Twenty- Fourth Annual

VEGF-A and VEGF receptors, Flt-1/FLT-1 (VEGFR-1) and Flk-1/KDR (VEGFR-2), are key regulators of tumor angiogenesis and tumor growth. The purpose of this study was to determine the anti-angiogenic and anti-tumor efficacy of vascular-targeting fusion toxin VEGF121/rGel in an orthotopic glioblastoma mouse model using non-invasive in vivo bioluminescence imaging (BLI), magnetic resonance imaging (MRI), and positron emission tomography (PET). Methods: Tumor-bearing mice were randomized into two groups and balanced according to BLI and MRI signals. PET imaging using Cu-DOTA-VEGF121/rGel was performed before VEGF121/rGel treatment. F-fluorothymidine (F-FLT) scans were performed before and after treatment to evaluate VEGF121/rGel therapeutic efficacy. In vivo results were confirmed with ex vivo histology and immunohistochemistry. Results: Logarithmic transformation of peak BLI signal intensity showed a strong correlation with MRI tumor volume (r = 0.89, n = 14). PET imaging using CuDOTA-VEGF121/rGel pre-treatment showed a tumor accumulation of 11.8 ± 2.3 %ID/g at 18 h post-injection (p.i.) and the receptor specificity of the tumor activity accumulation was confirmed by successful blocking of the uptake in the presence of excess amount of VEGF121. PET imaging using F-FLT showed significant decreases in tumor proliferation in VEGF121/rGel-treated mice compared with controls. Histologic analysis showed specific tumor neovasculature damage following four doses of VEGF121/rGel treatment, accompanied by a significant decrease in peak BLI tumor signal intensity. Conclusions: The results of this study suggest that future clinical multi-modality imaging and therapy using VEGF121/rGel may provide an effective means to prospectively identify patients who will benefit from VEGF121/rGel therapy and then stratify, personalize, and monitor treatment to obtain optimal survival outcomes. Funded by the Stanford Medical Scholars Research Program STEERABLE SHEATH FOR ENDOSCOPIC AND TRANSLUMENAL SURGERY Natalia Isaza, BS; Tom Low, MS; Pablo Garcia, MS; Sanjeev Dutta, MD Department of Surgery, Stanford University School of Medicine & SRI International Objective: The application of endoscopic tools for diagnostics and therapeutics is increasing. Intraluminal surgical procedures are enabled by technologies that provide more maneuverability and dexterity in smaller diameters. New clinical applications such as Natural Orifice Transgastric Endoscopic Surgery (NOTES) are forcing even more stringent requirements in the design of tools to navigate and perform surgical tasks. However, there are two key requirements which current technology has not addressed adequately: (1) the ability to navigate a sheath through a tortuous path without support; (2) the ability to rigidize the sheath on-demand in order to deliver and operate surgical tools. Methods and Results: We developed a novel technology to steer and rigidize a balloon-like sheath to reach a target in the peritoneal cavity or gastrointestinal tract. The technology is based on the ability to electrically alter the properties of a proprietary material embedded in the sheath. Selective changes to the elasticity of sheath regions cause controlled bending when internal pressure is applied. The design leaves the center of the sheath open for introduction of surgical tools. Conclusion: This steerable sheath may enable previously challenging endoscopic operations by providing unprecedented maneuverability and stiffness control. It is also potentially scalable to enable the design of smaller and more dexterous endoscopic tools than what is currently available. Funded by the Stanford Medical Scholars Research Program NOX AND ADMA CHANGES WITH FOCAL ISCHEMIA, AMELIORATION WITH THE CHAPERONIN GROEL Lijun Xu, Bingyin Wang, Kirandeep Kaur, Melanie F. Kho, John P. Cooke, Rona G. Giffard, Department of Anesthesia and Cardiovascular Center, Stanford University School of Medicine, Stanford, CA, USA Both nitric oxide and asymmetrical dimethylarginine (ADMA) play a critical role in the regulation of cerebral blood flow, though their neuroprotective and cytotoxic effects are still under investigation. In this study we evaluated whether NO and ADMA levels change in plasma, ischemic brain tissue and cerebral spinal fluid (CSF) in response to focal ischemia. Male S.D rats (280-320G) were subjected to 2 hrs MCAO using a suture. Venous blood samples were collected at baseline, after 2 hrs MCAO, and at 24 hrs reperfusion. CSF and tissue samples were taken at 24 hrs reperfusion. Plasma total nitrate/nitrite concentration (NOx) and ADMA were measured by ELISA kit. We found NOx levels in plasma, ischemic brain tissue, and cerebrospinal fluid (CSF) increased significantly 24h after 2h transient middle cerebral artery occlusion (MCAO) in rats. ADMA levels were unchanged in plasma, but decreased significantly in CSF 24h following MCAO. The CSF ADMA/NOx ratio decreased markedly following ischemia. Rats protected by expression of the chaperonin GroEL or its folding deficient mutant D87K had lower plasma NOx levels at 24h reperfusion. ADMA, NO, and their ratio in CSF correlates with extent of injury in the protected rats. A peripherally detectable biomarker that rises quickly and correlates with injury is still being sought in stroke. The possibility of using serum and CSF ADMA/ NO levels to markers of ischemic stroke is worth further study. Funded by the Stanford Medical Scholars Research Program. GENOMIC ANALYSES IDENTIFY ABNORMALITIES IN LIPID METABOLISM IN DERMATOMYOSITIS PATIENTS Bory Kea, Robert Pesich, Lorinda Chung, Patrick Brown, and David Fiorentino Institutions: School of Medicine, Stanford University; Dept. of Biochemistry, School of Medicine, Stanford University; Division of Immunology and Rheumatology, Department of Medicine, Stanford University; Dept. of Dermatology, School of Medicine, Stanford University Dermatomyositis (DM) is a complex autoimmune disease in which the characteristic skin inflammation is the only constant, defining feature. Despite this, the mechanism of cutaneous disease in DM has not been extensively investigated and remains poorly understood. We sought to identify gene expression patterns that could lead to novel hypotheses regarding pathogenesis of skin disease in DM. We used printed oligonucleotide DNA microarrays that represent nearly all human genes (44,544 70mer sets) to analyze the skin biopsies from 9 healthy controls and 12 adult patients with DM. We performed SAM (Statistical Analysis of Microarray) analysis to identify genes that discriminate between active skin disease and control subjects. With a false discovery rate of 5%, we found 207 and 355 genes whose expression was strongly increased or decreased, respectively, in DM relative to controls. Functional annotation clustering of these genes identified two major biological processes, as defined by Gene Ontology—that of lipid metabolism and host-pathogen interaction. These processes included a major signature for lipid metabolism (43 genes, p=2E13), steroid biosynthesis (10 genes, p=6E-10), and carboxylic acid metabolism (39 genes, p=8E-14), and to a lesser extent, biotic stimulus (54 genes, p= 3.7E-8), response to pest, pathogen, or parasite (31 genes, p=2E-7), and defense response (49 genes, p=9E-7). Our data are consistent with previous findings from muscle biopsies in DM indicating activation of type I interferon responsive genes, and we extend these findings to include the intriguing possibility that lipid metabolism contributes to the pathogenesis of skin disease in dermatomyositis. Funded by the Stanford Medical Scholars Research Program & Genentech THE EFFECTS OF DIGITAL DERMOSCOPY ON SKIN SELF-EXAMINATION IN PATIENTS AT INCREASED RISK FOR MELANOMA Layton, Christle. Pollit, Ricardo. Ngyuen, Josephine, MD. Susan Swetter, MD. Stanford Department of Dermatology. In recent decades, the rate of melanoma has been on the rise, becoming one of the most common preventable cancers. Melanoma prevention and education are aimed at decreasing its morbidity and mortality, especially for high-risk populations such as patients with atypical moles. This project's main objective is to evaluate the effect that digital dermoscopy imaging has on increasing skin self-examination (SSE) in patients who are at increased risk for melanoma based on abnormal mole phenotype (atypical mole syndrome and familial atypical mole-melanoma syndrome). SSE is the purposeful inspection of one's skin for new or changing moles. SSE is estimated to reduce melanoma mortality by 63%. Dermoscopy is a non-invasive technique that magnifies skin features and pigmented skin lesions that are not visible to the unaided eye. A study is being conducted at the Stanford Pigmented Lesion and Cutaneous Melanoma Clinic (PLCMC) using digital dermoscopy to identify melanoma and melanoma precursors. In addition, a patient survey is being conducted to examine whether digital dermoscopy improves the quality and frequency of SSE. Using the melanoma ABCDE (asymmetry, border irregularity, color variation, diameter, and evolution) criteria, patients are shown normal and abnormal mole features. Patients are surveyed before and after dermoscopy to evaluate if this intervention helps to educate high-risk patients about SSE and their ability to detect suspicious lesions for early melanoma detection. We hypothesize that an intervention focused on digitally imaging pigmented skin lesions will increase patients' self skin-examination and awareness of melanoma warning signs. This type of intervention may help to reduce melanoma morbidity and mortality in this high-risk population and lay the groundwork for using digital dermoscopy to help educate patients regarding normal and abnormal skin features. Funded by the Stanford Medical Scholars Research Program. References: 1. Howe HL, Wingo PA, Thun MJ, et al. Annual report to the nation on the status of cancer (1973 through 1998), featuring cancers with recent increasing trends. J Natl Cancer Inst 2001;93:824-842 2. Berwick M, Begg CB, Fine JA, Roush GC and Barnhill RL. Screening for cutaneous melanoma by skin self-examination. J Natl Cancer Inst. 1996;88:17-23. FACTORS ASSOCIATED WITH POOR FOLLOW-UP AMONG GLAUCOMA PATIENTS IN SOUTH INDIA Bradford W. Lee, Kuldev Singh (Stanford Medical School, Ophthalmology), Parthasarathi Sathyan (Aravind Eye Hospital, Coimbatore, India), Alan L. Robin (Johns Hopkins University, Baltimore, MD). Purpose: To determine the factors associated with poor attendance of follow-up glaucoma examinations (FGEs) among glaucoma patients in South India. Methods: This prospective case-control study enrolled 300 established patients with primary glaucoma who did or did not attend FGEs as advised in the past year at Aravind Eye Hospital. Responses regarding various factors hypothesized to be associated with poor attendance of FGEs were collected by oral questionnaire. Unadjusted and adjusted odds ratios were then calculated using step-wise multiple logistic regression. Results: The factors most associated with poor attendance of FGEs included: lower perceived importance of attending FGEs [Adj. OR—10.80, 4.40-26.50], non-use of glaucoma medications [Adj. OR—2.10, 1.10-4.00], and means-tested waiving of clinic fees for low-income patients (“free patients) [Adj. OR—3.10, 0.91-10.50]. Notable factors not significantly associated with FGE attendance included: severity of disease, convenience of transportation to clinic, and FGE-related expenses (direct and indirect). Conclusions: Despite the provision of free clinical services for low-income patients, having one’s clinic fees waived is still independently associated with poor attendance of FGEs. Lower perceived importance of attending FGEs and non-use of glaucoma medications are also associated with poor attendance of FGEs. Meanwhile, many factors traditionally believed to explain poor attendance of FGEs, such as inconvenient transportation to clinic and less severe disease, were found to have little to no effect in this study. These findings suggests that efforts to improve patient attendance of FGEs should focus on changing patients’ perceptions about the importance of attending regular FGEs, since even marginal differences in patients’ perceived importance of follow-up (“somewhat important” vs. “very important”) were associated with significant differences in FGE attendance. Furthermore, administering short questionnaires that elicit factors associated with poor follow-up may be a valuable means of identifying patients at greater risk for poor follow-up. These patients can then be counseled, educated, and treated appropriately in order to minimize disease progression and unnecessary glaucoma-induced vision loss. Acknowledgements: -Stanford Medical Scholars Research Program -Aravind Eye Care System -Jennifer Staple & UFS WHY WE SHOULD SCREEN ALL FOREIGN-BORN ASIAN AMERICAN ADULTS FOR HEPATITIS B: A CROSS-SECTIONAL STUDY OF 3,163 ASIANS IN CALIFORNIA Steven Y. Lin, Ellen T. Chang, and Samuel K. So Department of Surgery Asian Americans are at disproportionately high risk for liver disease due to their high prevalence of chronic hepatitis B virus (HBV) infection – a disease that, if undetected, is associated with a 25% chance of death from cirrhosis or liver cancer. Our objective was to study the prevalence of chronic HBV infection and hepatitis B vaccination among Asian adults. From 2001 to 2006, we provided free HBV serological screening to Asians in the San Francisco Bay Area. Participants completed a survey assessing hepatitis B vaccination status. The study was conducted in Asian communities in San Francisco, San Jose, Cupertino, Millbrae, Milpitas, Sunnyvale, and a screening clinic at Stanford Hospital. Among a volunteer sample of 3,163 Asian adults (age range: 18 to 101 years, median: 52.9 years), over 93% were foreign-born. The main outcome measures were seroprevalence of hepatitis B surface antigen (HBsAg) and surface antibody (HBsAb). Of 3,163 Asian adults screened, 8.9% were chronically infected with HBV. Alarmingly, 2 in 3 (65.4%) of those chronically infected were unaware that they were infected. Participants born in East Asia, Southeast Asia or the Pacific Islands (10.7% HBsAg-positive) were approximately 20 times more likely to be chronically infected than participants born in the U.S. (0.7% HBsAg-positive) (relative risk=19.4, 95% confidence interval: 2.6, 141.8). Of those who were not infected, 44.8% lacked protective antibodies against HBV and were susceptible to future infection. Only 12.0% of participants reported having been vaccinated against HBV. Of these individuals, 20.3% lacked protective antibodies and 5.2% were found to be chronically infected with HBV. Given the serious medical implications of this study, a strong public health response is needed. In support of the newly released Centers for Disease Control and Prevention national recommendations, we call for all foreign-born Asian adults to be screened for HBV – regardless of their vaccination status. Funded by the Asian Liver Center at Stanford University THE ROLE OF CSN5 IN UV-MEDIATED DNA DAMAGE Helen Liu, David J. Wong, Howard Y. Chang Stanford University School of Medicine The nucleotide excision repair (NER) pathway is essential for prevention of DNA damage and skin cancers induced by ultraviolet radiation. CSN5 is the catalytic subunit of the COP9 signalosome and a key regulator of the ubiquitin ligase activities of DDB2 and CSA complexes that mediate NER. The CSN5 gene resides on the long arm of human chromosome 8, which is frequently amplified in human squamous cell carcinomas (SCC). However, the role of CSN5 in ultraviolet-induced carcinogenesis remains unclear. Here we show that CSN5 protein is overexpressed in 60% of human SCCs. Enforced expression of CSN5 in epithelial cells, but not a catalytically inactive mutant, is sufficient to confer protection against apoptosis induced by UV radiation. To better define the roles of CSN5 in stratified epidermis in vivo, we generated K5-CSN5 and K5-CSN5(D151N) transgenic mice that express CSN5 or the catalytically inactive CSN5(D151N) in murine epidermis. The role of CSN5 on UV-induced apoptosis, DNA repair, and mutagenesis in our transgenic mouse models will be presented. These studies will also better define CSN5 as a potential therapeutic target for human epidermal neoplasia and lightregulated dermatoses. Funded by the Stanford Medical Scholars Research Program EPENDYMOMA INCIDENCE IS A FUNCTION OF GENDER AND AGE BUT NOT TIME: A SEER STUDY Courtney S McGuire, Kristen L Cobb and Paul G Fisher Health Research and Policy Epidemiology Neurology and Pediatrics Previous small studies disagree about how clinical risk factors influence ependymoma incidence, perhaps due to limited sample size. We aimed to show the relationship of incidence to gender, race, and tumor location by rigorous analysis of a cancer registry, and to determine incidence trends over the past three decades. Data were obtained from Surveillance Epidemiology End Results (SEER-9) from 1973 to 2003. Histologic site codes (ICD-O-3: 9391-9394) were used to define ependymomas. Differences in age-adjusted incidence rates were compared by confidence intervals in SEER*Stat 6.2. A multiplicative Poisson regression and joinpoint regression were performed to determine annual percentage change and look for sharp changes in incidence, respectively. 1402 subjects (798 males, 604 females; 1213 whites, 112 blacks) were identified. Incidence (/100,000) was significantly higher in males than females (males 0.227 +/standard error [SE] 0.029, females 0.166 +/SE 0.030). For children (18 years and younger), age at diagnosis differed significantly by tumor location, with mean ages of incidence for infratentorial 5.04 +/SE 0.41 years, supratentorial 7.77 +/SE 0.60 years, and spinal 12.16 +/SE 0.80 years. Between 1973 and 2003, there were no significant changes in incidence over time and no sharp changes at any one year. Males have higher incidence of ependymoma compared to females. A biologic explanation for this finding remains elusive. Ependymoma occurs at distinctly different locations at different ages, consistent with hypotheses postulating distinct populations of radial glia stem cells within the central nervous system. Contrary to prior research, ependymoma incidence does not appear to have increased over the past three decades. Funded by the Stanford Medical Scholars Research Program CURCUMIN EXPOSURE INDUCES G1 ARREST IN SACCHAROMYCES CEREVISIAE BY IRON STARVATION Steven C. Minear, Allyson O'Donnell, Guri Giaever, Corey Nislow, Tim A. Stearns, Martha Cyert. Dept. of Biological Sciences. Curcumin, a naturally occurring substance, shows great potential as a therapeutic compound. Clinical trials are underway using curcumin as an anti-cancer therapeutic, and curcumin treatment is proposed for many other diseases, including Alzheimers and atherosclerosis. Studies on curcumin activity report pleiotrophic downstream effects, however curcumin’s mechanism of action in vivo remains unclear. We show that curcumin impairs iron uptake in cells. This role for curcumin in modulating iron uptake helps explain previously described curcumin-related phenomenon. Using genomic screens, we identified mutations of non-essential genes in Saccharomyces cerevisiae that result in hypersensitivity to curcumin. Our analysis shows that curcumin influences the high-affinity copper and iron import pathways. Iron supplementation of media rescued all hypersensitive strains, while copper supplementation rescued only a subset of strains. Rescue of all mutants by iron, including those with defects in copper regulation, suggests that the primary function of curcumin is to starve cells of iron. This is not surprising since high-affinity iron import is copper-dependent. Curcumin exposure depletes cellular iron and elicits a transcriptional response consistent with iron starvation. Moreover, curcumin induces a G1 arrest, an effect alleviated by iron supplementation. We expanded our studies to include human tissue culture cells and found striking similarities between yeast and mammalian cellular responses to curcumin. Like yeast, human cell growth is inhibited when curcumin is present, and iron addition rescues this effect. Our novel findings link the previously reported iron chelation by curcumin to curcumin-induced cell cycle arrest, and begins to define the mechanism by which curcumin produces its wide variety of effects. The curcumin-induced iron starvation is conserved in yeast and human cells. Findings from our genetic screens in yeast can be used as a framework for further investigations in humans. Further exploration of curcumin’s iron-transport modulation will enable a more targeted therapeutic application of this compound. Funded by the Stanford Medical Scholars Research Program IN VIVO DELIVERY OF WNT PROTEINS BY LIPOSOMAL PACKAGING Nathan T. Morrell1, Jae-Beom Kim1, Philipp Leucht1, Derk ten Berge2, Roel Nusse2, and Jill A. Helms1 1:Department of Surgery, Division of Plastic and Reconstructive Surgery, 2: Department of Developmental Biology The Wnt family of lipoproteins exercises strong cell-biological effects of therapeutic value, but, until now, no method for in vivo delivery existed. We exploited the lipophilicity of Wnts by packaging purified Wnt3a protein into lipid vesicles and assessed its activity using both in vitro and in vivo assays. When purified Wnt3a was added to liposomes, 55% of input activity was detected when assessed via an in vitro reporter assay. Because Wnt proteins contain posttranslational lipid modifications, we assumed that, rather than being encapsulated, Wnt3a protein would be tethered to the liposomal surface where it could stimulate target cells. A series of experiments involving enzymatic cleavage and Western blot analyses revealed that 55% of the input protein was localized to the exo-liposomal surface where is could stimulate target cells; 15% of the input protein was localized to the endo-liposomal surface where it was unavailable to induce the Wnt signaling cascade. This packaging scheme allows us to conclude that liposomal packaging does not adversely affect Wnt3a activity. Finally, in vivo studies using reporter mice demonstrated that Wnt3a liposomes elicit a biological response at a concentration where purified protein does not. The delivery of Wnt3a liposomes to skeletal injury sites induced reporter activity and lead to a dramatic enhancement of tissue repair and regeneration. Taken together, liposomal packaging may turn purified Wnt protein into a powerful therapeutic reagent. Because the endogenous transport of Wnt proteins may occur through association with lipid vesicles, Wnt3a liposomes may represent a biomimetic strategy for the delivery of Wnts for the treatment of diseases, aid in regeneration, or enhancement of stem cell self-renewal and proliferation. Future experiments will aim to optimize this system for therapeutic benefit. Funding (for NTM) by the HHMI Research Training Fellowship and the Stanford Medical Scholars Research Program A UBIQUITOUS DOUBLE-FLUORESCENT CRE REPORTER MOUSE Mandar D. Muzumdar, Kazunari Miyamichi, Ling Li, Bosiljka Tasic, and Liqun Luo Howard Hughes Medical Institute and Department of Biological Sciences The Cre/loxP system has been used extensively for conditional mutagenesis in mice. In this system, Cre recombinase regulated by a tissue-specific and/or temporallyregulated promoter can excise essential loxP-flanked (“floxed”) genes via intrachromosomal recombination to generate conditional knockouts. Reporters of Cre recombinase activity are important to define the spatial and temporal extent of Cremediated recombination. Here we describe mT/mG, a double-fluorescent Cre reporter mouse that expresses membrane-targeted tandem dimer Tomato (mT) prior to Cre-mediated excision and membrane-targeted green fluorescent protein (mG) following excision. Homozygous mT/mG mice are viable and fertile, demonstrating minimal toxicity of the fluorescent markers. We show that reporter expression is ubiquitous, allowing visualization of fluorescent markers in live and fixed samples of all tissues examined. We further establish that mG expression is Cre-dependent and complementary to mT at single cell resolution. mT/mG is the first demonstration of tandem-dimer Tomato expression in vivo in mice. Our results suggest that tdTomato is a sufficiently bright and photostable red fluorescent protein for complementary use with existing green fluorescent protein lines. Both single-copy membrane-targeted markers outline cell morphology, highlight membrane structures, and permit visualization of fine cellular processes in vivo. mT/mG represents the first ubiquitously-expressed double-fluorescent Cre reporter mouse. We conclude that mT/mG will serve not only as a ubiquitous Cre reporter but also as a tool for lineage tracing, mosaic analysis, transplantation studies, and analysis of cell morphology in vivo. Funded by a Howard Hughes Medical Institute Research Training Fellowship for Medical Students and the Stanford Medical Scholars Research Program. PPARγ CONTROLS ALTERNATIVE MACROPHAGE ACTIVATION TO AMELIORATE OBESITY-INDUCED INSULIN RESISTANCE Justin I. Odegaard, Roberto R. Ricardo-Gonzalez, Matthew H. Goforth, Christine R. Morel, Vidya Subramanian, Lata Mukundan, Alex Red Eagle, Divya Vats, Frank Brombacher, Anthony W. Ferrante, Ajay Chawla 1 Division of Endocrinology, Metabolism and Gerontology, Department of Medicine, 2 Graduate Program in Immunology, 3 Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5103 4 Department of Medicine, Naomi Berrie Diabetes Center, Columbia University College of Physicians and Surgeons, New York, NY 5 Institute of Infectious Disease and Molecular Medicine, Division of Immunology, Health Science Faculty, University of Cape Town, Werhner Beit South, Cape Town, South Africa Obesity and insulin resistance, cardinal features of metabolic syndrome, are closely associated with a state of low-grade inflammation. In adipose tissue chronic overnutrition leads to macrophage infiltration, resulting in local inflammation that potentiates insulin resistance. Because macrophages also actively participate in the resolution of inflammation, we postulated that macrophage activation programs that terminate inflammation might ameliorate obesity-induced insulin resistance. In particular, the interleukin-4 (IL-4) driven program of alternative macrophage activation has been shown to dampen inflammation and enhance repair in tissues; however, its role in obesity and insulin resistance remains unknown. Using mice with macrophage-specific deletion of peroxisome proliferator activated receptor-γ (PPARγ), we show here that PPARγ is required for maturation of alternatively activated macrophages. Disruption of PPARγ in myeloid cells impairs alternative macrophage activation, thereby predisposing these animals to development of diet-induced obesity, insulin resistance, and glucose intolerance. Furthermore, gene expression profiling revealed that downregulation of oxidative phosphorylation gene expression in skeletal muscle and liver leads to decreased insulin sensitivity in these tissues. Together, our findings demonstrate that resident alternatively activated macrophages have a beneficial role in regulating nutrient homeostasis and suggest that macrophage polarization towards the alternative state might be a useful strategy for treating obesity and type 2 diabetes. This work was supported by grants made available to AC: NIH (DK062386 and HL076746), Astellas Foundation, Takeda Pharmaceuticals North America, Rockefeller Brothers Fund and by Goldman Philanthropic Partnerships. AC is a Charles E. Culpeper Medical Scholar. JIO was supported by Stanford MSTP and AHA fellowships, RRR by NRSA fellowship (AI066402), and LM by NIH Training grant (AI07290). THE ROLE OF BONE MARROW-DERIVED CELLS IN THE PROCESS OF HYPERTROPHIC SCAR FORMATION Shola B. Olorunnipa, Shahram Aarabi, and Geoffery C. Gurtner Hypertrophic scars occur following cutaneous wounding and result in severe functional and aesthetic defects. The pathophysiology of this process remains unknown and, as a result, treatment remains a challenge. Recent studies by our laboratory have demonstrated that mechanical stress applied to a healing wound is sufficient to produce hypertrophic scars in mice. These scars are histopathologically identical to human hypertrophic scars and show equivalently dramatic increases in volume and cellular density. The development of this novel murine model opens the door to further investigation of the underlying pathophysiology of hypertrophic scarring. Our study utilizes this model to investigate the contribution of cells derived from the bone-marrow to the formation of hypertrophic scars. Bone marrow from Green fluorescent protein (GFP) labeled mice was transplanted into irradiated C57BL/6 mice. Hypertrophic scars were generated on the dorsa of transplant recipient mice using a mechanical strain device. Normal scars were also generated on each mouse to serve as internal controls. The resulting scars were harvested and analyzed via fluorescent microscopy enabling us to visualize the contribution of bone-marrow derived cells to both hypertrophic scars and control wounds. Preliminary results based on histologic analysis suggest that the 25-fold increase in cellular density seen in murine hypertrophic scars can be attributed in part to greater recruitment of bone-marrow cells to the site of injury. Ongoing studies are utilizing cell markers and flow cytometry to determine the cellular phenotypes of these GFPpositive marrow-derived cells and to provide a quantitative measurement of their contribution to hypertrophic scars when compared to internal controls. Funded by the Stanford Medical Scholars Research Program OVERCOMING DELAYS IN CHILDBIRTH DUE TO HEMORRHAGE: A QUALITATIVE STUDY OF THE NON-PNEUMATIC ANTI-SHOCK GARMENT (NASG) IN NIGERIA Adeoti Oshinowo , Hadiza Galadanci, MD , Mohammed Awwal, MD , Oladosu Ojengbede, MD , Lyndsay McDonough, MPH , Elizabeth Butrick, MPH, MSW , Suellen Miller, PhD, CNM . (1) School of Medicine, Stanford University. (2) Obstetrics and Gynecology, Aminu Kano Teaching Hospital, Kano, Kano, Nigeria. (3) Obstetrics and Gynecology, Murtallah Mohamed Speciality Hospital, Kano, Kano, Nigeria. (4) Obstetrics and Gynecology, University College Hospital, College of Medicine, Ibadan, Nigeria. (5) School of Public Health, Tulane University. (6) Obstetrics, Gynecology and Reproductive Sciences, University of California, San Francisco. (7) OB/GYN and Reproductive Sciences, University of California, San Francisco. Background: Obstetric hemorrhage (OH) is the leading cause of maternal mortality due to delays in obtaining Emergency Obstetric Care. Women die during transports to facilities or while awaiting appropriate care in facilities. One strategy for decreasing MMR from OH is a first aid device, the Non-pneumatic Anti-Shock Garment (NASG), a low technology compression suit. The NASG is being pilot tested in Kano, Nigeria. Objectives: To understand provider, patient and family perceptions of the NASG in order to enhance its acceptability and decrease delays in application. Methods: 10 focus groups of 134 health care providers (doctors, nurse-midwives, nurses, and staff) and 6 in-depth, individual interviews of patients who survived severe OH and shock and/or their family members, were conducted and analyzed using grounded theory. Results and conclusions: Providers agreed that the NASG was easy to use, improved management of OH, but was difficult to fold correctly, and was not always the first thing providers would think of to apply in frantic emergency situations. Patients generally accept the NASG, but those who received less information about it before it was applied (they were unconscious at the time of application) who wake up in it are confused and uncomfortable with it; some try to remove it. There are clear policy implications from this study. As a new device, more training in pre-service education may help incorporate the NASG into providers' emergency response algorithm. Ante-natal and community education activities may help women and family members learn about the NASG as a life-saving device, not something to fear. Learning objectives: 1. Recognize obstacles to implementing a new technology to reduce maternal mortality associated with obstetric hemorrhage in Nigeria. 2. List training methods that will enhance providers incorporating a new technology into their emergency response algorithm. 3. List community outreach activities that can be conducted to improve families understanding of a new device to keep women alive during transport to emergency obstetrical care. My Acknowledgments: •Dr. Suellen Miller (my UC Berkeley Advisor) and Elizabeth Butrick for always bringing “light to my life”; Doug Oman, Alan Hubbard, and Maureen Lahiff for their statistical genius; Dr. Paul Hensleigh, for being my Stanford project advisor; Lyndsay McDonough and Dr. Aminu Isyaku for helping me navigate Kano; Women's Global Health Imperative; and the Stanford Medical Scholars Research Program funding. ENCODING SKELETAL MORPHOLOGY IN THE GENOME Luiz C. Pantalena Filho, Catherine Guenther, Christine Ham, David Kingsley Department of Developmental Biology, Stanford University There is great variation in the skeletal system of vertebrates. This variation has a strong genetic component. The skeletal system is also modular; in other words, it is possible to change one skeletal element and its shape, while allowing other elements to remain unaltered. The following experiments present a role for the gene Bone Morphogenetic Protein 5 (Bmp5) in this process. Isolation of Bmp5 cis-regulatory elements show that skeletal elements, such as ribs, can be further subdivided into compartments. They also allow the manipulation of skeletal morphology. By altering levels of BMP signaling in rib compartments during development, I have been able to alter both rib cross sectional shape and vertebrae-to-sternum trajectory in transgenic animals. In addition, I was able to detect impaired bone deposition, in a compartment-specific manner, in regulatory Bmp5 mutants lacking specific cisregulatory elements. Finally, I also describe experiments implicating the use of cisregulatory elements in fracture response. Therefore, these experiments show that Bmp5 cis-regulatory elements affect skeletal morphology. I have also added to the concept of compartmentalization of skeletal elements, which add to the modularity of the skeleton. Finally, I have shown that processes using Bmp5 to shape bone during development are re-capitulated during fracture healing. These experiments describe a method to encode skeletal morphology in the genome. Funded by the Stanford Medical Scholars Research Program EFFECT OF PULSE DURATION ON THE SIZE AND CHARACTER OF THE LESION IN RETINAL PHOTOCOAGULATION Yannis M. Paulus, ATul Jain, Michael W. Wiltberger, Dan E. Andersen, Phil Huie, Mark S. Blumenkranz, Daniel Palanker Department of Ophthalmology, Stanford University School of Medicine The recently-developed semi-automated Patterned Retinal Photocoagulation (PRP) system transmits multiple spots in a prearranged pattern, thus decreasing procedure time and increasing reproducibility from conventional PRP. To utilize this new technology in clinical practice, an assessment of laser parameters must be performed. The effects of laser beam size, power, and pulse duration from 1 to 100ms on the characteristics of ophthalmoscopically visible retinal coagulation lesions were systematically evaluated. A 532nm Nd:YAG laser was used to irradiate 36 retinas in Dutch-Belt rabbits with retinal beam sizes of 66, 132, and 330 μm. Lesions were clinically graded 1 minute after lesion placement, lesion size was measured by digital imaging, and lesion depth was assessed histologically at different time points. Retinal lesion size increased linearly with laser powers from 50 to 250 mW and logarithmically with pulse durations from 10 to 100 ms. Ophthalmoscopically visible retinal lesions obtained with 10 and 20 ms pulses were 3 and 2 times smaller than those produced by 100 ms exposures, respectively. The width of the therapeutic window, defined by the ratio of the threshold power for producing a rupture to that of a mild coagulation, decreased with decreasing pulse durations. For 330 μm retinal beam sizes, the therapeutic window declined from 5.4 to 3.7 as pulse duration decreased from 100 to 20 ms. At pulse durations of 1 ms, the therapeutic window decreased to unity, at which point rupture and a mild lesion were equally likely to occur. This data shows that at shorter pulse durations, the diameter of retinal lesions is smaller and less dependent on variations in laser power than at longer durations. The width of the therapeutic window, a measure of relative safety, increases with the beam size. Clinically, pulse durations of 10 to 20ms represent an optimal compromise between the favorable impact of speed, higher spatial localization, and reduced collateral damage on one hand, and sufficient width of the therapeutic window (>3), on the other. This may translate into less damage to nerve fiber layer, decreased choroidal swelling, and reduced pain due to decreased penetration of heat into the choroid, but this will need to be confirmed in clinical trials. Funded by the Stanford Medical Scholars Research Program, the Alcon Research Institute, and the Horngren and Miller Family Foundations. THE EFFECTS OF DIGITAL DERMOSCOPY ON SKIN SELF-EXAMINATION IN PATIENTS AT INCREASED RISK FOR MELANOMA Pollit, Ricardo. Layton, Christle. Ngyuen, Josephine, MD. Susan Swetter, MD. Stanford Department of Dermatology. In recent decades, the rate of melanoma has been on the rise, becoming one of the most common preventable cancers. Melanoma prevention and education are aimed at decreasing its morbidity and mortality, especially for high-risk populations such as patients with atypical moles. This project's main objective is to evaluate the effect that digital dermoscopy imaging has on increasing skin self-examination (SSE) in patients who are at increased risk for melanoma based on abnormal mole phenotype (atypical mole syndrome and familial atypical mole-melanoma syndrome). SSE is the purposeful inspection of one's skin for new or changing moles. SSE is estimated to reduce melanoma mortality by 63%. Dermoscopy is a non-invasive technique that magnifies skin features and pigmented skin lesions that are not visible to the unaided eye. A study is being conducted at the Stanford Pigmented Lesion and Cutaneous Melanoma Clinic (PLCMC) using digital dermoscopy to identify melanoma and melanoma precursors. In addition, a patient survey is being conducted to examine whether digital dermoscopy improves the quality and frequency of SSE. Using the melanoma ABCDE (asymmetry, border irregularity, color variation, diameter, and evolution) criteria, patients are shown normal and abnormal mole features. Patients are surveyed before and after dermoscopy to evaluate if this intervention helps to educate high-risk patients about SSE and their ability to detect suspicious lesions for early melanoma detection. We hypothesize that an intervention focused on digitally imaging pigmented skin lesions will increase patients' self skin-examination and awareness of melanoma warning signs. This type of intervention may help to reduce melanoma morbidity and mortality in this high-risk population and lay the groundwork for using digital dermoscopy to help educate patients regarding normal and abnormal skin features. Funded by the Stanford Medical Scholars Research Program. References: 1. Howe HL, Wingo PA, Thun MJ, et al. Annual report to the nation on the status of cancer (1973 through 1998), featuring cancers with recent increasing trends. J Natl Cancer Inst 2001;93:824-842 2. Berwick M, Begg CB, Fine JA, Roush GC and Barnhill RL. Screening for cutaneous melanoma by skin self-examination. J Natl Cancer Inst. 1996;88:17-23. LATERAL DISPLACEMENT IS AN INDICATOR OF STENT-GRAFT MIGRATION IN ENDOVASCULAR ANEURYSM REPAIR Benjamin Y. Rafii*, Oscar J. Abilez, and Christopher K. Zarins Department of Surgery, Division of Vascular Surgery Background and Purpose: Positional stability of stent-grafts (endografts) is important in the long-term durability of endovascular aortic aneurysm repair (EVAR). Longitudinal migration of endografts may lead to loss of fixation and development of endoleaks, potentially exposing the patient to continued risk of aneurysm rupture. Previous studies have described inadequate proximal (aortic neck) fixation and distal (iliac) fixation as predictors of subsequent longitudinal migration. However, the importance of lateral stability of the endograft within the aneurysm sac is unknown. The present study examines whether longitudinal migration of the endograft corresponds with lateral displacement, defined as significant movement of the midportion of the endograft within the transverse plane. Methods: A retrospective review of computed tomography scans taken immediately and one year postoperatively from 43 patients undergoing endovascular aneurysm repair at Stanford Hospital between 1998 and 2005 was conducted. The study population included 19 patients with ≥ 5 mm longitudinal migration at one year (migrators) and 24 patients with < 5 mm longitudinal migration at one year (nonmigrators). A novel measurement approach was employed to quantify absolute and percent lateral displacement of the endograft, using the vertebral body as an anatomical reference point. Multivariate data analysis was performed using JMP 6 statistical software. Results and conclusions: The mean longitudinal displacement in the migrator group was 8.1 ± 3.7 mm, with a range of 5.0 to 19.0 mm. The mean longitudinal displacement in the non-migrator group was 1.7 ± 1.6 mm, with a range of 0.0 to 4.0 mm. The mean absolute lateral displacement among migrators was 5.3 ± 5.8 mm, with a range of 0.0 to 21.0 mm. The mean absolute lateral displacement among nonmigrators was 3.4 ± 3.5 mm, with a range of 0.0 to 13.0 mm. There was a significant (p=0.008) correlation between longitudinal migration and absolute lateral displacement of the endograft relative to the vertebral body. When measured separately, there was a significant (p=0.0045) correlation between longitudinal migration and absolute lateral displacement among migrators; in contrast, there was no significant correlation between longitudinal migration and absolute lateral displacement among non-migrators (p=0.2). Taken together, these results suggest a relationship between longitudinal migration of the endograft and lateral displacement of the endograft in the transverse plane. This raises the possibility that positional instability in the transverse plane may contribute to longitudinal migration of the endograft over time. Future interventions or preventative measures may be directed toward lateral stabilization of the endograft within the aneurysm sac. Funded by the Stanford Medical Scholars Research Program CHARACTERIZATION OF PARENTAL PSYCHOPATHOLOGY IN ANOREXIA NERVOSA Sheila Ravi*, Sarah Forsberg and James Lock,MD,Ph.D Department of Psychiatry Although there are many studies that have characterized patients suffering from eating disorders, few have focused on characteristics of their parents. The current study aimed to both assess the levels of psychopathology displayed by the parents of adolescents suffering from anorexia nervosa (AN), and examine the relationship between specific adolescent eating disorder characteristics and parental psychopathology. 75 female adolescent subjects suffering from AN were administered the Eating Disorder Examination (EDE) and their parents were given the SCL-90-R questionnaire. Analysis showed that significant numbers of both fathers and mothers of anorexic adolescents suffer from sub-clinical and clinical levels of obsessive compulsive behaviors, hostility, depression, and anxiety as measured by the subscales of the SCL-90-R. Further analysis showed a significant relationship between hostility and depression scores among mothers and fathers, respectively, and the duration of their child’s illness. There were no significant relationships between other aspects of eating disorder symptom severity and parental psychopathology. While these results cannot delineate the direction of the relationship between parental psychopathology and adolescent eating disorders, it suggests that parental psychopathology may play a limited role in either the development or maintenance of AN. Funded by the Stanford Medical Scholars Research Program OPTIMIZATION OF FLEXOR TENDON TISSUE ENGINEERING: THE ROLE OF MECHANICAL FORCES Jonathan Riboh BS; Alphonsus Chong MD; Hung Pham BS; Michael Longaker MD, MBA; Chris Jacobs PhD; James Chang MD, FACS 1: Division of Plastic and Reconstructive Surgery, Department of Surgery 2: Department of Bioengineering Flexor tendon injuries are both frequent and devastating. Despite recent progress in operative and rehabilitative care, contractions, fibrous adhesions, and long-term disability are common. These challenges are magnified in severe trauma, where the amount of tendon lost exceeds the supply of autologous grafts. Tissue engineering promises to help address these issues. In this study we applied Functional Tissue Engineering (FTE) techniques to the regeneration of flexor tendons. FTE focuses on combining biological and mechanical stimuli to recreate environments in the laboratory that mimic those encountered in situ. The goals of this study were fourfold: to identify the best cell line for flexor tendon engineering, and to study the effects of mechanical forces on cell proliferation, collagen production and morphology. Four candidate cell lines were tested: epitenon tenocytes (E), tendon sheath fibroblasts (S), bone marrow-derived stem cells (bMSC), and adipoderived stem cells (ASC). These cells were first tested for their ability to adhere to extracellular matrix (fibronectin), an essential component for scaffold seeding. S and ASC adhered significantly better than the others. Cells were then subjected to 3 different strain regimens: Continuous Cyclic Strain (CCS: 8% elongation, 1 Hz, 100% duty cycle), Intermittent Cyclic Strain 1:2 (ICS 1:2: 4% elongation, 0.1 Hz, 33% duty cycle) and ICS 1:5 (4% elongation, 0.1 Hz, 17% duty cycle). CCS caused a decrease in cell proliferation in all four cell lines, and induced moderate levels of apoptosis. However, collagen I production doubled in E, S and ASC and increased 10x in bMSC. S and ASC had the fastest growth rates. Based on these experiments, only S and ASC were retained for further study. ICS (1:2 and 1:5) caused a 20% increase in cell proliferation in ASC, and caused a 35% increase in collagen I production in S. All forms of cyclic strain caused parallel alignment of cells, with parallel organization of their cyctoskeleton, as well as nuclear and cellular elongation. In order to better understand the response to mechanical strain, microarray analysis of the ASC transcriptome before and after cyclic strain was performed. In this study we identified ASC as the ideal candidate cell line for flexor tendon engineering, which is encouraging given the ease of isolation of these adult stem cells. Furthermore, we established the dose-dependent effects of cyclic strain on cell proliferation and collagen production. We are currently using our functional genomics data to understand the influence of cyclic strain on the differentiation of ASC into various mesenchymal tissues. Funded by the Stanford Medical Scholars Research Program POST-TRANSLATIONAL MODIFICATION OF TUMOR SUPPRESSORS AND THE METHYLATION OF RETINOBLASTOMA Louis Saddic, Or Gozani, and Julien Sage Sage Lab, CCSR South, Stanford University The retinoblastoma (RB) tumor suppressor controls cell cycle progression at the G1/S transition of the cell cycle. Deletion of RB is found in a wide range of human cancers, including pediatric retinoblastomas and osteosarcomas, and carcinomas of the breast, bladder, and prostate. Proper regulation of RB activity during normal cell cycle is critical to prevent the development of these tumors. Most of this regulation takes place through phosphorylation by cyclin-dependent kinases, but emerging evidence suggests that RB activity may be controlled by other kinases a well as by other forms of post-translational modification such as acetylation. Arginine and lysine methylation of histones has been initially implicated in the regulation of chromatin structure. Since then, the discovery that non-histone protein can also undergo methylation has extended the importance of this post-translational modification in cells. For instance, methylation of the p53 protein controls its stability and its tumor suppressor activity. Thus far, there is no evidence that RB activity is controlled by methylation events. However, based on the abundance of lysines on the RB protein and the presence of RB in high molecular weight complexes containing methyltransferases, I hypothesize that RB is methylated in mammalian cells and that this methylation affects RB’s ability to act as a regulator of the cell cycle. Preliminary results have identified a putative lysine residue of RB that is methylated by the lysine methyltransferase Set9. The importance of this modification and the identification of additional methylated residues by other methyltransferases are currently being investigated. I would like to thank Andrew Venteicher and Ruth Tennen in the Artandi lab for assisting me with biochemical assays, Ken Lau for Mass Spectrometry, and the entire Sage Lab for critical commentary and experimental assistance. Funded by the Stanford Medical Scholars Research Program & Howard Hughes Medical Institute. ASSESSING PREOPERATIVE AND POSTOPERATIVE VISUAL ACUITY IN PATIENTS RECEIVING FREE CATARACT SURGERY BY OPHTHALMOLOGISTS AT FOUR EYE CLINICS IN GHANA AND INDIA Jennifer B Staple and Dr. Peter Egbert. Department of Ophthalmology. The objective of this four-site interventional study is to assess visual outcome after community-based screening and hospital-based cataract extraction programs. The outcomes measures include retention at follow-up, surgical complications, individual visual acuity results, and program level outcomes (efficiency rates). A total of 991 patients receiving free surgery at eye clinics in Ghana and India were included in the study. The surgical techniques vary for the free surgery patients at each eye clinic due to cost-effectiveness, training, and equipment available. One eye clinic in India provided 618 Phacoemulsification cataract surgeries, another clinic in India provided 49 Small Incision Cataract Surgeries (SICS), one eye clinic in Ghana provided 105 SICS, and another provided 219 ECCE+IOL. The results assessed the postoperative acuity improvement as well as the percentage of operated eyes achieving postoperative functional acuity of equal or better than 20/40. This study analyzed patient outcomes, principally preand postoperative vision in the operated eye. Of the 618 patients receiving Phacoemulsification in the eye clinic in India, 50% (311) had a preoperative visual acuity of “Count Fingers” (CF) or worse, which is defined as a best acuity of viewing fingers held directly in front of the eyes. Of the patients receiving surgery, 87% (537) had a final postoperative visual acuity of 20/40 or better in the operated eye. At the eye clinic in India providing SICS, 84% (41) patients had visual acuity of CF or worse, and 88% (44) had a final postoperative acuity of 20/40 or better. At the clinic in Ghana providing SICS, 79% (83) started with visual acuity of CF or worse. Postoperatively, 37% (39) of the patients had a visual acuity of 20/40 or better, and 58% (61) patients had visual acuity of 20/50 or better. At the Ghanaian eye clinic providing ECCE+IOL surgery, 100% (219) had a preoperative visual acuity of CF or worse. Postoperatively, 5% (11) had visual acuity of 20/40 or better, and 14% (31) of the patients had visual acuity of 20/50 or better. Age, pre-operative acuity, and any significant complications were also documented and analyzed in comparison to the outcomes. This prospective study is important to assess the quality of outcomes of communitybased surgery programs in two countries with differing levels of equipment, environmental conditions, and training opportunities. The goal is to contribute data to the international ophthalmology community about outcomes of cataract surgeries in medically underserved regions of the world. Funded by the Stanford Medical Scholars Research Program THE MIGHTY MOUSE: UBIQUITOUS EXPRESSION OF TRI-FUSION IMAGING MULTIMODALITY (BIOLUMINESCENCE, FLUORESCENCE, PET) REPORTER GENE IN TRANSGENIC MOUSE Ricky T. Tong, Pritha Ray, and Sanjiv S. Gambhir Department of Radiology and Bioengineering Reporter genes are extremely useful in following the gene expression and cellular behavior in development and disease studies in mice. While bioluminescence and fluorescence reporter genes provide valuable information, they are not tomographic, quantitative nor do they have great tissue depth penetration. Similarly, PET reporter genes are especially useful for whole body imaging, but they lack the sensitivity that is provided by bioluminescence or fluorescence imaging system at superficial depths. This present study aims to create a transgenic mouse that will ubiquitously express a multimodality imaging reporter construct and can be imaged by the three most common imaging techniques (bioluminescence, fluorescence, PET) used in small animal imaging research. This transgenic mouse model will be crucial in stem cell, cancer, and tissue engineering research as this universal donor mouse can serve as the source of any cells or tissues for transplant experiments. The tri-fusion reporter vector harbors a bioluminescence reporter gene (a mutated thermo-stable firefly luciferase), a fluorescence reporter gene (a monomeric red fluorescence protein) and a positron emission tomography (PET) reporter gene (truncated herpes simplex virus type 1 sr39 thymidine kinase). We first test and confirm the activity levels of the tri-fusion protein in multiple cell lines. To create the transgenic mouse, we insert the plasmid into fertilized eggs and implant them in female mice. Since the tri-fusion reporter gene is driven by the chicken β-actin promoter, all cells of the transgenic mouse, as expected, produce strong bioluminescence, fluorescence, and PET signals when the proper substrate (PET/bioluminescence) or light (fluorescence) is provided. In conclusion, we have demonstrated that our transgenic mouse provides strong bioluminescence, fluorescence, and PET signals in all cells. The first and current application of this mouse is to examine the contribution of circulating stem cells in tumor development. By performing a parabiosis surgery, we are “stitching” a transgenic mouse with a wild type mouse such that the two mice share blood circulation. A tumor is implanted in the flank of the wild type mouse and the circulating cells from the transgenic mouse are monitored using one of the three imaging modalities. Similarly, solid organ transplant experiments (using the transgenic mouse as the donor mouse) will also be performed in the near future. Funded by the Stanford Medical Scholars Research Program BLOOD AND LYMPHOID IMMUNE RECONSTITUTION FOLLOWING ALLOGENEIC HEMATOPOIETIC CELL TRANSPLANTATION IN MICE Gabriel J. Tsao, Jessica A. Allen, Kathryn Logronio, Judith A. Shizuru. Division of Blood and Marrow Transplantation. Introduction: Allogeneic hematopoietic cell transplantation (HCT) is an established way to cure many hematologic malignancies and bone marrow (BM) failure states. Unfortunately, there remain considerable risks involved with the procedure, in particular, the potential for graft-versus-host disease (GVHD) and the post-transplantation immunocompromised state. While pharmacotherapy is used to prevent and control GVHD, a more effective approach is through the transplantation of purified hematopoietic stem cells (HSC) which are devoid of T cells. However, decreased engraftment rates, reduced blood counts and increased infectious complications have been observed following HSC transplants, suggesting that the risks of using such grafts outweigh the potential benefits. While it is generally believed that HSC grafts result in impaired immune function post-HCT, there are surprisingly few reports interrogating immune recovery in recipients of T cell reduced compared to unmanipulated BM grafts. Methods: We compared immune reconstitution in mice transplanted with unmanipulated BM and purified HSC grafts, including MHC-matched, MHC-mismatched, and haploidentical transplant pairs. Immune reconstitution was evaluated quantitatively with complete blood and lymph node (LN) cell counts and by phenotyping and immunohistochemical analysis of LN size and architecture. Qualitative function was assessed by lymphocyte proliferation assays to MHC-restricted peptides. Results: Peripheral blood reconstitution showed markedly increased white counts across all lineages in the BM as compared to the HSC transplants. However, lymphoid reconstitution as measured by LN cell counts, size and architecture was significantly improved in the purified HSC transplant groups, especially in the MHC-mismatched setting. Qualitative measures of immune function revealed that LN proliferative responses were significantly increased in the HSC compared to BM transplant groups. Using MHC-restricted peptides, we determined that T cell restriction was dictated by donor rather than host elements. No significant differences in T-regulatory cell populations were found in the blood or LNs in the transplanted recipient groups. Conclusions: Our results show that although HSC grafts initially reconstitute slower than BM grafts as measured by parameters in the peripheral blood, their lymphoid reconstitution is superior, both quantitatively and qualitatively. Furthermore, when donor and host are mismatched at the MHC, the peripheral T cells are restricted to the donor MHC type. These studies challenge two tenets in clinical HCT and in basic immunology: First, it is generally thought that T cell depletion and graft manipulation leads to impaired immune reconstitution. Our studies suggest that even subclinical GVHD can significantly impair immune function and that transplantation of highly purified grafts may lead to superior long-term immune function post-allogeneic HCT. Second, these studies directly show that hematopoietic lineage cells can determine the MHC restriction of peripheral T cells, traditionally understood to be mediated exclusively by thymic epithelium cells. We thank Dr. Joseph Lin for his helpful contributions to this project. This work was supported in part by the Stanford Medical Scholars Research Program and a Paul and Daisy Soros Fellowship. THE ROLE OF THE COMPLEMENT CASCADE IN GLAUCOMA Luis E Vazquez, Beth Stevens, Navid Nouri, Gareth R Howell, Simon WM John, Ben A Barres Department of Neurobiology, Stanford University School of Medicine; Stanford, California 94305 Howard Hughes Medical Institute, The Jackson Laboratory; Bar Harbor, Maine 04609 Glaucoma is the leading neurodegenerative cause of blindness worldwide. Blindness results from death of the retinal ganglion cells (RGCs) of the retina. Although important risk factors that render RGCs susceptible to death have been identified, the cause of their death remains unknown. We hypothesize that RGCs degenerate because they loose their connections (synapses) with the rest of the cells in the retina. This has indeed been demonstrated in other neurodegenerative diseases, such as Alzheimer’s disease. We took advantage of the DBA/2J mouse model of Glaucoma to test this hypothesis, and to get at the molecular underpinnings of Glaucoma. Here, we demonstrate that there is synapse loss in the retina of glaucomatous mice. Moreover, within the retina, the area most severely affected is the inner plexiform layer (IPL), which hosts the RGC synaptic terminals. More importantly, synapse loss seems to precede RGC death, supporting the idea that synapse loss may lead to RGC degeneration. In addition, we found that C1q, a complement protein known to be upregulated in Glaucoma, accumulates in the IPL around the time of synaptic elimination. Similar to the synaptic stain (PSD-95), the C1q stain is also punctate, suggesting that C1q protein may be decorating synapses and targeting them for destruction. This C1q accumulation was not observed in pre-Glaucoma or in control mice. These findings, together with extensive data obtained by Beth Stevens in the Barres lab, suggests that pathological C1q upregulation by RGCs results in accumulation of C1q protein around synapses, targeting them for destruction. This synapse elimination may underlie the RGC death observed in glaucomatous mice. We believe that our findings shed light on the pathophysiology of Glaucoma in humans, and may open new avenues for treatment of this disease. Funded by the Stanford Medical Scholars Research Program HOW DO CYP1B1 MUTATIONS CAUSE GLAUCOMA? Jack T. Wang, Jeffrey L. Goldberg, and Ben A. Barres. Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305-5125, and Bascom Palmer Eye Institute, University of Miami, Miami, Florida 33136 Glaucomas are a group of ophthalmic diseases marked by excavation of optic disc, progressive death of retinal ganglion cells (RGCs), and eventually visual field loss. It is thought that obstruction of aqueous humor drainage secondary to structural deformities in the trabecular meshwork raises intraocular pressure (IOP) in the anterior chamber, causing direct physical damage and consequently optic nerve degeneration and RGC death. However, high IOP is neither necessary nor sufficient for the onset and progression of glaucoma. Furthermore, in glaucoma the RGCs specifically degenerate while other retinal cell layers remain relatively intact. It is unclear what makes RGCs more susceptible to degeneration than other retinal cell types in glaucoma, though the highly dynamic transcriptome of developing RGCs suggests that a genetic, cell-autonomous event may influence RGCs’ viability and succeptibility to external insult. Furthermore, the early onset and developmental abnormalities seen in congenital glaucoma indicate that such genetic events may occur during development. To address the possible developmental and molecular changes that may predispose RGCs to cell death in glaucoma, I examined differential gene expressions by RGCs at various stages of development. I found high RGC expression and developmental regulation of CYP1B1, a gene linked to the disease loci for congenital glaucoma and encodes an enzyme that catalyzes the rate limiting step of all-trans retinoic acid (RA) synthesis. The gene is enriched in the cytoplasm of embryonic RGCs and there was a dramatic downregulation of CYP1b1 of almost 21.7 folds by developing RGCs. I further demonstrated that CYP1b1 significantly promotes RGC survival but not neurite outgrowth in both postnatal and embryonic ages upon CYP1b1 overexpression, and that siRNA knockdown of CYP1b1 transcript abolished the survival effect. Co-culturing RGCs transfected with CYP1b1 in RAcontaining medium was found to further enhance the survival effect of CYP1b1 compared to controls, although RA by itself was not sufficient to increase RGC viability. This indicates that CYP1b1 is sufficient and necessary to promote RGC survival during development putatively through a downstream RA-mediated process. The study is first to identify CYP1b1 in the neural retina, as its expression was previously unidentified in RGCs possibly due to the gene’s developmental downregulation. The results further suggest that CYP1b1 may normally be neuroprotective in the developing neural retina, and its mutations can lead to glaucoma by decreasing RGC survival through decreased RA production or RA-dependent mechanisms. This indicates that rather than an external problem of aqueous drainage, some cases of glaucoma may arise from an intrinsic, cell-autonomous cause and that CYP1b1 mutations lead to early-onset glaucoma by failing to support survival within RGCs. This work is supported by the Stanford Medical Scholars Research Program, the HHMI Medical Fellows Research Training Grant, and the NIH CNS Repair and Regeneration Grant (EY11310). A NOVEL ADJUVANT’S IMMUNOGENECITY IS NOT MEDIATED BY PLASMACYTOID DENDRITIC CELLS Thierry Giffon, Lena Winestone, David Lewis Department of Pediatrics, Division of Immunology and Transplantation Biology Each year, influenza A causes extreme morbidity and mortality, particularly in infants, the elderly, and the immunocompromised. The recent spread of pathogenic avian influenza A increases the likelihood of a human influenza pandemic. Current vaccines protect through the induction of neutralizing antibodies against hemagglutinin and neuraminidase surface proteins. This requires that the vaccine be closely matched in subtype to the virus that will be circulating six months after the start of production. However, a human pandemic would likely emerge and spread significantly more rapidly. It has been shown that the cytolytic T lymphocyte (CTL) response is essential for viral clearance from the respiratory tract. Furthermore, CTL are generally specific against internal proteins and have been shown in mice to be less dependent on viral subtype, and thus are more likely to protect against a previously unseen subtype. A unique adjuvant based on a complex of lipid carrier and non-coding DNA (CLDC) has been shown to stimulate a robust CTL immune response when administered with protein antigens. We hypothesized that the robust CTL response seen with CLDC is mediated by plasmacytoid dendritic cells (pDC) acting as antigen presenting cells. Thus, we examined the role that pDCs play in vivo in the protection provided by vaccination with CLDC. Two mice were injected with anti-plasmacytoid dendritic cell antigen 1 (PDCA-1) in order to ablate their pDC immune response. After the depletion was confirmed, at Day 1, both these 2 mice and 2 controls were administered CLDC with influenza vaccine. Twenty-eight days later both mice were sacrificed and their immune responses were quantified. ELISA was used to track IFN-gamma secretion in response to live virus and purified antigen at 4 time points. ELISA was also used to quantify IgG2c and IgG1 antibody responses at 2 and 4 week time points. In addition, hemagglutination-inhibition (HAI) antibody titers were quantified at these time points. In all cases, there was no significant difference between the immune response mounted by the controls and that mounted by the pDC-depleted mice. These experiments suggest that CLDC’s immunogenicity is mediated by a mechanism independent of pDCs. Future studies should attempt to identify the mechanism that mediates the increased immunogenicity that CLDC provides. Funded by the Stanford Medical Scholars Research Program