Bulfinch 370 | 617.643.1 cultydevelopment/orcd

ABSTRACTS

4th Annual MGH Research Fellows

Poster Celebration

Monday, June 8, 2009 3:00-5:00pm, Bulfinch Tent

Posters ~ Awards ~ Food & Drinks

MGH Center for Faculty Development 606 | www.massgeneral.org/fa

TABLE OF CONTENTS

Agenda ………………………………………………………………………3 Review Committee ………………………………………………………………4 Mass. General Postdoctoral Association ………………………………………5 Abstracts ………………………………………………………………………6 Author Index ………………………………………………………………………67 Notes ………………………………………………………………………69

2

AGENDA 10:00 am Posters go on display in Bulfinch Tent 3:00 pm Poster Celebration opens 3:30 pm Awards presentation Welcoming remarks: Tayyaba Hasan, PhD Mass General Postdoc Association Announcement of awards and summary of winning posters 4:00-4:30 pm Authors present at odd-numbered posters 4:30-5:00 pm Authors present add even-numbered posters Posters may remain on display until 6:30 pm

A note on award winning posters: At the time that this abstract book went to press, the awards had not yet been determined, therefore award-winning posters are not marked in this book. Poster numbers listed in the book and on poster boards were assigned randomly, and do not reflect any ranking that the poster received in review. Awards will be announced at the Celebration, and an addendum page listing the winning posters will be available after the awards ceremony.

3

REVIEW COMMITTEE

The 2009 Review Committee, made up of MGH faculty, worked very hard to select the award winning posters from among an excellent group of over 60 submissions. The group first reviewed the abstracts according to criteria based on those used in the review of NIH grants, and scored each poster on three categories: (1) Significance (does the study address an important problem?); (2) Approach (are the conceptual framework and methods appropriate for the project?); and (3) Innovation (does the project challenge existing paradigms or develop new methodologies?). Abstracts that scored in the top 50th percentile were selected for a final review. In the final review, the committee used the additional information in the posters to determine the award winners. We are extremely grateful to all members of the review committee for the thoughtful effort they put into reviewing each abstract and poster.

2009 Poster Celebration Review Committee David MacLaughlin, PhD, Co-Chair Anne Hart, MD

Hensin Tsao, MD, PhD, Co-Chair Robert Hasserjian, MD

Rajendra Badgaiyan, MD Samuel Rabkin, PhD

Murat Bastepe, MD, PhD Dushyant Sahani, MD

Gilles Benichou, MD Rex Neal Smith, MD

Giorgio Bonmassar, PhD Jose Teixeira, PhD

Andrew Cole, MD H. Shaw Warren, MD

Louis Ercolani, MD Xu Zhang, PhD

Giulia Fulci, PhD Randall Zusman, MD

Mukesh Harisinghani, MD

4

MGH POSTDOCTORAL ASSOCIATION (MGPA) The following members of the MGPA were extremely helpful in making the 2009 MGH Research Fellows Poster Celebration a success. The following MGPA Board members did much behind-the-scenes work to help with the many logistical details of this important event. Adnan Abu-Yousif, PhD (MGPA 2009 Co-Chair) Jonas Dyhrfjeld-Johnsen, PhD (MGPA 2009 Executive Assistant) Erik Hett, PhD (MGPA 2009 Co-Chair) Ulrike Hoffman, MD Sujeeve Jeganathan, PhD Albena Kantardzhieva, PhD Hilary Luderer, PhD Nadege Roche, PhD

APGM Mass General Postdoc  Association 

Work/LifeBalance  

Career  Development 

 Join the 

https://www2.massgeneral.org/mgpa

Postdoc  Advocacy 

Research Advancement 

Communication  & Networking 

Email: mgpa@partners.org

5

Poster #1

Abstract Title: Nanotechnology mediated mechanism-based combination therapy against metastatic pancreatic cancer. Prakash Rai, Sung Chang, Zhiming Mai, Bryan Spring, Daniel Neuman, Tayyaba Hasan, Wellman Center for Photomedine, Massachussets General Hospital, Boston, MA.

Purpose: We wanted to investigate the efficacy of a novel nanotechnology- based strategy to co-deliver a photosensitizer (PS) and an anti-angiogenic agent (Avastin) in a murine model of human pancreatic cancer (PanCa). The National Cancer Institute reported 37,680 new cases and 34,290 deaths from pancreatic cancer (PanCa) in 2008. PanCa is highly resistant to cytotoxic therapies including chemo- and radiotherapy and only 15-20% of patients are candidates for surgical intervention. Median survival following diagnosis is about 6 months. There is clearly a desperate need for developing new strategies to treat PanCa. Photodynamic therapy (PDT), a photochemistry based modality, has demonstrated promising results in treating PanCa. PDT relies on the activation of certain non toxic chemicals called photosensitizers (PS) with an appropriate energy/wavelength of light and often bypasses the resistance mechanism of chemotherapy and radiotherapy. Combination of PDT with anti-angiogenic therapy has been found to enhance treatment outcome.

Materials & Methods: Benzoporphyrin derivative monoacid (BPD) is a clinically approved PS used for treatment of age related macular degeneration. Avastin is a monoclonal antibody against vascular endothelial growth factor (VEGF) which has been approved for a variety of cancers. It blocks secreted VEGF but leaves the intracellular VEGF pool untouched. We investigated the effect of neutralizing intracellular VEGF using nanotechnology for co-delivery of Avastin and BPD. For this we explored the use of a new construct called “nanocells” in which the BPD was non-covalently trapped inside polymer nanoparticles and these, along with Avastin, were then encapsulated inside liposomes. We first investigated the phototoxic effects of these nano-constructs in vitro on AsPc-1 cells (derived from a human PanCa) following PDT using a standard MTT assay. We also evaluated the treatment response in vivo using orthotopic mouse models of PanCa in nude mice. Two weeks following treatment mice were sacrificed and we measured tumor weight, tumor volume. Lungs and iliac lymph nodes were collected and analyzed by an RT-PCR based assay to quantify metastases.

Results: In vitro, nanocells containing Avastin (NCA) improved BPD uptake and delivered Avastin intracellularly. NCA significantly enhanced the cytotoxicity following PDT in AsPc-1 cells. NCA based PDT also significantly improved the local and metastatic treatment response in mice that were orthotopically implanted with pancreatic tumors. Conventional delivered Avastin (extracellularly) in combination with PDT did not show any significant improvement.

Conclusion: Our results suggest the involvement of an internal VEGF signaling pathway that potentially plays an important role in cell survival following PDT. We propose a new paradigm for Avastin-based anti-VEGF therapy by combining intracellular delivery of Avastin with PDT using nanotechnology for the treatment of PanCa. Encapsulation of Avastin may drastically reduce the serious side effects associated with the drug which have recently come to light while significantly improving its efficacy. This could have a major clinical impact on diseases that are currently being treated with Avastin.

Author Contact: Prakash Rai, Wellman Center of Photomedicine, 617-726-6139, prrai@partners.org

6

Poster #2

Towards a Noninvasive Assessment of Valve Biology: Echocardiographic Measures of Mitral Leaflet Distensibility Catherine Szymanski (1,2), Mark D Hanschumacher (1), Emmanuel Messas (2), Harry C Judge (3), Miguel Chaput (1), Judy W Hung (1), Jacob P Dal-Bianco (1), Eleanor Morris (1), Jane E Marshall (1), Albert A Hagege (2), Robert A Levine (1) Affiliation

(1) Cardiology, Cardiac Ultrasound Laboratory, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

(2) Cardiology Department, European Hospital Georges Pompidou, INSERM U633, Faculte de Medecine Paris Descartes, Paris, France

(3) John Hopkins University, University School of Medicine, Baltimore, MD, USA Purpose: Changes in mitral valve (MV) elasticity or distensibility occur in disease and directly affect MV function, contributing to MV prolapse (MVP) or flail vs restricted coaptation of stiffer leaflets in functional mitral regurgitation (FMR) and MV stenosis (MS). Recent studies suggest MV distensibility may be modified to reduce MR, but distensibility has only been measured in excised MVs. Our aim was to test the feasibility of obtaining a noninvasive measure of MV distensibility in patients by measuring systolic change in anterior leaflet length (ALL) or anterior leaflet strain; and to test the hypothesis that these measures vary in diseases with known altered MV elasticity. Materials & Methods: ALL was quantified in a long-axis view standardized by 3D echo in 80 patients: 20 each with normal hearts, MVP, FMR and MS. Distensibility was measured as end-systolic (ES) ─ end-diastolic (ED) total ALL normalized to an ED reference; and alternatively as mid-leaflet strain measured by tracking echo features. Results: ALL was greater in all disease groups vs normal (p<.001). The maximum systolic increase in ALL relative to ED was 7.9 ± 7.4 % in normals vs >2-fold higher (17.6 ± 11.2 %) in MVP; it was 63-76% lower (2.9 ± 3.0 %, 1.9 ± 3.1 %) in FMR and MS, with comparable results for segmental AL strain (Table). Conclusion: Noninvasive echocardiographic measures of MV distensibility based on systolic changes in total length or segmental strain are feasible. Results are consistent with excised valve biomechanics, showing increased distensibility in MVP and decreased values in FMR and MS. Ultimately, these techniques have the potential to monitor response to new therapies that aim to improve MV biology and mechanics to reduce MR. arameter unit Norm MVP FMR MS

ALL systole mm. 26.6 ± 2.7 39.7 ± 5.6 * 32.7 ± 4.1 * 33.4 ± 5.0 *

ALL diastole mm. 24.5 ± 3.1 33.5 ± 3.2 * 32.0 ± 4.2 * 32.8 ± 4.9 *

ALL (sys-dia)/dia % 7.9 ± 7.4 17.6 ± 11.2 * 2.9 ± 3.0 * 1.9 ± 3.1 *

SegL (ES-ED)/ED % 9.1± 4.8 19.6 ± 10.9 * 3.2 ± 2.6 * 2.7 ± 3.7 *

* P < 0.05 vs. normals

Author Contact: Catherine Szymanski, Cardiology, Cardiac Ultrasound Laboratory, 617 724 1995, cszymanski@partners.org

7

Poster #3 SOCS3 Downregulates Hepatitis C Virus Replication though mTOR Pathways Run–Xuan Shao, Wenyu Lin, Lee F. Peng, Sabina Sabharwal, Woo Jin Chung, Jae Young Jang, Raymond T. Chung Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School. Boston, Massachusetts, 02114, USA Purpose: Chronic infection with HCV is associated with defects in interferon (IFN) signaling and activation. The suppressor of cytokine signaling 3 (SOCS3) is thought to block type I IFN signaling by impairing STAT1 activation. We and others have observed that levels of hepatic SOCS3 are significantly higher in chronic hepatitis C (CHC), particularly among persons who are nonresponders to subsequent IFN treatment. However, the effects of SOCS3 on HCV itself are not known. Given its putative role, we hypothesized that SOCS3 would be antagonistic to viral replication. We therefore sought to dissect the relationship between SOCS3 and HCV using HCV replication models. Materials & Methods: We used OR6 cells harboring a full-length HCV genotype 1b replicon expressing luciferase as a measure of HCV replication. A genotype 2a HCV full-length infection system was also established in Huh 7.5.1 cells using the JFH1 infectious strain. Levels of SOCS3 and HCV core protein were determined by Western blotting. HCV replication in OR6 cells was monitored by measuring Renilla luciferase activity (RLUs). HCV RNA was quantified by real time RT-PCR. We transiently transfected SOCS3 into OR6 cells and JFH1 infected Huh7.5.1 cells, and observed the impact on HCV replication. We also created stable SOCS3 knockdown using shRNAs in Huh7.5.1 cells, and assessed HCV replication. Results: SOCS3 protein expression was significantly lower in OR6 cells and JFH1 infected Huh7.5.1 cells. Surprisingly, when SOCS3 was overexpressed in OR6 cells, HCV replication was significantly lower than in mock-transfected cells by luciferase assay (rlus/cell viability, SOCS3: 1.6, control cells: 3.3, p=0.0012), and by RT-PCR (HCV/actin, SOCS3: 2.1, control cells: 5.1, P=0.028). Similarly, HCV core protein levels were significantly decreased. Moreover, in JFH1 infected Huh7.5.1 cells, SOCS3 overexpression also lowered HCV-RNA level (HCV/actin, SOCS3: 1.0, control cells: 2.4, P=0.043) and core protein. In contrast, SOCS3 knockdown in JFH1 infected cells using shRNA was associated with increased HCV core protein. Interestingly, IFN-induced P-STAT1 levels were not increased by overexpression of SOCS3 in OR6 or JFH-1 infected cells. Furthermore, inhibit mTOR could restores SOCS3’s inhibitory effects on HCV replication in JFH1-infected cells. Conclusion: Our data surprisingly suggest that SOCS3 actually suppresses HCV replication in an mTOR dependent manner, and does not impair IFN signaling in the context of HCV infection. They instead suggest a model by which increased levels of SOCS3 observed in chronic IFN nonresponders may reflect a compensatory host response to persistent infection and impaired defenses elsewhere in the innate antiviral pathway. Efforts to enhance SOCS3 function may be a useful strategy to control HCV infection. Author Contact: Run-Xuan Shao, GI Unite, 617-726-2061, rshao@partners.org

8

Poster #4

Ovarian Cancer Cells Migrate and Assemble In Vitro into Heterogenous Micronodules with a Differential Response to Treatment Imran Rizvi1,2, Jonathan Celli1, Conor L. Evans1, Adnan Abu-Yousif1, Daniel Neuman1, Bryan Spring1, Yupeng Tu1, Johannes de Boer1 and Tayyaba Hasan1, PhD 1Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA 02114 2Thayer School of Engineering, Dartmouth College, Hanover, NH 03755

Purpose: Survival rates associated with ovarian carcinoma (OvCa) have remained stagnant for decades, and the disease remains among the most fatal female cancers in the United States. Studding of the peritoneal cavity with metastatic implants is a leading cause of OvCa deaths, yet the underlying biology of tumor dissemination remains poorly understood. There is a critical need to develop better research models that recapitulate the complex architectural and biological cues of micrometastatic disease, and can serve as reliable platforms to evaluate the clinical efficacy of novel therapeutic regimens. The purpose of this study was to develop and characterize an in vitro 3D model for micrometastic OvCa as a reliable platform to evaluate new therapeutic strategies. Materials & Methods: We used three-dimensional (3D) cultures for micronodular OvCa as our in vitro research platform to study adherent micrometastatic disease. OvCa cells were seeded on Growth Factor Reduced Matrigel™ beds and spontaneously formed 3D acinar structures that more closely resemble in vivo tumor nodules than cells in monolayer. We characterized acinar growth and development using a range of optical imaging platforms including optical coherence tomography, a large-volume, 3D imaging technology, which allows us to non-perturbatively examine structural changes in the acini. This novel application of optical coherence tomography was complemented by confocal, multi-photon, and brightfield time-lapse microscopy to investigate assembly and growth dynamics, along with drug penetration and metabolic activity, in the micronodular tumors. We also evaluated treatment response to carboplatin, a chemotherapeutic agent frequently used to manage OvCa, and to photodynamic therapy (PDT), an emerging light-based treatment modality. Results: Using customized batch processing routines to analyze sequential images of 3D acinar growth, we report a marked heterogeneity in the size distribution and structural development of the acini. This divergence in acinar population emerges approximately five days after plating, and results from both migratory and proliferative events. At a fluence of 5 J/cm2, we observed a 13-fold increase in viability in PDT treated 3D cultures as compared to cells in monolayer. Similarly, OvCa cells were 7 times less sensitive to carboplatin in 3D cultures than in monolayer at a dose of 100µM. Treatment response in 3D micronodules following PDT did not appear to be size dependent, whereas a viable tumor core was observed in acini treated with carboplatin. Conclusion: Development of biologically relevant research platforms to complement, or replace, traditional model systems could significantly enhance our ability to conduct reliable mechanistic studies and predict the efficacy of new therapeutic strategies. The differential response observed in our 3D acini following treatment with carboplatin versus PDT could be attributed to heterogeneities in drug and light penetration as well as restoration of physiologically relevant architectural cues and cell signalling not present in monolayer cultures. These findings warrant further investigation of a complementary role for PDT to enhance the efficacy of chemotherapeutic agents, such as carboplatin, to treat advanced stage OvCa.

Author Contact: Imran Rizvi, Wellman Center for Photomedicine, 617-726-3991, IRIZVI@PARTNERS.ORG

9

Poster #5 Glycogen Synthase Kinase 3 is an Important Regulator of T Regulatory Cell Activity Jay A. Graham, M.D.1, Michael Fray1, Stephanie de Haseth1, Catharine M. Chase1, Robert B. Colvin, M.D.2, Joren C. Madsen, M.D., D.Phil.1, A. Benedict Cosimi, M.D.1, Gilles Benichou, Ph.D.1, and Alessandro Alessandrini, Ph.D.1. 1Department of Surgery, Transplantation Unit, 2Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, U.S.A. 02114 Purpose: Considerable interest has developed in designing allotransplant tolerance regimens involving the manipulation of regulatory T cells. Yet the mechanism by which regulatory T cells (Tregs) suppress the immune response is not well defined, nor are the signaling pathways involved in suppression fully understood. Recent data have shown that stabilization of beta-catenin of the Wnt pathway in Treg cells enhances their survival and protects against inflammatory bowel disease. Beta-catenin is phosphorylated by the kinase, glycogen synthase kinase-3beta (GSK-3beta), which targets it for ubiquitination and degradation. Inhibition of GSK-3beta by SB216763, a specific GKS-3beta inhibitor, leads to a significant increase and stabilization of beta-catenin levels. We show that SB216763 treatment potentiates Treg function, in part by prolonging cellular survival and FoxP3 expression in regulatory T cells. Materials & Methods: CD4+CD25- Effector T cell and CD4+CD25+ Regulatory T cell Isolation and Culture Single cell suspensions were prepared from B6 mice using standard methods. After a brief erythrocyte lysis using Red Blood Cell Lysing Buffer (Sigma-Aldrich), the cells were resuspended in degassed PBS with 0.5% BSA and 2mM EDTA. Separation of CD4+25- and CD4+CD25+ cells was accomplished with magnetic labeling using the MACs Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). CD4+25- cells were negatively selected with the flow through fraction, while the CD4+25+ cells were positively selected. The purity of fractions was confirmed by FACs analysis. Isolated CD4+CD25+ cells were cultured in vitro with and without SB216763 in complete media supplemented with IL-2. Cells counts were obtained using standard microscopic hemocytometry techniques. In Vitro Suppression Assay CD4+CD25+ Tregs were co-cultured with CD4+CD25- cells stimulated with anti-CD3 and anti-CD28 Dynabeads (Invitrogen) with and without SB216763. Proliferation was measured in triplicates by the incorporation of tritiated thymidine over the last 18-20 h of the coculture. All cells were cultured in complete RPMI medium. Flow Cytometry Fluorescence activated cell sorting (FACScan BD Biosciences; San Jose, CA) of spleen samples was performed on cells incubated with 0.5 μg antibody for 30 min at 4o C, with PE-Cy5 conjugated anti-mouse CD4 (L3T4). Cells were then washed and permeabilized with 0.2% Ipegal CA-630 (Sigma-Aldrich) and stained intracellularly with 0.5 μg antibody for 30 min at room temperature with Alexa-Fluro 488 conjugated anti-mouse Foxp3 (FJK-16s). All reagents used for staining were obtained from BD Pharmingen; San Diego, CA. Results: We show that SB216763 potentiates Treg function in vitro, as judged by increased suppression of Teff CD4+CD25- proliferation when Teff CD4+CD25-cells were incubated at a 1:1 ratio with Tregs in 5 µM SB216763 (86% inhibition vs 65% without the drug). Moreover, the Tregs in coculture with SB216763 had a slower diminishment in intracellular FoxP3 signal indicating a probability of enhanced suppression in vitro. Furthermore, regulatory T cells, grown in the presence of low levels of IL-2, exhibited a 40% decrease in cell number after three days in culture. Cells treated with GSK-3beta inhibitor exhibited a 19% drop in cell number during the same time period. Conclusion: Our results suggest GSK-3beta can be a target in developing therapeutics that will allow for the increased stabilization and function of T regs and hence allow the development of strategies in inducing allotransplant tolerance. Author Contact: Jay A. Graham M.D., Dept of Surgery, 617-726-6536, jgraham8@partners.org

10

Poster #6 Finasteride: promoter or repressor of initiated prostate epithelial cells? Yinong Niu, Christian R. Diaz, Wenhua Li, Rongbin Ge, Aria F. Olumi* Department of Urology, Massachusetts General Hospital, Harvard Medical School. Boston, Massachusetts, USA Purpose: Finasteride, a 5-alpha reductase inhibitor, is commonly used for treatment of benign prostatic hyperplasia (BPH), and more recently as a chemopreventive agent for prostate cancer. In a large randomized clinical trial Finasteride reduced the rate of low grade prostate cancer, however, the percentage of patients diagnosed with high grade disease increased. This has led to the controversy whether Finasteride facilitates the detection of high grade cancers, or whether Finasteride promotes development of high grade tumors. Here, we demonstrate that in initiated prostate epithelial cells, Finasteride does not promote cell death significantly and initiates molecular pathways associated with prostate cancer progression. Materials & Methods: Immunohistochemistry was performed to determine the relative expression level of 5-α reductase in human prostate tissues. RT-PCR was carried out to evaluate the 5-α reductase mRNA transcription in initiated and malignant prostate epithelial cells. BPH-1 and LNCaP cells were grown alone or in coculture transwells with fibroblasts in the presence or absence of Finasteride. Cell viability (MTS), phosphorylated Akt and phosphorylated ERK1/2 (Western blot) were evaluated. Results: In benign human prostate tissue, 5-α reductase type 2 was expressed at variable levels, and 5-α reductase 2 mRNA transcription was not detected in several initiated and malignant prostate epithelial cells. BPH-1 cells were resistant to Finasteride at very high doses. In addition, after Finasteride treatment, we found that in BPH1 and LNCaP cells, phosphorylated Akt and phosphorylated ERK1/2 were upregulated, two molecules which are commonly responsible for proliferation and tumor progression in prostate cancer. However, when BPH-1 cells were grown in co-cocultures with fibroblasts, Finasteride’s pro-apoptotic function was restored and it induced cell death in 30% of cells (p<0.05). In contrast to mono-cultures, expression of p-Akt and p-ERK1/2 were significantly repressed in co-cultures. Conclusion: Some prostate epithelial cells may be resistant to Finasteride due to lack of expression of 5 α reductase type 2 gene, leading to activation of molecular pathways that promote prostate epithelial proliferation. Fibroblasts restore the pro-apoptotic function of Finasteride in prostate epithelial cells, suggesting that the fibroblastic microenvironment plays an important role in the therapeutic effects of Finasteride. These studies have significant implications in the current therapeutic strategies for men with BPH and chemoprevention for prostate cancer. Author Contact: Yinong Niu , Department of Urology, 617-643-1953, Yniu@partners.org

11

Poster #7 Multimodal Nanoagents for the Detection and Treatment of Atherosclerosis Jason R. McCarthy, Jayeeta Bhaumik, Farouc A. Jaffer and Ralph Weissleder Center for Molecular Imaging Research and Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, Boston, MA Purpose: The sequelae of atherosclerotic vascular disease are the leading cause of mortality worldwide. It is therefore essential to be able to diagnose and treat complicated atherosclerotic segments prior to the onset of clinical symptoms. Research over the past decade demonstrates that inflammation and the innate immune response participate critically in the initiation and progression of atherosclerosis. In particular, macrophages contribute crucially to all stages of atherogenesis, from foam cell and fatty streak formation to the coordination of the inflammatory response leading to plaque rupture and thrombosis in advanced atherosclerotic lesions. Macrophages thus represent an important cellular target for atherosclerosis therapies. Thus, we have developed a magnetofluorescent nanoparticle functionalized with a potent near-infrared light acivated therapeutic agent (NILAT), 5,10,15-Tris[4-(N-D-glucopyranuronosylamido)phenyl-20-[4-(N-glutarylamido)phenyl-17,18-dihydroxychlorin (GPC). This theranostic nanoagent (TNA) is being used to target and locally eradicate tissue macrophages in lesions. Materials & Methods: A sugar-modified chlorin-based photosensitizer was developed in high yields, and conjugated to carboxymethyl polyvinyl alcohol (CMPVA) coated, amine modified iron oxide nanoparticles. Cellular uptake and therapeutic efficacy of these agents were tested in vitro in RAW 264.7 murine macrophages. Results: UV-vis absorption spectroscopy was utilized to quantitate photosensitizer conjugation to the nanoparticle surface. Cellular uptake of the theranostic nanoagents in RAW cells was confirmed by fluorescence microscopy. The phototoxicity of the TNA was found to be significantly greater than the known photosensitizer chlorin e6. When tested for phototoxicity in the absence of light, TNA displayed no cytotoxicity at any concentrations. Conclusion: We have synthesized a novel chlorin-based near-infrared light activated therapeutic agent based upon a glucose-modified chlorin. CMPVA coated iron oxide nanoparticles were readily functionalized with fluorescent dyes for imaging and NILAT for therapy. TNA displays enhanced uptake in murine macrophages and has an EC50 of 9 nM NILAT and 204 pM nanoparticle when irradiated at 650 nm. TNA did not show any cytotoxicity in the absence of light. Author Contact: Jayeeta Bhaumik, CMIR-MGH, 617-643-6855, jbhaumik@mgh.harvard.edu.

12

Poster #8

Uncovering a novel molecular mechanism for the attenuation of NFκB signaling: Evidence

for the transcriptional regulation of IκB kinase β (IKKβ)

Vasant Chellappa, Amy McQuay, Semir Beyaz, Sven Diederichs and Shiv Pillai

MGH Cancer Center and Harvard Medical School We describe here a novel molecular mechanism for the attenuation of NFκB signaling. Our results reveal that, activation of IκB kinase β (IKKβ), a key enzymatic component of the multi-subunit IκB kinase (IKK) complex results in the feedback inhibition of IKKβ synthesis. This feedback regulation event may represent a mechanism by which NFκB signaling is attenuated. Various upstream kinases capable of activating NFκB induce the attenuation of IKKβ mRNA expression, and consequently of IKKβ protein synthesis. Mutation of the critical T-loop activation motif (177Sxxx181S) serine residues in IKKβ renders the molecule resistant to the activation-induced attenuation of synthesis. These studies on the regulation of IKKβ have been experimentally demonstrated by the transient over-expression of an NFκB activating kinase, together with an epitope-tagged version of IKKβ (XPRESS-IKKβ) in HEK-293T cells and have been iteratively reproduced with a different epitope on IKKβ (V5-IKKβ). These studies have uncovered a novel molecular mechanism for the regulation of NFκB activity in cells at the level of the regulation of IKKβ synthesis. We are currently pursuing the idea that active IKKβ may induce an IKKβ specific micro RNA that attenuates IKKβ synthesis. This feedback mechanism may be crucial in attenuating inflammation and may be deregulated in certain cancers in which constitutive NFκB activation has been demonstrated.

13

Poster #9 The diagnostic role of multiparameter immophenotyping by Flow Cytometry in Multiple Myeloma: a new model Elisa Cannizzo1,2, Emanuele Bellio2, Michelle E. Dorn1, Craig Sadowski1, Janessa J. Bucci1, Mario Petrini2, Giovanni Carulli2, Frederic Preffer1

1Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston, USA 2Division of Hematology, Department of Oncology, Transplants and New Technologies in Medicine. University of Pisa, Pisa, Italy Purpose: Immunophenotypic studies on multiple myeloma (MM) have now been performed for more than 15 years. However, a careful review of the literature shows discrepancies in results regarding the exact phenotype and resultant clinical significance of abnormal plasma cells (aPC). Multiparameter flow cytometry (FC) represents an attractive approach in the detection of aPC due to its capacity to combine both an examination of both an aberrant immunophenotype as well as clonality studies. Due to the large numbers of cells amenable to analysis by flow cytometry, it may be additionally useful in the detection of minimal residual disease (MRD). Problems with such evaluation of plasma cells (PC) include those related to the frequent hemo-dilution of bone marrow aspirates (BMA) with peripheral blood (PB) as well as the lability of PC stored outside of the body. At this time the histologic examination of bone marrow remains the gold standard in the diagnosis of MM. We have developed an objective and reproducible new statistical diagnostic model that examines what correlation exists between the immunophenotype and clonality detected by FC and histology that define the diagnostic role of FC in MM. Materials & Methods: Fifty-five patients were enrolled in a pilot study for routine diagnostic analysis of MM; a minimum of 100 PC were analyzed for each patient sample. Table 1 shows the monoclonal antibody panel we used to study the immunophenotype of plasma cells (PC) utilizing a BD FACSCanto II. Results: Analysis of CD38, CD19 and CD10 expression, when applied to our model, resulted in optimal concordance with histology. Conclusion: This statistical model showed a correlation between FC and histological analyses. This statistical model represents a new objective and reproducible way to interpret the immunophenotype of PC and aPC and correlates this analysis with histological results. Our goal is to use this information to consolidate this model and test its applicability on a larger scale. Table 1 AmCyan FITC PE PerCP APC PeCy7 Pacific Blue APC-Cy7CD45 CD38 CD221 CD19 CD27 CD138 CD56 CD45 CD38 CD200 CD19 CD81 CD138 CD10 CD45 CD38 CD28 CD19 CD117 CD138 CD33 CD45 Cyto-Lambda Cyto-Kappa CD19 CD38 CD138 CD56 CD20 Author Contact: Elisa Cannizzo, MD, Pathology Department, 617-726-8487, ecannizzo@partners.org

14

Poster #10 Abstract Title: Pref-1 Predicts Marrow Adiposity and Low Bone Mass Authors: Pouneh Fazeli1, Miriam Bredella2, Madhusmita Misra1, Erinne Meenaghan1, Clifford Rosen3, Karen Miller1, Anne Klibanski1

Affiliation: 1Neuroendocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114; 2Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114; 3Maine Medical Center Research Institute, Portland, ME 04102 Purpose: The regulation of bone cell lineage is important in understanding states of low bone mass. Adipocytes and osteoblasts originate from a common progenitor human mesenchymal stem cell (hMSC), and bone marrow adipocytes may affect osteoblast differentiation. Low bone mineral density (BMD) and increased bone marrow fat are characteristic of anorexia nervosa (AN), a state of self-imposed starvation. Pref-1, a member of the epidermal growth factor-like family of proteins expressed in hMSCs, preadipocytes and bone, is a negative regulator of adipocyte and bone cell differentiation. Osteoblast-specific Pref-1 over-expression in a mouse model results in reduced BMD. We hypothesized that Pref-1 would be elevated in AN and would predict marrow fat and low BMD. Materials & Methods: We studied 30 females: 20 with AN and 10 controls of comparable age (26.8y + 1.5 vs. 29.2y + 1.7, respectively) and differing BMI by design (17.6 + 0.2 vs. 21.9 + 0.5). We measured Pref-1, leptin, an adipokine markedly decreased in AN, and BMD of the AP lumbar spine, lateral spine, and total hip by DEXA in all subjects. Bone marrow fat content by proton MR spectroscopy was measured in 10 women with AN and 10 controls. Results: Pref-1 was significantly higher (0.47 vs. 0.37 ng/mL, p=0.03) and log leptin lower (p=0.01) in AN as compared to controls. There was a negative correlation between log Pref-1 and BMD of the AP spine (R= -0.54, p=0.003) and lateral spine (R= -0.44, p=0.02) and a positive correlation between leptin and BMD of the AP spine (R=0.39, p=0.04) and hip (R=0.42, p=0.03) in all subjects. In AN, log Pref-1 was negatively correlated with BMD of the AP spine (R= -0.57, p=0.008) and lateral spine (R= -0.55, p=0.01) and log leptin was positively correlated with hip BMD (R=0.50, p=0.02), while in controls log leptin was negatively correlated with BMD of the AP spine (R= -0.8, p=0.0095) and hip (R= -0.72, p=0.029). There was a positive correlation between log Pref-1 and marrow fat of the proximal femoral metaphysis (R=0.53, p=0.018) and a negative correlation between leptin and L4 marrow fat (R= -0.45, p=0.046) in the entire group. Conclusion: These data support the hypotheses that Pref-1, a negative regulator of adipocyte and osteoblast differentiation, is 1) elevated in AN and 2) predicts low BMD and increased marrow fat. In contrast, leptin is a positive predictor of BMD in AN and a negative predictor of marrow fat. These data suggest that Pref-1 may contribute to the pathogenesis of low bone mass states by regulating the differentiation process of hMSCs into bone and fat. Author Contact: Pouneh Fazeli

Neuroendocrine Unit, Department of Medicine Phone: 617-726-1347 e-mail: pkfazeli@partners.org

15

Poster #11 Abstract Title: HIV gp120 Inhibits T Helper Cell Proliferation during Acute HIV Infection Authors: Jenna Rychert, Daryld Strict, James Robinson, Eric Rosenberg Affiliation: Massachusetts General Hospital, Tulane Medical School Purpose: CD4+ T helper (TH) cells are essential for an effective anti-viral immune response. In most untreated HIV+ individuals, however, TH cells fail to proliferate to HIV antigens, even during the earliest stages of infection. This suggests that events occurring during acute infection set the stage for immunologic impairment, likely contributing to the immune system’s failure to contain HIV replication. The HIV envelope glycoprotein (gp120) binds to CD4 to mediate entry into a cell. Gp120 is readily shed from virions and infected cells. Treatment of CD4+ T cells in vitro with gp120 prior to antigen stimulation results in loss of proliferation, similar to what is observed in vivo. We hypothesize that during acute infection, HIV-specific TH cell proliferation is lost due to high viremia and production of gp120. Materials & Methods: Plasma obtained from HIV+ subjects during primary infection was screened for gp120 by ELISA. Proliferation to HIV, CMV and VZV was measured using tritium incorporation. Viral load and CD4 T cell counts were performed by clinical laboratories at MGH. To detect antibodies that prevent the interaction between gp120 and CD4, PBMC were incubated with 4ug/ml rgp120 and serial 10 fold dilutions of plasma for 1 hour then the frequency of gp120+ cells was determined by flow cytometry. Results: Subjects who had detectable gp120 pre-seroconversion (shedders) maintained similar levels in follow up samples despite seroconversion and significant changes in viral load due to initiation of therapy. Furthermore, shedders were more likely than non-shedders to fail to proliferate to HIV antigens over the course of infection. We then asked whether subjects had antibodies that prevented the interaction between gp120 and CD4. We found that the majority of subjects, regardless of shedding did not have antibodies that prevented this interaction. Conclusion: Gp120 is detected in the plasma of a subset of HIV+ individuals and is associated with poor HIV-specific TH cell proliferation. Regardless of shedding, the antibody response against gp120 provides little protection against gp120 mediated inhibition of TH cell proliferation. Author Contact: Jenna Rychert

Infectious Disease 617-726-9404 JRychert@partners.org

16

Poster #12 Abstract Title: Skeletal resistance to teriparatide: Effects of escalating versus constant dose teriparatide in osteoporotic women Authors: Elaine W. Yu, MD1

Robert M. Neer, MD1

Jason Wyland, P.A.1 Amanda V. de la Paz, B.A.1 Melissa Davis, B.A.1 Joel S. Finkelstein, MD1

Affiliations: 1Massachusetts General Hospital Endocrine Unit Purpose: Skeletal resistance to the anabolic effects of PTH 1-34 (teriparatide) has been observed. For reasons that are currently unknown, bone turnover markers peak after several months of treatment and then begin to decline. We hypothesized that escalating teriparatide dosing over the course of 18 months could overcome this apparent teriparatide resistance. Materials & Methods: We conducted a randomized controlled trial of escalating dose (20 mcg daily for 6 months, 30 mcg daily for 6 months, 40 mcg daily for 6 months) versus constant dose (30 mcg daily for 18 months) teriparatide in 70 postmenopausal women with osteoporosis. Primary outcomes included bone markers N-terminal propeptide of type 1 procollagen (P1NP) and osteocalcin (OC), and bone mineral density (BMD) as assessed by dual-energy x-ray absorptiometry (DXA). Results: Bone marker and BMD data are currently available for 41 and 52 women, respectively. In the constant dose teriparatide group, mean P1NP and OC peaked by 12 months and then gradually declined. In the escalating dose teriparatide group, mean P1NP and OC demonstrated a slow gradual increase over 18 months. Area under the curve analysis demonstrated no significant difference in P1NP (p=0.10) or OC response (p=0.25) between the two groups. BMD increased in both groups, but there was no significant difference between the escalating and constant dose groups (PA spine: 8.6% vs. 6.7%, p=0.21; total hip 1.5% vs. 2.0%, p=0.69). Conclusion: In summary, both escalating and constant teriparatide dosing significantly increased P1NP, OC and BMD. However, escalating dosing was unable to provide additional benefit to overcome skeletal resistance to teriparatide. Author Contact: Elaine W. Yu, Endocrine Unit, 617-643-6353, ewyu@partners.org

17

Poster #13

Iodine-124 as a label for a study of slow pharmacokinetics by PET V. Belov, A. A. Bonab, A.J. Fischman, M. Papisov

Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114-2696 Purpose: With the growing number of biotechnology products entering preclinical and clinical studies, the capability of quantitative PET imaging plays an increasingly significant role. Imaging of slow pharmacokinetics (PK) requires labeling of the drug candidate with radionuclides that have long physical half-lives. Among all currently available positron emitters, 124I has the longest physical half-life (4.2 days). Although the presence of high energy � and single photon � components in the 124I emission is not an advantage, the long physical half-life, combined with the well investigated behavior of iodine in vivo, makes this isotope attractive for the long term (several days) studies. The objective of this research was to determine whether the properties of 124I as a positron emitter translate into data quality suitable for PK research. Materials & Methods: Imaging was carried out using MicroPET P4 (Siemens/Concorde Microsystems). Full width at half maximum (FWHM) and modulation transfer function (MTF) as characteristics of spatial resolution were studied using a line source, Ø=0.19 mm, in aqueous surrounding. A 51 mm diameter x 127 mm tall acrylic cylindrical phantom filled with water containing 2.26 mCi of 124I was used to evaluate the count-rate performance and sensitivity of coincidence detection. Model animal studies in rats and cynomolgus monkeys were carried out using four human recombinant proteins (enzyme replacement therapy candidates). The proteins were labeled with 124I, up to 5 mCi/mg. Results: The data show that the limiting transaxial and axial spatial resolutions (expressed as FWHM) were satisfactory in the center of the camera. The iterative reconstruction algorithm OSEM3D/MAP showed better results than the more conventional Filtered Back Projection (2.46 vs. 3.34 mm, and 3.24 vs. 3.62 mm). Some degradation of resolution in the periphery (5 cm radial offset) was observed in both transaxial (27.1%) and axial (26.2%) directions. Spatial resolution evaluated in terms of MTF was also better for OSEM3D/MAP, for high spatial frequencies (fine details of the object and sharp edges in the image) as well as for low frequencies (coarse details). A good linearity of the “true” coincidence count-rate was observed up to 1.2 mCi. Animal studies demonstrated excellent delineation and resolution of even very small organs (e.g., single lymph nodes in rats, Ø<1 mm). The quality of numerical data was appropriate for PK analysis over at least 8 days. Conclusion: The data suggest that 124I is an excellent label for quantitative investigation of slow pharmacokinetics by PET. Author Contact: Vasily Belov, PhD Department of Radiology, Division of Nuclear Medicine Phone: (617)371-4937 e-mail: vbelov@partners.org

18

Poster #14

CD133 distinguishes a tumorigenic population in primary human ovarian cancer Michael D. Curley, Vanessa A. Therrien, Christine L. Cummings, Petra A. Sergent, Carolyn R. Koulouris, Anne M. Friel, Drucilla J. Roberts, David T. Scadden, Bo R. Rueda, and Rosemary Foster At present, evidence is accumulating regarding the existence of unique populations of specialized tumor-initiating stem-like cells within various tumor types of distinct origins. These cancer stem cells (CSC), with characteristics reminiscent of normal stem cells, are thought to be responsible for driving tumor growth. We propose that ovarian cancers arise from CSC, and have used an in vivo serial transplantation model to identify specific cell surface markers associated with ovarian tumorigenicity that will permit distinction of these cells from the remaining tumor bulk. Our initial studies used cells derived from primary human ovarian tumors of serous, clear cell and endometrioid origin which were injected at limiting dilutions to determine whether all or only a fraction of tumor cells had the capacity to form tumors. These analyses determined that injection of at least 10,000 cells resulted in tumor formation and no tumors were generated following injection of fewer cells. These data indicated that only a fraction of tumor cells have the capacity for tumor formation, as has been shown for other solid tumor types, which is in agreement with the cancer stem cell hypothesis. This in vivo model also selects for primary human ovarian tumor cells with increased tumorigenic capacity, given that time to tumor formation decreased with successive serial transplant of these tumors in NOD/SCID mice despite fewer cells being injected. In an effort to identify these tumorigenic cells, we utilized flow cytometry techniques to screen these transplanted tumors for expression of cell surface markers that have been shown to demarcate tumorigenic populations in other cancer types. Our analyses indicated a wide range of expression of CD44, CD24 and EpCAM across these tumor types; however, CD133 was consistently expressed at a low level (<12.5%) in ovarian serous tumors, and surprisingly, at much higher levels (>65%) in ovarian clear cell tumors. These expression patterns were mirrored by immunohistochemical analyses of the same tumor subtypes. To determine if CD133 expression identified ovarian cancer cells that have an increased tumorigenic capacity, both serous and clear cell transplanted tumors were sorted by flow cytometry, and the resulting CD133+ and CD133- populations were injected subcutaneously into NOD/SCID mice. These in vivo tumorigenicity assays showed that CD133 identifies cells with an increased tumorigenic capacity, though CD133- cells still had the capacity to form tumors. Our data provide a reliable model for assaying ovarian cancer tumorigenicity in vivo and indicate that CD133 distinguishes a tumorigenic subpopulation in ovarian serous cancer that may prove a useful target for novel therapeutic strategies. Author contact: Michael Curley, Vincent Center for Reproductive Biology

Ph: 617-726-8865; Email; mdcurley@partners.org

19

Poster #15 Abstract Title: Phenotypic Effects of a Bipolar Liability Gene Among Individuals with Major Depressive Disorder Authors: Francesco Casamassima, MD1,2, Jie Huang, MD, MS, MPH3,4 , Maurizio Fava, MD2,4, Gary S. Sachs, MD2,4, Jordan W. Smoller, MD, ScD3,4, Giovanni B. Cassano, MD1, Lorenzo Lattanzi, MD1,, Jes Fagerness, BS3, Jonathan P. Stange, BA2, & Roy H. Perlis, MD, MSc2,3,4. Affiliation: 1 University of Pisa, Division of Psychiatry, Via Roma, 67 56100, Pisa, Italy 2 Department of Psychiatry, Massachusetts General Hospital, Boston, MA 02114 USA 3 Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114 USA 4 Department of Psychiatry, Harvard Medical School, Boston, MA 02114 USA Purpose: Variations in voltage-dependent calcium channel L-type, alpha 1C subunit (CACNA1C) gene have been associated with bipolar disorder in a recent meta-analysis of genome-wide association studies (Ferreira et al., 2008). The purpose of the study is to investigate the impact of these variations on Major Depressive Disorder. Materials & Methods: Caucasian non-Hispanic participants in the STAR*D study of treatment for depression for whom DNA was available (N = 1213) were genotyped at two single-nucleotide polymorphisms (SNPs) (rs10848635 and rs1006737) in the CACNA1C gene. We examined putative phenotypic indicators of bipolarity among patients with major depression and elements of longitudinal course suggestive of latent bipolarity. We also considered remission and depression severity following citalopram treatment. Results: The rs10848635 risk allele was significantly associated with lower levels of baseline agitation (p = 0.03; β = -0.09). The rs1006737 risk allele was significantly associated with lesser baseline depression severity (p = 0.04; β = -0.4) and decreased likelihood of insomnia (p = 0.047; β = -0.22). Both markers were associated with an increased risk of citalopram-emergent suicidality (rs10848635: OR = 1.29, p = 0.04; rs1006737: OR = 1.34, p = 0.02). Conclusion: In this exploratory analysis, treatment-emergent suicidality was associated with two risk alleles in a putative bipolar liability gene. Author Contact: Francesco Casamassima, Psychiatry, 857-366-2391, fcasamassima@partners.org

20

Poster #16 Amyloid deposition is associated with increased posterior cingulate activity during memory recall in asymptomatic older adults P. Vannini, J. O’Brien, D. Putcha, K. O’Keefe, M. Pihlajamäki, P. LaViolette, I. Hamdi, L. Wang, J.A. Becker, K.A. Johnson, R.A. Sperling. Massachusetts General Hospital /Brigham and Womens Hospital /Harvard Medical School Purpose: Cortical β-amyloid deposition is a major histopathological finding in Alzheimer’s disease (AD) and a likely contributor to the observed memory impairment. Although the past decade has seen remarkable advances in our understanding of the basic pathobiology of AD, we still lack a complete mechanistic account of exactly how the early accumulation of amyloid pathology relates to the emergent clinical syndrome of memory impairment in humans. This knowledge could not only give insights into the disease pathology during the early stages of the disease but also help in finding reliable biomarkers for individuals at high risk of impending clinical decline. Our previous multimodality studies, combining [11C]Pittsburgh Compound B (PiB) PET with functional MRI (fMRI) studies of associative memory, have suggested that amyloid deposition may be related to altered medial parietal activity during encoding in asymptomatic and minimally impaired older individuals. In this study, we sought to investigate the impact of fibrillar amyloid burden on memory related neuronal activation during retrieval processes, specifically cued recall, in clinically normal older individuals. Materials & Methods: Twenty-one older subjects (age=72.8±2.3; CDR=0) were scanned (GE 3T) while performing a face-name associative memory task. During retrieval runs, subjects were shown the face from previously learned face-name pairs, and asked to indicate if they knew the name associated with the face (cued recall). Each subject also underwent a PiB PET scan and amyloid burden was quantified using PiB distribution volume ratios (DVR) corrected for partial volume effects in the whole brain and in anatomic regions of interest (ROI) delineated from a high-resolution MP-RAGE sequence using FreeSurfer. Results: To investigate how amyloid deposition might be related to brain activation during cued recall, a whole brain (SPM2) multiple regression analysis (p<.001) was performed using individual posterior cingulate PiB DVR values, age and performance (% recalled stimuli) as regressors. A positive correlation was found in the medial parietal lobe, with a global maxima found in the right posterior cingulate region [x=6,y=-30,z=30](r=.63,p=.003), indicating that more amyloid is related to increased fMRI signal after controlling for age and performance. Conclusion: These findings demonstrate that high levels of amyloid deposition, regardless of age and test performance, are associated with increased activation in brain regions supporting memory retrieval. It remains unclear whether the increased activation during successful retrieval is compensatory or is related to local amyloid toxicity resulting in hyperactivity. Our findings may serve to elucidate the neural underpinnings of memory dysfunction seen in aging and neurodegenerative disease and lend further support for the idea that functional brain changes begin far in advance of asymptomatic AD. Author Contact: Patrizia Vannini, Department of Psychiatry; Gerontology Research Unit, phone:617 726 62 03, email: patrizia@nmr.mgh.harvard.edu

21

Poster #17 Abstract Title: The establishment of regulated, specific and powerful promoters Authors; Jianmin Huang, Lynne L. Levitsky, David B. Rhoads Affiliation: Pediatric Endocrinology Purpose: The transcription regulation is a fundamental mechanism for the control of gene expression. Selective gene expression by differentiated cells is mediated by cell- and tissue-specific transcription factors (TFs) interacting with promoter elements upstream of gene transcription start site. Engineering powerful and regulated native promoter(s) responsive to specific TFs would greatly reduce the potential risks of constitutive and viral promoters (such as CMV promoter), frequently used for gene transfer but can promote tumors. A novel approach to create powerful promoters able to drive expression of any desired genes has been developed in our laboratory. Materials & Methods: Hepatocyte nuclear factor HNF4α, expressed preferentially in pancreas, liver, kidney and gastrointestinal tissues, activates downstream genes by binding HNF4α responsive elements (H4RE) in target promoters. Two HNF4α-responsive promoters were cloned into the pGL3 luciferase reporter for manipulation and analysis. The mouse Hnf1α promoter, with one conserved H4RE, was modified in two ways. First, 3 additional H4REs were inserted adjacent to the native H4RE, each separated by 10bp (H4RE×4). Second, the 0.3kb fragment containing the H4RE×4 was duplicated, creating two H4RE×4 motifs separated by 300bp. Two modifications were also made to the human ApoCIII promoter, which has two conserved H4RE 620bp apart (proximal H4RE in reverse orientation). First, both H4RE were converted to forward H4RE×4 motifs. Second, the distance between the two modifs was reduced to 300bp by removal of half the intervening DNA. Activities and HNF4α responsiveness were compared by Luciferase reporter assays in HEK-293 cells. To assess the ability of powerful promoters to yield physiological levels of important proteins, coding sequences of rat HA-tagged HNF4α3, human HNF1α, and human insulin gene were fused to the mHnf1αdupH4RE×4 promoter in pGL3 (replacing Luciferase gene). These plasmids were transiently transfected to HEK-293 or Hep3B cells with or without co-transfected pcDNA-HNF4a3 expression plasmid and expression was measured by immunoblotting proteins extracted 48h later. Results: In HEK-293 cells, all Hnf1α promoter reporters displayed comparable basal activities. Co-transfection of HNF4α3 stimulated the native promoter 12.5-fold and the mHnf1αH4RE×4 promoter 5-fold further (62-fold on the baseline). Promoter activity increased slightly (65-fold) when the number of H4RE copies increased to 10 (H4RE×10), but sharply decreased in H4RE×16 promoter (both arranged in tandem separated each other by10bp spacers). However, duplicated mHnf1αdupH4RE×4 promoter activity was dramatically increased 1200-fold over the native promoter. Similar results were also obtained with the ApoCIII promoter reporters. Immunoblotting analysis showed that the duplicated H4RE×4 promoters under stimulation of both exogenous and endogenous HNF4α were able to drive readily-detectible protein expressions of genes following promoters. Conclusion: The number and spatial relationship of TF binding sites strongly affected transactivation potentials of the promoters examined. We have increased the number of copies of a TF response element and achieved an exponential increase in promoter activity, capable to achieving physiological expression levels. Author Contact: Jianmin Huang (Tel: 617-726-5792, email: jmhuang@partners.org)

22

Poster #19 PIC-ing Apart Ovarian Cancer: A PhotoImmunoConjugate-based Approach Adnan O. Abu-Yousif, Xiang Zheng, Bryan Q. Spring, and Tayyaba Hasan Wellman Center for Photomedicine, MGH, HMS

The high mortality associated with epithelial ovarian cancers is largely attributed to the

development of resistance to standard treatments. Overall response rates to systemic chemotherapy in platinum resistant disease are dismal, ranging from 12-25%, thus creating a pressing need for the development of target-based combinatorial therapies that are effective in resistant disease. Photodynamic therapy (PDT) is an emerging photochemistry-based molecular and biophysical modality for the treatment of various malignancies, and has proven effective at treating cancers that have developed resistance to chemo- and radiotherapies. Our group previously demonstrated that the combination of PDT and C225 proved to be well tolerated, effective, and synergistic in mice with a 33% cure rate in a disseminated murine model of ovarian cancer. These studies provide a sound mechanistic basis for rational design of combination therapeutic regimens using PDT and other front line therapies. In keeping with this approach, we designed a photoimmunoconjugate (PIC) that was created by coupling the photosensitizer benzoporphyrin derivative mono acid ring A (BPD) to the EGFR-targeting monoclonal antibody C225. This approach allows for simultaneous delivery of both agents, increases the selectivity of BPD, and could enhance the efficacy of both treatments. Confocal laser microscopy analysis of EGFR-expressing cells revealed that conjugation to C225 altered the cellular localization of BPD from the mitochondria to the lysosome. Furthermore, the PIC retained the monoclonal antibody’s target recognition and effectively blocked EGF-induced phosphorylation of the EGFR in OVCAR5 cells. The phosphorylation of Akt-1 and MAPK/ERK, two downstream molecules involved in growth arrest, chemosensitivity, and angiogenesis was also inhibited by BPD-C225 treatment. In addition, we have demonstrated increased uptake in tumor sites compared to that observed in normal tissues in vivo. Furthermore, we have utilized a fluorescent microendoscope to image PIC fluorescence in vivo and ex vivo demonstrating tumor specificity with the PIC. The uptake of the BPD-C225 PIC is sustained in tumor tissues longer than free BPD, yet is effectively cleared within 5 days. Our results suggest that photo-immunotargeting could be a useful dual strategy for the elective destruction of tumor cells, with this PIC directing the photosensitizer, BPD specifically to the target ovarian cancer cells, while retaining the receptor blocking function of the EGFR targeting monoclonal antibody, C225.

23

Poster #20 Effect of post-retrieval propranolol on psychophysiologic responding to subsequent smoking script-driven imagery and craving Gladys N Pachas, MD; A. Eden Evins, MD, MPH; Roger Pitman, MD, Massachusetts General Hospital and Harvard Medical School; and Scott Orr, PhD VA Medical Center, NH Purpose: Each year over 430,000 people in the US die from smoking-related illness. The global mortality toll is over 5 million annually and increasing. First line pharmacotherapies for smoking cessation at least double the smoking cessation rate over placebo. However, only 30% of people quit on a given smoking cessation attempt with effective pharmacotherapy and up to 90% relapse to smoking within the first year. Clearly, more effective treatments are needed for initiation and maintenance of tobacco abstinence. Animal studies show that post-reactivation propranolol blocks the reconsolidation of drug related memory. The purpose of this study is to evaluated, the effect of memory reconsolidation blockade with a single dose of propranolol prior to smoking cue exposure (memory reactivation) on psychophysiological response to smoking cues one week later, then to evaluate the effects of a series of six memory reactivation sessions with weekly single-dose propranolol on subjective experience of craving and smoking behavior. Materials & Methods: This is a randomized, double-blind, placebo-controlled trial of post-reactivation treatment with propranolol in treatment-seeking nicotine dependent participants designed to test the hypothesis that acute administration of the B-adrenergic blocker, propranolol, following retrieval of drug-associated memories will reduce the strength or salience of the memory through the process of memory reconsolidation blockade, and that this will result in less physiologic reactivity to smoking cues and, if repeated, will result in the reduction of cue-induced drug craving. The first phase involves administration of a single dose of either propranolol or placebo followed by reactivation of memories of the craving experience to smoke cigarettes during a smoking script preparation session in which participants describe salient aspects of their smoking experience that trigger intense tobacco craving. One week later, previously recorded scripts are played to activate participants’ memories of smoking and evoke craving and to assess physiologic activation to smoking-related scripts. The second phase of the study involves a smoking cessation attempt with 6 weekly memory reconsolidation blockade treatments on a platform of nicotine replacement therapy. Participants who are able to attain at least 7 days of abstinence begin a series of six weekly single doses of propranolol or placebo and memory reactivation sessions where we activate participant’s memories of smoking and evoke craving. Outcome measures include physiological response measures (skin conductance, heart rate and left corrugator electromyogram) and subjective experience of craving assessed weekly with a visual analog scale (VAS) and the validated Tiffany QSU Results: Phase 1: In the first 29 participants in this ongoing trial, there is evidence of a treatment (drug, placebo) by stimulus (smoking vs. neutral script) interaction, in which left corrugator electromyogram (EMG) activation during script-driven mental imagery one week after a single dose of propranolol was significantly smaller (p=0.009) in the propranolol vs. placebo subjects (mean 0.206 vs. 1.11 respectively). No significant treatment by stimulus interactions were found in the preliminary analysis for skin conductance and heart rate. Phase 2: In the first 14 subjects who finished second phase, there is evidence of a treatment (drug, placebo) by stimulus (smoking memory activation) interaction in which cue-induced smoking craving measured with tiffany QSU one week after the last dose of propranolol or placebo is smaller (p=0.006) in the propranolol vs. placebo group (mean 10.00 vs. 11.50 respectively) A trend in the same direction was observed with subjective experience of craving assessed with a VAS from visit 8 to 12. Conclusion: In this preliminary analysis, we found evidence that a single dose of propranolol reduces physiologic reactivity (EMG) to craving stimulated by script-driven imagery. There is also evidence of craving reduction after the series of propranolol; this supports the possibility that memory reconsolidation blockade with beta adrenergic blockade during memory reactivation reduces cue-induced drug craving; further information will be available once the study is completed. Author Contact: Gladys Pachas; Psychiatry; 617-643-1991; gpachas1@partners.org

24

Poster #21 A Novel Vascular Co-culture Model with a Biomimetic Internal Elastic Lamina

Eric B. Finkelstein, Gwen E. Owens, David M. Hoganson, Irina Pomerantseva, Joseph P. Vacanti, and Craig M. Neville

Department of Pediatric Surgery, Center for Regenerative Medicine, Massachusetts General Hospital,

Harvard Medical School, Boston, MA USA

Purpose: The internal elastic lamina (IEL) is a layer of extracellular matrix, largely elastin, which separates the intima (endothelial cell, EC) layer from the media (smooth muscle cell, SMC) layer of arteries. The IEL is a support element for the vessel, allowing contact and communication between ECs and SMCs. The IEL regulates interstitial flow to the media, forming a water impermeable barrier with flow only through numerous fenestrations. A variety of vascular diseases result in alterations of the IEL.

Drug-induced vascular injury (DIVI) is a poorly understood phenomenon often observed during preclinical testing of pharmaceutical compounds. Current screening protocols involve dosing rats with compounds, and assessing the extent of vascular injury, by looking for hemorrhage and other gross changes in the arteries of the mesenteric vascular bed. Our goal is to establish an in vitro model of the rat mesentery that includes functional EC and SMC layers separated by a synthetic IEL that can replicate the DIVI phenotype. This model will be used for high-throughput toxicology screening. We used polycarbonate membranes to model the IEL and have optimized EC-SMC co-culture conditions to establish a normal vascular phenotype.

Materials & Methods: To model the internal elastic lamina, ECs and SMCs were co-cultured on opposite sides of a polycarbonate membrane (Millipore) that was mounted on cell culture inserts and oxygen plasma treated. SMCs were seeded first and cultured for 1-7 days, at which point endothelial cells were added to the opposite side of the membrane. Culture conditions were optimized by varying the time between SMC and EC seeding, the time of co-culture and medium composition. The EC and SMC layers that formed during co-culture were assessed by immunostaining and electron microscopy in order to compare their structural architecture and phenotype to the native vasculature.

Results: The fenestrations of the natural internal elastic lamina of rat mesenteric arteries range in size from 2.3-13.1µm (mean 6.4µm) with a porosity of 11.1% [1]. For our studies, we have used a track-etched polycarbonate membrane with a 5-20% porosity and 10µm pore size, to model the internal elastic lamina. This membrane supports cell growth, and allows cell-cell contacts to be established, while maintaining the cells as distinct layers. Smooth muscle cells formed a multi-layered structure as they do in the media, while the endothelial cells formed an endothelium-like mono-layer. While the morphology of individual cell types grown in mono-culture and co-culture was similar, in co-culture, there was evidence of ECs aligning with SMCs on the opposing side of the membrane. Conclusion: We have developed a novel vascular co-culture model with a biomimetic internal elastic lamina. We have optimized cell culture conditions in this model and verified that ECs and SMCs in co-culture have normal structure and function. We are developing assays to facilitate high-throughput screens for assessment of DIVI by candidate compounds. We anticipate that this in vitro model will be used to study a number of vascular injuries in addition to those brought about by toxicity, such as naturally occurring atherosclerosis and vasculitis. With a biomimetic IEL to control intima-media interactions and interstitial flow, we will be able to model the in vivo injury condition more effectively. Author Contact: Eric B. Finkelstein, Ph.D., Pediatric Surgery/Center for Regenerative Medicine, 617-643-3377, ebfinkelstein@partners.org

25

Poster #22 Erlotinib chemoprevention of hepatocellular carcinoma in a rat model of cirrhosis Bryan C. Fuchs1, Tsutomu Fujii1, Gregory Y. Lauwers2, Christopher M. McGinn3, Suguru Yamada1, Toshihiko Kuroda1, Michael Lanuti3 and Kenneth K. Tanabe1 1Division of Surgical Oncology, 2Department of Pathology and 3Division of Thoracic Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114 Purpose: Hepatocellular carcinoma (HCC) is the sixth most common solid tumor worldwide and due to its poor prognosis it is the third leading cause of cancer-related death. Given the lack of successful treatment options for HCC, chemoprevention in high-risk patients has been proposed as an alternative strategy. Elevated levels of epidermal growth factor (EGF) in cirrhotic patients are associated with greater risk for development of HCC, and experimental overexpression of EGF in the liver in animal models induces transformation to HCC. Based on these findings, we address the hypothesis that blocking the EGF-EGF receptor (EGFR) pathway may be an effective chemoprevention strategy. Materials & Methods: An anchorage-independent growth assay was used to determine the effects of increasing concentrations of EGF on the in vitro transformation of normal human liver epithelial, THLE-5B, cells. Several EGFR tyrosine kinase inhibitors (AG1478, erlotinib, gefitinib, and lapatinib) were tested for their ability to inhibit this process at concentrations that are not cytotoxic as determined by MTT analysis. A rat model of diethylnitrosamine (DEN)-induced cirrhosis was used to examine the efficacy of erlotinib for inhibition of HCC formation in these cirrhotic livers. DEN (50 mg/kg) was administered weekly throughout the study while erlotinib (2 mg/kg) was administered 5 days per week for 6 weeks beginning at the onset of cirrhosis. At the end of the study, rats were sacrificed, livers were sectioned and stained to analyze disease progression and tumor nodules were counted. Results: EGF transforms THLE-5B cells to anchorage-independent growth in a dose-dependent manner. EGF-induced transformation was prevented by all the EGFR inhibitors at low concentrations (< 1 μM) that exhibit no cytotoxicity. The DEN-treated rat model closely resembles cirrhosis in humans. Specifically, liver cirrhosis and HCC were observed after 12 and 18 weeks, respectively. Gross observations were confirmed by H&E and trichrome staining. Further, erlotinib significantly (p < 0.01) prevented the development of HCC tumor nodules from on average 17 in vehicle controls (range 9-22) to on average 5 in erlotinib-treated animals (range 1-8) (Figure 1). Conclusion: EGF-EGFR inhibition is a potentially effective chemoprevention strategy against HCC. The majority of HCC cases arise in patients with cirrhosis, thereby defining a high-risk group to target for chemoprevention trials. Figure 1. Erlotinib reduces tumorigenesis in a rat model of cirrhosis and HCC.

Author Contact: Bryan C. Fuchs, Department of Surgery, 617-724-3839, bfuchs@partners.org

26

Poster #23 Dual-colored Fluorogenic Probes for Imaging Oxidative Stress Fangwei Shao, Jose-Luiz Figueiredo, Cory Siegel, Ralph Weissleder and Scott A Hilderbrand Center for Systems Biology/Center for Molecular Imaging Research, Masschusetts General Hospital. Harvard Medical School. Boston, Massachusetts, USA Purpose: Hypochlorous acid is one of the most powerful natural oxidants. Myeloperoxidase (MPO), a heme enzyme secreted by neutrophils and macrophages converts hydrogen peroxide, a relatively unreactive oxidant, to HOCl in the presence of chloride ions. As a reactive oxygen species (ROS), HOCl plays a role in the pathogenesis of diseases, including cancer, atherosclerosis and arthritis. There is a current unmet need for fluorogenic sensors that are HOCl specific and are capable of monitoring the production of this oxidant in vivo. Materials & Methods: The design, synthesis and characterization of two HOCl-responsiveprobes (CyClO-1 and CyClO-2) with fluorescence emission in the near infrared (NIR) are detailed in this study. Fluorescence and absorption spectra of both probes were observed to show the linear activation upon the titration of sodium hypochlorite. The selectivity of the probes was also investigated by screening the non-activated probes against a panel of biological ROS. Activation of the probes by MPO derived HOCl was studied by incubation of the probes in the presence of MPO and hydrogen peroxide. Results: The probes are prepared in a simple one step procedure in excellent yield. The first probe CyClO-1 contains a chloride moiety on the polymethine dye backbone, whereas CyClO-2 contains a carboxylic acid modified aryl thioether which serves as a potential bioconjugable handle. Both probes have excellent water solubility and NIR optical properties. In their reduced, non-activated forms, the sensors absorb and emit above 800 nm. Upon oxidation, a saturated C-C bond on the polymethine backbone of the sensors is oxidized to generate an unsaturated double bond. This oxidation results in a greater than 120 nm blue-shift in both the absorption and fluorescence emission spectra to 692 and 722 nm, respectively for CyClO-2. Fluorescence activation of greater than 40-fold is observed after treatment of CyClO-2 with HOCl. Both sensors demonstrate a linear response to increasing concentrations of HOCl. Fluorescence activation at 720 nm is also observed after addition of MPO and H2O2. The sensors are not activated by the MPO/H2O2 system in the presence of a MPO inhibitor, confirming that MPO-derived HOCl is the cause of oxidative activation. ROS screening demonstrates that the probes are activated only by HOCl and not other ROSs, such as hydrogen peroxide, superoxide, hydroxyl radical peroxynitrite and nitric oxide. Conclusion: Two novel fluorogenic probes have been developed as dual-color fluorescent probes for hypochlorous acid with high sensitivity and selectivity. The significant blue-shift in fluorescence emission upon oxidation by HOCl allows for independent monitoring of the probe distribution and activation via excitation of the probes at 750 and 640 nm, respectively.These agents are promising materials for application to in vivo imaging of MPO activity.

R

N

SO3H

N

SO3H

SO3HHO3S

HOClor MPO/H2O2

R

N

SO3H

N

SO3H

SO3HHO3S

R =Cl CyClO-1

S COOH CyClO-2

Author Contact: Fangwei Shao, Center for systems biology, 617-6436855, fshao@mgh.harvard.edu

27

Poster #24 Abstract Title: Protease inhibition causes luminal obliteration and increased ring thickening in the cricoid Authors: Efrain Martinez-Alvernia, MD*, Leila Mankarious, MD* Affiliation: *Massachusetts Eye and Ear Infirmary and Harvard Medical School. Purpose: Luminal expansion of the cricoid cartilage appears to be stunted by loss of luminal epithelium

(LE) and can be enhanced by exogenous transforming growth factor beta3 (TGFβ3). When both the LE

and perichondrium are disrupted, matrix metalloproteinase (MMP) signal within adjacent chondrocytes is

diminished but can be regenerated with exogenous TGFβ3. The pattern of loss of MMP signal with

growth stunting and present signal associated with luminal expansion suggests a role for these enzymes’

activity in normal subglottic expansion. Objective: To determine if MMP inhibition affects cricoid

expansion and by what mechanism in order to further define the mechanism of action of TGFβ3-induced

luminal expansion.

Materials & Methods: Subglottises from 20 neonatal mice were divided into: (A) 10 grown with a non-

selective MMP inhibitor, (B) 10 grown in basic medium alone. Outcome measurements included,

luminal cross section area, apoptosis, cell proliferation and identification of cleaved aggrecan fragments.

Results: Group A demonstrated statistically-significant luminal obliteration when compared to group B,

with apparent circumferential thickening of the cricoid ring, relatively decreased apoptosis, decreased

aggrecan cleavage fragments in the extracellular matrix (ECM) and increased chondrocyte proliferation.

Conclusion: MMP inhibition leads to significant ring wall thickening and luminal obliteration either due

to a relative loss of apoptosis, accumulation of ECM, increased chondrocyte proliferation or via a

combination of factors. Because TGFβ3 is known to increase luminal expansion and maintain MMP

signal in the cricoid cartilage, we hypothesize that is does so by influencing the above.

Author Contact: Efrain Martinez-Alvernia, ENT, 857 719 3917, eamartinez@partners.org or

mefrainaugusto@hotmail.com

28

Poster #25 Effects of Photodynamic Therapy on Tumor Host Microenvironment and Treatment Optimization Strategies Zhiming Mai, Nicolas Solban, Irene Georgakoudi, Sung K. Chang, William L. Rice, Charles P. Lin and Tayyaba Hasan Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School. Boston, Massachusetts, USA Purpose: Photodynamic therapy (PDT) is an approved treatment modality for oncologic conditions, and is in clinical trials for the treatment of prostate cancer (Pca). Although treatment outcomes with PDT are usually promising in terms of the local tumor shrinkage, and the improvement of survival and quality of life in patients, tumor regrowth and metastases often occur later when the treatment is used alone. To fully explore its benefits in clinical applications, it is important to understand how the tumor host microenvironment changes in response to the PDT. The aim of this study is to mechanistically investigate tumor host microenvironment alterations following PDT, and based on these findings to rationally design mechanisms-based effective combination treatments for prostate cancer. Materials & Methods: We focused the examination of PDT-elicited tumor host microenvironment alterations that had impacts on tumor regrowth and metastasis using a rat Pca MatLyLu (MLL) cell line. In particular, PDT-treated cells i.v. injected into rats at various time were optically monitored for their circulation kinetics on an in vivo flow cytometer. Altered expressions of angiogenic, adhesion and homing molecules were evaluated using molecular biology-based approaches. Finally, an experimental metastasis rat model was employed to study the effect of PDT conditions on the pulmonary metastasis. Results: In vivo flow cytometry showed a significant increase in the circulation kinetics of Pca cells treated with subcurative PDT doses when compared to control. In vivo flow cytometry is a cutting-edge technology that has been pioneered at the Wellman Center for Photomedicine. We were the first to demonstrate circulation kinetics of the living Pca cells following the PDT in vivo and in a real-time manner using this state-of-the-art system. An increase in circulating cancer cells following curative/palliative surgery of other cancers is well documented, and considered as an initial sign of distant metastases. PDT leaded to a temporally transient up-regulation of vascular endothelial growth factor (VEGF) following treatment in rats. VEGF is an angiogenic cytokine, which plays important roles in tumor regrowth and metastasis. Recent reports suggest that various cytotoxic therapies can potentially lead to a pro-angiogenic response, which correlates with poor long-term clinical outcomes. In vitro experiments demonstrated that subcurative PDT transiently decreased adhesion to the extracellular matrix protein collagen IV, by decreasing β1 integrin protein levels without affecting α5 integrin levels. In vivo, consistent with in vitro results, immunohistochemical and Western analysis of PDT-treated orthotopic prostate tumors showed a decrease in β1 integrin but not of α5 integrin. The decreased adhesion to the extracellular matrix protein collagen IV would help malignant cells detach from primary tumor mass into bloodstream and migrate to distant organs. Conclusion: This study suggests that there is no “silver bullet” monotherapeutics for the treatment of complex diseases such as cancer. Characterization of tumor host microenvironment will be helpful in understanding the mechanism of treatment responses and will provide knowledge to rationally design effective treatment regimens involving timely and specific interventions as part of combinatorial therapeutics. In particular, combining PDT with other treatment strategies aimed at suppression of VEGF molecules at the peak of their response following the PDT as well as normalization of the homing and the subsequent adhesion of circulating tumor cells will complement and enhance PDT for the effective control of local prostate tumor and distant metastasis. Here, we present a paradigm of how to obtain the knowledge through basic mechanistic studies towards rationally designing mechanisms-based combination treatments applicable to the effective clinical use. Author Contact: Zhiming Mai; Dermatology; 617-726-6995; zmai@partners.org

29

Poster #26 Abstract Title

Characterization of cell viability and proliferation in single bioprinted tissue engineering “building block”

F. Xu a,b, S. Moon a,b, U. Demirci a,ba Bio-Acoustic-MEMS in Medicine (BAMM) Laboratory, Center for Biomedical Engineering, Department of

Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA b Harvard-MIT Health Sciences and Technology, Cambridge, MA, USA. c Department of Bioengineering, Clemson University, Clemson, SC, USA

Purpose: ‘Building block’ approach based on the emerging cell printing technology for engineering complex 3D tissue/organ structures has the potential to solve several crucial limitations associated with the current tissue engineering methods, such as seeding cells with spatial control and handling multi-types of cells. In this study, we aim to characterize the single ‘building block’ (i.e. cell-laden collagen droplet) containing primary rat bladder smooth muscle cells (SMCs), including cell viability, cell proliferation and long-term culturing. Materials & Methods: The cells used in this study were primary SMCs isolated from rat bladder tissue. SMC suspensions were mixed with reconstituted collagen for printing. Images of the printed droplets were taken under a bright-field microscope at Days 0-5, respectively. Cell viability was characterized by using live/dead staining. Cell proliferation was characterized by the total number of cells within the printed droplets as a function of time. Results: The results showed that: (i) SMCs encapsulated in collagen ‘building blocks’ can survive the printing process with a high viability (>94%) under four different initial numbers of cells in ‘building block’ (7±2, 16±2, 26±3 and 37±3); (ii) after printing, SMCs can divide and proliferate in the ‘building blocks’, and larger initial number of cells gives more increase in number of cells, but the proliferation rates are similar; (iii) printed patch can be cultured for long term (>7 weeks) and was observed to form a tissue-like 3D construct. Conclusion: These results will supply a base for future high-throughput creation of 3D tissue models, which would enable the collection of large data sets to further our understanding in the clinical tissue/organ diseases. Author Contact: your name, department, phone, email Feng Xu, Postdoctoral Research Fellow Renal Department, BWH, Harvard Medical School; Harvard-MIT Health Science & Technology Phone: 617-768-8315, Email: fxu2@rics.bwh.harvard.edu, fengxu@mit.edu

30

Poster #27 Antiplatelet Agents increase risk of hemorrhagic stroke in individuals with cerebral amyloid angiopathy Alessandro Biffi MD, Amy Halpin BS, Amytis Towfighi MD, Katharina Busl MD, Kristin Schwab BA, Lynelle Cortellini MSc, Eric E. Smith MD MPH, Natalia S. Rost MD, Steven M. Greenberg MD PhD, Anand Viswanathan, MD PhD, Jonathan Rosand MD MSc Hemorrhagic Stroke Research Program, Department of Neurology, and Center for Human Genetic Research, Massachusetts General Hospital and Harvard Medical School, Boston MA, USA Purpose: Cerebral amyloid angiopathy (CAA) is a common pathological condition in the elderly, which can cause both symptomatic intracerebral hemorrhage (ICH) and asymptomatic microbleeds detected on MRI.. Because the risk of thrombembolic disorders also rises dramatically with aging, antiplatelet agents are commonly administered to individuals with CAA. We sought to determine whether use of antiplatelet agents increases risk of intracerebral hemorrhage (ICH) in patients diagnosed with cerebral amyloid angiopathy (CAA). Materials & Methods: Subjects were consecutive inpatients who qualified for the diagnosis of CAA following admission to the hospital for ICH from July 1994 to March 2006. Diagnosis of probable or possible CAA was assigned according to previously validated Boston Criteria. Baseline clinical, imaging and laboratory data were collected. Survivors of the initial ICH were followed prospectively for recurrent ICH and duration of antiplatelet agent as well as anticoagulant use. Cox proportional hazards model was used with antiplatelet agent and warfarin exposures as time-varying variables, adjusting for known predictors of ICH recurrence in CAA. These included APOE genotype, as well as neuroimaging markers (CT-defined white matter hypodensity and number of microbleeds on gradient-echo MRI). Results: 104 subjects were diagnosed with probable or possible CAA. Of these, 16 (15.4%) were exposed to antiplatelet after their initial ICH. Over a median follow-up time of 34.3 months (Interquartile Range: 15.1 – 57.6), 29 subjects experienced recurrent ICH. Baseline predictors of recurrence included a history of ICH prior to the index ICH at which they were enrolled (Hazard Ratio [HR] 4.8, p = 0.005) and number of microbleeds on MRI (2 – 4 or ≥ 5, HR 2.9 and 4.7 respectively, p value 0.04 and 0.001 respectively). Antiplatelet agents use was associated with ICH recurrence (HR 3.9, 95% Confidence Interval: 1.6 – 8.2, p = 0.022) when adjusting for baseline clinical predictors, with a 33% increase in 2-year hemorrhage recurrence rate. When adjusting for number of microbleeds on MRI (0, 1, 2 to 4 or ≥ 5 microbleeds) HR for antiplatelet use were 1.9 (p > 0.2), 3.2 (p = 0.004), 4.8 (p= 0.037) and 5.3 (p =0.048). Conclusion: Data from this prospectively followed longitudinal cohort demonstrate that that use of antiplatelet agents increase risk of ICH recurrence in individuals with CAA. This effect may rise with increasing number of microbleeds on MRI. Further studies will be necessary in order to clarify the decision to administer antiplatelet agents to patients with CAA.

Modified Kaplan-Meier plot of effect of antiplatelet agents on recurrent ICH in CAA

Author Contact: Alessandro Biffi, MD Neurology / CHGR, 617-643-3304 biffi@chgr.mgh.harvard.edu

31

Poster #28 Abstract Title: PET Studies on Burn Induced Insulin Resistance in Rabbits under Clamp Conditions Authors: Hongzhi Xu, Edward A. Carter, Yong-Ming Yu, Alan J. Fischman, Ronald G. Tompkins Institution: Shriners Burn Hospital, Boston, MA, 02114 Purpose: Burn injury (BI) is associated with the development of insulin resistance which complicates clinical management. Previous studies using PET imaging in a burn rabbit model have demonstrated that BI was associated with increased blood flow, oxygen consumption and FDG uptake in skeletal muscle. In the present study, the PET imaging studies have been conducted in burned rabbits under euglycemic insulin clamp (EIC) conditions. Materials & Methods: 12 male rabbits (2.5-4.6 kg, Millbrook Farms) were anesthetized, the dorsum shaven. 6 animals received full thickness burn injury (25% total body area) by merging the dorsal part into boiling water for 15 sec (B), 6 received sham burn (SB) in room temperature water. 3 days after BI, jugular vein and carotid artery catheters were surgically implanted after overnight fasting. After measuring fasting blood glucose (BG), an EIC status was maintained in each animal by a constant infusion of insulin (2.25 mU /kg/min, priming: 25 mU/kg), concomitantly with variable rate of 25% Dextrose infusion to “clamp” the BG at ~100mg% in both groups. PET imaging was conducted, with region of interest (ROI) in the both hind limbs to determine the rates of blood flow, O2 utilization, and FDG uptake of the leg muscles, distinctively, following inhalation of 15O2, and I.V. injections of 15O2H, or 18F-FDG tracers. At the same time of EIC and PET imaging, each animal then received a primed constant tracer infusion of [6,6,2H2]glucose with a targeted infusion rate of 1 μmol/kg/min, and a priming dose of 80 μmol/kg for 4 hrs. Results: 3 days after BI the fasting BG of the burned rabbits was higher than the sham animals (Table 1), indicating an insulin resistance state. Insulin reduced BG in both groups. However, to maintain the same BG, SB required more exogenous glucose (ExG) than the B (Table 1). PET imaging showed that under EIC blood flow to the skeletal muscle was higher in the B than SB (3.85 ± 0.40 vs. 2.24 ± 0.43ml/min/100g; N=6, P < 0.05). However, the rates of O2 consumption (41.84 ± 9.03 vs. 32.35 ± 4.45ml/min/kg; N=6, NS) and FDG utilization by the skeletal muscle (53.20 ± 14.52 vs. 45.51 ± 7.85 μmol/min/kg; N=6, NS) remained the same between B and SB. Isotopic tracer study showed that endogenous glucose production in fasting state (i.e. hepatic gluconeogenesis) was significantly increased in burn group (13.6 ± 1.7 vs. 9.1 ± 1.0 mg/kg/min; N=6, P< 0.05). Table 1. BG Levels before and after EIC and Dextrose Infusion Rates during EIC* Measurements B (n=6) SB (n=6) P values B vs. SB Fasting BG (mg %) 223 ±25 148 ±21 P<0.01 BG during EIC (mg %) 111±6 107±6 NS** Mean ExG Rates (mg/min/kg) 4.35±0.93 6.86 ±0.65 P<0.01 *Data are in Mean ± SE. ** NS: No Significant difference Conclusion: Insulin resistance starts to develop at early stage of burn injury. PET imaging combined with EIC and isotopic tracer study showed that at early stage of burn, the manifestation of insulin resistance is unsuppressed hepatic glucose production, rather than decreased glucose uptake in peripheral tissues. Author Contact: Hongzhi Xu, Department of Surgery, 6172793273, hxu3@partners.org

32

Poster #29 Abstract Title: Fulvestrant Anti-hormonal Resistance in Breast Cancer: A zinc finger-artificial transcription factor library approach Authors: Jeongeun Lee, Benjamin Wittner, Sridhar Ramaswamy, Toshi Shioda, Vijay Yajnik, Altan McDermott, Magdalena Eichtinger, Jason Lohmueller, Morgan Maeder, Keith Joung, Dennis Sgroi. Affiliation: MGH, Charlestown, MA Purpose: The purpose of this study is to identify molecular changes that are associated with fulvestrant resistance. Thereby, we will identify potential therapeutic target(s) for fulvestrant resistance. Materials & Methods: Using 25 different, well-characterized zinc finger domains, we created a retrovirus-based combinatorial library that consists of 390,625 different proteins each consisting of an array of four zinc fingers fused to the NF-κB p65 transcriptional activation domain. Using this zinc finger-artificial transcription factor (ZF-ATF) library, we will identify unique ZF-ATFs capable of inducing fulvestrant-resistance in hormone-dependent breast cancer cells.

MCF7-R73(unmodified)

cells

Retroviral Transduction ofZF-ATF Library

Pool of modifiedCells expressing Different ZF-ATFs

Fulvestrant selection

Identify Fulvestrant-Resistant cells

Fulvestrant selection

Fulvestrant selection

PCR rescue of ZF-ATFs responsible for

fulvestrant-resistance, convert to retrovirus

and transduce individual ZF-ATFsinto MCF7-R73 cells

Cells induced to fulvestrant resistance by individual

ZF-ATFs

Figure 1. Schematic representation of of ZF-ATF library approach Steps of the ZF-ATF library functional genomic approach. Retrovirus containing plasmids encoding the ZF-ATF library are used to transform MCF7-R73 cells. In each cell a different ZF-ATF is expressed and regulates unknown target genes in an unbiased manner, leading to phenotypic variation amongst transduced cells. Transduced cells are selected for fulvestrant resistance, ZF-ATFs conferring resistance are rescued by PCR, DNA encoding individual ZF-ATFs subcloned into a retroviral plasmid, retrovirus produced and MCF7-R73 cells transduced by retrovirus bearing individual ZF-ATFs.

Results: The generated six fulvestrant resistant clones showed growth in the presence of 100 nM fulvestrant and this resistance was specific to fulvestrant and dependent on the expression of a ZF-ATF. Comparative gene expression profile analysis identified 241 genes whose expression was commonly altered among all six fulvestrant-resistant clones. Interestingly, the expression of 240 of these 241 genes was down-regulated. Conclusion: These data demonstrate the successful identification of unique ZF-ATFs that confer fulvestrant resistance in hormonal-dependent breast cancer cells, and this system represents a unique model in which to study the mechanisms of fulvestrant-resistance. Author Contact: Jeongeun Lee, Molecular Pathology Department, 617-726-5692, jlee69@partners.org.

33

Poster #30 Intrapancreatic Cholangiocarcinoma (ICC): An attempt to define, appropriately stage and genetically explore a largely unknown disease Konstantinidis IT1, Deshpande V3, Hezel A2, Warshaw AL1, Lauwers GY3, Androutsopoulos V1, Fernandez Del-Castillo C1, Thayer SP1, Ferrone CR1. Departments of Surgery1, Medical Oncology2 and Pathology3. Massachusetts General Hospital. Boston. MA. Purpose: Our aim is to describe the patterns of peribiliary adenocarcinomas (PBA), explore the pathologic definition and genetic basis of ICC and determine if duodenal invasion and depth of invasion are related to prognosis. Materials & Methods: Retrospective review of clinicopathologic data and pathology slides of patients with CCA resected between 9/1990-5/2008 was performed. DNA was extracted from paraffin embedded tissues. The mutational profile of these tumors was constructed using an extended panel of mutations. Patients with PDAC served as controls for comparisons. Results: A total of 79 patients were identified. Two patterns of CBD involvement were recognized: In type 1 (n=43) the tumor was growing circumferentially and symmetrically around the CBD. In type 2 (n=36) the pattern of tumor growth was asymmetrical. Only 81% of type 1 and 69% of type 2 were characterized as ICC in the original pathology report. There was no difference in age, presentation, size, frequency of nodal metastasis, microinvasion of nerves and vessels between the two types. Type 2 was more frequent in women (77% vs 67%;p=0.01) and it was associated with high grade PanIN (42% for type 2 vs 9% for type I;p<0.001) whereas type 1 was associated with CBD dysplasia (44% for type 1 vs 14%;p=0.004). Only type 1 tumors had significantly better survival compared to PDAC patients (median survival 66mo for type 1, 29mo for type 2 and 19mo for PDAC;p<0.001). Complete genetic data were available for 19 patients. Mutated KRAS gene was found in all the type 2 tumors that were found to have mutations whereas it was found in only 25% of the type 1 tumors (p=0.05). Depth of invasion was measured from pathology slides in 72 patients (3T1, 8T2, 45T3 and 16T4). Tumor extension beyond the bile duct negatively impacts overall survival (OS) (T1 and T2 median survival was not reached, T3 35mo and T4 31mo). There was no significant difference in the size of T3 and T4 tumors (T3 1.97cm vs. T4 2.22cm;p=0.4). While pancreas invasion was associated with worse survival (T1/T2 not reached vs. T3 35mo;p=0.03) duodenal invasion did not further decrease survival (T3 35mo vs. T4 31mo;p=0.6). Median depth of invasion was 10mm. Patients with tumors invading more than 10mm had a worse median OS (for 10mm invasion or less median survival was not reached vs. 29mo;p=0.007) and more frequent perineural invasion (72% vs. 94%;p=0.01). After Cox regression analysis lymph node metastasis and >10mm of invasion, not duodenal invasion, were significant prognostic factors of OS. Conclusions: Peribiliary adenocarcinomas are a heterogeneous group of neoplasms that are characterized as ICC without formal criteria. Two patterns of tumor growth are recognized with histological, genetic and survival differences. Tumors growing symmetrically around the CBD are more likely to represent true ICC. The current AJCC staging does not correlate well with prognosis. Increasing depth of invasion (>10mm) rather than duodenal invasion is a negative prognostic factor for overall survival. Author Contact: Ioannis Konstantinidis. Surgery. Tel: 617-955-8839. ikonstantinidis@partners.org

34

Poster #31 Abstract Title: CDIP sensitizes cancer cells to a TNFα-mediated apoptotic cell fate Authors: Lauren Brown-Endres1, David Schoenfeld1, Takao Ide1, César Muñoz-Fontela2, Stuart A. Aaronson2 and Sam W. Lee1

Affiliations: 1Cutaneous Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA 2Department of Oncological Sciences, Mount Sinai School of Medicine, New York, NY, USA Purpose: To investigate the role of Cell Death Involved p53-target (CDIP) in TNFα-mediated cell fate decision-making processes. Materials & Methods: CDIP was ablated in several cancer cell lines by shRNA targeting and the effect of this on TNFα-mediated apoptosis was assessed by TUNEL assay. NF-kappa beta survival and apoptotic target gene expression were assessed by RT2 Profiler™ PCR arrays under conditions of TNFα-mediated survival versus apoptosis, and transcript half-life was determined under similar conditions after treatment with actinomycin D. Western blot analysis was also used to detect several members of the TNFα signalling pathway and assess whether CDIP expression could influence this pathway in a manner that was distinct from that of cells treated with TNFα alone. Results: We show that CDIP mediates sensitivity to TNFα-induced apoptosis, and that cancer cells with endogenous CDIP expression are inherently sensitive to TNFα killing. CDIP-mediated sensitivity to TNFα-induced apoptosis is dependent upon JNK, and occurs at the level of NF-kappa beta target gene expression. CDIP→TNFα signalling results in a program of NF-kappa beta target gene expression in which survival gene expression (i.e. SOD2, TRAF1, A1, cIAP) is suppressed, and IL-8 is up regulated. SOD2 suppression, together with JNK dependency, points to an important role for ROS in CDIP→TNFα-mediated apoptosis, which is substantiated by results demonstrating that inhibition of ROS completely blocks CDIP→TNFα-induced apoptosis. We provide evidence that IL-8 acts as a pro-apoptotic factor downstream of ROS, and suggest a model in which CDIP acts to suppress NF-kappa beta survival targets, allowing for ROS activation of JNK-dependent apoptosis, which is reinforced by IL-8. Conclusion: CDIP is an important sensitizing factor to TNFα-induced apoptosis, which has implications for TNFα-based cancer therapeutics, as well as autoimmune diseases resulting from excessive TNFα signaling. Author Contact: Lauren Brown-Endres, Ph.D. Dermatology-Cutaneous Biology Research Center Building 149 3rd floor Telephone 617-724-9958 Fax 617-726-4453 Email: lbrown17@partners.org

35

Poster #32 The Multimeric Structure of Polycystin-2 (TRPP2): Structural-Functional Correlates of Homo- and Hetero-multimers with TRPC1 Peng Zhang1, Ying Luo3, Bernard Chasan4, Silvia González-Perrett2, Nicolás Montalbetti2, Gustavo A. Timpanaro2, María del Rocío Cantero2, Arnolt J. Ramos1, Wolfgang H. Goldmann1, Jing Zhou3, and Horacio F. Cantiello1,2*

1Nephrology Division & Electrophysiology Core, Massachusetts General Hospital East, Charlestown, MA, 02129 and Department of Medicine, Harvard Medical School, Boston, MA, 02115 USA; 2Laboratorio de Canales Iónicos, ININCA, UBA-CONICET, Buenos Aires, 1122AAJ, Argentina; 3Renal Division, Department of Medicine, Brigham's and Women's Hospital, and Harvard Medical School, Boston, MA, 02115 USA; 4Physics Department, Boston University, Boston, MA, 02115 Purpose: Polycystin-2 (PC2, TRPP2), the gene product of PKD2, whose mutations cause autosomal dominant polycystic kidney disease (ADPKD), belongs to the superfamily of TRP channels. PC2 is a non-selective cation channel, with multiple subconductance states. In this report, we explored structural and functional properties of PC2 and whether the conductance substates represent monomeric contributions to the channel complex. Materials & Methods: The present study used the lipid bilayers system to study electrophysiological properties of PC2 and TRPC1 channels, and the atomic force microscopy (AFM) to study the structure of PC2 and TRPC1 homo- and hetero-multimeric complexes. Results: A kinetic analysis of spontaneous channel currents of PC2 showed that four intrinsic, non-stochastic sub-conductance states, which followed a staircase behavior, were both pH-, and voltage-dependent. To confirm the oligomeric contributions to PC2 channel function, heteromeric PC2/TRPC1 channel complexes were also functionally assessed by single channel current analysis. Low pH inhibited the PC2 currents in PC2 homomeric complexes, but failed to affect PC2 currents in PC2/TRPC1 heteromeric complexes. Amiloride, in contrast, abolished PC2 currents in both the homomeric PC2 complexes and the heteromeric PC2/TRPC1 complexes, thus PC2/TRPC1 complexes have distinct functional properties from the homomeric complexes. The topological features of the homomeric PC2-, TRPC1-, and heteromeric PC2/TRPC1 channel complexes, assessed by atomic force microscopy (AFM), were consistent with structural tetramers. TRPC1 homomeric channels had different average diameter and protruding height as compared to the PC2 homomers. The contribution of individual monomers to the PC2/TRPC1 hetero-complexes was easily distinguishable. Conclusion: The data support tetrameric models of both the PC2 and TRPC1 channels, where the overall conductance of a particular channel will depend on the contribution of the various functional monomers in the complex. Author Contact: Peng Zhang; Nephrology; 617-724-9215; pzhang3@partners.org

36

Poster #33

Abstract Title: Doxorubicin Loaded Polymeric Nanoparticulate Delivery System to Overcome Drug Resistance in Osteosarcoma

Michiro Susa1,2, Keinosuke Ryu1,2, Cao Yang1,2, Xianzhe Liu1,2, Francis J. Hornicek1,2, Henry Mankin2, Arun K. Iyer3, Mansoor M. Amiji3, and Zhenfeng Duan1,2

1 Department of Orthopaedic Surgery, Massachusetts General Hospital, Boston, MA 02114

2 Sarcoma Biology Laboratory, Center for Sarcoma and Connective Tissue Oncology, Massachusetts General Hospital, Boston, MA 02114

3Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University Boston, Massachusetts 02115 Purpose: Drug resistance is a primary hindrance for the efficiency of chemotherapy against osteosarcoma. Although chemotherapy has improved the prognosis of osteosarcoma patients dramatically after introduction of neo-adjuvant therapy in the early 1980’s, the outcome has since reached plateau to approximately 70% for 5 year survival. The remaining 30% of the patients eventually develop resistance to multiple types of chemotherapy. In order to overcome both the dose-limiting side effects of conventional chemotherapeutic agents and the therapeutic failure incurred from multidrug resistant (MDR) tumor cells, we explored the possibility of loading doxorubicin onto biocompatible, lipid-modified dextran-based polymeric nanoparticles and evaluated the efficacy. Materials & Methods: Doxorubicin was loaded onto a lipid-modified dextran based polymeric nano-system. The effect of various concentrations of doxorubicin alone or nanoparticle loaded doxorubicin on KHOS, KHOSR2, U-2OS, and U-2OSR2 cells and drug sensitivities was analyzed. Effects on drug retention, immunofluoresence, Pgp expression, and induction of apoptosis were also analyzed. Results: Dextran nanoparticles loaded with doxorubicin had a curative effect on multidrug resistant osteosarcoma cell lines by increasing the amount of drug accumulation in the nucleus via Pgp independent pathway. Nanoparticle loaded with doxorubicin also showed increased apoptosis compared to free doxorubicin. Conclusion: Lipid-modified dextran nanoparticles loaded with doxorubicin showed pronounced anti-proliferative effects against osteosarcoma cell lines. These findings may lead to new treatment options for MDR osteosarcoma Author Contact: Michiro Susa, Sarcoma Biology Laboratory, Center for Sarcoma and Connective Tissue Oncology, 617-724-2065, msusa@partners.org

37

Poster #34 Effective Use of Nanocell Drug Delivery System for Photodynamic Therapy Combination Treatment of c-MET Positive Pancreatic Cancer in Preclinical Mouse Model Lei Zak Zheng, Prakash Rai, Conor Evans, Bryan Spring and Tayyaba Hasan Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, Boston Purpose: Photodynamic therapy (PDT) is an effective treatment for several cancers clinically. In this preclinical study, we developed an innovative drug delivery vehicle, referred to as “nanocell”, and we intend to test if PDT combination treatment efficacy on an orthotopic model of c-MET positive pancreatic cancer can be significantly improved through nanocell co-delivery of PDT photosensitizer BPD and a c-MET tyrosine kinase inhibitor PHA-665752.

Fig 1a Fig 1b Materials & Methods: Firstly, we designed and synthesized 2 types of nanocells (NC1 and NC2) containing the photosensitizer BPD and a c-MET inhibitor PHA-665752. NC1 is composed of a PLGA-polymer nanoparticle core containing PHA, and an outside lipid bilayer envelope containing BPD (Fig 1a). NC2 is composed of a PLGA-polymer nanoparticle core containing BPD, with PHA in the outside lipid envelope (Fig 1b). Both NC1 and NC2 were tested on c-MET positive pancreatic cell line AsPC-1 for PDT/c-MET combo treatment effect in vitro. NC1, which showed a better in vitro effect, was further tested in vivo on AsPC-1 orthotopic model. Results: Firstly, we found that nanocells can co-deliver both BPD and PHA-665752 into cells (Fig 2a). In a cellular drug uptake study, we found that nanocells deliver more BPD into cells than standard clinical BPD delivery formulation does. The nanocells deliver PHA-665752 into the cells and are able to effectively inhibit c-MET phosphorylation. Secondly, we compared the in vitro PDT/c-MET combo treatment efficacy of nanocell delivery with conventional drug delivery and we found that nanocells offer synergistic PDT/c-MET combo treatment effect in killing cancer cells. Notably, when equal amounts of BPD and PHA-665752 are delivered onto cells together but not co-encapsulated, little treatment benefit was observed when compared with BPD alone, therefore suggesting the synergy of PDT/c-MET combo treatment comes from the unique drug delivery method. We also found out that NC1 is slightly better than NC2 in its treatment efficacy because NC1 delivers more BPD into the cells than NC2. Thirdly, we tested the NC1 PDT/c-MET combo treatment in a c-MET positive orthotopic pancreatic cancer model in vivo. Our results indicate that NC1 offers the most effective treatment efficacy in reducing local tumor burden, when compared with BPD alone and BPD + nanoparticle PHA-665752. Besides the superior treatment efficacy, NC1 also significantly reduced systemic toxicity of PHA-665752, and allowed more PHA-665752 to be administered. Conclusion: We have successfully developed the nanocell drug delivery system that co-delivers photosensitizer BPD and c-MET inhibitor PHA-665752 for the PDT/c-MET combination treatment of c-MET positive pancreatic cancer. We found that nanocell offered a significant improvement of PDT treatment efficacy compared to conventional PDT/c-MET combo treatment both in vitro and in vivo. Author Contact: Lei Zak Zheng, PhD, Wellman Center for Photomedicine, Thier 2-224, 40 Blossom St, Boston, Tel: 617-724-3734, E-mail: LZZHENG@partners.org

38

Poster #35 Multifactorial Analysis of Image-Guided Lymph Node Biopsy in Patients with Primary Prostate Cancer Tina Islam1,2, Veena R. Iyer1,2, Peter F. Hahn2, Debra A. Gervais2, Pari V. Pandharipande2, Mukesh G. Harisinghani1,2

1 Center for Molecular Imaging Research, Massachusetts General Hospital 2 Department of Radiology, Massachusetts General Hospital Purpose: To evaluate complication rate, diagnostic yield, sensitivity (S), specificity (SP), negative predictive value (NPV), positive predictive value (PPV) of image-guided lymph node biopsy (LNB) in patients (Pts) with prostate cancer (PCa) and relate LNB findings with serum tumor markers and histopathological findings. Materials & Methods: Reports of LNB in 103 Pts with PCa performed at our institution (2000-09) were reviewed. 98 Pts underwent CT-guided, 5 Pts US-guided LNB using standard techniques. Histological findings were categorized as benign, atypical, malignant for PCa, malignant for second Ca, non-diagnostic. Initial LNB were considered false negative (FN) if repeated LNB were positive, imaged nodal size increased, or decreased with therapy. They were deemed true negative (TN) if surgery was negative, imaged nodal size was stable ≥12 months, or regressed without therapy. Otherwise clinical follow-up ≥24 months was used. Inadequate follow-up included lost to follow-up or death without autopsy. Diagnostic yield was calculated by dividing the number of diagnostic LNB by number of performed LNB. Logistic regression analysis was used for relating PSA values (mean 15.7 ng/ml) and Gleason score (mean 7.4) to the histological result of LNB. Student’s t-test was used to compare mean PSA values for benign vs malignant LNB for the most common nodal stations. All analyses used Statistical Analysis Software. Results: Of 103 LNB 52 were malignant for PCa, 4 positive for a second Ca, 27 benign,13 non-diagnostic. For 7 no report was found. Diagnostic yield of LNB=86,46%. 52 LNB were considered true positive, 21 TN, 1 FN, 0 false positive, 5 lost to follow-up. S=0.98 (95% CI 0.899-0.9995), SP=1.0 (95% CI 0.838-1.0), NPV =0.95, PPV =1.0. A high PSA value prior to LNB and a high Gleason score related with a malignant LNB result (P=0.0114 and P=0.0262, coefficient logistic model 0.0431 and 0.6165). Consistently higher mean PSA values prior to LNB were found when a malignant node was biopsied along the iliac, obturator, paraaortic and inguinal drainage pathways. Complication rate was 0.97%. Conclusion: LNB is a safe and accurate procedure with good diagnostic yield. In PCa patients a high Gleason score and high PSA value are associated with nodal disease. Image-guided lymph node biopsy is useful in patients with poorly differentiated prostate cancer and high PSA value. Author Contact: Tina Islam, MD, PhD Center for Molecular Imaging Research Department of Radiology Massachusetts General Hospital Harvard Medical School 55 Fruit Street Boston, MA 02114, USA Islam.Tina@mgh.harvard.eduPhone: 617 724 4266 Fax: 617 726 4891

39

Poster #36

Epilepsy in vitro: Development of spontaneous, chronic epileptiform activity in organotypic

hippocampal slice cultures mirrors in vivo epileptogenesis

J. Dyhrfjeld-Johnsen1,2*, W. Swiercz1,2,Y. Berdichevsky1,3, H. Sabolek1,2, K. Staley1,2

1Harvard Medical School, Boston, MA. 2Dept. of Neurology, Massachusetts General Hospital, Boston, MA. 3Ctr. for Engineering in Medicine, Massachusetts General Hospital, Boston, MA

Purpose: The study of epileptogenesis and development of anti-epileptic drugs in vivo in animal models

is cost and labour intensive. While valuable insight has been gained from in vitro studies using acute

slices, these studies are hampered by additional tissue damage induced by the slicing procedure as well as

the limited ability of acute slices to sustain ictal (seizure-like) activity. We here describe the spontaneous

development of in vivo-like epileptiform activity in organotypic hippocampal slice cultures, which

becomes apparent when cultures are recorded in their growth medium. This activity furthermore responds

in a characteristic manner to treatment with standard anti-epilpetic drugs (AEDs). Materials & Methods: Organotypic hippocampal slice cultures were prepared from P8 Sprague-Dawley

rat pups as described by Gähwiler et al. (1981). Glial growth was inhibited by a 24 hour exposure to anti-

mitotic agents following 48 hours in culture. 350 µm slices were cultured in 50% Eagles basal media,

25% Hanks balanced salt solution and 25% horse serum in roller tubes. Field-potential recordings (1

hour) were performed after 7-30 days in vitro (DIV) under perfusion with O2/CO2-bubbled growth-

medium using a medium-filled glass-pipette placed in CA3 under IR-DIC visualization. Recorded activity

was analyzed using custom written software for event-detection.

Results: After 7 DIV, only multi-unit activity was recorded in slice cultures. However, after 14-17 DIV,

prominent interictal-like spike discharges were recorded in the majority of slice cultures while few

displayed ictal-like discharges. After 21-30 DIV, most slices displayed prolonged ictal-like discharges.

The incidence of ictal-like epileptiform activity as a function of slice culture age could be fitted to a

sigmoidal function with ictal-like activity occurring in 50% of cultures after 21 DIV. This developmental

profile mirrors recent data from experiments using chronic video-EEG monitoring in rats following kainic

acid induced status epilepticus (Williams et al., 2009). Washing in the standard AED Phenytoin (100 μM)

in the growth-medium converted ongoing ictal-like activity in slice cultures to inter-ictal spike discharges

as seen clinically.

Conclusion: Organotypic hippocampal slice cultures provide an excellent model system for the study of underlying mechanisms of epileptogenesis on the single cell and local network level. With an ictogenesis profile corresponding to in vivo recordings and physiological responses to standard AEDs, slice cultures offer a cost and labor effective model system also suited for optical imaging and pharmacological testing. Author Contact: Jonas Dyhrfjeld-Johnsen, Neurology, 714-374-5609, jdyhrfjeld-johnsen@partners.org

40

Poster #37

Optical Theranostics for Drug Resistant Skin and Soft Tissue Infection

Ulysses W. Sallum1, Xiang Zheng1, Josiah Gruber2, Sarika Verma1, Conor L. Evans1, Humra Athar1, rian Pogue2, Tayyaba Hasan1 B

1-Wellman Center for Photomedicine, Massachusetts General Hospital, Department of Dermatology Harvard Medical School, Boston, MA, 02114 2- Thayer School of Engineering, Dartmouth College, Hanover, NH, 03755

Purpose: The initial diagnosis and management of skin and soft tissue infections (SSTIs) is very challenging. In light of the continuous emergence of bacteria with resistance to conventional antibiotics, finding effective therapies has become increasingly difficult. We are developing light activated technologies that target drug resistance mechanisms. This technology is called photodynamic therapy (PDT). PDT uses light and non-toxic dyes (photosensitizers, PSs) to convert light energy into chemical energy that destroys a target, which in the current study is a drug resistant bacterium. Some light energy is also released by PSs as fluorescence affording them potential as beacons that guide us to sites of infection. We have engineered a PS molecule that exploits a bacterial drug resistance enzyme, ß-lactamase, to enable its photodynamic activity and is therefore named ß-lactamase enzyme-activated photosensitizer (ß-LEAP). We have recently demonstrated the specific nature of ß-LEAP PDT. In this study, we compared the ability of ß-LEAP to measure antibiotic susceptibility to that of a conventional antibiotic susceptibility test and revealed that a similar read out is obtainable in 1/60th the time.

Materials & Methods: A Bacillus cereus penicillinase system was used for study. We used the competitive substrate inhibition of ß-LEAP fluorescent product formation as a means to detect the relative affinity of different ß-lactam antibiotics to a ß-lactamase in whole bacterial cell suspensions. The results were then compared to those of a minimum inhibitory concentration (MIC) assay.

Results: With the exception of clavulanic acid, all ß-lactams yielded Ki’s with narrow confidence intervals. ß-LEAP hydrolysis by both purified enzyme and bacterial cultures was inhibited to the greatest extent by the cephalosporins, which had significantly lower Ki values than the five penicillins. Penicillin G and carbenicillin were the next most effective inhibitors and yielded identical Ki values. Ampicillin and amoxicillin were the least effective inhibitors of ß-LEAP hydrolysis and did not result in the inhibition of growth, indicating a broader range of sensitivity for the ß-LEAP diagnostic assay of bacterial cultures.

Conclusion: ß-LEAP has potential as a theranostic for drug resistant SSTIs. In vitro, ß-LEAP can detect effects of antibiotics outside the scope of the MIC assay while recapitulating the relative levels of inhibition observed for different antibiotics. We are currently developing the in vivo application of this technology. We anticipate translation of this technology to enable the detection of drug resistant bacteria, depth of bacterial tissue invasion, and tissue damage and to also provide an effective alternative therapy.

Principle of Theranostic Translation: Therapeutic & Diagnostic Combined (Q=quencher)

SSTI

Author Contact: Ulysses W. Sallum, Ph.D., Wellman Center for Photomedicine, USALLUM@partners.org, 617-724-5394

41

Poster #38

B lymphocytes down-regulate anti-CD45RB-induced tolerance in islet transplant model

Kang Mi Lee, Gaoping Zhao, Christian Schuetz, James Kim, Moh-Moh Lian, James Markmann

and Shaoping Deng

Induction of tolerance will facilitate the application of islet transplantation in type 1 diabetic

patients. Therapy with monoclonal antibody targeting CD45RB reliably induces allogenic

tolerance. Herein, we have assessed that utility of anti-CD45RB therapy to induce tolerance to

islet allograft and the role of B lymphocytes in anti-CD45 induced tolerance in this model. Anti-

CD45RB therapy induces long-term survival of BALB/c islet grafts in only 4 out of 10

immunocompetent C57BL/6 mice, but same therapy induces long-term survival in 8 out of 9 B-

cell-deficient C57BL/6 mice. After the removal of allograft and retransplantation of islets, the

long-term survival mice accept donor-specific islets but reject third-strain islets. These data

show that donor-specific tolerance has been induced in both recipients, and B lymphocytes are

not required for tolerance induction, but instead may contribute to the resistance to tolerance

induction. We also showed that in a transfer model the splenocytes from the tolerant mice

bearing the established islet graft were effective in down-regulating islet allograft rejection in the

immunocompetent recipient mice without any treatment. In conclusion, in the absence of B

cells, anti-CD45RB therapy can more effectively prevent islet allograft rejection and promote

tolerance induction. Therefore, anti-CD45RB treatment together with B cell deletion may be

more promising therapy for type 1 diabetic patients following islet transplantation.

42

Poster #39 In vitro Ovarian Cancer Growth and Treatment Response Dynamics Visualised with Time-Lapse OCT Imaging Conor L. Evans1 , Imran Rizvi1,2 , Tayyaba Hasan1 , and Johannes F. de Boer3 Author Affiliation: 1 Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School, 40 Blossom St, Boston, MA 02114 2 Thayer School of Engineering, Dartmouth College, 8000 Cummings Hall, Hanover, NH 03755 3 Department of Physics and Astronomy, Vrije Universiteit, De Boelelaan 1081, T-0.67, 1081 HV Amsterdam, The Netherlands Author Contact:Conor L. Evans, PhD, Wellman Center for Photomedicine, 617-726-1089 A major challenge in ovarian cancer research is the difficulty in observing the growth, development, and treatment of microscale lesions in vivo. Ovarian cancer metastasizes rapidly through an exfoliation process, where cancer cells are shed from the primary tumor into the peritoneal cavity to form micrometastatic lesions. These microscopic metastases, which grow along peritoneal surfaces, are exceedingly difficult to detect clinically with existing technologies and are therefore often missed in the diagnosis and treatment of ovarian cancer. As a result, little is known about the early development of these tumor nodules, and even less is understood regarding their structural and molecular responses to therapeutic regimens. Three-dimensional in vitro models can address many of the limitations of traditional animal and cell culture models as they are capable of recapitulating many of the cell-cell interactions needed to model tumor growth and can be developed for high-throughput experiments. An ovarian cancer overlay culture based on NIH OVCAR-5 cells generates acini that are similar in size and composition to in vivo micrometastatic nodules, and grow along the matrix surface, providing a powerful model for visualizing ovarian cancer tumor growth and treatment response. As the ovarian acini mature, however, their diameters can range up to 200 µm, beyond the imaging depth of standard confocal microscopes. By three weeks of tumor growth, acini can grow as large as 1 mm in diameter with complex internal structures. These large spheroids, which sit atop several hundred microns of gel matrix, become unfeasible to visualize even with advanced multiphoton microscopy systems. This lack of suitable imaging technology has made it difficult to visualize the important structural dynamics which occur in these complex tumor models. In this study, these limitations were overcome by employing ultrahigh-resolution optical coherence tomography (OCT) to visualize the growing acini. The optical analog of ultrasound, OCT is a highly sensitive, cross-sectional imaging technique capable of visualizing the microscopic details of biological tissues. With cellular resolution and penetration depths exceeding 1.5 mm, OCT is particularly apt for visualizing even the largest 3D nodules. Most importantly, OCT is a non-perturbative imaging technique. The standard methods for tumor spheroid imaging require fixation and staining, which are highly perturbative, destructive and can introduce artifacts. With OCT imaging, cells remain viable allowing for long-term, longitudinal three-dimensional visualization of acinar growth, development, and treatment. Ultrahigh resolution OCT was used to visualize the growing acini, observing a hollowing process occurring in the interior of the tumor nodules. In order to follow acinar growth longitudinally, we developed a time-lapse OCT system capable of automated volume acquisition for continuous multi-day imaging studies. Combined with data from multi-photon imaging of stained samples, time-lapse OCT elucidated the three-dimensional structural evolution of the tumor nodules. This time-lapse system was further applied to capture the dynamics of carboplatin treatment on the ovarian cancer models. The longitudinal structural movies show the breakdown of the microscopic ovarian tumor nodules in response to chemotherapy for the first time, revealing the survival of nodule cores in agreement with clinical findings of platinum drug therapeutics.

43

Poster #40 Abstract Title: F-box protein 10: a novel anti-apoptotic protein regulates TRAIL-induced apoptosis Authors: Rongbin Ge, Zongwei Wang, Aria F. Olumi. Affiliation: Urology department, Massachusetts General Hospital, Boston, MA Purpose: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces selective apoptotic death of human cancer cells while sparing normal human cells. Although TRAIL holds great promise as a potential chemotherapeutic agent, multiple tumor types are resistant to this agent. F-box protein 10 (FBXL10), the largest member of the human family of F-box proteins, is a substrate-recognition component of SCF ubiquitin-ligase complexes and transcriptional repressor of AP-1 family member proteins. However, the role of FBXL10 in apoptosis is poorly understood. Previously, we have demonstrated that the AP-1 protein, c-Fos, represses the anti-apoptotic molecule, c-FLIP(L), in order to prime cancer cells for TRAL-induced apoptosis. Here, we report a novel anti-apoptotic function of FBXL10 and show that downregulation of FBXL10 is essential to sensitize cancer cell to TRAIL-induced apoptosis through upregulation of c-Fos transcriptional activity in vitro and in vivo. Results: In TRAIL sensitive cancer cells, FBXL10 RNA and protein levels significantly decreased after treatment with TRAIL in a dose and time-dependent manner whereas c-Fos was upregulated in an inverse relationship. However, in TRAIL resistant cancer cells, FBXL10 and c-Fos levels were not affected. Expression level of FBXL11, a FBXL10 homologous protein, was not affected by TRAIL treatment. Silencing of FBXL10 by RNAi in TRAIL-resistant cancer cells induced upregulation of c-Fos and primed the cancer cells to undergo apoptosis. Moreover, co-transfection of FBXL10 and human c-Fos promoter repressed c-Fos promoter activity. However, FBXL10 did not repress c-Fos promoter activity when AP-1 binding sites on the c-Fos promoter were mutated. Furthermore, using the ChIP assay, we observed that FBXL10 protein directly binds the c-Fos promoter in the TRAIL sensitive cells, and after treatment with TRAIL the interaction between FBXL10 and c-Fos promoter is significantly reduced. Finally, in the orthotopic in vivo prostate cancer xenografts treated by a TRAIL receptor 2 agonist antibody, (Lexatumumab), significant downregulation of FBXL10 immunofluorescence was observed in the TRAIL sensitive xenografts, but not in the TRAIL resistant xenografts, further establishing the role of FBXL10 in TRAIL-induced apoptosis. Conclusion: We demonstrate that the FBXL10 protein has a novel anti-apoptotic function in regulating TRAIL induced apoptosis. c-Fos, which is necessary for priming prostate cancer cells to undergo apoptosis, is repressed by FBXL10 in TRAIL resistant prostate cancer cells. Differentiating molecular mechanisms between TRAIL sensitive and TRAIL resistant cancer cells will improve the efficacy of apoptotic therapies for prostate cancer patients. Author Contact: Rongbin Ge, Urology, 617-643-1956, rge1@partners.org

44

Poster #42 Abstract Title Two-thirds Reduction in CT Perfusion Relative Cerebral Blood Flow (using Delay Corrected Post rocessing Software) Optimally Correlates with Admission Diffusion Weighting Imaging Assessment of Core Infarction Lesion Volume in Acute Stroke Patients Shahmir Kamalian, Seyedmehdi Payabvash, Shervin Kamalian, Adnan Akbar, Karen L. Furie, Michael H. Lev Massachusetts General Hospital, Harvard Medical School, Boston Purpose: Infarct core lesion size at admission is one of the most important determinants of clinical stroke outcome. Our purpose was to determine the optimal CT perfusion (CTP) parameter and threshold to define the infarct core, using concurrent diffusion weighted imaging (DWI) as a reference standard. Materials & Methods: We evaluated 48 consecutive patients who had biphasic CTP acquired over 66s within 9 hrs of stroke onset, who underwent DWI with 3 hrs of CTP. CTP maps were created with both standard and delay-corrected software. Coregistered DWI lesions and a symmetric "mirror" ROI were segmented and transposed onto CTP maps. Relative values were calculated by dividing pixel values mean mirror ROI value for rCBV, rCBF and rMTT, respectiviely. Pixel level ROC curve analysis was performed for absolute and relative perfusion values. Optimal thresholds, sensitivities, and specificities for each parameter were calculated. The resulting lesion volumes using the optimal absolute and relative thresholds were additionally compared by linear regression. Results: More than 2.5 million pixels were analyzed. The areas under the ROC curves (AUC) for absolute and relative CBF using both non-delay and delay corrected software (range 0.85 - 0.88) were significantly higher (p<0.5) than those for CBV and MTT. Using delay versus non-delay corrected software, the optimal thresholds for absolute CBF were 5.2 and 4.8 ml/100gm/min, respectively, and for rCBF were 68% and 72% reduction, respectively. For absolute CBV these were 1.2 and 0.9 ml/100gm, and for rCBV these were 27% and 39% reduction. Of the optimal CBF measures, relative CBF, calculated using the delay corrected software, resulted in the highest correlation coefficient and closest slope to 1 (r:0.82, slope:0.75) when linear regression was performed between CTP and DWI admission lesion volumes. Absolute CBV had the lowest correlation coefficient for both types of the software. Conclusion: Relative CT-CBF, acquired with a 66 second biphasic acquisition protocol, and calculated using delay corrected post-processing software at a threshold of 68% reduction, provided optimal correlation with admission DWI lesion volumes in acute stroke patients. This may be important in the validation and standardization of acute stroke CTP. Author Contact: Shahmir Kamalian, Radiology/Neuroradiology, 617-643-3525, skamalian@partners.org

45

Poster #43

Abstract Title: Cyst-like tubers are associated with TSC2 and epilepsy in tuberous sclerosis complex Catherine J. Chu-Shore, MD1, Philippe Major, MD1, Maria Montenegro, MD1, Elizabeth Thiele, MD, PhD1 1Department of Neurology, Massachusetts General Hospital

Purpose: Tuberous sclerosis complex (TSC) is a genetic condition characterized by the presence of hamartomatous lesions in multiple organs, including tubers in the brain. Epilepsy affects approximately 85% of patients with TSC (Epilepsia 1993; 34: 651-657), although the mechanisms underlying epileptogenesis in this disease remains unknown. Cortical tubers are areas of disrupted cortical architecture thought to be due to abnormal cellular migration, proliferation, or differentiation during fetal development. Some cortical tubers are epileptic foci, while others appear to be physiologically quiescent. It is unknown whether or not variations in tuber morphology may account for this difference. Some tubers can develop cyst-like characteristics over time, evident on neuroimaging (Pediatr Radiol 2006; 36:498- 501). The objectives of this study were to (1) determine the frequency of cyst-like tubers in patients with TSC (2) determine whether cyst-like tubers correlate with TSC genotype and (3) determine whether cyst- like cortical tubers are associated with a history of infantile spasms, epilepsy, or refractory epilepsy. Materials & Methods: A retrospective chart review was performed of 173 patients with TSC. MRI images were evaluated for the presence of at least one cyst-like cortical tuber. Patient charts were then reviewed for genetic mutation, a history of infantile spasms, epilepsy, and epilepsy refractory to more than three medications. Results: 46% of patients had at least one cyst-like cortical tuber present on neuroimaging. Patients with a TSC2 mutation were more likely to have a cyst-like tuber than patients with TSC1 mutation (p=0.002) or patients with no mutation identified (NMI, p=0.039). Patients with at least one cyst-like cortical tuber were more likely to have a history of infantile spasms (p=0.00005), epilepsy (p=0.0038) and refractory epilepsy (p=0.0007) than patients without a cyst-like cortical tuber. Conclusion: Cyst-like cortical tubers are strongly associated with the TSC2 gene mutation and a more aggressive seizure phenotype in patients with TSC. Author Contact: Catherine Chu-Shore, MD, Dept of Neurology, 617 726-6540, cchushore@partners.org

46

Poster #44

Title: Adaptive Statistical Iterative Reconstruction (ASIR) Markedly Improves Signal- and Contrast-to-Noise Ratios in Low Dose Head CT Scanning Affiliation Shervin Kamalian, Otto Rapalino, Shahmir Kamalian, Seyedmehdi Payabvash, Michael H. Lev, Stuart R. Pomerantz Massachusetts General Hospital, Harvard Medical School, Boston Purpose: Radiation dose reduction in CT is limited by scan degradation from increased image noise. Adaptive Statistical Iterative Reconstruction (ASIR) is a time-efficient clinically-available CT reconstruction technique for reducing image noise in the creation of source CT images at the scanner. We evaluated its effect on quantitative measures of brain image quality at multiple ASIR levels. Materials & Methods: 40 consecutive noncontrast head CTs acquired on GEHC Discovery CT750HD, using our clinically-routine reduced-dose protocol (120 kV, 200 mAs) were included in this study. For each scan, images without ASIR reconstruction were analyzed and compared to images reconstructed at 5 different levels of ASIR (20%, 40%, 60%, 80% and 100%). Region-of-interest (ROI) measurements of CT attenuation (HU) were acquired over normal gray matter (GM) and white matter (WM). Ten scans had a hypodense subacute infarction over which additional ROIs were obtained (INF) and compared to contralateral normal tissue (NL). Signal-to-noise ratio (SNR), defined as mean attenuation over standard deviation, was calculated for each tissue region at each different ASIR level. Contrast-to-noise ratios (CNR) for GM-WM and INF-NL were calculated as the difference in mean attenuation for the two regions divided by the square root of the sum of their squared standard deviations. One-way variance analysis (Tukey's post hoc test) was used to determine statistical significance (p<0.05). Results: (See figure) For GM, WM, INF and NL regions, SNR increased from 8.64, 6.81, 4.41, and 7.56, respectively, without ASIR, to 14.98, 11.58, 7.26, and 10.94 at 100% ASIR (p <.05). GM-WM and INF-NL CNRs increased from 1.34 and 2.49 without ASIR, to 2.93 and 4.54 at 100% ASIR (p<.05). SNR and CNR improvements from ASIR can be attributed primarily to reduction in image noise, as progressive ASIR levels were not observed to raise HU attenuation. Conclusion: Adaptive Statistical Iterative Reconstruction is a tool that may enable significant radiation dose reduction of head CTs with preserved image quality by substantially reducing image noise. Author Contact: Shervin Kamalian, Radiology/Neuroradiology, 617-6433525, mkamalian@partners.org

47

Poster #45 Abstract Title: An Empirical Approach to Diagnostic Classification of Eating Disorders in Children and Adolescents Kamryn T. Eddy, Ph.D.,1 Ross D. Crosby, Ph.D.,2 Daniel le Grange, Ph.D.,3 & David B. Herzog, M.D.1

1 Massachusetts General Hospital and Harvard Medical School, Boston, MA; 2 Neuropsychiatric Research Institute, Fargo, ND; 3 University of Chicago Medical Center, Chicago, IL Purpose: The Diagnostic and Statistical Manual of Mental Disorders (DSM) is designed to guide clinical practice and research. Yet if the diagnostic system does not accurately categorize or even include the full range of psychopathology then its utility is limited. This appears to be particularly true for children and adolescents with eating disorders. For youth with eating disorders, it is not clear whether the current DSM-IV classification system accurately captures observed symptom presentations. Indeed, the majority of children and adolescents who seek treatment for eating disorders cannot be diagnosed with either of the major eating disorders: anorexia nervosa or bulimia nervosa. In preparation for the fifth edition of the DSM, empirical studies are needed to determine whether alternative classification schemes may be more valid. The purpose of the current study was to empirically derive eating disorder phenotypes in a clinical sample of children and adolescents using the statistical technique of latent profile analysis (LPA), and to directly compare these identified latent profile (LP) groups to the DSM-IV eating disorder categories. Materials & Methods: Eating disorder symptom data were collected via clinical interview from 387 youth (ages 7-19; mean 15.2 ± 2.3y) who were seeking treatment through a specialty Eating Disorders Program. These data were included in LPA, run using Latent Gold Version 4.5; general linear models, run using SPSS Version 17, were used to compare latent profile groups to DSM-IV-TR ED categories on pre- and post-treatment indices. Results: Three LPs were identified: LP1 (n=142) was characterized by normal weight, binge eating and purging; LP2 (n=131) was characterized by low weight, minimal eating disorder behaviors and cognitions; and LP3 (n=114) was characterized by low weight, excessive exercise, and extreme eating disorder cognitions. Across pre-treatment indices of eating disorder psychopathology, general psychopathology, and self-esteem, and post-treatment weight change, the LP groups were more differentiated than the DSM-IV categories (p<.001), suggesting that they were more clinically valid. Neither LP nor DSM-IV-TR categories predicted change in binge/purge behaviors. Conclusion: Identified LPs imperfectly resembled DSM-IV eating disorders. LP1 resembled DSM-IV bulimia nervosa; LP2 and LP3 each resembled anorexia nervosa, but were differentiated by severity of eating disorder cognitions and the presence of excessive exercise only in LP3. Validation analyses suggest these empirically-derived groups improve upon the current DSM-IV-TR categories. In children and adolescents, revisions to DSM-IV eating disorder categories may include the addition of a group of low weight individuals with minimal cognitive eating disorder symptoms. Future research should continue to examine the incremental validity and clinical utility of alternative classification schemes of eating disorders, particularly in youth, to inform DSM-V. Author Contact: Kamryn T. Eddy, Ph.D., Department of Psychiatry, 617-724-1744, keddy@partners.org

48

Poster #47 Abstract Title Sublethal temperatures increase DNA damage and cell death in distant non-heated cells Authors Martin Purschke, Rox Anderson and Dieter Manstein Affiliation Wellman Center for Photomedicine, Massachusetts General Hospital, Harvard Medical School. Boston, Massachusetts, USA Purpose: Direct heat exposure to cells causes protein degradation and DNA damage, which can lead to genetic alteration and cell death. But little is known about heat-induced effects on the surrounding tissue. After burns or laser surgery, loss of viability in the surrounding tissue has been explained by a temperature gradient due to heat diffusion. Therefore we tested the effects of heat exposure on surrounding unheated cells. Materials & Methods: Human fibroblasts cells (HFF1) and human skin samples were grown/cultured in a special transwell insert system. After defined heat-exposure within the temperature range of 37-65 ˚C for 10 min, heat and non-heated cells were combined and shared the medium for up to 72 h. Any cell-to-cell contact and heat diffusion was excluded by the experimental set up. The viability was assessed by using the MTT assay, DNA damage and apoptosis were analyzed morphologically after fluorescence staining (DAPI) of the DNA. Results: This study demonstrates that, in the absence of any direct heating, heat diffusion, or cell-to-cell contact, surrounding “bystander” cells, which share the medium with heat-exposed cells exhibit DNA damage (MN), apoptosis and loss of viability. We coin this phenomenon “active thermal bystander effect” (ATBE). The ATBE was induced by fibroblasts exposed for 10 minutes to a temperature range of 44 to 54 ºC (p < 0.025) with a maximum at around 48 ºC. The ABTE was not induced by cells heated to lethality above 55 ºC. Conclusion: Our data showed that heat exposed cells in the absence of any heat diffusion or cell to cell contact can generate in surrounding bystander cells loss of viability, apoptosis and DNA damage. The mediator for such process in not yet defined. The ATBE and its clinical implications for acute burn trauma, hyperthermic treatments and for laser or electrosurgical procedures deserves further investigation. Author Contact: Martin Purschke, Ph.D. Research Fellow Wellman Center for Photomedicine Massachusetts General Hospital 40 Blossom Street (BHX631) Boston, MA 02114 Tel.: 617-726-1591 Fax: 617-724-2075 Mobil: 617-816-1883 mpurschke@partners.org

49

Poster #48 Toll-like receptor 2 plays a critical role in cardiac dysfunction and high mortality during experimental sepsis

Lin Zou1, Yan Feng1, Yu-Jung Chen1, Rui Si1, Marielle Scherrer-Crosbie2, Shiqian Shen2, Fumito Ichinose1, Wei Chao1

1Anesthesia Center for Critical Care Research, Department of Anesthesia & Critical Care and 2Department of Medicine, Massachusetts General Hospital, Harvard Medical School Purpose: Sepsis has a prevalence of 750,000 cases each year in the United States. In severe cases such as septic shock, the mortality reaches more than 40%. It is well known that cardiac dysfunction represents a main feature of severe sepsis and contributes to its high mortality. Yet, our understanding of the molecular mechanisms controlling these critical events remains incomplete. Innate immune system such as Toll-like receptor 2 (TLR2) represents the first line of defense against microbial infection, but its role in cardiac dysfunction during sepsis is unknown.

Materials & Methods: Polymicrobial peritonitis, a clinically relevant model of sepsis, was generated by cecum ligation and puncture (CLP). Wild-type (WT) and TLR2-/- mice were divided into the sham and CLP groups. The sham animals underwent laparotomy but without CLP. One ml of normal saline was administered subcutaneously after surgical procedures. The cardiac function was assessed by 1) a pressure transducer catheter inserted into the left ventricles (LV) of isolated hearts (Langendorff model), 2) serial echocardiography in vivo, and 3) in vitro measurement of Ca2+ transients and sarcomere shortening in adult cardiomyocytes isolated from the normal and septic animals. TLR2 chimeric mice were generated by lethal irradiation and bone marrow reconstruction.

Results: Twenty-four hours after CLP, left ventricular (LV) contractile function of the isolated hearts was significantly depressed compared to the hearts isolated from the sham-operated mice as demonstrated by 35% reduction in LV developed pressure (LVDP), 30% in dP/dtmax, and 40% in dP/dtmin (P < 0.01). In comparison, mice deficient for TLR2 and subjected to the same experimental sepsis had much better preserved LV systolic and diastolic function with significantly improved LVDP and dP/dt. Mouse echocardiography revealed a time-dependent progressive reduction in LV contractile function in CLP mice as demonstrated by a significant decrease in the strain rate of the LV anterior wall, a relatively load-independent measurement of LV function. Strain rate was partially preserved in the TLR2-/- mice after CLP compared to WT mice. Adult cardiomyocytes isolated from CLP mice had depressed sarcomere shortening as well as Ca2+ transients compared to the cells form the sham mice. In contrast, TLR2 deficient cardiomyocytes had much better preserved sarcomere shortening following CLP. Importantly, the CLP model of sepsis led to a high mortality rate (64% at 48 hours after CLP, 91% at 96 hours and 2 weeks, n=11) in WT mice. In contrast, mice deficient for TLR2 showed dramatic reduction in the mortality rate following CLP (22% at 48 hours, 33% at 96 hours, and 56% at 2 weeks, n=9, P < 0.05, log-rank test). To delineate the specific contribution of TLR2 signaling from parenchymal tissues vs. bone marrow (BM)-derived circulating cells, we constructed chimeric TLR2 mice. Compared to the WT→WT (Donor→Recipient) chimeric control mice, the KO→WT mice (WT mice lacking TLR2 in their circulating blood cells) exhibited similar cardiac depression between 12 hrs and 48 hrs as measured by serial echocardiography (n = 9-12) and similar mortality rate within 14 days following CLP (n=9-11).

Conclusion: These data suggest that TLR2 signaling, particular that of parenchymal tissue, mediates cardiac dysfunction and contributes to the high mortality induced by polymicrobial intra-abdominal sepsis. Our study indicates that specific targeting of TLR2 signaling pathway may confer a novel therapeutic benefit in the clinical management of severe sepsis. Author Contact: Lin Zou, MD, Department of Anesthesia & Critical Care, Tel: 617-724-0157, Email: lzou3@partners.org

50

Poster #49

Cancellation of EEG and MEG signals generated by extended sources Jooman Han, Seppo Ahlfors Department of Radiology, Athinoula A. Martinos Center for Biomedical Imaging, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA Purpose: Magnetoencephalography (MEG) and electroencephalography (EEG) are non-invasive extra-cranial measurements of magnetic fields and electric potentials reflecting neural electrical activity in the human brain. In the case of widespread coherent activity in a cerebral cortex, significant amount of the signal cancellation is expected because of the folding of the cortical surface. In this study, we quantified the cancellation effect according to the size of spatial extent of locally coherent neural sources. Materials & Methods: We defined and calculated a measure of cancellation for each EEG and MEG signals generated by a locally extended current source patch. In this simulation, we employed a 306-channel MEG system (VectorView, Elekta Neuromag) and an EEG cap with 60 electrodes covering a whole scalp as a sensor configuration and a boundary element model with three compartments (brain, skull and scalp) as a head model. The sources were assumed to be circular patches of uniform activity on the cortical surface. The radii of the patches were varied from 1 mm to 50 mm and the locations were randomly selected on the cortical surface. Results: Figure 1 shows measures of cancellation effects. For both EEG and MEG, the cancellation index increased as the size of a patch or the orientation non-uniformity (average orientation of cortical surface on which a source patch was located) increased. It was noticeable that upper diagonal points found for MEG, but not for EEG, were related to the insensibility of MEG to radially oriented sources. Conclusion: Substantial cancellation in MEG and EEG signals was observed for spatially extended source activity. A systematic difference between MEG and EEG due to selective cancellation effects may help to better interpret simultaneously recorded MEG and EEG signals.

Figure 1 Cancellation index for EEG and MEG Author Contact: Jooman Han; Radiology, 617-694-6209; jooman@nmr.mgh.harvard.edu

51

Poster #50

A novel aortic perfusion cannula for reducing atheroemboli Anand Jagannath M.S., Jennifer K. White M.D., James Titus, Ryuichi Yoneyama M.D., Joren C. Madsen M.D., D.Phil, Arvind K. Agnihotri M.D. Department of Cardiac Surgery, Massachusetts General Hospital, Boston, MA Purpose: Atheroemboli formed during cardiopulmonary bypass (CPB) surgery can cause cerebrovascular events resulting in peri-operative stroke or post-operative cognitive defects. The high velocity jet from standard aortic perfusion cannulae can dislodge ascending aortic atheroma causing these emboli. This study describes the functional performance of a novel funnel-tip cannula designed to reduce atheroemboli by decreasing flow velocity and shear compared with standard aortic cannula and other velocity and shear-reducing designs. Materials & Methods: A funnel-tip cannula was constructed and compared to standard straight-tip cannulae and the Dispersion™ and Soft Flow™ cannulae. Pressure drop measurements were collected at 1-6 L/min flows. Velocity flow profiles were created using phase contrast magnetic resonance imaging. Absolute velocity was measured in a phantom aorta at 5 L/min flow. Each cannula was further studied in a synthetic model of an atherosclerotic aorta to determine the mass of dislodged particulate matter generated at 2, 3, and 5 L/min flows. Results: The funnel-tip cannula demonstrated significantly lower values (p < 0.05) in pressure drop (55 mm Hg), exit velocity (309 cm/s, 167 cm/s for center axis and wall respectively), and particulate dislodgement (0.15 ± 0.05 g) than other tested cannulae. The Soft Flow™ cannula generated the next lowest pressure drop but exhibited twice the exit velocity and particulate dislodgement of the funnel-tip cannula. The Dispersion™ cannula did not demonstrate a reduction in velocity or particulate dislodgement compared to the standard straight-tip cannulae. Conclusion: The results of this study suggest that a low-angled funnel-tip cannula has favorable flow characteristics warranting further investigation. Design development may reduce the risk of atheroemboli generation during CPB surgery. Author Contact: Anand Jagannath, Cardiac Surgery, (617) 726-7707, adjagannath@partners.org

52

Poster #51

Innate immune adaptor MyD88 contributes to ischemic myocardial injury and modulates neutrophil migration and chemokine receptor expression Yan Feng1, Lin Zou1, Rui Si1, Yasuko Nagasaka1, Shiqian Shen2, Wei Chao1 1Anesthesia Center for Critical Care Research, Department of Anesthesia & Critical Care and 2Department of Medicine, Massachusetts General Hospital, Harvard Medical School

Purpose: The innate immune response to ischemia-reperfusion (I/R) is, by far, the most common cause of myocardial inflammation and contributes to ischemic myocardial injury. Yet, the signaling mechanisms that control these critical events are incompletely understood. Our previous studies have demonstrated that MyD88, an adaptor molecule critical for innate immune signaling, may be essential for myocardial injury following ischemia as systemic deficiency of MyD88 leads to reduced myocardial infarction and improved cardiac function. The current study was aimed to delineate the specific contribution of cardiac and bone marrow-derived MyD88 in mediating ischemic cardiac injury and to explore the underlying mechanisms. Materials & Methods: Chimeric model: MyD88 chimeric mice were generated by lethal irradiation and bone marrow (BM) reconstruction. Two groups of chimeric mice were generated: KO→WT (Donor→Recipient) and WT→WT as the control for irradiation and BM reconstruction. Mouse I/R injury: After anesthesia and thoracotomy, the left coronary artery was ligated for 30 or 60 min and followed by 24 hours of reperfusion. At the end of reperfusion, the hearts were harvested and examined for ischemic area (air-at-risk, AAR) and infarct size by TTC staining. Multiplex cytokine immunoassay: Myocardial IL-1β, TNFα, IL-6, KC, MCP-1, MIP-1α, MIP-1β, MIP-2, and LIX were quantified using a fluorescent bead-based multiplex immunoassay. Neutrophil in vivo mobility assay: One ml of 4% thioglycollate was injected intraperitoneally (i.p.). Six hours later, peritoneal lavage was collected and leukocytes were counted. Neutrophils were labeled with anti-mouse Ly-6C Ab and counted by flow cytometry. Chemokine receptor expression: CXCR2 expression, critical for neutrophil mobility function, was measured by flow cytometry.

Results: There was a significant decrease in infarct sizes in KO compared to WT mice (22 ± 1% AAR vs. 37 ± 2%, p < 0.001, n = 6-9). Compared to the WT→WT control, KO→WT chimeric mice, which lacked MyD88 in their circulating blood cells but maintained intact MyD88 signaling in the heart, had significantly smaller MI sizes (20 ± 2%, p < 0.05, n = 8). Surprisingly, infarct sizes of WT→WT mice were also smaller compared to WT (28 ± 1%, p < 0.05, n = 8). I/R led to a dramatic inflammatory response in the heart. All of the 9 cytokines tested were increased following I/R compared to the sham controls. However, there was no significant difference between the 2 chimeric (WT→WT and KO→WT) and the WT animals suggesting neither irradiation nor lacking of MyD88 in circulating blood cells altered cytokine levels in the heart. To determine if alteration of neutrophil function might contribute to the reduced myocardial infarction in the MyD88 chimeric mice, we examined the neutorphil mobility and chemokine receptor expression. Neutrophil migration was dramatically decreased in KO mice compared to WT mice (3.5 x 106/cavity vs. 10.9 x 106/cavity, p < 0.01, n=3). Neutrophil mobilization also decreased in WT→WT and KO→WT chimeric mice (5.2 x 106, p < 0.05; 3.8 x 106, p < 0.01, respectively, n=6) suggesting that irradiation/BM reconstruction and/or MyD88 deficiency inhibited neutrophil function. Moreover, there was a significant decrease (> 50%) in CXCR2 expression on the peritoneal neutrophils of MyD88 KO mice (p < 0.001) but not in WT→WT mice. Importantly, compared to WT→WT control, CXCR2 expression on the neutrophils of KO→WT mice was also significantly decreased (p < 0.01, n=6).

Conclusion: Taken together, these findings support the hypothesis that the MyD88 signaling, particularly that in bone marrow-derived circulating inflammatory cell, plays a critical role in mediating myocardial I/R injury. MyD88 may mediate I/R injury partly by modulating the chemokine receptor CXCR2 expression and thus the migration of neutrophils.

Author Contact: Yan Feng, MD, Dept. of Anesthesia, Tel: 617-724-0157, Email: yfeng@partners.org

53

Poster #52 Retrospective Study of Depression in Amyotrophic Lateral Sclerosis (ALS) Nazem Atassi, MD., Kerrie Nelson, PhD., Yuvika Paliwal, MS., Merit Cudkowicz, MD. MSc. Neurology Clinical Trials Unit (NCTU), Massachusetts General Hospital, Harvard Medical School. Purpose: To determine the prevalence of depression and its effects on survival and disease progression in Amyotrophic Lateral Sclerosis (ALS) Materials & Methods: This is a retrospective study of 397 people with ALS who participated in one of two clinical trials. We included all 300 participants from the celebrex trial and 97 participants from the topiramate trial who were assigned to placebo. The presence and severity of depression was determined by patient’s report. Disease severity and progression measured by ALSFRS-R, the use of gastric feeding tube (G-tube) and survival were compared between the depressed (D) and non-depressed (ND) groups. Results: The prevalence of depression was 19% in the celebrex cohort and 22% in the topiramate placebo arm. There were no differences in average ALSFRS-R at baseline (celebrex p=0.5 and topiramate p=0.2) or in ALSFRS-R decline rate (celebrex p=0.42 and topiramate p=0.23) between D and ND groups. Average G-tube use was 20% in both groups in the celebrex study and was 14% (D) and 8% (ND) (p=0.4) in the topiramate study. Mortality rates in the celebrex study were 20% (D) and 19% (ND) (p=0.7) and were 43% (D) and 38% (ND) (p=0.53) in the topiramate study. Kaplan-Meier curves revealed no significant difference in survival. Conclusion: The prevalence of depression in our cohort was 19-22% and the presence of depression did not have an impact on survival or disease progression. Accurate measurement of depression in an ongoing study may reveal significance. Author Contact: Nazem Atassi, Department of Neurology, (617) 643-6114, NATASSI@PARTNERS.ORG

54

Poster #53 A mechanism for androgen-independent prostate cancer through modulation of androgen receptor signaling by the p21-activated kinase 6 (PAK6) Min Zhang, Michael Siedow, Gregory Saia, Arnab Chakravarti Department of Radiation Oncology, Massachusetts General Hospital/Harvard Medical School Purpose: Prostate cancer (PCa) is one of the most diagnosed and mortal cancers in the United States. The major clinical problem is the development of androgen-independent prostate cancer (AIPC) during androgen deprivation therapy. The underlying molecular mechanisms are poorly understood and represent a challenge to develop new therapies. p21-activated kinase 6 (PAK6) is a 75-kDa protein that was first identified in a yeast two-hybrid screen for novel androgen receptor (AR) interactors, but its functions have not been established. In this study, we explored the role of PAK6 in mediation hormone resistance in prostate cancer cells. Materials & methods: PAK6 expression was detected in androgen deprived prostate cancer cell line LNCaP. Short hairpin RNA (shRNA) was used to attenuate PAK6 expression in LNCaP cells, and apoptosis and cell cycle distribution were assessed in these cells. Given that AR can be phosphorylated by either the AKT or the mitogen-activated protein kinase (MAPK) pathway and becomes a ligand-independent AR, we investigated the effect of PAK6 on the cross-talks between AR signaling and AKT/MAPK pathways. Results: We found that PAK6 expression in LNCaP PCa cells was markedly increased when the cells were cultured for 8 weeks in steroid hormone depleted medium. Antagonism of PAK6 using short hairpin RNA (shRNA) significantly increased antiangogen bicalutamide-induced apoptosis and G1 arrest. These finds implied that PAK6 may play a role in the development of androgen independent prostate cancer. Knockdown of PAK6 in LNCaP cells diminished AKT phosporylation at Ser473 and Thr308 as well as p44/42 MAPK phophorylation at Thr202 and Tyr204, indicating that overexpression of PAK6 during androgen deprivation may activate AR signaling by phosporylation of both AKT and MAPK. Conclusion: Modulation of androgen receptor signaling by PAK6 is a potential mechanism involved in androgen independent prostate cancer development and progression. Our study provides a rational basis for the design of combined therapies, including PAK6 inhibitor, to further enhance the responsiveness of prostate cancer cells to androgen ablation therapy.

55

Poster #54

Abstract Title: A Retrospective Study of Fasting Blood Glucose and Lipid Levels in Psychiatric Inpatients with Psychotic vs Non-psychotic Major Depressive Disorder.

Authors: Nelson Tauro M.D.(1,3), John D. Matthews, M.D.(1,3), Elizabeth A. Davis, M.D.(1,3), Caleb J. Siefert, Ph.D.(2), Sarah L. Stone, M.P.H., Adrienne O. van Nieuwenhuizen, B.A.(1), Lawrence T. Park, M.D.(1,3), Kaloyan Tanev, M.D.(1,3), David W. Abramson, M.D.(1,3), Maurizio Fava, M.D.(1,3) Affiliations: 1. Department of Psychiatry, Massachusetts General Hospital, Boston. MA. 2. Psychological Evaluation and Research Laboratory, Boston. MA. 3. Harvard Medical School. Boston MA. Purpose: Elevated fasting blood glucose and lipid levels are two important risk factors for the development of cardiovascular disease. About 20-30% of patients with major depressive disorder have psychotic symptoms. Studies have shown that Psy-MD is a more severe disorder and is associated with poor response to pharmacological treatment, higher rates of relapse, and greater impairment in functioning compared with Non-Psy MD. There are no reports comparing glucose and lipid regulation between Non-Psy MD and Psy MD. This is the first published report assessing fasting glucose and lipid blood levels between these two groups. Materials & Methods: A retrospective chart review was conducted on patients admitted to the inpatient psychiatric unit at Massachusetts General Hospital for a period of one year with diagnoses of Psy MD or Non-Psy MD. Fasting glucose and lipid blood levels measured at the time of admission were compared between the two groups. Regression analysis was conducted with age, gender, and use of anti-psychotic medications on admission entered on the first block and Major Depressive Disorder with Psychosis (i.e. yes/no) entered on the second. Results: Inclusion of Major Depressive Disorder with Psychosis status made a significant contribution to the prediction of blood glucose levels beyond that predicted by age, gender, and use of anti-psychotic medications alone. Conclusion: Patients with a diagnosis of Major Depressive Disorder with psychosis have a disturbance in glucose metabolism which is independent of the antipsychotic medications they are on. These observations may be a manifestation of high cortisol, which will be discussed. Author Contact: Nelson Tauro, Department of Psychiatry, 617-724-9141, ntauro@partners.org

56

Poster #55

Microarray Analysis of CD8 T-Cell Responses during Acute HCV Infection

Lizeng Qin1, Nick Haining2, Brian Nolan1, Hesham Nawar1, Lia Lewis-Ximenez4, Raymond T Chang1, Arthur Y Kim3 and Georg M. Lauer1

Gastrointestinal Unit1 and Department of Infectious Diseases3, Massachusetts General Hospital; Department of Medical Oncology, Dana-Faber Cancer Institute2; all Harvard Medical School; Massachusetts; Instituo Oswaldo Cruz/Fiocruz, Rio de Janeiro, Brazil4

Purpose: Over 180 millions of people are infected with HCV worldwide, with only a small minority being able to control infection. The correlates of protection, especially the determinants of an effective HCV-specific T-cell response, remain undefined.

Materials & Methods: We identified a cohort of subjects with early acute HCV infection and varying clinical outcomes and determined their HCV-specific CD8 T-cell response using ELISpot with overlapping peptide libraries. Having defined the individual T-cell response we employed class I multimers to sort live HCV-specific CD8+ T-cells. RNA was extracted from the cells and analyzed on Affymetrix microarrays.

Results: We were able to analyze HCV-specific responses from 27 subjects, 15 with a

chronic evolution of acute HCV infection and 12 with spontaneously resolving infection. Microarray analysis identified a substantial number of genes that were differentially expressed in the two patient populations. Currently we further the data in correlation with additional clinical markers. We also have started to look in more detail at selected genes with a distinct expression profile in order to identify mechanisms involved in the failure of T-cell mediated viral control

Conclusion: Our preliminary results indicate that T-cell responses in acute HCV infection are distinct between subjects with self-limited versus persistent infection. Identifying mechanisms involved in T-cell failure will be critical for the development of HCV vaccines and immunotherapies.

Author Contact: Lizeng Qin, MD, PhD Gastrointestinal Unit, Warren 10 Ph: 617-643-6143 Email: lqin1@partners.org

57

Poster #56 Abstract Title Tract Based DTI Analysis of White Matter Pathways In Pediatric Multiple Sclerosis Authors: Mellekate Vishwas1, Tanuja Chitnis M.D2, Rudolph Pienaar1, Brian Healy3 Ph.D., Ellen Grant M.D1. Affiliation: Athinoula A Martinos Center for biomedical imaging, Charlestown, MA 1, Partners Pediatric Multiple Sclerosis Center , Massachusetts General Hospital for Children 2, Biostatistics center, MGH Boston, MA 3. Purpose: Diffusion tensor imaging (DTI) and fiber tracking using DTI assess tract-based changes through measures of mean apparent diffusion coefficient (ADC) and fractional anisotropy (FA) along fiber pathways. Objective: To compare ROI (regions of interest) and tract-based measures of mean ADC and mean FA in 3 types of white matter pathways: Inter-hemispheric, projection and intra-hemispheric fibers, in children with MS compared to normative healthy controls. We hypothesize that tract based measures will be as sensitive as ROI analysis for detecting white matter involvement. Materials & Methods: DTI data were analyzed for 10 children with established RRMS and compared with age, sex and imaging technique-matched controls. Datasets were reconstructed using Diffusion toolkit and analyzed using TrackVis. Fiber tracts were constructed using previously published standard placement of ROIs on color FA maps cross-referenced to B=0 T2 weighted images. A Wilcoxon signed rank test was used to compare the patients and the matched controls Results: In children with MS, mean ADC was higher (P<0.006) and mean FA was lower (P<0.004) in the ROIs used to segment midline corpus callosum, long association fibers and posterior limb of internal capsule compared to normal healthy controls. In pediatric MS, mean ADC values along the entire tract were significantly higher (P<0.006) and mean FA was significantly lower (P<0.001) in all the white matter tracts studied; Callosal fibers, Cortico-spinal tracts and long association fibers. Conclusion: Tract based measures of mean ADC and FA is as effective as ROI based methods at detecting functional system involvement. Both methods detected widespread tissue damage in children with RRMS, as reflected by an increase in the mean ADC and a decrease in the mean FA values of the three major white matter bundles studied. Tract based measures will allow future correlation with functional scores sub served by each functional pathway. Author Contact: Mellekate Vishwas, MBBS, MD. Phone: 970-481-5873 Email: Vishwas@nmr.mgh.harvard.edu

58

Poster #57 Abstract Title: Identification of novel mTOR signaling components by a genome-wide RNAi screen in pancreatic cancer cells. Authors: Papageorgiou A, Avruch J. Affiliation: Diabetes Research Laboratories; Department of Molecular Biology, Massachusetts General Hospital; Department of Medicine, Harvard Medical School. Purpose: Identify novel molecular components of the (mTOR) signaling pathway by a genome-wide RNAi screen. Specifically, I am seeking for RNAis that either inhibit or activate baseline phosphorylation levels of the ribosomal S6 protein (S6-P)-the mTOR target- in serum/nutrient-replete cells using pancreatic cancer cells as a model system. Materials & Methods: To identify novel components of the mTOR signaling pathway, I am using the Dharmacon SMARTpool siRNA genome-wide library available for screening at ICCB at the Harvard Medical School. It targets the whole human genome (21,176 genes) and has been successfully used in other screens. The advantage of using Mia-Paca 2 cells as a model system is that the mTOR/ PS6K/ PS6 pathway is constitutive active in these cells. mTORC1 phosphorylates and directly activates phosphoS6K which in turn phosphorylates the ribosomal protein S6. Therefore, I am using phosphorylation of ribosomal S6 protein (Ser 235/236) as detected by immunofluorescence (IF). Specifically, gene-specific RNAi pools are pre-aliquoted into 384-well plates, to which I subsequently dispense cells. Mia-Paca 2 cells are left unperturbed in serum-containing medium with RNAis for 72hr. I use the ImageXpress Microscope, which enables high content screening (available at ICCB) and allows detection based on fluorescence intensity and the metaexpress software program used for quantitative imaging. To identify and select for positive hits, I use a threshold of three standard deviations from baseline; i.e., cells treated with full medium and transfected with a non-specific RNAi construct against a scrambled sequence. Results: The z score, which is used in optimization and qualitative evaluation of high-throughput screening assays was calculated to be 0.69. So far, I have validated 61 of the strongest positive hits out of a total 85 hits obtained from the Dharmacon Kinase library. 70% of the hits were confirmed in two or more of the individual RNAi pools. Conclusion: Based on the above preliminary results, I expect a high rate of confirmed positive hits identified through this screen. Secondary assays will follow in which I will map out where the hits fit in the mTOR signalling pathway. Author Contact: Name: Angela Papageorgiou Department: Molecular Biology Phone: 713-898-4119 Or 617-726-6909 Email: angela@molbio.mgh.harvard.edu

59

Poster #58 Abstract Title: The Association between Parents’ and Children’s Use of Oral Health Services: Does Health Insurance Matter? Authors: Inyang A. Isong, MD, MPH, Katharine E. Zuckerman, MD, Karen A. Kuhlthau, PhD, James M. Perrin, MD Affiliation: Center for Child and Adolescent Health Policy, Massachusetts General Hospital, Boston, MA Purpose: Background: Several parental factors influence children’s utilization of oral health services. Some localized studies have shown that children’s dental utilization patterns correlate positively with those of their parents. Having health insurance might influence health-seeking behavior in general, and thus modify the relationship between parents’ and children’s dental utilization. Objective: To investigate associations between parents’ and children’s dental utilization patterns among a representative sample of US children. We additionally sought to assess if having health insurance modifies any relationship between parent and child dental utilization. Materials & Methods: We used the 2007 National Health Interview Survey to analyze a sample of children aged 2-17 years, matched with one parent. Using logistic regression, we tested the association between parents’ and children’s dental utilization. Stratified multivariable analyses tested if health insurance status modified this relationship. Results: Overall, 77% (n=4,634) of children and 64% (n = 3,785) of parents had a dental visit in the previous 12 months. Adjusting for socio-demographic and utilization variables, children were more likely to have a dental visit when their parents also had a dental visit. Children of uninsured parents had a 2.54 odds (95% CI 1.77, 3.66) of having a dental visit when their parents also had a dental visit, while children of insured parents had a 4.04 odds (95% CI 3.19, 5.11) of having a dental visit when their parents also had a dental visit. A test of interaction showed significant differences in the stratified odds ratios. Conclusion: Children of parents who do not use dental care have much lower rates of using dental care themselves. Parental health insurance coverage may play a role in reducing the impact of parental non-use of dental care on children’s underutilization of dental care. Comprehensive strategies to eliminate financial barriers that target parents and not just children may help address children’s underutilization of oral health services. Author Contact: Inyang A Isong, MD, MPH Center for Child and Adolescent Health Policy, Massachusetts General Hospital for Children 50 Staniford Street, Suite 901 Boston, MA 02114 Telephone: 617-643-6182 Fax: 617-726-1886 Email: iisong@partners.org

60

Poster #59 Purinergic receptors in mouse and rat epididymis : Role of luminal ATP and adenosine in V-ATPase activation Belleannee C.1, Da Silva N. 1, Shum W. 1, and Breton S. 1

1Center for Systems Biology, Program in Membrane Biology, Nephrology Division, Massachusetts General Hospital, Boston, MA 02114 In the epididymis, V-ATPase is expressed in clear cells and acidifies the lumen where sperm mature and

are stored. We showed that luminal alkaline pH triggers the apical membrane accumulation of V-ATPase

and that this process is calcium-dependent. However, the link between alkaline pH and intracellular

calcium is not completely elucidated. Some purinergic receptors are sensitive to pH, and we examined

their expression by RT-PCR in rat epithelial cells isolated by laser capture microdissection. mRNA

transcripts for P1 adenosine receptors (subtypes A1, A2B and A3) and P2 ATP receptors (subtypes P2X1,

P2X2, P2X4, P2X11, P2Y1 and P2Y2) were detected. Ecto-ATPase and alkaline phosphatase were also

observed, indicating potential degradation of ATP into adenosine in the epididymal lumen. In vivo

perfusion of rat and mouse epididymis showed that both ATP (600µM) and adenosine (300µM) induced

apical membrane V-ATPase recruitment even in the absence of an alkaline pH stimulus. BAPTA-AM

abolished the ATP-mediated effect, indicating the participation of calcium in this response. These data

show that ATP and adenosine are sufficient to cause the accumulation of V-ATPase in the apical

membrane of clear cells. The paracrine activation of purinergic receptors might, therefore, play a

significant role in the regulation of proton secretion in the male reproductive tract. This work is supported

by NIH grant HD40793.

61

Poster #60 Abstract Title: Regulation of Vacuolar H+-ATPase (V-ATPase) recycling via a RhoA and ROCKII dependent pathway in epididymal clear cells Authors: Winnie W.C. Shum, Nicolas Da Silva, Dennis Brown, and Sylvie Breton

Affiliation: Massachusetts General Hospital, Center for Systems Biology, Program in Membrane Biology and Division of Nephrology The V-ATPase in clear cells is important for luminal acidification in the epididymis, a process that is critical for sperm maturation and storage. We showed that proton secretion in these cells is regulated via V-ATPase recycling. We now find that both RhoA and its effector Rho-kinase ROCK II are enriched in clear cells, where they are located mainly in the sub-apical region, the intracellular structures and the basolateral membrane. In addition, a pool of F-actin was detected beneath the apical membrane and the basolateral membrane of clear cells using a pan-actin antibody or a phalloidin-TRITC on epididymis sections. In epididymal tubules perfused in vivo, agents known to stimulate V-ATPase membrane accumulation (e.g. 1 mM cpt-cAMP) significantly reduced the intensity of cortical F-actin staining using phalloidin-TRITC. The ROCK inhibitor Y27632 (10 µM) or the cell permeable Rho inhibitor, Clostridium C3 transferase (3.75 µg/ml) also induced apical membrane V-ATPase accumulation and significant decrease in the sub-apical versus cytosolic F-actin intensity. These results provide evidence that depolymerizaton of the actin cytoskeleton via inhibition of RhoA or its effector ROCK favors apical membrane V-ATPase accumulation. Thus, cAMP-dependent inhibition of RhoA and its downstream effector ROCKII might be part of the intracellular signaling cascade that is triggered upon agonist-induced apical membrane V-ATPase-dependent accumulation and subsequent activation V-ATPase-dependent proton secretion in clear cells. This research is supported by NIH grant DK38452. Author Contact: Winnie Shum, Center for Systems Biology, Program in Membrane Biology, 617-724-9695, Shum.Winnie@mgh.harvard.edu

62

Poster #61 Taxonomy Ontology Vocabulary Authoring Tool (TOVAT): Another step towards fully automated natural language processing software P Prakash MD, T Shultz, K Dreyer MD ((Massachusetts General Hospital, Harvard Medical School. Boston, MA) Purpose: Prior studies have described the high accuracy of natural language processing software (leximer) for determining presence or absence of clinically significant findings (96%) and imaging recommendations in a large number of radiology reports. This information helps in assessing the patterns in radiology practice and in education and research. However, leximer is limited for evaluating the clinical or pathological type of findings due to heterogeneity in nomenclature used by different radiologists. An intelligent system that can take a set of user queries regarding the type of location of the disease and identify the related reports is a key for automated retrieval of significant positive information from radiology reports. The purpose of our study was to build a software tool which will help in overcoming the limitation of Leximer. Materials and methods: We are building a software tool (TOVAT) that helps automate the retrieval of type and location of findings from radiology reports. TOVAT is a radiology dictionary with all synonyms, suffixes, prefixes and acronyms, built using Radlex as a guide. For example, (1) in normal human anatomy, heart can be represented by cardiac or cardio, therefore a prefix cardi* can be used to depict all the terms related to heart, where * represents any combination of alphabets; (2) in pathology, spond* can be used to depict any pathology related to spinal cord like spondylopathy, spondylosis, spondylolisthesis, etc. We are using TOVAT to train the NLP software (Leximer) and then analyze a subset of six million radiology reports. The trained NLP software will be used to identify the abnormal findings in different body regions (head, neck, chest, abdomen, pelvis, lower and upper extremities, blood vessels) and different modalities of radiology investigations (CT, MRI, USG, radiographs etc.). Results: Additional training of leximer with standardized nomenclature in TOVAT will help in overcoming the limitation of Leximer to identify the positive findings in radiology reports. TOVAT-trained leximer can be used to identify the prevalence of various radiological and pathological findings in radiology reports and guide the recommendations of radiology investigations in future medical practice. Conclusion: NLP trained with TOVAT can help determine the presence of clinically significant findings, as well as their type and location in radiology reports with improvements in search and clinical capabilities. Author Contact: Priyanka Prakash 25 New Chardon Street, Ph: 617-643-5647 Email: pprakash1@partners.org

63

Poster #62

Radiation dose reduction with Chest CT using adaptive statistical iterative reconstruction technique: Initial experience

Priyanka Prakash, MD, Mannudeep K. Kalra, MD, Subbarao Digumarthy, MD, Jo-Anne O. Shepard, MD ((Massachusetts General Hospital, Harvard Medical School. Boston, MA) Purpose: CT scan has become a primary imaging modality in radiology but it comes with large amount of radiation exposure. Continuous efforts are being made to cut down the radiation exposure in CT scans. Recently a new reconstruction technology, adaptive statistical iterative reconstruction technique, has been introduced to assist in radiation dose reduction. The purpose of our study is to assess radiation dose reduction and image quality for weight based chest CT examinations reconstructed with adaptive statistical iterative reconstruction (ASIR) technique. Materials and Methods: With local ethical committee approval, weight adjusted chest CT examinations were performed in 98 patients with ASIR and in 54 weight-matched patients with filtered back projection (FBP) on a 64-slice MDCT. Patients were categorized into three groups of <60 kg (n=32), 61-90 kg (n= 77), and >91 kg (n= 43) for weight based adjustment of automatic exposure control technique. Remaining scan parameters were held constant at 0.984:1 pitch, 120 kVp, 40 mm table feed per rotation, 2.5 mm section thickness. Patients’ weight, scanning parameters, CT dose index volume- CTDI vol, and dose length product – DLP, effective dose, and mean mAs were recorded. Image noise was measured in the descending thoracic aorta at the level of the carina. Two thoracic radiologists independently reviewed chest CT for subjective image noise images (1=too little, 3=too much), diagnostic acceptability (1=fully acceptable, 4=unacceptable), artifacts (1=none, 4=affecting diagnostic information) and critical reproduction of visually sharp anatomical structures. Data were analyzed using analysis of variance (ANOVA). Results: ASIR resulted in overall 28% dose reduction in comparison to FBP. Effective dose values were 6.5±1.7 (28.8% decrease), 7.2±1.6 (27.3% decrease) and 12.7±2.3 (26.8 % decrease) mSv, for <60, 61-90 and >91 kg weight groups with ASIR, compared to 9±2, 10±1.9, and 17±2 mSv with FBP respectively (p<0.0001).Despite dose reduction, there was less noise with ASIR (12.6±2.9) than with FBP (16.6 ± 6.2) (p<0.0001). Also, mean mAs also showed a comparable decrease of 29.2% (ASIR, 248±73; FBP, 343±88 mA), 25.9% (ASIR, 264±66; FBP, 348±87 mA) and 25.7% (ASIR, 481±120; FBP, 641±83 mA) for <60, 61-90 and >91 kg weight groups respectively. The two readers consistently found the lower dose ASIR images to have less or the same noise and artifacts as the FBP (p<0.0001). Also, the diagnostic acceptability and critical reproduction of visually sharp anatomical structures was comparable in the lower dose ASIR and standard dose FBP images. Conclusion: ASIR helps reduce chest CT radiation dose and improve image quality compared to the conventionally used FBP image reconstruction. Author Contact: Priyanka Prakash, Radiology, 25 New Chardon Street, Boston, MA, Ph: 617-643-5647, email: pprakash1@partners.org

64

Poster #63

Rho-kinase inhibition abolishes the vasoconstrictor response to hypoxia in the infant piglet pulmonary arterial circulation Naveen Balasundaram, Paraag Chowdhary, James Titus, Anand Jagannath, Kathleen Carillo, Jeff L Myers. Surgery, Massachusetts General Hospital, Boston, MA Purpose: We examined the effects of the rho-kinase inhibitor fasudil on the regional response of the infant pulmonary arterial (PA) circulation to hypoxia. Materials and Methods: 15 two week old piglets were instrumented to record PA pressure (PAP). Control animals (n=7) were exposed to hypoxia (FiO2 = 0.05). Treated animals (n=8) were infused with fasudil (5mg/Kg) before and during hypoxia. Input mean impedance (Zm, reflecting distal arteriolar vasoconstriction) and characteristic impedance (Zo, reflecting proximal arteriolar opposition to flow) were calculated.

Diameter (mm)

AoP (mmHg)

PAP (mmHg)

LAP (mmHg)

PA Flow (ml/min)

Zm (dynes

cm/ sec-5)

Zo (dynes

cm/ sec-5)

Control baseline 8.5 + 0.4 60 + 3.5 13.5 + 0.8 4.3 + 0.3 298 + 41 4070 +

459 610 +

113

Control Hypoxia 9.6 + 0.3* 66 + 7.2 28.2 +

1.4* 10.6 + 3.1*

418 + 59*

6622 + 1252* 850 + 57

Test baseline 10.7 + 0.6

54.9 + 5.1

13.5 + 0.4 4.6 + 0.8 334 + 37 3620 +

409 461 +

118

Test fasudil 10.5 + 0.6

40 + 0.4*

12.4 + 0.6 4.1 + 0.7 360 + 43 3092 +

366 374 + 54

Test fasudil+hypoxia

11.2 + 0.6

38.7 + 3.7*

16.3 + 1*† 5.2 + 0.7 441 + 44 3262 +

512 330 + 35

Mean + SEM *P < 0.05 v. baseline †P < 0.05 v. fasudil Results: Hypoxia caused a potent vasoconstrictor response distally (increased Zm). Zo was unchanged indicating no alteration of the proximal circulation. Pre-treatment with fasudil completely abolished the increase in Zm and PAP indicating that the distal vasoconstrictor response is rho-kinase dependent. Conclusion:Rho-kinase inhibitors may be an effective therapy for hypoxic pulmonary hypertension in infants, but may be limited by its effect on systemic blood pressure. Author Contact: Naveen Balasundaram, Dept of Pedi Cardiac Surgery, docnaveen@gmail.com/nbalasundaram@partners.org, 617-963-9149/617-726-7638

65

Poster #64

Increased cardiac output and pulmonary impedance contribute in varying amounts to increased pulmonary arterial pressure in acutely hypoxic piglets, depending on the degree of hypoxia Naveen Balasundaram, Paraag Chowdhary, James Titus, Anand Jagannath, Jeff L. Myers, Dept of Pediatric Cardiac Surgery, Massachusetts General Hospital Purpose: It is an accepted fact that the pulmonary vasculature undergoes vasoconstriction and increased impedance with hypoxia. However complete descriptions of all hemodynamic parameters including the changes in pulmonary flow, especially in the pediatric circulation have not been seen in recent literature. The purpose of the study is to demonstrate the effects that different levels of hypoxia have on all the pulmonary hemodynamic parameters in the infant circulation. Materials & Methods: 15 two week old piglets underwent high-fidelity instrumentation to measure their Pulmonary artery pressure, flow, left atrial pressure and aortic pressure. Blood gases and hematocrit were measured at regular intervals. After taking baseline readings, the first group (n=12) was exposed to 10% O2 and the other group (n=9) was exposed to 5% O2, and readings taken again. The Zm (input mean impedance) and Zo (characteristic impedance) were then calculated

Results: In the 5% group, there was a significant rise in the Zm, Zo, pressure, flow and diameter. In the 10% group, there were similar rises in Zo, flow, pressure and diameter. However Zm failed to rise. A comparison of the change in flow shows that the increase in pulmonary flow in the 10% group was much higher than in the 5% group. Conclusion: This data indicates that milder hypoxia may cause rise of pulmonary pressure based on effects of increased pulmonary flow and characteristic impedance (showing proximal vessel opposition to flow). It is only in more severe hypoxia, that the input mean impedance begin to rise and contribute in increasing proportion to the increase in pressure. Hence any increase in PA pressure seen in hypoxia cannot be immediately attributed to an increase in input mean impedance or hypoxic pulmonary vasoconstriction Author Contact: Naveen Balasundaram, Dept of Pediatric Cardiac Surgery, docnaveen@gmail.com/nbalasundaram@partners.org, 617-963-9149/617-726-7638

66

AUTHOR INDEX Author Name Poster # Page # Abu-Yousif, Adnan ............................................................ 19 23 Atassi, Nazem ............................................................ 52 54 Balasundaram, Naveen ............................................................ 63, 64 65, 66 Belleannee, Clemence ............................................................ 59 61 Belov, Vasily ............................................................ 13 18 Bhaumik, Jayeeta ............................................................ 7 12 Biffi, Allessandro ............................................................ 27 31 Brown-Endres, Lauren ............................................................ 31 35 Cannizzo, Elisa ............................................................ 9 14 Casamassima, Francesco ............................................................ 15 20 Chellappa, Vasant ............................................................ 8 13 Chu-Shore, Catherine ............................................................ 43 46 Curley, Michael ............................................................ 14 19 Dyhrjeld-Johnsen, Jonas ............................................................ 36 40 Eddy, Kamryn ............................................................ 45 48 Evans, Conor, L. ............................................................ 39 43 Fazeli, Pouneh ............................................................ 10 15 Feng, Yan ............................................................ 51 53 Finkelstein, Eric, B ............................................................ 21 25 Fuchs, Bryan, C. ............................................................ 22 26 Ge, Rongbin ............................................................ 40 44 Graham, Jay, A. ............................................................ 5 10 Han, Jooman ............................................................ 49 51 Huang, Jianmin ............................................................ 17 22 Islam, Tina ............................................................ 35 39 Isong, Inyang ............................................................ 58 60 Jagannath, Anad ............................................................ 50 52 Kamalian, Shahmir ............................................................ 42 45 Kamalian, Shervin ............................................................ 44 47 Konstantinidis, Ioannis ............................................................ 30 34 Lee, Jeongeun ............................................................ 29 33 Lee, Kang Mi ............................................................ 38 42 Mai, Zhiming ............................................................ 25 29 Martinez-Alvernia, Efrain ............................................................ 24 28 Niu, Yinong ............................................................ 6 11 Pachas, Gladys ............................................................ 20 24 Papageorgiou, Angela ............................................................ 57 59 Prakash, Priyanka ............................................................ 61, 62 63-64 Purschke, Martin ............................................................ 47 49 Qin, Lizeng ............................................................ 55 57 Rai, Prakash ............................................................ 1 6 Rizvi, Imran ............................................................ 4 9 Rychert, Jenna ............................................................ 11 16 Sallum, Ulysses ............................................................ 37 41 Shao, Fangwei ............................................................ 23 27 Shao, Run-Xuan ............................................................ 3 8

67

AUTHOR INDEX Author Name Poster # Page # Shum, Winnie ............................................................ 60 62 Susa, Michiro ............................................................ 33 37 Szymanski, Catherine ............................................................ 2 7 Tauro, Nelson ............................................................ 54 56 Vannini, Patrizia ............................................................ 16 21 Vishwas, Mellekate ............................................................ 56 58 Xu, Feng ............................................................ 26 30 Xu, Hongzhi ............................................................ 28 32 Yu, Elaine, W. ............................................................ 12 17 Zhang, Min ............................................................ 53 55 Zhang, Peng ............................................................ 32 36 Zheng, Lei ............................................................ 34 38 Zou, Lin ............................................................ 48 50

68

NOTES

69

70

71

We are grateful to Susan Riley of Bulfinch Temps, who stepped in and very efficiently helped with many details of the Poster Celebration, including the creation of this abstract book.

(617) 643-2589 thasanORCD@partners.org

Bulfinch 370F

Tayyaba Hasan, PhD Director

Ann Skoczenski, PhD Program Manager Bulfinch 360E (617) 643-1170 askoczenski@partners.org

Boston, MA 02114 55 Fruit Street, Bulfinch 370

Massachusetts General Hospital

www.massgeneral.org/facultydevelopment/orcd