TBI depression.The major limitation is the small sample size of this pilot study.
Future research will focus on increased sample size and more sensitive neuroimaging

1. Jorge RE, Robinson RG, Starkstein SE, Arndt SV: Depression and anxiety following
traumatic brain injury. J Neuropsychiatry Clin Neurosci. 1993 Fall;5(4):369-74.

2. Jorge RE, Robinson RG, Moser D, Tateno A, Crespo-Facorro B, Arndt S: Major depression
following traumatic brain injury.Arch Gen Psychiatry. 2004 Jan;61(1):42-50.

This work was supported by grant from the Johns Hopkins Neurobehavioral Research
Unit/General Clinical Research Center
Depressive Symptoms and Platelet Aggregation after Acute Coronary Syndrome

Heather L. Rogers, Medical and Clinical Psychology, Uniformed Services University of the Health
Sciences, Bethesda, MD, Roy C. Ziegelstein, James A. Fauerbach, Gina M. Magyar-Russell, and
Marlene S. Williams, Johns Hopkins University School of Medicine, Baltimore MD

Depression at the time of an acute coronary syndrome (ACS) independently predicts
subsequent cardiac death. Since some investigators have found that depressed individuals
show increased platelet activation, we assessed depressive symptoms (using the Beck
Depression Inventory [BDI], the Patient Health Questionnaire-9 [PHQ-9], and the Beck
Hopelessness Scale [BHS]) and platelet aggregation in a pilot study of 24 patients admitted
with ACS. Platelet aggregation was assessed using standard light transmission in platelet-rich
plasma and results were expressed as EC50, slope, and extent of aggregation using adenosine
diphosphate (ADP) and serotonin (5HT) as agonists. PHQ-9 scores were negatively correlated
with ADP EC50 (r=-0.43, p<0.10). ACS patients scoring 8 or more on the PHQ exhibited
enhanced platelet aggregation, as demonstrated by significantly lower EC50 to ADP vs. those
with PHQ scores < 8 (p<0.01). ACS patients with BDI scores of 19 or more tended to exhibit
enhanced platelet aggregation to ADP vs. those who scored < 19 (p<0.10). ACS patients with
BHS scores of 14 or more also tended to exhibit enhanced platelet aggregation to ADP vs.
those with BHS scores < 14 (p<0.10). BDI scores tended to be associated with increased 5HT-
induced platelet aggregation (r=0.41, p<0.10). ACS patients with higher BHS scores also
tended to have greater 5HT-induced platelet aggregation than those with lower BHS scores
(p<0.10). Results were similar after excluding individuals on antidepressant medications
and/or clopidogrel (n=13). These results suggest that depressive symptoms are related to
enhanced platelet aggregation in individuals with ACS. In particular, both BDI and PHQ
measures of depressive symptoms predicted platelet aggregation responses to ADP. Beck
depressive symptoms and Beck hopelessness were associated with platelet aggregation
responses to 5HT. These preliminary data suggest that platelet activation may, in part, link
depression to cardiac events after ACS.
Toll-like Receptor-9 Suppression in Plasmacytoid Dendritic Cells Following IgE-dependent
Activation is Mediated by Autocrine TNF-a

John T. Schroeder, Kristin L. Chichester, Anja P. Bieneman

Background: Functional significance for the ag2 variant of the high affinity IgE receptor
(FceRI) reportedly expressed on human dendritic cell (DC) subtypes remains poorly
understood. Studies show that immature plasmacytoid dendritic cells (pDC) secrete large
quantities of TNF-a and IL-6 when directly stimulated with anti-IgE antibody. This mode of
activation, however, reduces TLR9 expression in pDCs and their ability to mount an IFN-a
response when subsequently activated with oligodeoxynucleotide containing CpG.

Objective: To investigate the mechanism(s) underlying this IgE-dependent suppression of
TLR9 and innate immune responsiveness in pDCs by focusing on autocrine cytokine

Methods: pDCs were isolated from blood using BDCA-4 selection. Cytokine responses to anti-
IgE antibody- and/or CpG- dependent stimulation were measured using ELISA. TLR9
expression was determined using quantitative RT-PCR and Western blotting. Results: The
time required for down-regulating TLR9 expression in pDCs following anti-IgE stimulation
correlated with the induction and duration of TNF-a secreted by these cells. Pretreatment of
pDCs with recombinant TNF-a (but not IL-6 or IL-10) markedly suppressed TLR9 expression.
Functional response to CpG (i.e. IFN-a induction) was also inhibited with TNF-a pretreatment
(IC50 = ~200 pg/ml). Finally, an antibody that neutralizes TNF-a activity completely restored
TLR9 expression during anti-IgE stimulation and significantly improved IFN-a secretion upon
subsequent activation with CpG.

Conclusions: Autocrine TNF-a secretion resulting from IgE/FceRI-dependent activation plays a
critical role in suppressing TLR9-dependent responses in pDCs that normally promote Th1

Clinical Implications: IgE crosslinking on immature pDCs impairs innate immune function in
these cells by suppressing IFN-a production.
Evidence for the ANCA-Associated Vasculitis Index of Damage (AVID)

Philip Seo, Peter A. Merkel, Ulrich Specks, Gary S. Hoffman, Carol A. Langford, Robert Spiera,
Cees G. Kallenberg, Anthony M. Turkiewicz, E. William St. Clair, John H. Stone,
for the OMERACT-Vasculitis Workshop and the RAVE Research Group

Johns Hopkins University, Boston University, Mayo Clinic, Cleveland Clinic Foundation,
Hospital for Special Surgery, University Medical Centre Groningen, University of Alabama,
Duke University

Purpose: Because Wegener’s granulomatosis (WG) and microscopic polyangiitis (MPA) are
clinically distinct from other forms of primary systemic vasculitis, the Vasculitis Damage Index
(VDI) may not capture forms of damage peculiar to the AAV. We sought to develop a new
instrument that is specific for these diseases.

Methods: Through consensus of investigators in the United States and Europe, we
developed the AAV Index of Damage (AVID). Both AVID and the VDI have been applied to all
patients enrolled in the Rituximab for ANCA-associated Vasculitis (RAVE) trial, a randomized,
placebo-controlled trial of patients with WG or MPA.

Results: The baseline data for the first 66 patients enrolled in the RAVE trial are available for
analysis. All patients are ANCA-positive, meet the Chapel Hill Consensus Conference
definitions of WG or MPA, and have clinically severe disease. The mean VDI score at baseline
was 1.6 (95% confidence interval (CI): -0.2, 3.4), and ranged from 0 to 8; 39.4% of patients had
VDI scores of 0. The mean AVID score was 3.5 (95% CI: -0.4, 7.4), and ranged from 0 to 17;
34.6% of patients had AVID scores of 0. The most common form of damage, chronic rhinitis
or crusting, was detected with equal frequency by the VDI and AVID (22.7%). Hypertension
and sensory neuropathy were present in 15.2% of patients at the time of enrollment. Other
common forms of damage included sensory neuropathy (13.6%), non-nephrotic range
proteinuria (12.1%), conductive hearing loss (12.1%), and chronic sinusitis (12.1%).

Conclusions: This study provides preliminary evidence that a disease-specific approach to the
assessment of damage in patients with WG and MPA may reduce underreporting. The range
of AVID scores was more than twice the range of VDI scores, implying that the AVID index
may discriminate better between different levels of damage for these diseases.
S. H. Shippey, R. E. Gutman, L. H. Quiroz, V. L. Handa
Johns Hopkins Bayview Medical Center, Baltimore, MD.

OBJECTIVE: To identify trends among practice patterns in the management of cystoceles
among members of the American Urogynecologic Society (AUGS)

METHODS: A survey was designed to assess the prevalence of vaginal versus abdominal
approaches to cystocele repair, midline plication, paravaginal repair, use of different graft
materials in cystocele repairs, and experience with complications related to the use of graft
materials. The survey was delivered to AUGS physician members whose e-mail addresses
appeared in the directory on the AUGS website. Respondents were directed to a website
where they could complete a 22-question survey. This research was found by the Johns
Hopkins IRB to be exempt from formal review. The study was also reviewed by the chairman
of the AUGS Research Committee.

RESULTS: Of 963 surveys, 72 could not be delivered and there were 315 respondants (35%).
Over half of respondents had completed residency at least 10 years earlier; 38% had
completed fellowships in either female urology or urogynecology; 47% practiced in an
academic setting. For the treatment of primary cystocele 77% of respondents had used
midline vaginal plication in the past year; only 40% had used this approach for management
of recurrent cystocele. For recurrent cystocele, the most commonly reported approach was
vaginal paravaginal repair with graft placement (50%). At the time of abdominal sacral
colpopexy, most considered the anterior vaginal sacropexy graft sufficient to address
cystocele. At the time of uterosacral suspension, midline plication was the most common
approach to address cystocele. 86% of respondents stated that they had used grafts to
reinforce cystocele repairs in the last year. The most commonly used graft material was
synthetic mesh (67%); other graft materials used included xenografts (47%), allografts (19%),
and autologous fascia (4%). Almost half of respondents had used a minimally invasive
transobturator technique for repairing cystoceles. Both dysparunia and graft erosion were
reported more commonly by respondents who reported using synthetic grafts than those
using biologic grafts (79% vs 67% and 77% vs 48%). Similar percentages of respondents
reported having to treat synthetic and biologic graft erosions with excision in the operating
room (26% and 21 %).

CONCLUSIONS: Practice patterns vary for cystocele repair. Our results suggest that the
surgical approach is influenced by whether the cystocele is primary or recurrent and whether
a concomitant apical procedure is performed. Also, large majority of respondents reported
utilizing grafts to surgically manage cystoceles. The wide variety of surgical approaches is
likely a reflection of the absence of a clearly-defined “best practice” for cystocele repair and
underscores the need for rigorous surgical trials.
Inter-Site Consistency at a Multi-Site.

Shultes-von-Schlageter M, Park E, Tucker PM. Psychiatry Clerkship. Academic Psychiatry 2006
30: 356-359.

This study examines the effects of clinical site assignment within a multiple-site psychiatry
clerkship program on the convergent outcome of the National Board of Medical Examiners
(NBME) subject examination. NBME scores, controlled for baseline pre-clerkship knowledge
base as measured by second year human behavior scores, were compared for inpatient and
outpatient clinical clerkship sites. No effect of clinical site assignment on NBME scores was
demonstrated, suggesting that inter-site consistency occurred for the performance
outcomes measured. Similar assessments can help other clerkship directors monitor clinical
sites for quality of their educational programs, which is increasingly important as medical
schools expand their clinical sites to community-based programs.
The Role of Anesthetic Induced Depth of Hypnosis (AIDH) in Postoperative Delirium (PD)

Frederick E. Sieber, M.D., Khwaja Zakriya, M.D., Simon Mears, M.D., Ph.D., Alan Gottshalk,
M.D., Ph.D., Constantine Lyketsos, M.D., Ph.D.
Anesthesiology and Critical Care Medicine, Johns Hopkins Bayview Medical Center,
Baltimore, Maryland

Introduction: The etiology of the postoperative delirium (PD) seen in elderly populations
appears to be multifactorial, and thought to involve the interaction of precipitating factors
with patient vulnerability factors1. Previous studies examining the role anesthesia plays in PD
have not controlled for degree of sedation. In this randomized trial in elderly patients
undergoing hip fracture repair we tested the hypothesis that minimizing anesthetic induced
depth of hypnosis (AIDH) during spinal anesthesia is associated with a lower incidence of PD.
Methods: After obtaining IRB approval and informed consent 49 hip fracture patients
underwent spinal anesthesia with propofol sedation. AIDH was assessed by monitoring the
Bispectral index. Patients were randomized to one of two intraoperative sedation levels: a
targeted sedation level resulting in either a bispectral index ~50 (n=24) or a bispectral
index~80 (n=25). To control for baseline vulnerability factors, randomization included group
stratification based on mini-mental status exam (MMSE) and age. To control for precipitating
factors, in both groups, intraoperative blood pressure and postoperative pain were managed
via algorithm. Patients were assessed daily for PD in-hospital using confusion assessment
method (CAM). Between group data were compared with Chi-square or Fishers exact tests.
Results: The two groups were similar in age and prevalence of dementia (chi square). There
were 11 patients with dementia in each experimental group. Bispectral index was clearly
different between the two experimental groups, at levels targeted by treatment
assignment.[table1]The incidence of PD in the Bispectral index~50 and Bispectral index~80
groups was notably different (rate effect size = 0.56, exact p =0.076).[table2]
Discussion: This randomized experiment suggests that the incidence of PD after regional
anesthesia might be reduced by as much as 50% if minimal sedation is applied. This novel
approach might also lead to better cognitive outcomes in high risk elderly patients. If this
finding can be replicated in a larger RCT, it would lead to a major change in geriatric
anesthesia practice and intraoperative management, especially in patients with dementia.

1. Inouye SK. Delirium in older persons. N Engl J Med. 2006;354:1157-1165.

D. Siluk1,2*, R.V. Oliveira1*, M.E. Rodriguez-Rosas1*, S. Ling1*, A. Bos1*, L. Ferrucci1*, I.W. Wainer1*
 Intramural Research Program of NIH, National Institute on Aging, Baltimore, USA,
  Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdańsk,

  The aim of this study was to examine the effect of vitamins A and E supplementation on
plasma concentrations. The patients included in the study were part of the on-going
Baltimore Longitudinal Study of Aging conducted by National Institute on Aging, NIH. For this
purpose we developed a fast (15 min) and simple HPLC method for the determination of all-
trans-retinol (vitamin A) and alpha-, gamma- and delta-tocopherols (vitamin E) in human
plasma. The assay utilizes 0.2 ml of plasma to which internal standard solution is added. The
vitamins were extracted using liquid-liquid procedure and analyzed on a Zorbax Eclipse XDB-
C18 column using a step gradient with polar organic mobile phase and variable wavelength
fluorescence detection. The method has been already applied to 374 samples. The plasma
concentration ranges determined for all studied vitamins were in agreement with ranges
reported in the literature. It was found that when men and women were compared, women
had a significantly higher plasma concentration of alpha-tocopherol, men had a significantly
higher concentration of vitamin A and delta-tocopherol, while there was no significant
gender difference in gamma-tocopherol concentrations. A subgroup of 35 patients was also
analyzed with relation to high, low and no vitamin E supplementation. There was a reverse
relationship between the alpha-tocopherol intake and the plasma concentrations of gamma-
and delta-tocopherols. The relation of plasma vitamin concentrations to clinical outcomes
will be discussed.

Danuta Siluk1,2*, Donald Mager3, Naomi Gronich1, Darrell Abernethy1, Irving W.Wainer1
1. Laboratory of Clinical Investigation, Gerontology Research Center, National Institute on
Aging, NIH, Baltimore, MD, USA
2. Department of Biopharmaceutics and Pharmacodynamics, Medical University of Gdańsk,
3. Department of Pharmaceutical Sciences,University at Buffalo, SUNY, Buffalo,          NY,

Purpose. Development and validation of a method for the simultaneous determination of R-
and S-propranolol and R- and S-hyoscyamine in human plasma and it application to the
analysis of samples from a clinical study.

Methods. Sample preparation was performed by solid-phase extraction of 2 mL of human
plasma using Oasis MCX cartridges (Waters) and the enantioselective separations were
achieved using a Chirobiotic V chiral stationary phase (Astec). The chromatography was
carried out using gradient elution with a mobile phase composed of methanol:acetic
acid:triethylamine which was varied from 100:0.05:0.04 to 100:0.05:0.1 (v/v/v) over 30 min
and delivered at a flow rate 1 mL/min. The internal standard was R,S-(D7)-propranolol and the
analytes were quantified using a single quadrupole mass spectrometer employing APCI
interface operated in the positive ion mode with single ion monitoring.

Results. The enantioselective separation factors, α, were 1.15 and 1.07 for S-and R-propranolol
and R- and S-hyoscyamine, respectively. The standard curves were linear from 0.25-200 ng/mL
for R- and S-propranolol and 0.5-50.0 ng/mL for R- and S-hyoscyamine with coefficients of
determination (r2) ranging from 0.9977 to 0.9999. The intra- and inter-day precision and
accuracy were ≤13.2% and ≤10.2%, respectively. The mean recoveries for R- and S-propranolol
were 85.5% and 84.6% and for R- and S-hyoscyamine 79.9% and 81.5%, respectively. The
method was applied to the analysis of plasma samples obtained after the i.v. administration
of racemic propranolol and atropine. The results from the initial 7 subjects indicate that R-
propranolol was cleared faster than S-propranolol (as expected), while S-hyoscyamine was
cleared faster than R- hyoscyamine, 0.054 L×min-1
×kg-1 and 0.044 L×min-1×kg-1 (p = 0.041).

Conclusions. The results demonstrated that the method is sensitive, precise accurate and
specific and can be applied to the simultaneous determination of propranolol and atropine
enantiomers. This is the first reported assay for the direct determination of R- and S-
hyoscyamine in human plasma.
In Search of Neuroprotective Hormetic Phytochemicals

T. G. Son*, S. Camandola, R. Telljohann, D. Hyun, D. Jo, Q. Yu, N. H. Greig and M. P. Mattson

Laboratory of Neurosciences, National Institute on Aging Intramural Research Program, Baltimore,

     The nervous system is of fundamental importance in the adaptive responses of organisms to
   many types of stress (hormesis), including exposures to environmental “toxins”. Although
   antioxidant properties have been suggested as the basis of the health benefits of
   phytochemicals, emerging findings suggest a quite different mechanism of action. Many
   phytochemicals normally function as toxins that protect the plants against insects and other
   damaging organisms. However, at the relatively low doses consumed by humans and other
   mammals these same “toxic” phytochemicals may activate adaptive cellular stress response
   pathways that can protect cells against a variety of adverse conditions. Hormesis-based
   mechanisms of action of phytochemicals such as resveratrol, curcumin and sulforaphane have
   been proposed (TINS 2006; 29:632-9.). We have developed neural cell culture screens to
   identify novel phytochemicals that, at low doses, activate cytoprotective stress response
   pathways including those involving Nrf2 - ARE, SIRT1 – FOXO3 and NF-κB. In this study, we
   screened a panel of biopesticides and identified plumbagin as a potent activator of the Nrf-2 –
   ARE pathway. Low concentrations (nanomolar) of plumbagin induced the expression of the
   Nrf2/ARE-responsive antioxidant genes HO-1 and NQO1. In addition, pretreatment with
   plumbagin protected neurons against oxidative stress-induced death. Experiments are currently
   in progress to identify other neurohormetic phytochemicals and to determine their efficacy as a
   neuroprotective agent in animal models of neurodegenerative conditions.

Key words: Neurotoxicity, Neuroprotection, Stress resistance, Phytochemical, Hormesis.
The ROMK (Kir1.1) Potassium Channel Is Present in Mammalian Urinary Tract Epithelia and

David A. Spector,1 Qing Yang,1 Leonid Klopouh,1 Edward J. Weinman,2 Deborah A. Steplock,2
Rajatsubhra Biswas,2 Jie Liu,3 James B. Wade.3 1Div of Renal Medicine, Johns Hopkins Bayview
Medical Center; 2Div. of Nephrology; 3Dept. of Physiology, Univ. of Maryland School of Med,
Baltimore, MD.

The mammalian urinary tract (UT) is usually considered to function solely as a transit and
storage site for urine produced by the kidneys. However, in-vivo data supports the notion
that UT epithelia may modify urine by vectoral transport of solutes, including potassium,
across UT epithelial cell membranes- possibly via specific membrane transporters. We
examined the expression, localization and regulation of the secretory potassium channel
ROMK (Kir.1.1) in ureter, bladder, and other tissues of Wistar rats and beagle dogs by
immunoblotting, immunochemistry, and RT-PCR. Two polyclonal antibodies to the C
terminus of ROMK, one raised in chicken (LC35, Wade et al), one in rabbits (Alomone Labs),
were used as probes. Both probes identified similar ~ 48-53 kDa bands characteristic of
ROMK in immunoblots of homogenized bladder, ureter, renal cortex and medulla and in UT
epithelial cell scrapings and bladder smooth muscle homogenates in both rats and dogs.
Bands were ablated when the antibodies were preabsorbed with the immunizing peptide.
By immunocytochemistry both probes strongly localized ROMK to the apical (luminal)
membrane of the UT epithelial cells and less intensely to UT smooth muscle cells. ROMK was
similarly identified by immunoblots and staining of muscle in other tissues: aorta, heart, large
and small intestine, and striated muscle. RT-PCR demonstrated ROMK mRNA in renal tissues,
UT epithelial cells and smooth muscle, and in other tissues containing muscle in both species.
In groups of rats subjected to 5%, 0.8%, or 0% dietary potassium for 5-7 days there were no
apparent differences in expression (in immunoblots of homogenates of whole UT organs or
bladder smooth muscle) or localization of ROMK in bladder. Thus we show for the first time
that ROMK is present in UT epithelia and many muscle tissues. In mammalian urinary tract
ROMK may participate in transepithelial potassium transport and thereby modify final urine
potassium concentration.
High urea and creatinine concentrations and urea transporter B in mammalian urinary tract

David A. Spector,1 Qing Yang,1 and James B. Wade2

Division of Renal Medicine, The Johns Hopkins University School of Medicine, and 2Department
of Physiology, University of Maryland School of Medicine, Baltimore, Maryland

Although the mammalian urinary tract is generally held to be solely a transit and storage
vehicle for urine made by the kidney, in vivo data suggest reabsorption of urea and other
urine constituents across urinary tract epithelia. To determine whether urinary tract tissue
concentrations are increased as a result of such reabsorption, we measured urea nitrogen
and creatinine concentrations and determined whether urea transporter B (UT-B) was
present in bladder, ureter, and other tissues from dogs and rats. Mean urea nitrogen and
creatinine concentrations in dogs and rats were three- to sevenfold higher in urinary tract
tissues than in serum and were comparable to those in renal cortex. In water-restricted or
water-loaded rats, urea nitrogen concentrations in bladder tissues fell inversely with the state
of hydration, were proportional to urine urea nitrogen concentrations, and were greater than
the corresponding serum urea nitrogen concentration in every animal. Immunoblots of rat
and dog urinary tract tissues demonstrated the presence of UT-B in homogenates of bladder
and ureter, and immunocytochemical analysis localized UT-B to epithelial cell membranes.
These findings are consistent with the notion that urea and creatinine are continuously
reabsorbed from the urine across the urothelium, urea in part via UT-B, and that urine is thus
altered in its passage through the urinary tract. Urea reabsorption across urinary tract
epithelia may be important during conditions requiring nitrogen conservation and may
contribute to pathophysiological states characterized by high blood urea nitrogen, such as
prerenal azotemia and obstructive uropathy.

urothelial transport; bladder; ureter
Alterations in Central Dopamine Receptor Activity Before and After Gastric Bypass Surgery

K.E. Steele, T. Magnuson, M.A. Schweitzer, A. Kumar, H. Kuwabawa, J. Galecki, J. Brasic, M.
Alezander, W. Ye, and D.F. Wong.

Background:While bariatric surgery has proved highly successful at producing sustained
weight loss, variability in treatment response persists. A better understanding of the
pathophysiology of appetite and obesity may improve patient selection and management.
Research into feeding behavior and satiety has focused on the role of dopamine in reward-
based behaviors. Specifically, positron-emission computed tomography (PET) has
demonstrated reduced brain dopamine receptor availability in obese subjects compared to
controls. This may be due to a primary deficiency in dopamine receptors or to secondary
dopamine receptor down regulation. We performed a preliminary study to investigate
dopamine D2 receptor activity in obese patients before and after laparoscopic Roux-en Y
gastric bypass (LGBP).

Methods: 5 female patients ages 20 to 38 years old with a mean body mass index of 45
underwent PET with C11 raclopride injection. Five regions of interest were studied: ventral
striatum, anterior and posterior putamen, and caudate. Repeat PET was performed at six
weeks following LGBP. D2 receptor binding was compared within subjects pre and post
surgery. Baseline D2 binding was also compared to historical nonobese controls.

Results: No significant difference in D2 binding was seen between the obese subjects and
non-obese controls. D2 receptor availability appeared to increase 6 weeks after gastric
bypass surgery. The increase in receptor availability appeared roughly proportional to the
amount of weight lost.

Conclusion: Brain dopamine D2 binding appears to increase following LGBP. This preliminary
finding needs to be replicated in a larger population, but suggests that diminished D2 binding
in the obese may be due to D2 receptor downregulation. Changes in dopamine receptor
binding may play an important role in centrally mediated appetite suppression and resultant
weight loss after LGBP.
Increased Fatness and Reduced Fitness are Associated with Aortic
Stiffness in Persons with Type 2 Diabetes and Hypertension

Kerry J. Stewart, EdD; Brian H. George, MS; Kristina Potrekus, MS;
Anita C. Bacher; MSN, Harry A. Silber, MD, PhD, Nae-Yuh Wang; PhD,
Edward P. Shapiro, MD, Pamela Ouyang, MD. Johns Hopkins University
School of Medicine, Baltimore MD.

Background: Large artery stiffness, thought to be a marker of
cardiovascular disease, is accelerated in persons with diabetes and
hypertension. While reduced fatness and increased fitness are
associated with lower BP, we tested the hypothesis that increased
fatness and reduced fitness are each independently associated with
greater aortic stiffness. Methods: Sedentary subjects (M=60; F=29),
mean (SD) age 57.1 (5.8) years, were examined at baseline as part of
an exercise training study to reduce BP in persons with type 2
diabetes. Subjects were taking their usual medical therapy. For BP
eligibility, subjects were required to have SBP between 120-159 and/
or DBP between 80-99 mm Hg during 2 consecutive weekly visits. BP was
the mean of the screening visits and a visit after qualification for
the study. Aortic stiffness was assessed by carotid-to-femoral pulse
wave velocity (PWV), using ultrasound probes simultaneously at each
of these sites. General fatness was assessed by dual-energy x-ray
absorptiometry (DEXA) and expressed as percent body fat. Abdominal
fatness was assessed by magnetic resonance imaging at the level of
the umbilicus and the areas for total, subcutaneous, and visceral fat
were measured. Maximal oxygen uptake was obtained on a treadmill. The
associations of PWV with BP, oxygen uptake, and abdominal fat were
determined by stepwise regression analysis with adjustment for age
and gender. Results: PWV was 922.9 (300.0) msec, SBP was 126.9 (13.2)
mm Hg, DBP was 71.8 (8.8) mm Hg, pulse pressure (PP) was 55.0 (10.4)
mm Hg, percent body fat was 35.0 (6.5) %, and maximal oxygen uptake
was 21.7 (5.0) ml/kg/min. In the final model, the variance in PWV was
accounted for by increased PP, 8.9%; increased abdominal visceral fat
accounted for an additional 6.2%; increased percent body fat, an
additional 3%; and decreased maximal oxygen uptake, an additional
5.6%. Conclusions: These data bring to light an independent
relationship of aortic stiffness with increased fatness and reduced
fitness. Though further work is needed to elucidate the mechanisms
linking aortic stiffness, fatness, and fitness, these findings
support the need for a therapeutic approach for reducing aortic
stiffness that considers the potential benefits of weight reduction
and exercise.
Role of Mast Cells in the Development of Pulmonary Allergic Inflammation of SHP-1
Deficient Mice

Sun-Young Oh, Ph.D., Tao Zheng, M.D., John Schroeder, Ph.D., Becky Vonakis, Ph.D., Oksoon
Choi, Ph.D., Zhou Zhu, M.D., Ph.D.
Johns Hopkins Asthma and Allergy Center, Baltimore, MD

Rationale: Viable motheaten (mev) mice are deficient in phosphatase SHP-1 and have
inflammatory disorders involving multiple organs, including the lung. We observed that mev
mice develop a spontaneous pulmonary inflammation that is largely a Th2 dominated allergic
response. However, the cell types that initiate this process are not clear. We hypothesized
that SHP-1 deficiency leads to mast cells dysfunction and increased production and release of
Th2 cytokines and proinflammatory mediators.

Methods: We determined histamine content and release by cells isolated from lung and
spleen; mast cells in the lung; cytokine release by splenocytes after stimulation with anti-
mast cells (BMMCs).

Results: Compared with WT mice, cell suspensions from both lung and spleen of mev mice
showed significantly increased amount of histamine content and spontaneous release of
histamine; the number of mast cells in the lung parenchyma of mev mice was significantly
increased; more importantly, many of these cells appeared to be degranulating; Th2
cytokines IL-4 and IL-13 were highly increased in spleen cells of mev mice after stimulation
with anti-Fc                                 -         -10 did not change; the release of
hexosaminidase, IL-4, IL-13, and TNF-                                       mev mice were
also significantly increased.

Conclusions: These results demonstrate that SHP-1 deficiency leads to increased mast cell
accumulation in the lung and mast cell production and release of mediators and cytokines
that favor Th2 inflammation, suggesting that SHP-1 plays a critical role in regulating allergic
inflammatory response through mast cells.

(Funding: This work was partially supported by NIH Grant RO1HL074095 and an American
Lung Association Research Grant to ZZ.)
Corticotropin releasing factor receptor 1 (CRF-R1) in the ventral tegmental area is
preferentially encoded and translated by dopaminergic neurons

Patricia Tagliaferro, Hui-Ling Wang and Marisela Morales
Cellular Neurobiology Research Branch, NIDA-IRP, NIH, DHHS, Baltimore, MD, USA.

We previously suggested the ventral tegmental area (VTA) as a site for interactions between
stress and the mesocorticolimbic dopamine system, and showed that corticotropin releasing
factor (CRF) axon terminals establish synapses with dopamine (DA) neurons in the VTA. As
biological activity of CRF is exerted by its interactions with two CRF receptors (CRF1 and
CRF2), we investigated whether these receptors were present in specific VTA cells. By
radioactive in situ hybridization we found cellular expression of CRF1 receptor mRNA but not
CRF2 receptor mRNA in the VTA. By double in situ hybridization, we found that nearly 70% of
all neurons expressing CRF1 receptor mRNA co–expressed tyrosine hydroxylase (TH, marker
for dopaminergic neurons in the VTA) mRNA, and constitute 20% of the total population of
TH positive cells. To determine the subcellular distribution of CRF1 receptor protein within the
VTA, we performed CRF-R1 immunolabeling of the VTA and electron microscopy analysis.
CRF1 receptor immunolabeling was observed in the cytoplasm and plasma membrane of
dendrites, including plasma membrane in the synaptic region. By double CRF1 receptor and
TH labeling we determined that cytoplasmic and plasmalemmal CRF1 receptor labeling was
more frequently detected in TH immunopositive dendrites (66%) than in those lacking TH
labeling (34%).

In summary, we showed that a subset of VTA dopamine neurons encodes and synthesizes
CRF1 receptors distributed in the cytoplasm and plasma membrane. The presence of CRF1
receptor protein in the plasma membrane of VTA dopamine neurons offers a molecular
mechanism for interactions between CRF and the mesocorticolimbic dopamine system.
The Metabolic Syndrome is associated with Circulating Markers of Endothelial Dysfunction
in the Multi-Ethnic Study of Atherosclerosis (MESA)

Dhananjay Vaidya1, Moyses Szklo1, Mary Cushman2, Paul Holvoet3, Hossein Bahrami1, Nancy S.
Jenny2, Pamela Ouyang1.
 Johns Hopkins University, Baltimore, MD, 2University of Vermont, Colchester, VT, 3Katholieke
Universiteit Leuven, Belgium

Components of the Metabolic Syndrome (MetSyn) are associated with endothelial
dysfunction, an early change in atherosclerosis. We examined whether circulating biomarkers
of endothelial dysfunction were associated with MetSyn in MESA.
Methods: We studied a random sample of 568 women and 427 men from MESA with
assessment of baseline MetSyn (NCEP-ATP-III) status and measurements of plasma soluble
thrombomodulin (sTM), soluble intercellular adhesion molecule-1 (sICAM-1), soluble E-
selectin (E-sel) and CD-40 ligand (CD40L). Linear regression models for log(markers) adjusted
for race, sex, total reported weekly physical activity, smoking, blood pressure and lipid-
lowering medication use, and prevalent MetSyn were estimated. Using these coefficients we
show marker levels for persons with or without MetSyn, standardized to the average
characteristics of the study sample: i.e age (59 years), sex ratio (43% male), race ratio (46%
white, 10% Chinese, 21% Black, 23% Hispanic), average physical activity (25 hours/wk), and who
were not on BP/lipid medication. We assessed interaction by sex and race and tested
individual MetSyn components for independent associations.
Results: Prevalent MetSyn was associated with higher levels of all four markers (Table) with
no interaction by sex or race. The metabolic components of abdominal obesity, low HDL-
cholesterol, prehypertension/hypertension and impaired fasting glucose/diabetes were each
associated with two of the markers independently of the other four metabolic components.
Conclusion: MetSyn and some of its components are associated with high circulating levels of
markers of endothelial dysfunction. Future studies must investigate if endothelial
dysfunction mediates the progression to atherosclerosis among persons with metabolic
abnormalities of MetSyn.

Table: Standardized levels (95% Confidence Intervals) for markers of endothelial
dysfunction, and metabolic components independently associated with the markers
Marker                  MetSyn (95%CI)       No MetSyn (95%CI) p-value Ind. Comp*

sTM (ng/mL)             36.0 (34.0 to 38.0) 32.0 (30.8 to 33.3) <0.001     2, 3
sICAM-1 (ng/mL)         287 (278 to 297)     266 (260 to 273)     <0.001   1, 2, 4
eSEL (ng/mL)            55.7 (52.7 to 58.9) 45.0 (43.3 to 46.8) <0.001     1, 3, 4

CD40L (ng/mL)           3.58 (3.32 to 3.87) 3.23 (3.06 to 3.41)   0.019    none
*Independently associated metabolic components (p<0.05): 1-Abdominal obesity, 2- low
HDL-cholesterol, 3-prehypertension/hypertension, 4-impaired fasting glucose/diabetes
Brief cocaine abstinence induced by voucher and cash-based incentives

Ryan Vandrey, George Bigelow, and Maxine Stitzer, Behavioral Pharmacology Research Unit

Goods-based contingency management interventions (e.g., those using vouchers or prizes as
incentives) have demonstrated efficacy in reducing cocaine use, but cost has limited
dissemination to community clinics. Recent research suggests that development of a cash-
based contingency management approach may improve treatment outcomes while reducing
operational costs of the intervention. However, the clinical safety of providing cash-based
incentives to substance abusers has been a concern. The present 16-week study compared
the effects of goods-based versus cash-based incentives worth $0, $25, $50, and $100 on
short-term cocaine abstinence in a small sample of cocaine-dependent methadone patients
(N = 12). A within-subject design was used; a 9-day washout period separated each of 8
incentive conditions. Higher magnitude ($50 and $100) cash-based incentives (checks)
produced greater cocaine abstinence compared with the control ($0) condition, but a
magnitude effect was not seen for goods-based incentives (vouchers). A trend was observed
for greater rates of abstinence in the cash-based versus goods-based incentives at the $50
and $100 magnitudes. Receipt of $100 checks did not increase subsequent rates of cocaine
use above those seen in control conditions. The efficacy and safety data provided in this and
other recent studies suggest that use of cash-based incentives deserves consideration for
clinical applications of contingency management, but additional confirmation in research
using larger samples and more prolonged periods of incentive delivery is needed.
Brain Derived Neurotrophic Factor Regulates Energy Metabolism and Autonomic Activity

R. Wan *, M. Brown, A. Cheng, T. Brown, D. Mager†, and M. Mattson
Laboratory of Neuroscience, Gerontology Research Center, National Institute on Aging,
Baltimore, MD 21224
† Department of Pharmaceutical Sciences, University at Buffalo, SUNY, Buffalo, NY 14260

Brain derived neurotrophic factor (BDNF) is a member of the neurotrophin family that is
widely expressed in the developing and adult nervous system, where it is believed to play
important roles in regulating cell survival and synaptic plasticity. Here we employed BDNF
heterozygous knockout (BDNF+/-) mice to investigate the role of BDNF in the regulation of
glucose metabolism and autonomic nervous system (ANS) function. A telemetric recording
system was used to monitor heart rate, core body temperature and general activity. Osmotic
pumps were used to continuously infuse either vehicle or BDNF (0.6 or 1.2 ug/day) into the
lateral ventricle of the brain. In both wild-type (WT) and BDNF+/- mice, the infusion of BDNF
significantly suppressed food intake and body weight, and decreased blood glucose levels
dose-dependently with quantitatively greater effects in BDNF+/- mice. BDNF+/- mice exhibit
an elevated heart rate compared to WT mice. However, continuous long-term central
infusion of BDNF elevated heart rate and body temperature and reduced locomotor activity
in both WT and BDNF+/- mice, with BDNF+/- mice being more sensitive. Our findings suggest
that BDNF is involved in the regulation of autonomic function in the adult mouse. We are
currently working to elucidate the effects of BDNF signaling on activities of parasympathetic
and sympathetic neurons that innervate the heart, and the possible role of BDNF as a
mediator of the effects of exercise and dietary energy restriction on the ANS.
Histone Acetylation and the Role of Corepressor in Negative-Regulation of TSHα-Subunit
Gene by thyroid hormone

Dongqing Wang, Xianmin, Xia, Alexis Oetting, Paul Yen

The hypothalamic/pituitary/thyroid axis is tightly-regulated by circulating thyroid hormone
(TH) levels via negative-regulation of thyroid stimulating hormone (TSH) synthesis in the
pituitary gland. Currently the molecular mechanisms involved in negative regulation of
transcription by T3 are poorly characterized. To understand the mechanism of how T3
negatively-regulates gene transcription, our lab established a permanently-transfected cell
line that stably expressed luciferase under the control of the TSH-α promoter in rat pituitary-
derived GH3 cells. These cells exhibited a strong dose-dependent decrease in transcriptional
activity in response to T3. Surprisingly, the histone deactylase (HDAC) inhibitor, trichostatin A
(TSA), decreased luciferase activity to a similar level as T3. Moreover, two peptide inhibitors
of histone acetyltransferase activity led to an increase in transcriptional activity. These
findings suggested that histone acetylation may be involved in the negative-regulation of
TSH α-subunit gene expression. Accordingly, we performed chromatin immunoprecipitation
(ChIP) assays to examine histone acetylation and co-factor recruitment to the TRE of the
TSHα promoter. We observed that both T3 and TSA led to increased histone H3 acteylation.
Interestingly, HDAC, NCoR, SMRT association with TSHα promoter was decreased by T3
without any changes in SRC-1 recruitment. In summary, our findings suggest that T3
induction of histone acetylation mediated by decreased corepressor association with TSHα
promoter may play an important role in the negative-regulation of TSH synthesis in the
The laterodorsal tegmental nucleus have three distinct sub-populations of neurons:
cholinergic, glutamatergic and GABAergic

Hui-Ling Wang, Patricia Tagliaferro and Marisela Morales
National Institute on Drug Abuse, Intramural Research Program, Cellular Neurophysiology.
5500 Nathan Shock Drive, Baltimore, MD. 21224.

The laterodorsal tegmental (LDTg) nucleus provides cholinergic innervations to several brain
areas including the ventral tegmental area (VTA). Neurons containing choline acetyl
                                   -amino butyric acid (GABA) have been detected in the
LDTg. From immunolabeling studies, it has been concluded that many of the LDTg
cholinergic neurons may be glutamatergic or GABAergic. Here, we show that these
cholinergic neurons do not display glutamatergic or GABAergic phenotypes.

Glutamate is selectively transported by vesicular glutamate transporters (VGluT1 or VGluT2)
into synaptic vesicles in axon terminals of glutamatergic neurons. We combined in situ
hybridization [to detect mRNA encoding VGluT1, VGluT2 or glutamic acid decarboxylase
(GAD, synthesizing enzyme of GABA)] and immunohistochemistry (to detect ChAT) to
determine whether LDTg cholinergic neurons express VGluT1, VGluT2 or GAD. We did not
find VGluT1 mRNA within the LDTg, but detected many neurons with VGluT2 mRNA or GAD
mRNA. The number of ChAT positive neurons co-expressing VGluT2 (13 ChAT/VGluT2 out of
2011 ChAT) or GAD (18 ChAT/GAD out of 761 ChAT) was minimal. Moreover, in the VTA we
found axon terminals containing either VGluT2 or vesicular acetylcholine transporter (used as
marker for cholinergic inputs from LDTg), but so far we haven’t detected both transporters
within the same axon terminal.

We conclude that the LDTg cholinergic neurons are not glutamatergic or GABAergic, and
suggest that excitatory cholinergic effects on targeting areas, like the VTA, are determined
by the nature of the postsynaptic cholinergic receptors as opposed to the co-release of
acetylcholine and glutamate from the same axon terminal.
RY-023, a selective inverse agonist at the benzodiazepine binding site on GABA-
receptor, improves performance in a delayed-match-to-sample task in rhesus monkeys.

Michael R. Weed, Ph.D. *, Terry Clayton **, and James M. Cook, Ph.D. **
* Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of
** Department of Chemistry, University of Wisconsin-Milwaukee

Alzheimer’s disease (AD) and Mild Cognitive Impairment (MCI) are among many diseases
that impair cognitive function and reduce the quality of life of those afflicted. Cholinergic
therapeutics for AD or MCI may be effective in less than half of those treated; therefore,
development of new strategies for improving cognitive function in AD or MCI patients would
result in a significant improvement in quality of life for a great many patients. Inverse
agonists of the benzodiazepine (Bz) binding site on the gamma-aminobutyric acid (GABA)
type A (GABAA) receptor-complex (GABAA-BzR) of the alpha-                             -BzR)
enha                                                                 -BzR inverse agonists are
also effective in rodent models of AD (e.g. they reverse scopolamine-induced memory
                          -                                                                   -
BzR therapeutics promising for AD. However, few studies of the cognitive effects of
           -BzR inverse agonists have been conducted in primates.
Two adult male rhesus monkeys, 8-9 kg, were trained on a variation of the CANTAB delayed
matching to sample (DMS) test of memory for visual stimuli. Sessions consisted of 40 trials,
10 at each with delays of 2, 30, 60 and 90 seconds with each delay presented in a random
order throughout the session. Stimuli consisted of photographs of over 900 objects adapted
from commercial photo-object software (Photo Clip Art 150,000 by Hemera Technologies,
Inc.). The CANTAB software selected the stimuli randomly without replacement from the
total 900 without using the same images in the last 100 trials (including previous sessions).
Therefore, the stimuli were trial-unique for a given day, and were not likely to have been
used within the last two sessions. The two monkeys were administered RY-23 (0.17 – 1
mg/kg) or its vehicle 20 min prior to the beginning of the DMS session. Performance after
vehicle tended to be delay-dependent with longer delays resulting in a lower percentage of
correct. Administration of RY-23 increased the percentage of correct trials in both monkeys
for both short and long delays, including when averaged across the session. There were no
systematic effects on latency to respond with any dose of RY-23. Importantly there were no
adverse effects at doses (1 mg/kg) that suppressed responding. These data demonstrate that
the selective GABAAa5-BzR inverse agonist RY-23 enhances performance in the DMS task,
consistent with an improvement of memory.
A peptide inhibitor targeting the regulatory subunit of PI 3-kinase, p55PIK, is effective in
human thyroid cancer therapy

Xianmin Xia1, Guihua Wang2, Aiwu Cheng3, Xuelai Luo2, Jianping Gong2, Junbo Hu2, Paul M.

1Division of Endocrinology, Johns Hopkins Bayview Medical Center, 4940 Eastern Ave.,
Baltimore MD 21224, USA; 2Department of Surgery, Tongji Medical School, Huazhong
University of Science and Technology, Wuhan, China; 3Laboratory of Neuroscience, GRC, NIA,
5600 Nathan Shock Dr., Baltimore, MD 21224, USA.

Some aggressive thyroid cancers, particularly papillary and follicular variants, medullary
thyroid, and anaplastic, respond poorly to conventional radio- or chemotherapy and novel
therapies are urgently needed for these malignancies. Class IA phophatidylinositol 3 kinase
(PI 3-kinase)-mediated signaling is important in the proliferation and progression of many
cancers, including thyroid cancer. Previously, we showed that p55PIK, a regulatory subunit of
PI 3-kinase, specifically interacts with retinoblastoma protein (Rb) through p55PIK’s unique
amino-terminus. Moreover, this interaction mediates cell cycle progression stimulated by
growth factors. To study this interaction site as a potential drug target for cancer therapy,
we generated an adenovirus construct (Ad-N24-GFP) expressing the 24 N-terminal amino acid
residues of p55PIK (N24). The proliferation of several thyroid cancer cells tested such as ARO,
WRO, NPA, FTC 133 and FTC 236 was significantly inhibited by the infection of Ad-N24-GFP. In
cells that express Rb, N24 induced cell cycle arrest at G0/G1 phase. Interestingly, in FTC 236, a
human thyroid cancer cell line, that does not express Rb, N24 strongly inhibited cell
proliferation by blocking cell cycle transition at both S and G2/M phases. These data strongly
suggest that specific PI 3-kinase isoforms regulate cell cycle progression at different
checkpoints by Rb-dependent and –independent mechanisms. Our results also showed that
FTC 236 cells infected with Ad-N24-GFP had markedly decreased tumor xenograft growth in
nude mice and the growth of tumor xenografts of FTC 236 cells decreased significantly when
Ad-N24-GFP administered intra-tumorally in nude mice, demonstrating the potential for viral-
mediated delivery of N24 peptide or novel agents that target specific subunit isoforms of PI
3-kinase for the treatment of thyroid cancers.
Gene expression atlas of the mouse central nervous system: impact and interactions of age, energy
intake and gender

Xiangru Xu1, Ming Zhan2, Wenzhen Duan1, Vinayakumar Prabhu2 , Randall Brenneman1, William Wood2,
Jeff Firman2, Huai Li2, Peisu Zhang1, Carol Ibe1, Alan B. Zonderman2, Dan L. Longo3, Suresh Poosala2,
Kevin G. Becker2 and Mark P. Mattson1,4

The structural and functional complexity of the mammalian central nervous system (CNS) is
organized and modified by complicated molecular signaling processes that are poorly understood.
We measured transcripts of 16,896 genes in 5 CNS regions from cohorts of young, middle age and
old male and female mice that had been maintained on either a control diet or a low energy diet
known to retard aging. Each CNS region (cerebral cortex, hippocampus, striatum, cerebellum and
spinal cord) possessed its own unique transcriptome fingerprint that was independent of age,
gender and energy intake. Less than 10% of genes were significantly affected by age, diet or gender,
with most of these changes occurring between middle and old age. The transcriptome of the spinal
cord was the most responsive to age, diet and gender, while the striatal transcriptome was the least
responsive. Gender and energy restriction had particularly robust influences on the hippocampal
transcriptome of middle-aged mice. Prominent functional groups of age- and energy-sensitive genes
were those encoding proteins involved in DNA damage responses (Werner and telomere-associated
proteins), mitochondrial and proteasome functions, cell fate determination (Wnt and Notch
signaling) and synaptic vesicle trafficking. Mouse CNS transcriptomes responded to age, energy
intake and gender in regional distinctive manner. The systematic transcriptome dataset also provides
a window into mechanisms of age-, diet- and sex-related CNS plasticity and vulnerability.

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